Construction Of Mycobacterium Tuberculosis Rv0927c Gene Deletion Mutants And Its Biological Characteristics

Tuberculosis(TB)is a contagious disease caused by Mycobacterium tuberculosis complex(MTBC).It has high incidence rate and mortality rate,and seriously threatens human health and social economic development.Based on the global attention to tuberculosis and the urgent need for effective vaccines,the gene knockout technology provides great convenience for the research of Mycobacterium tuberculosis(MTB).In this study,MTB H37Ra was used as the host bacteria and Rv0927c gene was used as the research object.The gene deletion strain was constructed by using specific transduction technology,and identified its biological characteristics.Meanwhile,the technology platform of MTB gene deletion was constructed,and the method of MTB gene deletion was improved,so as to gain insight into the pathogenic mechanism of MTB.The upstream and downstream fragments of the target gene were amplified by homologous recombination technique mediated by temperature sensitive phage,and connected with the target fragment of pYUB004S plasmid to construct the recombinant plasmid.Then the recombinant pYUB004S plasmid was ligated with the phAE159 vector.The ligation product was packaged by phage and transformed into Mycobacterium smegmatis(MS)mc2155 at 30℃ to induce the recombinant phage.After that,the phage infection experiment was carried out at 37℃,and the positive mutant strain was screened by resistant plate containing 75 μg/mL Hygromycin B(Hyg).Finally,the mutant was identified by PCR and sequencing.The results showed that Rv0927c gene deletion strain was successfully obtained.Also,the Rv0927c gene complement strain was successfully constructed by using pMV261 vector.The deletion of Rv0927c gene had no significant effect on the growth rate,colony morphology and bacterial length of H37Ra.Compared with the wild strain and the complementary strain,the ability to tolerate the peroxide and surfactant SDS was improved under the stress(P<0.05).Also,the deletion strain had weak biofilm formation ability,but did not change the resistance phenotype of H37Ra.In addition,in vitro,the results of NF-κB pathway test showed that Rv0927c gene deletion can can promote NF-κB pathway(P<0.05).In addition,Rv0927c did not affect the expression level of TNF-α and IL-2 in bovine peripheral blood mononuclear cell.The results of intracellular proliferation test showed that the number of bacteria in the deletion strain was always lower than that in the wild strain and complementary strain,and the difference was extremely significant at 36 h(P<0.0001),indicating that Rv0927c gene was conducive to the survival of MTB in macrophages.Generally speaking,H37Ra Rv0927c gene deletion strain was successfully constructed,which provided ideas for further understanding of gene function of MTB.