艰难梭菌谷氨酸脱氢酶的表达及抗原性分析

摘要

Objective To construct prokaryotic expression vectors for glutamate dehydrogenase(GDH)of Clostridium difficile(CD),and express recombinant GDH in Escherichia coli,and identify its antigenicityed.Methods The entire gene of GDH was cloned from ATCC43255 genome DNA.The recombinant antigens were expressed in E.coli with IPTG induction and purified by Ni-NATBeads.The antigenicity was detected using CD Qick Chek Complete dual-antigen EIA. Results Prokaryotic expression vectors of CD GDH were constructed successfully.The antigen could be identified by specific anti-GDH antibodies.Conclusion The GDH antigen can be used to prepare corresponding antibodies,which facilitate the development of immunoassay for CD GDH.%目的：构建艰难梭菌（CD）谷氨酸脱氢酶（GDH）的原核表达载体，表达重组蛋白并鉴定其抗原活性。方法以 CD 的 ATCC43255菌株基因组 DNA 为模板，通过 PCR 法扩增 GDH 全长基因片段，构建原核表达载体并转化到大肠杆菌中，IPTG 诱导表达 GDH 蛋白，并用 Ni 柱进行纯化。利用 CD 抗原检测试剂盒对 GDH 抗原区段的抗原性进行鉴定。结果构建的原核表达载体 pET-28a／GDH 可在大肠杆菌中成功表达，获得的 GDH 抗原能与相应抗体产生特异性反应。结论原核表达的 CD 的 GDH 抗原具有很好的抗原活性，为进一步制备相应抗体并建立CD 的 GDH 抗原的免疫检测方法奠定了基础。