Eukaryotic expression, protein purification and biological effects research of human CS1-Fc fusion protein.
- Author:
Ruzhang CHEN
1
;
Xitong WANG
1
;
Yanchen LI
1
;
Jimin GAO
1
Author Information
- Publication Type:Journal Article
- Keywords: CAR-T; CS1-Fc fusion protein; eukaryotic expression; human signaling lymphocyte activation family 7 (SLAMF7/CS1); multiple myeloma
- From: Chinese Journal of Biotechnology 2020;36(1):122-132
- CountryChina
- Language:Chinese
- Abstract: Signaling lymphocyte activation family 7 (SLAMF7/CS1) is a cell surface glycoprotein that is highly expressed in multiple myeloma cells. CS1 is a sensitive and specific biomarker for multiple myeloma. CAR-T cell immunotherapy is a new method for the treatment of multiple myeloma. CS1 CAR-T cell immunotherapy has good effect on relapsed refractory multiple myeloma. To detect the expression efficiency of CS1 CAR on CS1 CAR-T cells and to find an auxiliary means to CAR-T cell immunotherapy, we prepared a CS1-Fc fusion protein. First, the extracellular domain of CS1 was amplified from the existing plasmid by PCR and ligated with human IgG1-Fc fragment by overlap extension PCR. The recombinant fragment was ligated into pMH3 eukaryotic expression vector. After restriction enzyme digestion and DNA sequencing, the pMH3-CS1-Fc-his recombinant plasmid was successfully constructed. The recombinant plasmid was transfected into Chinese hamster ovary cell (CHO-S) by liposome. The expression of the CS1-Fc fusion protein in CHO-S cells was identified by flow cytometry after G418 pressure screening. Next, the CS1-Fc fusion protein was purified by nickel column. Western-blot analysis showed that molecular weight of the fusion protein was about 70 kDa was identified by Western blotting. The CS1-Fc fusion protein couldeffectively detect the expression rate of CS1 CAR and promote the activation, proliferation andcytokines secretion of the CS1 CAR-T cells. The results will lay the experimental foundation for the in vitro detection and potentiation of CAR-T cells in multiple myeloma treated with CS1 CAR-T cell.
