CN113846067A - Method for producing chicken Marek's virus strain and application thereof - Google Patents

Method for producing chicken Marek's virus strain and application thereof Download PDF

Info

Publication number
CN113846067A
CN113846067A CN202111248458.5A CN202111248458A CN113846067A CN 113846067 A CN113846067 A CN 113846067A CN 202111248458 A CN202111248458 A CN 202111248458A CN 113846067 A CN113846067 A CN 113846067A
Authority
CN
China
Prior art keywords
marek
strain
virus
disease
chicken
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202111248458.5A
Other languages
Chinese (zh)
Inventor
祁小莉
方丽华
钱旭东
张萍
唐堂
施庆才
郦晓琼
叶东红
仇婧
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qyh Biotech Co ltd
Original Assignee
Qyh Biotech Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Qyh Biotech Co ltd filed Critical Qyh Biotech Co ltd
Priority to CN202111248458.5A priority Critical patent/CN113846067A/en
Publication of CN113846067A publication Critical patent/CN113846067A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16311—Mardivirus, e.g. Gallid herpesvirus 2, Marek-like viruses, turkey HV
    • C12N2710/16334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16311—Mardivirus, e.g. Gallid herpesvirus 2, Marek-like viruses, turkey HV
    • C12N2710/16351—Methods of production or purification of viral material

Abstract

The invention relates to the field of biological products for livestock, in particular to a method for producing a chicken Marek's virus strain and application thereof. The method comprises the following steps: culturing the chicken embryo fibroblast for 22-24 hours at the temperature of 36.5-39 ℃; then inoculating a working strain, and continuously culturing at 36.5-39 ℃ until 70% of cells are diseased; the cell growth of the chick embryo fibroblast adopts an F10-199 nutrient solution, and the density is 120-180 ten thousand/ml. The chicken Marek's disease virus CVI988-Rispens strains produced by the specific method have small batch difference and high virus plaque number, have the same prevention effect compared with foreign similar vaccines, realize the purposes of large culture area, space saving and high yield in limited space, simultaneously ensure the purity, safety and favorable immunogenicity, and reduce the risks of artificial pollution, uncontrollable quality and the like.

Description

Method for producing chicken Marek's virus strain and application thereof
Technical Field
The invention relates to the field of biological products for livestock, in particular to a method for producing a chicken Marek's virus strain and application thereof.
Background
Marek's disease is a highly contagious neoplastic disease of chickens caused by Marek's Disease Virus (MDV) of the herpesviridae family, and is characterized by lymphoproliferation and tumorigenesis, and by mononuclear cell infiltration into peripheral nerves, gonads, various organs of the iris, muscles and skin. Can infect and damage immune organs of the chicken, cause immunosuppression, cause the immune response and resistance of the body to the vaccine to be reduced, cause the complication and secondary infection of other various diseases, and cause huge economic loss.
The effective method for preventing the disease is vaccine immunization, the HVT freeze-dried vaccine can not effectively control the disease, and the type I liquid nitrogen vaccine has better effect than the HVT and wide application range. CVI988-Rispens seedling-making strain is a strain introduced into the Netherlands, and the traditional production process usually adopts a rotary bottle adherent culture mode, has the advantages of simple structure, less investment, mature technology and the like, but also has the defects of high pollution risk, poor controllability, high labor intensity, large occupied space, high energy consumption, high water consumption and the like. The method for preparing and producing the Marek's disease type I liquid nitrogen vaccine by using the cell factory has the advantages of easiness in large-scale production, capability of well maintaining the stability of a virus antigen, high yield, small batch difference and the like, and the produced vaccine can effectively control and prevent the Marek's disease.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a method for producing a chicken Marek's disease vaccine strain and application thereof.
In a first aspect, the present invention provides a method of producing a chicken marek's virus strain comprising: culturing the chicken embryo fibroblast for 22-24 hours at the temperature of 36.5-39 ℃; then inoculating a working strain, and continuously culturing at 36.5-39 ℃ until 70% of cells are diseased;
the cell growth of the chick embryo fibroblast adopts an F10-199 nutrient solution, and the density is 120-180 ten thousand/ml.
Further, the F10-199 solution includes:
HAM' S/F-10 and 199 medium.
Further, the method comprises:
preparing chicken embryo into chicken embryo fibroblast in the form of cell suspension;
culturing the chicken embryo fibroblast for 22-24 hours at the temperature of 36.5-39 ℃; then inoculating a working strain, and continuously culturing at 36.5-39 ℃ until 70% of cells are diseased;
collecting all cells, centrifuging at 2-8 ℃ at 2000-3000 r/min for 10-20 minutes, and collecting precipitates.
Further, the working strain is Marek's disease virus serotype I CVI988-Rispens strain.
Further, the culture is carried out in a cell factory, and the inoculation amount of the working strain is 1500-2000 PFU/cm2(ii) a And/or the density of the chick embryo fibroblast is 140-160 ten thousand per mL.
In a second aspect, the invention provides a marek's disease vaccine comprising a marek's disease strain produced by the method.
Further, the marek's disease vaccine comprises: marek's virus strain, 199 nutrient solution, neonatal bovine serum and cryoprotectant prepared by the method of any one of claims 1 to 5;
wherein the volume ratio of the 199 nutrient solution, the newborn bovine serum and the cryoprotectant is (70-90) to (5-20).
Further, the number of virus plaques contained in the Marek's disease vaccine is not less than 3200 PFU/feather; and/or the cryoprotectant is one or more of glycerol, OriGen CD-50 (55% dimethyl sulfoxide, 5% dextran 40 mixed solution) or dimethyl sulfoxide; and/or, the 199 nutrient solution comprises: HAM' S/F-10 and 199 medium.
Dextran is an extracellular cryoprotectant, DMSO has high permeability and is an intracellular protectant, the dextran and the DMSO jointly protect the activity of cells, the cells are prevented from reforming ice crystals when being thawed, the activity of the cells is prevented from being influenced, and the immunogenicity of vaccines can be better protected.
The invention further provides the use of the method for increasing the production efficiency of Marek's virus strains; the Marek's virus strain is preferably a Marek's disease virus CVI988-Rispens strain.
The invention further provides the use of such a method for increasing the immunogenicity of a marek's virus strain; the Marek's virus strain is preferably a Marek's disease virus CVI988-Rispens strain.
As a preferred embodiment, the present invention provides a method for preparing a marek's disease vaccine comprising:
(1) preparing a chicken embryo fibroblast suspension:
selecting 10-15 days old chick embryos, sterilizing and clearing, adding 0.25-0.35% of pancreatin solution, digesting in a water bath kettle at 38.5-39.0 ℃ for 20-30 min, stopping digestion, removing supernatant, and keeping a precipitate; adding 5-10% serum of Dahan liquid dispersed cells into the precipitate, diluting to 40-60 mL of each embryo, and filtering to obtain chicken embryo fibroblast suspension for subsequent processes;
(2) inoculation of
Carrying out asynchronous virus receiving or synchronous virus receiving;
the asynchronous virus receiving comprises the following steps:
subpackaging the chick embryo fibroblast suspension in a cell factory, wherein each layer is 150-250 mL, the density is 140-160 ten thousand/mL, culturing at 36.5-39.0 ℃ for 22-24 hours to form a compact cell monolayer, inoculating a working seed virus to the chick embryo fibroblast monolayer which grows well, standing and culturing in a greenhouse at 36.5-39.0 ℃ for 48-72 hours, and harvesting pathological cells when pathological changes reach more than 70%;
the synchronous virus receiving comprises the following steps:
uniformly mixing the working seed virus with the prepared cell suspension, subpackaging in a cell factory, wherein each layer is 150-250 mL, the density is 140-160 ten thousand per mL, and when the pathological change reaches more than 70%, the pathological change cells are harvested;
(3) harvesting:
discarding nutrient solution, fully contacting cells with 15-25 mL/layer of EDTA-pancreatin digestive juice, collecting virus cells, stopping digestion, centrifuging at 2-8 ℃ for 10-20 minutes at 2000-3000 r/min, and discarding supernatant after centrifugation;
(4) seedling preparation and subpackaging:
collecting the precipitated cells, adding the frozen stock solution, and uniformly mixing; the formula of the frozen stock solution comprises 199 nutrient solution with 70-85% of the volume of the prepared seedlings, newborn bovine serum with 10-20% of the volume of the prepared seedlings and dimethyl sulfoxide with 5-10% of the volume of the prepared seedlings;
(5) cooling:
and (3) storing the vaccine in liquid nitrogen when the temperature of the vaccine is reduced to-80 to-120 ℃.
The invention has the following beneficial effects:
the invention relates to a method for producing a Marek's disease virus CVI988-Rispens strain by using a cell factory. Compared with the traditional bottle-rotating process, the vaccine produced by the process has small batch difference and high virus plaque number, has the same prevention effect as the foreign similar vaccines, realizes the purposes of large culture area, space saving, high yield in limited space, ensures good purity, safety and immunogenicity, reduces risks of artificial pollution, uncontrollable quality and the like, and has the advantages of saving space and improving the yield.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
This example provides a method for preparing a marek's disease vaccine, wherein a marek's disease virus CVI988-Ri spens strain is prepared by an asynchronous method, which comprises the following steps:
(1) preparation of chick embryo fibroblast monolayer:
selecting 10-day-old chick embryos with clear blood vessels and good development, disinfecting air chamber parts with 75% of alcohol and 4% of iodine tincture, taking out the chick embryos aseptically, removing heads and internal organs, washing with Hankel's solution for 2-3 times, and shearing the embryo bodies into 1-3 mm3The tissue blocks are washed by Hank's solution for 3 times, added with 0.25% pancreatin solution for 2 times, digested in a water bath kettle at 38 ℃ for 30min, then the pancreatin solution is discarded, washed by milk Han's solution for 2-3 times (enzyme removal), slightly kept stand and the supernatant is discarded. Adding appropriate amount of 5% serum emulsion to disperse cells, adding appropriate amount of 5% serum emulsion to dilute after dispersion, making into 150 ten thousand/mL cell suspension, shaking thoroughly, and sampling filtrate for cell counting. The cell suspension was dispensed into a cell factory, 150mL of each layer was sent to a greenhouse for culture, and cultured in F10-199 solution (including HAM' S/F-10 and 199 media, purchased from HyClone and Gibco, respectively) at 37 ℃ for 24 hours to form a dense cell monolayer.
(2) Inoculation:
discarding cell growth liquid, mixing 199 maintaining liquid and working seed poison, and inoculating with inoculum size of 2000PFU/cm2150mL of each layer is inoculated to a well-grown chick embryo fibroblast monolayer, the chick embryo fibroblast monolayer is placed in a greenhouse at 37 ℃ for static culture for 48 to 72 hours, and when the pathological changes reach more than 70 percent, pathological change cells are harvested.
(3) Harvesting:
discarding the nutrient solution, fully contacting cells with 20 mL/layer of EDTA-pancreatin digestive juice, collecting the cells into a sterile centrifuge tube after the virus cells fall off, adding 200mL of 199 nutrient solution to stop digestion, performing sterile inspection, and separating the cells into centrifuge bottles; centrifuging at 2-8 ℃ at 2000r/min for 10 minutes, discarding the supernatant after centrifugation, and collecting the precipitated cells.
(4) Seedling preparation and subpackaging:
collecting the precipitated cells, adding 199 nutrient solution with the volume of 80% of the prepared seedlings, newborn bovine serum with the volume of 10% of the prepared seedlings and dimethyl sulfoxide serving as a cryoprotectant with the volume of 10% of the prepared seedlings, shaking up, sampling for performing sterile inspection before harvesting, counting the living cells, quantitatively subpackaging the obtained mixture into sterile ampoules, and sealing, wherein the number of virus plaques contained in each ampoule is not less than 3200 PFU/feather.
(5) And (3) programmed cooling:
and (3) carrying out programmed cooling by using a programmed cooling instrument, and storing the vaccine in liquid nitrogen when the temperature of the vaccine is reduced to-80 to-120 ℃.
Example 2
This example provides a method for preparing a marek's disease vaccine, wherein a marek's disease virus CVI988-Ri spens strain is prepared by a synchronization method, which comprises the following steps:
(1) preparing a chicken embryo fibroblast suspension:
selecting 10-day-old chick embryos with clear blood vessels and good development, disinfecting air chamber parts with 75% of alcohol and 4% of iodine tincture, taking out the chick embryos aseptically, removing heads and internal organs, washing with Hankel's solution for 2-3 times, and shearing the embryo bodies into 1-3 mm3The tissue blocks are washed by Hank's solution for 3 times, added with 0.25% pancreatin solution for 2 times, digested in a water bath kettle at 38 ℃ for 30min, then the pancreatin solution is discarded, washed by milk Han's solution for 2-3 times (enzyme removal), slightly kept stand and the supernatant is discarded. Adding appropriate amount of 5% serum emulsion to disperse cells, adding appropriate amount of 5% serum emulsion to dilute after dispersion, making into 150 ten thousand/mL cell suspension, shaking thoroughly, and sampling filtrate for cell counting.
(2) Inoculation:
mixing the seed virus with the prepared cell suspension, and inoculating with an inoculum size of 2000PFU/cm2And subpackaging in a cell factory, wherein each layer is 150mL, conveying to a greenhouse, standing and culturing at 37 ℃ in an F10-199 solution, and harvesting diseased cells when the diseased cells reach more than 70%.
(3) Harvesting:
discarding the nutrient solution, fully contacting cells with 20 mL/layer of EDTA-pancreatin digestive juice, collecting the cells into a sterile centrifuge tube after the virus cells fall off, adding 200mL of 199 nutrient solution to stop digestion, performing sterile inspection, and separating the cells into centrifuge bottles; centrifuging at 2-8 ℃ at 2000r/min for 10 minutes, discarding the supernatant after centrifugation, and collecting the precipitated cells.
(4) Seedling preparation and subpackaging:
collecting the precipitated cells, adding 199 nutrient solution with the volume of 80% of the prepared seedlings, newborn bovine serum with the volume of 10% of the prepared seedlings and dimethyl sulfoxide serving as a cryoprotectant with the volume of 10% of the prepared seedlings, shaking up, sampling for performing sterile inspection before harvesting, counting the living cells, quantitatively packaging the virus plaques with the number not less than 3200 PFU/feather, and sealing the bottles.
(5) And (3) programmed cooling:
and (3) carrying out programmed cooling by using a programmed cooling instrument, and storing the vaccine in liquid nitrogen when the temperature of the vaccine is reduced to-80 to-120 ℃.
Test example 1
The yield of Marek's disease virus CVI988-Rispens strain in example 1 above was 600 ten thousand feathers.
The yield of Marek's disease virus CVI988-Rispens strain in example 2 above was 600 ten thousand feathers.
This experimental example carried out purity test, safety test (against 3 Marek's cell virus, 1 domestic vaccine) and efficacy evaluation (against 1 foreign Marek's vaccine) on the Marek's disease type I liquid nitrogen vaccine (CVI988-Rispens strain) produced in example 2 for purity, safety and immune effect.
(1) Purity test
And (4) carrying out sterility, mycoplasma and exogenous virus inspection according to the current Chinese veterinary pharmacopoeia. The results are shown in tables 1, 2 and 3.
TABLE 1 Chicken Marek's disease type I liquid nitrogen vaccine (CVI988-Rispens strain) sterility test results
Figure BDA0003321887360000051
Note: "-" indicates sterile growth.
TABLE 2 Mycoplasma test results for Marek's disease chicken type I liquid nitrogen vaccine (CVI988-Rispens strain)
Figure BDA0003321887360000061
Note: "-" indicates negative; "+" indicates positive.
TABLE 3 test results of exogenous viruses of Marek's disease type I liquid nitrogen vaccine (CVI988-Rispens strain) of chickens
Figure BDA0003321887360000062
Note: "-" indicates that the ELISA result of the chicken anemia virus test is less than or equal to 0.35, the result is judged to be negative, the ELISA result of the avian leukemia virus test is less than 0.3, the result is judged to be negative, or the result of the avian reticuloendotheliosis virus test does not generate specific green fluorescence.
As can be seen from the above tests in conjunction with tables 1, 2, and 3: the sterility test, mycoplasma test and exogenous virus test of 3 batches of chicken Marek's disease type I liquid nitrogen vaccines (CVI988-Rispens strains) are all negative, and the vaccines are proved to be pure.
(2) And (3) safety inspection: 120 SPF chicks of 1 day old were divided into 6 groups of 20, 20 each, and 1-5 groups were inoculated neck subcutaneously with Z4(1000 PFU/one), SB1(1000 PFU/one), CVI988-Rispens (30000 PFU/one), HVT (1000 PFU/one), RB1B (1000 PFU/one), and another group of CEF suspensions were used as controls. 10 chickens were taken from each test group 9 days after inoculation, weighed, dissected, weighed for bursa of fabricius, examined for splenomegaly necrosis and thymus atrophy, and scored according to 0-3 (0-normal, 3-splenomegaly necrosis/thymus severe atrophy), the remaining test chickens were continuously raised to 90 days old, dissected for all test chickens, examined for MD lesions, dissected for dead chickens within the test period, and examined for MD lesions.
TABLE 4 safety test of chicken Marek's disease type I liquid nitrogen vaccine (CVI988-Rispens strain)
Figure BDA0003321887360000071
Note: the relative weight is 87.9-110.7g (average 95g) of the test chicken (10 days old) in the control group, and the numerical value of the test group is the relative percentage of the control group.
As can be seen from table 4: when 1-day-old SPF chickens were immunized with 10-fold doses of CVI988/Rispens vaccine, the test chickens did not lose weight and the immune organs such as bursa of Fabricius, spleen, thymus, etc. were not damaged. At 90 days of age, no clinical symptoms of MD and no pathological changes were observed. The results indicate that the CVI988/Rispens vaccine is very safe.
(3) Evaluation of efficacy: immunopotency tests were performed on 2 sources of SPF chickens with the marek's disease type 1 (CVI988/Rispens) live vaccine of example 2 and other 2 MD vaccines (bivalent vaccine type 2+3 and HVT lyophilized vaccine) from SPF chicken farms of qianyao south tokyo bio-pharmaceutical factories and SPF chicken farms of beijing laboratory animals, respectively.
The dead chickens from 1-day-old immunization to 7-day-old challenge period and the dead chickens within 1 week after challenge are nonspecifically dead, the dead chickens are deducted from the total number of the test chickens in each treatment group, the dead chickens after 1 week of challenge are subjected to one-by-one autopsy, the change of the general tumor and the change of immune organs such as bursa of fabricius, thymus, spleen and the like are recorded, the pathological materials are collected for histological examination when being suspicious, all the surviving chickens are killed at 70 days, the autopsy is carried out, the change of the general tumor and the change of immune organs such as bursa of fabricius, thymus, spleen and the like are recorded, and the pathological materials are collected for histological examination when being suspicious.
As can be seen from the results in tables 5 and 6 below: the bivalent 2+3 vaccine and the HVT freeze-dried vaccine are set as the control in the efficacy evaluation test, and the test data shows that the protective power of the CVI988/Rispens vaccine is the highest among the three.
TABLE 5 vaccine efficacy evaluation test
Figure BDA0003321887360000072
Figure BDA0003321887360000081
TABLE 6 evaluation of vaccine efficacy
Figure BDA0003321887360000082
Note: (ii) test 1※SPF chickens derived from Qianyaoqian biological pharmaceutical factory; test 2※※SPF chickens from the Peking center of laboratory animals SPF chicken farm
(MD + chicken is a chicken with a gross MD tumor and a gross MD tumor which are suspicious and are confirmed by histology to be the chicken with MD + chicken which dies within 1 to 3 weeks after being attacked by super-strong virus and has no obvious tumor but highly atrophic thymus, bursa of fabricius and spleen
③MD+(T ═ MD) positive chicken cumulative number/experimental challenge chicken
Calculation and statistical analysis of Protection Index (PI)
PI ═ PI (toxicity attacking control group MD positive rate-immune group MD positive rate)/toxicity attacking control group MD positive rate × 100
The significance of the difference of PI is tested by chi-square analysis, and the difference is represented by different letters on the right.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (10)

1. A method of producing a chicken marek's virus strain comprising: culturing the chicken embryo fibroblast for 22-24 hours at the temperature of 36.5-39 ℃; then inoculating a working strain, and continuously culturing at 36.5-39 ℃ until 70% of cells are diseased;
the cell growth of the chick embryo fibroblast adopts an F10-199 nutrient solution, and the density is 120-180 ten thousand/ml.
2. The method of claim 1, wherein the F10-199 solution comprises:
HAM' S/F-10 and 199 medium.
3. The method according to claim 1 or 2, comprising:
preparing chicken embryo into chicken embryo fibroblast in the form of cell suspension;
culturing the chicken embryo fibroblast for 22-24 hours at the temperature of 36.5-39 ℃; then inoculating a working strain, and continuously culturing at 36.5-39 ℃ until 70% of cells are diseased;
collecting all cells, centrifuging at 2-8 ℃ at 2000-3000 r/min for 10-20 minutes, and collecting precipitates.
4. The method of any one of claims 1-3, wherein the working strain is Marek's disease virus serotype I CVI988-Rispens strain.
5. The method according to any one of claims 1 to 4, wherein the culturing is carried out in a cell factory and the inoculum size of the working strain is 1500 to 2000PFU/cm2(ii) a And/or the density of the chick embryo fibroblast is 140-160 ten thousand per mL.
6. A marek's disease vaccine comprising a marek's disease strain produced by the method of any one of claims 1-5.
7. The Marek's disease vaccine of claim 6, comprising: marek's virus strain, 199 nutrient solution, neonatal bovine serum and cryoprotectant prepared by the method of any one of claims 1 to 5;
wherein the volume ratio of the 199 nutrient solution, the newborn bovine serum and the cryoprotectant is (70-90) to (5-20).
8. The Marek's disease vaccine of claim 7, comprising viral plaques of no less than 3200PFU per plume; and/or the cryoprotectant is one or more of glycerol, OriGen CD-50 or dimethyl sulfoxide.
9. Use of a method according to any one of claims 1 to 5 for increasing the efficiency of production of a marek's virus strain; the Marek's virus strain is preferably a Marek's disease virus CVI988-Rispens strain.
10. Use of a method according to any one of claims 1 to 5 for increasing the immunogenicity of a marek's virus strain; the Marek's virus strain is preferably a Marek's disease virus CVI988-Rispens strain.
CN202111248458.5A 2021-10-26 2021-10-26 Method for producing chicken Marek's virus strain and application thereof Pending CN113846067A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111248458.5A CN113846067A (en) 2021-10-26 2021-10-26 Method for producing chicken Marek's virus strain and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111248458.5A CN113846067A (en) 2021-10-26 2021-10-26 Method for producing chicken Marek's virus strain and application thereof

Publications (1)

Publication Number Publication Date
CN113846067A true CN113846067A (en) 2021-12-28

Family

ID=78983056

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111248458.5A Pending CN113846067A (en) 2021-10-26 2021-10-26 Method for producing chicken Marek's virus strain and application thereof

Country Status (1)

Country Link
CN (1) CN113846067A (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050202045A1 (en) * 2001-06-14 2005-09-15 Schering-Plough Veterinary Corporation Recombinant avian herpesvirus useful in vaccine production
CN102000328A (en) * 2010-11-23 2011-04-06 北京市兽医生物药品厂 Method for manufacturing Marek's disease vaccine by utilizing cell factory
CN103127496A (en) * 2011-11-30 2013-06-05 普莱柯生物工程股份有限公司 III type turkey herpes virus freeze-drying vaccine
CN103157107A (en) * 2011-12-14 2013-06-19 普莱柯生物工程股份有限公司 Chicken Marek's disease vaccine produced by using continuous passage cell line and production method
CN103977400A (en) * 2014-05-29 2014-08-13 南京创启生物科技有限公司 Method for producing marek disease live vaccine of chicken by using cell line
CN113373107A (en) * 2021-01-29 2021-09-10 上海生物制品研究所有限责任公司 Method for producing virus by preparing passage chick embryo cells in large scale

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050202045A1 (en) * 2001-06-14 2005-09-15 Schering-Plough Veterinary Corporation Recombinant avian herpesvirus useful in vaccine production
CN102000328A (en) * 2010-11-23 2011-04-06 北京市兽医生物药品厂 Method for manufacturing Marek's disease vaccine by utilizing cell factory
CN103127496A (en) * 2011-11-30 2013-06-05 普莱柯生物工程股份有限公司 III type turkey herpes virus freeze-drying vaccine
CN103157107A (en) * 2011-12-14 2013-06-19 普莱柯生物工程股份有限公司 Chicken Marek's disease vaccine produced by using continuous passage cell line and production method
CN103977400A (en) * 2014-05-29 2014-08-13 南京创启生物科技有限公司 Method for producing marek disease live vaccine of chicken by using cell line
CN113373107A (en) * 2021-01-29 2021-09-10 上海生物制品研究所有限责任公司 Method for producing virus by preparing passage chick embryo cells in large scale

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
L F LEE等: "Monoclonal antibodies with speci{city for three dixerent serotypes of Marek\'s disease viruses in chickens", vol. 130, no. 130, pages 320 - 508 *
周煜等: "鸡马立克氏病Ⅰ型CVI98/Rispens活疫苗(基础种毒)毒力返强测定" *
国家药典委员会: "《生产科研资料汇编1981-1982》", 中国医药科技出版社, pages: 183 *
徐兆强: "鸡马立克氏病二价活疫苗"双欣立克"的研制" *
马明等: "细胞工厂生产鸡马立克氏病二价活疫苗（CVI988+FC126株）工艺的优化", vol. 37, no. 37 *

Similar Documents

Publication Publication Date Title
CN108148803B (en) Grass carp muscle tissue cell line and application thereof
CN109913404B (en) Preparation method of chicken infectious bursal disease virus live vaccine
CN102220287A (en) Avian infectious bronchitis cold adaptation attenuated vaccine strain and application thereof
CN109679927B (en) Swine senna valley virus, preparation method of pig senna valley virus inactivated vaccine, pig senna valley virus inactivated vaccine and application
CN109097340B (en) Avian adenovirus, quadruple vaccine and preparation method thereof
CN102000328B (en) Method for manufacturing Marek's disease vaccine by utilizing cell factory
CN106075429B (en) Goat pox, sheep pox and aphtha triple cell attenuated vaccine and preparation method and application thereof
CN103143007B (en) Method for preparing infectious bursal disease virus live vaccine by utilizing passage chicken embryo fibroblast
CN105664150A (en) Newcastle disease virus, avian influenza virus and avian adenovirus triple inactivated vaccine
CN109207436A (en) One plant of 4 type aviadenovirus strain of I group and its application
CN111643659A (en) Rabbit hemorrhagic disease virus baculovirus vector vaccine and preparation method thereof
CN113846067A (en) Method for producing chicken Marek's virus strain and application thereof
CN103977400B (en) Method for producing marek disease live vaccine of chicken by using cell line
CN103127496B (en) Type III herpes turkey virus freeze dried vaccine
CN103060356A (en) Duck plague virus deficient in g E gene transfer vector and constructing method thereof
CN114107171A (en) Goose retinal epithelial cell line and construction method and application thereof
CN106139141B (en) Sheep pox and orf bivalent cell attenuated vaccine and preparation method and application thereof
CN112126628B (en) Goat pox virus propagation method, goat pox live vaccine, preparation method and application thereof
CN103157107A (en) Chicken Marek's disease vaccine produced by using continuous passage cell line and production method
CN102380093B (en) Method for producing newcastle disease living vaccines by utilizing passage fibroblasts
CN102978167A (en) Method for preparing nephropathogenic avian infectious bronchitis viruses and live vaccines by using cell lines
CN103146653B (en) Infectious bursal disease virus chick embryo fibroblast adapted strain H11 and application thereof
CN114350601B (en) Cherry valley duck fibroblast line and construction method and application thereof
CN109750004A (en) One plant of LN plants of Raccoon dog parvovirus enteritis virus, Raccoon dog parvovirus enteritis inactivated vaccine and preparation method thereof
CN117586965A (en) Production method of chicken Marek's disease vaccine

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination