Wang Jing, Wang Hui, Wang Chao, Yuan Yuan, Cui Yan, Ye Jing, Li Qing
Department of Pathology and Pathophysiology, Fourth Military Medical University, Xi'an, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2012 Aug;28(8):837-40.
To isolate and identify single chain Fv (scFv) antibodies against breast cancer from a constructed human phage display library.
Recombinant phages specific for breast cancer cells were enriched after four-round screening with MCF-7 cells. We selected the antigen-positive ones from the enriched clones by phage ELISA. The positive clones were used to infect E.coli HB2151 to express soluble scFv antibody. The antigen binding activity of the soluble antibodies was detected by Western blotting.
The specific antibodies against MCF-7 cells were enriched after four rounds of affinity selection. SDS-PAGE and Western blotting showed a band at relative molecular mass 30 000 Da, which indicated soluble antibodies were present. ELISA analysis revealed that soluble antibodies had the affinity to a human breast cancer cell line MCF-7 but not to other cancer cell line, which demonstrated scFv could react specifically with breast cancer cells.
We constructed a scFv phage library against human breast cancer with high capacity by phage display technology. The scfv was demonstrated successfully to be expressed in E.coli HB215 and have a specificity to breast cancer. Our findings may provide an alternative approach, and a basis for further studies on diagnosis and therapy of breast cancer.
从构建的人噬菌体展示文库中分离并鉴定抗乳腺癌的单链Fv（scFv）抗体。
用MCF-7细胞进行四轮筛选后，富集对乳腺癌细胞具有特异性的重组噬菌体。通过噬菌体ELISA从富集的克隆中筛选出抗原阳性克隆。将阳性克隆用于感染大肠杆菌HB2151以表达可溶性scFv抗体。通过蛋白质印迹法检测可溶性抗体的抗原结合活性。
经过四轮亲和选择后，富集到了针对MCF-7细胞的特异性抗体。SDS-PAGE和蛋白质印迹显示在相对分子质量30 000 Da处有一条带，表明存在可溶性抗体。ELISA分析显示可溶性抗体对人乳腺癌细胞系MCF-7有亲和力，而对其他癌细胞系无亲和力，这表明scFv能与乳腺癌细胞特异性反应。
我们通过噬菌体展示技术构建了一个高容量的抗人乳腺癌scFv噬菌体文库。已成功证明scfv能在大肠杆菌HB215中表达，并对乳腺癌具有特异性。我们的研究结果可能为乳腺癌的诊断和治疗提供一种替代方法及进一步研究的基础。
