人血清淀粉样蛋白A的重组表达及免疫学检测标准品的初步研究

Recombinant Expression of Human Serum Amyloid A and its Application as Standard Material for Immunoassay

构建人SAA1原核表达载体,建立SAA1蛋白大肠杆菌表达条件和纯化工艺,评价重组SAA1蛋白在制备免疫学检测标准物质中的应用前景。采用同源重组的方法构建表达载体,在N端加入6×His和SUMO标签帮助SAA1蛋白的可溶性表达和纯化;在小量体系中评价SUMO-SAA1蛋白的可溶性表达后,用镍亲和柱和阴离子交换层析方法纯化蛋白,用蛋白酶切除SUMO标签后再通过镍亲和柱层析方法获得高纯度人SAA1重组蛋白;用西门子N-Latex SAA试剂评价重组蛋白的免疫反应性和稳定性,并与SAA国际标准物质进行比较研究。结果表明SUMO-SAA1在大肠杆菌中大部分为可溶性表达且表达量高,产物纯度可达90%以上,稳定性良好并与SAA国际标准物质具有类似的免疫反应性。利用SUMO标签的助溶作用,成功在大肠杆菌中表达了人SAA1重组蛋白,该蛋白适合作为制备SAA1免疫检测的参考标准物质的原材料。

This study aims to obtain human SAA1 recombinant protein by constructing human SAA1 prokaryotic expression vector,expressing human SAA1 recombinant protein in E. coli and establishing its purification procedure. Moreover,the application of SAA1 recombinant in the preparation of SAA1 standard material for immuoassay is also evaluated. SAA1 expression vector was constructed by homologous recombination,with 6 × His and SUMO tag at the N terminal of SAA1 protein. After assessing the solubility in E. coli,SAA1 protein was purified by Ni^2＋affinity column and anion-exchange chromatography. The stability and immuno-reactivity were measured by Simens N-Latex SAA reagent,and compared with WHO SAA 1 st international standard. Results showed that SUMOSAA1 was highly expressed in E. coli in soluble form. Purified SAA1 protein had high purity（〉90%）,good stability and immunoreactivity similar to WHO SAA 1 st international standard. Recombinant human SAA1 is succesfully expressed in E. coli with the assisstance of SUMO tag. The purified SAA1 recombinant protein is suitable as the key material of standard material,calibrator and control for SAA1 immunoassay.