摘要
目的探索原核表达系统生产的T细胞免疫球蛋白及黏蛋白结构域蛋白-1-Fc(Tim-1-Fc)融合蛋白能否为Tim-1蛋白模拟品。方法大肠杆菌合成Tim-1-Fc融合蛋白,以体外蛋白质结合实验测定其能否与Tim-4蛋白结合。通过荧光激活的细胞分选(FACS)检测磷脂酰丝氨酸(PS)与膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)间的结合情况,间接探明该蛋白与PS的结合。结果体外蛋白相互作用实验证实Tim-1-Fc融合蛋白可以结合Tim-4蛋白。FACS数据分析表明无论有无过氧化氢诱导,Tim-1-Fc组凋亡细胞FITC信号平均强度(231.16±8.27、263.45±7.91)均高于对照组(190.80±5.09、203.29±10.89,t=-7.200、7.739,P<0.01),差异有统计学意义。结论原核表达版本的Tim-1-Fc融合蛋白可能具备在体外模拟Tim-1蛋白的作用。
Objective To investigate whether T-cell immunoglobulin and mucin-containing protein-1-Fc(Tim-1-Fc)fusion protein derived from prokaryotic system is competent as a Tim-1-mimicking agent.Methods After fusion protein Tim-1-Fc was synthesized by E.coli,an in vitro protein-protein interaction assay was performed to determine whether it could interaction with Tim-4.Its phosphatidylserine(PS)-binding ability was elucidated indirectly by the interaction between PS and Annexin V-fluoresceine isothiocyanate(FITC),which was analyzed via fluorescence activated cell sorting(FACS).Results Protein-protein interaction assay validated that Tim-1-Fc could bind to Tim-4 in vitro.Analysis of FACS data showed that in the absence and presence of H2O2 induction,average intensities of FITC signal from apoptotic cells in Tim-1-Fc-treated group were statistically higher than those in PBS-treated group(231.16±8.27,263.45±7.91,t=-7.200,7.739,P<0.01).Conclusion Prokaryotic version of Tim-1-Fc should be competent to mimic Tim-1 in vitro.
作者
李强
李尧
宋京海
Li Qiang;Li Yao;Song Jinghai(Department of General Surgery,Beijing Hospital,National Centre of Gerontology,Institute of Geriatric Medicine,Chinese Academy of Medical Sciences,Beijing 100730,China;Department of Hepatobiliary and Pancreatic Surgery,Shougang Hospital,Peking University)
出处
《中华实验外科杂志》
CAS
北大核心
2022年第10期1899-1902,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金(81671581)。
