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[Construction, expression and purification of a mammalian secretory recombinant fusion protein rPC].
- Source :
-
Sheng wu gong cheng xue bao = Chinese journal of biotechnology [Sheng Wu Gong Cheng Xue Bao] 2020 May 25; Vol. 36 (5), pp. 969-978. - Publication Year :
- 2020
-
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.
- Subjects :
- Animals
CTLA-4 Antigen genetics
Cell Proliferation
HEK293 Cells
Humans
Programmed Cell Death 1 Receptor genetics
Protein Binding
Protein Domains genetics
Lung Neoplasms metabolism
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins isolation & purification
Recombinant Fusion Proteins metabolism
Subjects
Details
- Language :
- Chinese
- ISSN :
- 1872-2075
- Volume :
- 36
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Sheng wu gong cheng xue bao = Chinese journal of biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 32567280
- Full Text :
- https://doi.org/10.13345/j.cjb.190405