Constuction Of Pesudotyped Virus Bearing The Porocine Reproductive And Respiratory Syndrome GP5 Protein

In this study,the glycoprotein GP5,GP6 gene of PRRSV were amplified by RT-PCR and cloned into PGEM-T vector and sequenced.The genetic variance analysis,respectively 97.4%and 96.0%with domestic JX0612 nucleotide and amino acid identity.This result shows that the PRRSV HN/06 may be popular with the domestic highly pathogenic strain of JX0612 which have a certain degree of relevance.The GP5,GP5-GP6,GP5/6 genes were cloned into eukaryotic expression vector pcDNA3.0,constructed pcDNA-GP5,pcDNA-GP5-GP6,pcDNA-GP5/6,respectively transfered 293T cell with calcium phosphate method,collecting cells after 48h, incubated for 1h at 4(?) with 1∶50 PRRSV anti-serum.After three washes with PBS/FCS,the cells were incubated with fluorescein-labelled goat anti-swine IgG for 1h at 4(?),subsequently subjected to flow cytometry using a FACSCalibur.The results showed that the expression quantity of coexpresses recombinant plasmid pcDNA-GP5/6 was the highest,achieved 48.5%;Next pcDNA-GP5 gene expression Achieved 33%;The series recombinant plasmid pcDNA-GP5-GP6 had not nearly examined the expression,only7.3%.A single 10cm diameter dish of subconfluently grown 293T cells was transfected withpHIT111,pHIT60 and env(envelope protein gene) carrying plasmids via calcium phosphate transfection.Supernatants were harvested 48 h post-transfection and centrifuged(3500 r·min^-1,10min) to remove cellular debris,incubated for 1h at 4(?) with 1∶50 GP5 anti-serum.Western blot confirmed GP5 protein was expression in the surface of this virus particles.Through the pseudotypes particles infected Marc-145 PAM and the other different cells,with the MuLV-G for the control,detection the report gene,the result shows that GP5,GP6 genes had a certain relation with the virus infected.Meanwhile this indicates that PRRSV GP5-M protein heterodimer influence transporting and positioning of GP5 protein.After tests showed that the M proteinmediated protein in the cells GP5 expressed GP5 than a single expression of high.Through infection of the pilot found by the M protein mediated E protein leave the infectious virus particles than a single GP5 of infectious viral particles at high.In our reseach,we have shown that GP5 proteins are expressed at the pseudotype virus surface,and that the pseudotype virus can infect its host cell.This indicates that the host cell surface includes an acceptor of GP5.So we may in the future screen of antiviral drugs block the PRRSV from entry.The PRRSV MuLV pseudotype virus system we have described provides both a novel method for the study of the invasion mechanism of PRRSV,and screening of antiviral drugs.