降钙素原单克隆抗体的制备及鉴定应用

The preparation of Procalcitonin monoclonal antibodiy and appraisal application

目的表达和纯化降钙素原(PCT)基因的融合蛋白,制备PCT融合蛋白的鼠源性单克隆抗体。方法根据GenBank数据库中降钙素原氨基酸序列设计引物,从TT细胞中扩增出降钙素原的全长片段,将其克隆至pEASYE1原核表达载体,构建重组表达质粒pEASY-E1-PCT;利用大肠杆菌BL21(DE3)感受态细胞转化阳性克隆质粒中,以IPTG作为诱导剂诱导表达融合蛋白,通过His镍离子亲和层析纯化目的蛋白,纯化的目的蛋白免疫BALB/c小鼠,获取单克隆抗体并纯化,ELISA法检测抗体效价及交叉鉴定,制备的单抗与市售的降钙素原检测试剂盒进行对比。结果成功构建了pEASY-E1-PCT原核表达载体,获得2株可以稳定分泌特的单抗,经鉴定这两株抗体效价高、特异性好,与市售检测试剂盒检测结果一致。结论成功构建了原核表达载体pEASY-E1-PCT,获得了高纯度降钙素原PCT蛋白,制备并获得了2株特异的单克隆抗体,制备单抗与商品试剂盒对血清样本检测结果基本一致,为进一步研究降钙素原的生物学功能及试剂盒开发提供实验基础。

Objective To express and purify Procalcitonin gene fusion protein,and to produce the monoclonal antibody of procalcitonin;Methods Design primers according to procalcitonin original amino acid sequences in GenBank database,the recombinant expression plasmid of pEASY-E1-PCT,we reconstructed using TT cell cDNA as template.The fusion protein of pEASY-E1-PCT was expressed in BL21.Express the fusion protein induced by IPTG as inducers,Nickel column purification of proteins and used as immunogen,obtain monoclonal antibodies and purification.The specificity of antiserum against mouse PCT was characterized by ELISA,PCT mAb and commodities of procalcitonindetection kits were compared.Results The recombinant expression plasmids of pEASY-E1-PCT,we reconstructed and the fusion protein of pEASY-E1-PCT was expressed and purifeid,two strains of PCT mAb were screened,ELISA results demonstrated two mAbs had high titer and specificity,and the results,The mAb test result is consistent with the Commodity kits.Conclusion The prokaryotic expression vector was constructed successfully pEASY-E1-PCT,obtained the high purity calcitonin original PCT proteins,received two strains specific mAb,the mAb and commodity kit for detecting serum samples results are basically identical,which can provide the experimental basis for the further study of procalcitonin biological functions and development kit.