BHK-21细胞中猪乙型脑炎病毒动态增殖的研究  

Study on dynamic proliferation of porcine Japanese encephalitis virus in BHK-21 cells

作　　者：张福良[1] 马圣明 邢月腾 刘芳 曹金泽 ZHANG Fuliang;MA Shengming;XING Yueteng;LIU Fang;CAO Jinze(School of Biology and Food Engineering,Anyang Institute of Technology,Anyang 455000,China)

机构地区：[1]安阳工学院生物与食品工程学院,河南安阳455000

出　　处：《黑龙江畜牧兽医》2023年第5期75-79,136,137,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：安阳市科技计划项目(2021C01NY044);安阳工学院博士科研项目(BSJ2022037)。

摘　　要：为了研究猪乙型脑炎病毒(Japanese encephalitis virus, JEV)在仓鼠肾细胞(BHK-21细胞)中的增殖情况,试验首先建立了实时荧光定量PCR方法,并检测该方法的敏感性、特异性和重复性,然后利用建立的实时荧光定量PCR方法和病毒半数组织感染量(TCID_(50))方法测定并比较猪JEV在BHK-21细胞中的增殖规律。结果表明:建立的猪JEV实时荧光定量PCR检测方法的扩增效率为99.3%,敏感性、特异性和重复性良好,检测敏感性可以达到1.0×10^(1)copies/μL;TCID_(50)方法测定的细胞悬液和细胞培养上清液中的病毒含量分别为1.0×10^(5.44)TCID_(50)/0.1 mL和1.0×10^(4.86)TCID_(50)/0.1 mL,实时荧光定量方法测定的病毒粒子数量分别为1.0×10^(7.50)copies/μL和1.0×10^(5.60)copies/μL,两种方法在细胞悬液中出现峰值的时间为感染病毒后60小时,在细胞培养上清液中为72小时。说明猪JEV在BHK-21细胞中增殖的最佳收毒时间是感染病毒后60小时。In order to study the proliferation of Japanese encephalitis virus(JEV) in hamster kidney cells(BHK-21 cells), a real-time fluorescence quantitative PCR method was first established. The experiment firstly established the real-time fluorescence quantitative PCR method and tested the sensitivity, specificity and reproducibility of the method, and then determined and compared the proliferation pattern of porcine JEV in BHK-21 cells using the established real-time fluorescence quantitative PCR method and TCID_(50)method. The results showed that the amplification efficiency of the established porcine JEV real-time fluorescence quantitative PCR method was 99.3%;the sensitivity, specificity and reproducibility were good and the detection sensitivity could reach 1.0×10^(1) copies/μL. The virus contents in the cell suspension and cell culture supernatant determined by TCID_(50)method were 1.0×10^(5.44)TCID_(50)/100 μL and 1.0×10^(4.86)TCID_(50)/100 μL, respectively;the number of virus particles determined by real-time fluorescence quantitative method was 1.0×10^(7.5)copies/μL and 1.0×10^(5.6)copies/μL, respectively. The time of peak in the cell suspension for both methods was 60 h after infection with the virus and 72 h in the cell culture supernatant. The results indicated that the optimal time for harvesting porcine JEV proliferated in BHK-21 cells was 60 h after infection with the virus.

关 键 词：猪乙型脑炎病毒 BHK-21细胞 实时荧光定量PCR TCID_(50) 增殖曲线 最佳收毒时间 

分 类 号：S852.659.6[农业科学—基础兽医学]