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目的制备一种新型的膜联蛋白V,用异硫氰酸荧光素进行标记,观察它对外露磷脂酰丝氨酸(PS)红细胞的荧光染色情况。方法带有金属螯合位点的膜联蛋白V通过毕赤酵母的培养和甲醇诱导表达获得,膜联蛋白V粗品通过离子交换和硫酸铵沉淀得到纯化产物。在硼酸盐缓冲液完成膜联蛋白V的异硫氰酸荧光素标记,标记产物经过离子交换进行纯化。红细胞经过戊二醛处理后PS外露于细胞表面。在荧光显微镜下观察异硫氰酸荧光素标记的膜联蛋白V对外露PS红细胞的结合。结果毕赤酵母重组表达的新型膜联蛋白V经过离子交换和硫酸铵沉淀后得以纯化和浓缩。经过异硫氰酸荧光素进行标记后观察到它对外露PS红细胞的结合。结论毕赤酵母系统重组表达的膜联蛋白V具有结合外露PS红细胞的能力,可以应用于细胞凋亡的体外检测。
Objective To prepare a novel Annexin V labeled with fluorescein isothiocyanate and observe the fluorescence staining of exposed phosphatidylserine (PS) erythrocytes. Methods Annexin V with metal chelation sites was obtained by cultivation with Pichia pastoris and expression by methanol. The crude product of Annexin V was purified by ion exchange and ammonium sulfate precipitation. Complementing fluorescein isothiocyanate labeled with annexin V in borate buffer, the labeled product is purified by ion exchange. Erythrocytes treated with glutaraldehyde (PS) were exposed on the cell surface. The binding of fluorescein isothiocyanate labeled annexin V to exposed PS red blood cells was observed under a fluorescence microscope. Results The recombinant Annexin V recombinantly expressed in Pichia pastoris was purified and concentrated by ion exchange and ammonium sulfate precipitation. After labeling with fluorescein isothiocyanate, its binding to exposed PS red blood cells was observed. Conclusions Annexin V recombinantly expressed in Pichia pastoris has the ability to bind with exposed PS red blood cells and can be used for in vitro detection of apoptosis.