密码子优化的RHDV2双VP60基因重组杆状病毒构建及表达蛋白免疫原性分析

doi:10.16431/j.cnki.1671-7236.2024.02.023

Abstract

Abstract: 【Objective】 This study was aimed to optimize the preparation strategy for virus-like particle (VLP) vaccine of Rabbit hemorrhagic disease virus 2 (RHDV2), investigate the immunogenicity of the optimized VLP vaccine on domestic rabbits, and provide new insights for the development of a cost-effective, high-yield RHDV2 novel vaccine.【Method】 RHDV2 VP60 full gene was synthesized based on the codon preference of insect cells, and the dual VP60 gene was inserted into the eukaryotic vector pFastBac^TM Dual.The recombinant plamid was transformed into Escherichia coli DH10Bac competent cell carrying Bacmid plasmid, constructing the recombinant Baculovirus Bacmid-VP60-VP60 with dual VP60 genes.The Sf9 insect cells were transfected and the expression of the recombinant Baculovirus Bacmid-VP60-VP60 was verified through Western blotting, indirect immunofluorescence assay (IFA) and transmission electron microscopy (TEM).The recombinant protein antigen prepared by the optimized strategy was combined with aluminum hydroxide adjuvant at a 9∶1 ratio to prepare the VLP inactivated vaccine.The protective effect of the optimized VLP vaccine was evaluated through safety testing, minimum immunization dose and duration of immunity.【Result】 The recombinant Baculovirus Bacmid-VP60-VP60 was successfully constructed.The results of Western blotting showed that the recombinant Baculovirus transfected into Sf9 cells expressed RHDV2 VP60 protein about 60 ku in size.IFA identification results showed that Sf9 cells infected with the recombinant Baculovirus produced a large amount of yellow-green fluorescence, indicating a massive expression of VP60 protein in Sf9 cells.TEM observation revealed that the VP60 protein folded into VLP approximately 40 nm in size, presenting a spherical and smooth surface structure.The VLP vaccine prepared using the optimized strategy exhibited good safety and immunogenicity in rabbits, with a minimum immunization dose of 0.5 mL and an immunity duration of over 210 days.【Conclusion】 A recombinant Baculovirus containing codon-optimized dual VP60 gene was constructed and successfully expressed RHDV2 VP60 protein in insect cells.The protein-prepared VLP vaccine exhibited robust immunogenicity in rabbits.

Key words: Rabbit hemorrhagic disease virus 2 (RHDV2); virus-like particle (VLP); codon bias; recombinant Baculovirus; immunogenicity

CLC Number:

S852.65⁺9.1

YU Jifeng, XIE Jing, HUANG Yong, XIAO Lu, LIN Yi, CAO Ye, YE Yonggang, WEI Yong, WU Xuejing, LI Jiangling, KANG Runmin. Construction of Codon-optimized RHDV2 Dual VP60 Recombinant Baculovirus and Immunogenicity Analysis of the Expressed Protein[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(2): 668-677.

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Construction of Codon-optimized RHDV2 Dual VP60 Recombinant Baculovirus and Immunogenicity Analysis of the Expressed Protein

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