H5N1亚型禽流感病毒核蛋白NP的原核表达及抗体制备

Prokaryotic expression of NP protein of influenza A virus H5N1 and preparation of polyclonal antibody

目的制备H5N1亚型禽流感病毒NP重组蛋白和抗体,为研制含NP组分的人禽流感亚单位疫苗和建立评价方法提供检测抗原和抗体。方法以H5N1亚型禽流感病毒A/chicken/Hubei/489/2004(H5N1)NP基因cDNA克隆质粒pMD-NP为模板,PCR扩增获得NP基因抗原区片段,克隆到原核表达载体pET-28a,构建重组质粒pET-ΔNP,转化大肠杆菌BL21-CondonPlus(DE3)-RIL,IPTG诱导重组蛋白表达。采用Ni亲和层析方法纯化NP重组蛋白,免疫家兔,收集免疫血清,ELISA测定抗体效价;间接免疫荧光检测所制备抗体与真核细胞表达NP蛋白的反应性。结果与结论成功的制备了高效价兔抗NP抗体,抗体效价在105以上,该抗体能与原核和真核系统中表达的NP蛋白特异性反应,为研制以NP蛋白为抗原组分的人禽流感亚单位疫苗和建立评价方法奠定了基础。

The nucleoprotein(NP)gene fragment of an avian influenza A virus H5N1(A/chicken/Hubei/489/2004)was amplified by PCR from pMD-NP plasmid template (GenBank No AY770081),and then was cloned into the prokaryotic expression vector pET-28a to generate a recombinant plasmid pET-NP. This recombinant plasmid was transformed to E.coli BL21-condon plus(DE3)-RIL,and the truncated NP fusion protein with a hexahistidine tag was expressed by the induction with IPTG. Rabbits were immunized with the purified NP protein and ...