人源性抗乳腺癌单链抗体库的筛选与初步鉴定

目的:本研究旨在已构建的大容量人源性抗乳腺癌噬菌体单链抗体库的基础上,筛选出高亲和力的特异性单链抗体(scFv)并对抗体基本特性进行初步鉴定。方法:以人乳腺癌细胞系MCF-7为靶标,经过4轮淘洗,筛选出高亲和力的特异性抗乳腺癌scFv,并对其结构序列进行分析;通过ELISA和Western blot方法,鉴定scFv的亲和力和特异性,以及其蛋白的基本表达情况。结果:成功构建具有高亲和力的抗乳腺癌单链抗体库,获得scFv的长度约为750 bp,ELISA证实所得抗体对乳腺癌细胞具有良好的亲和力和高度的特异性,IPTG诱导表达及Western blot结果显示,scFv为相对分子质量(Mr)30 000的可溶性蛋白。结论:本研究在已构建的大容量抗乳腺癌单链抗体库的基础上,筛选获得了高亲和力的抗乳腺癌单链抗体库。研究结果为进一步获得可应用于临床诊断和治疗的乳腺癌靶向性抗体奠定了良好的基础。

Isolation and identification of single chain Fv antibodies against breast cancer from a human phage display library

AIM: To isolate and identify single chain Fv(scFv) antibodies against breast cancer from a constructed human phage display library.METHODS: 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.RESULTS: 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.CONCLUSION: 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.