摘要
目的构建人类白细胞抗原（HIA）DR4（＊0005）真核表达载体，并使其在小鼠成纤维细胞系中得到稳定表达。方法从含且HLA-DRA全长eDNA的质粒中扩增HLA．DRA的开放读码框（ORF）序列，通过酶切将载体pIRES2-EGFP中的增强绿色荧光蛋白（EGFP）切除，将HLA-DRAORF插入到载体中；采用RT-PCR方法从HLA．DR4（＊04051阳性人外周血单个核细胞中克隆出HLA-DRB1＊0405ORF序列，将其插入pIRESrDRct载体的多克隆位点处，构建出真核表达载体pIRES，DRccβ＊0405。对构建的载体进行限制性内切酶鉴定和测序。采用脂质体转染方法将表达载体转入小鼠成纤维细胞系DAP2．3中，G418抗性筛选，并进行单克隆扩增。通过流式细胞术对细胞克隆进行鉴定，筛选出高表达HLA．DR4（＊0005）的细胞株，并采用激光共聚焦显微镜观察H1A-DR4（＊0405）在小鼠成纤维细胞中的表达情况。结果酶切鉴定和测序证实目的基因插入正确且序列与Genbank一致。流式细胞术筛选得到了高表达HLA-DR4（＊0005）的稳定转染细胞株，阳性率达到74．65％。激光共聚焦显微镜观察证实HLA-DR4（＊0405）分子在小鼠成纤维细胞系得到了表达，主要分布于胞膜及胞质内。结论成功构建出真核表达载体pIRES2-DRαβ1＊0405，并在小鼠成纤维细胞系中获得稳定表达，为进一步开展HM-DRB1＊0405的功能研究和转基因鼠模型的建立奠定了基础。
Objective To construct the eukaryotic expression vector which carries the genes of human leukocyte antigen （HLA） DR4 alpha chain and beta chain （＊0405）, and investigate the stable expression of HLA-DR4 （＇0405） molecules in the mouse fibroblasts. Methods HLA-DRA chain open reading frame （ORF） was obtained by PCR from a plasmid which carries a full cDNA sequence of alpha chain. HLA-DRBI＊0405 ORF sequence was generated by RT-PCR method from total RNA of the peripheral blood mononuclear cells which express the HLA-DR4 （＊0405） molecules. The two genes were inserted into pIRES2-EGFP expression vector after enhanced green fluorescent protein （EGFP） fragment was cut off by enzyme digestion. The inserted sequences were confirmed by enzyme digestion and sequencing. Then the constructed pIRES2-DRαβ＊0405 was transfected into mouse fibroblast cell line DAP2.3 and the stable cell lines were obtained by G418 selection and flow cytometry. The expression and intracellular distribution of HLA-DR4（＊0405） was observed with laser confocal scanning microscope. Results The sequences inserted into the pIRES2 vector were the target sequence confirmed by Genbank. One stable cell line highly expressing HLA-DR4 （＊0405） （74.65%） was obtained by fluorescence activated cell sorter （FACS） analysis. Furthermore, HLA-DRg（＊0405） molecules could be stably expressed in the membrane and cytoplasm of DAP2.3 cells. Conclusions We succeed in constructing the eukaryotic expression vector pIRES2-DRαβ＊0405, and confirm that the heterodimeric HLA-DR4 （＊0405） molecules could be stably expressed in DAP2.3 cells. We also obtain one cell line expressing HLA-DR4 （＊0405） stably. It provides the possibility to establish the ＊0405 transgenic mice model.
出处
《中华临床免疫和变态反应杂志》
2007年第1期8-13,130,共7页
Chinese Journal of Allergy & Clinical Immunology
基金
国家自然科学基金(30430290)
国家高技术研究发展计划项目(863项目)(2006AA02Z4D0)
北京市自然科学基金(7052044)
关键词
人类白细胞抗原
真核表达
类风湿关节炎
human leukocyte antigen
eukaryotie expression
rheumatoid arthritis
