节点文献
可溶性HLA-B54/IgG1Fc二聚体融合蛋白的构建表达及功能鉴定
Construction and functional test of the divalent HLA-B54/IgG1Fc fusion protein
【摘要】 目的构建HLA-B54/IgG1真核表达载体,稳定转染至721.221细胞中,获得HLA-B54/IgG1Fc二聚体融合蛋白。方法通过RT-RCR我们获得了HLA-B54的胞外段与人IgG1的CH1-CH2-CH3的cDNA,将HLA-B54与IgG1重链的恒定区插入到质粒pcDNA3.1(+)中形成pcDNA3.1(+)-HLA-B54/IgG1Fc重组基因。为了获得稳定转染,用电穿孔法将质粒pcDNA3.1(+)-HLA-B54/IgG1Fc转染到721.221细胞中,大量收集通过G418筛选的721.221阳性克隆株的无血清的上清,通过PEG20000浓缩得到可溶性HLA-B54/IgG1Fc二聚体融合蛋白,双抗体夹心ELISA和Western-blot(利用MHCⅠ类特异性抗体、人IgG的Fc段特异性抗体)来鉴定融合蛋白。结果限制性内切酶酶切鉴定及序列测定等证实了成功构建了pcDNA3.1+[HLA-B54/IgG1Fc]重组质粒。ELISA和Western-blot结果表明:HLA-B54/IgG1Fc融合基因能够在721.221细胞中表达。融合蛋白由预期的HLA-B54的胞外段与IgG1的Fc段两个部分组成。结论二聚体融合蛋白HLA-B54/IgG1的构建有助于理解HLA-B54限制性的T细胞反应。
【Abstract】 Objective Construct an eukaryotic vector which contains HLA-B54/IgG1 gene,and transfer it into cell721.221 to obtain HLA-B54/IgG1 protein.Methods The cDNA were obtained from extracellular domains of HLA-B54 and IgG1 CH1-Hinge-CH2-CH3 domains of human by using RT-PCR,transfer them into plasmid pcDNA3.1(+) to form pcDNA3.1(+) [HLA-B54/IgG1] plasmid.The HLA-B54/IgG1Fc gene was transfected into 721.221 cell line by electroporation for stable transfection.Dimeric HLA-B54/IgG1Fc fusion protein was collected in serum-free media,which contained high expressing clones(selected under G418),and got concentrated protein through PEG20000.,and identified the protein by using sandwich ELISA and Western blotting.Results The recombinant plasmid pcDNA 3.1(+)-HLA-B54/IgG1Fc was confirmed by double digestion of restriction endonucleases analysis and DNA sequencing.The results from sandwich ELISA and Western blot by specific antibody indicated that the HLA-B54/IgG1Fc hybrid gene expressed in 721.221 cells.The HLA-B54/IgG1Fc fusion protein consists with the expected extracellular domains of HLA-B54 and the Fc region of IgG1.Conclusions The construction of HLA-B54/IgG1Fc fusion protein is useful in understanding the HLA-B54-restricted T-cell response.
【Key words】 Divalent HLA-B54/IgG1 molecule; Eukaryotic expression; Intact conformation;
- 【文献出处】 齐齐哈尔医学院学报 ,Journal of Qiqihar University of Medicine , 编辑部邮箱 ,2013年24期
- 【分类号】R392.1
- 【下载频次】89