摘要
研究旨在克隆徐淮山羊过氧化物酶体激活增殖受体(PPAR)基因的cDNA,并通过EGFP融合蛋白对该基因产物亚细胞水平定位。采用RT-PCR方法克隆徐淮山羊脂肪组织PPAR基因cDNA,并构建含有增强型绿色荧光蛋白(EGFP)报告基因的融合表达载体pEGFP-C1-PPAR,聚乙烯亚胺(PEI)介导基因转染NIH-3T3细胞,48h后荧光倒置显微镜下观察基因表达产物的分布,RT-PCR检测mRNA在体外水平的表达。结果表明:首次成功克隆出徐淮山羊PPAR基因的全序列cDNA,大小为1428bp,GenBank登录号为GU082382;构建了融合表达载体pEGFP-C1-PPAR;RT-PCR检测mRNA表达明显;EGFP-PPAR融合蛋白定位在NIT-3T3细胞质中。体外克隆的徐淮山羊PPAR基因cDNA,在单细胞水平上可表达于细胞质中,结果为进一步研究PPAR的生物学功能奠定基础。
The study aimed to clone cDNA of peroxisome proliferator-activated receptor（PPAR） gene of Xuhuai goat,and analysis sub-cellular localization of the expression product through EGFP fusion protein.cDNA of PPAR gene of Xuhuai goat was cloned and submitted to GenBank;fusion expression vector named pEGFP-C1-PPAR which containing the enhanced green fluorescent protein（EGFP） was constructed;NIH-3T3 cells were transfected pEGFP-C1-PPAR through polyethylene imine（PEI） and then observed under inverted microscope after 48 h,mRNA expression level in vitro was detected by RT-PCR.The results showed cDNA of PPAR gene was first successfully cloned in Xuhuai goat,and the sequence size was 1 428 bp,GenBank accession number was GU082382;fusion expression vector pEGFP-C1-PPAR was successfully constructed;mRNA expression level in vitro was significantly;EGFP-PPAR fusion protein located at cytoplasm of NIH-3T3 cells.In conclusion,the in vitro cloning cDNA of PPAR gene of Xuhuai goat could be expressed in the cytoplasm at the single cell level,which provided basis for further study on the biological functions of PPAR.
出处
《中国畜牧杂志》
CAS
北大核心
2011年第7期6-10,共5页
Chinese Journal of Animal Science
基金
转基因生物新品种培育重大专项(2008ZX08008-003
2009ZX08008-003B)
