摘要
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 express recombinant Mycobacterium tuberculosis Ag85a-ESAT6 fusion protein in prokaryotic cells,purify the expressed product and determine its biological activity.Methods Ag85a-ESAT6 fusion gene fragment was inserted into vector pET-28a（＋）,and the constructed recombinant plasmid pET-28a-Ag85a-ESAT6 was transformed to E.coli BL21（DE3）for expression under induction of IPTG.The denaturalized expressed product was purified by Ni-agarose gel chromatography and renaturalized,then used for in vitro stimulation of spleno-lymphocytes of mice immunized with five kinds of recombinant vaccinia viruses expressing Ag85a and ESAT6 of M.tuberculosis.The proliferation of splenic lymphocytes was determined by MTT method.Results Both restriction analysis and sequencing proved that recombinant plasmid pET-28a-Ag85a-ESAT6 was constructed correctly.The expressed recombinant fusion protein,with a relative molecular mass of about 41 000,mainly existed in a form of inclusion body and contained 55.65% of total somatic protein.After purification and renaturation,the protein reached a purity of more than 95% and an expression level of about 1.2 g/L fermentation liquid,and showed good reactogenicity.As compared with those immunized with empty vaccinia virus or physiological saline,the proliferation activities of spleno-lymphocytes of mice immunized with the five kinds of recombinant vaccinia viruses increased significantly after co-culture with Ag85a-ESAT6 fusion protein（P 〈0.01）.Conclusion Ag85a-ESAT6 fusion protein was successfully expressed by using prokaryotic system and showed biological activity after purification and renaturation.
出处
《中国生物制品学杂志》
CAS
CSCD
2010年第11期1153-1157,共5页
Chinese Journal of Biologicals
基金
"艾滋病和病毒性肝炎等重大传染病防治"科技重大专项基金(2008ZX10003-013)
