摘要
构建硬酯酰辅酶A去饱和酶1(Stearoyl-CoA Desaturase l,SCD1)真核表达质粒pcDNA3.1(+)/SCD1,转染鹅原代肝细胞验证其对细胞中脂肪代谢的影响。根据鹅SCD1基因设计带有酶切位点引物,RT-PCR扩增SCD1基因,克隆至pMD-19T载体,亚克隆至pcDNA3.1载体,构建重组真核表达载体pCDNA3.1(+)/SCD1。酶切及序列测定测序正确后转染鹅原代培养肝细胞,荧光定量PCR检测SCD1基因的表达,并用油红O染色检测细胞中脂滴情况。结果:成功构建SCD1基因真核表达载体,转染鹅原代培养肝细胞后SCD1基因获得了表达,与脂肪代谢相关基因SREBP表达上调,同时检测到细胞中脂滴明显增多。结论:构建了重组质粒pCDNA3.1(+)/SCD1,并在鹅原代培养细胞中获得表达,为进一步研究SCD1基因在鹅肝脏脂肪代谢中的作用奠定基础。
In order to investigate the influence of goose stearoyl-CoA desaturase 1 （SCD1） on fat metabolism,a eukaryotic expression vector pcDNA3.1 （＋）/SCD1 was constructed and the recombinant protein was expressed in goose primary hepatocyte.SCD1ORF was amplified by RT-PCR from goose liver samples.The eukaryotic expression vector pcDNA3.1 （＋）/SCD1 was constructed by introducing SCD1 DNA fragment into the sites of EcoR Ⅰ and Xho Ⅰ of pcDNA3.1（＋）.The plasmid pcDNA3.1（＋）/SCD1 was transfected into goose primary hepatocyte with lipofectin method.The expression of SCD1 was detected and lipid metabolism genes SREBP was up regulated by real-time PCR.Restrictive endonuclease assay and sequence analysis verified the successful construction of the recombinant vector pcDNA3.1 （＋）/ SCD1.The transcription of SCD1 mRNA was detected in transfected goose primary hepatocyte and the lipid droplet was increased.This study provides a foundation for further study on function of SCD1 gene on goose fat metabolism.
出处
《上海交通大学学报（农业科学版）》
2014年第2期5-9,共5页
Journal of Shanghai Jiaotong University(Agricultural Science)
基金
江苏省研究生科研创新计划项目(CXZZ12_0912)
国家自然科学基金项目(31072021)
关键词
鹅
SCD1基因
原代肝细胞
真核表达
goose
SCD1 gene
primary hepatocyte
eukaryotic expression
