中国动物传染病学报2024，Vol.32Issue(6)：17-24,8.
重组猫IFN-α的质粒构建、真核表达及体外抗病毒活性分析
Plasmid Construction and Eukaryotic Expression of FeIFN-α and Its Antiviral Activity in vitro
摘要
Abstract
This article was to explore the in vitro antiviral effect of feline IFN-α(FeIFN-α)in MDCK and F81 cells and provide data for subsequent in vivo experiments.The FeIFN-α sequence deposited in GenBank(NP_001027000.1)with addition of the 6×His tag cloned to PUC57 vector and sub-cloned to pcDNA3.1 to get the eukaryotic expression plasmid for transfection into 293T cells.The FeIFN-α expression was detected by Western blotting.Preliminary experiments were performed to determine the optimal dilution ratio by infecting cells with vesicular stomatitis virus-green fluorescent protein(VSV-GFP)and examining the antiviral activity of FeIFN-α by crystal violet staining.The antiviral activity was detected by fluorescence observation and flow cytometry.Finally,interferon stimulation to genes(ISG15,MX1,OAS1)controlled by FeIFN-α was detected by relative fluorescence quantitative qPCR.The results showed successful expression of the FeIFN-α with biological activity and significant inhibition of VSV-GFP infection in F81 and MDCK cells.The qPCR results also showed that FeIFN-α effectively induced the increase of ISG15,MX1 and OAS1 genes in F81 and MDCK cells.This research provided a reference for subsequent antiviral experiments in dogs and cats.关键词
猫IFN-α/构建与表达/抗病毒活性/干扰素刺激基因Key words
FeIFN-α/construction and expression/antiviral activity/interferon stimulating gene分类
农业科技引用本文复制引用
李沛恒,邹万成,陈竞,张国庆,崔春梅,蒋嵘,金宁一,李昌..重组猫IFN-α的质粒构建、真核表达及体外抗病毒活性分析[J].中国动物传染病学报,2024,32(6):17-24,8.基金项目
中国医学科学院与健康科技创新工程项目(2020-I2M-5-001) （2020-I2M-5-001）
