抗胃癌单链抗体免疫毒素的制备及活性检测

摘要

目的对抗胃癌单链抗体免疫毒素基因重组质粒进行表达，进而获得有活性的单链抗体免疫毒素。方法应用IPTG诱导大肠杆菌表达系统进行单链抗体免疫毒素基因重组质粒的表达，并对产生的不溶性包涵体进行纯化，经变性、复性后，观察其生物学活性。结果诱导表达了约66KD的蛋白，分子量符合预计大小，以包涵体的形式存在于菌体中。经包涵体纯化、变性、复性，使其折叠为正确的空间结构，经检测可与胃癌细胞MGC803特异结合，并有一定的细胞毒性。结论获得了可与胃癌细胞系MGC803特异结合，并有一定的细胞毒性的抗胃癌单链抗体免疫毒素。%Objective　To obtain the active anti-carcinoma ScFv immunotoxin.Methods　In E.coli the anti-carcinoma ScFv immunotoxin recombinant plasmid was induced by IPTG to express the ScFv immunotoxin in inclusion bodies,and the latter were succesively purified、denatured and renatured.By competitive inhibition ELISA and colorimetric MTT assay，the activity of anti-carcinoma ScFv immunotoxin was determined.Results　The anti-carcinoma ScFv immunotox recombinant plasmid expressed the ScFv immunotoxin in inclusion.Its molecular weight was about 66KD.The ScFv immunotoxin in inclusion was refolded to form correct structure through denaturation and renaturation showed specific and cytotoxic to gastric cancer cell line MGC803.Conclusions　The resultant anti-carcinoma ScFv immunotoxin is of strict specificity and cytotoxicity to gastric cancer cell line MGC803.