Preparation Of Monoclonal Antibody Against GP5 And N Protein And Novel Subunit Vaccine Of Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus

Porcine respiratory and reproductive syndrome (PRRS) is an infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which is characterized by seriously reproductive failure in pregnant pig and respiratory symptom in piglets with high mortality. It was recognised in America in 1987 for the first time and now has spread worldwide and caused significant economic losses to universal pig industry.GP5 is the foremost immunogenic protein of PPRSV and could induce the generation of strongest neutralizing antibody. But lots of available vaccines constructed basing on natural ORF5 gene could hardly inducing earlier and stronger antibody level because of the covering effect of epitope A to epitope B. However, appropriate modification on ORF5 gene may reinforce the immunogenicity of GP5. On the other hand, protein N is also highly immunogenic as extremely conservative protein. The antibodies against protein N was produced at initial stage after PPRSV infected pig and lasting for a long time, thus protein N became one of the target antigen proteins that was generally acknowledged optimum for PPRS diagnosis.Plant virus has advantage of high expression level and safety as a noval vector. The coat protein of plant virus has the ability to self-assemble into virus-like particles (VLPs), displaying foreign epitopes on the saface of virus-like particles was shown to be highly immunogenic and could induce specific antibodies in vivo, which made it a promising platform of noval vaccine research.This study is mainly aimed at preparation of monoclonal antibodies against Gp5 and N protein as well as native sub unit vaccine to a highly pathogenic PRRSV WUH3 strain which was recently isolated by our laboratory. The main contents include:1. Preparation of monoclonal antibody against GP5The cDNA fragment of ORF5 without signal sequence was acquired via amplification by RT-PCR using extracted genomic RNA of highly pathogenic PRRSV WUH3 strain as template and a pair of specific primers as downstream and upstream primers, and the target fragment size is about 540bp. The construction of prokaryotic expression vector pKG-ORF5 was completed by insertion of target gene fragment into pGEX-KG expression vector dealing with restriction endonucleases BamHl and XhoI. Mice was immunized using inclusion body expressed in E.coli to prepare monoclonal antibody. After test by indirect-ELISA and inditification by Western blot, three positive hybridomas were acquired finally and namely 2G8,3G8,1F12, and the ELISA titer of ascetic fluids is 100×2.2. Preparation of monoclonal antibody against protein NBoth the plasmids pET-30a and pGEX-KG-ORF7 were digested with restriction endonucleases BamHI and HindⅢand constructed prokaryotic expression vector pET30a-ORF7 after recycling and ligation. Immunization of mice with purified prokaryotic expression protein obtained 8 positive hybridomas in total after test by ELISA, respectively named 1A2,1D9,1F6,2C7,3B9,3H2,4D2 and 4E2. Ascetic fluids of all the hybridomas were proved to be positive expect 1D9 and 4E2 by Western blot with eukaryoticly expressed protein N as antigen. The ELISA titer of ascetic fluids is 100×29.3. Research on novel subunit vaccineWe choose the Gp5 N-terminal 22-66 amino acids excluding the signal sequence but including neutralizing epitope B and overlaying covering A, according to the genetic feature of ORF5 of highly virulent PRRSV WUH3 strain. At the same time, we modified GP5 epitope by inserting a PADRE between epitope A and B, replacing epitope A with B, eliminating the glycosylation sites (N30, N34, N35 and N51) at the both ends of the neutralizing epitope and changing the codons of ORF5 to preferable ones for swine. After that, the modified GP5 epitope added at the C-termination of PapMV CP by linker was artificially synthesized and cloned to expression vector pET-30a to express fusion protein in E.coli. The expressed fusion protein was demonstrated to have biologic activity with western blotting and form virus-like particles(VLPs) after observed with electron microscopy. The detection of antibody result indicated that there was not obvious change in the neutralizing antibody level after immunization mice with the fusion protein above.