EXPRESSION OF HA5-1 GENE CODING FOR SUBUNIT OF HEMAGGLUTININ (HA) ANTIGEN OF INFLUENZA VIRUS AlH5Nl IN ESCHERlCHlA COLl

Abstract

Highly pathogenic avian influenza is a transmission disease which spreads rapidly and results in highly lethal proportion of infected avian. This disease is caused by influenza A virus that belongs to the family Orthomyxoviridae with several different subtypes. Nowadays, 16 subtypes have been identified; most of them cause infection in birds and possibly in human. Our aim is to construct an influenza vaccine for poultry based on recombinant HA derived from influenza virus HSNI. In order to do so, cloned ha5-/ gene coding for a subunit of the strain AIH5Nl influenza virus hemagglutinin (HA), lacking a native signal peptide was fused to a bacterial gene trx coding for thioredoxin in the expression vector pET22trx. The novel vector was transformed into E. coli BL21 for surveying the expression level of the gene in E. coli. As expected, recombinant fusion protein TrxHA5-1 of 57 kDa was expressed at high level in E. coli BL21 under the regulation of T7 lac promoter with inducer IPTO 0.5 mM. However, only small amount of recombinant proteins was soluble (\0%). At 22, 28, 30, 37"e, amounts of the HAS-I synthesized by recombinant strains reached to 78, 110, 126, 142 mg/l after 4 hours of cultivation in induction medium. However, protein produced at 25°C had the highest solubility and occupied about 50% of total proteins and at 30°C, expression level of the recombinant proteins was reduced (about 20% oftolal protein). The chimeric protein was successfully purified by his-tag affinity column under denaturizing and redenaturation conditions. The TrxHaS-1 expressed in the bacteria was recognized by polyclonal HA antibodies against H5N 1 vaccine strain in the chicken sera