目的 克隆SD大鼠脑红蛋白(Ngb)基因至真核表达载体pAcGFP1-C1,构建表达质粒pAcGFP1-C1-Ngb,并转染至人SH-SY5Y细胞表达Ngb蛋白.方法 提取大鼠肝脏组织总RNA,采用逆转录聚合酶链式反应(RT-PCR)方法扩增大鼠Ngb基因,与双酶切的pAcGFP1-C1连接,从而构建pAcGFP1-C1-Ngb真核表达质粒.转染SH-SY5Y细胞,并用Western blot的方法鉴定Ngb蛋白在SH-SY5Y细胞中的表达.结果 获得SD大鼠Ngb基因全长序列和重组真核表达载体pAcGFP1-C1-Ngb.Western blot结果显示,转染pAcGFP1-C1-Ngb后的SH-SY5Y细胞Ngb基因有稳定的表达.结论 成功地克隆了SD大鼠Ngb基因、构建了pAcGFP1-C1-Ngb真核表达载体,并在人SH-SY5Y细胞中获得Ngb蛋白的稳定表达,为进一步研究Ngb基因在细胞中的功能奠定了基础.%Objective To clone SD rat Ngb gene,recombine eukaryotic expression vector pAcGFP1-C1-Ngb,and measure its expression level in human SH-SY5Y cells. Methods The total RNA was extracted from SD rat brain. The cDNA encoding Ngb was obtained by RT-PCR. Sequence was confirmed by sequencing and BLAST,and then it was inserted in the eukaryotic expression vector pAcGFP1-C1,as pAcGFP1-C1-Ngb. The recombinant plasmid pAcGFP1-C1-Ngb was indentified by restriction endonuclease and sequencing, and then trans-fected into human SH-SY5Y cells by Iipofectamine 2000 transfection reagent. The level of Ngb in SH-SY5Y cells was detected by western blotting. Results Ngb gene was obtained by RT-PCR. The recombinant eukaryotic expression vector pAcGFP1-C1-Ngb was successfully constructed. The Ngb gene was successfully expressed in transfected SH-SY5Y cells. Conclusion The successful construction and expression of Ngb recombinant plasmid has set a foundation for further function investigation.
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