摘要
为优化草鱼(Ctenopharyngodon idella)鳔细胞(CiSB)细胞悬浮培养工艺,以提高基因Ⅱ型草鱼呼肠孤病毒(grass carp reovirus,GCRV)含量,利用Cytodex 1微载体悬浮培养系统规模化培养CiSB和Ⅱ型GCRV,对CiSB细胞初始接种密度、搅拌转速和微载体浓度等工艺参数进行摸索和优化。结果显示,在CiSB细胞贴壁期,以转速30 r·min^(-1)、每静置30 min搅拌2 min的间歇搅拌方式培养最佳,4 h后细胞贴附率可达96%以上;在微载体密度2 g·L^(-1)、细胞初始接种密度2×10^(5)个·mL^(-1)、搅拌速度30 r·min^(-1)的条件下,初始培养血清浓度设定为10%,培养3 d后更换50%的培养基并使血清浓度达到5%,可获得最佳的细胞生长效能。CiSB经消化转移放大至1 L,微载体上细胞贴附均匀、生长良好。接种Ⅱ型GCRV至规模化培养的CiSB细胞,拷贝数最高达6.2×10^(5)拷贝数·μL^(-1)。研究结果可为草鱼出血病疫苗的规模化生产工艺研究提供依据。
Grass carp(Ctenopharyngodon idella)is one of the most important cultured fish species in China,it is appreciated by the majority of breeders and consumers for its fast growth rate,diverse feed sources and delicious meat.Grass carp hemorrhagic disease caused by grass carp reovirus genotypeⅡ(GCRVⅡ)has seriously hindered the development of the grass carp farming industry with a long onset season,wide epidemic scope,rapid transmission and high mortality rate.Vaccination is the most effective method for the control of viral diseases.However,viral vaccine production requires a large-scale culture of the host cells.In the current race for virus vaccine production,microcarrier and suspension culture systems are amongst the most promising techniques available.As the microcarrier bead has a large surface area for cell attachment,it can produce a larger number of cells than the conventional monolayer culture.The current challenge in GCRVⅡ only induces a few fish cells proliferating and does not produce cytopathic effects(CPE),making it difficult to obtain high titers of the virus.Grass carp swim bladder cells(CiSB)are sensitive to GCRVⅡ and can be used in the proliferation of GCRVⅡ.To optimize the large-scale cell culture of grass carp swim bladder cells(CiSB)for improving the antigenic content of GCRVⅡ,the Cytodex 1 microcarrier suspension culture system was chosen to cultivate CiSB cells and propagate GCRVⅡ.At the same time,the related indicators of proliferation dynamics were determined.The initial distribution of attached cells was recognized as the most critical stage for evaluating attachment efficiency.Suitable intermittent stirring could improve the contact time and also the attachment rate.During the apposition period,a speed of 30 r·min^(-1) and stirring for 2 min every 30 min were the best parameters for CiSB cell growth,and the cell apposition rate could reach more than 96% after 3 h.Next,in the cell proliferation stage,the conditions such as the initial inoculation density,stirring speed,microcar
作者
杨玉茹
王英英
王庆
周文礼
尹纪元
石存斌
YANG Yuru;WANG Yingying;WANG Qing;ZHOU Wenli;YIN Jiyuan;SHI Cunbin(College of Fisheries,Tianjin Agricultural University,Tianjin 300384,China;Key Laboratory of Fishery Drug Development of Ministry of Agriculture and Rural Affairs,Key Laboratory of Aquatic Animal Immune Technology of Guangdong Province,Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510380,China)
出处
《海洋渔业》
CSCD
北大核心
2022年第5期577-588,共12页
Marine Fisheries
基金
国家重点研发计划(2019YFD0900103)
中国水产科学研究院中央公益性科研院所基本科研业务费专项(2020XT0403)
广东省基础与应用基础研究基金(2020A1515010327)
国家大宗淡水鱼产业技术体系(CARS-45)
广东省现代农业产业技术体系创新团队建设专项资金(2022KJ150,2022KJ119)。
关键词
草鱼鳔细胞(CiSB)
Ⅱ型草鱼呼肠孤病毒
微载体培养
工艺优化
grass carp swim bladder cells(CiSB)
genotype Ⅱ grass carp reovirus
microcarrier culture
technology optimization
