《中国畜牧兽医》 ›› 2019, Vol. 46 ›› Issue (3): 924-930.doi: 10.16431/j.cnki.1671-7236.2019.03.034

• 基础兽医 • 上一篇    下一篇

NO自由基对猪圆环病毒2型复制的体外抑制作用研究

李基棕1,2, 张冬霞1, 李丽1, 薛涛1, 刘传敏2   

  1. 1. 临沂大学药学院, 临沂 276000;
    2. 江苏省农业科学院兽医研究所, 兽医诊断检测重点实验室, 农业部兽用生物制品工程技术重点实验室, 南京 210014
  • 收稿日期:2018-06-26 出版日期:2019-03-20 发布日期:2019-03-20
  • 通讯作者: 薛涛, 刘传敏 E-mail:xuetao@lyu.edu.cn;lcm1028@126.com
  • 作者简介:李基棕(1986-),男,湖南怀化人,博士,副研究员,研究方向:动物传染病防治和诊断技术,E-mail:lijizong22@sina.com
  • 基金资助:

    山东省自然科学基金(ZR2016CP08);国家自然科学基金项目(31502099、31702272);中国博士后科学基金(2015M581755);江苏省博士后科学基金(1501161B);江苏省自然科学基金(BK2017059)

The Free Radical of Nitric Oxide Inhibits Porcine Circovirus Type 2 Replicion in vitro

LI Jizong1,2, ZHANG Dongxia1, LI Li1, XUE Tao1, LIU Chuanmin2   

  1. 1. School of Pharmacy, Linyi University, Linyi 276000, China;
    2. Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Key Laboratory of Veterinary Diagnosis Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
  • Received:2018-06-26 Online:2019-03-20 Published:2019-03-20

摘要:

试验旨在探讨NO自由基(NO·)对猪圆环病毒2型(PCV2)复制的抑制作用。硝普钠(SNP)和抗坏血酸(VC)联合加入培养基中可产生NO·,而单独使用SNP产生NO的形式为NO+。本研究采用MTT法测定药物的细胞毒性,通过Griess反应测定供体药物产生NO的水平。分别用SNP、VC、SNP+VC和对照药物乙酰青酶胺(NAP)处理PK-15细胞,6 h后接种1 MOI的PCV2病毒液,72 h后收获培养上清和细胞,通过间接免疫荧光试验(IFA)、Western blotting分析、病毒效价和实时荧光定量PCR试验检测病毒增殖水平。结果显示:SNP、VC和SNP+VC对PK-15细胞的最大安全浓度分别为500、62.5和31.25 μmol/L;60 μmol/L SNP和30 μmol/L SNP+30 μmol/L VC两种供体产生的NO量极显著高于非药物处理组和对照药物NAP处理组(P<0.01);与阳性对照组相比,60 μmol/L SNP+30 μmol/L VC处理组中的PCV2毒价和DNA拷贝数极显著下降(P<0.01),且PCV2 Cap蛋白的表达也受到明显抑制,而SNP和VC单独处理组上述指标无显著变化(P>0.05)。以上结果表明,SNP+VC产生的NO·对PCV2增殖有显著抑制作用,SNP产生的NO+不影响PCV2增殖,NO体外抑制PCV2增殖主要是通过NO·实现。

关键词: NO; NO自由基(NO·); 猪圆环病毒2型(PCV2); 抑制

Abstract:

This study was conducted to investigate the inhibitory effect of NO· on the replication of porcine circovirus type 2 (PCV2).NO was generated from SNP or SNP in the presence of ascorbate (VC).SNP+VC generates mainly NO· in culture medium while NO+ was the major product of SNP alone.PK-15 cells were pretreated with SNP,VC,SNP+VC or control drug (NAP) for 6 h,respectively,then infected with PCV2 (1 MOI) and incubated with the above mentioned drugs for additional 72 h.After incubation,culture medium and cells were collected.IFA,Western blotting,TCID50 and QPCR assay methods were performed to determine the viral titers.The results showed that the highest concentration of SNP,VC and SNP+VC in treated cells were 500,62.5 and 31.25 μmol/L,respectively.The production of NO in 60 μmol/L SNP or 30 μmol/L SNP+30 μmol/L VC treated cells were higher than that in untreated cells and NAP treated cells (P<0.01).Virus titers and PCV2 DNA copies from 30 μmol/L SNP+30 μmol/L VC treated group were significantly lower than that from infected control (P<0.01).In addition,The expression of viral Cap protein from the sample treated with 30 μmol/L SNP+30 μmol/L VC also obviously decreased in relation to those from infected control samples.However,almost no antiviral activities of SNP or VC alone were indicated during PCV2 infection.In conclusion,inhibition of NO on PCV2 replication was mediated by NO·,rather than NO+.

Key words: NO; NO·; porcine circovirus type 2 (PCV2); inhibition

中图分类号: