Construction And Expression Of DNA Vaccine And Prokaryotic Expression Vector Of Influenza A Virus HA And Preparation Of Polyclonal Antibody Against HA

Influenza virus is the pathogen of influenza, which is a kind of highly contagious disease with the first incidence rate among all of the infectious diseases. In 20th century, 4 pandemic ever swept the global, just the 1918 Spanish influenza pandemic led to the death of at least 20 million people worldwide. Severity of influenza epidemics is a formidable public health challenge as well as significant socio-economic burden. The indulge in willful persecution of the avian influenza from 1997 has been waking people as alarm bell. Till now people could not control the disease due to the high variability of influenza virus in spite of the rapid progress of the modern scientific technology. DNA vaccine has become the focus of widespread interest, not only because of the novelty and simplicity, but also because of its efficacy to induce broad-spectrum immunity. 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.The hemagglutinin (HA) is the major surface glycoprotein of influenza virus that mediates the attachment and penetration of the virus into host cells. The HA is the major surface antigen of influenza virus, which can induce the production of neutralizing antibodies to neutralize the infectivity of the virus. Therefore, the hemagglutinin play a important role in diagnose and control influenza.In order to study HA gene DNA vaccine, the HA gene was amplified from Influenza virus A/New Caledonia/20/99(H1N1) by RT-PCR, and subcloned into eukaryotic expressing vector pVAX1 which contains the CMV promoter and enhancer.To validate the expression of pVAX1/HA vector in eukaryotic cells, we transfected the vector into 293T cells, and the expression of HA was detected at 48 hours after transfection by immunofluorescence staining. As negative controls, the cells that transfected with empty pVAXl vector and the untransfected 293T cell showed no fluorescence. These experiments indicated that DNA vaccine can expresse HA in eukaryotic cells.Preparation of antibody against HA protein is essential for the further study such as the tissue distribution and immunogenicity of HA after injection of HA DNA vaccine into organisms. So the prokaryotic expression vector pET-28a(+)/HA was constructed and expressed HA protein in the E.coli BL21 induced by IPTG, then the HA recombinant protein with poly-His tag in the N-terminal was purified by gel filtration chromatography(GFC). Polyclonal antibody against HA was generated by immunizing New Zealand rabbit with purified His-tagged HA protein, and its titer was tested by indirect ELISA, the result showed that the polyclonal antibody against HA protein had good immunoreactivity. Our study provides important experimental material and useful data for further development of vaccines and diagnostic reagents against influenza.