Eukaryotic expression, protein purification and biological effects research of human CS1-Fc fusion protein

CHEN Ruzhang1 WANG Xitong1 LI Yanchen1 GAO Jimin1

(1.School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China 325035)

【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. The CS1-Fc fusion protein could effectively detect the expression rate of CS1 CAR and promote the activation, proliferation and cytokine 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.

【Keywords】 human signaling lymphocyte activation family 7 (SLAMF7/CS1); CS1-Fc fusion protein; eukaryotic expression; CAR-T; multiple myeloma;

【DOI】

【Funds】 National Natural Science Foundation of China (No. 81573110) Research Foundation of National Health Commission of the People’s Republic of China (No. WKJ-ZJ-1928) Science and Technology Major Projects of Wenzhou (No. ZJ2017014) Technology Innovation and Application Development Major Projects of Chongqing (No. cstc2019jscx-msxm X0431)

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(Translated by HE Z)

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This Article

ISSN:1000-3061

CN: 11-1998/Q

Vol 36, No. 01, Pages 122-132

January 2020

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Article Outline

Abstract

  • 1 Materials and methods
  • 2 Results and analysis
  • 3 Discussion
  • References