Pilot Study Of Gene Vaccine Of Helicobacter Pylori Urea Channel Protein (UreI)

Helicobacter pylori (H. pylori) is a major pathogen of chronic active gastritis and peptic ulcer, and plays an important role in the pathogenesis of gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. H. pylori is unique in its ability to colonize the normal human stomach by acid acclimation. It has been suggested that surface urease is responsible for neutralizing the acidic environment of the organism by generating a cloud of NH3. Urea addition activates cytoplasmic urease at medium pH values of <6.2. NH3 produced by the hydrolysis of urea freely diffuses from the cytoplasm into the periplasm and elevates the pH toward 6.2 . Hence, Urease and urea influx through UreI have been shown to be essential for gastric colonization and for acid survival . H. pylori persistence infection was related with H. pylori UreI significantly.If suppression UreI,urea transmembrane transport would be inactivated.It was probably that H.pylori was unable to colonize human stomach.Therefore it woulde be extensive prospect to make use of H. pylori vaccine with UreI antigen.This study utilized gene recombination engineering to construct the prokaryotic/eukaryotic expression vector and be expressed in E.coli BL21/cos-7,and studied the immunity of the recombination protein and mechanism of H.pylori.The fist part: Construction, expression and antigenic study of vaccine candidate with urea channel protein (UreI) of human Helicobacter pylori. 1.Construction prokaryotic vector pET32a(+)/UreI and expression recombinant fusion protein in E.coli BL21.Objective: To construct the prokaryotic fusion expression vector of the urea channel protein gene (UreI) of Helicobacter pylori pET32a(+)/UreI and be expressed in E.coli BL21.Methods: The target gene encoding UreI was amplified from Hp DNA by PCR, digested by restricted endonuclease enzyme of Hind III, KpnI simultaneously, and inserted into prokaryotic expression vector pET32a(+) digested by corresponding restricted endonuclease enzyme. The recombinant plasmid was used to select, identify by restricted endonuclease enzyme digestion and sequence analysis and transform, meanwhile expressed in E.coli BL21 by IPTG. The recombinant fusion protein was purified by 6his marked Ni-TEDTM kit and analysed by Western blot.Results: Restriction endonuclease analysis, sequencing showed that the target gene was found to be 585 base pairs and had been inserted in to recombinant vector. As compared with gene HP AM417609 reported by GenBank, cloned UreI gene sequence was completely coincidence and composed of 585 bp. SDS-PAGE analysis showed that the recombinant plasmid pET32a(+)/UreI could be expressed in E.coli BL21, its relative molecule mass of expressed product was 130 KD, After purified with Ni-NTA agarose resin,the purity of recombinant fusion protein was about 95%. Western blot result showed that the recombinant fusion protein could be recognized by anti-6His monocloned antibody, suggesting that this fusion protein have good reactionogenicity.2.Construction prokaryotic expression vector of the urea channel protein gene (UreI) of Helicobacter pylori pQE30/UreI and expression in E.coli BL21.Objective:To construct the prokaryotic expression vector pQE30/UreI,and be expressed in E.coli BL21.Methods: The target genes UreI were acquired from the prokaryotic expression vector pET32a(+)/UreI by Hind III , Kpn I digestion simultaneously. pQE30 was aequalesly digestion by Hind III,Kpn I. The objective genes and pQE30 were extracted out of agarose electrophoresis with gel kit,and connected by T4 ligase.The recombinant plasmid was used to select, transform, meanwhile expressed in E.coli BL21 by IPTG. The recombinant protein was purified by 6his marked Ni-TEDTM kit and analysed by Western blot.Results: Restriction endonuclease analysis confirmed that was the target gene and had been inserted in to recombinant vector. SDS-PAGE analysis showed that the recombinant plasmid pQE30/UreI could be expressed in E.coli BL21, its relative molecule mass of expressed product was 21 KD, After purified with Ni-NTA agarose resin,the purity of recombinant protein was about 90%. Western blot result showed that the recombinant protein could have good reactionogenicity.The second part: Construction and expression recombinant eukaryotic vector of urea channel protein (UreI) of human Helicobacter pylori . Objective: To construct the recombinant eukaryotic expression vector pEGFP-N1/UreI and be expressed in COS-7 cell.Methods:The target genes UreI were amplified from the prokaryotic expression vector pET32a(+)/UreI by PCR.UreI and pEGFP-N1 was by Hind III,Kpn I digested simultaneously and was connected by T4 ligase,and then transfected, expressed in cos-7 cell. The expressive product of recombinant eukaryotic vector in COS-7 were analysed by Western blot.Results: The recombinant eukaryotic vector pEGFP-N1/UreI was constructed successfully. Western blot analysis showed that recombinant eukaryotic vector could be expressed in COS-7 cel1.