Study On The Effect Of Secondary Structural Protein Of PRRSV By Pseudotype System

Porcine reproductive and respiratory syndrome is caused by PRRSV, single strand , normal chain,envelope and no-segmented RNA viruses that belongs to the virales Nidovirales, Arteriviridae,Arterivirus. PRRSV is an important pathogen of pigs which has severely hazarded swine the industry. It primarily not only causes respiratory failure in susceptible sows , but also causes respiratory problems in piglets.The retroviral envelope protein can be exchanged for envelope proteins from non-related viruses,a process called pseudotyping.Pseudotyped viruses with heterogenic glycoprotein incorporated into retroviral particles were proved to be a safe viral entry model,which only go through a single cycle infection and acquired the host range of the parent viruses where the glycoprotein were derived,thus they could facilitate the research on viral entry mechanism,viral tropism,neutralization antibody analysis,and receptor identification.Nowdays,mostly studies about molecular biology of PRRSV is its major structural protein, especially the GP5 of PRRSV, In our laboratory,we had constructed the MuLV-GP5 by pseudotype system,but the study on the secondary structural protein is more less. In this study,the secondary glycoprotein GP2,GP3and GP4 gene of PRRSV were amplified by RT-PCR and cloned into eukaryotic expression vector pcDNA3.0 to gengerate the expressing plasmid pcDNA-GP2,pcDNA-GP3 and pcDNA-GP4, Respectively transfered 293T cell with liposome method,and the plasmid pcDNA3.0 transfered 293T cell as negative control,the plasmid pcDNA-GP5(constructed by our lab) transferred 293T cell as positive control. collecting cells after 48h, the cells were incubated with specificness polyclonal anti-PRRSV serum and fluorescein-labelled goat anti-swine IgG, respectively. Using a flow cytometry to detect the protein expression quantity.The results showed that the expression quantity of negative control achieved 0.7%, the expression quantity of positive control pcDNA-GP5 was the highest ,achieved 30.4%,the recombinant plasmid pcDNA-GP2,pcDNA-GP3 had a small quantity expression,each expression were achieved 4.9%,5.4% and 7.6%.293T cells were co-transfected with the recombinant plasmid,pHIT60(has the structural genes of MuLV) and pHIT111(has the retroviral genome,containing LacZ as a reporter) plasmids to generate pseudotying virus. The retroviral supernatants were harvested 48 hours post-transfection and centrifuged to remove cellular debris,and used in western-blot ,the results indicated that the GP2,GP3 and GP4 were not expressed in the concentrated supematant fluid, the secondary structural protein could not integrate into the surface of virus particles. Through the transfected supernatants infected Marc-145,PAM and the other different target cells, and parallel transfections were carried out with supernatants produced in absence of a viral envelope and with the vesicular stomatitis virus(VSV)G proteins,which is known to efficiently pseudotype MuLV. The results of infection assays indicated that the transfected supernatants could not detect the expression of reporter gene. The results also indicated that those supernatants had no infection. So the alone secondary structural protein of PRRSV unable construct the infectious MuLV pseudotype virus.