Tudy On The Generation Of Monoclonal Antibodies Against GP5Protein And The Establishment Of Diagnostic Method And DNA Vaccine Of PRRSV

PRRS is first reported in the United States and now it spreads throughout the world leading to the large loss of the global swine industry, so it is importent to detect and prevent the disease. There are many methods to detect this disease and it can be classfied two kinds:the detecting of the antigens and antibodies. The detecting of the antigens includes PCR and the real time PCR. The detecting of the antibodies includes IFA、 IPMA、SVN and EILSA. Compared to the other methods, ELISA is simpler to perform, time-saving, sensitive and suitable for large scale surveys of PRRSV infection and is useful to evaluate the efficiency of various vaccines against PRRSV. Immunization is the main method to prevent and control this disease. In the present, the available vaccines to prevent PRRS are mainly attenuated and inactivated vaccines. Although attenuated vaccine is effective to prevent pigs from PRRSV infection, there exist dangers of reversion to a virulent strain. In contrast, inactived vaccine is safe, but it needs repeatedly to be injected and its effect is not stable, possibly causing the failure of vaccining. It is urgent to develop more safe and effective vaccine to prevent and control PRRS. The GP5protein coded by ORF5is the major envelope protein of PRRSV and it is the main protective immunogen of PRRSV inducing the production of neutralizing antibodies which appears in the late stage of PRRSV infection. So it becomes the focus of current research of vaccine and detection against PRRSV. Because of this, the following researchs were explored.1. The expression of ORF5gene of PRRSV WUH3strain in E.coliBecause intact ORF5gene can’t be expressed or expressed in a low level in E.coli, a pair of primers was designed to amplify the ORF5gene out of the signal peptide. The modified fragment of ORF5gene was cloned into prokaryotic expression vector pGEX-KG to construct prokaryotic expression plasmid pGEX-KG-ORF5. After induction by IPTG, a high expression of fusion protein was obtained. The fusion protein GST-GP5has a molecular weight of38kDa, mainly in inclusion bodies form.2. The preparation of monoclonal antibody against GP5protein of PRRSV WUH3strainThe prokaryotic expression product of PRRSV GP5protein was purified and used as the immunogen to immunize4-week female BALB/c mice. By fusion and indirect ELISA screening,4strains of monoclonal antibody against GP5protein was obtained, named4A11、3D10、1E9and1H11. Mice were injected with4strains of hybridoma for production of ascites in large quantity. The constructed eukaryotic expression plasmid PCAGGS-HA-SynORF5was transinfected into Hela cells and the expressed protein was collected. Western blotting and IFA confirmed that4A11and1H11strains of monoclonal antibody reacted specifically with eukaryotically expressed GP5protein.3. Development of PRRSV GP5-ELISA for GP5specifical antibodies detectionThe fusion protein GST-GP5was used to detect the PRRSV GP5specifical antibodies.We firstly coated the monoclonal antibody GST and then added the GST-GP5protein to develope PRRSV GP5-ELISA for GP5specifical antibodies detection. The suitable concentration of the monoclonal antibody GST and GST-GP5protein was determined by titration test. The results indicated that the optimal dilution of the monoclonal antibody GST is1:10000and the optimal concentration of GST-GP5protein is2μg/mL. Comparison between GP5-ELISA and imported PRRSV antibodies diagnosis kit IDEXX showed the two methods had86.1percent agreement by detecting208serum samples.4. Construction and immunogenicity of PRRSV DNA vaccineThe eukaryotic expression plasmid PCAGGS-HA-APCH1-SynORF5PCAGGS-HA-SynORF5、PCAGGS-HA-APCH1were constructed by SynORF5and APCH1cloned to the eukaryotic expression vetor PCAGGS-HA. In order to study whether the APCH1protein could enhance the immunogenicity of GP5protein, The plasmid PCAGGS-HA-APCHl-SynORF5、PCAGGS-HA-SynORF5PCAGGS-HA-APCH1and control plasmid PCAGGS-HA were used to separately immunize BALB/c mice. The results showed that co-expression of GP5and APCH1could visually enhance the immunogenicity of GP5(P<0.05), but donot significantly increase the level of neutralizing antibodies to PRRSV (P>0.05).6weeks after the first immunizition, the spleen lymphocytes of mice were separated and treated with inactivated PRRSV. The IFN-y ELISA kit was used to detect the expression level of IFN-y. The result showed that PCAGGS-HA-APCHl-SynORF5can induce higher level of IFN-y than PCAGGS-HA-SynORF5(P＜0.05). These results suggested that APCH1could enhance the immunogenicity of PRRSV DNA vaccine.