基础医学与临床 ›› 2010, Vol. 30 ›› Issue (5): 534-537.

• 技术与方法 • 上一篇    下一篇

钙滴定法测定99mTc定点标记膜联蛋白V的亲和力

蔡炯 李方 牛娜 王世真   

  1. 中国医学科学院北京协和医院核医学科 中国医学科学院北京协和医院核医学科
  • 收稿日期:2009-09-01 修回日期:2009-11-24 出版日期:2010-05-05 发布日期:2010-05-05
  • 通讯作者: 李方

The measurement of affinity of site-specific labeled annexin V with 99mTc by calcium titration

Jiong CAI, Fang LI, Na NIU, Shi-zhen WANG   

  1. Department of Nuclear Medicine, PUMC Hospital Department of Nuclear Medicine, PUMC Hospital
  • Received:2009-09-01 Revised:2009-11-24 Online:2010-05-05 Published:2010-05-05
  • Contact: Fang LI,

摘要: 目的 测定一种新型99mTc定点标记膜联蛋白V与磷脂酰丝氨酸外翻红细胞的结合亲和力。 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;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。

关键词: 膜联蛋白V, 亲和力, 99mTc, 毕赤酵母, 磷脂酰丝氨酸

Abstract: 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 with sodium glucoheptonic acid and SnCl2. The radioactive annexin V was added at wide range of concentration 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 lower protein to cells ratio as 33.4. Conclusion The annexin V expressed recombinantly in pichia pastoris show higher affinity to PS exposed erythrocytes.

Key words: annexin V, affinity, 99mTc, pichia pastoris, phosphatidylserine