Antigenicity Of The Novel Proteins Identified By Proteogenomics From Mycobacterium Tuberculosis And Their Application On The Diagnosis Of Tuberculosis

Objectives Mycobacterium tuberculosis(MTB)clinical isolates are slow to grow,difficult to culture and easy to be cross-contaminated by other mycobacteria,and lacking highly specific,ultra-sensitive biomarkers,which bring to difficulties in clinical accurate differential diagnosis and timely treatment of tuberculosis.Therefore,it is necessary to establish new rapid,accurate,specific and sensitive TB detection technologies.New effective strategy is the necessary prerequisite and basis to eventual control of the spread of tuberculosis.Based on large-scale proteogenomics of MTB H37 Rv,we found and verified22 MTB missing genes,among which half of them belong to MTB specific genes.The purpose of this study is to screen novel MTB specific antigens or combination in serologic detection,and to provide technical support for rapid diagnosis and kit development of tuberculosis in the future.Methods Firstly,we cloned all the novel genes and established an E.coli heterologous expression system for selecting highly soluble expression vector based on four different vectors of p GEX-4T-2,p ET-28 a,p ET-32 a,and p MAL-c2 X.After codon optimizating and target protein inducing by isopropyl-β-D-thiogalactoside(IPTG),the soluble expressed fusion protein was purified by affinity chromatography.Secondly,mass spectrometry was performed to verify the purified target protein.Thirdly,Bepipred Linear Epitope Prediction v2.0,Bio Sun and Dlbepitope software were used to predict the trusted epitopes of B cells for these new encoding products.Finally,purified and verified proteins were evaluated their antigenicity and applied valued in TB diagnosis by ELISA technology.Results 1 Among the four different vectors,the pMAL-c2 X was an ideal vector because of its higher soluble expression and signal interference in ELISA detection.The purified proteins were verified by mass spectrometry.2 Theoretical prediction found that these new proteins have new B cell confident epitopes by Bepipred Linear Epitope Prediction v2.0,Bio Sun and Dlbepitope softwares.3 The preliminary serological comparison found that p MAL-c2 X empty showed lower signal interference in ELISA detection,which was suitable for subsequent serological antigenic screening of these fusion-expressed proteins.Two potential antigens of TB38.76 and TB26.88 were screened after clinical sample detection.Antigen TB38.76 had a sensitivity of 68.33% and a specificity of 72.5% for tuberculosis patients.TB38.76 had a sensitivity of 90% and a specificity of 55% for patients with positive sputum culture.Antigen TB26.88 had a sensitivity of 80% and a specificity of 50% for patients with positive sputum culture.4 Depending on the detecting values,we found TB26.88,TB38.76,38 k Da and commercial kit(Shang Hai Rong Sheng Kit)had complementary in TB patient discrimination and could effectively improve diagnostic efficiency.Conclusions 1 Using E.coli heterologous expression system,based on pGEX-4T-2,p ET-28 a,p ET-32 a,p MAL-c2 X four vectors successfully constructs 22 MTB new gene recombinant plasmids,and the p MAL-c2 X is an ideal vector with higher soluble expression.2 From 22 new antigens finds 2 potential new antigens TB26.88 and TB38.76,which are complementary to known antigens and commercial kits for the diagnosis of tuberculosis,for the optimization of future combined antigens and kit development provide a scientific basis.Figure12;Table18;Reference 116...