SEDL基因及其突变体真核表达载体的构建与鉴定

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。

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

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.