Isolation And Characterization Of Variant Strains Of PRRSV And Development Of Diagnostic Method And Gene Vaccine

Porcine reproductive and respiratory syndrome(PRRS)is often called blue ear disease, which is characterized by respiratory disease and reproductive failure in sows and high motality in piglets. The aetiological agent, porcine reproductive and respiratory syndrome virus(PRRSV),belongs to the member of arteriviruses, a group of small, enveloped, positive-strand RNA virus. The high morbidity and high mortality rate of the pig " ardent fever " syndrome induced by the highly pathogenic PRRSV mutants has caused enormous economic losses in pig industry in South China since May in 2006. Accurate and reliable detection technology and safe and efficient vaccine are the key to prevent and control the PRRS. This research is aim to develop a detection methods of highly pathogenic blue-ear disease etiology and serological, to find out the role of pigs prothymosin (ProTa) as a molecular adjuvant, and the feasibility of constructing attenuated Salmonella live vector vaccine carrying PRRSV-GP5 gene. The initial results obtained are as follows:1. Isolation and Identification of SCMS08 Strain of Porcine reproductive and respiratory syndrome virusA Mutant PRRSV of Sichuan SCMS08 strain was separated by using Marc-145 cells and its biological characteristics were studied. The hypervariable region of NSP2 and the GP5 genome were sequenced. The results showed that it's highest homology with JXA1 strain. Their nucleotide and deduced amino acid identity was 99.7%, 99.8% and 99.2%,99.3%, respectively.2. Development and application of Multiplex RT-PCR assay techniques for Different Subtype of PRRSV and Phylogenetic analysis of PRRSV in SichuanEstablished and optimized for rapid and simultaneous detection of European strain, classical and Highly pathogenic subtype of Porcine reproductive and respiratory syndrome virus (PRRSV). Based on the variation sequences of NSP2 genes of European strain,classical and Highly pathogenic subtype of PRRSV,3 sets of specific primers were designed and the multiplex RT-PCR assay was established. The method was used to detect the 76 clinical samples. Among 76 clinical samples, PRRSV positive percentage was 60%, PRRSV European strain positive percentage was 0, PRRSV highly pathogenic strain positive percentage was 53.9%. The hypervariable regions of NSP2 gene of 9 PRRSV positive samples were cloned and sequenced. The results showed that they were high homology among the nine PRRSV strains, and were closest relative with JXA1 strains.The were highly pathogenic PRRSV NSP2 mutant strains. The highly pathogenic PRRSV in Sichuan might be imported directly from Jiangxi.3. Identification of the B cell dominant epitopes of PRRSV NSP2 Hypervariable region and construction of an indirect ELISA methodThe secondary structure and epitope of NSP2 Hypervariable region of SCMS08 strain were predicted and analyzed by bioinformatic methods. According to the forecast results,4 sets of primers were designed,and the NSP2 hypervariable area were Sub-cloned. Four prokaryotic expression vectors were successfully constructed, and 4 fusion protein was highly expressed induced by IPTG The 4 fusion protein was about 63,35,26,27ku, showed by SDS-PAGE,and could react with PRRSV-positive sera by Western blot. The NSP2-4 fusion protein respond weakly with PRRSV-positive serum. A strong epitope was found in NSP2 gene of PRRSV SCM strain. The four expressed protein was purified by a Ni-chelated chromatography. The indirect ELISA based on the four protein(NSP2-1,NSP2-2,NSP2-3,NSP2-4) was developed for detecting PRRSV antibody. The PRRSV positive percentage was 84.6%, 77.4%,21.3%,52.1% for PRRSV classical strains, and 100%,100%,77.6%,63.2% for PRRSV highly pathogenic strains.The results indicated that NSP2-1-ELISA was specific, sensitive and suitable for routine diagnosis of PRRS. It's expected to develop an diagnosis which could distinguish between vaccine and wild virus by optimizing the location of the epitope.4. Cloning and Expression of Porcine Prothymosin a GeneThe porcine Prothymosin a cDNA fragment was amplified from kidney by reverse transcription-polymerase chain reaction (RT-PCR), and sequenced. pET32-mProTa was constructed by gene rearrangement, then it was transformed into E. coli Rosetta 2 (DE3).The expression of the target protein was induced with IPTG and purified. The expressed recombinant ProTa was identified by SDS-PAGE and Western blotting. The complete open reading frame (ORF) of ProTa gene includes 333bp and encodes 109aa. The recombinant prokaryotic expression vector pET32-mProTawas constructed successfully and expressed stably in E. coli Rosetta 2 (DE3). At about 12 070 of molecular weight, there was an induced protein band. MTT essay confirmed that it can enhance lymphocyte proliferation obviously. pEGFP-ProT a was constructed by gene rearrangement. The plasmid was transfected into Vero cells with lipofectamine. The expressed EGFP was observed by fluorescent microscope. The eukaryotic expression vector pEGFP-ProT a was constructed successfully. Green fluorescence mainly located on cell nucleus of transfected cells by fluorescent microscope. The recombinant expression vector pEGFP-ProT a has been successfully constructed and ProT a gene can be expressed in Vero cells. This study lays a foundation for further research into the function of porcine ProTαgene.5. Immunology study on porcine ProTa and PRRSV-GP5 combined vaccine mediated by Attenuated SalmonellaEukaryotic expression vectors pCI-GP5, pCI-ProTa and pCI-GP5-ProTa were constructed respectively. The pCI-GP5, pCI-ProTa and pCI-GP5-ProTa recombinant plasmids were transfected into the Vero cells. RT-PCR and indirect immunofluorescence assay showed that the target genes were transcribed and highly expressed in the Vero cells. The pCI-GP5-ProTa, pCI-GP5, pCI-ProTa, and pCI-GP5 combined with pCI-ProTa recombinant plasmids were intramuscularly injected into BALB/c mice. The results showed that the humoral and cellular immunity of the pCI-GP5-ProTa, pCI-GP5 combined with pCI-ProTa experimental groups were higher than the pCI-GP5 experimental group. It demonstrated that the ProTa highly enhanced the immunogenicity of DNA vaccine as an immunologic adjuvant. Then the pCI-GP5-ProTa was transformed into the attenuated Salmonella C500, and the new vaccines attenuated Salmonella C500 carrying pCI-GP5-ProTa were successfully constructed. The mucosal immunity and lymphocyte proliferation significantly increased in mice by using the new vaccines.