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应用噬菌体抗体库技术制备全人源抗滋养层细胞表面抗原-2(Trop-2)特异性Fab抗体片段.抗体库经细胞筛选和固相抗原筛选,获得特异性的阳性克隆.阳性载体经核酸序列分析后,构建工程菌,经IPTG诱导表达,SDS-PAGE和Western blot分析,呈现28ku和32ku大小的两条蛋白质条带.Fab分子经流式细胞术、细胞免疫荧光检测,结果表明,Fab能够与BxPc3细胞膜蛋白特异性结合,而与NIH3T3细胞不结合.免疫共沉淀与质谱分析结果表明,该Fab分子能够与Trop-2蛋白特异性结合.免疫组化显示,该抗体可结合胰腺癌细胞膜蛋白,在细胞培养液中加入Fab,能够抑制BxPc3细胞的生长.以上研究结果提示,该抗体有望成为胰腺癌临床影像诊断或治疗的候选分子.
The phage antibody library technology was used to prepare a full-length Trop-2 specific Fab antibody fragment.Antibody library was screened by cell selection and solid phase antigen to obtain specific positive clones Positive carriers were detected by nucleic acid After sequence analysis, the engineered bacteria were constructed and induced by IPTG, SDS-PAGE and Western blot analysis showed two protein bands of 28ku and 32ku.The Fab molecules were detected by flow cytometry and immunofluorescence.The results showed that Fab Which could specifically bind to BxPc3 membrane protein but not to NIH3T3 cells.The results of co-immunoprecipitation and mass spectrometry showed that this Fab molecule could specifically bind to Trop-2 protein.Immunohistochemistry showed that this antibody could bind to membrane of pancreatic cancer cells Protein, adding Fab into cell culture medium can inhibit the growth of BxPc3 cells.The above results suggest that the antibody is expected to become a candidate for clinical imaging diagnosis or treatment of pancreatic cancer.