Cloning of Alpha-Bungarotoxin Gene and Its Prokaryotic Expression as a Non-fusion Protein

On the basis of the reported amino acid sequence of alpha-bungarotoxin(α-BGT),DNA sequence of α-BGT was deduced and fourteen partially complementary oligonucleotides were designed and synthesized.A plasmid carrying the coding region of α-BGT was obtained by primer extension,PCR and ligation with pMD-18-T.The target fragment was digested with XbaⅠ and EcoRⅠ,recovered and ligated with pET28a(+).The resultant expression vector was transformed into BL21(DE3),BL21(DE3)Codon plus,and BL21(DE3)plysS,respectively.Recombinant α-BGT was expressed in BL21(DE3) and was analyzed by 15% Tris/tricine SDS-PAGE.The result showed that the recombinant protein,mostly found in inclusion bodies,accounted for 11.98% of the total bacterial lysate.The expression capacity could be increased to 16.28% by optimizing expression conditions.Western blotting results showed that the expressed protein had similar immunogenicity with the natural α-BGT protein purified from the venom of Krait Bungarus spp.In vivo toxicity assay of purified and renatured proteins in mice showed that LD_(50) was about 1.28 μg/g.