[Construction of gene deletion mutant and compensation strains in and its effects on Caco-2 cells].

To analyze the effects of gene on invasion and intracellular proliferation of Caco-2 cells and in order to provide insight into the function of that gene and the underlying mechanism of caused infection. Functional verification of gene deletion mutant and compensation strain. The deletion mutant strain was constructed through a suicide plasmid-mediated homologous recombination. The compensation plasmid constructed by cloning the coding sequence of by PCR into plasmid pBAD33 was mobilized into the deletion mutant by conjugation and the pBAD33 was introduced into wild strains and deleted mutant strains as control. The relative expression of mRNA was detected by quantitative reverse transcription PCR. In order to analyze the virulence of against Caco-2 cells, Caco-2 cells was cocultured with wild type carrying gene, the deletion mutant strain and compensation strain respectively. The expression level of mRNA and the the number of after Caco-2 cells intervention were compared between the three groups by LSD- test, and the invasion rate was compared by χ test. The expression level of mRNA in wild type was set as unit "1", the deletion mutant strain was "0.00", and the compensation strain was "2.60" (LSD-=1.11, =0.31; LSD-=-1.77, =0.13; LSD- =-2.88, =0.03), which confirmed the successful construction of the deletion mutant strain and the compensation strain. The invasion experiment results of the above three strains on Caco-2 cells showed that the invasion rate of wild strain was 0.23%, the invasion rate of deleted mutant strain was 0.16%, and the invasion rate of compensation strain was 0.16%, with no statistical significance (χ=1.13, =0.570). By comparing the number of at different time points after Caco-2 cells intervention, it was discovered that the number of in wild strains (6.50×10 CFU/ml) and compensation strains (7.25×10 CFU/ml) was significantly increased than that in deletion mutant strain (1.90×10 CFU/ml) after 16 h coculture (LSD-=7.95, =0.00; LSD-=-1.27, =0.25; LSD- =-9.22, =0.00). It is not considered that gene can affect the invasion of on Caco-2 cells, but the gene can promote the reproduction of in Caco-2 cells.