CD83重组蛋白的原核表达、复性纯化及多克隆抗体制备  

Prokaryotic expression,renaturation,purification and polyclonal antibody preparation of recombinant CD83

作　　者：马宁 李兴杰 姜鸿宇 徐国锋 王星 张宗德(指导) MA Ning;LI Xingjie;JIANG Hongyu;XU Guofeng;WANG Xing;ZHANG Zongde(Inflammation&Allergic Diseases Research Unit,the Affiliated Hospital of Southwest Medical University,Luzhou 646000,China)

机构地区：[1]西南医科大学附属医院炎症与变态反应实验室,泸州646000 [2]西南医科大学基础医学院,泸州646000

出　　处：《中国免疫学杂志》2022年第20期2506-2511,共6页Chinese Journal of Immunology

基　　金：国家自然科学基金项目(82000016);泸州市人民政府-西南医科大学科技战略合作项目(00022690);西南医科大学自然科学基金项目(00031586,00031294)资助。

摘　　要：目的:利用原核表达系统表达、纯化CD83蛋白,制备CD83多克隆抗体。方法:构建CD83胞外结构域的原核表达质粒,利用TOP10F’菌株表达,使用IPTG诱导并寻找最佳条件;采用0.3%SKL将包涵体溶解变性,透析复性后通过GST亲和层析纯化GST-CD83蛋白;通过QuickAntibody免疫佐剂免疫SD大鼠制备CD83多克隆抗体,ELISA分析抗体的反应活性和特异性。结果:成功构建重组质粒pGEX-2T-CD83(Met1-Arg133),获得最佳诱导条件:0.2 mmol/L IPTG、25℃、O/N。成功获得CD83重组蛋白,免疫大鼠后获得了特异性及活性较高且效价为1∶104~1∶105的多克隆抗体。结论:获得的CD83重组蛋白及多克隆抗体,为后期深入开展CD83蛋白及CD83抗体的免疫抑制特性研究提供支持。Objective:To obtain CD83 protein using prokaryotic expression system,and to produce CD83 polyclonal antibodies.Methods:The prokaryotic expression plasmid of CD83 extracellular domain was constructed,which was expressed by Top10F’,induced by IPTG and searched for the optimal conditions.The inclusion body was dissolved and denaturated by 0.3%SKL.After dialysis renaturation,GST-CD83 protein was purified by GST affinity chromatography system.The CD83 polyclonal antibody was producted by immunizing SD rats with QuickAntibody system,and the reactivity and specificity were analyzed by ELISA.Results:The recombinant plasmid pGEX-2T-CD83(Met1-Arg133)was successfully constructed,and the optimal induction conditions were as follows:0.2 mmol/L IPTG,25℃,O/N.The recombinant CD83 protein was successfully obtained.After immunization in SD rats,polyclonal antibodies with high specificity,high activity and high titer of 1∶104~1∶105were obtained.Conclusion:The recombinant protein and the polyclonal antibody of CD83 will provide support for further research on the immunosuppressive properties of CD83 protein and CD83 antibody.

关 键 词：CD83重组蛋白 原核表达 包涵体复性 多克隆抗体 

分 类 号：R392.12[医药卫生—免疫学]