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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 .
Objective To express the fusion protein of single chain antibody e2 3 (sc Fv) / Pseudomonas exotoxin active fragment PE40 immunotoxin in Escherichia coli expressing p185 of the antigen oncogene c-erb B-2, which is a fusion protein of breast cancer, gastric cancer And other c-erb B-2 overexpression in malignant tumors.Methods The nuclei encoding the signal peptide at the 5 ’end of e2 3 (sc Fv) PE40 gene cloned in the eukaryotic expression vector p L NCX were removed, The recombinant fusion protein was cloned into the prokaryotic expression vector pGEX-4T.The results of sequence analysis showed that the anti-p185 e2 3 (sc Fv) / PE40 sequence was correct and the fusion gene was induced by IPTG After 4h of expression, a neo-protein band appeared at Mr90 0 0 0 by SDS-polyacrylamide gel electrophoresis analysis, accounting for about 15% of the total bacterial protein.Conclusion The recombinant protein was successfully transformed and expressed in prokaryotes e2 3 (sc Fv) / PE40 fusion protein.