刘京;龚铮;年悬悬;邓涛;周蓉;张国梅;李雪丹;张哲罡;张家友;杨晓明;

• 1:武汉生物制品研究所病毒性疫苗研究二室
• 2:国家联合疫苗工程技术研究中心
• 3:中国生物技术股份有限公司

摘要(Abstract)：

利用真核表达系统稳定表达HA重组蛋白并评价其免疫原性及攻毒保护效果，为重组流感亚单位疫苗的研制提供更多的理论基础。本研究以H1N1型流感病毒HA胞外区域序列为目的基因构建重组质粒KS001-HA并转染至CHO细胞得到表达重组蛋白rHA的单克隆细胞株，通过离子交换层析法纯化rHA。将rHA与佐剂联合免疫小鼠以评估免疫原性，并对免疫后的小鼠进行攻毒，监测小鼠存活率和体重变化，检测其肺组织病毒载量，并分析肺组织的病理变化。重组质粒KS001-HA在CHO细胞中稳定整合并分泌表达rHA蛋白，其相对分子质量约70 kD，经PNGaseF酶处理后，蛋白大小变为60kD。纯化后rHA蛋白纯度>95%。加强免疫后小鼠血清抗体效价显著增强，佐剂配伍免疫效果优于rHA单独免疫，其中HA203佐剂配伍组15、30、60μg剂量组免疫效应显著高于阳性对照组H1N1疫苗原液组。攻毒后HA203佐剂配伍组无小鼠死亡，小鼠体重平均下降率低于10%，肺组织肺泡结构清晰，仅见肺泡壁轻度增厚，伴随少量的炎性细胞浸润，仅检测到少量呈灶性分布的棕黄色颗粒。本研究成功构建了重组质粒KS001-HA，于CHO细胞中表达，并筛选出稳定表达目的蛋白的单克隆细胞株。rHA蛋白以单体形式存在且糖基化丰富。rHA蛋白独自作用小鼠产生的免疫原性较低且不能保护小鼠抵抗H1N1型流感病毒的攻击，rHA蛋白联合佐剂免疫小鼠后具有较好的免疫原性，以HA203佐剂效果最好。HA蛋白配伍HA203佐剂可以诱导小鼠产生特异性体液免疫保护小鼠抵御H1N1型流感病毒的攻击，减少炎性细胞浸润，有效抑制病毒的复制。

关键词(KeyWords)： 流感亚单位疫苗;重组蛋白血凝素;免疫原性

Abstract:

Keywords:

基金项目(Foundation): 国家重大科学工程建设项目计划（项目号：2016Z09106003-008）,题目：四价流感病毒裂解疫苗的研制

作者(Authors): 刘京;龚铮;年悬悬;邓涛;周蓉;张国梅;李雪丹;张哲罡;张家友;杨晓明;

DOI: 10.13242/j.cnki.bingduxuebao.004243

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