人肝脏凝血因子Ⅺ的原核表达及其兔多克隆抗体的制备

Preparation of rabbit polyclonal antibodies against the human liver coagulation factor Ⅺ

目的利用原核表达系统表达人肝脏凝血因子Ⅺ(coagulation factorⅪ,F11)蛋白,并制备F11的兔多克隆抗体。方法从cDNA文库中筛选并扩增F11基因,将其分别与带有HIS标签的pET-32a及GST标签pGEX-4T-1载体连接,获得表达质粒,将表达质粒转入BL21菌中表达F11融合蛋白(分别含有HIS、GST-Tag),通过SDS-PAGE进行鉴定。获得的F11融合蛋白经纯化后作为免疫原及检测原,制备兔多克隆抗体,Western blot分析获得的兔多克隆抗体的特异性。结果成功构建pET-32a-F11、pGEX-4t-1-F11表达质粒,获得两种标签的融合蛋白。将重组蛋白纯化后作为免疫原免疫兔,取得了能与重组蛋白特异性结合的兔血清。结论成功制备了F11重组蛋白及F11兔多克隆抗体,为进一步研发F11诊断试剂打下了基础。

Objective To express recombinant coagulation factor Ⅺ（ F11） using prokaryotic expression system,and to prepare rabbit polyclonal antibodies against F11. Methods F11 gene was obtained from the cDNA library,and connected with pET-32 a and pGEX-4T-1 vector to get the expression plasmid respectively. F11 fusion protein was expressed by putting the expression plasmid into BL21,and identified by SDS-PAGE. Rabbits were immunited with the purified F11 fusion protein for the preparation of polyclonal antibodies. The specificity of rabbit polyclonal antibodies was identified by Western blot. Results Recombinant plasmids pET-32a-F11 and pGEX-4T-1-F11 were successfully constructed. And F11 rabbit serum identified corresponding recombinant proteins after the purified fusion protein as immunogen to immune rabbit. Conclusion Rabbit polyclonal antibodies against recombinant F11 protein are prepared successfully,which lays a foundation for further development of F11 diagnostic reagents.