Construction And Immune Efficacy Of HA Gene Expression Against HAPIV

Avian Influenza (AI) is one of fatal infectious diseases caused by influenza A virus in avian.Further more there is new situation that many subtypes avian influenza viruses (AIV) are coexistent. Vaccines are still the main means to prevent AI. But traditional vaccines can't play a efficient role to prevent the AI for the characteristic of AIV for the variation and more subtypes. So it's an important question how to design and study new vaccines,especially vaccines. Based on genetic analysis of an avian influenza virus AIV H5HA gene was cloned and H7HA gene was synthesized. Then DNA recombinant was constructed for the prevention of H5,H7 subtype influenza virus. After the mouse was inoculated with the recombinant the indexes of humoral and cell immunity were detected. This study established the base for prevention of multitude influenza viruses. The more information as follows:In present study one full-length hemagglutinin (HA) genes was amplified from H5N1 avian influenza virus isolates by RT-PCR. The gene was cloned into pUCm-T vector. The recombinant plasmid were sequenced.By restriction enzymes digestion and sequence analysis the results showed the gene fragments was obtained successfully.The length of complete open reading frame of the gene was 1707bp. H7N1 AIV HA gene was synthesized from A/African starling/983/79(H7N7) cDNA template. The completely open reading frame including 1704bp encode 568 amino acids. After inserting H5,H7HA into pVAX1 the recombinant plasmid pVAX1-H57 was constructed. Then its antigenicity was detected by IFA in Hela cells. The Western-blot IFA results showed that these recombinant plasmids could expressed the object proteins. These results identified that it's good antigenicity.BALB/c mice which was used as mammal experimental model were innoculated by co-expressed DNA recombinant strain as before. The humoral and cell immunity level were detected. The results showed that special antibody aiming at H5,H7 subtype could be generated in group of pVAX1-H57 and antibody titer (1:1600～1:6400) were lower than that of inactivated vaccination group (1:12800) but is all higher than that of control group (p<0.01) . The antibody titer against H57HA in group pVAX1-H57 is 1:6400 and 1:3200 which was higher than other groups (p<0.05). The result from flow cytometry after statistics analyse indicated that contrast with the inactivated vaccination group pVAX1 group and PBS group the mice which immuned with pVAX1-H57 could found the significant elevation of T lympholeukocyte subgroup CD4+ and CD8+In addition ratios of all groups were stabile between 1.5-2.0 which indicate there is no abnormalities during the immune response. The results of ELISPOT detection for number of IFN-γblot showed the number (83.597) of groups pVAX1-H57 was higher (p<0.05) than inactivated vaccination group (39.857). And the difference between control group were quiet.The results above indicated that the recombinant DNA and fowl pox virus which were constructed above have good immunogenicity. The study had established the foundation for the multivalent vaccine of influenza viruses.