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目的:利用基因工程技术原核表达并纯化结核分枝杆菌α晶体蛋白(Acr)。方法:以结核分枝杆菌H37Rv株基因组DNA为模板,通过PCR方法扩增Acr的编码基因,以pCold为载体构建重组质粒,再转化到表达宿主菌大肠杆菌BL21(DE3)中,用IPTG诱导表达,经SDS-PAGE和Western印迹分析和纯化该表达产物。结果:构建了具有正确基因序列的Acr重组表达质粒,重组Acr在大肠杆菌BL21(DE3)中经低温诱导得到可溶性表达;分别用6×His的单克隆抗体和16-kDa单克隆抗体对表达产物进行Western印迹分析,结果显示在相对分子质量约19 000处均有特异性条带,与预计大小吻合;纯化后蛋白纯度达90%,浓度达0.8 mg/mL。结论:表达了重组可溶性Acr,为深入研究该蛋白的生物学、免疫学活性奠定了基础。
OBJECTIVE: To prokaryotic express and purify Mycobacterium tuberculosis alpha crystal protein (Acr) using genetic engineering technique. Methods: The genomic DNA of Mycobacterium tuberculosis H37Rv strain was used as template to amplify the gene encoding Acr by PCR. The recombinant plasmid pCold was constructed and transformed into E. coli BL21 (DE3) expressing host strain and induced by IPTG The expression product was analyzed and purified by SDS-PAGE and Western blotting. Results: The Acr recombinant plasmid with the correct gene sequence was constructed. The recombinant Acr was induced by low temperature in E. coli BL21 (DE3) to obtain the soluble expression. The recombinant protein was expressed by using 6 × His monoclonal antibody and 16-kDa monoclonal antibody Western blot analysis showed that the specific molecular weight was about 19 000, which was consistent with the expected size. The purified protein had a purity of 90% at a concentration of 0.8 mg / mL. Conclusion: The expression of recombinant soluble Acr lays the foundation for further study of the biological and immunological activity of this protein.