Szymczak-Workman Andrea L, Vignali Kate M, Vignali Dario A A
Cold Spring Harb Protoc. 2012 Feb 1;2012(2):251-4. doi: 10.1101/pdb.prot067884.
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. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector. This protocol describes the use of recombinant polymerase chain reaction (PCR) to connect multiple 2A-linked protein sequences. The final construct is subcloned into an expression vector.
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，可在单个载体内高效、按化学计量产生离散的蛋白质产物。使用传统方法表达两个以上基因存在几个局限性，最明显的是蛋白质表达不平衡和载体尺寸大。2A肽序列的使用缓解了这些问题。它们很小（18 - 22个氨基酸），并且具有不同的氨基末端序列，这最大限度地减少了同源重组的机会，并允许在单个载体内使用多个不同的2A肽序列。重要的是，置于2A肽序列之间的基因分离率接近100%，这允许基因按化学计量和一致地表达，而不管其在载体内的放置顺序如何。本方案描述了使用重组聚合酶链反应（PCR）连接多个2A连接的蛋白质序列。最终构建体亚克隆到表达载体中。
