| Avian influenza is a viral seriously disease of poultry by type A of influenza virus, and often causes enormous losses of economy. It is a catastrophic disease of the poultry industry, especially highly pathogenic avian influenza by H5 and H7 subtype avian influenza virus. Hemagglutinin (HA) is an important protective antigen of avian influenza virus, so HA gene is the first candidate gene for DNA vaccine. We have studied the HA DNA vaccine in this paper that contains mainly three parts.(1) Construction arid expression of avian influenza virus HA gene eukaryotic vectors. Hemagglutinin gene is an important gene of protective antigen of Avian Influenza Virus (AIV). In order to study HA gene vaccine, the H5 gene of AIV, amplified by PCR, was subcloned into eukaryotic expressing vectors pcDNA4/HisMax and pRc/CMV. The recombinant plasmids were transfected into Hela cell by Tfx?20 Transfection Reagent, Superfect Transfection Reagent, and electroporation respectively. The expressed HA was examined and identified by Western Blotting and Hemagglutination Assay. The results showed that HA was expressed precisely in Hela cells and had bioactivity. We found three special bands which were HA> HA1 > HA2 by Western Blotting, which were identical to those of AIV. The quantity of expressed HA from pC4H5, transfected by superfect transfection reagent, was approximately 8 times as much as that from pCMVHS. These results suggest that pC4H5 is a highly efficient eukaryotic expressing vector.(2) Immunized and challenged assays in mice of HA DNA vaccine. Four groups of BALB/c female mice (4 weeks old) were immunized with pC4H5 and pCMVHS of eukaryotic expressing plasmids by two injections, 3 weeks apart, of SOjig/mouse doses of the plasmid DNA into the right quadriceps muscle under light anesthesia (50mg/Kg, Sodium Pentobarbital). Two of the four groups were inserted into the muscle with a pair of electrode needles with 5 mm apart to cover the DNA injection sites and electric pulse were delivered using an electric pulse generator (ECM830; BTX; San Diego). Four pulses of 100V each were delivered to each injection site at the rate of one pulse per 300ms, each pulse lasting for 40 ms. Then four pulses of the opposite polarity were applied. In the 5lh week, the mice immunized by the electroporation and the im injections of plasmid DNA, were anesthetized and infectedwith H5 subtype avian influenza virus[10 50% egg-lethal dose (ELDso)], by intranasal application of 20uL of the virus suspension. We did Abs assays, lymphocyte cell proliferation assays, CTL assays, the ratio assays of CD4+/CDg+, IFN-y and IL-2 assays during the experiment. The group of mice were immunized by electroporation produced anti-HA IgGAbs in the 2nd week, whereas, the group of mice immunized by im produced anti-HA IgG Abs in the 4th and 5th week, so electroporation is better than im. Lymphocyte cell proliferation assays and CTL assays indicated that cell immune of mice was activated in the 4th week and more activated after challenge. The ratio of CD4+/CDg+ and cellar factors (IFN-y and IL-2) in serum of mice were strengthened with the increase of immunization times. Two groups of mice immunized with two eukaryotic expressing vectors were not significant (p>0.05), while those between experiment and control groups were significant, which indicated that these HA DNA vaccines could activate immune system of mice.Mice of control groups died in the 9th and 10th day after challenge, and always could get avian influenza virus from lungs of mice of control groups during the experiment. Mice immunized by im or electroporation were all surviving, and we could not get avian influenza virus from lungs of mice immunized by electroporation in the 5th ,7th ,16th day after challenge. We could get virus from lungs of mice immunized by im in the 5th day after challenge, and get virus from lungs of part of mice immunized by im in the 7th day after challenge, while we could not get virus from lungs of mice immunized by im in the 16l day after challenge, wh... |
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