摘要
构建猪IFN-β重组真核表达质粒pcDNA3.1-PoIFN-β,转染悬浮培养的CHO细胞,进行上清分泌表达;通过CHO细胞表达系统,成功分泌表达和制备纯化的rPoIFN-β。通过CCK-8试验,分析rPoIFN-β对细胞的毒性,结果显示纯化的rPoIFN-β对细胞无明显毒性;采用VSV/PK-15系统检测rPoIFN-β抗病毒活性效价,结果显示其抗病毒活性效价为4.54×10^(7)U/mg;制备的rPoIFN-β可显著抑制O型和A型FMDV在PK-15细胞的复制,表现出较强的抗病毒活性;通过分析rPoIFN-β对细胞ISGs的诱导作用,发现rPoIFN-β能够显著刺激12种ISGs的表达。研究结果表明,本试验制备纯化的rPoIFN-β具有较低的细胞毒性和较高的抗病毒活性效价,为口蹄疫防控制剂的开发和研究提供了基础数据。
In this study,the porcine IFN-β recombinant eukaryotic expression plasmid pc DNA3.1-Po IFN-β was constructed,and the suspension cultured CHO cells were transfected for supernatant secretion expression,and the purified r Po IFN-β was successfully secreted and prepared by the CHO cell expression system.Through the CHO cell expression system,the purified r Po IFN-β was successfully secreted and prepared.The cytotoxicity of r Po IFN-β was analyzed by CCK-8 test,we found that the purified r Po IFN-β showed no apparent cytotoxicity to PK-15 cells.Antiviral activity assay for r Po IFN-β was performed and evaluated by standard procedures in VSV/PK-15(virus/cell) test system,the biological activity of r Po IFN-β was 4.54×10^(7)U/mg.The prepared r Po IFN-β significantly inhibited virus replication of the two serotype of FMDV and showed strong antiviral activity.By analyzing the induction effect of r Po IFN-β on cellular ISGs,and r Po IFN-β greatly induced the 12 ISGs production in PK-15 cells.The results showed that the purified r Po IFN-β prepared in this experiment has low cytotoxicity and high antiviral activity titer,which provides basic data for the development and research of FMD prevention and control agents.
作者
王凌云
郝荣增
茹毅
常辉
杨洋
李亚军
张丽伟
卢炳州
刘艳红
龚真莉
井波
齐萌
郑海学
WANG Ling-yun;HAO Rong-zeng;RU Yi;CHANG Hui;YANG Yang;LI Ya-jun;ZHANG Li-wei;LU Bing-zhou;LIU Yan-hong;GONG Zhen-li;JING Bo;QI Meng;ZHENG Hai-xue(College of Animal Science and Technology,Tarim University,Aral 843300,China;National Foot-and-Mouth Disease Reference Laboratory/State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2023年第2期143-149,共7页
Chinese Veterinary Science
基金
甘肃省科技重大专项(21ZD3NA001)
中国农业科学院所级重点任务(CAAS-ASTIP-JBGS-20210401)
中国农业科学院兰州兽医研究所所级基本科研业务费项目(1610312021013)。
关键词
猪IFN-β
CHO细胞
蛋白表达
抗病毒活性
口蹄疫病毒
porcine interferonβ
CHO cells
expression of protein
antiviral activity
foot-andmouth disease virus
