球形幽门螺杆菌vacA、cagA基因克隆及序列分析
发布时间:2018-02-28 13:13
本文关键词: 球形幽门螺杆菌 cagA基因 vacA基因 克隆 表达 出处:《安徽理工大学》2005年硕士论文 论文类型:学位论文
【摘要】:目的 克隆球形H.pylori vacA、cagA基因,并进行序列测定及表达,研究球形H.pylori的致病性,并为抗H.pylori疫苗的研制奠定实验基础。 方法 采用亚抑菌浓度的氨苄青霉素诱导幽门螺杆菌标准株NCTC11637产生球形变异,收集球形H.pylori。从球形幽门螺杆菌基因组DNA中特异扩增出vacA、cagA基因片段,扩增的目的片段经纯化回收后克隆到pMD-18T中,转化入大肠杆菌JM109。阳性重组质粒用PCR及双酶切鉴定,并进行序列测定。采用亚克隆技术,用BamH Ⅰ和Sac Ⅰ从重组质粒pMD-18T-vacA、pMD-18T-cagA上切下vacA、cagA基因,插入表达载体pET32a(+)质粒,转化大肠杆菌BL21,阳性重组子经双酶切及PCR扩增鉴定。重组质粒pET32a(+)-vacA、pET32a(+)-cagA转化大肠杆菌,IPTG诱导表达后进行SDS-PAGE电泳和凝胶扫描定量分析。 结果 从球形幽门螺杆菌基因组DNA中分别扩增出3888bp、3444bp的vacA、cagA基因,构建重组质粒pMD-18T-vacA、pMD-18T-cagA及pET32a(+)-vacA、pET32a(+)-cagA,双酶切及PCR扩增均筛选出含目的片段的阳性克隆。测序结果显示,球形H.pylori与已公布的H.pylori vacA、cagA基因序列的同源性分别达99.8%、99.7%。推测氨基酸同源性分别达99.3%、99.2%。vacA、cagA基因在大肠杆菌中经诱导表达后分别获得约156kD、148kD的蛋白,VacA蛋白含量占全菌体蛋白含量的15.5%。 结论 成功地对球形幽门螺杆菌vacA、cagA基因进行体外扩增及构建原核重组质粒pMD-18T-vacA、pMD-18T-cagA、pET32a(+)-vacA、pET32a(+)-cagA,并经酶切及序列分析所验证,证明球形幽门螺杆菌含有完整的vacA、cagA基因,并且能在大肠杆菌中获得高效表达。球形H.pylori含有vacA、cagA基因并能体外克隆和表达,可能与其潜在的致病性有关。
[Abstract]:Objective to clone, sequence and express Vaca cagA gene of spherical H.pylori, to study the pathogenicity of H.pylori and to lay an experimental foundation for the development of anti-H.pylori vaccine. Methods the subinhibitory concentration of ampicillin was used to induce the spherical variation of Helicobacter pylori standard strain NCTC11637. The Vaca cagA gene fragment was specifically amplified from the genomic DNA of Helicobacter pylori. The amplified fragment was purified and recovered and cloned into pMD-18T and transformed into E. coli JM109.The positive recombinant plasmid was identified by PCR and double enzyme digestion and sequenced. Vaca cagA gene was cut off by BamH 鈪,
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