表达传染性法氏囊病病毒VP2蛋白的重组复制缺陷型HAdV-5的构建及鉴定

【目的】采用AdEasy系统构建能表达传染性法氏囊病病毒（Infectious bursal disease virus，IBDV） VP2蛋白的重组复制缺陷型人5型腺病毒（Human adenovirus serotype-5，HAdV-5），以期为IBDV活载体疫苗的研发提供新的思路。【方法】通过同源重组将VP2基因克隆至穿梭质粒pAdTrack-GOI构建重组穿梭质粒pAdTrack-VP2；PmeⅠ酶线性化pAdTrack-VP2后热击转化大肠杆菌BJ5183-AD-1感受态细胞，利用菌内同源重组构建重组腺病毒质粒pAd-VP2；将pAd-VP2经PacⅠ酶线性化和纯化后转染HEK293A细胞，包装表达VP2蛋白的重组复制缺陷型HAdV-5（rAd5-VP2），将鉴定正确的重组病毒感染非复制型细胞（Vero、CEF和DF1细胞）并分析目的蛋白的表达情况。【结果】将rAd5-VP2通过HEK293A细胞扩大培养，测得第10代病毒的滴度为7.9×10⁹ IFU/mL；RT-PCR结果显示，VP2基因在重组腺病毒中能稳定翻译；Western blotting和间接免疫荧光试验均检测到VP2蛋白的表达，蛋白分子质量为41 ku；将rAd5-VP2感染Vero、CEF和DF1细胞也检测到VP2蛋白的表达，表明HAdV-5有作为IBDV活载体疫苗研发的可能性。【结论】本研究构建了重组复制缺陷型HAdV-5，该毒株能感染部分哺乳动物细胞和家禽细胞并能检测到目的蛋白的表达。

Construction and Identification of Recombinant Replication-deficient HAdV-5 Expressing VP2 Protein of Infectious Bursal Disease Virus

【Objective】 The purpose of this study was to construct recombinant replication-deficient Human adenovirus serotype-5 (HAdV-5), that could express the VP2 protein of Infectious bursal disease virus (IBDV) by using the AdEasy system, in order to provide a new idea for the development of IBDV live vector vaccine.【Method】 By homologous recombination, VP2 gene was cloned into the shuttle plasmid pAdTrack-GOI, and finally the recombinant shuttle plasmid named pAdTrack-VP2 was constructed.After linearization of pAdTrack-VP2 by PmeⅠ enzyme, it was transformed into E.coli BJ5183-AD-1 competent cells by heat shock.The recombinant Adenovirus plasmid named pAd-VP2 was constructed by homologous recombination in bacteria.After that, pAd-VP2 was digested with PacⅠ enzyme, and the purified product was transfected into HEK293A cells to package the recombinant replication-deficient HAdV-5 (rAd5-VP2) expressing VP2 protein.The identified recombinant viruses were infected with non-replicating cells such as Vero cells, CEF cells and DF1 cells, and the expression of the target protein was analyzed.【Result】 rAd5-VP2 was expanded by HEK293A cells and the titer of the 10th generation Adenovirus was 7.9×10⁹ IFU/mL.RT-PCR showed that VP2 gene could translate stably in recombinant Adenovirus.Both Western blotting and indirect immunofluorescence assays detected the expression of VP2 protein with a molecular weight of 41 ku.The expression of VP2 protein was also detected in Vero cells, CEF cells and DF1 cells infected with recombinant Adenovirus, indicating that HAdV-5 has the potential to be developed as a live vector vaccine for IBDV.【Conclusion】 Recombinant replication-deficient HAdV-5 was constructed, which could infect some mammalian cells and poultry cells, and the expression of the target protein could be detected.