【摘要】 旨在克隆Marc-145细胞蛋白激酶R(PKR)基因，对其进行遗传特性及表达研究。利用RT-PCR克隆获得Marc-145细胞PKR基因，并对其核苷酸序列及推导的氨基酸序列进行同源性与遗传进化分析；将该基因分别插入pEGFP-C1与pEGFP-N1多克隆位点，构建增强型绿色荧光蛋白(EGFP)融合真核重组质粒，转染HEK293细胞，通过荧光显微镜观察、Western blot检测EGFP标签，开展PKR融合表达研究。结果显示，成功克隆获得Marc-145细胞PKR基因，编码区为1 653 bp,推导编码550个氨基酸，与选取的灵长类及哺乳动物参考物种PKR基因核苷酸序列同源性在72.2%～99.8%之间，氨基酸序列同源性在55.4%～99.5%之间，与豚鼠及其供体物种非洲绿猴(Cercopithecus aethiops)同源性分别为最低、最高；成功构建了EGFP-PKR融合质粒，但荧光蛋白观察及Western blot检测表明，仅C端融合质粒成功表达。结果表明，成功克隆了Marc-145细胞PKR基因并进行了融合表达，为进一步开展猪繁殖与呼吸综合征病毒(PRRSV)在感染细胞与细胞内复制增殖时PKR所发挥的作用、PRRSV与细胞固有免疫抗病毒互作机制等研究奠定了基础。更多还原

【Abstract】 The aim is to clone protein kinase R(PKR) gene of the Marc-145 cell and study its genetic characteristics and fusion expression.The PKR gene of Marc-145 cells was cloned by RT-PCR,and the nucleotide sequences and deduced amino acid sequences were analyzed for homology and genetic evolution.The gene was inserted into the multiple cloning sites of pEGFP-C1 and pEGFP-N1 vector respectively to construct the eukaryotic expression plasmids fused with EGFP tag, the recombinant plasmids were transfected into HEK293 cells, and the PKR fused with EGFP tag was detected by fluorescence microscopy and Western blot, respectively.The results showed that the PKR gene of Marc-145 cells was successfully cloned with a coding region of 1 653 bp, deduced to encode 550 amino acids, the nucleotide and the amino acid sequence homologies with primates and mammals were from 72.2% to 99.8%,55.4% to 99.5%,respectively, and the homologies with guinea pig and its donor species-Cercopithecus aethiops were the lowest and highest respectively.The EGFP-PKR fusion plasmid was successfully constructed, but only the C-terminal fusion plasmid was successfully expressed through the fluorescent microscopy and Western blot detection.In this study, the PKR gene of Marc-145 cells was successfully cloned and expressed, which laid a foundation for further research on the role of PKR in PRRSV infection in Marc-145 cell and the interaction mechanism of PRRSV and cellular innate immunity.更多还原