幽门螺杆菌Ⅳ型分泌系统cagM基因的克隆、表达及抗体制备  

Cloning and expression cagM gene of Helicobacter pylori type Ⅳ secretion system and antibody preparation

作　　者：田树伟[1] 邵世和[2] 韩军[1] 黄河[1] 黄世腾[2] 

机构地区：[1]江苏大学生命科学研究院 [2]江苏大学基础医学与医学技术学院病原生物学研究室,江苏镇江212013

出　　处：《江苏大学学报（医学版）》2010年第3期239-243,共5页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(30870096);江苏省高校自然科学基金资助项目(08KJB3120001)

摘　　要：目的：构建表达幽门螺杆菌（Helicobacter pylori,H.pylori）NCTC 11637 IV型分泌系统cagM（HP0537）全长编码基因的原核表达载体,分析其抗原性并制备抗体。方法：应用PCR技术从H.pylori基因组扩增cagM基因片段,TA克隆后测序分析,并构建表达载体pET-28a（＋）-cagM,转化E.coliBL21,IPTG诱导表达,SDS-PAGE及鉴定表达蛋白,表达蛋白以Ni2＋-NTA柱进行纯化,用软件分析其抗原性后,免疫家兔制备多克隆抗体,ELISA检测多克隆抗体效价。结果：测序结果表明cagM基因全长1 131 bp（基因库登录号为GU269568）,编码376个氨基酸,与基因库中已知菌株J99,26695和G27的氨基酸同源性为98%~99%;诱导表达后经SDS-PAGE检测表明,在43 700处发现一条新生条带,Ni2＋-NTA柱纯化后为单一条带,软件分析表明其抗原性较强,免疫家兔制备的多克隆抗体效价为1∶1.6×105。结论：首次成功克隆并表达了H.pyloripNCTC 11637cagM基因,并制备了其抗体,为以后进一步研究其生物学功能以及H.pylori相关疾病的检测与治疗奠定了基础。Objective： To construct prokaryotic expression vector for expressing cagM（HP0537） of Helicobacter pylori（H.pylori） NCTC 11637 of Ⅳ type secretion system,to analyze its antigenicity and to produce antibodies.Methods： PCR technology was used to amplify cagM from the H.pylori genome.Then TA cloning was used for sequence analysis.We constructed expression vector pET-28a（＋）-cagM,and transformed it to E.coli BL21.IPTG was used to induce expression.After SDS-PAGE for identification of expressed protein and Ni2＋-NTA column for purification,used software analysis of its antigenicity,and prepared antibody by immunization of rabbits.At last,ELISA was used to detect polyclonal antibody titers.Results： cagM gene sequencing results showed that the full-length was 1 131 bp（GenBank accession number： GU269568）,encoding 376 amino acids.Compared with strains J99,26695 and G27,its amino acid homology was 98%~99%.After the induced expression and SDS-PAGE analysis,in the 43 700 position found a new band,and Ni2＋-NTA column purified was a single band.The software analysis showed that the antigenicity was stronger.The prepared rabbit polyclonal antibody titer was 1∶1.6 × 105.Conclusion： For the first time successfully cloned and expressed the H.pylori NCTC 11637 cagM genes,and prepared its antibody,which provide a foundation for further study its biological function,as well as the detection and treatment of Helicobacter pylori-related diseases research in the future.

关 键 词：幽门螺杆菌 Ⅳ型分泌系统 cagM基因 抗体 

分 类 号：R378.99[医药卫生—病原生物学]