(19) (CA) APPLICATION FOR CANADIAN PATENT (12)

Size: px
Start display at page:

Download "(19) (CA) APPLICATION FOR CANADIAN PATENT (12)"

Transcription

1 MUM - ram., OPIC mem CIPO 71 OFFICE DE LA PROPRIETP - 1 CANADIAN INTELLECTUAL INTELLECTUELLE DU CANADA PROPERTY OFFICE Ottawa Hull K1A 0C9 (21) (Al) 2,164,297 (86) 1994/06/03 (43) 1994/12/22 6 (51) Int.C1. C12N 15/86; C12N 15/62; C12N 5/10; CO7K 19/00; CO7K 14/08; A61K 39/12; A61K 39/295 (19) (CA) APPLICATION FOR CANADIAN PATENT (12) (54) Mengovirus as a Vector for Expression of Foreign Polypeptides (72) Altmeyer, Ralf - France ; Van Der Werf, Sylvie - France ; Girard, Marc - France ; Palmenberg, Ann C. - U.S.A. (71) Institut Pasteur - France ; (30) (US) 08/090, /06/03 (57) 54 Claims Notice: This application is as filed and may therefore contain an incomplete specification. I*. Industrie Canada Industry Canada OPIC - CIPO 191 Canada.

2 [ CORRECTED VERSION* PCT CORRECTED VERSION** (51) International Patent Classification 5 : C12N 15/86, CO7K 14/085, 14/16, 14/145, A61K 39/12, 39/125, 39/21, 39/205, 39/295 (21) International Application Number: PCT/US94/06177 (22) International Filing Date: 3 June 1994 ( ) WORLD INTELLECTUAL PROPERTY ORGANIZATION International Bureau INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) A3 (11) International Publication Number: WO 94/29472 (43) International Publication Date: 22 December 1994 ( ) (74) Agents: TURNER, John, B. et al.; Finnegan, Henderson, Farabow, Garrett & Dunner, 1300 L Street, N.W., Washington, DC (US). (30) Priority Data: 08/090,531 3 June 1993 ( ) US (81) Designated States: CA, JP, US, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, 1E, IT, LU, MC, NL, FT, SE). (60) Parent Application or Grant (63) Related by Continuation US 08/090,531 (CIP) Filed on 3 June 1993 ( ) (71) Applicant (for all designated States except US): INSTITUT PASTEUR [FR/FR]; Rue du Docteur-Roux, F Paris Cedex 15 (FR). Published With international search report. With an indication in relation to a deposited microorganism furnished under Rule 13bis separately from the description. Date of receipt by the International Bureau: 12 December 1994 ( ) (88) Date of publication of the international search report: 16 February 1995 ( ) (72) Inventors; and (75) Inventors/Applicants (for US only): ALTMEYER, Ralf [DE/FR]; 12, part de Beam, F Saint-Cloud (FR). VAN DER WERF, Sylvie [FR/FR]; 112, all& de la Pointe Genete, F Gif-Sur-Yvette (FR). GIRARD, Marc [FR/FR]; 6, rue Cesar-Franck, F Paris (FR). PALMENBERG, Ann, C. [US/US]; 5010 Manor Cross, Madison, WI (US). (54) Title: MENGOVIRUS AS A VECTOR FOR EXPRESSION OF FOREIGN POLYPEPTIDES (57) Abstract This invention relates to attenuated recombinant mengoviruses expressing a heterologous amino acid sequence. The recombinant mengoviruses can express amino acid sequences comprising epitopes of various viral pathogens and are useful as live vaccines for humans and animals against these pathogens. Consequently, this invention also relates to vaccines comprising these recombinant mengoviruses or proteins thereof, cells infected with the recombinant mengoviruses, nucleic acid derived from the recombinant mengoviruses, and methods of inducing an immune response. The invention is exemplified by a recombinant mengovirus comprising amino acids of gp120 of the MN isolate of HIV-I. * (Referred to in PICT Givette No. 06/1995, Section II) * *(Referred to in PCI. Gazette No. II/1995, Section II)

3 WO 94/ PCT/US94/ Description MENGOVIRUS AS A VECTOR FOR EXPRESSION OF FOREIGN POLYPEPTIDES CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part application of United States application Serial No. 08/090,531, filed June 3, The entire disclosure of this application is relied upon and expressly incorporated by reference herein. BACKGROUND OF THE INVENTION This invention relates to mengoviruses modified to contain a nucleic acid encoding one or more foreign polypeptides for immunological and non-immunological purposes. Modified mengoviruses of this invention can be recombinant viruses and/or chimaeric viruses. Mengovirus is a picornavirus belonging to the genus cardiovirus. While the natural host for mengovirus is the mouse, with infection resulting in acute murine meningoencephalitis, mengovirus has a wide host range. In addition to the mouse, mengovirus is also able to infect various animal species including pigs, elephants, and primates including humans. The picornaviruses are a family of pathogenic viruses. Examples of picornaviruses include rhinovirus responsible for the common cold, poliovirus, foot and mouth disease virus (FMDV), Coxsackie viruses, hepatitis A virus, and murine cardioviruses including mengovirus and encephalomyocarditis virus. Picornaviruses have a non-enveloped capsid containing a small positive sense RNA genome. The capsids of all picornaviruses are composed of a 60 subunit protein shell having 5:3:2 icosahedral symmetry with each subunit containing four nonidentical polypeptide chains (VP1, VP2, VP3, and VP4). The shell encapsulates a single copy of the positive sense RNA genome. The three dimensional structure of mengovirus has been determined to atomic resolution by

4 WO 94/ PCT/US94/ X-ray crystallography. Luo et al., Science 235: (1987). The viral genome of mengovirus is a positive stranded RNA molecule of about 7,800 nucleotides in length. The genome is polyadenylated at its 3' end and covalently linked to a small viral polypeptide VPg at its 5' end. The mengoviral genome has been cloned in the form of a complementary DNA (cdna) molecule. The genome includes a single open reading frame encoding a viral polyprotein. The viral proteins are located within the polyprotein in the order L-Pl-P2-P3 from the N to the C terminal end of the polyprotein. The polyprotein is processed by a series of cleavage events to give rise to all structural and nonstructural proteins. The details of this processing are reviewed by Ann C. Palmenberg, Proteolytic Processing of Picornaviral Polyprotein 44 Ann. Rev. Microbiol. 603 (1990), which is incorporated herein by reference. L designates a leader polypeptide that is present in cardio and aphthoviruses. P1 is a precursor to the structural proteins VP1, VP2, VP3, and VP4, which are also identified as 1D, 1B, 1C, and 1A, respectively. P2 and P3 are precursors to the non-structural viral proteins required for the replication of the viral RNA and the processing of the polyprotein. The viral RNA is infectious. That is, upon its introduction into permissive cells it is able to initiate a complete viral multiplication cycle regenerating infectious virus. RNA transcripts synthesized in vitro by an RNA polymerase from the full-length viral cdna were also shown to be infectious. See Duke et al., J. Virol. 63:1822 (1989). The murine cardioviruses, such as mengovirus and encephalomyocarditis virus, and aphthoviruses can be distinguished from other positive strand RNA viruses by the presence of long homopolymeric poly(c) tracts within their 5' noncoding sequences. Although the length, generally bases, and sequence discontinuities, e.g. uridine residues, that sometimes disrupt the homopolymeric sequence have served to characterize natural viral isolates, the exact biological

5 WO 94/ PCT/US94/06177 function of the poly(c) region is not clear. cdna-mediated truncation of the mengovirus poly(c) tract attenuates the pathogenicity of this virus in mice. See Duke et al., Nature 343:474 (1990). An attenuated strain of mengovirus, vm16, has been described by Duke et al., Attenuation of Mengovirus through Genetic Engineering of the 5' Non-coding Poly(C) Tract, Nature 343:474 (1990), which is incorporated herein by reference, and by Duke and Palmenberg, Cloning and Synthesis of Infectious Cardiovirus RNAs Containing Short, Discrete Poly(C) Tracts, J. Virol. 63:1822 (1989), which is also incorporated herein by reference. This attenuated strain contains a deletion in the poly(c) tract of the 5' non-coding region of its genome. This attenuated strain protects mice from a challenge with virulent mengovirus and encephalomyocarditis virus (EMCV). The advent of recombinant DNA technology has permitted the development of live recombinant vaccines. There exists a need in the art for suitable vectors by which polypeptides and/or epitopes or antigens of human or animal pathogens can be incorporated resulting in modified live viruses that can be used in vaccines. SUMMARY OF THE INVENTION This invention helps satisfy the needs in the art by providing, inter alia, a viable modified attenuated mengovirus where a structural or non-structural protein of the mengovirus comprises a heterologous amino acid sequence, a fusion protein of the viable modified mengovirus, a permissive cell infected with the viable modified mengovirus, a recombinant nucleic acid (RNA or DNA) comprising the fulllength sequence of the modified mengovirus, a vaccine, and a method of inducing an immune response. This invention relates, inter alia, to a viable modified mengovirus wherein the modified mengovirus is an attenuated strain and comprises a heterologous nucleotide sequence. An embodiment of this invention relates to a viable modified mengovirus where modified mengovirus is an attenuated strain

6 WO 94/ PCT/US94/ and comprises a heterologous nucleotide sequence coding for a heterologous peptide or protein. In a further embodiment, the viable modified mengovirus is an attenuated strain having a mutation or a deletion in the poly (C) tract of the 5' non-coding region of the genome of the mengovirus. In particular, an embodiment of this invention relates to a viable recombinant mengovirus where the recombinant mengovirus is an attenuated strain having a deletion in the poly(c) tract of the 5' non-coding region of the mengovirus genome, and where the leader polypeptide of the recombinant mengovirus is full-length and comprises a heterologous amino acid sequence. In one specific embodiment of this invention the recombinant mengovirus contains amino acids of gp120 of the MN isolate of HIV-I inserted after amino acid 6 of the leader polypeptide. Another embodiment of this invention relates to a fusion protein comprising a full-length leader polypeptide of an attenuated mengovirus strain into which a heterologous amino acid sequence is inserted. In a further embodiment, this invention relates to permissive cells infected with a recombinant mengovirus of this invention. In specific embodiments the permissive cells are HeLa, VERO, BHK21, and P815 cells. An additional embodiment of this invention relates to a recombinant nucleic acid molecule (RNA or DNA) comprising a mengovirus nucleic acid sequence and a heterologous nucleic acid sequence. Preferably, the heterologous sequence is inserted within the mengovirus sequence encoding the full:- length leader polypeptide. More preferably, the recombinant nucleic acid molecule comprises the full-length attenuated mengovirus sequence and the heterologous sequence inserted within the full-length leader polypeptide sequence. In yet another embodiment, this invention relates to a viral genome of a recombinant mengovirus of this invention. Another embodiment of this invention relates to vaccines comprising a recombinant mengovirus of this invention. In

7 WO 94/ PCT/US94/06177 specific embodiments the vaccines comprise the recombinant mengovirus in admixture with a pharmaceutically acceptable carrier. An additional embodiment of this invention relates to a method of inducing an immune response comprising administering a recombinant mengovirus of the invention via a parenteral or oral route to an organism such as a human or animal, in which an immune response is to be induced. The invention also concerns immunogenic compositions. Such compositions comprise the recombinant mengovirus in admixture with a pharmaceutically acceptable carrier. A further embodiment of this invention relates to a viable recombinant mengovirus of this invention further comprising protease cleavages sites between a heterologous amino acid sequence and the leader polypeptide. In a specific embodiment of this invention, the protease cleavage site is a protease 3C cleavage site. In yet another embodiment, this invention relates to a permissive cell infected with a viable recombinant mengovirus of this invention, where the permissive cell expresses a heterologous amino acid sequence in native form. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic depiction of the organization of the mengovirus genome. Figure 2 is a plasmid map of pm16 depicting several restriction sites, the location of the T7 promoter (arrow), and the sequences derived from pbluescribe M13(+) (designated as pbs, thin line) and the cdna sequences derived from mengovirus (heavy line). Figure 3 is a plasmid map of p05156s. Figure 4 is a plasmid map of pmra-1. The heavy line refers to DNA sequences from mengovirus. The thin line refers to sequences from pbluescribe M13(+). The arrow refers to the T7 promoter. Various restriction sites are identified. The nucleotide numbering system refers to the position of nucleotides in the recombinant plasmid.

8 WO 94/ PCT/US94/ Figure 5 is a plasmid map of pmra-2. The heavy line refers to DNA sequences derived from mengovirus. The thin line refers to sequences from pbluescribe M13(+). The arrow refers to the T7 promoter. The stippled line refers to the sequence of ilgp120-vcn. Various restriction sites are identified. The nucleotide numbering system refers to the position of nucleotides in the recombinant plasmid. Figure 6 is a plasmid map of pmra-3. The heavy line refers to DNA sequences derived from mengovirus. The thin line refers to sequences from pbluescribe M13(+). The arrow refers to the T7 promoter. The stippled line refers to the sequence of Agp120-VCN. Various restriction sites are identified. The nucleotide numbering system refers to the position of nucleotides in the recombinant plasmid. Figure 7 is a diagram of the vmln450 gene organization and the sequence of the recombinant L protein. Amino acids (aa) derived from mengovirus are written in plain letters, while amino acids derived from HIV gp120 are written in bold letters. Amino acids derived from linkers are underlined. Figure 8 depicts the results of a plaque assay. Fig. 8a is the plaque phenotype of vm16 and Fig. 8b is the plaque phenotype of vmln450 stained after 72 hours. Figure 9 depicts Reverse Transcription PCR (RT-PCR) results. RT-PCR was performed with oligonucleotide pairs M- VDW-1/3'VCN for lanes a-e and with oligonucleotide pairs M- VDW-1/M-1094 for lanes g-k. The templates used for the reactions were a) vmln450 RNA, b) vm16 RNA, c) negative control, d) pm16, e) pmra-3, g) vmln450 RNA, h) vm16 RNA, i) negative control, j) pm16, and k) pmra-3. Lane f) contains bacteriophage lambda DNA cut with RIndIII. Figure 10 depicts the sequence of the bogp120 -VCN region at the DNA level (plus strand) and the oligonucleotides used to sequence vmln450 RNA derived PCR products. The boxed sequence is restriction site Ncol. Figure 11 is a 12% SDS-PAGE gel of cytoplasmic extracts of a) mock infected HeLa cells, c) vm16 infected HeLa cells, e) vmln450 infected HeLa cells. Immunoprecipitations of

9 WO 94/ '64297 PCT/US94/ cytoplasmic extracts using MAb50.1 are shown in lane b) for mock infected cells, lane d) for vm16 infected cells, and lane f) vmln450 infected cells. Figure 12A depicts the results of an ELISA assay for sera obtained from mice infected with vmln450, vm16, and a virus free control using gp160 MN-LAI as an antigen. Figure 12B depicts the results of an ELISA assay for sera obtained from Balb/c mice infected with vmln450, vm16, and a virus free control using gp160 LAI as an antigen. The reactivity of Balb/c sera 2 weeks after a first immunization (filled bars) and 2 weeks after a second immunization (stippled bars) with gp160 LAI are shown. Titers are given as reciprocal values of serum dilution giving an O.D. at 490 nm of 1. Figure 12C depicts the results of an ELISA assay for sera obtained from CBA mice infected with vmln450, vm16, and a virus free control using gp160 LAI as an antigen. The reactivity of CBA sera 2 weeks after a first immunization (filled bars) and 2 weeks after a second immunization (stippled bars) with gp160 LAI are shown. Titers are given as reciprocal values of serum dilution giving an O.D. at 490 nm of 1. Figure 12D depicts the results of an ELISA assay for sera obtained from Cynomolgus monkeys infected with vmln450, vm16, and a virus free control using gp160 LAI as an antigen. The reactivity of Cynomolgus monkey preimmune sera (filled bars) and sera 4 weeks after immunization (stippled bars) with gp160 LAI is shown. Titers are given as reciprocal. values of serum dilution giving an O.D. at 490 nm of 1. Figure 13 is a diagram of the mengovirus polyprotein showing a protease 3C cleavage site at the L-VP4 junction. Figure 14 is a diagram of the polyprotein of the recombinant mengovirus vmqg-1 showing the amino acid sequence of the,&gp120-qg-l junction for vmqg-1 and vm16. Figure 15 is a flow diagram of the procedure used to construct pmra-5.

10 WO 94/ PCT/US94/06177 Figure 16 depicts the nucleic acid sequence of pm16. The viral sequences are indicated with Us, i.e. as an RNA sequence, and the plasmid sequences are indicated with Ts, i.e. as a DNA sequence. pm16 is a DNA plasmid. Figure 17 depicts the nucleic acid sequence of pm16-1. The first base of this sequence is the first viral base. The viral sequences are indicated by Us, i.e. as an RNA sequence, and the plasmid sequences are indicated by Ts, i.e. as a DNA sequence. pm16-1 is a DNA plasmid. pm16-1. Figure 18 is a comparison of the sequence of pm16 and Figure 19 depicts the construction of pmln450. The cdna sequence encoding amino acids 299 to 445 of HIV-IMN gpi20 was inserted between amino acids 5 and 6 of the L polypeptide in pm16 cdna at the beginning of the viral polyprotein open reading frame (A). The sequence of the resulting fusion protein, Agp120-L is shown in (B). Leader amino acids are represented in normal type and gp120 amino acids in bold characters. Additional residues encoded by the DNA linkers are underlined. Figure 20 depicts the plaque phenotype of vm16 (A) and vmln450 (B) viruses. Parental and recombinant viral plaques formed on HeLa cell monolayers were stained after 72 h incubation at 37 C. Each well has a diameter of 3.5 cm. Figure 21 depicts the expression of Lgp120-L in vmln450 infected cells. Mock (lanes A, B), vm16 (lanes C, D) and vmln450 (lanes E, F) infected HeLa cells were labelled with 35 S-methionine. Cytoplasmic extracts were prepared at 7 h postinfection and analyzed by 12% SDS-PAGE as described previously (12). Some samples (lanes B, D, F) were immunoprecipitated (13) with MAb 50.1 at 2/.1g/m1 before loading on the gel. The migration of Mengovirus marker proteins is indicated. Figure 22 depicts the Construction of plcmg4. Figure 22a is a portion of the protein sequence and corresponding DNA sequence or pmcs. Figure 22b is a portion of the protein sequence and corresponding DNA sequence of plcmg4. The

11 WO 94/ PCT/US94/ sequence of the Leader peptide region at the beginning of the Open Reading Frame is displayed. Restriction sites are indicated. The cdna sequence coding for the LCMN NP sequence (boxed sequence) was inserted between the sites SnaBl and Nhel of the plasmid pmcs. Underlined sequences result from DNA linkers. Figure 23 depicts the plaque phenotype of vm16 and vlcmg4 virus resulting from transfection of HeLa cells. Cells were grown in 3.5cm wells and coloured after 48 hours. Figure 24 depicts a double-stranded oligonucleotide containing restriction sites XhoI, SnaBI, and NheI. Figure 25 depicts the protein sequence and cdna sequence of the L-coding region of pm16. The position of the XhoI site is indicated. Figure 26 depicts the L-coding region of PMCS. The new restriction sites are indicated. Non-mengovirus amino acids resulting from DNA linkers are boxed. Figure 27 depicts cytoplasmic extracts and radioimmunoprecipitations. Lane 1 is a radioimmunoprecipitation of vmg-24 infected cytoplasmic extracts with monoclonal antibody RV2-22C5. Lane 2 is a cytoplasmic extract of vmg-5-24 infected cells. Lane 3 is a radioimmunoprecipitation (mab RV2-22C5) of vm16 infected cells. Lane 4 is a cytoplasmic extract of vm16 infected cells. Lane 5 is a radioimmunoprecipitation (mab RV2-22C5) of mock infected cells. Lane 6 is a cytoplasmic extract of mock infected cells. DETAILED DESCRIPTION OF THE INVENTION In order that the invention described and claimed herein may be more fully understood, the following detailed description of embodiments of this invention is provided. Within this description various terms of art are employed. These terms are generally used in their ordinary and well recognized sense. Various terms that are employed throughout this description are defined infra. As used herein, the term "recombinant virus" refers to a genetically modified virus. A recombinant virus can comprise

12 WO 94/ PCT/US94/ protein or nucleic acid from at least one other organism. Thus, a recombinant virus can refer to a virus expressing a non-structural heterologous polypeptide as well as viruses comprising a heterologous polypeptide as a structural element. Moreover, a recombinant virus can be a chimaeric virus. As used herein, the term "attenuated strain" refers to a strain with reduced disease-producing ability and/or pathogenicity. As used herein, the term "genome" refers to the nucleic acid comprising all the genes of a species. The nucleic acid making up the genome may be RNA or DNA depending on the nature of the species. For example, the genome of picornaviruses or other RNA viruses is made up of RNA, while the human genome is made up of DNA. As used herein, the term "heterologous" refers to a substance not naturally found in a given species. For example, the term "heterologous amino acid sequence" when used with reference to a specific virus refers to an amino acid sequence not found in that virus, e.g., the proteins of that virus. As used herein, the term "nucleotide or nucleic acid sequence" refers to a linear series of nucleotides connected by covalent bonds between the 3' and 5' carbons of adjacent nucleotides. A nucleotide or nucleic acid sequence may be an RNA sequence or a DNA sequence. As used herein, the term "amino acid sequence" refers to a linear series of amino acids connected by covalent bonds. As used herein, the term "fusion protein" refers to a protein comprising at least two amino acid sequences, where one of the amino acid sequences is not normally found together in nature with the other amino acid sequence(s). For example, a mengovirus fusion protein can comprise a mengovirus amino acid sequence covalently linked to a heterologous amino acid sequence. As used herein, the term "epitope" refers to a configuration of amino acids in a protein, where the

13 WO 94/ PCT/US94/ configuration of amino acids is associated with an immune response. For example, an epitope can be defined by an antigenic motif that is recognized by an antibody and that can induce an immune response. An epitope may be, but is not limited to, a linear sequence of amino acids. As used herein, the term "permissive cell" refers to c.1 cell that can be productively infected with a virus. Thus, a permissive cell to mengovirus, is a cell that can be infected by mengovirus. As used herein, the term "recombinant nucleic acid molecule" refers to a hybrid nucleotide sequence (RNA or DNA) comprising at least two nucleotide sequences placed together by in vitro manipulation or a clone thereof. As used herein, the term "cdna" refers to complementary DNA. In the case of organisms whose genome is comprised of DNA, the cdna is complementary to mrna or a fragment thereof. In the case of organisms whose genome is comprised of RNA, the cdna is complementary to the genome of the organism or a fragment thereof. As used herein, the term "polypeptide" refers to a linear series of amino acids connected one to the other by peptide bonds. The term "polypeptide" includes but is not limited to proteins. As used herein, the term "expression" refers to the process of producing a polypeptide from a structural gene. As used herein, the term "polyprotein" refers to a covalently linked linear series of amino acids comprising more than one protein. In some cases, proteins constituting a polyprotein can be released by endoproteolytic cleavage by a specific protease. The genomic organization of mengovirus is shown in Figure 1. This figure depicts mapping of the viral polypeptides to the genome as well as various intermediates in the processing of the polyprotein to mature components of the virus. The poly(c) region is also identified in Figure 1. As described by Duke et al., supra, and Duke and Palmenberg,

14 WO 94/29472 PCT/US94/ supra, deletions in this region are associated with an attenuated phenotype. Consequently, plasmids containing cdna of the genome of mengovirus with mutations, e.g., substitutions and deletions, in the poly(c) region can be used as the source of mengovirus DNA for the construction of various embodiments of this invention relating to recombinant mengoviruses exhibiting an attenuated phenotype. In preferred embodiments of this invention, a suitable source of mengovirus nucleic acid is plasmid pm16. pm16 has been deposited at the Collection Nationale de Cultures de Micro-organismes (C.N.C.M.) in Paris, France on June 2, 1993 under accession number A partial plasmid map of pm16 is shown in Figure 2 and the sequence of pm16 is shown in Figure 16. This plasmid encodes a mutated poly(c) tract of C 13 UC 10, but otherwise comprises a DNA sequence corresponding to the full-length genome of mengovirus inserted between the EcoRI and BamHI restriction sites of the double-stranded replicative form vector pbluescribe M13(+). Consequently, this plasmid contains the mengovirus cdna downstream from the T7 promoter. In other embodiments of this invention, a suitable source of mengovirus nucleic acid is pm16-1. pm16-1 has also been deposited at the C.N.C.M. on June 2, 1993 under accession number The sequence of pm16-1 is shown in Figure 17. In other embodiments of this invention other plasmids may be used as a source of mengovirus nucleic acid. For example, pm18, encoding a C 8 poly(c) tract, or pm19, encoding a C 12 poly(c) tract or a plasmid containing a complete deletion of the poly(c) tract can be used. If an attenuated phenotype is not required pmwt, encoding the wild type poly(c) tract C 5 ec 10, can be used. As each of these plasmids contains DNA complementary to the mengovirus genome outside the poly(c) tract, one plasmid can be constructed from another mengovirus plasmid (or wild type mengovirus DNA) by various in vitro manipulations. One possibility is the replacement of the EcoRV - AvrII restriction fragment

15 WO 94/ PCT/US94/ containing the poly(c) tract from one plasmid with the appropriate EcoRV - AvrII fragment of the plasmid to be constructed. Once a vector encoding an attenuated mengovirus genome in DNA form has been obtained, a heterologous nucleotide sequence encoding an amino acid sequence to be expressed by the recombinant mengovirus can be inserted within the coding region of the mengovirus genome. In specific embodiments of this invention the nucleic acid sequence codes for a heterologous antigen or epitope. The site at which the heterologous nucleotide sequence is inserted can be at a restriction site. In specific embodiments of this invention, the site is at the NCol restriction site encompassing nucleotide 729 of the mengovirus genome. When the heterologous nucleotide sequence is inserted at a restriction site, the mengovirus DNA vector is restricted with the appropriate enzyme to cleave the DNA vector. The heterologous DNA sequence is then ligated to the restricted mengovirus vector to produce a recombinant DNA molecule comprising the mengovirus genome -- now including the heterologous nucleotide sequence. When the heterologous nucleotide sequence is not inserted at a restriction site, one of ordinary skill in the art can select a restriction fragment of the DNA vector comprising the insertion site. A synthetic DNA fragment can then be synthesized that corresponds to the selected restriction fragment but additionally includes the heterologous nucleotide sequence inserted at the desired site. The synthetic DNA fragment can then be inserted in place of the selected restriction fragment in the DNA vector to generate a recombinant DNA molecule comprising a recombinant mengovirus genome cdna. The heterologous nucleotide sequence can be prepared in a variety of ways. For example, the sequence may be obtained by specifically cleaving cdna encoding the heterologous polypeptide to be expressed by the recombinant mengovirus.

16 WO 94/ PCT/US94/06177 For example, this may be accomplished using appropriate restriction enzymes. Alternatively, the heterologous nucleotide sequence can be chemically synthesized using methods well known in the art. Recombinant mengoviruses and proteins or expression products thereof that comprise a desired heterologous polypeptide, e.g., an antigen or epitope, can be obtained by generating an RNA transcript from the recombinant DNA molecule comprising a heterologous nucleotide sequence inserted into the recombinant mengovirus genome. For example, in the case of pm16 and pml6-1 an RNA transcript can be produced in vitro using T7 RNA polymerase. Alternative promoters and corresponding polymerases can be substituted. Aliquots of the transcription mixture can be used to transfect permissive cells. For example, the DEAE Dextran, calcium phosphate, poly-ornithin, electroporation, and synthetic transfection agents can be used to transfect mammalian cells such as HeLa, VERO, BHK21, and P815. The production of progeny viruses can be monitored by microscopy, and the viruses can be released by well known methods of cellular disruption, for example, freezing and thawing. In specific embodiments of this invention, the heterologous polypeptide is inserted within the leader polypeptide (L). It was determined that insertion of the foreign epitope at 6 amino acids from the N terminus of the mengovirus polyprotein does not render L non-functional and thus does not interfere with the multiplication of the virus either in vitro or in vivo. Other insertion sites may be chosen within the viral genome for insertion at positions where the insert does not interfere with functions that are important for the viral life cycle. If there are no restriction sites at a suitable insertion site they can be introduced, e.g. by site directed mutagenesis. Thus in principle, all restriction sites and other locations within the genome can be envisaged for insertion with the exception of well defined functional areas, e.g. the catalytic triad of 3C. Preferred sites

17 WO 94/ PCT/US94/ include non-structural regions of the genome, e.g., P2, P3 and/or regions corresponding to the N- or the C- terminus of viral proteins. In order to produce a fusion protein comprising L and a foreign polypeptide, such as an antigen or epitope, the foreign nucleotide sequence is inserted into the mengovirus cdna within the L polypeptide coding sequence in such a way as to conserve reading frame. The recombinant virus can then express the foreign sequences as part of the viral polyprotein. The polyprotein is processed, inter alia, to the form of a fusion protein comprising the L polypeptide with the heterologous polypeptide inserted therein. The fusion protein can then be obtained from the cytoplasm of infected cells. In terms of this invention the heterologous DNA sequence inserted into a mengovirus vector can encode any amino acid sequence. In certain embodiments of this invention the amino acid sequence comprises a heterologous epitope. In these embodiments, the heterologous amino acid sequence can comprise several foreign epitopes or a single foreign epitope, or it can define an epitope together with other amino acids, e.g. mengovirus amino acids. In other embodiments, the amino acid sequence can consist essentially of a heterologous epitope. By way of example, amino acid sequences of various embodiments of this invention are listed in Table I.

18 WO 94/29472 Cd.* A t PCT/US94/06177 Table I PATHOGEN PROTEIN SUBDOMAIN AMINO ACIDS 1/Hepatitis B Virus S / Streptococcus type 24 M 12 N terminal spec. type 5 M type 6 M as type 19 M 3/ Influenza virus NS1 HA2 4/ Plasmodium 93kd blood falciparium stage protein 5/ Schistosoma Immunodominant mansoni Calcium binding 6/ Hepatitis C Virus proteins (CaBP's) C El , , / Borrelia OSP A burgdoferi 8/ HTLV-1 Env / Chlamydia spec. MOMP variable domain (Outer membrane IV protein) 10/ HIV-I, HIV-2, glycoprotein, e.g. V3 loop SIV gp120 (PND) 11/TGEV S S In embodiments of this invention the heterologous DNA sequence to be inserted is generally in the range of about 700-1,100 bases. Sequences given in Table I are examples of heterologous sequences for the construction of recombinant mengovirus of the invention. Recombinant mengoviruses containing heterologous sequences of species not given in the Table can be constructed as indicated herein. In cases where the heterologous sequence is too large to be expressed in. mengovirus a set of recombinant mengoviruses each expressing a part of the given protein can be constructed. The heterologous DNA sequence of this invention can encode more than one polypeptide. The DNA sequences encoding the polypeptides can be directly linked to each other or they can be separated by a joining sequence. In specific

19 WO 94/ PCT/US94/06177 embodiments, these joining sequences can encode a cleavage site. In embodiments of this invention the L polypeptide of the recombinant virus comprises a segment of gp120 of HIV-I. In specific embodiments, the L polypeptide comprises amino acids of gp120 of the MN strain of HIV-I. In more specific embodiments, amino acids of gp120 of the MN strain of HIV-I are inserted after amino acid 6 of the L polypeptide. This HIV sequence comprises sequences coding for the V3 loop, which constitutes the principal neutralization determinant (PND) and sequences downstream involved in binding of the gp120 molecule to the CD4 receptor. The resulting recombinant mengovirus expresses an HIV-I gp120 - mengovirus L fusion protein that was recognized by HIV-I specific antibodies and induced anti HIV-I antibodies in animals. The HIV-I gp120 - mengovirus L fusion protein also induced a gp120 - specific cytotoxic immune response in animals. In terms of antigenicity, an L fusion protein comprising a foreign epitope can retain the ability to induce and bind antibodies directed to the native protein sequence. Hence, the strategy could be applied to any foreign protein that exhibits antigenic properties of interest. Consequently, in cases of single well-defined and short epitopes, the construction of recombinant mengoviruses, containing these epitopes in a larger protein, is the strategy of choice. Recombinant viruses of this invention have been shown to induce antibodies in mice and cynomolgus monkeys that bind to the protein and/or neutralize the pathogen from which the foreign sequences are derived. Therefore, the induction of an immune response in other animal species susceptible to mengovirus, such as humans, is predicted based on the in vitro and in vivo results obtained. Thus, a protective immune response may be elicited by recombinant viruses of this invention. For example, a recombinant virus expressing sequences of the G protein of rabies can be engineered in

20 WO 94/ PCT/US94/ accordance with this invention for use as a vaccine in animals, including mice. Similarly, a recombinant virus expressing sequences from the glycoprotein of HTLV-1 could be obtained for use in macaques, other primates, or humans. The antigenicity and immunogenicity of proteins comprising foreign epitopes expressed by the recombinant mengoviruses can be improved in various ways: the size of the heterologous nucleotide sequence encoding the foreign epitope can be increased to express larger segments of the foreign antigen (or the whole antigen) up to a maximum size of about 350 amino acids, the foreign antigen can be expressed in native form rather than as a fusion protein, and selective targeting of the foreign antigen to appropriate cell compartments can be achieved to allow post-translational modifications, such as glycosylation, that can be important for the antigenicity and/or immunogenicity of the fusion protein. In embodiments of this invention, the recombinant mengovirus can express multiple sequences of one protein. In addition, the recombinant mengovirus can comprise multiple sequences from different proteins. Thus, this invention makes it possible to immunize or induce an immune response against multiple pathogens at the same time. In certain embodiments of this invention a heterologous polypeptide can be expressed in native form by including protease cleavage sites between the amino acid sequence of the heterologous polypeptide and the mengovirus sequences. In preferred embodiments of this invention mengovirus protease 3C cleavage site is used. The endogenous mengovirus protease 3C is responsible for most cleavages of the mengovirus polyprotein. For example protease 3C mediates the cleavage between the L-peptide and VP4(1A) by specifically cleaving precursor protein L-P1-2A at a Q-G amino acid linkage yielding free L-peptide and P1-2A. Figure 13 is a depiction of the mengovirus genome with this protease 3C cleavage site indicated. The amino acid sequence in Figure 13 is a sufficient substrate for cleavage

2.1.2 Characterization of antiviral effect of cytokine expression on HBV replication in transduced mouse hepatocytes line

2.1.2 Characterization of antiviral effect of cytokine expression on HBV replication in transduced mouse hepatocytes line i 1 INTRODUCTION 1.1 Human Hepatitis B virus (HBV) 1 1.1.1 Pathogenesis of Hepatitis B 1 1.1.2 Genome organization of HBV 3 1.1.3 Structure of HBV virion 5 1.1.4 HBV life cycle 5 1.1.5 Experimental models

More information

Chapter 18: Applications of Immunology

Chapter 18: Applications of Immunology Chapter 18: Applications of Immunology 1. Vaccinations 2. Monoclonal vs Polyclonal Ab 3. Diagnostic Immunology 1. Vaccinations What is Vaccination? A method of inducing artificial immunity by exposing

More information

Virological Methods. Flint et al. Principles of Virology (ASM), Chapter 2

Virological Methods. Flint et al. Principles of Virology (ASM), Chapter 2 Virological Methods Flint et al. Principles of Virology (ASM), Chapter 2 Overview The most commonly used laboratory methods for the detection of viruses and virus components in biological samples can be

More information

CUSTOM ANTIBODIES. Fully customised services: rat and murine monoclonals, rat and rabbit polyclonals, antibody characterisation, antigen preparation

CUSTOM ANTIBODIES. Fully customised services: rat and murine monoclonals, rat and rabbit polyclonals, antibody characterisation, antigen preparation CUSTOM ANTIBODIES Highly competitive pricing without compromising quality. Rat monoclonal antibodies for the study of gene expression and proteomics in mice and in mouse models of human diseases available.

More information

INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE Q5B

INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE Q5B INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE ICH HARMONISED TRIPARTITE GUIDELINE QUALITY OF BIOTECHNOLOGICAL PRODUCTS: ANALYSIS

More information

Chapter 9. Biotechnology and Recombinant DNA Biotechnology and Recombinant DNA

Chapter 9. Biotechnology and Recombinant DNA Biotechnology and Recombinant DNA Chapter 9 Biotechnology and Recombinant DNA Biotechnology and Recombinant DNA Q&A Interferons are species specific, so that interferons to be used in humans must be produced in human cells. Can you think

More information

Classic Immunoprecipitation

Classic Immunoprecipitation 292PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Classic Immunoprecipitation Utilizes Protein A/G Agarose for Antibody Binding (Cat.

More information

Aviva Systems Biology

Aviva Systems Biology Aviva Custom Antibody Service and Price Mouse Monoclonal Antibody Service Package Number Description Package Contents Time Price Customer provides antigen protein $6,174 Monoclonal package1 (From protein

More information

KMS-Specialist & Customized Biosimilar Service

KMS-Specialist & Customized Biosimilar Service KMS-Specialist & Customized Biosimilar Service 1. Polyclonal Antibody Development Service KMS offering a variety of Polyclonal Antibody Services to fit your research and production needs. we develop polyclonal

More information

RNA Viruses. A Practical Approac h. Alan J. Cann

RNA Viruses. A Practical Approac h. Alan J. Cann RNA Viruses A Practical Approac h Alan J. Cann List of protocols page xiii Abbreviations xvii Investigation of RNA virus genome structure 1 A j. Easton, A.C. Marriott and C.R. Pringl e 1 Introduction-the

More information

Recombinant DNA & Genetic Engineering. Tools for Genetic Manipulation

Recombinant DNA & Genetic Engineering. Tools for Genetic Manipulation Recombinant DNA & Genetic Engineering g Genetic Manipulation: Tools Kathleen Hill Associate Professor Department of Biology The University of Western Ontario Tools for Genetic Manipulation DNA, RNA, cdna

More information

Recombinant DNA and Biotechnology

Recombinant DNA and Biotechnology Recombinant DNA and Biotechnology Chapter 18 Lecture Objectives What Is Recombinant DNA? How Are New Genes Inserted into Cells? What Sources of DNA Are Used in Cloning? What Other Tools Are Used to Study

More information

Anti-ATF6 α antibody, mouse monoclonal (1-7)

Anti-ATF6 α antibody, mouse monoclonal (1-7) Anti-ATF6 α antibody, mouse monoclonal (1-7) 73-500 50 ug ATF6 (activating transcription factor 6) is an endoplasmic reticulum (ER) membrane-bound transcription factor activated in response to ER stress.

More information

Biotechnology and Recombinant DNA

Biotechnology and Recombinant DNA Biotechnology and Recombinant DNA Recombinant DNA procedures - an overview Biotechnology: The use of microorganisms, cells, or cell components to make a product. Foods, antibiotics, vitamins, enzymes Recombinant

More information

CHAPTER 8 IMMUNOLOGICAL IMPLICATIONS OF PEPTIDE CARBOHYDRATE MIMICRY

CHAPTER 8 IMMUNOLOGICAL IMPLICATIONS OF PEPTIDE CARBOHYDRATE MIMICRY CHAPTER 8 IMMUNOLOGICAL IMPLICATIONS OF PEPTIDE CARBOHYDRATE MIMICRY Immunological Implications of Peptide-Carbohydrate Mimicry 8.1 Introduction The two chemically dissimilar molecules, a peptide (12mer)

More information

Biotechnology and Recombinant DNA (Chapter 9) Lecture Materials for Amy Warenda Czura, Ph.D. Suffolk County Community College

Biotechnology and Recombinant DNA (Chapter 9) Lecture Materials for Amy Warenda Czura, Ph.D. Suffolk County Community College Biotechnology and Recombinant DNA (Chapter 9) Lecture Materials for Amy Warenda Czura, Ph.D. Suffolk County Community College Primary Source for figures and content: Eastern Campus Tortora, G.J. Microbiology

More information

STANDARD 2 Students will demonstrate appropriate safety procedures and equipment use in the laboratory.

STANDARD 2 Students will demonstrate appropriate safety procedures and equipment use in the laboratory. BIOTECHNOLOGY Levels: 11-12 Units of Credit: 1.0 CIP Code: 51.1201 Prerequisite: Biology or Chemistry Skill Certificates: #708 COURSE DESCRIPTION is an exploratory course designed to create an awareness

More information

Essentials of Real Time PCR. About Sequence Detection Chemistries

Essentials of Real Time PCR. About Sequence Detection Chemistries Essentials of Real Time PCR About Real-Time PCR Assays Real-time Polymerase Chain Reaction (PCR) is the ability to monitor the progress of the PCR as it occurs (i.e., in real time). Data is therefore collected

More information

European Medicines Agency

European Medicines Agency European Medicines Agency July 1996 CPMP/ICH/139/95 ICH Topic Q 5 B Quality of Biotechnological Products: Analysis of the Expression Construct in Cell Lines Used for Production of r-dna Derived Protein

More information

ELISA BIO 110 Lab 1. Immunity and Disease

ELISA BIO 110 Lab 1. Immunity and Disease ELISA BIO 110 Lab 1 Immunity and Disease Introduction The principal role of the mammalian immune response is to contain infectious disease agents. This response is mediated by several cellular and molecular

More information

1.Gene Synthesis. 2.Peptide & Phospho-P. Assembly PCR. Design & Synthesis. Advantages. Specifications. Advantages

1.Gene Synthesis. 2.Peptide & Phospho-P. Assembly PCR. Design & Synthesis. Advantages. Specifications. Advantages 1.Gene Synthesis Assembly PCR Looking for a cdna for your research but could not fish out the gene through traditional cloning methods or a supplier? Abnova provides a gene synthesis service via assembly

More information

Custom Antibodies Services. GeneCust Europe. GeneCust Europe

Custom Antibodies Services. GeneCust Europe. GeneCust Europe GeneCust Europe Laboratoire de Biotechnologie du Luxembourg S.A. 6 rue Dominique Lang L-3505 Dudelange Luxembourg Tél. : +352 27620411 Fax : +352 27620412 Email : info@genecust.com Web : www.genecust.com

More information

Title: Mapping T cell epitopes in PCV2 capsid protein - NPB #08-159. Date Submitted: 12-11-09

Title: Mapping T cell epitopes in PCV2 capsid protein - NPB #08-159. Date Submitted: 12-11-09 Title: Mapping T cell epitopes in PCV2 capsid protein - NPB #08-159 Investigator: Institution: Carol Wyatt Kansas State University Date Submitted: 12-11-09 Industry summary: Effective circovirus vaccines

More information

CHAPTER 6: RECOMBINANT DNA TECHNOLOGY YEAR III PHARM.D DR. V. CHITRA

CHAPTER 6: RECOMBINANT DNA TECHNOLOGY YEAR III PHARM.D DR. V. CHITRA CHAPTER 6: RECOMBINANT DNA TECHNOLOGY YEAR III PHARM.D DR. V. CHITRA INTRODUCTION DNA : DNA is deoxyribose nucleic acid. It is made up of a base consisting of sugar, phosphate and one nitrogen base.the

More information

DNA Fingerprinting. Unless they are identical twins, individuals have unique DNA

DNA Fingerprinting. Unless they are identical twins, individuals have unique DNA DNA Fingerprinting Unless they are identical twins, individuals have unique DNA DNA fingerprinting The name used for the unambiguous identifying technique that takes advantage of differences in DNA sequence

More information

restriction enzymes 350 Home R. Ward: Spring 2001

restriction enzymes 350 Home R. Ward: Spring 2001 restriction enzymes 350 Home Restriction Enzymes (endonucleases): molecular scissors that cut DNA Properties of widely used Type II restriction enzymes: recognize a single sequence of bases in dsdna, usually

More information

Common Course Topics Biology 1414: Introduction to Biotechnology I

Common Course Topics Biology 1414: Introduction to Biotechnology I Common Course Topics Biology 1414: Introduction to Biotechnology I Assumptions Students may be enrolled in this course for several reasons; they are enrolled in the Biotechnology Program, they need a science

More information

VLLM0421c Medical Microbiology I, practical sessions. Protocol to topic J10

VLLM0421c Medical Microbiology I, practical sessions. Protocol to topic J10 Topic J10+11: Molecular-biological methods + Clinical virology I (hepatitis A, B & C, HIV) To study: PCR, ELISA, your own notes from serology reactions Task J10/1: DNA isolation of the etiological agent

More information

CCR Biology - Chapter 9 Practice Test - Summer 2012

CCR Biology - Chapter 9 Practice Test - Summer 2012 Name: Class: Date: CCR Biology - Chapter 9 Practice Test - Summer 2012 Multiple Choice Identify the choice that best completes the statement or answers the question. 1. Genetic engineering is possible

More information

Viruses. Viral components: Capsid. Chapter 10: Viruses. Viral components: Nucleic Acid. Viral components: Envelope

Viruses. Viral components: Capsid. Chapter 10: Viruses. Viral components: Nucleic Acid. Viral components: Envelope Viruses Chapter 10: Viruses Lecture Exam #3 Wednesday, November 22 nd (This lecture WILL be on Exam #3) Dr. Amy Rogers Office Hours: MW 9-10 AM Too small to see with a light microscope Visible with electron

More information

Structure and Function of DNA

Structure and Function of DNA Structure and Function of DNA DNA and RNA Structure DNA and RNA are nucleic acids. They consist of chemical units called nucleotides. The nucleotides are joined by a sugar-phosphate backbone. The four

More information

Transfection-Transfer of non-viral genetic material into eukaryotic cells. Infection/ Transduction- Transfer of viral genetic material into cells.

Transfection-Transfer of non-viral genetic material into eukaryotic cells. Infection/ Transduction- Transfer of viral genetic material into cells. Transfection Key words: Transient transfection, Stable transfection, transfection methods, vector, plasmid, origin of replication, reporter gene/ protein, cloning site, promoter and enhancer, signal peptide,

More information

From DNA to Protein. Proteins. Chapter 13. Prokaryotes and Eukaryotes. The Path From Genes to Proteins. All proteins consist of polypeptide chains

From DNA to Protein. Proteins. Chapter 13. Prokaryotes and Eukaryotes. The Path From Genes to Proteins. All proteins consist of polypeptide chains Proteins From DNA to Protein Chapter 13 All proteins consist of polypeptide chains A linear sequence of amino acids Each chain corresponds to the nucleotide base sequence of a gene The Path From Genes

More information

Antibody Function & Structure

Antibody Function & Structure Antibody Function & Structure Specifically bind to antigens in both the recognition phase (cellular receptors) and during the effector phase (synthesis and secretion) of humoral immunity Serology: the

More information

Chapter 20: Biotechnology: DNA Technology & Genomics

Chapter 20: Biotechnology: DNA Technology & Genomics Biotechnology Chapter 20: Biotechnology: DNA Technology & Genomics The BIG Questions How can we use our knowledge of DNA to: o Diagnose disease or defect? o Cure disease or defect? o Change/improve organisms?

More information

About Our Products. Blood Products. Purified Infectious/Inactivated Agents. Native & Recombinant Viral Proteins. DNA Controls and Primers for PCR

About Our Products. Blood Products. Purified Infectious/Inactivated Agents. Native & Recombinant Viral Proteins. DNA Controls and Primers for PCR About Our Products Purified Infectious/Inactivated Agents ABI produces a variety of specialized reagents, allowing researchers to choose the best preparations for their studies. Available reagents include

More information

HCS604.03 Exercise 1 Dr. Jones Spring 2005. Recombinant DNA (Molecular Cloning) exercise:

HCS604.03 Exercise 1 Dr. Jones Spring 2005. Recombinant DNA (Molecular Cloning) exercise: HCS604.03 Exercise 1 Dr. Jones Spring 2005 Recombinant DNA (Molecular Cloning) exercise: The purpose of this exercise is to learn techniques used to create recombinant DNA or clone genes. You will clone

More information

Transcription and Translation of DNA

Transcription and Translation of DNA Transcription and Translation of DNA Genotype our genetic constitution ( makeup) is determined (controlled) by the sequence of bases in its genes Phenotype determined by the proteins synthesised when genes

More information

CONTENT. Chapter 1 Review of Literature. List of figures. List of tables

CONTENT. Chapter 1 Review of Literature. List of figures. List of tables Abstract Abbreviations List of figures CONTENT I-VI VII-VIII IX-XII List of tables XIII Chapter 1 Review of Literature 1. Vaccination against intracellular pathogens 1-34 1.1 Role of different immune responses

More information

THE His Tag Antibody, mab, Mouse

THE His Tag Antibody, mab, Mouse THE His Tag Antibody, mab, Mouse Cat. No. A00186 Technical Manual No. TM0243 Update date 01052011 I Description.... 1 II Key Features. 2 III Storage 2 IV Applications.... 2 V Examples - ELISA..... 2 VI

More information

Production of antigens and antibodies in plants: alternative technology?

Production of antigens and antibodies in plants: alternative technology? Production of antigens and antibodies in plants: alternative technology? George Lomonossoff John Innes Centre Norwich, UK ECOPA, Alicante 29 th Sept. 2006 Why use Plants as Biofactories? Produce large

More information

Custom Antibody Services

Custom Antibody Services Custom Antibody Services Custom service offerings DNA sequence Plasmid Peptide Structure Protein Peptide Small molecule Cells Spleen Lymphocytes Antigen Preparation Immunization Fusion & Subcloning Expansion

More information

STUDIES ON SEED STORAGE PROTEINS OF SOME ECONOMICALLY MINOR PLANTS

STUDIES ON SEED STORAGE PROTEINS OF SOME ECONOMICALLY MINOR PLANTS STUDIES ON SEED STORAGE PROTEINS OF SOME ECONOMICALLY MINOR PLANTS THESIS SUBMITTED FOR THE DEGREB OF DOCTOR OF PHILOSOPHY (SCIENCE) OF THE UNIVERSITY OF CALCUTTA 1996 NRISINHA DE, M.Sc DEPARTMENT OF BIOCHEMISTRY

More information

QUANTITATIVE RT-PCR. A = B (1+e) n. A=amplified products, B=input templates, n=cycle number, and e=amplification efficiency.

QUANTITATIVE RT-PCR. A = B (1+e) n. A=amplified products, B=input templates, n=cycle number, and e=amplification efficiency. QUANTITATIVE RT-PCR Application: Quantitative RT-PCR is used to quantify mrna in both relative and absolute terms. It can be applied for the quantification of mrna expressed from endogenous genes, and

More information

How many of you have checked out the web site on protein-dna interactions?

How many of you have checked out the web site on protein-dna interactions? How many of you have checked out the web site on protein-dna interactions? Example of an approximately 40,000 probe spotted oligo microarray with enlarged inset to show detail. Find and be ready to discuss

More information

The Biotechnology Education Company

The Biotechnology Education Company EDVTEK P.. Box 1232 West Bethesda, MD 20827-1232 The Biotechnology 106 EDV-Kit # Principles of DNA Sequencing Experiment bjective: The objective of this experiment is to develop an understanding of DNA

More information

IIID 14. Biotechnology in Fish Disease Diagnostics: Application of the Polymerase Chain Reaction (PCR)

IIID 14. Biotechnology in Fish Disease Diagnostics: Application of the Polymerase Chain Reaction (PCR) IIID 14. Biotechnology in Fish Disease Diagnostics: Application of the Polymerase Chain Reaction (PCR) Background Infectious diseases caused by pathogenic organisms such as bacteria, viruses, protozoa,

More information

2007 7.013 Problem Set 1 KEY

2007 7.013 Problem Set 1 KEY 2007 7.013 Problem Set 1 KEY Due before 5 PM on FRIDAY, February 16, 2007. Turn answers in to the box outside of 68-120. PLEASE WRITE YOUR ANSWERS ON THIS PRINTOUT. 1. Where in a eukaryotic cell do you

More information

Why use passive immunity?

Why use passive immunity? Vaccines Active vs Passive Immunization Active is longer acting and makes memory and effector cells Passive is shorter acting, no memory and no effector cells Both can be obtained through natural processes:

More information

Custom Antibody Services

Custom Antibody Services prosci-inc.com Custom Antibody Services High Performance Antibodies and More Broad Antibody Catalog Extensive Antibody Services CUSTOM ANTIBODY SERVICES Established in 1998, ProSci Incorporated is a leading

More information

HiPer RT-PCR Teaching Kit

HiPer RT-PCR Teaching Kit HiPer RT-PCR Teaching Kit Product Code: HTBM024 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 4 hours Agarose Gel Electrophoresis: 45 minutes Storage Instructions: The

More information

1 2 3 4 5 6 Figure 4.1: Gel picture showing Generation of HIV-1subtype C codon optimized env expressing recombinant plasmid pvax-1:

1 2 3 4 5 6 Figure 4.1: Gel picture showing Generation of HIV-1subtype C codon optimized env expressing recombinant plasmid pvax-1: Full-fledged work is in progress towards construction and cloning of codon optimized envelope with subsequent aims towards immunization of mice to study immune responses. 1 2 4 5 6 Figure 4.1: Gel picture

More information

2. True or False? The sequence of nucleotides in the human genome is 90.9% identical from one person to the next. False (it s 99.

2. True or False? The sequence of nucleotides in the human genome is 90.9% identical from one person to the next. False (it s 99. 1. True or False? A typical chromosome can contain several hundred to several thousand genes, arranged in linear order along the DNA molecule present in the chromosome. True 2. True or False? The sequence

More information

Basics of Immunology

Basics of Immunology Basics of Immunology 2 Basics of Immunology What is the immune system? Biological mechanism for identifying and destroying pathogens within a larger organism. Pathogens: agents that cause disease Bacteria,

More information

Lecture 8. Protein Trafficking/Targeting. Protein targeting is necessary for proteins that are destined to work outside the cytoplasm.

Lecture 8. Protein Trafficking/Targeting. Protein targeting is necessary for proteins that are destined to work outside the cytoplasm. Protein Trafficking/Targeting (8.1) Lecture 8 Protein Trafficking/Targeting Protein targeting is necessary for proteins that are destined to work outside the cytoplasm. Protein targeting is more complex

More information

Viral Infection: Receptors

Viral Infection: Receptors Viral Infection: Receptors Receptors: Identification of receptors has come from expressing the gene for the receptor in a cell to which a virus does not normally bind -OR- By blocking virus attachment

More information

Name Date Period. 2. When a molecule of double-stranded DNA undergoes replication, it results in

Name Date Period. 2. When a molecule of double-stranded DNA undergoes replication, it results in DNA, RNA, Protein Synthesis Keystone 1. During the process shown above, the two strands of one DNA molecule are unwound. Then, DNA polymerases add complementary nucleotides to each strand which results

More information

VETERINARY SERVICES MEMORANDUM NO. 800.90

VETERINARY SERVICES MEMORANDUM NO. 800.90 August 5, 1998 VETERINARY SERVICES MEMORANDUM NO. 800.90 Subject: To: Guidelines for Veterinary Biological Relative Potency Assays and Reference Preparations Based on ELISA Antigen Quantification Veterinary

More information

MAB Solut. MABSolys Génopole Campus 1 5 rue Henri Desbruères 91030 Evry Cedex. www.mabsolut.com. is involved at each stage of your project

MAB Solut. MABSolys Génopole Campus 1 5 rue Henri Desbruères 91030 Evry Cedex. www.mabsolut.com. is involved at each stage of your project Mabsolus-2015-UK:Mise en page 1 03/07/15 14:13 Page1 Services provider Department of MABSolys from conception to validation MAB Solut is involved at each stage of your project Creation of antibodies Production

More information

PRACTICE TEST QUESTIONS

PRACTICE TEST QUESTIONS PART A: MULTIPLE CHOICE QUESTIONS PRACTICE TEST QUESTIONS DNA & PROTEIN SYNTHESIS B 1. One of the functions of DNA is to A. secrete vacuoles. B. make copies of itself. C. join amino acids to each other.

More information

Recombinant DNA Technology

Recombinant DNA Technology Recombinant DNA Technology Dates in the Development of Gene Cloning: 1965 - plasmids 1967 - ligase 1970 - restriction endonucleases 1972 - first experiments in gene splicing 1974 - worldwide moratorium

More information

Expression and Purification of Recombinant Protein in bacteria and Yeast. Presented By: Puspa pandey, Mohit sachdeva & Ming yu

Expression and Purification of Recombinant Protein in bacteria and Yeast. Presented By: Puspa pandey, Mohit sachdeva & Ming yu Expression and Purification of Recombinant Protein in bacteria and Yeast Presented By: Puspa pandey, Mohit sachdeva & Ming yu DNA Vectors Molecular carriers which carry fragments of DNA into host cell.

More information

Rapid Acquisition of Unknown DNA Sequence Adjacent to a Known Segment by Multiplex Restriction Site PCR

Rapid Acquisition of Unknown DNA Sequence Adjacent to a Known Segment by Multiplex Restriction Site PCR Rapid Acquisition of Unknown DNA Sequence Adjacent to a Known Segment by Multiplex Restriction Site PCR BioTechniques 25:415-419 (September 1998) ABSTRACT The determination of unknown DNA sequences around

More information

PCR was carried out in a reaction volume of 20 µl using the ABI AmpliTaq GOLD kit (ABI,

PCR was carried out in a reaction volume of 20 µl using the ABI AmpliTaq GOLD kit (ABI, Supplemental Text/Tables PCR Amplification and Sequencing PCR was carried out in a reaction volume of 20 µl using the ABI AmpliTaq GOLD kit (ABI, Foster City, CA). Each PCR reaction contained 20 ng genomic

More information

Alison Stewart 11/12/06 Prokaryotic Cells, Eukaryotic cells and HIV: Structures, Transcription and Transport Section Handout Discussion Week #7

Alison Stewart 11/12/06 Prokaryotic Cells, Eukaryotic cells and HIV: Structures, Transcription and Transport Section Handout Discussion Week #7 Alison Stewart 11/12/06 Prokaryotic Cells, Eukaryotic cells and HIV: Structures, Transcription and Transport Section Handout Discussion Week #7 Compare and contrast the organization of eukaryotic, prokaryotic

More information

Molecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION

Molecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION Molecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION Susan Carson Heather B. Miller D.Scott Witherow ELSEVIER AMSTERDAM BOSTON HEIDELBERG LONDON NEW YORK OXFORD PARIS SAN DIEGO SAN

More information

Final Review. Aptamers. Making Aptamers: SELEX 6/3/2011. sirna and mirna. Central Dogma. RNAi: A translation regulation mechanism.

Final Review. Aptamers. Making Aptamers: SELEX 6/3/2011. sirna and mirna. Central Dogma. RNAi: A translation regulation mechanism. Central Dogma Final Review Section Week 10 DNA RNA Protein DNA DNA replication DNA RNA transcription RNA Protein translation **RNA DNA reverse transcription http://bass.bio.uci.edu/~hudel/bs99a/lecture20/lecture1_1.html

More information

Competitive PCR Guide

Competitive PCR Guide Lit. # L0126 Rev. 8/99 Competitive PCR Guide Table of Contents I. What is Competitive PCR? A. Difficulties of Quantitative Analysis in Normal PCR... 2 B. Principle of Competitive PCR... 3 C. Competitive

More information

The Use of Antibodies in Immunoassays

The Use of Antibodies in Immunoassays TECHNICAL NOTE The Use of Antibodies in Immunoassays Introduction Structure of an IgG Antibody Immunological reagents are the backbone of every immunoassay system. Immunoassays can be utilized to quantitatively

More information

Molecular Cloning, Product Brochure

Molecular Cloning, Product Brochure , Product Brochure Interest in any of the products, request or order them at Bio-Connect. Bio-Connect B.V. T NL +31 (0)26 326 44 50 T BE +32 (0)2 503 03 48 Begonialaan 3a F NL +31 (0)26 326 44 51 F BE

More information

The immune response Antibodies Antigens Epitopes (antigenic determinants) the part of a protein antigen recognized by an antibody Haptens small

The immune response Antibodies Antigens Epitopes (antigenic determinants) the part of a protein antigen recognized by an antibody Haptens small The immune response Antibodies Antigens Epitopes (antigenic determinants) the part of a protein antigen recognized by an antibody Haptens small molecules that can elicit an immune response when linked

More information

Understanding the immune response to bacterial infections

Understanding the immune response to bacterial infections Understanding the immune response to bacterial infections A Ph.D. (SCIENCE) DISSERTATION SUBMITTED TO JADAVPUR UNIVERSITY SUSHIL KUMAR PATHAK DEPARTMENT OF CHEMISTRY BOSE INSTITUTE 2008 CONTENTS Page SUMMARY

More information

Viral Replication. Viral Replication: Basic Concepts

Viral Replication. Viral Replication: Basic Concepts Viral Replication Scott M. Hammer, M.D. Viral Replication: Basic Concepts Viruses are obligate intracellular parasites Viruses carry their genome (RNA or DNA) and sometimes functional proteins required

More information

4. DNA replication Pages: 979-984 Difficulty: 2 Ans: C Which one of the following statements about enzymes that interact with DNA is true?

4. DNA replication Pages: 979-984 Difficulty: 2 Ans: C Which one of the following statements about enzymes that interact with DNA is true? Chapter 25 DNA Metabolism Multiple Choice Questions 1. DNA replication Page: 977 Difficulty: 2 Ans: C The Meselson-Stahl experiment established that: A) DNA polymerase has a crucial role in DNA synthesis.

More information

Protein Purification and Analysis

Protein Purification and Analysis Protein Purification and Analysis Numbers of genes: Humans ~40,000 genes Yeast ~6000 genes Bacteria ~3000 genes Solubility of proteins important for purification: 60-80% soluble, 20-40% membrane Some proteins

More information

Protein Expression. A Practical Approach J. HIGGIN S

Protein Expression. A Practical Approach J. HIGGIN S Protein Expression A Practical Approach S. J. HIGGIN S B. D. HAMES List of contributors Abbreviations xv Xvi i 1. Protein expression in mammalian cell s Marlies Otter-Nilsson and Tommy Nilsso n 1. Introduction

More information

RevertAid Premium First Strand cdna Synthesis Kit

RevertAid Premium First Strand cdna Synthesis Kit RevertAid Premium First Strand cdna Synthesis Kit #K1651, #K1652 CERTIFICATE OF ANALYSIS #K1651 Lot QUALITY CONTROL RT-PCR using 100 fg of control GAPDH RNA and GAPDH control primers generated a prominent

More information

The role of IBV proteins in protection: cellular immune responses. COST meeting WG2 + WG3 Budapest, Hungary, 2015

The role of IBV proteins in protection: cellular immune responses. COST meeting WG2 + WG3 Budapest, Hungary, 2015 The role of IBV proteins in protection: cellular immune responses COST meeting WG2 + WG3 Budapest, Hungary, 2015 1 Presentation include: Laboratory results Literature summary Role of T cells in response

More information

specific B cells Humoral immunity lymphocytes antibodies B cells bone marrow Cell-mediated immunity: T cells antibodies proteins

specific B cells Humoral immunity lymphocytes antibodies B cells bone marrow Cell-mediated immunity: T cells antibodies proteins Adaptive Immunity Chapter 17: Adaptive (specific) Immunity Bio 139 Dr. Amy Rogers Host defenses that are specific to a particular infectious agent Can be innate or genetic for humans as a group: most microbes

More information

TOOLS sirna and mirna. User guide

TOOLS sirna and mirna. User guide TOOLS sirna and mirna User guide Introduction RNA interference (RNAi) is a powerful tool for suppression gene expression by causing the destruction of specific mrna molecules. Small Interfering RNAs (sirnas)

More information

PRODUCTION AND QUALITY CONTROL OF MEDICINAL PRODUCTS DERIVED BY RECOMBINANT DNA TECHNOLOGY

PRODUCTION AND QUALITY CONTROL OF MEDICINAL PRODUCTS DERIVED BY RECOMBINANT DNA TECHNOLOGY PRODUCTION AND QUALITY CONTROL OF MEDICINAL PRODUCTS DERIVED BY RECOMBINANT DNA TECHNOLOGY Guideline Title Production and Quality Control of Medicinal Products derived by recombinant DNA Technology Legislative

More information

Optimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a

Optimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a Optimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a Ryan S. Stowers, 1 Jacqueline A. Callihan, 2 James D. Bryers 2 1 Department of Bioengineering, Clemson University, Clemson,

More information

Aviva Systems Biology

Aviva Systems Biology Aviva Custom Antibody Services and Prices Rabbit Polyclonal Antibody Service Package Number Description Package Contents Time Price Polyclonal package 1 (From protein to antiserum) Polyclonal package 2

More information

pcas-guide System Validation in Genome Editing

pcas-guide System Validation in Genome Editing pcas-guide System Validation in Genome Editing Tagging HSP60 with HA tag genome editing The latest tool in genome editing CRISPR/Cas9 allows for specific genome disruption and replacement in a flexible

More information

Guidance for Industry

Guidance for Industry Guidance for Industry Interpreting Sameness of Monoclonal Antibody Products Under the Orphan Drug Regulations U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation

More information

Recombinant DNA Unit Exam

Recombinant DNA Unit Exam Recombinant DNA Unit Exam Question 1 Restriction enzymes are extensively used in molecular biology. Below are the recognition sites of two of these enzymes, BamHI and BclI. a) BamHI, cleaves after the

More information

ab185916 Hi-Fi cdna Synthesis Kit

ab185916 Hi-Fi cdna Synthesis Kit ab185916 Hi-Fi cdna Synthesis Kit Instructions for Use For cdna synthesis from various RNA samples This product is for research use only and is not intended for diagnostic use. Version 1 Last Updated 1

More information

Genetics Module B, Anchor 3

Genetics Module B, Anchor 3 Genetics Module B, Anchor 3 Key Concepts: - An individual s characteristics are determines by factors that are passed from one parental generation to the next. - During gamete formation, the alleles for

More information

Biopharmaceutical Process Evaluated for Viral Clearance

Biopharmaceutical Process Evaluated for Viral Clearance Authored by S. Steve Zhou, Ph.D. Microbac Laboratories, Inc., Microbiotest Division The purpose of Viral Clearance evaluation is to assess the capability of a manufacturing production process to inactivate

More information

JIANGSU CARTMAY INDUSTRIAL CO.,LTD www.labfurniture.asia mail: info@labfurniture.asia

JIANGSU CARTMAY INDUSTRIAL CO.,LTD www.labfurniture.asia mail: info@labfurniture.asia The basic layout, the main functions and instrumentation concept of micro Inspection Division laboratory, 1, Virology Laboratory 1. Functions: for the city to monitor the prevalence of HIV disease, dealing

More information

Chapter 2 Antibodies. Contents. Introduction

Chapter 2 Antibodies. Contents. Introduction Chapter 2 Antibodies Keywords Immunohistochemistry Antibody labeling Fluorescence microscopy Fluorescent immunocytochemistry Fluorescent immunohistochemistry Indirect immunocytochemistry Immunostaining

More information

Your partner in immunology

Your partner in immunology Your partner in immunology Expertise Expertise Reactivity Reactivity Quality Quality Advice Advice Who are we? Specialist of antibody engineering Covalab is a French biotechnology company, specialised

More information

Analysis of the adaptive immune response to West Nile virus

Analysis of the adaptive immune response to West Nile virus Analysis of the adaptive immune response to West Nile virus Jonathan Bramson (PI) Robin Parsons Alina Lelic Galina Denisova Lesley Latham Carole Evelegh Dmitri Denisov Strategy for characterizing T cell

More information

Chapter 20: Biotechnology

Chapter 20: Biotechnology Name Period The AP Biology exam has reached into this chapter for essay questions on a regular basis over the past 15 years. Student responses show that biotechnology is a difficult topic. This chapter

More information

Application Guide... 2

Application Guide... 2 Protocol for GenomePlex Whole Genome Amplification from Formalin-Fixed Parrafin-Embedded (FFPE) tissue Application Guide... 2 I. Description... 2 II. Product Components... 2 III. Materials to be Supplied

More information

Ms. Campbell Protein Synthesis Practice Questions Regents L.E.

Ms. Campbell Protein Synthesis Practice Questions Regents L.E. Name Student # Ms. Campbell Protein Synthesis Practice Questions Regents L.E. 1. A sequence of three nitrogenous bases in a messenger-rna molecule is known as a 1) codon 2) gene 3) polypeptide 4) nucleotide

More information

Protein immunoblotting

Protein immunoblotting Protein immunoblotting (Western blotting) Dr. Serageldeen A. A. Sultan Lecturer of virology Dept. of Microbiology SVU, Qena, Egypt seaas@lycos.com Western blotting -It is an analytical technique used to

More information

Bacterial Transformation and Plasmid Purification. Chapter 5: Background

Bacterial Transformation and Plasmid Purification. Chapter 5: Background Bacterial Transformation and Plasmid Purification Chapter 5: Background History of Transformation and Plasmids Bacterial methods of DNA transfer Transformation: when bacteria take up DNA from their environment

More information

2. The number of different kinds of nucleotides present in any DNA molecule is A) four B) six C) two D) three

2. The number of different kinds of nucleotides present in any DNA molecule is A) four B) six C) two D) three Chem 121 Chapter 22. Nucleic Acids 1. Any given nucleotide in a nucleic acid contains A) two bases and a sugar. B) one sugar, two bases and one phosphate. C) two sugars and one phosphate. D) one sugar,

More information

Custom Antibodies & Recombinant Proteins

Custom Antibodies & Recombinant Proteins Custom Antibodies & Recombinant Proteins INTRODUCTION Custom services to meet your research and development requirements Improvements in health, medicine and diagnostics over the past century can be largely

More information