Date of Graduation

12-2013

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Biomedical Engineering

Advisor/Mentor

Kaiming Ye

Committee Member

Douglas D. Rhoads

Second Committee Member

Sha Jin

Keywords

Biological sciences, Influenza, Iron oxide nanoparticle, Subunit vaccine

Abstract

Influenza is a common infectious disease resulting from a frequently mutated RNA virus. Vaccination is currently the most effective method to prevent people from seasonal or pandemic influenza. The production of traditional egg-based influenza vaccine is time-consuming and provides limited effect against new strains. Therefore, it is necessary to develop a rapid method to produce influenza vaccines. We proposed a novel influenza vaccine based on the E.coli expression system. Hemagglutinin (HA) is the major target surface protein of influenza virus for vaccine development. In this study, we sub-cloned the HAs encoding gene into an E. coli expression vector; the signal peptide sequence, the transmembrane and cytoplasmic domains of the whole HA of H5N1 (A/Vietnam/1203/2004) were removed. Expression of recombinant HAs fused with a C-terminal His-tag was investigated and confirmed through SDS-PAGE and Western blot assay. After being purified under denaturing conditions using NTA-Ni affinity chromatography, HAs were dialyzed for refolding. We obtained concentrated recombinant HAs from bacterial cultures at a yield of 250 µg/ 500 ml. Finally, animal studies revealed the production of anti-HA antibodies in mice immunized with different doses of the recombinant HAs. We also compared the adjuvant effects of iron oxide nanoparticle (IONs) and selected commercial adjuvants. These results suggest that this system has the potential to be a new method for the mass production of influenza vaccines at low cost. More efforts are going to be focused on the adjuvant effect of IONs in future work.

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