Molecular Cloning of Clostridium Perfringens type B Vaccine Strain Beta Toxin Gene in E. coli

摘要

Clostridium perfringens is a gram-positive, obligate anaerobic bacterium, which is widely distributed in the environment. C. perfringens is subdivided to 5 groups (types A to E), based on its four major toxin (alpha, beta, epsilon and iota). C. perfringens type B beta toxin causes inflammation and bloody necrotic enteritis. Type B related enterotoxaemia is a major problem of veterinary sciences. The aim of the present study was to molecular cloning and sequencing of C. perfringens type B vaccine strain beta toxin gene. Genomic DNA was extracted using phenol-chloroform method. Beta toxin sequence was retrieve from GenBank and using oligo software, appropriate primers were designed. Using Pfu DNA polymerase, beta toxin gene was amplified and after purification it was ligated into pJET1.2/blunt cloning vector. The ligation product was transformed using E. coli/TOP10 competent cells and the recombinant pJETβ clones were chosen on LB-amp. pJETβ recombinant plasmid was extracted from recombinant bacterium host and was sequenced using universal primers. Sequencing and BLAST and phylogenetic analysis of cpb showed over 99% identity to other previously deposited cpb in the GenBank.