Cloning and Expression of the Spy Gene of Streptococcus pyogenes in Origami Susceptible Cells as a Vaccine Candidate

Subject Areas : Molecular Microbiology

Mina pourtavakol ¹ , Sasan Saki ² , Behrouz Shojaei Saadi ³ , Rezvan Naemi ⁴

1 - Department of Biology, School of Basic Science, Islamic Azad University, Arak, Iran
2 - Department of Medical Laboratory Science, School of Medicine, Islamic Azad University, Arak, Iran
3 - Department of biology, School of Basic Science, Islamic Azad University, Arak, Iran
4 - MSc, Department of Microbiology, Faculty of Basic sciences, Shahid Rajaei Teacher Training University, Tehran, Iran.

Received: 2024-06-04 Accepted : 2024-08-26 Published : 2024-09-10

Keywords: Streptococcus pyogenes, Spy gene, pET-26b plasmid, cloning.,

Abstract :

Introduction: Streptococcus pyogenes (Group A , GAS ) is responsible for a wide range of infections. Additionally, acute rheumatic fever and acute glomerulonephritis can occur following the Group A infections. Numerous studies have shown that the Spy gene is associated with increased immunogenicity and severe infections. The aim of this study was to clone and express the Spy gene of Streptococcus pyogenes in Origami susceptible cells to develop a potential vaccine for this infection in the future. 

Methods: In this descriptive cross-sectional study, 100 throat swabs were collected from patients visiting clinics in Tehran under sterile conditions. After transfer to the laboratory, strains isolation was performed using microbiological methods. Following DNA extraction and PCR testing to detect Spy gene, cloning was carried out in Escherichia coli Origami, the pET-26b plasmid. Colony selection, colony PCR, and Real-Time PCR were performed to confirm the accuracy of cloning. 

Results: Out of 12 Streptococcus pyogenes strains, 11 isolates carried the Spy gene. Enzymatic digestion of the pET-26b plasmid and cloning into the Escherichia coli Origami expression vector were successful. Additionally, the Ct value of the cloned cells compared to the internal control without cloning indicated successful expression of the gene. 

Conclusion: The results of this study indicate appropriate expression of the Spy gene using the cloning method in the Escherichia coli Origami vector and pET-26b plasmid, confirmed by Real-Time PCR. 

References:

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