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脂肪甘油三酯脂酶载体构建

发表时间:2015-06-15  浏览量:1689  下载量:544
全部作者: 李松涛,栗晓霞,吕琳,李颖,孙长颢
作者单位: 哈尔滨医科大学公共卫生学院
摘 要: 目的:研究旨在构建脂肪甘油三酯脂酶(adipose triglyceride lipase,ATGL)基因的表达载体。方法:调取C57BL/6小鼠ATGL基因的cDNA文库,并使用高保真Taq酶克隆出含有相应酶切位点的ATGL编码区全序列,并通过基因测序验证序列准确性。得到的目的基因片段利用T4链接酶接入pBluescriptⅡ质粒中,并再一次通过基因测序确定重组质粒连接的准确性。建好的质粒继而转染HEK293细胞,检测细胞中ATGL的mRNA和蛋白水平。结果:PCR产物双酶切和基因测序确定连接正确,细胞中ATGL的mRNA和蛋白表达量显著增高。结论:成功构建了C57BL/6小鼠ATGL基因的表达载体,建好的载体可用于ATGL基因功能的后续研究。
关 键 词: 预防医学与卫生学其他学科;脂肪甘油三酯脂酶;C57BL/6小鼠;载体构建
Title: Construction of adipose triglyceride lipase expression vector
Author: LI Songtao, LI Xiaoxia, LÜ Lin, LI Ying, SUN Changhao
Organization: Public Health College, Harbin Medical University
Abstract: Objecive: To construct the expression vector of adipose triglyceride lipase (ATGL). Methods: we retrieved the cDNA library of the ATGL gene of C57BL/6 mice and cloned the coding region containing the corresponding restriction sites of the whole sequence of ATGL by using high-fidelity Taq enzyme, and then verified the accuracy of the sequence by gene sequencing. we connected the target gene and pBluescriptⅡplasmid through T4 ligase, determined by gene sequencing accuracy to make sure that the sequence was correct and then transfected the HEK293 cell with recombinant plasmid and detected the express level of ATGL mRNA and protein. Results: The PCR products of enzyme digestion and gene sequence are coincided with expectation. ATGL mRNA and protein expression in the cells are significantly increased. Conclusion: The results show that the ATGL recombinant expression vector is constructed successfully, which can be used to study the following function of ATGL gene.
Key words: other subjects of preventive medicine and hygienic; adipose triglyceride lipase; C57BL/6 mice; construction of expressing vector
发表期数: 2015年6月第11期
引用格式: 李松涛,栗晓霞,吕琳,等. 脂肪甘油三酯脂酶载体构建[J]. 中国科技论文在线精品论文,2015,8(11):1204-1208.
 
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