牛分支杆菌热休克蛋白65基因的分子克隆和表达

以牛型分枝杆菌Vallee菌株基因组DNA为模板,应用PCR方法扩增热休克蛋白65(Hsp65)基因,PCR产物经纯化与EcoRⅠ/SaⅠ双酶切后与经同样处理的原核表达载体pGEX-6P-1进行连接,转化感受态细胞BL21中,筛选和构建了原核重组表达质粒pGEX-H65,核苷酸序列测定验证其正确性.以1 Mol/LIPTG诱导,进行SDS-PAGE电泳.结果表明,Hsp65基因表达的融合蛋白GST-H65裂解为两部分;即64 kD和22 kD,两个蛋白分子量相加与预测的86 kD(HsP60 kD GST26 kD)相符.牛分支杆菌热休克蛋白HSP65的成功表达为其功能的研究打下基础.

The cloning and expression of Hsp65 gene from Mycobacterium bovis

The gene encoding for protein Hsp65 was amplified from M. Boris Vallee strain chromosomal DNA by PCR technique. PCR product was cloned into pGEX-6p-1 expressing vector. Plasmid DNA was extracted and digested with enzymes and sequenced to confirm its rightness. The right recombinant plasmids were transformed into E. Coli. BL21 strain. Bacterial lysates prepared from1 mmol/1 IPTG induced cultures were loaded directly SDS-PAGE. The result shows that there are two bands with apparent MW of64 kD and 22 kD, with the summation 86 kD which is equivalent to the summation of Hsp(60 kD) and GST(26 kD), which implies the fusion protein GST-Hsp65 cleavaged into two parts. In conclusion, we obtained the recombinant pGEX-H65 expressing vector which contains Hsp65 gene and can be expressed successfully.