猪肺炎支原体脂蛋白LppB的克隆表达及其编码氨基酸的生物学分析  

Prokaryotic expression of Mycoplasma hyopneumoniae LppB and its biological characteristics

作　　者：刘威[1] 袁芳艳[1] 周丹娜[1] 刘泽文[1] 杨克礼[1] 段正赢[1] 郭锐[1] 蔡行[1] 刘保亮 吴修竹 肖少波 田永祥[1] 

机构地区：[1]湖北省农业科学院畜牧兽医研究所动物胚胎与分子育种湖北省重点实验室,湖北武汉430064 [2]华中农业大学农业微生物学国家重点实验室,湖北武汉430064

出　　处：《中国兽医学报》2017年第12期2275-2280,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31502069;31170160);湖北省农业科学院青年科学基金资助项目(2015NKYJJ14);湖北省农业科学院竞争性计划资助项目(2016jzxjh030);湖北省农业科技创新中心资助项目(2015-620-004-001)

摘　　要：本研究旨在获得猪肺炎支原体脂蛋白LppB的表达产物,分析其编码氨基酸的生物学特性,为进一步研究脂蛋白LppB的功能奠定基础。试验构建重组表达质粒pET30a-LppB,通过快速定点突变试剂盒将LppB基因中1 068,1 353,1 578nt处的A突变为G,使改造后的LppB基因能在大肠杆菌系统中正确表达,IPTG诱导重组蛋白的表达并进行蛋白纯化。SDS-PAGE分析结果显示,IPTG诱导表达后获得一条特异性蛋白条带,相对分子质量约为70 000,与预期蛋白大小一致。可溶性分析结果表明目的蛋白主要以可溶的形式表达于上清中;Western blot鉴定结果表明纯化的蛋白能与抗6×His标签的单抗发生特异性反应。采用DNAStar Protean模块对脂蛋白LppB进行了二级结构、蛋白质骨架柔性区域、抗原指数的预测,并确定了B细胞抗原优势表位的可能分布。The study aimed to obtain the expression products of Mycoplasrna hyopneumoniae lipoprotein LppB, and analyze its biological characteristics. Briefly, the LppB gene was obtained by PCR and constructed recombinant plasmid pET30a-LppB. The codon TGA（located in 1 068,1 353, and 1 578 nt） were mutated into TGG by using site-directed mutagenesis kit, so as to ensure the correctness of lipoprotein LppB expression in Escherichia coli. The expression of recombinant protein was induced by IPTG and the protein was purified by Ni-NTA agarose. The SDS-PAGE results showed that the LppB fusion protein was mainly expressed as a soluble protein with approximately 70 000, which was consistent with the expected protein size. The results of Western blot showed that the purified protein could react to mouse anti-His monoclonal antibody. Furthermore, the secondary structure, backbone flexibility, antigenicity, surface aeessibility of the LppB protean were analyzed using DNAStar Protean software, and the potential distribution of the dominant epitope of B-cells was predicted.

关 键 词：猪肺炎支原体 脂蛋白 密码子改造 二级结构 

分 类 号：S852.62[农业科学—基础兽医学]