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

摘要/Abstract

摘要： 【目的】试验旨在优化兔出血症病毒2型(Rabbit hemorrhagic disease virus 2,RHDV2)病毒样颗粒(virus-like particle,VLP)疫苗的制备策略,探究RHDV2 VLP疫苗对家兔的免疫原性,为低成本、高产量RHDV2新型疫苗研发提供新思路。【方法】根据昆虫细胞的密码子偏好性优化合成RHDV2 VP60全基因,将双VP60基因插入真核载体pFastBac^TM Dual,转化携带Bacmid质粒的大肠杆菌DH10Bac感受态细胞,构建含双VP60基因的重组杆粒Bacmid-VP60-VP60,转染Sf9昆虫细胞,通过Western blotting、间接免疫荧光试验(IFA)及透射电镜对重组杆状病毒Bacmid-VP60-VP60进行表达验证;将优化策略制备的重组蛋白抗原与氢氧化铝佐剂按照9∶1比例制备VLP灭活疫苗,通过安全性检验、最小免疫剂量、免疫持续期等评估优化策略制备的RHDV2 VLP疫苗的保护效果。【结果】试验成功构建重组杆粒Bacmid-VP60-VP60。Western blotting鉴定结果显示,重组杆状病毒转染Sf9细胞表达出大小约60 ku的RHDV2 VP60蛋白。IFA鉴定结果显示,感染重组杆状病毒的Sf9细胞产生了大量的黄绿色荧光,表明重组杆状病毒在Sf9细胞中大量表达VP60蛋白。透射电镜观察结果显示,VP60蛋白折叠成VLP,大小为40 nm左右,呈现球形结构,表面光滑。优化策略制备的RHDV2 VLP疫苗对家兔具有良好的安全性和免疫原性,最小免疫剂量为0.5 mL/只,免疫持续期可达210 d以上。【结论】试验构建了含有密码子优化的双VP60基因的重组杆状病毒,并在昆虫细胞中成功表达RHDV2 VP60蛋白,该蛋白制备的VLP疫苗对家兔具有良好的免疫原性。

关键词: 兔出血症病毒2型(RHDV2); 病毒样颗粒(VLP); 密码子偏好性; 重组杆状病毒; 免疫原性

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

中图分类号:

S852.65⁺9.1

于吉锋, 谢晶, 黄勇, 肖璐, 林毅, 曹冶, 叶勇刚, 魏勇, 吴学婧, 李江凌, 康润敏. 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[J]. 中国畜牧兽医, 2024, 51(2): 668-677.

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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