H7N9亚型禽流感病毒血凝素蛋白的真核表达及抗原性的鉴定

Eukaryotic expression of H7N9 subtype avian influenza virus hemagglutinin and identification of its antigenicity

目的:通过哺乳动物细胞表达出禽源H7N9流感表面HA蛋白,并鉴定其抗原性。方法:通过引物设计,利用融合PCR方法扩增得到H7N9禽流感HA基因,以p CAGGS为表达载体构建重组质粒p CAGGS-H7-thrombin。测序正确后将重组质粒转染293T细胞。经72 h后收获上清,通过镍柱和凝胶层析柱纯化后得到目的蛋白。Western blot和ELISA结果鉴定纯化的H7蛋白的抗原性。结果:293T细胞表达的上清经HisTrapTMHP纯化后过Superdex 200 10/300 GL分子筛,在洗脱体积达到11 m L左右出峰,由出峰位置判断其为H7三聚体。Western blot检测在60 ku左右有一条阳性条带,ELISA检测其对抗H7N9小鼠阳性血清有很高的反应值。试验成功表达禽流感病毒H7N9的H7蛋白,并且针对H7N9的阳性血清有较高的灵敏性,为进一步研究H7N9亚单位疫苗奠定了基础。

This study aims to express avian influenza virus （AIV） H7N9 surface glycoprotein H7 in mammalian cells and identify its anti- genicity. Firstly, we amplified HA gene fragment of AIV H7N9, and cloned into pCAGGS expression vector, thus obtaining plasmid pCAGGS-H7-thrombin. Then, the recombinant plasmid was transformed into 293T cells after sequencing. The supernatant was harvested af- ter 72 hours and the target protein was purified using nickel column and gellayer column. At last, the antigenieity of H7 was identified by Western blot and ELISA. The results showed that after the supernatant was purified through HistrapTM HP and Superdex 200 GL, H7 protein was successfully obtained. The molecular weight was demonstrated to be about 60ku by Western blot. ELISA result showed H7 had high re- sponse values with the positive sera from mice infected with H7N9. This study provides a foundation for further research on H7N9 subunit vaccine.