抗人卵巢癌抗体可变区cDNA的克隆与ScFv的构建和表达及放射免疫显像

目的:为研究小分子抗体作为导向载体的作用,制备抗人卵巢癌单克隆抗体COC183B2的单链抗体(single chain Fv,ScFv),并进行放射免疫显像(radioimmunoimaging,RⅡ).方法:利用PCR技术从COC183B2杂交瘤细胞中扩增COC183B2重链可变区基因(VH)和轻链可变区基因(VL),并将其克隆入高效表达载体pTHA90中.重组子转化大肠杆菌TOP10,IPTG诱导表达ScFv融合蛋白,经改进氯化亚锡法进行99mTc标记,在卵巢癌裸鼠皮下瘤模型上进行RⅡ.结果:(1)ScFv融合蛋白以包涵体形式获高效表达;(2)改进氯化亚锡法成功进行小分子抗体ScFv 99mTc的标记,标记率达98%;(3)应用99mTc标记ScFv融合蛋白进行RⅡ,肿瘤显像早,图像清晰.结论:应用单链抗体ScFv可改善RⅡ.

Variable region gene cloning, ScFv construction, expression and radioimmunoimaging of anti-human ovarian carcinoma McAb

Objective: To study the function of smaller molecular antibody as targeting vehicles, prepare single chain Fv (ScFv) fragment of monoclonal antibody (McAb) COC183B 2 for radioimmunoimaging (RII). Methods: Heavy chain variable region (V H) gene and light chain variable region (V L) gene were obtained from hybridoma cell line COC183B 2 by PCR method, and cloned into high level expression vector pTHA90 by gene recombination techniques. The recombinant was transformed into E.coli. TOP 10 and induced with IPTG. The expressed ScFv protein and normal mouse IgG (nMIgG) were labeled with 99m Tc by an improved pretining method and then injected into nude mice bearing human ovarian carcinoma by tail veins for RII. Results: (1) The ScFv protein was expressed in a high level as inclusion bodies. (2) The labeling ratio was more than 98% by an improved pretining method. (3) The images of 99m Tc ScFv were obtained at 1.5, 5 and 20 hours later which showed clear localization of the tumor. Conclusion: The expressed ScFv protein was much better than the intact McAb for RII.