摘要目的:构建4种重组季节性流感病毒血凝素(hemagglutinin, HA)，并在小鼠模型中评价其免疫原性。方法:将4种季节性流感病毒Wisconsin(H1N1)、Darwin(H3N2)、Austria(B/Victoria lineage，BV)和Phuket(B/Yamagata lineage，BY)株HA蛋白编码序列进行密码子优化合成，构建重组质粒，经过转化和转染获得重组杆状病毒，SDS-PAGE和Western blot鉴定表达的重组HA蛋白。重组HA蛋白经镍柱亲和层析纯化，与Al(OH) 3或AddaVax佐剂配伍后肌肉免疫BALB/c小鼠。间接ELISA和血凝抑制试验检测小鼠体液免疫应答，ELISPOT检测小鼠细胞免疫应答，微量中和抗体试验检测小鼠血清抗体效价。采用 t检验或非参数秩和检验对数据进行统计学分析。 结果:PCR扩增和琼脂糖凝胶电泳证实重组杆粒构建正确。Western blot验证了重组蛋白H1N1-HA、H3N2-HA、BV-HA和BY-HA正确表达，SDS-PAGE结果显示4种重组HA蛋白纯度均>95%。3针免疫后，与AddaVax佐剂配伍的重组H1N1-HA、H3N2-HA和BV-HA蛋白组总IgG水平高于同种亚型免疫组且差异均有统计学意义( P均<0.05)。4种重组HA蛋白混合配伍AddaVax佐剂组的IFN-γ、IL-2和IL-4分泌水平比四价流感病毒裂解疫苗组高且差异有统计学意义( P均<0.01)。微量中和抗体试验检测结果显示，四价流感病毒裂解疫苗组效价为1∶225，而4种重组HA蛋白混合与AddaVax佐剂配伍组效价可达1∶1 200。 结论:本研究成功表达了4种重组季节性流感病毒HA蛋白，与AddaVax佐剂配伍免疫小鼠有较好的免疫原性。

abstractsObjective:To construct four recombinant hemagglutinin (HA) antigens from seasonal influenza viruses and evaluate their immunogenicity in mouse models.Methods:HA coding sequences from four seasonal influenza virus strains Wisconsin (H1N1), Darwin (H3N2), Austria (B/Victoria lineage, BV) and Phuket (B/Yamagata lineage, BY) were optimized and synthesized, and then used to construct four recombinant plasmids. Recombinant baculoviruses were obtained through transformation and transfection. The expression of recombinant HA antigens was identified by SDS-PAGE and Western blot. The recombinant HA antigens were purified by nickel column affinity chromatography and intramuscularly administered to BALB/c mice after formulation with Al(OH) 3 or AddaVax adjuvant. Humoral immune responses were assessed by indirect ELISA and hemagglutination inhibition test, while cellular immune responses were evaluated by ELISPOT. Microneutralization test was used to detect the titers of serum antibodies in mice. Statistical analysis was performed using t test or non-parametric rank sum test. Results:PCR amplification and agarose gel electrophoresis confirmed the correct construction of the recombinant bacmids. Western blot showed verified the successful expression of the four recombinant antigens (H1-HA, H3N2-HA, BV-HA, and BY-HA). SDS-PAGE results showed that the purity of all four recombinant HA antigens exceeded 95%. After three-dose immunization, the total IgG levels in mice immunized with the recombinant H1N1-HA, H3N2-HA, or BV-HA formulated with AddaVax adjuvant were higher than those in the corresponding groups immunized with the same recombinant antigen alone (all P<0.05). The secretion levels of IFN-γ, IL-2, and IL-4 in the group receiving the mixture of all four recombinant HA antigens formulated with AddaVax adjuvant were higher than those in the group immunized with a commercial quadrivalent split influenza vaccine (all P<0.01). Results of the microneutralization test showed that the antibody titer in the quadrivalent split influenza vaccine group was 1∶225, whereas the titer in the group immunized with the mixture of four recombinant HA antigens formulated with AddaVax adjuvant could reach up to 1∶1 200. Conclusions:In this study, four recombinant seasonal influenza virus HA antigens are successfully expressed and demonstrated good immunogenicity in mice when formulated AddaVax adjuvant.