表达量高、血凝活性好的H3N2流感病毒血凝素(HA)蛋白疫苗的筛选研究

Screening of Hemagglutinin(HA)Protein Vaccine with High Expression and Good Hemagglutination Activity for H3N2 Influenza Virus

目的:筛选表达量高、血凝活性好的H3N2流感病毒血凝素(HA)重组蛋白。方法:以A/MINNESOTA/41/2019(H3N2)毒株的HA为基础,将HA胞外区N端融合GP67信号肽,C端融合三聚化基序,分别构建HA胞外区全长及近膜区截短2个氨基酸的HA重组杆状病毒,并构建相应的不含三聚化基序的HA重组杆状病毒;经昆虫细胞表达,Western blot鉴定,亲和层析纯化后分析重组蛋白的血凝效价及稳定性。结果:四种HA重组蛋白均获得有效表达,其中HA胞外区全长融合三聚化基序的重组蛋白(HA-T)表达水平最高,经Strep tag亲和层析获得高度纯化,产量高达30 mg/L,Ethylene glycolbis交联分析显示为稳定的HA三聚体形式,血凝活性分析显示HA-T的活性最高,血凝效价为29,动态光散射显示HA-T在4℃放置3个月性质稳定。结论:HA-T表达量高、稳定性好、血凝活性高且易于纯化,研究结果为流感重组蛋白疫苗的研发策略提供了参考。

Objective:To screen the recombinant hemagglutinin(HA)protein with high expression and good hemagglutination activity for H3N2 influenza virus.Methods:Based on the HA ectodomain gene of A/MINNESOTA/41/2019(H3N2)strain,GP67 signal sequences and trimerization motifs were respectively fused to the N-and C-terminals of the full-length HA ectodomain gene and the near transmembrane domain two residues truncated gene.The recombinant genes were expressed in baculovirus system.At the same time,the corresponding recombinant HA proteins without containing trimerization motifs were generated.The expressions of these recombinant HA proteins were identified by Western blot.The recombinant proteins were purified by affinity chromatography.Then,the hemagglutination titer and stability of the recombinant proteins were analyzed.Results:Four kinds of HA recombinant proteins were effectively expressed,among which the expression level of HA ectodomain full-length fusion trimerization motif(HA-T)was the highest,and the yield of it was up to 30 mg per liter suspension medium purified by Strep tag affinity chromatography.The cross-linking analysis showed that HA-T was in the form of stable trimer of HA.In addition,the hemagglutination activity analysis showed that HA-T had the highest activity,and its hemagglutination titer can reach 29.Dynamic light scattering showed that it was stable at 4℃for 3 months.Conclusions:HA-T has the advantages of high expression,good stability,high hemagglutination activity and easy purification.The research provides a useful reference for the development strategy of HA recombinant protein vaccines.