文档介绍：pGEX┐e23(scFv)PE40融合基因的构建及其在大肠杆菌中的表达
作者:王慧,韩苇,颜真,张英起
【关键词】单链抗体
关键词: 单链抗体,免疫毒素,基因表达
摘要:目的在大肠杆菌中表达抗原癌基因c-erbB-2表达产物p185的单链抗体e23(scFv)/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-erbB-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pLNCX中的e23(scFv)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pGEX-4T中表达. (scFv)/,经SDS-聚丙烯酰胺凝胶电泳分析,在Mr 90000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p185e23(scFv)/PE40融合蛋白.
INTRODUCTION
The c-erbB-2oncogene is located in17q21,and en-codes185ku(p185)transmembrane receptor(HER2)that belongs to the epidermal growth fac-tor receptor family and has intrinsic tyrosine kinase activity
[1-4] .HER-2gene is activated by amplifica-tion and overexpression of its protein product,which leads to its constitutive activation resulting in unregulated cell growth,then processes tumorigenic or transforming activity
[5-6] .It has been reported that HER2is overexpressed in25%~30%of breast cancers,urinary bladder cancer and gastric carcino- study of breast cancer suggests that c-erbB-2has a direct role in the pathogenesis and clini-cal aggressiveness of c-erbB-2overexpressing tu-mors
[7,8] .The patients with c-erbB-2positive have higher risks of metastasis and recurrence,shorter non-tumor life span and total life span
[9-13] .The anti-p185humanized monoclonal antibody Herceptin can effectively kill several c-