human NF-κB miRNA干扰载体的构建

目的:设计并构建针对胰腺癌基因治疗中最关键的靶基因人细胞核因子κB(human NF-κB)的miRNA干扰载体。方法:根据靶基因设计并合成miRNA oligo(引物设计软件),将oligo退火成双链,然后用载体构建试剂盒进行重组克隆,将双链的miRNA oligo插入到miRNA表达载体中,构建miRNA质粒,转化入感受态细胞DH5α,从转化平板分别挑取克隆,用载体通用引物进行菌落PCR筛选,筛选得到的阳性克隆进行测序,以验证重组克隆中插入片段序列是否与设计的oligo序列一致。结果:经过比对,重组克隆中插入片段序列与设计的oligo序列完全一致。结论:成功构建了针对靶基因human NF-κB的miRNA干扰载体,为进一步研究奠定了基础。

Human NF-κB miRNA interference vector construction

Objective: Design and construction for the gene therapy of pancreatic carcinoma is the key target genes human nuclear factor(NF)-κB miRNA interference vector.Methods: According to the design and synthesis of target genes(miRNA oligo primer design software),oligo annealing twin chain,then the vector construction kit for recombinant clones,double chain miRNA oligo inserted into expression vector construction of miRNA,miRNA plasmid,transformed into competent cells from DH5α conversion plate respectively,were cloned,use carrier universal primers of colony PCR screening,screening positive clones were sequenced,to verify the recombinational cloning insertion sequence and oligo sequence alignment design.Results: After alignment,recombinant clone insert fragment sequences and oligo sequences identical design.Conclusion: We have successfully constructed aiming at target genes human NF-κB miRNA interference carrier,laid the foundation for further study.