Analysis Of Biological Characteristics Of Recombinant Mycobacterium Smegmatis Ms＿Rv1767 And Its Effect On Macrophages

Tb is a human and animal infectious disease caused by MTB infection.Pathogens spreads infection in humans and livestock by aerosols.Macrophages are the main target cells of MTB,which can protect themselves from MTB infection through autoimmunity.MTB can also evade host defense through its own immune escape mechanism.Studies have found that this mechanism is related to the virulence genes of MTB.Analysis showed that Rv1767 gene was conserved in pathogenic mycobacteria,which may be a pathogenicity related gene.Using p MV261 as an expression plasmid,Mycobacterium smegmatis(Ms)positive strain Ms＿Rv1767 with Rv1767 was heterogeneally expressed in order to investigate the basic biological characteristics of the gene and the effects of Ms＿Rv1767 on RAW264.7 mouse macrophages.1.Construction and identification of recombinant Mycobacterium smegmatis Ms＿Rv1767Bioinformatics analysis showed that Rv1767 protein is conserved in pathogenic mycobacteria,without signal peptide and transmembrane structure,stable and poor hydrophobicity.With the help of p MV261 shuttle expression plasmid,the Rv1767 gene was transformed into Mycobacterium smegmatis,and the construction of recombinant strain Ms＿Rv1767 was verified by acid-fast staining and liquid PCR.The Rv1767 protein induced by Myc labeled antibody was successfully heterologous expressed in Mycobacterium smegmatis.2.Biological characteristics analysis of recombinant strain Ms＿Rv1767In vitro growth dynamics test showed that Rv1767 could significantly reduce the growth rate of the strain,colony morphology observation showed that the recombinant bacteria colony had no significant change,and the determination of biofilm formation ability showed that the expression of Rv1767 protein could reduce the biofilm formation ability of the recombinant strain.In vitro stress experiments showed that the expression of Rv1767 protein could change the adaptability of the recombinant strain to adverse conditions.MIC and Spot test showed that the sensitivity of the recombinant strain Ms＿Rv1767 to some anti-tuberculosis drugs changed.3.The effect of recombinant strain Ms＿Rv1767 on RAW264.7 mouse host macrophagesMouse macrophages were infected with recombinant strain,by calculating the number of bacteria invading cells and their survival probability in cells,it was found that the expression of Rv1767 protein improved the invasion ability,but weakened the intracellular survival ability.CCK-8 assay showed that the cell viability was significantly reduced under the infection of the strain.Intracellular cholesterol content determination and oil red O staining showed that the secretion of lipids by recombinant strains increased.By means of ELISA and RT-qPCR,it was found that Rv1767 protein affected the expression of inflammatory cytokines at the protein and transcriptional levels.Flow cytometry showed that Rv1767 protein inhibited apoptosis under bacterial strain infection.In summary,the results of this study showed that the expression product of Rv1767 gene reduced the growth rate of the recombinant strain but did not change its colony morphology,weakened the adaptability of the recombinant strain to adverse conditions and its sensitivity to some anti-tuberculosis drugs,enhanced its invasion ability to macrophages,increased the secretion level of lipids by cells,reduced the survival ability and apoptosis level of macrophages,and reduced the secretion level of macrophages to early pro-inflammatory cytokines.The experimental data show that the Rv1767 gene may be a cell wall-related virulence gene that regulates growth.