| Influenza virus (IV) is one of the deadliest human diseases and it can make human respiratory infections. Humanity has repeatedly suffered influenza pandemics (1918H1N1sub type,1957H2N2sub type,1968H3N2and1977H1N1type). So far the human race is still unable to conquer the flu virus. Fast detection and vaccination is the most economic and effective method to control and eliminate influenza virus. Using Real-time fluorescence quantitative polymerase chain reaction (PCR) detected flu viruses has the advantage of fast, high specificity, but is expensive, not suitable for wide use. The current vaccines used include attenuated vaccines, inactivated vaccine, cracking vaccine, and so on. These vaccines can elicit the protective antibody in the body, but still have some shortcomings, such as attenuated vaccines has the potential of virulence reversion and the later of two are difficult to stimulate the cellular immune responses. Thus, it is needed to develop the rapid detection method for influenza virus and novel effective vaccines for influenza virus infections.Many published articles were confirmed that the protective effects of influenza virus glycoprotein antigen, among the glycoprotein antigens, hemagglutinin (HA) function is quite important. HA has immunogenicity and HA antibodies can counteract with flu viruses, and it is the major protective antigen. The target gene the experiment in the study is HA gene of A/California/05/2009H1N1influenza virus which published by GenBank. The HA gene of A/California/05/2009H1N1influenza virus from GenBank date base was fully synthesized, and inserted into pET30a+to construct the recombinant prokaryotic expression plasmid pET-HA. Then the recombinant plasmid was transformed into E. coli BL21(DE3) and the recombinant bacteria induced by IPTG to express the desired HA fusion protein. Western blot analysis showed that the HA fusion protein could specifically react with the positive serum from patients infected by influenza A (H1N1) virus specially. Using this expressed fusion HA protein, an indirect enzyme-linked immunosorbent assay was established. It optimal coating antigen was1μg/well of HA fusion protein, and the suitable dilution titer of serum sample was form1:80to1:640. Furthermore,300human sera collected from hospital were tested by this ELISA, the results showed that the positive sera is74, and coined well with that of the commercialized kit.The synthesized HA gene of A/California/05/2009H1N1influenza virus was further cloned into the transfer vector pFastBacl and transformed DH5a to get the recombinant plasmid pFastBac-HA. The recombinant plasmid pFastBac-HA was then transformed into the strain E. coil DH10Bac, which included a shuttle vector, that is Bacmid. After transportation, the HA gene of A/California/05/2009H1N1influenza virus was integrated into Bacmid, identified by PCR, the positive recombinant named as Bacmid-HA. And then, the recombinant Bacmid-HA was transected into Sf9insect cells with liposome2000. The expression of HA protein in transected cells was indentified by the IFA, SDS-PAGE and Western blot analysis. The HA protein expressed from transfected insect cells had bioactive, its HA titers was1:16in the hemagglutination assay. BALB/c mice were immunized with100μg of HA protein via subcutaneous injection, the sera were collected after14day of the second immunized. The ELISA results showed that there was a significant difference between HA immunized group and the blank control group (p<0.05). Their HI titer was1:16in hemagglutination inhibition assay. Furthermore, the results enzyme linked spot(ELISPOT) assay showed that the number of IL-4secreting cells was much more than that of IFN-y secreting cells in splenocytes of immunized mice with the expressed HA protein, and indicated that the immune responses induced in vivo was tendency to Th-2. |
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