山东医药Issue(14)：11-14,4.DOI:10.3969/j.issn.1002-266X.2014.14.004
蛋白激酶C受体1过表达载体、干扰载体的构建及鉴定
Construction and identification of over-expression vector and RNA interference vectors of receptor for activated C-kinase 1
摘要
Abstract
Objective To construct and identify the over-expression and interference eukaryotic expression vectors of receptor for activated C-kinase 1 (RACK1).Methods The RACK1 gene full-length reading frame was amplified from the human hepatoma cell line Huh-7.5.1 by RT-PCR.Then we amplified the corresponding CDS by nested PCR and cloned it into PIRES2-EGFP.The monoclone was identified by PCR and DNA sequencing .At the same time, we designed and syn-thesized complementary DNA sequences of 2 pairs of short hairpin structure and a pair of negative control sequence with hu-man RACK1 gene.After annealing, they were linked into restriction enzyme digested RNAi-Ready pSIREN-RetroQ-Zs-Green vector , and then identified by enzyme digestion analysis and DNA sequencing .The constructed over-expression vec-tor and interference vectors were transfected into HUVEC cell line by liposome .Results The size of two pairs of primers amplified by PCR sequences was completely true .A total of 954 nt oligonucleotides were successfully inserted into the vec-tor PIRES2-EGFP.The inserted sequences of RNA interference vectors pRetroQ /RACK1-1, pRetroQ/RACK1-2 and pRet-roQ/HK were consistent with our expectations .The EGFP and GFP could be seen in the transfected HUVEC cell line by fluorescence microscopy .Conclusion The over-expression vector and siRNA expression vectors of RACK 1 are successful-ly constructed , and can be introduced into HUVEC cell line .关键词
蛋白激酶C受体1/过表达载体/干扰载体/RNA干扰Key words
receptor for activated C-kinase 1/over-expression vector/interference vector/RNA interference分类
生物科学引用本文复制引用
张丽,贾雄飞,牛华,郑瑞,张望龙,毛小琴..蛋白激酶C受体1过表达载体、干扰载体的构建及鉴定[J].山东医药,2014,(14):11-14,4.基金项目
云南省应用基础研究计划项目（2011FB216）。 （）
