The Prelininary Research On Porcine Reproductive And Respiratory Syndrome Virus GP5on The Relationship Between Cell Apoptosis And Viral Replication

Porcine reproductive and respiratory syndrome （PRRS） is an acute and highlycontagious enteric disease of swine caused by PRRSV.since the emergence of PRRS,it hasbecome one of the economically important swine diseases worldly and designated as statutoryreport of animal epidemics by the world organization for animal health（OIE）.In our country,it was classed II infectious disease. At present the pathogenic mechanism of PRRSV is notclear. Based on PRRSV GP5major structural protein and truncated GP5function as the goal,to determine the PRRSV GP5protein subcellular localization, the cell proliferation, cell cycle,cell apoptosis and second infection of virus, and explore preliminary regulation mechanism ofantiviral cytokine.The results are as follows:1. PRRSV ORF5or truncated ORF5genes were successfully amplified and cloned intoeukaryotic piggyBac expression vector, the recombinant plasmid pPB-GP5, pPB-GP584-96,pPB-GP597-119and piggyBac vector were transfected into Marc-145cell using Lipofectamine2000, screened by purocine, the size of GP5or truncated GP5genes PCR amplification was603bp,564bp and534bp. Westernblot tests showed GP5or truncated GP5protein wereapproximately22.1ku,20ku,19.6ku,respectively.2. The subcellular localization of the GP5or trucated GP5proteins was investigated byconfocal fluorescence microscopy. The results showed that the total length of GP5andGP584-96, GP597-119protein distributed through the whole Marc-145cells.3. The GP5s protein expression effects on cell growth and cell cycle were investigatedby CCK-8and flow cytometry assay. comparing with the control cell line Marc-145s, PRRSVGP5and truncated GP584-96protein expression does not affect the cell growth,but GP597-119protein expression significantly inhibits the cell growth and cell cycle were arrested at G2/Mphase.4. To determine whether PRRSV GP5or trucated GP5proteins expression could inducecell apoptosis. Real-time fluorescent quantitative PCR detection showed that PRRSV GP5 protein significantly increase the anti-apoptosis Bcl-2gene expression, Bax/Bcl-2ratio lessthan1, but the truncated GP5protein expression and the Bax, Bcl-2gene expression weredecreased by GP584-96,GP597-119, and Bax/Bcl-2ratio greater than1;Westernblot resultsindicated that compared with PRRSV infection positive control group, PRRSV GP5andtruncated GP584-96,GP597-119protein expression can not activated caspase–3; DNA ladderresults showed that comparing with PRRSV infection positive control group, PRRSV GP5and truncated GP584-96,GP597-119protein expression were not nduced cell genome broken.5. To determine whether GP5or trucated GP5proteins expression influence on PRRSVSD16secondary infection,with related the anti-viral cytokine interferons α,β,γ expression. theindirect immunofluorescence test （IFA）, absolute quantitative PCR,viral replication titer（TCID50）and Westernblot results showed that PRRSV infection can be obviously promotedthe virus infection during the early stage. With anti-viral cytokine interferons γ geneexpression declined obviously, and the Marc-145-GP584-96significantly inhibited PRRSVsecondary infection with interferons βgene increase,it was also checked with the differentPRRSV strains （VR-2332, JXA1, TA-4, SD16）respectively.Conclusion：PRRSV GP5proteins expression could induce cell apoptosis，but promote virusinfection...