CN111514288A - Preparation method of canine distemper and canine parvo bivalent inactivated vaccine - Google Patents
Preparation method of canine distemper and canine parvo bivalent inactivated vaccine Download PDFInfo
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Classifications
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- 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
- A61P31/14—Antivirals for RNA viruses
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
-
- 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
-
- 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
- A61K2039/70—Multivalent vaccine
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- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14311—Parvovirus, e.g. minute virus of mice
- C12N2750/14334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
- C12N2760/18434—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Virology (AREA)
- Pharmacology & Pharmacy (AREA)
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- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Molecular Biology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
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- Epidemiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a preparation method of a canine distemper and canine parvo bivalent inactivated vaccine, which comprises the following steps: preparing virus liquid, namely inoculating canine distemper virus seeds to a single-layer Vero cell, harvesting virus liquid when cytopathic effect reaches 80%, and repeatedly freezing and thawing to obtain the canine distemper virus liquid; inoculating canine parvovirus virus seeds to F81 cells, harvesting virus liquid when cytopathic effect reaches more than 90%, and repeatedly freezing and thawing to obtain canine parvovirus liquid; inactivating viruses, namely adding a formaldehyde solution into the canine distemper virus solution to inactivate the canine distemper virus to obtain a canine distemper virus inactivation solution; adding a formaldehyde solution into the canine parvovirus solution for inactivation to obtain a canine parvovirus inactivation solution; preparing a vaccine, namely mixing the canine distemper virus inactivation solution and the canine parvovirus inactivation solution according to the ratio of (1-2): 1 to obtain a mixed solution, and then adding aluminum hydroxide to obtain the bivalent inactivated vaccine. The canine distemper and canine parvo bivalent inactivated vaccine prepared by the method has excellent and stable efficacy and good immune effect.
Description
Technical Field
The invention relates to a preparation method of a canine distemper and canine parvo bivalent inactivated vaccine.
Background
The dogs are widely used and can be used as experimental dogs, military dogs, police dogs, pet dogs and the like, but the probability of death from Canine Distemper Virus (CDV) and Canine Parvovirus (CPV) is high, particularly when the dogs are young, the two viruses are infectious among dogs, once infected, the two viruses usually cost a lot but have a little effect, and the canine distemper and canine parvoinactivated vaccines in the current market are unstable in quality, and have the defects of immune failure, short immune time, multiple adverse reactions and the like.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art and provide a preparation method of the canine distemper and canine parvo bivalent inactivated vaccine.
In order to solve the technical problems, the technical scheme of the invention is as follows: a preparation method of a canine distemper and canine parvo bivalent inactivated vaccine comprises the following steps:
preparing virus liquid, namely inoculating canine distemper virus seeds to a single-layer Vero cell, harvesting virus liquid when cytopathic effect reaches 80%, and repeatedly freezing and thawing to obtain the canine distemper virus liquid; inoculating canine parvovirus virus seeds to F81 cells, harvesting virus liquid when cytopathic effect reaches more than 90%, and repeatedly freezing and thawing to obtain canine parvovirus liquid;
inactivating viruses, namely adding a formaldehyde solution into the canine distemper virus solution to inactivate the canine distemper virus to obtain a canine distemper virus inactivation solution; adding a formaldehyde solution into the canine parvovirus solution for inactivation to obtain a canine parvovirus inactivation solution;
preparing a vaccine, namely mixing the canine distemper virus inactivation solution and the canine parvovirus inactivation solution according to the ratio of (1-2): 1 to obtain a mixed solution, and then adding aluminum hydroxide to obtain the bivalent inactivated vaccine.
Further, the canine distemper virus seed is 11 th generation virus seed of a seed batch for producing the CDV-L strain;
and/or the canine parvovirus virus seed is the 10 th generation seed of the seed batch produced by the canine parvovirus CPV-L strain.
Further, the Vero cell is a 25 th generation cell of a Vero working cell bank;
and/or the F81 cells are the 25 th generation cells of the F81 cell bank.
Further, in the process of preparing the virus liquid, inoculating the canine distemper virus seed liquid with the volume concentration of 2-3% to the single-layer Vero cells; and/or inoculating the canine parvovirus seed solution with the volume concentration of 1-2% to F81 cells.
Further, in the preparation process of the virus liquid, after the canine distemper virus seeds are inoculated to the single-layer Vero cells, the cells are cultured for 4-5 days at 35 ℃, the virus liquid is harvested when the cytopathic effect reaches 80%, and after repeated freeze thawing is carried out for 3 times, cell fragments are removed through filtration, so that the canine distemper virus liquid is obtained;
and/or inoculating canine parvovirus virus seeds to F81 cells, culturing overnight at 37 ℃, replacing cell maintenance liquid, continuously culturing for 5 days at 37 ℃, harvesting virus liquid when cytopathic effect reaches more than 90%, repeatedly freezing and thawing for 3 times, and filtering to remove cell fragments to obtain the canine parvovirus liquid.
Further, in the virus inactivation process, adding a formaldehyde solution into the canine distemper virus solution to prepare a virus solution with the volume concentration of formaldehyde being 0.1%, and placing the virus solution in an environment at 4 ℃ for at least 24 hours, wherein the virus solution is uniformly mixed for multiple times to obtain the canine distemper virus inactivation solution;
and/or adding a formaldehyde solution into the canine parvovirus solution to prepare a virus solution with the formaldehyde volume concentration of 0.4%, and placing the virus solution in an environment at 37 ℃ for at least 48h, and uniformly mixing for a plurality of times to obtain the canine parvovirus inactivated solution.
Furthermore, the poison price of the canine distemper virus liquid is more than or equal to 105.3TCID50Per ml; the poison value of the canine parvovirus fluid is more than or equal to 106.0TCID50/ml。
Further, in the bivalent inactivated vaccine, the content of aluminum hydroxide is 600-1000 mu g/ml.
Further, the canine distemper virus inactivation solution is respectively 100、10-1、10-2Diluting, inoculating to Vero cells in single layer at 35 deg.C and 5% CO in an amount of 3% of the cell culture solution2Culturing and observing in an incubator, continuously transmitting for 3 generations, and generating no CPE;
canine parvovirus inactivation solution is respectively 100、10-1、10-2After dilution, the cells were inoculated into F81 cells in an amount of 2% by volume based on the cell culture medium of F81 cells, and the cells were incubated at 37 ℃ and 5% CO2Culturing and observing in an incubator, continuously transmitting for 3 generations, and generating no CPE.
Further, the storage temperature of the canine distemper virus liquid and the canine parvovirus liquid is-20 ℃; the storage temperature of the canine distemper virus inactivation liquid and the canine parvovirus inactivation liquid is 2-8 ℃.
After the technical scheme is adopted, the canine distemper and canine parvovirus bivalent inactivated vaccine prepared by the preparation method disclosed by the invention is excellent and stable in efficacy, free of any adverse reaction and good in immune effect after the canine is inoculated, integrates the canine distemper and canine parvovirus inactivated vaccine, can be used for simultaneously immunizing canine distemper viruses and canine parvoviruses only by inoculating one needle to a canine, and is simple in preparation method.
Detailed Description
In order that the present invention may be more clearly understood, the following detailed description of the present invention is given with reference to specific examples.
1 Material
1.1 seed poison for production
The 11 th generation virus seed in the seed batch is produced by the canine distemper virus CDV-L strain, and the 10 th generation virus seed in the seed batch is produced by the canine parvovirus CPV-L strain.
1.2Vero cells, F81 cells
The Vero cell adopts the 25 th generation cell of a Vero working cell bank;
f81 cells were obtained from the 25 th generation of F81 working cell bank.
1.3 raw and auxiliary materials
Cell culture solution DMEM was purchased from Beijing Tianxin and Biotechnology Limited, newborn bovine serum was purchased from Wuhan Sanli biotechnology Limited, trypsin was purchased from Gibco corporation, control glass bottles were purchased from Megaku (Dengyang) novel medicinal packaging material Limited, butyl rubber plugs were purchased from Jiangyin Wei culvert glass product factories, and aluminum-plastic composite caps were purchased from Yangxin packaging material Limited in Jingjiang.
Cell growth liquid: comprises 10 percent of newborn bovine serum and 90 percent of cell culture solution by volume percentage;
cell maintenance solution: comprises 2 percent of newborn bovine serum and 98 percent of cell culture solution by volume percentage.
1.4 adjuvants
Aluminum hydroxide adjuvant, available from Jilin Zhengyu Bio-products GmbH.
2. Method of producing a composite material
2.1 preparation and examination of Virus solution for preparing vaccine
2.1.1 preparation of Canine distemper Virus solution
Taking 1 bottle of Vero cells growing to a compact monolayer, digesting the Vero cells by using trypsin, mixing the Vero cells digested by the trypsin and a cell growth solution according to the volume ratio of 1:3, subpackaging the Vero cells and a cell growth solution into 3 bottles of 15L rotary bottles, and culturing at 37 ℃. And (3) inoculating the canine distemper virus liquid with the volume concentration of 2-3% into a Vero cell 15L rotary bottle after the Vero cell grows to a single layer, culturing for 4-5 days at 35 ℃, harvesting the virus liquid when the cytopathic effect reaches 80%, repeatedly freezing and thawing for 3 times, filtering to remove cell fragments to obtain the canine distemper virus liquid, and storing the canine distemper virus liquid at the temperature below-20 ℃. Samples were taken for virus content determination and sterility testing. Wherein the solvent of the canine distemper virus venom is cell maintenance fluid.
2.1.2 preparation of Canine parvovirus fluid
Taking 1 bottle of F81 cells growing to a compact monolayer, digesting with trypsin, mixing the F81 cells digested with the trypsin with a cell growth solution according to a volume ratio of 1:3, subpackaging the mixture into 3 bottles of 15L rotary bottles, inoculating a canine parvovirus seed solution with a volume concentration of 1-2% into the F81 cells of 15L rotary bottles, culturing overnight at 37 ℃, replacing a cell maintenance solution, continuously culturing for 5 days at 37 ℃, harvesting a virus solution when cytopathic effect reaches more than 90%, repeatedly freezing and thawing for 3 times, filtering and removing cell fragments to obtain a canine parvovirus solution, and storing the canine parvovirus solution at a temperature of-20 ℃ or below. Samples were taken for virus content determination and sterility testing. Wherein, the solvent of the canine parvovirus virus fluid is cell maintenance fluid.
2.2 inactivation
2.2.1 Canine distemper Virus inactivation
Adding a formaldehyde solution into the canine distemper virus solution to prepare a virus solution with the formaldehyde volume concentration of 0.1%, placing at 4 ℃ for at least 24 hours, uniformly mixing for multiple times in the period to obtain a canine distemper virus inactivation solution, sampling the canine distemper virus inactivation solution for inactivation identification, and storing the canine distemper virus inactivation solution at 2-8 ℃.
2.2.2 Canine parvoVirus inactivation
Adding a formaldehyde solution into the canine parvovirus liquid to prepare a virus liquid with the formaldehyde volume concentration of 0.4%, placing the virus liquid at 37 ℃ for at least 48 hours, uniformly mixing for many times in the period to obtain a canine parvovirus inactivated liquid, sampling the canine parvovirus inactivated liquid for inactivation and identification, and storing the canine parvovirus inactivated liquid at 2-8 ℃.
2.3 inspection of the semi-finished product
2.3.1 determination of the toxicity value
2.3.1.1 Canine distemper virus toxic value determination
Canine distemper virus liquid is indicated as "attached 1CDV virus TCID50Determination of Standard operating procedure "carry out toxicity test of not less than 105.3TCID50/ml。
2.3.1.2 CPV Virus titer determination
The virus solution before inactivation is indicated as "appendix 2CPV virus TCID50Determination of Standard operating procedure "carry out toxicity test of not less than 106.0TCID50/ml。
2.3.2 sterility test
The sterile growth is carried out according to the appendix of the current Chinese veterinary pharmacopoeia.
2.3.3 inactivation assay
2.3.3.1 Canine distemper Virus inactivation test
Canine distemper virus inactivation solution (10)0、10-1、10-2Diluting, inoculating to Vero cells in single layer at 35 deg.C and 5% CO in an amount of 3% of the cell culture solution2Culturing and observing in an incubator, continuously transmitting for 3 generations, and generating no CPE.
2.3.3.2 Canine parvovirus inactivation test
Canine parvovirus inactivation solution according to the formula 100、10-1、10-2After dilution, the cells were inoculated into F81 cells in an amount of F81 cells2% of the cell culture medium volume, at 37 ℃ and 5% CO2Culturing and observing in an incubator, continuously transmitting for 3 generations, and generating no CPE.
2.4 preparing and packaging
And (3) under the aseptic condition, mixing the canine distemper virus inactivation liquid and the canine parvovirus inactivation liquid which are qualified in the inspection according to a certain ratio (1-2): 1, adding aluminum hydroxide (to a final concentration of 600-1000 mug/ml), fully and uniformly mixing, quantitatively subpackaging 5 ml/bottle into a tubular glass bottle, adding a butyl rubber plug, rolling an aluminum-plastic combined cover and labeling.
2.5 inspection of finished products
2.5.1 Properties
And mixing to obtain a light pink suspension.
2.5.2 load
The inspection is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the regulations are met.
2.5.3 sterility test
The test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and no bacteria grow.
2.5.4 measurement of Formaldehyde content
The quality standard is met by inspection according to the appendix of the current Chinese veterinary pharmacopoeia.
2.5.5 determination of aluminum hydroxide content
The concentration is 600-1000 mug/ml by titration detection.
2.5.6 Security check
5 healthy and susceptible dogs (CDV SN is not higher than 1:4, CPV SN is not higher than 1:4 or HI is not higher than 1:8) with the age of 45-60 days are injected with 2 vaccine heads (2ml) per subcutaneous for 21 days. The vaccinated dogs were normal both locally and systemically (mental, appetite, body temperature and faeces etc.).
2.5.7 efficacy test one of the following methods is optional.
5 healthy mice (CDV SN not higher than 1:4, CPV SN not higher than 1:4 or HI not higher than 1:8) with 19-21 days old are injected with 0.1 head (0.1ml) of vaccine per mouse subcutaneously. 14 days later, blood was collected from 5 control mice under the same conditions, and serum was separated and tested for CDV SN antibody and CPV HI antibody. And (5) determining that the CDV SN in the serum of the control mouse is not higher than 1:4, the CPV HI is not higher than 1:8, the CDV SN in the serum of the immune mouse is not lower than 1:64, and the CPV HI is not lower than 1:32, namely the control mouse is qualified).
5 healthy and susceptible dogs (CDV SN is not higher than 1:4, CPV SN is not higher than 1:4 or HI is not higher than 1:8) with the age of 45-60 days are injected with 1 part (1ml) of vaccine per dog subcutaneously. After 21 days, blood was collected from 5 control dogs under the same conditions, and serum was separated and tested for CDV SN antibody and CPV HI antibody. And (4) determining that the CDV SN of the control dog is not higher than 1:4, the CPV HI is not higher than 1:8, the CDV SN of the immune dog is not lower than 1:64, and the CPV HI is not lower than 1:32, namely the control dog is qualified.
CDV neutralizing antibody titers (CDV SN) were determined according to the "standard procedures for determining CDV serum neutralizing antibodies of Note 3".
CPV hemagglutination inhibition potency (CPV HI) was determined according to "standard procedures for 4 CPV microaerophilic hemagglutination and hemagglutination inhibition assay".
3. Results of efficacy testing
3.1 mouse assay
0.1ml of vaccine is injected subcutaneously for ten batches of prepared vaccine, namely, 0.1ml of vaccine is injected subcutaneously for 19-21 days old healthy mice; the control was not immunized and efficacy testing was performed. The results all meet the standard, and the specific results are shown in the table 1-1.
TABLE 1-1 mouse potency test results
3.2 Canine test
The ten batches of the prepared vaccines are subjected to efficacy test by using 45-60-day-old healthy susceptible dogs and injecting 1.0ml of vaccine per dog subcutaneously, and the results all meet the standard, and the specific results are shown in tables 1-2.
TABLE 1-2 Canine efficacy test results
Note 1CDV Virus TCID50Standard procedure for determination
1. The cells are subcultured one day before inoculation, the number of the cells is as large as possible, the cells reach 90% confluence on the next day, and the inoculation operation is preferably carried out within 12-24 hours. And (3) blowing and beating the cells in a liquid storage tank, uniformly mixing the cells to prepare the cells with proper cell density, spreading the cell suspension into a 96-well plate cell culture plate by using a liquid transfer device, and adding 100 mu l of cell suspension into each well. The plates were incubated at 37 ℃ with 5% CO2Incubate in an incubator for about 24 hours.
2. And continuously diluting the virus liquid in the sterilized small tubes by 10 times, namely, sucking 100 mu l of virus liquid by using a liquid transfer gun, adding the virus liquid into the first small tube filled with 900 mu l of DMEM liquid, uniformly pumping, sucking 100 mu l of virus liquid, transferring the virus liquid into the 2 nd tube filled with 900 mu l of DMEM liquid, uniformly pumping, and adding the virus liquid into the 3 rd tube. This was done in series to form a series of 10 x virus dilutions.
3. The complete medium in the 96-well plate grown to a monolayer of cells was aspirated off, and the diluted virus solution was inoculated into the 96-well plate. Starting from the virus solution with the highest virus dilution factor, the virus solutions are inoculated from high to low in sequence, 100 mu l of virus solution is added into each well, and 8 wells are inoculated with each dilution of virus solution.
4. The virus solution was added to the plates at 35 ℃ with 5% CO2Incubate in incubator, observe and record Cytopathic (CPE) condition day by day. The process usually takes 5 to 7 days.
5. Virus titer calculations were performed according to the Reed-Muench method. See Table appendix 1.
Distance ratio ═ (greater than 50% percent-50%)/(greater than 50% percent-less than 50% percent)
lg TCID50Logarithm of CPE virus dilution + logarithm of distance ratio × dilution factor greater than 50%
The logarithm of the above result is the virus content TCID50/0.1ml。
Table Note 1 example of calculation of Virus Titers by Reed-Muench method
Distance ratio ═ (greater than 50% percent-50%)/(greater than 50% percent-less than 50% percent)
＝(88.89％-50％)/(88.89％-11.11％)
＝0.5
lg TCID50Logarithm of CPE virus dilution + logarithm of distance ratio × dilution factor greater than 50%
＝4+0.5×1
＝4.5
The logarithm of the above result is found to be 104.5TCID50/0.1ml。
Note 2CPV Virus TCID50Standard procedure for determination
1. When the cells grow to a monolayer, the cells are passaged, blown and evenly mixed in a liquid storage tank, and cell suspension with proper cell density is prepared.
2. Continuously diluting the virus liquid by 10 times in a sterilization tube, namely sucking 100 mu l of virus liquid by using a liquid transfer gun, adding the virus liquid into a tube filled with 900 mu l of DMEM, blowing and uniformly mixing, sucking 100 mu l of virus liquid, transferring the virus liquid into a 2 nd tube filled with 900 mu l of DMEM, blowing and uniformly mixing, and adding the virus liquid into a 3 rd tube. This was done in series to form a series of 10 x virus dilutions.
3. Synchronously inoculating each diluted virus solution into a 96-well plate with 8 wells, adding each F81 cell suspension and each virus solution with 100 μ l, and performing inoculation at 37 deg.C and 5% CO2The incubator was incubated overnight, the maintenance solution was replaced, and observation was continued for 5 days under the same incubation conditions. Cytopathic (CPE) condition was observed and recorded day by day. The process usually takes 5 to 7 days.
4. Virus titer calculations were performed according to the Reed-Muench method. See Table appendix 2.
Distance ratio ═ (greater than 50% percent-50%)/(greater than 50% percent-less than 50% percent)
lg TCID50Logarithm of CPE virus dilution + logarithm of distance ratio × dilution factor greater than 50%
Logarithm of the above resultI.e. the virus content TCID50/0.1ml。
Table Note 2 example of calculating Virus Titers by Reed-Muench method
Distance ratio ═ (greater than 50% percent-50%)/(greater than 50% percent-less than 50% percent)
＝(62.5％-50％)/(62.5％-0％)
＝0.2
lg TCID50Logarithm of CPE virus dilution + logarithm of distance ratio × dilution factor greater than 50%
＝5+0.2×1
＝5.2
The logarithm of the above result is found to be 105.2TCID50/0.1ml。
Note 3 standard procedures for CDV serum neutralizing antibody determination
The antibody titer in serum is detected by adopting a fixed virus-diluted serum endpoint method.
1. Cell: cells were passaged and seeded in 96-well plates. The number of cells should preferably reach 90% full at 24 hours. The virus inoculation should be carried out for 12-24 hours.
2. Virus: taking out virus from low temperature refrigerator, thawing, diluting according to its titer, and diluting to 100TCID50/0.1ml。
3. Serum: the serum to be detected is firstly put into a 56 ℃ water bath kettle to be heated and inactivated for 30 minutes so as to destroy complement and other thermolabile nonspecific virucidal factors. Subsequently, the serum was serially diluted 2-fold.
4. Feeling is as follows: quantitative virus liquid and blood serum with different dilutions in the same volume are taken into a 1.5ml sterilized centrifuge tube, and the sterilized centrifuge tube is blown by a pipette and evenly mixed, and then is placed at 37 ℃ for 1 hour.
5. Inoculation: the cell culture medium in the 96-well plate was aspirated and the virus-serum mixture or the virus or serum of the control group was rapidly inoculated into a monolayer of cells in the 96-well plate. The inoculation is carried out in sequence from low to high starting from the mixture with the lowest serum dilution factor, 100 mul of virus-serum mixture is added into each well, and 4 wells are inoculated with each dilution mixture.
6. The plates were incubated at 35 ℃ with 5% CO2Incubate in incubator, observe and record Cytopathic (CPE) condition day by day. The process usually takes 4 to 5 days.
7. The titer of the antibody in the serum was calculated according to the Reed-Muench method. See appendix 3.
Distance ratio ═ (greater than 50% percent-50%)/(greater than 50% percent-less than 50% percent)
Serum antibody titer ═ log of serum dilution greater than 50% protection rate + distance ratio x log of dilution factor
And (4) calculating the antilog of the values to obtain the neutralizing antibody titer SN of the serum to be detected.
TABLE APPLICATION 3 example of fixed Virus dilution serum
The dilution of serum which can protect 50% of the cultured cells or experimental animals is between 10 according to the Reed-Muench method-1.2And 10-1.8In the meantime. Distance ratio (83-50)/(83-40) 0.77
Serum antibody titer ═ log of serum dilution greater than 50% protection rate + distance ratio x log of dilution factor
＝-1.2+0.77×(-0.6)
＝-1.2-0.46
＝-1.66
An inverse logarithm of-1.66 of 1/46, i.e. a 1:46 dilution of the serum to be tested, protects 50% of the cultured cells from the appearance of CPE.
Note 4 CPV micro hemagglutination and hemagglutination inhibition test standard operation procedure
First, material preparation
1、PBS(0.015mol/L，pH6.5)
Mother liquor A: 0.2mol/L NaH2PO4. Weighing NaH2PO427.8g, dissolved in distilled water to a constant volume of 1000 ml.Stored at 4 ℃ for later use.
Mother liquor B: 0.2mol/L Na2HPO4. Weighing Na2HPO4·7H2And O53.65 g, dissolving in distilled water, and fixing the volume to 1000 ml. Stored at 4 ℃ for later use.
0.015mol/L PBS (pH6.5): mother liquor A68.5 ml and mother liquor B31.5 ml were mixed and checked by acidimeter to determine pH as 6.5. 75ml of the mixed solution was taken, 8.5g of NaCl was added thereto, and after dissolution, the volume was adjusted to 1000ml with distilled water.
2. 1% fresh pig Red blood cells (containing 1% healthy rabbit serum)
5-10 ml of blood of healthy pigs of 2-3 months old is taken, added with equivalent sterilized PBS and mixed evenly, centrifuged at 1500r/min for 10 minutes, and the precipitate is taken and washed for 2 times by the same method. And finally, taking 1ml of erythrocyte sediment and 1ml of healthy rabbit serum (inactivated), adding into 98ml of sterilized PBS, and uniformly mixing. Storing at 4 deg.C for use.
Second, micro hemagglutination test
1. On a 96-well microplate, 25. mu.l of PBS is added to each well from the 1 st well to the 12 th well or a desired multiple well by a pipette, a sample to be detected (25. mu.l) is sequentially diluted in series from the 1 st well to the last well by the pipette, and 25. mu.l of liquid in the pipette is discarded (if the titer is higher, the dilution can be previously 10 times).
2. Adding 25 mul of 1% pig red blood cell suspension into each hole, arranging red blood cell control holes without the sample to be detected, immediately shaking uniformly on a microplate oscillator, standing for 40-60 minutes at 2-8 ℃, and judging the result when the red blood cells in the control holes are obviously button-shaped. The judgment results are shown in Table 4-1.
Epiboly 4-1 micro hemagglutination test
3. And a result judgment method comprises the following steps: based on the amount of hemagglutination, 100% agglutination is determined as #; if 75% of the agglutination is +++; the concentration of 50% of the agglutination is ++; 25% agglutination was judged as +; it was judged as-without aggregation. The samples were judged to be positive when + + or more were present. The highest dilution of antigen in the positive wells was used as the endpoint. The hemagglutination valence (HA) of sample No. 2015001 as in the above example was 1: 64.
Third, micro hemagglutination inhibition test
1. Preparation of hemagglutinin working solution
1.1 measurement of hemagglutinin titer
96-well microplate method: and (3) diluting the hemagglutinin into different multiples by using PBS, adding 25 mu l of 1% pig red blood cell suspension, adding 25 mu l of PBS, shaking uniformly on a shaker, standing at the temperature of 2-8 ℃ for 40-60 minutes, and judging the result when the red blood cells in the control holes are obviously button-shaped. The highest dilution of the positive wells was used as the endpoint.
1.2, preparation and inspection of hemagglutinin working solution
If the hemagglutination valency assay results in a ratio of 1:1024 (for example), then 4 hemagglutination units (i.e. 4HAU) are 1024/4-256 (i.e. 1: 256).
1.3, 4 preparation of HAU hemagglutinin
Adding 1.0ml of hemagglutinin into 9.0ml of PBS to obtain 1:10 dilution, and adding 1.0ml of the 1:10 dilution into 24.6ml of PBS to obtain the final concentration of 1: 256.
1.4, inspection
To check whether the hemagglutination price of 4HAU was accurate, 1: 256-fold dilutions prepared were added to 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0ml PBS in amounts of 1.0ml, respectively, to give final dilutions of 1:2, 1:3, 1:4, 1:5, 1:6 and 1: 7. Then taking 25 mul from each dilution, adding 25 mul of PBS, adding 25 mul of 1% pig red blood cell suspension, uniformly mixing, placing the hemagglutination plate at 2-8 ℃ for 40-60 minutes, if the prepared antigen liquid is 4HAU, giving out an agglutination end point by 1:4 dilution; if 4HAU is higher than 4 units, 1:5 or 1:6 may be the end point, and if lower, 1:2 or 1:3 may be the end point. The dilution of hemagglutinin should be adjusted appropriately according to the test result to ensure that the working solution is 4 HAU.
2. Hemagglutination inhibition assay
96-well microplate method: serially diluting the serum to be detected by 2 times by using PBS, adding 25 mu l of antigen liquid containing 4HAU into each hole, setting PBS and virus contrast, fully oscillating, placing at room temperature for at least 30 minutes or at 2-8 ℃ for at least 60 minutes, adding 25 mu l of 1% pig red blood cell suspension, placing at 2-8 ℃ for 40-60 minutes, and judging the result when the red blood cells in the contrast holes are obviously button-shaped. See the attached note 4-2 in the table.
TABLE ADIPTIONS 4-2 hemagglutination inhibition assay
3. Method for determining hemagglutination inhibition result
Based on the amount of non-agglutinated blood cells, 100% non-agglutinated blood cells are judged as #; if 75% is not agglutinated, the product is judged to be +++; the 50% non-agglutinated state is judged as ++; 25% of the total amount of the ingredients is judged to be plus; the result was found to be-after agglutination. The samples were judged to be positive when + + or more were present. The highest dilution of serum in positive wells was used as the endpoint for determination. The hemagglutination inhibition titer (HI) of sample No. 2015005 as in the above example was 1: 64.
The canine distemper and canine parvovirus bivalent inactivated vaccine prepared by the preparation method disclosed by the invention is excellent and stable in efficacy, free of any adverse reaction after the canine is inoculated, good in immune effect, and capable of immunizing canine distemper viruses and canine parvoviruses simultaneously only by inoculating one injection, and the preparation method disclosed by the invention is simple.
The above embodiments are described in further detail to solve the technical problems, technical solutions and advantages of the present invention, and it should be understood that the above embodiments are only examples of the present invention and are not intended to limit the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A preparation method of a canine distemper and canine parvo bivalent inactivated vaccine, which is characterized by comprising the following steps of,
the method comprises the following steps:
preparing virus liquid, namely inoculating canine distemper virus seeds to a single-layer Vero cell, harvesting virus liquid when cytopathic effect reaches 80%, and repeatedly freezing and thawing to obtain the canine distemper virus liquid; inoculating canine parvovirus virus seeds to F81 cells, harvesting virus liquid when cytopathic effect reaches more than 90%, and repeatedly freezing and thawing to obtain canine parvovirus liquid;
inactivating viruses, namely adding a formaldehyde solution into the canine distemper virus solution to inactivate the canine distemper virus to obtain a canine distemper virus inactivation solution; adding a formaldehyde solution into the canine parvovirus solution for inactivation to obtain a canine parvovirus inactivation solution;
preparing a vaccine, namely mixing the canine distemper virus inactivation solution and the canine parvovirus inactivation solution according to the ratio of (1-2): 1 to obtain a mixed solution, and then adding aluminum hydroxide to obtain the bivalent inactivated vaccine.
2. The method for preparing the canine distemper and canine parvo bivalent inactivated vaccine according to claim 1,
the canine distemper virus seed is 11 th generation virus seed of a seed batch for producing a CDV-L strain;
and/or the canine parvovirus virus seed is the 10 th generation seed of the seed batch produced by the canine parvovirus CPV-L strain.
3. The method for preparing the canine distemper and canine parvo bivalent inactivated vaccine according to claim 1,
the Vero cell is the 25 th generation cell of a Vero working cell bank;
and/or the F81 cells are the 25 th generation cells of the F81 cell bank.
4. The method for preparing the canine distemper and canine parvo bivalent inactivated vaccine according to claim 1,
in the preparation process of the virus liquid, the canine distemper virus seed liquid with the volume concentration of 2-3% is inoculated to a single-layer Vero
A cell; and/or inoculating the canine parvovirus seed solution with the volume concentration of 1-2% to F81 cells.
5. The method for preparing the canine distemper and canine parvo bivalent inactivated vaccine according to claim 4,
in the preparation process of the virus liquid, after the canine distemper virus seeds are inoculated to a single-layer Vero cell, the cell is cultured for 4-5 days at 35 ℃, the virus liquid is harvested when the cytopathic effect reaches 80%, and after repeated freeze thawing is carried out for 3 times, cell fragments are removed through filtration, so that the canine distemper virus liquid is obtained;
and/or inoculating canine parvovirus virus seeds to F81 cells, culturing overnight at 37 ℃, replacing cell maintenance liquid, continuously culturing for 5 days at 37 ℃, harvesting virus liquid when cytopathic effect reaches more than 90%, repeatedly freezing and thawing for 3 times, and filtering to remove cell fragments to obtain the canine parvovirus liquid.
6. The method for preparing the canine distemper and canine parvo bivalent inactivated vaccine according to claim 1,
adding a formaldehyde solution into the canine distemper virus solution in the virus inactivation process to prepare a virus solution with the volume concentration of formaldehyde being 0.1%, and placing the virus solution in an environment at 4 ℃ for at least 24h, wherein the virus solution is uniformly mixed for a plurality of times in the period to obtain the canine distemper virus inactivation solution;
and/or adding a formaldehyde solution into the canine parvovirus solution to prepare a virus solution with the formaldehyde volume concentration of 0.4%, and placing the virus solution in an environment at 37 ℃ for at least 48h, and uniformly mixing for a plurality of times to obtain the canine parvovirus inactivated solution.
7. The method for preparing the canine distemper and canine parvo bivalent inactivated vaccine according to claim 1,
the titer of the canine distemper virus liquid is more than or equal to 105.3TCID50Per ml; the poison value of the canine parvovirus fluid is more than or equal to 106.0TCID50/ml。
8. The method for preparing the canine distemper and canine parvo bivalent inactivated vaccine according to claim 1,
in the bivalent inactivated vaccine, the content of the aluminum hydroxide is 600-1000 mu g/ml.
9. The method for preparing the canine distemper and canine parvo bivalent inactivated vaccine according to claim 1,
canine distemper virus inactivation solution is respectively 100、10-1、10-2Diluting, inoculating to Vero cells in single layer at 35 deg.C and 5% CO in an amount of 3% of the cell culture solution2Culturing and observing in an incubator, continuously transmitting for 3 generations, and generating no CPE;
canine parvovirus inactivation solution is respectively 100、10-1、10-2After dilution, the cells were inoculated into F81 cells in an amount of 2% by volume based on the cell culture medium of F81 cells, and the cells were incubated at 37 ℃ and 5% CO2Culturing and observing in an incubator, continuously transmitting for 3 generations, and generating no CPE.
10. The method for preparing the canine distemper and canine parvo bivalent inactivated vaccine according to claim 1,
the storage temperature of the canine distemper virus liquid and the canine parvovirus liquid is-20 ℃;
the storage temperature of the canine distemper virus inactivation liquid and the canine parvovirus inactivation liquid is 2-8 ℃.
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