真核表达载体pcDNA3.1-myc-his(-)B的改构与应用

目的:对真核表达载体pcDNA3.1-myc-his(-)B进行改构,以简化目的基因克隆的步骤。方法:用事先设计好的2条寡核苷酸链互相退火,替换pcDNA3.1-myc-his(-)B的多克隆位点中NotⅠ与HindⅢ间的部分;利用改构的真核表达载体,分别构建亲环蛋白A、胆绿素还原酶B和过氧化氢酶基因的重组真核表达载体,瞬时转染293T细胞后,用His标签抗体验证其表达。结果:测序结果证实已将预想的序列替换至pcDNA3.1-myc-his(-)B的多克隆位点中相应位置,改构为质粒pcDNA3.1(-)reconstruct;利用改构的真核表达载体表达了相应的目的基因。结论:改构的真核表达载体pcDNA3.1(-)reconstruct可以简化融合myc和His标签的目的基因克隆的过程,并且增加了限制性位点的可选择性。

Modification and Application of Eukaryotic Expression Vector pcDNA3.1-myc-his(-)B

Objective：Modifying eukaryotic expression vector pcDNA3.1-myc-his （-）B in order to simplify the cloning of target gene.Methods：Two designed oligonucleotides were annealing each other,and the DNA fragment between NotⅠ and HindⅢ in the multiple clone site of pcDNA3.1-myc-his（-）B was replaced by the product.By utilizing this modified vector,the recombined eukaryotic expression vector of gene cyclophilin A,biliverdin reductase B and catalase were successfully constructed.To test if they can be exogenous expressed,after transiently transfecting 293T with the recombined vectors,the total protein of these transfected cells were Western-blotted with anti-His tag antibody separately.Results：Sequencing results suggested that designed sequence has successfully replaced the corresponding location of the multiple clone site of pcDNA3.1-myc-his （-）B,and the modified vector was named pcDNA3.1 （-）reconstruct.Three above-mentioned target genes were all successfully exogenously expressed by utilizing this vector.Conclusion：The modified eukaryotic expression vector pcDNA3.1（-）reconstruct can effectively simplify the cloning of myc epitope and His tag fused target genes,as well as the selection of their available restriction sites.