人源性抗血小板噬菌体抗体库的构建

Construction of human immunoglobulin combinatorial library and screening of phage antibodies to platelet

目的 应用噬菌体表面呈现表达系统构建一个经血小板主动免疫的人抗体组合文库 ,并筛选与人血小板结合的噬菌体抗体 (Fab片段 )。方法 从一反复输注血小板并出现血小板输注无效的患者的外周血分离淋巴细胞 ,提取mRNA ,逆转录合成cDNA ,用半巢式PCR扩增重链Fd和κ轻链基因。依次将PCR产物插入载体pComb3相应的位点 ,构建噬菌体抗体Fab组合文库。并以人血小板膜蛋白包被 96孔板 ,对抗体库进行 3轮吸附 洗脱 扩增的亲和选择 ,获得与人血小板膜抗原结合的噬菌体抗体 (Fab片段 )库。结果 半巢式PCR有效地扩增出重链Fd和κ轻链基因 ,以此构建了噬菌体呈现文库。通过特异性筛选 ,获得可与人血小板特异结合的Fab噬菌体抗体次级库。结论 构建的人抗体组合文库通过筛选得到与人血小板特异结合的噬菌体抗体 (Fab片段 )次级库。其对抗血小板抗体药物研究、与血小板相关抗体有关的疾病研究、血小板血型抗原表位研究、进而进行血小板血型分型研究等 。

Objective Human immunoglobulin combinatorial library was generated by using phage surface display expression system, and phage antibodies (Fabs) to platelet were screened.Methods:PBMC were separated from a patient who Were transfusion refractory to platelet. mRNA was isolated and cDNA was synthesized by reverse transcriptase.The immunoglobulin heavy chain Fd and the light chain κ genes were amplified by half nested PCR and the PCR products were cloned into the expression vector pCome3 respectively.The immunoglobulin combinatorial library was constructed and screened by 3 rounds of affinity selection.Results Human Immunoglobulin Combinatorial Library was successfully constructed.The specific phage antibodies were highly enriched after 3 rounds of biopanning selection against platelet membrane proteins.Conclusion The antibody library and human monoclonal antibodies to platelet may be useful as molecular tools to study the anti platelet drugs, platelet related diseases, epitope of human platelet antigens.