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

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。

Abstract:

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.