华北理工大学基础医学院,河北省慢性疾病基础医学重点实验室;传染病溯源预警与智能决策全国重点实验室,国家卫生健康委医学病毒和病毒病重点实验室,中国疾病预防控制中心病毒病预防控制所;
体外构建表达水痘-带状疱疹病毒(Varicella-zoster virus,VZV)糖蛋白E(Glycoprotein E,gE)的复制缺陷型人26型重组腺病毒疫苗,并进行重组病毒免疫原性的初步评价。利用同源重组的方法将gE原始信号肽序列替换成tPA组织纤溶酶原激活剂(Tissue plasminogen activator,tPA)信号序列(tPA-gE),将目的片段基因(tPA-gE)连接到腺病毒载体Ad26-empty得到重组腺病毒质粒Ad26-tPA-gE。线性化的重组腺病毒质粒转染至HEK293A细胞进行病毒包装与扩增,利用氯化铯密度梯度离心法纯化病毒,基于腺病毒六邻体蛋白质测定病毒滴度,Western blot验证重组腺病毒中tPA-gE表达情况,电镜检测重组病毒大小与形态。重组病毒(Ad26-tPA-gE)通过肌肉注射免疫C57BL/6N小鼠,第8周后下颌采血通过间接ELISA检测小鼠血清中特异性的抗gE的抗体效价评价体液免疫,ELISPOT检测小鼠脾淋巴细胞分泌IFN-γ及IL-2情况观察细胞免疫。纯化得到滴度为7.2×10~(10)ifu/mL的重组腺病Ad26-tPA-gE, Western blot验证重组病毒感染的细胞能够正常表达gE,电镜检测病毒大小均一,形态完整。重组腺病毒疫苗Ad26-tPA-gE可成功诱导小鼠产生抗gE特异性IgG、IgG1和IgG2a抗体,以及淋巴细胞分泌IFN-γ和IL-2。成功构建了表达VZV gE的复制缺陷型重组腺病毒,免疫C57BL/6N小鼠后有效激活体液及细胞免疫反应,为后续基于腺病毒载体的带状疱疹疫苗研究提供基础。
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基本信息:
DOI:10.13242/j.cnki.bingduxuebao.004648
中图分类号:R392-33
引用信息:
[1]杨梦瑶,柴鹏弟,章青等.表达水痘-带状疱疹病毒gE蛋白的复制缺陷型重组腺病毒的构建[J].病毒学报,2025,41(01):48-58.DOI:10.13242/j.cnki.bingduxuebao.004648.
基金信息: