结核分枝杆菌H37Rv基因glcB的克隆、表达及酶活性的实验研究

Cloning,expression and characterization of gene glcB from Mycobacterium tuberculosis H37Rv

目的应用大肠杆菌BL21(DE3)pLysS表达有活性的结核分枝杆菌H37Rv分泌蛋白GlcB(80.4kDa),为早期结核病的诊断以及新的抗结核药物的研制提供理论依据。方法应用聚合酶链反应(PCR)扩增结核分枝杆菌的基因glcB(Rv1837c),并与克隆载体PCR4BLUNT-TOPO连接,DNA测序后,经酶切将glcB与表达载体pET23b连接,并转化至大肠杆菌BL21(DE3)pLysS中表达目的蛋白GlcB。IPTG诱导表达,破菌,离心,采用Ni2+亲和层析法纯化上清液中的目的蛋白,经聚丙烯酰胺凝胶电泳(SDS-PAGE)结合考马斯亮蓝染色检测重组蛋白的表达及纯度;同时应用抗6个组氨酸的单克隆抗体(monoclonal antibody,mAb)进行固定化蛋白质免疫学测定(Western blot),并进行了酶活性的分析。结果PCR产物序列正确,GlcB可在大肠杆菌BL21(DE3)pLysS中高效表达,可溶性好。经SDS-PAGE结合考马斯亮蓝染色及Western blot检测证实,表达的重组蛋白纯度高,与预期的GlcB大小一致,约80kDa。活性分析提示该重组蛋白具有苹果酸合成酶的功能,且酶活性为5.5μmol/(min·mg),与相关的文献报道一致。结论结核分枝杆菌H37Rv分泌蛋白GlcB能够在大肠杆菌中高效表达,且具有酶活性,有望为新一代抗结核药物的研制及结核早期诊断提供依据。

This study is aimed to get purified and active recombinant protein GlcB of Mycobacterium tuberculosis H37Rv by expression in Escherichia coli （E. coli）, and provide the basis for development of the early diagnosis of tuberculosis and the new anti-tuberculosis drugs. The gene encoding GlcB was amplified by PCR and the PCR product was ligated with the cloning vector PCR4BLUNT-TOPO. After DNA sequencing, the gene was ligated with pET23b. The expressing plasmid, pET23b glcB, was transformed into E. coli BL21 （DE3） pLysS to express the recombinant GlcB. The expression of protein was induced by IPTG. While the recombinant protein GlcB was purified by NTA-Ni- agarose purification system, and the enzymatic activity was detected. The results showed that the PCR product was correct and most of the expressed recombinant protein was soluble. Purified protein showed one clear band about 80 kDa on SDS-PAGE followed by Commassie blue staining, which was con- sistent with the predicted H37Rv GlcB, and was confirmed by Western blot analysis. The enzymatic activity was 5.5μmol/（min · mg）, consistent with those reported. All these results suggest that the soluble secreted recombinant protein GlcB can be ef- fectively expressed in E. coli and possess a high enzymatic activity. Therefore, it provided the basis for the study on the new anti-tuberculosis drugs and the early diagnosis for tuberculosis.