重组α2-HS糖蛋白的原核表达、纯化及免疫学活性分析

目的:原核表达并纯化具有生物学活性的重组α2-HS糖蛋白(AHSG)。方法:提取人肝癌细胞株HepG2细胞总RNA,逆转录得cDNA,PCR扩增出目的基因,与pMD18-T载体连接后,转化至大肠杆菌DH5α中,大量扩增后提取克隆质粒,连接至原核表达载体pEP-30a(+)中,转化BL21(DE3),经IPTG诱导蛋白表达,通过镍离子亲和层析的方法纯化重组蛋白,采用SDS-PAGE电泳、免疫印迹杂交法对纯化产物进行分析鉴定。结果:成功构建了AHSG原核表达系统BL21(pET30a-AHSG),最佳诱导条件为80μmol/LIPTG诱导3h;诱导表达的蛋白相对分子质量约54600,主要以不溶性的包涵体形式存在,纯化后的重组蛋白质量浓度最高达2.3g/L,经免疫印迹杂交鉴定有抗原性。结论:成功构建了AHSG原核表达系统,该系统可高效表达重组AHSG蛋白,该蛋白具有良好的抗原性。

Prokaryotic expression,purification and immunological analysis of recombinant alpha2-Heremans-Schmid glycoprotein

Aim:To express and purify recombinant alpha2-Heremans-Schmid glycoprotein(AHSG) with biological activity.Methods:The total RNA from HepG2 cells was extracted and cDNA was synthesized.AHSG gene was amplified by RT-PCR method,then cloned on pMD18-T vector.The constructed clone plasmid was transformed into E.coli DH5α,then the fragments of AHSG acquired from that were ligated with pET-30a(+) vector.The recombinant AHSG protein was expressed after the constructed expression plamid was tranformed into E.coli BL21(DE3) and purified by Nickel ion affinity chromatography.The purified products were identified and analyzed by SDS-PAGE and Western blot,and the concentration of purified protein was measured.Results:The prokaryotic expression system BL21(pET30a-AHSG) was constructed successfully.The most appropriate condition for the expression of target protein was 80 μmol/L IPTG for 3 h.A fusion protein approximately 54 600 was yielded,mainly existing in the form of insoluble inclusion bodies.The highest concentration of recombinant protein purified by Nickel ion affinity chromatography was up to 2.3 g/L.The recombinant protein had antigenicity identified by Western blot.Conclusion:The prokaryotic expression system of AHSG and protein purification system have been successfully constructed and the purified AHSG recombinant protein is highly antigenic and immunogenic.