艰难梭菌二元毒素A的原核表达及免疫原性分析

目的构建艰难梭菌二元毒素(Cdt)A的原核表达载体,表达并纯化His-CdtA重组蛋白,分析其免疫原性。方法以RT 027型艰难梭菌为模板,采用聚合酶链反应(PCR)扩增cdtA的全长序列,导入大肠埃希菌BL21感受态细胞,诱导His-CdtA重组蛋白表达,并用纯化后的蛋白免疫小鼠,分析小鼠产生的抗体效价。结果成功构建pET-28b-cdtA原核表达载体,诱导并纯化出高浓度的His-CdtA重组蛋白,免疫小鼠后产生高效价的抗CdtA抗体。结论纯化的His-CdtA重组蛋白免疫小鼠产生了高效价的抗CdtA抗体,为后续制备抗CdtA单克隆抗体及建立CdtA的实验室检测方法学奠定了基础。

Prokaryotic expression and immunogenicity analysis of Clostridium difficile binary toxin A

Objective To construct a prokaryotic expression vector of Clostridium difficile binary toxin(Cdt) A,to express and purify His-CdtA recombinant protein,and to analyze its immunogenicity. Methods The fulllength sequence of cdtA was amplified by polymerase chain reaction(PCR) using RT027 Clostridium difficile as template,which was introduced into Escherichia coli BL21 competent cells. The His-CdtA recombinant protein was induced,and the mice were immunized with the purified protein for analyzing the antibodies produced by the mice. Results The prokaryotic expression vector pET-28b-cdtA was successfully constructed,and a high concentration of His-CdtA recombinant protein was induced and purified. After immunizing mice,high-titer antibody of CdtA recombinant protein was produced. Conclusions The mice immunized with the purified His-CdtA recombinant protein produce high-titer anti-CdtA antibodies,which lay a foundation for the subsequent preparation of monoclonal antibodies against CdtA and the methodology of CdtA laboratory detection.