摘要
目的:克隆功能未知的人类新基因C17orf62的长编码序列(C17orf62-L),分析其在细胞系中的表达情况,研究其在细胞内定位情况及其对细胞活力的影响。方法:利用GenBank中的数据,通过聚合酶链反应(polymerasechain reaction,PCR)从人的cDNA文库中克隆C17orf62编码187个氨基酸的长编码序列C17orf62-L,运用生物信息学分析其结构特性。通过逆转录PCR(reverse transcription PCR,RT-PCR)分析C17orf62在细胞系中的表达情况。经激光共聚焦显微镜观察C17orf62-L的亚细胞定位情况。使用流式细胞检测以及Western blot实验,分析C17orf62-L对细胞活力的影响及其机制。结果:生物信息学分析显示,C17orf62-L具有信号肽切割位点并包含跨膜结构域。RT-PCR证明C17orf62-L在多种细胞系广泛表达;激光共聚焦实验证实,C17orf62-L广泛分布于胞质,并且与高尔基体部分共定位。功能分析发现,C17orf62-L可诱导细胞死亡,且可通过多聚ADP核糖聚合酶(poly ADP-ribose polymerase,PARP)切割发挥作用。结论:C17orf62是一个新的人类细胞死亡诱导基因。
Objective：To isolate the long coding sequence of human novel gene C17orf62 which is named as C17orf62-L by us,and analyze its effects on cell viability,subcellular localization,and expression profile in multiple cell lines.Methods： RT-PCR（reverse transcription polymerase chain reaction）was used to clone C17orf62-L which encoded 187 amino acids from human multi-tissue cDNA library.We used bioinformatics analysis to identify structural characteristics of C17orf62-L,and RT-PCR to detect its expression.By Laser Scanning Confocal Microscopy we identified its subcellular localization of C17orf62-L.Furthermore,flow cytometry experiment was used to validate whether overexpression of C17orf62-L could influence cell phenotypes and Western blot was used to study related mechanisms.Results： C17orf62-L was cloned and constructed into the pcDNA-and pEGFP-expression plasmids.C17orf62-L had signal peptide and transmembrane domain.C17orf62-L was widely expressed in multiple cell lines and was validated partial co-localization with Golgi apparatus.Functional studies showed C17orf62-L could induce cell death accompanied with rising of cleaved PARP（poly ADP-ribose polymerase）.Conclusion： Human C17orf62 is a novel cell death inducing gene.
出处
《北京大学学报（医学版）》
CAS
CSCD
北大核心
2011年第2期168-172,共5页
Journal of Peking University:Health Sciences
基金
国家高技术研究发展计划专项经费(2006AA02A305)
科技部“重大新药创制”科技重大专项项目(2009ZX09503-004)资助~~
