幽门螺杆菌重组vacA-ctxB蛋白的基因克隆与表达  

Gene cloning and expression of VacA-ctxB recombinant protein to Helicobacter pylori

作　　者：张任飞[1] 杨致邦[1] 栗俊杰[1] 夏丽君[2] 李昌庆[3] 陈瀑[4] 

机构地区：[1]重庆医科大学基础医学院病原生物学教研室,重庆400016 [2]核工业四一六医院检验科,四川成都610051 [3]成都市第六人民医院检验科,四川成都610051 [4]重庆医科大学附属第一医院检验科,重庆400022

出　　处：《中国微生态学杂志》2008年第6期562-565,共4页Chinese Journal of Microecology

摘　　要：目的构建幽门螺杆菌(H.pylori)vacA毒性片段与霍乱毒素B亚单位(ctxB)基因的原核表达载体,并诱导表达VCTB重组蛋白,为制备防治H.pylori感染的口服疫苗奠定基础。方法以H.pylori基因组DNA为模板,PCR扩增vacA毒性片段基因,克隆至质粒pQE30中,获得重组质粒pQE30-vacA。再以pET32(a)+-ctxB质粒为模板PCR扩增ctxB目的基因并插入pQE30-vacA中,构建含双基因的表达质粒pQE-vctB。克隆至大肠埃希菌Top10,并在DH5α中诱导表达。SDS-PAGE分析表达结果,Ni-NTA柱纯化后Western blot鉴定其抗原性,免疫家兔后ELISA法检测血清中VacA和CtxB抗体鉴定其免疫原性。结果vacA的DNA片段为723 bp左右。ctxB基因的DNA片段为372 bp左右,与预计长度相符合。测序结果vctB融合基因由1092 bp组成,编码364个氨基酸残基的多肽,与基因文库相符。表达蛋白VCTB经SDS-PAGE分析,相对分子量为40 000,与预期的一致;表达量约占菌体总蛋白的20%,提纯后SDS-PAGE分析可见单一条带,纯度可达92%以上。Western blot鉴定能与抗VacA人血清发生特异性反应,ELISA测定能与抗ctxB兔血清发生特异性反应。结论含vctA和ctxB融合基因的表达载体构建成功,并在大肠埃希菌DH5α中表达了重组蛋白质VCTB,表达蛋白具有良好的抗原性和免疫原性,可用于制备口服疫苗。Objective To lay a foundation for preparation of prophylaxis vaccine and therapy of H. pylori infection, the fusion gene of H. pylori vacA and cholera toxin subunit B （ctxB） was constructed and VCTB recombinant protein was expressed. Methods The vacA toxic subunit gene was amplified using PCR and template of DNA genome from H. pylori, and cloned into plasmid pQE30 to acquire plasmid pQE30-vacA. The ctxB gene was amplified, using pET32 （a）^＋ -ctxB as the template and inserted into pQE30-vacA to construct the expressing plasmid pQE-vctB containing vacA and ctxB genes, pQE-vctB was cloned into E. coli Top10, then translated into E. coli DH5α to be induced and expressed. VCTB recombinant protein was analyzed by SDS-PAGE after purified by Ni^2＋ -NAT chromatography. Its antigenicity was identified by Western blot. To confirm its immunogenicity,the antibodies of VacA and CtxB in the serum were detected by ELISA after immunizing by the VCTB recombinant protein. Results The vacA gene sequenced as about 723 bp and ctxB gene sequenced as about 372 bp were consistent with the anticipated length of DNA. The vctB fusion gene sequenced as 1092 bp by the sequencing was in conformit with the genebank, and encoded polypeptides of 364 amino acid residues. The expression product was about 20% of total cell protein, and its relative molecular weight was 40 000, consistent with the anticipated weight analysed by SDS-PAGE. A single strap was clear after recombinant protein was puritied and the purity of recombinant protein was above 92% by SDS-PAGE. It was confirmed that VCTB recombinant protein could react specifically with VacA antibody in human serum identified by Westem blot and with CtxB antibody in rabbit serum detected by ELISA. Conclusion The vector containing vacA and ctxB fusion gene of H. pylori was constructed successfully, and VCTB recombinant protein was expressed in E. coli DH5α. VCTB fusion protein expressed can be used to prepare oral vaccine for their better antigenicity and immunogenicity.

关 键 词：幽门螺杆菌 细胞空泡毒素 霍乱毒素B亚单位 融合蛋白 表达 

分 类 号：R392.33[医药卫生—免疫学]