Study On Recombinant Mycobacterium Smegmatis With Mycobacterium Tuberculosis EIS Gene And Its Intracellular Survival Test

Objective:One-third of the world's population is infected with MTB,but only <10% of these are active tuberculosis, most of them are latently infected. A series of problems,such as latent infection, recurrence, term treatment and drug-resistent of MTB main due to persistent MTB. As the host immune system falters, the persistent bacterium returns to replication mode, which leads to the recurrence of active infection and threatens to human health.Hence,study on the mechanism of intracellular survival,and search for a possible way to inhibit and kill persistent MTB,is important to control and cure tuberculosis.There are many researches about the mechanism of intracellular survival MTB. However, the exact pathogenesis of how internalized MTB persists in phagocytic cells for such a long time remains unclear,that is also the reason why we can't find effective ways for curring latent MTB. With the elucidation of the complete genome sequence of MTB and the development of genetic techniques, additional genes and protein products of the pathogen have been identified as potentially playing roles in mycobacterial pathogenesis. But which one(s) is(are) responsible for the pathogenesis need further experiment to demonstrate.Today,a number of methods have been proposed to detect active intracellular pathogens, but the time that is required to obtain susceptibility results for MTB using classical methodologies is still too long due to its characteristic slow growth,which might requires several days or weeks of incubation. Development of a rapid and accurate method to diagnose latent MTB infection is essential for improving prevention and treatment of tuberculosis.Flow cytometry is a previous technique in the setting of the clinical laboratory, which gives reliable results within a few hours,is considered.The eis gene from pathogenicity H37Rv has been identified in this study. We cloned eis gene into an Escherichia coli-Mycobacteria shuttle plasmid pMV261 to constract a recombinant plasmid named pMV-eis. Then it was transformed into M.S by electroporation. After cultivation,positive colonies were picked out and identified by restriction map,SDS-PAGE and Western blot. Flow cytometry was used to test intracellular survival of M.S transformants during infection of the human macrophage line U937, to develop a method for rapid detection of latent MTB infection. After 2 h phagocytosis period,extracellular bacteria was washed away, and then U937 macrophages were incubated at 37°C in 5% CO₂ for some time. At various times, the macrophages were lysed and intracellular bacterias were harvested for test. The results by flow cytometry were compared to the ones by agar proportion method.Methods:1.Primers with proper restriction sites were designed accrodding to the nucleotide sequence of eis gene(GeneID: 885903). MTB genomic DNA was isolated from H37Rv strains. And eis gene was amplified by using them.2. The eis fragment was treated with BamHI and HindIII, ligated in the shuttle kanamycin-resistant plasmid pMV261, which yielded a recombinant plasmid named pMV-eis.Then pMV-eis was transformed in E. coli DH5αfor propagation. Insert sequences were controlled by restriction map and sequencing.3. Electroporated competent M.S mc²155 with approximately 1μg of plasmid DNA pMV-eis. Positive transformants were selected by growth on LB plates with kanamycin. The recombiant M.S was identified by restriction map and PCR.And the expressing product was detected by SDS-PAGE and western blotting.4. Study on intracellular growth test of recombiant M.S: M.S that contained the pMV-eis plasmid was prepared and added to the well that contained U937 cells at an MOI of 10.At 4, 12, 24 and 48 h after infection, cells were collected and lysed by 0.1%Triton-X to release surviving intracellular bacteria. M.S that contained pMV261 was included in every assay as a negative control. Lysates were used to be detected by flow cytometry and agar proportion method.Results:1. The eis gene was cloned in pMV261, which yielded a recombinant plasmid named pMV-eis.Sequencing and homologous analysis veryfied the cloned eis gene, and its nucleotide and deduced aminoacid sequences was equaled to the related gene in GenBank (GeneID:885903).2. Plasmid pMV-eis was transformed into M.S by electroporation. Individual positive colonies were picked out on LB agar that contained 30μg/ml kanamycin.The growth curve of the recombinant M.S was consistent with that of the wild-type strain, suggesting the absence of Eis protein toxicity against M.S.SDS-PAGE and Western blot result showed that relative molecular mass of expression product was about 42KD.3. Recombinant M.S infected U937 cells at an MOI of 10.At some time, intracellular bacteria were detected by flow cytometry after staining FDA.Results demonstrated that: intracellular survival of M.S recombinants exhibited significantly lower mortality rates than control at 12, 24 and 48h post-infection.4. At 12, 24 and 48 h after infection, the number of CFUs for intracellular viable recombinant M.S was significantly more than control, which was similar to the results by flow cytometry. And there was no significant difference at 4 h to 48 h post-infection by the proportion method or by flow cytometry.Conclusions:1. The shuttle plasmid pMV-eis was successfully constracted.2. The plasmid pMV-eis was transformed into M.S by electroporation, recombinant M.S was successfully constracted.3. SDS-PAGE and Western blot showed that a 42KD protein was effectively expressed and had biological activity in rM.S4. Flow cytometry is a rapid, accurate and convenient method, and can be used for quick detect survival of mycobacterium in macrophages...