大鼠野生型Gadd45γ基因和Gadd45γshRNA真核表达质粒的构建及鉴定  

Construction and identification of Gadd45γ gene and its shRNA eukaryotic expression vector

作　　者：车楠[1] 邱文[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594-599,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金资助(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);南京医科大学自然科学基金(08NMUZ002)

摘　　要：目的:构建大鼠野生型Gadd45γ基因和其特异性短发卡状小干涉RNA(shRNA)真核表达质粒,并观察其在大鼠肾小球系膜细胞(GMC)中过表达及沉默Gadd45γ基因的情况。方法:用DNA重组技术将针对大鼠Gadd45γ基因的cds区序列或针对其不同位点所设计的4对shRNA序列分别克隆到真核表达质粒pcDNA3.1/HA或pGCsi.U6.neo.GFP中。在酶切分析及序列测定正确后,用GenEscortTMⅢ转染试剂将上述两种质粒分别转染至大鼠GMCs中,用免疫细胞化学和Western blot检测HA-Gadd45γ融合蛋白的表达及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两种重组质粒均构建正确。免疫细胞化学和Western blot分析表明构建的pcDNA3.1/Gadd45γ质粒在大鼠GMCs中能够表达;Gadd45γshRNA-3具有最佳沉默效率。结论:成功构建了大鼠野生型Gadd45γ基因和其特异性的shRNA真核表达质粒,该实验结果为进一步研究Gadd45γ基因的生物学功能奠定了基础。Objective：To construct Gadd45γ and its shRNA expression vectors and assess their function on rat glomerular mesangial cell （GMC）. Methods：The cds area of Gadd45γ and four 19-21 bp reverse repeated motifs targeting of Gadd45γ gene were synthesized and cloned into eukaryotic expression plasmid pcDNA3.1/HA and pGCsi.U6.neo.GFP. After being screened and confirmed,the recombinant plasmids were transfeeted into GMCs,then the levels of Gadd45γ protein in rat GMCs were measured using Western blot and immunocytochemistry to prove its expression and find out the optimal shRNA. Results：It was verified by partial nucleotide sequencing and restriction endonuclease digestion thai the constructed eukarvotie vectors were correct. The results by Western blot and immunocytochemistry showed that the constructed peDNA3.1/Gadd45γ plasmid could express correctly,anti the optimal shRNA which could effectively silence the target gene,was Gadd45γ shRNA-3. Conclusion：The pcDNA3.1/Gadd45γ plasmid and its shRNA were constructed successfully. These data provide the foundation for studying biological functions of Gadd45γ gene in the future.

关 键 词：Gadd45γ SHRNA 肾小球系膜细胞 

分 类 号：R392.12[医药卫生—免疫学]