The Expression And Evaluation For Serodiagnosis Of MPT63Protein Of Mycobacterium Tuberculosis

Mycobacterium tuberculosis which causes tuberculosis is a pathogenic bacterium that bring about the highest mortality of chronic infections. The population growth, flow to speed up and multiple drug-resistant strains emerging, make tuberculosis patient number rise again in the worldwide. In order to control and prevent TB, the early detection and accurate medication is very important. Serological diagnosis is primary mean of laboratory diagnosis of tuberculosis. Finding stable and efficient tuberculosis antigens is the key. MPT63is in the pathogenic strains of MTB. The expression is one of third of secreted proteins of Mycobacterium tuberculosis.In order to evaluate the potential of this immunodominant antigen in serodiagnosis of TB,in this present study, we aim to insert the sequence of MPT63into expression plasmids, then induce them to expression in Rosetta cells. The expression products were then purified, in the meantime,the immunoreactions of this antigens were analyzed and defined. These set of experiments had thus paved the way for the further utilization of recombinant peptide in the serodiagnosis of tuberculosis in the future.DNA encoding MPT63antigen was amplified by PCR using a pair of primers which were designed with Primer Premier5.0. The MPT63gene was inserted into the prokaryotic express-ion vector pET-30a with the6X His-tag in the N-terminal. The recombinant pET30a-mpt63expression vector was transferred into Rosetta. The positive clone was screened by means of PCR. IPTG induced the expression of protein at28℃,2.0mmol/L,after5hours could reach the highest amount. Solubility analysis showed that the MPT63existed in the form of soluble protein in the E. coli. Western blotting revealed good antigenicity of MPT63.Tuberculosis can be rapidly and timely diagnosed with serological methods. Combination of recombinant M. tuberculosis fusion protein can improve diagnosis sensitivity and specificity. ELIS A results showed the sensitivity and specificity of recombinant fusion protein Rv3425(16.67%,95.83%), Mtb81(20.83%,95.83%)and MPT63(18.75%,97.92%). The sensitivity and specificity of combination of recombinant fusion protein Rv3425, Mtb81, MPT63are37.5%and91.67%.