| Objective:Helicobacter pylori(Helicobacter pylori, H. pylori) is a facultative intracellular bacterium that invasion the gastric epithelium may be one of the mechanisms of pathological changes of gastric mucosa, but its mechanism invasion is not clear.Studies have shown that β1 integrin(integrin β1) is known to H. pylori invasion cell receptor, cag I, cag L and cag A can bind to it. Previous studies in our laboratory have demonstrated that cag L and cag A were associated with the H.pylori invasion function.It has been proved that cag I and cag L are important virulence genes of H.pylori type IV secretion system, it also has been shown that cag I and cag L interact to form a functional complex. Therefore, the present study is to explore whether cag I correlated with H. pylori invasion and the invasion cell function have difference among cag I,cag L and cag A. The research aim to explore the H. pylori invasion pathogenic role and mechanism to provide some reference value and new ideas.Method:1. Constructed H. pylori NCTC11637 cag I deletion strain According to NCBI H. pylori J99 genomic sequence, primers were designed to obtain the upstream gene and downstream gene, using the gene homologous recombination principle kanamycin resistance gene(kana R) connected between the cag L and cag H by PCR, and co-inserted into p Bluescript SK Ⅱ(-) vector to construct the recombinant plasmid with kanamycin resistance marker. The recombinant plasmids were transformed into standard strain NCTC11637 containing complete cag I gene,using kanamycin out the NCTC11637 cag I deletion strain, and by microscopic observation, urease and identification of the DNA and RNA to obtain the correct deletion strain.2. Constructed H. pylori NCTC cag L/I deletion strain According to NCBI H. pylori J99 genomic sequence, primers were designed to obtain the upstream gene and downstream gene, using gene homologous recombination method kanamycin resistance gene(kana R) connected between the cag N and cag H, and co-inserted into p Bluescript SKⅡ(-) vector to construct the recombinant plasmid with a kanamycin resistance marker. The recombinant plasmids were transformed into standard strain NCTC11637 containing complete cag L and cag I genes, using kanamycin out the NCTC11637 cag I deletion strain, and by microscopic observation, urease and identification of the DNA and RNA to obtain the correct deletion strain.3. The expression of cag I, cga L and cag A H. pylori NCTC11637Δcag I, NCTC11637Δcag L(this laboratory has been constructed)and NCTC11637Δcag A(constructed by our laboratory) as the experimental group,NCTC11637 as control group,RNA were extracted, and reverse transcribed into c DNA, c DNA as a template, using q RT-PCR method the expression of cag I, cag L and cag A genes were measured.4. The protective invasion assay of gentamicin H. pylori NCTC11637Δcag I, NCTC11637Δcag L(constructed by laboratory),NCTC11637Δcag L/I, NCTC11637Δcag A(constructed by laboratory) and NCTC11637, infected AGS(MOI = 50):a group of cells wash and lysis after the infection 2h, bacterial lysates were coated culture plates for colony counting after incubation, the number of bacteria adherent cells is calculated adhesion rate(the number of bacteria adhered cells / the number of bacteria infected cells × 100%);other cells simultaneously infected 2h, by gentamicin protection invasion assay for another 2h, cells were lysed bacterial lysates coated culture plates, colony counting after incubation, this is the number of bacteria invade cells, to calculate the invasion adhesion ratio(the number of bacteria invade cells / the number of bacteria adherent cells × 100%).5. Statistical analysis Using SPSS 22.0 nonparametric rank sum test for statistical analysis.Results:1. H. pylori NCTC11637Δcag I and NCTC11637Δcag L/I were successfully constructed.2. The expression of cag I, cag L and cag A in NCTC11637Δcag I、NCTC11637Δcag L and NCTC11637Δcag A In NCTC11637Δcag I, the expression of cag I, cag L,cag A were 0.00179±0.00044,0.00385±0.00324,0.93830±0.02763, compared with NCTC11637,the expression of cag I and cag L in NCTC11637Δcag I were reduced,the expression of cag A was invariant;in NCTC11637Δcag L,the expression of cag I,cag L,cag A were0.97391±0.35477,0.00261±0.00204,0.79419±0.126,compared with NCTC11637,the expression of cag L in NCTC11637Δcag L was reduced,the expression of cag I and cag A were invariant;in NCTC11637Δcag A, the expression of cag I,cag L,cag A were1.00621±0.47831, 1.00233±0.50291, 0.000012±0.000008, compared with NCTC11637,the expression of cag A in NCTC11637Δcag A was reduced,the expression of cag I and cag L were invariant.3. The protective invasion assay of gentamicin The invasion adhesion ratio of NCTC11637Δcag I, NCTC11637Δcag L,NCTC11637Δcag L/I, NCTC11637Δcag A and NCTC11637 on AGS cells were:7.88±0.82%,5.88±1.52%,5.43±1.54%,4.62±1.29%,20.41±5.77%, analyzed using nonparametric rank sum test, the invasion adhesion ratio of NCTC11637Δcag I,NCTC11637Δcag L, NCTC11637Δcag L/I and NCTC11637Δcag A were significantly lower than that of NCTC11637(P<0.05);the invasion adhesion ratio of NCTC11637Δcag I with NCTC11637Δcag L and NCTC11637Δcag L / I had no significant difference(P> 0.05); the invasion adhesion ratio of NCTC11637Δcag L with NCTC11637Δcag L / I had no significant difference(P> 0.05); the invasion adhesion ratio of NCTC11637Δcag L with NCTC11637Δcag A had no significant difference(P> 0.05).Conclusion:1. cag I influence the expression of cag L in gene transcription level.2. cag I was associated with H.pylori cell invasive function.3. cag I, cag L and cag A have no difference in the invasive function, however cag A may play a major role. |
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