Preparation and immune activity analysis of H5N1 subtype avian influenza virus recombinant protein-based vaccine

• Published in: Poultry science Vol. 88; no. 8; pp. 1608 - 1615
• Main Authors: Xie, Q.M, Ji, J, Du, L.Q, Cao, Y.C, Wei, L, Xue, C.Y, Qin, J.P, Ma, J.Y, Bi, Y.Z
• Format: Journal Article
• Language: English
• Published: Oxford, UK Poultry Science Association 01.08.2009; Oxford University Press
• Subjects: Animals; Antibodies, Monoclonal; Bacterial Outer Membrane Proteins - genetics; Chickens; culture media; disease control; disease diagnosis; Escherichia coli; Escherichia coli Proteins - genetics; Hemagglutination Inhibition Tests; hemagglutinins; Hemagglutinins - genetics; Hemagglutinins - immunology; immune response; Influenza A virus; Influenza A virus H5N1; Influenza A Virus, H5N1 Subtype - immunology; Influenza in Birds - immunology; Influenza Vaccines - immunology; microbial genetics; plasmid vectors; Plasmids - genetics; Plasmids - immunology; post-translational modification; protein conformation; protein folding; protein synthesis; recombinant antigens; Recombinant Proteins; recombinant vaccines; vaccination; vertebrate viruses; viral antigens; viral proteins; recombinant protein-based vaccine; H5N1 subtype avian influenza virus; immune activity; hemagglutinin
• ISSN: 0032-5791; 1525-3171
• DOI: 10.3382/ps.2009-00092

Avian influenza is a severe disease among farmed poultry and free-living birds and a constant threat to the commercial chicken industry around the world. Hemagglutinin (HA) is the major immunogen on the envelope of influenza A virus and is the predominant inducer of neutralizing antibody. To obtain the bioactive antigen proteins in large quantities, a new protein expression vector pBCX was constructed, which is based on the pET32a vector. The HA gene of the H5N1 subtype of avian influenza virus (AIV) was inserted into the pBCX vector and expressed efficiently in Escherichia coli BL21 (DE3). Fused expression of the exogenous gene and msyB produced a 97-kDa msyB-HA fusion protein. Sodium dodecyl sulfate-PAGE combined with scanning analysis demonstrated that the msyB-HA fusion protein accounted for 29.5% of the total bacterial protein, 90.5% being soluble. The msyB-HA fusion protein was purified with nondenaturing 50% Ni-NTA column chromatography, and the result showed that 24 mg of purified msyB-HA fusion protein could be obtained from 1 L of induced expression bacterial culture medium. The comparative results in the present study showed that pBCX was superior to pET32a as a protein expression vector. Western blotting showed the recombinant msyB-HA (rHA) to have better antigenic activity, which may be the result from the better posttranslation protein modification and folding in the pBCX expression system. With the rHA fusion protein as antigen, we successfully prepared and screened specific monoclonal antibodys against the H5N1 subtype AIV, which indicated that the rHA had antigen epitopes and biofunctions. The immune test confirmed that the rHA protein vaccine could also induce high neutralizing antibodies, and the AIV challenge test proved that the rHA protein-based vaccine could prevent the corresponding infection. This study demonstrates that the recombinant HA protein produced by the pBCX expression system could be used as a recombinant protein-based vaccine and has potential for further development for diagnosis.