Mycobacterium Tuberculosis Rv0685 Protein Expression, Purification And Antibody Detection Protein Chip Preparation And Application

Objective:1.Using genetic engineering technique to clone,express,purify Rv0685 protein of Mycobacterium tuberculosis, analyzing it's specificity by Western blotting, preparing protein chip, and authenticating it's specificity.2.We prepared protein chip of five antigens, including ESAT6,CFP10,M16,M38 which were prepared by ourself and LAM which was bought, used these chips to detect 130 bacillary phthisis blood serum samples and 50 blood serum samples of health people, analyzed the sensitivity,specificity and constituent ratio of the five antibodies, these experiments are for establishing antigen foundation to develop protein chip test kits which can detect antibody of Mycobacterium tuberculosis.Methods:1.Designning and synthesizing primer base on Rv0685 gene sequence of Mycobacterium tuberculosis standard column which is publicated in GenBank; We obtained Rv0685 antigen gene fragment by PCR, and the Mycobacterium tuberculosis standard column was template; Conjunctting expression vector pQE80 and Rv0685 gene fragment cutted by restriction enzyme, and transforming the recombinant plasmid into E.coli DH5a, then selecting positive clone to detect it's sequence, transformming the correct recombinant plasmid into prokaryotic expression vector E.coli M15, inducing the Rv0685 protein with optimization temperature and IPTG concentration, analyzing the expression modality of the protein by SDS-PAGE, purified the recombinant protein with HisTrap FF, analyzing it's specificity by Western blotting. The protein chip is prepared by spotting Rv0685 protein of five concentration gradient on glass slides, then analyzed it's specificity further with clinical sample.2. The protein chip is prepared by spotting ESAT6 CFP10 M16 M38 and LAM antigens on glass slides,use these chips to detect 130 bacillary phthisis blood serum samples and 50 blood serum samples of health people, analyze the sensitivity specificity and constituent ratio of the five antibodies.Conclusion:1. We obtained the correct Rv0685 gene fragment and recombinant plasmid pQE80-Rv0685, definited the optimization temperature and IPTG concentration, obtained the Rv0685protein, It can bind with sera of tuberculosis patient, and the consequence of protein chip is on investigation. The sensitivity of the protein chip is 90.8%(118/130) and the specificity is 90%(45/50),the detection rate of LAM is 91.5% which is the highest. The sensitivity and specificity of the protein chip which can be used to auxiliary diagnose bacillary phthisis,that is prepared by ESAT6 CFP10 M16 M38 and LAM antigens are higher,so these antigens can be used to develop protein chip test kits which can detect antibody of Mycobacterium tuberculosis.