抗肿瘤双特异免疫导向治疗制剂CAtin的表达及活性分析  

Expression and Activity Experiment of Anti-tumor Bispecific Immunological Directing Agent CAtin

作　　者：李霄[1,2] 金宁一[2] 李昌[2] 田明尧[2] 陈漉[2,3] 贾鹏[1,2] 刘立明[2,4] 刘妍[2,3] 高鹏[2,3] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]吉林大学军事医学科学院,全军基因工程实验室,长春130062 [3]吉林大学第一临床医院,长春130021 [4]吉林农业大学,动物科技学院,长春130001

出　　处：《高等学校化学学报》2009年第4期682-686,共5页Chemical Journal of Chinese Universities

基　　金：国家“八六三”计划(批准号:2007AA021004);吉林省科技发展计划(批准号:20060566);重大新药创制科技重大专项(批准号:2008ZXJ09003-014)资助

摘　　要：结合生物信息学方法与已知癌胚抗原(Carcinoembryonic antigen,CEA)特异性单链抗体(Single chainFv fragment,scFv)核苷酸序列,经分子设计和密码子优化后,通过化学方法合成CEA二硫键稳定性单链抗体(Disulfide stabilized single chain Fv fragments,scdsFv)基因片段.将凋亡素基因(Apoptin)通过一段柔性连接肽(Linker)连接在CEA scdsFv基因下游,并克隆入大肠杆菌表达载体质粒pET28a,转化BL21感受态菌后经异丙基-β-D-硫代半乳糖苷(Isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导,表达融合蛋白CAtin.SDS-PAGE和Western-Blot分析表明,目的蛋白得到良好表达,经条件优化后表达量最高可达44.1 mg/L.融合蛋白经分步洗涤法和谷胱甘肽对表达的目的蛋白进行初步纯化和复性后,利用人肝癌细胞(HCC)对所制备融合蛋白进行亲和力测定、细胞结合活性测定和特异性细胞杀伤活性分析.结果显示,所制备融合蛋白不仅能够有效地与上述肿瘤细胞结合,并对其具有明显的杀伤活性,表明成功制备了具有特异性识别和特异性杀伤活性的双特异抗肿瘤免疫导向制剂.Combining the bioinformatics methods and the analysis of the nucleotide sequences of anti-carcinoembryonic antigen （CEA） single chain Fv fragment （scFv）, the anti-CEA disulfide stabilized single chain fragments（scdsFv） was chemical synthesized after molecular designing and codon optimizing. Apoptin gene was linked downstream to anti-CAE sedsFv via soft linker peptide and cloned into the prokaryotic expression vector pET28a. Then the recombinant plasmid was transferred into Escherichia coli strain BL21. After induce expression by isopropyl β-D-1-thiogalactopyranoside（IPTG） , the protein expression was analyzed by SDS-PAGE and Western-Blot. The results show that target protein CAtin is well expressed and the maximal production is 44. 1 mg/L. Human hepatoma carcinoma cell （HCC） was used to detecting antibody affinity, cellular binding activity and cellular killing activity after the purification and renaturation of the CAtin by washing and glutathione methods. The results indicate that CAtin can bind and suppress the cells effectively. The work presents here suggest that CAtin is an anti-tumor bispecific immunological directing agent which has both specific recognition activity and specific killing activity.

关 键 词：癌胚抗原 二硫键稳定性单链抗体 凋亡素基因 原核表达 

分 类 号：O629.73[理学—有机化学] Q511[理学—化学]