人CS1-Fc融合蛋白的真核表达、蛋白纯化及生物学效应鉴定

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

摘要:

人信号淋巴细胞激活分子F7 (SLAMF7/CS1) 是一种细胞表面糖蛋白，在多发性骨髓瘤细胞中高度表达。已有研究表明CS1是多发性骨髓瘤较为灵敏且特异的生物标志物。CAR-T细胞免疫疗法是治疗多发性骨髓瘤的新方法，其中CS1 CAR-T细胞免疫疗法针对复发性难治性多发性骨髓瘤有较好的疗效。为了检测CS1 CAR-T细胞上CS1 CAR的表达效率和探寻CAR-T细胞免疫疗法的辅助手段，文中制备了一种CS1-Fc融合蛋白。首先利用PCR技术从已有质粒中扩增得到CS1的胞外段序列，再通过重叠延伸PCR与人IgG1-Fc段相连。将重组片段连接至pMH3真核表达载体上，经酶切鉴定和DNA测序后，将重组质粒pMH3-CS1-Fc-his转染至中国仓鼠卵巢细胞 (CHO-S)。经G418加压筛选和流式细胞术鉴定，证实CS1-Fc融合蛋白在CHO-S细胞中获得了表达。利用镍柱对CS1-Fc融合蛋白进行纯化，经Western blotting鉴定，融合蛋白的分子量约为70 kDa。流式细胞术和细胞计数分析结果显示，CS1-Fc融合蛋白能有效检测CS1 CAR的表达效率，证实了CS1-Fc融合蛋白对CS1 CAR-T细胞具有活化、促增殖及促分泌细胞因子的作用。本研究结果为多发性骨髓瘤细胞免疫治疗CAR-T细胞的体外检测和效能强化奠定了实验基础。

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.