中国兽医科学2025，Vol.55Issue(10)：1350-1356,7.DOI:10.16656/j.issn.1673-4696.2025.0185
蓝舌病毒血清1型VP5蛋白的截短表达及其单克隆抗体的制备
Truncated expression of VP5 protein of bluetongue virus serotype 1 and preparation of its monoclonal antibodies
摘要
Abstract
This study used the amino acid sequence of VP5 protein from the bluetongue virus serotype 1(BTV-1)reference strain in GenBank.After deleting amino acids 1-79 at the N-terminus and codon-optimizing the gene according to Escherichia coli codon bias,the synthetic truncated gene en-coding BTV-1 VP5Δ79aa was cloned into the expression vector pET-28a-sumo to construct the recombinant plasmid pET-sumo-VP5Δ79aa.The recombinant plasmid was transformed into BL21(DE3)competent cells.Then the expression was induced with 0.5 mmol/L IPTG at 37 ℃ and 16 ℃,respectively.Solubility analy-sis was performed.SDS-PAGE results indicated that the recombinant protein was predominantly expressed as inclusion bodies in E.coli.The recombinant VP5Δ79aa protein was purified according to the Ni-NTA affinity chromatography,obtaining a single target band of the expected molecular weight.After refold-ing through urea gradient dialysis,the purified recombinant VP5Δ79aa protein was used to immunize fe-male BALB/c mice(6-to 8-week-old).Splenocytes from immunized mice were fused with myeloma cells to generate hybridoma cell lines stably secreting monoclonal antibodies(McAbs).Two McAbs(4A10 and 5H11)demonstrating strong reactivity and specificity were identified through indirect ELISA,immunofluo-rescence,and Western-blot analyses.Both of the McAbs reacted with the truncated VP5Δ79aa and full-length recombinant VP5 proteins and specifically recognized the native VP5 protein in cells in-fected with BTV-1.This study establishes a foundation for further exploration of the biological func-tions and antigenic epitope identification of BTV VP5 protein.关键词
蓝舌病毒/VP5蛋白/截短表达/单克隆抗体Key words
bluetongue virus/VP5 protein/truncated expression/monoclonal antibody分类
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刘学春,户鑫兵,宋昱庆,孟凡华,田占成,关贵全,殷宏,独军政..蓝舌病毒血清1型VP5蛋白的截短表达及其单克隆抗体的制备[J].中国兽医科学,2025,55(10):1350-1356,7.基金项目
国家重点研发计划项目(2021YFD1800500,2024YFD1800100) （2021YFD1800500,2024YFD1800100）
甘肃省基础研究创新群体项目(22JR5RA024) （22JR5RA024）
甘肃省科技厅项目(22CX8NA011) （22CX8NA011）
国家肉牛牦牛产业技术体系专项(CARS-37) （CARS-37）
中国农业科学院科技创新工程项目(CAAS-ASTIP-2021-LVRI) （CAAS-ASTIP-2021-LVRI）
