【摘要】 本文构建特异性抗肝癌 (HCC )噬菌体单链抗体 (ScFv )库并观察其靶向性。应用噬菌体表面呈现技术 ,从经HCC细胞免疫的小鼠脾淋巴细胞构建特异性抗HCC噬菌体ScFv库 ,应用HCC细胞和正常肝细胞筛选。制备游离ScFv,以正常肝细胞作为对照 ,应用ELISA、点杂交和免疫组织化学方法以及流式细胞仪 (FCM )检测ScFv与HCC细胞的特异性结合能力。结果显示抗体库库容为 2 14× 10 6 ;核酸序列分析显示ScFv基因结构正确 ;游离ScFv相对分子质量为 3 2 0 0 0 ;ScFv抗HCC效价为 1∶16;点杂交和免疫组织化学技术检测显示HCC细胞被染成棕黄色 ,阴性对照、空白对照未着色 ;FCM检测发现ScFv具有与HCC细胞特异性结合的能力。特异性抗HCC噬菌体ScFv具有与HCC结合的靶向特异性。更多还原

【Abstract】 Attempt was made to prepare the phage display of the single chain fragment (ScFv) library against hepatocellular carcinoma (HCC) for the observation of its tropism in vitro By means of gene engineering and phage display techniques to construct ScFv library,HB2151 cells were transfected with ScFv gene against HCC by phages to express free ScFv The affinity of soluble antibodies against HCC cells was detected by immunohistochemical methods and flow cytometery,ELISA and dot blot technique with normal liver cells as control It was found that the content of phage display ScFv library was about 2 14×10 6,and the ScFv gene structure discovered by nucleotide sequence analysis was correct The relative molecular mass of free ScFv was about 32 000,and the ScFv anti HCC titre was 1∶16,the HCC antigens were stained to be brown yellow color as shown by dot blot and immunohistochemical studies By flow cytometry study,the ScFv was found to be conjugated with HCC specifically It concludes from the above observations that the phage display ScFv against HCC had specific affinity with HCC antigens.更多还原