Darier病ATP2A2基因A68E突变的定点诱变及其突变载体构建  

Site-directed Mutagenesis and Recombinant Rxpression Plasmid Construction of a ATP2A2 Mutation (A68E) in Darier Patients

作　　者：史丙俊[1] 江阳[1] 薛梅[1] 刁庆春[1] 胡刚[2] 冯捷[2] 

机构地区：[1]重庆市中医院(重庆市第一人民医院)皮肤性病科,重庆400011 [2]西安交通大学医学院第二附属医院皮肤科,陕西西安710004

出　　处：《中国皮肤性病学杂志》2013年第1期29-32,共4页The Chinese Journal of Dermatovenereology

摘　　要：目的利用大引物PCR技术对发现的中国人Darier病ATP2A2基因A68E突变进行体外定点诱变,并构建携带有突变基因的真核表达载体。方法首先设计三条引物(包括两条外侧正向和反向引物以及内部突变引物),通过2轮PCR扩增,扩增出含有所需突变位点的片段(ATP2A2-A68E),然后将突变片段克隆入pGEM-T载体中,通过双酶切后进行连接,将目的片段重组至真核表达载体pcDNA3.1中。结果用大引物PCR法成功构建ATP2A2基因A68E突变片段,测序结果显示了ATP2A2基因上203位碱基C已变为A,此突变导致ATP2A2基因第68位密码子由丙氨酸变为谷氨酸。结论 pcDNA3.1-ATP2A2-A68E突变体的成功构建,为进一步进行该突变体的结构与功能的研究奠定了基础。Objective To perform site-directed mutagenesis of a new ATP2A2 mutation （A68E） identified in a Chinese family with Darier disease based on the megaprimer polymerase chain reaction method, and construct the re- combinant expression plasmid pcDNA3. 1-ATP2A2-A68E. Methods The site-directed mutagenesis of ATP2A2 was created by the megaprimer polymerase chain reaction method. Three oligonucleotide primers were designed according to the sequence of ATP2A2 cDNA including two flanking primers, which were up- stream and downstream of the mutation site, and one mutagenic primer. Mutagenesis was achieved by a two- step PCR. The amplified fragments from the second PCR which contained the mutation site were subcloned into the pGEM-T Vector, then the fragments containing the mutation site was obtained after cut by restriction enzyme and was inserted into the same restriction site of pcDNA3.1-ATP2A2. Results ATP2A2-A68E mu- tation fragment was constructed successfully by megaprimer PCR DNA sequencing. Direct sequence analyses displayed a C to A transition at position 203. The mutation results in a alanine （GCA） to glutamate （GAA） substitution at position 68. Conclusion The successfully constructed recombinant expression plasmid pcD- NA3.1-ATP2A2-A68E seems to provide the molecular basis for future research into the mechanisms of path- ogenesis in Darier disease.

关 键 词：大引物PCR ATP2A2 毛囊角化病 定点诱变 PCDNA3 1 

分 类 号：R751[医药卫生—皮肤病学与性病学]