[0036] Example 1
[0037] This embodiment provides a method for preparing a quadruple vaccine for Newcastle disease, avian influenza, infectious bursal disease and avian adenovirus disease. figure 1 It is the flow chart of the preparation method, and the specific steps include:
[0038] (1) Source of poison (bacteria) species
[0039] Chicken Newcastle disease virus La Sota strain, H9 subtype avian influenza virus HB01 strain, recombinant E. coli Rosetta(DE3)-rVP2 strain expressing infectious bursal disease virus VP2 protein, expressing avian adenovirus (group I, 4 The recombinant E. coli Rosetta(DE3)-rFiber2 strain of Fiber2 protein was identified, kept and supplied by Wuhan Keqian Biological Co., Ltd.
[0040] Among them, the H9 subtype avian influenza virus HB01 strain has the preservation number of CCTCC NO: V202017, which was deposited in the Chinese Type Culture Collection of Wuhan University on January 7, 2020, and the preservation address is: Wuhan University, Wuhan, China; postcode 430072, classified and named It is: avian influenza virus (H9 subtype) HB01 strain.
[0041] The H9 subtype avian influenza virus HB01 strain has agglutinating effect on chicken red blood cells (that is, it has hemagglutination), and its hemagglutination can be specifically inhibited by avian influenza (H9 subtype) positive serum. The strain is genotyped It belongs to the avian influenza H9N2, which is in line with the characteristics of the H9 subtype avian influenza virus.
[0042] (2) Preparation of poison (bacteria) for production
[0043] 1. Preparation of virus seed for the production of chicken Newcastle disease virus La Sota strain: use sterile physiological saline as 10 4 Double dilution, 10-day-old SPF chicken embryos were inoculated into the allantoic cavity, 0.1ml per embryo. The chicken embryos that died 72 to 120 hours after the inoculation and had obvious disease scars were selected, and the chicken embryo allantoic fluid was harvested and packed in sterilized containers. The allantoic fluid, which has been tested for sterility and has an agglutinating value of 1% chicken red blood cells not less than 1:512 (micro method), is mixed, quantified, and frozen and stored. Indicate the date of harvest, the number of poison seeds, etc.
[0044] 2. Preparation of virus seed for the production of H9 subtype avian influenza virus HB01 strain: The virus seed was diluted 104 times with sterile normal saline, and 10-day-old SPF chicken embryos were inoculated into the allantoic cavity, 0.1ml per embryo. For the chicken embryos 60 to 120 hours after the inoculation, the chicken embryo allantoic fluid was harvested and packed in a sterilized container. The chicken embryo liquid tested for sterility and the agglutination value of 1% chicken erythrocytes is not less than 1:256 (micro method) is mixed, quantified, and frozen and stored. Indicate the date of harvest, the number of poison seeds, etc.
[0045] 3. Preparation of the recombinant Escherichia coli Rosetta(DE3)-rVP2 strain expressing the VP2 protein of infectious bursal disease virus: the freeze-dried Rosetta(DE3)-rVP2 strain was streaked in LA medium ( Incubate at 37℃ for 15 hours, pick and inoculate typical colonies that meet the standard in LB medium (containing 10μg/ml Kana), culture at 37℃ in a shaker at 200r/min until OD600nm is about 1.0. As a first-level seed bacteria, the bacteria solution should be stored at 2~8℃ for no more than 14 days; take the first-level seed bacteria solution of Rosetta(DE3)-rVP2 strain and inoculate it in LB medium (containing 10μg/ ml Kana), cultured in a shaker at 37°C at 200r/min to an OD600nm of about 1.0, and used as secondary seed bacteria after harvest. Bacteria solution should be stored at 2~8℃ for no more than 7 days.
[0046] 4. Preparation of recombinant Escherichia coli Rosetta(DE3)-rFiber2 strain expressing avian adenovirus (group I, type 4) Fiber2 protein: streaking the freeze-dried Rosetta(DE3)-rFiber2 strain in LA Culture medium (containing 10μg/ml Kana), culture at 37℃ for 15 hours, pick and inoculate typical colonies that meet the standard into LB medium (containing 10μg/ml Kana), culture at 37℃ shaker at 200r/min until OD600nm is about 1.0 Harvested, as a first-level seed bacteria, the bacteria liquid should be stored at 2~8℃ for no more than 14 days; take the first-level seed liquid of Rosetta(DE3)-rFiber2 strain and inoculate it in LB medium (containing 10μg/ ml Kana), cultured in a shaker at 37°C at 200r/min to an OD600nm of about 1.0, and used as secondary seed bacteria after harvest. Bacteria solution should be stored at 2~8℃ for no more than 7 days.
[0047] (3) Preparation of virus liquid for vaccine production
[0048] 1 Preparation of virus solution for chicken Newcastle disease virus vaccine production
[0049] 1.1 Inoculation Take the virus seeds for production and use sterile physiological saline as 10 4 Double dilution, inoculate 9-11-day-old susceptible chicken embryos in the allantoic cavity of each embryo, 0.1ml per embryo, seal the pinhole, and continue incubating at 36-37°C without turning the eggs.
[0050] 1.2 Incubation and observation After inoculation, the eggs are irradiated once a day. The embryos that die within 48 hours are discarded. The embryos that die within 48 hours are taken out at any time and stored at 2-8°C until 120 hours. After 120 hours, regardless of whether the chicken embryos are dead or not, all are taken out, the air chamber is upright, and it is placed at 2~8℃ to cool for 4~24 hours.
[0051] 1.3 Harvest Take out the chilled chicken embryos, sterilize the egg shells, and harvest chicken embryo allantoic fluid separately, put several chicken embryos into a group, put them in the same sterile container, take samples for red blood cell agglutination test, and agglutinate 1% chicken red blood cells The price should be no less than 1:512 (micro method), and at the same time, sterility test should be carried out in accordance with the appendix of the current "Chinese Veterinary Pharmacopoeia". It should be grown aseptically and stored below -15°C for later use.
[0052] 1.4 Concentration of virus liquid The chicken embryo allantoic liquid of chicken Newcastle disease virus La Sota strain is coarsely filtered to remove large particles of impurities in the virus liquid, and then concentrated to 1/4 of the original volume by an ultrafiltration concentration system, and the concentrated virus liquid is sampled Then, determine the virus content (after the concentrated virus solution is diluted by 4 times, the virus content per 0.1ml should be ≥10 8.0 EID 50 After concentration, the virus solution should be stored at 2~8℃.
[0053] 1.5 Inactivation of the virus solution Add the tested chicken embryo allantoic solution to the formaldehyde solution to make the final concentration of the formaldehyde solution 0.2%. Stir while adding to make it fully mixed, and inactivate it at 37°C for 20 hours. Store at 2~8℃ after live.
[0054] Preparation of virus liquid for vaccine production of 2H9 subtype avian influenza virus
[0055] 2.1 Inoculation Take the virus seeds for production, dilute 104 times with sterile normal saline, inoculate 9-11-day-old susceptible chicken embryos in the allantoic cavity of each embryo, 0.1ml per embryo, seal the pinhole, and continue at 36-37°C Incubate without turning the eggs.
[0056] 2.2 Incubation and observation After inoculation, the eggs are irradiated once a day. The embryos that die within 24 hours are discarded, and the embryos that die after 24 hours are taken out at any time and stored at 2-8°C until 96 hours. After 96 hours, regardless of whether the chicken embryos are dead or not, all are taken out, the air chamber is upright, and it is placed at 2~8℃ to cool for 4~24 hours.
[0057] 2.3 Harvest Take out the cooled chicken embryos, sterilize the egg shells, and harvest the chicken embryo allantoic fluid. Several chicken embryos are divided into a group and placed in the same sterilized container. Samples are taken for agglutination test of 1% chicken red blood cells. The price should not be less than 1:256 (micro method), and the sterility test should be carried out in accordance with the appendix of the current "Chinese Veterinary Pharmacopoeia". It should be grown aseptically and stored below -15°C for later use.
[0058] 2.4 Concentration of virus liquid The chicken embryo allantoic liquid of the H9 subtype avian influenza HB01 strain is coarsely filtered to remove large particles of impurities in the virus liquid, and then concentrated to 1/4 of the original volume by an ultrafiltration concentration system, and the concentrated virus After sampling the solution, determine the virus content (after 4-fold dilution of the concentrated virus solution, the virus content per 0.1ml should be ≥10 7.0 EID 50 After concentration, the virus solution should be stored at 2~8℃.
[0059] 2.5 Virus liquid inactivation Add the tested chicken embryo allantoic liquid to the formaldehyde solution, the final concentration of the formaldehyde solution is 0.2%, stir while adding to make it fully mixed, inactivate at 37 ℃ for 24 hours, inactivate Store at 2~8℃ for later use.
[0060] 3 Preparation of infectious bursal disease virus VP2 protein antigen
[0061] 3.1 Bacterial culture and induction The production strains were inoculated into synthetic medium (containing 10μg/ml Kana) at a volume ratio of 2%, cultured in a fermenter at 36~37℃, stirred at 200r/min, and dissolved oxygen (DO ) Is 10%, pH 7.2~7.3. After culturing under this condition until OD600nm reaches about 60, add isopropylthiogalactoside (IPTG) to a final concentration of 0.1mmol/L, and induce culture at 16℃ for 14 hours Then collect the fermentation broth.
[0062] 3.2 Extraction of the antigen. After the culture is completed, the bacteria are collected by centrifugation. After resuspending and washing 3 times in PBS buffer (pH7.4, 0.01M), add 10ml of PBS buffer (pH7.4, 0.01M) per gram of wet weight of the bacteria. M) Resuspend the bacterial cells under pressure, and centrifuge to collect the supernatant.
[0063] 3.3 Purification of protein antigens Purify VP2 protein by ion exchange method. The purified rVP2 protein is filtered and sterilized through a 0.22μm pore filter and stored at 2-8°C.
[0064] 4 Preparation of avian adenovirus (group I, type 4) Fiber2 protein antigen
[0065] 4.1 Bacterial culture and induction The secondary seeds are inoculated into synthetic medium (containing 10μg/mlKana) at a volume ratio of 2%, cultured in a fermenter at 36~37℃, stirring speed is 200r/min, and dissolved oxygen (DO) is 10%, pH 7.2-7.3, cultured under this condition until OD600nm reaches about 60, add isopropylthiogalactoside (IPTG) to a final concentration of 1.0mmol/L, and induce culture at 36~37℃6 Collect the fermentation broth after hours.
[0066] 4.2 Extraction of the antigen. After the culture is completed, the bacteria are collected by centrifugation. After resuspending and washing 3 times in PBS buffer (pH7.4, 0.01M), add 10ml PBS buffer (pH7.4, 0.01M) per gram of wet weight of the bacteria. M) Resuspend the bacterial cells under pressure, and centrifuge to collect the supernatant.
[0067] 4.3 Protein antigen purification The target protein is purified by His tag affinity chromatography. The purified Fiber2 protein is filtered and sterilized by a 0.22μm pore filter and stored at 2-8°C.
[0068] (4) Preparation of vaccine
[0069] 1 Oil phase preparation Take 94 parts of white oil for injection (in milliliters), add 1 part of aluminum stearate (in grams), stir while adding until it is completely transparent, and then add 6 parts of Spen-80 (In milliliters), sterilize at 121°C for 30 minutes after mixing, and cool to room temperature for later use.
[0070] 2 Aqueous phase preparation. Dilute the inactivated chicken Newcastle disease virus, H9 subtype avian influenza virus, infectious bursal disease virus VP2 protein and avian adenovirus Fiber2 protein to the required concentration for the vaccine and according to 1:1:1: 1 Volume ratio is fully mixed, and the antigen content of chicken Newcastle disease virus in the antigen solution after mixing is 10 8.0 EID 50 /0.1ml, H9 subtype avian influenza virus antigen content is 10 7.0 EID 50 /0.1ml, the titer of infectious bursal disease virus VP2 protein antigen is 1:16, and the antigen content of avian adenovirus Fiber2 protein is 50μg/0.1ml. Take 96 parts of the mixed antigen solution, add 4 parts of sterilized Tween-80, and stir for 20-30 minutes to completely dissolve the Tween-80.
[0071] 3 Emulsification Inject 2 parts of the oil phase into the emulsification tank, stir slowly while slowly adding 1 part of the water phase, after the addition, perform shear emulsification.
[0072] 4 Dispense Pack the emulsified vaccine in a quantitative manner and seal it. Label and store at 2~8℃.
