大鼠ATF3基因shRNA真核表达质粒的构建及鉴定  

Construction and identification of the eukaryotic vector expressing shRNA targetting rat ATF3 gene

作　　者：任琎[1] 姜孝明[1] 夏梅[1] 赵聃[1] 王迎伟[1] 

机构地区：[1]南京医科大学微生物与免疫学系,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2009年第5期600-604,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金资助项目(30772016);教育部博士点专项科研基金(20060312005);江苏省教育厅自然科学基金(08KJB310004);南京医科大学校级重点项目(08NMUZ002)

摘　　要：目的:构建针对大鼠ATF3(activating transcription factor3,ATF3)基因的特异性短发卡RNA(small hairpinRNA,sh RNA)真核表达质粒。方法:用DNA重组技术将针对大鼠ATF3基因不同位点所设计的5个shRNA序列克隆到真核表达质粒pGenesil-1中。在酶切分析及序列测定后,用脂质体将5个shRNA分别转染至大鼠肾小球系膜细胞(glomerular mesangial cells,GMC),随后用Western blot检测蛋白的相对表达量来筛选最佳沉默效率的shRNA。结果:限制性酶切及核酸序列分析证明重组质粒构建正确。Western blot证实在重组质粒中,shATF3-1具有最佳的沉默效率。结论:成功构建了针对ATF3基因的shRNA的真核表达质粒,并且该shRNA可以特异性地降低大鼠肾小球系膜细胞ATF3的蛋白表达。Objective：To construct shRNA expression vectors targeting of rat ATF3 gene specifically. Methods：Five 19-21 bp shRNAs targeting of rat ATF3 gene were synthesized and cloned into eukaryotic expression plasmid pGenesil-1. After being digested by restriction endonuclease and sequencing confirmed,the recombinant plasmids were transfected into rat glomerular mesangial ceils （GMC） ,and then the level of ATF3 protein was measured by Western blot to select the optimal shRNA. Results：It was verified by partial nucleotide sequencing and restriction endonuclease digestion that the eukaryotic vectors expressing shRNA targeting of rat ATF3 gene were successfully constructed. The most optimal shRNA which could effectively silence the target genes was shATF3-1. Conclusion：The eukaryotic vector with the ability of specifically knocking down rat ATF3 gene was constructed successfully.

关 键 词：ATF3 SHRNA 肾小球系膜细胞 

分 类 号：R392.12[医药卫生—免疫学]