【作者】 周林福；

【导师】 方平楚； 严杰；

【作者基本信息】 浙江大学 ， 微生物学， 2001， 硕士

【摘要】 幽门螺杆菌（Helicobacter pylori, Hp）是广泛感染于人群胃、十二指肠的革兰染色阴性的微需氧菌，已确认是慢性胃炎和消化性溃疡的主要致病因子，并被WHO列为人类第一类致癌原（Group I cancinogen），认为Hp的感染与胃癌和胃粘膜相关淋巴组织（MALT）淋巴瘤发生有关。防治Hp感染是大幅度降低慢性胃病和胃癌发病的关键。许多抗生素虽能杀灭Hp，但所有治疗方案均存在疗程长、副作用大、费用昂贵、细菌易产生耐药性以及不宜在人群中大面积推广使用等缺点。目前认为最有前景的防治措施是开发安全、有效的Hp疫苗。由于Hp培养条件极其苛刻，生长缓慢，极易污染，以人工培养的方法获得大量Hp培养物十分困难，在Hp培养物中提取纯化蛋白的量也微乎其微。因此，通过大肠杆菌等基因工程菌获得Hp重组蛋白是研制疫苗的最佳方法。据文献报道，Hp细胞毒素相关蛋白（CagA）等具有强免疫原性和免疫保护性，霍乱毒素B亚单位（CTB）具有良好的粘膜免疫性剂作用。为此，本实验室通过原位PCR的方法获取Hp CagA和CTB基因，构建了Hp CagA以及CTB的重组质粒，并进行了重组蛋白的表达，为Hp口服疫苗的研制奠定了基础。1.Hp CagA和CTB基因的获取 合成Hp NCTC11637 CagA和霍乱弧菌古典生物型CTB基因的全长引物，两端设计与载体匹配的酶切位点，分别经原位PCR，获取Hp CagA和CTB的全长基因片段。2.CagA-pGEMEX-1、CTB-pGEMEX-1重组质粒的构建 以Sac1、Xho1双酶切CagA全长片段，以Xho1和Apa1双酶切CTB；酶切产 20（）浙江大学硕士学位论文物与同样酶切的 PGEMEl用 T4 DNA连接酶作粘端连接，得到 CagA－PGEMEX－l、crsPcliulEix－互重组质粒。以盯 和酶切方法筛选鉴定重组质粒。后者经测序证实序列已完整无误的插入PGEMEX－l载体。3．CagA－pGEffeX－l、CTB－pGEMEX－二重组质粒在大肠杆菌N109DE3中的表达 CagArpGEMEX－l、CTB－pGEMEX－l重组质粒转化至JM109DE3菌株后，从基因和蛋白质水平对CagA－pGEMEX－l、L7B－pGEMEX－1进行检测。基因检测仍用原位PCR法，结果发现转化了 CagA－PGEMEX－豆 的菌株出现一条 3．8 Kb的阳性条带，转化了CTB－pGEMEX－l的菌株出现一条0．4 Kb左右的阳性条带。重组质粒转化菌株用IPTG诱导后，行全菌SDS－PAGE，结果出现预计的融合蛋白条带：转化了CagA－pGEMEX－1的菌株出现了150 kDa左右的蛋白条带，经Destern blot证实有Hp NCTCll637CagA的反应原性；转化了CTB－pGEMEX－1的菌株出现一条35 kDa左右的蛋白条带。 结论：1．成功构建了重组质粒CagA－PGEMEX－1、C’rB－PGEMEX刁，其中后者经序列分析证实对码、序列无误。2．SDS－PAGE证实经重组质粒CagA－…EMEX－l、L7B－灿EMEX－互转化的大肠杆菌川109DE3能够成功表达CasA、CTB融合蛋白。3．免疫印迹法证实重组质粒CagAngEMEX－1转化的大肠杆菌JM109DE3表达的融合CagA蛋白具有免疫反应性。更多还原

【Abstract】 Helicobacter pylori (H. pylori, Hp) is a raicroaerophilic spiral-shaped Gram-negative bacterium that colonizes universally the gastric lumen of humans. H. pylori was identified as the cause of chronic active gastritis and peptic ulcer disease in humans and is considered to be a risk factor for the development of gastric adenocarcinoma and MALT lymphoma. And it has been categorized as a group I carcinogen by WHO. It is vital to cure and prevent Hp infection in decreasing the occurrence of the gastritis and peptic ulcer disease. A lot of antibiotics can kill Hp, but all treatment methods have shortages, such as long treatment period, side effects, high cost, and the increasing problem of antibiotic resistance, which make these therapies impractical on a large scale. The development of a safe and effective vaccine against Hp which confers long term protective immunity is the best strategy. Hp is very difficult to culture, grow slowly, and is easy to be polluted, so it is impossible to obtain a large amount of Hp. The protein we can extract from Hp is trace. Thus, it is the best strategy to acquire recombinant protein with bio-engineering methods. As literatures reported, Hp cytotoxin associated gene product A (CagA) has strong reactinogenicity and immuno protection and Cholera toxin subunit B (CTB) is a good mucosal adjuvant. In our research, we acquire CagA and CTB gene with in situ PCR and construct recombinant plasmids. And at last we express these plasmids in E. coli, which lay a good foundation for the research of Hp vaccine.1.Acquisition of CagA and CTB genes:By in situ polymerase chain reaction (PCR) method, CagA was amplified from Hp chromosomal DNA and CTB from Vibrio cholerae. Additional features of the gene were added including the terminal restriction endonuclease sites matched to the vector pGEMEX-1.2. Constructions of recombinant plasmids CagA-pGEMEX-1 and CTB-pGEMEX-1:CagA gene was digested with Sacl and Xhol; CTB gene was digested with Xholand Apal; With T4 DNA ligase, digested products were ligated with the same2001digested pGEMEX-1, and recombinant plasmids CagA-pGEMEX-UCTB-pGEMEX-1 were constructed. The recombinant plasmids were identified with PCR and enzyme digestion, and were confirmed by sequencing at last. 3. Expression of fusion proteins in E. coli J11109DE3:After the E. coli JM109DE3 was transformed with recombinant plasmids CagA-pGEMEX-1 >CTB-pGEMEX-l, they were detected at the gene and protein level. The E. coli JM109DE3 transformed recombinant plasmids CagA-pGEMEX-1 could be found a 3. 8kb DNA band with PCR. The E. coli JM109DE3 transformed recombinant plasmids CTB-pGEMEX-1 could be found a 0.4kb DNA band with PCR.Induced with IPTG, an about 150KD additional protein band, the fusion protein expressed in E.coli JM109DE3 transformed recombinant plasmids CagA-pGEMEX-1, was demonstrated by SDS-PAGE. 35KD additional protein band, the fusion protein expressed in the strain transformed recombinant plasmids CTB-pGEMEX-1 was also demonstrated. The good reactinogenicity of fusion CagA was identified with Western blot.Conclusions:1.The high fidelity recombinant plasmids CagA-pGEMEX-1, CTB-pGEMEX-1 have been successfully constructed, and the later has been confirmed by sequencing.2. The recombinant plasmids CagA-pGEMEX-1, CTB-pGEMEX-1 can express fusion protein in E. coli JM109DE3.3.Fusion CagA expressed in E.coli JM109DE3 has good reactinogenicity.更多还原