Construction Of Eukaryotic Green Fluorescent Protein Expression Vector Carrying CagA Of Helicobacter Pylori

Background and Objective:The Helicobacter pylori correlate closely with the occurrence of chronic gastritis,peptic ulcer, gastric cancer and mucosa-associated lymphoid tissue lymphadenoma. Helicobacter pylori have two bacteria plant types:type-I and type- II.The criteria of this division based on that,the type-I should include both cytotoxin- associated gene A and Vacuolating cytotoxin antigen; while,the type- II should include none of them.The bacteria plant type- I include a special gene segment,about 40kb.This segment is called Cag Virulence Island,which charges for the coding CagA virulence factors. From 1997 to 1998,the sequence of the whole gene group of the Helicobacter pylori bacteria plants 26695 and J99 were detected,therefore people could make a comparison between the gene tissue structures of two type- I bacteria plants,and know the main features of them. Type- I bacteria plant is assumed to be the signified cause of peptic ulcer and gastric tumor. So the research on the CagA gene becomes the hotspot of the Helicobacter pylori investigation.Meanwhile,the current treatment for Helicobacter pylori infection is based on antibiotics with a proton pump inhibitor or antacid,which however has several disadvantages,such as side effect of antibiotics, emergence of antibiotic-resistance strains,high racurrence rate. Vaccination is a way to eradicate Helicobacter pylori infection,but no safe and effective vaccine has been developed. The objective of this study is to construct fluorescent expression vector pEGFP-C3-CagA and induce the vector to express in eukaryotic cells, to supply a basis for the research on the recombinant gene engine vaccine of Helicobacter pylori, and to provid a reference to the potential antigen for developing type- I bacteria plant identification kit.Method:The target gene primers of CagA were designed according to the sequence GenBank.in order to facilitate cloning and ensure the exactness of insert direction, incision enzyme points were designed in the primers. Then PCR was applied to amplified the CagA gene from the Helicobacter pylori bacteria plant. After this, the target gene was linked into pGEM-T easy plasmid, which was transformed into engineering bacteria JM109. Then, a-complementation test and PCR were utilized to select the positive colony, and detect the gene sequence of the target gene. The result was compared with the pre-known CagA gene sequence in GenBank; Then the CagA was cut from pGEM-T easy-CagA, and linked with green fluorescent expression vector, pEGFP-C3, which were transformed into engineering bacteria JM109.And the positive clone was select by Kanamycin fastness and PCR. Finally, the recombined plasmid pEGFP-C3-CagA was transfected in gastric carcinoma cell strain BGC823 by lipoplast method. Observe the expressed pEGFP-C3-CagA under fluorescent microscope.Result:1. Amplification of target gene: CagA gene was obtained by PCR with the Hp bacteria culture as template, and it contents 523bp added its incision enzyme point.2. The recombined plasmid pGEM-T-CagA was constructed successfully. The homology in nucleotide acid of CagA gene was 100% between the sequencing result of the target gene and in GenBank.3. The recombined plasmid pEGFP-C3-CagA was constructed correctly, which was expressed stably after it was transfected in eucaryon cells.Conclusions:1. The gene order of CagA conservative region was obtained with PCR from eukaryotic expression. 2. The enhanced green fluorescent protein eukaryotic expression vector carrying CagA was constructed correctly, which was expressed stably in gastric carcinoma cell strain BGC823.3. The eukaryotic expression of CagA was constructed successfully. It may supply a basis for the study of recombination gene vaccine of Helicobacter pylori, and may also provide the potential antigen for developing type-I Hp bacteria plant diagnostic reagents.