中国饲料Issue(9)：30-35,6.DOI:10.15906/j.cnki.cn11-2975/s.2024120011-04
牛病毒性腹泻病毒受体蛋白CD46的原核表达及纯化
Prokaryotic expression and purification of bovine viral diarrhea virus receptor protein CD46
摘要
Abstract
This study aimed to construct a prokaryotic expression system for CD46,enabling the efficient expression and purification of CD46-His fusion protein in Escherichia coli.The CD46 gene was ligated with the pET-30a(+)vector to construct a recombinant expression plasmid,which was then transformed into E.coli BL21(DE3)for protein expression.Inclusion bodies were harvested through ultrasonication and high-speed centrifugation.The protein was denatured using 8 mol urea solution and refolded with arginine-containing renaturation buffer to obtain soluble CD46 protein.Further purification was achieved using anion exchange chromatography with HitrapTM and molecular sieve HiLoadTM 16/600 Superdex 200 pg on the AKTA purification system to obtain highly homogeneous and pure protein products.The results showed that pET30a-CD46 recombinant plasmid was successfully constructed and expressed.Soluble CD46 protein was obtained from inclusion bodies using 8 mol urea denaturation and arginine renaturation methods,with a denaturation efficiency of 8.5％.After purification by ion exchange chromatography and molecular sieve,the final CD46 protein purity exceeded 95％,with a yield of 28.51％.Compared with traditional eukaryotic expression systems,this method offers advantages of shorter cycle time,lower cost,and higher yield.This study provides a simple and efficient method for large-scale production of CD46 protein,laid the groundwork for further research into BVDV infection mechanisms and the development of diagnostic reagents.It also offered a prokaryotic expression and purification protocol for subsequent CD46 crystallization studies.Moreover,this cost-effective prokaryotic expression strategy could serve as a reference for the expression and purification of other membrane proteins.关键词
CD46/BVDV/原核表达系统/蛋白质纯化/蛋白质变性Key words
CD46/BVDV/prokaryotic expression system/protein purification/urea denaturation分类
农业科技引用本文复制引用
张晓敏,杨续金,韩晓东..牛病毒性腹泻病毒受体蛋白CD46的原核表达及纯化[J].中国饲料,2026,(9):30-35,6.基金项目
国家自然科学基金项目(32060039) （32060039）
