绿原酸体外抗新型鸭呼肠孤病毒作用的研究  

Study on Anti-Novel duck reovirus Effect of Chlorogenic Acid In vitro

作　　者：潘力 马秀丽[1] 黄中利[1] 李桂明[1] 唐亮 于可响[1] 林树乾[1] PAN Li;MA Xiu-Li;HUANG Zhong-Li;LI Gui-Ming;TANG Liang;YU Ke-Xiang;LIN Shu-Qian(Institute of Poultry,Shandong Academy of Agricultural Sciences,Jinan 250023,China;College of Animal Science and Technology,Shandong Agricultural University,Tai'an 271018,China)

机构地区：[1]山东省农业科学院家禽研究所,济南250023 [2]山东农业大学动物科技学院,泰安271018

出　　处：《农业生物技术学报》2020年第4期754-760,共7页Journal of Agricultural Biotechnology

基　　金：山东省自然科学基金(ZR2019MC022);国家重点研发计划(2017YFC1702700);济南自主培养创新团队项目(2019GXRC025);中央引导地方科技发展专项资金(鲁财教指[2017]77号);山东省农业科学院学科团队建设项目(CXGC2018E11);山东省现代农业产业技术体系家禽首席岗位项目(SDAIT-13-011-01);山东省农业科学院农业科技创新工程项目(CXGC2016A10);山东省农业科学院科技创新工程学科团队专项经费(CXGC2018E11);山东省重点实验室开放课题(SDPDI201809)。

摘　　要：由新型鸭呼肠孤病毒(Novel duck reovirus, NDRV)引起的NDRV病是困扰我国养鸭业的主要疾病之一。绿原酸(chlorogenic acid, CHA)作为许多中药的主要成分,在抗病毒方面发挥着重要作用。本研究拟明确CHA在NDRV感染幼仓鼠(Mesocricetus auratus)肾细胞系BHK-21不同阶段的抗病毒作用效果,为CHA抗NDRV的临床治疗提供参考依据。首先根据NDRV的S3基因序列(GenBank No. KJ879932)设计引物,建立NDRV实时荧光定量PCR (real-time fluorescent quantitative PCR, qPCR)检测方法;然后采用细胞增殖-毒性检测试剂盒测定CHA的细胞毒性,确定CHA在BHK-21细胞上的最大安全浓度;最后进行抗病毒试验,组Ⅰ向BHK-21细胞接种病毒NDRV,然后加入不同浓度的CHA,定期收集细胞冻融液;组Ⅱ的病毒吸附方式与组Ⅰ相同,用药前病毒NDRV先在BHK-21细胞上增殖10 h,定量检测增殖10 h后的病毒载量作为基值;以病毒增殖10 h为实验起始点,分别加入不同浓度的CHA,后续流程与组Ⅰ相同;以上两组均设置阴性和阳性对照。定期观察细胞病变,并将收集的样品进行NDRV的q PCR检测。结果显示,本研究建立的用于NDRV检测的qPCR方法最低检测限为92.5拷贝/μL;CHA在BHK-21细胞上的最大安全浓度为128μg/mL;CHA能在体外抑制NDRV增殖,存在明显的剂量效应关系。本研究可为NDRV的相关机制研究和实际应用提供基础资料。Novel duck reovirus(NDRV) disease is a serious infectious disease for poultry in China.Chlorogenic acid(CHA) is a main component of many traditional Chinese medicines, and plays an important role in antiviral research. Exploring the antiviral effect of CHA on NDRV which might provide a basis for the clinical treatment of NDRV to some extent. Firstly, based on S3 gene sequence(GenBank No. KJ879932) of NDRV, the method of real-time fluorescent quantitative PCR(qPCR) was established. Secondly, maximum safe concentration of CHA on baby hamster(Mesocricetus auratus) kidney cell line BHK-21 cells was determined by CCK-8 kit. Finally, the anti-virus test was performed. The experiment was divided into 2 groups. Group Ⅰ : NDRV were inoculated in BHK-21 cells before different concentrations of CHA were added. Then the freeze-thaw solution of cells was collected periodically. Group Ⅱ : The method of virus adsorption was the same as that of groupⅠ. Before treatment, the virus were proliferated on BHK-21 cells for10 h, and the viral load after 10 h proliferation was quantitatively detected as the base value. After 10 h of virus proliferation, different concentrations of CHA were added, and the follow-up procedure was the same as that of Group Ⅰ. Negative and positive control were set in both groups. And the remaining experiment procedures were the same as those in groupⅠ. The cytopathic effects of each group were observed regularly,and the collected samples were quantitatively detected by qPCR. The results showed the lowest detectable viral nucleic acid of the method was 92.5 copies/μL, and the maximum safe concentration of CHA on BHK-21 cells was 128 μg/mL. The effect of anti-NDRV of CHA indicated that different concentrations of CHA could inhibit the proliferation of NDRV in vitro, and this inhibitory effect had an obvious dose-effect relationship. This study could provide basic data for the study of NDRV mechanism and its practical application.

关 键 词：新型鸭呼肠孤病毒 绿原酸 抗病毒 qPCR 

分 类 号：S83[农业科学—畜牧学]