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Construction of the Cell Line Chinese Hamster Ovary Expressing High-level Recombinant Darbepoetin Alfa (NESP)

Author: WuJianYong
Tutor: SunXiangMingï¼›YiXiaoPing
School: East China University of Science and Technology
Course: Biochemical Engineering
Keywords: Long-acting EPO Site Integration CHO cells
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 53
Quote: 0
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Abstract


This article first pFRT / lacZeo plasmid transfected CHO-K1 cells get the cell lines CHO/FRT2 sites with high activity via galactosidase activity detection screening password by site-directed mutagenesis of NESPl ( patent reported sequence ) sequence the sub-optimization to give NESP2, NESP3, and constructed expression vector pcDNA5/FRT/NESP . The expression vector the pOG44 plasmid containing the FLP gene of genetically CHO/FRT2 cells obtained the cell lines CHO/FRT2/NESP efficient expression of recombinant NESP : the NESP1 , NESP2 NESP3 expression levels were 786 IU / ml , 1315 IU / ml, 1029 IU / ml, indicating that after codon optimization NESP2 having a higher protein expression levels . Further, the carrier pcDNA5/FRT/NESP2 transfected CHO / FRT cells , ELISA detection result Showing NESP2 the expression of galactosidase activity level trend confirmed galactosidase as a screening indicator protein , can be used to screen effective protein expression in the hot zone . Thereafter, the blue gel affinity chromatography, gel filtration, ion exchange chromatography purified recombinant CHO/FRT2/NESP2 cell culture supernatant to obtain a certain amount of preliminary purification of recombinant NESP protein samples . By SDS-PAGE electrophoresis and Western blot analysis confirmed that , relative to the prototype of EPO , NESP has a higher molecular weight . Isoelectric focusing analysis results also further confirm the NESP ratio EPO has a higher sialic acid abundance , a higher degree of glycosylation .

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