原发性开角型青光眼致病基因MYOC真核表达质粒的构建及其在COS-7细胞中的表达

目的构建原发性开角型青光眼(POAG)致病基因MYOC的真核表达质粒,并在COS-7细胞中表达MYOC蛋白。方法用RT-PCR法扩增人眼组织(角膜缘)MYOC基因cDNA,纯化回收后,克隆入pGEM-T载体,再亚克隆入真核表达质粒pEGFP-N3,构建重组表达质粒pEGFP-N3-MYOC,转染COS-7细胞,用荧光显微镜观察MYOC蛋白在COS-7细胞中的表达,Western blot分析MYOC蛋白分泌特点。结果经酶切和DNA测序鉴定,证实重组表达质粒pEGFP-N3-MYOC构建正确,荧光显微镜观察MYOC蛋白能在COS-7细胞中表达,并且定位在细胞质中,而绿色荧光蛋白分布在整个细胞内。Western blot结果显示,MYOC蛋白能分泌到细胞外。结论已成功构建MYOC基因真核表达质粒,并能在COS-7细胞中表达MYOC蛋白,为进一步研究POAG发病机制奠定了基础。

Construction of Eukaryotic Expression Vector for Pathogenic Gene MYOC of Primary Open Angle Glaucoma and Its Expression in COS-7 Cells

Objective To construct the eukaryotic expression vector for pathogenic gene MYOC of primary open angle glaucoma(POAG) and express in COS-7 cells. Methods MYOC cDNA was amplified from human eye tissue(cornea limbus) by RT-PCR, purified and cloned into vector pGEM-T, then subcloned to eukaryotic expression vector pEGFP-N3. Transfect COS-7 cells with the constructed recombinant plasmid pEGFP-N3-MYOC, observe the expression of MYOC protein under fluorescent microscope and identify the expressed product by Western blot. Results Both restriction analysis and DNA sequencing proved that recombinant plasmid pEGFP-N3-MYOC was constructed correctly. Fluorescent microscopy showed that MYOC protein was expressed and located in cytoplasm, while green fluorescent protein(GFP) was distributed in the whole cells. Western blot revealed that expressed MYOC protein could be secreted to the extracellular region. Conclusion The eukaryotic expression vector for MYOC gene was successfully constructed and expressed in COS-7 cells, which laid a foundation of further study on pathogenic mechanism of POAG.