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SEDL基因及其突变体真核表达载体的构建与鉴定 被引量：1

Construction and identification of eukaryotic expression vector of human SEDL gene and its mutants
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摘要 目的构建SEDL基因及其突变体与增强型绿色荧光蛋白(EGFP)表达载体的融合表达质粒pEGFP-C3-SEDL并获得表达。方法分别提取X连锁迟发性脊柱骨骺发育不良(SEDT)患者和正常对照外周血淋巴细胞RNA,RT-PCR方法扩增SEDL基因cDNA,双酶切后克隆至pEGFP-C3空载体,构建表达质粒pEGFP-C3-SEDL。双酶切和DNA测序鉴定后,转染COS-7细胞,通过流式细胞仪和荧光显微镜观察重组蛋白表达情况。结果 DNA测序显示重组真核表达载体pEGFP-C3-SEDL构建成功,SEDL基因c.370-371ins A突变位点被成功克隆到突变体重组质粒中。荧光倒置显微镜观察证实重组质粒均能在细胞内进行蛋白表达。结论 SEDL基因及其突变体真核表达载体的成功构建为其进一步研究SEDL基因突变致SEDT的分子机制奠定了基础。 Objective： To construct and identify eukaryotic expression vector of human SEDL gene and its mutants. Methods： Total RNA from blood lymphocytes was extracted using Trizol reagent and RT - PCR experiments were performed to amplify SEDL cDNA. Amplified products were digested with Hind Ⅲ and BamHI and cloned into plasmid pEGFP - C3. Recombinant vector pEGFP - C3 -SEDL was confirmed by double enzyme digestion experiment and DNA sequencing, respectively. COS -7 cells were transfected with plasmid DNA using Lipofectamine reagent. Recombinant expression was evaluated by fluorescence microscopy. Results： Recombinant vector pEGFP - C3 - SEDL was successfully constructed. And the insertion mutation c. 370 - 371insA of SEDL gene was also cloned into recombinant plasmid. The SEDL gene was expressed successfully in transfected COS -7 cells. Conclusion： Construction of eukaryotic expression vector of human SEDL gene and its mutants might contribute to study the mechanism of Spondyloepiphyseal dysplasia tarda caused by SEDL mutation.
作者 夏欣一 周鑫 崔英霞 魏莉 戈一峰 姚兵 李晓军 黄宇烽
机构地区 南京军区南京总医院解放军检验医学研究所中心实验科
出处 《中国优生与遗传杂志》 2010年第5期7-9,35,共4页 Chinese Journal of Birth Health & Heredity
基金 江苏省科技厅生殖健康研究技术服务平台项目(BM2008151)
关键词 SEDL基因 绿色荧光表达载体 迟发性脊柱骨骺发育不良 SEDL gene EGFP expression vector Spondyloepiphyseal dysplasia tarda
分类号 R681.1 [医药卫生—骨科学]
  • 相关文献

参考文献12

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二级参考文献23

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共引文献3

同被引文献20

  • 1麻宏伟,姜俊,吕峻峰,郭然,牛国辉.X-连锁迟发性脊椎骨骺发育不良SEDL基因剪接受体突变导致潜在剪接位点激活(英文)[J].中华医学遗传学杂志,2005,22(3):251-253. 被引量：6
  • 2Fiedler J,Le Merrer M,Mortier G,et al.X-linked spondyloepiphyseal dysplasia tarda:Novel and recurrent mutations in 13 European families[J].Hum Mutat,2004,24(1):103.
  • 3Bar-Yosef U,Ohana E,Hershkovitz E,et al.X-linked spondyloepiphyseal dysplasia tarda:A novel SEDL mutation in a Jewish Ashkenazi family and clinical intervention considerations[J].Am J Med Genet A,2004,125(1):45-48.
  • 4Gedeon AK,Tiller GE,Le Merrer M,et al.The molecular basis of X-linked spondyloepiphyseal dysplasia tarda[J].Am J Hum Genet,2001,68(6):1386-1397.
  • 5Gedeon AK,Colley A,Jamieson R,et al.Identification of the gene (SEDL) causing X-linked spondyloepiphyseal dysplasia tarda[J].Nat Genet,1999,22(4):400-404.
  • 6Shaw MA,Brunetti-Pierri N,Kádasi L,et al.Identification of three novel SEDL mutations,including mutation in the rare,non-canonical splice site of exon 4[J].Clin Genet,2003,64(3):235-242.
  • 7Xiao C,Zhang S,Wang J,et al.A single nucleotide deletion of 293delT in SEDL gene causing spondyloepiphyseal dysplasia tarda in a four-generation Chinese family[J].Mutat Res,2003,525(1-2):61-65.
  • 8Guo H,Xu X,Wang K,et al.A novel RNA-splicing mutation in TRAPPC2 gene causing x-linked spondyloepiphyseal dysplasia tarda in a large Chinese family[J].J Genet,2009,88(1):87-91.
  • 9Xiong F,Gao J,Li J,et al.Noncanonical and canonical splice sites:a novel mutation at the rare noncanonical splice-donor cut site (IVS4+1A》G) of SEDL causes variable splicing isoforms in X-linked spondyloepiphyseal dysplasia tarda[J].Eur J Hum Genet,2009,17(4):510-516.
  • 10Shi YR,Lee CC,Hsu YA,et al.A novel nonsense mutation of the sedlin gene in a family with spondyloepiphyseal dysplasia tarda[J].Hum Hered,2002,54(1):54-56.

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中国优生与遗传杂志
2010年 第5期

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