Effective Expression Of Glycoprotein5of PRRSV And Establishment Of Indirect ELISA For Detection Of Antibody Against Glycoprotein5

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious andinfectious disease caused by porcine reproductive and respiratory syndrome virus（PRRSV）.PRRS is characterized by high incidence and mortality rate and commonly known as “blueear disease”. PRRSV is divided into two genotypes, the European (type1virus) and theAmerican (type2virus), which is represented by Lelystad virus (LV) and VR-2332respectively. Most of the PRRSV isolated from China belongs to American type. Studies haveshown that, glycoprotein5of PRRV (American) that is encoded by ORF5gene, comprised by200Amino acids residues and containing several neutralizing epitopes is an enveloped protein,is one of the key immunogenic proteins of PRRSV and is the leading target for thedevelopment of the genetic engineering vaccines against PRRS. However, there is still nocommercial ELISA kit for the detection of antibody against GP5. And it is impossible to geteffective assessment about the level of neutralizing antibody after immunization. To solve theproblem, we carried out this study.To get efficient expression of recombinant protein GP5, this study amplified ORF5geneencoding the glycoprotein5from the genome RNA of PRRSV (SD-JN, American type) byRT-PCR, and get the amplification of the gene fragment of ORF5-1and ORF5-2thatencoding the GP5protein truncated signal peptide and the hydrophobic region by two pairs ofspecific primers. In this study, ORF5-1and ORF5-2were connected into the prokaryoticexpression vector pET-32a (+), which was transformed into E.coli BL21(DE3)pLysS.Comparing the induced results under different IPTG concentration, induced time andtemperature by test, this study optimized the induced conditions for the expression of GP5protein. The purified recombinant protein GP5was obtained through affinity chromatographywith His tag. Its immunogenicity was identified by Western blot test, used monoclonal ascitesantibody of mouse anti-GP5. Results of the experiment, the ORF5gene was amplified, andthe signal peptide that was located in amino acid of N1-N31and the hydrophobic structurethat was located in amino acids of N61-N125was learned by predicated systerm. And therecombinant plasmid pET-32a-GP5was constructed for the prokaryotic expression of GP5protein, and the best induced condition of E.coli BL21-GP5was assured:0.1mM IPTG andinduced in37℃for6hours.97.75mg recombinant protein of GP5can be extracted from perliter induced engineering bacteria in this study. And the result of Western blot test shows thatthe recombinant protein GP5has a good immunogenicity.To establish the iELISA method for detecting the antibody against PRRSV GP5in the pig serum, this study used the purified GP5protein as antigen coated in ELISA reaction plate.Analyzing the value of P/N in OD450under different concentration of antigen-coated, dilutionratio of serum, packet buffer, diluted ratio of enzyme-labeled antibody, and time of colordeveloping by test, this study get the optimum conditions of iELISA. And by detecting theOD450value of30negative serum samples, this study draws the standard of determination. Inthis study,163serum samples of swine were detected compared with the method of Westernblot and negative serum of PRRSV and positive serum of PCV2, CSFV, PRV and PPV weredetected by the iELISA. Results of experiment show that the optimal reaction conditions ofthe iELISA established in this study: the antigen-coated concentration is200ng/hole, theserum sample dilution ratio is1:80, the optimum packet buffer is phosphate buffer, theoptimum blocking solution is2.5%DM/PBST, the optimum iELISA antibody dilution ratio is1:8000, and the optimum reaction time of color developing is15minutes. This method doesnot react with positive serum of PCV2, CSFV, PRV and PPV, and negative serum of PRRSV.Compared with Western blot test, the coincidence rate is93.16%. All of the result showsiELISA established in this study having good specificity and a higher degree of accuracy.