一种多基因串联共表达载体的构建

为了构建一种可用于串联共表达多个基因的原核表达载体,通过PCR引入点突变,对商业载体pET-22b的酶切位点进行定向改造,设计独特的XbaⅠ/SpeⅠ模块。获得改造成功的载体pET-m22b,测序结果显示启动子、核糖体结合位点、多克隆位点及终止子均位于XbaⅠ和SpeⅠ两酶切位点间。选取不同来源的基因进行串联组合及表达鉴定,四个基因成功组合于同一载体中,表达效果良好,各基因表达效率不受明显影响。研究构建的载体pET-m22b,使多基因的共表达更加便捷,为蛋白复合物的表达与纯化、代谢通路的异源重建及其优化等研究奠定了良好的基础。

Construction of a Vector Suitable for the Tandem Coexpression of Multiple Genes by a Single Plasmid

To develop an robust strategy for the tandem repeat co-expression of multiple genes in one plasmid, the point mutations were introduced into commercial expression vector pET-22b by the overlap PCR. After the mutation, the restriction enzyme site XbaⅠ was moved to the upstream of T7 promoter and a SpeⅠ site was introduced to the downstream of T7 terminator. The whole expression cassette including the T7 promoter, ribosome binding sites, multiple cloning site, T7 terminator were covered by the XbaⅠ-SpeⅠrestriction fragment. Restriction digestion and sequencing indicate that the mutated expression vector pET-m22b was constructed successfully. The four genes of different sources were choose for the coepxression by the pET-m22b. The SDS-PAGE shown that the four genes were highly co-expressed and without any affection on each other. The results indicated an efficient co-expression vector suitable for the tandem combination expression of multiple genes in single plasmid was constructed, which will make the co-expression of many genes more easy and will also greatly facilitate the expression and purification of multi-subunit protein complex, the rapid heterogenesis re-construction and optimization of biochemical pathways etc.