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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7.2×1010ifu/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。
Abstract：This study constructed a replication-deficient recombinant adenovirus vaccine expressing glycoprotein E(gE) of varicella-zoster virus(VZV) in vitro and conducted a preliminary evaluation of its immunogenicity.Using homologous recombination, the original signal peptide sequence of gE was replaced with the tissue plasminogen activator(tPA) signal sequence(tPA-gE), and the target fragment gene(tPA-gE) was ligated to the adenovirus vector Ad26-empty to obtain the recombinant adenovirus plasmid Ad26-tPA-gE. The linearized recombinant adenovirus plasmid was transfected into HEK293A cells for virus packaging and amplification. The virus was purified using cesium chloride density gradient centrifμgation, and virus titers were determined based on adenovirus hexon protein. Western blot was analysis verified the expression of the tPA-gE protein in the recombinant adenovirus, and electron microscopy confirmed the size and morphology of the recombinant virus.C57BL/6N mice were immunized with Ad26-tPA-gE via intramuscular injection. Eight weeks post-immunization, mandibular blood samples were collected to evaluate humoral immunity by determining specific anti-gE antibody titers in mouse serum using indirect ELISA. ELISPOT assays were performed to assess the secretion of IFN-γ and IL-2 by splenic lymphocytes, providing insights into cellular immunity. The purified recombinant adenovirus Ad26-tPA-gE had a titer of 7.2×1010 ifu/mL. Western blot analysis confirmed the normal expression of gE in cells infected with the recombinant virus, and electron microscopy revealed uniform size and intact morphology. The recombinant adenovirus vaccine Ad26-tPA-gE successfully induced the production of gE-specific IgG, IgG1, and IgG2a antibodies, along with the secretion of IFN-γ and IL-2 by lymphocytes. This study successfully constructed a replication-deficient recombinant adenovirus expressing VZV gE, which effectively activated humoral and cellular immune responses in C57BL/6N mice after immunization. These findings provide a foundation for future research on adenovirus-based herpes zoster vaccines.
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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: