H7N9禽流感病毒HA2基因真核表达与免疫原性初步研究

Recombinant eukaryotic expression and preliminary immunogenic characterization of HA2 gene of avian influenza virus H7N9

目的利用真核表达载体构建H7N9禽流感病毒血凝素刺突茎部(HA2)的真核表达质粒,在293F细胞中表达HA2蛋白,并初步探讨其免疫原性。方法根据pMD18-T-HA中的序列设计H7N9禽流感病毒HA2扩增引物,在下游引物中引入胰酶酶切位点;目的基因经特异性酶切位点克隆入自身带有Fc标签的pFUSE-IgG1-Fc1载体,构建重组质粒HA2-Fc;将重组质粒转染293F细胞,通过间接免疫荧荧光(IFA)和免疫印迹法(WB)鉴定HA2蛋白的表达和免疫原性。结果成功构建H7N9禽流感病毒HA2基因真核表达质粒HA2-Fc,并在293F细胞中表达出分子量大约为50kDa的重组蛋白。IFA和WB显示该蛋白与抗H7N9病毒鼠多抗具有良好的免疫反应。结论成功构建表达HA2亚单位的真核表达系统,重组蛋白具有较高的免疫原性,为筛选H7N9广谱疫苗候选分子、广谱中和抗体,及深入研究其致病机理和免疫机制奠定基础。

Objective To develop a eukaryotic expression vector of stalk part of avian influenza H7N9 HA gene;lto expressand study immunogenity of HA2 recombinant protein. Methods Primers were designed to amplify HA2 gene from pMD18-T-HA vector,trypsin cleavage site was introduced in reverse primer. PCR products were subjected by double digestion and clonedinto pFUSE-IgG1-Fel with Fc tag to construct recombinant HA2-Fc expression vector,which was transfected into 293F cellsfor transient expression of HA2 fusion protein. Immunofluorescence assay and Western blot were used to test HA2 recombinantprotein expression and its immunogenity. Results The HA2-Fc recombinant expression vector was successfully constructed,which expressed a 50 kDa recombinant fusion protein in 293F cells. HA2-Fc recombinant protein showed good immune re-sponse with multiclonal mouse anti-H7N9 antibodies by IFA and Western Blot. Conclusion The eukaryotic expression rectorof HA2-Fc fusion protein was successfully constructed , the expressed recombinant protein showed high immunogenity ,whichlaid foundation for further research on development of universal subunit vaccines, screening broad neutralizing antibodies andpathogenic and immunoresponse mechanisms of H7N9.