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目的重组表达牛支原体P81膜蛋白,为研制牛支原体疾病诊断试剂提供研究基础。方法根据GenBank发表的牛支原体P81基因的编码序列,利用点突变试剂盒基于重叠延伸PCR原理设计5对引物,将P81基因的第165、690、1 455、1 674位编码色氨酸的TGA密码子分别突变为TGG,并连接到表达载体pET-32a(+),转化DH5α感受态细胞后经IPTG诱导表达目的蛋白,Western blot检测其反应原性。结果成功扩增出4处TGA突变为TGG的牛支原体新疆分离株P81基因,大小与预期值2 112bp一致。重组表达载体转化菌经IPTG 37℃诱导8h,表达分子质量单位(Mr)约94×103的目的蛋白,与预期相符。Western blot检测该蛋白能被抗P81多克隆抗体识别。结论成功构建重组牛支原体P81膜蛋白基因表达载体,获得的目的蛋白具有良好反应原性,为研发牛支原体病免疫诊断试剂奠定了基础。
Objective To recombinantly express the P81 membrane protein of Mycoplasma bovis, and provide the basis for the research of the diagnostic reagent of Mycoplasma bovis. Methods According to the coding sequence of P81 gene of M. bovis published in GenBank, five pairs of primers were designed based on the overlap extension PCR using the point mutation kit. The TGA codes encoding the tryptophan at position 165,690,1 455,1 674 of P81 gene Were subcloned into TGG and ligated into the expression vector pET-32a (+), transformed into DH5α competent cells and induced by IPTG to express the target protein. Western-blot was used to detect the reactogenicity. Results Four strains of P81 gene of M. bovine Mycoplasma Xinjiang were successfully amplified, which was consistent with the expected value of 11212 bp. The recombinant expression vector transformed cells were induced by IPTG at 37 ° C for 8h, and the target protein with a molecular mass unit (Mr) of about 94x103 was expressed, in line with the expectation. The protein was detected by Western blot with anti-P81 polyclonal antibody. Conclusion The P81 membrane protein gene expression vector of recombinant Mycoplasma bovis is successfully constructed. The obtained target protein has good reactionogenicity, which lays the foundation for the development of immunodiagnostic reagents for M. bovis.