基于CPB细胞扩增体系的鳜弹状病毒增殖条件的优化  

Study on optimal proliferation condition of Siniperca chuatsi rhabdovirus in CPB cells

作　　者：罗霞[1] 付小哲[1] 李宁求[1] 林强[1] 黄志斌[1] 张悠[1] LUO Xia;FU Xiaozhe;LI Ningqiu;LIN Qiang;HUANG Zhibin;ZHANG You(Key Laboratory of Fishery Drug Development,Ministry of Agriculture;Key Laboratory of Aquatic Animal Immune Technology,Guangdong Province Pearl River Fishery Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510380,China)

机构地区：[1]中国水产科学研究院珠江水产研究所,农业部渔用药物创制重点实验室,广东省水产动物免疫技术重点实验室,广东广州510380

出　　处：《水产学报》2018年第9期1481-1488,共8页Journal of Fisheries of China

基　　金：广东省渔港建设和渔业产业发展专项(粤财农〔2017〕17号);广东省省级科技计划项目(2016B020234003);广东省省级科技计划项目(2016A020210021)

摘　　要：为获知鳜弹状病毒(Siniperca chuatsi rhabdovirus,SCRV)QY株在体外培养细胞中最适增殖条件,以鳜脑细胞系(Chinese perch brain cell line,CPB)为增殖体系,采用实时荧光定量PCR(quantitative real-time PCR,q RT-PCR)技术所测病毒拷贝数为判断指标,比较同步接毒和异步接毒2种接种方式以及病毒接种量、血清浓度等培养条件对病毒增殖的影响,确定SCRV-QY株在CPB细胞中的最适增殖条件。结果显示,单位体积病毒增殖量方面,异步接毒法优于同步接毒法,以感染复数(multiplicity of infection,MOI)为10将病毒接种长满单层的CPB细胞中,28°C吸附1.5 h,用含2%胎牛血清的L15培养液于28°C培养时,病毒增殖量最高,为5.59×10^(10)拷贝/mL;而从单位成本所获病毒量考虑,同步接毒法筛选出的4种增殖条件单位成本所获的病毒产量优于异步接毒法,其中当MOI=0.03、胎牛血清终浓度为6%时,同步接种对数生长中期的CPB细胞,28°C恒温培养70 h后收获病毒液,单位成本所获病毒量最高,为4.88×10^(13)个拷贝/元。综上所述,本研究以病毒增殖量和培养基成本作为考量,优化SCRV-QY株的增殖条件,可为SCRV疫苗低成本、规模化生产提供理论依据。The optimal proliferation condition of Siniperca chuatsi rhabdovirus(SCRV)in CPB cells was determined through detecting the viral copy numbers by Real-Time Reverse Transcription PCR(qRT-PCR).Two inoculation methods including synchronous and asynchronous inoculation and different culture conditions such as the amount of inoculated virus,FBS concentration of the culture medium were investigated in this study.The virus was harvested when the CPE reached 80%and then RNA was extracted for qRT-PCR detection.Results showed that all the above factors had obvious influence on the SCRV proliferation in vitro.When compared with the viral yields in unit volume,asynchronous inoculation method was superior to the synchronous inoculation.The highest amount of virus(5.59×1010 copies/mL)were obtained when cultured in L-15 medium containing 2%fetal bovine serum at 28°C for 48 h with MOI=10 and inoculation for 1.5 h.While the viral yields with unit cost was compared,4 proliferation conditions with synchronous inoculation method were singled out which were superior to asynchronous inoculation.The highest viral yields per unit cost(4.88×1013 copies/yuan)were obtained when SCRV was inoculated to the CPB cells at the middle-log phase with a MOI=0.03 and cultured with L-15 medium containing 6%fetal bovine serum and harvested after culturing at 28°C for 70 h.In conclusion,viral yield and the medium cost were studied as the assessment criteria for SCRV proliferation in CPB cells in this paper,and it provided the theoretical basis for SCRV vaccine production with low cost through the comprehensive viral yield analysis of the medium cost.

关 键 词：鳜弹状病毒 鳜脑细胞 接毒方法 增殖条件 实时荧光定量PCR 

分 类 号：S941.4[农业科学—水产养殖]