SEDL基因及其突变体真核表达载体的构建与鉴定  

Construction and identification of eukaryotic expression vector of human SEDL gene and its mutants

作　　者：夏欣一[1] 周鑫[1] 崔英霞[1] 魏莉[1] 戈一峰[1] 姚兵[1] 李晓军[1] 黄宇烽[1] 

机构地区：[1]南京军区南京总医院解放军检验医学研究所中心实验科,江苏南京210002

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)

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.

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 

分 类 号：R681.1[医药卫生—骨科学]