Preliminary Construction Of Mtb Examination Assay And Screening Of Its Host-Pathogen Interaction Proteins

Tuberculosis(TB)is an ancient chronic zoonosis caused mainly by Mycobacterium tuberculosis(Mtb).Not only does it still cause huge economic losses,but also seriously threatens human health.Mycobacterium tuberculosis have several secreted proteins that are abundantly expressed in host cells.Understanding the role played by these secreted proteins in the pathogenesis of this disease will help us develop a new tuberculosis vaccine.The situation of tuberculosis in China is very serious and traditional diagnosis methods can no longer meet clinical needs,and there is no single test method that can balance sensitivity and specificity.This study is based on the diagnosis of tuberculosis and the target of anti-tuberculosis vaccines.1.Develop three Raw264.7 cell lines stably expressed ESAT6,MPT64 and TAP tag ZZtag-3×Flag by CRISPR/Cas9 system.In this experiment,we successfully ampilify following fragments: Neomycin,ZZtag,TEV cleaverage site,3×Flag,mCherry,Arm1 and Arm2.Then those fragments are fused by overlap PCR to a complete plasmid named pMD18T-TAP.Six plasmids named pMD18T-TAP,pMD18T-TAP-ESAT6,pMD18T-TAP-CFP10,pMD18T-TAP-HspX,pMD18T-TAP-MPT64 and pMD18T-TAPAg85 B were constructed successfully which were used for TAP.And then we developed three Raw264.7 cell lines individually stably expressed ESAT6,MPT64 and TAP tag ZZtag-3×Flag by CRISPR/Cas9 system.And ESAT6's interacting protein Serine/arginine repetitive matrix protein 1 was screened by TAP-MS,which also need following experiment to identify this interaction.2.This study aims to develop a set of programs(TB-LAMP,MDA-PCR and HCR)for the detection of Mycobacterium tuberculosis H37 Ra.Our experiment use IS6110 as the target gene to amplify H37 Ra.The results show that TB-LAMP and MDA-PCR can get the positive amplification results when there are dozens of H37 Ra.Our experiment first attempt to improve traditional HCR method for the detection of H37 Ra and get the positive results.Then we target more gens(IS6110,CFP10,GryB)to detect H37 Ra by HCR and more positive signals are got.In this study,three methods(TB-LAMP,MDA-PCR and HCR)were established to detect IS6110 gene.Meanwhile,it is the first time that MDA-PCR and HCR are used to detect Mycobacterium tuberculosis H37 Ra.