Cloning and sequencing of the etx gene from Clostridiumperfringens type D strains isolated from patients with antibioticassociateddiarrhoea (AAD)

سال انتشار: 1401
نوع سند: مقاله کنفرانسی
زبان: انگلیسی
مشاهده: 69

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شناسه ملی سند علمی:

MEDISM23_334

تاریخ نمایه سازی: 16 مهر 1401

چکیده مقاله:

Background and Aim : C. perfringens is divided into seven types (A, B, C, D, E, F, and G) basedon the production of different toxins (alpha, beta, epsilon, iota, enterotoxin, necrotic enteritis toxinB-like). Of these, alpha, beta, and specially enterotoxin are of major importance in humanmedicine, and the others are commonly involved in veterinary medicine. The epsilon toxin (ETX)of type D, hitherto unknown association with human disease, is mainly responsible forenterotoxemia in small ruminants. This study was performed to characterize the etx gene sequenceof the recently C. perfringens type D strains isolated from Iranian AAD cases.Methods : C. perfringens type D isolates (N= ۱۵), recently recovered from human AAD patients,were used in this study. Plasmid DNA of the isolates was extracted. For genetic analysis of the etxgene, its entire sequence was synthesized with the designed specific primers. The amplified genewas digested with EcoRI and XhoI, then ligated into pET-۲۶b(+), and electroporated into E. coliDE۳ strain. The recombinant strains were obtained by screening the Kanamycin-resistant clonesand investigated for the presence of etx gene by PCR and sequencing. The etx gene sequenceswere manually aligned according to their nucleotides and the deduced amino acid sequences.Phylogenetic analyses were conducted using MEGA X, and the phylogenetic tree was generatedusing the maximum likelihood (ML) approach.Results : The etx gene from human type D isolates was synthesized. The expected size of theamplified fragment corresponded to ۱۰۰۶ bp. These were cloned in the pET-۲۶b(+) vector and thepET-۲۶b-? plasmid (۶۲۲۷ bp) was constructed. These results illustrated that this plasmid had beenconstructed successfully in E. coli.Conclusion : The present study reports the first sequencing analyses of the etx-positive C.perfringens type D isolates associated with AAD disease. The etx sequences showed highsimilarity, with >۹۷-۹۹% identity to sequences available in the GenBank database and the etxsequence from C. perfringens type D vaccinal strain (CN۴۰۹).

کلیدواژه ها:

Cloning ، Sequencing ، Clostridium perfringens Type D ، etx Gene ، Antibioticassociateddiarrhoea (AAD)

نویسندگان

Mojtaba Alimolaei

Kerman branch, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Kerman, Iran

Mehrdad Shamsaddini Bafti

Kerman branch, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Kerman, Iran

Shirin Soltani

Kerman branch, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Kerman, Iran