幽门螺杆菌vacA-ctxB融合基因原核表达载体的构建  

Construction of vacA-ctxB prokaryotic expression vectors to Helicobacter pylori

作　　者：张任飞[1] 杨致邦[1] 周侠[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]成都市核工业四一六医院检验科,成都610051 [3]成都市第六人民医院检验科,成都610051 [4]重庆医科大学附属第一医院检验科,重庆400022

出　　处：《重庆医科大学学报》2009年第1期37-40,共4页Journal of Chongqing Medical University

摘　　要：目的:构建幽门螺杆菌(Helicobacterpylori,H.pylori)致细胞空泡毒素抗原(Vacuolating cytotoxin antigen A,vacA)毒性片段与霍乱毒素(Cholerae toxin,Ctx)B亚单位(ctxB)基因的原核表达载体,为进一步表达VCTB重组蛋白,制备防治H.pylori感染的口服疫苗奠定基础。方法:通过比较国内H.pylori代表株的vacA基因毒性亚单位,找出高度同源性片段,按基因文库中提供的vacA基因和ctxB基因序列设计引物。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。以pET32(α)+-ctxB质粒为模板PCR扩增ctxB目的基因。再将纯化的ctxB基因片段插入pQE30-vacA中,构建含双基因的表达质粒pQE-vctB,构建的表达质粒pQE-vctB转化大肠杆菌Top10,培养后,提取纯化重组质粒pQE-vctB,内切酶双酶切鉴定。结果:扩增的vacA DNA片段约为723bp,ctxB基因的DNA片段约为372bp,与预计长度相符合。构建的表达质粒pQE-vctB酶切鉴定,与插入pQE30的目的基因片段相符。测序结果vctB融合基因由1092bp组成,编码364个氨基酸残基的多肽,与基因文库相符。结论:含vacA和ctxB融合基因的原核表达载体构建成功,为进一步表达VCTB重组蛋白奠定了基础。Objective： To lay a foundation of expressing VCTB fusion protein and preparation the oral vaccine for prophylaxis and therapy of H.pylori infection, the prokaryotic expression vectors contained the fusion gene vacA toxic subunit of H.pylori and cholera toxin subunit B （ctxB） was constructed. Methods： The high homogeneity gene fragment was found in comparing VacA toxic subunit gene of domestic typical H.pylori strains. The primers were designed according to vacA and ctxB sequences in the gene bank.VacA toxic subunit gene was amplified by PCR as the template of DNA genome of H.pylori and cloned into plasmid pQE30, which was plasmid pQE30-vacA. The ctxB gene was amplified by PCR as the template of pET32（ α）^＋-ctxB plasmid. The purified ctxB gene was inserted into pQE30-vacA to construct expressing plasmid pQE-vctB of containing vacA and ctxB genes, pQE-vctB was transformed into prokaryotic E.coli Top10. The recombinant plasmid of bacteria cultivated was extracted and purified, and cutted with the incision enzyme to evaluated. Results： The sequence of vacA gene amplified was about 723 bp and the sequence of ctxB gene was about 372 bp.They were consistented with the anticipated length of the DNA. The expressing plasmid pQE-vctB was conformitied with objective gene inserted into pQE30, vctB fusion gene sequenced as 1 092 bp by the sequencing was conformitied with the genebank,and encodes polypeptides of 364 amino acid residues. Conclusion： The prokaryotic expression vectors contained vacA and ctxB fusion gene was constructed successfully and lay a foundation of expression VCTB fusion protein.

关 键 词：幽门螺杆菌 细胞空泡毒素 霍乱毒素B亚单位 载体 

分 类 号：R378.2[医药卫生—病原生物学]