抗大肠癌噬菌体人单链抗体的初步鉴定及序列分析

目的：对抗大肠癌细胞的单克隆噬菌体单链抗体进行初步鉴定和测序分析。方法：采用细胞ELISA，免疫组化，DNA序列测定和计算机分析方法，对5个单克隆噬菌体抗体（CH273，CH205，CH209，CHA12，CH723）进行初步鉴定和序列分析。结果：5个抗体均对人大肠癌细胞、人胚肾上皮细胞和其它某些人肿瘤细胞反应，也与人正常肝细胞有弱阳性反应，但不与鼠源性的癌细胞和正常细胞反应。细胞免疫组化进一步证实了ELISA结果的正确性。大肠癌免疫组化对大肠癌组织有特异性的结合反应，而不与正常大肠组织反应。测序结果为CH273 ScFv全长732bp；V，D，J分别属于VH3－30－D1－26－JH3－linker-V1-13-JL2,GenBank序号为AY028777和AY028996；CH205全长366bp,V,D,J分别VH1－46－D6－13－JH3，GenBank序号为AF359365；CH209，CHA12和CH723的ScFv基因完全相同，全长723bp，其VH－DH－JH与CH273 ScFv基因中的VH－DH－JH完全一致，V，D，J分别属于VH3－30－D1－26－JH3－linker-L2-Jκ2，GenBank序号为AF363774。结论：噬菌体抗体具有结合人大肠癌组织和细胞的活性，为进一步开发临床应用人源抗肿瘤抗体和小分子抗体片段奠定基础。

Primary characterization and sequence analysis of anti-colorectal cancer phage fusion antibodies]

OBJECTIVE: To identify 5 phage fusion antibodies against colorectal cancer from in vitro immunized phage library and analyze their sequences. METHODS: Cell ELISA, immunohistochemistry, DNA sequencing and computer analysis were employed. RESULTS: Five clones of phage antibodies were tested by cell ELISA, and all of them reacted to human colorectal cancer cell lines, human embryo kidney endothelial cell line and some tumor cell lines, but not to mouse-original cell lines. They also reacted weakly to human hepatic cell lines. The binding specificity of the phage antibodies for colorectal cancer cells was confirmed by immunohistochemistry with cultured cells and colorectal carcinoma and colon tissue sections. They reacted to colorectal carcinoma cell lines, human embryo kidney endothelial cell lines and nasopharyngeal carcinoma cell lines. CH273 reacted specifically to colorectal cancer cells in human colorectal carcinoma sections but not to any of the cells in human colon sections. The 5 clones were further analyzed after their DNA sequencing. The sequences of CH723, CH209 and CHA12 were identical. The lengths of CH273, CH205 and CH723 were 732 bp, 366 bp and 723 bp, respectively. The VDJ regions of CH273, CH205 and CH723 belonged to VH3-30-D1-26-JH3-linker-V1-13-JL2, VH1-46-D6-13-JH3 and VH3-30-D1-26-JH3-linker-L2-J kappa 2, respectively. CONCLUSIONS: Phage antibodies binding to colorectal tissues and cells are confirmed, on which human anti-tumor ScFv and VH fragments may be further developed and applied to clinical therapy.