含有分裂型内含肽DnaE基因的表达盒设计、合成及高效表达载体的构建

目的:建立一种简便、快捷的基因从头设计、优化与合成策略,进行含有分裂型内含肽DnaE基因的表达盒全合成并构建高效表达载体。方法:以免费软件GeneDesign 3.0为主要平台,同时结合Tandem Repeats Finder、UNAFold等不同生物信息学软件,对含有DnaE基因、合适酶切位点的表达盒进行设计与分段合成;合成寡核苷酸片段通过重叠PCR进行组装与克隆。结果:利用建立的设计流程,合成了大小为44～64 nt的14段寡核苷酸片段,通过重叠PCR,实现了14段寡核苷酸片段的一次性组装,经过克隆、酶切鉴定、序列分析得到了序列完全正确的表达载体。结论:建立了一套有效的、基于免费软件的基因从头设计与合成的策略,构建了可以用于环肽小分子文库表达与筛选的表达载体。

Artificial Design, Chemical Synthesis of the Expression Cassette Containing Split Mini Intein DnaE and its Application for the High-Level Expression Vector Construction

Objective： To develop an robust strategy for the de novo gene design,optimization and synthesis,to synthesize the expression cassette containing the split mini intein DnaE,and to construct Escherichia coli high-lev-el expression vector using the designed expression cassette.Methods： By the combination of free software GeneDesign,Tandem Repeats Finder and UNAFold,the expression cassette containing DnaE gene,restriction sites was designed,optimized and chemical synthesized.The synthetic oligonucleotides were one-step assembled by over-lap PCR.Results： The expression cassette was divided into 14 oligonucleotides with length ranging from 44 to 64 nts.The synthetic 14 oligonucleotides were one-step assembled by overlap PCR and cloned into expression vector pET-28a.Conclusion： An efficient de novo gene design,optimization approach was developed by the combination of free online bioinformatics software.A high-level expression vector suiting for the circular peptide library construction and screening was constructed.