OVERPRODUCTION OF M2 ECTODOMAIN (M2E) FUSION PROTEIN IN ESCHERICHIA COLI BL21(DE3) AS UNIVERSAL AVIAN INFLUENZA VACCINE CANDIDATE FOR BIRDS

• Main Author: Pertela, Gian
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/47167
• Institution: Institut Teknologi Bandung

Avian influenza (AI) virus constantly mutate in their genomes naturally. These changes cause AI vaccines for poultry to be less protective so the incidence of deadly AI virus infections in poultry still occurs. Ectodomain of M2 protein (M2e) is one of the most conserved protein domains in AI viruses. Therefore, it is suitable as a candidate of universal AI vaccines. The small size of M2e causes low immunogenicity of this protein. The aim of this study was to obtain an immunogenic M2e protein by increasing the size of M2e protein using flagellin from Salmonella pullorum as fusion protein. The recombinant DNA sequence was constructed as M2e tetramer gene fused with the flagellin gene (fla.4xm2e51). DNA fla.4xm2e51 was inserted into the pET-SUMO plasmid and transformed into Escherichia coli. Overproduction of the Fla.4xM2e51 fusion protein in E. coli BL21(DE3) was carried out at 37°C with various IPTG concentrations (0.1, 0.5, and 1 mM). Fla.4xM2e51 protein was purified using a nickel column (Ni-NTA) and eluted using 250 mM imidazole. The results showed that the fla.4xm2e51 DNA was successfully constructed in pET-SUMO which was confirmed by specific DNA amplification, restriction analysis, and alignment of the fla.4xm2e51 DNA sequence in the plasmid. Fla.4xM2e51 protein was successfully expressed at 0.1 mM IPTG induction and was purified with a nickel column. The Fla.4xM2e51 protein was susceptible to degradation, therefore optimization of the purification process needs to be performed to obtain a more pure and stable protein at higher amount.