同步接毒条件下鳜传染性脾肾坏死病毒在CPB细胞中的最适增殖条件  

Study on optimal proliferation conditions of infectious spleen and kidney necrosis virus of Siniperca chuatsi in CPB cells with synchronous inoculation

作　　者：罗霞[1] 付小哲[1,2] 李宁求[1,2] 林强[1,2] 张悠[1] 黄志斌[1] 

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 [2]淡水水产健康养殖湖北省协同创新中心,湖北武汉430070

出　　处：《水产学报》2015年第11期1712-1720,共9页Journal of Fisheries of China

基　　金：广东省教育部产学研结合专项(2012B091100164);国家科技支撑计划(2012BAD25B02);国家自然科学基金(31202032)~~

摘　　要：为了获知鳜传染性脾肾坏死病毒(ISKNV)同步接毒最适增殖条件,通过检测不同血清浓度、病毒接种量、细胞状态等培养条件下的病毒拷贝数,确定鳜脑组织细胞(CPB)同步接种ISKNV的最适体外增殖条件。结果表明,上述各因素对ISKNV的增殖量均有明显影响。其中选取处于对数生长中期的CPB细胞,胰酶消化后按照病毒感染复数(MOI)为0.2同步接种ISKNV,培养液中胎牛血清终浓度为6%时,28℃恒温培养9 d后收获,所得病毒拷贝数最多,为2.54×108拷贝/m L。将所测得病毒拷贝数折算成培养基成本进一步分析表明,按照上述相同条件进行接毒,每元人民币培养基所得病毒量也最高,为4.24×1011拷贝。综上所述,本研究基于病毒增殖量及培养基成本对病毒增殖条件进行优化,可为低成本ISKNV疫苗的生产提供理论依据。The optimal proliferation conditions of infectious spleen and kidney necrosis virus（ ISKNV） in CPB cells with synchronous inoculation method were confirmed through detecting the viral copy numbers.The virus which proliferated with different FBS concentration,amount of inoculated virus,cell state,and viral harvest time was harvested and then DNA was extracted for q PCR detection. Results showed that all of the above factors had obvious influence on the ISKNV proliferation in vitro. It would obtain the highest amount of virus（ 2. 54 × 10^8 copies / m L） under the conditions that the MOI was 0. 2,the virus was inoculated to the CPB cells cultured for 48 h at the middle-log phase and cultured with L-15 medium containing 6% fetal bovine serum and harvested after culturing at 28 ℃ for 9 days. The average cost of viral yield per yuan and per day was further analyzed. The results indicated that if the culture medium cost was only considered,it would obtain the highest viral yield（ 4. 24 × 10^11copies） per yuan as the above method; however,if it was as far as possible to shorten the time of cell culture and virus culture for saving time cost,it would obtain the highest viral yield per day（ 3. 64 × 10^7copies） under the conditions that the MOI was 4,virus was inoculated to the CPB cells cultured for 24 h at the early-log phase and cultured with L-15 medium containing 10%fetal bovine serum and harvested after culturing at 28 ℃ for 4 days. In conclusion,this paper will provide the theoretical basis for ISKNV vaccine production with lowcost through the comprehensive viral yield analysis of the medium cost and culture time cost.

关 键 词：鳜 传染性脾肾坏死病毒 鳜脑细胞 同步接毒 荧光定量PCR 增殖条件 

分 类 号：S941[农业科学—水产养殖]