Study On Mechanisms Of Overexpression PPS Effects On Mycobacterium Tuberculosis Pathogenicity

Objective: Construct mycobacterium tuberculosis International Standard virulent strain H37 Rv bacterial strain that pup/mpa/dop/paf A gene overexpresses(Hereinafter referred to as the H37 Rv strain),mycobacterium tuberculosis International Standard Avirulent strain H37 Ra bacterial strain that pup/mpa/dop/paf A gene overexpresses(Hereinafter referred to as the H37 Ra strain) and Mycobacterium tuberculosis isolates strains with prevalence in Xinjiang region that pup/mpa/dop/paf A gene overexpresses(Hereinafter referred to as the XJ strain). Explore the effect of Mycobacterium tuberculosis Pup, Dop, Paf A and Mpa gene in Pup-proteasome system overexpression reacts at the pathogenicity of clinical Mycobacterium tuberculosis isolates strains with prevalence in Xinjiang region.Methods: Extract N/med tuberculosis bacili-e.coli recombinant plasmid PMV361 pup,PMV361mpa,PMV361 dop and PMV361 paf A which the earlier stage of this research group constructs,Further respectively shift Recombinant shuttle-plasmid PMV361 pup,PMV361mpa,PMV361 dop and PMV361 paf A to H37 Rv bacterial strain, H37 Ra bacterial strain and XJ strain competent cell by Using electricity transformation method,and then respectively construct H37 Rv bacterial strain that Pup gene overexpresses(Hereinafter referred to as the H37Rv::Pup strain),H37 Rv bacterial strain that Mpa gene overexpresses(Hereinafter referred to as the H37Rv:: Mpa strain), H37 Rv bacterial strain that Dop gene overexpresses(Hereinafter referred to as the H37Rv:: Dop strain), H37 Rv bacterial strain that paf A gene overexpresses(Hereinafter referred to as the H37Rv:: paf A strain); H37 Ra bacterial strain that Pup gene overexpresses(Hereinafter referred to as the H37Ra::Pup strain),H37 Ra bacterial strain that Mpa gene overexpresses(Hereinafter referred to as the H37Ra:: Mpa strain), H37 Ra bacterial strain that Dop gene overexpresses(Hereinafter referred to as the H37Ra:: Dop strain), H37 Ra bacterial strain that paf A gene overexpresses(Hereinafter referred to as the H37Ra:: paf A strain); XJ bacterial strain that Pup gene overexpresses(Hereinafter referred to as the XJ::Pup strain), XJ bacterial strain that Mpa gene overexpresses(Hereinafter referred to as the XJ:: Mpa strain), XJ bacterial strain that Dop gene overexpresses(Hereinafter referred to as the XJ:: Dop strain), XJ bacterial strain that paf A gene overexpresses(Hereinafter referred to as the XJ:: paf A strain); Make use of H37 Rv bacterial strain, H37 Ra bacterial strain and XJ bacterial strain that pup/mpa/dop/paf A gene overexpresses,Which QRT- PCR detection and identification constructs.Respectively use built Pup/Mpa/Dop/paf A gene overexpression of mycobacterium tuberculosis International Standard Avirulent strain H37 Ra bacterial strain,mycobacterium tuberculosis International Standard virulent strain H37 Rv bacterial strain and XJ bacterial strain to infect macrophages,At the same time with mycobacterium tuberculosis International Standard Avirulent strain H37 Ra bacterial strain, mycobacterium tuberculosis International Standard virulent strain H37 Rv bacterial strain and XJ bacterial strain infected macrophages group as control group,make use of flow cytometry to detect apoptosis rate of each infected macrophage when the infected model is copied successfully after 1h,6h,12 h and 24 h,and analyze its time photograph sex change.At the same time,respectively use built Pup/Mpa/Dop/paf A gene overexpression of mycobacterium tuberculosis International Standard Avirulent strain H37 Ra bacterial strain,mycobacterium tuberculosis International Standard virulent strain H37 Rv bacterial strain and XJ bacterial strain to infect macrophages,At the same time with mycobacterium tuberculosis International Standard Avirulent strain H37 Ra bacterial strain, mycobacterium tuberculosis International Standard virulent strain H37 Rv bacterial strain and XJ bacterial strain infected macrophages group as control group, make use of flow cytometry to detect apoptosis rate of each infected macrophage when the infected model is copied successfully after24 h and 96 h,and analyze its time photograph sex change. Cells of each group respectively for bacterial culture of cracking, detect the strains in infected macrophages proliferation,testing each strain infected macrophages by flow cytometry method by CFU colony counting after rate.Results: 1、respectively shift Recombinant shuttle-plasmid PMV361 pup, PMV361 mpa, PMV361 dop and PMV361 paf A to H37 Rv bacterial strain, H37 Ra bacterial strain and XJ strain competent cell by Using electricity transformation method, Make use of H37 Rv bacterial strain, H37 Ra bacterial strain and XJ bacterial strain that pup/mpa/dop/paf A gene overexpresses,Which QRT- PCR detection and identification constructs,2、Detect the apoptosis condition of the mycobacterium tuberculosis infecting macrophages by use of Annexin V-FITC/PI double-staining method.the results are as following: Use H37Ra: : Pup; H37Ra: : Mpa; H37Ra: : Dop; H37Ra: : paf A strains to infect macrophages group respectively,The apoptosis rate of the bacteral strain infecting macrophage in 1 h, 6 h, 12 h and 24 h appears rising trend,;The above strains infected macrophages group compared with the group H37 Ra strains infected macrophages,The apoptosis rate of each strain infected macrophage, at the same point in time the apoptosis rate decreases;Use H37Rv::Pup; H37Rv::Mpa; H37Rv::Dop; H37Rv::paf A strains to infect macrophages group respectively,The apoptosis rate of the bacteral strain infecting macrophage in 1 h, 6 h, 12 h and 24 h appears rising trend,;The above strains infected macrophages group compared with the group H37 Rv strains infected macrophages,The apoptosis rate of each strain infected macrophage, at the same point in time the apoptosis rate decreases,.H37Rv: : Pup; H37Rv: : Mpa; H37Rv: : Dop; H37Rv: : paf A strains infected macrophages group respectively, and H37Ra: : Pup; H37Ra: : Mpa; H37Ra: : Dop; H37Ra: : paf A strains infected macrophages group compared respectively,The apoptosis rate of each strain infected macrophage, at the same point in time the apoptosis rate decreases,. XJ::Pup; XJ::Mpa; XJ::Dop; XJ::paf A strains infected macrophages group respectively, The apoptosis rate of each strain infected macrophage, at the same point in time the apoptosis rate decreases.3、respectively use built Pup/Mpa/Dop/paf A gene overexpression of mycobacterium tuberculosis International Standard Avirulent strain H37 Ra bacterial strain,mycobacterium tuberculosis International Standard virulent strain H37 Rv bacterial strain and XJ bacterial strain to infect macrophages,At the same time with mycobacterium tuberculosis International Standard Avirulent strain H37 Ra bacterial strain, mycobacterium tuberculosis International Standard virulent strain H37 Rv bacterial strain and XJ bacterial strain infected macrophages group as control group, make use of flow cytometry to detect apoptosis rate of each infected macrophage when the infected model is copied successfully after24 h and 96 h,and analyze its time photograph sex change. The results test: the expression of the colony count of the above strains was higher than that of control group,the proliferation in the macrophage obviously enhanced.Conclusions: 1. Successfully construct H37 Rv bacterial strain,H37 Ra bacterial strain and Mycobacterium tuberculosis isolates strains with prevalence in Xinjiang region that pup/mpa/dop/paf A gene overexpresses,2. Different appoptosis rate of mycobacterium tuberculosis infect macrophages is related to the expression of MTB pup/mpa/dop/paf A gene;3. The overexpression of Pup/Mpa/Dop/paf A gene can significantly enhance the toxicity and pathogenicity of H37 Ra bacterial strain、H37Rv bacterial strain and Mycobacterium tuberculosis isolates strains with prevalence in Xinjiang.