Construction And Expression Of Helicobacter Pylori FlaA And FlaB Flagellin Genes And Immunological Identification Of The Fusion Protein

Helicobacter pylori appears to be causally associated with active and chornic gastritis,as well as peptic and duodenal ulcers,and also be associated with carcinoma of the stomach.The prevalence of H.pylori infection is more than 50% in most country and area. At the present, antibiotic therapy is usually used to eradicate H.pylori, However, there are a lot of problems such as H.pylori re-infection, side effects and high costs of the drugs during the antibiotic therapy. Inoculating the vaccine of H.pylori is the most valid.measure that prevent and control the H.pylori infection. The genetic engineering vaccine should be the maior direction of H.pylori vaccine development.The flagella is necessary for colonization and persistence by H.pylori, can make the H.pylori have strong motility in mucous environment and induce IL-8 secrete and strengthen inflammation of the position by H.pylori infect. The flagellin consist of FlaA and FlaB protein by flagella gene flaA and flaB coding.In this study, The flaA and flaB gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR.The flaA and flaB gene nucleotide sequence of the target DNA amplification fragment were sequenced after T-A cloning. The recombinant expression vector pET32a(+)-flaA-BL21DE3 and pET32a(+)-flaB-BL21DE3 were constructed. The expression of FlaA and FlaB fusion protein in E.coli BL21DE3 induced by IPTG at different dosages was examined by SDS-PAGE. Western blot using commercial antibody against whole cell of H.pylori as well as immunodiffusion assay using self-prepared rabbit antiserum against FlaA and FlaB fusion protein were applied to determine antigenicity of the fusion protein. ELISA was used to detect the antibody against FlaA and FlaB in sera of 125 patients infected with H.pylori and to examine FlaA and FlaB expression of 98 clinical isolates of H.pylori. The FlaA and FlaB fusion protein will act as antigens for H.pylori genetic engineering vaccine and detection kit.MATERIALS AND MTEHODS1.Culture of H.pyloriClinical isolate Y06 and NCTC11637 of H.pylori were smeared on H.pylori selective Columbia agar plate containing 10% of sheep blood and antibiotic additive. The plate was cultivated under microacrobic conditions for 5d. The bacterium with gram-negative, curve seagull-like shape, positive for rapid urease and oxidase assay and immunoactive to the antiserum of H.pylori as well as its colony on the plate with tiny and semi-pellucid was identified as H.pylori.2. Preparation of DNA templateGenomic DNA of H.pylori strain Y06 and NCTC11637 was extracted by routing phenol-chloroform method, DNase-free RNase digestion and phenol-chloroform extraction. The DNA template was solved in TE buffer and its concentration and purity were determined by ultraviolet spectrophotometry.3.Polymerase Chain ReactionOligonucleotide primers were designed to amplify the whole sequence of flaA and flaB gene from H.pylori strain Y06 based on the published corresponding genome sequence. The sequence of flaA sense primer with an endonuclease site of EcoR V: 5'-CCGGATATCATGGCTTTTCAGGTCAA-3'. The flaA sequence of antisense primer with an endonuclease site of Xho I: 5'-CCGCTCGAGAAACTAAGTTAAAAGCC-3'. The sequence of flaB sense primer with an endonuclease site of EcoR I: 5'-CCGGAATTCATGAGTTTTAGGATAAA-3'. The flaB sequence of antisense primer with an endonuclease site of Xho I: 5'-CCGCTCGAGCTGTTATTGTAAAAGCC-3'.The total volume per PCR was 100 ul containing 250 nmol/L each the primers, 100 ng DNA template. The parameters for PCR: 94℃5min, ×1; 94℃308, 52℃30S, 72℃90S, ×10; 94℃30S, 52℃30S, 72℃100S (10S addition for the each of the following cycles), ×15; 72℃ 10min, × 1. The results of PCR were observed under UV light after electrophoresis in 1.5% agarose pre-stained with ethidium bromide.4.T-A Cloning, sequencing and Construction of Expression VectorThe target amplification DNA fragment of flaA and flaB gene was cloned into pUCm-T vector (pUCm-T-flaA and pUCm-T-flaB) by using the T-A Cloning Kit according to the ma...