| p E G F P - N 3 - L P L重组真核表达载体的构建及在V e r o细胞中的表达 |
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| 引用本文： | 赵佳福,许厚强,乜玉丽,陈 祥,张 勇.p E G F P - N 3 - L P L重组真核表达载体的构建及在V e r o细胞中的表达[J].西南农业大学学报,2012,34(4):040-045. |
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| 作者姓名： | 赵佳福 许厚强 乜玉丽 陈 祥 张 勇 |
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| 作者单位： | 高原山地动物遗传育种与繁殖省部共建教育部重点实验室 贵州省动物遗传育种与繁殖重点实验室 贵州大学 |
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| 基金项目： | 国家科技部转基因生物新品种培育科技重大专项子项目(2009ZX08009-139B);贵州省动物转基因研究平台建设基金资助项目(Z093159);贵州大学研究生创新基金资助项目(校研农2010021) |
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| 摘 要： | 目的:构建贵州白香猪LPL基因与增强型绿色荧光蛋白基因的融合表达载体pEGFP-N3-LPL,转染Vero细胞,观察重组质粒的表达.方法:采用HindⅢ和BamHⅠ两种限制性内切酶获得LPL基因编码区片段和pEGFP-N3线型载体,将目的片段与该载体连接、转化、挑取阳性克隆,进行菌落PCR、双酶切及测序鉴定.使用Lipofectamine2000将重组质粒转染Vero细胞,观察细胞中绿色荧光蛋白的表达并对其进行mRNA检测.结果:成功构建重组真核表达载体pEGFP-N3-LPL,经mRNA检测,实验组在1 500bp处出现特异性条带,说明重组载体已在Vero细胞中获得表达.结论:本试验构建的真核细胞表达载体pEGFP-N3-LPL,为进一步阐明LPL基因与肌内脂肪沉积间的生物学作用机制奠定了基础.
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| 关 键 词： | 脂蛋白酯酶 pEGFP-N3载体 Vero细胞 真核表达 荧光检测 |
C o n s t r u c t i o no fR e c o m b i n a n tE u k a r y o t i cE x p r e s s i o nV e c t o r
o fp E G F P - N 3 - L P Lw i t hE f f i c i e n tE x p r e s s i o n s i nV e r oC e l s |
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| ZHAO Jia-fu,XU Hou-qiang,NIE Yu-li, CHEN Xiang,ZHANG Yong.C o n s t r u c t i o no fR e c o m b i n a n tE u k a r y o t i cE x p r e s s i o nV e c t o r
o fp E G F P - N 3 - L P Lw i t hE f f i c i e n tE x p r e s s i o n s i nV e r oC e l s[J].Journal of Southwest Agricultural University,2012,34(4):040-045. |
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| Authors: | ZHAO Jia-fu XU Hou-qiang NIE Yu-li CHEN Xiang ZHANG Yong |
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| Institution: | Key Laboratory of Animal Genetics,Breeding and Reproduction in the Plateau Mountainous Region,Ministry of Education,Guizhou Key Laboratory of Animal Genetics,Breeding and Reproduction,Guizhou University,Guiyang 550025,China |
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| Abstract: | Objective: To construct the recombinant eukaryotic vector of pEGFP-N3-LPL,which carries the fusion gene of Guizhou Baixiang pig LPL gene and EGFP,and to transfer it into Vero cells so as to observe its expression.Methods: The LPL encoding fragments and pEGFP-N3 linear vector were obtained with the restriction enzymes of Hind Ⅲ and BamHⅠ,the target fragment was bound to the vector,and then the positive clones were screened to carry on colony PCR,double digestion and sequencing.The recombinant plasmid was transferred into Vero cells with Lipofectamine 2000.The expression of green fluorescent protein was observed and detected by mRNA detection.Results: The recombinant eukaryotic expression vector pEGFP-N3-LPL was successfully constructed.A 1500bp specific band appeared in the experimental group by mRNA detection,thus suggesting that recombinant vector was successfully expressed in Vero cells.Conclusion: The eukaryotic expression vector of pEGFP-N3-LPL has been successfully constructed,which lays a foundation for further elucidating the biological mechanism of LPL gene and intramuscular fat deposition. |
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| Keywords: | lipoprotein lipase pEGFP-N3 vector Vero cell eukaryotic expression fluorescence detection |
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