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

doi:10.16431/j.cnki.1671-7236.2022.12.002

Abstract

Abstract: 【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.

Key words: infectious bursal disease; VP2 protein; recombinant replication-deficient Human adenovirus serotype-5

CLC Number:

S852.65^＋7

XU Ting, XIONG Ting, LI Linyu, LIU Yufu, WEN Lianghai, XIE Wenting, YANG Zekun, CHEN Ruiai. Construction and Identification of Recombinant Replication-deficient HAdV-5 Expressing VP2 Protein of Infectious Bursal Disease Virus[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4524-4534.

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

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