CN109750004A - One plant of LN plants of Raccoon dog parvovirus enteritis virus, Raccoon dog parvovirus enteritis inactivated vaccine and preparation method thereof - Google Patents
One plant of LN plants of Raccoon dog parvovirus enteritis virus, Raccoon dog parvovirus enteritis inactivated vaccine and preparation method thereof Download PDFInfo
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Abstract
One plant of LN plants of Raccoon dog parvovirus enteritis virus, Raccoon dog parvovirus enteritis inactivated vaccine and preparation method thereof, belong to veterinary biologics field.Of the invention LN plants of one plant of Raccoon dog parvovirus enteritis virus, the strain are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number on December 7th, 2018 are as follows: CGMCC NO.16627.Its effective active composition of Raccoon dog parvovirus enteritis inactivated vaccine of the invention is LN plants of Raccoon dog parvovirus enteritis virus.The present invention prepares Raccoon dog parvovirus enteritis inactivated vaccine using the full suspension technology of cell; Raccoon dog parvovirus strong virus attack can be resisted; protective rate is 91.7% or more; racoon dog viral enteritis can effectively be prevented; Effective immune period 6 months; it is undesirable to solve effect existing for existing vaccine immunity recoon dog, using the complexity of process existing for spinner culture vaccine, period length, unstable quality, the problem that differences between batches are big, side reaction is high.
Description
Technical field
The invention belongs to veterinary biologics technical fields, and in particular to one plant of LN plants of Raccoon dog parvovirus enteritis virus,
Raccoon dog parvovirus enteritis inactivated vaccine and preparation method thereof.
Background technique
Raccoon dog parvovirus enteritis is a kind of acute, highly contagious disease, has become seriously endanger racoon dog in recent years
One of the Important Infectious Diseases of sub- aquaculture.Raccoon dog parvovirus enteritis in 1984 occurs for the first time in Heilongjiang Province, China.Clinic is main
It to be increased with body temperature, up to 40 degree or more；Spirit is depressed, vomiting, and appetite stimulator or abolish are intended to increase yearningly；Violent diarrhea, discharge are mixed
Have blood, mucus watery stool or be mixed with the loose stools of intestinal mucosa, some appearance casts are just etc..Recoon dog Canine Parvovirus Enteritis is year after year
It grows in intensity, it is domestic at present not yet specifically for the vaccine of Raccoon dog parvovirus enteritis, Mink Parvovirus is mostly used greatly
Enteritis inactivated vaccine or canine parvovirus attenuated vaccine are immunized, although antigen has certain cross immunity to protect in immunology
Shield, but immune effect is not ideal enough.
No matter what preparation process used is all spinner culture in addition, traditional inactivated vaccine or attenuated vaccine, cultivated
Journey very complicated, large labor intensity, is taken a lot of work arduously at period length, and unstable quality, and differences between batches are big, and pair is anti-after injecting animal
It should be higher.Therefore, safe and effective Raccoon dog parvovirus enteritis vaccine is developed, is the urgent need in industry and market, is had wide
Wealthy market application value, is of great significance to prevention and control recoon dog or even fur-bearing animal Canine Parvovirus Enteritis.
Summary of the invention
In order to solve to mostly use Mink Parvovirus Enteritis inactivated vaccine or canine parvovirus attenuated vaccine to racoon dog greatly at present
It is undesirable that son carries out immune existing immune effect, and it is complicated using process existing for spinner culture vaccine, the period is long, time-consuming takes
The problem that power, unstable quality, differences between batches are big, side reaction is high, the present invention provide one plant of Raccoon dog parvovirus enteritis virus LN
Strain, Raccoon dog parvovirus enteritis inactivated vaccine and preparation method thereof.
Used technical solution is as follows in order to solve the technical problem by the present invention:
Of the invention LN plants of one plant of Raccoon dog parvovirus enteritis virus, during which has been preserved on December 7th, 2018
State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number are as follows: CGMCC NO.16627.
A kind of microbial inoculum of the invention, effective active composition are above-mentioned LN plants of one plant of Raccoon dog parvovirus enteritis virus.
A kind of Raccoon dog parvovirus enteritis inactivated vaccine of the invention, effective active composition are that one plant of above-mentioned racoon dog is tiny
LN plants of viral enteritis virus.
A kind of preparation method of Raccoon dog parvovirus enteritis inactivated vaccine of the invention, comprising the following steps:
Step 1: suspension cell is recovered
It recovers to F81-TY plants of cells；
Step 2: suspension cell passes on
F81-TY strain cell after step 1 is recovered is expanded culture with low serum suspending nutrient solution, obtains F81-TY
Strain cell culture fluid；
Step 3: viral shaking flask domestication
Culture domestication is carried out to LN plants of Raccoon dog parvovirus enteritis virus, filters out the Raccoon dog parvovirus for the culture that suspends
Property enteritis virus produce seed culture of viruses；
Step 4: virus multiplication culture
Raccoon dog parvovirus enteritis virus production seed culture of viruses after step 3 is tamed is seeded to the F81-TY that step 2 obtains
In strain cell culture fluid, virus liquid is harvested after proliferation；
Step 5: seedling
Raccoon dog parvovirus enteritis inactivated vaccine is prepared using the virus liquid that step 4 obtains.
As preferred embodiment, according to Step 3: Step 1: Step 2: Step 4: the sequence of step 5 carries out epidemic disease
Seedling preparation.
As preferred embodiment, detailed process is as follows for step 1:
The low full suspension cell of serum of the F81-TY strain of liquid nitrogen cryopreservation is taken, quick-thawing is placed in 37 DEG C of water-baths and is inoculated in and contain
In the shaking flask for having low serum suspending nutrient solution 400mL, serum-concentration 1-5% is placed in 37 DEG C, 60rpm, 5% (v/v) CO2It shakes
Bed culture 72 hours, reaches 8.0 × 10 to cell density5A/mL or more, when and cell viability >=95%, after being recovered
F81-TY plants of cells.
As preferred embodiment, detailed process is as follows for step 2:
(1) the F81-TY strain cell after step 1 being recovered is in the ratio for being 1:4 with low serum suspending nutrient solution volume ratio
It is inoculated in the shaking flask equipped with low serum suspending nutrient solution, in 37 DEG C, 60rpm, 5% (v/v) CO2Shaking table culture 72 hours；To
Suspension cell density is not less than 7.5 × 105A/mL when and cell viability >=90%, obtains the cell suspending liquid of Shake flask grown；
(2) cell suspending liquid of Shake flask grown is directly inoculated into the bioreactor of working volume 5L, is inoculated with volume
For 1000-2000mL, low serum suspending nutrient solution is added to 5L, three road gas of bioreactor parameter setting, respectively air,
Oxygen and carbon dioxide, revolving speed 60-75rpm, temperature are 37 DEG C, PH 7.1, and after culture 36 hours, dissolved oxygen is set as
50%, the cell culture fluid cell density after continuing culture 36 hours is not less than 7.5 × 105A/mL, and cell viability >=
90%；
(3) it takes cell culture fluid 3.5L obtained in step (2) to be transferred in the bioreactor of working volume 10L, mends
Add low serum suspending nutrient solution to 10L, three road gas of bioreactor parameter setting, respectively air, oxygen and carbon dioxide,
Revolving speed is 75-85rpm, and temperature is 37 DEG C, PH 7.1, and dissolved oxygen 70% carries out trypan blue staining after continuing culture 72 hours
Counting and viability examination, when the viable cell density detected is not less than 7.5 × 105To get to for connecing malicious cell when a/mL
Culture solution；
(4) the bioreactor remaining cell culture solution 1.5L of working volume 5L is added into low serum suspending nutrient solution extremely
5L continues to cultivate, continuously cultivate as cell seed according to step (2) and step (3), obtains F81-TY plants of cell culture fluids.
As preferred embodiment, detailed process is as follows for step 3:
(1) cell suspending liquid of the Shake flask grown obtained in (1) of step 2 is compared to the ratio for 1:3 or 1:4 according to passage
Passage, synchronous LN plants of Raccoon dog parvovirus enteritis virus of inoculation, connecing toxic dose is 5-10% (v/v)；
(2) in 37 DEG C, 5% (v/v) CO2Shaking table culture 48 hours, its virus titer of every 12 hours sample detections and blood clotting
Potency detects the provirus sensibility under the conditions of suspension cell, is denoted as F1 generation；
(3) F1 generation virus liquid is repeated to connect poison according to step (1), meets the 5-10% that toxic dose is Cell suspension volumes,
By step (2) CMC model, its virus titer of every 12 hours sample detections and hemagglutinative titer are denoted as F2 generation, are seeded to repeatedly
In F8 generation, connecing toxic dose is 5% (v/v), detects each generation virus titer and hemagglutinative titer, since F5 generation, viral level and blood
Solidifying potency is stable 10 respectively7.0TCID50/ ml, HA 210, obtain raw for the Raccoon dog parvovirus enteritis virus for the culture that suspends
Seed culture of viruses is produced, is saved in -20 degree.
As preferred embodiment, detailed process is as follows for step 4:
In the Raccoon dog parvovirus after synchronous access step 3 domestication of the 5-10% ratio of F81-TY plants of cell culture fluid volumes
Enteritis virus produces seed culture of viruses, condition of culture are as follows: three road gas of bioreactor parameter setting, respectively air, oxygen and dioxy
Change carbon, revolving speed 40-50rpm, temperature is 37 DEG C, PH 6.8, dissolved oxygen 40-50%；Sampling in every 12 hours carries out trypan blue dye
Color method cell count and vigor inspection after cultivating 72-96h, are lower than 5 × 10 to viable cell density4A/mL or cell activity≤
When 50%, virus liquid is aseptically harvested, and pass through blood coagulation tests, TCID50Virus titer is detected, seedling virus is obtained
Liquid, viral level TCID50It is 106.7/ ml, HA-HI test HA are 210。
As preferred embodiment, detailed process is as follows for step 5:
(1) it inactivates
It is added in the ratio that inactivator beta-propiolactone and virus liquid volume ratio are 1:1500 into the virus liquid that step 4 obtains
Inactivator beta-propiolactone stirs 30min, mixes, 4 DEG C of inactivation 48h, intermittent stirring 3-5 times, each 20min, 37 DEG C of hydrolysis 3-
6h, 4 DEG C of preservations obtain inactivation liquid；
(2) inspection of semifinished product
Steriling test: steriling test is carried out to the virus liquid sampling that step 4 harvests；
Inactivation is examined: to the resulting inactivation liquid of step (1), being inoculated with 4 bottles of normal cats kidney passage cells respectively at 37 DEG C, often
Bottle inoculation 0.4mL, sets and cultivates at 37 DEG C, discard MEM culture medium afterwards for 24 hours, add new MEM culture medium, after continuing culture 3 days again
Lesion should not occur in passage 1 time, cell, harvest culture solution, and feminine gender should be presented in hemagglutination test, determine that inactivation is qualified；
(3) it emulsifies and matches seedling
Liquid will be inactivated and aluminium hydroxide gel is the ratio mixing of 9:1 (v/v) by volume, and added by the 0.01% of final volume
Enter thimerosal, then sterile saline is added by the 50% of final volume, emulsification mixes, and adjusts pH value with sterilizing sodium bicarbonate and is
7.2-7.4 obtains Raccoon dog parvovirus enteritis inactivated vaccine.
The beneficial effects of the present invention are:
1, one plant isolated out of clinical onset recoon dog enteron aisle of the present invention to recoon dog safety, immunogenicity is good, disease
Poison is proliferated stable Raccoon dog parvovirus enteritis vaccine strain, name are as follows: LN plants of Raccoon dog parvovirus enteritis virus, microorganism protects
Hiding number is deposit number are as follows: CGMCC NO.16627.
2, it is prepared for inactivated vaccine for recoon dog Canine Parvovirus Enteritis viral prevalence strain (LN plants) for the first time, for preventing
Recoon dog Canine Parvovirus Enteritis improves the protective rate of recoon dog viral enteritis, solves in the past by mink viral enteritis
Inactivated vaccine or canine viral enteritis attenuated live vaccines are to the hypodynamic defect of recoon dog viral enteritis cross protection.
3, suspension process prepares antigenic compound, virus multiplication and harvest time to suspension process using not related
Property.Present invention discover that low serum suspends, application of the F81-TY plants of cells of culture in the production of Raccoon dog parvovirus enteritis virus, makes
Target virus fast breeding receives malicious time more traditional rolling bottle technique and is advanced by the production cycle for shortening the antigenic compound for 24 hours.
Suspend application of the F81-TY plants of cells of culture in the production of Raccoon dog parvovirus enteritis antigen protein compound, and it is thin to be expected to termination racoon dog
The rolling bottle epoch of minor illness viral enteritis antigen protein compound.
4, the present invention is by the adjustment of experiment parameter, and repeatedly suspend passage and attenuation, filters out one plant and adapts to low serum suspension
The Raccoon dog parvovirus adapted strain of F81-TY plants of cell culture is used for the production of Raccoon dog parvovirus enteritis antigen protein compound, solution
Virus prepared by traditional attached cell of having determined is not susceptible in suspension cell technique, is suspension process in racoon dog the drawbacks of proliferation
Application in the preparation of Canine Parvovirus Enteritis antigen protein compound solves technological difficulties.
5, the present invention selects F81-TY plants of cell lines of low serum suspension that the adherent F81 cell of tradition is replaced to prepare Raccoon dog parvovirus
Property enteritis antigen protein compound, solve because between rolling bottle adherent cell growth bottle, differences between batches are big due to lead to product quality batch
Between the big problem of difference, and provide advantage for downstream purification technique in future, ensure that product quality stable homogeneous.
6, the present invention selects F81-TY plants of cell lines of low serum suspension that the adherent F81 cell of tradition is replaced to prepare Raccoon dog parvovirus
Property enteritis antigen protein compound, reduce serum-concentration, while saving production cost, reduce product because of serum factor band
The side reaction come, improves the safety of product.
7, the present invention selects F81-TY plants of cell lines of low serum suspension that the adherent F81 cell of tradition is replaced to manufacture Raccoon dog parvovirus
Property enteritis antigen protein compound, improve the type and concentration of inactivator, and noresidue after inactivator hydrolysis, ensure that production
While quality, the generation of clinical side reaction caused by reducing because of inactivator.
8, the present invention selects F81-TY plants of cell lines of low serum suspension that the adherent F81 cell of tradition is replaced to prepare Raccoon dog parvovirus
Property enteritis antigen protein compound, reduces the usage amount of culture medium, and the amount of the virus liquid of harvest is exactly the culture base unit weight consumed,
And the amount of the culture medium of traditional rolling bottle technique consumption is 2 times for harvesting virus liquid.Production cost is saved.
9, the present invention is inactivated, is emulsified using the Raccoon dog parvovirus enteritis virus antigen of the full suspension technology preparation of cell
And Raccoon dog parvovirus enteritis inactivated vaccine is made with techniques such as seedlings.The experiment proved that the Raccoon dog parvovirus enteritis inactivates epidemic disease
Seedling can resist Raccoon dog parvovirus strong virus attack, and protective rate can effectively prevent the generation of racoon dog viral enteritis 91.7% or more,
Effective immune period 6 months.It is proposed of the invention provides new selection for the prevention and control of Raccoon dog parvovirus enteritis.
Detailed description of the invention
Fig. 1 is F81-TY plants of low serum suspension culture cell photo (100 ×) for 24 hours.
Fig. 2 is low serum suspension F81-TY plants of 48h cell photos (100 ×) of culture.
Fig. 3 is low serum suspension F81-TY plants of 72h cell photos (100 ×) of culture.
Fig. 4 is that LN plants of malicious photos of 48h receipts of F81-TY plants of inoculation Raccoon dog parvovirus enteritis virus are cultivated in the suspension of low serum
(100×)。
Specific embodiment
Of the invention LN plants of one plant of Raccoon dog parvovirus enteritis virus are that applicant separates out of clinical onset recoon dog enteron aisle
Obtain, name are as follows: LN plants, the strain to recoon dog safety, immunogenicity is good, virus multiplication is stable.The strain is in 2018
On December 7, in is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number are as follows:
CGMCC NO.16627, depositary institution address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 (grind by Chinese Academy of Sciences microorganism
Study carefully institute), depositary institution's postcode is 100101.
A kind of microbial inoculum of the invention, effective active composition are above-mentioned LN plants of one plant of Raccoon dog parvovirus enteritis virus.
A kind of Raccoon dog parvovirus enteritis inactivated vaccine of the invention, effective active composition are that one plant of above-mentioned racoon dog is tiny
LN plants of viral enteritis virus.
The present invention is by the way of sensitivity F81 attached cell continuous passage and full suspension process, the racoon dog that falls ill from market
Separation, clone purification Raccoon dog parvovirus in sub- enteron aisle, obtain one plant to racoon dog safety, immunogenicity is good, virus multiplication is stable
Raccoon dog parvovirus enteritis vaccine strain.Raccoon dog parvovirus antigen binding improvement is prepared using the full suspended cell culture technic of F81-TY
Beta-propiolactone inactivation, emulsification and prepare Raccoon dog parvovirus enteritis inactivated vaccine with techniques such as seedlings.A kind of racoon dog of the invention is thin
The preparation method of minor illness viral enteritis inactivated vaccine, mainly comprises the steps that
Step 1: suspension cell is recovered
It recovers to F81-TY plants of cells；
Step 2: suspension cell passes on
F81-TY strain cell after step 1 is recovered is expanded culture with low serum suspending nutrient solution, obtains F81-TY
Strain cell culture fluid；
Step 3: viral shaking flask domestication
Culture domestication is carried out to LN plants of Raccoon dog parvovirus enteritis virus, filters out the Raccoon dog parvovirus for the culture that suspends
Property enteritis virus produce seed culture of viruses；
Step 4: virus multiplication culture
Raccoon dog parvovirus enteritis virus production seed culture of viruses after step 3 is tamed is seeded to the F81-TY that step 2 obtains
In strain cell culture fluid, virus liquid is harvested after proliferation；
Step 5: seedling
Raccoon dog parvovirus enteritis inactivated vaccine is prepared using the virus liquid that step 4 obtains.
It can also be according to upper hand Step 3: Step 1: Step 2: Step 4: the sequence of step 5 carries out vaccine preparation.
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
Experimental method and reagent used in the following example are unless otherwise instructed conventional method and conventional examination
Agent.
One, material
1, F81-TY plants of cells: i.e. the F81-TY plants of low full suspension cell of serum grinds biotechnology Limited Liability by Ji Linte
Corporation is standby and saves.
2, dehydrated medium is purchased from Zhengzhou Ai Ke Biotechnology Co., Ltd, title: the dedicated low serum of F81 cell, which suspends, to be trained
Support backbone powder, article No.: F-6011-D.
3, newborn bovine serum (hereinafter referred to as serum): Inner Mongol Wei Kesheng Biotechnology Ltd., title: newborn
Cow's serum, lot number: 20170830.
4, low serum suspending nutrient solution, preparation process are as follows:
(1) the dedicated low serum suspension medium dry powder of the F81 cell of 50L/ packet specification is taken to be dissolved in water for injection, gained
The final volume of culture medium be 50L, the volume of water for injection is the 80-90% of final volume, is stirred to clarify.
(2) by solution sodium hydroxide solution obtained in step (1) or hydrochloric acid solution tune PH to 6.8-7.0, and with note
It penetrates and liquor capacity is supplemented to final volume 50L with water.
(3) it after solution obtained in step (2) to be added to the newborn bovine serum of final volume 1-5% volume, is filtered with 0.2 micron
Film filtration sterilization, packing is spare, saves in 2-8 DEG C.
5, feline kidney cells (F81): by Jilin Special Research Biological Technology Co., Ltd.'s preservation.
Two, instrument and equipment
Carbon dioxide incubator, Panasonic (MCO-230AICUVL-PC)；
PCR instrument (BIO-RAD T100Thermal)；
Centrifuge, HITACHICT-16RX type；
Suspending cell culture bottle (500mL；1000mL), purchased from Zhengzhou love section biology；
Magnetic stirring apparatus is purchased from USABELLCO company；
Bioreactor is purchased from the neat will (BC-5L in Guangzhou；BC-10L).
The separation and identification of LN plants of 1 Raccoon dog parvovirus enteritis virus of embodiment
1, virus purification
(1) pathological material of disease is handled
The pathological material of disease (Hebei Luannan area) of acquisition is diluted with same amount of normal saline, mixes well concussion, 3000rpm centrifugation
30min takes supernatant, be placed in -20 DEG C of refrigerators freeze it is spare.
(2) virus purification
Treated pathological material of disease is diluted with MEM nutrient solution according to the volume ratio of 1:2, synchronizes that be inoculated in adherent cat kidney thin
One bottle of born of the same parents (F81), 37 DEG C of cultures for 24 hours, change nutrient solution into cell maintenance medium (the MEM culture solution containing 2% serum), observe day by day
Cytopathy cultivates 4-5 days receipts poison, and continuous 4 generation of blind passage detects culture HA characteristic, and the positive freezes spare.
2, viruses indentification
(1) target viral is found in Electronic Speculum observation, is shown as Raccoon dog parvovirus enteritis virus through PCR sequencing post analysis；Tool
There are the ability of agglutination pig erythrocyte, HA valence 29；Viral level are as follows: 106.5TCID50/ml；It is insensitive to organic solvents such as ether；
After purification through Plaque Clone, one plant of pure Raccoon dog parvovirus enteritis virus (LN plants) is obtained, after positive serum neutralizes, inoculation
Feline kidney cells (F81) are cultivated 3 days, and cell is without lesion.
(2) exogenous virus and detection of mycoplasma be as the result is shown: without canine coronavirus, adenovirus and rotavirus；Mycoplasma
It is detected as feminine gender.
3, preservation: by above-mentioned separation and identify LN plant of one plant of Raccoon dog parvovirus enteritis virus, name are as follows: LN plants,
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on December 7th, 2018, preservation is compiled
Number are as follows: CGMCC NO.16627, depositary institution address are as follows: (Chinese Academy of Sciences is micro- for Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Biological study institute), depositary institution's postcode is 100101.
The preparation (suspension process) of 2 Raccoon dog parvovirus enteritis inactivated vaccine of embodiment
1, suspension cell is recovered
The low full suspension cell of serum of the F81-TY strain of liquid nitrogen cryopreservation is taken, quick-thawing is placed in 37 DEG C of water-baths and is inoculated in and contain
In the shaking flask for having low serum suspending nutrient solution 400mL, serum-concentration 1-5% is placed in 37 DEG C, 60rpm, 5% (v/v) CO2It shakes
Bed culture 72 hours, reaches 8.0 × 10 to cell density5A/mL or more, when and cell viability >=95%, after being recovered
F81-TY plants of cells.
2, suspension cell passes on
F81-TY strain cell after recovery that step 1 obtains is amplified into culture with low serum suspending nutrient solution, is obtained
F81-TY plants of cell culture fluids.
Its concrete operations engineering is as follows:
(1) the F81-TY strain cell after the recovery for obtaining step 1 is 1:4 according to low serum suspending nutrient solution volume ratio
Ratio be inoculated in the shaking flask equipped with low serum suspending nutrient solution, in 37 DEG C, 60rpm, 5% (v/v) CO2Shaking table culture 72 is small
When；It is not less than 7.5 × 10 to suspension cell density5A/mL, when and cell viability >=90%, the cell for obtaining Shake flask grown is outstanding
Supernatant liquid；
(2) cell suspending liquid of Shake flask grown is directly inoculated with here will BC-7L type (working volume 5L) bioreactor
In, inoculation volume is 1000-2000mL, add low serum suspending nutrient solution to 5L, three road gas of bioreactor parameter setting,
Respectively air, oxygen and carbon dioxide, revolving speed 60-75rpm, temperature are 37 DEG C, PH 7.1, will be molten after culture 36 hours
Oxygen is set as 50%, and the cell culture fluid cell density after continuing culture 36 hours is not less than 7.5 × 105A/mL, and cell is living
Power >=90%；
(3) cell culture fluid 3.5L obtained in step (2) is taken to be transferred to Qi Zhi BC-14L type (working volume 10L) biology
In reactor, low serum suspending nutrient solution is added to 10L, three road gas of bioreactor parameter setting, respectively air, oxygen
And carbon dioxide, revolving speed 75-85rpm, temperature are 37 DEG C, PH 7.1, dissolved oxygen 70% continues (process after culture 72 hours
In every 24 hours observation cellular morphologies, see attached drawing 1, Fig. 2, Fig. 3) carry out trypan blue staining counting and viability examination, work as detection
The viable cell density arrived is not less than 7.5 × 105To get to the cell culture fluid that can be used as connecing poison when a/mL；
(4) BC-7L type (working volume 5L) bioreactor remaining cell culture solution 1.5L is added into low serum suspension training
Nutrient solution continues to cultivate, continuously cultivate as cell seed, obtain F81-TY plants of cell trainings to 5L according to step (2) and step (3)
Nutrient solution.
3, viral shaking flask domestication
Raccoon dog parvovirus enteritis virus LN plants isolated to embodiment 1 carry out culture domestication, and it is outstanding to filter out adaptation
The Raccoon dog parvovirus enteritis virus of floating culture.
Its concrete operations engineering is as follows:
(1) cell suspending liquid of the Shake flask grown obtained in (1) of step 2 is compared to the ratio for 1:3 or 1:4 according to passage
Passage, by the Raccoon dog parvovirus enteritis virus seed culture of viruses of adherent feline kidney cells (F81) culture proliferation, (embodiment 1 is divided for synchronous inoculation
From LN plants of Raccoon dog parvovirus enteritis virus), connect toxic dose be 5-10% (v/v)；
(2) in 37 DEG C, 5% (v/v) CO2Shaking table culture 48 hours, its virus titer of every 12 hours sample detections and blood clotting
Potency detects the provirus sensibility under the conditions of suspension cell, is denoted as F1 generation；
(3) F1 generation virus liquid is repeated to connect poison according to step (1), connecing toxic dose is cell suspending liquid (in (1) of step 2
The cell suspending liquid of the Shake flask grown of acquisition) volume 5-10%, by step (2) CMC model, every 12 hours sample detections its
Virus titer and hemagglutinative titer are denoted as F2 generation, are seeded to F8 generation repeatedly, and connecing toxic dose is 5% (v/v), detect each generation disease
Toxic effect valence and hemagglutinative titer, since F5 generation, viral level and hemagglutinative titer are stable 10 respectively7.0TCID50/ ml, HA 210,
The Raccoon dog parvovirus enteritis virus for obtaining the culture that adapts to suspend produces seed culture of viruses, saves in -20 degree.
4, virus multiplication culture
Raccoon dog parvovirus enteritis virus production seed culture of viruses after step 3 domestication is seeded to the F81-TY strain that step 2 obtains
In cell culture fluid, venom is harvested after proliferation.
Its concrete operations engineering is as follows:
The F81-TY strain cell culture fluid that can be used as connecing poison that step 2 obtains is taken, according to F81-TY plants of cell culture liquids
Raccoon dog parvovirus enteritis virus after synchronous access step 3 domestication of long-pending 5-10% ratio produces seed culture of viruses, condition of culture are as follows: raw
Object reactor parameter sets three road gases, respectively air, oxygen and carbon dioxide, revolving speed 40-50rpm, and temperature is 37 DEG C,
PH is 6.8, dissolved oxygen 40-50%；Sampling in every 12 hours carries out trypan blue staining cell count and vigor inspection, cultivates 44-
After 48h, it is lower than 5 × 10 to viable cell density4When a/mL or cell activity≤50%, virus liquid is aseptically harvested, and
Pass through blood coagulation tests, TCID50Virus titer is detected, obtains seedling with venom (observing cellular morphology when harvest, see attached drawing 4),
Viral level TCID50It is 106.7/ ml, HA-HI test HA are 210。
5, seedling: the seedling obtained using step 4 prepares Raccoon dog parvovirus enteritis inactivated vaccine with venom.
Its concrete operations engineering is as follows:
(1) it inactivates
Add according to the ratio that inactivator beta-propiolactone and virus liquid volume ratio are 1:1500 into the virus liquid that step 4 obtains
Enter inactivator beta-propiolactone, stir 30min, mixes, 4 DEG C of inactivation 48h, intermittent stirring 3-5 times, each 20min, 37 DEG C of hydrolysis 3-
6h, 4 DEG C of preservations obtain inactivation liquid.
(2) inspection of semifinished product
Steriling test: the virus liquid for first harvesting step 4 samples, and presses " Chinese veterinary pharmacopoeia " (third portion in 2005) annex 15
Page carries out, and answers asepsis growth.
Inactivation is examined: taking the resulting inactivation liquid of above-mentioned steps (1), it is thin to be inoculated with 4 bottles of normal cats kidney passages respectively at 37 DEG C
Born of the same parents, every bottle of inoculation 0.4mL, set and cultivate at 37 DEG C, discard MEM culture medium afterwards for 24 hours, add new MEM culture medium, continue culture 3
It is passed on again after it 1 time, lesion should not occur in cell, harvest culture solution, and feminine gender should be presented in hemagglutination test, determine that inactivation is qualified.
(3) it emulsifies and matches seedling
Liquid will be inactivated and aluminium hydroxide gel is the ratio mixing of 9:1 (v/v) by volume, and added by the 0.01% of final volume
Enter thimerosal, then sterile saline is added by the 50% of final volume, fully emulsified mixing adjusts pH value with sterilizing sodium bicarbonate
For 7.2-7.4, Raccoon dog parvovirus enteritis inactivated vaccine is obtained.
The preparation (suspension process) of 3 Raccoon dog parvovirus enteritis inactivated vaccine of embodiment
Adjust embodiment 2 in specific steps tandem, i.e., according in embodiment 2 step 3,1,2,4,5 sequence
It carries out, other parts are all the same.
Compared with 4 two kinds of vaccine preparation techniques of embodiment (rolling bottle technique is with suspension process)
It carries out the culture of Raccoon dog parvovirus respectively using spinner culture and suspension culture method, carries out HA potency and disease respectively
Malicious content TCID50Measurement.
1, high serum F81 rolling bottle attached cell poison: culture of the virus in rolling bottle, by 1 bottle of MEM culture medium culture to 72h
And the rolling bottle F81 cell of fine and close single layer is covered with, culture solution is discarded, is washed through PBS, after pancreatin digestion, is collected with digestion terminate liquid
Cell is inoculated in the 1000mL cell culture fluid containing 8-10% newborn bovine serum that filtration sterilization is added in advance in 1:3 ratio
In, while by the synchronous inoculation Raccoon dog parvovirus of 1.5-3%, upper favourable turn, hot-house culture under the conditions of being placed in 37 DEG C, revolving speed 7- after mixing
14 turns/min；68-72h is cultivated after connecing poison, when cytopathy reaches 75-85%, cell seine is frozen in harvest virus when the shape of island
HA potency and TCID are carried out after melting50Detection.According to said method prepare three batches of seedling venom, lot number be respectively ZP201801,
ZP201802、ZP201803。
2, F81-TY plants of bioreactor suspension cell poison of low serum suspension: method prepares three batches of seedlings and uses with embodiment 2
Venom, lot number are respectively XF201804, XF201805, XF201806.The results are shown in Table 1.
1 distinct methods of table carry out Raccoon dog parvovirus culture sample testing result
Two kinds of cell culture modes prepare the Comprehensive Comparison of viral antigen, as a result such as the following table 2.
2 two kinds of cell culture modes of table prepare the Comprehensive Comparison result of viral antigen
| Comparison project | Spinner culture | The full culture that suspends |
| Cell growth pattern | It is adherent | It is complete to suspend |
| Production technology difficulty | It is complicated | Simply |
| Cell density | 1.0*106A/mL | 1.5*106A/mL |
| Serum-concentration | 8%~10% | 1~5% |
| Semi-finished product virus titer | 106.50TCID50 | 106.73TCID50 |
| Semi-finished product HA-HI test | 27~28 | 29~210 |
| Purifying process difficulty | It is medium | Simply |
| Side reaction degree | It is low | It is extremely low |
| Differences between batches | In | It is low |
| Production efficiency | It is low | It is high |
| Artificial demand | It is more than 20 people | 8-10 people |
| Overall cost | In | It is low |
Viral antigen is prepared using F81 cell spinner bottle adhere-wall culture.The cell culture stage generally requires that higher concentration is added
Newborn bovine serum (8-10%) is needed to discard culture solution after cell covers with single layer, be washed through PBS, is used instead after pancreatin digestion new
The cell maintenance medium that raw bovine serum concentration is 2-5%, not only technique is cumbersome, and since operation clinker degree difference causes cell to pass
It is different for growing state quality, malicious restrovirus proliferation speed difference is connect, the stable homogeneous of product quality is finally influenced.In addition,
The use of high concentration serum not only increases production cost, and foreign protein is isolated and purified in work in the later period and is very difficult in serum
It removes, side reaction is big after inoculation.In addition, there may be exogenous virus and mycoplasma in serum, the bio-safety wind of product is increased
Danger.Since rolling bottle cell grows limited area, it is unfavorable for cell and amplifies culture, and need enough greenhouses and favourable turn, land occupation is more,
It is artificial more.
Viral antigen is prepared using the low serum suspension F81-TY plants of cells of culture of bioreactor.First, the degree of automation
Height needs personnel few, and can be using low serum (1-5%) culture medium with industrial value, antigen cost comparative advantages
Significantly.Second, low serum, which suspends to cultivate, can greatly reduce the heat source substances such as foreign protein in serum, reduce product later-period purification
Difficulty and the occurrence probability of side reaction make product with more safety.Third, suspend F81-TY plants of cells of culture, cell Proliferation
Well, it is easy to amplify culture, the receipts malicious time shifts to an earlier date compared with other training methods, shortens the production cycle, while reducing labor intensity.
4th, reduce culture medium usage amount, synchronize connect malicious mode allow the virus liquid of harvest amount be exactly consume the amount of culture medium, and other
The amount of technique consumption culture medium is 2 times for harvesting virus liquid.5th, no matter HA potency or TCID50All it is slightly above spinner culture
Technique reaches producting rule requirement.6th, the viral antigen compound of this method preparation is safe and effective, to racoon dog viral enteritis
Protective effect is played, there is the application of preferable market and promotion prospect.
5 Raccoon dog parvovirus enteritis inactivated vaccine safety testing of embodiment
Example 2 examines qualified Raccoon dog parvovirus enteritis inactivated vaccine to carry out hindlimb muscle inoculation negative antibody racoon dog
(60-70 age in days) sets up single dose, single dose repetition and overdose (5 times) immunoprophylaxis group, while setting up saline control
Group.It is observed continuously after vaccine inoculation 14 days, the indexs such as record racoon dog body temperature, appetite, the state of mind and main clinic symptoms.Test knot
Fruit is shown in Table 3.Show that recoon dog indices are normal through vaccine safety verification experimental verification, does not occur any clinical symptoms, illustrate epidemic disease
Seedling is to racoon dog safety.
3 Raccoon dog parvovirus enteritis inactivated vaccine safety test result of table
| Group | Racoon dog number | Temperature changing | Clinical symptoms | It is inoculated with local reaction | Morbidity and death toll |
| Single dose | 10 | Normally | Nothing | Nothing | 0 |
| Single dose repeats | 10 | Normally | Nothing | Nothing | 0 |
| Overdose | 10 | Normally | Nothing | Nothing | 0 |
| Control | 5 | Normally | Nothing | Nothing | 0 |
Note: vaccine lot number 20180901.
6 Raccoon dog parvovirus enteritis inactivated vaccine of embodiment is compared with similar vaccine is to the immune efficacy of racoon dog
Group 1: the Raccoon dog parvovirus enteritis inactivated vaccine through examining qualification prepared by embodiment 2.
Group 2: Mink Parvovirus Enteritis inactivated vaccine (MEVB plants) available on the market.
Above two vaccine difference hindlimb muscle inoculation negative antibody racoon dog (60-70 age in days), every inoculation 1ml are set simultaneously
Vertical saline control group.It takes a blood sample within 21 days after immune, detects Serum HI antibody potency, while virulent oral using 100 ID50
Poison is attacked, is observed 15 days.
Test result is shown in Table 4.Compared it is found that organize 1 it is immune after in 21 days serum HI antibody level be higher than group 2；Two groups of epidemic diseases
After seedling Immunization, group 1 can get 100% protection, and 2 protective rates of group are 60%.Therefore, the Raccoon dog parvovirus prepared by the present invention
The immune efficacy of property enteritis inactivated vaccine is substantially better than Mink Parvovirus Enteritis inactivated vaccine (MEVB available on the market
Strain).
4 Raccoon dog parvovirus enteritis inactivated vaccine of table is compared with similar vaccine is to the immune efficacy of racoon dog
Generation phase and duration test is immunized in 7 Raccoon dog parvovirus enteritis inactivated vaccine of embodiment
Prepared by Example 2 is examined qualified Raccoon dog parvovirus enteritis inactivated vaccine hindlimb muscle to be inoculated with antibody yin
Property racoon dog (3-4 monthly age), be inoculated with 20, every immunizing dose is 1ml, 7d, 14d, 21d, 60d, 120d, 180d after immune
Blood sampling, detection Serum HI antibody potency are selected 12 recoon dogs and control group 3 at random, are taken orally respectively in immune 120d, 180d
100 ID50 are virulent to carry out attacking poison, observes 15d, measures the vaccine immunity duration.Test result shows that immune rear 7d starts to produce
Raw antibody, 21d can produce the HI antibody (1:32) for reaching immunoprotection.Vaccine immunity 120d obtains 100% through intensity attack and protects
180d is immunized in shield, obtains 91.7% (11/12) protection through strong virus attack, illustrates that Raccoon dog parvovirus enteritis inactivated vaccine is immune
Phase reaches 180d or more.
By above-mentioned it is demonstrated experimentally that the Raccoon dog parvovirus enteritis inactivated vaccine being prepared by full suspension process is exempted from
21d after epidemic disease recoon dog can produce the HI antibody level (>=1:32) for reaching immune effect, and 180d after being immunized can effectively protect recoon dog
From the virulent attack of parvovirus, protective rate is 91.7%, therefore the Raccoon dog parvovirus enteritis that the present invention is prepared is gone out
Live vaccine can effectively prevent the generation of Raccoon dog parvovirus enteritis with effective protection racoon dog from virulent attack.With good
Market application value.
The invention discloses one plant of LN plants of Raccoon dog parvovirus enteritis virus, Raccoon dog parvovirus enteritis inactivated vaccine and its
Preparation method, those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular
It is that all similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this
Invention.Product of the invention is described by preferred embodiment, and related personnel can obviously not depart from the present invention
Hold, product as described herein be modified in spirit and scope or appropriate changes and combinations, carrys out implementation and application skill of the present invention
Art.
Claims (10)
1. one plant LN plants of Raccoon dog parvovirus enteritis virus, which is characterized in that during the strain has been preserved on December 7th, 2018
State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number are as follows: CGMCC NO.16627.
2. a kind of toxic agent, effective active composition is LN plants of one plant of Raccoon dog parvovirus enteritis virus described in claim 1.
3. a kind of Raccoon dog parvovirus enteritis inactivated vaccine, effective active composition is that one plant of racoon dog described in claim 1 is tiny
LN plants of viral enteritis virus.
4. the method for preparing Raccoon dog parvovirus enteritis inactivated vaccine as claimed in claim 3, which is characterized in that including following step
It is rapid:
Step 1: suspension cell is recovered
It recovers to F81-TY plants of cells；
Step 2: suspension cell passes on
F81-TY strain cell after step 1 is recovered is expanded culture with low serum suspending nutrient solution, is obtained F81-TY plants thin
Born of the same parents' culture solution；
Step 3: viral shaking flask domestication
Culture domestication is carried out to LN plants of Raccoon dog parvovirus enteritis virus, filters out the Raccoon dog parvovirus intestines for the culture that suspends
Scorching virus production seed culture of viruses；
Step 4: virus multiplication culture
It is thin that Raccoon dog parvovirus enteritis virus production seed culture of viruses after step 3 is tamed is seeded to the F81-TY strain that step 2 obtains
In born of the same parents' culture solution, virus liquid is harvested after proliferation；
Step 5: seedling
Raccoon dog parvovirus enteritis inactivated vaccine is prepared using the virus liquid that step 4 obtains.
5. the preparation method according to claim 4, which is characterized in that according to Step 3: Step 1: Step 2: Step 4:
The sequence of step 5 carries out vaccine preparation.
6. the preparation method according to claim 4, which is characterized in that detailed process is as follows for step 1:
The low full suspension cell of serum of the F81-TY strain of liquid nitrogen cryopreservation is taken, quick-thawing in 37 DEG C of water-baths is placed in and is inoculated in containing low
In the shaking flask of serum suspending nutrient solution 400mL, serum-concentration 1-5% is placed in 37 DEG C, 60rpm, 5% (v/v) CO2Shaking table training
It supports 72 hours, reaches 8.0 × 10 to cell density5A/mL or more, when and cell viability >=95%, the F81- after being recovered
TY plants of cells.
7. the preparation method according to claim 4, which is characterized in that detailed process is as follows for step 2:
(1) the F81-TY strain cell after step 1 being recovered is inoculated in low serum suspending nutrient solution volume ratio for the ratio of 1:4
In the shaking flask equipped with low serum suspending nutrient solution, in 37 DEG C, 60rpm, 5% (v/v) CO2Shaking table culture 72 hours；Wait suspend
Cell density is not less than 7.5 × 105A/mL when and cell viability >=90%, obtains the cell suspending liquid of Shake flask grown；
(2) cell suspending liquid of Shake flask grown is directly inoculated into the bioreactor of working volume 5L, inoculation volume is
1000-2000mL adds low serum suspending nutrient solution to 5L, three road gas of bioreactor parameter setting, respectively air, oxygen
Gas and carbon dioxide, revolving speed 60-75rpm, temperature are 37 DEG C, PH 7.1, and after culture 36 hours, dissolved oxygen is set as 50%,
Cell culture fluid cell density after continuing culture 36 hours is not less than 7.5 × 105A/mL, and cell viability >=90%；
(3) it takes cell culture fluid 3.5L obtained in step (2) to be transferred in the bioreactor of working volume 10L, adds low
Serum suspending nutrient solution is to 10L, three road gas of bioreactor parameter setting, respectively air, oxygen and carbon dioxide, revolving speed
For 75-85rpm, temperature is 37 DEG C, PH 7.1, and dissolved oxygen 70% carries out trypan blue staining counting after continuing culture 72 hours
And viability examination, when the viable cell density detected is not less than 7.5 × 105To get to for connecing malicious cell culture when a/mL
Liquid；
(4) the bioreactor remaining cell culture solution 1.5L of working volume 5L is added into low serum suspending nutrient solution to 5L, pressed
Continue to cultivate according to step (2) and step (3), continuously be cultivated as cell seed, obtains F81-TY plants of cell culture fluids.
8. preparation method according to claim 7, which is characterized in that detailed process is as follows for step 3:
(1) cell suspending liquid of the Shake flask grown obtained in (1) of step 2 is compared to the ratio biography for 1:3 or 1:4 according to passage
In generation, synchronous LN plants of Raccoon dog parvovirus enteritis virus of inoculation, connecing toxic dose is 5-10% (v/v)；
(2) in 37 DEG C, 5% (v/v) CO2Shaking table culture 48 hours, its virus titer of every 12 hours sample detections and hemagglutinative titer,
The provirus sensibility under the conditions of suspension cell is detected, F1 generation is denoted as；
(3) F1 generation virus liquid is repeated to connect poison according to step (1), the 5-10% that toxic dose is Cell suspension volumes is met, by step
Suddenly (2) CMC model, its virus titer of every 12 hours sample detections and hemagglutinative titer are denoted as F2 generation, are seeded to F8 repeatedly
In generation, connecing toxic dose is 5% (v/v), detects each generation virus titer and hemagglutinative titer, since F5 generation, viral level and blood clotting
Potency is stable 10 respectively7.0TCID50/ ml, HA 210, obtain the Raccoon dog parvovirus enteritis virus production for the culture that suspends
Seed culture of viruses is saved in -20 degree.
9. the preparation method according to claim 4, which is characterized in that detailed process is as follows for step 4:
In the Raccoon dog parvovirus enteritis after synchronous access step 3 domestication of the 5-10% ratio of F81-TY plants of cell culture fluid volumes
Virus production seed culture of viruses, condition of culture are as follows: three road gas of bioreactor parameter setting, respectively air, oxygen and carbon dioxide,
Revolving speed is 40-50rpm, and temperature is 37 DEG C, PH 6.8, dissolved oxygen 40-50%；Sampling in every 12 hours carries out trypan blue staining
Cell count and vigor inspection after cultivating 72-96h, are lower than 5 × 10 to viable cell density4A/mL or cell activity≤50%
When, virus liquid is aseptically harvested, and pass through blood coagulation tests, TCID50Virus titer is detected, seedling virus liquid is obtained,
Its viral level TCID50It is 106.7/ ml, HA-HI test HA are 210。
10. the preparation method according to claim 4, which is characterized in that detailed process is as follows for step 5:
(1) it inactivates
Inactivation is added into the virus liquid that step 4 obtains in the ratio that inactivator beta-propiolactone and virus liquid volume ratio are 1:1500
Agent beta-propiolactone stirs 30min, mixes, 4 DEG C of inactivation 48h, intermittent stirring 3-5 times, each 20min, 37 DEG C of hydrolysis 3-6h, and 4 DEG C
It saves, obtains inactivation liquid；
(2) inspection of semifinished product
Steriling test: steriling test is carried out to the virus liquid sampling that step 4 harvests；
Inactivation is examined: to the resulting inactivation liquid of step (1), being inoculated with 4 bottles of normal cats kidney passage cells respectively at 37 DEG C, every bottle connects
Kind 0.4mL, sets and cultivates at 37 DEG C, discard MEM culture medium afterwards for 24 hours, add new MEM culture medium, passes on again after continuing culture 3 days
1 time, lesion should not occur in cell, harvest culture solution, and feminine gender should be presented in hemagglutination test, determine that inactivation is qualified；
(3) it emulsifies and matches seedling
Liquid will be inactivated and aluminium hydroxide gel is the ratio mixing of 9:1 (v/v) by volume, and sulphur is added by the 0.01% of final volume
Willow mercury, then sterile saline is added by the 50% of final volume, emulsification mixes, and adjusting pH value with sterilizing sodium bicarbonate is 7.2-
7.4, obtain Raccoon dog parvovirus enteritis inactivated vaccine.
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| CN114317405A (en) * | 2021-12-21 | 2022-04-12 | 广东省华晟生物技术有限公司 | Serum-free full-suspension culture type F81 cell line and construction method and application thereof |
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|---|---|---|---|---|
| CN108690834A (en) * | 2018-05-31 | 2018-10-23 | 中国农业科学院特产研究所 | Raccoon dog parvovirus strain and its application, Raccoon dog parvovirus inactivated vaccine and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108690834A (en) * | 2018-05-31 | 2018-10-23 | 中国农业科学院特产研究所 | Raccoon dog parvovirus strain and its application, Raccoon dog parvovirus inactivated vaccine and preparation method thereof |
Non-Patent Citations (1)
| Title |
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| 丁尚红: "貉细小病毒研究概述", 《吉林畜牧兽医》 * |
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|---|---|---|---|---|
| CN114317405A (en) * | 2021-12-21 | 2022-04-12 | 广东省华晟生物技术有限公司 | Serum-free full-suspension culture type F81 cell line and construction method and application thereof |
| CN114317405B (en) * | 2021-12-21 | 2023-08-29 | 广东省华晟生物技术有限公司 | Serum-free full-suspension culture type F81 cell line and construction method and application thereof |
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