诱生型一氧化氮合酶抗体的制备与初步应用  

Preparation and application of the specific antibody against iNOS

作　　者：许祥裕[1] 朱敏生[1] 潘英[1] 沈月[1] 朱东亚[2] 朱有华[1] 

机构地区：[1]南京军区军事医学研究所,江苏南京210002 [2]中国药科大学,江苏南京210009

出　　处：《中国生化药物杂志》2001年第4期169-172,共4页Chinese Journal of Biochemical Pharmaceutics

基　　金：国家自然科学基金资助项目 ( 396 70 85 6 )

摘　　要：目的制备诱生型一氧化氮合酶 (iNOS)特异性抗体并应用于iNOS检测。方法将iNOSN端片段装入原核表达载体pET 2 8a(+) ,在大肠杆菌BL2 1中表达 ,两步法纯化目的蛋白 ,使用纯化蛋白制备iNOS多克隆抗体。获得的抗血清用于小鼠巨噬细胞及小鼠脑缺血模型中iNOS的检测。结果得到具有较高表达量的融合蛋白 ,经两步纯化获得iNOSN端融合蛋白纯品 ,将此蛋白免疫家兔 ,得到iNOS多克隆抗体 ,Westernblot分析表明该抗体不与nNOS、eNOS交叉反应 ,而能检测组织、细胞中的iNOS。PurposeThe aim is to prepare specific antibody against iNOS and to use it in detecting iNOS.Methods The fragment of iNOS(1 196AA)was inserted in pET 28a(+) plasmid and the combinant construction was transformed into BL 21 E.coli. Adopt two steps to purify the expressed protein: 1. Purification with a His.Bind TM column. 2. Recovery from SDS PAGE gel. The antibody was raised by immunizing rabbits with purified protein, and used in detecting iNOS in macrophages that induced by TNF and in murine ischemic brain. ResultsThe fine purified target protein was obtained by two step purification. A polycolonal antibody was raised in the rabbit against the purified recombinant protein. Western blot analysis revealed that it did not cross react with nNOS and eNOS,and could detect the iNOS expressed in murine macrophages and murine ischemic brain. ConclusionThe antibody against iNOS that was prepared by iNOS N terminal fusion protein had good specificity and reactivity.

关 键 词：诱生型一氧化氮合酶 重组蛋白 特异性抗体 制备 应用 

分 类 号：R392－33[医药卫生—免疫学] Q503[医药卫生—基础医学]