摘要
利用对血栓部位活化血小板α 颗粒膜蛋白GMP 140有亲和特异性的单克隆抗体SZ5 1的血栓部位导向作用 ,并利用金属硫蛋白的高金属结合能力 ,在基因工程水平构建分子量适中的单链抗体血栓导向显像剂 .1分子单抗SZ5 1单链抗体 (ScFv SZ5 1)与 1分子小鼠金属硫蛋白 (MT Ⅰ )cDNA的重组基因拼接产物 ,在大肠杆菌菌株BL2 1(DE3)pLysS中进行发酵表达 ,重组蛋白HLMT表达量为 4 0mg L发酵液 .该重组蛋白以包涵体形式存在于菌体中 .通过一种有效的包涵体制备方法可获得目的蛋白含量为 84 %的包涵体沉淀 .在优化的条件进行了包涵体的变复性 ,复性液先后经过Q SepharoseFF阴离子交换层析和SephadexG 5 0凝胶过滤层析进行纯化 .SDS PAGE鉴定了重组蛋白纯度达 95 %以上 .HPLC进一步证明了最终纯化产品的均一性 .质谱测定HLMT分子量为 384 31 3,与理论值基本相符 .等电聚焦电泳测定pI为 3 0 .Western印迹结果表明 ,HLMT中单链抗体部分具有与活化血小板α 颗粒膜蛋白GMP 140结合特异性 .原子吸收分光光度计法 (AAS)证明 ,HLMT中金属硫蛋白保持了原金属结合活性 .
A novel recombinant imaging protein directing thrombus was developed as potential thrombus detection agent. A recombinant gene was constructed with one mole of SZ51 single chain fragments of variable region(ScFv), which reacted specifically to the α granule membrane protein GMP 140 on the surface of activated platelets during thrombus forming, and one mole of MT which was fused to C terminus as labeling moiety. It was expressed in \%E.coli\% BL21(DE3)pLysS. The recombinant products named HLMT were accumulated as cytoplasmic inclusion bodies and then refolded \%in vitro\%. Several factors influencing the refolding activity were examined and a way of efficient recovery of active protein was established. The refolding solution was applied to Q Sepharose FF ion exchange chromatography and Sephadex G 50 chromatography. The purity determined by SDS PAGE was over 95%. The homogeneity of the purified protein was proved by HPLC. Mass spectrometry showed that the molecular weight of HLMT was 38431 3. The p I was 3 0. The atomic absorption spectra photometry (AAS) proved that the HLMT had the same metal binding capacity as metallothionein. Western blotting showed that the HLMT had the activity of anti human activated platelets as the intact antibody SZ51. HLMT was expected to be a fast, effective targeting and imaging agent in the clinical thrombus detection.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2001年第6期781-785,共5页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家九五攻关项目 (No.96 C0 2 0 1)资助~~
关键词
血栓导向显像剂
金属硫蛋白
单链抗体
分离纯化
性质
imaging agent directing thrombus, metallothionein, ScFv, isolation and purification, characterization
