人源程序性死亡配体PD-L2蛋白的原核表达与抗血清制备  

Prokaryotic expression and antiserum preparation of human programmed ligand PD-L2 protein

作　　者：杨娟娟 刘照 夏菁潞 罗岭 俞博彤 孟春[1] YANG Juanjuan;LIU Zhao;XIA Jinglu;LUO Ling;YU Botong;MENG Chun(College of Biological Science and Technology,Fuzhou University, Fuzhou,Fujian 350116,China)

机构地区：[1]福州大学生物科学与工程学院,福建福州350116

出　　处：《福州大学学报（自然科学版）》2018年第3期438-444,共7页Journal of Fuzhou University(Natural Science Edition)

基　　金：国家自然科学基金资助项目(31700656);福建省自然科学基金资助项目(2017J05050);福州大学本科生科研训练计划(SRTP)资助项目(23119;23125);福州大学贵重仪器设备开放测试基金资助项目(2017T025)

摘　　要：利用原核表达系统获得包涵体表达形式的重组蛋白h PD-L220~123(Ig V结构域)和h PD-L220~208(Ig V结构域和Ig C结构域);将重组蛋白利用Ni-NTA柱亲和层析纯化与复性后,作为免疫原免疫Balb/c雌性小鼠制备鼠抗人的多抗血清,经ELISA法检测其效价均达到1∶106;Western-blot实验结果表明,制备的多抗血清均能特异性识别h PD-L2蛋白.以制备的多抗血清进行IHC实验评估与鉴定,结果显示:以h PD-L220~123重组蛋白为免疫原制备的多抗血清阳性较弱且定位模糊,以h PD-L220~208重组蛋白为免疫原制备的多抗血清具有很强的膜定位且背景清晰.The recombinant proteins h PD-L220-123（ the major antigenic epitope region of human PDL2） and h PD-L220-208（ the extracellular domain of human PD-L2） were expressed in inclusion bodies.The two recombinant proteins were purified by Ni-NTA agarose resin and refolded. Then,the rat antiserums against the recombinant proteins of h PD-L220-123 and h PD-L220-208 were prepared with titers of1 ∶ 106 measured by indirect ELISA. Western-blot experiments demonstrated that the prepared antiserums can specifically recognize h PD-L2 protein. Finally,lung cancer tissues with high level expression of h PD-L2 were used to assay the location of the prepared antiserums by IHC experiments,the results indicating that the membrane localization of the antiserum prepared by antigen of h PD-L220-123 protein was blur and weak; the membrane localization of the antiserum prepared by antigen of h PD-L220-208 protein had a specific localization,with less background and non-specific staining.

关 键 词：人源PD-L2蛋白 主要抗原表位区 原核表达 抗血清 

分 类 号：Q511[生物学—生物化学]