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目的从已成功建立的前列腺癌噬菌体抗体库中筛选抗人前列腺癌单链抗体(ScFv),并分析其生物学特性。方法以良性前列腺增生(BPH)细胞与噬菌体抗体库共孵育,去除抗体库中能够与BPH细胞结合的噬菌体抗体,再以前列腺癌细胞系DU145与去除非特异性结合后的噬菌体抗体库共孵育,通过“吸附-洗脱-扩增”对前列腺癌的噬菌体抗体库进行3轮的富集。采用流式细胞术筛选出对人前列腺癌细胞系DU145高亲合力的阳性克隆。阳性噬菌体克隆经亚克隆并转染大肠杆菌TG1,表达出可溶性ScFv抗体。采用流式细胞术、免疫荧光法分析其生物学特性。结果以前列腺癌细胞系DU145为抗原,筛选出19个噬菌体抗体,经测定C11与DU145细胞亲合力最高,亚克隆至原核表达载体表达纯化为C11 ScFv抗体。流式细胞术和免疫荧光法检测结果均显示,C11ScFv抗体与前列腺癌细胞DU145和PC3结合,与BPH细胞不结合。结论通过噬菌体展示技术筛选到前列腺癌细胞系高亲合力的可溶性ScFv抗体,为进一步研究抗体的靶向性治疗奠定了基础。
Objective To screen anti-human prostate cancer single chain antibody (ScFv) from the established prostate cancer phage antibody library and analyze its biological characteristics. Methods BPH cells were incubated with the phage antibody library to remove the phage antibody that binds to BPH cells in the antibody library and then incubated with the prostate cancer cell line DU145 and the phage antibody library with nonspecific binding “Adsorption - Elution - Amplification ” Prostate cancer phage antibody library for three rounds of enrichment. Flow cytometry was used to screen out positive clones positive for human prostate cancer cell line DU145. Positive phage clones were subcloned and transfected into E. coli TG1 to express soluble ScFv antibodies. Using flow cytometry, immunofluorescence analysis of its biological characteristics. RESULTS: Nineteen phage antibodies were screened for the prostate cancer cell line DU145. The highest affinity of C11 was found between DU145 cells and C11, and subcloned into prokaryotic expression vector to express C11 ScFv antibody. Flow cytometry and immunofluorescence assay showed that C11ScFv antibody binds to prostate cancer cells DU145 and PC3 and not to BPH cells. Conclusions The high avidity soluble ScFv antibody of prostate cancer cell line was screened by phage display technique, which laid a foundation of further research on the targeted therapy of antibody.