| Coxsackie B3 virus is important human pathogens, which can cause a wide range of infectious diseases, increasing incidence in recent years, the infection in animals have also have been reported. The 25% of Human viral myocarditis are caused by the Coxsackie virus infection, and CVB3 is the most common pathogen. At present, the conventional methods of detection of CVB3 serum antibody have neutralization tests,Western blot. But these methods have some disadvantages, which is wasting time or operating complicated. not suitable for the detection of the basic level detection. Therefore, set up a simple, good repeatability Coxsackie B3 virus serum antibody detection method has great significance. We establish a method of ELISA serum antibody detection through Coxsackie B3 virus from Sichuan golden monkey. and a variety of animals were tested in serum..Coxsackie virus B3 belong to a small RNA virus is an Enterovirus Branch. Regardless of the genome segments for the single positive-strand RNA, containing P1,P2 and P3 areas. And the P1 area contains one of VP4, VP2, VP3 and VP1 genes of five structures. The VP1 protein is the major surface antigen of the virus, and is closely related to virus virulence, host cells involved in the virus-specific adsorption and assembly of progeny virus, and are induced by the primary antibody Protein.Thus, this study use the VP1 gene sequences which belong to the Coxsackie B3 virus from Sichuan golden monkey to design a pair of specific primers. Obtained the VP1 gene. Then, insert the VP1 to the expression vector of the pET-28a (+).the successful construction of the expression vector pET-28a (+)-VP1. After, by construction of the expression of the recombinant plasmid into the host bacteria E.coil Medium, IPTG induced expression. Separately by the expression product of the SDS-PAGE and Western-blotting detection, results show that: the expression of E. coli can be highly efficient target gene, IPTG induction concentration of 1.0 mmol / L, when the induction time is best for 5h, and the expressed protein has a good immunogenicity. On this basis, to set up the ELISA, using the expression of VP1 protein as coating antigen. Further optimization of ELISA conditions to determine the detection of serum antibodies to Coxsackie B3 virus detection method of indirect ELISA conditions: coating concentration of 3.125μg / ml, 37℃2h, 4℃overnight; closed solution of 5% choose skim milk, 4℃overnight; The serum diluted 80 times, 37℃and incubated under the conditions of 1h; merchandise 4000 two-fold diluted anti-, 37℃reaction of 1h, the termination of reaction in order to read the OD value, OD490 values> 0.39 as positive. By blocking cross-reaction tests and tests confirmed that the method established specific good and Coxsackie B1, B2 and B4 virus, such as no cross-reaction occurred. And, using the indirect ELISA method set up for children, golden monkey, dogs, pigs and tigers serum was tested and found that CVB3 infection in children in general, the overall antibody positive rate was 46%; golden monkey and dogs there are in the CVB3 infection, the positive rate of 12% and 28.5%; and pigs and tigers do not detect serum antibodies to CVB3. |
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