Screening And Verification The Biomaker Of Tuberculosis Based On Biomimeitc Affinity Chromatography

Tuberculosis,caused by the M.tuberculosis,is a chronic infectious disease.A clinical diagnostic system for tuberculosis based on imaging,bacteriology,molecular biology and immunology has been established.Unfortunately,the clinical diagnosis for tuberculosis patients have not improved since the limitation of the methods,and more than one third patients are detected with false results.To increase the rate of clinical diagnostic tests for tuberculosis,the new biomarkers finding are urgent needed.The proteomics of the serum from tuberculosis patients and culture filtrate proteins of M.tuberculosis is investigating using biomimetic affinity chromatography in conjunction with LC-MS/MS analysis,respectively.In order to find suitable biomimetic affinity ligands for specific adsorption Ig G in serum of human-beings and culture filtrates proteins from M.tuberculosis H₃₇Rv stain,a library with 1.0×10⁴ ligands was constructed.Then,the biomimetic affinity chromatography media A115-94 was selected out from 200 distinct ligands which were randomly taken out from the library with 1.0×10⁴ ligands.The Ig G in serum of tuberculosis patients is adsorbed on the two columns of A115-94,then two kinds of antigens,culture filtrate proteins and cytoplasmic proteins of M.tuberculosis H37Rv,was used to immune affinity chromatography,respectively.Finally,195 adsorption proteins of MTB were detected with specific immune affinity with Ig G of tuberculosis patients using MS analysis.'Knowing yourself as well as the enemy‘is critical for the biomarkers of tuberculosis selection,6 biomimetic affinity media with distinct physicochemical property were selected from 30 kinds of ligands.The sample of MTB-CFP was divided into 7 overlapping fractionations using these 6 biomimetic affinity media.And,541 proteins of MTB were identified using MS analysis including 61 first-time identifications.The biomarkers confirmation was also accomplished based on the results of the above identifications.40 MTB proteins were tried to express in E.coli host cells according to the proteins abundant using MS analysis,as well as the properties and functions of them using bioinformatics analysis.18 MTB proteins were obtained with more than 90%purification,of which,6 antigens with more sensitivity and specificity than the commercial serological biomarker of Psts-I/38k Da according to the detection in 212 sera of tuberculosis patients and 64 sera of healthy control individuals.Finally,a technical platform of identification MTB proteins in sera of tuberculosis patients is established.Of which,two Bi AC media of At-23 and A115-94 were selected and used to fractionate albumin and immunoglobulin respectively,prior to the LC-MS/MS analysis.After screening the proteomic database of MTB complexity,4 antigens were identified with 2 different peptides in fractionations of tuberculosis patients.Meanwhile,3 antigens(Fbp A,Fbp B and Bfr B)were verified by western blotting assay in sera of tuberculosis fractionations.Biomimetic affintiyt chromatogtaphy in conjunction with LC-MS/MS analysis is an effective method for screening the serological biomarkers for other diseases.This strategy can also be used for investigation with others proteomic profiling of pathogenic bacteria.