转染siat7e基因的MDCK细胞悬浮性状分析与质控  

Analysis and quality control of suspension characteristics of MDCK cells transfected with siat7e gene

作　　者：王革[1] 马超[1] 刘晓凡[1] 李薇[1] 刘鹏[1] 魏然[1] 

机构地区：[1]兰州生物制品研究所有限责任公司,甘肃兰州730046

出　　处：《中国生物制品学杂志》2016年第1期75-78,共4页Chinese Journal of Biologicals

摘　　要：目的分析转染siat7e基因的MDCK细胞(MDCK-siat7e)的悬浮特性,并建立相关质控方法。方法将MDCKsiat7e细胞接种无血清培养基BD001,37℃,130 r/min振摇培养11 d,显微镜观察不同培养时间细胞形态,并检测细胞生长浓度、活力及代谢状况。提取MDCK-siat7e细胞的m RNA,采用RT-PCR法扩增siat7e基因片段并测序。通过Real time PCR法检测不同代次(原代、10、20、30代)MDCK-siat7e细胞siat7e基因的表达量。结果 MDCK-siat7e细胞在无血清培养基中呈悬浮生长状态,细胞接种培养初期生长较慢,但活力较好;然后细胞生长经停滞期进入生长期,细胞生长加快且活力大于98%,细胞浓度达峰点后生长进入平台期;培养后期细胞浓度降低且活力下降。原代与传代细胞siat7e基因序列测定结果与Gen Bank公布的ST6Gal Nac V序列(AJ507292.1)一致。MDCK-siat7e细胞传至30代,仍可表达siat7e,表明外源siat7e基因整合入MDCK细胞基因组中,并随MDCK细胞传代持续地转录和表达。结论本实验对siat7e基因的定性及表达量的分析可用于MDCK-siat7e细胞悬浮性的检测,为其作为疫苗用细胞基质的质量监控提供了依据。Objective To analyze the suspension characteristics of MDCK cells transfected with siat7 e gene and develop the method for quality control. Methods MDCK-siat7 e cells were inoculated into serum-free medium BD001, cultured at37 ℃ by shaking at a speed of 130 r / min for 11 d, observed for morphology at various time points by microscopy, and determined for concentration, vigor and metabolism. The m RNA of MDCK-siat7 e cells were extracted, with which siat7 e gene was amplified by RT-PCR and sequenced. The expression levels of siat7 e gene in MDCK-siat7 e cells of various passages（primary, 10, 20 and 30） were determined by real-time PCR. Results MDCK-siat7 e cells grew in suspended state in free-serum medium, which grew slowly but showed high vigor at early stage of culture. However, in exponential growth phase after lag phage, the cells grew more quickly, of which the vigor was more than 98%, and the concentration reached a peak value then entered into a plateau phage. At the late stage of culture, both the concentration and vigor of cells decreased. The sequencing results of siat7 e gene in primary and subcultured cells were consistent with that of ST6 Gal Nac V（AJ507292. 1） in Gen Bank. The siat7 e gene was still expressed in MDCK-siat7 e cells after subculture to passage 30, indicating than the siat7 e gene was integrated to the genome of MDCK cells and transcribed and expressed continuously with the subculture. Conclusion Analysis of suspension characteristics and expression level of siat7 e gene might be used for the determination of suspensibility of MDCK-siat7 e cells, which provided a basis for the monitor of quality of the cells as a matrix for vaccine.

关 键 词：MDCK-siat7e细胞 悬浮 siat7e基因 

分 类 号：Q26[生物学—细胞生物学]