Cloning, Expression and Purification of Recombinant HMW1 , HMW2 and Hia as a Fusion protein for Vaccine Candidate of NontypeableHaemophilus influenza

Ghasem Abbaszadeh-Goudarzi^1,2, Kazem Abbaszadeh-Goudarzi^1,2, Seyed Davar Siadat^2,4٭, Ladan Teimoori-Toolabi^1,5, Saeid Bouzari⁶, Sassan Rezaie³, Mahdi Davari^2,4 and M.Reza Khorramizadeh^7,1,٭

¹Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of medical Sciences, Tehran, Iran.

²Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran.

³Department of Medical Mycology and Parasitology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

⁴Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran.

⁵Department of Molecular Medicine, Biotechnology Research Center; Pasteur Institute of Iran, Tehran, Iran.

⁶Department of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran.

⁷Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran.

Corresponding Author E-mail: khoramza@tums.ac.ir

DOI : http://dx.doi.org/10.13005/bbra/2381

ABSTRACT: NontypeableH.influenzae (NTHi) is an important pathogen in children causing otitis media. However,there is no vaccine against NTHi-induced diseases. HMW1,HMW2 and Hia  are adhesin proteins of NTHi-strains and have potentiality to provide protection against NTHi-infections. The aim of present study was to construct recombinant HMW1-HMW2-Hia as a fusion protein and evaluate immune responses against recombinant fusion protein as vaccine candidates antigens. Binding domain of hmw1 gene (1080 bp) was amplified by PCR from genomic DNA and cloned into PET-28a vector,this vector was already modified by the insertion of a fragment named hmw2-hia into multiple cloning sites, and then was confirmed by colony-PCR, enzymatic digestion and gene sequencing. To express recombinant fusion protein, PET-28a-hmw1-hmw2-hia vector was transformed into competent BL12(DE3). Expressed protein was purified by affinity chromatography. BALB/c mice were subcutaneously immunized by purified protein in combination with Freund’s adjuvant. Serum antibody responses and functionality of antibodies were determined by ELISA and SBA, respectively. In immunized group of Balb/c mice, the serum IgG responses was significantly increased against recombinant fusion protein in comparison with control, and also the antisera have shown strongly bactericidal activity against NTHi strain. The results have shown that recombinant fusion protein could induce the immune system to produce antibodies which was funtional to kill NTHi-strains. Based on the observation, the antisera have the possibility to provide protection against infections caused by NTHi.

KEYWORDS: NTHi; fusion Protein; Expression Vector; affinity chromatography

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