Realize The Expression The Adhesion Fimh In The Out Membrance Protein Of Helicobacter Pylori

Helicobacter pylori is the only known pathogenic bacteria with the abililty to colonize stablely in stomach of human, and it can cause serious stomach illness as gastritis, ulcers and even stomach cancer, so the World Health Organization has listed it as class I carcinogen. The infection of H. pylori is up to 50% worldly, but the traditional method is restricted due to the cost, side effects and drug-resistant strains. The development of vaccines for the prevention or treatment of H. pylori is of great significance, but the evaluation systems have some flaws, and the mice mode has instability phenomenon. In this work, type I fimbriae lectin-like FimH adhesion of Salmonella typhimurium was introduced into H. pylori to enhance the adhesion properties of the gut mucosa. A fimH gene or its N-terminal lectin domain fimH(ld)’was knocked into H. pylori 26695 genome, downstream of outer membrane protein gene lpp20 or omp18, in order to realize the surface display of the FimH adhesin or its N-terminal lectin domain FimH(Ld).In part one of the studies, the gene targeting technology in H. pylori genome was optimized. Culture medium, selective pressure of antibiotics, competent cell concentration, incubation time, as well as the length of homologous arm were investigated. The results showed that it was better for bovine serum in supporting the growth of H. pylori than sheep blood. The kanamycin resistance was more suitable for selecting the transformants and avoiding false positives than chloramphenicol resistant. The concentration of competent cell should be at least 109cfu/ml, and be better at concentration of 1010cfu/ml. H. pylori competent cells also needed a recovery time for about 24 hours after electric shock and before growth on selective plate. The length of homologous arm within a range of 300bp to 800bp was not a significant factor for homologous recombination efficiency. We also reported here for the first time that transferring the recombinants from old selective plate where was full of lysates of dead bacteria to a new selective plate could significantly enhance the growth of recombinants and improve the transformation efficiency.Four gene knocked-in vectors were constructed, and they were called p-Lpp20-FimH, p-Lpp20-FimH(Ld), p-Ompl8-FimH and p-Omp18-FimH(Ld) respectively. The plasmids were introduced into H. pylori by electroporation, and fimH or fimH(ld) were integrated into lpp20 pylori by electroporation, and JimH or fimH(ld) were integrated into lpp20 or ompl8 gene in-frame through homologous recombination. Transformants containing fimH or fimH(ld) gene were selected on campylobacter agar base containing 10% bovine serum as well as 25ug/mL kanamycin, and verified by PCR amplification of the fimH gene or fimH(ld) from H. pylori 26695 chromosomal DNA. Cross primers were also used to verify the lpp20-fimH, lpp20-fimH(ld), omp18-fimH, ompl8-fimH(ld) in H. pylori chromosomal DNA. The mRNA of H, pylori transformants were extracted and transcripts of lpp20-fimH, lpp20-fimH(ld), ompl8-fimH, ompl8-fimH(ld) were verified by reverse transcription PCR. The fimH gene was also cloned into prokaryotic expression vector pET-22b, then introduced into Escherichia coli BL21(DE3) and FimH inclusion body was obtained finally. Recombinant protein was puried by washing them three times using 5M Urea. Anti-FimH serum antibodies were generated by immunization of white rabbit with FimH inclusion bodies. Blood serum antibody titer against FimH was up to 10,000. Subcellular fractionation was performed to obtain the outer membrane proteins of H. pylori transformants. Samples of outer membrane fractions were run on SDS-PAGE gels and transferred to a nitrocellulose membrane to be analyzed by Western blotting. Lpp20-FimH and Lpp-FimH(Ld) fusion proteins were detected with anti-FimH serum antibodies, but Omp18-FimH and Omp18-FimH(ld) were difficult to be detected. So Lpp20-FimH and Lpp-FimH(Ld) were expressed in outer membrane of H. pylori successfully. Samples of outer membrance fractions were analyzed for function by dot hybirdzation. Lpp20-FimH, Lpp-FimH(Ld), Omp18-FimH and Omp18-FimH(ld) fusion proteins were detected to have the activity to combine with mannose.