牛分支杆菌HBHA基因的克隆及原核表达  

Cloning and Prokaryotic Expression of Mycobacterium bovis HBHA Gene

作　　者：赵子惠[1] 温峰琴[1] 项海涛[1] 曾巧英[1] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《动物医学进展》2015年第10期5-9,共5页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(31260616);甘肃农业大学伏羲杰出人才项目;现代农业(奶牛)产业技术体系专项经费项目(CARS-37)

摘　　要：应用PCR技术扩增牛分支杆菌肝素结合血凝素(HBHA)基因并原核表达牛分支杆菌HBHA基因,利用在线分析软件对该基因序列及编码蛋白序列进行生物信息学分析。构建原核表达载体pET28a(+)-HBHA,并转化至大肠埃希菌BL21(DE3),经0.6mmol/L IPTG诱导目的蛋白表达,采用镍离子亲和层析纯化目的蛋白,SDS-PAGE及Western blot检测表达结果。结果表明,牛分支杆菌HBHA基因完整的ORF全长600bp,编码199个氨基酸。HBHA蛋白为碱性、亲水性蛋白质,含有12个潜在的磷酸化位点,存在抗原表位,亚细胞定位主要存在于细胞核及线粒体中,蛋白空间结构以α-螺旋、无规则卷曲、β-折叠为主。成功构建了原核表达载体pET28a(+)-HBHA并在大肠埃希菌中得到了高效表达,分子质量大小为28ku,主要以可溶性形式存在。说明牛分支杆菌HBHA蛋白是一种抗原指数较高的亲水性蛋白,在原核系统能高效的表达,为进一步研究提供了理论基础。The study cloned and prokaryotic expressed the Mycobacterium bovis HBHA gene and conducted biological analysis.The Mycobacterium bovis HBHA gene was amplified by PCR and the biological infor-mation analysis was performed with an online tool and cloned into the prokaryotic expression vector pET-28a（＋）.The constructed recombinant plasmid was transformed into competent Escherichia coli BL21 （DE3）and the target protein was expressed by 0.6 mmol/L IPTG induction.The expressed protein was purified by Ni-sepharose affinity chromatography and analyzed by SDS-PAGE and Western-blot.The cloned ORF of Mycobacterium bovis HBHA gene consisted of 600 nucleotides.Mycobacterium bovis HBHA was a basic hydrophilic protein,which comprised 12 potential phosphorylation sites.The antigenicity was observed.HBHA mainly distributed in the nucleus and the main structures included α-helix,random coil andβ-extension.Prokaryotic expression vector pET28a（＋）-HBHA was successfully constructed,which could induced the 28 ku recombinant protein of HBHA in the prokaryotic expression system.The Myco-bacterium bovis HBHA was a basic and hydrophilic protein with antigenicity.It was well expressed in pro-karyotic expression system.

关 键 词：牛分支杆菌 肝素结合血凝素基因 生物信息学分析 原核表达 

分 类 号：S852.618[农业科学—基础兽医学]