人HNA-3a基因的克隆及其在HEK-293细胞的表达  

Cloning of human HNA-3a gene and its expression in HEK-293 cells

作　　者：陈丽[1] 周浩[1] 何宏天 管政[1] CHEN Li;ZHOU Hao;HE Hong-tian;GUAN Zheng(Department of Blood Transfusion,The First Affiliated Hospital of Bengbu Medical College,Bengbu Anhui 233004,China)

机构地区：[1]蚌埠医学院第一附属医院输血科,安徽蚌埠233004

出　　处：《蚌埠医学院学报》2019年第1期6-8,13,共4页Journal of Bengbu Medical College

基　　金：蚌埠医学院自然科学基金重点项目(BYKY1720ZD)

摘　　要：目的:构建人HNA-3a基因全长的真核表达载体,探索一种检测抗人HNA-3a抗体的实验方法,观察HNA-3a蛋白表达情况。方法:参照GeneBank中人HNA-3a基因序列,设计并合成引物,采用RT-PCR方法反转录HNA-3a基因外显子片段全长,定向克隆到pEGFP-N3载体中,然后采用Lipofectamine^(TM) 2000试剂将含目的基因的pEGFP-N3表达载体转染至HEK-293细胞中进行稳定表达,并以免疫荧光和蛋白质免疫印迹法(WB)鉴定目的基因的表达情况。结果:免疫荧光和免疫印迹结果显示,目的基因在HEK-293细胞中获高效表达。结论:成功构建了高效表达HNA-3a蛋白的真核表达载体,为检测人血浆中抗HNA-3a抗体奠定了基础。Objective:To construct an eukaryotic expression vector containing full-length sequences of human HNA-3a gene,explore an experimental method for detecting human anti-HNA-3a antibody,and observe the protein expression of HNA-3a.Methods:The primers were designed and synthesized according to human HNA-3a gene sequences in Genebank.The full-length of HNA-3a gene was reversely transcribed using RT-PCR,and cloned into pEGFP-N3 vector.The pEGFP-N3 expression vector containing human HNA-3a gene was transfected into the HEK-293 cells for transient expression using Lipofectamine TM 2000 reagent,and the expression of the target gene was identified by Western blotting.Results:The eukaryotic expression vector containing the full-length sequences of human HNA-3a gene was successfully constructed,and the target gene was highly expressed in HEK-293 cells.Conclusions:The eukaryotic expression vector with high expression of HNA-3a protein is successfully constructed,which lays the foundation for the detection of the anti-HNA-3a antibody in human plasma.

关 键 词：基因表达 克隆 HNA-3a HEK293细胞 

分 类 号：Q343[生物学—遗传学]