POAG相关基因MYOC cDNA的克隆及MYOC/GFP融合蛋白在真核细胞的表达与定位  

Clone of POAG related MYOC gene cDNA and expression and localization of MYOC/GFP fusion protein in eukaryotic cell

作　　者：王建文[1] 孙慧敏[1] 季建[1] 李筱荣[1] 郑建秋[1] 唐广贤 吕建华 王庆瑛 

机构地区：[1]天津医科大学眼科中心,天津市300070 [2]邢台眼科医院,河北省邢台市054001

出　　处：《眼科新进展》2006年第8期581-584,共4页Recent Advances in Ophthalmology

摘　　要：目的克隆原发性开角型青光眼(primaryopenangleglaucoma,POAG)相关基因MYOCcDNA,研究其编码的蛋白质在真核细胞系中的表达和定位。方法用RT-PCR法,从人眼组织(角膜缘、睫状体)中扩增MYOCcDNA,克隆入载体pGEM-T,然后酶切,定向克隆到真核表达质粒pEGFP-N3中,构建pEGFP-N3-MYOC重组表达质粒,然后用限制性内切酶消化和DNA测序鉴定,最后通过脂质体包埋转染法,用pEGFP-N3-MYOC和pEGFP-N3质粒转染Hela和COS-7细胞,用荧光显微镜观察它们在Hela和COS-7细胞内的表达和定位。结果经酶切和DNA序列测定,证实重组质粒构建正确,荧光显微镜观察MYOC/GFP融合蛋白能在Hela和COS-7细胞中表达并且定位在细胞质中,而GFP分布在整个细胞内。结论成功克隆POAG相关基因MYOCcDNA,并且MYOC/GFP融合蛋白只表达在Hela和COS-7细胞质中,这为进一步研究POAG发病机制奠定了基础。Objective To clone the sequence of the cDNA of POAG related MYOC gene and study the expression and localization of MYOC/GFP fusion protein in eukaryotic cell lines. Methods POAG related MYOC gene cDNA was amplified from eye tissue （cornea limbus, ciliary body） by RT-PCR. The cDNA was cloned into plasmid pGEM-T, then pGEM-T was digested by restriction enzyme, and the products were cloned into eukaryotic expression plasmid pEGFP-N3 for constructing recombinant plasmicl pEGFP-N3-MYOC. Finally pEGFP-N3-MYOC and pEGFP-N3 plasmids were transfected into Hela and COS-7 cell lines by lipofectin. The accuracy of pEGFP-N3-MYOC was confirmed by restriction enzyme digestion and DNA sequencing. The expression and localization of MYOC/GFP fusion protein and GFP protein were observed under fluorescence microscope. Results By using restriction enzymes digesting and DNA sequencing, recombinant plasmid pERFP-N3-MYOC was constructed correctly. MYOC/GFP fusion protein and GFP protein were expressed in transfected Hela and COS-7 cells. MYOC/GFP fusion protein was located in cell plasma, but GFP protein was expressed in entire cell. Conclusion POAG related MYOC gene is cloned successfully, and the MYOC/GFP fusion protein can be expressed in Hela and COS-7 cells and locate in cell plasma, which lay the foundation for further studying the mechanism of POAG.

关 键 词：基因 MYOC RT—PCR 重组质粒 青光眼 

分 类 号：R775[医药卫生—眼科]