牛病毒性腹泻病毒受体蛋白CD46的原核表达及纯化OA

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.