Cloning, Expression And Primary Application Of Mycobacterium Tuberculosis 16ku And 38ku Protein

It has been over a century since Koch discovered Mycobacterium tuberculosis (MTB) in1882, but it is still a serious problem to the public health. Tuberculosis is listed as one of the focal control diseases by WHO. It is caused by MTB, which is the main pathogenic bacteria to human being (and animals) among the Mycobacterium, with a high morbidity, up to 90%. The tendency of MTB infection has increased sharply in recent years, with around 8 to 10 million new cases each year, which makes MTB recognized as "the first killer". As a result, the research on prevention, treatment and diagnose of MTB has become an urgent need.All the times, the diagnosis of tuberculosis depends on fast acid stain of sputum and culture of MTB, as well as the clinical symptom and chest X-ray. But these methods lack sensitivity due to the typical growth characteristic of MTB, especially in diagnosing the extropulmonary tuberculosis. Although currently there are clinical available screening technique such as ELISA, ELISAPOT, most of the blocking antigens are roughly made, such as polymerizing OT, PPD, BCG, semi-glycolipids antigen, which make the tresult of method unstable, and lead to clinical difficulties in the diagnosis of extropulmonary and culture-negtive pulmonary tuberculosis.Ivanyi, screening the antigens with tuberculosis monoclonal antibody, has got the finding that only the antigens 16ku protein and 38ku protein take the specific antigen epi-position, which proved to be good specificity. 38ku protein, a lipoprotein and main antigen of MTB, which displays better than other single antigen in evocation to delayed hypersensitivity in Guinea pig, has good sensibility and specificity in serological test of TB. 16ku protein is a kind small heat shock protein and also one of the predominant membrane protein in MTB, which is owned only by MTB. Moreover, 16ku not only can induce immunologic reaction mediated by T cell, but also hold the activity of molecular chaperone, which plays a key role in survival and stability of MTB. In order to enhance the the specificity and sensitivity of the current diagnostic agent, we make researches on the genes of MTB 16ku and 38ku protein, obtaining recombination protein antigen through gene engineering technology. This is not only for applying to the diagnosis, but also lay primary foundation for the future gene engineering bacterium. So the main contents of our research are: the gene cloning and prokaryotic expression of MTB 16ku and 38ku protein, the identification and purification of the expressed products and the establishment of ELASA on MTB 16ku and 38ku protein. 1. Cloning and expression of MTB 16ku and 38ku protein in E.coli16ku and 38ku protein genes were amplified by PCR from MTB H37Rv strain genome and cloned into vector pGEX-4T-2. After positive clones was sequenced, recombinant plasmids pGEX-16 and pGEX-38 were transformed into E.coli respectively, induced by IPTG, to express GST-16 and GST-38 fusion protein whose relative molecular mass(RMM) is 42ku and 64ku and whose product detected by thin-layer scanning accounts for 42% and 18%respectively in the whole protein. We made Western blotting with anti-MTB antibody in rabbits, which demonstrated that a specific combined colored zone is in 42ku and 62ku on the trans-membrane. What has happened proves that the fusion proteins can combined with anti-TB antibody specifically. Fusion proteins were eluted from GSTrap FF affinity chromatography after thrombin cleavage. the purified protein products were obtained whose RMM are 16ku and 38ku respectively.2. the expressive identification and purification of the fusion proteinsUse correctly sequencing recombinant plasmid pGEX-16 to transform E. coli DH5 α. At the IPTG final concentration 0.3mmoL.L~-1, a low inducing temperature of 28℃, we can get the soluble expression of 16ku protein. Use recombinant plasmid pGEX-38 to transform the highly expressed E. coli BL21. At the IPTG final concentration 0.5mmoL.L~-1, inducing temperature of 30℃, we can get the soluble expression of 38ku protein. The gain of the soluble protein offer convenient for the next step of purifying the fusion protein, and keeps integrated activity for the further application.Fusion proteins were eluted from GSTrap FF affinity chromatography after thrombin cleavage. The purified protein products were obtained whose RMM are 16ku and 38ku respectively.3. Primary establishment of ELASA on MTB protein16ku and 38kuFrom articles, we have chosen the polystyrene board from abroad to be the solid carrier, then diluted and coated the system of 16ku and 38ku purified proteins. With testing antibody IgG from sheep marked HRP and developer TMB, we set up indirect ELISA for testing 16ku and 38ku proteins of MTB. In addition, we also test the sensitivity, specificity and accuracy of such method.