利用杆状病毒颗粒在哺乳动物细胞中表达一种分泌蛋白。

Expression of a secreted protein in mammalian cells using baculovirus particles.

作者信息

Jardin Barbara Ann, Elias Cynthia B, Prakash Satya

机构信息

Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering, Artificial Cells and Organs Research Center, McGill University, Montreal, QC, Canada.

出版信息

Methods Mol Biol. 2012;801:41-63. doi: 10.1007/978-1-61779-352-3_4.

DOI:10.1007/978-1-61779-352-3_4

PMID:21987246

Abstract

There are many methods presently available to produce recombinant proteins in mammalian systems. The BacMam system is a simple straightforward method which overlaps two well-established technologies, namely the BEVS insect cell system and the transduction of mammalian cells in vitro. This chapter describes a method for the study of gene expression in mammalian cells in a series of simple steps. Protocols outlined include the design and construction of the recombinant baculovirus, cell culture techniques required to maintain both insect and mammalian cells, generation of baculovirus stocks, and methods to obtain maximal and reproducible gene expression in mammalian cells. Currently available statistical techniques using factorial design of experiment to optimize conditions for recombinant protein in vitro are outlined. Then details with respect to process scale-up in disposable bioreactors are included.

摘要

目前有许多方法可用于在哺乳动物系统中生产重组蛋白。杆状病毒表达载体（BacMam）系统是一种简单直接的方法，它结合了两种成熟的技术，即杆状病毒表达载体（BEVS）昆虫细胞系统和体外哺乳动物细胞转导技术。本章通过一系列简单步骤描述了一种研究哺乳动物细胞中基因表达的方法。概述的实验方案包括重组杆状病毒的设计与构建、维持昆虫细胞和哺乳动物细胞所需的细胞培养技术、杆状病毒储备液的制备，以及在哺乳动物细胞中获得最大且可重复的基因表达的方法。概述了目前使用实验因子设计来优化体外重组蛋白条件的统计技术。随后还包括了一次性生物反应器中工艺放大的详细内容。