Cloning and Expression of alpha-Bungarotoxin Gene.

ABSTRACT

The genetic engineering methods to produce the snake neurotoxin obviously have potential advantages over the traditional methods of extracting neurotoxin from snakes. This work was to study the expression and the feasibility of scaled production of snake neurotoxin in the routine expression systems such as E.coli with the -bungarotoxin as an example. First, on the basis of the reported amino acid sequence of alpha-bungarotoxin, DNA sequence of -bungarotoxin was deduced and four partially complementary oligonucleotide fragments were designed. The coding region of -bungarotoxin was obtained by renaturing the DNA fragments, nick filling-in, ligation and PCR. The coding region of -bungarotoxin was cloned into plasmids pGEX-2T, named pDZ04, and transformed E.coli BL21 in order to study the expression of alpha-bungarotoxin gene. The results of SDS-PAGE analysis showed that the recombinant plasmid pDZ04 could express efficiently in BL21, and the fusion protein took up about 30%-40% of total bacterial protein. Finally, The biological activity of the recombinant alpha-bungarotoxin and the fusion protein was studied with the natural alpha-bungarotoxin purified from the snake venom as control, ELISA results showed that they had similar antigenicity.