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

Q. M. Xie^*,1, J. Ji^*,1, L. Q. Du^*, Y. C. Cao^,2, L. Wei, C. Y. Xue, J. P. Qin^*, J. Y. Ma^* and Y. Z. Bi^*,

^* College of Animal Science, South China Agricultural University, Guangzhou 510642, People’s Republic of China; State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-Sen University, Guangzhou 510006, People’s Republic of China; and Institute of Animal Husbandry and Veterinary Medicine, Beijing Municipal Academy of Agriculture and Forestry Sciences, Beijing 100097, People’s Republic of China

² Corresponding authors: qmx{at}scau.edu.cn and caoych{at}mail.sysu.edu.cn

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.

Key Words: H5N1 subtype avian influenza virus • hemagglutinin • recombinant protein-based vaccine • immune activity

¹ The first 2 authors contributed equally to this work.