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. 结果 序列测定表明.改建后的抗p185e23（scFv）/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-ma.The 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-erbB-2positive cell lines and dramatically inhibit the growth of xenograft overexpressing c-erbB-2in nude mouse.As a re-sult，Herceptin had been approved by FDA of U S A to be used in the first line chemotherapy as an ad-junctive agent in c-erbB-2positive metastasis breast cancers in September，1998
［14］ .In our study，we re-constructed a single-chain chimeric immunotoxin termed e23（scFv）PE40.In e23（scFv）PE40，the variable domain of the light chain of mAb e23was attached through a peptide linker to the variable do-main of the heavy chain，which in turn was fused to domainⅡandⅢof PE.Then we expressed this immunotoxin successfully in a prokaryotic fusion protein expression vector pGEX-4T.
MATERIALS AND METHODS
Materials pLNCX-e23（scFv）PE40that consisted of e23，an anti-p185single chain variable fragment（scFv）and PE40was presented by Professor Yang An-Gang.Our laboratory preserved pBluescprit cloning vector and pGEX-4T fusion protein expres-
sion vector.Wizard TM Plus Miniprep DNA Purifica-tion System was bought from Promega Company.NucleoTrap Gel Purification Kit was bought from Clontech Company.Restriction endonuclease and enzymes used in DNA cloning were obtained from Takara.Anti-PE40serum was the product of Sig-ma.Other agents were qualified for laboratory use.Methods Construction of fusion gene To delete the signal peptide coding sequence of pLNCX-e23（scFv）PE40，4fragments of the VL5′sequence of e23（scFv）were designed and synthesized by Shanghai Sangon Biotechnology limited Company and an EcoRI recognition site was inserted into the begin-ning of the adapter.The sequences were shown be-low: EcoRI initiation code
DISSUSSION
prokaryote，which can be used in breast，gastric and other c-erbB-2positive cancers as a adjunctive a-gent.The expression of GST-fusion e23（scFv）PE40present a feasible way to get e23（scFv）PE40.
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Biography:WANG Hui（female，born in1974in Wuzhong City，Ningxia Hui Autonomous Region，China）
graduated from Fourth Military Medical University in1996and now a master degree candi-date in Biotechnology Center，Fourth Military Medical University，directed by Prof ZHANG Ying-Qi and Assoc-prof Correspondence to YAN Zhen:Tel.+86 29 3374773