| Influenza viruses are the most important causative agent of respiratory tract infections. Every time during the influenza epidemics, excess mortality is observed. For example, more than 20 million peple died in 1918. Influenza virus can infect equine, swine, avian and human, and can be transmitted from one to another. For example, avian influenza viruse of the H5N1 subtype in 1997 were transmitted from chickens to man in Hong Kong. Infections caused by the H5N1 subtype were proved to be fatal for six out of eighteen identified cases. It is a good methed to use vaccines to prevent influenza. Inactivated influenza vaccine (Ivac) is used in the world widely. Another vaccine approach, which has been successfully applied, is the use of attenuated viruses (Livac) or subunit vaccine (Subvac). DNA vaccine is a new kind of vaccines to prevent influenza. Today, in many laboratories in the world, DNA vaccine of influenza is in experiment. It has showed great efficacy in the experiment of animals.DNA vaccine can stimulate B-cells, T-helper cell and cytotoxic T lymphocytes (CTL), induce specific humoral and cellular immune response. DNA vaccine is considered as an important research by NIH and WHO. The research of hemagglutinin gene is an important subject of DNA vaccine of influenza. Hemagglutinin is encoded by the segment 4 of genome RNA of influenza A virus. Influenza virus hemagglutinin can stimulate protective antibody in humun and anmimals bodies. The anti-HA antibody show greatneutralizing effect. The neutralizing effect of anti-HA antibody can be attributed to either the prevention of viral entry into susceptible cells by the blocking of virus binding , or their acting on later stages of viral replication. HA have two residues, that are HAl and HA2. The HAl recognizes receptors of target cells.We cloned HAl of hemagglutinin and insert the gene into eukaryotic expressing vector, then we transfected the recombinant plasmid to eukaryotic cells to see if they could expess HAl.The aims of this work are to explore gene engineering vaccine and DNA vaccine.In our experiment, influenza virus in allantoic fluid of embryonic egg was purified by SDGC or AR-RBC. The antibody specific for influenza virus was produced by immunizing rabbits with the purified virus. Virus RNA was extracted from allantoic fluid of chicken embryo inoculated with influenza A virus A/PR/8/34(HlNl). The HAl gene was amplified by RT-PCR with specific primers. The amplified DNA fragment was cloned into eukaryotic expressing vector pcDNA3.1(+). Then E. coli competent cell was transformed with recombinant plasmid and positive clones were selected. By restriction endonucleases identification, PCR and sequencing analysis, the recombinant plasmid of HAl, pcDNA3.1/HAl, was successfully constructed. The HEK293 cell was transfected by pcDNA3.1/HAl. The HAl expression in HEK293 cell was tested by immunofluorescence. The cells were incubated with rabit anti-virus serum, followed by FITC-labelled goat anti-rabit IgG.By AR-RBC, the hemagglutination titer of the purified influenza virus was 1:2 560. Hemagglutination inhibition titer of the serum of rabits immunized with the purified virus was beyond 1:1 280. The recombinant plasmid was identifid by restriction endonucleases identification and PCR. About 1 .Okb band was observed in the electrophoresis through endonucleases digestion or PCR. By sequencing analysis and Blast, the recombinant plasmid of HAl was 98% align to HAl sequence in GenBank. In immunofluorescence assay, the HA gene was successfully expressed from plasmid pcDNA3.1/HAl. So the recombinant plasmid was successfully constructed.In our study, we cloned HAl gene of influenza A virus, and we constructed eukaryotic expressing plasmid pcDNA3.1/HAl.The plasmid could express HAl in eukaryotic cells. Our research is a base for further research of gene engineering vaccine and DNA vaccine. |
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