Development Of Indirect Elisa Based On VP1Protein For Detecting Antibody And Inactived Vaccine Of Encephalomycoarditis Virus

The encephalomyocarditis virus (EMCV) is a single-stranded positive RNA virus belonging to the genus Cardiovirus of the family Picornaviridae. It can naturally infect a wide range of vertebrate species including insects, reptiles, rodents and primates. Of all domestic animals, pigs are the most sensitive to EMCV. Natural infections with EMCV can cause fatal myocarditis in young piglets or reproductive failure in sows. In this report, an indirect ELISA based on VP1protein was developed for detecting antibody to EMCV and the inactived vaccine of EMCV were prepared and the immunogenicity was examined in mice and piglets.1Prokaryotic expression and purification of EMCV VP1proteinSpecific primers for EMCV structural protein VP1gene were designed, and the full-length of VP1gene sequence was amplified by RT-PCR. The sequence was cloned into the prokaryotic expression vector pET-32a and the recombinant plasmid pET-32a-VP1was constructed and identified by restriction enzyme digestion and sequencing. After the pET-32a-VP1was transformed into E.coli competent cells (BL21), the VP1protein was expressed stably by inducetion with IPTG. And the bacterial concentration, IPTG concentration and time of employed inducer (IPTG) were selected for expression. The optimization condition was1.0mM of IPTG inducing by8h. The recombinant VP1protein was purified with affinity chromatography. SDS-PAGE and Western-blot results showed that the protein had a good antigencity.2Development and preliminary application of indirect ELISA with the recombinant VP1protein for detecting antibody to EMCVAn indirect enzyme-linked immunosorbent assay for detecting EMCV VP1specific antibodies was developed based on the recombinant protein VP1expressed in E.coli. The optimal antigen concentration for coating was2.0μg/mL, while the optimal dilution of serum was1:50and the incubation time was1h. The dilution of conjugate was1:10000, and the reaction time was30min. When OD450S≧0.40and P/N≧2.1, the result of the serm sample was positive and OD450≦0.30was negative. The method had no cross reaction with other positive serum, such as PCV2, PRRSV, CSFV and PRV, showing high specification. The results of epidemiological survey with the method showed that EMCV infection rate was13.0%in jiang su province.3Selection of stabilizer for the key components of indirect ELISAVarious stabilizers for antigen, serum, washing liquid were selected in the study by diction of the storage time of the components at4℃. The results showed that coated antigen can be stored at4℃for12months after incubated45mins with the stabilizer A (10%(NH4)2SO4in0.01M Tris-HCL). Serum and washing liquid can be stored at4℃for more than12months treated with thimerosala (final concentration0.1‰). An evaluation of SPA-HRPand TMB substrate solution was conducted. The results showed that their inter and intra coefficients of variation were less than10%. The results proved that the various protective agents can extend the shelf time of each key component in the ELISA. It laid an important material foundation for the development of indirect ELISA kit.4Study on inactived vaccine of encephalomyocarditis virusFive inactived EMCV vaccines by mixing EMCV antigen inactived with BEI with five kinds of adjuvants ISA201, ISA15A, GEL,1313and CP940were prepared. Mice (n=35) aged6weeks were divided into7groups:Groups1-5were vaccined with the5inactivated vaccines via s.i., respectively. Group6was inoculated the inactivated EMCV antigen without adjuvant. Group7was injected with2%DMEM as negative control. Booster vaccination was performed after an interval of3weeks. The levels of antibodys to EMCV were detected by ELISA and neutralizing assay at3and6weeks post inoculation. And lymphocyte proliferation were detected at6weeks post inoculation. Meanwhile, the mice were challenged with vindent strain EMCV NJ08at6weeks post primary vaccination. The results showed that the ELISA antibody titers in the five vaccines groups were more than1:1600. Especially in the groups of ISA201. ISA15A and GEL, the antibody titers were more than1:3200. And the neutralizing antibody titers in the vaccined groups were similer to each other, being1:16. The stimulation index of lymphocyte proliferation were above3.0in the GEL, ISA15A groups, which being higher than those in other groups. The protection rates in the ISA15A, ISA201and GEL was90%. The21-days-old piglets inoculated with the ISA15A adjuvant vaccine showed no clinical signs during the period of observation. And the neutralizing antibody levels were1:128in piglets at3weeks post booster vaccination. It indicated that inactivated EMCV vaccine with ISA15A adjuvant was safe and could provide protective effencity against EMCV challenge.