抗人卵巢癌单域抗体V_H 基因的克隆、表达及放射免疫显像

 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COC1 83B2 单域抗体(重链可变区 ,VH) ,并进行放射免疫显像 (RII) .利用PCR技术从COC1 83B2 杂交瘤细胞中扩增COC1 83B2 的VH 基因片段 ,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VH 融合蛋白 ,经改进氯化亚锡法进行锝 ( 99mTc)的标记 ,在卵巢癌裸鼠皮下瘤模型上进行RII.结果表明 :( 1 )VH 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VH 融合蛋白99mTc的标记 ,标记率达 96%以上 ;( 3)用99mTc标记VH 融合蛋白进行RII,肿瘤显像早 ,图像清晰 .说明99mTc可用于小分子抗体标记 ,应用单域抗体VH 可改善RII

Cloning,Expression and Radioimmunoimaging of Single Domain(V_H)Gene of Anti-human Ovarian Carcinoma McAb

To study the function of small molecule antibody as targeting vehicles,the heavy chain variable region(V H) fragment of monoclonal antibody (McAb) COC183B 2 was prepared for radioimmunoimaging(RII).V H gene was amplified from hybridoma cell line COC183B\-2 by PCR,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 V H 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 vein for RII.The results showed that (1) the V H protein was expressed in a high level as inclusion bodies;(2)The labeling ratio was more than 96% by an improved pretining method;(3)The images of 99m Tc\|V H protein were obtained at 1.5,5 and 20 hours later,which showed clear localization of the tumor. 99m Tc can be used to label small molecule antibodies.The expressed V H protein was much better than the intact McAb for RII.