动物细胞双顺反子高效稳定表达系统建立  

Establishment of a Stable Bicistronic System in Animal Cells with high Efficiency

作　　者：黄立强[1] 孙奋勇[2] 林珊莉[1] 吴翠玲[1] 

机构地区：[1]广州生物工程中心 [2]暨南大学生物工程研究所

出　　处：《中国生物工程杂志》2004年第10期52-55,共4页China Biotechnology

基　　金：国家"8 63"计划资助项目 (2 0 0 2AA2Z3 44E) ;广州市科技攻关计划资助项目 (2 0 0 2 Z 0 0 1 0 1)

摘　　要：建立稳定高效的动物细胞表达系统对于许多重组蛋白质药物的工业化生产有着十分重大的意义。用PCR扩增得到dhfr基因 ,克隆入载体pIRESneo3基因以替换原有的neor 基因 ,并将hbFGF作为报告基因插入其多克隆位点 ,得到重组子pIRESdhfr hbFGF ,转导CHO细胞后 ,MTX浓度梯度筛选稳定表达hbFGF的细胞株 ,ELISA与WesternBlot检测培养基中hbFGF的分泌表达。结果发现 1 0、1 0 0、1 0 0 0nmol LMTX组均检测到hbFGF的表达 ,其中表达量最高的一株细胞传代 2 0次后表达量仍能维持较高的水平。The establishment of a stable animal cells expression system with high efficiency is of great importance for industrialized production of various recombinant drug. dhfr gene,obtained by PCR,was used to replaced the neo r gene of the vector pIRESneo3 to produce a new vector named pIRESdhfr,hbFGF as a reporter gene,was inserted into the MCS of pIRESdhfr,the recombinant pIRESdhfr\|hbFGF was transducted into CHO-dhfr\+-,after screening with MTX,the secretory expression of hbFGF was tested by ELISA and Western Blot.In 10,100,1000 nmol/L MTX groups,hbFGF was expressed with high efficiency persisting a long time.So,bicistronic expression system can help to improve the expression level of a gene with interest in a stable manner.

关 键 词：FGF 稳定表达 IRES 基因 转导 MTX 细胞株 动物细胞 表达系统 重组蛋白质 

分 类 号：Q786[生物学—分子生物学] Q813.1