B/Yamagata系流感病毒血凝素(HA)蛋白疫苗的筛选研究

Screening of Hemagglutinin(HA)Protein Vaccine for Influenza Viruses B/Yamagata Lineage

目的:筛选表达量高且具有血凝抑制活性的B/Yamagata系流感病毒血凝素(HA)重组蛋白。方法:以B/Singapore/INFTT-16-0610/2016的HA为基础,在HA胞外区C末端分别融合GCN4pLL三聚化基序、GCN4pLL-半胱氨酸及GCN4pLL-七鳃鳗可变淋巴细胞受体-B(VLR-B)抗体C端的肽段序列,分别获得HA突变体基因BY-T、BY-LLc及BY-PLc;分别插入pFastBac1,采用杆状病毒昆虫细胞表达系统进行表达。表达鉴定后,Strep-Tactin亲和层析纯化,然后鉴定纯化突变体蛋白的寡聚程度及血凝活性。突变体蛋白免疫小鼠,检测免疫血清HA特异性Ig G抗体和血凝抑制(HAI)抗体水平。结果:各HA突变体均能够有效表达,BY-T突变体表达水平最高,易于纯化,纯度好,呈多聚化形式存在;BY-LLc及BY-PLc表达水平次之,呈高聚化形式存在。三种HA突变体蛋白的血凝活性相当,均可有效诱发的HA特异性结合的Ig G抗体和HAI抗体。结论:HA蛋白胞外区C端融合寡聚化基序GCN4pLL的突变体易于纯化,具有血凝活性,可诱发小鼠产生HA特异性抗体和HAI抗体。研究为B/Yamagata系流感病毒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 level and hemagglutinin inhibitory activity for B/Yamagata influenza virus.Methods:Based on the HA of B/Singapore/INFTT-16-0610/2016,the peptide sequences of GCN4pLL trimerization motif,GCN4pLL-cysteine,and GCN4pLL-the sequences from C-terminus of lamprey variable lymphocyte receptor(VLR)-B antibodies were fused to C-terminus of HA ectodomain,to obtain HA mutant genes BY-T,BY-LLc,and BY-PLc.They were inserted into pFastBac1 separately and expressed using baculovirus expression vector system.After expression identification,Strep-Tactin affinity chromatography purification was performed to purify the mutant proteins followed by identification of the degree of oligomerization and hemagglutination activity of the purified mutant proteins.Anti-HA IgG titer and hemagglutination inhibition(HAI)antibody titer in sera of mice immunized with HA mutants were detected.Results:All HA mutants were effectively expressed.The BY-T mutant had the highest expression level,was easy to purify,had good purity,and existed in a polymeric form.The expression levels of BY-LLc and BY-PLc are secondary and exist in a highly polymerization form.The hemagglutination activities of the three HA mutant proteins were comparable,and all of them could effectively induce anti-HA IgG and HAI antibodies.Conclusion:The HA protein ectodomain mutant fused with oligomeric motif GCN4pLL at the C-terminus is easy to purify and has hemagglutination activity,which can induce anti-HA IgG and HAI antibodies in mice.The results provide a reference for the development strategy of recombinant protein vaccines against influenza virus B/Yamagata lineage.