Study On The Expression Of ORF5 Gene Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome Virus(PRRSV) is the causative agent ofPorcine Reproductive and Respiratory Syndrome(PRRS),a newly recognized pig disease ofeconomical importance,characterized by reproductive failure(abortions,stillbirths,mummiedfetuses)and respiratory problems in any age. We isolated Guangdong strain of PRRSV andsequenced ORF5 gene. We constructed recombinant plasmid ORF5-pBAD/g â…¢ andexpressed in E coli. We constructed recombinant plasmid PAdCMV-ORF5 and identified it.(1) Expression in prokaryote system According to the complete sequence of American PRRSV,a pair of primers that haverestrict enzyme sites Kpnâ… and Xbaâ… were designed. A 602bp gene fragment was amplifiedfrom the pMD-ORF5 by PCR and inserted into the pBAD-gâ…¢C Plasmid. A ORF5-pBAD/gâ…¢C recombinant plasmid was constructed and transformed inio the TOP10. A fusionProtein,weight 22 kDa,was induced and expressed with l mmol/L IPTG. Western blotshowed that it could react with the antibody against 6 Histine.(2) Expression in eukaryote system According to the complete sequence of American PRRSV,a pair of primers that haverestrict enzyme sites Kpnâ… and Xbaâ… were designed. A 602bp gene fragment was amplifiedfrom the pMD-ORF5 by PCR. The pAdTrack-CMV vector and the PCR product (ORF5 gene)were all digested by the two restriction enzymes Kpnâ… and Xbaâ… , then pAdTrack-CMVvector and ORF5 gene were linked together with T4 DNA ligase. The ligation mixture wastransformed into competent E.coli DH5a preparaed with Calcium Chloride method. Theplasmid DNAs of some kanamycin resistant colonies were identified by PCR analysis andrestriction analysis. And the resultant plasmid was linearized with Pme I and subsequentlycotransfected into the E.Coli BJ5183 together with the backbone plasmid pAdEasy. Aftertheir homogeneous recombination, then screened the positive recombinant adenovirusgenome plasmid DNA, the recombinant adenovirus genome DNA was digested with Pacâ… and transfected into 293 cells with Lipofectamine to package the recombinant adenovirus.The recombinant adenovirus carrying ORF5 gene was screened under fluorescent microscopyand identified by PCR analysis.