Avian influenza virus belongs to the influenza virus genus A influenza virus of the family Orthomyxoviridae, influenza A virus host is extremely wide. the pathogenicity of influenza A viruses and strains and hosts have a lot to do with different strains of influenza A viruses on the same host or the same strain of the same host pathogenicity varies greatly. the H9N2 subtype AIV is the most widely involved in our country, the morbidity and mortality rate is low, but the harm can not be ignored. The virus in addition to causing avian respiratory diseases, but also violates the reproductive system of poultry, so that the egg production of laying hens fell sharply or egg production can not reach the peak: broiler infection growth is slow, reduce the meat ratio; H9 subtype AIV easy and other viral diseases (such as chicken Newcastle disease, etc.) or bacterial diseases (such as E. coli disease, etc.) mixed infection, can cause a higher mortality rate. Canine kidney epithelial cells (Madin-Darby canine kidney cells (MDCK)) are highly susceptible to influenza viruses, making them one of the best hosts for influenza vaccine production. In this paper, the MDCK-10, which has been successfully suspended and domesticated in the pre-laboratory, was validated for stability and evaluated for virus production, and then to animal experiments. From the development of shaking flask small test process, to the pilot test and large-scale production in bioreactors to carry out a full range of process mapping, so as to update the current stage of the production of chicken embryo as raw materials for the production of the inherent process, for the domestic low pathogenic avian influenza production enterprises to carry out the full-suspension serum-free large-scale production of the data base.

   Suspended MDCK-10 cells in the exponential growth phase were inoculated in Corning shaker flasks at a density of about 1.5×10⁶cells/mL, filled with 20-30% of the total volume of the flasks, and cultured in an incubator at 37℃, 5% CO₂, and 130 r/min; the subsequent successive passages were carried out by diluting the cell sap with fresh medium to 1.5×10⁶ cells/mL, and the cells were passaged every 48h. The subsequent successive passaging was performed by diluting the cell solution to 1.5×10⁶ cells/mL with fresh medium and passaging every 48h. After 40 generations of consecutive cell passages, the cell status was observed and data were summarized to support the stability of cell passages in continuous dilution.

   Suspension MDCK-10 on the tank 14L glass bioreactor, culture volume 6L, density increase to 8-10 × 10⁶cells/mL, according to the fresh medium and cell solution volume ratio of 1:1 dilution of cell solution, respectively, inoculation of H9F6 strain, H9C1 strain, H9C2 strain, H9CD strain at the ratio of one ten-thousandth of the culture volume, trypsin addition concentration of 10 μg / mL, control Reactor parameters: temperature: 37°C; speed: 60 rpm; pH 7.15; DO: 60%. The viral solution was collected after 48-72h of incubation, and samples were taken to determine the viral titer after freezing and thawing once. A 37% formaldehyde solution was added at a final concentration of 0.1% to 37°C for 24h of inactivation, during which time it was stirred every 4h. Inoculation of chicken embryos to determine the inactivation effect: inoculated in 9-11 days old SPF chicken embryos, each chicken embryo inoculation 0.1mL, incubated at 37 ℃. 24h photo-embryo, non-specific death should not exceed 1. 24h after the embryo twice a day, observation for 5 days. The hemagglutination value of all embryos was determined separately, and no hemagglutination should occur. The inactivation was judged to be complete when the hemagglutination price was measured in all the embryos and transmitted blindly for one generation, and no hemagglutination was observed.

   The four inactivated antigens (H9F6, H9C1, H9C2 and H9CD strains) were diluted 2-fold and 4-fold with sterilized saline, and after dilution, 6% Tween-80 was added to them respectively, and the aqueous phase and the oil phase were white oil. According to the ratio of water phase: oil phase is 1:3 emulsification seedling production.

  （1）Preparation of inactivated cellular vaccines

     Cellular inactivated vaccines of the H9CD strain were prepared according to methods 05 and 06.

  （2）Preparation of embryotoxic inactivated vaccine

     The H9CD strain was diluted 10-4-10-5 with sterilized saline and inoculated into 10-11 days old susceptible chicken embryos via allantoic fluid at 0.1 ml per embryo.The chicken embryos were selected to be dead within 48-120h after inoculation and survived for 120h, and then cooled at 2-8℃ for 24h, harvested chicken embryos' allantoic fluid, and inactivated according to the method of 05 and inactivation test, and then emulsified according to the method of 06 to make the vaccine.

  （3）Immunization of SPF chickens

     The 21-day-old SPF chickens were divided into 2 groups of 5 birds each, the first group was immunized with inactivated cytotoxic vaccine of H9CD strain, the second group was immunized with inactivated vaccine of H9CD strain, 0.2 ml/bird, and the other group was set up as a blank control group of 5 birds, which was not immunized. The serum was collected on the 14th and 21st day after immunization, and the antibody potency of H9 serum was determined.