Construction Of Orphan Response Regulator Rv0818 Gene Deletion Mutant And Its Preliminary Functional Study In Mycobacterium Tuberculosis

Two-Component Systems are important for Mycobacterium tuberculosis to survive in the extreme environment in macrophage, such as hypoxia, nitric oxide and nutritional deficiency. Little is known about the function and its stream signal of Rv0818——the orphan response regulator involved in Mycobacterium tuberculosis Two-component systems. We constructed Rv0818 deletion strain by specialized transduction. The purpose of this study is to investigate the change of phenotype of the mutants, such as morphological changes, resistance to NO and intracellular viability. This study provides an experimental basis for the pathogenesis of tuberculosis, and developed a candidate gene deletion mutant for the research on tuberculosis vaccine. 1 Construction and identification of gene deletion strainsThe Rv0818 homologous recombination region was inserted into the mycobacteriophage genome. The positive clones grown on Hyg resistant plate were identified by PCR and sequencing. The results showed that the target gene was replaced by resistant region, and the gene deletion strain was constructed successfully. 2 Comparison of morphological characteristicsThe morphological characteristics of wild and mutant strains grown on plate were compared. The results showed that deletion strains growth on solid plate formed fewer serpentine cords, which is associated with individual size, growth rate and the wall structure of the strain, indicating the attenuate of mutants. 3 Comparison of NO tolerancesThe growth of wild type and mutant under NO stimulation were compared. The results showed that the wild type and the mutant were inhibited by high concentration of NO. While the wild type recovered from the inhibition more quickly, indicating that the lack of Rv0818 effected the adaptability of the strain to the environment. 4 Comparison of cell viabilityThe phagocytosis rate of wild type and mutant were compared. The results showed that the mutant was easier to be swallowed by macrophages. The survival rate was compared after 24h/48h/72 h infections. The results showed that the viable count of mutant was unchanged, while the viable count of the wild type increased 3 times in 72 h,indicating that the lack of Rv0818 decreased the viability of the strains in vivo. 5 The changes of Rv0818 downstream gene expressionThe expression of several gene of mutant was compared to wild type. The results showed that the genes involved in NO resistance were up regulated, and the metabolism related genes was significantly down regulated in the lack of Rv0818, verifying the role of Rv0818 in transcriptional regulation. 6 Preparation of rabbit polyclonal antibody against Rv0818The rabbit anti Rv0818 polyclonal antibody was prepared and tested by Western Blot. The results showed that the antibody could be used in the further study of Rv0818.