摘要
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p ET-28a-SAA1,采用Ca Cl2法制备BL21（DE3）感受态,热激转化法将p ET-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p H 7.0,IPTG浓度0.4m M,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。
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）,according to BL21（DE3） gene fragment of Escherichia coli expressed preference for design and synthesis of SAA1 protein,the expression vector of p ET-Pet28a-SAA1 was constructed,the preparation of Escherichia coli BL21 by Ca Cl2 method（DE3） electrocompeten,expression vector p ET-28a-SAA1 into Escherichia coli BL21 by heat shock transformation method（DE3）. The optimization of expression conditions of recombinant protein,the optimal expression conditions：temperature 30,time 6h,medium speed 180r/min,p H 7,IPTG concentration of 0.4 m M,and then the expression of recombinant SAA1 protein after induced expression of protein,via using SDS-PAGE electrophoresis appraisal,Ni column purification,dialysis,and concentrated preparation for concentration of 8.09mg/ml,the purity of 85% SAA1 protein of high purity. 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.
作者
宋英林
赵玉环
褚春旭
张司晨
张晓
SONG Yinglin ZHAO Yuhuan CHU Chunxu ZHANG Sichen ZHANG Xiao(Hospital of Changchun University of Science and Technology, Changchun 130022 School of Life Science and Technology, Changchun University of Science and Technology, Changchun 130022)
出处
《长春理工大学学报（自然科学版）》
2016年第6期134-137,143,共5页
Journal of Changchun University of Science and Technology(Natural Science Edition)
关键词
人血清淀粉样蛋白A1
原核表达
胶体金免疫层析
定量检测
human serum amyloid A1
prokaryotic expression
colloidal gold immuno chromatography
quantitative detection
