| Avian influenza, or"bird flu,"is an infectious disease caused by influenza A viruses, which normally lead to serious disease and mortality in domestic poultry industry. Since 1997, a novel Influenza A (H5N1) virus– also called HPAIV–has attracted the current global attention with its outbreak world- wide in poultry and crossed species barrier to infect human. By May 26th, 2007, there were a total of 307 confirmed human cases of the H5N1 virus infection, 186 of which were fatal, the death rate is up to 60.6%, according to the World Health Organization (WHO). There though no evidence that this new viruses have acquired the ability of transmitted between human, but the widespread nature of H5N1 in birds and the likelihood of mutations over time raise the opportunities of genetic reassorted between avian and human influenza virus, and will product a pandemic strain. If this happened, millions people will be killed in the next pandemic.Since the specific vaccine for humans which effective against avian influenza has not yet been approved, and the H5N1 viruses have a high resistance to most commercially available drugs which include oseltamivir (commercially named "Tamiflu", a neuraminidase inhibitor) and amantadine (M2 protein inhibitor), so the development of new effective drugs is in concerned.In this study, we have immunized health horses with H5N1 strain derived from mammals, and established a immune program by examined antibodies of horses blood. The hyper-immunized horse serum has a higher HI antibody titers which is 1:2560 and a higher SN antibody titers which is 1:5120, and also contained a little cross reaction to H1, H3, H7 and H9 hemagglutinin antigens. We then inoculated serum to mice to investigate the prophylaxis and treatment efficacy, test results demonstrated that 0.2ml serum can provide 100% protection against 10 LD50 viruses 4-7 days before challenge, and 0.2ml serum can also provide 100% protection against 5 LD50 viruses 24h after challenge. The comparison result of survival rate, lung index and viruses titers in lung demonstrated hyper-immunized serum is better to Tamiflu.In the coming study, we explored a optimized product processes of antibody segments which includes IgG extraction and purification, digestion by pepsin and purification with ion exchange chromatography. On the consequence, the purity of F(ab')2 produced by optimized processes is up to 90% which is higher than traditional products. Mice injected with 0.2mg-0.4mg F(ab')2 24h after viruses inoculation could obtained 80% protection against 3 LD50 viruses challenge. The comparison result of survival rate, lung index and viruses titers in lung demonstrated it is better to Tamiflu.To prepare an prefer monoclonal antibody for immunotoxin construction, BALB/c mice were immunized with AIV A/tiger/Harbin/2002(H5N1), hybridoma cell lines which secreting anti-AIV HA mAbs were developed through cell fusion, screening and cloning. By indirect ELISA and Western-blotting assay, we selected an specifically anti-HA mAbs which named 3A13, the indrected IFA test confirmed that the mAbs could adhesion to the infectious cell membrane to act as a vector of immunotoxin anti AIV infected cell.Using genetic engineering techniques ,we then coloned HA-ScFv gene by linked the VH and VL gene of 3A13 with a linker, and inserted it in pPE which contained PE40 expressing box to constructed the expression vector pPET-HAScFv- PE40. Then the chimeric protein pHAScFv-PE40 was constructed successfully and expressed in E. coli BL21 (λDE3). Cytotoxicity of recombinant toxin to AIV infectious cells was analyzed with MTT, and the results demonstrated that fusion proteins could destroy the infectious MDCK cells with 56.1% cytotoxicity, and could inhibit the AIV replication with 10 times decreased viral copy compare to viral control. All our research would provide a new method for control next human avian influenza pandemic.
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