| Bovine viral diarrhea mucosal disease was a contagious disease, which can infect all ages of cattle, existing all over the world. This disease can cause patient high fever, oral and nasal mucosal bleeding or mucus oversecretion, reduction of milk production even off production, pregnant cow abortion or stillbirth, featus anomalies and other symptoms. BVDV has serological cross-reactions with CSFV, and BDV. The EO gene, which exists in the BVDV structural proteins, can serve as diagnostic antigen or subunit vaccine in genetic engineering, because of EO gene’s high conservative property and immunogenicity to some extent.A pair of specific primers was designed according to the published BVD V-EO gene sequences in this article. Oregon C24V standard poison was inoculated in MDBK cells proliferation and the virus solution was collected, then RNA was extracted from the virus solution. A921bp EO gene was amplified by RT-PCR, with RNA as template. By the establishment of pMD18-T-E0cloning vector, DNA sequencing was made after PCR amplification and restriction endonuclease. Subsequently, the recombinant plasmid vector pET28a-E0which contain expression EO Proteinwas translated into express bacterium BL21(DE3), the restructuring EO protein was expressed through training and induction. The western blot showed that the molecular weight of the purified recombinant EO protein is about34kD in size, and the purified recombinant EO protein proved to be active biologically, can react with BVDV-positive serum.With EO protein purified as the coating antigen, the experiment showed that the best antigen concentration was1.23μg/hole, the optimal serum dilution was1:160. Specificity test and repeatability test results showed that indirect ELISA method is better in specificity, variability of the method is smaller. Compared with the commercialization of BVDV antibody detection kit, the detection sensitivity of ELISA method is88.5%, specificity of that is82.1%. |
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