一种多基因串联共表达载体的构建  

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

作　　者：何彰华[1,2] 王洋[2] 赵珺[2] 刘晓杰[2] 张丽华[2] 王东[2] 师明磊[2] 黄芬[1] 尤平[1] 赵志虎[2] 

机构地区：[1]陕西师范大学生命科学学院,西安710062 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《中国生物工程杂志》2011年第1期40-45,共6页China Biotechnology

基　　金：国家重大新药创制关键技术平台资助项目(2008ZXJ09007-01)

摘　　要：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。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.

关 键 词：表达载体 共表达 串联组合 代谢通路组装 

分 类 号：Q78[生物学—分子生物学]