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。
Objective Construct an eukaryotic vector which contains HLA-B54/IgGI gene, and transfer it into ce11721. 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/IgG11 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-B5g/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.
出处
《齐齐哈尔医学院学报》
2013年第24期3596-3598,共3页
Journal of Qiqihar Medical University
