摘要
对一种哺乳动物的非贴壁细胞Bal b/c近交系小鼠肥大细胞瘤细胞株P815进行了G418敏感浓度及甘油耐受性的测定;并通过磷酸钙法用含HBVS基因的质粒pRc/CMVS与含筛选标记基因neo的质粒pSV_2-neo进行了共转染。经G418筛选,获得了G418抗性细胞P851-S,在G418选择压力下,已连续筛选80余天,传代12次;斑点杂交和ELISA实验分别表明P815-S细胞内有HBVS基因的存在,培养上清中有HBsAg的表达。
Bal b/c inbred mice mastocytoma cell line P815 (a kind of non-attaching-wall mammalian cells) was tested to determine its sensitive concentration to G418 and whether it could tolerate glycerine; then, with calcium phosphate method, P815 cells were cotransfected by a plasmid (pRc/CMVS) containing HBV-S gene and a plasmid (pSV2-neo) containing neomycin-recistance gene (marker gene). By selection with G418, G418 resistant cells (P815-S) were obtained, and were already selected under the pressure of G418 for more than 80 days, 12 generations continuously. The results of dot-blot and hybnding and ELISA showed that HBV-S genes exist in P815-S cells; HBVS proteins are secreted into culture medium of P815-S cells. These results indicated that cotransfection was successful.
出处
《生物技术通讯》
CAS
1998年第1期28-31,共4页
Letters in Biotechnology
关键词
共转染
质粒
表达
非贴壁细胞
甘油“休克”
cotransfection
plasmid
expression
non-attaching-well cells
glycerine 'shock
