Development Of Trivalent Genetic Engineering Live Veter Vaccine Against Porcine Japanese Encephalitis, Porcine Parvovirus And Pseudorabies

Pseudorabies virus (PRV),Porcine parvovirus (PPV) and Porcine Japanese Encephalitis virus(JEV) are three major viral pathogens, causing the swine reproductive syndrome about stillbirth, mummification, embryonic death, infertility (SMEDI).At present, these three virus often cause mixed infection or secondary infection in clinic, which are accompanied with immunosuppressive viral diseases including Classical swine fever virus(CSFV), Porcine reproductive and respiratory syndrome virus(PRRSV) and Porcine Circovirus type 2 (PCV2), as well as bacterial diseases etc. It makes clinic symptoms of swine diseases more complex and complicated, increases the difficulty of treatment and prevention work, leads to great lost of values in swine industry. Therefore, in order to save the labor, predigest immunity program, reduce the immune-interfering, diminish the immunized swine, and distinguish the vaccination from wild virus infection, we work to develop a sort of safe, effective, steady and convenient novel vaccine, which can prevent and control three diseases simultaneously, which was proved an effective approach decreasing swine SMEDI and correlative diseases. Combining the gene-deleted PRV was as a live-viral vector and PPV VP2 was as a virus-like particle vector apply to develop the novel animal vaccine,which provide a new way of the study, In this research, we carried through a serological and epidemiology investigation of porcine pseduorabies and Japanese encephalitis in some pig farms of Sichuan province in order to gather its popular present condition; A bivalent DNA vaccine were developed to chimera a cordon optimized multi-epitope gene e containing critical epitopes of the JEV envelope and PPV VP2 gene against Porcine Japanese Encephaliti and porcine parvovirus; trivalent genetic engineering live vector vaccine against Porcine Japanese Encephalitis, porcine parvovirus and Pseudo rabies were constructed by the way of homologous recombination, using PRV SA215 as a live vector. The main research contents are presented as the following:1,Seroepidemiology survey of porcine pseduorabies and Japanese encephalitis in some representative pig farms of Sichuan provinceDuring the two years from 2009 to 2010, about 1177 pig'sera samples received from 27 typical different-scale pig farms of 18 areas of Sichuan province,were detected with the IDEXX PRV gE-ELISA kit which can distinguish the wild-type virus infection.The serological data were analysed statisticall and systematically.The results showed that the average PRV wild-type virus positve rate was 8.2% of all samples,negative pig farms was 59.3%, the northeast area of Sichuan has the highest positive rate was 18.8%, the positive rate of boar was the highest,which was 7.4%, the positive rate of small-scale pig farms which the quantity of broadsow was lower than 100 was highest,which was 52.7%.We had detected the PRV immune antibody of pig'sera samples received from 14 pig farms of 10 areas of Sichuan province, by using IDEXX PRV gB-ELISA kit.The results show the gB antibody positive rates of different pig farms has great difference range from 66.7% to 100%, the average positve rate of 14 pig farms was 87%. This result indicate that the immunity of PRV in Sichuan was irregular. During the two years from February 2009 to October 2010, We had detected pig'sera samples received from 21 typical pig farms of 14 areas of Sichuan province,by using JEV antibody indirect ELISA kit. Then,the serological data were analysed statisticall and systematically.The results show JEV antibody's total postive rate of all detected pigs was 75.1%, he east area of Sichuan has the highest positive rate was 92.1%,the JEV antibody positive rate went up in April by degrees,followed in May the second highest serum antibody positive rate reaching 92.1% .Then,826 serum samples that was JEV antibody positive when using the JEV ELISA kit were classified according to different sort of swinery.we detected JEV antigen of the sample that were random sampling about 20% of each classification by using JEV specificity RT-PCR.The result show that the positive rate of replacement gilt was the highest,which was 6.5%.The results indicate the JEV infection is existence in Sichuan province.2,Design of JEV E gene multi-epitope e gene and its prokaryotic expressionThe antigen epitopes of E protein of JEV were analyzed on base of bioinformatics methods and reported references,and then five B-cell epitopes,one T-cell epitopes from E protein and a general helper T-cell epitope (PADRE) that were induted for enhancing cellullar immunologic response were selected.Selecting suitable linkers, each epitope were paialleled by this linkers according to different order.We selected the optimization group of multiepitope-tandems. The analysis for the tandems gene indicated possessing good bioinformatical characteristics.The e gene was synthesized and cloned into the prokaryotic expression vector pET-32a to construct a recombinant plasmid pET-e, fusion protein was successfully expressed induced by IPTG. The results of Western-blotting showed that the expressed fusion protein occurred specific reaction with proper positive serum of JEV,suggesting that JEV multi-epitopes peptide possesing immunogenicity.The results will lay a foundation for follow-up working, in favour of the development of the genetic engineering vaccine.3,The research on effect of the Glycine-rich region deleted PPV VP2 to the VLPsThe coding regions which rich contain 9 successive glycine in the N-terminal of PPV VP2 protein are cleavage sites of VP3 protein.But these regions are easily missed in the regular PCR amplification.In order to research on the effect of the deletion to he form of PPV VLPs, and the proper site for the inserted foreign gene in the VP2 VLPs, the VP2 eukaryotic expression vector pCI-△VP2 with the certain regions deletion was constructed,with entire VP2 as control. Transfection to the Vero cells was mediated by liposome,for its expression was observed by bioinformatics technology,SDS-PAGE and Western-blotting,Indirect immunofluorescence, Positive staining of scanning microscopy and Negative staining of immunoelectron microscopy.In the further step,the Kunming mice were immunized with this DNA vaccine by intramuscular injection.Specific humoral and cellular immune response elicited in mice were detected by ELISA test,T-lymphocyte proliferation assay and FACS. The result showed that the deleted VP2 and the entire one could both be self assembled, and showed the similar HA activity to the entire virion.The strong level of specific humoral and excellent cellular immune response was showed.It was shown that the deleted glycine-rich region can not effect the assembling and immunogenicity of VLPs.The deleted VP2 could be used to research on the PPV VLPs vaccine and antigen transferring vector, as the new approach to the VLPs research development in vector and modification,and the new theoretical evidence for the relationship between the structure of VP2 gene and function of protein.4,Construction of chimeric eukaryotic expression plasmid pCI-Ve containing codon optimzied JEV multi-epitope gene e and PPV VP2 gene as bivalent DNA vaccine against Porcine Japanese Encephalitis and Porcine parvovirusIn order to improve the expression level of heterogenous gene.we re-designed and artificially synthesized the JEV multi-epitope gene e which containing critical epitopes of the JEV envelope by adopting the codons preferentially used in mammalian without any changing of the amino acid sequences.Synthetic codon optimzied gene e was cloned into the 5'terminal of PPV VP2,constructing the chimeric gene Ve.Then, JEV multi-epitope gene e and chimeric gene Ve was cloned into commercial eukaryotic expression vector pCI-neo respectively to construct the the recombinant plasmids pCI-e and DNA vaccine pCI-Ve against Porcine Japanese Encephalitis and porcine parvovirus. Then,the eukaryotic expression recombinant plasmids pCI-e and pCI-Ve were transfected into Vero cells mediated by liposome, for their expression was observed by SDS-PAGE and Western-blotting,Indirect immunofluorescence, microscopy observation of positive staining. The result showed that the gene e and gene Ve were expressed successfully and the expressed proteins possessed immunogenicity and obvious bioactivity. Virus-like particles (VLPs) were observed through electronmicroscope in vero cell transfected with pCI-Ve,suggesting that the epressed protein of Ve gene in eukaryotic cells may self-assemble and form chimeric VLPs.5,Study on the immune effect of the novel DNA vaccine against JEV and PPV 4 to 6-week-old healthy Kunming mice were intramuscular inoculated with 100μg of recombinant plasmid pCI-e, pCI-VP2 and pCI-Ve per mice respectively,and given boost inoculation for one times with 2-week interval.We detected humoral and cellular immune responses of immuned mice,as well as production of specific anti-JEV neutralizing antibodies.We evalaute the immunoprotection of pCI-Ve with JEV challeged.The result indicate that the recombinant plasmids pCI-Ve induce good humoral and cellular immune responses,it provided 75% protective rate against intraperitoneal challenge with a lethal dose of JEV, suggesting pCI-Ve have potential to become a novel DNA vaccine preventing from JEV and PPV. 6,Development of trivalent genetic engineering live veter vaccine against Porcine Japanese Encephalitis, Porcine parvovirus and PseudorabiesChimeric gene Ve which containing JEV multi-epitope e gene by codon optimzied and PPV VP2, were inserted into the multiple clone sites of universal transfer vector pPI-2.EGFP of PRV SA215 that was constructed by our laboratory previously, to construct the transfer plasmid pPI-Ve.Then,we constructed the recombinant pseudorabies virus SA215(G) throug the method of the homologous recombination reaction that Ve take the place of parentstrain PRV SA215. PRV SA215(G) was identified by Western-blotting, PCR, observation of green fluorescence and cytopathogenic effect,the result indicate our PRV SA215(G) were constructed successfully. Furthermore, CEF in Vell cells and the virion of electron microscopic had no significant deviation compared with that of the parentstrain SA215 and that the insertion of foreign genes Ve had few influence on the biological characteristics of recombinant virus.But,by electron microscopic,we observed not only the same virion as PRV SA215,but also the slightly bigger than PPV virus-like particles formed by VP2 gene expressing and self-assembling in Vero cell culture. It is presumed that Ve gene was expressed in PRV and self-assembled chimeric VLPs. Piglet immunized with recombinant PRV SA215(G) at a dose of 106.0 TCID per piglet by intramuscular injection,was induced effective JEV,PPV and PRVspecial humoral immunoresponse, as well as stimulated good cellular immuneresponse of Th1 and Th2 type that the T helper cell involved in this process tending to the Th1 type mainly. The neutralizing antibody response elicited by PRV SA215(G) was similar to by PRV SA215.These results suggest that the recombinant PRV SA215(G)could be suitable candidate vaccine strain for developing a novel genetic live veter vaccine to combating JEV,PPV and PRV in the pig industry.