摘要
参考GenBank中蓝舌病毒血清1型(BTV-1)标准参考株VP5蛋白氨基酸序列,缺失N端1~79位氨基酸,按照大肠杆菌偏好性进行基因密码子优化后,合成编码BTV-1 VP5截短蛋白的基因VP5Δ79aa,将其克隆到表达载体pET-28a-sumo上,构建了重组质粒pET-sumo-VP5Δ79aa。将该重组质粒转化到BL21(DE3)感受态细胞中,用0.5 mmol/L IPTG分别在37℃和16℃条件下进行诱导表达和可溶性分析,经SDS-PAGE检测,重组VP5Δ79aa蛋白主要以包涵体形式表达。用Ni-NTA亲和层析法纯化重组VP5Δ79aa蛋白,得到与预期大小相符的单一目的条带。纯化后的重组VP5Δ79aa蛋白经不同浓度梯度的尿素透析复性后,将其免疫雌性BALB/c小鼠(6~8周龄),取免疫后脾细胞与骨髓瘤细胞融合,获得能稳定分泌单克隆抗体的杂交瘤细胞株。经间接ELISA、免疫荧光和Western-blot分析,筛选得到2株反应性和特异性良好的单克隆抗体4A10和5H11,其不仅能与重组VP5Δ79aa截短蛋白以及重组全长VP5蛋白反应,而且能特异识别BTV-1感染细胞中的天然VP5蛋白,这为深入研究BTV VP5蛋白的生物学功能及其抗原表位鉴定奠定了基础。
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 encoding 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 analysis 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 refolding through urea gradient dialysis,the purified recombinant VP5Δ79aa protein was used to immunize female 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,immunofluorescence,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 infected with BTV-1.This study establishes a foundation for further exploration of the biological functions and antigenic epitope identification of BTV VP5 protein.
作者
刘学春
户鑫兵
宋昱庆
孟凡华
田占成
关贵全
殷宏
独军政
LIU Xuechun;HU Xinbing;SONG Yuqing;MENG Fanhua;TIAN Zhancheng;GUAN Guiquan;YIN Hong;DU Junzheng(State Key Laboratory for Animal Disease Control and Prevention/College of Veterinary Medicine of Lanzhou University/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,China;Gansu Province Research Center for Basic Disciplines of Pathogen Biology,Lanzhou 730046,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University,Yangzhou 225009,China)
出处
《中国兽医科学》
北大核心
2025年第10期1350-1356,共7页
Chinese Veterinary Science
基金
国家重点研发计划项目(2021YFD1800500,2024YFD1800100)
甘肃省基础研究创新群体项目(22JR5RA024)
甘肃省科技厅项目(22CX8NA011)
国家肉牛牦牛产业技术体系专项(CARS-37)
中国农业科学院科技创新工程项目(CAAS-ASTIP-2021-LVRI)。
关键词
蓝舌病毒
VP5蛋白
截短表达
单克隆抗体
bluetongue virus
VP5 protein
truncated expression
monoclonal antibody
