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悬浮培养HEK-293 N3S细胞生产重组腺病毒Ad-GFP的实验研究
引用本文:田博,吴彬,张群伟,毕建进,王澜,朱宝珍,耿越,吴祖泽.悬浮培养HEK-293 N3S细胞生产重组腺病毒Ad-GFP的实验研究[J].生物工程学报,2007,23(5):915-918.
作者姓名:田博  吴彬  张群伟  毕建进  王澜  朱宝珍  耿越  吴祖泽
作者单位:1. 山东师范大学生命科学学院,济南,250014
2. 军事医学科学院放射与辐射医学研究所实验血液学实验室,北京,100850
基金项目:国家高技术研究发展计划(863计划)
摘    要:利用5L生物反应器悬浮培养HEK-293N3S细胞生产携带绿色荧光蛋白基因的重组腺病毒(recombinant adenovirus-greenfluorescent protein,Ad-GFP),为规模化生产腺病毒基因药物建立一种稳定可行的生产工艺。复苏的种子细胞进行逐级放大最后接入5L搅拌式生物反应器中,采用含5%胎牛血清(FBS)的DMEM/F12培基灌流培养293N3S细胞,当细胞密度达到(2~4)×106个/mL时感染Ad-GFP,48h后收获细胞,经两步氯化铯超速离心获得纯化的Ad-GFP。采用紫外分光光度计比色法和高压液相色谱法(HPLC)测定病毒颗粒数和纯度,采用组织培养半数感染剂量(TCID50)法检测腺病毒的感染滴度。连续培养10~12d,细胞密度可达到(2~4)×106个/mL左右,纯化的Ad-GFP感染滴度和颗粒数分别为1.0×1011IU/mL和1.68×1012VP/mL,比活性为6.0%,A260/A280比值为1.33,产品纯度达到99.2%。建立了5L生物反应器悬浮培养293N3S细胞生产重组腺病毒Ad-GFP的生产工艺,对携带其他基因的重组腺病毒药物生产具有一定的指导意义。

关 键 词:悬浮培养  重组腺病毒  HEK-293  N3S细胞  生物反应器
文章编号:1000-3061(2007)05-0915-04
修稿时间:2007-01-05

Producing Recombinant Adenovirus Encoding Green Fluorescent Protein (Ad-GFP) by Suspension Cultured HEK-293 N3S Cells
TIAN Bo,WU Bin,ZHANG Qun-Wei,BI Jian-Jin,WANG Lan,ZHU Bao-Zhen,GENG Yue and WU Zu-Ze.Producing Recombinant Adenovirus Encoding Green Fluorescent Protein (Ad-GFP) by Suspension Cultured HEK-293 N3S Cells[J].Chinese Journal of Biotechnology,2007,23(5):915-918.
Authors:TIAN Bo  WU Bin  ZHANG Qun-Wei  BI Jian-Jin  WANG Lan  ZHU Bao-Zhen  GENG Yue and WU Zu-Ze
Institution:Academy of Life Science, Shandong Normal University, Jinan 250014, China;Institute of Radiation Medicine, Academy of Military Medical Science, Beijing 100850, China;Institute of Radiation Medicine, Academy of Military Medical Science, Beijing 100850, China;Institute of Radiation Medicine, Academy of Military Medical Science, Beijing 100850, China;Institute of Radiation Medicine, Academy of Military Medical Science, Beijing 100850, China;Institute of Radiation Medicine, Academy of Military Medical Science, Beijing 100850, China;Academy of Life Science, Shandong Normal University, Jinan 250014, China;Institute of Radiation Medicine, Academy of Military Medical Science, Beijing 100850, China
Abstract:Adenovirus vectors are one of the most promising gene transfer systems. They are of great value for gene therapy because these vectors achieve temporal high-level transgene expression and high gene transfer efficiency. To meet increasing needs of adenovirus vectors for gene therapy programs, parallel development of efficient, scalable and reproducible production processes is required. Perfusion cultivation of 293 cells is one of the most commonly used methods to produce adenovirus vectors and it is suitable for industrialized production specially. Experimental studies had been carried out to produce recombinant adenovirus containing the green fluorescent protein gene (Ad-GFP) by perfusion cultivation of HEK-293 N3S cells in a 5L stirring bioreactors. Perfusion rate was 1-2 volume/day. To infect the 293 N3S cells with Ad-GFP at the density of (2-4) x 10(6) cells/ ml. The time of collecting cells was 48 hours post infection. After three rounds of freeze/thaw and centrifugation, the crude viral lysates were stored at--80 degrees C until use. Then to get the Ad-GFP products by 2 x CsCl-gradient purification. The purity of the products was determined by the A260/A280 ratio and a high performance liquid chromatography (HPLC) assay. The infective titer was determined by a TCID50 assay. The culture term was 10-12 days. The infectious titer, the number of virus particle and the ratio of infectious titer to virus particle for the product were 1.0 x 10(11) IU/mL, 1.68 x 10(12) VP/mL and 6.0% IU/VP respectively. The A260/A280 ratio was 1.33, and the purity determined by HPLC was 99.2%. The cell specific productivity was around 1000 IU/cell. By perfusion cultivation of 293 N3S cells in a 5L stirring bioreactors, we established the production process for Ad-GFP, which paves a way to produce other recombinant adenovirus for gene therapy.
Keywords:suspension culture  recombinant adenovirus  HEK-293 N3S cells  bioreactor
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