从scFv噬菌体抗体库中筛选抗GPⅡb／Ⅲa单链抗体

Production of monoclonal anti-GP lI b/HI a scFv antibodies from scFv phage libraries

目的从scFv噬菌体库中获取人源化特异性Anti—GPⅡb／Ⅲa单克隆scFv抗体。方法对Tomlinson I＋JscFv文库进行3次淘洗，富集特异性的抗GPⅡb／Ⅲa抗体。通过酶联免疫吸附试验（ELISA）和双脱氧终止法基因测序及同源性对比，检测出人源化的抗GPⅡb／Ⅲa单克隆抗体。结果在3次淘洗后，得到了抗GPⅡb／Ⅲa单克隆噬菌体抗体，阳性克隆的获取率在95．6％以上；ELISA和基因测序筛选出25种不同的全长抗GPⅡb／Ⅲa噬菌体抗体，这些基因序列与人免疫球蛋白可变区基因同源性达到89％以上；分泌性抗体ELISA检测提示这些抗体顺利表达了蛋白并特异性结合GPⅡb／Ⅲa，其中15种scFv对GPIIb／ma有更强的阳性反应。结论人源化的特异性抗-GPⅡb／Ⅲa scFv能通过噬菌体展示技术快速有效地获得。

Objective To screen monoclonal anti-GPⅡb/Ⅲa antibodies from scFv phage libraries and obtain specific monoclonal anti-GPⅡb/Ⅲa scFv. Methods Specific anti-GPⅡb/Ⅲa scFv antibodies were enriched by three rounds of selection from Tomlinson I ＋ J libraries. By using polyclonal and mono- clonal phage enzyme linked immunosorbent assay （ELISA） and gene sequencing by bideoxy chain termina- tion, full-length specific monoclonal anti-GPⅡb/Ⅲa antibodies were picked out and their gene sequences were obtained. Results Twenty-five different full-length monoclonal scFv phage fragments were obtained after three rounds of panning and their gene sequences were identified, and positive rate of monoclones was above 95.6% ; homology comparison with variable regions of human immunoglobulin gene showed the simi- larity was above 89% ; the result of soluble scFv ELISA showed that these specific scFv could be expressed smoothly, and 15 full-length monoclonal scFv antibodies were stronger positive than the other in these scFv. Conclusion Antibody phage display was a rapid and effective method to obtain Anti-GPⅡb/Ⅲa scFv fragements.