利用大肠埃希菌分泌表达乙型肝炎病毒核心抗原

Secretory expression of hepatitis B virus core antigene by using Eschrichia coli

目的:研究乙型肝炎病毒核心(C)基因在大肠埃希菌(E.coli)中的表达,以期获得分泌表达的具有良好免疫反应性和特异性的HBcAg。方法:构建重组质粒pET-3c-cAg、pET-30a-cAg、pGEX-4T-1-cAg,转入E.coli BL21(DE3),IPTG诱导表达HBcAg。SDS-PAGE、ELISA和Western blot对表达产物进行分析。结果:限制性内切酶酶切和DNA序列分析证实HBV C基因正确克隆到E.coli各表达载体中。ELISA和Western blot分析表明,以pET-3c和pET-30a为表达载体能在E.coli分泌表达HBcAg,而以pGEX-4T-1为载体只能胞内表达,其中以pET-3c为载体的分泌表达量最高,培养上清中其效价为1∶128;用分泌表达的HBcAg作为诊断抗原检测抗-HBc阴阳性血清及国家参考品,符合率为100%。结论:成功获得HBcAg在E.coli的分泌表达,且其具有较高的特异性和免疫反应性。

Objective：To study the expression of hepatitis B virus core gene in Eschrichia coli（E.coli） and to obtain secretory expressed recombinant HBcAg with good immunoreactivity and specificity.Methods： Recombinant plasmids,pET-3c-cAg,pET-30a-cAg and pGEX-4T-1-cAg were constructed.They were transformed into E.coli BL21（DE3） using the calcium chloride transformation method.The E.coli transformants induced by IPTG to express the recombinant HBcAg（rHBcAg）.The expressed products were analysed by SDS-PAGE,ELISA and Western blot.Results： The analysis of restriction enzymes and DNA sequencing proved that HBV core gene had already been cloned to each E.coil expression plasmid.ELISA and Western blot analysis indicated that the secretion of HBcAg was expressed by the pET-3c and pET-30a as carrier in E.coli.pGEX-4T-1 as carrier can only be intracellular expression and the secretory expression of pET-3c as the carrier is the highest,with the titer in culture supernatant is 1∶ 128.The secreted HBcAg was utilized to detect anti-HBc in serum specimens and the anti-HBc standard panel.The coincidence rate was 100%.Conclusion： The secreted HBcAg with good immunoreactivity and specificity was successfully expressed in E.coli.