结核分枝杆菌Hsp65的表达、纯化与鉴定

目的克隆、表达和纯化结核分枝杆菌热休克蛋白65(Heat Shock Protein 65),为研究结核病诊断技术提供基础。方法用PCR方法从结核分枝杆菌H37Rv基因组中分两段扩增出hsp65基因,将上下游基因片段分别连接进入pMD18-T载体中,序列测定正确后,通过基因内部的单一酶切位点进行连接。将完整的hsp65基因克隆到pProEX HTa载体并在大肠杆菌DH5α中诱导表达,表达产物经SDS-PAGE和Western-blot分析后,用Ni-NTA亲和层析柱进行纯化。获得的纯化蛋白与10例临床可疑结核病人血清进行Western-blot分析。结果获得的结核分枝杆菌Hsp65基因序列与GenBank公布的一致;表达蛋白相对分子质量为65 kDa,与文献报道相同;Western-blot结果显示,在相对分子量约65 kDa处与鼠抗MTB Hsp65 mAb特异性结合带;纯化的目的蛋白与8例病人血清有特异性结合条带,2例为阴性,该结果与临床诊断一致。结论成功表达、纯化和鉴定了Hsp65蛋白,为其在结核病诊断研究中的应用奠定了基础。

Expression, purification and identification of Hsp-65 protein from Mycobacterium tuberculosis

To obtain the purified Hsp-65 protein from Mycobacterium tuberculosis for use in clinical diagnosis of tuberculosis,two parts of the Hsp-65 genomic DNA of M.tuberculosis strain H37Rv,the upper and lower fragments,were amplified by PCR and the PCR products were inserted to vector pMD18-T accordingly and then sequenced.After correct sequencing,they were ligated to the single endonuclease digested gene locus.The whole hsp65 gene fragment digested by EcoR1 and Hind III was subcloned into expression vector pProEX-Hta and then expressed in E.coli DH5a.The expressed protein was identified by SDA-PAGE and Western blot analysis with monoclonal antibody against Hsp65 and sera from clinical suspected cases with tuberculosis,and the recombinant fusion protein His-6 was purified by Ni-NTA purification system.It was shown that the sequences of PCR products were identical to those of the Hsp-65 gene in GenBank.The relative molecular mass of 65 kDa idendified by SDS-PAGE analysis was consistent to that reported previously.As revealed by Western blotting,a specific binging band could be detected at site where relative molecular mass of 65 kDa located with the mouse anti-Hsp65 monoclonal antibody.Meanwhile,this purified protein could react with sera of 8 cases of suspected tuberculosis,but other 2 cases shoowd negative result.It is apparent that the recombinant Hsp65 protein was successfully expressed and purified,and it would be helpful for the further studies in the diagnosis and treatment of tuberculosis.