Preparation Of The Monoclonal Antibody Against Rv0199and Rv3802c Protein From Mycobacterium Tuberculosis

Bovine tuberculosis is a general term for a variety of disease caused by Mycobacterium tuberculosiscomplex Mycobacterium bovis is a major pathogen of this disease. Each other spread the disease betweenhumans and animals can infect the host is very wide, especially in developing countries, the eradication ofthe disease has been a problem. The disease each year causing serious economic losses in worldwide andthe OIE classified it as a Class B animal disease. Rv0199protein is a protein of pathogenic mycobacteria invivo infection must to subvert macrophages acidified defense mechanism, thus ensuring long-term survivalof pathogenic mycobacteria in the host body. The Rv3802c protein involved in the M. tuberculosis cell wallof the building, and to play a role in mycolic acid synthesis and resistance to antibiotics. Therefore, for theRv0199and Rv3802c protein has a very important significance.In this study, by PCR using primers designed from Mycobacterium tuberculosis standard strain H37Rvgenome amplified rv0199and rv3802c gene. Two genes will be the double digestion by a specificrestriction enzymes are respectively connected to the expression vector pET-28b and transformed intoBL21(DE3) competent, abundantly expressed by IPTG induction, sonication, respectively, according to thetwo the nature of the protein (Rv0199inclusion body expression, Rv3802c soluble expression), usingNi-NTA affinity column chromatography (Rv3802c) and denatured refolding (Rv0199) purified. Rv3802cprotein purification were obtained, and the size of40kDa; the Rv0199protein the size of25kDa. Thepurified protein by SDS-PAGE and Western-blot analysis, proved to having high purity and goodimmunogenicity.In order to better research further two proteins, in this experiment was also prepared Rv0199andRv3802c a monoclonal antibody. The two proteins were used to immunize female BALB/c mice, by fusionafter screening, respectively, to obtain a stable secretion of the hybridoma strain3802E6and0199H5.3802E6and0199H5mice by intraperitoneal injection was prepared by ascites, and ascites purified certifiedfor two monoclonal antibody subclasses are IgG1, κ light chain are chain. Two monoclonal antibodies inWestern-blot and indirect ELISA test to prove its corresponding antigen reacts only react onlyMycobacterium tuberculosis lysate good specificity for further research on the Rv0199and Rv3802cprotein function laid a good basis.