一种真核细胞分泌型重组融合蛋白rPC的构建、表达与纯化  

Construction,expression and purification of a mammalian secretory recombinant fusion protein rPC

作　　者：李春春[1] 谢雨琼 曹江[1] 邵吉民[2] Chunchun Li;Yuqiong Xie;Jiang Cao;Jimin Shao(Clinical Research Center,the 2nd Affiliated Hospital,Zhejiang University School of Medicine,Hangzhou 310009,Zhejiang,China;Department of Pathology and Pathophysiology,Zhejiang University School of Medicine,Hangzhou 310058,Zhejiang,China)

机构地区：[1]浙江大学医学院附属第二医院临床研究中心,浙江杭州310009 [2]浙江大学医学院病理学与病理生理学系,浙江杭州310058

出　　处：《生物工程学报》2020年第5期969-978,共10页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.81172516)资助。

摘　　要：抑制性免疫检查点PD-1或CTLA-4靶向治疗药物已用于肿瘤的临床治疗,但单一靶点药物会有耐药发生,联合使用同时封闭多个靶点可提高疗效,因此拟构建一个可封闭多个靶点的新型重组蛋白。首先设计并合成了一个由人类PD-1和CTLA-4两个受体的胞外功能域组成并且C端带6´His标签的分泌型重组融合蛋白rPC编码序列,插入真核细胞表达载体pLVX-IRES-ZsGreen1,稳定转染HEK293细胞,收集细胞培养上清,以亲和方法纯化重组蛋白rPC,通过实时荧光定量PCR检测多个人类肿瘤细胞系中PD-1配体PD-L1、PD-L2和CTLA-4配体CD80、CD86的表达,以选择相对高表达的细胞,利用细胞免疫荧光染色方法检验rPC与肿瘤细胞的结合能力,并用CCK-8法检测rPC是否对肿瘤细胞的生长有影响。结果表明,重组融合蛋白rPC可由稳定转染表达载体的HEK293细胞表达并分泌,纯化后的rPC可以与PD-1和CTLA-4配体表达相对较高的肺癌细胞NCI-H226结合,并且rPC处理对其生长并无直接影响,与预期一致。成功获得的重组融合蛋白rPC可用于进一步的体内外功能研究,也为今后研发新型多靶点肿瘤免疫治疗蛋白药物打下了坚实的基础。Drugs targeting immune checkpoint are used for cancer treatment,but resistance to single drug may occur.Combination therapy blocking multiple checkpoints simultaneously can improve clinical outcome.Therefore,we designed a recombinant protein rPC to block multiple targets,which consists of extracellular domains of programmed cell death protein 1(PD-1)and cytotoxic T lymphocyte-associated antigen 4(CTLA-4).The coding sequence was inserted into expression vector and stably transfected into HEK293 cells.The culture supernatant was collected and rPC was affinity-purified.Real-time quantitative PCR was used to evaluate the expression levels of ligands for PD-1 and CTLA-4 in several human cancer cell lines.The binding of rPC with cancer cells was examined by immunofluorescence cell staining,the influence of rPC on cancer cell growth was assayed by CCK-8.The results showed that rPC could be expressed and secreted by stably transfected HEK293 cells,the purified rPC could bind to lung cancer NCI-H226 cells which have high levels of ligands for PD-1 and CTLA-4,no direct impact on cancer cell growth could be observed by rPC treatment.The recombinant protein rPC can be functionally assayed further for developing novel immunotherapeutic drugs for cancer.

关 键 词：重组融合蛋白 PD-1 CTLA-4 免疫检查点 

分 类 号：R730.51[医药卫生—肿瘤]