The Expression Of Hc Fraction Gene Of Botulinum Neurotoxin Serotype A In Escherichia Coli And Pichia Pastrois

Botulinum Neurotoxin Serotype A produced by the Clostridium botulinum, are the most lethal substances known .A single molicule can abolish the function of a nerve cell.The toxins exert their action by blocking the release of the neurotransmitter , acetylcholie, at the neuromuscular junction, The blockage of BONT/A makes neuromuscular weakness and flsccid paralysis, and lead to respiratory failure and death.Botulinum is a serious public heath problem in developing countries around the world.The high AT content of the native gene is very difficult to express , We designed a new He gene by reverse the codon used for E.coli and yeast, then, The He gene was cloned into the expression vector PQE-60. and transformed E.coli Origami strain. Induced with IPTG. The expression content is about 6% of the totle bacterial protein. The production of expression is purified , The purity is above 90%.He gene was also cloned into the expression vector pPIC9K of yeast. The vector constructed included the promoter which is AOXl(the enzyme alcohol oxidase)and kanamycin resistance. The vector recombined was clectransformed yeast GS115 strain after linearized. Make the gene inserted into chromsome of yeast. We got a strain yeast which high secreted the protein recombined by selection His+,G418 screening, PCR identified. The production of expression is our interest protein through SDS-PAGE and Western blot assay.This paper is a study on the expression of the protective gene of BONT/A in the prokaryotic and eukaryotic . It indicates the possibility to produce the recombinant protein in quantities. It has also laid down a good foundation for the further research on vaccine or antibody of BONT/A.