重组人免疫球蛋白D-Fc片段蛋白的构建  

Construction of recombinant human immunoglobulin D-Fc fragment protein

作　　者：张静 陈文生[1] 胡晓曦 黄琼[1] 吴育晶[1] 魏伟[1] Zhang Jing;Chen Wensheng;Hu Xiaoxi(Institute of Clinical Pharmacology,Anhui Medical University,Key Laboratory of Anti-inflammatory and Immune Medicine,Ministry of Education,Collaborative Innovation Center of Anti-inflammatory and Immune Medicines,Hefei 230032)

机构地区：[1]安徽医科大学临床药理研究所抗炎免疫药物教育部重点实验室抗炎免疫药物安徽省协同创新中心,合肥230032

出　　处：《安徽医科大学学报》2019年第4期514-519,共6页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81330081;81673444;81603121);安徽医科大学博士科研资助项目(编号:XJ201629)

摘　　要：目的构建重组人免疫球蛋白D-Fc(IgD-Fc)片段蛋白,检测IgD受体(IgDR)在健康人CD4^+T细胞和T淋巴瘤细胞株Jurkat、MOLT-4上的表达情况,检测IgD-Fc与IgD竞争结合CD4+T细胞表面IgDR的能力。方法采用PCR法得到人IgD-Fc目的基因,经原核蛋白表达和层析纯化,制备出重组人IgD-Fc片段蛋白。流式细胞术检测IgDR在健康人CD4+T细胞和T淋巴瘤细胞株Jurkat、MOLT-4上的表达情况,以及IgD、重组人IgD-Fc片段蛋白与CD4+T细胞表面IgDR的竞争结合情况。结果成功构建含IgD-Fc基因片段的原核表达载体p ET28a(+)/IgD-Fc,得到重组人IgD-Fc片段蛋白。CD4+T细胞及T淋巴瘤细胞株表面IgDR与IgD亲和力相近,重组人IgD-Fc片段蛋白可与IgD竞争性结合IgDR。结论重组人IgD-Fc片段蛋白工艺成熟稳定,可与IgD竞争性结合IgDR,可能成为针对IgD水平升高的自身免疫性疾病的新型治疗药物。Objective To recombine human immunoglobulin D-Fc(IgD-Fc)fragment protein,to detect the expression of IgD receptor(IgDR)on CD4^+T cells of healthy controls and Jurkat,MOLT-4 T lymphoma cell lines,and to observe the ability of IgD-Fc competing with IgD to bind to IgDR on the surface of CD4^+T cells.Methods IgD-Fc target gene was extracted by PCR method,and the recombinant human IgD-Fc fragment protein was obtained through the expression of prokaryotic protein and chromatography purification.Flow cytometry was used to detect the expression of IgDR on CD4^+T cells of healthy controls and Jurkat,MOLT-4 T lymphoma cell lines,and to study the ability of IgD-Fc competing with IgD to bind to IgDR on CD4^+T cells.Results The prokaryotic expression vector pET28 a(+)/IgD-Fc containing target gene fragment was constructed,and the recombinant human IgD-Fc fragment protein was obtained,the affinity of IgD binding to IgDR on the surface of CD4^+T cells and T lymphoma cell lines was similar,IgD-Fc recombinant protein could competing with IgD to binding with IgDR.Conclusion The technology of recombinant human IgD-Fc fragment protein is mature and stable,recombinant human IgD-Fc fragment protein can competitively block IgD binding to IgDR,and may become a novel therapeutic treatment for autoimmune diseases with elevated IgD levels.

关 键 词：免疫球蛋白D IgDR 自身免疫性疾病 蛋白纯化 

分 类 号：R967[医药卫生—药理学]