金钱白花蛇β-环蛇毒素可溶性原核表达及纯化方法建立OA

Objective:To study the β-bungarotoxin from the source animal of the Chinese banded krait(Bungarus multicinctus Blyth),and to establish soluble expression and purification methods for the PLA2 and Kunitz genes of β-bungarotoxin through gene synthesis and cloning.Methods:Based on the genomic information of Bungarus multicinctus obtained previously,the A1,B2,B2(C55R),and PILP3 genes of β-bungarotoxin were selected and synthesized.Subcloning was performed to construct the prokaryotic ex-pression vectors Pet-28a-A1 and Pet-28a-B2,which were then transformed into E.coli BL21(DE3)competent cells.First,the solu-ble expression conditions were investigated using B2 as the research target.Second,SDS-PAGE gel electrophoresis was used to detect protein expression and to examine the expression of genes under different induction conditions.Finally,purification was carried out u-sing affinity chromatography.Results:Soluble prokaryotic expression and purification methods were established,and soluble recombi-nant proteins of A1,B2,B2(C55R),and PILP3 were obtained using these methods.Protein purification was performed using a His affinity chromatography column,and SDS-PAGE gel electrophoresis confirmed the identity of the A1,B2,B2(C55R),and PILP3 proteins from Bungarus multicinctus.Conclusion:The establishment of soluble expression and purification methods for β-bungarotoxin,as well as the expression and purification of A1,B2,B2(C55R),and PILP3 proteins,lays a foundation for further research on the functions of β-bungarotoxin in Bungarus multicinctus.