牛分枝杆菌MPB83基因的原核表达及免疫活性分析

Expression and Immunogenicity Analysis of MPB83 Gene From Mycobacterium bovis

利用PCR技术,以牛型分枝杆菌(M.bovis)Vallee菌株的全基因组DNA为模板,扩增出了一条600bp的MPB83基因片段,将其克隆至pMD18T载体中,经核苷酸序列测定确证后,KpnI/EcoRI双酶切,然后亚克隆到原核表达载体pET30a的相同酶切位点,构建表达质粒pETMPB83,将鉴定的重组质粒转化大肠杆菌BL21(DE3),IPTG诱导后SDSPAGE检测表达情况,重组质粒pETMPB83在30kDa处有一特异表达带,与预计大小相符。经Ni柱纯化后,Westernblot检测纯化蛋白具有免疫活性,用纯化的该蛋白进行动物(兔)接种制备抗血清,用Westernblot和ELISA检测该抗血清的效价和特异性,结果表明特异性较好。

The gene encoding protein MPB83 was amplified from Mycobacterium bovis chromosome DNA by PCR. The purified PCR products was cloned initially into pMD18-T vector, the plasmid DNA was digested with enzymes （KpnI/EcoRI） , and subcloned into pET30a （ ＋ ） expression vector. The recombinant plasmid DNA was digested with enzymes （KpnL/EcoRI）. Plasmids containing the right inserted fragments were retransformed into E. coli BL21 （DE3） strain, and bacterial lysates prepared from 1 mmol/L IPTG induced culture were loaded directly SDS-PAGE. The recombinant pET-MPB83 produced an apparent band with the molecular weight of 30kDa. The pET-MPB83 was purified by Ni-NTA His-Tag Protein Purified Kit. The recombinant pET-MPB83 product has immunocompetence by Western blot. Rabbits was inoculated with the purified pET-MPB83 to prepare anti-serum. Western blot and ELISA were used to analyze the specificity and appetency of the anti-serum. In conclusion, the recombinant pET-MPB83 expressed product with apparent molecular weight of 30kDa; the purified pET-MPB83 has been obtained by Ni-NTA His-Tag Protein Purified Kit; the purified recombinant pET- MPB83 product has immunogenicity by Western blot. Anti-serum prepared from rabbits inoculated with the purified pET-MPB83 has high appetency by Western blot and ELISA.