Nano propolis composite adjuvant for poultry and application of nano propolis composite adjuvant

Abstract

The invention discloses a nano propolis composite adjuvant for poultry and application thereof, and belongs to the field of veterinary drugs. The nano propolis composite adjuvant for poultry is prepared by compounding a propolis ethanol extract, an immunopotentiator and a surfactant. The nano propolis composite adjuvant for poultry provided by the invention has the characteristics of no toxic or side effect, extremely low residual quantity and good immune effect, and can be used as an adjuvant of a poultry inactivated vaccine and a diluent of a live vaccine; the nano propolis composite adjuvant for poultry can be prepared into vaccines with enveloped viruses such as Newcastle disease viruses and avian influenza viruses, and the prepared vaccines have good stability and have the excellent characteristic of inducing cellular immunity and humoral immunity at the same time.

Description

Technical Field

[0001] The invention belongs to the field of veterinary medicine, and specifically relates to a nano propolis composite adjuvant for poultry and application thereof. Background Art

[0002] Vaccine immunization is one of the effective measures to prevent animal diseases. Its purpose is to induce the body to produce an immune response against special pathogenic microorganisms through immunization, including live attenuated vaccines, inactivated vaccines and subunit vaccines, etc., to prevent special diseases. Live attenuated vaccines can stimulate the body to produce comprehensive systemic immunity and local immunity, and the immunity is long-lasting, but this type of vaccine has the risk of spreading and virulence. Inactivated vaccines are prepared by inactivating pathogenic microorganisms through physical and chemical methods. This type of vaccine induces a weak immune response in the body, requires a large immune dose, and has a high production cost. Therefore, immune adjuvants are needed to enhance its immune effect. Especially in recent years, with the continuous development of weak immunogenic vaccines such as genetically engineered subunit vaccines, recombinant vaccines, and synthetic polypeptide vaccines, due to the high purity of their antigens, they need the assistance of immune adjuvants. However, the original immune adjuvants can no longer meet the needs of the rapid development of new vaccines. It is particularly urgent to develop new immune adjuvants with high immune activity and low side effects.

[0003] Propolis adjuvant refers to the propolis solution obtained after propolis is extracted with ethanol. The propolis solution contains a variety of immune active substances such as flavonoids, phenolic acids and their esters. It is a natural and efficient immunopotentiator that can improve the activity of phagocytes, promote the increase of the number of white blood cells in the body and the production of specific antibodies, and enhance the body's immune function. Most of the currently developed propolis adjuvants are ethanol extracts of propolis. The vaccine prepared with this adjuvant and enveloped viruses has low antibody titer after use. It may be that some component in the propolis adjuvant destroys the outer membrane antigen of the enveloped virus; in addition, the vaccine prepared by mixing propolis ethanol extract as an adjuvant with inactivated bacterial antigens produces precipitation during storage. The precipitation gathers at the bottom of the vaccine bottle and is not easy to disperse. It needs to be shaken vigorously when used, which brings inconvenience to use. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a nano-propolis composite adjuvant for poultry and its application. The nano-propolis composite adjuvant for poultry provided by the present invention has the characteristics of no toxic side effects, extremely low residue, and good immune effect, and can be used as an adjuvant for poultry inactivated vaccines and a diluent for live vaccines; the nano-propolis composite adjuvant for poultry can be prepared into vaccines with enveloped viruses such as Newcastle disease virus and avian influenza virus, and the prepared vaccine has good stability and has the excellent characteristics of inducing cellular immunity and humoral immunity at the same time.

[0005] The first aspect of the present invention provides a nano-propolis composite adjuvant for poultry, which is composed of the following components in volume percentage: 10% to 30% propolis ethanol extract, 30% to 45% immunopotentiator, and 30% to 50% surfactant; the propolis dry matter content in the propolis ethanol extract is 50 mg / mL to 70 mg / mL.

[0006] Furthermore, the immunopotentiator is a mixture of immunopotentiator A and immunopotentiator B; the mixing volume ratio of immunopotentiator A to immunopotentiator B is 5-8:1.

[0007] Furthermore, the immunopotentiator A is white oil or vitamin D, the immunopotentiator B is a mixture of Mycobacterium phlei extract and dimethyldioctadecylammonium bromide, or the immunopotentiator B is saponin.

[0008] Furthermore, the mixing volume ratio of the Mycobacterium phlei extract to dimethyl dioctadecyl ammonium bromide is 100:1-30.

[0009] Furthermore, the Mycobacterium phlei extract is obtained by lysing inactivated Mycobacterium phlei cells under 800-1000 bar conditions and then centrifuging at 11000 rpm-13000 rpm for 25 min-35 min.

[0010] Furthermore, the lysis process needs to be repeated twice.

[0011] Furthermore, the saponin is Quillaja saponin A or Quillaja saponin QS-21.

[0012] Furthermore, the surfactant is polyglycerol fatty acid ester, Span-80 and polyoxyethylene hydrogenated castor oil.

[0013] Furthermore, the volume ratio of the polyglycerol fatty acid ester, Span-80 and polyoxyethylene hydrogenated castor oil is 7-9:3-5:2.

[0014] The second aspect of the present invention provides a use of the above-mentioned poultry nano-propolis composite adjuvant in the preparation of poultry inactivated vaccine, subunit vaccine or live vaccine diluent.

[0015] The third aspect of the present invention provides a nano-propolis composite adjuvant vaccine for poultry, wherein the vaccine comprises the above-mentioned nano-propolis composite adjuvant for poultry.

[0016] Furthermore, the vaccine also includes inactivated antigens or subunit antigens of pathogenic microorganisms that infect poultry.

[0017] Furthermore, the antigen is Newcastle disease virus, avian influenza virus or Haemophilus paragallinarum.

[0018] Furthermore, the volume ratio of the antigen to the poultry nano-propolis composite adjuvant is 2-4:1.

[0019] Preferably, the volume ratio of the antigen to the poultry nano-propolis composite adjuvant is 3:1.

[0020] In summary, compared with the prior art, the present invention has the following beneficial effects: The invention provides a nano-propolis composite adjuvant for poultry, which consists of a propolis ethanol extract, an immunopotentiator and a surfactant. The propolis ethanol extract is rich in bioactive ingredients such as flavonoids and terpenes, and can effectively regulate the immune system of poultry, stimulate the proliferation of immune cells, and enhance specific and nonspecific immune functions. The addition of the immunopotentiator further strengthens the immune response, and synergizes with the propolis extract to significantly enhance the resistance of poultry to pathogens and improve the immune effect of vaccines. The surfactant added to the nano-propolis composite adjuvant for poultry can reduce the surface tension of the nano-propolis composite adjuvant for poultry, form a stable nano-scale emulsion structure, and improve the stability of vaccine preservation. The nano-scale preparation greatly increases the specific surface area, so that the combination of the adjuvant and immune cells is more efficient, thereby improving the bioavailability.

[0021] By adopting a specific non-ionic surfactant polyoxyethylene hydrogenated castor oil, Span-80 and polyglycerol fatty acid ester compound system, a stable nano-scale emulsion structure is formed, which effectively prevents precipitation and aggregation after the propolis extract is mixed with inactivated bacterial antigens, ensuring that the vaccine has good stability during storage and does not produce precipitation.

[0022] By adding vitamin D, white oil, dimethyl dioctadecyl ammonium bromide, saponin or Mycobacterium phlei extract, the poultry nano-propolis composite adjuvant can synergize with propolis ethanol extract to activate the multi-pathway response of the poultry immune system, significantly increase the antibody titer against enveloped viruses (such as avian influenza virus and Newcastle disease virus), prevent propolis components from destroying the viral outer membrane, and enhance the immune effect of the vaccine.

[0023] The nano propolis composite adjuvant for poultry provided by the invention has the characteristics of no toxic side effects, extremely low residue, good immune effect and good safety, and can be used as an adjuvant for poultry inactivated vaccines and a diluent for live vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the appearance of propolis ethanol extract. From left to right, the two bottles of samples are distilled water and propolis ethanol extract.

[0025] Figure 2 This is the absorption spectrum of propolis ethanol extract.

[0026] Figure 3These are the measurement results of the particle size and particle size distribution of the nano propolis composite adjuvant prepared in Example 1.

[0027] Figure 4 These are the measurement results of the particle size and particle size distribution of the nano propolis composite adjuvant prepared in Example 2.

[0028] Figure 5 These are the measurement results of the particle size and particle size distribution of the nano propolis composite adjuvant prepared in Example 3.

[0029] Figure 6 These are the stability test results of the nano-propolis composite adjuvant vaccine for poultry, wherein Figure A, Figure B, Figure C, Figure D, Figure E and Figure F are the stability test results of the nano-propolis composite adjuvant vaccine for poultry prepared in Example 4, Example 5, Example 6, Comparative Example 1, Comparative Example 2 and Comparative Example 3, respectively. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0031] In some embodiments of the present invention, propolis was purchased from Shandong Ludu Biotechnology Co., Ltd., Span 80, polyglycerol fatty acid esters, polyoxyethylene hydrogenated castor oil and Tween 80 were all purchased from Croda, UK, polyoxyethylene hydrogenated castor oil was purchased from BASF, Germany, vitamin D was purchased from Shanxi Jinyang Pharmaceutical Excipients Co., Ltd., white oil was purchased from Mobil, Quillaja saponin A, Quillaja saponin QS-21 and dimethyl dioctadecyl ammonium bromide (DDA) were purchased from Sigma aldrich, and other chemical reagents were of analytical grade and purchased from Sinopharm Group.

[0032] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0033] Most of the propolis adjuvants currently developed are directly ethanol extracts of propolis. The antibody titer of vaccines prepared with this adjuvant and enveloped viruses is low after use. It may be that some component in the propolis adjuvant destroys the outer membrane antigen of the enveloped virus. In addition, vaccines prepared by mixing propolis ethanol extract as an adjuvant with inactivated bacterial antigens produce precipitation during the storage process. The precipitate accumulates at the bottom of the vaccine bottle and is not easy to disperse. It needs to be shaken vigorously when used, which brings inconvenience to use.

[0034] The present invention provides a nano-propolis composite adjuvant for poultry and its application. The present invention obtains a nano-propolis composite adjuvant for poultry by compounding a propolis ethanol extract, an immunopotentiator and a surfactant, characterizes the properties of the nano-propolis composite adjuvant for poultry, and detects the safety of the nano-propolis composite adjuvant for poultry; the present invention further prepares a nano-propolis composite adjuvant vaccine for poultry using the nano-propolis composite adjuvant for poultry, and detects the properties, safety and antibody titer after immunization of the vaccine; it is found that the nano-propolis composite adjuvant for poultry of the present invention can be prepared into a vaccine with enveloped viruses such as Newcastle disease virus and avian influenza virus, and the prepared vaccine not only has good stability, but also can significantly improve the antibody titer against enveloped viruses.

[0035] Example 1: A method for preparing a nano-propolis composite adjuvant for poultry, comprising the following preparation steps: S1. Preparation of propolis ethanol extract The propolis ethanol extract was prepared with reference to GB / T 24283-2018 Propolis. The specific preparation steps were as follows: the crude propolis was frozen at -15°C for 36 hours, then ground and sieved with a freezing grinder, 95% v / v ethanol was added to the sieved crude propolis, the weight ratio of the sieved crude propolis to 95% v / v ethanol was 1:4, extracted at 37°C for 60 hours, cooled, filtered, centrifuged to remove the sediment, and then ultrafiltered and concentrated to obtain the pure propolis ethanol extract.

[0036] The invention carries out property inspection on the propolis ethanol extract, and determines the pH value, relative density, oxidation time, dry matter content and total flavonoid content of the propolis ethanol extract.

[0037] Test of Propolis Ethanol Extract: (1) Property inspection like Figure 1 As shown, the prepared propolis ethanol extract is placed in a colorless transparent container. Under natural light conditions, the propolis ethanol extract is observed to be a brown or dark brown, shiny, transparent liquid with a unique aromatic odor of propolis.

[0038] like Figure 2 As shown, the propolis ethanol extract solution was scanned in the wavelength range of 300-600nm, and the results showed that the propolis ethanol extract solution had a maximum absorption peak at 490nm.

[0039] Take an appropriate amount of this product and test the propolis ethanol extract sample by light scattering method according to the detection method in "General Rules 0982 of the Pharmacopoeia of the People's Republic of China (2020 Edition) (Part IV)". The particle size should be less than 100nm.

[0040] After testing, the particle sizes of three batches of samples (propolis ethanol extract Lot. 2021001, propolis ethanol extract Lot. 2021002, propolis ethanol extract Lot. 20210033) were all below 100 nanometers, meeting the standards.

[0041] (2) pH determination According to the pH determination method in Appendix 3101 of the "Pharmacopoeia of the People's Republic of China (2020 Edition)" (Volume III), the pH value of propolis ethanol extract samples should be measured and should be within the range of 6.0~7.0.

[0042] The results are shown in Table 1. The pH values ​​of the three batches of samples were in the range of 6.2 to 6.6, which met the standards.

[0043] Table 1 pH value of propolis ethanol extract (3) Relative density determination According to the relative density determination method in Appendix 0601 of the "Pharmacopoeia of the People's Republic of China (2020 Edition)" (Part 1), the propolis ethanol extract samples should be measured within the range of 0.75~0.95.

[0044] The results are shown in Table 2. The relative density values ​​of the three batches of samples are in the range of 0.80~0.86, which meets the standard.

[0045] Table 2 Relative density of propolis ethanol extract (4) Oxidation time The time it takes for the purple-red potassium permanganate solution to fade away is used to indicate the content of reducing substances in propolis. The propolis ethanol extract samples are tested in accordance with the detection method for propolis oxidation time in the National Standard of the People's Republic of China (GB / T 24283-2018). The oxidation time should be ≤22 seconds.

[0046] The results are shown in Table 3. The oxidation time of the three batches of samples is in the range of 18 to 20, which meets the standard.

[0047] Table 3 Oxidation time of propolis ethanol extract (5) Dry matter content of propolis ethanol extract Take the propolis ethanol extract solution, weigh the weight of the dry matter after ethanol volatilization, obtain the weight of the ethanol extract, and calculate its percentage of the sample weight.

[0048] The results are shown in Table 4. The propolis dry matter content of the three batches of samples was in the range of 55~62mg / mL, which met the standard.

[0049] Table 4 Dry matter content of propolis ethanol extract (6) Determination of total flavonoid content According to the National Standard of the People's Republic of China (GB / T 24283-2018) for propolis, the total flavonoid content in propolis was determined by spectrophotometric colorimetry. The total flavonoid content in 100 ml of propolis should be 20-40 mg.

[0050] The results are shown in Table 5. The total flavonoids content in propolis of the three batches of samples was in the range of 29-35 mg / 100 mL, which was in line with the standard.

[0051] Table 5 Total flavonoid content of propolis ethanol extract S2. Extraction of Mycobacterium phlei extract: Mycobacterium phlei ( Mycolicibacterium phlei ) (purchased from Shenzhen Aiyi Doctor Biotechnology Co., Ltd.) was inoculated into 2% glycerol-supplemented nutrient broth medium (purchased from Qingdao Haibo Biotechnology), cultured at 37°C for 72 hours with aeration, and heated at 100°C for 30 minutes to inactivate the bacterial solution. The inactivated bacterial solution was centrifuged at 8000rpm for 30 minutes to obtain the bacterial cells, which were resuspended with physiological saline and centrifuged twice to remove impurities. The bacterial weight was calculated, and the solution was resuspended with physiological saline to a concentration of 10%. The bacterial cells were broken with a high-pressure homogenizer (model SCIENTZ-150A, Ningbo Xinzhi Biotechnology Co., Ltd.) at 900 bar, repeated twice, and the broken bacterial cells were centrifuged at 12000rpm for 30 minutes, and the supernatant was collected as the extract of Mycobacterium phlei.

[0052] S3. Preparation of nano-propolis composite adjuvant for poultry 30 mL of white oil, 20 mL of Span-80, 10 mL of polyglycerol fatty acid ester and 5 mL of polyoxyethylene hydrogenated castor oil were mixed evenly, and then 20 mL of the propolis ethanol extract prepared in step S1 was added and mixed evenly, and then 0.1 g of Quillaja saponin A was added, and finally the volume was made up to 100 mL with water for injection, stirred at 800 rpm for 20 min, fully mixed and emulsified, and filtered and sterilized with a 0.22 μm filter to obtain a nano-propolis composite adjuvant for poultry.

[0053] Example 2: A method for preparing a nano-propolis composite adjuvant for poultry, comprising the following preparation steps: 30 mL of vitamin D, 20 mL of Span-80, 10 mL of polyglycerol fatty acid ester and 5 mL of polyoxyethylene hydrogenated castor oil were mixed evenly, and then 20 mL of the propolis ethanol extract prepared in step S1 of Example 1 was added and mixed evenly, and then 0.1 g of Quillaja saponin QS-21 was added, and finally the volume was made up to 100 mL with water for injection, stirred at 800 rpm for 30 min, fully mixed and emulsified, and filtered and sterilized with a 0.22 μm filter to obtain a poultry nano propolis composite adjuvant.

[0054] Example 3: A method for preparing a nano-propolis composite adjuvant for poultry, comprising the following preparation steps: 30 mL of vitamin D, 20 mL of Span-80, 10 mL of polyglycerol fatty acid ester and 5 mL of polyoxyethylene hydrogenated castor oil were mixed evenly, and then 20 mL of the propolis ethanol extract prepared in step S1 of Example 1 was added and mixed evenly, and then 0.1 g of DDA and 5 mL of the Mycobacterium phlei extract prepared in step S2 of Example 1 were added, and finally the volume was supplemented to 100 mL with water for injection, stirred at 800 rpm for 30 min, fully mixed and emulsified, and filtered and sterilized with a 0.22 μm filter to obtain a nano propolis composite adjuvant for poultry.

[0055] The present invention tested the particle size, safety and other indicators of the nano-propolis composite adjuvant for poultry prepared in Examples 1 to 3, and the test results are as follows.

[0056] (1) Determination of adjuvant particle size and particle size distribution According to the detection method provided in "General Rules 0982 of the Pharmacopoeia of the People's Republic of China (2020 Edition) (Volume 4), the particle size and particle size distribution of the poultry nano-propolis composite adjuvant prepared in Examples 1 to 3 of the present invention.

[0057] Test results such as Figure 3 , Figure 4 and Figure 5 As shown, the particle sizes of the nano-propolis composite adjuvant for poultry prepared in Examples 1 to 3 are 145-221 nm, 145-255 nm and 126-294 nm, respectively, all within the range of 100-300 nm, indicating that the nano-propolis composite adjuvant for poultry prepared in the present invention is a nano-scale product.

[0058] (2) Sterility testing According to the inspection method for semi-finished products in Appendix 3306 Sterility Inspection Method of the "Pharmacopoeia of the People's Republic of China (2020 Edition)" (Volume III), the nano-propolis composite adjuvant for poultry prepared in Examples 1 to 3 was subjected to sterility inspection.

[0059] The results are shown in Table 6, and no bacteria grew.

[0060] Table 6 Sterility test of nano-propolis composite adjuvant Note: TG stands for thioglycollate fluid medium, GA stands for casein agar medium, TSB stands for tryptic soy broth, and - stands for sterile growth.

[0061] (3) Safety inspection The safety test was carried out on the nano-propolis composite adjuvant for poultry prepared in Examples 1 to 3.

[0062] Mouse test: 10 female mice weighing 18-22 g were randomly divided into a blank control group and a test group. Each mouse in the blank control group was intramuscularly injected with 0.5 mL of sterile saline. Each mouse in the test group was intramuscularly injected with 0.5 mL of the nano-propolis composite adjuvant solution for poultry prepared in Examples 1 to 3 and observed for 7 days. The animals were weighed before and after the test.

[0063] Guinea pig method test: 4 guinea pigs, weighing 250-350 g, were randomly divided into a blank control group and a test group. Each guinea pig in the blank control group was intramuscularly injected with 1.0 mL of sterile saline. Each guinea pig in the test group was injected with 1.0 mL of the nano-propolis composite adjuvant solution for poultry prepared in Examples 1 to 3, and observed for 7 days.

[0064] The test results are shown in Tables 7 to 10. After the injection of the avian nano-propolis composite adjuvant solution, all mice and guinea pigs survived the observation period without abnormal reactions, and there was no swelling, necrosis, or abscess at the injection site. At the end of the observation period, the weight of each mouse and guinea pig increased significantly, and there was no significant difference between the blank control group and the control group, indicating that the adjuvant is safe in mice and guinea pigs.

[0065] Table 7 Safety test in mice Table 8 Safety test on guinea pigs Table 9 Safety test in mice Table 10 Safety test in guinea pigs Example 4: A method for preparing a nano-propolis composite adjuvant vaccine for poultry, comprising the following preparation steps: S1. Preparation of Newcastle disease virus solution 1. Preparation of Newcastle disease virus antigen solution The strain used in production is the LaSota strain of Newcastle disease virus, which is identified, stored and supplied by the China Veterinary Drug Administration.

[0066] (1) Inoculation: Take the production strain and dilute it with sterile saline for 10 3 Times, inoculate 10-day-old susceptible chicken embryos into the allantoic cavity, inoculate each embryo with 0.1 mL, seal the pinhole after inoculation, and continue incubation at 37°C.

[0067] (2) Incubation and observation: After the chicken embryos are inoculated, they are irradiated once every 24 hours, and chicken embryos that die within 48 hours are discarded. Thereafter, they are irradiated once every 24 hours, and dead embryos are removed at any time. After 96 hours, all embryos, dead or not, are removed, with the air chamber facing upward, and placed at 4°C for 12 hours.

[0068] (3) Harvest: Take out the cooled chicken embryos and harvest the chicken embryo fluid (collect live embryos first, then dead embryos). Place the harvested chicken embryo fluid in a sterilized container and sample to determine the HA titer. HA titers below 1:256 should be discarded. Perform sterility testing in accordance with the current "Chinese Veterinary Pharmacopoeia" and should be sterile. Store the harvested chicken embryo fluid below -15°C before inactivation.

[0069] (4) Concentration: After the harvested chicken embryo virus liquid is centrifuged at 2-8°C to remove the residue and other particulate impurities in the chicken embryo liquid, it is concentrated by ultrafiltration concentrator until the HA titer is not less than 1:2048, and the concentration is stopped. At the same time, sterility test is carried out in accordance with the current "Chinese Veterinary Pharmacopoeia", and it should grow aseptically. A sample is retained for the detection of the toxicity, and the concentrated chicken embryo virus liquid is immediately inactivated.

[0070] (5) Inactivation: Place concentrated chicken embryo virus liquid of Newcastle disease into an inactivation tank, add 10% formaldehyde solution, start the mixer to mix thoroughly, and the final concentration of formaldehyde is 0.1%. After adding the formaldehyde solution, place it into another inactivation tank to prevent the virus adhering to the tank mouth from contacting the inactivator. Inactivate at 37℃ for 16 hours (start timing when the temperature in the tank reaches 37℃, and start the mixer to stir continuously), then take it out and store it at 4℃.

[0071] 2. Newcastle disease virus antigen liquid test (1) HA titer: Take Newcastle disease virus antigen liquid and measure it according to the appendix of the current "Chinese Veterinary Pharmacopoeia". The HA titer shall not be less than 1:1024.

[0072] (2) Virus content: The concentrated chicken embryo virus solution taken out before inactivation was diluted 10 times in series, and 10 -7 , 10 -8 , 10 -9Three dilutions were used, and five 10-day-old SPF chicken embryos were inoculated into each allantoic cavity, with 0.1 mL inoculated into each embryo. The cells were incubated at 37°C. The chicken embryos that died before 48 hours were discarded, and the chicken embryos that died within 48 to 120 hours were taken out at any time, and the chicken embryo fluid was harvested. The cells were observed for 120 hours, and the HA titer of both dead and live embryos should be measured. The HA titer of embryos not less than 1:128 was considered infected, and the EID was calculated according to the Reed-Muench method. 50 Virus content per 0.1mL ≥108.3 EID 50 It can be used to make seedlings.

[0073] (3) Sterility test: Take the inactivated concentrated chicken embryo virus liquid and conduct a sterility test according to the appendix of the current "Chinese Pharmacopoeia of Veterinary Medicine". It should be sterile.

[0074] (4) Inactivation test: Take 5 10-day-old SPF chicken embryos, inoculate the inactivated virus solution into the allantoic cavity, inoculate 0.2 mL per embryo, and continue to incubate at 37°C. Photograph the embryos twice a day and observe for 120 hours. The non-specific death of chicken embryos should not exceed 1. Measure the HA titer of all chicken embryo fluids, which should be negative, and blindly propagate for one generation. Observe for 120 hours and measure the HA titer. If there is still no hemagglutination, it is considered to be completely inactivated.

[0075] S2, avian influenza virus (H9 subtype) antigen liquid 1. Preparation of avian influenza virus (H9 subtype) fluid The avian influenza virus (H9 subtype) JY strain was isolated, identified, stored and supplied by Shandong Ludu Biotechnology Co., Ltd.

[0076] (1) Inoculation: Take the production strain and dilute it with sterile saline for 10 3 Times, inoculate 10-day-old susceptible chicken embryos into the allantoic cavity, inoculate each embryo with 0.1 mL, seal the pinhole after inoculation, and continue incubation at 37°C.

[0077] (2) Incubation and observation: After the chicken embryos are inoculated, they are irradiated once every 24 hours, and chicken embryos that die within 48 hours are discarded. Thereafter, they are irradiated once every 24 hours, and dead embryos are removed at any time. After 96 hours, all embryos, dead or not, are removed, with the air chamber facing upward, and placed at 4°C for 12 hours.

[0078] (3) Harvest: Take out the cooled chicken embryos and harvest the chicken embryo fluid (collect live embryos first, then dead embryos). Place the harvested chicken embryo fluid in a sterilized container and sample to determine the HA titer. HA titers below 1:256 should be discarded. Perform sterility testing in accordance with the current "Chinese Veterinary Pharmacopoeia" and should be sterile. Store the harvested chicken embryo fluid below -15°C before inactivation.

[0079] (4) Concentration: After the harvested chicken embryo virus liquid is centrifuged at 2-8°C to remove the residue and other particulate impurities in the chicken embryo liquid, it is concentrated by ultrafiltration concentrator until the HA titer is not less than 1:2048, and the concentration is stopped. At the same time, sterility test is carried out in accordance with the current "Chinese Veterinary Pharmacopoeia", and it should grow aseptically. A sample is retained for the detection of the toxicity, and the concentrated chicken embryo virus liquid is immediately inactivated.

[0080] (5) Inactivation: Place concentrated chicken embryo virus solution of avian influenza virus (H9 subtype) into an inactivation tank, add 10% formaldehyde solution, start the mixer to mix thoroughly, and the final concentration of formaldehyde is 0.1%. After adding the formaldehyde solution, place it into another inactivation tank to prevent the virus adhering to the tank mouth from contacting the inactivator. Inactivate at 37℃ for 16 hours (start timing when the temperature in the tank reaches 37℃, start the mixer to stir continuously), then take it out and store it at 4℃.

[0081] 2. Avian influenza virus (H9 subtype) antigen liquid test (1) HA titer: Take the concentrated chicken embryo virus liquid and measure it according to the appendix of the current "Chinese Pharmacopoeia of Veterinary Medicine". The HA titer must be no less than 1:1024 before it can be used for vaccine production.

[0082] (2) Virus content: The concentrated chicken embryo virus solution before inactivation was diluted 10 times in series and 10 -6 , 10 -7 and 10 -8 3 dilutions, 5 10-day-old SPF chicken embryos were inoculated into each allantoic cavity, 0.1 mL was inoculated into each embryo, and the cells were incubated at 37°C. The chicken embryos that died before 24 hours were discarded and the embryos were observed twice a day. The chicken embryos that died within 24-120 hours were taken out at any time, and the chicken embryo fluid was harvested and observed for 120 hours. The HA titer of all dead and live embryos should be measured. The HA titer of not less than 1:16 was considered infected, and the EID was calculated according to the Reed-Muench method. 50 , virus content per 0.1mL ≥107.7 EID 50 It can be used to make seedlings.

[0083] (3) Sterility test: Take the inactivated concentrated chicken embryo virus liquid and conduct a sterility test according to the appendix of the current "Chinese Pharmacopoeia of Veterinary Medicine". It should be sterile.

[0084] (4) Inactivation test: Take 5 10-day-old SPF chicken embryos, inoculate the inactivated virus solution into the allantoic cavity, inoculate 0.2 mL per embryo, and continue to incubate at 37°C. Photograph the embryos twice a day and observe for 120 hours. The non-specific death of chicken embryos should not exceed 1. Measure the HA titer of all chicken embryo fluids, which should be negative, and blindly propagate for one generation. Observe for 120 hours and measure the HA titer. If there is no hemagglutination, it is considered to be completely inactivated.

[0085] S3. Preparation of Nano-propolis Composite Adjuvant Vaccine for Poultry The Newcastle disease virus antigen solution prepared in step S1 and the avian influenza (H9 subtype) antigen solution prepared in step S2 were mixed in a volume ratio of 1:1 to form an antigen phase.

[0086] The antigen phase and the poultry nano-propolis composite adjuvant prepared in Example 1 were stirred and mixed evenly in a volume ratio of 3:1 to obtain a Newcastle disease-avian influenza (H9 subtype) bivalent inactivated vaccine, namely, poultry nano-propolis composite adjuvant vaccine, with 250 mL in each bottle.

[0087] Example 5: A method for preparing a nano-propolis composite adjuvant vaccine for poultry, comprising the following preparation steps: The antigen phase prepared in step S3 of Example 4 and the poultry nano-propolis composite adjuvant prepared in Example 2 were stirred and mixed evenly in a volume ratio of 3:1 to obtain a Newcastle disease-avian influenza (H9 subtype) bivalent inactivated vaccine, i.e., poultry nano-propolis composite adjuvant vaccine, with 250 mL in each bottle.

[0088] Example 6: A method for preparing a nano-propolis composite adjuvant vaccine for poultry, comprising the following preparation steps: The antigen phase prepared in step S3 of Example 4 and the poultry nano-propolis composite adjuvant prepared in Example 3 were stirred and mixed evenly in a volume ratio of 3:1 to obtain a Newcastle disease-avian influenza (H9 subtype) bivalent inactivated vaccine, i.e., poultry nano-propolis composite adjuvant vaccine, with 250 mL in each bottle.

[0089] Comparative Example 1: A method for preparing a nano-propolis composite adjuvant vaccine for poultry, comprising the following preparation steps: The antigen phase prepared in step S3 of Example 4, physiological saline and the propolis ethanol extract prepared in step S1 of Example 1 were uniformly mixed in a volume ratio of 75:20:5 to obtain a Newcastle disease-avian influenza (H9 subtype) bivalent inactivated vaccine, i.e., a nano-propolis composite adjuvant vaccine for poultry, with 250 mL in each bottle.

[0090] Comparative Example 2: A method for preparing a nano-propolis composite adjuvant vaccine for poultry, comprising the following preparation steps: S1. Preparation of nano-propolis composite adjuvant for poultry: 30 mL of white oil, 20 mL of Span-80 and 15 mL of Tween-80 were mixed evenly, and then 20 mL of the propolis ethanol extract prepared in step S1 of Example 1 was added and mixed evenly, and then 0.1 g of Quillaja saponin A was added, and finally the volume was made up to 100 mL with water for injection, stirred at 800 rpm for 30 min, fully mixed and emulsified, and filtered and sterilized with a 0.22 μm filter to obtain the nano-propolis composite adjuvant for poultry.

[0091] S2. Preparation of nano-propolis composite adjuvant vaccine for poultry: The antigen phase prepared in step S3 of Example 4 and the nano-propolis composite adjuvant for poultry prepared in step S1 of this comparative example are stirred and mixed evenly in a volume ratio of 3:1 to obtain a Newcastle disease-avian influenza (H9 subtype) bivalent inactivated vaccine, i.e., nano-propolis composite adjuvant vaccine for poultry, with 250 mL in each bottle.

[0092] Comparative Example 3: A method for preparing a nano-propolis composite adjuvant vaccine for poultry, comprising the following preparation steps: S1. Preparation of nano-propolis composite adjuvant for poultry: 30 mL of vitamin D, 20 mL of Span-80 and 15 mL of Tween-80 were mixed evenly, and then 20 mL of the propolis ethanol extract prepared in step S1 of Example 1 was added and mixed evenly, and then 0.1 g of Quillaja saponin QS-21 was added, and finally the volume was made up to 100 mL with water for injection, stirred at 800 rpm for 30 min, fully mixed and emulsified, and filtered and sterilized with a 0.22 μm filter to obtain a poultry nano-propolis composite adjuvant.

[0093] S2. Preparation of nano-propolis composite adjuvant vaccine for poultry: The antigen phase prepared in step S3 of Example 4 and the nano-propolis composite adjuvant for poultry prepared in step S1 of this comparative example are stirred and mixed evenly in a volume ratio of 3:1 to obtain a Newcastle disease-avian influenza (H9 subtype) bivalent inactivated vaccine, i.e., nano-propolis composite adjuvant vaccine for poultry, with 250 mL in each bottle.

[0094] The present invention tests the properties and other indicators of the poultry nano-propolis composite adjuvant vaccine prepared in Examples 4 to 6 and Comparative Examples 1 to 3, and the results are as follows: The results of the property inspection and filling inspection are as follows: Figure 6 As shown, the appearance of the poultry nano-propolis composite adjuvant vaccine of Examples 4 to 6 is a light yellow, uniform, translucent suspension; while the appearance of the poultry nano-propolis composite adjuvant vaccine of Comparative Example 1 is light yellow, and precipitation occurs after being placed at 4°C for 2 weeks; the poultry nano-propolis composite adjuvant vaccines of Comparative Examples 2 and 3 are stratified after being placed at 4°C, which does not meet the property inspection standards of the composite vaccine, so the vaccines of Comparative Examples 2 and 3 are not further subjected to subsequent viscosity, formaldehyde residue, filling volume, safety and efficacy inspection tests.

[0095] (1) The viscosities of the poultry nano-propolis composite adjuvant vaccines prepared in Examples 4 to 6 and Comparative Example 1 were all less than 20 cP, the residual formaldehyde content was less than 0.05%, and the filling volume was between 251.3 mL and 252.0 mL.

[0096] (2) Sterility test: After testing, the poultry nano-propolis composite adjuvant vaccines prepared in Examples 4 to 6 and Comparative Example 1 all grew sterile.

[0097] (3) Safety test: 14-day-old SPF chickens were injected with 1 mL of the poultry nano-propolis composite adjuvant vaccine prepared in Examples 4 to 6 and Comparative Example 1 at the subcutaneous points of the neck to conduct a safety test of the vaccine, and a blank control group without treatment was set up. The chickens were observed for 14 days after the vaccine injection. During the observation period, the spirit, feeding and drinking of all the test chickens were normal. After 14 days, they were killed, and no abnormalities were found in all organs. There was no unabsorbed vaccine at the injection site, and there was no swelling or ulceration. The results showed that the poultry nano-propolis composite adjuvant vaccine prepared in Examples 4 to 6 and Comparative Example 1 showed good safety.

[0098] (4) Efficacy test: Use 21-day-old SPF chickens to test the efficacy of the poultry nano-propolis compound adjuvant vaccine.

[0099] After immunizing 10 SPF chickens with the poultry nano-propolis composite adjuvant vaccine prepared in Examples 4 to 6, the HI antibody titer of the Newcastle disease virus in chickens was in the range of 1:16 to 1:64, and the average values ​​were 1:29.9, 1:27.9 and 1:26, respectively, reaching the protection standard of the Newcastle disease vaccine; while after immunizing SPF chickens with the vaccine prepared in Comparative Example 1, the HI antibody titer of the Newcastle disease virus in chickens was in the range of 1:2 to 1:8, and the average value was 1:4, which did not reach the protection standard of the Newcastle disease vaccine. By statistical t-test analysis, there was no significant difference between Examples 4 to 6, and there was no significant difference between Example 4 and Example 5. p The value is 0.773, and the difference between Example 4 and Example 6 p The value is 0.535, and the difference between Example 5 and Example 6 p The value is 0.764. In addition, the p The values ​​were all less than 0.001, indicating that there was a significant difference between the immune effects of the nano-propolis composite adjuvant vaccine for poultry prepared in Examples 4 to 6 and the nano-propolis composite adjuvant vaccine for poultry prepared using propolis ethanol extract in Comparative Example 1. In addition, the HI antibody titer of the blank control group was ≤1:2 (Table 11).

[0100] Table 11 HI antibody titer of Newcastle disease virus in chickens after vaccination Note: The same letters in the table represent no significant difference between the groups, different letters represent significant difference between the groups, *** represents p <0.001.

[0101] After immunizing SPF chickens with the poultry nano-propolis composite adjuvant vaccine prepared in Examples 4 to 6, the HI antibody titer of avian influenza (H9) virus was in the range of 1:64 to 1:256, and the average values ​​were 1:104, 1:97 and 1:147, respectively, reaching the protection standard of avian influenza (H9) vaccine; while after immunizing SPF chickens with the vaccine prepared in Comparative Example 1, the HI antibody titer of avian influenza (H9) virus was in the range of 1:1 to 1:4, and the average value was 1:3, which did not reach the protection standard of avian influenza (H9) vaccine; by statistical analysis t test, there was no significant difference between Examples 4 to 6, and there was no significant difference between Example 4 and Example 5. p The value is 0.79, and the difference between Example 4 and Example 6 p The value is 0.105, and the difference between Example 5 and Example 6 p The value is 0.06. In addition, the p The values ​​were all less than 0.001, indicating that there was a significant difference between the immune effects of the poultry nano-propolis composite adjuvant vaccine prepared in Examples 4 to 6 and the poultry nano-propolis composite adjuvant vaccine prepared using propolis ethanol extract in Comparative Example 1. In addition, the HI antibody titer of the blank control group was ≤1:2 (Table 12).

[0102] Table 12 HI antibody titers of avian influenza (H9) after vaccination Note: The same letters in the table represent no significant difference between the groups, different letters represent significant difference between the groups, *** represents p <0.001.

[0103] Example 7: A method for preparing a nano-propolis composite adjuvant vaccine for poultry, comprising the following preparation steps: S1. Preparation of vaccine solution for chicken infectious coryza (type A) The bacteria used for production and testing are Haemophilus paragallinarum type A C-Hpg-8 strain, which is identified, stored and supplied by the China Veterinary Drug Administration.

[0104] Seed preparation for production: Take the strain and streak it on a chicken broth agar plate. After culturing at 37°C in an environment containing 5% CO2 for 16 to 18 hours, select typical colonies with strong fluorescence and inoculate them into the yolk sac of 5-day-old chicken embryos. Continue incubating at 37°C, collect the yolk fluid of chicken embryos that died within 30 hours as the primary seeds, and store them at -20°C.

[0105] Take the infected chicken embryo yolk fluid, streak it on a chicken broth agar plate, and culture it at 37°C for 16 hours in the presence of 5% CO2. Select typical colonies with strong fluorescence and inoculate them into chicken broth medium, and culture it at 37°C for 16 hours as secondary seeds.

[0106] Add the secondary seed liquid into a semi-synthetic culture medium (1000 mL of the semi-synthetic culture medium contains 5 g of polypeptone, 30 g of casein peptone, 15 g of sodium glutamate, 5 g of yeast extract powder, 3 g of glucose, 15 g of sodium chloride, and the remainder of distilled water. Adjust the pH to 7.2-7.4, and add 5% of inactivated healthy chicken serum and 0.8% of 0.5% coenzyme I before use). Culture at 37°C for 18 hours, shake the culture bottle twice, and confirm that there is no contamination from foreign bacteria after a pure test. Add 0.05% formaldehyde solution and store at 2°C.

[0107] Collect the bacteria by centrifugation or concentrate them with a hollow fiber ultrafilter, and then make a suspension with pH 7.2 phosphate buffer so that each 1 mL contains at least 5 billion bacteria (use the national standard for biological products "China Bacterial Turbidity Standard" for comparison, and assume that the standard tube contains 2.8 billion / mL of Haemophilus paragallinarum). After passing the purity test, inactivate it.

[0108] 0.15% formaldehyde solution and 0.01% thimerosal were added to the concentrated solution of vaccine culture solution according to the volume ratio, sterilized at 2°C for 7 days, and the sterile growth was tested and used as vaccine antigen.

[0109] Semi-finished product inspection: Sterility test: Take the inactivated concentrated vaccine culture liquid and conduct a sterility test in accordance with the appendix of the current "Chinese Pharmacopoeia of Veterinary Medicine", and find no sterile growth.

[0110] Test results of bacterial solution for seedling preparation: After 18 hours of culture, two batches of bacterial solution (batch 001 and culture batch 002) were randomly selected for testing. The harvest volume of culture solution of culture batch 001 and culture batch 002 was 2,700mL and 11,000mL, respectively, and the number of viable bacteria was 12×10 8 CFU / mL and 18×10 8 CFU / mL, and the total bacterial counts were 56×10 8 CFU / mL and 58×10 8 CFU / mL. The bacterial solution was centrifuged and then resuspended in PBS. The harvest volume of concentrated bacterial solution of culture batch 001 and culture batch 002 was 180mL and 730mL, respectively. The total bacterial count (turbidity bacterial count) was 840×10 8 CFU / mL and 870×10 8 CFU / mL, meeting the requirements for seedling production.

[0111] The inactivation test results of the concentrated bacterial solution used for seedling production: After the concentrated bacterial solution for seedling production was inactivated with formaldehyde solution, it was inoculated with semi-synthetic liquid culture medium and solid culture medium, and no sterile growth was observed after culture.

[0112] Results of sterility test of semi-finished bacterial solution for seedling production: According to the sterility test of semi-finished products in the appendix of the current "Chinese Pharmacopoeia of Veterinary Medicine", the concentrated bacterial solution for seedling production that was inactivated by formaldehyde solution was inoculated into fluid thioglycollate medium (Fluid Thioglycollate Medium, TG), casein agar medium (Peptone from casein Agar Medium, GA), and trypticase soy broth (Trypticase Soy Broth, TSB) for sterility test of the semi-finished products, and all showed sterile growth.

[0113] S2. Add the nano-propolis composite adjuvant prepared in Example 3 and the inactivated chicken infectious coryza antigen prepared in step S1 of this example into a mixing tank at a ratio of 1:3, turn on the stirring motor of the mixing tank and stir for 40 minutes until the mixture is uniform.

[0114] Comparative Example 4: A method for preparing a nano-propolis composite adjuvant vaccine for poultry, comprising the following preparation steps: The propolis ethanol extract prepared in step S1 of Example 1, the inactivated chicken infectious coryza antigen prepared in step S1 of Example 7 and physiological saline were added into a mixing tank in a volume ratio of 5:75:20, and the stirring motor of the mixing tank was turned on and stirred for 40 minutes until the mixture was uniformly mixed.

[0115] The present invention further tests the relevant traits of the poultry nano-propolis composite adjuvant vaccine prepared in Example 7 and Comparative Example 4, and the results are as follows: (1) Vaccine property inspection and packaging inspection The nano-propolis composite adjuvant vaccines for poultry prepared in Example 8 and Comparative Example 4 are both uniform, light yellow, translucent water-soluble suspensions; the viscosities are 30.2 cP and 36.3 cP, respectively; the formaldehyde content is below 0.04%, and the filling amount meets the standard requirements.

[0116] (2) Sterility test results The nano-propolis compound adjuvant vaccine for poultry was tested for sterility according to the method in the appendix of the current "Chinese Pharmacopoeia of Veterinary Medicine", and the results showed no sterile growth.

[0117] (3) Safety test results Eight 70-day-old SPF chickens were used, and each chicken was subcutaneously injected with 1.0 mL of avian nano-propolis compound adjuvant vaccine. The chickens were observed for 14 consecutive days, and no local or systemic adverse reactions caused by the avian nano-propolis compound adjuvant vaccine were found, and the results were judged to be qualified.

[0118] (4) Results of efficacy test Twenty SPF chickens of 70 days old were randomly divided into three groups, with 8 chickens in each group of test group 1 and test group 2, and 4 chickens in the blank control group. Each chicken in the test group 1 was subcutaneously injected with 0.5 mL of the nano-propolis composite adjuvant vaccine for poultry in Example 8, and each chicken in the test group 2 was subcutaneously injected with 0.5 mL of the nano-propolis composite adjuvant vaccine for poultry in Comparative Example 4, with no treatment as the blank control group; one month later, 0.2 mL of C-Hpg-8 bacterial solution (the number of viable bacteria was 5.0×10 5 CFU), and observed for 14 days. All 4 chickens in the blank control group were sick, showing swelling of the suborbital sinus and the surrounding area on one or both sides, and runny nose or tears; 1 chicken in the 8 chickens in the test group 2 was sick, and the other 7 chickens showed no clinical symptoms; while the 8 chickens in the test group 1 did not show any clinical symptoms, and all were protected; indicating that the immune effect of the nano-propolis composite adjuvant vaccine for poultry prepared in Example 8 is stronger than that of the nano-propolis composite adjuvant vaccine for poultry prepared in Comparative Example 4.

[0119] It should be noted that when the claims of the present invention involve a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiment and all changes and modifications that fall within the scope of the present invention.

[0120] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A nano-propolis composite adjuvant for poultry, characterized in that: The nano propolis composite adjuvant for poultry is composed of the following components in volume percentage: 10% to 30% of propolis ethanol extract, 30% to 45% of immunopotentiator, and 30% to 50% of surfactant; the propolis dry matter content in the propolis ethanol extract is 50 mg / mL to 70 mg / mL.

2. The nano-propolis composite adjuvant for poultry according to claim 1, characterized in that: The immunopotentiator is a mixture of immunopotentiator A and immunopotentiator B; the mixing volume ratio of the immunopotentiator A and immunopotentiator B is 5-8:1; the immunopotentiator A is white oil or vitamin D, and the immunopotentiator B is a mixture of Mycobacterium phlei extract and dimethyldioctadecyl ammonium bromide; or the immunopotentiator B is saponin.

3. The nano propolis composite adjuvant for poultry according to claim 2, characterized in that: The mixing volume ratio of the Mycobacterium phlei extract to dimethyl dioctadecyl ammonium bromide is 100:1-30.

4. The nano propolis composite adjuvant for poultry according to claim 1, characterized in that: The Mycobacterium phlei extract is obtained by lysing inactivated Mycobacterium phlei cells under 800-1000 bar conditions and centrifuging at 11000 rpm-13000 rpm for 25-35 min; the saponin is Quillaja saponin A or Quillaja saponin QS-21.

5. The nano propolis composite adjuvant for poultry according to claim 1, characterized in that: The surfactants are polyglycerol fatty acid ester, Span-80 and polyoxyethylene hydrogenated castor oil.

6. The nano-propolis composite adjuvant for poultry according to claim 5, characterized in that: The volume ratio of the polyglycerol fatty acid ester, Span-80 and polyoxyethylene hydrogenated castor oil is 7-9:3-5:

2.

7. Use of the poultry nano-propolis composite adjuvant according to claim 1 in preparing poultry inactivated vaccine, subunit vaccine or live vaccine diluent.

8. A nano-propolis composite adjuvant vaccine for poultry, characterized in that: The vaccine comprises the nano-propolis composite adjuvant for poultry according to claim 1 and inactivated antigens or subunit antigens of pathogenic microorganisms that infect poultry.

9. The vaccine according to claim 8, characterized in that The antigen is Newcastle disease virus, avian influenza virus or Haemophilus paragallinarum.

10. The vaccine according to claim 9, characterized in that The mixing volume ratio of the antigen and the poultry nano-propolis composite adjuvant is 2-4:1.

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