Establishment And Application Of Prrsv RT-PCR Detection Method And Construction Of Pam Line Stably Expressing Gp5of SD16Strain

Porcine alveolar macrophages (PAM) were separated from20lung samples of pigslaughterhouse in Yangling. The ORF7gene of porcine reproductive and respiratorysyndrome virus (PRRSV) SD16strain was amplified by RT-PCR method using the specificprimers. Recombinant plasmid pMD18-T-N were constructed and the method of PRRSVdetection was established. Practical application shows that this detection method has goodspecificity, sensitivity and reproducibility. Two of Twenty clinical samples were positive ofPRRSV by this detection method.According to the genome sequence of PRRSV SD16strain, a pair of primer for GP5amplification was synthesized. GP5gene was amplified by method of RT-PCR and clonedinto the plasmid pMD18-T. Pick a single clone, to determine the sequence after bacteria PCRand enzyme cutting detection,Sequencing results show that the whole length of PRRSV SD16ORF5gene is603base pairs, encoding200amino acids as a complete open reading frame.The plasmid pPB-GP5for transfection was constructed. It takes the PAM cells as thetarget cell for transfection,and the PAM stably expressing PRRSV GP5was obtained withfluorescent labeling and antibody screening.Immunofluorescence assay (IFA), cell growthcurve and western blot were used for cell line identification. Green fluorescence could beobserved in a part of PAM cells, which means the plasmid pPB-GP5were induced into thesome cells correctly. Specific red fluorescence could be observed in IFA, which means theGP5protein express in cells correctly. A specific band of25.0ku could be found in themembrane of the western blot, which consistent with prediction and mean that stablytransfected cell lines capable of expressing GP5was obtained. The growth curves of theobtained cell line and PAM, which means no obvious damage for the plasmid transfection.Overall,these studies have three results, firstly the PAM was obtained, and then themethod for PRRSV detection was established, finally, this method has application value forPRRSV clinical testing. Then the recombinant plasmid pPB-GP5was transferred into PAM cells. The cell line PAM-GP5stably expressing PRRSV GP5was obtained with fluorescentlabeling and antibody screening. The cell line was used to study PRRSV SD16strain etiologymolecular biology. The research for the study of the pathogenesis of PRRS and GP5effects isof great significance.