Construction And Clinical Application Of Mycobacterium Tuberculosis Fusion Protein TB10.4-MPT64

Objective: To construct Mycobacterium tuberculosis fusion protein TB10.4-MPT64,and to investigate its serological diagnostic value and immunogenicity.Methods: The target gene was introduced into pET-30 a vector by molecular cloning method,and the recombinant plasmid PET-30A-TB10.4-MPt64 was constructed,and then transferred into Escherichia coli BL21 to induce the expression of Mycobacterium tuberculosis fusion protein TB10.4-MPT64.The purified Mycobacterium tuberculosis fusion protein TB10.4-MPT64 was obtained.The value of TM protein in the clinical application of TB serology was explored by ELISA test after the enzyme label plate coated with Mycobacterium tuberculosis fusion protein TB10.4-MPT64.The antibody expression types in mouse serum were detected by ELISA.The immunogenicity of TM protein vaccine was investigated by flow cytometry.Results: The recombinant plasmid pET-30a-TB10.4-MPT64 was successfully constructed and transferred into Escherichia coli BL21 to induce the expression of target protein.The prokaryotic expression conditions of Mycobacterium tuberculosis fusion protein TB10.4-MPT64 were as follows: The recombinant plasmid pET-30a-TB10.4-MPT64 could be expressed as soluble protein after ITPG induction at 15℃.The Mycobacterium tuberculosis fusion protein TB10.4-MPT64 existed in the supernatant of the ultrasonic crushing bacterial solution,and its expression level increased with the extension of time.At0.2mmol/L ITPG,the recombinant plasmid was able to obtain a better induced expression concentration.Finally,the recombinant Mycobacterium tuberculosis fusion protein TB10.4-MPT64 with a purity greater than 80% was obtained by ITPG induction and Ni column affinity chromatography.Checkerboard titration was used to determine the optimal coating antigen concentration and serum dilution concentration of Mycobacterium tuberculosis fusion protein TB10.4-MPT64 for serum detection of tuberculosis patients.Under the optimal concentration condition corresponding to the maximum P/N,2μg/100μl was the optimal coating concentration of antigen.1:100 was the most suitable serum dilution concentration.Statistical analysis was conducted to explore the role of recombinant Mycobacterium tuberculosis fusion protein TB10.4-MPT64 as antigen in the rapid detection of tuberculosis.Using the mean absorbance of OD450 nm +2 times standard deviation as cut-off value of healthy human serum,OD450nm>cut-off value as positive value,OD450 nm.Conclusion: Mycobacterium tuberculosis fusion protein TB10.4-MPT64 was prepared.This protein has been found to be a potential test antigen for TB diagnosis.The novel subunit vaccine based on the protein was preliminarily explored to induce memory immune response,which laid the foundation for the subsequent development and improvement of vaccine and immunogenicity research.