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目的:在大肠杆菌中表达并纯化出具有良好生物学活性的卡介苗HSP70(BCG HSP70)蛋白。方法:利用PCR技术扩增出BCG HSP70的编码基因,将其克隆到pMD18-T载体,测序分析。再将该目的基因亚克隆至pET28a表达载体,转化入大肠杆菌BL21(DE3),获得正确的阳性克隆子后,用IPTG诱导表达。纯化表达产物,SDS-PAGE及Western blot鉴定目的蛋白,并通过脾增生实验检测其生物学活性。结果:扩增的BCG HSP70基因经序列测定与GenBank公布的序列完全一致。表达产物经SDS-PAGE分析,在相对分子质量(Mr)约70000处有表达条带。Western blot结果证实纯化产物在Mr约70000处可见特异性条带。蛋白纯度约96.5%。脾细胞增生试验显示,该蛋白能够明显刺激鼠脾细胞增殖。结论:成功表达并纯化了具有良好生物学活性的BCG HSP70,为进一步研究BCG HSP70及BCG的功能奠定了基础。
OBJECTIVE: To express and purify the BCG HSP70 protein with good biological activity in Escherichia coli. Methods: The coding gene of BCG HSP70 was amplified by PCR, cloned into pMD18-T vector and sequenced. The target gene was then subcloned into pET28a expression vector and transformed into E. coli BL21 (DE3). After correct positive clones were obtained, expression was induced by IPTG. The expressed product was purified and the target protein was identified by SDS-PAGE and Western blot. The biological activity of the protein was detected by spleen proliferation assay. RESULTS: The amplified BCG HSP70 gene was sequenced exactly as published in GenBank. The expressed product was analyzed by SDS-PAGE and expressed at a molecular weight of about 70,000 (Mr). Western blot results confirmed that the purified product in Mr about 70000 visible bands. The protein purity is about 96.5%. Spleen cell proliferation test showed that the protein can significantly stimulate the proliferation of murine splenocytes. CONCLUSION: BCG HSP70 with good biological activity was successfully expressed and purified, which laid the foundation for further study on the function of BCG HSP70 and BCG.