Cloning And Expression Of The Complete Hp CagA Gene And Cholera Toxin Subunit B Gene

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.