通过抑制PDHK3的表达降低乳酸水平增加CHO细胞抗体表达量

Effect of PDHK3 Gene Expression Inhibition on lactate Level and Antibody Expression of CHO Cells

目的:通过抑制乳酸产生相关基因丙酮酸脱氢酶激酶3(PDHK3)的表达,降低CHO细胞系的乳酸表达,增加CHO细胞系蛋白表达量。方法:针对PDHK3序列设计特定的shRNA质粒,在CHO细胞系构建过程中将含有此shRNA的质粒与表达抗体蛋白的质粒一同转染至宿主细胞中,经过抗生素筛选后进行批次补料生产抗体蛋白。测定乳酸及蛋白产量,同时检测PDHK3基因的mRNA表达水平及shRNA质粒中的neomycin基因拷贝数。结果:利用此方法分别转染的不同抗体蛋白分子,PDHK3基因的mRNA水平降低了65.0%~76.0%,批次补料过程乳酸含量显著降低,同时相应的抗体蛋白表达量提升了18.6%~233.0%。结论:利用shRNA方法抑制乳酸相关的PDHK3基因的表达,可以有效降低乳酸表达,同时提高目的蛋白的表达。

Objective:We Aim to decrease lactate level and increase the protein titer of the CHO cells by reducing the expression of pyruvate dehydrogenase kinases3.Methods:Designed specific shRNA plasmids targeting pyruvate dehydrogenase kinase 3(PDHK3)sequence,and transfected them with expression protein plasmids into host cells during CHO cells construction process.After selection with antibiotics,produced antibody by fed-batch.The lactate and antibody productivity were tested.At the meanwhile,the cells were assayed for the PDHK3 mRNA expression level as well as the copy number of the neomycin gene in shRNA plasmids.Results:Different antibody protein molecules were transfected with this method,the PDHK3 mRNA expression level decreased by 65.0%~76.0%,thus the lactate production significantly reduced,as well as the productivity of the corresponding antibody proteins were increased by 18.6%to 233.0%.Conclusion:This study showed that downregulating PDHK3 expression by shRNAmethodcanreduced the lactate level and increased the antibody production.