The Metabolomic Analysis Of RAW264.7 Cells Infection By Recombinant BCG-Rv3874 And Recombinant BCG-Rv3875

Bacillus Calmette Guerin(BCG)is a live attenuated vaccine against tuberculosis that has been in widespread use for decades because of its safety and cost advantages.As time goes by,BCG also exposed the shortcomings of low immunity,regional differences and age limit.Improving the existing BCG to enhance its immune efficacy is an important research direction.Compared with mycobacterium tuberculosis,BCG lost a gene sequence called RD region,which was divided into 16 regions(RD1-RD16)containing genes related to the virulence and immunogenicity of mycobacterium tuberculosis.It is considered to be an effective strategy to enhance the immune potency of BCG by transforming immunogenicity related genes into BCG.In this study,the target gene was amplified according to the RD-1 region gene of Mycobacterium tuberculosis H37 Rv and the recombinant pMV-261 shuttle plasimd was constructed.The recombinant plasimd was introduced into competent BCG by electric-transformation method,and a total of 8 rBCG were obtained after purification.To observe the changes of cell activity and the expression of inflammatory factors in mouse RAW264.7 macrophages infected by r BVG-Rv3874/ rBCG-Rv3875 / BCG.Then detection of lipid composition in mouse RAW264.7 macrophages that normal cell or infected of BCG / rBCG-Rv3874 / rBCG-Rv3875 through the LC-MS method,and performed preliminary metabolomics analysis.The results obtained in this study are as follows:1.Successfully constructed 8 recombinant plasmids of pMV261-Rv3871,pMV261-Rv3872,pMV261-Rv3873,pMV261-Rv3874,pMV261-Rv3875,pMV261-Rv3877,pMV261-Rv3878,pMV261-Rv3879 c.2.The recombinant plasmid was transformed into the competent BCG strain by electric transformation.Successfully constructed 8 recombinant BCGs of rBCG-Rv3871,rBCG-Rv3872,rBCG-Rv3873,rBCG-Rv3874,rBCG-Rv3875,rBCG-Rv3877,rBCG-Rv3878,rBCG-Rv3879 c.Mapping the growth curves of each recombinant BCG,determination of logarithmic growth phase and the CFU.3.The macrophages cell RAW264.7 growth by inhibition infected of BCG/rBCG-Rv3874/rBCG-Rv3875.The RAW264.7 macrophages cell infected of rBCG-Rv3874 / rBCG-Rv3875 can upregulate the expression of proinflammatory cytokines in cells,increasing the level of apoptosis,but the proinflammatory effect was lower than that of BCG,IFN-? expression was down-regulated.4.Extract all fat from macrophages cell RAW264.7 that infected of BCG / rBCG-Rv3874 /rBCG-Rv3875.A total of 1450 components were detected by LC-MS method.The infection ofBCG / rBCG-Rv3874 / rBCG-Rv3875 on RAW264.7 macrophages resulted in significant changes in 213,357,372 lipid components respectively than normal RAW264.7 macrophages.This study showed that rBCG-Rv3874 and rBCG-Rv3875 could induce more intense metabolic changes on RAW264.7 macrophages compared with BCG,These changes are related to the introduction of Rv3874 and Rv3875 genes.The cell infected of rBCG-Rv3875 have more changes than rBCG-Rv3874,suggesting that RAW264.7 macrophages have a stronger response to Rv3875 gene.This study have great value for studying the function of gene in RD-1 region of Mycobacterium tuberculosis and screening of rBCG.