TIGIT-Fc融合蛋白的表达以及对巨噬细胞功能的影响

Immunoregulation effect of TIGIT-Fc on macrophages

目的:构建携带小鼠TIGIT-Fc融合基因的慢病毒载体,稳定整合基因至小鼠H22细胞,制备并纯化TIGIT-Fc融合蛋白,探讨其对巨噬细胞功能的影响。方法:利用分子生物学方法将小鼠TIGIT胞外段与人IgG3 Fc段基因融合,构建分泌型TIGIT-Fc载体,再重组入慢病毒载体,感染小鼠H22细胞,接种至C57BL/6小鼠腹腔产生腹水,收集腹水后通过蛋白A柱纯化获得TIGIT-Fc融合蛋白,以此干预LPS处理的巨噬细胞,检测其分泌IL-10的水平。结果:TIGIT-Fc蛋白在H22细胞中以分泌形式表达,经过纯化后的TIGIT-Fc融合蛋白可在体外作用于高表达PVR受体的成熟巨噬细胞,促进其分泌抗炎症因子IL-10。结论:TIGIT可负调节成熟巨噬细胞的功能,促进其分泌IL-10。

Objective： To prepare the production of TIGIT-Fe fusion protein using H22 cells stably integrated the gene by lentivirus vector, and to explore the immunoregulatory effect on maerophages by TIGIT-Fe. Methods： T1GIT-Fc fusion gene were constructed by molecular cloning. The fusion gene was then subeloned to plasmids contained the secretion signaling peptide. The secrected TIGIT-Fe fusion gene was inserted into the lentivirus backbone vector. The purified lentivirus vector was the used to infect the murine H22 cell line. TIGIT-Fc protein was purified by protein A column from the aseites of H22-injeeted C57BL/6 mice. Macrophages stimulated by lipopolysaecharide （LPS） was challenged to TIGIT-Fc treatment or control. Cytokine levels was then detected by ELISA. Results： TIGIT-Fc protein was purified from the aseites of H22-injected mice. PVR was upregulated in LPS-treated macrophages. IL-10 level was upregulated in TIGIT-Fc treated maerophages. Conclusion： TIGIT-Fc promotes the mature macrophages to secrete anti-inflammatory eytokine IL-10.