Repository of Research and Investigative Information

Repository of Research and Investigative Information

Ilam University of Medical Sciences

Cloning and expression of the Streptococcus pyogenes hyaluronidase gene in E.coli

(2012) Cloning and expression of the Streptococcus pyogenes hyaluronidase gene in E.coli. Australian Journal of Basic and Applied Sciences. pp. 95-99. ISSN 19918178 (ISSN)

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Official URL: https://www.scopus.com/inward/record.uri?eid=2-s2....

Abstract

Background: extracellular hyaluronidase of Streptococcus pyogenes is a spreading factor (hyaluronate lyase) which is composed of 868 amino acid protein with a molecular size of 99636 Da, the aims of cloning and expressing extracellular hyaluronidase of Streptococcus pyogenes in E.coli Methods: the hyal A gene was inserted in pTz57R/T vector. Subsequently, the vector was purified and than digested using SacIcyrillic, ukrainian and BamHIcyrillic, ukrainian restriction endonuclease. After inserting in pET32a vector, the recombinant plasmid was introduced in E.coli DH5α and transferred in the E.coli BL21 (DE3) pLysS bacteria, to monitor its expression by using IPTG and Protein analysis was carried out by SDS-PAGE electrophoresis. The new recombinant protein antigenicity was evaluated by Immunoblot analysis. Results: PCR product was a fragment with about 2608 bp, and high similarity with gene bank accession number af218838. Recombinant protein in expression vector pET32a was produced by 1mM IPTG. Several bands were seen in SDS-PAGE. The 113 kDa band belongs to Hyl A recombinant protein with His -tag, and other bands were 50-113 kDa in molecular weight range. This recombinant protein in western blotting was confirmed by using serum from mouse immunized with supernatant culture of S. pyogenes. Conclusion: several molecular weight of recombinant protein possibly is due to heavy weight of Hyl A protein or having two start codon in hylA gene. In other hands, western blotting results indicate that all bands of this recombinant protein have antigenic property, and can be used for diagnostic assay.

Item Type: Article
Keywords: Extracellular Hyaluronidase Recombinant
Page Range: pp. 95-99
Journal or Publication Title: Australian Journal of Basic and Applied Sciences
Journal Index: Scopus
Volume: 6
Number: 2
ISSN: 19918178 (ISSN)
Depositing User: مهندس مهدی شریفی
URI: http://eprints.medilam.ac.ir/id/eprint/1604

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