Studies On Bi-linked Genetic Engineering Marker Vaccine Against JEV And PRV

Japanese Encephalitis (JE) and Pseudorabies are the main causes of infectious reproductive failure in swine, and make significant economic losses in swine industry. In addition, JE is a serious mosquito-borne viral disease of major Public health importance in Asia and Australia.Pigs are considered the main vertebrate host and represent an important amplifier and reservoir for JEV. Therefore prevention and control of JE in swine can benefit for both swine production and 'human being' public health. At present, besides biosecurity measures, vaccination is the most effective measure to prevent and control these diseases. However, there is no JEV strain targeted specially to swine vaccination, so there are problems in both inactivated and attenuated JEV vaccines used currently. In addition, the stresses caused by administrating vaccination frequently deteriorate the performance of pigs. In practice, multivalent vaccines are preferred. For the development of a bi-linked vaccine, So, E and PrM genes of JEV were amplified, cloned and sequenced. The recombinant pseudorabies viruses were constructed and the biological characteristics were evaluated. To differentiate infections of wild JEV and the recombinant viruses, an ELISA was developed with the NS1 protein expressed in prokaryotic cells as coating antigen.1. Cloning and sequencing of PrM, E, NS1 and NS3 genes of JEVFour pairs of primers were synthesized based on the nucleotide acid sequence of JEV CQRC-1 strain, and the appropriate restriction enzyme sites were introduced into the primers. PrM, E, NS1 and NS3 genes of JEV were generated by RT-PCR, then were cloned into pMD18-T simple vector, and designated PrM-T, E-T, NS1-T and NS3-T. PrM, E, NS1 and NS3 of strain CQRC-1 shared 96-97%, 96-97%, 98-99% and 98-99% nucleotide identities with strains SA14,JaGAr01 and P3, respectively.2. Construction of recombinant pseudorabies viruses expressing PrM or E protein of Japanese encephalitis virusThe transfer plasmids pPI-2.EGFP.PrM and pPI-2.EGFP.E were constructed by inserting the PrM and E gene of JEV into the multiple clone sites of universal transfer vector pPI-2.EGFP, respectively. The recombinant pseudorabies viruses were rescued by co-transfecting the pPI-2.EGFP.PrM and pPI-2.EGFP.E with the genome of SA215, respectively. The recombinant pseudorabies viruses were identified by fluorescence demonstration, PCR test and southern blotting, then named SA215 (E) and SA215 (F). The result of western blot analysis demonstrated the E and PrM protein could be expressed in recombinant PRV strains. The results showed that the recombinant pseudorabies viruses were successfully constructed.3. Biological characteristics and genetic stability of the recombinant viruses SA215 (E) and SA215 (F)Comparison of cytopathogenic effect, plaque form and one step growth curve in Vero cell and the virion characteristics between SA215(E), SA215(F) and SA215 confirmed no effect on SA215 biological characteristics after inserting PrM or E gene into genome of SA215. After six successive passages in Vero cell, the observation of fluorescence and PCR detection results indicated that E and PrM genes had been stably inserted into the genome of the recombinant viruses, and that the recombinant viruses SA215(E) and SA215(F) genetic stability were high.4. Evaluation of the immunogenicity and safety of two bi-linked genetic engineering vaccinesTo evaluate the safety and immunogenicity of the two bi-linked genetic engineering vaccines, Balb/c mice aged 6 weeks, piglets aged 1 day and rabbits weighted 1.5kg that were serologically negative to both pseudorabies virus and Japanese encephalitis virus were selected. The results confirmed that both of SA215 (E) and SA215 (F) are safe to mice, rabbits and piglets. All mice challenged with lethal dose of virulent JEV were protected by SA215(E), by contrast, only half of the mice were protected by SA215(F).The rabbits vaccinated with them could acquire protect immune against lethal dose challenge of the virulent PRV strain Fa. Furthermore, the specific immunological responses could be induced in vaccinated animals. SA215 (E) and SA215 (F) elicited neutralization antibody to JEV and JEV-specific CTL activity. But those of SA215 (E) were higher than that of SA215 (F). Neutralization antibody titer in the mice vaccinated two doses with SA215 (E) or SA215 (F) reached 1:20 and 1:4, respectively.The above results revealed that the recombinant strain SA215 (E) could be potentially used as bi-linked marker vaccine against JEV and PRV in swine industry.5. Development of indirect ELISA with NS1The recombinant plasmids NS1-T and NS3-T were digested with BamH I/HindⅢand KpnI/EcoRI to generate NS1 and NS3 genes, and subcloned into the prokaryotic expression vector pET-32a(+), respectively. The recombinant plasmids were transformed into E.coli BL21. The transferred bacteria were induced by IPTG. The expression of NS1 protein was optimized with proper inducing conditions of 0.4 mmol/L IPTG, 4 hours and 37℃. Using the purified fusion protein as coating antigen, the optimal concentration of NS1 for coating of plate was 10μg/ml; the dilution of sera was 1:40; the best blocking solution was 1% BSA; serum sample for detection should be incubated for 90min; the working concentration of HRP-SPA was 1:2000; HRP-SPA should be incubated for 60min at 37℃; the substrate for ELISA was incubated at RT for 10min. Following the determination of condition of ELISA, the threshold value of ELISA was 0.152. Serological results by the indirect enzyme-linked immunosorbent assay (ELISA) indicated that the assay could differentiate infection with vaccination, and could be used as accompanied method to the surveillance of the SA215 (E) in this experiment.