Detection of cd NA End Sequence of Porcine Fibrinogen Like Protein2 and It s Structure Analysis

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1 HER EDI TA S ( Beijing) 25 (1) :17 21,2003 FGL 2 cd NA 1,Anand Ghanekar 2,Matthew Chan 2,Ming Feng Liu 2, 1,Gary Levy 2 (1., ;2., ) : FGL 2 cdna 2 32 P dctp cdna DNA ;cdna (rapid amplification of cdna end,race) RNA,,, Advantage 2 PCR ; FGL 2 3 PCR, FGL 2 3, DNA Advantage 2 PCR ; PCR DNA,RACE, DNA PCR FGL 2 3 :RACE ; PCR ; FGL2 :Q953 :A : (2003) Detection of cd NA End Sequence of Porcine Fibrinogen Like Protein2 and It s Structure Analysis L IU Hao 1,Anand Ghanekar 2,Matthew Chan 2,Ming Feng Liu 2,L IU Yong2Feng 1,Gary Levy 2 (1. Organ Transplant U nit, First of Filiated Hospital, China Medical U niveristy, S henyang China, ; 2. M ultiorgan Transplant Program, Toronto General Hospital, U niversity of Toronto,621 U niversity A ve, N U , Toronto, ON, Canada, M5 G 2 C4) Abstract :The purpose of this study is to detect the end sequence of porcine FGL 2 gene cdna and make a preliminary analysis of it s structure. Porcine DNA library was screened by a cdna probe labeled with radioactive isotope 2 32 P dctp. Rapid amplification of cdna end ( RACE) : Retroverse transcription product of total RNA extracted from normal porcine tissue was used as the template,gene specific primers were designed and advantage 2 polymerase mix was used in PCR,of which using porcine genomic DNA as the template :forward primer was designed according to the acquired consensus re2 gion of human and pig FGL 2 3 sequences while reverse primer was designed from human FGL 2 3 end downstream se2 quence ; TA cloning. Screening library failed to get any specific positive clone ; the specific transcription initiation site and first poly A signal were successfully detected by RACE reaction although it fails again to detect the second poly A signal. The unknown sequence of porcine FGL 2 3 end including the second poly A signal was successfully detected by PCR using genomic DNA as the template. RACE reaction can be applied as an effective method to detect the specific transcription ini2 tiation and termination sites. Using advantage 2 polymerase mix instead of regular DNA polymerase may significantly im2 prove the sensitivity,accuracy and specificity of PCR reaction. If it met particular difficulties in the regular screening of DNA li2 brary,pcr reaction utilizing primers designed from the known consensus sequence and genomic DNA as template may be consid2 ered as an appropriate alternative. Key words :RACE ; PCR ; FGL2 : ; : : ( ),,,,, Tel : ,E2mail com

2 18 HER EDI TA S ( Beijing) (fibrinogen like protein, F GL2), Xa, [1 ] [2 ], [3 ], ( hyperacute rejection, HAR), ( human complement decay accelerating factor hdaf, CD55) [4 ] ( membrane cofactor protein, MCP,CD46) (membrane at2 tack complex inhibition factor,macif,cd59) [5 ], (acute vascular rejection,avr) (delayed xenograft rejection,dxr),, [6 ] FGL 2, FGL 2,, FGL 2 DNA, ( ) FGL 2 3, 3, cdna D NA FGL 2 3, 1 : 5 - A TA TAA GCG A GT GTC CCC TGA TT - 3 ; 1 : 5 - TTT TCC TCA GTG A TG A GT GAA CC : 5 - CAA AAA TGC A GC TTT CCC CA T T - 3 ; 2 :5 - GGA GTG CAC TTT TGA TCA CA G AAA - 3 DNA (Q IA GEN Genomic DNA Mini Kit for Cultured Cell Q IA GEN,www. qiagen. com), DNA PCR ( PTC Programmable Thermal Controller), PCR 1 %, DNA PCR DNA (Q IAquick Gel Extraction Kit Q IA GEN) 2 32 P dctp ( rediprime II random prime labeling system code RPN 1633 AMERSHAM,www. amersham. com), 1 2, DNA (Lambda Library User Manual P T CLON2 TECH,www. clontech. com) ex2 presshyb hybridization solution CLON TECH P T FGL 2 cd NA ( SMAR T RACE cdna Amplification Kit CLON TECH PT3269-1) RNA ( RNeasy Mini Kit Q IA2 GEN), PowerScript TM RTPCR cdna,, FGL 2 GSP ( gene specific primer) N GSP(nested gene spe2 cific primer) : GSP1 : 5 - CGC CAT GTT CTG GTG AAG TTG GTG CT - 3 ;N GSP1 :5 - CCC CCA TGG TCT CCA TGT CAC AGT AA - 3 ; GSP2 : 5 - AAG CTG AAG CTG TCG AAC TGG TGC TG - 3 ; N GSP2 : 5 - CCA AGG AAG AGA TCG ACG GGC TTC AG - 3 ; GSP3 : 5 - CAA TTG GAC CCC TAC CCT GCA AAT GT - 3 ;N GSP3 :5 - TGT GCC ACA GGT GCA ACC CAA AAA - 3 ; GSP4 :5 - TGG TAG GTA TGG GTG GGG AAT GAA GCA C - 3 (polydt) 5 - AAG CAG TGG TAT CAA CGC AGA GTA C( T) 30 N - 1 N - 3 (N = A, C,G,or T ; N - 1 = A, G,or C) UPM ( universal primer mixture) 5 - CTA ATA CGA CTC ACT ATA GGG CAA GCA GTG GTA TCA ACG CAG AGT - 3, PCR ( PE GeneAmp Systems 2400/ 9600), TOUCH DOWN, : 1 :94 5s,72 3min,5 ; 2 :94 5s,70 10s,72 3min,5 ; 3 :94 5s,68 10s,72 3min,25,PCR, TA PCR TOPO ( TOPO TA Cloning Kit INVITROGEN www. invitro2

3 1 : FGL 2 cdna 19 gen. com),, DNA (plasmid miniprep kit QIAGEN),, ( 1) 1 FGL 2 cdna ( RACE) DNA,,,, Northern RNA, 1. 5kb 4. 3kb,. polya1, polya2, RNA, cdna GSP1 NGSP1, UPM 5 RACE, bp,, 5 4 C PowerScript GSP2 NGSP2 GSP3 NGSP3 GSP4 polydt, 3 RACE, bp, ; GSP3 NGSP3,GSP4 650bp, A 27 DNA RACE, bp ; 2256bp Fig. 1 Rapid amplif ication of the cdna end of porcine FGL 2 gene 1. 3 FGL 2 3 D NA - PCR FGL 2 3 PCR 5 - AA T A GG A TA CCA AA T GTA AA T G - 3 ; FGL TGG TGT TCC TCT A TT TGC CTC T - 3 ; DNA advan2 tage 2 (CLON TECH) PCR, PCR TA, DNA ( 2) 2 DNA : 1 2 RACE : GSP1 PCR 890bp, N GSP1 830bp, FGL 2 GSP2 1540bp,

4 20 HER EDI TA S ( Beijing) N GSP2 1100bp GSP3 N GSP3 GSP4 650bp ( 3) FGL 2 3 DNA PCR : PCR 1233bp ( 3), 3 2 FGL 2 3 DNA PCR FGL 2,,, FGL 2 3 PCR ; FGL 2 3 ; DNA PCR, 1233bp Fig. 2 PCR reaction of porcine FGL 2 3 end sequence using genomic DNA as template 3 PCR RACE : GSP1 890bp ; N GSP1 830bp ; GSP2 1540bp ;N GSP2 - GSP4 650bp FGL bp ; GSP3 N GSP3 ; DNA PCR, 1230bp Fig. 3 Agarose gel electrophe resis of PCR product,, FGL 2, [7 ] (http :/ / www. ncbi. nlm. nih. Gov/ BLAST/ ),, DNA, 3, DNA (Midi Lamb2 da DNA Kit Q IA GEN),, DNA, Southern,, DNA ( pbluescript II Phagemid Vector STRA TA GEN E), (One Shot TOP10 Competent Cell INV ITRO GEN),, FGL 2 FGL 2 RNA FGL 2cDNA Northern, 1. 5kb 4. 3kb, FGL 2 RNA 1, 2,, RACE RNA, RTPCR cdna, cdna GSP,, PCR, PCR cdna3 : (1) 23 28bp ; (2) GC 50 % 70 %; (3) 70 ; (4), 3, ; (5) PCR 2kb, Primer 3 OU TPU T PCR (http :/ / www2genome. wi. mit. edu/ cgi2bin/ primer/ primer3-www. cgi), NETPrimer (http :/ / www. premier biosoft. com/ net primer/ netprlaunch/ netprlaunch. Html)

5 1 : FGL 2 cdna 21, PCR, TOUCHDOWN [8 ],PCR 70, 3 RACE, 5 RACE, PowerScript RTPCR cdna, RNA5, 3 5 polyc, polyc RNA 5 SMART II A Oligonucleotide 5 - AAG CAG TGG TAT CAA CGC AGA GTA CGC GGG- 3 3 polyc, 5 15 AAG CAG TGG TAT CAA CGC AGA GT UPM, PCR, cdna PCR RNA5, GSP1 N GSP1, PCR PCR PCR, 2256,, 3 5, FGL 2 3 PCR, FGL 2 3, DNA PCR, PCR TA, DNA,,5 100 %,, FGL 2 3 AA TAAA cdna, PCR Ad2 vantage 2, : (1) TITAN IUM Taq DNA, N, 5,, ; (2),, ; (3), DNA,, PCR,, [9 ],,,, PCR, 3kb, 18kb [10 ] ( References) : [1 ] Levy G A,Liu M F,Ding J W, et al. Molecular and functional analysis of the human prothrombinase gene ( HFGL 2) and its role in viral hepatitis[j ]. American Journal of Pathology,2000,156 : [2 ] Clark D A,Ding J W, Yu G,Levy G A, Gorczynski R M. Fgl2 prothrombinase expression in mouse trophoblast and decidua trig2 gers abortion but may be countered by OX - 2[J ]. Mol Hum Re2 prod,2001,7 (2) : [ 3 ] Dorling A, Lechier R I. Disordered thromboregulation after xenografting. Current opinion in organ transplantation [ M ]. Lip2 pincott Williams & Wilkins Inc,2001,6 : [ 4 ] Carrington C A,dos Santos Cruz G. Effect of cell surface concentration of human DAF on transgenic pig aortic endothelial cells on the degree of protection afforded against human complement deposition[j ]. Xeno2 transplantation,2001,8 (2) : [5 ] Diamond L E,Quinn C M,Martin M J,Lawson J, Platt J L,Lo2 gan J S. A human CD46 transgenic pig model system for the study of discordant xenotransplantation [ J ]. Transplantation, 2001, 71 (1) : [6 ] Cowan P J, Aminia A,Barlow H, et al. Renal xenografts from triple transgenic pigs are not hyperacutely rejected but cause coag2 ulopathy in non - immunosuppressed baboons [ J ]. Transplanta2 tion,2000,69 : [ 7 ] Yuwaraj S,Ding J W,Liu M F, et al. Genomic characterization, localization,and functional expression of FGL 2,the human gene encoding fibroleukin :a novel human procoagulant [J ]. Genomics, 2001,71 : [8 ] Johnson J R,Clabots C. Improved repetitive - element PCR fin2 gerprinting of Salmonella enterica with the use of extremely ele2 vated annealing temperatures[j ]. Clin Diagn Lab Immunol,2000, 7 (2) : [ 9 ] Kellogg D E,Rybalkin I,Chen S,Mukhamedova N,Vlasik T,Siebert P,Chenchik A. Taqstart antibody : Hotstart PCR facilitated by a neu2 tralizing monoclonal antibody directed against [J ]. BioTechniques,1994,16 : Taq DNA polymerase [ 10 ] Cheng S,Fockler C,Barnes W M, Higuchi R. Effective amplifica2 tion of long targets from cloned inserts and human genomic DNA [J ]. Proc Natl Acad Sci USA,1994,91 :