摘要
本研究以IBV Beaudette株基因组为模板获得N基因片段,构建重组原核表达质粒pET-32a-His-IBV-N,在大肠杆菌BL21(DE3)诱导表达,纯化后获得带有His标签的N重组蛋白.将其与等体积的弗氏完全佐剂混匀乳化后免疫BALB/c小鼠3次,收集血清,得到鼠源抗N蛋白多克隆抗体.利用间接免疫荧光试验和Western blot鉴定多克隆抗体的特异性.结果显示,N蛋白多克隆抗体能特异性识别转染表达的Flag-N蛋白和IBV感染表达的N蛋白,说明此抗体能识别N蛋白的线性表位和空间表位,可应用于Western blot和间接免疫荧光实验.本研究为进一步研究N蛋白的功能和IB的诊断提供了技术支撑.
Abstract
The objective of this study was to prepare the murine-derived polyclonal antibodies(pAbs)against infectious bronchitis virus(IBV)N protein.The N gene was amplified by RT-PCR using IBV Beaudette genome as template and the amplified fragment was used for construction of the recombinant prokaryotic expression plasmid pET-32a-His-IBV-N.This plasmid was transformed into E.coli BL21(DE3)to express His-tagged recombinant protein N.BALB/c mice were immunized three times with the purified His-N protein emulsified in the same volume of Freund's complete adjuvant and the serum samples were collected for the following study.The specificity of the anti-N pAbs was examined in indirect immunofluorescence assay(IFA)and Western blot.The results showed that the murine anti-N pAbs specifically recognized both plasmid expressed N protein and virus expressed N protein.The availability of anti-N pAbs provided the tool for study of the biological function of N protein and offered the reagent for IB diagnosis.
基金项目
国家自然科学基金面上项目(32172834)
科技部十四五揭榜挂帅项目(2021YFD1801104)
江苏省人兽共患病学重点实验室资助项目(R2007)
国家科技期刊平台
NSTL
万方数据