基础医学与临床 ›› 2007, Vol. 27 ›› Issue (12): 1381-1384.

• 研究论文 • 上一篇    下一篇

艰难梭菌细胞毒素B功能区的原核表达及免疫原性

刘红升 张清华 蒋知新 姜泊   

  1. 南方医科大学 北京解放军305医院 北京解放军305医院 南方医科大学南方医院消化病研究所
  • 收稿日期:2006-12-07 修回日期:2007-04-06 出版日期:2007-12-25 发布日期:2007-12-25
  • 通讯作者: 刘红升

High expression and Immunogenicity of clostridium difficile

Hong-sheng LIU, Qing-hua ZHANG, Zhi-xin JIANG, Bo JIANG   

  1. Southern Medical University
  • Received:2006-12-07 Revised:2007-04-06 Online:2007-12-25 Published:2007-12-25
  • Contact: Hong-sheng LIU,

摘要: 目的 纯化已表达艰难梭菌(CD)细胞毒素B羧基末端受体结合区(CD3)的基因,并检测其免疫原性。方法PCR克隆目的基因到表达载体pET22b(+),重组质粒转化到E.coliBL21(DE3),经IPTG诱导表达,利用金属螯合色谱层析方法纯化后,SDS-PAGE方法对纯化蛋白进行分析。并检测其免疫反应性。结果成功表达的是分子质量约为71 300的重组蛋白,占菌体总蛋白的34%,可溶性表达占上清的22.7%,包涵体占沉淀的25.7%。重组蛋白纯化后可溶性蛋白浓度为0.781 g/L。并与抗毒素B抗体具有良好的免疫原性。结论: 成功克隆了CD3基因,构建的重组质粒可高效表达CD3重组蛋白, 为CD相关性疾病的诊断及后期制备疫苗,提供了有力的保障。

Abstract: Objective To perform expression and to detect immunogenicity of Clostridium difficile Toxin B receptor binding zone(CD3). Methods The clostridium difficile toxin B C terminal repeated gene was amplified by PCR and cloned into the prokaryotic expression vector pET 22b(+), and there combined plasmid pET 22b(+)CD3 was transformed into E.colistrainBL21(DE3).There combined vector was confirmed by digestion with EcoRI/XhoI and sequencing. The E.colistrainBL21(DE3) containing pET22b(+)CD3 was induced with IPTG and analyzed with SDS PAGE. At last metal chromatography method for purification of the recombinant protein, this can be analyzed using the SDS-PAGE method. Results A 71.3 ku protein was acquired after inducing with IPTG and thin layer scanning suggested that CDTBR occupied 34% of the total bacterial protein, 22.7% of the supernatant and 24.9% of the inclusion body. The purifying the recombinant protein is 0.781g/ml by Coomassie brilliant blue G-250 chromatometry method。Conclusions The high expression and purification of clostridium difficile toxin B receptor gene lay a foundation for the further study on CD3 function and clostridium difficile vaccine.