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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 prokaryotic HCV E2 protein and obtain anti-E2 polyclonal antibody. Methods E2 gene fragment of 831bp (384 ~ 661aa) was amplified from HCV genome sequence by PCR and cloned into prokaryotic expression vector pET32a (+). IPTG induced the expression of HCVE2 protein. The protein was detected by SDS-PAGE and Western Blot The purified fusion protein was purified by Ni-NTA coupled agarose adsorption column. The purity of the purified protein was> 90% by TLC and Bradford method. The polyclonal antibody was prepared by immunization of New Zealand rabbits with the expressed E2 protein. The antibody was detected by indirect ELISA Titer, Western blotting was used to detect antibody specificity. Results The prokaryotic expression of HCV envelope protein E2 New Zealand rabbit immunized with polyclonal antibody titer of 1:1280 or more, can specifically recognize the E2 protein. Conclusion The prokaryotic expression vector of HCVE2 gene was constructed successfully. The polyclonal antibody prepared by prokaryotic expression of HCV envelope protein E2 can specifically recognize E2 protein. It laid the foundation for the further study of HCVE2 protein function and HCV receptor.