Construction, expression and purification of a mammalian secretory recombinant fusion protein rPC.
- Author:
Chunchun LI
1
;
Yuqiong XIE
1
;
Jiang CAO
1
;
Jimin SHAO
2
Author Information
- Publication Type:Journal Article
- Keywords: CTLA-4; PD-1; immune checkpoint; recombinant fusion protein
- MeSH: Animals; CTLA-4 Antigen; genetics; Cell Proliferation; HEK293 Cells; Humans; Lung Neoplasms; metabolism; Programmed Cell Death 1 Receptor; genetics; Protein Binding; Protein Domains; genetics; Recombinant Fusion Proteins; genetics; isolation & purification; metabolism
- From: Chinese Journal of Biotechnology 2020;36(5):969-978
- CountryChina
- Language:Chinese
- 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.
