结核分枝杆菌ESAT-6蛋白的表达及纯化研究

目的构建表达结核分枝杆菌ESAT-6蛋白的表达系统，建立表达及纯化方法，为其应用打下基础。方法根据ESAT-6的基因序列设计引物，从结核菌基因组DNA中扩增基因，酶切后插入表达载体，测序证实后转入大肠杆菌JM109菌株，经IPTG诱导表达，产物经谷胱甘肽-琼脂糖凝胶法纯化。结果ESAT基因产物大小、酶切片断大小和载体的大小与设计一致、ESAT-6基因测序的结果与目的基因完全符合。表达纯化的蛋白的大小与蛋白质分子量标准比较一致。结论构建结核分枝杆菌ESAT-6蛋白的表达载体成功，纯化出目的蛋白。

Construction,expression and purification of Mycobacterium tuberculosis early secreting antigenic target-6

Objective To construct the expression system for ESAT - 6 of M. tuberculosis and establish methods for its expression and purification. Methods Primers containing endoneaclease cleavage sites were designed according to M. tuberculosis esat - 6 sequence and the esat - 6 gene was amplified from the genomics . After endonuclease digestion, esat - 6 was inserted into the expressing vector of p - GEX - 6P - 1 to construct ESAT- 6 expressing vector E6 - pG. ESAT - 6 was prepared with IPTG induction and analyzed by SDS - PAGE. Various conditions were tested for maximal production of ESAT - 6. Results The vector E6 - pG was comfirmed by endonuclease digestion and DNA sequencing. The ESAT - 6 was demonstrated by SDS - PAGE. It was found that maximal preparation of ESAT - 6 could be obtained at 37℃ with 0.5umol/mL IPTG for culturing 3h. Pure target protein has been obtained with the protocol we developed. Conclusion E6 - pG was proved to express ESAT - 6 of M tubeculosis sucessfully.