摘要
目的测定一种新型99mTc定点标记膜联蛋白V与磷脂酰丝氨酸(PS)外翻红细胞的结合亲和力。方法通过毕赤酵母的培养和甲醇诱导表达获得带有金属螯合位点的膜联蛋白V;用超滤的方法纯化膜联蛋白V;用葡庚糖酸钠和氯化亚锡还原将99mTc定点标记于膜联蛋白V氨基末端。采用不同浓度钙离子滴定放射性膜联蛋白V结合磷脂酰丝氨酸外翻红细胞的亲和力。结果在不同的蛋白质分子/细胞比值下测定出的半数标记蛋白结合细胞的钙离子浓度是不同的,在低蛋白质分子/细胞比值下测定出99mTc-膜联蛋白V对细胞的亲和力pK=33.4。结论毕赤酵母系统重组表达的膜联蛋白V具有较高的结合外露PS红细胞的能力。
Objective To derermine the affinity of site-specific labeled annexin V with ^99mTc to phosphatidylserine（PS） exposed erythrocytes.Methods The annexin V fused with a metal chelating binding site was obtained from Pichia Pastoris culture and methanol induction expression.The annexin V was purified from the culture supernatant crude product by ultrafiltration.The annexin V was conjugated with ^99mTc site-specifically through sodium glucoheptonic acid and SnCl2.The radioactive annexin V was added to determine its affinity to PS exposed erythrocytes.Results The calcium concentration at which half of the protein is bound to cells（EC50） is changed with varying ratio of protein to cells.The affinity was determined at a low protein/cell ratio as 33.4.Conclusion The annexin V recombinantly expressed in Pichia Pastoris shows high affinity to PS exposed erythrocytes.
出处
《基础医学与临床》
CSCD
北大核心
2010年第5期534-537,共4页
Basic and Clinical Medicine
基金
北京协和医院临床重点课题
霍英东肺癌专项基金
