牛病毒性腹泻-黏膜病病毒E_0-E_2融合基因的构建及其在卡介苗中的表达

Construction and Expression in BCG of E_0-E_2 Fusion Gene of Bovine Viral Diarrhea-Mucosal Disease Virus

为提高牛病毒性腹泻-黏膜病病毒基因工程疫苗的免疫效力,运用重叠延伸PCR法将E0、E2截短基因进行融合后亚克隆至PMV361载体上,重组质粒经酶切和PCR鉴定后电转化入BCG中。构建的重组卡介苗经45℃热诱导2 h后,进行SDS-PAGE分析和Western blotting检测。结果表明:E0-E2融合基因在卡介苗中获得了表达,目的蛋白大小为66.5 kD,且具有反应原性。该重组卡介苗的构建为牛病毒性腹泻-黏膜病和结核病的防制奠定了基础。

To improve the immune efficacy of genetic engineering vaccine for bovine viral diarrhea- mucosal disease virus, this study used overlap extension PCR method to fuse Eo and E2 truncated gene and and subclone them to PMV361 vector. The recombinant plasmid was electroporated into BCG after restriction enzyme digestion and PCR identification. The recombinant BCG was heat-induced at 45 ℃ for 2 h, and the product was analyzed by SDS-PAGE and Western blotting. The results showed that the E0-E2 fusion gene was expressed in the BCG, and the target protein was 66.5 kD, which had reactionogenicity. The construction of the recombinant BCG laid a foundation for preventing bovine viral diarrhea-mucosal disease and tuberculosis.