摘要
为探索牙鲆弹状病毒(HIRRV)对敏感宿主牙鲆肾脏原代细胞的敏感性,本研究分别采用组织块移植法和胰酶消化法对牙鲆(Paralichthys olivaceus)肾脏细胞进行分离和原代培养。研究显示,采用组织块移植法进行培养后,会出现较多的成纤维样细胞,而采用胰酶消化法进行培养后,细胞呈现上皮细胞样且形态完整,7天后细胞的汇合率可达80%。采用胰酶消化法对牙鲆肾细胞进行原代培养,传代后进行病毒敏感性实验,实验表明牙鲆肾细胞接种HIRRV后,第3天出现了明显的CPE,病毒滴度达1×105.8 TCID50/mL;RT-PCR可从感染3天的细胞培养物中检测到HIRRV的特异性mRNA;利用抗HIRRV单克隆抗体结合免疫荧光试验可从感染24h的细胞中检测到特异性荧光信号,信号分布在细胞膜和细胞质中。研究结果表明,基于胰酶消化法建立的牙鲆肾脏原代细胞对HIRRV较敏感,HIRRV可成功侵染该细胞并在细胞内实现快速增殖。本研究为牙鲆弹状病毒的致病机制研究提供了材料和实验基础。
To explore the susceptibility of the cultured primary kidney cells of Japanese flounder(Paralichthys olivaceus)to hirame rhabdovirus(HIRRV),the primary kidney cells of Japanese flounder were isolated and cultured using tissue transplantation and trypsinization techniques,respectively.The results showed that the morphology of the kidney cells was mainly fibroblast-like as were observed with tissue transplantation method,while they were epithelial-like as were observed with trypsinization method.The cell confluence rate was 80%at 7 days post-culture.The virus susceptibility tests were carried out after cell passage.Viral-induced cytopathic effect was observed,and the viral titer reached about 10 5.8 TCID 50/mL at 3 days post HIRRV infection.RT-PCR detected the mRNA of HIRRV in infected cells.Using monoclonal antibody against HIRRV and indirect immune fluorescent techniques,specific fluorescence signal was detected in the infected cells at 24 h post infection.The signal located in cell membrane and cytoplasm.Our findings suggested that the kidney cells were susceptible to HIRRV;the virus could successfully infect and proliferate rapidly in the cells.The primary kidney cell culture of Japanese flounder was established in this study,which will provide useful materials for the pathogenic mechanism studies of HIRRV.
作者
张家林
唐小千
绳秀珍
邢婧
战文斌
ZHANG Jia-Lin;TANG Xiao-Qian;SHENG Xiu-Zhen;XING Jing;ZHAN Wen-Bin(Laboratory of Pathology and Immunology of Aquatic Animals,College of Fisheries,Ocean University of China,Qingdao 266003,China)
出处
《中国海洋大学学报（自然科学版）》
CAS
CSCD
北大核心
2018年第1期19-24,共6页
Periodical of Ocean University of China
基金
国家自然科学基金项目(31672685
31672684
3142295)
海洋渔业科学与食物产出过程功能实验室开放基金项目资助~~
关键词
牙鲆
原代细胞培养
胰酶消化法
牙鲆弹状病毒
敏感性
Paralichthys olivaceus
primary cell culture
trypsinization
HIRRV
susceptibility
