幽门螺杆菌BabA蛋白N段的基因克隆、表达纯化及体外粘附活性评价  

Gene Cloning,Expression,Purification,and in vitro Adhesion Activity Determination of Amino-terminal Fragment of Helicobacter pylori BabA Protein

作　　者：高涵[1] 薛利军[2] 刘小北[2] 冒晓蓓[2] 任丽丽[2] 耿建[2] 戴婷婷[2] 于锋[1] 褚晓源[2] 

机构地区：[1]中国药科大学药学院,南京211198 [2]南京军区南京总医院,南京210002

出　　处：《中国生物工程杂志》2013年第3期34-40,共7页China Biotechnology

基　　金：国家自然科学基金资助项目(30901733)

摘　　要：目的:克隆幽门螺杆菌BabA蛋白的N段(氨基酸1-437位)基因(BabA1),构建其原核表达质粒,表达并纯化获得携带6×His标签的BabA1融合蛋白,评价融合蛋白的体外粘附活性。方法:以幽门螺杆菌J99菌株基因组DNA为模板,通过PCR扩增获得BabA1基因片段,并定向克隆至原核表达载体pET-32a(+)。经酶切及测序分析正确后,将重组质粒pET32a-BabA1转化大肠杆菌BL21(DE3)菌株,进行IPTG诱导表达,并对表达产物进行镍柱纯化、SDS-PAGE和Westernblot鉴定。采用菌落计数法评价BabA1融合蛋白在体外的细胞粘附活性。结果:成功扩增了BabA1基因,构建了pET32a-BabA1原核表达质粒,并通过优化该表达系统的最适诱导和纯化条件,获得了具有活性的6×His-BabA1融合蛋白,后者能显著增强大肠杆菌BL21(DE3)在体外对胃癌MFC细胞的粘附。结论:成功诱导表达并纯化获得了有粘附活性的6×His-BabA1融合蛋白,为进一步研究其免疫活性和生物学功能奠定了基础。Objective： To clone the gene of amino-terminal fragment of Helicobacter pylori BabA protein （ BabA1 ） and construct a prokaryotic expression plasmid. A fusion protein of 6譎is-BabA1 was then acquired and evaluated its adhesion activity in vitro. Methods： BabA1 gene was amplified by PCR with genomic DNA of H. pylori J99 strain as the template and inserted into pET-32a（＋）vector. The pET32a-BabA1 plasmid was confirmed through restriction enzyme analysis and DNA sequencing and then transformed E. coli BL21 （DE3） cell. The 6譎is-BabA1 protein was induced by IPTG, purified through a Ni2＋-NTA column, and identified by SDS-PAGE and Western blot. Then a bacterial colony count assay was adopted to determine the cellular adhesion activity of 6譎is-BabA1. Results： BabA1 gene was correctly cloned and recombinant pET32a-BabA1 plasmid was obtained. The 6譎is-BabA1 protein was efficiently induced and successfully purified under optimized conditions. Moreover, 6譎is- BabA1 protein enhanced the adhesion of E. coli BL21 （DE3） strain to gastric cancer MFC cells in vitro. Conclusion： The acquired 6譎is-BabA1 protein has significant adhesion activity, which facilitates further studies on its immunological and biological functions.

关 键 词：幽门螺杆菌 BabA1 原核表达 蛋白纯化 粘附 

分 类 号：Q78[生物学—分子生物学]