The Effect And Mechanisms Of Mycobacterium Tuberculosis CFP-10 And Esat-6 Collaborative On The Host Cell

Mycobacterium tuberculosis is a long-term parasitic bacteria within the host cell and can cause tuberculosis. CFP-10 and ESAT-6, two small molecules secreted proteins, are virulence factors of Mycobacterium tuberculosis and play important roles in the process of Mycobacterium tuberculosis infection and pathogenicity. During the process of Mycobacterium intracellular parasites in the infected host, the mechanical of the CFP-10 and ESAT-6 in the infected host is still unclear. The CFP-10-PEGFP-N1 and ESAT-6-PEGFP-N1 eukaryotic expression vector were constructed in the earlier stage, then the vectors were used to transfect the eukaryotic cells and the effect of two virulence factors alone on apoptosis of host were observed.Object: Based on the previous studies, the present study is to build the CFP10-PDs Red1-N1 with red fluorescent reporter gene and the ESAT-6-PEGFP-N1 with green fluorescent reporter gene by genetic engineering technology and then the genes were used to co-transfect eukaryotic cells. The cell models of CFP-10 gene and ESAT-6 gene expression in the same cell were obtained and then the effects of virulence factors CFP-10 and ESAT-6 of Mycobacterium tuberculosis on the expression of apoptosis-associated genes was also investigated.Methods: The CFP-10 gene was obtained by double digestion of CFP-10-PEGFP-N1 eukaryotic recombinant plasmid and then the gene was inserted into PDs Red1-N1 eukaryotic expression vector, thus CFP10-PDs Red1-N1 eukaryotic recombinant expression vector with the red fluorescent reporter gene was constructed. The groups were shown in the following:1. PEGFP-N1 group2. PDsRed1-N1 group3. PEGFP-N1 and PDs Red1-N1 double transfection group4. CFP-10-PDsRed1-N1 group5. ESAT-6-PEGFP-N1 group6. CFP-10-PDsRed1-N1 and ESAT-6-PEGFP-N1 double transfection group7. Liposome group8. Blank control group The 293 FT of eukaryon cell was co-transfected with plasmid group by Lipofectamine TM2000. Then the fluorescence level was observed by inverted fluorescence microscope, the CFP-10 protein and ESAT-6 protein expression level was observed by using Western Blot method detection, the co-localized situation of CFP-10 protein and ESAT-6 protein in the transfected cells was observed by laser confocal microscopy analysis and the dynamic expression level of four apoptosis-associated genes including Bcl-2, Bax,Caspase-8, Caspase-3 in untransfected and transfected cells was observed by real-time quantitative PCR.Results: A sequence CFP10-PDs Red1-N1 eukaryotic recombinant expression vector was obtained. The transfected 293 FT cells with CFP-10 gene and ESAT-6 gene can emit red fluorescence and green fluorescence, respectively, with the aid of inverted fluorescence microscope, indicating that eukaryotic co-expression cell model both CFP-10 gene and ESAT-6 gene of Mycobacterium tuberculosis virulence factors was built successfully. The fusion protein of the PDSRED-CFP-10 and PEGFP-ESAT-6 was expressed in transfected cells with the aid of Western Blot test. By confocal laser microscopy technical, it was found that red fluorescence and green fluorescence of transfected 293 FT cells with empty vector PEGFP-N1 and PDsRed1-N1 group were mainly distributed in the cytoplasm. Some region of the transfected cell shows yellow with the mixture of red and green fluorescent and others remains red fluorescence, indicating that the distribution of the red fluorescence is different from that of the green fluorescence. It was found that yellow fluorescence of transfected 293 FT cells with vector CFP-10-PDs Red1-N1 and ESAT-6-PEGFP-N1 group was also mainly distributed in the cytoplasm. All cytoplasm regions of the transfected cell shows yellow with the mixture of red and green fluorescent, indicating that the distribution of the red fluorescence is the same as that of the green fluorescence. The dynamic expression level of four apoptosis-related gene including Bcl-2, Bax, Caspase-8 and Caspase-3 in untransfected and transfected cells by real-time quantitative PCR showed the increase rate of the Bcl-2 gene expression for 24 h is higher than that of the Bax gene compared with transfection for 12 h in the cell of CFP-10-PDsRed1-N1 and ESAT-6-PEGFP-N1 double transfection group,and the trend of the Bax / Bcl-2 ratio variation decreased, especially for 24 h. At the same time, Caspase-8 and Caspase-3 gene expression generally decreased as a function of time in the cell of CFP-10-PDs Red1-N1 and ESAT-6-PEGFP-N1 double transfection group, and a bigger reduction of the gene expression for 24 h was shown than that of 12 h.Conclusion: CFP-10-PDsRed1-N1 eukaryotic recombinant expression vector can be expressed correctly in eukaryotic cells and the cell model by genes expression of virulence factors in the same cell was successfully constructed. The Expression of CFP-10 gene and ESAT-6 gene in the same cell have no effect with each other, and the distribution of the protein product are same in the cell, indicating that these two virulence factors may work in the cell with a complex form. The results obtained with the aid of Real-time quantitative PCR for the apoptosis-related genes showed that the gene expression level of the Bcl-2、Bax、Caspase-8 and Caspase-3 can be influenced and the relative ratio of the Bax / Bcl-2was reduced by the CFP-10 / ESAT-6 complex. The reduction of Caspase-8 and Caspase-3gene expression in the cell of CFP-10-PDs Red1-N1 and ESAT-6-PEGFP-N1 double transfection group are more similar with that of PEGFP-N1 and PDsRed1-N1 double transfection group.