小鼠PD-1及其配体PD-L1胞外区基因的原核表达及亲和性  

Prokaryotic expression of murine PD-1 and its ligand PD-L1 extracellular domain gene and their affinity

作　　者：吕开绩 陈旭东[1] 程开[1] 王路得 朱进顺 Kamara Saidu 张丽芳[2] 朱冠保[1] LYU Kaoi;CHEN Xudong;CHENG Kai;WANG Lude;ZHU Jinshun;KAMARA Saidu;ZHANG Lifang;ZHU Guanba(Department of Gastroenterological Surgery,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou,325015;Instiute of Molecular Virology and Immunology,Department of Medical Microbiology and Immunology,Wenzhou Medical University,Wenzhou,325035)

机构地区：[1]温州医科大学附属第一医院胃肠外科,浙江温州325015 [2]温州医科大学分子病毒与免疫研究所微生物学与免疫学教研室,浙江温州325035

出　　处：《温州医科大学学报》2018年第6期401-407,412,共8页Journal of Wenzhou Medical University

基　　金：国家自然科学基金资助项目(81372447);浙江省自然科学基金资助项目(Y2100660)

摘　　要：目的:制备小鼠PD-1胞外区(mPD-1)与其配体PD-L1胞外区(mPD-L1)原核表达蛋白,并制备mPDL1兔多克隆抗体,在体外对mPD-L1和mPD-1的结合亲和性进行验证。方法:通过分子克隆方法分别构建重组质粒pET21a/mPD-L1和p GEX4T-1/mPD-1,并通过原核表达系统制备相应的重组蛋白并纯化,通过Western blot进行鉴定;将纯化的mPD-L1蛋白免疫健康日本大耳白兔制备兔多克隆血清抗体,用ELISA、Western blot分析得到的多克隆抗体的效价和特异性,并且将该多克隆抗体作为免疫荧光的阳性对照。最后应用ELISA和免疫荧光验证mPD-1与其配体mPD-L1蛋白的亲和性。结果:SDS-PAGE和Western blot结果显示原核表达的重组蛋白与预期大小一致,mPD-L1和mPD-1分别为25 kDa和40 kDa。经mPD-L1蛋白免疫后的日本大耳白兔能够产生特异性抗体,抗体效价在免疫后第6周达到高峰,Western blot检测结果显示多克隆血清可特异性识别mPD-1。ELISA结果表明,原核表达的mPD-L1蛋白能够和mPD-1蛋白结合,实验组OD450值明显高于对照组。免疫荧光结果显示,在小鼠PD-L1阳性细胞B16(小鼠黑色素瘤细胞株)的胞膜区出现绿色荧光团块。结论:利用原核细胞表达并纯化后获得mPD-L1和mPD-1蛋白,并在体外成功验证了mPD-L1和mPD-1之间的结合特性,为后期利用mPD-1和PD-L1的生物学特性研究提供了基础。Objective： To prepare mouse PD-1（mPD-1） and its ligand PD-L1（mPD-L1） protein by prokaryotic expression system and mPD-L1 rabbit polyclonal antibody to verify in vitro the binding affinity of mPDL1 and mPD-1. Methods： The recombinant plasmids p ET21 a/mPD-L1 and p GEX 4 T-1/mPD-1 were constructed respectively by molecular cloning. Then the recombinant protein of PD-L1 and PD-1 was purified by Ni-NTA affinity chromatography and GST Agarose beads before it was confirmed by SDS-PAGE and Western blot. The purified mPD-L1 extracellular domain protein was immunized with healthy Japanese white rabbits to prepare rabbit polyclonal serum antibody. The titer and specificity of the obtained polyclonal antibody were analyzed by ELISA and Western blot, and the polyclonal antibody was used as positive control in immunofluorescence. Results：SDS-PAGE and Western blot results showed the expected size of the prokaryotic expression of the recombinant protein and mPD-L1 and mPD-1 were 25 kDa and 40 kDa respectively Japanese white rabbits immunized with mPD-L1 protein could produce specific antibodies with its titer peaked at 6 weeks after immunization. Western blot also showed that polyclonal serum could specifically recognize mPD-1. ELISA results showed that prokaryotic mPD-L1 protein could bind mPD-1 protein, with the experimental group OD450 reading significantly higherthan the control group. Immunofluorescence results showed that green fluorescent mass seen in the membrane area of B16 cell（mouse melanoma cell） line which expressed PD-L1. Conclusion： mPD-L1 and mPD-1 proteins obtained after prokaryotic expression and then purified, and successfully verified the binding characteristics between mPD-L1 and mPD-1 in vitro, which provided the basis for the later study of the biological characteristics of mPD-1 and PD-L1.

关 键 词：mPD-1 mPD-L1 原核表达 干扰素 ELISA 免疫荧光 小鼠 

分 类 号：R392.7[医药卫生—免疫学]