The Study Of The Clone, Nucleus Response And Immunogenicity Of The CagA Gene Of Helicobacter Pylori And The Effect On The Apoptosis Of Gastric Epithelial Cells

Part 1 The study of clone,nucleus response and immunogenicity of the cagA gene of Helicobacter pyloriObjective:This study is aimed to clone the specific cagA gene segment of the gastric cancer idiotype Helicobacter pylori(H.pylori), establish the system of prokaryotic expression and identify the immunogenicity of sequences of recombination signal.And it also hoped to set the foundation for picking out the strain in H.pylori correlated to gastric cancer and direction therapy in clinical.Methods:Select the sixth type cagA fragment which was conceivable to be related to gastric canser in earlier research.It's identified as AAG09884 in GenBank.Then the the cagA gene segment was optimized designed and composition.The incision enzyme sites on both ends of this gene were increased separately.The synthesized cagA gene was cut from the puc57-cagA plasmid and then was carried by expression vector pET32a to transformed into the host bacterium(type) BL21(DE3).Then the positive cloned pET32a-cagA was selected by receptivity of aniline and colony PCR.The Host Bacterium with pET32a-CagA were affected to express fusion protein by IPTG.The expression of the CagA protein was analyzed by SDS-PAGE gel electrophoresis and the antigenicity of the fusion protein was examined by Western-blot.Results:1.Target gene synthesis:Design and synthesize cagA gene(AAG09884),which is 678 bp long,and encodes 228 amino acid (figure 1-1)。The result of sequence determination shows that the sequence of synthesis cagA gene was the same as the one that we designed.2.This study construct the plasmidof prokaryotic expression of the pET32a-cagA successfully.The result of sequencing of the target cagA gene and homology of the CagA proteinum are 100%the same as what we contrivance.3.The Host Bacterium BL21(DE3)contained pET32a-cagA is able to express CagA fusion protein after the induction of IPTG.And the result of SDS-PAGE gel electrophoresis shows that the molecular weight of fusion protein is the same as what we expected (45KD).4.The result of Western-blot test shows that the fusion protein is able to be combined with antibody of the whole bacterium of anti-H. pylori.Conclusion:1.The study synthesizes the segment of cgaA gene of H.pylori successfully.The analysis of sequence of amino acid shows that its homology is 100%the same as the AAG09884 of GeneBank.2.This study construct the prokaryotic expression of pET32a-CagA plasmid successfully.And it also attain CgaA fusion protein whose molecular weight is about 45KD by induct the IPGT into host bacterium(type).And the protein that we get also has the antigenicity of CgaA protein.Part 2 The expression of H.pylori cagA gene in gastric epithelial cellls and its effect on the apoptosis of gastric epithelial cellsObjective:To Form eukaryotic expression vectors containing cagA gene and be expressed in gastric epithelial cellls(GES-1).To observe the situation of its expression and localization,and to study the effect of the cagA gane to the apoptosis of GES-1 Preliminarily.Method:Cut the compositive cagA gene from puc57—cagA, copy it into the Green Fluorescent Protein expression vector(pEGFP-C1 and pCDNA3.1-his/myc-A).Transfect each Recombinant expression vector into gastric epithelial cells(GES-1)with liposome and use the fluorescent microscope to observe its expression and localization in GES-1 cells,to use real-time quantitative PCR and Western-blot to examine the expression of cagA gene in GES-1 cells.And observe the effect of cagA gene to celled apoptosis by flow cytometry.Results:1.The study success to form eukaryotic expression plasmid of pEGFP-C1-cagA and pCDNA3.1 -his/myc-A-cagA.The result of the sequence of goal genes was completely consistent with the one tentative planned.2.Green Fluorescent Protein expression plasmid (pEGFP-C1-cagA)was transected into GES-1 and expressed widely spread in both cytoplasm and nucleus.3.After the expression plasmid (pCDNA3.1 -his/myc-A-cagA)was transected,CagA gene were expressed in GES-1 cells.4.By using flow cytometry analyzed pCDNA3.1 -his/myc-A-cagA which have transected,the CagA Protein can promote the apoptosis of gastric epithelioid cells(GES-1).Conclusion:1.Establishing eukaryotic expression vectors for H.pylori cagA gene sequence Successfully,and acquireing expression in gastric epithelial cells type GES-1,which express widely spread in both cytoplasm and nucleus.2.The cagA gene might have the effect on promoting the apoptosis of gastric epithelial cell(GES-1)by Flow cytometry of transforming...