Prokaryotic Expression Of Mycobacterium Tuberculosis Rv1984c And ESAT-6 Gene And Evaluation Of Their Immunodianostic Value

TB (tuberculosis, TB) is currently the number one killer infectious diseases. Our country is only inferior in the global 22 TB high burden country in occupies 2nd.The commonly used examination tuberculosis bacillus ramosus method, mainly depends upon the phlegm smear, the bacilliculture, the tuberculin (PPD) and so on examination technologies, time-consuming is long, the sensitivity and the specificity are low, causes to leak frequently examines and misdiagnoses, thus has delayed and the treatment opportunity, also brings the huge economic burden to the patient. Therefore, establishes fast, sensitive, the simple early diagnosis method is the question which the present domestic and foreign examination tuberculosis continues to solve. The immunity diagnosis takes one of its prevention and control early diagnosis technologies, in recent years obtained a more widespread attention.ELISA (Enzyme-linked Immunosorbent Assay, ELISA) is simple, fast, easy, and no special precision instruments, as the detection method has been widely used in clinical tests and scientific research. Efficient enzyme catalysis and the antigen or antibody immunoassay combining antigen antibody reaction cascade, and then decomposed by the enzyme substrate color and sensitive detection of trace amounts of specific antigen in serum or antibody. The dominant antigen and enzyme-linked immunosorbent assay combined to build fast, simple and accurate diagnosis by serological immunity more and more attention.This article selects RD the area code gene esat-6 and Rv1984, increases the goal fragment using PCR from tuberculosis bacillus ramosus standard H37Rv, and clones separately it to expression vector pET30a and pET28a, the construction reorganization expresses the material particle and transforms to the E.coil BL21(DE3) expression strain, after IPTG induction expression, carries on the analysis with SDS-PAGE, chooses the best expression condition and the soluble analysis. With purified Rv1984c, ESAT-6 and ESAT-6-CFP-10-16KD recombinant protein as coating antigen, the serum of the TB immunological detection. Mixture of single antigen and antigen sensitivity and specificity of diagnosis of tuberculosis, TB diagnostic kit for further provide the basis for the development.Result1. Rv1984c and esat-6 gene were amplified from H37Rv of mycobacterium tuberculosi DNA genome by PCR.the same size of target gene 576bp and 288bp respectively. The recombinant plasmid by enzyme digestion, PCR and sequencing compared by NCBI the compliance rate of 100%.2. We express recombinant pET28a-Rv1984c and pET30a-ESAT-6 fusion protein of approximately 23KD and 17.1KD respectively in size after induced by IPTG. pET28a-Rv 1984c dissolve the inclusion body as dissolvable protein. pET30a-ESAT-6 is dissolvable protein.3. pET28a-Rv 1984c and pET30a-ESAT-6 by Ni-NTA affinity purification got the recombinant protein purity of 97.4% and 51.2% respectively.4. We set up indirect ELISA method that recombinant protein pET28a-Rv1984c and pET30a-ESAT-6, ESAT-6-CFP-10-16KD as the coated antigen detect TB patients, non-tuberculous respiratory disease patients and healthy people serum.The result of the sensitivity was 46.8%,70.21% and 88.37%, specificity of 76.71%,90.4% and 97.4%, Cross-reactivity rate of 10%,40% and 15% respectively.