摘要
为探究抑制性受体CD300a在牛Ⅰ型疱疹病毒(bovine herpesvirus 1,BHV-1)免疫调控机制中的作用,分离鉴定了1株BHV-1内蒙古株,并建立CD300a基因的荧光定量PCR方法,检测BHV-1感染牛单核巨噬细胞后0,6,12,24,48,72 h 6个时间点以及空白对照组CD300a基因的转录变化。将β-actin基因作为内参,使用双标准曲线法计算攻毒组与对照组CD300a基因各时间段的相对表达量,分析CD300a在攻毒前、后的表达差异。结果显示,BHV-1分离株在MDBK细胞上出现典型细胞病变效应,测得其病毒滴度为107.23TCID50/mL。本研究建立的CD300a基因荧光定量PCR检测方法,CD300a基因和β-actin基因标准品的Ct值与模板浓度之间均具有良好的线性关系,溶解曲线均为特异单峰,最低检测限均为10拷贝/μL,具有良好的灵敏性,可以准确检测出BHV-1攻毒后巨噬细胞内的CD300a基因表达量。经计算,攻毒组CD300a表达量均高于对照组,其中,6,12,24 h的表达差异倍数显著高于0,72 h(P<0.05),表达差异倍数在12 h时达到最高,且显著高于其他各组(P<0.05),表达差异倍数从6 h时开始巨幅增长,12 h达到最大,48 h开始趋于稳定。结果表明CD300a基因作为巨噬细胞表面的抑制性受体,表达量在BHV-1入侵时会显著变化,推测CD300a基因为协助病毒进入宿主细胞或免疫逃逸也相应增多,在抗病毒免疫机制中起到一定作用。
In order to explore the role of inhibitory receptor CD300a in the immune regulation mechanism of bovine herpesvirus 1(BHV-1),an Inner Mongolia strain of BHV-1 was isolated and identified,and a fluorescence quantitative PCR method of CD300a gene was established to detect the transcriptional changes of CD300a gene at 0,6,12,24,48 and 72 h after BHV-1 infection in bovine mononuclear macrophages and the blank control group.Usingβ-actin gene as the internal reference,the relative expression of CD300a gene in each time period between the challenge group and the control group was calculated by double standard curve method,and the difference of CD300a expression before and after challenge was analyzed.The results showed that the BHV-1 isolate showed typical cytopathic effect in MDBK cells,and the virus titer was 107.23TCID50/mL.The fluorescence quantitative PCR detection method of CD300a gene established in this study showed that the Ct values of both CD300a gene andβ-actin gene standards had good linearity with the template concentration,the dissolution curve was a specific single peak,and the lowest detection limit was10copies/μL.It had good sensitivity and could accurately detect the expression of CD300agene in macrophages after BHV-1challenge.According to the calculation,the expression of CD300ain the challenge group was higher than that in the control group,and the expression difference multiple at6,12and 24hwas significantly higher than that at 0,72h,and reached the highest at 12h,which was significantly higher than that in other groups(P<0.05).The expression difference multiple increased significantly at 6h,reached the maximum at 12h,and became stable at 48h.The results showed that the expression of CD300agene,as an inhibitory receptor on the surface of macrophages,changed significantly during BHV-1invasion.It is speculated that CD300agene plays a certain role in antiviral immune mechanism because it assists the virus to enter host cells or immune escape.
作者
王旭芬
安冬
侯琳
赵润涛
张贺
赵洋
张志丹
周伟光
WANG Xufen;AN Dong;HOU Lin;ZHAO Runtao;ZHANG He;ZHAO Yang;ZHANG Zhidan;ZHOU Weiguang(Key Laboratory of Clinical Diagnosis and Treatment of Animal Diseases of Ministry of Agriculture,College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot O10018,China;Hailar Disease Prevention and Control Center,Hulunbuir,Inner Mongolia 021000,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2023年第2期253-259,共7页
Chinese Journal of Veterinary Science
基金
内蒙古自治区科技重大专项资助项目(2021ZD0013)
内蒙古自治区自然科学基金资助项目(2019MS3050)。
