| The recombinant expression E.cole BL21-pGEXsM2 which contains which include deletion of the transmembrance segment of M2 was induced in the final concentration of 0.6mmol/LIPTG at 37℃, and acquired soluble fusion protein; Western blot assays suggested that the protein could react with the monoclonal antibody of M2 14C2, the protein was 35 KD, proved it having good immunological activity; the expressed protein were purified by Glutathione Sepharose 4B, a great of target protein and a little of GST protein were obtain, the titer of M2 was 5.6mg/ml.Indirect-enzyme linked immuno- sorbent assay ( Indirect-ELISA) was developed by using the purified fusion protein of M2 as antigen. Our date show that the titer of incubating antigen were 50μg/ml; the suitable concentration of detected sera was 1:200;working concentration of goat anti-chicken IgG-HRP antibody was 1:20000; the fusion protein didn't react with the positive sera of chicken experimentally infected with ND,IB,IBD and MD; M2-specific antibody were detected in the positive sera of M2 which were diluted to 1:1600.After being added to adjuvant, the purified M2 protein was prefabricated as subunit vaccine. Sixty-five SPFchickens separate into 4 groups: HA-vaccinated group, M2-vaccinated group, HA+M2-vaccinated group and control group. HA-vaccinated group were immunized 30μg HA protein, M2-vaccinated group were immunized 30μg M2 protein, HA+M2-vaccinated group were immunized 30μg HA protein and 30μg M2 protein, per chicken were immunized again after two weeks. Preimmune and after immuned, we got the blood and sera.,then detected M2 and HA antibody.The chickens were challenged with H5N1 or H7N1 virus by the three weeks after the second immunized. As a resuilt, M2-vaccinated group and HA+M2-vaccinated group produced M2-special antibody,twe weeks after twice immunization, M2-specific antibody titer was 25600 in M2-vaccinated group and was 51200 in HA+M2-vaccinated group. M2-specific antibody titer was 6400 eight weeks after twice immunization. HA-vaccinated group and HA+M2-vaccinated group produced HI antibody, one week after twice immunization, HI antibody titer was 26.8, the HI antibody titer didn't exhibit evident difference betweent HA+M2-vaccinated and the HA-vaccinated group.By H5N1 lethal challenge assay, HA+M2-vaccinated and HA-vaccinated induced 100% protection, M2-vaccinated induced 20% protection. By H7N1 lethal challenge assay, HA+M2-vaccinated and M2-vaccinated induced 10% protection and the control groups were all killed in a week. |
