CN108949700A - A kind of JS14-2 plants of goat haemadsorption virus 1 and its application - Google Patents
A kind of JS14-2 plants of goat haemadsorption virus 1 and its application Download PDFInfo
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Abstract
The invention belongs to veterinary biologics field, discloses a kind of JS14-2 plants of goat haemadsorption virus 1 and its preparing the application in inactivated vaccine.It isolates and purifies after obtaining goat haemadsorption virus 1 JS14-2 Strain, by virus liquid through centrifugation, formalin-inactivated, adjuvant emulsion is added and prepares inactivated vaccine.The present invention provides a kind of Strain that can be used for preparing goat haemadsorption virus 1 inactivated vaccine, which belongs to domestic popular strain, and virulence is strong, and antigenicity is good；The strain has preferable growth characteristics on MDBK cell, can obtain stable high titre and (be more than or equal to 107 TCID50/ mL, HA-HI test are more than or equal to 28)；Vaccine preparation method is perfect, and immunogenicity is good with immune protective effect；Low using immunizing dose, highly-safe, immune duration is long, greatlys save production and use cost, is conducive to effective prevention and control disease in clinical practice.
Description
Technical field
The invention belongs to veterinary biologics field, it is related to a kind of JS14-2 plants of goat haemadsorption virus 1 and its application.
Background technique
Goat haemadsorption virus 1 (Caprine parainfluenza virus 3, CPIV3) is one identified recently
Kind Paramyxoviridae Respirovirus member, to have the RNA virus of cyst membrane, main infection goat and sheep.The virus is main
By respiratory infectious, cause respiratory disease, will lead to when stress, mix or other cause of diseases of secondary infection obvious
Clinical symptoms, cause serious respiratory disease, lead to higher morbidity and mortality.Epidemiological study shows the country
There are higher viral infection rates for flock of sheep.The virus is newly discovered new cause of disease, both at home and abroad without to the effective prevention and control of the virus
The report of method.It there is no the vaccine and diagnostic reagent that can prevent the infection of goat haemadsorption virus 1.Therefore, it develops and is directed to me
The inactivated vaccine of state's prevalence strain and to join seedling, attenuated live vaccines accordingly extremely urgent.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of JS14-2 plants of goat haemadsorption virus 1, strain tool
There are stronger virulence and good immunogenicity.In addition, for the correlation that there is no at present for goat haemadsorption virus 1 vaccine
Report provides a kind of utilization bovine kidney cells MDBK (abbreviation MDBK cell) virus of proliferation preparation goat haemadsorption virus 1 inactivation
The method of vaccine.
The present invention filters out pathogenic strong from the goat haemadsorption virus 1 prevalence strain of China's prevalence and has higher
JS14-2 plants of the goat haemadsorption virus 1 (Caprine parainfluenza virus 3, JS14-2) of immunogenicity, should
The classification naming of virus is goat haemadsorption virus 1 (Caprine parainfluenza virus 3, CPIV3), Latin
Scientific name is Caprine parainfluenza virus 3, has been deposited in China typical culture collection center, address is in China
Wuhan Wuhan University, postcode 430072, deposit number: CCTCC NO:V201832, preservation date: on June 26th, 2018.The disease
Strain voluntarily acquires one plant of strain having compared with strong virus force and very high immunogenicity of acquisition in Jiangsu Province from inventor.
The present invention also provides a kind of JS14-2 plants of goat haemadsorption virus 1 of isolation and purification method, the method packets
Identification morbidity sheep is included, the nose swab sample of acquisition morbidity sheep is added antibiotic in the nose swab sample, is incubated for the sample
And be centrifuged, it takes supernatant through membrane filtration degerming, is inoculated in bovine kidney cells monolayer cell culture, and observe cytopathy, until going out
Now stable cytopathy.
Further, the present invention provides a kind of JS14-2 plants of goat haemadsorption virus 1 of isolation and purification methods, wherein
The antibiotic is effective to gram-positive bacteria and negative bacterium.
Further, the present invention provides a kind of JS14-2 plants of goat haemadsorption virus 1 of isolation and purification methods, wherein
The virus is repeatedly passed on during observation cytopathy.
Further, the present invention provides a kind of JS14-2 plants of goat haemadsorption virus 1 of isolation and purification methods, wherein
The antibiotic is mycillin.
Further, described the present invention provides a kind of JS14-2 plants of goat haemadsorption virus 1 of isolation and purification method
Method further includes carrying out plaque purification to the virus being separated to.
Further, described the present invention provides a kind of JS14-2 plants of goat haemadsorption virus 1 of isolation and purification method
Method further includes carrying out plaque purification at least once to the virus being separated to.
Further, described the present invention provides a kind of JS14-2 plants of goat haemadsorption virus 1 of isolation and purification method
Method further includes being diluted purifying before carrying out plaque purification to the virus being separated to.
Further, described the present invention provides a kind of JS14-2 plants of goat haemadsorption virus 1 of isolation and purification method
Method further includes carrying out RT-PCR and sequence analysis to the viral RNA after purification.
Further, described the present invention provides a kind of JS14-2 plants of goat haemadsorption virus 1 of isolation and purification method
Method further includes carrying out pure property inspection to virus after purification.
The present invention also provides a kind of method for preparing goat haemadsorption virus 1 JS14-2 vaccine, the method includes
Preparation goat haemadsorption virus 1 JS14-2 attacks strain, and inactivates to the strain of attacking, and by after inactivation virus with spit
Temperature -80 is uniformly mixed, and water phase is made, then injection white oil is uniformly mixed with Si Ben -80, and oily phase is made, and oil is mutually mixed with water phase
Emulsification is closed, can be prepared by goat haemadsorption virus 1 vaccine.
The present invention also provides a kind of methods for preparing goat haemadsorption virus 1 JS14-2 vaccine, wherein preparing goat
It includes that goat haemadsorption virus 1 JS14-2 strain is seeded to bovine kidney cells that haemadsorption virus 1 JS14-2, which attacks strain, is being gone out
Cell suspension, and multiple multigelation are now collected after obvious cytopathy, are centrifuged, the supernatant of collection is goat parainfluenza virus
3 type JS14-2 attack strain.
The present invention also provides a kind of method for preparing goat haemadsorption virus 1 JS14-2 vaccine, the method is also wrapped
It includes and the strain of attacking after inactivation is detected, it is determined whether inactivation is complete.
The present invention also provides a kind of methods for preparing goat haemadsorption virus 1 JS14-2 vaccine, wherein strain of attacking against each other
Whether inactivate complete detection to be seeded to bovine kidney cells by the strain of attacking after inactivating and pass on after multigelation, observation is thin
Born of the same parents lesion is realized.
A kind of goat haemadsorption virus 1 JS14-2 vaccine, is prepared using method as described above.
A kind of goat haemadsorption virus 1 JS14-2 therapeutic agent comprising vaccine as described above and its with pharmaceutically
The drug that acceptable antigen, immunologic adjuvant, carrier or auxiliary material combination are constituted, such as inactivated vaccine, live vector seedling, monovalent seedling, multivalence
Seedling or connection seedling etc..
It is generated as attack strain in induction the present invention also provides a kind of JS14-2 plants of above-mentioned goat haemadsorption virus 1
Purposes in goat parainfluenza.
Mountain is generated as attack strain induction the present invention also provides a kind of JS14-2 plants of above-mentioned goat haemadsorption virus 1
Sheep parainfluenza animal model is being established for evaluating the purposes in goat haemadsorption virus 1 vaccine effect.
It is used for as attack strain in foundation the present invention also provides a kind of JS14-2 plants of above-mentioned goat haemadsorption virus 1
The evaluation method in the animal model of goat haemadsorption virus 1 vaccine effect is evaluated, the method includes to goat parainfluenza
Viral JS14-2 plants of 3 type attacks the animal progress lesion observation after poison.
It is used for as attack strain in foundation the present invention also provides a kind of JS14-2 plants of above-mentioned goat haemadsorption virus 1
The evaluation method in the animal model of goat haemadsorption virus 1 vaccine effect is evaluated, the method includes to goat parainfluenza
The animal that viral JS14-2 plant of 3 type is attacked after poison carries out RT-PCR and detects viremia virusemia and toxin expelling.
The present invention also provides a kind of JS14-2 plants of above-mentioned goat haemadsorption virus 1 to diagnose goat parainfluenza virus in preparation
Application in the reagent of malicious 3 type virus associated diseases.
The utility model has the advantages that of the invention JS14-2 plants of goat haemadsorption virus 1 have stable biological characteristics, have compared with
Strong is pathogenic, can be used for establishing and attacks poison morbidity model.JS14-2 plants of goat haemadsorption virus 1 have good immunogene
Property, antibody detection method can be established with it.With JS14-2 plants of the goat haemadsorption virus 1 oil adjuvant killed vaccine prepared safety
Reliably, the antibody of higher level can be generated after goat immune, the antibody duration reaches 12 months, and attacks poison to homologous seed culture of viruses
With good protecting effect, the symptom of immune rear flock of sheep is significantly reduced with toxin expelling, can effectively prevent goat parainfluenza virus 3
The prevalence of type.
Detailed description of the invention
For preferably the present invention will be described, the following drawings is provided, provided attached drawing is merely to illustrate the present invention,
Rather than it limits the invention:
Fig. 1 is that the Electronic Speculum of goat haemadsorption virus 1 JS14-2 strain virus particle is observed；
Fig. 2 is the JS14-2 plants of inactivated vaccine immuning effect test each group HI antibody titer variations of goat haemadsorption virus 1
Situation；
Fig. 3 is JS14-2 plants of inactivated vaccine immune antiboidy duration detection cases of goat haemadsorption virus 1.
Specific embodiment
Below in conjunction with specific embodiment and Figure of description, the present invention is further illustrated, rather than to the present invention
It is limited.
Separation, purifying and the identification of JS14-2 plants of 1 goat haemadsorption virus 1 of embodiment
1 virus purification
2013, there is the disease based on respiratory symptom, table in many places Deng Di scale goat in China Jiangsu Anhui
It is now spiritual depressed, cough, stream slurries or dense property nose liquid.The nose swab sample of acquisition morbidity goat is goat pair through Testing and appraisal
3 type of influenza virus.Then continue to carry out a lot of similar cases in Jiangsu pathogeny detection again, acquisition morbidity sheep nose swab sample
Product, -80 DEG C of preservations.
Mycillin, 37 DEG C of incubations are added in the nose swab sample that will test as the positive morbidity goat of parainfluenza virus
30min, 12000r/m are centrifuged 20min, take supernatant through 0.22 μm of membrane filtration degerming, are inoculated in bovine kidney cells (MDBK) and (are purchased from
China Veterinery Drug Inspection Office) monolayer cell culture, in 37 DEG C, 5%CO2Culture is protected from light in cell incubator, observation is thin daily
Born of the same parents' lesion (CPE).When blind passage to 2nd generation, 48h starts to generate obvious cytopathy (CPE) after connecing poison, goes out after reaching for the 3rd generation
Now stable cytopathy.
2 viral purifications
The 3rd generation virus being separated to is made into 10 times of gradient dilutions with nutrient solution.Then it is inoculated in respectively and covers with MDBK cell
24 porocyte culture plates of single layer, each dilution are inoculated with 2 holes, and every hole is inoculated with 100 μ L.It concurrently sets and does not connect the negative right of poison
According to.37 DEG C of absorption 1h discard virus liquid, and prepared agar 0.5mL is added in every hole, and 37 DEG C are cultivated 5-7 days, neutral red staining 2h
It cultivates and passed on for 2 generations in the MDBK cell that plaque is inoculated on 96 orifice plates in picking hole, collect the culture for cytopathy occur,
A plaque purification is carried out again, and is passed on and expanded culture purified strain.
3RT-PCR and sequence are analyzed
Above-mentioned viral RNA after purification is extracted, is carried out using Beijing Quan Shi King Company One step RT-PCR detection kit
RT-PCR expands virus M gene, 20 μ L of RT-PCR total volume, wherein 2 × R-Mix Buffer, 10 μ L, upstream and downstream primer each 0.5
0.4 μ L of μ L (F:5 '-AGTGATCTAGATGATGATCCA-3 ', R:5 '-GTTATTGATCCAATTGCTGT-3 '), E-Mix,
4 μ L of RNA template, no Rnase water complement to 20 μ L.Amplification program: 45 DEG C of reverse transcription 40min；94℃ 5min；94 DEG C of 30s, 50
DEG C 30s, 72 DEG C of 30s, 35 circulations；72℃ 10min.Recycling PCR product send biotech firm to be sequenced, and carries out BLAST comparison
Analysis is confirmed that it is CPIV3, which is named as JS14-2 plants of goat haemadsorption virus 1.
RT-PCR expands JS14-2 plants of goat haemadsorption virus 1 of major antigen gene HN, and carries out sequencing analysis, with
Reported goat haemadsorption virus 1 isolated strain is compared, and there are many places changes for the amino acid sequence of the gene order and coding
It is different, it is analyzed by software prediction, the antigenicity enhancing of the HN albumen of coding, immunogenicity are more preferable.
The coagulation analysis of embodiment 2
The 3rd generation of separation and the after purification HA-HI test of the 3rd generation goat haemadsorption virus 1 virus are measured by hemagglutination test.
Different virus liquids is subjected to 2 times of doubling dilutions on 96 hole blood-coagulation-boards, 1% guinea pig red blood cells, 37 DEG C of incubations are then added
45min, using highest dilution that red blood cell can be made to be aggregated completely as the HA-HI test of virus.As the result is shown: hemagglutination test measures
Its HA-HI test is up to 256.
The observation of 3 viral morphology of embodiment
JS14-2 plants of cell culture 200mL of goat haemadsorption virus 1 are taken, in 1 2000r/min centrifugation 5min removal
Cell fragment, supernatant take precipitating PBS to dissolve overnight, obtain viral suspension through 40 000r/min ultracentrifugation 2h.It will be sick
Malicious suspension is loaded on copper mesh, after 2% phosphotungstic acid negative staining, observes viral particle morphology under transmission electron microscope.Such as Fig. 1 institute
Show, it is seen that largely have the virion of cyst membrane, irregular form is presented in virus, and size is about 100-150nm.
The pathogenicity and infection morbidity model of JS14-2 plants of 4 goat haemadsorption virus 1 of embodiment
1. attacking the preparation of seed culture of viruses
Goat haemadsorption virus 1 Strain JS14-2 is taken to be seeded to MDBK cell, inoculum concentration MOI=0.1,37 DEG C of suctions
Culture solution is abandoned after attached 2h, the DMEM culture medium for containing 2% fetal calf serum is added, and 37 DEG C are cultivated 5 days to the obvious cytopathy of cell appearance
Cell suspension is collected after change, multigelation 3 times, 10000r/min centrifugation, supernatant is JS14-2 plants of goat haemadsorption virus 1
Viral seed culture of viruses.The HA-HI test that hemagglutination test measures JS14-2 plants of goat haemadsorption virus 1 is 256, and presses Reed-Muench
Method calculates TCID50It is 10-7/mL.Pure property inspection, virus liquid are carried out according to existing " Republic of China Veterinary Pharmacopoeia " annex
It should be without varied bacteria growing.JS14-2 plants of goat haemadsorption virus 1 have good growth characteristics in MDBK cell, with first front range
Sheep haemadsorption virus 1 isolated strain compares the virus liquid that can more rapidly obtain stable high titre.
2 animal packets and virus infection program
Selection CPIV3 antigen and antibody test are 2-3 monthly age health goat 10 of feminine gender, are randomly divided into and attack malicious group
(CC) and control group (NC), every group 5；Attack JS14-2 plants of goat haemadsorption virus 1 of the seed culture of viruses of poison group inoculation after purification
(106TCID50/ mL), collunarium 2mL.Control group is inoculated with MDBK cells and supernatant by same dose and method.Attack two components after poison
Circle raising, is observed 14 days, observes and records the clinical symptoms (cough and nasal discharge etc.) of each group goat daily.Attack the 1st after poison,
3, the nose swab and blood serum sample of 5,7,9,11,14 days acquisition each group goats, -80 DEG C of preservations.RT-PCR detect viremia virusemia with
Toxin expelling.It attacks after poison to cut open for 14 days and enters the observation of row gross anatomy lesion.
3.RT-PCR detects viremia virusemia and toxin expelling
The above-mentioned viral RNA attacked in poison group and control group goat nose swab sample is extracted, RT-PCR expands M genetic test row
Malicious situation, 20 μ L of RT-PCR total volume, wherein 2 × R-Mix Buffer, 10 μ L, each 0.5 μ L (F:5 '-of upstream and downstream primer
AGTGATCTAGATGATGATCCA-3 ', R:5 '-GTTATTGATCCAATTGCTGT-3 '), E-Mix 0.4 μ L, 4 μ of RNA template
L, no Rnase water complement to 20 μ L.Amplification program: 45 DEG C of reverse transcription 40min；94℃ 5min；94 DEG C of 30s, 50 DEG C of 30s, 72
DEG C 30s, 35 circulations；72℃ 10min.PCR product electrophoresis detection in 1.2% Ago-Gel is taken after reaction,
And it photographs to record.The sample for choosing RT-PCR test positive carries out virus purification on MDBK cell, observes cytopathy.
It the results are shown in Table 1.
1 viremia virusemia of table and toxin expelling detection case
Note :-, it is negative；+, it is positive
4 clinical symptoms and dissect lesion
The results are shown in Table 2 for clinical symptoms, attacks poison group and starts within goat the 3rd day after attacking poison nasal mucus, cough, 5-9 occur
There is more apparent respiratory symptom in it, shows as poor spirit, stream serosity or purulence nasal mucus, cough；Control group is without obvious
Clinical symptoms.RT-PCR detects viremia virusemia, and the results are shown in Table 2 with toxin expelling situation, attacks poison group goat the 3-9 days after infection
Viremia virusemia, the 3-11 days detectable nasal cavity toxin expellings can be detected.The detection of control group goat clinical sample is feminine gender.It will inspection
It surveys to send sequencing company to be sequenced after positive band recycling, expanding strain as the result is shown is CPIV3 JS14-2 plants.
Dissect result is visible to attack poison group Lung in Goats and apparent hyperplasia, consolidation occurs, part of the surface have serous effusion, with
Wall of the chest adhesion.Other organs have no obvious lesion.Control group lung tissue has no obvious Pathologic changes.
RT-PCR, which detects positive nose swab sample, can generate stable cytopathy through the separation of MDBK cell, collect passage disease
Poisons RT-PCR is detected as JS14-2 plants of CPIV3.
JS14-2 plants of 2 goat haemadsorption virus 1 of table attacks clinical symptoms after poison
Note :-, normally；+, slightly；++, moderate；+++, severe
JS14-2 plants of blood clottings of embodiment 5CPIV3 inhibit (HI) antibody test
First carry out the HA titre detection of virus (referring to embodiment 4).Antigen is diluted, 4 HA units of its content are made.96
Each 25 μ L physiological saline of Kong Zhongjia of hole V-type micro-reaction plate.Add 25 μ L serum in the first hole, continuous 2 times times are carried out after mixing
Than dilution, last 1 hole is drawn 25 μ L after mixing and is discarded.4 unit antigen controls are set simultaneously and only add the feminine gender of guinea pig red blood cells
Control, sets 37 DEG C of 40min.Every hole adds 25 μ L, 1% guinea pig red blood cells liquid, sets 37 DEG C of 40min.Completely inhibit the antibody of agglutination
Highest extension rate is hemagglutination inhibition antibody (HI) titre of serum.Antibody titer is more than or equal to 16 (24) it is the positive, be less than etc.
In 8 (23) it is feminine gender.The result shows that can produce HI antibody (Fig. 2, Fig. 3) after vaccine immunity.
JS14-2 plants of inactivated vaccine preparations of embodiment 6CPIV3
JS14-2 plants of seeds culture of viruses of goat haemadsorption virus 1 that will be prepared in embodiment 4, with final concentration of after centrifugal treating
0.1% formalin-inactivated 16h, the virus liquid after inactivation are placed in 2-8 DEG C and save backup.Virus liquid sample after inactivation is tieed up with cell
It holds liquid and makees 10 times of dilutions, be seeded to the MDBK cell of 90% convergence degree, 37 DEG C of absorption 1h are added after cell maintenance medium culture 5d instead
Virus liquid is harvested after multiple freeze thawing 3 times, according to the above method 2 generation of blind passage on MDBK cell.MDBK cell does not occur cytopathy,
Show that inactivation of virus is complete.
Injection white oil is mixed with Si Ben -80 according to volume ratio for 94:6, is mixed well, oily phase is made in high pressure sterilization.
Inactivation of viruses liquid is uniformly mixed with Tween-80 according to volume ratio for 96:4, water phase is made.By oily phase and water phase according to volume ratio
For 2:1 mixing, goat haemadsorption virus 1 JS14-2 inactivated vaccine strain is made in emulsification.Qualified vaccine will be emulsified and be sub-packed in sterilizing
In vaccine bottle afterwards, 2-8 DEG C of preservation.
Embodiment JS14-2 plants of inactivated vaccine immuning effect tests of 7 CPIV3
1. test grouping and sampling
The negative goat 15 of 2-3 monthly age health is selected, be randomly divided into three groups, every group 5: (VC is first immunized Immunization group
After attack poison), attack malicious control group (CC, not Immunization), blank control group (NC, not immune not attack poison).VC group is spaced 14d neck
Only, front and back blood sampling detection HI antibody respectively is immunized in the vaccine prepared in the immune embodiment 5 twice of intramuscular injection, each 1ml/.?
VC and CC group sheep is carried out to attack poison (10 within 14 days after secondary immunity7TCID50/ mL, 2ml/ head, collunarium), NC group by same dose and
Method is inoculated with MDBK cells and supernatant.Clinical symptoms, the 1st, 3,5,7,9,11,14 day acquisition each group mountain are observed daily after attacking poison
The nose swab and blood serum sample of sheep, -80 DEG C of preservations.RT-PCR detects viremia virusemia and nasal cavity toxin expelling.It attacks after poison 14 days (14dpc)
It cuts open and kills, carry out pathological observation.
2.HI antibody and the detection for attacking each index after poison
Hemagglutination inhibition antibody detection after immune is carried out by embodiment 5.As a result as shown in Figure 2: 14d can be detected after immune
To HI antibody, HI antibody titer is 27.2±0.8；Antibody titer increases rapidly after secondary immunity, reaches 212.2±0.5；Remaining two groups anti-
Body is feminine gender.Show that inactivated vaccine has good immune effect.CC group antibody generates rapidly after attacking poison, reaches at 14 days
28.8±1.5, show to attack poison success.VC group HI antibody titer maintains previous level variation little.
Start within CC group 3 days the clinical symptoms such as appearance has a running nose, coughs, discharge of eye increases, CC group clinical symptoms after attacking poison
Obviously, there is serosity/purulence nasal mucus, at most continue to the 14th day.VC group has 2 (2/5) to occur slight stream nose at the 5-7 days
It is obvious slight (table 3) that tears symptom, degree and duration relatively attack poison group.There is obvious toxin expelling at 3-11 days in CC group whole (5/5),
4/5 there is viremia virusemia at 5-9 days；VC group has 2 (2/5) of short duration toxin expelling (table 4) occurred at 5,7 days.
Dissect observes Histopathologic changes, and hyperplasia, consolidation and wall of the chest adhesion phenomenon occur in CC group lungs.Immunization
Group lungs are normal.The above results show that inactivated vaccine has preferable immune protective effect.
Table 3 attacks clinical symptoms situation after poison
Note :-, normally；+, slightly；++, moderate；+++, severe
Table 4 attacks malicious restrovirus mass formed by blood stasis and toxin expelling situation detects
Embodiment JS14-2 plants of inactivated vaccine safety testings of 8 CPIV3
Selection CPIV3 feminine gender 2-3 monthly age health lamb 20 be only randomly divided into 4 groups (single multiple dose group, doubling dosage group,
Single multiple dose repeating groups and control group).JS14-2 plants of CPIV3 of single multiple dose group, the musculi colli injection preparation of embodiment 6 go out
Live vaccine, 1mL/ is only；Doubling dosage group, musculi colli inject inactivated vaccine, and 2mL/ is only；Single multiple dose repeating groups, head exempt from 1mL/
Only, it is primary according to head to exempt from same procedure duplicate injection within 14 days after head exempts from；PBS buffer solution is immunized in control group.After injection observation whether there is or not
Allergic reaction and transient thermal response；Measurement rectal temperature daily, and observe the feelings such as each group goat mental status, drinking-water and feed
Condition；It touches and checks every goat injection site, if having the local inflammation reactions such as red and swollen heat pain；Respectively after immune preceding and immune
It weighs within 21 days, immune group and nonimmune group of relatively average opposite daily gain have no significant difference；It is cutd open after 28 days immune and kills examination
Goat is tested, checks injection site vaccine absorbing state and pathological change.
It is observed continuously after immune, each group lamb state of mind, feeding, drinking-water are normal as the result is shown, and it is anti-not occur allergy
It answers and transient thermal response, touch inoculation position does not find swelling；Immune group of weighing and nonimmune group of weight are without significant difference.
After 28 days immune, each group test sheep cut open killing, observes vaccine absorbing state, as a result injection site absorbs good, no oedema,
The pathological changes such as exudation and bleeding.Therefore, JS14-2 plants of inactivated vaccines of 3 type of goat parainfluenza are safe to 2-3 monthly age lamb.
The detection of 9 immune antiboidy duration of embodiment
Healthy lamb 20 of 2-3 monthly age are selected, are randomly divided into 2 groups, every group 10.1st group of carry out vaccine immunity, interval
The 14d musculi colli injecting immune CPIV3JS14-2 strain inactivated vaccine that twice prepared by embodiment 6, each 1ml/ is only；2nd group is
Blank control group.0 after first immunisation (- 0.5 month) and secondary immunity, take a blood sample according to embodiment 5 within 1,2,3,5,6,7,9,12 months
Method detects HI antibody level in serum.
As a result as shown in Figure 3: HI antibody can reach 2 after goat haemadsorption virus 1 inactivated vaccine first immunisation8.1 ±0.7, January reaches peak value 2 after secondary immunity11.3±0.8, then slowly decline, is all larger than 64 (2 in 9 months interior antibody titres6), until
Still maintain within 12 months the positive (25.1±1).Illustrate inactivated vaccine immune duration of the present invention up to 12 months.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (10)
1. a kind of goat haemadsorption virus 1 strain is named as JS14-2 plants of goat haemadsorption virus 1, deposit number are as follows:
CCTCC NO:V201832,
Preservation date are as follows: on June 26th, 2018, preservation address: China, Wuhan, Wuhan University, in China typical culture collection
The heart.
2. a kind of JS14-2 plants of goat haemadsorption virus 1 of isolation and purification method, which is characterized in that the method includes identifying
Fall ill sheep, acquisition morbidity sheep nose swab sample, antibiotic is added in the nose swab sample, be incubated for the sample and from
The heart takes supernatant through membrane filtration degerming, is inoculated in bovine kidney cells monolayer cell culture, and observes cytopathy, until occurring steady
Fixed cytopathy carries out plaque purification to the virus being separated to, and carries out pure property inspection to virus after purification.
3. a kind of goat haemadsorption virus 1 JS14-2 therapeutic agent comprising JS14-2 strain and its with it is pharmaceutically acceptable
Antigen, the vaccine composition that constitutes of immunologic adjuvant, carrier or auxiliary material combination, optionally inactivated vaccine, live vector seedling, monovalent seedling,
Multivalence seedling or connection seedling.
4. a kind of method for preparing goat haemadsorption virus 1 JS14-2 therapeutic agent, which is characterized in that the method includes preparations
Goat haemadsorption virus 1 JS14-2 strain, and the strain is inactivated, and the virus after inactivation is mixed with Tween-80
It closes uniformly, water phase is made, then injection white oil is uniformly mixed with Si Ben -80, be made oily phase, oily emulsification mutually mixed with water,
Prepare goat haemadsorption virus 1 vaccine.
5. a kind of method for preparing goat haemadsorption virus 1 JS14-2 vaccine, which is characterized in that the method includes preparing mountain
Sheep haemadsorption virus 1 JS14-2 strain, wherein being inoculated with to whether strain inactivates complete detection by the strain after inactivating
It is passed on to bovine kidney cells and after multigelation, observes cytopathy to realize.
6. a kind of goat haemadsorption virus 1 JS14-2 therapeutic agent, is prepared using method as claimed in claim 4.
7. JS14-2 plants of goat haemadsorption virus 1 as described in claim 1 include goat haemadsorption virus 1 in preparation prevention
Purposes in the therapeutic agent of viral associated diseases.
8. JS14-2 plants of goat haemadsorption virus 1 as described in claim 1 generate goat pair stream in induction as attack strain
Purposes in sense.
9. JS14-2 plants of goat haemadsorption virus 1 as described in claim 1 generate goat parainfluenza as attack strain induction
Animal model is being established for evaluating the purposes in goat haemadsorption virus 1 vaccine effect.
10. JS14-2 plants of goat haemadsorption virus 1 as described in claim 1 in preparation diagnosis goat haemadsorption virus 1 disease
Purposes in the reagent of malicious associated diseases.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112280748A (en) * | 2020-09-30 | 2021-01-29 | 内蒙古大学 | Sheep-derived sheep parainfluenza virus type 3 vaccine strain, vaccine composition prepared from vaccine strain, and preparation method and application of vaccine composition |
| CN112280748B (en) * | 2020-09-30 | 2022-12-30 | 内蒙古大学 | Sheep-derived sheep parainfluenza virus type 3 vaccine strain, vaccine composition prepared from vaccine strain, and preparation method and application of vaccine composition |
| CN113736799A (en) * | 2021-09-17 | 2021-12-03 | 江苏省农业科学院 | Goat parainfluenza virus type 3 infectious cDNA cloning construction method and application thereof |
| CN113736799B (en) * | 2021-09-17 | 2024-02-06 | 江苏省农业科学院 | Construction method and application of goat parainfluenza virus 3-type infectious cDNA clone |
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