Corruption Clostridium Alpha Toxin Gene Cloning And Expression

Clostridium septicum was the first anaerobic pathogenic organism cultured by Pasteur in 1877, which can lead to many animal diseases, such as ovine braxy, malignant circulating hydroncus of ovine, gangrenosis dermatitis of chicken and turkey. There are also some reports about infection of cattle, pigs and deer. The characteristic of disease led by clostridium septicum are high paroxysm speed and death rate, which endangers aquaculture so much.Strict anaerobes is divided and appraised with normal medium and anaerobic culture and temporarily named HeB01 in this article. According to the published nucleotide sequences, alpha-toxin gene of clostridium septicum was amplified and linked to pUCm-T vector. The result of sequences determination and homologies analysis showed that comparing the isolate virus with AB08346, AB08347, AB08348 and AB08349 we found that their nucleotide sequences homologies were 99.5%, 99.7%, 99.6%å'Œ 99.6% respectively, which means alpha-toxin gene is very conservative. After that alpha-toxin gene was directional cloned to prokaryotic expression vector pQE30 and recombined plasmid pQE30- α was constructed. After recombined bacteria M15(pQE30- α ) which came from transformation of acceptor bacteria M15 was induced with IPTG, exceptional protein bands was seen with SDS-PAGE analysis. The alpha-toxin proteins were detected successfully by ELISA and western blot, which seems to be immunogenic crucially. After the bacterin made from alpha-toxin proteins immunes mice, they could resist attack of alpha-toxin, which shows that these recombined bacteria will hopeful be regard as selected production bacteria of clostridium septicum gene engineering toxoid vaccine.