2A肽切割的验证

Verification of 2A peptide cleavage.

作者信息

Szymczak-Workman Andrea L, Vignali Kate M, Vignali Dario A A

出版信息

Cold Spring Harb Protoc. 2012 Feb 1;2012(2):255-7. doi: 10.1101/pdb.prot067892.

Abstract

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. The easiest and most effective way to assess 2A cleavage is to perform transient transfection of 293T cells (human embryonic kidney cells) followed by western blot analysis, as described in this protocol. 293T cells are easy to grow and can be efficiently transfected with a variety of vectors. Cleavage can be assessed by detection with antibodies against the target proteins or anti-2A serum.

摘要

用于多基因递送的可靠多顺反子载体的需求处于生物医学技术的前沿。现在可以使用2A肽连接的多顺反子载体从单个开放阅读框（ORF）表达多种蛋白质。这些小序列克隆在基因之间时，通过2A肽序列内的新型“切割”事件，能够在单个载体内高效、按化学计量产生离散的蛋白质产物。评估2A切割的最简单、最有效的方法是按照本方案所述，对293T细胞（人胚胎肾细胞）进行瞬时转染，然后进行蛋白质印迹分析。293T细胞易于培养，并且可以用多种载体高效转染。可以通过用针对靶蛋白的抗体或抗2A血清进行检测来评估切割情况。