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目的原核表达、纯化重组结核杆菌Ag85a-ESAT6融合蛋白,并检测其生物活性。方法将Ag85a-ESAT6融合基因片段插入pET-28a(+)载体中,构建重组质粒pET-28a-Ag85a-ESAT6,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物在变性条件下经Ni-琼脂糖凝胶层析柱纯化复性后,在体外对经5种不同类型的表达结核杆菌Ag85a和ESAT6的重组痘苗病毒免疫的小鼠脾淋巴细胞进行刺激,以MTT法检测脾淋巴细胞的增殖反应。结果重组质粒pET-28a-Ag85a-ESAT6经双酶切及测序鉴定证明构建正确;表达的重组融合蛋白相对分子质量约41000,主要以包涵体形式表达,表达量占菌体总蛋白的55.65%;纯化复性的重组融合蛋白纯度达95%以上,表达量约为1.2g/L发酵液,且具有良好的反应原性;与空痘苗病毒或生理盐水免疫的小鼠相比,5种重组痘苗病毒免疫的小鼠脾淋巴细胞与Ag85a-ESAT6融合蛋白共培养后,增殖活性明显升高(P<0.01)。结论成功利用原核系统表达了结核杆菌Ag85a-ESAT6融合蛋白,纯化复性的Ag85a-ESAT6融合蛋白具有生物学活性。
Objective To prokaryotic express and purify recombinant Mycobacterium tuberculosis Ag85a-ESAT6 fusion protein and test its biological activity. Methods Ag85a-ESAT6 fusion gene fragment was inserted into pET-28a (+) vector to construct recombinant plasmid pET-28a-Ag85a-ESAT6. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG. The expressed product was renatured on a Ni-Sepharose column under denaturing conditions and then stimulated in vitro on mouse spleen lymphocytes immunized with five different types of recombinant vaccinia virus expressing Mycobacterium tuberculosis Ag85a and ESAT6, Detection of proliferation of splenic lymphocytes by MTT assay. Results The recombinant plasmid pET-28a-Ag85a-ESAT6 was confirmed by double enzyme digestion and sequencing. The recombinant fusion protein was constructed with the relative molecular mass of 41000 and mainly expressed in inclusion bodies, accounting for 55.65% of the total bacterial proteins. Purified renaturation of recombinant fusion protein purity of more than 95%, the expression amount of about 1.2g / L fermentation broth, and has good reactionogenicity; compared with empty vaccinia virus or saline immunized mice, five kinds of recombinant vaccinia After co-cultured with Ag85a-ESAT6 fusion protein, the splenic lymphocytes of virus-immunized mice were significantly increased (P <0.01). Conclusion The mycobacterium tuberculosis Ag85a-ESAT6 fusion protein was successfully expressed in prokaryotic system, and the purified and refolded Ag85a-ESAT6 fusion protein was biologically active.