CAJ | 학술논문

为了优化中国仓鼠卵巢细胞（ CHO）表达体系，建立高表达CHO细胞株筛选方法，将外源基因表达载体上抗性筛选基因表达的新霉素磷酸转移酶（ NPT）的261位氨基酸天冬氨酸突变成甘氨酸。经G418筛选，转染含突变型NPT表达载体的细胞存活率显著低于转染含野生型NPT表达载体的细胞存活率。以绿色荧光蛋白-抗体IgG1 Fc结构域融合蛋白为报告基因，验证了突变后新霉素磷酸转移酶对抗生素G418抗性减弱。经G418持续加压培养3周后，转染含突变型NPT表达载体的细胞中EGFP的表达量显著高于转染含野生型NPT表达载体的细胞，表明其具有筛选出高表达单克隆细胞株的潜力。
위료우화중국창서란소세포（ CHO）표체체계，건립고표체CHO세포주사선방법，장외원기인표체재체상항성사선기인표체적신매소린산전이매（ NPT）적261위안기산천동안산돌변성감안산。경G418사선，전염함돌변형NPT표체재체적세포존활솔현저저우전염함야생형NPT표체재체적세포존활솔。이록색형광단백-항체IgG1 Fc결구역융합단백위보고기인，험증료돌변후신매소린산전이매대항생소G418항성감약。경G418지속가압배양3주후，전염함돌변형NPT표체재체적세포중EGFP적표체량현저고우전염함야생형NPT표체재체적세포，표명기구유사선출고표체단극륭세포주적잠력。
To optimize Chinese hamster ovary (CHO)expression system and establish a process of screening CHO cell lines with high productivity;neomycin-phosphotransferase (NPT)expressed by the resistance marker gene on the expression vector was mutated with amino acid D at 261 changed to G.After selection by culturing with G418;the survival rate of CHO cells bearing mutant-NPT was significantly lower than that of the cells bear-ing wide type NPT.An enhanced green fluorescent protein (EGFP)was genetically linked to the N terminus of the IgG1 Fc fragment part to generate an EGFP-Fc fusion protein regarded as a report gene;which verified that the resistance of mutant-NPT to G418 was weakened.By comparing fluorescence assay of EGFP intensity in stable transfections after selection with the same concentration of G418 for 3 weeks;mutant-selected pools expressed more exogenous protein than the WT-selected pools.Therefore;the ratio of high producers in a transfected cell population greatly increased.