大鼠Pdcd4基因真核载体的构建与表达

Construction and Expression of Rat Pdcd4 Eukaryotic Vector

目的构建大鼠程序性细胞死亡因子4(programmed cell death 4,Pdcd4)基因真核表达载体,为进一步研究该基因的功能提供条件。方法提取E3大鼠肺组织总RNA,RT-PCR扩增大鼠Pdcd4基因全长cDNA;经过双酶切、连接等反应,构建pEGFP-C1-Pdcd4重组表达载体。将该载体转染大鼠巨噬细胞系NR8383细胞,通过RT-qPCR与Western blot法检测NR8383细胞Pdcd4基因的表达。结果RT-PCR扩增的大鼠Pdcd4全长cDNA为1410bp;pEGFP-C1-Pdcd4重组真核表达载体中插入片段与NCBI GenBank文库中大鼠Pdcd4 cDNA的序列完全一致;倒置荧光显微镜下观察转染后NR8383细胞中绿色荧光蛋白的表达确定转染成功;RT-qPCR与Western blot法检测NR8383细胞中Pdcd4基因的表达水平明显上调。结论成功构建pEGFP-C1-Pdcd4重组真核表达载体,为进一步研究Pdcd4基因的作用机制奠定基础。

Objective To construct eukaryotic vector of rat programmed cell death 4(Pdcd4)for further function study.Methods Total RNA was isolated from E3 rat lung tissue.Then Pdcd4 DNA fragments were obtained by PCR amplification with specific primers after reverse transcription.After digestion with HindⅢand XhoⅠthe fragments were ligated into pEGFP-C1 vector cleaved with the same restriction endonucleases to generate pEGFP-C1-Pdcd4 recombinant vector.The integrity of constructed vector was confirmed by PCR and the sequencing analysis.NR8383 cells,a rat macrophage cell line,were transfected with pEGFP-C1-Pdcd4 recombinant plasmid or pEGFP-C1 empty vector.Twenty-four hours after the transfection,cells were harvested and Pdcd4 was detected by RT-qPCR and Western blot.Results Pdcd4 cDNA contains 1410bp.The recombinant eukaryotic expression vector pEGFP-C1-Pdcd4 was determined by PCR and was consistent with the theoretical value.No mutated base sequence was found by sequencing analysis.Pdcd4 was upregulated markedly after recombinant plasmid transfection.Conclusion The recombinant eukaryotic vector pEGFP-C1-Pdcd4 is successfully constructed.