Serotype 4 avian adenovirus strain, its vaccine, vaccine composition and multi-unit vaccine and application

Abstract

The present invention relates to the field of biomedicine, and in particular to a serotype 4 avian adenovirus strain, a vaccine, a vaccine composition, a multi-unit vaccine and applications thereof. The serotype 4 avian adenovirus strain of the present invention has a deposit number of CGMCC No.45070. The Hexon gene sequence of the strain of the present invention is shown in SEQ ID NO:1. The present invention also relates to a vaccine, a vaccine composition and a multi-unit vaccine prepared using the strain. The strain of the present invention has high pathogenicity, with a lethality rate of 100% for 21-day-old SPF chickens, and the vaccine and vaccine composition prepared using the strain have good immune protection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a serotype 4 avian adenovirus strain, a vaccine, a vaccine composition and a multi-unit vaccine thereof and applications thereof. Background Art

[0002] Fowl Adenovirus (FAdV) belongs to the genus Adenovirus of the family Adenoviridae and is divided into 5 species (AE) and 12 serotypes. Virus isolation and identification found that the highly pathogenic serotype 4 FAdV is currently widely prevalent in domestic chickens.

[0003] Fowl adenovirus 4 (FAdV4) serotype causes hydropericardium-hepatitis syndrome (HHS) in chickens, which mainly infects chickens aged 2-4 weeks. Typical lesions include pericardial effusion, hepatic enlargement and bleeding, kidney swelling with urate deposition, spleen necrosis, etc. HHS can occur in broiler chickens, three-yellow chickens, white-feathered broilers and laying hens, with an acute onset, rapid spread and high mortality rate.

[0004] The use of effective vaccines is the key to controlling chicken pericardial effusion-hepatitis syndrome and an effective way to reduce its harm. The current vaccine against serotype 4 FAdV has the defect of weak protection. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a serotype 4 avian adenovirus strain, a vaccine, a vaccine composition and a multi-component vaccine and applications thereof.

[0006] The invention provides a serum type 4 avian adenovirus strain, the preservation number of which is CGMCC No.45070.

[0007] Optionally, the Hexon gene sequence of the strain is shown in SEQ ID NO:1.

[0008] The invention provides application of the serum type 4 avian adenovirus strain in a medicine or reagent for preventing poultry pericardial effusion-hepatitis syndrome; the poultry include chickens and ducks.

[0009] The invention provides application of the serum type 4 avian adenovirus strain in preparing an inactivated vaccine.

[0010] The present invention also provides a vaccine, which contains an immunizing amount of the above-mentioned serotype 4 avian adenovirus strain or an antigen of its culture; the antigen of the serotype 4 avian adenovirus strain or its culture is an inactivated antigen.

[0011] Optionally, the inactivated antigen is derived from chicken embryo;

[0012] Preferably, the titer of the inactivated antigen is greater than 10 5 EID50 / mL; more preferably 10 5 ~10 5.5 EID50 / mL.

[0013] The present invention also provides a vaccine composition, which contains an immunizing amount of the above-mentioned serotype 4 avian adenovirus strain or an antigen of its culture and a pharmaceutically acceptable carrier, wherein the antigen of the serotype 4 avian adenovirus strain or its culture is an inactivated antigen; preferably, the pharmaceutically acceptable carrier includes an adjuvant; more preferably, the adjuvant is a mixture of white oil, Siben-80 and aluminum stearate.

[0014] The invention also provides application of the vaccine or the vaccine composition in preparing medicine for preventing avian adenovirus infection.

[0015] Optionally, the application is the use of the vaccine or the vaccine composition in the preparation of a medicament for preventing pericardial effusion syndrome in avian species, wherein the avian species include chickens and ducks.

[0016] The present invention also provides a multi-combination vaccine, which contains the vaccine or the vaccine composition and further includes one or more of the following antigens: Newcastle disease virus antigen, avian influenza virus antigen, infectious bronchitis virus antigen, infectious bursal disease virus antigen, egg drop syndrome virus antigen, avian reovirus antigen, Escherichia coli antigen, Avibacterium paragallinarum antigen, Mycoplasma synoviae, Mycoplasma gallisepticum antigen, Pasteurella multocida antigen, Marek's virus antigen, avian encephalomyelitis virus antigen, or infectious laryngotracheitis virus antigen.

[0017] The technical solution provided by the embodiment of the present invention has the following advantages compared with the prior art:

[0018] The present invention obtains a highly pathogenic serotype 4 avian adenovirus strain by separation, and inoculates 9-day-old SPF chicken embryos with 10 4.399 When EID50 is reached, the mortality rate is 100%; for 21-day-old SPF chickens, the dose of the poison is 10 4.575 At EID50, the mortality rate reaches 100%.

[0019] The present invention studies its immunogenicity and obtains an inactivated vaccine with good immune effect and strong protection. After comparison, it is found that its protection is higher than that of existing vaccines, providing technical support for effectively preventing diseases caused by FAdV-4.

[0020] Instructions for strain preservation

[0021] The serum type 4 avian adenovirus strain of the present invention was deposited on January 12, 2022 at the General Microbiology Center of the China Culture Collection Administration Committee in the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.45070. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the Hexon gene PCR electrophoresis diagram of the strain of the embodiment of the present invention (M: DNA Marker, 1: SDLC202011, 2: positive control SDLC201601, 3: negative control)

[0023] Figure 2 The Hexon gene evolution tree of the strains of the present invention (● is the strain of the present invention)

[0024] Figure 3 The figure shows the survival of 21-day-old SPF chickens artificially infected with the strain of the embodiment of the present invention. DETAILED DESCRIPTION

[0025] In order to more clearly understand the above-mentioned objects, features and advantages of the present disclosure, the scheme of the embodiments of the present invention will be further described below.

[0026] The meanings of the terms used in the embodiments of the present invention are as follows:

[0027] The term "antigen" as used in the present invention includes inactivated antigens, live attenuated whole virus antigens, subunit antigens or synthetic peptide antigens.

[0028] The term "culture" used in the present invention refers to the subcultures of different generations of the virus. Those skilled in the art know that only slight variations may occur in the gene sequence between different generations.

[0029] The term "vaccine composition" used in the present invention refers to a pharmaceutical composition containing immunogenicity of serotype 4 avian adenovirus, which can induce, stimulate or enhance the immune response of chickens against serotype 4 avian adenovirus. The vaccine composition includes an immunizing amount of attenuated whole virus antigen, inactivated antigen, subunit antigen or synthetic peptide antigen of serotype 4 avian adenovirus strain.

[0030] The term "inactivated antigen" as used herein, also referred to as inactivated antigen, refers to a suspension of inactivated viruses used as antigens to produce immunity. Inactivated antigens can be easily produced using known methods. For example, whole virus inactivated antigens can be obtained by treating viruses with formaldehyde solutions.

[0031] As used herein, the term "combination vaccine" is used to refer to a vaccine prepared from a virus mixture of the serotype 4 avian adenovirus of the present invention and at least one different virus.

[0032] As used herein, the term "prevent" means that the symptoms of infection or disease associated with serotype 4 avian adenovirus are blocked or delayed.

[0033] The term "adjuvant" used in the present invention refers to a substance added to the composition of the present invention to increase the immunogenicity of the composition. Known adjuvants include, but are not limited to: (1) inorganic adjuvants, such as aluminum hydroxide, alum, aluminum phosphate, etc.; (2) oils, such as peanut oil emulsified adjuvant, mineral oil, vegetable oil, lanolin, etc.; (3) artificial synthetic adjuvants, double-stranded polyinosinic acid, cytidylic acid, double-stranded polyadenylic acid; (4) Freund's adjuvant, which is divided into Freund's incomplete adjuvant and complete Freund's adjuvant; (5) biological adjuvants are bacteria or their products, which themselves have immunogenicity, such as mycobacteria (tuberculosis, bacillus Calmette-Guérin), Corynebacterium parvum, Bordetella pertussis, Gram-negative bacilli endotoxin, etc.

[0034] As used herein, the term "prevent" means that the symptoms of infection or disease associated with serotype 4 avian adenovirus are blocked or delayed.

[0035] The present invention relates to a serotype 4 avian adenovirus strain, with the deposit number: CGMCC No. 45070, which was deposited on January 12, 2022 at the General Microbiology Center of the Chinese Academy of Sciences Institute of Microbiology, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain has high pathogenicity and can inoculate 9-day-old SPF chicken embryos with a dose of 10 4.399 When EID50 is reached, the mortality rate is 100%; for 21-day-old SPF chickens, the dose of the poison is 10 4.575 When EID50, the lethality rate reaches 100%. Specifically, the Hexon gene sequence of the strain is shown in SEQ ID NO: 1. The sequence was compared with other adenovirus reference sequences on NCBI to create a phylogenetic tree, confirming that the isolated strain is serotype 4 avian adenovirus.

[0036] The embodiments of the present invention relate to the use of the serotype 4 avian adenovirus strain in drugs or reagents for preventing or diagnosing poultry pericardial effusion-hepatitis syndrome; wherein poultry include chickens and ducks, mainly chickens, and specific types include broilers, three-yellow chickens, white-feather broilers and laying hens, etc.

[0037] The present invention relates to the use of the serotype 4 avian adenovirus strain in the preparation of an inactivated vaccine. The present invention studies the immune activity of the strain and finds that it is a highly virulent strain and is particularly suitable for the preparation of an inactivated vaccine.

[0038] The embodiment of the present invention relates to a vaccine, which contains an immunizing amount of the above-mentioned serotype 4 avian adenovirus strain or an antigen of its culture; specifically, the antigen of the serotype 4 avian adenovirus strain or its culture is an inactivated antigen. The embodiment of the present invention proposes an inactivated vaccine, and the research and development process of the inactivated virus vaccine is mainly to culture the virus using chicken embryos, and then kill the infectious virus by physical or chemical methods while maintaining the integrity of its antigen particles, so that it loses pathogenicity but retains antigenicity. It has the technical advantages of safe use, easy storage, and no pollution risk, and is easy to make into a combined vaccine or a multivalent vaccine.

[0039] As an implementation method of an embodiment of the present invention, the inactivated antigen used in the vaccine is from chicken embryos; that is, the strain is inoculated on chicken embryos to multiply the virus, and then the allantoic fluid is collected as the virus stock solution, and the inactivated antigen is obtained after inactivation. Other culture and amplification methods can also be used. Specifically, the inactivation method uses a 0.5v / v‰ to 1.5v / v‰ formaldehyde solution at 37°C for 12 hours to 36 hours, preferably a 1v / v‰ formaldehyde solution at 37°C for 24 hours.

[0040] As an implementation method of the present invention, the titer of the inactivated antigen is greater than 10 before inactivation. 5 EID50 / mL.

[0041] As an implementation method of the present invention, the titer of the inactivated antigen is 10 5 ~10 5.5 EID50 / mL.

[0042] As an implementation of an embodiment of the present invention, the vaccine preparation method comprises at least the following steps:

[0043] S1. Virus proliferation:

[0044] The virus liquid is obtained by inoculating and multiplying chicken embryos; the specific conditions are preferably: inoculate 9-day-old SPF chicken embryos with serum type 4 avian adenovirus strain, 200 μL per embryo. Incubate at 37°C, illuminate the embryos twice a day, and discard dead embryos within 24 hours; take out dead chicken embryos in time after 24 hours and store them at 1-4°C, take out all chicken embryos after 72 hours, store them at 1-4°C overnight, collect allantoic fluid, filter, and the filtrate is the virus liquid.

[0045] S2, inactivation:

[0046] The inactivation is carried out by formaldehyde inactivation; the specific conditions are preferably: adding the above virus liquid to a 1v / v‰ formaldehyde solution and placing it in a 37°C constant temperature shaking box for inactivation for 24 hours.

[0047] After sterility testing, the inactivated virus solution can be used as a vaccine, or other adjuvants can be further added to prepare a vaccine composition.

[0048] The embodiment of the present invention also relates to a vaccine composition, which contains the antigen of the above-mentioned strain or its culture and a pharmaceutically acceptable carrier; the antigen of the serotype 4 avian adenovirus strain or its culture is an inactivated antigen.

[0049] As an implementation of an embodiment of the present invention, the pharmaceutically acceptable carrier includes an adjuvant.

[0050] As an implementation method of the present invention, the adjuvant is a mixture of white oil, Siben-80 and aluminum stearate.

[0051] The vaccine composition of the present invention may further include other agents added to the composition of the present invention, including drugs, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants and preservatives.

[0052] Preferably, the immunostimulant includes α-interferon, β-interferon, γ-interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and interleukin 2 (IL2). To prepare such a composition, methods known in the art can be used.

[0053] As an implementation method of an embodiment of the present invention, the method for preparing the vaccine composition at least includes the step of emulsifying the inactivated virus liquid:

[0054] The inactivated virus solution and Tween-80 were mixed in a volume ratio of 96:4, shaken and mixed to prepare the water phase. White oil, Span-80, and aluminum stearate were mixed in a weight ratio of 47:3:1, heated and dissolved until transparent, and cooled to room temperature to prepare the oil phase. The prepared oil phase and water phase were mixed in a volume ratio of 5:2, stirred and mixed in a high-speed homogenizer, and emulsified to prepare a vaccine composition.

[0055] The embodiment of the present invention also relates to the use of the above vaccine or the above vaccine composition in the preparation of a drug for preventing avian adenovirus infection, and the application is the use of the vaccine or the vaccine composition in the preparation of a drug for preventing pericardial effusion syndrome.

[0056] The present invention also relates to a combined vaccine, comprising the above vaccine or the above vaccine composition, and one or more of the following antigens:

[0057] Newcastle disease virus antigen, avian influenza virus antigen, infectious bronchitis virus antigen, infectious bursal disease virus antigen, egg drop syndrome virus antigen, avian reovirus antigen, Escherichia coli antigen, Avibacterium paragallinarum antigen, Mycoplasma synoviae, Mycoplasma gallisepticum antigen, Pasteurella multocida antigen, Marek's disease virus antigen, avian encephalomyelitis virus antigen, or infectious laryngotracheitis virus antigen.

[0058] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0059] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0060] The strains used in the following examples were all provided by the Institute of Animal and Poultry Disease Prevention and Control Technology of Liaocheng University.

[0061] Example 1

[0062] This example is used to illustrate the separation and identification process of the virus strains in the examples of the present invention.

[0063] 1.1 Virus Isolation

[0064] Aseptically take the liver of a suspected HHS-sick chicken. Aseptically take the liver of a sick chicken and put it into a sterilized centrifuge tube, add an equal amount of PBS (containing 1wt% penicillin-streptomycin double antibody), grind it with a high-throughput tissue grinder, and centrifuge it at 4°C, 12000r / min for 10min. Take the supernatant and inoculate it into 9-day-old SPF chicken embryos, 200 μL / piece, incubate at 37°C, discard the dead embryos within 24 hours, and then illuminate the embryos once every 12 hours. Take out the dead chicken embryos in time and put them in a 4°C refrigerator overnight to collect the allantoic fluid. All the chicken embryos that have not died for 72 hours are taken out and frozen to death in a refrigerator, and the allantoic fluid is collected aseptically.

[0065] 1.2 Virus identification

[0066] The collected allantoic fluid was used to extract viral DNA, and the Hexon gene fragment of avian adenovirus was amplified by PCR using the primers shown in SEQ ID NO: 1 and SEQ ID NO: 2. The specific primer sequences are shown in Table 1:

[0067] Table 1

[0068] Serial number name Nucleotide sequence SEQ ID NO:2 H1 tggacatgggggcgaccta SEQ ID NO:3 H2 aagggattgacgttgtcca

[0069] The PCR reaction system was: 2 μL DNA template, 1 μL each of upstream and downstream primers (20 μM), 12.5 μL PCR Mix, and 8.5 μL ddH2O.

[0070] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s; annealing at 55°C for 30 s; extension at 72°C for 90 s; repeating 34 cycles starting from the second step; extension at 72°C for 10 min; and storage at 4°C.

[0071] The PCR products were electrophoresed on 1% agarose gel. Figure 1 shown.

[0072] The PCR product was sent to Shanghai Biotech Co., Ltd. for sequence determination. The sequencing result was shown in SEQ ID NO: 1. It was compared with other adenovirus reference sequences on NCBI to create an evolutionary tree. The result is shown in Figure 2 The results showed that the isolated strain was serotype 4 avian adenovirus.

[0073] The nucleotide sequence of SEQ ID NO:1 is:

[0074]

[0075] PCR or RT-PCR methods are used to detect Marek's disease virus, infectious laryngotracheitis virus, avian influenza virus, Newcastle disease virus, infectious bronchitis virus, egg drop syndrome virus, infectious bursal disease virus, etc. to exclude the possibility of virus strains being contaminated by other pathogens.

[0076] The isolated strain was deposited at the General Microbiology Center of China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on January 12, 2022, and was named serotype 4 avian adenovirus SDLC202011 strain, with the deposit number: CGMCC No.45070.

[0077] Example 2

[0078] This example is used to illustrate the pathogenicity of the virus strains of the examples of the present invention.

[0079] 1. Chicken embryo half infectious dose EID 50 Determination of

[0080] Take the SDLC202011 strain virus solution and dilute it to 10 with normal saline. -1 ~10 -7 Different dilutions were used, and 6 SPF chicken embryos were inoculated with 200 μL for each dilution. The embryos were inoculated with 200 μL and incubated at 37°C. A normal saline control group was also set up, and dead embryos within 24 hours were discarded. The embryos were photographed twice a day thereafter, and the dead chicken embryos were taken out in time and placed in a 4°C refrigerator overnight to collect the allantoic fluid. After incubation for 96 hours, all the chicken embryos were taken out, placed in a 4°C refrigerator overnight to collect the allantoic fluid.

[0081] The collected allantoic fluid was subjected to viral DNA extraction and PCR detection, and the detection method was the same as in Example 1. The virus titer was calculated by the Reed-Muench method based on the PCR results to be 10 5.098 EID 50 mL -1 .

[0082] 2. Pathogenicity test on SPF chicken embryos

[0083] Take 10 9-day-old SPF chicken embryos, and inoculate 200 μL of SDLC202011 virus solution into the allantoic cavity of each chicken embryo. Set up 5 controls, and inject 200 μL of saline into each embryo. Observe the embryos twice a day, discard dead embryos within 24 hours, and remove dead embryos in time for autopsy.

[0084] Crack open the eggshell of the dead chicken embryo, take out the embryo with tweezers, rinse off the blood stains on the embryo with water, put it in a glass culture dish for dissection, cut along the abdominal wall with small scissors, and observe whether there are any lesions in the liver, heart, kidneys and other organs of the embryo.

[0085] All the chicken embryos inoculated with the virus solution died on the 8th day, with a mortality rate of 100%. The main lesions found in the autopsy were: milky white fluid in the pericardium of some, hemorrhage spots in the liver, and yellow tinge in some livers.

[0086] 3. Pathogenicity test on 21-day-old SPF chickens

[0087] Twenty 21-day-old SPF chickens were randomly divided into two groups: the experimental group had 10 chickens, and the leg muscle was injected with 300 μL of virus solution per chicken (about 10 4.575 EID 50 ), and the control group of 10 chickens, which were injected with 300 μL of normal saline into the leg muscle. The chickens were isolated and raised in an isolator, with free access to food and water, and the spirit, morbidity and mortality of the chickens were observed daily.

[0088] 1-2 days after infection, the chickens had no abnormal eating and drinking. On the 3rd day, 9 chickens in the test group died. 1-2 hours before death, the chickens became depressed, stood still with their necks shrunk, and their feathers became messy. The death peak was reached. On the 4th day, the last chicken also died, with a mortality rate of 100%. There was no death in the control group. The survival of chickens after artificial infection is shown in Figure 3 .

[0089] The main lesions of the chickens that died of the virus infection were found in the timely autopsy: different degrees of effusion in the pericardium, severe effusion was dark yellow, and the pericardial effusion of some chickens was jelly-like; the liver was swollen and yellowish, with different degrees of bleeding spots on the surface of the liver; the kidneys were swollen and appeared like mottled kidneys due to urate deposition; the spleen was swollen; and some lungs had bleeding spots. No obvious lesions were found in the organs of the control group.

[0090] Example 3

[0091] This example is used to illustrate the preparation and detection of vaccines and vaccine compositions according to the present invention:

[0092] 1. Virus proliferation

[0093] Inoculate 9-day-old SPF chicken embryos with 200 μL of serotype 4 avian adenovirus strain SDLC202011, and inoculate a total of 45 eggs. Incubate at 37°C, illuminate the embryos twice a day, and discard dead embryos within 24 hours; remove dead chicken embryos in time and place them in a 4°C refrigerator. Take out all the chicken embryos after 72 hours, place them in a 4°C refrigerator overnight, collect the allantoic fluid, place it in a conical flask, and filter it with 8 layers of gauze. The filtrate is the virus fluid.

[0094] 2. Optimization of the best inactivation conditions

[0095] Take 3 portions of virus liquid, 100 mL each, add 0.5 v / v‰, 1 v / v‰, and 1.5 v / v‰ formaldehyde solution respectively, and place them in a 37°C constant temperature shaking box for inactivation. Inoculate chicken embryos with the virus liquid inactivated for 12 hours, 24 hours, and 36 hours in each group to detect whether the virus liquid is completely inactivated.

[0096] According to the test, the virus liquid can be inactivated by 1v / v‰ formaldehyde for 24 hours.

[0097] 3. Inactivation of the virus liquid of the strain of the present invention

[0098] The SDLC202011 strain virus liquid was added with 1v / v‰ formaldehyde solution and placed in a 37°C constant temperature shaking box for inactivation for 24 hours.

[0099] 3.4 Sterility test of inactivated virus solution

[0100] Take 1 mL of the inactivated virus solution and add it to 49 mL of thioglycollate medium for 72 hours, then transplant it to glucose peptone water medium and casein agar medium for 5 days to observe whether there is bacterial growth. If there is no bacterial growth, the next step of the test can be carried out.

[0101] 3.5 Emulsification of inactivated virus solution

[0102] The inactivated virus solution was mixed with Tween-80 at a volume ratio of 96:4, and shaken to mix well to prepare an aqueous phase.

[0103] White oil, Span-80 and aluminum stearate were mixed in a weight ratio of 47:3:1, heated and dissolved until transparent, and cooled to room temperature to prepare an oil phase.

[0104] The prepared oil phase and water phase were mixed in a volume ratio of 5:2, placed in a high-speed homogenizer for stirring and emulsification to prepare a vaccine.

[0105] 4. Inspection of inactivated vaccines

[0106] 4.1 Vaccine dosage form testing

[0107] Drop the vaccine on the surface of a plate filled with distilled water and observe the vaccine diffusion. If the oil drop does not spread, it is an oil-in-water type (W / O), and if it spreads in a cloud-like manner, it is an oil-in-water type (O / W).

[0108] The inactivated vaccine of the embodiment of the present invention is a white milky liquid, which does not diffuse when dropped into water, and is a water-in-oil vaccine.

[0109] 4.2 Vaccine stability testing

[0110] Take 10 mL of vaccine, centrifuge at 3000 rpm for 15 min, and observe whether there is stratification or antigen precipitation.

[0111] No stratification or oil-water separation occurred after centrifugation.

[0112] 4.3 Determination of vaccine viscosity

[0113] Use a 1mL glass pipette with an upper inner diameter of 2.7mm and a lower inner diameter of 1.2mm to draw up 1mL of the finished vaccine at about 25°C, and record the time required to vertically release 0.4mL.

[0114] The time required for the vaccine to naturally drip 0.4 mL vertically from a 1 mL pipette is 2 seconds, which is within the specified range of vaccine viscosity.

[0115] 4.4 Sterility testing of vaccines

[0116] The inactivated vaccine was inoculated into nutrient broth, ordinary agar, blood agar and other culture media, cultured at 37°C for 24 hours, and observed for bacterial growth.

[0117] No bacteria grew in any culture medium.

[0118] 4.5 Vaccine safety testing

[0119] The prepared inactivated vaccine was injected subcutaneously into the neck of five 21-day-old SPF chickens, 1 mL per chicken, and the chickens were observed for local or systemic adverse reactions. After two weeks of observation, an autopsy was performed.

[0120] The vaccinated chickens showed no adverse reactions, the vaccine was well absorbed, and no muscle damage was found at the vaccination site.

[0121] Example 4

[0122] This example is used to illustrate the protective effect of the vaccine in Example 3 on chickens:

[0123] 1. Experimental animals and experimental protocols

[0124] 60 SPF chickens were randomly divided into 6 groups: 3 groups were experimental groups, each of which was inoculated with 500 μL of the inactivated vaccine of Example 3 at two points under the skin of the neck; the other 3 groups were control groups, each of which was injected with 500 μL of physiological saline at two points under the skin of the neck;

[0125] The mice in each group were kept in isolation and had free access to food and water.

[0126] Three weeks later, the experimental group 1 and the control group 1 were intraperitoneally injected with 300 μL of SDLC202011 virus solution per animal (approximately 10 4.575 EID 50 ),

[0127] The experimental group 2 and the control group 2 were intraperitoneally injected with 300 μL of SDLC201601 virus solution per animal (about 10 3.9EID 50 ),

[0128] The experimental group 3 and the control group 3 were intraperitoneally injected with 300 μL of HuB201701 virus solution per animal (about 10 3.13 EID 50 ) ; intraperitoneal injection of SDLC202011 virus solution 300μL / mouse (about 10 4.58 EID 50 ).

[0129] Observe and record the incidence and mortality of chickens every day for two weeks.

[0130] 2. The results of the vaccine's immune protection against SPF chickens are shown in Table 2:

[0131] Table 2

[0132]

[0133]

[0134] Among them, the SDLC201601 strain virus and the HuB201701 strain virus were recorded in the master's degree thesis "Development of inactivated vaccine and yolk antibody of avian adenovirus type 4 group I", author Yin Guozheng, Liaocheng University, Master's electronic journal publication information: Year and issue: 2020 No. 01. Preserved in the Institute of Animal and Poultry Disease Prevention and Control Technology of Liaocheng University.

[0135] According to the experimental records in Table 2, according to the experimental data of the control group, the virulence of the strain of the present invention is higher than that of other strains. According to the experimental data of the experimental group, the vaccine prepared by the present invention has a good protective effect against multiple strains. The immune protection of the inactivated vaccine provided by the present invention against 3 different strains reaches 100%, and has the potential for commercial vaccine development.

[0136] Referring to the method of this embodiment (the difference is that other commercially available vaccines are inoculated during the immunization process, and the inoculation amount refers to the recommended amount in the instructions), the protective effect of the vaccine of the embodiment of the present invention on chickens is compared with that of the existing commercially available vaccines. The protective effect of the vaccine of the embodiment of the present invention is higher than that of the existing commercially available vaccines.

[0137] The above description is only a specific embodiment of the present disclosure, so that those skilled in the art can understand or implement the present disclosure. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure will not be limited to the embodiments described herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein. Sequence Listing <110> Liaocheng University <120> Serotype 4 avian adenovirus strain, its vaccine, vaccine composition and multi-unit vaccine and application <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1248 <212> DNA <213> avian adenovirus <400> 1 cgccataccg gctcccagct gctgggaaca gtcgctacgt caacttccac atccaagtgc 60 cccaaaaatt cttcgccatc aaaaacctgc tgctgctctc cggctcgtac acctacgagt 120 gggtgctgcg caaagacccc aacatgatcc tccaatccag tctgggcaac gacctgcgcg 180 ccgacggcgc cagcatcatc tacaacgagg tgaacctcat ggccaacttc atgcccatgg 240 atcacaacac cagtaaccag ctcgagctga tgctgagaaa cgccaccaac gatcagacct 300 tcgtggacta cctggggagcc aaaaacgctc tatactcggt gcccgcgggc tccaccgccc 360 tcaccatcaa cattcccgct cgcacctggg aggggatgcg cgggtggtcc ttcactcgca 420 tcaaggcggc cgagacgcct cagctgggcg cccagtacga cgtcaacttc aagtactcgg 480 gcagcatcgc ctactcagat ggaggcttct acctctcgca caccttccgt aacatgagca 540 tcctcttcga cacgtccatc aactggccgg gcaacgaccg gttgctcacg cctaacatgt 600 tcgagatcaa gcgctcggtg gcgctcgaca ccgagggctt caccatgagc cagtgcgaca 660 tcaccaagga ctggtacctg atccagatgg ccacgaacta caacttcgtc tataacggct 720 atcgattctg gcccgatcgt cagtacttcc actacgactt cctgcgaaat ttcgacccca 780 tgacgcgcca gggacccaac ttcgcattgc ccggcctctt cgacctcgtg tcttacaccc 840 ctaccacgga caacagcgga cagcagccta atcaggaagc cgtgcgcaac aactctgggt 900 ttatcgcccc ccgctcctgg cccgtctgga gcgctcacca gggcgagagc tggcccgcca 960 actggccgta cccgctctgc ggtcagcagg ccatccaacc cgcacaggtc ctcagctaca 1020 agaagttcct ctgcgacaac tacctgtgga ccatcccgtt cagttccgac tttatgtaca 1080 tgggcgaact gacagatctg ggccagaacc ccatgtacac caacaactcg cacagcatgg 1140 tcatcaactt cgagctcgac cccatggatg atcccactta cgtgtacatg ctctatggcg 1200 tgttcgacac cgttagggtc aaccagcccg aacgtaacgt gcagcata 1248 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <400> 2 tggacatggg ggcgaccta 19 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <400> 3 aagggattga cgttgtcca 19

Claims

1. A serotype 4 avian adenovirus strain, with the accession number being: CGMCC No.45070; The Hexon gene sequence of the strain is shown in SEQ ID NO:

1.

2. Use of the serotype 4 avian adenovirus strain as claimed in claim 1 in the preparation of a drug or reagent for preventing poultry pericardial effusion-hepatitis syndrome; the poultry include chickens and ducks.

3. Use of the serotype 4 avian adenovirus strain as claimed in claim 1 in the preparation of inactivated vaccines.

4. A vaccine, comprising an immunizing amount of an antigen of the serotype 4 avian adenovirus strain or its culture as claimed in claim 1; the antigen of the serotype 4 avian adenovirus strain or its culture is an inactivated antigen.

5. The vaccine according to claim 4, characterized in that The inactivated antigen is derived from chicken embryo.

6. The vaccine according to claim 5, characterized in that The titer of the inactivated antigen is greater than 10 before inactivation. 5 EID50 / mL.

7. The vaccine according to claim 6, characterized in that The titer of the inactivated antigen is 10 before inactivation. 5 ~10 5.5 EID50 / mL.

8. A vaccine composition, comprising an immunizing amount of an antigen of the serotype 4 avian adenovirus strain or its culture as claimed in claim 1 and a pharmaceutically acceptable carrier, wherein the antigen of the serotype 4 avian adenovirus strain or its culture is an inactivated antigen.

9. The vaccine composition according to claim 8, characterized in that The pharmaceutically acceptable carrier includes an adjuvant.

10. The vaccine composition according to claim 9, characterized in that The adjuvant is a mixture of white oil, Siben-80 and aluminum stearate.

11. Use of the vaccine according to any one of claims 4 to 7, or the vaccine composition according to any one of claims 8 to 10, in the preparation of a medicament for preventing avian adenovirus infection.

12. The use according to claim 11, characterized in that: The application is the application of the vaccine or the vaccine composition in the preparation of a drug for preventing pericardial effusion syndrome in birds, and the birds include chickens and ducks.

13. A combined vaccine, characterized in that: A vaccine according to any one of claims 4 to 7, or a vaccine composition according to any one of claims 8 to 10, further comprising one or more of the following antigens: Newcastle disease virus antigen, avian influenza virus antigen, infectious bronchitis virus antigen, infectious bursal disease virus antigen, egg drop syndrome virus antigen, avian reovirus antigen, Escherichia coli antigen, Avibacterium paragallinarum antigen, Mycoplasma synoviae, Mycoplasma gallisepticum antigen, Pasteurella multocida antigen, Marek's disease virus antigen, avian encephalomyelitis virus antigen, or infectious laryngotracheitis virus antigen.

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