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通过真核表达系统表达并纯化流感病毒H1N1血凝素(Haemagglutinin,HA)茎部，将其与不同佐剂联合免疫小鼠，评价其免疫原性及攻毒保护效果。本研究以密码子优化且结构修饰的甲型流感病毒A/Brisbane/59/2007(H1N1)的HA茎部为目的基因构建真核表达质粒pcDNA3.1‐MiniHA(H1)，转染HEK‐293T细胞，通过镍离子亲和层析柱纯化Mini‐HA。将Mini‐HA分别与Al(OH)3、Al(OH)_3+CPG ODN、AddaS03?三种佐剂联合免疫小鼠，通过ELISA、ELISPOT和细胞内因子染色进行体液与细胞免疫检测，以H1N1‐PR8进行攻毒保护试验，监测小鼠存活率、体重变化、肺病毒载量以及肺组织病理变化，评估其保护效果。结果显示，Mini‐HA表达形成三聚体结构，Mini‐HA各组初次免疫后即可检测到针对HA茎部及全长HA的特异性IgG，加强免疫后抗体滴度显著增高；Mini‐HA联合Al(OH)_3+CPG ODN佐剂可以刺激Th1型细胞因子分泌，联合AddaS03?佐剂可以刺激Th1/Th2型细胞因子分泌；攻毒保护试验中，Mini‐HA联合Al(OH)_3+CPG ODN或AddaS03?佐剂组小鼠存活率达100%，体重下降低于5%，其中Mini‐HA联合AddaS03?佐剂组肺组织结构完整，仅见小部分肺泡腔代偿性扩张以及少量淋巴细胞浸润。本研究成功纯化了HA茎部蛋白Mini‐HA，联合佐剂免疫小鼠具有良好的免疫原性，以AddaS03?佐剂效果最好，可以诱导产生特异性体液与细胞免疫应答，保护小鼠抵御H1N1‐PR8流感病毒的攻击，有效抑制病毒复制、减少肺组织病理损伤。
Abstract：The influenza virus hemagglutinin stem region (Mini‐HA) was produced using a eukaryotic expression system,then co‐immunized with different adjuvants in mice to evaluate its immunogenicity and protective efficacy.Plasmid pcDNA3.1‐MiniHA (H1) was constructed using the target gene sequence of the HA stem region from A/Brisbane/59/2007 (H1N1) with codon optimization and structural modification.HEK‐293T cells were transfected with the plasmid,and Mini‐HA protein was purified from the culture supernatant using nickel affinity chromatography.Mice were co‐immunized with Mini‐HA and three different adjuvants,namely Al(OH)3,Al(OH)_3+CPG ODN,and AddaS03?.Humoral and cellular immune responses were assessed through ELISA,ELISPOT,and ICS.Furthermore,protective efficacy was evaluated through challenge experiments with A/PR/8/34 (H1N1),monitoring survival rates,body weight changes,lung virus titers,and lung histopathological alterations.The results revealed that specific IgG antibodies against both the HA stem region and the full‐length HA were detected after the initial immunization,and antibody titers significantly increased following booster immunizations.Mini‐HA combined with Al(OH)_3+CPG ODN stimulated the secretion of Th1‐type cytokines,while combined with AddaS03?induced both Th1 and Th2‐type cytokine secretion.In the challenge experiments,mice co‐immunized with Mini‐HA and either Al(OH)_3+CPG ODN or AddaS03?adjuvants provided 100%protection from lethal challenge,with less than a 5%mean weight loss.Specifically,co‐immunization with Mini‐HA and AddaS03?exhibited intact lung tissue structure,with only minor compensatory alveolar dilation and a small amount inflammatory cell infiltration.In this study,the HA stem protein Mini‐HA was successfully purified and formed a trimer structure.Mice immunized with Mini‐HA combined with adjuvant had good immunogenicity,and AddaS03?adjuvant had the best effect,which could induce specific humoral and cellular immune responses,protect mice against the attack of H1N1PR8,effectively inhibit virus replication,and reduce pathological damage to lung tissue.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.004449
中图分类号:R392
引用信息:
[1]王瑭琪,韩瑞雯,程雪婷,等.甲型流感病毒H1N1血凝素茎部亚单位疫苗与不同佐剂组合在小鼠中诱导的免疫应答与保护[J].病毒学报,2024,40(01):1-12.DOI:10.13242/j.cnki.bingduxuebao.004449.
基金信息:
国家重点研发计划（项目号：2022YFC2304100）,题目：新冠与流感等大型风险病原体多肽疫苗的研制;国家重点研发计划（项目号：2022YFC2303401）,题目：潜在高危新病毒风险识别的新技术体系研究;国家重点研发计划（项目号：2021YFA1201003）,题目：抗病毒纳米药物的体内外功能评价; 国家自然科学基金（项目号：82041041）,题目：自然感染和疫苗接种人群中的广谱保护性免疫应答规律;国家自然科学基金（项目号：82061138008）,新型冠状病毒感染与疫苗接种后的免疫保护相关机制-基于类器官与非人灵长类动物模型、新冠疫苗受试者与患者临床样本的多维度研究~~
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