稳定表达MRG15基因的人晶状体上皮细胞株的建立  

Establishment of human lens epithelial cell strains with stable expression of MRG15

作　　者：张自峰[1] 张健[2] 周健[1] 惠延年[1] 惠玲[3] 刘新平[2] 药立波[2] 王雨生[1] 崔志利[1] 

机构地区：[1]第四军医大学西京医院眼科全军眼科研究所,中国陕西省西安市710032 [2]第四军医大学基础部生物化学与分子生物学教研室,中国陕西省西安市710032 [3]中国陕西省西安市北方医院眼科兵工眼科中心,710043

出　　处：《国际眼科杂志》2006年第3期555-557,共3页International Eye Science

基　　金：中国国家自然科学基金资助项目(No.30000185);第四军医大学博士学位论文课题资助项目(No.2004012);第四军医大学西京医院科技创新基金资助项目(No.XJCX05M15)~~

摘　　要：目的:克隆人15号染色体上死亡相关因子(MORF4-re-latedgeneonchromosome15,MRG15)基因的编码区全长,构建真核表达载体,建立稳定表达MRG15的人晶状体上皮细胞株,为MRG15在年龄相关性白内障(agerelatedcataract,ARC)发生中相关功能的研究奠定基础。方法:采用RT-PCR由人ARC晶状体前囊膜标本中扩增MRG15的编码区全长,克隆至pMD18-T载体并测序,结果正确后亚克隆至真核表达载体pcDNA3.1(+)中,采用脂质体法对培养人晶状体上皮细胞进行稳定转染,挑取单克隆,用Westernblot进行MRG15表达的鉴定。结果:测序证实,由ARC晶状体前囊膜中扩增出的MRG15编码区全长序列正确。重组质粒pcDNA3.1(+)-MRG15经酶切后释放出与理论长度相符的片段。培养人晶状体上皮细胞稳定转染挑取单克隆后,经Westernblot检测,30个克隆中有19个MRG15的表达量明显增高。结论:克隆了人MRG15的编码区全长,成功构建了真核表达载体pcDNA3.1(+)-MRG15,并建立了稳定表达MRG15的人晶状体上皮细胞株。AIM： To clone the full length of human MRG15 （MORF4-related gene on chromosome 15） coding sequence, construct its eukaryotic expressing vector, and establish human lens epithelial cell strains of MRG15, for the investigation of function of MRG15 in age related cataract （ARC）. METHODS： The MRG15 coding region was amplified from anterior lens capsule of ARC by RT-PCR. The PCR product was cloned into vector pMD18-T, sequenced and then subcloned into pcDNA3.1 （＋） vector. The recombined plasmid was transfected into cultural human lens epithelial cells with lipofectin. Clone cells were got after selected by G418 and further screened by Western blot. RESULTS： Human MRG15 coding region was successfullyamplified from anterior lens capsule of ARC, and expected fragment was digested from the recombined plasmid pcDNA3.1（＋） -MRG15. Detected by Western blot, 19 out of 30 stable cell strains showed significantly increased expression of MRG15. CONCLUSION： Human MRG15 coding region, can be successfully cloned and constructed into pcDNA3.1 （＋）, and its stable expression cell strains were established.

关 键 词：MRG15基因 年龄相关性白内障 晶状体上皮细胞 克隆 

分 类 号：R776.1[医药卫生—眼科]