抗前列腺癌细胞特异抗体库的构建及特异结合抗体的筛选

Construction of the Antibody Library and Selection of the Antibodies Specifically Binding to the Prostate Carcinoma Cells

目的:获得前列腺癌的噬菌体呈现型单链抗体库,筛选与前列腺癌特异结合的抗体,为前列腺癌的诊断和治疗奠定基础。方法:用两种恶性程度较高的前列腺癌细胞PC3,DU145膜蛋白混合物免疫Balb/c小鼠。取脾脏提取总RNA,用RT-PCR分别扩增抗体重、轻链可变区基因(VH和VL),经Linker连接形成ScFv基因片段,将ScFv基因片段与噬菌粒载体pCANTAB5E的连接产物转化大肠杆菌TG1。用辅助噬菌体M13K07进行超感染,获得重组噬菌体抗体。以恶性程度低的前列腺癌细胞LNCap为对照,用PC3细胞对重组噬菌体抗体库进行五轮筛选后,随机挑取克隆,经phage-ELISA筛选特异性结合PC3细胞的ScFv。结果:用所构建的库容量为3.5×106的单链抗体库筛选特异结合抗体,得到一个与PC3细胞特异结合的噬菌体-单链抗体。结论:本研究所构建的抗体库中可筛选到与前列腺癌细胞结合特异性较好的抗体,可用于前列腺癌的诊断和治疗中。

Objective: To obtain phage-displayed ScFv library directly against prostate carcinoma cells, and select antibodies binding to prostate carcinoma cells specifically, so as to lay a foundation for developing diagnostic agents and clinical therapies of prostate carcinoma. Methods: Balb/c mice were immunized i. p . with purified membrane protein mixture of prostate carcinoma cells PC3, DU145. mRNA was isolated from the spleens of immunized mice, heavy and light chain genes ( VH and VL) of antibody were amplified separately by RT-PCR and assembled into ScFv gene with a specially constructed linker DNA. , the ScFv gene was ligated into the phagemid vector pCANTAB 5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13K07 helper phage to yield recombinant phage. After five rounds of panning with PC3 cells, the positive clones were selected with the ELISA from the enriched phages. Results: A ScFv library of 3. 5 × 106 was obtained and one phage-ScFv which can bind specifically PC3 cells was found. Conclusions: A prostate carcinoma specific antibody was identified , which paves a way for study of prostate carcinoma.