Amplification and Cloning of alfa-Toxin Gene from Clostridium Perfringenes Bacterium in E. coli

• Main Authors: Hosseinali Sasan, Mahin Rasani, Samaneh Karamooz, ali esmaeelizadeh, Masoud Asadi Fouzi, Ahmad AyatollahiMehrgardi
• Format: Article
• Language: fas
• Published: Shahid Bahonar University of Kerman 2020-02-01
• Series: مجله بیوتکنولوژی کشاورزی
• Subjects: alpha-toxin; clostridium perfringenes; cloning
• Online Access: https://jab.uk.ac.ir/article_2524_5f10468a62aa99ea88feb4f959b8be4b.pdf

<strong>Objective</strong> The aim of this study was to amplify and cloning complete, as well as the primary part of the α-toxin gene from Clostridium perfringens with the size of 1100 and 750 bps, in <em>E. coli</em> using the expression vector pET28a.   <strong>Materials and Methods</strong> Genomic DNA was extracted using Kiagen kit. Forward and reverse primers were designed in the lab, as the <em>HindIII</em> and <em>XhoI</em> enzymes rows were added in the initial regions of the oligonucleotides according to the cloning region of the pET28a plasmid. The genes of interests were amplified by PCR and cloned in the pET28a vector using the heat-shock method. Extraction of plasmid DNA was done using alkaline lysis method.   <strong>Results</strong> The results showed the PCR products, clear and unique bands for the complete, as well as the initial portion of the desired genetic components in accordance with the expected sizes. In addition, by using single and double enzyme digestion experiments with <em>HindIII</em> and <em>XhoI</em> enzymes, cloning of alpha toxin genes in host bacteria and production of recombinant bacteria was confirmed.   <strong>Conclusions</strong> Due to the successful cloning of alpha toxin genes in expression host bacteria done by this work, it can be said that primers designed in this study are highly specific for the α-toxin gene amplification. These primers can be also used as markers for the identification and classification of Clostridium perfringens bacteria. Also, by development construction of genetically engineered recombinant vaccines, it is possible to control and cope with important pathogens such as Clostridium perfringens bacteria.