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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 gene encoding human MRG15 (MORF4-related-gene on chromosome15) and construct a fluorescent expression vector to detect its expression in human lens epithelial cells. To study the relationship between MRG15 and age-related cataract (ARC) The relevance of laying the foundation. Methods The gene encoding MRG15 was amplified from human ARC anterior capsule by RT-PCR, cloned into pMD18-T vector and sequenced. The result was correct and subcloned into pEGP-N2. After identification, the human lens epithelial cells were transiently transfected by liposomes and the localization of MRG15-pEGP-N2 was detected by fluorescence microscopy. Results Sequencing confirmed that the sequence and the reading frame of the MRG15 gene amplified from the anterior lens capsule of human ARC were all correct. The recombinant plasmid MRG15-pEGP-N2 was cleaved to produce a fragment corresponding to the expected theoretical length. Liposomes were transfected 24 h after the observed mainly in cultured human lens epithelial cells in the nucleus. Conclusion The coding sequence of human MRG15 was cloned and the fluorescent eukaryotic expression vector MRG15-pEGP-N2 was successfully constructed and expressed in cultured human lens epithelial cells.