中国免疫学杂志2011，Vol.27Issue(4)：347-351,5.DOI:10.3969/j.issn.1000-484X.2011.04.014
幽门螺杆菌cag4蛋白的原核表达、纯化及鉴定
Prokaryotic expression,purification and identification of cag4 in Helicobacter pylori
摘要
Abstract
Objective :To construct a recombination prokaryotic system of cag pathogenicity island protein 4 (cag4 ) for expression and punfica-tion of the fusion protein .Methods :PCR technique was used to amplify cag4 gene encoding lytic transglycosylase in CagPAI of heli cobacter pylori NCTC11637 .By TA cloning and restriction enzyme digested ,we constructed prokaryotic expression plasmid pET-28a-cag4 and transformed the plasmid into E . coli BL21 (DE3 ) for heterogenesis expression after sequencecl .SDS-PAGE and Westem blot were performed after induced by IPTG in vitro .We punfied and collected the recombinant protein by using Ni2+-NTA columns under denaturation condition . The renaturation of the recombinant protein was performed via dialysis treatment .Besides these ,we isolated peptidoglycan from Micrococcus lysodedcticus by SDS boiling method .The enzyme activity of recombinant protein was detected by zymogram analysis and the effect of pH values by turbidimetric analysis .Results :The cag4 gene was obtained successfully . E .coli BL21 strains which harbored the recombinant plasmid could express cag4 fusion protein .The inducing conditions of prokaryotic expression were optimized .The expressing protein was really identical to cag4 pmtein by Westem blot .The enzyme activity ,which defined as the rate of degraded (△A·min-1mg -1 ) ,was higher at pH 6 .0 group than other groups (pH5 .0 and 7 .0 ) .Conclusion :The cag4 in Helicobacter pylori possesses the enzyme activity of lytic transglycosvlase .关键词
幽门螺杆菌cag4蛋白/原核表达/纯化/融合蛋白分类
医药卫生引用本文复制引用
王文凯,钟桥,谢立苹,邵世和..幽门螺杆菌cag4蛋白的原核表达、纯化及鉴定[J].中国免疫学杂志,2011,27(4):347-351,5.基金项目
本文受国家自然科学基金项目(30870096)、江苏省高校自然科学基金项目(08KJB310001)和江苏大学临床医学科技发展基金项目(JLY20080051)资助 （30870096）
