摘要
旨在克隆徐淮山羊脂蛋白酯酶(LPL)基因的cDNA,并通过EGFP融合蛋白实现该基因亚细胞水平的表达定位。本研究通过RT-PCR方法克隆徐淮山羊LPL基因cDNA,并初步进行生物信息学分析,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-LPL。聚乙烯亚胺(PEI)介导pEGFP-C1-LPL转染NIH-3T3细胞,48h后在荧光倒置显微镜下观察,并用RT-PCR方法检测LPLmRNA在细胞内的表达。结果,成功克隆出山羊LPL基因1 530bp长的cDNA,完整阅读框大小为1 437bp,共编码478个氨基酸,GenBank登录号为GU082383。信号肽区域预测结果表明LPL蛋白含有一小段信号肽序列,其可能性为100%。信号肽酶切割位置在第23和24个氨基酸之间,其可能性达到65.9%。成功构建融合表达载体pEGFP-C1-LPL,并且RT-PCR显示转染后LPL在mRNA水平上表达明显。EGFP-LPL融合蛋白定位在NIT-3T3细胞外和细胞膜部分。结果表明,首次成功克隆出徐淮山羊LPL基因cDNA;重组质粒pEGFP-C1-LPL构建成功,并在NIH-3T3细胞中明显表达。
The aims of the study were to clone cDNA of lipoprotein lipase（LPL） gene of Xuhuai goat,and analyze the sub-cellular localization of the expression product through EGFP fusion protein.cDNA of LPL gene of Xuhuai goat was cloned by RT-PCR.Preliminary analysis of bioinformatics was implemented.Fusion expression vector named pEGFP-C1-LPL containing the enhanced green fluorescent protein（EGFP） was constructed.pEGFP-C1-LPL were transfected into NIH-3T3 cells through polyethylene imine（PEI） and then observed under fluorescence inverted microscope after 48 h.The mRNA expression level in vitro was detected by RT-PCR.cDNA of LPL gene was successfully cloned in Xuhuai goat,and the sequence size was 1 530 bp,and the complete open reading frame size was 1 437 bp encoding 478 amino acids.GenBank accession number was GU082383.Signal peptide region prediction results showed that LPL protein contained a short signal peptide with a possibility of 100%,and the signal peptidase cleavage site was between the 23rd and the 24th amino acids with a possibility of 65.9%.Fusion expression vector pEGFP-C1-LPL was successfully constructed,and mRNA expression level in vitro was significant.EGFP-LPL fusion protein was located at the cell membrane and outside of NIH-3T3 cells.The result show that LPL gene of Xuhuai goat was first cloned successfully.Recombinant plasmid pEGFP-C1-LPL was successfully constructed and expressed in cells significantly.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2011年第6期772-778,共7页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
转基因生物新品种培育重大专项(2008ZX08008-003
2009ZX08008-003B)
