Fusion Strategy of Influenza A-H5N1 Virus M2e Epitope DNA Sequence to Hepatitis B Virus HBsAg-S Gene

摘要

In order to make a broad vaccine for influenza-A virus, a conserve epitope must be considered. M2 is a conserve transmembran protein in all strain of influenza-A virus. Therefore, in this experiment we used 69 bp synthetic nucleotides from extracellular domain of M2 from H5N1 as an epitope (M2e) for influenza-A vaccine. A vaccine may usually more expose to immune system if they are made on a virus-like particles (VLPs). It was shown in many papers that the HBsAg-S protein is able to make a self assemble VLP. Therefore the synthetic M2e DNA was fused to a HBsAg-S gene, which encodes a small envelope protein of the Hepatitis B virus (HBsAg-S protein), which in turn creating a new VLP with M2e on it. This fusion replaced IPQ epitope of the HBsAg-S with M2e from M2 protein of the H5N1 virus. The fusion of M2e and HBsAg-S was conducted using asymmetric PCR. The single stranded DNA of M2e was produced with primers containing HBsAg-S gene sequence. Next, this single-stranded M2e was applied as mega primer for 'fusion PCR', which will fuse the M2e and part of the HBsAg-S fragment. A ～680 bp DNA band (fusion of M2e and HBsAg-S fragment) is generated from the 'fusion PCR'. Alignment between hypothetic fusion sequence and the result was compared with native HBsAg-S sequence. The analysis shows that the IPQ epitope from HBsAg-S was totally replaced by the M2e.