Kininogen D5和TRAIL融合蛋白的克隆表达及生物学活性的研究  

Cloning,Fusion Expression and Biological Characterization of Kininogen D5 and TRAIL

作　　者：宗英[1] 孙铭娟[1] 董晓毅[1] 高云[1] 王梁华[1] 焦炳华[1] 

机构地区：[1]第二军医大学基础部生物化学和分子生物学教研室,上海200433

出　　处：《药物生物技术》2008年第1期1-5,共5页Pharmaceutical Biotechnology

基　　金：上海市现代生物与新药产业发展基金(024319115)

摘　　要：用重组PCR技术得到Kininogen D5和TRAIL的融合编码序列,将该DNA片段克隆到原核表达载体pMAL-c2,重组质粒转化大肠杆菌BL21,IPTG诱导蛋白表达,得到MBP-KT和MBP-TK,经AmyloseResin亲和层析柱层析,得到初步纯化的融合蛋白。结果表明MBP-KT对胰腺癌细胞1990有明显的杀伤作用其作用的ED50为20 ng/ml,而MBP-TK对1990的抑制作用不明显;同时,MBP-KT和MBP-KD5对内皮细胞ECV304有明显的抑制作用,MBP/KT作用于ECV304的ED50为0.1μg/ml,MBP/KD5作用于ECV304的ED50为9.5μg/ml,但是MBP-TK和TRAIL几乎无作用,表明融合蛋白KT既具抗肿瘤作用又有抗新生血管的作用。本研究为进一步开发靶向性杀伤肿瘤药物奠定了基础。The fused gene of Kininogen D560148 and TNF-related apoptosis-inducing ligand（TRAIL114-281） were amplified by PCR, and was inserted into pMAL-c2 vector. The recombinant plasmid pMAL-KT and pMAL-TK were transformed into E. coli BL21. After IPTG induction, the recombinant MBP-KT and MBP-TK proteins in partially purified forms were obtained respectively. The recombinant products were tested for their cytotoxic or cytostatic activities against SW1990 and ECV304 cells. The results showed that MBP-KT plays a significant cytotoxicity against SW-1990 cells, but MBP-TK had no evident effect. Meanwhile, it exhibited also a growth inhibitory effect on proliferation of ECV304 cells. The results suggested that fusion protein MBP-KT had an antiproliferative effect on cancer cells as well as an inhibition effect on angiogenesis. This research provided the foundations of the target therapy in cancer treatment.

关 键 词：激肽原 肿瘤坏死因子相关凋亡诱导配体 增殖抑制 血管生成抑制因子 

分 类 号：R966[医药卫生—药理学]