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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。
OBJECTIVE: To clone full-length coding region of the MORF4-re-lated gene on chromosome 15 (MRG15) gene and construct a eukaryotic expression vector to establish a human lens epithelial cell line stably expressing MRG15 for MRG15 in the age-related The study of related functions in the development of agerelatedcataract (ARC) lays a foundation. Methods: The full-length coding region of MRG15 was amplified from human ARC lens by RT-PCR and cloned into pMD18-T vector and sequenced. The results were correct and then subcloned into eukaryotic expression vector pcDNA3.1 (+) The liposome method was used to stably transfect cultured human lens epithelial cells. The single clones were picked and the expression of MRG15 was confirmed by Western blotting. RESULTS: Sequencing confirmed that the full-length MRG15 coding region amplified from the anterior capsule of the ARC lens was correct. Recombinant plasmid pcDNA3.1 (+) - MRG15 after digestion and the release of the theoretical length of the fragment. After culture of human lens epithelial cells stably transfected with monoclonal antibody, the expression of 19 MRG15 in 30 clones was significantly increased by Western blot. CONCLUSION: The full-length coding region of human MRG15 was cloned. The eukaryotic expression vector pcDNA3.1 (+) - MRG15 was successfully constructed and a human lens epithelial cell line stably expressing MRG15 was established.