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document
Preparation and immune activity analysis of H5N1 subtype avian influenza virus recombinant protein-based vaccine . 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 .
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Intervention_Pharmacological
Sodium dodecyl sulfate-PAGE
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Intervention_Physical
scanning analysis
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Outcome_Physical
antigenic activity
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Participant_Condition
H5N1 subtype avian influenza virus recombinant protein-based vaccine
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Participant_Condition
pBCX vector