Cloning of Streptococcal pyogenes Exotoxin, A gene in E. coli
Subject Areas : Genetics of MicroorganismsElham Siasi 1 , Mitra Salehi 2 , Simin Mirjalili 3
1 - 1. Department of Microbiology, Collage of science, North Tehran Branch, Islamic Azad University, Tehran, Iran.
2 - 1. Department of Microbiology, Collage of science, North Tehran Branch, Islamic Azad University, Tehran, Iran
3 - Department of Biotechnology, Collage of science, North Tehran Branch, Islamic Azad University., Tehran, Iran
Keywords: Exotoxin A, Streptococcus pyogenes, speA gene, E. coli XL1Blue, Cloning,
Abstract :
Aim and Background: Exotoxin A is one of the toxins which produce by group a streptococcuses. This Toxin is a protein soluble substance which causes scarlet fever and different kinds of infections as a super antigen. This toxin has antigenic property and special anti-toxins can neutralize it. In this research, was studied production of Streptococcus pyogenes exotoxin A recombinant protein in E. coli by cloning. Materials and methods: At the first, genome of the bacteria was extracted and used PCR. Then agarose gel electrophoresis was performed for detection of size and quality of the resulting amplicons. According to TA Cloning kit protocol, the target gene was inserted in to the PTG19 Cloning vector and the recombinant plasmid was inserted in to the E. coli XL1Blue bacteria. Eventually checking the sequencing data obtained from cloned gene in the vectore compared with the refrenece sequenece obtained from the NCBI database, confirm the success of the gene cloning by its correct sequence. Results: The results showed that PCR product is a fragment with a length of about 708 bais pairs. This is for the first time that the exotoxin A gene was cloned by the TA Cloning in the PTG19 Cloning vector and the E. coli XL1Blue host cell. The results of sequencing were confirmed presence of exotoxine A gene, speA. Conclusion: The speA gene was amplified and correct cloning was confirmed by TA Cloning with PTG19 vector and E. coli XL1Blue host cell.
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