HCV胞膜蛋白E2基因的融合表达、纯化及多克隆抗体制备

The Fusion Expression and Purification of HCV Envelop Protein E2 and Preparation of Its Polyclonal Antibody

目的原核表达HCV胞膜蛋白E2,获得抗E2多克隆抗体。方法利用PCR方法从HCV基因组序列中扩增出831bp(384～661aa)的E2基因片段并按读框克隆到原核表达载体pET32a(+),IPTG诱导HCVE2蛋白表达,SDS-PAGE和WesternBlot法检测蛋白表达,Ni-NTA偶联的琼脂糖吸附柱纯化融合蛋白,薄层扫描及Bradford法检测纯化蛋白的纯度>90%;用表达的E2蛋白免疫新西兰兔制备多克隆抗体,利用间接ELISA法检测抗体效价,WesternBlot法检测抗体特异性。结果用原核诱导表达出HCV胞膜蛋白E2免疫新西兰兔后获得多克隆抗体的效价在1∶1280以上,能特异性识别E2蛋白。结论成功构建HCVE2基因的原核表达载体,利用原核表达HCV胞膜蛋白E2制备的多克隆抗体能特异性识别E2蛋白。为进一步开展HCVE2蛋白功能和HCV受体的研究奠定了基础。

Objective To express the envelop protein E2 of HCV and preparation of its polyclonal antibody. Methods The 831bp （384-661aa） fragment of HCV envelope proteins E2 gene was amplified by polymerase chain reaction （PCR） and cloned into the expression vector pET32a. Induced by IPTG, the soluble protein of interest could be obtained. The protein was purified by metal chelate affinity chromatography. The titer and specificity of polyclonal antibody were detected by indirect ELISA assay and Western blotting. Results The polyclonal antibody was induced by HCV E2 protein. The thin layer scanning showed that its purity reached at least 90%. The titer of antiserum was as high as 1： 1280, and the very high specificity was detected by Westem blotting. Conclusion Through IPTG induction and metal chelate affinity chromatography purification, the soluble HCV E2 protein with high purity and its specific antibody have been obtained. This study settles a foundation for investigating the function of HCV envelope proteins and studying the interaction of HCV envelope proteins with their receptors on the surface of cells.