抗癌抗生素力达霉素与抗IV型胶原酶单链抗体的基因工程组装融合蛋白  

AN ENGINEERED AND ASSEMBLED FUSION PROTEIN OF ANTITUMOR ANTIBIOTIC LIDAMYCIN AND scFv ANTIBODY DIRECTED AGAINST TYPE IV COLLAGENASE

作　　者：李顺强[1] 江敏[1] 甄永苏[1] 

机构地区：[1]中国医学科学院,中国协和医科大学医药生物技术研究所,北京100050

出　　处：《药学学报》2000年第7期488-491,共4页Acta Pharmaceutica Sinica

基　　金：国家重点基础研究规划资助项目!(G1998051104)

摘　　要：目的　以IV型胶原酶为靶点 ,研制兼具有抗肿瘤侵袭转移和强烈杀伤肿瘤细胞的新型单抗导向药物。方法　力达霉素 (LDM)分子由力达蛋白 (LDP)和力达发色团 (LDC)两部分组成。从基因工程转化菌中提取融合蛋白 (LDP Fv)并与LDC组装重建 ,制备高效抗癌抗生素力达霉素与单链抗体的组装融合蛋白 (LDM Fv)。通过ELISA、酶谱分析、Boyden小室体外侵袭实验及克隆形成测定 ,观察其生物学活性。结果　LDM Fv能特异性与肿瘤细胞的IV型胶原酶结合并抑制其活性 ,LDP Fv有阻断肿瘤细胞侵袭的能力 ,但无细胞毒性 ,加入LDC制成的组装融合蛋白LDM Fv对肿瘤细胞有强烈的杀伤作用 ,并明显强于丝裂霉素和阿霉素。结论　LDM Fv可能成为一种新型高效。AIM To develop a novel, highly potent antitumor immunoconjugate of smaller molecular size by linking lidamycin (also called C1027), an antibiotic with extremely potent cytotoxicity to cancer cells, to an scFv antibody directed against type IV collagenase. METHODS Lidamycin (LDM) molecule consists of an enediyne chromophore (LDC) and a protein (LDP) of 10 5 ku. The two moieties of the molecule can be separated and an active molecule can be reconstituted. The novel assembled fusion protein, an antitumor immunoconjugate, was manufactured through genetic engineering and assembled molecule reconstitution. Firstly, a recombinant fusion protein LDP Fv was obtained from genetically engineered E.coli JM109 pKFL. Secondly, the assembled fusion protein LDM Fv was prepared by adding LDC to LDP Fv. RESULTS By 12% SDS PAGE the E. coli expressed fusion protein LDP Fv showed a distinct band of approximately 37 ku accounting for about 8% of the total protein. LDP Fv, which was non cytotoxic, retained the immunoreactivity of the original monoclonal antibody directed against type IV collagenase. LDV Fv significantly reduced the activity of type IV collagenase in highly metastatic human lung carcinoma PG cells and inhibited cell invasion in dose dependent manner. After molecule reconstitution, the assembled fusion protein LDM Fv showed extremely potent cytotoxicity to PG cells with an IC 50 value of 9 5×10 -15 mol·L -1 by clonogenic assay. CONCLUSION The engineered and assembled fusion protein LDM Fv is a highly effective antitumor immunoconjugate with much smaller molecular size than those ever reported.

关 键 词：Ⅳ型胶原酶 抗肿瘤抗生素 力达霉素 LDM-FV 

分 类 号：R979.4[医药卫生—药品]