Establishment Of Double Antibody Sandwich ELISA And Study On Genetic-engineering Vaccines Against Highly Pathogenic PRRSV

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases, which has caused great losses to the swine industry worldwide. It has been a problem for global swine industry since it was first reported in United States in 1987. Highly pathogenic PRRSV emerged in June 2006 in HuBei, HuNan, JiangXi and AnHui provinces of china. It has caused inestimable economic losses to our swine industry. Accurate, dependable detective technology and safe, effective vaccines are the keys to prevent and control the disease, also the focus of the study on the disease.In this study, ORF7 gene of PRRSV was expressed efficiently in E.coli as soluble proteins; Monoclonal antibodies against N protein of PRRSV were prepared; Double antibody sandwich ELISA assay was developed and applied for detecting PRRSV antigen, and the Monoclonal antibodies were used as coasting antibodies. The purpose is to establish an accurate, reliable, convenient and rapid detective method. In this study, genetic-engineering vaccines were constructed in which pseudorabies viruses and baculoviruses were used as vectors, and then the immune efficacy and immunoprotection of these vaccines were studied.1. Cloning and expressing of PRRSV ORF7 gene in Escherichia coliformSpecific primers were designed according to the nucleotide sequence of PRRSV WUH1 strain, and then the complete coding region of ORF7 gene was amplified by RT-PCR. PCR product of ORF7 gene was cloned into pGEM-T easy vector to generate the recombinant plasmid pGEM-T-N. There was not any mutation on ORF7 gene by sequencing. And then ORF7 gene was cloned into pET-30a to construct plasmidpET-30a-N. This plasmid was transformed to E.coli BL21 and N protein was expressed after induction with IPTG. The protein was expressed as a soluble protein which was verified by SDS-PAGE, and it was specific to standard positive serum of PRRSV, which indicated the protein possessed immunological activity.2. Preparing Monoclonal antibodies against PRRSV N proteinBalb/c mice were immuned with purified N protein, and then the immunized spleen cells were prepared to fuse with myeloma cells SP2/0. The fused cells were double selected by indirect ELISA assay in which N protein and PRRSV were both coasted as antigens, and then they were cloned by limiting dilution assay. Two hybridomas N3H12 and N4D2 secreting higher titer of MAbs than other hybridomas, were used in the next study. The two hybridomas were injected into Balb/c mice to prepare ascetic fluid, and the ELISA titer is 100x214and 100×215.3. Development and application of Double antibody sandwich ELISA assay for detecting PRRSV antigenPRRSV WUH1 strain, CH-la strain and Marc-145 cells were coasted as antigens, two MAbs exchanged with each other as coating antibodies and enzyme lablled antibodies. The result of double antibody sandwich ELISA showed that N4D2 was suitable as coating antibodies and N3H12 was suitable as enzyme lablled antibodies.The optical coating concentration of N4D2 was 2μg/mL and the best working concentration of enzyme lablled antibodies was 1:1000 which was determined by checkerboard titration. The best working condition is:coasting for fourteen hours at 2～8 ℃, blocking for an hour at 37℃,incubating coasting antibodies and antigens at 37℃ for an hour, incubating antigens and enzyme lablled antibodies at 37℃ for an hour, substrate reaction time is fifteen minutes.This assay showed strong specificity, good repeatability and high sensibility. The result of comparative examination between ELISA kit and RT-PCR kit indicated there was a high positive correlation rate (84.6%), negative correlation rate (86.4%) and total correlation rate (85.8%).The 127 sera used in comparative examination were collected from different farms.4. Study on the recombinant genetic-engineering vaccines against PRRSV based on rPRV-GP5m-MThe recombinant virus rPRV-GP5m-M was used as vaccine vector, two PRV recombinants rPRV-PRRSV-2, rPRV-PRRSV-3 were generated. Two expression cassettes CMV-ORF5-polyA(CMV:Cytomegalovirus immediate-early promoter; ORF5:ORF5 gene of highly pathogenic PRRSV; polyA:SV40 Late polyA) and CMV-ORF2m-polyA (ORF2m:modified ORF2 gene of PCV2) were inserted into gG point of rPRV-GP5m-M to generate rPRV-PRRSV-2. rPRV-PRRSV-3 was constructed to insert CMV-SynORF5-polyA(express synthetic ORF5 gene of highly pathogenic PRRSV), CMV-ORF4-polyA(express ORF4 gene of highly pathogenic PRRSV), CMV-ORF5R-2-polyA(express ORF5 gene of highly pathogenic PRRSV and modified ORF2 gene of PCV2 as fusion protein) into gG point of rPRV-GP5m-M. Western blot using standard PCV2 positive sera was carried out to demonstrate correct expression of foreign genes in rPRV-PRRSV-2 and rPRV-PRRSV-3. Comparable PRV-specific humoral immune responses can be detected in the mice immunized with recombinant virus. However, the neutralizing antibodies and ELISA antibodies against PRRSV induced by these PRV reeombinants were distinctive. rPRV-PRRSV-3 can induce stronger neutralizing antibodies and cellular immunity.5. Construction and immune effect of recombinant baculovirusAcMNPV can’t replicate in mammalian cells, so it’s very safe to express aline genes in mammalians. In this study, a modified recombinant baculovirus expressing VSV-G in the viral envelope was used as virus vector to escape the complement system.Therefore, Four expression cassettes CMV-ORF3-polyA, CMV-ORF4-polyA, CMV-ORF5-polyA and CMV-ORF6-polyA were inserted to the downstream of VSV-G in turn to construct recombinant baculovirus BV-CMV-3456. Mice were injected to intramuscular with different doses of recombinant baculovirus BV-CMV-3456 to characterize the induction of antigen-specific immune response. The injected dose was ranging from 107 to 109pfu. The results showed that BV-CMV-3456 can induce stonger immune response than BV-CMV-EGFP, and the immune response was dose reliable.