CN107299088A - B plants of infectious bovine rhinotrachetis virus IBRV and its application - Google Patents
B plants of infectious bovine rhinotrachetis virus IBRV and its application Download PDFInfo
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Abstract
The invention discloses B plants of infectious bovine rhinotrachetis virus IBRV and application, B plants of the infectious bovine rhinotrachetis virus IBRV of the present invention has the advantages that immunogenicity is good, cultural character is good, the infectious bovine rhinotrachetis virus (B plants of IBRV) of the present invention is inoculated with MDBK cells, harvest culture, after being inactivated with β propiolactone (β propiolactone), the commercialization Montanide with match BIC Corp of FranceTMInfectious bovine rhinotrachetis virus inactivated vaccine is made in ISA15AVG adjuvant mixing and emulsifyings.Experiment is proved, it can prevent the disease as caused by infectious bovine rhinotrachetis virus using the immune negative ox of wean health of the infectious bovine rhinotrachetis virus inactivated vaccine for preparing of the present invention, and this vaccine has the advantages that safety, generation antibody is fast, duration of immunity is lasting.
Description
Technical field
The invention belongs to biological field, more specifically, belong to biovaccine field, be related to a kind of infectious bovine rhinotrachetis
Virus and its application, more particularly to one plant voluntarily isolated IBRV-B plants of infectious bovine rhinotrachetis virus and by the disease
The inactivated vaccine that strain is prepared.
Background technology
Infectious bovine rhinotracheitis virus (Infectious bovine rhinotracheitisvirus, IBRV) can draw
Play ox and occur acute, hot, contagious disease, show as the upper respiratory tract and tracheal mucosa inflammation, expiratory dyspnea, rhinorrhea etc.
Symptom, can also cause purulence born of the same parents property vulvitis, vaginitis, balanitis, conjunctivitis, miscarriage, young bovine meningitis, mastitis, hysteritis
Etc. symptom.Early 1950s find the disease in Colorado earliest, and other subsequent each states also find the disease in succession.
Madm in 1956 etc. isolates virus from infected cattle first, and Huck confirms that IBRV belongs to herpesviral for (1971), at present in generation
Criticality is popular.China in 1980 finds the disease from New Zealand import kind ox first, and report is with the presence of this virus first, and the disease is made
Into larger economic loss.
Current vaccine includes inactivated vaccine, Attenuate vaccine, subunit seedling and gene delection mark seedling.There are two kinds of vaccines in Australia
Listing is Rhinogard (V155 plants) and IBEPUR (subunit vaccine), and 2,000,000 ox the sixth of the twelve Earthly Branches is had more than through inoculation in Australia
.Various traditional vaccines and marker vaccine (to distinguish immune group and nonimmune group) can obtain in overseas, and Australia is often
The vaccine for other cause of diseases is often used in combination, i.e., prevents and treats ox respiratory disease syndrome (BRD) jointly using multivalence seedling.
Domestic market there is no the related vaccines of independent research to be used for the sick preventing and treating.
The present inventor was separated to one plant of infectious bovine rhinotrachetis virus in 2015 from Beijing cattle farm, warp
Laboratory qualification is velogen strain, and the strain after domestication clone is named as IBRV-B plants.Using IBRV-B plants, ox infectiousness is carried out
The development work of rhinotracheitis virus inactivated vaccine, screening cultivates that immunogenicity is good, the Niu Chuanran of titer plateaus
Property IBRV-B plants of rhinotracheitis virus, have selected the MDBK cells of the most suitable virus multiplication and meet manufacture vaccine inactivation
Agent, adjuvant and other conditions.To the production technology of product, security, protective rate, immune programme for children, minimum dose, antibody duration
Experiment has all been carried out with storage life etc. to determine, and has obtained substantial amounts of test data, it was demonstrated that this vaccine safety is effective.It is basic herein
On, scale up test is carried out, sampled inspection all meets the quality standard drafted, and carried out safe examination to pilot product
Test and potency test, its result also confirms that this vaccine can effectively prevent the ox as caused by infectious bovine rhinotrachetis virus to breathe
Tract disease, on the basis of laboratory test, pilot-scale production, the present invention have developed infectious bovine rhinotrachetis virus IBRV-B
Strain inactivated vaccine, its result also confirms that this vaccine can effectively prevent the bovine respiratory as caused by infectious bovine rhinotrachetis virus
Disease, further demonstrates every quality standard of this vaccine.
The content of the invention
An object of the present invention is to provide that a kind of immunogenicity is good, the infectious bovine rhinotrachetis virus of titer plateaus
Strain；
The second object of the present invention is to provide application of the described infectious bovine rhinotrachetis virus strain in vaccine is prepared；
The third object of the present invention is to provide a kind of epidemic disease prepared by described infectious bovine rhinotrachetis virus strain
Seedling；
The fourth object of the present invention is to provide described vaccine should in prevention infectious bovine rhinotrachetis medicine is prepared
With.
In order to achieve the above object, present invention employs following technological means：
The present inventor was separated to one plant of infectious bovine rhinotrachetis virus in 2015 from Beijing cattle farm, warp
Laboratory qualification is velogen strain, and the strain after domestication is named as IBRV-B plants.The IBRV-B strains of the present invention are from the typical ox of generation
Separation is obtained in infectious bovine rhinotracheitis virus infected cattle, therefore the propagation compared with the other strains being separated on cell
Titre is high, and the immunogenicity to ox is good.As vaccine seed culture of viruses after MDBK passages, effect inspection is IBRV-B strains the 7th with strong poison
~10 generations.The strain can cause MDBK cells more than 90% to infect.
A kind of infectious bovine rhinotrachetis virus of the present invention, is the 7th generation Strain after being passed on MDBK cells, life
Entitled infectious bovine rhinotrachetis virus IBRV-B plants, Classification And Nomenclature is infectious bovine rhinotrachetis virus, is deposited in China micro-
Biological inoculum preservation administration committee common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Chinese science
Institute of microbiology of institute, its culture presevation, which is numbered, is：CGMCC No.14294, preservation date is on 06 15th, 2017.
Gone out present invention also offers described infectious bovine rhinotrachetis virus in preparation infectious bovine rhinotrachetis virus
Application in live vaccine.
A kind of infectious bovine rhinotrachetis virus inactivated vaccine of the present invention, it is characterised in that contain the present invention after inactivation
IBRV-B plants of described infectious bovine rhinotrachetis virus.
In infectious bovine rhinotrachetis virus I inactivated vaccines of the present invention, it is preferred that described ox infectiousness nose
IBRV-B plants of bronchitis virus is inactivated using following methods：0.2% beta-propiolactone is inactivated 24 hours under the conditions of 4 DEG C,
Then room temperature continues to inactivate 24 hours.
In the present invention, it is preferred to, described is characterized in that containing the ox infectiousness nose of the present invention after inactivation
Bronchitis virus inactivated vaccine is by the commercialization Montanide of 15% (v/v) match BIC Corp of FranceTMISA15AVG is helped
Obtained after agent and 85% (v/v) IBRV-B plants of emulsifications of the infectious bovine rhinotrachetis virus inactivated with beta-propiolactone.
In the present invention, emulsifying process is the commercialization Montanide using match BIC Corp of FranceTMISA15AVG is helped
Agent, it is degerming after can be directly used for vaccine emulsification preparation；Same batch of virus liquid of inactivation is thawed before emulsification and mixed, sterile indoor
Refiner is stirred with 200r/min, then according to aqueous phase antigen and the volume proportion of oil phase adjuvant 8.5: 1.5；Emulsification procedure
It is stirred for first aqueous phase is put into emulsifier, is then slowly added into oil phase adjuvant, is continuously stirred 30 minutes with 800r/min；
Produce obtained vaccine and belong to oil-in-water formulation, for stable interface and elimination bubble, obtain optimum emulsification effect, it is necessary to static 30
Dispensed after minute.
Further, prevention is being prepared after being characterized in that containing inactivation present invention also offers described inactivated vaccine
Infectious bovine rhinotrachetis virus of the present invention caused by disease biological products in purposes.
Especially, described disease is the respiratory disease that healthy ox occurs.
Brief description of the drawings
Fig. 1 is IBRV-B plants of negative staining sem images of infectious bovine rhinotrachetis virus
Fig. 2 is the fluoroscopic examination knot that IBRV-B plants of infectious bovine rhinotrachetis virus is cultivated 48 hours on MDBK cells
Really；
Fig. 3 is the one step growth curve after IBRV-B plants of infection MDBK cells of infectious bovine rhinotrachetis virus.
Embodiment
Below by experiment and the present invention will be further described in conjunction with the embodiments, it should be understood that these embodiments
The purpose of illustration is only used for, protection scope of the present invention is never limited in.
The separation of IBRV-B plants of 1 infectious bovine rhinotrachetis virus of embodiment and culture identification
1 seed culture of viruses is originated and standard
According to new biological product requirements of customs declaration, the substantial amounts of test data obtained with reference to the present invention, reference《Chinese veterinary drug
Allusion quotation》(version in 2005), is identified with seed culture of viruses seedling, and seed culture of viruses standard in Trial Regulation (draft) now is carried out into following say
It is bright.
1.1 seeds culture of viruses are originated
The seed culture of viruses for manufacturing this product is IBRV-B plants of infectious bovine rhinotrachetis virus, is the present inventor in 2015 from hair
Infected cattle is voluntarily separated and obtained after domestication, because this strain is divided from occurring typical infectious bovine rhinotrachetis virus infected cattle
From acquisition, and the viral titer compared with the other strains being separated on cell is high, and the immunogenicity to ox is good, so
It is selected to be used for the production of vaccine.As vaccine seed culture of viruses after MDBK passages, effect inspection is IBRV-B strains the 7th~10 with strong poison
Generation.The seed culture of viruses is the 7th generation Strain after being passed on MDBK cells, is deposited in China Committee for Culture Collection of Microorganisms
Common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of institute of the Chinese Academy of Sciences, its strain
Deposit number is：CGMCC No.14294, preservation date is on 06 15th, 2017.
1.2 seeds culture of viruses (CGMCC No.14294) standard
1.2.1 electron microscopic morphology
IBRV-B plants of particle almost sphericals, there is cyst membrane, in solid and hollow two kinds of forms, its diameter be about 150nm~
194nm, average 170nm, nucleocapsid is in the regular dodecahedron of axle cubic symmetry, and outward appearance is in hexagon, and cyst membrane thickness is asymmetric, shape
State is not of uniform size, and surface has villiform projection (see Fig. 1).
1.2.2 viral level
Seed culture of viruses is made into continuous 10 times with DMEM nutrient solutions to be serially diluted, each dilution factor takes 100 μ l to add 96 hole cell trainings
Support in plate, each dilution factor makees 8 repetitions, is subsequently added MDBK cells, per the μ l of hole 100, (cell content is with 3 × 105/ ml), and
If normal cell culture is compareed, 37 DEG C are put, containing 5% CO2Cultivated in incubator, cytopathy (CPE), cell circle are observed day by day
Contracting, is gathered into grape cluster sample group, and cavity is formed on cell monolayer and is then judged to CPE.Observation 7 days, records the hole of cytopathy
Number, the TCID50 of virus is calculated according to Reed-Muench methods.As a result：Every milliliter of virus liquid is not less than 1011.0TCID50。
1.2.3 virulence
Viral generation is the generation cell toxicant of infectious bovine rhinotrachetis virus IBRV-B strains the 7th~10；It is per milli to attack toxic agent amount
10 should be not less than by rising viral level7.0TCID50；Program is healthy negative calf 4, Intranasal sprays inoculation IBRV-B strains the 7th~
10 generation cell toxicant 10ml, viral level is respectively 1 × 108.0TCID50；Criterion is the body temperature rise since the 2nd day, is held
It is continuous to increase to over 40.0 DEG C of average 5d, different degrees of clinical symptoms are shown, spiritual depressed, the sticky nose of suppurating property is shown as
Liquid, schneiderian membrance redness has hickie, and eyes secretion purulent secretion, clinical symptoms continue 9d or so.Cut open inspection lesion is concentrated mainly on
Lung, the increase of thymic lymphoma knot, tracheorrhagia, other organs are without obvious naked eyes lesions visible.Pathological section result shows, lungs
In apostematosa pneumonia, have in fibrinous exudate pulmonary edema, the destruction of bronchiole tube wall, tube chamber and be full of inflammatory cell, lung tissue
Destruction, it is seen that a large amount of inflammatory cells and necrosis region.In lungs, tracheae and site tissue separates virus near the eyes.
1.2.4 immunogenicity
By seed culture of viruses, synchronously inoculation MDBK cells are bred, and harvest virus liquid, after being inactivated with beta-propiolactone, plus
MontanideTMOil-in-water type inactivated vaccine is made in ISA15 AVG adjuvant mixing and emulsifyings, with the susceptible wean calf (IBRV of health
Neutralize antibody titers are not higher than 1: 2) 5, this code seedling 2ml is pressed in intramuscular injection, if compareing ox 5 rather, Intranasal sprays connect
Plant IBRV-B plants of each 10ml (107.0TCID50), attack to cut open for 14 days after poison and kill all oxen.Control group should at least 4 there is ox infectiousness
The typical clinical symptom of rhinotracheitis infection, immune group should at least 4 head protections.
1.2.5 it is pure to examine
The 10th~11 generation of primordial seed batch, basic bacteria batch to IBRV-B plants of the infectious bovine rhinotrachetis virus of foundation
In 12nd~21 generation and the production generation of seed lot the 22nd~25, have carried out bacterium, mould, mycoplasma and have examined, and to the generation of the 11st, 12 and 24
Secondary seed culture of viruses has carried out the inspection of exogenous virus, as a result shows, the primordial seed that the present invention is set up batch, basic bacteria are criticized, production kind
Son batch pollutes without bacterium, mould, mycoplasma and exogenous virus, pure.
1.2.6 it is specific
Seed culture of viruses is diluted to 200TCID50/0.1ml with DMEM liquid, the anti-infectious bovine rhinotrachetis virus with inactivation is special
Specific serum equivalent is mixed, and is put 37 DEG C and is neutralized 1 hour, synchronous well-grown 3 bottles of MDBK cells of inoculation (cell content with 3 ×
105/ ml), while setting virus control, normal cell controls and negative serum control, put 37 DEG C, containing 5% CO2In incubator, training
Support 5~7.Cytopathy (CPE) all should be occurred without by neutralizing virus group and normal cell controls group；Virus control group and negative blood
CPE all should occur in clear control group.
1.2.7 seed culture of viruses uses generation
It is continuous on MDBK cells to pass for 40 generations by original IBRV-B plants isolated of infectious bovine rhinotrachetis virus
(i.e. the generation of continuous passage 33 on the basis of the strain for being CGMCC No.14294 is numbered in culture presevation), determine every generation virus
Vaccine is made in TCID50, selection the 15th, 20,25,30,35,40 generations virus after inactivating respectively, be inoculated with 2~4 monthly age calves, 14 days
Carry out attacking poison afterwards.As a result show, virus is passed on MDBK cells, after the 7th generation (including the 7th generation virus, i.e. culture presevation are compiled
Number Strain for being CGMCC No.14294) each generation viral level is stable, is not less than 1011.0TCID50, and neutralization titer
More than 32, the standard for making vaccine has been reached.Challenge test shows, the 15th, 20,25, the vaccine for preparing of 30 generations virus
Immune cattle to the protectiveness of IBRV-B plants of attacks without significant difference, calf health survival；It is prepared by the 35th and 40 generations virus
Poison is attacked after vaccine immunity respectively 2 and 1 death, and 4 calves of control group have different degrees of infectious bovine rhinotrachetis
Viral infection symptoms.According to result above, to ensure immunogenicity and security that production seed culture of viruses is good, present invention determine that disease
The passage highest number of times of poison is in 27 generations, and original seed culture of viruses was 7~11 generations, and basic seed culture of viruses was the 12nd~21 generation, and seed culture of viruses highest is used in production
Passage number was limited within 3 generations (the 22nd~25 generation).
1.2.8 the determination of virus seed conservation phase
IBRV-B plants of wet poisons of infectious bovine rhinotrachetis virus are stored at -20 DEG C and -70 DEG C, when difference after preservation
Between sampling carry out neutralization titer and TCID50 and determine, as a result show that wet poison freezes 18 months in -20 DEG C, in -70 DEG C of preservations 30
Malicious valency starts to be decreased obviously during the moon；Lyophilized poison is stored at -20 DEG C and -70 DEG C, and neutralization titer is carried out in annual sampling after preservation
Determined with TCID50.As a result it is declined slightly in -20 DEG C of viral potency for preserving 36 months, preserves 48 months viral neutralization effects
Valency and TCID50 values are decreased obviously, and 48 months viral neutralization titers and TCID50 are preserved at -70 DEG C and are not become significantly
Change, preserve 60 months and decline substantially.In view of other unfavorable factors existed when virus is preserved, it is ensured that viral storage life is reliable
Property, so IBRV-B plants of wet poisons of infectious bovine rhinotrachetis virus are set to 12 months in -20 DEG C of storage life, at -70 DEG C
Storage life is set to 24 months；IBRV-B plants of lyophilized poison of infectious bovine rhinotrachetis virus are set to 36 in -20 DEG C of holding time
Individual month；Lyophilized poison is 48 months in -70 DEG C of holding time.
2 Cells for production
2.1 cell derived
MDBK cells, purchased from Chinese Academy of Sciences's Shanghai cell bank, by Institute of Animal Sciences, Chinese Academy of Agricultural Sciences
Preserve.
The selection of 2.2 cells
The present invention has selected continuous cell line MDBK to carry out the propagation of virus in the incubation of virus, and virus is by virus
It is morning lesion time on 0.01 inoculation MDBK cells, viral titer highest, MDBK cell infections Niu Chuanran to infect value (MOI)
Property rhinotracheitis virus IBRV-B strains 48 hours as shown in Fig. 2 IBRV-B plants of infection MDBK of infectious bovine rhinotrachetis virus are thin
One step growth curve such as Fig. 3 after born of the same parents.Under the conditions of such a, the virus titer highest of 48 hours harvests.
2.3 the foundation of cell line
Seedling selects MDBK cells with cell line.Scope control is passed in 40~55 generations, the present invention establishes initial cell
Storehouse, basal cell storehouse and working cardial cell storehouse.Wherein master cell bank, which is divided into, fills 55 bottles；Basal cell storehouse dispenses 370 bottles；Work is thin
Born of the same parents storehouse, which is divided into, has filled 350 bottles.The cell of each generation is all pressed《Chinese veterinary pharmacopoeia》Annex is tested, should without bacterium, mould,
The pollution of mycoplasma and exogenous virus.
The identification of 2.4 cell lines
According to existing《Chinese veterinary pharmacopoeia》" production, inspection cell standard " regulation relevant in attached sheet is tested, accorded with
Standardization.It is specifically shown in " certified test report of MDBK cell lines ".
The preparation of the inactivated vaccine of embodiment 2
The preparation technology flow of IBRV-B plants of infectious bovine rhinotrachetis virus is according to commercialization ripe at present
It is prepared by vaccine preparation technology.From the point of view of 3 batches of vaccines and 5 batches of vaccine quality inspection results of pilot-scale production prepared by laboratory, the work is used
Vaccine product steady quality prepared by skill flow, effect inspection result complies fully with formulated quality standard.
The preparation of 1 virus liquid
In virus propagation process, several factors can influence the propagation of cell and virus.By a series of comparative tests, choosing
It is 10% with cell culture fluid serum-concentration, maintaining liquid serum-concentration is 2%, stabilization, the virus liquid of high titre can be bred；
Experiment is compared to the pH value of nutrient solution, cell concentration, viral inoculum dose, receipts malicious time etc. in addition, disease is as a result shown
The control of cell maintenance medium pH value is that 0.01 inoculation MDBK is thin between 7.2~7.4, by virus infection value (MOI) during poison breeding
Born of the same parents, the optimal harvest time of virus are 48~72 hours, can obtain the virus liquid of high viral level.
It is in the present embodiment 10% from serum-concentration in cell culture fluid, pH value is controlled in 7.3, maintaining liquid (commodity
Name be DMEM, purchased from Hyclone companies) in serum-concentration be 2%, by virus infection value (MOI) be 0.01 inoculation Niu Chuanran
Property the generation Strain of rhinotracheitis virus IBRV-B strains the 7th (culture presevation numbering is CGMCC No.14294) to plant poison thin in MDBK
Born of the same parents, virus harvest time are 48 hours.
2 inactivation technologies
Select final concentration of 0.1%, 0.2%, 0.3%, 0.4% formalin and it is final concentration of 0.1%, 0.2%,
0.3%th, 0.4% beta-propiolactone carries out inactivation comparative test to infectious bovine rhinotrachetis virus, and result of the test shows,
0.2% beta-propiolactone can be by infectious bovine rhinotrachetis virus complete inactivation, moreover, experiment after 24 hours under the conditions of 4 DEG C
As a result show that damage of the beta-propiolactone to cell is smaller；Infectious bovine rhinotrachetis virus adds 0.3% formalin, through 37
DEG C inactivation also can reach within 48 hours complete inactivation virus purpose；But formaldehyde has strong and stimulating, if remained in vaccine free
Formaldehyde enter animal body, adverse reaction can be produced.It is thus final to determine to be gone out under the conditions of 4 DEG C with 0.2% beta-propiolactone
Infectious bovine rhinotrachetis virus living is after 24 hours, and room temperature inactivates 24 hours beta-propiolactones with hydrolysed residue.
3 emulsifying process
Using France match BIC Corp commercialization MontanideTM ISA15AVG adjuvants, it is degerming after can be directly used for epidemic disease
It is prepared by seedling emulsification；Same batch of virus liquid of inactivation is thawed before emulsification and mixed, is stirred in sterile indoor refiner with 200r/min
Uniformly, then according to aqueous phase antigen and the volume proportion of oil phase adjuvant 8.5: 1.5；Emulsification procedure is that aqueous phase first is put into emulsifier
In be stirred, be then slowly added into oil phase adjuvant, continuously stirred 30 minutes with 800r/min；Produce obtained vaccine and belong to water bag
Oil-type, for stable interface and elimination bubble, obtains optimum emulsification effect, it is necessary to be dispensed after static 30 minutes.
4 inspection of semifinished product quality standards
4.1 steriling test
By existing《Chinese veterinary pharmacopoeia》Annex is tested, and answers asepsis growth.
4.2 viral levels are determined
Seed culture of viruses is made into continuous 10 times with DMEM nutrient solutions to be serially diluted, each dilution factor takes 100 μ l to add 96 hole cell trainings
Support in plate, each dilution factor makees 8 repetitions, is subsequently added MDBK cells, per the μ l of hole 100, (cell content is with 3 × 105/ ml), and
If normal cell culture is compareed, put 37 DEG C, cultivate in the CO2 incubators containing 5%, cytopathy (CPE), cell circle are observed day by day
Contracting, is gathered into grape cluster sample group, and cavity is formed on cell monolayer and is then judged to CPE.Observation 7 days, records the hole of cytopathy
Number, the TCID50 of virus is calculated according to Reed-Muench methods.As a result：Every milliliter of virus liquid is not less than 1011.0TCID50。
4.3 inactivations are examined
The virus liquid after inactivation is taken, 3 bottles of (cell contents of MDBK cells are inoculated in the ratio of virus liquid and growth-promoting media 1: 10
With 3 × 105/ ml), 37 DEG C of culture observations 5 days, to without lesion person's generation of blind passage 2 again, while setting virus control.As a result acellular disease
Become.3 batches of semi-finished product of Laboratory Production are qualified.
5 on product inspection quality standard
5.1 safety verification standards
In order to ensure the security of vaccine, the present invention has successively been carried out " to small white mouse to 5 batches of vaccines prepared by laboratory
Safety testing ", " safety being inoculated with to the inoculation of calf single dose, single dose repeated inoculation, disposable overdose (2 multiple dose)
Property experiment ", " to the inoculation of finished cattle single dose, single dose repeated inoculation, disposable overdose (2 multiple dose) be inoculated with security
Experiment ", the security to the inoculation of cow in calf single dose, single dose repeated inoculation, disposable overdose (2 multiple dose) inoculation are tried
Test ".Result of the test shows：After each immune animal single dose inoculation, single dose repeated inoculation, disposable heavy dose of inoculation, animal
State is good, and feeding drinking-water is normal, is as good as paradoxical reaction；To the breeding function of cow in calf also without obvious shadow after vaccine inoculation
Ring.Experiment proves that vaccine is safe above.As long as being summed up by the experimental observation present invention after vaccine inoculation in 21 days not
There are the phenomenons such as local redness, ulcer, erosion and neuratorphy, feeding reduction, would not occur later because vaccine inoculation
Caused by adverse reaction, it is possible to prove the security of vaccine.Therefore, provided in Trial Regulation (draft)：With 2~June
(not high 1: 2) 2 of neutralize antibody titers, each deep intramuscular injection vaccine 4ml separately takes condition identical 2 to the age susceptible calf of health
Head ox is not inoculated with as control, Continuous Observation 14 days.Observation period planted agent occur without it is any local as caused by vaccine injection or
Systemic adverse reactions.
The formulation of 5.2 efficacy test standards
5.2.1 antibody titer protects correlation test with Immunization
By 1 batch of qualified infectious bovine rhinotrachetis virus inactivated vaccine 151101 of safety testing, according to various dose
0.5ml/ parts, 1ml/ parts, 2ml/ parts, 4ml/ parts respectively be immunized 2~6 monthly ages healthy calf (neutralizing antibody is not high
In 2) 5 intervals immune 2 times, non-immunized controls group 3 on the 14th, two exempt from 14 later with 107.0TCID50/ml ox infectiousness noses
The generation of bronchitis virus IBRV-B strains the 10th strong poison 4ml nasal cavities internal spraying inoculation, to determine that neutralizing antibody titers are protected with Immunization
The correlation of shield.Result of the test shows, when serum neutralize antibody titers are being not less than 1: 32, and calf attacks malicious protective rate can 80%
More than.When thus determining that vaccine potency is examined, test stone is secondary immunity after 14 days, and its serum neutralize antibody titers is not less than
1∶32。
5.2.2 minimum immune dosage and minimum dosage experiment
By 3 batches of qualified infectious bovine rhinotrachetis virus inactivated vaccines 151101 of safety testing, 151102 and
151103, the health at 2~6 monthly ages is immunized respectively according to 1ml/ parts of various dose, 2ml/ parts, 4ml/ parts, 6ml/ parts
Calf (IBRV neutralizing antibodies are not higher than 2), neutralizing antibody detection is carried out in second of immune blood sampling in latter 14 days.As a result show, with
2ml/ doses be immunized calf after 28 days neutralize antibody titers average value be not less than 1: 32, and 4ml/ part above dose immunizations
Calf after 28 days neutralize antibody titers be not less than 1: 64.According to neutralize antibody titers with attacking poison protection correlation test result,
It is determined that when infectious bovine rhinotrachetis virus neutralize antibody titers reached more than 1: 64 on 28th after immune, ox can be protected completely
The attack of infectious bovine rhinotracheitis Virus.In view of potency that may be present during the preservation, transport and use of vaccine
Reduction factor.The minimum immune dosage of infectious bovine rhinotrachetis virus inactivated vaccine (IBRV-B plants) is defined as by the present invention
2ml/ parts, dosage is defined as 4.0ml/ parts.
5.2.3 immune guinea pig and immune cattle neutralizing antibody correlation test
By 3 batches of infectious bovine rhinotrachetis virus inactivated vaccines 151101 of Laboratory Production, 151102 and 151103 points
Other immune guinea pig and calf, carry out neutralize antibody titers detection, analyze the neutralizing antibody correlation of cavy and calf.As a result table
Bright, when 2ml/ heads are immunized in immunized guinea pigs 0.5ml/, calf, its serum neutralize antibody titers is not less than 1: 32, two after 28 days
Person has obvious parallel relation.Experiment results proved can replace target animals (ox) to carry out efficacy test with experimental animal cavy.
Therefore, according to result above, the efficacy test standard of this product is set to by the present invention：
With body weight 350g~400g adult guinea pigs (IBRV neutralize antibody titers are not higher than 1: 2) 5, each intramuscular injection vaccine
0.5ml, after 28 days, together with control cavy 4, takes a blood sample, determines IBRV neutralize antibody titers in the lump.Compare cavy HI antibody titers
1: 2 should be not higher than, immune guinea pig should at least there are 4 antibody response occur, and neutralize antibody titers should be not less than 1: 32.
With the 3 monthly ages susceptible calf (IBRV neutralize antibody titers are not higher than 1: 2) 5 of health, each deep intramuscular injection vaccine
2ml, after 28 days, together with control ox 4, takes a blood sample, determines IBRV neutralize antibody titers in the lump.Compareing ox HI antibody titers should not be high
In 2, antibody response should all occur in note seedling ox, and neutralize antibody titers should be not less than 1: 32.
6 antibody dynamic regularities are tested with immune duration
By 3 batches of qualified infectious bovine rhinotrachetis virus inactivated vaccines 151101 of safety testing, 151102 and
151103 difference immune health calves, pregnant ox and Adult Bovine.Further to grasp immune efficacy and immune duration, formulate and close
The immune programme for children of reason, it is ensured that immune cows keep high and lasting antibody level, and antibody test is carried out to immune cattle different time,
To grasp its antibody dynamic regularity.
Result of the test shows that 2ml/ parts are immunized in cows, and booster immunization is once, in its serum and anti-after 28 days after 2~3 weeks
Body is not less than 1: 32, and antibody peak is in 4~6 months after being immunized, and antibody level is gradually reduced thereafter, to 12 months still up to 1:
32, to ensure the immune protective effect of vaccine, it is 10 months to determine its immune duration.
The determination of 7 immune programme for children
According to antibody dynamic regularity, duration of immunity of the cows after immune, in combination with clinically infectious bovine rhinotrachetis disease
The actual conditions of poison infection, present invention determine that the immune programme for children of infectious bovine rhinotrachetis virus inactivated vaccine (IBRV-B plants)
For：It is annual to be immunized twice.Immune every time is 2ml/ (Adult Bovine is doubled).
The storage life of 8 vaccines
3 batches of inactivated vaccines (151101,151102 and 151103) storage life that the present invention is prepared to laboratory is tried
Test research, 3 batches of vaccines preserve under the conditions of 2~8 DEG C to 9,12,15,18 months, it is separately sampled to its property in each period
Shape, security and immune efficacy detection.As a result show, 3 batches of vaccines preserved under the conditions of 2~8 DEG C 18 months characters do not occur it is bright
Aobvious change, steriling test and security all meet the requirement of quality standard.151101 batches of vaccines are preserved 18 months in efficacy test
It is 1: 16 to have a cavy HI antibody titer after sample immune guinea pig；151102 and 151103 batches of vaccines preserve 18 months samples and exempted from
It is 1: 16 to have a head of cattle serum neutralize antibody titers after epidemic disease ox, but the average neutralizing antibody level of immune cattle is 1: 32, it is contemplated that
The potency loss that vaccine is caused during transport and use, the present invention is preserved under the conditions of the storage life of vaccine is set into 2~8 DEG C
15 months.
9 are compared with the immune efficacy of domestic similar commercial seedling
3 batches of vaccines 151101,151102 and 151103 that laboratory is manufactured experimently and commercial available vaccines A (lot numbers：20151011)
With B (lot numbers：20150905) 350g~400g healthy guinea pigs and 5~6 monthly age healthy calfs, its serum after 28 days, are immunized respectively
Neutralize antibody titers are not less than 1: 32, reach that attacking poison protection requires that vaccine neutralizing antibody average value prepared by laboratory is slightly higher
In commercially available A vaccines, hence it is evident that higher than commercially available B vaccines.
Respectively before immune, it is immune after 3,6,9,12 months venous blood collection separation serum, detect antibody production.As a result
Show：After immune in 6 months, the vaccine of laboratory trial-production and commercially available A and B vaccine immunities ox, neutralize antibody titers are 1:
More than 32, it can reach that attacking poison protection requires.And commercially available B vaccine immunities ox neutralizing antibody declines substantially after 6 months.
The adverse reactions such as abscess, incrustation are not present in the inoculation position for injecting 3 batches of vaccines 151211,151212 and 151213,
And there is more than 2% above-mentioned adverse reaction in commercially available A and B vaccine immunizations position.
The preferred embodiments of the present invention are the foregoing is only, are merely illustrative for the purpose of the present invention, and it is nonrestrictive；
Those of ordinary skill in the art understand that can carry out many to it in the spirit and scope that the claims in the present invention are limited changes
Become, modification, or even equivalent change, but fall within protection scope of the present invention.
Claims (6)
1. one plant of infectious bovine rhinotrachetis virus, it is characterised in that the infectious bovine rhinotrachetis virus is named as ox biography
IBRV-B plants of metachromia rhinotracheitis virus, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its
Culture presevation is numbered：CGMCC No.14294.
2. the infectious bovine rhinotrachetis virus described in claim 1 is in infectious bovine rhinotrachetis virus inactivated vaccine is prepared
Application.
3. a kind of infectious bovine rhinotrachetis virus inactivated vaccine, it is characterised in that containing described in the claim 1 after inactivation
Infectious bovine rhinotrachetis virus.
4. inactivated vaccine as claimed in claim 3, it is characterised in that described inactivation refers to pass the ox described in claim 1
Metachromia rhinotracheitis virus inactivate 24 hours using 0.2% beta-propiolactone under the conditions of 4 DEG C, then 24 hours water of room temperature inactivation
Solve the beta-propiolactone of residual.
5. the inactivated vaccine described in claim 3 or 4 is preparing prevention disease as caused by infectious bovine rhinotrachetis virus
Application in biological medicine.
6. application as claimed in claim 5, it is characterised in that the breathing problem that described disease occurs for the negative ox of health.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102049043A (en) * | 2009-11-04 | 2011-05-11 | 天津瑞普生物技术股份有限公司 | Infectious bovine rhinotracheitis vaccine and preparation method thereof |
| CN102337250A (en) * | 2011-05-24 | 2012-02-01 | 深圳市青珊瑚科技有限公司 | Infectious bovine rhinotracheitis virus strain and preparation method and application thereof |
| CN104928260A (en) * | 2015-06-01 | 2015-09-23 | 何洪彬 | Infectious bovine rhinotracheitis virus IBRV-JN03 isolate and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102049043A (en) * | 2009-11-04 | 2011-05-11 | 天津瑞普生物技术股份有限公司 | Infectious bovine rhinotracheitis vaccine and preparation method thereof |
| CN102337250A (en) * | 2011-05-24 | 2012-02-01 | 深圳市青珊瑚科技有限公司 | Infectious bovine rhinotracheitis virus strain and preparation method and application thereof |
| CN104928260A (en) * | 2015-06-01 | 2015-09-23 | 何洪彬 | Infectious bovine rhinotracheitis virus IBRV-JN03 isolate and application thereof |
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