Transformation of Dunaliella salina by Agrobacterium tumefaciens for the Expression of the Hemagglutinin of Avian Influenza Virus H5.

Inkar Castellanos-Huerta,Gabriela Gómez-Verduzco,Isidro Fernández-Siurob,Gilberto Velázquez-Juárez,Bernardo Bañuelos-Hernández,Guadalupe Ayora-Talavera,Víctor Manuel Petrone-García,Guillermo Tellez-Isaias

doi:10.3390/microorganisms10020361

Abstract

Avian influenza (AI) is one of the main threats to the poultry industry worldwide. Vaccination efforts are based on inactivated, live attenuated, and recombinant vaccines, where the virus hemagglutinin (HA) is the main component of any vaccine formulation. This study uses Dunaliella salina to express the AIV HA protein of an H5 virus. D. salina offers a system of feasible culture properties, generally recognized as safe for humans (GRAS), with N-glycosylation and nuclear transformation by Agrobacterium tumefaciens. The cloning and transformation of D. salina cells with the H5HA gene was confirmed by polymerase chain reaction (PCR). SDS-PAGE and Western blot confirmed HA5r protein expression, and the correct expression and biological activity of the HA5r protein were confirmed by a hemagglutination assay (HA). This study proves the feasibility of using a different biological system for expressing complex antigens from viruses. These findings suggest that a complex protein such as HA5r from AIV (H5N2) can be successfully expressed in D. salina.

Highlights

The expression of a protein from total soluble protein (TSP) was confirmed in D. salina pellets from the transfected cells (Figure 1b)
We have shown the successful expression of soluble H5HA protein from avian influenza subtype H5N2 in a microalgae system using D. salina as a host species
Based on the results of Western blot (WB) and reactivity to monoclonal and polyclonal antibodies directed against the H5HA, the findings suggest the proper expression of the H5rD protein

Summary

Introduction

Avian influenza (AI) is one of the most critical illnesses in the poultry industry worldwide [1]. AI is defined as a systemic disease ranging from clinically undetected to severe with a high mortality depending on the virus subtype [2]. The AI virus (AIV), a member of the Orthomyxoviridae family, is the etiologic agent of AI [3]. Hemagglutinin (HA) and neuraminidase (NA) are the major glycoproteins found on the surface of the influenza virus [4], though HA is the most abundant (up to 10-fold more than NA) [5]. HA is a trimeric elongated rod-shaped protein with three monomers (75 kDa) connected in a trimer (225 kDa) with a length of 10–14 nm and a 4–6 nm diameter.

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