Animal Health and Infectious Diseases

Animal Health and Infectious Diseases

Cloning of the Matrix Protein (M1) Gene of H9N2 Avian Influenza Virus into the pTZ57R/T Vector

Document Type : Original Article

Authors
Shirin Mohammadipour 1
Hadi Tavakkoli 2
Saideh Afrisham 1
Sheyda Sharabiani 3
Masoumeh Abed 3
Mohammad Amir Esmaeili 4
Ghadir shahbazi 5
1 Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran
2 Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran
3 Department of Biology, Faculty of Basic Sciences, Islamic Azad University, Rasht Branch, Rasht, Iran
4 DVM, Faculty of Veterinary Medicine, Islamic Azad University, Urmia Branch, Urmia, Iran
5 Department of Food Hygiene, Faculty of Veterinary Medicine, Islamic Azad University, Shabestar Branch, Shabestar, Iran
10.22034/jahid.2026.2092554.1075
Abstract
Abstract
Background and Aim: The matrix protein 1 (M1) is one of the most conserved structural proteins among different influenza virus subtypes and plays a critical role in viral assembly, replication, and pathogenicity. Due to its high degree of conservation, the M1 gene represents an attractive target for molecular studies and diagnostic assay development. The present study aimed to clone a fragment of the M1 gene of the H9N2 avian influenza virus into the pTZ57R/T cloning vector.
Methods: Following propagation of the H9N2 avian influenza virus in embryonated chicken eggs, viral RNA was extracted from harvested allantoic fluid. The target M1 gene fragment was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The amplified product was purified and subsequently ligated into the pTZ57R/T plasmid vector. Recombinant plasmids were transformed into Escherichia coli XL1-Blue competent cells and propagated for plasmid amplification. Recombinant clones were identified and confirmed using standard molecular techniques.
Results: Successful amplification, cloning, and propagation of the M1 gene fragment were achieved. The pTZ57R/T vector in combination with E. coli XL1-Blue provided an efficient system for cloning and maintenance of the target gene. The recombinant construct remained stable and yielded sufficient quantities of plasmid DNA for subsequent molecular applications.
Conclusion: The successful cloning of the M1 gene of the H9N2 avian influenza virus provides a valuable genetic resource for future molecular investigations. The recombinant construct may serve as a suitable template for the development of diagnostic assays and further studies on the molecular biology and pathogenesis of influenza A viruses.
Keywords
H9N2 subtype
M1 protein
ptz57R/T plasmid
cloning
Subjects
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Animal Health and Infectious Diseases
Volume 3, Issue 1
June 2026
Pages 91-97