摘要
目的利用阳性重组菌XL1-Blue-Pcomb3 表达出人源性大肠癌Fab段噬菌体原始库,固相化人大肠癌组织及细胞的相关抗原后对其进行筛选,鉴定筛选后的抗体库与人大肠癌有无特异性的结合活性。方法PCR鉴定XL1-Blue- Pcomb3重链Fd段和资链的插入率,表达出人大肠癌Fab段原始库。然后提取3例用于构建原始抗体库的致敏大肠癌组织抗原,13例非致敏大肠癌组织抗原及3种体外培养的大肠癌细胞株LoVo、HT-29、LS-174T的抗原,利用3组混合抗原分别对原始抗体库进行3轮筛选,将筛选后的3个三级库等体积混和后通过ELISA及免疫组化鉴定其与人大肠癌组织及细胞是否具有特异性的结合活性,取胃癌、食管癌及正常肠粘膜组织和胃癌、肝癌细胞进行对照研究。结果Fd片段及资链的插入率分别为40%和70%,Fd片段及资链均插入质粒Pcomb3的重组率为28%,Fab段基因库库容为2.1×106,3组大肠癌混合抗原分别对原始库进行3轮筛选后,抗体库均得到了不同程度的富集,ELISA及免疫组化显示富集后的抗体库对人大肠癌组织及体外培养的大肠癌细胞均有较特异性的结合活性。结论利用噬菌体抗体库技术筛选到了人大肠癌相关Fab段抗体群,且筛选后的抗体群与人大肠癌抗原有较特异性的结合活性。
Objective To express the original human Fab antibody phage display library with positive recombined bacterium XL1-blue-Pcomb3 and identify its specific binding activity with colorectal cancer cells in vitro after screening with human colorectal cancer-related antigens. Method The recombination rate of Fd fragment of the heavy chain and insertion of κchain of the antibodies was determined with PCR, and the original Fab library was expressed. The antigens were extracted from 3 sensitized colorectal cancer tissues previously used for construction of the original Fab library and from 13 non-sensitized colorectal cancer tissues, along with the antigens from LoVo, HT-29 and LS-174T cells cultured in vitro. The original Fab antibody library was screened with the 3 groups of mixed antigens derived in preceding procedure and 3 tertiary Fab antibody libraries were obtained, which were then mixed in equal volume for subsequent tests of binding activity with human colorectal cancer tissues and cells in vitro using enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining. Specimens of gastric and esophageal carcinomas and normal intestinal mucosa, together with liver cancer cells and gastric cancer cells were utilized as control. Result The recombination rate of Fd and κchain were 40% and 70% respectively, and the rate of their simultaneous insertion into Pcomb3 vector was 28%. The capacity of library for Fab fragment genes was 2.1×10 6 , and the original antibody libraries screened with the 3 groups of mixed antigens were enriched to varied degrees, which all displayed relatively specific binding activity with human colorectal cancer tissue and cells in vitro. Conclusion Colorectal cancer-related antibody Fab fragments are obtained through screening phage display library, which show relatively specific binding activity with human colorectal cancer tissues and cells.
出处
《第一军医大学学报》
CSCD
北大核心
2002年第8期678-683,共6页
Journal of First Military Medical University
基金
广东省自然科学基金(010643)
