基于同源重组和反向PCR扩增技术构建BRD7溴区结构域缺失突变体

Construction of bromodomain-deleted BRD7 mutation vector based on homologous recombination and reverse PCR amplification

目的:利用同源重组和反向PCR扩增技术构建溴区包含蛋白7(bromodomain-containing protein 7,BRD7)的溴区结构域(bromodomain)缺失的真核表达载体。方法:设计一对反向PCR引物,以预先构建好的p IRES2-EGFP-3Flag/BRD7质粒为模板,利用高保真酶的高保真性和较长的延伸能力,反向扩增出BRD7溴区结构域缺失突变体(p IRES2-EGFP-3Flag/BRD7△brd)的线性DNA片段,然后转化大肠杆菌,利用大肠杆菌同源重组修复能力即可自身环化,而不需要经连接酶连接或其他处理即可得到p IRES2-EGFP-3Flag/BRD7△brd重组表达质粒,通过测序和Western印迹进一步对该缺失突变体的序列和蛋白质表达性能进行验证。结果:通过酶切鉴定、测序和Western印迹证实p IRES2-EGFP-3Flag/BRD7△brd构建成功。结论:利用PCR反向扩增和同源重组技术,可以简便快速地构建p IRES2-EGFP-3Flag/BRD7△brd突变体,实验成本较低,并且发现质粒模板浓度会影响载体的同源重组效率,这种改进的技术可广泛应用到基因突变体的构建。

Objective: To construct a eukaryotic expression vector of bromodomain-containing protein 7(BRD7) with deletion of bromodomain(BRD7△brd) using the homologous recombination andreverse PCR amplification techniques.Methods: The linear DNA fragments of bromodomain-deleted mutation of BRD7(pI RES2-EGFP-3Flag/BRD7△brd) were amplified by one pair of reverse PCR primers using high-fidelity enzyme, and then these fragments were transformed into E.coli to obtain the eukaryotic expression vector expressing BRD7△brd protein based on homologous recombination and plasmid cyclization.Results: Bromodomain-deleted clones were identified by digestion with restrictive enzymes, and then the sequence and protein expression were further confirmed by sequencing and Western blot assays. The results suggest that pI RES2-EGFP-3Flag/BRD7△brd was successfully constructed.Conclusion: We establish a simple and quick method to construct plasmids with pI RES2-EGFP-3Flag/BRD7△brd using reverse PCR amplification and homologous recombination techniques. We also found that the concentration of template in PCR reaction system is one of the critical factors that affect the rate of homologous recombination. Of all, this improved technique could be widely used in the construction of gene mutations.