ENO1多克隆抗体的制备与初步鉴定

Preparation and primary identification of polyclonal antibody against fusion expressed Enolase1

目的制备抗Enolase 1的兔多克隆抗体(pAb)并进行特异性鉴定。方法以人宫颈癌细胞系HeLa细胞cDNA为模板,构建重组表达质粒pET-32a-ENO1。His-ENO1融合蛋白在大肠杆菌中高效表达,纯化后作为免疫原制备兔多克隆抗体。采用ELISA、Western blot和免疫组化法鉴定抗ENO1兔多克隆抗体针对重组蛋白和天然蛋白的特异性。结果 His-ENO1融合蛋白在相对分子质量约29 000处呈现明显表达条带。Western blot鉴定表明,制备的抗ENO1兔多克隆抗体可特异性地识别ENO1重组蛋白和不同肿瘤细胞系HeLa、MCF7、HepG2和K562中的ENO1蛋白。免疫组化结果表明该多抗可特异性识别肾组织中的ENO1蛋白。结论成功制备出兔抗人ENO1抗血清,为进一步研究ENO1的功能奠定了基础。

In this study, we aimed to prepare rabbit polyclonal antibody （pAb） against enolasel （ENO1） and characterize the antibody for functional studies. ENO1 fragment was amplified from human cervical cancer cell line HeLa, and then used to construct recombinant expression vector pET-32a-ENO1. His-tagged ENO1 fusion protein was expressed in E. coli and purified for immunization. Rabbit pAb against ENO1 was obtained and identified by ELISA, Western blot, and immunohistochemistry. Data indicated that the His-ENO1 fusion protein was highly expressed in E. coli with molecular weight of 29 000 Da; Western blot analysis showed the pAb could specifically recognize ENO1 in different human tumor cell lines of HeLa, MCF7, HepG2 and K562. In immunohistochemistry assay, the pAb could specifically react to ENO1 in kidney tissue. In this study, polyclonal antibodies against human ENO 1 are successfully prepared, which will provide a useful tool for further study of ENO 1.