Preparation method of chicken new branch and abortion quadruple vaccine

Abstract

The invention relates to the field of preparation of poultry vaccines, in particular to a preparation method of a chicken newcastle disease and avian influenza combined vaccine, which comprises the following steps: respectively inoculating susceptible chick embryos with Newcastle disease virus, infectious bronchitis virus and avian influenza virus, and harvesting infected chick embryo liquid to obtain first virus liquid, second virus liquid and third virus liquid; inoculating the susceptible duck embryo with the egg drop syndrome virus, and harvesting infected duck embryo liquid to obtain a fourth antigen; and concentrating the harvested first virus liquid, second virus liquid, third virus liquid and fourth antigen, inactivating, and mixing and emulsifying with an adjuvant. The vaccine prepared by the invention does not generate any adverse reaction to chickens, is high in experimental safety, can induce high-level neutralizing antibodies in immunized chickens, and can effectively reduce the incidence of Newcastle disease virus, infectious bronchitis virus, egg drop syndrome and avian influenza.

Description

Technical Field

[0001] The present invention relates to the field of poultry vaccine preparation, and in particular to a new branch flow reduction quadruple vaccine for chickens 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 always been in a dominant position in poultry and livestock production. The healthy development of the chicken farming industry is one of the important ways to increase farmers' income and one of the important sources to ensure residents' meat consumption. The development of the chicken farming industry is an important way to promote Chinese farmers' income growth and wealth and the transfer of surplus rural labor. The stable supply of chicken products has become an important link in solving farmers' income growth and national economy and people's livelihood. However, the high incidence and extremely harmful infectious diseases of chickens are a stumbling block that seriously hinders the healthy development of the chicken farming industry, such as avian influenza, Newcastle disease, and infectious bronchitis.

[0003] Avian influenza (Avian influenza) first appeared in Italy in 1878 and was later confirmed to be caused by the influenza A virus. Since then, Avian influenza has not only caused huge economic losses to the livestock industry but also posed a serious threat to human health and life. Currently, cases of human infection with the H5N1 subtype of avian influenza virus have occurred in several countries. The World Health Organization has confirmed that avian influenza viruses can be transmitted from person to person under certain conditions. Therefore, developing a new, effective, and safe avian influenza vaccine has become a key task for virologists. Currently, the main method of preventing the disease is vaccination. Conventional inactivated vaccines are ineffective against the disease, while attenuated vaccines pose potential biosafety issues. Therefore, the development of a new, effective, inexpensive, and safe vaccine is imperative. With the rapid development of molecular biology techniques, researchers are continuously researching new genetically engineered 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 Coronavirus family. It often causes respiratory symptoms in growing chickens. Some strains can also cause kidney and reproductive damage, resulting in decreased egg production and even death. Infected chickens suffer stunted growth, increased feed consumption, reduced egg production and quality, and increased mortality, causing significant economic losses to the poultry industry. It is characterized by difficulty breathing, rales, coughing, open-mouth breathing, and sneezing. There are no significant breed differences in the disease's infectivity. Chickens of all ages are susceptible, but symptoms are more pronounced in birds under five weeks of age, with a mortality rate of 15-19%. The disease is most common from late autumn to late spring, but is most severe in winter. It is a major disease affecting the poultry industry, primarily affecting the respiratory tract. Pale yellow, translucent serous and mucous exudates can be seen in the nasal cavity, trachea, and bronchi. With a longer course of the disease, this becomes a cheesy substance and forms emboli. The air sacs may be turbid or contain cheesy exudates. Egg-laying hens experience congestion, bleeding, or deformation of the follicles; the oviducts become short, thick, and hypertrophic, with localized congestion and necrosis. Infected chicks suffer permanent damage to the oviducts, and they generally cannot lay eggs as adults. In addition to respiratory lesions, nephrotic bronchitis can also cause enlarged and pale kidneys, dilated renal tubules due to urate deposits, a mottled kidney pattern, and thickened ureters due to urate deposits. Urate deposits resembling a layer of white frost can also be seen on the surface of the heart and liver. Sometimes, inflammation and bleeding symptoms can be seen in the bursa of Fabricius.

[0005] Newcastle disease (ND), also known as Asian fowl plague and pseudofowl plague, is an acute, septicemic, and highly contagious disease that occurs in domestic and wild birds, including chickens, pigeons, quail, and turkeys. It is a notifiable infectious disease specified by the World Organization for Animal Health (OIE). Its etiology is the Newcastle disease virus (NDV), which belongs to the Paramyxoviridae, Paramyxovirinae, and Rubulavirus genus. ND not only causes severe economic losses to the poultry industry but also severely impacts international trade in poultry and poultry-related products. Consequently, countries around the world have implemented stringent prevention and control measures to combat the disease. Since the 1990s, ND has become increasingly severe in vaccinated chicken flocks in my country, characterized by a significant decrease in egg production, low mortality, and high antibody titers. This atypical ND has severely compromised chicken production performance and caused significant losses to the poultry industry.

[0006] Egg Drop Syndrome (EDS) is a viral infectious disease that causes a decrease in egg production in laying hens or breeder hens. When infected with the EDS virus, laying hens fail to reach peak egg production or experience a significant drop in egg production, potentially by 20% to 30%. This severely impacts the egg-laying industry, causing significant economic losses to farmers and businesses, and is extremely harmful. Vaccination is the primary preventative measure against this disease. If all hens develop antibodies before laying, egg production will not be affected.

[0007] Avian Encephalomyelitis (AE), also known as epidemic tremor, is a viral infectious disease caused by a virus (AEV) that primarily harms chicks under four weeks of age. It attacks the central nervous system, causing non-suppurative encephalitis in chicks. Symptoms include movement disorders, head and neck tremors, and hind digit paralysis. Characterized by sudden onset and unpredictable duration, AE can cause chick losses and reduced egg production in hens, making it one of the major global threats to the poultry industry. Since its first reports in my country in the mid-1980s, AE has spread throughout the country, causing significant losses to the poultry industry. Currently, there is no effective treatment for this disease, making preventive immunization even more crucial.

[0008] Currently, most vaccines used on the market are single-antigen vaccines, with fewer combining vaccines containing multiple antigens. However, these vaccines have limited effectiveness in preventing and controlling disease. Furthermore, the overuse of antibiotics has led to the emergence of drug-resistant strains, rendering drug treatment ineffective and wasteful. Antibiotic residues also make it difficult for meat animals to be exported. Summary of the Invention

[0009] Based on this, the present invention proposes a preparation method of a Newcastle disease-reducing quadruple vaccine for chickens, which reduces the incidence of Newcastle disease, infectious bronchitis, avian influenza, and chicken egg drop syndrome, and improves the survival efficiency of chickens and eggs.

[0010] According to one aspect of the present invention, there is provided a method for preparing a new branch flow-reducing quadruple vaccine for chickens, comprising the following steps:

[0011] Inoculating susceptible chicken embryos with Newcastle disease virus, infectious bronchitis virus and avian influenza virus respectively, harvesting infected chicken embryo fluids to obtain a first virus fluid, a second virus fluid and a third virus fluid;

[0012] Egg drop syndrome virus is inoculated into susceptible duck embryos, and infected duck embryo fluid is harvested to obtain the fourth antigen;

[0013] The harvested first virus liquid, second virus liquid, third virus liquid and fourth antigen are concentrated, inactivated and then mixed with adjuvants and emulsified to prepare the product.

[0014] Furthermore, the adjuvant includes an aqueous phase adjuvant and an oil phase adjuvant;

[0015] The aqueous phase adjuvant is 10%-15% of the total mass of the first virus solution, the second virus solution, the third virus solution, and the fourth antigen;

[0016] The oil phase adjuvant is 1-3 times the total mass of the first virus liquid, the second virus liquid, the third virus liquid and the fourth antigen.

[0017] Furthermore, the aqueous phase adjuvant comprises the following components in parts by mass:

[0018] Vitamin E, 5-15 parts;

[0019] Sucrose, 5-10 parts;

[0020] Levamisole, 2-10 parts;

[0021] antibiotics, 1-5 parts;

[0022] Casein, 8-12 servings.

[0023] Furthermore, the oil phase comprises the following components in parts by mass:

[0024] 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.

[0025] Furthermore, the preparation method of the first virus liquid comprises the following steps:

[0026] The freeze-dried attenuated strain of Newcastle disease virus was reconstituted, serially passaged and inoculated into chicken embryos for 9 generations, and then inactivated and ultrafiltrated and concentrated 5 times using a 30KD membrane package to obtain the first virus solution.

[0027] Furthermore, the preparation method of the second virus liquid comprises the following steps:

[0028] After inoculating chicken embryos with the attenuated H120 strain of avian infectious bronchitis virus, the embryo fluid is harvested to obtain a virus expansion culture fluid, which is then freeze-dried and compounded to obtain the second virus fluid.

[0029] Furthermore, the preparation method of the third virus liquid includes the following steps:

[0030] After the avian influenza virus is inoculated into the allantoic cavity of chicken embryos for passage, the chicken embryo fluid that is not dead and infected 72 hours after inoculation is harvested and inactivated, and the chicken embryo fluid with an agglutination titer of 1% chicken red blood cell suspension of not less than 1:512 is mixed to obtain the third virus fluid.

[0031] Furthermore, the preparation method of the fourth antigen comprises the following steps:

[0032] The genomic DNA of egg drop syndrome was extracted and used as a template to perform PCR amplification using specific primers targeting the Penton gene. The PCR product was recovered to obtain the target Penton fragment.

[0033] The Penton target fragment was connected to pMD18-T to construct a recombinant cloning plasmid, which was transformed into Escherichia coli DH5α competent cells, and the recombinant plasmid pMD18-T / Penton of the positive clone was screened.

[0034] The recombinant plasmid pMD18-T / Penton and the pCold I expression vector were double-digested, Penton and pCold I were ligated, the ligation product was transformed into Escherichia coli DH5α competent cells, and the recombinant plasmid pCold I / Penton of the positive clone was screened.

[0035] The recombinant plasmid pCold I / Penton was transformed into competent cells BL21, spread on LB solid medium containing ampicillin, cultured overnight at 37°C, and spotted in LB liquid medium containing ampicillin. After overnight culture at 37°C, the cells were transferred to LB liquid medium at a ratio of 1:100 and continued to be cultured. When the OD600 reached approximately 0.6, IPTG was added, and the culture was induced at 25°C for 4 hours. The bacterial liquid was collected, centrifuged and the supernatant was discarded, and the cells were resuspended at a ratio of 1 g of bacterial slurry: 9 ml of PBS. The resuspended cells were ultrasonically disrupted, and the supernatant was collected after disruption by centrifugation. The Penton recombinant protein was obtained after purification as the fourth antigen.

[0036] It can be seen from the above technical solutions that the preparation method of the chicken new branch flow reduction quadruple vaccine provided by the present invention has the following beneficial effects:

[0037] The present invention proposes vaccines against Newcastle disease, infectious bronchitis, egg drop syndrome, and avian influenza. The research and preparation of these vaccines address the current lack of effective prevention for these diseases in my country. They can effectively address the high incidence of diseases such as Newcastle disease virus, infectious bronchitis virus, egg drop syndrome, and avian influenza, and the resulting loss of egg production. The method provided by the present invention for preparing inactivated vaccines is simple and easy to operate. The prepared vaccines have good antigenicity, high safety, and no adverse effects on the external environment, making them easy to pass safety assessments.

[0038] The vaccine prepared by the present invention will not produce any adverse reactions to chickens, has high experimental safety, and can induce high levels of neutralizing antibodies in the bodies of immunized chickens, indicating that the immunization of chickens can effectively reduce the incidence of Newcastle disease virus, infectious bronchitis virus, chicken egg drop syndrome, and avian influenza.

[0039] The vaccine prepared by the present invention has good safety, with no local or systemic adverse reactions attributable to the vaccine. Analysis of properties, safety, and efficacy data during shelf life testing revealed no significant differences between the quadruple vaccine and similar single-dose vaccines, demonstrating stability and effectiveness.

[0040] The invention has the advantages of being able to simultaneously prevent Newcastle disease virus, infectious bronchitis virus, egg drop syndrome, and avian influenza. It can induce a synergistic immune effect through the combination of multiple immunogens, produce a specific immune effect, reduce the frequency of injections, change the situation where at least two injections are needed to prevent the above diseases, reduce the cost of immunization, reduce the stress response of animals, and the quadruple inactivated vaccine has a high titer and content, is convenient and fast to immunize, avoids the adverse reactions of multiple vaccinations, and reduces breeding costs. Compared with current vaccines, it can immunize more serotypes with a single injection, achieving the purpose of "one injection to prevent multiple diseases", is easy to use, reduces stress on chickens, and does not have the hidden danger of toxin shedding, and is safe and reliable.

[0041] The present invention utilizes multiple adjuvants in an organic and coordinated manner, leveraging their maximum advantages to create an optimal formulation, namely, an immunostimulatory complex. This allows for better immune adjuvant efficacy, and when used in combination with an antigen, it exhibits superior immune efficacy, low toxicity, and low cost. It can also reduce post-immunization side effects such as redness, swelling, and fever at the immunization site, without affecting the spirits or feeding habits of the immunized poultry, effectively enhancing the immune efficacy and production performance of the immunized poultry. DETAILED DESCRIPTION

[0042] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0043] Currently, most commercially available vaccines for Newcastle disease virus, infectious bronchitis virus, egg drop syndrome, and avian influenza are single-antigen vaccines, with fewer combining these four antigens. However, these vaccines offer limited effectiveness in disease prevention. Furthermore, the overuse of antibiotics has led to the emergence of drug-resistant strains, rendering drug treatment ineffective and wasteful. Antibiotic residues also pose a challenge for exporting meat animals.

[0044] In view of some existing problems, according to one aspect of the present invention, a method for preparing a new branch flow reduction quadruple vaccine for chickens is provided, comprising the following steps:

[0045] Inoculating susceptible chicken embryos with Newcastle disease virus, infectious bronchitis virus and avian influenza virus respectively, harvesting infected chicken embryo fluids to obtain a first virus fluid, a second virus fluid and a third virus fluid;

[0046] Egg drop syndrome virus is inoculated into susceptible duck embryos, and infected duck embryo fluid is harvested to obtain the fourth antigen;

[0047] The harvested first virus liquid, second virus liquid, third virus liquid and fourth antigen are concentrated, inactivated and then mixed with adjuvants and emulsified to prepare the product.

[0048] The present invention proposes vaccines against Newcastle disease, infectious bronchitis, egg drop syndrome, and avian influenza. The research and preparation of these vaccines address the current lack of effective prevention for these diseases in my country. They can effectively address the high incidence of diseases such as Newcastle disease virus, infectious bronchitis virus, egg drop syndrome, and avian influenza, and the resulting loss of egg production. The method provided by the present invention for preparing inactivated vaccines is simple and easy to operate. The prepared vaccines have good antigenicity, high safety, and no adverse effects on the external environment, making them easy to pass safety assessments.

[0049] The vaccine prepared by the present invention will not produce any adverse reactions to chickens, has high experimental safety, and can induce high levels of neutralizing antibodies in the bodies of immunized chickens, indicating that the immunization of chickens can effectively reduce the incidence of Newcastle disease virus, infectious bronchitis virus, chicken egg drop syndrome, and avian influenza.

[0050] The vaccine prepared by the present invention has good safety, with no local or systemic adverse reactions attributable to the vaccine. Analysis of properties, safety, and efficacy data during shelf life testing revealed no significant differences between the quadruple vaccine and similar single-dose vaccines, demonstrating stability and effectiveness.

[0051] The invention has the advantages of being able to simultaneously prevent Newcastle disease virus, infectious bronchitis virus, egg drop syndrome, and avian influenza. It can induce a synergistic immune effect through the combination of multiple immunogens, produce a specific immune effect, reduce the frequency of injections, change the situation where at least two injections are needed to prevent the above diseases, reduce the cost of immunization, reduce the stress response of animals, and the quadruple inactivated vaccine has a high titer and content, is convenient and fast to immunize, avoids the adverse reactions of multiple vaccinations, and reduces breeding costs. Compared with current vaccines, it can immunize more serotypes with a single injection, achieving the purpose of "one injection to prevent multiple diseases", is easy to use, reduces stress on chickens, and does not have the hidden danger of toxin shedding, and is safe and reliable.

[0052] The present invention utilizes multiple adjuvants in an organic and coordinated manner, leveraging their maximum advantages to create an optimal formulation, namely, an immunostimulatory complex. This allows for better immune adjuvant efficacy, and when used in combination with an antigen, it exhibits superior immune efficacy, low toxicity, and low cost. It can also reduce post-immunization side effects such as redness, swelling, and fever at the immunization site, without affecting the spirits or feeding habits of the immunized poultry, effectively enhancing the immune efficacy and production performance of the immunized poultry.

[0053] Furthermore, adjuvants include aqueous adjuvants and oily adjuvants;

[0054] The aqueous adjuvant is 10%-15% of the total mass of the first virus solution, the second virus solution, the third virus solution, and the fourth antigen;

[0055] The oil phase adjuvant is 1-3 times the total mass of the first virus liquid, the second virus liquid, the third virus liquid and the fourth antigen.

[0056] Furthermore, the aqueous phase adjuvant includes the following components in parts by mass:

[0057] Vitamin E, 5-15 parts;

[0058] Sucrose, 5-10 parts;

[0059] Levamisole, 2-10 parts;

[0060] antibiotics, 1-5 parts;

[0061] Casein, 8-12 servings.

[0062] Furthermore, the oil phase includes the following components in parts by mass:

[0063] 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.

[0064] Furthermore, the preparation method of the first virus liquid includes the following steps:

[0065] The freeze-dried attenuated strain of Newcastle disease virus was reconstituted, serially passaged and inoculated into chicken embryos for 9 generations, and then inactivated. The virus was concentrated 5 times by ultrafiltration using a 30KD membrane package to obtain the first virus solution.

[0066] Furthermore, the preparation method of the second virus liquid includes the following steps:

[0067] After inoculating chicken embryos with the attenuated H120 strain of avian infectious bronchitis virus, the embryo fluid is harvested to obtain a virus expansion culture fluid, which is then freeze-dried and compounded to obtain a second virus fluid.

[0068] Furthermore, the preparation method of the third virus liquid includes the following steps:

[0069] After the avian influenza virus is inoculated into the allantoic cavity of chicken embryos for passage, the inactivated chicken embryo fluid of chickens that are not dead and infected 72 hours after inoculation is harvested and mixed with the chicken embryo fluid with an agglutination titer of 1% chicken red blood cell suspension of not less than 1:512 to obtain the third virus fluid.

[0070] Furthermore, the preparation method of the fourth antigen comprises the following steps:

[0071] The genomic DNA of egg drop syndrome was extracted and used as a template to perform PCR amplification using specific primers targeting the Penton gene. The PCR product was recovered to obtain the target Penton fragment.

[0072] The Penton target fragment was connected to pMD18-T to construct a recombinant cloning plasmid, which was transformed into Escherichia coli DH5α competent cells, and the recombinant plasmid pMD18-T / Penton of the positive clone was screened.

[0073] The recombinant plasmid pMD18-T / Penton and the pCold I expression vector were double-digested, Penton and pCold I were ligated, the ligation product was transformed into Escherichia coli DH5α competent cells, and the recombinant plasmid pCold I / Penton of the positive clone was screened.

[0074] The recombinant plasmid pCold I / Penton was transformed into competent cells BL21, spread on LB solid medium containing ampicillin, cultured overnight at 37°C, and spotted in LB liquid medium containing ampicillin. After overnight culture at 37°C, the cells were transferred to LB liquid medium at a ratio of 1:100 and continued to be cultured. When the OD600 reached approximately 0.6, IPTG was added, and the culture was induced at 25°C for 4 hours. The bacterial liquid was collected, centrifuged and the supernatant was discarded, and the cells were resuspended at a ratio of 1 g of bacterial slurry: 9 ml of PBS. The resuspended cells were ultrasonically disrupted, and the supernatant was collected after disruption by centrifugation. The Penton recombinant protein was obtained after purification as the fourth antigen.

[0075] Furthermore, the harvested first virus liquid, the second virus liquid, the third virus liquid, and the fourth antigen are concentrated, inactivated, and then mixed with an adjuvant and emulsified to prepare:

[0076] The prepared first virus solution, second virus solution, third virus solution and fourth antigen were clarified by removing large particles and concentrated by ultrafiltration to 1 / 4 of the original volume;

[0077] Add 10% formaldehyde solution to the concentrated first virus solution, second virus solution, third virus solution, and fourth antigen to a final concentration of 0.2%, shake well while adding, and inactivate at 37°C for 16 hours;

[0078] The first virus solution, the second virus solution, the third virus solution, the fourth antigen and the aqueous phase adjuvant were mixed evenly in an appropriate sterile container, and then mixed with the oil phase adjuvant. The mixture was stirred at 10,000 r / min on a shearing machine for 10 minutes to complete the preparation.

[0079] Example: Preparation of samples.

[0080] Inoculating susceptible chicken embryos with Newcastle disease virus, infectious bronchitis virus and avian influenza virus respectively, harvesting infected chicken embryo fluids to obtain a first virus fluid, a second virus fluid and a third virus fluid;

[0081] The freeze-dried attenuated strain of Newcastle disease virus was reconstituted, serially passaged and inoculated into chicken embryos for 9 generations, and then inactivated. The virus was concentrated 5 times by ultrafiltration using a 30KD membrane package to obtain the first virus solution.

[0082] After inoculating chicken embryos with the attenuated H120 strain of avian infectious bronchitis virus, the embryo fluid is harvested to obtain a virus expansion culture fluid, which is then freeze-dried and compounded to obtain a second virus fluid.

[0083] After the avian influenza virus is inoculated into the allantoic cavity of chicken embryos for passage, the inactivated chicken embryo fluid of chickens that are not dead and infected 72 hours after inoculation is harvested and mixed with the chicken embryo fluid with an agglutination titer of 1% chicken red blood cell suspension of not less than 1:512 to obtain the third virus fluid.

[0084] The genomic DNA of egg drop syndrome was extracted and used as a template to perform PCR amplification using specific primers targeting the Penton gene. The PCR product was recovered to obtain the target Penton fragment.

[0085] The Penton target fragment was connected to pMD18-T to construct a recombinant cloning plasmid, which was transformed into Escherichia coli DH5α competent cells, and the recombinant plasmid pMD18-T / Penton of the positive clone was screened.

[0086] The recombinant plasmid pMD18-T / Penton and the pCold I expression vector were double-digested, Penton and pCold I were ligated, the ligation product was transformed into Escherichia coli DH5α competent cells, and the recombinant plasmid pCold I / Penton of the positive clone was screened.

[0087] The recombinant plasmid pCold I / Penton was transformed into competent cells BL21, spread on LB solid medium containing ampicillin, cultured overnight at 37°C, and spotted in LB liquid medium containing ampicillin. After overnight culture at 37°C, the cells were transferred to LB liquid medium at a ratio of 1:100 and continued to be cultured. When the OD600 reached approximately 0.6, IPTG was added, and the culture was induced at 25°C for 4 hours. The bacterial liquid was collected, centrifuged and the supernatant was discarded, and the cells were resuspended at a ratio of 1 g of bacterial slurry: 9 ml of PBS. The resuspended cells were ultrasonically disrupted, and the supernatant was collected after disruption by centrifugation. The Penton recombinant protein was obtained after purification as the fourth antigen.

[0088] The culture medium comprises 97-99% by volume of low-glucose DMEM or DMEM / F12 solution, 1-3% of newborn calf serum, and an appropriate amount of antibiotics, and the pH value is adjusted to 7.2-7.4.

[0089] The harvested first virus liquid, second virus liquid, third virus liquid and fourth antigen are concentrated, inactivated and then mixed with adjuvants and emulsified to prepare the product.

[0090] The first virus liquid, the second virus liquid, the third virus liquid and the fourth antigen are concentrated, inactivated and then mixed with adjuvants and emulsified to prepare:

[0091] The prepared first virus solution, second virus solution, third virus solution and fourth antigen were clarified by removing large particles and concentrated by ultrafiltration to 1 / 4 of the original volume;

[0092] Add 10% formaldehyde solution to the concentrated first virus solution, second virus solution, third virus solution, and fourth antigen to a final concentration of 0.2%, shake well while adding, and inactivate at 37°C for 16 hours;

[0093] The first virus solution, the second virus solution, the third virus solution, the fourth antigen and the aqueous phase adjuvant were mixed evenly in an appropriate sterile container, and then mixed with the oil phase adjuvant. The mixture was stirred at 10,000 r / min on a shearing machine for 10 minutes to complete the preparation.

[0094] Samples 1-4 were prepared by the above method:

[0095] Sample 1:

[0096] The aqueous adjuvant is 10% of the total mass of the first virus solution, the second virus solution, the third virus solution, and the fourth antigen. The aqueous adjuvant includes the following components in parts by mass:

[0097] Vitamin E, 15 parts; sucrose, 5 parts; levamisole, 10 parts; antibiotics, 1 part; casein, 8 parts.

[0098] The oil phase adjuvant is 3 times the total mass of the first virus solution, the second virus solution, the third virus solution, and the fourth antigen. The oil phase includes the following components in parts by mass:

[0099] 80 parts of high molecular weight polyacrylic acid, 2 parts of trehalose, 10 parts of sorbitol, and 3 parts of aluminum stearate were used as sample 1.

[0100] Sample 2:

[0101] The aqueous adjuvant is 12% of the total mass of the first virus solution, the second virus solution, the third virus solution, and the fourth antigen. The aqueous adjuvant includes the following components in parts by mass:

[0102] Vitamin E, 5 parts; sucrose, 10 parts; levamisole, 2 parts; antibiotics, 5 parts; casein, 12 parts.

[0103] The oil phase adjuvant is twice the total mass of the first virus solution, the second virus solution, the third virus solution, and the fourth antigen. The oil phase includes the following components in parts by mass:

[0104] 85 parts of high molecular weight polyacrylic acid, 5 parts of trehalose, 1 part of sorbitol, and 5 parts of aluminum stearate were used as sample 2.

[0105] Sample 3:

[0106] The aqueous adjuvant is 14% of the total mass of the first virus solution, the second virus solution, the third virus solution, and the fourth antigen. The aqueous adjuvant includes the following components in parts by mass:

[0107] Vitamin E, 8 parts; sucrose, 7 parts; levamisole, 4 parts; antibiotics, 2 parts; casein, 10 parts.

[0108] The oil phase adjuvant is twice the total mass of the first virus solution, the second virus solution, the third virus solution, and the fourth antigen. The oil phase includes the following components in parts by mass:

[0109] 90 parts of high molecular weight polyacrylic acid, 3 parts of trehalose, 4 parts of sorbitol, and 3 parts of aluminum stearate were used as sample 3.

[0110] Sample 4:

[0111] The aqueous adjuvant is 15% of the total mass of the first virus solution, the second virus solution, the third virus solution, and the fourth antigen. The aqueous adjuvant includes the following components in parts by mass:

[0112] Vitamin E, 12 parts; sucrose, 8 parts; levamisole, 6 parts; antibiotics, 4 parts; casein, 11 parts.

[0113] The oil phase adjuvant is 1 times the total mass of the first virus solution, the second virus solution, the third virus solution, and the fourth antigen. The oil phase includes the following components in parts by mass:

[0114] 80 parts of high molecular weight polyacrylic acid, 1 part of trehalose, 8 parts of sorbitol, and 5 parts of aluminum stearate were used as sample 4.

[0115] Comparative Example 1:

[0116] Commercially available avian influenza vaccines.

[0117] Comparative Example 2:

[0118] Commercially available Newcastle disease virus vaccines.

[0119] Comparative Example 3:

[0120] Commercially available infectious bronchitis virus vaccines.

[0121] Comparative Example 4:

[0122] Commercially available triple vaccines for Newcastle disease, infectious bronchitis and avian influenza.

[0123] Experimental example:

[0124] Normal saline was used as the blank control group.

[0125] To measure the compatibility of the vaccines provided by the present invention with in ovo inoculation of chicken embryos, 0.1 ml of each of the samples 1-4 of the present invention and the formulations in the comparative example were injected into the amniotic cavity of 18-day-old fertilized eggs using an Intelliject syringe (Avitech) and a 17.5 cm, 18-gauge needle.

[0126] Toxicity was measured as the number of chicks hatched after injection, compared with the number of chicks hatched after injection with 0.1 ml of saline (PBS). The results are shown in Table 1.

[0127] Table 1: Hatching rates after vaccination

[0128] dose Number of eggs injected Number of hatched chicks incubation% Experimental Group 1 0.1ml 100 82 82 Experimental Group 2 0.1ml 100 85 85 Experimental Group 3 0.1ml 100 87 87 Experimental Group 4 0.1ml 100 88 88 Comparative Example 1 0.1ml 100 83 83 Comparative Example 2 0.1ml 100 87 87 Comparative Example 3 0.1ml 100 81 81 Comparative Example 4 0.1ml 100 57 57 Blank control group 0.1ml 100 91 91

[0129] As shown in Table 1, the observation results after the vaccine was inoculated into chicken embryos showed that the vaccine provided by the present invention has good biocompatibility. The vaccine prepared by the present invention does not cause any adverse reactions in chickens, and the experimental safety is high.

[0130] 2. Determination of vaccine immune protection:

[0131] 300 chicks aged 5-7 weeks were selected, with 50 in each group. 0.5 ml of the vaccine of groups 1-4 was subcutaneously inoculated in the neck of each group, and 0.5 ml of normal saline was subcutaneously inoculated in the neck of the infected control group and the blank control group. Two weeks later, a second immunization was performed using the same dosage route. Four weeks after the second immunization, pathogens such as Newcastle disease virus, avian influenza, infectious bronchitis virus, adenovirus and Mycoplasma synoviae were mixed and subcutaneously infected in the neck of the chickens in the experimental group and the infected control group. The blank control group was not treated. The chickens were observed for 30 days and the protection rate was determined.

[0132] Disease judgment criteria: chickens have runny nose, cough, lack of energy, loss of appetite, joint inflammation, abnormal walking, etc. The observation results are shown in Table 2:

[0133] Table 2: Animal experiment results (%)

[0134]

[0135]

[0136] The present invention proposes an inactivated vaccine against Newcastle disease virus, infectious bronchitis virus, egg drop syndrome, and avian influenza. The research and preparation of the vaccine solves the current defect in my country that these diseases cannot be effectively prevented, and can effectively solve the current high incidence of Newcastle disease virus, infectious bronchitis virus, egg drop syndrome, and avian influenza, which causes a sudden drop in egg production in chickens.

[0137] The vaccine of the present invention has high potency, good immunogenicity, and can protect against Newcastle disease virus, infectious bronchitis virus, egg drop syndrome, and avian influenza. The vaccine prepared by the present invention has good safety, with no local or systemic adverse reactions attributable to the vaccine. Analysis of properties, safety, and efficacy data during shelf life testing revealed no significant differences between the vaccine provided by the present invention and single vaccines of similar products, demonstrating stability and effectiveness.

[0138] The present invention proposes vaccines against Newcastle disease, infectious bronchitis, egg drop syndrome, and avian influenza. The research and preparation of these vaccines address the current lack of effective prevention for these diseases in my country. They can effectively address the high incidence of diseases such as Newcastle disease virus, infectious bronchitis virus, egg drop syndrome, and avian influenza, and the resulting loss of egg production. The method provided by the present invention for preparing inactivated vaccines is simple and easy to operate. The prepared vaccines have good antigenicity, high safety, and no adverse effects on the external environment, making them easy to pass safety assessments.

[0139] The vaccine prepared by the present invention will not produce any adverse reactions to chickens, has high experimental safety, and can induce high levels of neutralizing antibodies in the bodies of immunized chickens, indicating that the immunization of chickens can effectively reduce the incidence of Newcastle disease virus, infectious bronchitis virus, chicken egg drop syndrome, and avian influenza.

[0140] The invention has the advantages of being able to simultaneously prevent Newcastle disease virus, infectious bronchitis virus, egg drop syndrome, and avian influenza. It can induce a synergistic immune effect through the combination of multiple immunogens, produce a specific immune effect, reduce the frequency of injections, change the situation where at least two injections are needed to prevent the above diseases, reduce the cost of immunization, reduce the stress response of animals, and the quadruple inactivated vaccine has a high titer and content, is convenient and fast to immunize, avoids the adverse reactions of multiple vaccinations, and reduces breeding costs. Compared with current vaccines, it can immunize more serotypes with a single injection, achieving the purpose of "one injection to prevent multiple diseases", is easy to use, reduces stress on chickens, and does not have the hidden danger of toxin shedding, and is safe and reliable.

[0141] The present invention utilizes multiple adjuvants in an organic and coordinated manner, leveraging their maximum advantages to create an optimal formulation, namely, an immunostimulatory complex. This allows for better immune adjuvant efficacy, and when used in combination with an antigen, it exhibits superior immune efficacy, low toxicity, and low cost. It can also reduce post-immunization side effects such as redness, swelling, and fever at the immunization site, without affecting the spirits or feeding habits of the immunized poultry, effectively enhancing the immune efficacy and production performance of the immunized poultry.

[0142] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a new branch of the blood flow reduction quadruple vaccine for chickens, characterized in that: The steps include: Inoculating susceptible chicken embryos with Newcastle disease virus, infectious bronchitis virus and avian influenza virus respectively, harvesting infected chicken embryo fluids to obtain a first virus fluid, a second virus fluid and a third virus fluid; Egg drop syndrome virus is inoculated into susceptible duck embryos, and infected duck embryo fluid is harvested to obtain the fourth antigen; The harvested first virus liquid, second virus liquid, third virus liquid and fourth antigen are concentrated, inactivated and then mixed with adjuvants and emulsified to prepare the product.

2. The preparation method according to claim 1, characterized in that The adjuvants include aqueous adjuvants and oily adjuvants; The aqueous phase adjuvant is 10%-15% of the total mass of the first virus solution, the second virus solution, the third virus solution, and the fourth antigen; The oil phase adjuvant is 1-3 times the total mass of the first virus liquid, the second virus liquid, the third virus liquid and the fourth antigen.

3. The preparation method according to claim 2, characterized in that The aqueous phase adjuvant comprises the following components in parts by mass: Vitamin E, 5-15 parts; Sucrose, 5-10 parts; Levamisole, 2-10 parts; antibiotics, 1-5 parts; Casein, 8-12 servings.

4. The preparation method according to claim 2, 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.

5. The preparation method according to claim 1, characterized in that The preparation method of the first virus liquid comprises the following steps: The freeze-dried attenuated strain of Newcastle disease virus was reconstituted, serially passaged and inoculated into chicken embryos for 9 generations, and then inactivated and ultrafiltrated and concentrated 5 times using a 30KD membrane package to obtain the first virus solution.

6. The preparation method according to claim 1, characterized in that The preparation method of the second virus liquid 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 a virus expansion culture fluid, which is then freeze-dried and compounded to obtain the second virus fluid.

7. The preparation method according to claim 1, characterized in that The preparation method of the third virus liquid comprises the following steps: After the avian influenza virus is inoculated into the allantoic cavity of chicken embryos for passage, the chicken embryo fluid that is not dead and infected 72 hours after inoculation is harvested and inactivated, and the chicken embryo fluid with an agglutination titer of 1% chicken red blood cell suspension of not less than 1:512 is mixed to obtain the third virus fluid.

8. The preparation method according to claim 1, characterized in that The preparation method of the fourth antigen comprises the following steps: The genomic DNA of egg drop syndrome was extracted and used as a template to perform PCR amplification using specific primers targeting the Penton gene. The PCR product was recovered to obtain the target Penton fragment. The Penton target fragment was connected to pMD18-T to construct a recombinant cloning plasmid, which 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 pCold I expression vector were double-digested, Penton and pCold I were ligated, the ligation 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 medium containing ampicillin, cultured overnight at 37°C, and spotted in LB liquid medium containing ampicillin. After overnight culture at 37°C, the cells were transferred to LB liquid medium at a ratio of 1:100 and continued to be cultured. When the OD600 reached approximately 0.6, IPTG was added, and the culture was induced at 25°C for 4 hours. The bacterial liquid was collected, centrifuged and the supernatant was discarded, and the cells were resuspended at a ratio of 1 g of bacterial slurry: 9 ml of PBS. The resuspended cells were ultrasonically disrupted, and the supernatant was collected after disruption by centrifugation. The Penton recombinant protein was obtained after purification as the fourth antigen.

9. The preparation method according to claim 1, characterized in that The first virus liquid, the second virus liquid, the third virus liquid and the fourth antigen are concentrated, inactivated and then mixed with adjuvants and emulsified to prepare: The prepared first virus solution, second virus solution, third virus solution and fourth antigen were clarified by removing large particles and concentrated by ultrafiltration to 1 / 4 of the original volume; Add 10% formaldehyde solution to the concentrated first virus solution, second virus solution, third virus solution, and fourth antigen to a final concentration of 0.2%, shake well while adding, and inactivate at 37°C for 16 hours; The first virus liquid, the second virus liquid, the third virus liquid, the fourth antigen and the aqueous phase adjuvant were mixed uniformly in an appropriate sterile container, and then mixed with the oil phase adjuvant. The mixture was stirred at 10000 r / min on a shearing machine for 10 minutes to complete the preparation.

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