动物细胞双顺反子高效稳定表达系统建立

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neo^r 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10、100、1000nmol LMTX组均检测到hbFGF的表达 ,其中表达量最高的一株细胞传代20次后表达量仍能维持较高的水平。因此利用双顺反子表达系统可以实现外源基因在动物细胞中的高效稳定表达 。

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

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.