[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 (alpha-BGT), DNA sequence of alpha-BGT was deduced and fourteen partially complementary oligonucleotides were designed and synthesized. A plasmid carrying the coding region of alpha-BGT was obtained by primer extension, PCR and ligation with pMD-18-T. The target fragment was digested with Xba I and EcoR I, recovered and ligated with pET28a(+). The resultant expression vector was transformed into BL21 (DE3), BL21 (DE3) Codon plus, and BL21 (DE3) plysS, respectively. Recombinant alpha-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 alpha-BGT protein purified from the venom of Krait Bungarus spp. In vivo toxicity assay of purified and renatured proteins in mice showed that LD50 was about 1.28 microg/g.