| Encephalomyocarditis is an acute infectious disease whose major clinical symptoms are encephalitis,myocarditis and myocardial surrounding inflammatory on pigs even mammals and the primates which was caused by Encephalomyocarditis virus(EMCV).The disease is an anthropozoonosis and is of great importance in public health. In recent years, it has been concerned by the domestic and foreign scholars. One of the most popular topics is the VP1 gene, for the protein it expressed is at the surface of the viral particle and has the strongest antigenicity VP1 protein can stimulates the organism to produce the polyclonal antibodies which are the main composition of virus receptor's binding site, and its amino acid sequence is closely related to viral pathogenicity.Foster the EMCV virus(F0) offered from ATCC with BHK-21 cells, extract nucleic acid from the original(F0), F4, F8, F12, F16 and F20, then amplify VP1 gene, construct cloning vector, The process of sequencing and joining is completed by the ShengGong biological engineering by carrier (Shanghai) Co., LTD. For standard strains with F0, use DNAstar to compare and analyze, the results indicate that the mutation rate of VP1 gene is 0.3%-1.6%, after subailture each generation the allogenicity between VP1 genes and virus F0 gene is 98.4%-99.7%, The amino acid mutation 0.7%-1.7%, the allogenicity between VP1 and virus F0 gene is 98.3%-99.3%. The mutation rate is very low,accords with the demands of Pharmacopoeia of People's Republic of China in 2010. The result indicates that the genetic characteristics of the VP1 gene are very stable after being cultured continuously in BHK-21 cells with EMCV and this method can be used in inoculation and cultivation during vaccine production.Design primer to amplify VP1 gene of F0, optimize its expression conditions, the purpose protein was expressed solublely and its molecular weight is about 56kD. Use Western Blotting to analyse, the result showed that this protein can bind with EMCV antiserum specifically, it proves it has good biological activity and specificity. Coating with EMCV-VP1, label inactivated EMCV virus with horse radish peroxidase(HRP), establish double antigen sandwich ELISA to test antibody, through its within snd between-batch precision and thermal stability the reagent research indicates that the CV is less than 10%, accords with sandwich Elisa box of requirements, and it has good stability and high specificity. Use this kit to test 1475 medical human serum EMCV-Ab from two areas in Gansu province and 1309 pig serum from Gansu province, the results indicate that the normal people positive rate is 16.7% and 17% in this two regions. The result indicate the 3-month pig EMCV-Ab serum positive is 56.4%, less than 3-month pig blood (70.7%), accord with reports in the literature. Also illustrate that there exists EMCV infection in Gansu province. Conclusion: EMCV can grow stably in BHK-21 cells, the mutation of VP1 gene is very low after passage. soluble expressed protein has good biological activity and specificity, used it for double antigen sandwich Elisa to test antibody, apply this method to test normal people and pig serum in Gansu. This method has good stability,elaboration and specificity. After detection, the results show that it exists EMCV infection in human and pig in Gansu province and the infection rate is high among pig. For EMCV is an anthropozoonosis virus, there may exists cross-infection between species, it needs paying more attention to in the future research. |
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