【摘要】 以牛型分枝杆菌基因组DNA为模板,PCR方法扩增mpb64基因,纯化PCR产物并与 pDM18-T载体连接、转化,经酶切及核苷酸序列鉴定为正确后,酶切产物亚克隆到原核表达载体 pET30a(+)的KpnI／EcoRI位点,构建重组表达质粒pET30a+-mpb64,转化到大肠杆菌DE3内,以 IPTG进行诱导,终浓度为1mmol／L,诱导产物进行SDS-PAGE电泳。结果表明,PCR方法成功扩增 出mpb64基因,核苷酸序列测定验证了其正确性,重组表达质粒表达的pET30a+-mpb64融合蛋白 相对分子量为30.4kDa,与实测相符。牛分枝杆菌pET30a+-mpb64的成功表达为牛结核病的诊断 及新型疫苗的研究奠定了基础。更多还原

【Abstract】 The gene encoding protein MPB64 was amplified from Mycobacterium bovis chromosomal DNA by using PCR. The purified PCR products was cloned initially into pMD18T vector, afterwards transformed into DH5a strain and plasmid DNA was digested with enzymes, then subcloned into pET30a ( + ) expressing vector. The recombinant plasmid DNA was digested with enzymes ( KpnI/EcoRI). Plasmids containing the right inserted were retransformed into E. coli BL21 (DE3) strain, bacterial lysates prepared from Immol/L IPTG induced cultured were loaded directly SDS - PAGE. The recombinant pET64 producted with apparent molecular weight of 30.4kDa. In conclusion, recombinant pET30a( + ) containing mpb64 specific fragment was obtained.更多还原