膜联蛋白V的异硫氰酸荧光素标记和应用研究

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 带有金属螫合位点的膜联蛋白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.

The Conjugation of Fluorescein Isothiocyanate to Annexin V and its Application.

Objective To prepare a novel annexin V and conjugate it with fluorescein isothiocyanate and test their binding ability to phosphatidylserine(PS)exposed erythrocytes.Methods The annexin V fused with an metal chelating binding site was obtained from pichia pastoris culturing and methanol induction expression.The annexin V was purified from the culture supernatant crude product by ion-exchange chromatography and ammonium sulfate precipitation.The annexin V was conjugated with fluorescein isothiocyanate in bori...