结核分枝杆菌小分子热休克蛋白16．3蛋白的原核表达及功能检测

Prokaryotic expression and function detection of Mycobacterium tuberculosis Hsp16. 3

目的进行结核分枝杆菌小分子热休克蛋白MTB Hsp16.3原核表达载体的构建、表达、纯化并初步观察其生物学效应。方法提取临床H37Rv分离株基因组DNA,PCR扩增Hsp16.3基因,将其重组到原核表达载体Pet28a中,构建原核表达载体Pet28a-Hsp16.3,进行双酶切及测序鉴定。将测序正确的重组质粒转化至E.coli BL21(DE3)中,经IPTG诱导表达后,对表达产物进行SDS-PAGE检测,同时通过镍柱纯化试剂盒纯化Hsp16.3,测定纯化后蛋白浓度,并进行Western blot法检测。将不同浓度纯化后蛋白作用小鼠腹腔巨噬细胞,实时定量PCR(qRT-PCR)检测巨噬细胞IL-10和IFN-γ的表达,同时设定空白对照组及阳性对照组。结果成功构建重组质粒Pet28a-Hsp16.3,并在E.coli BL21(DE3)中获得成功表达,通过镍柱纯化系统得到纯化Hsp16.3融合蛋白。qRT-PCR检测结果显示,不同浓度纯化后Hsp16.3蛋白作用小鼠腹腔巨噬细胞,可促进IFN-γ的产生而抑制IL-10的产生。结论成功克隆、表达和纯化了MTB Hsp16.3蛋白,Hsp16.3能促进小鼠腹腔巨噬细胞产生IFN-γ,抑制IL-10的产生。

Objective To construct a prokaryotic expression vector of Mycobacterium tuberculosis （MTB） small heat shock protein Hsp16.3, express and purify the fusion protein of His-Hsp16.3, and identify its function. Methods Hsp16.3 gene was amplified from H37Rv DNA by PCR, and then cloned into prokaryotic expression vector Pet28a. The positive recombinant plasmid Pet28a-Hsp16.3 was selected and identified by double enzyme digestion and sequencing. Then the recombinant plasmid of correct sequence was transformed into E. coil BL21 （ DE3）, and induced by IPTG. The expressed recombinant protein was analyzed by SDS-PAGE and Western blotting; the fusion protein was purified by Hislink spin protein purification system, and then detected for its concentration; real-time quantitative PCR （qRT-PCR） was used to determine the expressions of IL-10 and IFN-γ. in mouse peritoneal macrophages treated by the purified fusion protein of different concentrations. Meanwhile, the blank control group and positive control group were set up and compared. Results Double enzyme digestion and sequencing showed that the recombinant plasmid Pet28a-Hsp16.3 was successfully constructed, and SDS-PAGE and Westem blotting revealed that the fusion protein was expressed in E. coil BL21 （DE3）. Moreover, qRT-PCR showed that the purified fusion protein when added to the mouse peritoneal macrophage cells could significantly promote the produce of IFN-γ and restrain the expression of IL-10. Conclusion MTB Hsp16.3 was successfully cloned, expressed and purified. His-Hsp16.3 could significantly promote the produce of IFN-y and restrain the expression of IL-10.