艰难梭菌毒素A的可溶性表达及抗原性分析

Soluble expression and antigenicity analysis of toxin A of Clostridioides difficile

目的 实现艰难梭菌(Clostridioides difficile,C.difficile)毒素A(toxin A, TcdA)的可溶性表达并鉴定其抗原特异性。方法 使用生物信息学软件对TcdA受体结合域进行B细胞表位分析,选择优势抗原表位区段进行基因合成。将合成的基因连接到表达载体pET-28a和pET-32a中,构建重组质粒pET-28a/TcdA和pET-32a/TcdA,转化到BL21(DE3)感受态细胞中进行表达,并对诱导温度进行优化。采用镍柱纯化融合蛋白,然后用肠激酶酶切获得目的蛋白。最后使用艰难梭菌检测试剂盒检测蛋白的抗原性。结果 选择TcdA受体结合区域优势抗原表位区段2 231~2 708 aa进行表达。成功表达pET-28a/TcdA和pET-32a/Trx-TcdA重组蛋白。其中pET-28a/TcdA为包涵体形式。pET-32a/Trx-TcdA实现了部分可溶性表达,并且优化诱导温度(18℃)后,可溶性表达量进一步提高。表达的pET-32a/Trx-TcdA融合蛋白纯度较好,产量约为4.5 mg/L,经酶切后获得几乎没有冗余氨基酸的TcdA。经检测,TcdA蛋白能被相应的抗体特异性识别。结论 实现了TcdA蛋白可溶性表达且有很好的抗原性,可用于后续抗体制备,为建立艰难梭菌感染免疫学检测方法奠定了基础。

Objective To obtain toxin A(TcdA) of Clostridioides difficile(C. difficile) expressed in solubility in prokaryotic system and to detect its antigenic specificity. Methods The B-cell epitope of the receptor binding region of toxin A was analyzed by IEDB online software, and the dominant antigen epitope fragment was selected for gene synthesis. The target gene was cloned into vector pET-28 a and pET-32 a to construct the recombinant plasmids pET-28 a/TcdA and pET-32 a/TcdA, and then transformed into BL21(DE3) for expression. The induction temperature was optimized. After purified by Ni-NTA affinity chromatography, the fusion protein was digested with enterokinase to obtain the target protein. The antigenicity of the TcdA was detected by C. diff Qick Chek Complete dual-antigen EIA. Results The dominant epitope region 2 231~2 708 aa of TcdA receptor-binding domain was selected for expression. The fusion proteins pET-28 a/TcdA and pET-32 a/Trx-TcdA were successfully expressed. The pET-28 a/TcdA protein was expressed as inclusion bodies. The pET-28 a/TcdA protein was partly expressed in the soluble fraction, and the soluble expression was further increased after optimizing the induction temperature(18 ℃). After purification, the expression of pET-32 a/Trx-TcdA protein was about 4.5 mg/L. The purified fusion protein was digested to obtain TcdA with almost no redundant amino acids. The result of the kit showed that TcdA protein could be specific identified by anti-toxin A antibody. Conclusion The soluble expression of TcdA protein, and its good antigenicity can be used for subsequent antibody preparation, which provided a foundation for the establishment of immunological methods for C. difficile infection.