New tributary glandular slide quintuplet inactivated vaccine and preparation method thereof

Abstract

The invention relates to the field of poultry vaccine preparation, in particular to a new tributary glandular smooth quintuplet inactivated vaccine and a preparation method thereof.The vaccine is prepared from, by mass, 2-3 parts of a water phase and 3-5 parts of an oil phase; wherein the water phase is prepared from the following components in parts by mass: 10 to 25 parts of adjuvant; 10-20 parts of a first antigen containing inactivated avian influenza virus; 10-20 parts of a second antigen containing inactivated infectious bronchitis virus; 10-20 parts of a third antigen comprising an inactivated Newcastle disease virus; 10 to 20 parts of a fourth antigen containing inactivated chicken mycoplasma synoviae; the composition comprises 10-20 parts of a fifth antigen of inactivated fowl adenovirus, and the total parts are 100. The vaccine prepared by the invention does not generate any adverse reaction to chickens, is high in experimental safety, and can induce high-level neutralizing antibodies in immunized chickens, which indicates that the immunized chickens can effectively resist attack of Newcastle disease virus, avian influenza, infectious bronchitis virus, adenovirus and mycoplasma synoviae.

Description

Technical Field

[0001] The present invention relates to the field of poultry vaccine preparation, and particularly relates to a new combined inactivated vaccine against avian influenza, Newcastle disease, infectious bronchitis, infectious laryngotracheitis and egg drop syndrome and a preparation method thereof. Background Art

[0002] As an extension of the agricultural industry, chicken farming not only occupies a very important position in China's traditional breeding industry, but also has been in a dominant position in the production of livestock and poultry. The healthy development of the chicken farming industry is one of the important ways to increase farmers' income and also an important source to ensure residents' meat consumption. Developing the chicken farming industry is an important way to promote the increase of farmers' income and the transfer of rural surplus labor in China. The stable supply of chicken products has become an important link in solving farmers' income increase and national economy and people's livelihood. However, highly prevalent and extremely harmful chicken infectious diseases are serious stumbling blocks to the healthy development of the chicken farming industry, such as avian influenza, Newcastle disease, and infectious bronchitis, etc.

[0003] Avian influenza was first discovered in Italy in 1878 and was later confirmed to be caused by type A influenza virus. Since then, avian influenza has not only brought huge economic losses to the livestock industry, but also seriously threatened human health and life. At present, the situation of human infection with H5N1 subtype avian influenza virus has occurred in many countries. The World Health Organization has confirmed that the avian influenza virus can spread from person to person under certain conditions. Therefore, developing new, highly efficient and safe avian influenza vaccines has become an important task for virologists. At present, it is mainly through vaccination to prevent the occurrence of this disease. Conventional inactivated vaccines have poor immune effects against this disease, while live attenuated vaccines have potential biosafety problems. Therefore, it is imperative to develop new, highly efficient, inexpensive and safe vaccines. With the rapid development of molecular biology technology, scholars have been continuously conducting research on new genetic engineering influenza vaccines.

[0004] Infectious bronchitis (IB) is an acute, highly contagious and economically significant viral disease of chickens caused by the infectious bronchitis virus (IBV) of the Coronaviridae family. It often causes respiratory symptoms in growing chickens, and some strains of infection can also lead to damage to the kidneys and reproductive system, resulting in a decrease in egg production or even death. Infected chickens suffer from growth retardation, increased feed consumption, decreased egg production and egg quality, and increased mortality, causing huge economic losses to the chicken industry. Its characteristics are dyspnea, rales, coughing, open-mouth breathing, and sneezing. There are no obvious breed differences in chickens infected with this disease. Chickens of all ages are susceptible, but the symptoms are more obvious in chickens within 5 weeks of age, and the mortality rate can reach 15-19%. The disease is more common from late autumn to late spring of the following year, but is most severe in winter. It is one of the main diseases endangering the chicken industry, and the main lesions are in the respiratory tract. In the nasal cavity, trachea, and bronchi, there are visible light yellow translucent serous and mucous exudates, which turn into caseous substances and form plugs after a slightly longer course of the disease. The air sacs may be turbid or contain caseous exudates. In laying hens, the ovarian follicles are congested, hemorrhagic or deformed; the oviduct is short, thick, hyperemic and necrotic locally. When chicks are infected with this disease, the damage to the oviduct is permanent and they generally cannot lay eggs when they grow up. In addition to the respiratory organ lesions in nephropathogenic bronchitis, the kidneys are enlarged and pale, the renal tubules are dilated due to urate deposition, the kidneys are mottled, and the ureters are thickened due to urate deposition. There is also a layer of white frost-like urate deposition on the surfaces of the heart and liver. Sometimes, inflammatory and hemorrhagic symptoms can be seen in the bursa of Fabricius.

[0005] Newcastle Disease (ND), also known as Asian fowl plague, pseudo-fowl plague, etc., is an acute, septicemic and highly contagious infectious disease that occurs in poultry such as chickens, pigeons, quails, turkeys and wild birds. It is one of the notifiable diseases stipulated by the World Organization for Animal Health (OIE). Its pathogen is the Newcastle Disease Virus (NDV). NDV belongs to the Paramyxoviridae family, Paramyxovirinae subfamily, and Rubulavirus genus. Newcastle Disease not only causes serious economic losses to the poultry industry, but also seriously affects the international trade of poultry and poultry-related products. Therefore, all countries in the world have taken very strict prevention and control measures against the occurrence and spread of this disease. Since the 1990s, the occurrence of Newcastle Disease in vaccinated chicken flocks in China has gradually become serious. The main characteristics are a significant decrease in egg production, low mortality, and high antibody titers. This atypical Newcastle Disease seriously harms the production performance of chickens and causes huge losses to the poultry industry.

[0006] Mycoplasma synoviae (MS), also known as infectious synovitis of chickens, is an acute or chronic infectious disease of chickens, turkeys, etc. caused by Mycoplasma synoviae. Infectious synovitis of chickens was first reported by Olson et al. (1964). This disease mainly affects the synovial fluid of joints and tendon sheaths, and is characterized by swelling of joints, tendon sheaths and foot pads, etc. Infection of chicken flocks with this disease can lead to obvious lameness, retarded growth and development, and downgrading of carcasses, etc. This disease can be vertically transmitted, resulting in the disease of chicks hatched from the eggs produced during the disease period of breeding chickens. The infection of Mycoplasma synoviae has spread throughout the world and is distributed worldwide. It mainly affects commercial broilers, laying hens and breeding chickens. This disease can occur throughout the year, but is more likely to occur in winter and spring. The infection of Mycoplasma synoviae is generally more common in chickens aged 4 to 16 weeks. The incidence rate is generally 5% - 15%, and the mortality rate is about 1% - 10%. Since the development of this disease is slow and the course of the disease is long, once it infects a chicken flock, it is very difficult to eradicate, so it spreads in the chicken flock for a long time, resulting in low feed utilization rate, retarded growth and development, increased culling rate, decreased egg production, etc. When this pathogen exists in a chicken flock, mixed infections are likely to occur, aggravating the condition and increasing the mortality rate, causing serious economic losses. Mycoplasma synoviae is of the S type in the serological classification of avian mycoplasmas. There is only 1 serotype of Mycoplasma synoviae. DNA-DNA hybridization tests have shown that there are almost no differences between different strains. At present, the main prevention of Mycoplasma synoviae infection in clinical practice is the appropriate administration of drugs, which plays a certain role in preventing this disease. However, for those that have already shown typical clinical symptoms, the effect of applying drugs is not obvious. Not only does it pose a risk of drug residues affecting food safety, but it also cannot eradicate Mycoplasma synoviae in chickens, and it is very easy to develop drug resistance, increasing the difficulty of controlling this disease. Since this disease can be vertically transmitted, once it occurs, the eggs of the diseased chickens should not be used. Vaccination is a more effective preventive measure for controlling Mycoplasma synoviae disease. Therefore, it is particularly important to screen Mycoplasma synoviae strains with strong virulence and good immunogenicity and prepare vaccines.

[0007] Through the epidemiological investigation of group I avian adenovirus, the incidence rate of this disease in chicken flocks in China is relatively high. It can be transmitted through both horizontal and vertical routes and shows an increasing trend year by year. The range of infected hosts is also getting wider and wider. White - feather broilers, meat - breeding chickens, laying hens and yellow - feather chickens can all be infected and diseased. Especially after 2010, the incidence has shown an increasing trend and it is prevalent throughout the country. Many group I avian adenoviruses can replicate in healthy poultry, and the symptoms are very mild or do not show infection symptoms. However, group I avian adenovirus type 4 is an exception and can directly cause chicken flocks to get sick. The main lesions are pericardial effusion and enlargement of the liver and kidneys. This disease first occurred in the United States in 1963, and then appeared successively in various parts of the world. It is a common infectious pathogen of poultry and wild birds all over the world.

[0008] At present, most of the vaccines used on the market are single vaccines, and there are few combined vaccines containing multiple antigens. However, this has relatively limited effects in disease prevention and control. Moreover, due to the abuse of antibiotics, drug-resistant strains have emerged, rendering drug treatment ineffective and wasteful; the residues of antibiotic drugs also pose problems for meat animals in passing export inspections. Summary of the Invention

[0009] Based on this, the present invention proposes a new combined inactivated vaccine against Newcastle disease, infectious bronchitis, avian influenza, and infectious synovitis of chickens and its preparation method, which can reduce the incidence of Newcastle disease, infectious bronchitis, avian influenza, and infectious synovitis of chickens and improve the survival efficiency of chicks and chicken eggs.

[0010] According to one aspect of the present invention, there is provided a new combined inactivated vaccine against Newcastle disease, infectious bronchitis, avian influenza, and infectious synovitis of chickens, which comprises the following components in parts by mass:

[0011] An aqueous phase, 2 - 3 parts, and an oil phase, 3 - 5 parts;

[0012] Among them, in the aqueous phase, the following components are included in parts by mass:

[0013] An adjuvant, 10 - 25 parts;

[0014] A first antigen containing inactivated avian influenza virus, 10 - 20 parts;

[0015] A second antigen containing inactivated infectious bronchitis virus, 10 - 20 parts;

[0016] A third antigen containing inactivated Newcastle disease virus, 10 - 20 parts;

[0017] A fourth antigen containing inactivated Mycoplasma synoviae of chickens, 10 - 20 parts;

[0018] A fifth antigen containing inactivated avian adenovirus, 10 - 20 parts, with a total of 100 parts.

[0019] Furthermore, the adjuvant comprises the following components in parts by mass:

[0020] Vitamin E, 5 - 15 parts;

[0021] Levamisole, 2 - 10 parts;

[0022] A divalent inorganic salt, 1 - 10 parts;

[0023] Tween - 80, 1 - 5 parts;

[0024] Albumin, 8 - 12 parts;

[0025] Potassium sorbate, 4 - 6 parts.

[0026] Furthermore, the oil phase comprises the following components in parts by mass:

[0027] 80 - 90 parts of high molecular weight polyacrylic acid, 1 - 5 parts of trehalose, 1 - 10 parts of sorbitol, 3 - 5 parts of aluminum stearate.

[0028] Furthermore, the divalent inorganic salt is selected from one or two of manganese gluconate, calcium gluconate, calcium aspartate, zinc gluconate, ferrous gluconate and calcium chloride.

[0029] According to one aspect of the present invention, there is provided a preparation method for preparing the above-mentioned vaccine, comprising the following steps:

[0030] Prepare the first antigen, the second antigen, the third antigen, the fourth antigen and the fifth antigen respectively;

[0031] Mix the first antigen, the second antigen, the third antigen, the fourth antigen and the fifth antigen with an adjuvant to prepare an aqueous phase;

[0032] Prepare an oil phase;

[0033] Take 3 - 5 parts of the oil phase and add it to an appropriate sterile container, start the shearing machine and stir at 10000 r / min for 10 minutes, then add 2 - 3 parts of the aqueous phase, and then stir at 15000 r / min for 5 minutes to complete the preparation.

[0034] Furthermore, the preparation method of the first antigen comprises the following steps:

[0035] After inoculating chicken embryos by allantoic cavity with avian influenza virus and subculturing, harvest the allantoic fluid of the inoculated chicken embryos that have not died after 72 hours of inoculation, inactivate it, and mix the allantoic fluid with a hemagglutination titer of not less than 1:512 of 1% chicken red blood cell suspension to obtain the first antigen.

[0036] Furthermore, the preparation method of the second antigen comprises the following steps:

[0037] After inoculating chicken embryos with the attenuated strain H120 of infectious bronchitis virus of chickens, harvest the embryo fluid to obtain a virus-expanded culture solution, and after freeze-drying and reconstitution, obtain the second antigen.

[0038] Furthermore, the preparation method of the third antigen comprises the following steps:

[0039] Redissolve the freeze-dried attenuated strain of Newcastle disease virus of chickens, continuously subculture and inoculate chicken embryos for 9 generations, inactivate it, and use a 30KD membrane package to ultrafiltrate and concentrate the virus by 5 times to obtain the third antigen.

[0040] Furthermore, the preparation method of the fourth antigen comprises the following steps:

[0041] The Mycoplasma synoviae was added to the Mycoplasma synoviae medium for chickens with a pH value of 7.6 - 7.8 at a ratio of 1:10, and cultured at a constant temperature of 37°C until the logarithmic growth phase, during which the bacterial solution changed from red to yellow, and then the bacterial solution was collected;

[0042] The bacterial solution was concentrated 50 - fold, and after concentration, 10% formaldehyde solution was added to make the final concentration of formaldehyde 0.2%, and it was fully mixed. Under the oscillation speed of 180 r / min, it was inactivated at a constant temperature of 37°C for 24 hours to obtain the fourth antigen.

[0043] Furthermore, the preparation of the fifth antigen containing inactivated avian adenovirus includes:

[0044] Extract the genomic DNA of avian adenovirus. Using this as a template, specific primers targeting the Penton gene were used for PCR amplification, and the PCR product was recovered to obtain the Penton target fragment;

[0045] The Penton target fragment was ligated with pMD18 - T to construct a recombinant cloning plasmid, and the recombinant cloning plasmid was transformed into competent Escherichia coli DH5α cells to screen the recombinant plasmid pMD18 - T / Penton of positive clones.

[0046] The recombinant plasmid pMD18 - T / Penton and the pColdⅠ expression vector were respectively double - digested, the digested Penton and pColdⅠ were ligated, and the ligation product was transformed into competent Escherichia coli DH5α cells to screen the recombinant plasmid pCold I / Penton of positive clones.

[0047] The recombinant plasmid pCold I / Penton was transformed into competent cells BL21, coated on LB solid medium containing ampicillin, cultured overnight at 37°C, picked into LB liquid medium containing ampicillin, cultured overnight at 37°C, then transferred to LB liquid medium at a ratio of 1:100 for continued culture. When the OD600 reached about 0.6, IPTG was added, induced at 25°C for 4 h, and then the bacterial solution was collected. After centrifugation, the supernatant was discarded, resuspended according to the ratio of 1 g bacterial pellet:9 ml PBS, the resuspended bacteria were ultrasonically disrupted, and after disruption, the supernatant was taken by centrifugation and purified to obtain the Penton recombinant protein as the fifth antigen.

[0048] As can be seen from the above technical solutions, the new - type Newcastle - bronchitis - avian influenza - adenovirus - Mycoplasma synoviae pentavalent inactivated vaccine and its preparation method provided by the present invention have the following beneficial effects:

[0049] The present invention provides an inactivated vaccine against Newcastle disease virus, avian influenza, infectious bronchitis virus, adenovirus, and Mycoplasma synoviae in chickens. The research and preparation of this vaccine solve the defect that the disease cannot be effectively prevented in China at present, and can effectively solve the problems of high incidence of Newcastle disease virus, avian influenza, infectious bronchitis virus, adenovirus, and Mycoplasma synoviae in chickens and the resulting sudden drop in egg production. The process of preparing the inactivated vaccine with Newcastle disease virus is simple and convenient to operate. The prepared vaccine has good antigenicity and high safety, has no adverse effects on the external environment, and is easy to pass the safety evaluation.

[0050] The vaccine prepared by the present invention will not cause any adverse reactions to chickens, has high experimental safety, and can induce a high level of neutralizing antibodies in the immunized chickens, indicating that the immunized chickens can effectively resist the attack of Newcastle disease virus, avian influenza, infectious bronchitis virus, adenovirus, and Mycoplasma synoviae.

[0051] The vaccine prepared by the present invention has good safety and no local or systemic adverse reactions caused by the vaccine. Through the analysis of the data of character, safety test, and potency test in the shelf-life test, the results show that there is no significant difference between the five-in-one vaccine and the single vaccines of the same type, and both are stable and effective.

[0052] The advantages of the present invention are that it can prevent diseases caused by Newcastle disease virus, avian influenza, infectious bronchitis virus, adenovirus, and Mycoplasma synoviae at the same time, can induce a synergistic immune effect through the combination of multiple immunogens, produce a specific immune effect, reduce the injection frequency, change the situation that at least two injections are required to prevent the above diseases, reduce the immunization cost, reduce the stress response of animals, and the five-in-one inactivated vaccine has a high titer content, is convenient and fast for immunization, avoids the adverse reactions of multiple vaccinations, and reduces the breeding cost. Compared with the current vaccines, it can immunize more serotypes with a single injection, achieving the goal of "preventing multiple diseases with one injection", is easy to use, reduces the stress of poultry, and there is no hidden danger of spreading the virus, and is safe and reliable.

[0053] By using a variety of adjuvants, the organic combination and coordination of albumin, potassium sorbate, vitamin E, and levamisole, and the compounding of high molecular weight polyacrylic acid, trehalose, sorbitol, and aluminum stearate, the maximum advantage of the adjuvant can be exerted, forming the best formula, that is, forming an immune stimulating complex, better exerting the immune adjuvant effect, having a good immune effect when combined with the antigen, low toxicity, and low cost. It can reduce the side effects of redness, swelling, and fever at the immunization site after immunization, does not affect the spirit and feeding of poultry after immunization, and effectively improves the immune effect and production performance of immunized poultry. Detailed implementation mode

[0054] To make the objectives, technical solutions and advantages of the present invention more clear and understandable, the following provides a further detailed description of the present invention in conjunction with specific embodiments.

[0055] Currently, most of the Newcastle disease virus, avian influenza, infectious bronchitis virus, adenovirus and Mycoplasma synoviae used in the market are single vaccines, and there are few combined vaccines containing these five antigens. However, the effect in terms of disease prevention is relatively limited. Moreover, due to the abuse of antibiotics, drug-resistant strains have emerged, rendering drug treatment ineffective and wasteful; the residues of antibiotic drugs also pose a problem for meat animals when exporting.

[0056] According to some existing problems, in one aspect of the present invention, a new combined inactivated vaccine against Newcastle disease virus, avian influenza, infectious bronchitis virus, adenovirus and Mycoplasma synoviae is provided, which comprises the following components in parts by mass:

[0057] An aqueous phase, 2 - 3 parts, and an oil phase, 3 - 5 parts;

[0058] Among them, in the aqueous phase, the following components are included in parts by mass:

[0059] An adjuvant, 10 - 25 parts;

[0060] A first antigen containing inactivated avian influenza virus, 10 - 20 parts;

[0061] A second antigen containing inactivated infectious bronchitis virus, 10 - 20 parts;

[0062] A third antigen containing inactivated Newcastle disease virus, 10 - 20 parts;

[0063] A fourth antigen containing inactivated Mycoplasma synoviae, 10 - 20 parts;

[0064] A fifth antigen containing inactivated avian adenovirus, 10 - 20 parts, with a total of 100 parts.

[0065] The ratio of the aqueous phase to the oil phase can be adjusted according to the actual situation to ensure that the aqueous phase can be wrapped by the oil phase and fully emulsified.

[0066] The present invention provides an inactivated vaccine against Newcastle disease virus, avian influenza, infectious bronchitis virus, adenovirus and Mycoplasma synoviae. The research and preparation of this vaccine solve the defect that the disease cannot be effectively prevented in China at present, and can effectively solve the high incidence of Newcastle disease virus, avian influenza, infectious bronchitis virus, adenovirus and Mycoplasma synoviae and the loss of sudden drop in egg production of chickens. Using Newcastle disease virus to prepare the inactivated vaccine has a simple process, convenient operation, good antigenicity and high safety of the prepared vaccine, has no adverse effects on the external environment, and is easy to pass the safety evaluation.

[0067] The vaccine prepared by the present invention will not cause any adverse reactions to chickens, has high experimental safety, and can induce a high level of neutralizing antibodies in the immunized chickens, indicating that the immunized chickens can effectively resist the attacks of Newcastle disease virus, avian influenza, infectious bronchitis virus, adenovirus, and Mycoplasma synoviae.

[0068] The vaccine prepared by the present invention has good safety and no local or systemic adverse reactions caused by the vaccine. Through the analysis of the data of character, safety test, and potency test in the shelf-life test, the results show that there is no obvious difference compared with the single vaccines of the same kind of products, and the pentavalent vaccine is stable and effective.

[0069] The advantages of the present invention are that it can prevent diseases caused by Newcastle disease virus, avian influenza, infectious bronchitis virus, adenovirus, and Mycoplasma synoviae at the same time. It can induce a synergistic immune effect through the combination of multiple immunogens, produce a specific immune effect, reduce the injection frequency, change the situation that at least two injections are required to prevent the above diseases, reduce the immunization cost, reduce the stress response of animals, and the inactivated pentavalent vaccine has a high titer content, is convenient and fast for immunization, avoids the adverse reactions of multiple vaccinations, and reduces the breeding cost. Compared with the current vaccines, it can immunize more serotypes with a single injection, achieve the purpose of "preventing multiple diseases with one injection", is easy to use, reduces the stress of poultry, and there is no hidden danger of spreading the virus, and it is safe and reliable.

[0070] Furthermore, the adjuvant includes the following components by mass parts:

[0071] Vitamin E, 5 - 15 parts;

[0072] Levamisole, 2 - 10 parts;

[0073] Divalent inorganic salt, 1 - 10 parts;

[0074] Tween - 80, 1 - 5 parts;

[0075] Albumin, 8 - 12 parts;

[0076] Potassium sorbate, 4 - 6 parts.

[0077] Furthermore, the oil phase includes the following components by mass parts:

[0078] High molecular weight polyacrylic acid 80 - 90 parts, trehalose 1 - 5 parts, sorbitol 1 - 10 parts, aluminum stearate 3 - 5 parts.

[0079] Furthermore, the divalent inorganic salt is selected from one or two of manganese gluconate, calcium gluconate, calcium aspartate, zinc gluconate, ferrous gluconate, and calcium chloride.

[0080] By adopting a variety of adjuvants, the organic combination and coordination of albumin, potassium sorbate, vitamin E, and levamisole, and the compounding of lanolin and polyglycerol fatty acid ester can give full play to the maximum advantages of the adjuvant, form the best formula, that is, form an immunostimulating complex, better exert the immunoadjuvant effect, have a good immunization effect when combined with the antigen, have low toxicity and low cost. It can reduce the side effects of redness, swelling, and fever at the immunization site after immunization, will not affect the spirit and feeding of poultry after immunization, and effectively improve the immunization effect and production performance of immunized poultry.

[0081] According to one aspect of the present invention, there is provided a preparation method for preparing the above-mentioned vaccine, including the following steps:

[0082] Respectively prepare the first antigen, the second antigen, the third antigen, the fourth antigen, and the fifth antigen;

[0083] Mix the first antigen, the second antigen, the third antigen, the fourth antigen, and the fifth antigen with the adjuvant to prepare an aqueous phase;

[0084] Prepare an oil phase;

[0085] Take 3-5 parts of the oil phase and add it to an appropriate sterile container, start the shearing machine and stir at 10000 r / min for 10 minutes, then add 2-3 parts of the aqueous phase, and then stir at 15000 r / min for 5 minutes to complete the preparation.

[0086] The present invention provides an inactivated vaccine against Newcastle disease virus, avian influenza, infectious bronchitis virus, adenovirus, and Mycoplasma synoviae in chickens. The research and preparation of this vaccine solve the defect that the disease cannot be effectively prevented in China at present, and can effectively solve the high incidence of Newcastle disease virus, avian influenza, infectious bronchitis virus, adenovirus, and Mycoplasma synoviae in chickens and the loss of sudden drop in egg production of chickens. Using Newcastle disease virus to prepare the inactivated vaccine has a simple process, convenient operation, good antigenicity and high safety of the prepared vaccine, has no adverse effects on the external environment, and is easy to pass the safety evaluation.

[0087] The vaccine prepared by the present invention will not cause any adverse reactions to chickens, has high experimental safety, and can induce a high level of neutralizing antibodies in the immunized chickens, indicating that the immunized chickens can effectively resist the attack of Newcastle disease virus, avian influenza, infectious bronchitis virus, adenovirus, and Mycoplasma synoviae.

[0088] The vaccine prepared by the present invention has good safety and no local or systemic adverse reactions caused by the vaccine. Through the analysis of the data of character, safety test, and potency test in the shelf-life test, the results show that there is no obvious difference compared with the single vaccine of the same kind of products, and the pentavalent vaccine is stable and effective.

[0089] The advantages of the present invention are that it can prevent diseases caused by Newcastle disease virus, avian influenza, infectious bronchitis virus, adenovirus, and Mycoplasma synoviae in chickens simultaneously. It can induce a synergistic immune effect through the combination of multiple immunogens, generate specific immune responses, reduce the injection frequency, change the situation where at least two injections are required to prevent the above diseases, reduce the immunization cost, lower the stress response of animals, and the pentavalent inactivated vaccine has a high titer content, is convenient and fast for immunization, avoids the adverse reactions of multiple vaccinations, and reduces the breeding cost. Compared with the current vaccines, it can immunize more serotypes with a single injection, achieving the goal of "preventing multiple diseases with one injection", is easy to use, reduces the stress of poultry, and there is no hidden danger of virus dispersion, being safe and reliable.

[0090] Further, the preparation method of the first antigen includes the following steps:

[0091] After inoculating chicken embryos by allantoic cavity with avian influenza virus and passing them on, harvest the allantoic fluid of the inoculated chicken embryos that have not died after 72 hours and inactivate it, and mix it with the allantoic fluid of chicken embryos with a hemagglutination titer of 1% chicken red blood cell suspension not less than 1:512 to obtain the first antigen.

[0092] Further, the preparation method of the second antigen includes the following steps:

[0093] After inoculating chicken embryos with the attenuated strain H120 of infectious bronchitis virus of chickens, harvest the embryo fluid, obtain the virus expanded culture solution, and lyophilize and reconstitute it to obtain the second antigen.

[0094] Further, the preparation method of the third antigen includes the following steps:

[0095] Reconstitute the freeze-dried attenuated strain of Newcastle disease virus of chickens, continuously passage and inoculate chicken embryos for 9 generations, inactivate it, and ultrafilter and concentrate the virus 5 times using a 30KD membrane package to obtain the third antigen.

[0096] Further, the preparation method of the fourth antigen includes the following steps:

[0097] Add Mycoplasma synoviae to the chicken Mycoplasma synoviae medium with a pH value of 7.6 - 7.8 at a ratio of 1:10, and culture it at a constant temperature of 37°C until the logarithmic growth phase, so that the bacterial liquid changes from red to yellow, and collect the bacterial liquid;

[0098] Concentrate the bacterial liquid 50 times, add 10% formaldehyde solution after concentration, make the final concentration of formaldehyde 0.2%, mix well, and inactivate it at a constant temperature of 37°C for 24 hours at an oscillation speed of 180 r / min to obtain the fourth antigen.

[0099] Further, the chicken Mycoplasma synoviae medium includes: phosphate buffer, glucose, hydrolyzed lactalbumin, coenzyme I, arginine hydrochloride, L-cysteine hydrochloride, MEM, yeast extract powder, 1% phenol red, and trehalose; the auxiliary component includes penicillin.

[0100] Further, the preparation of the fifth antigen comprising inactivated avian adenovirus includes:

[0101] Extract the genomic DNA of avian adenovirus (Group I, Type 4). Using this as a template, perform PCR amplification with specific primers targeting the Penton gene, and recover the PCR product to obtain the Penton target fragment.

[0102] Ligate the Penton gene with pMD18-T to construct a recombinant cloning plasmid. Transform the recombinant cloning plasmid into competent Escherichia coli DH5α cells, screen for positive clones and identify them by PCR. Send the positive clone bacterial solution that has been successfully identified by PCR for sequencing. Name the recombinant plasmid with correct sequencing as pMD18-T / Penton.

[0103] Perform double digestion on the recombinant plasmid pMD18-T / Penton and the pColdⅠ expression vector respectively. Ligate the digested Penton and pColdⅠ, transform the ligation product into competent Escherichia coli DH5α cells, screen for positive clones and identify them by PCR and double digestion. Send the positive clone bacterial solution that has been successfully identified for sequencing. Name the recombinant plasmid with correct sequencing as pCold I / Penton.

[0104] Transform the recombinant plasmid pCold I / Penton into competent cells BL21, spread it on LB solid medium containing ampicillin, culture overnight at 37°C. Pick colonies into LB liquid medium containing ampicillin, culture overnight at 37°C and then transfer them to LB liquid medium at a ratio of 1:100 for continued culture. When the OD600 reaches about 0.6, add IPTG, induce for 4 h at 25°C, collect the bacterial solution, discard the supernatant after centrifugation, resuspend according to the ratio of 1 g of bacterial pellet: 9 ml of PBS, ultrasonically disrupt the resuspended bacteria, centrifuge after disruption and take the supernatant, and obtain the Penton recombinant protein after purification as the fifth antigen.

[0105] The technical solution of the present invention will be described in detail through the following preferred embodiments. It should be noted that the specific embodiments below are only for illustration and do not limit the present invention.

[0106] Preparation of antigen:

[0107] Preparation of the first antigen: The virus seed was appropriately diluted with sterile PBS and inoculated into the allantoic cavity of SPF chicken embryos at 0.1 ml per embryo, and then incubated at 36 - 37°C. The allantoic fluid of the infected chicken embryos that did not die 72 hours after inoculation was harvested and placed in a sterile container; the allantoic fluids that were tested sterile and had a hemagglutination titer of not less than 1:512 against 1% chicken red blood cell suspension were mixed, quantitatively sub-packed to obtain the first antigen containing avian influenza virus; the first antigen was introduced into an inactivation tank, and 10% formaldehyde solution was metered in and mixed thoroughly. The final concentration of the formaldehyde solution was 0.2%, and it was inactivated at 37°C for 16 hours to obtain the first antigen containing inactivated avian influenza virus.

[0108] Preparation of the second antigen: Infectious bronchitis virus was inoculated into chicken hepatocytes at an inoculation amount of 1%; it was cultured in an incubator at 37°C and 5% CO2. When CPE appeared in more than 80% of the cells, the virus fluid was harvested; the harvested virus fluid was concentrated 2 - 3 times with an ultrafiltration concentrator under the condition of 2 - 8°C to obtain a concentrated solution; the concentrated solution was introduced into an inactivation tank, and 10% formaldehyde solution was metered in and mixed thoroughly. The final concentration of the formaldehyde solution was 0.2%, and it was inactivated at 37°C for 16 hours to obtain the second antigen containing inactivated infectious bronchitis virus.

[0109] Preparation of the third antigen: The hemorrhagic follicles were aseptically collected from laying hens showing abnormal egg production, homogenized by a conventional method and repeatedly frozen and thawed 4 times, centrifuged at 12,000 r / min for 10 min at 4°C, and the supernatant was aseptically aspirated and stored frozen for later use; the virus strain in the supernatant was continuously passaged through SPF chicken embryos for 4 generations, and the obtained SPF chicken embryo-adapted virus strain was the Newcastle disease virus strain. The Newcastle disease virus strain was inoculated into SPF chicken embryos, and the allantoic fluid of the SPF chicken embryos that died after inoculation was collected as the third antigen containing inactivated Newcastle disease virus.

[0110] Preparation of the fourth antigen: Mycoplasma synoviae was added to the chicken Mycoplasma synoviae medium with a pH value of 7.6 - 7.8 at a ratio of 1:10 and cultured at a constant temperature of 37°C until the logarithmic growth phase, during which the bacterial liquid changed from red to yellow, and then the bacterial liquid was collected; the bacterial liquid was concentrated 50 times, and 10% formaldehyde solution was added after concentration to make the final concentration of formaldehyde 0.2% and mixed thoroughly. It was inactivated at a constant temperature of 37°C for 24 hours at an oscillation speed of 180 r / min to obtain the fourth antigen.

[0111] The chicken Mycoplasma synoviae medium includes: phosphate buffer, glucose, hydrolyzed milk protein, coenzyme I, arginine hydrochloride, L-cysteine hydrochloride, MEM, yeast extract powder, 1% phenol red, and trehalose; the auxiliary component includes penicillin.

[0112] Preparation of the fifth antigen: The genomic DNA of avian adenovirus (group I, type 4) was extracted and used as a template. Specific primers targeting the Penton gene were used for PCR amplification, and the PCR product was recovered to obtain the Penton target fragment.

[0113] The Penton gene was ligated with pMD18-T to construct a recombinant cloning plasmid. The recombinant cloning plasmid was transformed into competent Escherichia coli DH5α cells. Positive clones were screened and identified by PCR. The positive clone bacterial solution that was successfully identified by PCR was sent for sequencing. The recombinant plasmid with correct sequencing was named pMD18-T / Penton.

[0114] The recombinant plasmid pMD18-T / Penton and the pColdⅠ expression vector were respectively double digested. The digested Penton and pColdⅠ were ligated. The ligation product was transformed into competent Escherichia coli DH5α cells. Positive clones were screened and identified by PCR and double digestion. The positive clone bacterial solution that was successfully identified was sent for sequencing. The recombinant plasmid with correct sequencing was named pCold I / Penton.

[0115] The recombinant plasmid pCold I / Penton was transformed into competent cell BL21 and spread on LB solid medium containing ampicillin, and cultured overnight at 37°C. Single colonies were picked into LB liquid medium containing ampicillin and cultured overnight at 37°C. Then, they were transferred to LB liquid medium at a ratio of 1:100 and continued to be cultured. When the OD600 reached about 0.6, IPTG was added, and after induction at 25°C for 4 h, the bacterial solution was collected. After centrifugation, the supernatant was discarded, and resuspended at a ratio of 1 g of bacterial pellet: 9 ml of PBS. The resuspended bacteria were ultrasonically disrupted, and after disruption, the supernatant was taken by centrifugation. After purification, the Penton recombinant protein was obtained to get the fifth antigen.

[0116] The first antigen, the second antigen, the third antigen, the fourth antigen and the fifth antigen were mixed to obtain the mixed antigen for the examples.

[0117] Example: Preparation of samples.

[0118] The mixed antigen was mixed with adjuvant to prepare the aqueous phase;

[0119] The oil phase was prepared;

[0120] Three parts of the oil phase were taken and added to a proper sterile container, and stirred at 10000 r / min for 10 minutes by a shearing machine. Then, two parts of the aqueous phase were added, and stirred at 15000 r / min for 5 minutes to complete the preparation.

[0121] Sample 1:

[0122] The five-inactivated vaccine was prepared by the above method. The difference was that the aqueous phase used included: 10 parts of adjuvant; 20 parts of the first antigen; 20 parts of the second antigen; 20 parts of the third antigen; 20 parts of the fourth antigen; 10 parts of the fifth antigen.

[0123] The adjuvant includes: 5 parts of vitamin E; 2 parts of levamisole; 1 part of manganese gluconate; 5 parts of Tween-80; 11 parts of albumin; 4 parts of potassium sorbate.

[0124] The oil phase includes: 80 parts of high molecular weight polyacrylic acid, 2 parts of trehalose, 10 parts of sorbitol, and 3 parts of aluminum stearate. As Sample 1.

[0125] Sample 2:

[0126] The five-inactivated vaccine is prepared by the above method. Different from it, the aqueous phase used includes: 15 parts of adjuvant; 15 parts of the first antigen; 15 parts of the second antigen; 15 parts of the third antigen; 20 parts of the fourth antigen; 20 parts of the fifth antigen.

[0127] The adjuvant includes: 8 parts of vitamin E; 6 parts of levamisole; 7 parts of calcium aspartate; 3 parts of Tween-80, 12 parts of albumin; 6 parts of potassium sorbate.

[0128] The oil phase includes: 85 parts of high molecular weight polyacrylic acid, 5 parts of trehalose, 1 part of sorbitol, and 5 parts of aluminum stearate. As Sample 2.

[0129] Sample 3:

[0130] The five-inactivated vaccine is prepared by the above method. Different from it, the aqueous phase used includes: 25 parts of adjuvant; 10 parts of the first antigen; 10 parts of the second antigen; 15 parts of the third antigen; 20 parts of the fourth antigen; 20 parts of the fifth antigen.

[0131] The adjuvant includes: 10 parts of vitamin E; 10 parts of levamisole; 10 parts of zinc gluconate; 1 part of Tween-80, 10 parts of albumin; 5 parts of potassium sorbate.

[0132] The oil phase includes: 90 parts of high molecular weight polyacrylic acid, 3 parts of trehalose, 4 parts of sorbitol, and 3 parts of aluminum stearate. As Sample 3.

[0133] Sample 4:

[0134] The five-inactivated vaccine is prepared by the above method. Different from it, the aqueous phase used includes: 25 parts of adjuvant; 20 parts of the first antigen; 20 parts of the second antigen; 10 parts of the third antigen; 10 parts of the fourth antigen; 15 parts of the fifth antigen.

[0135] The adjuvant includes: 15 parts of vitamin E; 4 parts of levamisole; 6 parts of calcium chloride; 2 parts of Tween-80, 8 parts of albumin; 4 parts of potassium sorbate.

[0136] The oil phase includes: 80 parts of high molecular weight polyacrylic acid, 1 part of trehalose, 8 parts of sorbitol, and 5 parts of aluminum stearate. As Sample 4.

[0137] Control 1:

[0138] Commercially available Newcastle disease, infectious bronchitis, and avian influenza triple vaccine.

[0139] Comparative Example 2:

[0140] Commercially available Newcastle disease virus vaccine.

[0141] Comparative Example 3:

[0142] Commercially available Mycoplasma synoviae vaccine.

[0143] Comparative Example 4:

[0144] Commercially available adenovirus vaccine.

[0145] Comparative Example 5:

[0146] Commercially available Newcastle disease, infectious bronchitis, and avian influenza triple vaccine;

[0147] and commercially available Mycoplasma synoviae vaccine;

[0148] Commercially available adenovirus vaccine.

[0149] Experimental Example:

[0150] Normal saline was used as the blank control group.

[0151] In order to measure the survival of vaccinated chickens after inoculation with the vaccine provided by the present invention, 0.1 ml of each of the samples 1-4 of the present invention and the preparations in the comparative examples was injected into the amniotic cavity of 18-day fertilized eggs using an Intelliject syringe (Avitech) and a 17.5 cm, 18-gauge needle.

[0152] Toxicity was measured as the number of hatched chicks after injection, compared with the number of hatched chicks after injection with 0.1 ml of normal saline PBS. The results are shown in Table 1.

[0153] Table 1: Hatching rate after vaccination

[0154] Dose Number of injected eggs Number of hatched chicks Hatching % Experimental group 1 0.1ml 20 12 60％ Experimental group 2 0.1ml 20 14 70％ Experimental group 3 0.1ml 20 13 65％ Experimental group 4 0.1ml 20 12 60％ Control group 1 0.1ml 20 14 70％ Control group 2 0.1ml 20 12 60％ Control group 3 0.1ml 20 15 75％ Control group 4 0.1ml 20 13 65％ Control group 5 0.1ml 20 3 15％ Blank control group 0.1ml 20 14 70％

[0155] As shown in Table 1, the observation results after inoculating the vaccine into chicken embryos indicate that the vaccine provided by the present invention has good biocompatibility. The vaccine prepared by the present invention does not cause any adverse reactions to chickens, and the experimental safety is high.

[0156] 2. Determination of vaccine immune protection:

[0157] Select 300 chicks at the age of 5 - 7 weeks, with 50 chicks in each group. Subcutaneously inoculate 0.5 ml of the vaccines of experimental groups 1 - 4 into the necks of the chicks in groups 1 - 4 respectively, and subcutaneously inoculate 0.5 ml of normal saline into the necks of the infected control group and the blank control group. After 2 weeks, boost immunize with the same dose and route. 4 weeks after the boost immunization, mix the pathogens such as Newcastle disease virus, avian influenza virus, infectious bronchitis virus, adenovirus, and Mycoplasma synoviae of chickens, etc. subcutaneously inoculate the chickens in the experimental groups and the infected control group into the necks, and do not treat the chickens in the blank control group. Observe for 30 days and determine the protection rate.

[0158] Disease onset judgment criteria: The chicks have runny nose, cough, listlessness, loss of appetite, joint inflammation, abnormal walking, etc. The observation results are as follows:

[0159] Table 2: Results of animal experiments

[0160] Virus challenge rate Number of diseased Protection rate % Experimental group 1 50 3 94％ Experimental group 2 50 3 94％ Experimental group 3 50 3 94％ Experimental group 4 50 2 96％ Infection control group 50 50 0 Blank control group 50 0 100％

[0161] The present invention provides an inactivated vaccine against Newcastle disease virus, avian influenza virus, infectious bronchitis virus, adenovirus, and Mycoplasma synoviae of chickens. The research and preparation of this vaccine solve the defect that the disease cannot be effectively prevented in China at present, and can effectively solve the problems of high incidence of Newcastle disease virus, avian influenza virus, infectious bronchitis virus, adenovirus, and Mycoplasma synoviae of chickens at present and the loss of sudden drop in egg production of chickens.

[0162] The vaccine of the present invention has a high titer, good immunogenicity and can resist the attacks of Newcastle disease virus, avian influenza virus, infectious bronchitis virus, adenovirus, and Mycoplasma synoviae of chickens. The vaccine prepared by the present invention has good safety, and no local and systemic adverse reactions caused by the vaccine have occurred. In the storage period test, through the analysis of the data of character, safety test, and potency test, the results are compared with the single vaccines of similar products, and there is no obvious difference in the pentavalent vaccine, and they are all stable and effective.

[0163] The above - mentioned specific embodiments have further detailed the purpose, technical solution and beneficial effects of the present invention. It should be understood that the above - mentioned are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A novel tributary glandular slide pentavalent inactivated vaccine, characterized in that: The components include the following in parts by mass: Water phase, 2-3 parts, and oil phase, 3-5 parts; The aqueous phase includes the following components by weight: Adjuvant, 10-25 parts; Contains the first antigen of inactivated avian influenza virus, 10-20 copies; Contains a second antigen of inactivated infectious bronchitis virus, 10-20 copies; Contains the third antigen of inactivated Newcastle disease virus, 10-20 copies; Contains the fourth antigen of inactivated Mycoplasma synoviae, 10-20 copies; Contains the fifth antigen of inactivated avian adenovirus, 10-20 copies, totaling 100 copies.

2. The novel tributary gland-slip pentavalent inactivated vaccine according to claim 1, characterized in that: The adjuvant comprises the following components in parts by mass: Vitamin E, 5-15 parts; Levamisole, 2-10 parts; Divalent inorganic salt, 1-10 parts; Tween-80, 1-5 parts; albumin, 8-12 parts; Potassium sorbate, 4-6 parts.

3. The novel tributary gland-slip pentavalent inactivated vaccine according to claim 1, characterized in that: The oil phase comprises the following components in parts by mass: 80-90 parts of high molecular weight polyacrylic acid, 1-5 parts of trehalose, 1-10 parts of sorbitol, 3-5 parts of aluminum stearate.

4. The novel tributary gland-slip pentavalent inactivated vaccine according to claim 1, characterized in that: The divalent inorganic salt is selected from one or two of manganese gluconate, calcium gluconate, calcium aspartate, zinc gluconate, ferrous gluconate and calcium chloride.

5. A method for preparing the vaccine according to any one of claims 1 to 4, characterized in that: The steps include: preparing a first antigen, a second antigen, a third antigen, a fourth antigen and a fifth antigen respectively; Mixing the first antigen, the second antigen, the third antigen, the fourth antigen and the fifth antigen with an adjuvant to prepare an aqueous phase; preparing an oil phase; Take 3-5 parts of the oil phase and add it to a suitable sterile container. Start the shearing machine and stir at 10000 r / min for 10 minutes. Then add 2-3 parts of the water phase and stir at 15000 r / min for 5 minutes to complete the preparation.

6. The preparation method according to claim 5, characterized in that: The method for preparing the first antigen comprises the following steps: After the avian influenza virus is inoculated into the allantoic cavity of chicken embryos for passage, the first antigen is obtained by mixing the inactivated chicken embryo fluid of chickens that are not dead and infected 72 hours after inoculation with the chicken embryo fluid of 1% chicken red blood cell suspension with an agglutination value of not less than 1:

512.

7. The preparation method according to claim 5, characterized in that: The method for preparing the second antigen comprises the following steps: After inoculating chicken embryos with the attenuated H120 strain of avian infectious bronchitis virus, the embryo fluid is harvested to obtain the virus expansion culture fluid, which is freeze-dried and then compounded to obtain the second antigen.

8. The preparation method according to claim 5, characterized in that: The preparation method of the third antigen comprises the following steps: The freeze-dried virus of the weak strain of Newcastle disease virus was reconstituted, and after being continuously passaged and inoculated into chicken embryos for 9 generations, the virus was inactivated and ultrafiltered and concentrated 5 times using a 30KD membrane package to obtain the third antigen.

9. The preparation method according to claim 5, characterized in that: The preparation method of the fourth antigen comprises the following steps: Adding Mycoplasma synoviae to a Mycoplasma synoviae culture medium with a pH value of 7.6 to 7.8 at a ratio of 1:10, culturing at 37°C until the logarithmic growth phase, and making the bacterial solution change from red to yellow, and collecting the bacterial solution; The bacterial solution was concentrated 50 times, and after concentration, 10% formaldehyde solution was added to make the final formaldehyde concentration 0.2%, and the mixture was fully mixed and inactivated at 37°C for 24 hours at an oscillation speed of 180 r / min to obtain the fourth antigen.

10. The preparation method according to claim 9, characterized in that: The preparation and harvest of the fifth antigen containing inactivated avian adenovirus comprises: Extract genomic DNA of avian adenovirus, use it as a template, use specific primers for Penton gene to perform PCR amplification, and recover PCR products to obtain Penton target fragment; The Penton target fragment was connected to pMD18-T to construct a recombinant cloning plasmid, and the recombinant cloning plasmid was transformed into Escherichia coli DH5α competent cells, and the recombinant plasmid pMD18-T / Penton of the positive clone was screened. The recombinant plasmid pMD18-T / Penton and the pColdⅠ expression vector were double-digested, Penton and pColdⅠ were connected after the digestion, the connection product was transformed into Escherichia coli DH5α competent cells, and the recombinant plasmid pCold I / Penton of the positive clone was screened. The recombinant plasmid pCold I / Penton was transformed into competent cells BL21, spread on LB solid culture medium containing ampicillin, cultured overnight at 37°C, picked in LB liquid culture medium containing ampicillin, and cultured overnight at 37°C, then transferred to LB liquid culture medium at a ratio of 1:100 for further culture. When OD600 reached about 0.6, IPTG was added, and the bacterial liquid was collected after induction at 25°C for 4 hours. The supernatant was discarded after centrifugation, and the cells were resuspended at a ratio of 1 g bacterial mud: 9 ml PBS. The resuspended cells were ultrasonically disrupted, and the supernatant was obtained by centrifugation after disruption. The Penton recombinant protein was obtained after purification as the fifth antigen.