Cloning of Clostridium perfringens Iota Toxin Gene in Escherichia coli

Document Type: Original Articles

Authors

1 Department of Biology, Islamic Azad University, Urmia branch, Urmia, Iran

2 Department of Biology, Islamic Azad University,Urmia branch, Urmia, Iran

3 Specialized Clostridia reseach laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Alborz, Karaj, Iran.

Abstract

Iota toxin is produced by Clostridium perfringens type E. This toxin causes antibiotic-associated enterotoxemia in lambs and calves. Iota toxin is a binary toxin that has two components including Ia (the enzyme component) and Ib (the binding component). Ib binds to the surface receptor of target cells and translocate Ia into the cytosol of cells. The aim of this study was to clone toxigenic epitope of iota a gene in E. coli strain Top10. In this study, the phenol–chloroform method was used for the extraction of the whole genomic DNA. The toxigenic epitope of iota a gene was amplified by polymerase chain reaction (PCR). The PCR product was ligated into the pTZ57R/T vector cloning site. Then, based on the TA-cloning method, the product was cloned in competent E. coli strain Top10. Colony PCR was used to screen bacterial colonies transformed with recombinant plasmids. The presence of 446-bp fragment on agarose gel showed that the toxigenic epitope of iota a gene of C. perfringens has been cloned in E. coli strain Top10.

Keywords

Main Subjects


Article Title [French]

Clonage du gène iota-toxine du Clostridium perfringens chez

Abstract [French]

Iota-toxine est produite par le Clostridium perfringens de type E. Cette toxine provoque une entérotoxémie associée aux antibiotiques chez les agneaux et les veaux. L'iota-toxine est une toxine binaire qui a deux composants dont Ia (le composant enzymatique) et Ib (le composant de liaison). Ib se lie au récepteur de surface des cellules cibles et translocation Ia dans le cytosol des cellules. Le but de cette recherche était le clonage de l'épitope toxigénique du gène iota a dans la souche d’E. coli Top10. Dans ce travail de recherche, la méthode phénol-chloroforme a été utilisée pour l'extraction de l'ADN entier. L'épitope toxigénique de iota est un gène amplifié par PCR. Le produit de PCR a été ligaturé dans le clonage du vecteur pTZ57R / T et a été ensuite cloné dans E. colistrain Top10 selon la méthode de clonage TA,. L’analyse PCR des colonies a été utilisée pour cribler des colonies bactériennes qui ont été transformées avec des plasmides recombinants. La présence d'un fragment de 446 pb sur legel d'agarose a montré que l'épitope toxigénique de iota, un gène de C. perfringens, a été cloné dans la souche d’E. coli Top10.

Keywords [French]

  • Clonage
  • Clostridium perfringens
  • iota-toxine
  • E. coli
  • TA-clonage
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