Large-Scale Transfection of Mammalian Cells
Large-Scale Transfection of Mammalian Cells
复制标题
DOI：
10.1007/978-1-61779-352-3_2
复制
发表时间：
2012-01-01
期刊：
影响因子：
--
通讯作者：
Wurm, Florian M.
中科院分区：
文献类型：
--
作者：
Baldi, Lucia;Hacker, David L.;Wurm, Florian M.
哺乳动物细胞的大规模转染允许获得中等（毫克至克）量的重组蛋白（r-蛋白）用于基础或临床研究。在这篇文章中，我们描述了一个1升的转染使用聚乙烯亚胺（PEI）的DNA递送到人胚肾（HEK-293）细胞培养在无血清悬浮液中，以产生重组，人单克隆抗体，产量高达约1克/升，在10天的过程。该方法基于在高细胞密度（20 x 10（6）个细胞/mL）下通过向培养物中直接添加DNA和PEI进行的DNA递送步骤。随后，在存在丙戊酸（VPA）（一种组蛋白脱乙酰酶抑制剂）的情况下，将细胞稀释20倍，用于10天生产期。还描述了质粒纯化、酶联免疫吸附测定（ELISA）抗体定量和蛋白A亲和纯化的方法。
The large-scale transfection of mammalian cells allows moderate (milligram to gram) amounts of recombinant proteins (r-proteins) to be obtained for fundamental or clinical research. In this article, we describe a one-liter transfection using polyethyleneimine (PEI) for DNA delivery into human embryonic kidney (HEK-293) cells cultivated in serum-free suspension to produce a recombinant, human monoclonal antibody that yields up to about 1 g/L in a 10-day process. The method is based on a DNA delivery step performed at high cell density (20 x 10(6) cells/mL) by direct addition of DNA and PEI to the culture. Subsequently, the cells are diluted 20-fold for the 10-day production phase in the presence of valproic acid (VPA), a histone deacetylase inhibitor. The methods for plasmid purification, antibody quantification by enzyme-linked immunosorbent assay (ELISA), and affinity purification with protein A arc also described.