pGEX-e23(scFv) PE40融合基因的构建及其在大肠杆菌中的表达(英文)

Construction and expression of pGEX-e23 (scFv) PE40 in Escherichia coli

目的 在大肠杆菌中表达抗原癌基因 c- erb B- 2表达产物 p185的单链抗体 e2 3(sc Fv) /假单孢菌属外毒素活性片段 PE40免疫毒素的融合蛋白 ,为乳腺癌、胃癌等多种 c- erb B-2呈过量表达的恶性肿瘤的免疫治疗奠定基础 .方法 去除克隆在真核表达载体 p L NCX中的 e2 3 (sc Fv) PE40基因 5′端编码信号肽的核苷酸序列 ,并将改建后的融合基因克隆到原核融合表达载体 p GEX- 4T中表达 .结果 序列测定表明 .改建后的抗 p185 e2 3(sc Fv) /PE40序列正确 .融合基因经IPTG诱导表达 4h后 ,经 SDS-聚丙烯酰胺凝胶电泳分析 ,在Mr90 0 0 0处出现一条新生蛋白带 ,表达量约占菌体总蛋白的15 % .结论 成功改建并在原核中表达了抗 p185 e2 3(sc Fv) /PE40融合蛋白 .

AIM To express a immunotoxin consisting of a single chain variable fragment (scFv) specific for human oncogene c- erb B-2 product, p185 and Pseudomonas exotoxin (PE40), named e23 (scFv) PE40, in Escherichia coli which could be used as an adjuvant therapy agent in breast cancer, gastric cancer and other malignant tumor overexpressing c- erb B-2. METHODS e23 (scFv) PE40 fusion gene for prokaryotic expression was constructed and inserted into a prokaryotic fusion protein expression vector pGEX-4T and expressed in Escherichia coli DH5α. RESULTS The DNA sequence of e23 (scfv) PE40 fusion gene was correct. A new anticipated M r 90 000-protein band appeared on SDS-PAGE gel induced by IPTG and amounted to 15% of the total bacterial protein. CONCLUSION Fusion gene of e23 (scFv) PE40 has been successfully constructed and expressed in Escherichia coli DH5α.