Mycobacterium Tuberculosis Rv1987, TB7.7 Protein's Prokaryotic Expression, Purification and Identification

Mycobacterium tuberculosis is a pathogen of tuberculosis and has relatively high epideictic and mortality among human. It is the second most life-threatening infectious disease second only to AIDS，so it is very important for the prevention and control of tuberculosis. In our experiment, RV1987 and TB7.7 recombinant plasmids of mycobacterium tuberculosis were constructed. The specific protein RV1987 and TB7.7 of mycobacterium tuberculosis were prokaryotic expressed, purified and identified in Escherichia coli. Our experiment lay a sound foundation for the subsequent serological diagnosis of tuberculosis as to obtain the high-purity target protein. Methods: use the genome of H37Rv as template to amplify the target gene and cloned it into prokaryotic expression vector, then transformed it into E. coli C43 (DE3) strain. After that, go through induced, expressed and purified in order to obtain recombinant protein.Results: the recombinant plasmid of RV1987 protein and TB7.7 protein was successfully constructed and expressed to obtain purified products, which were purified by affinity chromatography with a purity greater than 90%. The concentrations of RV1987 and TB7.7 proteins were 413 microns /mL and 1693 microns /mL. Rv1987 protein was expressed in inclusion body. TB7.7 protein was expressed in both supernatant and inclusion body, and the expression in supernatant was slightly higher than In inclusion body. Western Blot showed that all the above proteins could react with the positive serum of tuberculosis and had good reactivity. Conclusion: Escherichia coli is the most common system for gene engineering to express heterogenous proteins and widely used by researchers because of its relatively high expression efficiency. The experimental results showed that we obtained by prokaryotic expression and purification of two specific protein has good response to the original, in the subsequent experiments is obtained by testing several protein specificity and sensitivity, we can on the basis of the existing stimulus antigen screen is suitable for our country crowd IGRAs stimulus antigen, as rapid auxiliary diagnosis of tuberculosis (TB), thus improve the serum antibody detection of TB case detection and reduce the misdiagnosis rate.