pGEX┐e23scFvPE40融合基因的构建及其在大肠杆菌中的表达.doc

pGEXe23（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. 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 domainandof 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 VL5sequence 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 We intended to express e23（scFv）PE40in 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. REFERENCES: 1Tang ZQ，Zhang YS，Corbley M J，Tong TJ.Cell aging of hu-man diploid fibroblasts is associated with changes in responsive-ness to epidermal growth factor and changes in HER-2expres-sion J.Mech Aging Dev，1994;73:57-67. 2Philip GK，Sun US.Therapy of an animal model of human gas-tric cancer using a combination of anti-erbB-2monoclonal anti-bodies J.Cancer Res，1992;52:2771-2776. 3Michael FP，Carlos CC.Expression of the HER2/neu proto-oncogene in normal human adult and fetal tissues J.Onco-gene，1990;5:953-962. 4Chen SY，Yang AG.Potent antitumour activity of a new class of tumor-specific killer cells J.Nature，1997;385:78-80.5Jiang K，Tong TJ.Research on the Relationship between erbB-2and the Malignant Phenotype as well as Proliferation Regulation of Gastric Cancer Cells J.Zhongguo Shengwu Huaxue Yu Fenzi Shengwu Xuebao（Chin J Biochem Mol Biol），1998;14:457-462. 6Clay BS.Target Toxin as Anticancer Agents J.Cancer Sup-pl，1994;74:1006-1011. 7Jiang K，Tong TJ.Progress in the research of c-erbB-2proto-oncogene J.Natl Med J Chin，1996;76:158-160. 8Wang WG，Tong TJ.The expression of c-erbB-2on apoptotic process J.Zhongguo Shengwu Huaxue Yu Fenzi Shengwu Xuebao（Chin J Biochem Mol Biol），1998;14:290-293. 9Zhao ZG，Zhang LL.Amplification of the oncogene c-erbB-2in transitional cell carcinoma of bladder J.Di-si Junyi Daxue Xuebao（J Fourth Mil Med Univ），2000;21（4）:482. 10Bi F，Zhang XY，Fan DM，Hui HX，Wanh CJ.Computer de-sign of anti-c-erbB-2ribozymes J.Di-si Junyi Daxue Xuebao（J Fourth Mil Med Univ），1997;18（1）:6-8. 11Hao XB，Zhang YH，Qiu FH，Zhang LC，Han XZ，Yin Y，Tao QY，Hao XK.Influence of c-erbB-2gene on growth properties of mouse fibroblast J.Di-si Junyi Daxue Xuebao（J Fourth Mil Med Univ），1997;18（1）:23-25. 12Bi F，Zhang XY，Hui HX.Construction and identification of the eucaryotic expression vector PDOR-AE of c-erbB-2antisense RNA J.Di-si Junyi Daxue Xuebao（J Fourth Mil Med Univ），1997;18（6）:593-595. 13Jin Y，Yang LJ，Tipoe GL.The quantitative observation of overexpression of Neu/c-erbB-2in benign and malignant epithe-lia of cheek mucosa J.Di-si Junyi Daxue Xuebao（J Fourth Mil Med Univ），1996;17（6）:462-266. 14Joan A，Jose B.Trastuzumba，a humanized anti-HER2mono-clonal antibody，for the treatment of breast cancer J.Drug Today，1999;35（12）:931-946. Biography:WANG Hui（female，born in1974in W