[1]冯磊,吴培培,褚轩,等.稳定表达人TMPRSS2蛋白质悬浮生长MDCK细胞系的构建[J].江苏农业学报,2015,(05):1084-1090.[doi:doi:10.3969/j.issn.1000-4440.2015.05.022]
 FENG Lei,WU Pei-pei,CHU Xuan,et al.Establishment of suspension-cultured MDCK cell line expressing type II transmembrane serine protease(TMPRSS2)[J].,2015,(05):1084-1090.[doi:doi:10.3969/j.issn.1000-4440.2015.05.022]
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稳定表达人TMPRSS2蛋白质悬浮生长MDCK细胞系的构建()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2015年05期
页码:
1084-1090
栏目:
畜牧兽医·水产养殖
出版日期:
2015-10-31

文章信息/Info

Title:
Establishment of suspension-cultured MDCK cell line expressing type II transmembrane serine protease(TMPRSS2)
作者:
冯磊吴培培褚轩陈丽王伟峰侯继波
(江苏省农业科学院/国家兽用生物制品工程技术研究中心,江苏南京210014) 
Author(s):
FENG LeiWU Pei-peiCHU XuanCHEN LiWANG Wei-fengHOU Ji-bo
(Jiangsu Academy of Agricultural Sciences/National Research Center of Engineering and Technology for Veterinary Biologicals, Nanjing210014, China)
关键词:
MDCK细胞稳定转染跨膜型丝氨酸蛋白酶禽流感病毒
Keywords:
MDCK cellstable transfectiontransmembrane serine proteaseavian influenza virus
分类号:
Q813.1+1
DOI:
doi:10.3969/j.issn.1000-4440.2015.05.022
文献标志码:
A
摘要:
为了构建能稳定表达人Ⅱ型跨膜型丝氨酸蛋白酶(TMPRSS2)的重组MDCK细胞,并考察该重组细胞株对禽流感H9亚型病毒的增殖能力,采用基因工程的方法,将TMPRSS2基因克隆入真核表达载体中获得重组表达载体pIRES-TMPRSS2。采用25 000线性PEI进行悬浮生长MDCK细胞的转染操作,经两轮G418抗性筛选获得能够稳定表达该蛋白质的重组MDCK-Sus-TMPRSS2细胞。RT-PCR、Western blot以及间接免疫荧光检测(IFA)的结果显示,TMPRSS2已在该重组细胞株中正常表达。该重组细胞的悬浮比生长速率达到0.438 d-1;在3 L反应器中,最大细胞密度可达到1 ml 6.83×106个。在对6株禽流感H9亚型病毒的连续盲传试验中,经MDCK-Sus-TMPRSS2细胞盲传的子代病毒可以获得较高的血凝效价和半数组织感染滴度。
Abstract:
To establish recombinant MDCK cells expressing type II transmembrane serine protease(TMPRSS2) and to investigate the influence of the MDCK cells on the replication of avian influenza virus, the gene of TMPRSS2 was cloned into the eukaryotic expression vector to generate recombinant pIRES-TMPRSS2. The recombinant plasmid was transfected into MDCK-Sus cells by linear 25 000 polyethyleneimine (PEI) by means of single-cell suspension culture. The recombinant MDCK-Sus-TMPRSS2 cell clone was achieved by two rounds of resistance selection to G418 solution. Western blot and IFA test revealed that TMPRSS2 protein was expressed and was located on cell membrane. The specific suspension growth rate and the maximum cell density of recombinant MDCK-Sus-TMPRSS2 cells reached 0.438 d-1 and 1 ml 6.83×106 cells. Six strains of avian influenza virus subtype H9 were adapted to 15 passages in recombinant MDCK-Sus-TMPRSS2 cells, and acquired much higher HA titers and 50% tissue culture infective doses(TCID50) than those in parental MDCK-Sus cells.〖JP〗

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备注/Memo

备注/Memo:
收稿日期:2015-02-14 基金项目:农业部公益性行业(农业)科研专项经费项目( 201303046);江苏省农业科技自主创新基金项目[SCX(13)5036] 作者简介:冯磊(1979-),男,江苏南通人,博士,副研究员,从事兽用生物制品工程技术研究。(Tel)025-84392068; (E-mail)fenglnt@hotmail.com 通讯作者:侯继波,(E-mail)houjibo@jaas.ac.cn
更新日期/Last Update: 2015-10-31