幽门螺杆菌外膜蛋白基因omp22克隆、表达和鉴定  

CLONING,EXPRESSION AND IDENTIFICATION OF OUT MEMBRANE PROTEIN omp22 GENE OF HELICOBACTER PYLORI

作　　者：黄学勇[1] 李永红[2] 段广才[3] 范清堂[3] 郗园林[3] 

机构地区：[1]河南省疾病预防控制中心,郑州450016 [2]广西壮族自治区疾病预防控制中心,南宁530028 [3]郑州大学公共卫生学院流行病学教研室,郑州450001

出　　处：《广西医科大学学报》2010年第5期678-680,共3页Journal of Guangxi Medical University

基　　金：河南省医学创新人才基金资助项目(No.2000-84);河南省科技厅科技攻关项目基金资助课题(No.0424410035)

摘　　要：目的:克隆幽门螺杆菌外膜蛋白基因omp22,构建幽门螺杆菌omp22基因与麦芽糖结合蛋白基因融合表达载体,并进行诱导表达,鉴定融合蛋白免疫原性,为幽门螺杆菌疫苗研究提供依据。方法:利用PCR技术从Helicobacter pylori郑州分离株MEL-Hp27染色体DNA上扩增omp22基因,并将其克隆到表达载体pMAL-c2X中,转化大肠杆菌(E.coli TB1),用IPTG诱导目的基因表达,SDS-PAGE方法对表达产物进行分析,Western blot鉴定其免疫原性。结果:PCR方法扩增的omp22基因长度约为540 bp。经酶切鉴定,插入到表达载体的基因片段与预期目的DNA片段相一致;SDS-PAGE结果显示表达产物分子量约为22 ku,融合蛋白的表达量约占全菌总蛋白的26%。结论:成功克隆并构建了omp22基因高效原核表达系统,为幽门螺杆菌基因工程组分疫苗的研究奠定基础。Objective:To clone and express the omp22 gene of Helicobacter pylori and to identify the immunogenicity of expressed protein in order to build a basis for the investigation work of vaccine and diagnostic antigen.Methods:omp22 gene of the Helicobacter pylori MEL-Hp27 was amplified by PCR,and inserted into expression vector pMAL-c2X.The positive recombinants were transferred into E.coli TB1,the genetically engineered bacteria including pMAL-c2X-omp22 plasmids were induced by IPTG,and the expression products were analyzed by SDS-PAGE and Western blot.Results:omp22 gene of 540bp was obtained from the Helicobacter pylori strains MEL-HP27 genome DNA.The gene segment inserted into the recombinant vector was identified by using restriction enzyme digestion.Plasmid pMAL-c2X-omp22 could express a specific approximative 65ku fusion protein(omp22 22ku and MBP 42.5ku) in E.coli TB1,and the fusion protein accounted for 26% of the total protein of recombinant bacterial.Conclusion:omp22 gene has been cloned and a prokaryotic high expression system for omp22 gene successfully constructed,which will help to develop gene recombinant vaccine against Helicobacter pylori infection.

关 键 词：幽门螺杆菌 外膜蛋白基因 克隆 融合表达载体 酶切鉴定 HELICOBACTER PYLORI MEMBRANE PROTEIN omp22基因 麦芽糖结合蛋白基因 融合蛋白 蛋白免疫原性 表达产物 SDS-PAGE 转化大肠杆菌 原核表达系统 方法 组分疫苗 诱导表达 疫苗研究 扩增 

分 类 号：R[医药卫生]