人MRG15基因荧光真核表达载体的构建及其在培养人晶状体上皮细胞的表达

Construction and expression of eukaryotic fluorescent expressing vector of human MRG15 gene in cultural human lens epithelial cells

目的克隆人MRG15(MORF4-related-gene on chromosome15)的编码基因,构建荧光表达载体,并检测其在培养人晶状体上皮细胞中的表达,为研究MRG15与年龄相关性白内障(age related cataract,ARC)的相关性奠定基础。方法用RT-PCR的方法从人ARC的晶状体前囊膜中扩增MRG15的编码基因,克隆至pMD18-T载体并测序,结果正确后亚克隆至荧光真核表达载体pEGP-N2中,酶切鉴定正确后采用脂质体法,瞬时转染培养人晶状体上皮细胞,荧光显微镜下检测MRG15-pEGP-N2的定位。结果测序证实,从人ARC晶状体前囊膜中扩增出的MRG15编码基因的序列及读框全部正确。重组质粒MRG15-pEGP-N2经酶切后产生与理论预期长度相符的片段。脂质体法转染后24h,观察到其主要在培养人晶状体上皮细胞的细胞核中表达。结论克隆了人MRG15的编码基因,成功构建了荧光真核表达载体MRG15-pEGP-N2,并可在培养人晶状体上皮细胞中表达。

Objective To clone the coding sequence of human MRG15 (MORF4-related gene on chromosome 15), construct and express its eukaryotic fluorescent expressing vector in cultural human lens epithelial cells, for the investigation of the relationship between MRG15 and age related cataract (ARC). Methods The MRG15 coding region was amplified from anterior lens capsule of ARC by RT-PCT. The PCR product was cloned into pMDl8-T vector,sequenced and then subcloned into vector pEGFP-N2. The recombined vector was transfected into cultural human lens epithelial cells with lipofectin, and the localization of MRG15-pEGFP-N2 was observed through fluorescence microscopy. Results Human MRG15 was successfully amplified from anterior lens capsule of ARC,and expected fragment was digested from the recombined vector, MRG15-pEGFP-N2. It was observed that MRG15 was localized within the nucleus of cultural human lens epithelial cells 24 hours after transfection. Conclusion Human MRG15 coding region, cloned from anterior lens capsule of ARC,was successfully constructed into pEGFP-N2,and can be expressed in cultural human lens epithelial cells.