| Summary: | 碩士 === 國立成功大學 === 分子醫學研究所 === 95 === Helicobacter pylori exploits blood group antigen adhesin (BabA) to interact with human gastric Lewis b (Leb) antigen and determines the colonization density in stomach of patients. Recently, SabA adhesin (sialyl acid-binding adhesin) of H. pylori has found to interact with the sialylated Lewix x antigen (sLex), which is expressed after chronic inflammation induced by H. pylori infection. For patients with weak or non-Leb expression, SabA adhesin may interact with sLex to maintain H. pylori colonization on gastric surface. The aim of first part in this study was to understand the relationship among H. pylori infection and expression of sLex under inflammation of MKN45 cells, and determine whether sabA gene involved in the expression of sLex antigen. Fresh MKN45 cells were treated with the filtrate of HP238 for 48 h, and the expression of sLex antigen screened by FACS Calibur was 20% higher than control. However, the filtrate of 100 mg H. pylori outer membrane protein (OMP) extract didn’t stimulate the expression of sLex antigen. Inserting the different CT repeats of wild-type sabA allele with cat cassette were to create different CT repeats of sabA mutants by homologous recombination. Three mutant strains with different CT repeats (J99, HP307 and HP258) and one without CT repeat, HP649 were confirmed by Southern blot analysis and Western blot analysis. Both filtrates of wild-type and sabA mutants treated MKN45 cells for 48 h stimulated the expression of sLex antigen without significant difference.
The aim of second part was to evaluate the role of SabA adhesin in H. pylori adhesion to MKN45 cells. Four wild-type strains (J99, HP307, HP258 and HP649) were incubated in different pH conditions for 4 h and then the expression of SabA protein was analyzed by Western blotting. The expression of SabA protein was not affected in different pH conditions. Inserting the babA alleles of 4 wild-type and mutants (J99, Hp307, HP258 and HP649) with km cassette to create babA mutants and babA-sabA double mutants by homologous recombination. Only wild-type and sabA mutant of strain HP258 were transformed to babA mutant and babA-sabA double mutant successfully and confirmed by Southern blot analysis and Western blot analysis. MKN45 cells infected with wild-type strains and mutants for 1 h after cell pretreated with the filtrate of RPMI 1640 or HP238 for 48 h and then the adhesion of bacteria was screened by FACS Calibur. No difference was found between wild-type strain and mutant H. pylori adhesion to MKN45 cells. The sabA gene (1.9 kb) without the signal sequence and a small fragment (247 bps) of sabA gene, which can distinguish sabA from sabB, were constructed into pET-30b vector and transformed to E. coli BL21. The recombinant SabA (r-SabA) protein was expressed and purified by Ni2+ column chromatography. The r-SabA proteins were injected to rabbit to develop the SabA antibody. Wild-type bacteria (J99, HP307, HP258 and HP649) pretreated with different dilution of anti-r-SabA antibody and then infected MKN45 cells after cell pretreated with filtrate of HP238 for 48 h. The result indicated anti-rSabA antibody had no effect in H. pylori adhesion.
Base on the above results, we find that H. pylori component can stimulate the expression of sLex antigen under inflammation of MKN45 cells whereas sabA gene and OMP of H. pylori do not involve in the expression of sLex antigen. In addition, SabA doesn’t affect H. pylori adhere to MKN45 cells, and anti-r-SabA antibody has no effect in H. pylori adhesion.
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