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目的对自行研制的可诱导共刺激分子-Ig融合蛋白的理化性质和体内生物学活性进行分析鉴定。方法采用HCl水解法、Edman法、胰酶消化MALDI-TOF-MS质谱法分别测定该融合蛋白的氨基酸组成、N末端15个氨基酸序列、肽谱等理化性质,并通过CFSE标记体内检测淋巴细胞增殖反应实验研究其抑制同种淋巴细胞增殖的生物学活性。结果氨基酸组成分析测得样品的氨基酸组成与ICOS-Ig理论值基本一致。蛋白N末端15个氨基酸序列为EINGSANYEM-FIFHN,与ICOS-Ig理论值一致。MALDI-TOF-MS质谱测定共获得6个与理论预测值相符的肽段。ICOS-Ig可以明显抑制同种T细胞的体内增殖反应。结论所研制的可诱导共刺激分子-Ig融合蛋白结构表达正确且具有体内抑制同种淋巴细胞增殖的活性,为该融合蛋白的质量标准研究奠定了基础。
OBJECTIVE To analyze the physicochemical properties and in vivo biological activity of self-developed co-stimulatory molecule-Ig fusion protein. Methods The amino acid composition, N-terminal 15 amino acid sequence and peptide profile of the fusion protein were determined by the method of HCl hydrolysis, Edman’s method and trypsin digestion MALDI-TOF-MS, respectively. Physiochemical properties of the fusion protein were detected by CFSE. The reaction experiments investigated its biological activity of inhibiting allogeneic lymphocyte proliferation. Results Amino acid composition analysis of the sample amino acid composition and ICOS-Ig theoretical value is basically the same. The 15 amino acid sequence of the protein is EINGSANYEM-FIFHN, which is consistent with the theoretical value of ICOS-Ig. MALDI-TOF-MS mass spectrometry obtained a total of 6 theoretical predictions consistent with the peptide. ICOS-Ig significantly inhibited the proliferation of allogeneic T cells in vivo. Conclusion The constructed co-stimulatory molecule-Ig fusion protein has the correct structure and inhibits the proliferation of allogeneic lymphocytes in vivo, which lays the foundation for the study of the quality standard of the fusion protein.