Molecular cloning of Clostridium septicum vaccine strain alpha toxin gene in E. coli

Clostridium septicum a Gram positive anaerobic bacterium produces several toxins including alpha, beta, gamma and delta. C. septicum alpha toxin is lethal and is responsible for a serious disease known as gas gangrene. The aim of the present study was to molecular cloning and sequencing of C. septicum vaccine strain alpha toxin gene. Genomic DNA was extracted using standard phenol and chloroform extraction method, and the target gene was amplified through PCR by specific primers. Quality and quantity of PCR product was evaluated using agarose gel electrophoresis and confirmed with spectrophotometry. The PCR product was purified and was ligated in pJET۱.۲blunt cloning vector and was used for E. coli/TOP۱۰ competent cells transformation. pJETαsep recombinant plasmid was purified and sequenced using universal primers. Sequencing and BLAST analysis of csa showed over ۹۹% identity to other previously deposited csa in the GenBank. The csa sequence was deposited in the GenBank under accession number JN۷۹۳۹۸۹. E. coli/TOP۱۰/pJETαsep as a recombinant bacterium could be used for purifying of recombinant csa gene and its expression in the suitable prokaryotic hosts.

کلیدواژه ها:

Clostridium septicum ، vaccine strain ، alpha toxin gene (csa) ، Cloning ، Sequencing