重组免疫毒素TRAIL DR5/ScFv-PE25的构建及活性检测  

Construction and Activity Evaluation for Recombinant Immunotoxin TRAIL DR5/ScFv-PE25

作　　者：胡小波[1] 谢聪[2] 张彩平[1] 龙石银[1] 曹朝晖[1] 

机构地区：[1]南华大学药学与生物科学学院生物技术系,湖南衡阳421001 [2]云南农业大学基础与信息工程学院,昆明650201

出　　处：《微量元素与健康研究》2016年第3期1-4,共4页Studies of Trace Elements and Health

基　　金：湖南省自然科学基金项目(14JJ3104);湖南省衡阳市科技局项目(2014KS22);南华大学博士启动基金项目(2014XQD40)

摘　　要：目的:构建绿脓杆菌外毒素PE融合抗TRAIL DR5单链抗体基因TRAIL DR5/Sc Fv的原核表达载体,诱导表达,并检测其生物学功能。方法:根据TRAIL DR5单链抗体HGS-ETR2的氨基酸序列,优化密码子,合成全长的编码序列作为模板,PCR技术扩增TRAIL DR5/Sc Fv基因,基因重组技术将两段TRAIL DR5/Sc Fv与PE25基因连接,组成TRAIL DR5/Sc Fv-PE25融合基因。重组质粒转化E.coli BL21(DE3),离子交换层析和亲和层析技术纯化重组蛋白。WST-8技术检测细胞毒性。结果:成功构建了重组免疫毒素基因TRAIL DR5/Sc Fv-PE25,重组蛋白经纯化后,每升发酵液可得到4.95 mg融合蛋白(纯度95%),该融合蛋白具有抑制A431/H9和Panc 3.014癌细胞增殖的作用。结论:成功表达并纯化TRAIL DR5/Sc Fv-PE25重组免疫毒素,检测其生物学活性,为TRAIL DR5阳性癌细胞的靶向治疗研究奠定了基础。Objective：To construct recombinant immunotoxin TRAIL DR5/ScFv- PE25 and to explore its biological properties. Methods： According to the amino acids sequence of TRAIL single- chain antibody （HGS - ETR2）, its related DNA sequence as template, TRAIL DRS/ScFv gene was amplified by PCR. TRAIL DRS/ScFv fused with PE25 gene, a truncated form of pseudomonas exotoxin A （PEA）, via a flexible peptlde was constructed. The immunotoxin fusion protein （TRAIL DRS/ScFv- PE25） was expressed in E. coil strain BL21 （DE3） and purified. The cell cytotoxlclty of the immunotoxin to A431/H9 and Panc 3. 014 cell line was detected by WST - 8. Results： The recombinant immunotoxin TRAIL DR5/ScFv- PE25 was constructed successfully. The induced expression product existed in a form of inclusion body and the purity was above 95 % after purification. The recombinant immunotoxin TRAIL DRS/ScFv- PE25can inhibits A431/H9 can Panc 3.014 cell growth and prolification, Conclusion： Recombinant immunotoxin TRAIL DR5/ScFv- PE25 in this study is a candidate cancer therapeutic agent which can selectively bind and significantly inhibit the growth of the cancer cells with TRAIL DR5 expression.

关 键 词：肿瘤坏死因子相关凋亡诱导配体死亡受体5 绿脓杆菌外毒素A 免疫毒素 单链抗体 

分 类 号：Q786[生物学—分子生物学] R730.5[医药卫生—肿瘤]