人血清淀粉样蛋白A1原核表达及胶体金检测方法的建立

通过胶体金免疫层析技术建立一种特异、便捷、快速的SAA1定量检测新方法.利用大肠杆菌BL21(DE3)原核表达人血清淀粉样蛋白A1(SAA1),根据BL21(DE3)表达偏好性设计并合成SAA1蛋白基因片段,构建表达载体pET-28a-SAA1,采用CaCl2法制备BL21(DE3)感受态,热激转化法将pET-28a-SAA1转入到BL21(DE3).经表达条件优化,获得重组蛋白最佳诱导表达条件:温度30℃,时间6h,转速180rpm/min,培养基pH 7.0,IPTG浓度0.4mM,诱导表达后目的蛋白经SDS-PAGE电泳鉴定,Ni柱纯化、透析、浓缩从而获得浓度为8.09mg/ml,纯度为85%的SAA1.同时,结合胶体金标记技术,建立了SAA1胶体金免疫层析试纸检测方法,线性范围0.16~500μg/ml,最低检测限0.16μg/ml,该研究为临床体外诊断SAA1快速检测提供技术基础.

Prokaryotic Expression and Colloidal Gold Detection Method of Human Serum Amyloid A1

To develop a specific,rapid,and convenient immune-chromatography assay (ICA) to quantitative detect the SAA1. Using Escherichia coli BL21 (DE3) prokaryotic expression system of human serum amyloid A1 (SAA1),accord-ing to BL21 (DE3) gene fragment of Escherichia coli expressed preference for design and synthesis of SAA1 protein, the expression vector of pET-Pet28a-SAA1 was constructed,the preparation of Escherichia coli BL21 by CaCl2 meth-od (DE3) electrocompeten,expression vector pET-28a-SAA1 into Escherichia coli BL21 by heat shock transformation method (DE3). The optimization of expression conditions of recombinant protein,the optimal expression conditions:tem-perature 30,time 6h,medium speed 180r/min,pH 7,IPTG concentration of 0.4 mM,and then the expression of recombi-nant SAA1 protein after induced expression of protein,via using SDS-PAGE electrophoresis appraisal,Ni column purifi-cation,dialysis,and concentrated preparation for concentration of 8.09mg/ml,the purity of 85% SAA1 protein of high pu-rity. At the same time,combining with colloidal gold marked technology,established the SAA1 colloidal gold immune chromatography test method,the linear is in the range of 0.16~500μg/ml,the minimum detection line 0.16μg/ml. The study provides a technical basis for the clinical diagnosis of SAA1 in vitro rapid detection and diagnosis.