Preparation method of chicken infectious bursal disease virus live vaccine

Abstract

The invention belongs to the technical field of preparation of chicken vaccines, and particularly relates to a preparation method of a chicken infectious bursal disease virus live vaccine, which comprises the following steps: step 1, preparing chicken embryo fibroblasts, selecting a chicken embryo, taking down the body part of the chicken embryo, separating the fibroblasts, inoculating the fibroblasts into a serum-free culture medium, and culturing the fibroblasts; 2, virus culture: selecting chicken infectious bursal disease with moderate virulence and good immunogenicity; the chicken infectious bursal disease virus live vaccine is prepared by mixing the B87 strain, the CA strain and the CF strain, three variant strains can be prevented at the same time through one-time inoculation, the prevention range of the chicken infectious bursal disease virus is expanded, meanwhile, the inoculation frequency can be reduced, and the problem that the growth health and growth rate of chickens are affected due to frequent inoculation is solved; the serum-free culture medium is adopted to culture the chick embryo fibroblasts, so that the subsequent operation of harvesting the cells is saved, the culture mode is simple and convenient, and the labor cost and the pollution risk are reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of chicken vaccine preparation, and particularly relates to a preparation method of a live vaccine against infectious bursal disease virus of chickens. Background Art

[0002] Infectious bursal disease (IBD) is an acute, highly contagious, lympholytic infectious disease of chickens and turkeys caused by infectious bursal disease virus (IBDV), mainly infecting young chickens aged 3 - 6 weeks. The virus mainly attacks immature B lymphocytes or B lymphoblast precursors in the bursa of Fabricius of chicks, causing severe and long-term immunosuppression in the chicken flock, reducing the body's ability to respond to vaccines, leading to immune failure, or causing secondary infections of other viral or bacterial diseases, thereby increasing the mortality and culling rate of the chicken flock. There is still no effective treatment for this disease, and immunization has always been the best choice for preventing and controlling IBD.

[0003] The infectious bursal disease virus of chickens has mutated and differentiated into different strains over a long time. Most of the existing live vaccines against infectious bursal disease virus of chickens can only prevent and control specific strains, resulting in young chickens needing to be frequently inoculated with a variety of different vaccines, which is likely to affect the growth health and growth rate of young chickens. Moreover, during the preparation process of the live vaccine against infectious bursal disease virus of chickens, its culture method is complicated, requiring a large amount of manual operation, and is likely to be contaminated by exogenous viruses, resulting in a relatively high production cost. Summary of the Invention

[0004] To solve the problems raised in the above background art, the present invention provides a preparation method of a live vaccine against infectious bursal disease virus of chickens, which has the characteristics of good epidemic prevention effect and convenient preparation.

[0005] To achieve the above object, the present invention provides the following technical solution: A preparation method of a live vaccine against infectious bursal disease virus of chickens, comprising the following steps:

[0006] Step 1: Preparation of chicken embryo fibroblasts. Select a chicken embryo, remove the trunk part of the chicken embryo, isolate fibroblast cells and inoculate them into a serum-free medium for fibroblast cell culture;

[0007] Step 2: Virus culture. Select B87 strain, CA strain, and CF strain of infectious bursal disease of chickens with moderate virulence and good immunogenicity, and inoculate them into different chicken embryo fibroblasts for proliferation culture respectively;

[0008] Step 3: Virus isolation and purification. Collect the cell culture supernatant and cell lysate, separate and remove impurities from them, and conduct purification. At the same time, detect whether there are exogenous viruses in the virus;

[0009] Step 4: Vaccine preparation. Mix the inactivated and purified virus stock solutions of strain B87, strain CA, and strain CF. Select 95 parts of the mixed stock solution and 5 parts of the sterilized oil adjuvant, and mix them thoroughly.

[0010] Step 5: Vaccine storage. Freeze-dry the vaccine under vacuum to form a dry powder vaccine.

[0011] Preferably, for the cell culture, the operation is carried out in a sterile environment. The fibroblast cells are separated by trypsin digestion method, and its culture medium is placed in an incubator at 34 - 37°C and 4 - 7% CO2 for culture.

[0012] Preferably, when inoculating the cells, select chicken embryo fibroblast cells in good condition, inoculate an appropriate amount of virus, and make the virus evenly distributed.

[0013] Preferably, the virus proliferation culture time is 24 - 36 h, and the temperature is 35 - 38°C.

[0014] Preferably, for virus separation and impurity removal, the centrifugation method is used, and for virus purification, the density gradient centrifugation method is used to purify the virus.

[0015] Preferably, the detection of exogenous viruses can be carried out by PCR detection method.

[0016] Preferably, after the vaccine is prepared, inoculate the vaccine into young chickens at 9 - 12 days old, and observe whether it causes abnormal reactions or diseases.

[0017] Preferably, the vaccine is stored in a refrigerator at a temperature of 2 - 8°C.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1. For the live vaccine against infectious bursal disease virus of chickens in the present invention, it is prepared by mixing strain B87, strain CA, and strain CF. One inoculation can simultaneously prevent three mutant strains, expanding the prevention range for infectious bursal disease virus of chickens. At the same time, it can reduce the number of inoculations, avoid frequent vaccination, and the problem of affecting the growth health and growth rate of chickens. The chicken embryo fibroblast cells are cultured using a serum-free medium, saving the subsequent operations of harvesting cells. Its culture method is simple and convenient, reducing the labor cost and pollution risk. Specific embodiments

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0021] Example 1

[0022] The present invention provides the following technical solutions: a preparation method of an infectious bursal disease virus live vaccine, comprising the following steps:

[0023] Step 1: Preparation of chicken embryo fibroblasts. Select chicken embryos, take the trunk parts of the chicken embryos, isolate fibroblast cells and inoculate them into a serum-free medium for fibroblast cell culture;

[0024] Step 2: Virus culture. Select infectious bursal disease viruses with moderate virulence and good immunogenicity, namely B87 strain, CA strain, and CF strain, and inoculate them into different chicken embryo fibroblasts for proliferation culture respectively;

[0025] Step 3: Virus isolation and purification. Collect the cell culture supernatant and cell lysate, separate and remove impurities therefrom, and perform purification. At the same time, detect whether there are exogenous viruses in the virus;

[0026] Step 4: Vaccine preparation. Mix the inactivated and purified virus stocks of B87 strain, CA strain, and CF strain, select 95 parts of the mixed stock solution, and 5 parts of sterilized oil adjuvant, and mix them well;

[0027] Step 5: Vaccine storage. Freeze-dry the vaccine under vacuum to make a dry powder vaccine.

[0028] Specifically, for cell culture, the operation is carried out in a sterile environment. The fibroblast cells are separated by trypsin digestion method, and the culture medium is placed in an incubator at 34°C and 4% CO2 for culture.

[0029] Specifically, when inoculating cells, select chicken embryo fibroblasts in good condition, inoculate an appropriate amount of virus, and make the virus evenly distributed.

[0030] Specifically, the virus proliferation culture time is 24 h and the temperature is 35°C.

[0031] Specifically, centrifugation method is used to separate and remove impurities from the virus, and density gradient centrifugation method is used to purify the virus.

[0032] Specifically, the detection of exogenous viruses can be carried out by PCR detection method.

[0033] Specifically, after the vaccine is prepared, inoculate the vaccine into 9-day-old chicks and observe whether it causes abnormal reactions or diseases.

[0034] Specifically, the vaccine is stored in a refrigerator at a temperature of 2°C.

[0035] Example 2

[0036] The present invention provides the following technical solutions: a method for preparing a live vaccine against infectious bursal disease virus of chickens, comprising the following steps:

[0037] Step 1: Preparation of chicken embryo fibroblasts. Select chicken embryos, remove the trunk parts of the chicken embryos, isolate fibroblast cells and inoculate them into a serum-free medium for fibroblast cell culture;

[0038] Step 2: Virus culture. Select infectious bursal disease viruses of chickens with moderate virulence and good immunogenicity, namely B87 strain, CA strain, and CF strain, and inoculate them into different chicken embryo fibroblasts for proliferation culture respectively;

[0039] Step 3: Virus isolation and purification. Collect the cell culture supernatant and cell lysate, separate and remove impurities therefrom, and conduct purification, and simultaneously detect whether there are exogenous viruses in the virus;

[0040] Step 4: Vaccine preparation. Mix the inactivated and purified virus stocks of B87 strain, CA strain, and CF strain, select 95 parts of the mixed stock solution, and 5 parts of the sterilized oil adjuvant, and mix them thoroughly;

[0041] Step 5: Vaccine storage. Conduct freeze-drying of the vaccine to prepare a dry powder vaccine.

[0042] Specifically, for cell culture, the operation is carried out in a sterile environment. The fibroblast cells are separated by trypsin digestion method, and the culture medium thereof is placed in an incubator at 36 °C and 5% CO2 for culture.

[0043] Specifically, when inoculating cells, select chicken embryo fibroblasts in good condition, inoculate an appropriate amount of virus, and make the virus evenly distributed.

[0044] Specifically, the virus proliferation culture time is 30 h and the temperature is 37 °C.

[0045] Specifically, centrifugation method is used for virus separation and removal of impurities, and density gradient centrifugation method is used for virus purification.

[0046] Specifically, PCR detection method can be used for detection of exogenous viruses.

[0047] Specifically, after the vaccine is prepared, inoculate the vaccine into 11-day-old chicks and observe whether there are abnormal reactions or diseases.

[0048] Specifically, the vaccine is stored in a refrigerator at a temperature condition of 5 °C.

[0049] Example 3

[0050] The present invention provides the following technical solutions: a method for preparing a live vaccine against infectious bursal disease virus of chickens, comprising the following steps:

[0051] Step 1: Preparation of chicken embryo fibroblasts. Select a chicken embryo, remove the trunk part of the chicken embryo, isolate fibroblast cells and inoculate them into a serum-free medium for fibroblast cell culture;

[0052] Step 2: Virus culture. Select chicken infectious bursal disease virus strains B87, CA, and CF with moderate virulence and good immunogenicity, and inoculate them into different chicken embryo fibroblast cells for proliferation culture respectively;

[0053] Step 3: Virus isolation and purification. Collect the cell culture supernatant and cell lysate, separate and remove impurities from them, and conduct purification. At the same time, detect whether there are exogenous viruses in the virus;

[0054] Step 4: Vaccine preparation. Mix the inactivated and purified virus stocks of strains B87, CA, and CF. Select 95 parts of the mixed stock solution and 5 parts of sterilized oil adjuvant, and mix them thoroughly;

[0055] Step 5: Vaccine storage. Conduct freeze-drying on the vaccine to make a dry powder vaccine.

[0056] Specifically, for cell culture, operate in a sterile environment, use trypsin digestion method to isolate fibroblast cells, and place their culture medium in an incubator at 37°C and 7% CO2 for culture.

[0057] Specifically, when inoculating cells, select chicken embryo fibroblast cells in good condition, inoculate an appropriate amount of virus, and make the virus evenly distributed.

[0058] Specifically, the virus proliferation culture time is 36h and the temperature is 38°C.

[0059] Specifically, centrifugation method is used to separate and remove impurities from the virus, and density gradient centrifugation method is used to purify the virus.

[0060] Specifically, PCR detection method can be used to detect exogenous viruses.

[0061] Specifically, after the vaccine is prepared, inoculate the vaccine into 12-day-old chicks and observe whether it causes abnormal reactions or diseases.

[0062] Specifically, the vaccine is stored in a refrigerator at a temperature of 8°C.

[0063] Finally, it should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a live vaccine of infectious bursal disease virus of chickens, characterized in that: The following steps are involved: Step 1: Preparation of chicken embryo fibroblasts: select chicken embryos, remove the trunk of the chicken embryos, separate the fibroblasts and inoculate them in serum-free medium for fibroblast culture; Step 2: virus culture, select chicken infectious bursal disease B87 strain, CA strain, and CF strain with moderate virulence and good immunogenicity, and inoculate them into different chicken embryo fibroblasts for proliferation culture; Step 3: Virus separation and purification: collect cell culture supernatant and cell lysate, separate and remove impurities, purify, and detect whether there is exogenous virus in the virus; Step 4: Vaccine preparation: Mix the inactivated and purified B87 strain virus stock solution, CA strain virus stock solution and CF strain virus stock solution, select 95 parts of the mixed stock solution and 5 parts of the sterilized oil emulsion adjuvant, and mix them thoroughly; Step 5: Vaccine storage: freeze-vacuum-dry the vaccine to make a dry powder vaccine.

2. The method for preparing the live vaccine of infectious bursal disease virus of chicken according to claim 1, characterized in that: The cell culture is carried out under a sterile environment, and the fibroblasts are separated by trypsin digestion. The culture medium is placed in a 37° C., 5% CO 2 incubator for culture.

3. The method for preparing the live vaccine of infectious bursal disease virus of chicken according to claim 1, characterized in that: When inoculating cells, chicken embryo fibroblasts in good condition are selected, an appropriate amount of virus is inoculated, and the virus is evenly distributed.

4. The method for preparing the live vaccine of infectious bursal disease virus of chicken according to claim 1, characterized in that: The virus proliferation culture time is 24-36 hours, and the temperature is 35-38°C.

5. The method for preparing the live vaccine of infectious bursal disease virus of chicken according to claim 1, characterized in that: The virus separation and impurity removal adopts centrifugation, and the virus purification adopts density gradient centrifugation to purify the virus.

6. The method for preparing the live vaccine of infectious bursal disease virus of chicken according to claim 1, characterized in that: The detection of the exogenous virus can be carried out by using PCR detection method.

7. The method for preparing the live vaccine of infectious bursal disease virus of chicken according to claim 1, characterized in that: After the vaccine is prepared, it is inoculated into 9-12 day old chickens to observe whether it causes abnormal reactions or diseases.

8. The method for preparing the live vaccine of infectious bursal disease virus of chicken according to claim 1, characterized in that: The vaccine was stored in a refrigerator at a temperature of 2-8°C.