新型肝癌免疫毒素A54-PE40KDEL的构建表达与活性鉴定  

Construction,expression and functional characterization of a novel anti-hepatocarcinoma immunotoxin A54-PE40KDEL

作　　者：虞丽平[1] 韩红辉[1] 石凌超[1] 杜冰[1] 张小平[1] 周忠良[1] 钱旻[1] 

机构地区：[1]华东师范大学生命科学学院,上海200062

出　　处：《中国免疫学杂志》2009年第4期346-350,共5页Chinese Journal of Immunology

基　　金：上海市科委生物医药领域重点科技攻关专项(074319104);上海市科技发展基金重点项目(04JC14033)资助

摘　　要：目的:构建新型肝癌免疫毒素A54-PE40KDEL的原核表达载体,诱导表达后检测其生物活性。方法:PCR扩增绿脓杆菌外毒素APE40KDEL基因插入到pET32a(+),然后与合成的含有肠激酶酶切位点和肝癌特异性结合肽A54的核酸序列连接,构建重组表达载体pET32a(+)-A54-PE40KDEL,其经鉴定后转化E.coliBL21表达。超声破菌,上清液经Ni+亲和层析纯化,肠激酶酶切,再次Ni+离子亲和层析后得到免疫毒素A54-PE40KDEL。MTT和间接免疫荧光技术检测其对不同细胞株的细胞毒性及靶向性。结果:重组质粒序列正确,经Westernblot证实表达产物含Mr约58000的目的蛋白。A54-PE40KDEL能够与肝癌细胞特异性结合,且对肝癌细胞的毒性作用与对照相比有极显著性差异(P<0.01)。结论:成功构建了A54-PE40KDEL原核表达载体;获得的新型肝癌免疫毒素A54-PE40KDEL在体外与肝癌细胞具有一定结合特异性及杀伤活性,为肝癌导向治疗提供了一条可行的途径。Objective：To construct the prokaryotic expressing system of a novel anti-hepatocareinoma immunotoxin A54-PF40KDEL, and to characterize its functions. Methods： The gene of pseudomonas exotoxin A PE40KDEL was amplified by PCR and then inserted into plasmid pET32a （ ＋ ）. The synthesized sequence encoding for hepatocarcinoma-specific binding peptide A54 and the sequence for recognition site of enterokinase protease was in-frame ligated with recombinant pET32a （ ＋ ）-PE40KDEL. The recombinant plasmid pET32a （ ＋ ）-A54- PF40KDEL was confirmed and then transformed into E. coli BL21 for expression. The fusion protein was purified from soluble proteins of the transformed bacteria by Immobilized Metal-Chelated affinity chromatography and digested by enterokinase protease to release the recombinant immunotoxin A54-PF40KDEL. The specific binding ability and cytotoxicity of the recombinant immunotoxin to different cell lines were evaluated by MTT assay and indirect immunofluorescence. Results：The recombinant plasmid was confirmed by DNA sequencing. It was showed by Western blot analysis that target protein was expressed with a molecular weight of 58 kD. The purified A54-PE40KDEL displayed selective binding and cytotoxic effect to liver cancer cell compared with other control cell lines（ P 〈 0.01 ）. Conclusion：The recombinant vector was constructed successfully. After expression and purification of A54-PF40KDEL, it is proven to be specifically cytotoxic to liver cancer cells and has potential applications in target therapy of hepatocelluar carcinoma.

关 键 词：免疫毒素 肝癌 绿脓杆菌外毒素A 特异性结合肽 

分 类 号：R392.11[医药卫生—免疫学]