结核分枝杆菌分泌蛋白ESAT-6、CFP10基因的克隆、鉴定及其原核表达

以人型结核分枝杆菌 H37RV株基因组 DNA为模板 ,应用 PCR法对 ESAT- 6和 CFP10基因进行扩增 ,产物经纯化后与载体 PMD18- T连接、转化及酶切鉴定 ,亚克隆到原核表达载体 PGEX- 6 P- 1,构建原核重组表达质粒 ,转化入大肠杆菌 BL2 1中 ,以 1mmol/L IPTG诱导 ,进行 SDS- PAGE电泳。结果表明 ,ESAT- 6和 CFP10基因表达的融合蛋白相对分子质量分别为 32 0 0 0和 36 0 0 0 ,与实测相符。重组结核杆菌分泌蛋白 ESAT- 6和 CFP10的成功表达为结核病诊断及重组疫苗的构建打下了基础

Cloning,Identification and Expression of ESAT-6,CFP10 Gene from Mycobacterial tuberculosis

The gene encoding protein ESAT-6, CFP10 was amplified from M.tuberculosis H37RV chromosomal DNA by using PCR. PCR product was cloned initially into PMD18-T vector, then transformed into E.coli. JM83 strain, and plasmid DNA was digested with enzymes. Then cloned into PGEX-6P-1 expressing vector. Plasmid DNA was extracted and digested with enzymes. Plasmids containing the right inserted were retransformed into E.coli. BL21 strain. Bacterial lysates prepared from 1 mmol/L IPTG induced cultures were loaded directly SDS-PAGE. Upon induction, the recombinant PGEX-6P-1-ESAT-6 and PGEX-6P-1-CFP10 producted with apparent Mr of 32 000 and 36 000. In conclusion, we obtained recombinant PGEX-6P-1 vector containing ESAT-6 and CFP10 specific fragment.