~99TC^m-HYNIC-Annexin V的制备及其在健康小鼠体内的分布

Preparation of 99~Tc^m-HYNIC-Annexin V and its biodistribution study in normal mice

目的制备人膜联蛋白 V(Annexin V),应用联肼尼克酰胺(HYNIC)偶联后进行^(99)Tc^m 标记,评价^(99)Tc^m-HYNIC-Annexin V 在健康小鼠体内的分布。方法采用基因工程方法构建 Annexin V 的原核载体并诱导其表达,与自行合成的双功能螯合剂 HYNIC 偶联,并进行^(99)Tc^m 标记。测定^(99)Tc^m-HYNIC-Annexin V 的标记率、放化纯,并研究其在健康小鼠体内的生物分布特性。结果 Annexin V在大肠杆菌中获得稳定表达,经十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)及 Western Blot-ting 检测,得到纯度较高的蛋白质。Annexin V 经 HYNIC 偶联后,其^(99)Tc^m 标记率可达90%左右,放化纯>90%。小鼠体内分布结果表明,^(99)Tc^m-HYNIC-Annexin V 血液清除迅速,主要从肾脏和肝脏排泄。结论 Annexin V 可在原核生物中稳定表达。其与 HYNIC 偶联后,^(99)Tc^m 标记率和放化纯较高,方法简单,条件温和。

Objective To prepare ^99Tc^m labeled Annexin V ,conjugated with hydrazinonictinamide （HYNIC） and to evaluate its biodistribution in normal mice. Methods Plasmid of human Annexin V was constructed by gene engineering method and was expressed in E. coll.. HYNIC was prepared and used as bifunctional chelate agent for ^99Tc^m labeling. The radiochemical purity, radiolabeling yield and biodistribution of ^99Tc^m-HYNIC-Annexin V were studied. Results The coding sequence of human Annexin V gene was successfully cloned into the expression vector, and highly expressed in E. coli, . Highly purified protein could be obtained and confirmed by Western Blotting and sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）, The radiochemical purity was over 90% , and the radiolabeling yield reached 90%. The biodistribution in normal mice showed it quickly removed from blood and excreted mainly through kidney and liver. Conclusions Annexin V could be stably expressed in E, coli, and high radiochemical purity and radiolabelling yield gained when conjugated with HYNIC and then labeled with ^99Tc^m. It is an easy and smooth labeling method.