CN107496913A - Brickpox, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method - Google Patents
Brickpox, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method Download PDFInfo
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- CN107496913A CN107496913A CN201710834383.6A CN201710834383A CN107496913A CN 107496913 A CN107496913 A CN 107496913A CN 201710834383 A CN201710834383 A CN 201710834383A CN 107496913 A CN107496913 A CN 107496913A
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Abstract
The invention provides a kind of brickpox, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method, resulting vaccine immunity sow, the more anti-effect of a pin can be played, in the market has been filled up and has lacked brickpox, pig parvoviral, the blank of swine influenza virus triple inactivated vaccine at present.Position is immunized the process employs specific immunologic adjuvant, after vaccine immunity will not cause the red and swollen side reaction generated heat, and not interfere with the spirit and feeding situation of immune rear pig, effectively improve the immune effect and production performance of immune pig.Immune efficacy is suitable compared with each single antigen vaccine in the prior art, and protective rate is more than 80%.Meanwhile the inventive method effectively improves brickpox, pig parvoviral, the stability of swine influenza virus triple inactivated vaccine quality, overcome that trivial operations during spinner culture, working strength are big, the shortcomings of easily polluting.
Description
Technical field
The present invention relates to technical field of vaccines, further to the technology of preparing of multiple vaccines, and in particular to a boar is red
Poison, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method.
Background technology
Brickpox (Swine erysipelas, SE) is by erysipelothrix ruhsiopathiae (Brysipelothrix
Rhusiopathiae a kind of acute type, hot infectious disease caused by), the red heat that is otherwise known as disease (red fever), it is called " sparking
Print ".Clinically using high fever, fash and arthritis as principal character.Brickpox is widely current in all over the world, and China is in 20th century
The eighties is multiple, and with swine fever, swine plague and the three big infectious diseases for claiming Chinese pig industry, brickpox is classified as into two class epidemic diseases in China
Disease.The disease more than 20 years of silence, however, in recent years, on more ground such as China Guangxi, Guangdong, Sichuan, Fujian, Jiangxi, Anhui, Hunan
There is the generation of brickpox, and the trend for thering is morbidity to increase.The generation again of brickpox and popular its reason of summarizing may be：
Bacillus rhusiopathiae suis has extensive host's property, and wherein sick pig and the pig that carries disease germs is the main infection sources；Bacillus rhusiopathiae suis is to extraneous ring
The resistance in border is strong, has potential latency；Raiser abandons immune swine erysipelas vaccine, non-advantage serotype brickpox for many years
Bacillus passes through the evolution and variation of decades, virulence enhancing；With swine fever, pig epidemic diarrhea, pig blue-ear disease, haemophilus parasuis
Disease, swine flu etc. occur together, and have aggravated this sick generation and prevalence.
Pig parvoviral is one of main pathogens for causing sow breeding difficulty, and first farrowing sow tire pig can be caused dead
Production, mummy tire, body early embryo are dead and sterile.The disease betides all over the world extensively, and is in region on most of pig farms
It is popular.The disease is often felt with some other virus, such as the virus mixing of pig circular ring virus, porcine reproductive and respiratory syndrome virus
Dye, induce pmws.Clinically it is found that polytype pig parvoviral, including PPV1,
PPV2, PPV3, PPV4, pig class bocavirus (Porcine boca-like virus, PBo-likeV), pig bocavirus
(PBoV).Research data shows that PPV3 mixes sense with pig circular ring virus with porcine reproductive and respiratory syndrome virus and PBo-likeV
The probability of dye is higher.
Swine flu, full name are pig influenza, are a kind of acute, high degree in contact of the pig as caused by influenza A
Respiratory infectious disease, clinically to occur suddenly, have difficulty in breathing, cough, generate heat, exhaustion, and untreated and rapid rehabilitation
It is characterized.In endemicity infectious disease, swine flu clinically shows unobvious or is difficult to distinguish with other respiratory diseases.
Although swine flu can recovery occurs without treatment, after swinery taint with SIV, can usually increase swinery to the susceptible of other respiratory pathogenses
Property, add and secondary or mixed infection probability occurs, so as to which the prdc of pig (PRDC) occur, exhale swinery
It is increasingly complex to inhale tract disease.The prdc (PRDC) of live pig is related to a variety of cause of diseases, such as swine influenza virus
(SIV), porcine reproductive and respiratory syndrome virus (PRRSV), mycoplasma hyopneumoniae (M.hyopneumonia), pig circular ring virus
(PCV), pasteurella multocida (P.multocida), Streptococcus suis (Streptococcus suis), haemophilus parasuis
(Haemophilus parasuis)。
The content of the invention
It is contemplated that the technological deficiency for prior art, there is provided a kind of brickpox, pig parvoviral, swine influenza virus
Triple inactivated vaccine preparation method, with solve the vaccine product of prior art be difficult to once be inoculated with can be immunized simultaneously it is above-mentioned
The technical problem of a variety of cause of diseases.
Another technical problem to be solved by the present invention is that brickpox antigen, PPV Antigen Using, swine flue antigen
In same vaccine during mixture, it is difficult to ensure the immune effect of antigen.
To realize above technical purpose, the present invention uses following technical scheme：
Brickpox, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method, comprise the following steps：
1) the type C43-5 bacterial strains of bacillus rhusiopathiae suis 2 are cultivated, living bacterial liquid is prepared；
2) antigen is dense in the type C43-5 living bacterial liquids of bacillus rhusiopathiae suis 2 for obtaining step 1) using dilution or by the way of centrifuging
Degree is adjusted to 109-1010CFU/mL；
3) step 2) is adjusted to the type C43-5 bacterium solutions of bacillus rhusiopathiae suis 2 after concentration using formaldehyde to be inactivated；
4) pig parvoviral BJ-2 strains are bred, venom living is prepared；
5) pig parvoviral BJ-2 strain venom in step 4) is inactivated using formaldehyde；
6) swine influenza virus H1N1 strains are bred, venom living is prepared；
7) swine influenza virus H1N1 strain venom in step 6) is inactivated using formaldehyde；
8) the pig parvoviral BJ-2 strains poison of bacillus rhusiopathiae suis C43-5 bacterium solutions, step 5) inactivation after step 3) is inactivated
Liquid, the swine influenza virus H1N1 strains venom of step 7) inactivation are with (1~2):(1~2):The volume ratio of (1~2) mixes, that is, obtains
The antigenic solution of mixing；
9) the hybrid antigen solution that step 8) obtains is mixed with adjuvant, that is, obtains brickpox, pig parvoviral, swine flu
Viral triple inactivated vaccine.
Preferably, pig parvoviral BJ-2 strain virus content >=10 in the step 4) venom living7.0TCID50。
Preferably, swine influenza virus H1N1 content >=10 in the step 6) venom living7.0TCID50。
Preferably, adjuvant described in step 9) is SEPPIC IMS 1313N VG adjuvants.
Preferably, step 1) specifically includes following operation：By the 1%-2% inoculating strains of culture medium total amount, press simultaneously
1%-2% adds cracking blood cell whole blood, or 0.1% Tween-80 and 0.5% glucose, mixes, 37 DEG C of fermentations in fermentation tank,
Dissolved oxygen amount sets 10%-15%, adjusts pH with sodium hydroxide, pH is set as 7.5-7.6, ferments 10-11 hours.
Preferably, the step 3) inactivation is to add 0.3% formalin by bacterium solution volume, 37 DEG C of inactivations 18~24 are small
When, during which every stirring in 2~3 hours once, 3~5 minutes every time.
Preferably, step 4) specifically includes following operation：IBRS-2 cells are inoculated into containing nutrient solution and microcarrier
Reactor in, and cell is well mixed with microcarrier, starts and attach program, make cell attachment on microcarrier；Cell connects
Kind of density is ten thousand/mL of 10-20, and microcarrier used is Cytodex series microcarriers, and the use density of microcarrier is 2-10g/L, institute
It is stirring type bioreactor with bioreactor, bioreactor volume is 14L；Bioreactor setup parameter is：
PH7.0-7.3,36-37 DEG C of temperature, dissolved oxygen 30%-50%, mixing speed 40-60rpm；When cell density reaches 200-500
Ten thousand/mL, is replaced with viral maintaining liquid, is inoculated with pig parvoviral, and virus inoculation amount be 0.01-0.1MOI, viral maintaining liquid for containing
The DMEM of 2% serum, pH value 7.1-7.4, bioreactor set virus breeding parameter as 36-37 DEG C of temperature, dissolved oxygen 35-
60%th, mixing speed 40-60rpm.
Preferably, the step 5) inactivation is in sterile chamber, formalin is added until wherein into virus liquid
Concentration of formaldehyde is 0.3% (w/w), then acts at 37 DEG C 48 hours, during which shakes 3-4 times.
Step 6) specifically includes following operation：By mdck cell with 1 × 105-2×105/ mL inoculum concentration is inoculated into reaction
In device, and cell is well mixed with microcarrier, starts and attach program, make cell attachment on microcarrier；Microcarrier used is
Cytodex series microcarriers, the use density of microcarrier is 2-10g/L, and bioreactor used is stirring type bioreactor,
Bioreactor volume is 14L；Bioreactor setup parameter is：PH7.0-7.2,33 DEG C -37 DEG C of temperature, dissolved oxygen 30%-
40%th, mixing speed 40-60rpm；When 80%-90% is paved with cell on carrier, it is replaced with containing 2 μ g/mL pancreatin serum-frees
MEM culture mediums, be inoculated with swine influenza virus H1N1, virus inoculation amount is MOI 0.002-0.005, continues to cultivate.
Preferably, the step 7) inactivation is in sterile chamber, formalin is added until wherein into virus liquid
Concentration of formaldehyde is 0.3% (w/w), then acts at 37 DEG C 48 hours, during which shakes 3-4 times.
In above technical scheme, the SEPPIC IMS 1313N VG adjuvants, refer to it is being produced by SEPPIC companies,
Model IMS 1313N VG vaccine adjuvant, can buy from market.
The invention provides a kind of brickpox, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method, gained
The vaccine immunity sow arrived, the more anti-effect of a pin can be played, filled up in the market lack at present brickpox, pig parvoviral,
The blank of swine influenza virus triple inactivated vaccine.The process employs specific immunologic adjuvant, position is immunized not after vaccine immunity
The red and swollen side reaction generated heat can be caused, the spirit and feeding situation of immune rear pig is not interfered with, effectively improve immune pig
Immune effect and production performance.Immune efficacy is suitable compared with each single antigen vaccine in the prior art, and protective rate is 80%
More than.
In the preparation method, bacillus rhusiopathiae suis prepares bacterium solution, pig parvoviral, swine influenza virus by the way of fermentation
Using microcarrier suspension culture mode propagative viruses, dissolved oxygen in vaccine antigen growth course, pH conditions are strictly controlled, is ensured
The immunogenicity of antigen is homogeneous between batch.With the production work of traditional brickpox inactivated vaccine, porcine parvovirus inactivated vaccines
Skill is compared, the life of brickpox, pig parvoviral, the fermented and cultured of swine influenza virus triple inactivated vaccine and microcarrier suspension culture
Production. art effectively improves brickpox, pig parvoviral, the stability of swine influenza virus triple inactivated vaccine quality, overcomes and turns
Trivial operations in bottle incubation, working strength is big, the shortcomings of easily polluting.
Embodiment
The embodiment of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
It is will not be described in detail in following examples to belonging to known structure or function.
Approximating language used in following examples can be used for quantitative expression, show do not changing the feelings of basic function
Under condition quantity can be allowed to have certain variation.Therefore, it is accurate that the numerical value corrected with the language such as " about ", " left and right " is not limited to this
Numerical value is in itself.In certain embodiments, the numerical value for " about " representing to allow its amendment is in the scope of positive and negative 10 (10%)
Interior change, such as, " about 100 " represent can be any numerical value between 90 to 110.In addition, " the about first numerical value arrives
In the statement of second value ", the first and second numerical value two values are at about corrected.In some cases, approximating language
May be relevant with the precision of measuring instrument.
In addition to being defined, technology used and scientific terminology have and art technology people of the present invention in following examples
The identical meanings that member is commonly understood by.
Embodiment 1
A kind of brickpox, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method, its step are as follows：
It is prepared by 1 production seed
It is prepared by 1.1 bacillus rhusiopathiae suises production seed
1.1.1 first order seed is bred
After bacillus rhusiopathiae suis freeze-drying lactobacillus is broken seal, streak inoculation is in blood agar inclined-plane after adding martin's bouillon dilution, and 37
DEG C culture 24 hours, rules in 10% serum Martin agar plate, cultivates 24-36 hours, and it is some to select the homogeneous bacterium colony of typical case, connects
Kind blood agar slant, 37 DEG C are cultivated 24 hours, as first order seed.
1.1.2 secondary seed is bred
First order seed is taken to be inoculated in martin's bouillon, 37 DEG C are cultivated 24 hours, are inoculated in substantial amounts of meat liver peptic digest soup
Or in martin's bouillon, cultivate 24 hours, by existing《Chinese veterinary pharmacopoeia》Annex is purely examined, and two level kind is used as after qualified
Son.
1.2 pig parvovirals produce seminal propagation
1.2.1 cell is bred
Young porcine kidney cell (IBRS-2) is recovered, adds cell nutrient solution, 37 DEG C of culture 24-48 hours, generation is good
Passed on after good cell monolayer.Net nutrient solution is abandoned during passage, adds EDTA- pancreatin digestive juices, is acted on several minutes, digestion is scattered
Cell suspension uniformly is made.Then it is sub-packed in Tissue Culture Flask, puts 37 DEG C and cultivate 24 hours, by existing《Chinese veterinary pharmacopoeia》It is attached
Record carries out sterile, mycoplasma and exogenous virus is examined, and should meet regulation.
1.2.2 seed culture of viruses is bred
By well-grown individual layer IBRS-2 cells, growth-promoting media is removed, changes with the cell maintenance medium containing 5% seed culture of viruses, puts
37 DEG C are continued to cultivate, daily to observe cytopathy situation, cytopathy occur in 36-72 hours, when lesion reaches more than 90%
When can harvest.Quantitative separating freezen protective or adds suitable stabilizer in bottle, preserves after vacuum freezedrying.Indicate harvest
Date, quantity, seed culture of viruses algebraically etc..
1.3 swine influenza viruses produce seminal propagation
1.3.1 cell is bred
Dog kidney passage cell (MDCK) is recovered, adds cell growth medium (92%-94%MEM liquid, the small ox bloods of 6%-8%
Clearly, pH 70-7.2), spinner culture, cell is passed on when covering with 90-100%.Net nutrient solution is abandoned during passage, adds EDTA-
Pancreatin digestive juice, act on several minutes, digestion, which is uniformly dispersed, is made cell suspension.Then it is sub-packed in Tissue Culture Flask, puts 37 DEG C
Culture 24 hours, by existing《Chinese veterinary pharmacopoeia》Annex carries out sterile, mycoplasma and exogenous virus is examined, and should meet regulation.
1.3.2 seed culture of viruses is bred
Well-grown mdck cell is taken, removes growth-promoting media, poison amount inoculation swine influenza virus is connect by 0.2%-0.5%
H1N1,37 DEG C of continuous cultures are put, observe cell growth status, 36-48 hour harvesting cultures daily, freeze thawing harvests three times
Virus.Quantitative separating freezen protective or adds suitable stabilizer in bottle, preserves after vacuum freezedrying.Indicate harvest date, number
Amount, seed culture of viruses algebraically etc..
It is prepared by 2 antigen for vaccine
2.1 seedlings are prepared with bacillus rhusiopathiae suis antigen
2.1.1 prepared by bacterium solution
Secondary seed is inoculated with by the 1%-2% of culture medium total amount, while cracking blood cell whole blood is added by 1%-2%, or
0.1% Tween-80 and 0.5% glucose, mix, 37 DEG C of fermentations in fermentation tank, dissolved oxygen amount setting 10%-15%, use hydrogen-oxygen
Change sodium regulation pH, pH and be set as 7.5-7.6, ferment 10-11 hours.Purely inspection and count plate are carried out after the completion of fermentation, is pressed
It is existing《Chinese veterinary pharmacopoeia》Annex is tested, should be pure.
2.1.2 somatic antigen concentration adjusts
Qualified bacterium solution will be examined to adjust bacterial concentration according to count results, adjusted to 109-1010CFU/mL；By existing
OK《Chinese veterinary pharmacopoeia》Annex is tested, should be pure.
2.1.3 somatic antigen inactivates
Qualified bacterium solution will be examined, by bacterium solution volume plus 0.3% formalin, 37 DEG C inactivate 18~24 hours, during which every
Every stirring in 2~3 hours once, 3~5 minutes every time, sampled after inactivation, by existing《Chinese veterinary pharmacopoeia》Annex is tested, should
Without bacterial growth.
2.2 seedlings are prepared with PPV Antigen Using
2.2.1 seedling cell culture
IBRS-2 cells are inoculated into the reactor containing nutrient solution and microcarrier, and above-mentioned cell is mixed with microcarrier
Close uniformly, start and attach program, make cell attachment on microcarrier.Cell-seeding-density is ten thousand/mL of 10-20, microcarrier used
For Cytodex series microcarriers, the use density of microcarrier is 2-10g/L, and bioreactor used is stirring-type biological respinse
Device, bioreactor volume are 14L.Bioreactor setup parameter is：PH7.0-7.3,36-37 DEG C of temperature, dissolved oxygen 30%-
50%th, mixing speed 40-60rpm.
2.2.2 virus inoculation and culture
When above-mentioned seedling cell density reaches ten thousand/mL of 200-500, viral maintaining liquid is replaced with, is inoculated with pig parvoviral,
Virus inoculation amount is 0.01-0.1MOI, and viral maintaining liquid is the DMEM containing 2% serum, pH value 7.1-7.4, and bioreactor is set
It is 36-37 DEG C of temperature to determine virus breeding parameter, dissolved oxygen 35-60%, mixing speed 40-60rpm.After inoculation at regular intervals
The microcarrier in bioreactor is taken, with micro- sem observation cytopathy situation, and cell detachment situation, and detects sample
TCID50.Viral level reaches 107.0TCID50/ mL can harvest virus liquid.
2.2.3 virus liquid inactivates
Qualified virus liquid hybrid filtering will be examined the mixing of side edged, to make in the formalin in sterile chamber, adding 40%
Formalin concentration is 0.3% in mixed liquor, then puts 37 DEG C and acts on 48 hours, during which shakes 3-4 times, sample, press after inactivation
It is existing《Chinese veterinary pharmacopoeia》Annex is tested, should be without bacterial growth.
2.3 seedlings are prepared with swine flue antigen
2.3.1 seedling cell culture
By mdck cell 1 × 105-2×105/ mL is inoculated into reactor, and above-mentioned cell is well mixed with microcarrier,
Start and attach program, make cell attachment on microcarrier.Microcarrier used is Cytodex series microcarriers, the use of microcarrier
Density is 2-10g/L, and bioreactor used is stirring type bioreactor, and bioreactor volume is 14L.Bioreactor
Setup parameter is：PH7.0-7.2,33 DEG C -37 DEG C of temperature, dissolved oxygen 30%-40%, mixing speed 40-60rpm.
2.3.2 virus inoculation and culture
When 80%-90% is paved with cell on carrier, it is replaced with viral maintaining liquid and (contains 2 μ g/mL pancreatin serum-frees
MEM), swine influenza virus H1N1 is inoculated with, virus inoculation amount is MOI 0.002-0.005.Biology is taken after inoculation at regular intervals
Microcarrier in reactor, with micro- sem observation cytopathy situation, and cell detachment situation, and detect sample TCID50.Virus
Content reaches 107.0TCID50/ more than mL can harvest virus liquid.
2.3.3 virus liquid inactivates
Qualified virus liquid hybrid filtering will be examined the mixing of side edged, to make in the formalin in sterile chamber, adding 40%
Formalin concentration is 0.3% in mixed liquor, then puts 37 DEG C and acts on 48 hours, during which shakes 3-4 times, sample, press after inactivation
It is existing《Chinese veterinary pharmacopoeia》Annex is tested, should be without bacterial growth.
The preparation of 3 vaccines
By the type C43-5 bacterial strains of bacillus rhusiopathiae suis 2 of inactivation, the pig parvoviral BJ-2 of inactivation, the swine influenza virus of inactivation
H1N1 by volume 1:1:1, mixed antigen mixed liquor by volume 1:1 adds SEPPIC IMS 1313N VG, in room temperature
Under, 3 minutes in the spiral agitator (500rpm) of low-shearing force, prepare stabilization of vaccines emulsion.
4 steriling tests
Above-mentioned vaccine is taken by existing《Chinese veterinary pharmacopoeia》Annex is tested, should be without bacterial growth.
The vaccine safety Journal of Sex Research prepared in the embodiment 1 of embodiment 2
Vaccine in Example 1, head part (2mL/ heads part), the 1.5-2 months after 5 part wean of intramuscular injection are indicated by label
(swine fever neutralizing antibody is negative, pig parvoviral HI antibody titers for age pig<8) 5, control group 5 is not immunized, and observes 21 day by day
Day, should be without the locally or systemically adverse reaction because caused by vaccinating.
It the results are shown in Table 1.
The safety research result of table 1
Safety testing result is shown：Brickpox, pig parvoviral, swine influenza virus triple inactivated vaccine are to weanling pig
Safety.
The vaccine potency of embodiment 3 is studied
1 experimental design
1.1 vaccine immunity
Vaccine prepared by Example 1, head part, every part 2mL, 1-2 after 1 part wean of intramuscular injection are indicated by label
Month more than body weight 20kg healthy susceptible pig 15, two exempt from after head exempts from 21, immunizing dose and method is same exempts from, while set pair
According to group 15, it is not immunized.
Buy commercially available brickpox inactivated vaccine (C43-5), porcine parvovirus inactivated vaccines (CP-99), swine influenza virus
H1N1 hypotype inactivated vaccines, respectively by 1-2 month body weight 20kg after its specification injecting method and injection dosage injection wean with
On healthy susceptible each 5 parts of pig.Vaccine is grouped as follows shown in table.
The vaccine immunity of table 2 is grouped
1.2 attack poison
3 weeks after immune, brickpox, pig parvoviral, swine influenza virus triple inactivated vaccine and brickpox inactivated vaccine are exempted from
Epidemic disease group and each 5 of control group, the mixing bacterium solution of each strong toadstool of the type of bacillus rhusiopathiae suis 1 for being injected intravenously 1MLD and 2 types (each 1 plant)；
4 weeks after immune, brickpox, pig parvoviral, swine influenza virus triple inactivated vaccine and porcine parvovirus inactivated vaccines immune group
5 each with control group, each collunarium attacks the 10 of malicious 4mL5.0TCID50/ mL venom；4 weeks after immune, brickpox, pig parvoviral,
Swine influenza virus triple inactivated vaccine and swine influenza virus H1N1 hypotype inactivated vaccine immune groups and each 5 of control group, each collunarium
Velogen strain H1N1 corresponding with intratracheal injection 1mL, every part 108.0TCID50.Attack poison and be grouped as follows table.
Table 3 attacks malicious packet
1.3 efficacy test results
Bacillus rhusiopathiae suis, pig parvoviral, swine influenza virus are observed 14 after attacking poison, record immune group and control group body
The clinical symptoms such as temperature, breathing, body weight and animal dead head number.As a result see the table below.
The efficacy test result of table 4
Embodiment 4
Brickpox, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method, comprise the following steps：
1) the type C43-5 bacterial strains of bacillus rhusiopathiae suis 2 are cultivated, living bacterial liquid is prepared；
2) antigen is dense in the type C43-5 living bacterial liquids of bacillus rhusiopathiae suis 2 for obtaining step 1) using dilution or by the way of centrifuging
Degree is adjusted to 109CFU/mL；
3) step 2) is adjusted to the type C43-5 bacterium solutions of bacillus rhusiopathiae suis 2 after concentration using formaldehyde to be inactivated；
4) pig parvoviral BJ-2 strains are bred, venom living is prepared；
5) pig parvoviral BJ-2 strain venom in step 4) is inactivated using formaldehyde；
6) swine influenza virus H1N1 strains are bred, venom living is prepared；
7) swine influenza virus H1N1 strain venom in step 6) is inactivated using formaldehyde；
8) the pig parvoviral BJ-2 strains poison of bacillus rhusiopathiae suis C43-5 bacterium solutions, step 5) inactivation after step 3) is inactivated
Liquid, the swine influenza virus H1N1 strain venom of step 7) inactivation are with 1:2:1 volume ratio mixes, that is, the antigenic solution mixed；
9) the hybrid antigen solution that step 8) obtains is mixed with adjuvant, that is, obtains brickpox, pig parvoviral, swine flu
Viral triple inactivated vaccine.
On the basis of above technical scheme, meet following condition：
Pig parvoviral BJ-2 strain virus content is 10 in the step 4) venom living7.0TCID50。
Swine influenza virus H1N1 contents are 10 in the step 6) venom living7.0TCID50。
Adjuvant described in step 9) is SEPPIC IMS 1313N VG adjuvants.
Embodiment 5
Brickpox, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method, comprise the following steps：
1) the type C43-5 bacterial strains of bacillus rhusiopathiae suis 2 are cultivated, living bacterial liquid is prepared；
2) antigen is dense in the type C43-5 living bacterial liquids of bacillus rhusiopathiae suis 2 for obtaining step 1) using dilution or by the way of centrifuging
Degree is adjusted to 1010CFU/mL；
3) step 2) is adjusted to the type C43-5 bacterium solutions of bacillus rhusiopathiae suis 2 after concentration using formaldehyde to be inactivated；
4) pig parvoviral BJ-2 strains are bred, venom living is prepared；
5) pig parvoviral BJ-2 strain venom in step 4) is inactivated using formaldehyde；
6) swine influenza virus H1N1 strains are bred, venom living is prepared；
7) swine influenza virus H1N1 strain venom in step 6) is inactivated using formaldehyde；
8) the pig parvoviral BJ-2 strains poison of bacillus rhusiopathiae suis C43-5 bacterium solutions, step 5) inactivation after step 3) is inactivated
Liquid, the swine influenza virus H1N1 strain venom of step 7) inactivation are with 2:1:1 volume ratio mixes, that is, the antigenic solution mixed；
9) the hybrid antigen solution that step 8) obtains is mixed with adjuvant, that is, obtains brickpox, pig parvoviral, swine flu
Viral triple inactivated vaccine.
Embodiments of the invention are described in detail above, but the content is only presently preferred embodiments of the present invention,
It is not intended to limit the invention.All all any modification, equivalent and improvement done in the application range of the present invention etc., all should
Within protection scope of the present invention.
Claims (10)
1. brickpox, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method, it is characterised in that including following step
Suddenly：
1) the type C43-5 bacterial strains of bacillus rhusiopathiae suis 2 are cultivated, living bacterial liquid is prepared；
2) antigen concentration is adjusted in the type C43-5 living bacterial liquids of bacillus rhusiopathiae suis 2 for obtaining step 1) using dilution or by the way of centrifuging
It is whole to 109-1010CFU/mL；
3) step 2) is adjusted to the type C43-5 bacterium solutions of bacillus rhusiopathiae suis 2 after concentration using formaldehyde to be inactivated；
4) pig parvoviral BJ-2 strains are bred, venom living is prepared；
5) pig parvoviral BJ-2 strain venom in step 4) is inactivated using formaldehyde；
6) swine influenza virus H1N1 strains are bred, venom living is prepared；
7) swine influenza virus H1N1 strain venom in step 6) is inactivated using formaldehyde；
8) bacillus rhusiopathiae suis C43-5 bacterium solutions, the pig parvoviral BJ-2 strains venom of step 5) inactivation, step after step 3) is inactivated
The swine influenza virus H1N1 strains venom of rapid 7) inactivation is with (1~2):(1~2):The volume ratio of (1~2) mixes, that is, is mixed
Antigenic solution；
9) the hybrid antigen solution that step 8) obtains is mixed with adjuvant, that is, obtains brickpox, pig parvoviral, swine influenza virus
Triple inactivated vaccine.
2. brickpox according to claim 1, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method, its
It is characterised by pig parvoviral BJ-2 strain virus content >=10 in the step 4) venom living7.0TCID50。
3. brickpox according to claim 1, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method, its
It is characterised by swine influenza virus H1N1 content >=10 in the step 6) venom living7.0TCID50。
4. brickpox according to claim 1, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method, its
It is SEPPIC IMS 1313N VG adjuvants to be characterised by adjuvant described in step 9).
5. brickpox according to claim 1, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method, its
It is characterised by that step 1) specifically includes following operation：By the 1%-2% inoculating strains of culture medium total amount, while add by 1%-2%
Enter to crack blood cell whole blood, or 0.1% Tween-80 and 0.5% glucose, mix, 37 DEG C of fermentations in fermentation tank, dissolved oxygen amount setting
10%-15%, pH is adjusted with sodium hydroxide, pH is set as 7.5-7.6, ferments 10-11 hours.
6. brickpox according to claim 1, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method, its
Be characterised by that the step 3) inactivation is to add 0.3% formalin by bacterium solution volume, 37 DEG C inactivate 18~24 hours, during which every
Stir once within 2~3 hours, 3~5 minutes every time.
7. brickpox according to claim 1, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method, its
It is characterised by that step 4) specifically includes following operation：IBRS-2 cells are inoculated into the reactor containing nutrient solution and microcarrier
It is interior, and cell is well mixed with microcarrier, start and attach program, make cell attachment on microcarrier；Cell-seeding-density is
Ten thousand/mL of 10-20, microcarrier used are Cytodex series microcarriers, and the use density of microcarrier is 2-10g/L, and biology used is anti-
It is stirring type bioreactor to answer device, and bioreactor volume is 14L；Bioreactor setup parameter is：PH7.0-7.3, temperature
36-37 DEG C of degree, dissolved oxygen 30%-50%, mixing speed 40-60rpm；
When cell density reaches ten thousand/mL of 200-500, viral maintaining liquid is replaced with, is inoculated with pig parvoviral, virus inoculation amount is
0.01-0.1MOI, viral maintaining liquid are the DMEM containing 2% serum, pH value 7.1-7.4, and bioreactor, which sets virus breeding, joins
Number is 36-37 DEG C of temperature, dissolved oxygen 35-60%, mixing speed 40-60rpm.
8. brickpox according to claim 1, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method, its
It is in sterile chamber to be characterised by the step 5) inactivation, and formalin is added until wherein concentration of formaldehyde is into virus liquid
0.3% (w/w), then act at 37 DEG C 48 hours, during which shake 3-4 times.
9. brickpox according to claim 1, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method, its
It is characterised by that step 6) specifically includes following operation：By mdck cell with 1 × 105-2×105/ mL inoculum concentration is inoculated into reaction
In device, and cell is well mixed with microcarrier, starts and attach program, make cell attachment on microcarrier；Microcarrier used is
Cytodex series microcarriers, the use density of microcarrier is 2-10g/L, and bioreactor used is stirring type bioreactor,
Bioreactor volume is 14L；Bioreactor setup parameter is：PH7.0-7.2,33 DEG C -37 DEG C of temperature, dissolved oxygen 30%-
40%th, mixing speed 40-60rpm；
When 80%-90% is paved with cell on carrier, the MEM culture mediums containing 2 μ g/mL pancreatin serum-frees, Pigs Inoculated stream are replaced with
Influenza Virus H1N1, virus inoculation amount are MOI 0.002-0.005, continue to cultivate.
10. brickpox according to claim 1, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method, its
It is in sterile chamber to be characterised by the step 7) inactivation, and formalin is added until wherein concentration of formaldehyde is into virus liquid
0.3% (w/w), then act at 37 DEG C 48 hours, during which shake 3-4 times.
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