原发性开角型青光眼致病基因小鼠MYOC Pro356Leu突变型质粒构建及鉴定  

Construction and identification of primary open angle glaucoma disease MYOC Pro356Leu mutant plasmid in mouse

作　　者：李春梅[1] 卓业鸿[1] 陈梦飞[1] 孙雪荣[1] 祁影[1] 葛坚[1] 

机构地区：[1]中山大学中山眼科中心眼科学国家重点实验室,广州510060

出　　处：《Eye Science》2010年第1期57-61,共5页眼科学报（英文版）

基　　金：中国国家自然科学基金项目资助(30600695/C030309)

摘　　要：目的:构建小鼠MYOC Pro356Leu突变型质粒及鉴定。方法:1.将含有红重组酶基因的质粒pKD46电击入RP23-180F15 BAC克隆。2.利用PCR扩增pStart-K载体,将PCR产物电穿孔入RP23-180F15 BAC红色重组细菌的MYOC基因组片段。3.PCR扩增mMyoc AfeⅠ片段,亚克隆mMyoc AfeⅠ片段进入pBluescript载体。4.设计两条寡核苷酸将Pro356Leu(C to T at codon 356)导入pBS_mMyoc AfeⅠ克隆。5.通过酶切及连接酶反应将pBS_mMyoc AfeⅠmut clone导入pStart-K_mMyoc构建成pStart-K_mMyoc mut克隆。结果:成功构建小鼠MYOC Pro356Leu突变型质粒pStart-K_mMyoc mut克隆,并经酶切及测序证实。结论:应用红重组技术及pStart-K载体克隆大片段DNA是有效的DNA克隆方法,我们所构建的MYOC Pro356Leu突变型质粒将为研究MYOC基因在原发性开角型青光眼发病中的功能和作用提供实验依据。Purpose：To construct and identify the plasmid of mutant MYOC Pro356Leu in mouse. Methods：1.Red recombinantinase-expressing plasmid pKD46 was transferred to RP23-180F15 BAC clone by electroporation.2.The pStart-K vector was amplified by PCR and the PCR product was electroporated into the bacteria containing the mMYOC.3.PCR amplification of the mMyoc AfeⅠ fragment and subcloningand then subcloned it into pBluescript vector.4.Two oligonucleotides was designed and introducing the Pro356Leu（C to T at codon 356） into the pBS_mMyoc AfeⅠ clone. 5.pBS_mMyoc Afe I mut clone was introduced to pStart-K_mMyoc by digestion and ligation reaction. Results：pStart-K_mMyoc mut clone,the mouse MYOC Pro356Leu mutant plasmid was constructed and identified well by restriction enzyme digest and sequenceing.Conclusion：It is a good method to construct a long DNA fragment clone by red recombinase and pStart-K vector.The constructed Mouse MYOC Pro356Leu plasmid will be very helpful for the studies on the function and biologic effects of MYOC gene in primary open angle glaucoma.

关 键 词：原发性开角型青光眼 MYOCPro356Leu突变 质粒构建 

分 类 号：R775[医药卫生—眼科]