CN106591242A - Canine parvovirus strain CPV-YH and applications thereof - Google Patents
Canine parvovirus strain CPV-YH and applications thereof Download PDFInfo
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Abstract
The invention provides a canine parvovirus strain CPV-YH and applications thereof, and belongs to the technical field of microbes. The preservation number of the provided canine parvovirus strain CPV-YH is CGMCC No.12990. The invention also provides applications of the canine parvovirus strain CPV-YH in the preparation of canine parvovirus vaccines and canine parvovirus diagnostic reagents. Based on the canine parvovirus strain CPV-YH, the invention also provides a vaccine composition for preventing and/or treating diseases caused by canine parvovirus and a reagent for detecting a canine parvovirus strain and/or diagnosing diseases caused by canine parvovirus. A novel content and direction are provided for the research on the variation of epidemic virus; a novel strain is provided for the canine parvovirus vaccine development; and a high quality raw material and choice are provided for the vaccine development.
Description
Technical field
The invention belongs to microbial technology field, and in particular to one plant of canine parvovirus poison strain CPV-YH and its application.
Background technology
Canine parvovirus disease (Canine Parvovirus disease, CPD) is also known as canine infectious enteritis or canine viral
Enteritis, is a kind of acute of dog, contact, the lethal biography caused by Canine Parvovirus (Canine parvovirus, CPV)
Catch an illness.Clinical manifestation is characterized with acute hemorrhagic entertitis and apyetouses myocarditiss.The disease can occur throughout the year, different
The dog of age, sex and kind is susceptible, but is mainly in pup.To fall ill, anxious, the course of disease is short, infectiousness is strong, mortality rate it is high based on
Want feature.It is to endanger one of canine farming infectious disease the most serious.
American scholars Eugster in 1977 and Nairn are at first from first observed in the dog feces for suffer from hemorrhagic enteritiss to class
Like feline panleucopenia viruses sample granule.Next year, almost separate in the U.S., Europe and Australia simultaneously and obtain CPV.In order to be different from
Be separated to from healthy dogs feces by Binn et al. within 1967 the superfine small viruses of dog (Minute virus of canine,
MVC, is traditionally also CPV-1), and it is named as the type of Canine Parvovirus 2 (CPV-2).Nineteen eighty-two, China beam scholar wise man etc. report earliest
The prevalence of the official confirmation such as similar Canine parvovirus infection enteritis, next year, Xu Hankun disease.
CPV is a kind of without cyst membrane, single-stranded, DNA viruses, and Parvoviridae (Parvoviridae) is belonged in classification, tiny
Chordopoxvirinae (Parvovirinae), parvovirus category (Parvovirus).So far, CPV only one of which serotype, but not
With the difference of antigenicity between strain, this species diversity is referred to as antigenic drift.The neutralizing antigenic site of Canine Parvovirus is located at VP2
On, the change of several critical bases and aminoacid thereon may result in antigenicity, host range and it is coagulation change, Mara
The research such as B confirms that CPV-2 is similar to RNA viruses, shows high genetic heterogeneity.But the DNA viruses as CPV occur
The sequence variation that so fast antigen is substituted, and it is rarely found that legacy is replaced completely.At present, Canine Parvovirus are by anti-
Original drift produces new mutant 3 hypotypes：CPV-2a、CPV-2b、CPV-2c.
The CPV separation strains of China there is also genovariation phenomenon, and CPV-2 was once popular in the early stage eighties in 20th century, but
Based on CPV-2a, CPV-2a and CPV-2b is extensive and deposits for the CPV being separated to after 1986.Except find CPV-2, CPV-2a,
Outside CPV-2b mutants, it was found that the 2 new variants for producing of further evolving on the basis of CPV-2a, its definite biology
Learn meaning unclear.Result is very serious when the natural reservoir (of bird flu viruses) drift occurred by virus occurs, because this virus can
With by innate immunity and non-adaptive host group's wide-scale distribution.Due to speed of mutation quickly, showing going out for Clinical practice
The immune effect of live vaccine and attenuated vaccine all declines in various degree, and existing vaccine can not fully meet control and prevention of disease and market
Consumption demand.
Therefore, in order to understand and grasp the variation situation of currently a popular strain, so as to more effectively prevention and control dog is tiny
Virosiss, this area is needed badly and isolates new CPV strains, for developing new canine parvovirus vaccine.
The content of the invention
The problems such as demand can not being met is declined according to the above-mentioned existing vaccine effect of this area objective reality, and is needed badly point
From the demand of new strain, the invention provides one plant of new canine parvovirus poison strain CPV-YH, and the Strain is preparing epidemic disease
Purposes in terms of Seedling, viral diagnosis reagent.
Technical scheme is as follows：
One plant of Canine Parvovirus (Canine parvovirus) strain CPV-YH, its preserving number is CGMCC NO.12990.
The whole genome sequence of the canine parvovirus strain CPV-YH is as shown in SEQ ID NO.1.
Purposes of the canine parvovirus strain CPV-YH in terms of canine parvovirus vaccine is prepared.
The purposes is characterized in that, with attenuation, and/or weak poison, and/or inactivation the canine parvovirus strain CPV-
Active substances of the YH as the vaccine.
Purposes of the canine parvovirus strain CPV-YH in terms of Canine Parvovirus diagnostic reagent is prepared.
The purposes includes, with described canine parvovirus strain CPV-YH as immunogen immune animal acquisition antibody；Or,
Specific primer is designed according to the whole genome sequence of the canine parvovirus strain CPV-YH.
Described Canine Parvovirus (Canine parvovirus) strain CPV-YH is carried out passing on propagation method, it is special
Levy and be, the strain described in claim 1 is inoculated in host cell, cultivated and/or passed on, it is preferable that the place
Chief cell adopts A-72 cells.
A kind of method for preparing canine parvovirus vaccine, is characterized in that, to the Canine Parvovirus (Canine
Parvovirus) strain CPV-YH be attenuated and/or inactivation become it is weak poison or without poison strain.
The method for preparing the antibody of anti-dog parvovirus, comprises the following steps：Using the Canine Parvovirus (Canine
Parvovirus) strain is used as immunogen immune animal.
What said method was prepared has the antibody of identification and/or binding activity to Canine Parvovirus.
A kind of vaccine combination for preventing and/or treating Canine Parvovirus associated diseases, it is characterised in that including subtracting
Poison, and/or it is weak poison, and/or inactivation the canine parvovirus strain CPV-YH.
The vaccine combination also includes the pharmaceutical carrier that can mix with the canine parvovirus poison strain CPV-YH；And/or,
Pharmaceutically acceptable adjuvant.
A kind of reagent for detecting Canine Parvovirus and/or diagnosis canine parvovirus, it is characterised in that include with the dog
Parvovirus strain CPV-YH is the antibody that immunogen is obtained；Or, the full-length genome sequence based on the canine parvovirus strain CPV-YH
The specific primer of row design.
The reagent is also including the conventional antibody in checkout and diagnosis field；And/or, other of checkout and diagnosis field are routinely tried
Agent.
The present invention is separated to one plant of new canine parvovirus Strain from morbidity dog group, is named as CPV-YH, and right
The Strain has carried out biological deposits, and its preserving number is CGMCC No.12990.
It should be noted that in order to obtain above-mentioned new canine parvovirus Strain, inventor to tradition using CRFK cells,
Mdck cell, FK-81 cells carry out virus isolation procedure and are improved, and from the inoculation of A-72 cells after pathological material of disease process, pass through
The Canine Parvovirus success rate of this kind of cell separation is improved.Separated virion is carried out negative staining electron microscope observation by the present invention, is tied
Fruit shows：It can be seen that virion is rounded or hexagon, without cyst membrane, 20～22nm of diameter.
Further, inventor is tested by A-72 passages and susceptible animal, has been carried out biological characteristicses and has been tested such as
Virus multiplication, virus TCID50Determine, this animal Orthogonal Rotational Regressive Tests, result of the test confirm the strain breed on A-72 cells soon, drip
Degree is high, pathogenicity is strong, and CPV-YH Strain is good virulent strain.
The present invention carries out full genome nucleoside to Canine Parvovirus CPV-YH strains with nearly 20 plants of CPV separation strains of domestic and international report
Acid homology comparative analysiss.Strain CPV-LZ2 separation strains homology highest 99% detached with domestic Lanzhou in 2011.With category
In a branch, single pass-through nucleotide alignments have found there is certain gene mutation phenomenon.Analysis result confirms the present invention
Separated strain is China's CPV-2a types, and the Major Epidemic strain of CPV can be used as the seed culture of viruses for preparing the tiny vaccine of dog.
Therefore, the present invention is claimed first the Canine Parvovirus (Canine that above-mentioned preserving number is CGMCC NO.12990
Parvovirus) strain CPV-YH.Meanwhile, it is tiny in preparation dog that the present invention is also claimed the canine parvovirus strain CPV-YH
Purposes in terms of viral vaccine.Because the canine parvovirus strain CPV-YH that the present invention is provided is that one plant of pathogenicity is extremely strong, cell increases
Fast virulent strain is grown, the technical staff of vaccine preparation field is when the present invention is seen, it is possible to tiny using the new dog of its exploitation
Viral vaccine, therefore, the behavior that CPV-YH Strain is used in terms of vaccine development is both fallen within this by any scale, any mode
The protection domain of invention.And those skilled in the art can utilize CPV-YH Strain, using any of vaccine development side
Developing the canine parvovirus vaccine, for example, in one embodiment of the invention, the vaccine development mode is concrete for method
For, with attenuation, and/or it is weak poison, and/or inactivation the canine parvovirus poison strain CPV-YH as the vaccine activity
Material.Wherein it is attenuated and inactivates and adopt this area conventional meanses, such as but not limited to, application for a patent for invention 98800846.7 is provided
The method of attenuating of virus " ".
On the other hand, the present invention is also claimed the canine parvovirus poison strain CPV-YH and is preparing Canine Parvovirus diagnosis
Purposes in terms of reagent.The technical staff of Viral diagnosis or diagnostic field take the present invention CPV-YH strains when, Ke Yiyong
It develops Canine Parvovirus diagnosis or detectable, and any scale, any type of CPV-YH Strain by the present invention are used for
The patent right of the present invention is all invaded in behavior in terms of the diagnosis of exploitation Canine Parvovirus or detectable.Those skilled in the art can be with
Using the CPV-YH Strain of the present invention, the present invention is developed by any conventional research method of Viral diagnosis diagnostic field
Described diagnostic reagent, for example, in one embodiment of the invention, the concrete mode of diagnostic reagent exploitation is, including
Following steps：Antibody is obtained with described canine parvovirus poison strain CPV-YH as immunogen immune animal.Or using another
Plant concrete mode：By described canine parvovirus poison strain CPV-YH itself by sequencing, gone out by bioinformatics means analysis
The specific sequence region of its mutation, designs specific primer, obtains Canine Parvovirus and/or the dog of a kind of PCR-based detection
The diagnostic reagent of tiny disease.
Additionally, the present invention is also based on the CPV-YH Strain, there is provided one kind is used to preventing and/or treating canine parvovirus
The vaccine combination of malicious associated diseases, it includes poison be attenuated, and/or weak, and/or inactivation the canine parvovirus poison strain
CPV-YH.In some specific embodiments, the vaccine combination also includes can be with the canine parvovirus poison strain CPV-YH
The pharmaceutical carrier of mixing, and/or, pharmaceutically acceptable adjuvant.
Present invention also offers a kind of reagent for detecting Canine Parvovirus virulent strain and/or diagnosis canine parvovirus, its
Including the antibody obtained as immunogen with the canine parvovirus poison strain CPV-YH.Further, the reagent also includes detection
The conventional antibody of diagnostic field.The reagent still further comprises other conventional reagents in checkout and diagnosis field.
By bioinformatic analysis, the CPV-YH of the present invention is one plant of brand-new canine parvovirus strain；And by cell
Pass on and susceptible animal experiment, it was confirmed that CPV-YH growth rates are fast, titre is high, pathogenicity is strong, are one plant of good virulent strain,
Have wide range of applications, counteracting toxic substances model not only can be set up in research field, can with production field be used for develop canine parvovirus
Malicious novel vaccine, or prepare Canine Parvovirus checkout and diagnosis test kit.The present invention is not only to solve and grasp currently a popular strain
Variation situation provides new content and direction, while meeting canine parvovirus field needs new strain needed for vaccine development badly
Screening requirements, provide the raw material of new high-quality and select space for vaccine development.
The preservation information of Canine Parvovirus (Canine parvovirus) the strain CPV-YH is as follows：
Culture presevation title：CPV-YH
Preserving number：CGMCC No.12990
Classification And Nomenclature：Parvovirus
Depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center
Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date：On September 21st, 2016
Description of the drawings
Fig. 1：(A is normal A-72 cells to Canine Parvovirus CPV-YH Strain separation and Culture；There is disease for inoculation pathological material of disease in B
The cell of change).
Fig. 2：Canine Parvovirus CPV-YH Strain morphology electron microscopes (amplify 20000 times).
Fig. 3：The evolution of amino acid tree of Canine Parvovirus CPV-YH Strain VP2 albumen.
Fig. 4：Test dog survival curve.
Specific embodiment
In conjunction with specific embodiments further describing the present invention, advantages of the present invention and feature can be more clear with description
Chu.It should be understood that the embodiment is only exemplary, any restriction is not constituted to the scope of the present invention.People in the art
Member to the details of technical solution of the present invention and form it should be understood that can enter without departing from the spirit and scope of the invention
Row modification is replaced, but these modifications or replacement each fall within protection scope of the present invention.Experiment used in following embodiments
Method if no special instructions, is conventional method；Material, reagent used etc., if no special instructions, commercially obtain
Arrive.
The source and record source of biomaterial
Embodiment 1 separates the pathological material of disease (intestinal tissue) of canine parvovirus virulent strain (CPV-YH) employing of the present invention from Beijing
The morbidity dog of certain pet clinic；
Embodiment 1 and the A-72 cells of the inoculation of embodiment 2 are commercially available；
(HA potency is for 1 for the tiny control strain CPV type strains of dog that the multiplication capacity contrast experiment of embodiment 3 adopts:2560)
From China Animal Disease Control And Prevention Center；
The experiment level beasle dog that the animal Orthogonal Rotational Regressive Tests of embodiment 4 are adopted is purchased from Beijing Marshall Biotechnology Co., Ltd；
The DH5 α competent cells that the connection conversion of embodiment 5 is used are commercially available.
Embodiment 1：The separation of canine parvovirus virulent strain (CPV-YH)
1 material and method
1.1 material
1.1.1 pathological material of disease gathers the morbidity dog from Beijing pet clinic.
1.1.2 cell A-72, Anheal Laboratories Co., Ltd's preservation, cultivate according to a conventional method, can
It is commercially available.
1.2 separation method
1.2.1 the separation of virus
Take without putrid and deteriorated intestinal tissue, with sterile scissors intestinal segment, scraping enteral film and intestinal contentses are longitudinally cut off, use
DMEM ((chain) mycin 2000IU/ml containing green grass or young crops) is rinsed and diluted, 4000r/min centrifugation 20min, takes supernatant, 0.22 μm of micropore of Jing
After membrane filtration is degerming, puts -70 DEG C and save backup.
1.2.2 the culture of virus
3% content of virus-culturing fluid accesses the inoculation A-72 cells of monolayer, and 37 DEG C of absorption 1h are added and contained 2% calf
The DMEM maintaining liquids of serum, 37 DEG C of static gas wave refrigerators, while setting normal cell controls.Observation daily whether there is cytopathy, if nothing
CPE, then harvest viral, multigelation 3 times in culture 5d, and continuous blind passage to 5 generations are still considered as feminine gender without pathological changes.There is cytopathy
Harvest virus liquid during up to 80%, rearmounted -70 DEG C of Jing -20 DEG C of freeze thawing 2 times is saved backup.
1.2.3 continuous passage culture, 2% content of virus-culturing fluid accesses the inoculation A-72 cells of monolayer, 37 DEG C of suctions
Attached 1h, adds the DMEM maintaining liquids containing 2% calf serum, 37 DEG C of static gas wave refrigerator 5d.Second culture 72h, observes pathological changes.
2 results and conclusion
There is obvious cytopathy (CPE) after pathological material of disease inoculation A-72 cells, the cells show for infecting virus expands for cell,
It is rounded, and has " drawing in the net " phenomenon, start shedding off then, sees Fig. 1：(A is normal A-72 cells；B is that inoculation pathological material of disease pathological changes occurs
Cell).Detached virus is further passed on, expanding propagation, put -70 DEG C of preservations, to the species for identifying isolated viral.This is implemented
The isolated Strain of example is named as CPV-YH, and send preservation by one of individual plant, and its preserving number is CGMCC
No.12990。
Embodiment 2：The identification of canine parvovirus virulent strain (CPV-YH)
1st, virus-specific detection
F1～the F4 of the separation strains multigelation 3 times for causing CPE is taken for cell culture,
2nd, virus titer is determined
The F4 of the separation strains multigelation 3 times for causing CPE is taken for cell culture, is allocated as virus with maintaining liquid to do 10-1
～10-9It is serially diluted, during 96 porocyte culture plates for covering with A-72 cell monolayers are seeded in respectively, each dilution factor inoculation 6
Hole, per hole 0.1ml, 37 DEG C of absorption 1h add maintaining liquid, put 5%CO237 DEG C of cultures of incubator, while setting only add the thin of maintaining liquid
Born of the same parents compare, and continuous observation of cell pathological changes on the 7th simultaneously record result, and by Reed-MuenchShi methods the TCID of isolated strain is calculated50。
As a result show：Separate TCID of the strain virus on A-72 cells50For 108.4/ ml, shows the sense on A-72 cells
Dye power is very strong.
3rd, negative staining electron microscope observation
Jing after cultivating 5～7d, sick cell collects supernatant, and high speed centrifugation removes cell debriss, then 20000r/min hypervelocity
Centrifugation 2h, with concentration, purified viruses, purified viruses adsorption acts on 2.5min on 200 mesh copper mesh under room temperature condition,
Gently adsorb surplus liquid with filter paper, then the dyeing of phosphotungstic acid negativity, is observed using transmission electron microscope.
Electron microscopic observation result shows：Can be observed a large amount of virion, virion is rounded or hexagon, without cyst membrane,
20～22nm of diameter.See Fig. 2：Canine Parvovirus CPV-YH Strain morphology electron microscopes (amplify 20000 times).
Embodiment 3：(HA potency is 1 for Canine Parvovirus CPV-YH strains and CPV type strains:2560) multiplication capacity contrast
The Canine Parvovirus that the tiny CPV type strains of the dog of this Laboratories Accession and embodiment 1 and/or embodiment 2 are provided
CPV-YH strains are inoculated with respective sensitive cellss, determine each viral median tissue cell infection amount (TCID50), to determine two plants
The multiplication capacity of viral cell in vitro.
Median tissue cell infection amount measurement result shows：Canine Parvovirus CPV-YH strains are in the titre of A-72 cells
108.4/ ml, is significantly higher than the 10 of CPV type strains5.4/ ml, this result explanation CPV-YH strain multiplication capacity in vitro is significantly larger than
CPV type strains.Virus titer measurement result is specifically shown in Table 1.
The Canine Parvovirus CPV-YH strains of table 1 and CPV 4#/2007 plant TCID50 contrast tables
Embodiment 4：Animal Orthogonal Rotational Regressive Tests of Canine Parvovirus virulent strain (CPV-YH) and application thereof
1. materials and methods
1.1 test strains：CPV-YH separation strains (A-72) F4 generations, TCID50For 10-8.4/ml；
1.2 experimental animal：Experiment level 10 (5 female 5 is male) random number 1#～10# of beasle dog, purchased from Beijing agate, this is biological
Technology Co., Ltd., 23 ages in days are introduced simultaneously with bitch after birth, Jing after isolated rearing wean, dog excrement Jing PCR detection methods mirror
Determine the items such as CPV, CDV, CCV, CRV, CAV-1 and CPI and be feminine gender, dog serum menses coagulate inhibition test (HI) detection CPV and resist
Body is equal<1:20.
1.3 artificial challenge：Artificial challenge's (age in days counteracting toxic substances of pup 60) is carried out after validation test animal is qualified, except control dog
(8#～10#) outward, only (1#～7#) carries out oral administration to remaining dog by 2ml/ dosage.
1.4 clinical observation：Feed at regular time and quantity daily, examine and itemized record body temperature, diet desire, the mental status
Deng clinical manifestation, and taken a blood sample the next day after counteracting toxic substances (anticoagulation and dog serum sample), the collection of fecal specimens.
1.5 test in laboratory：CPV, CDV, CCV, CRV, CAV-1 and CPI are carried out to fecal specimens with PCR method
Detection of antigen；Dog serum sample CPV antibody is detected with HI methods；Go forward side by side promoting the circulation of blood routine examination.
1.6 pathology cut open inspections：Cut open inspection, the pathology change of each histoorgan of itemized record in after death 6 hours by dead dog
Change, and record the picture concerned for preserving typical cytopathic.
2. experimental result
2.1 clinical symptoms
2.1.1 body temperature：After infection CPV, raise after 3# dog body temperature falls before again in addition to, other dogs only slightly raise, its
Middle 4# dogs body temperature raises more (more than 39.5 DEG C), and in addition to 3#, 4#, only body temperature is increased to maximum to remaining dog after infecting 3～5
Value.
2.1.2 appetite：After infection there is different degrees of loss of appetite to each dog in the same day, and after infection the 4th day stops
Only take food, only 4# dogs the 5th day and the 9th day a small amount of drinking water of taking food respectively after infection.
2.1.3 other symptoms：Occur within the 4th day different degrees of spiritual depressed, abnormal defecation after infection, occur soft
Just, Mucous Stool, and with symptoms of emesis.
2.2 death curve after counteracting toxic substances
Control dog (8#～10#) is all survived, and all 7 counteracting toxic substances pups are dead in 10 days after inoculation, and fatality rate is
100%.Survival curve figure is drawn according to death condition, Fig. 3 is seen.
2.3 test in laboratory：
2.3.1 PCR testing results：After infection CPV can be detected in the dog fecal specimens of collection in 12 hours, be gone out earliest
Existing CPV it is positive be 2#, 3#, 6#, 7# dog, CPV is always the positive in fecal specimens afterwards, until after death cut open inspection enteral
Tolerant detection is also positive.
2.3.2 HI laboratory test results：After infection 6 days, start CPV antibody occur in serum,
2.3.3 routine blood test testing result：There is rising in various degree in RBC number, hemoglobin, packed cell volume,
This dewatering symptom with dog only matches；And all dogs only before death the phase occur numeration of leukocyte drastically decline, it is average red
Phenomenon of the Cell Hb Concentration less than normal value.
2.3 cut open inspection pathological changes：Counteracting toxic substances dog cut open inspection finds that different degrees of dewatering symptom only occurs in each dog, and small intestinal is sent out
Raw hyperemia, even congestion, large area necrosis region, mesentery is congested, congestion, and gastric has a large amount of whites or yellow mucus, mucosa to have
Petechia, liver showed edema, bleeding, necrosis, splenomegaly, congestion, renal pelvis fusion or cortex, medullary substance regional boundary limit it is unclear, it is pulmonary venous pleonaemia, bad
Extremely, pericardium is thickened, and pancreas enlargement, has the pathological changes such as petechia.
3. conclusion
3.1 conclusion CPV-YH separation strains are the highly pathogenic strains of CPV, can be used to set up canine parvovirus disease counteracting toxic substances model；
The CPV-YH separation strains can also be used to develop new canine parvovirus vaccine, comprise the steps：With attenuation,
And/or the canine parvovirus poison strain CPV-YH of weak malicious, and/or inactivation is as the active substance of the vaccine.
On the other hand, the CPV-YH separation strains can also be used to exploitation detection Canine Parvovirus and/or diagnosis dog is tiny
The reagent of disease, including：Antibody is obtained as immunogen immune animal with described canine parvovirus poison strain CPV-YH, the antibody is
Can be used for by Ag-Ab association reaction, detect and whether infect in subjects Canine Parvovirus or whether with dog
Tiny disease.Can also be by the antibody and the conventional antibody of this area, and the agent combination of Protein Detection field routine is formed
Complete detection Canine Parvovirus and/or diagnosis canine parvovirus test kit.
Or, by described canine parvovirus poison strain CPV-YH itself by sequencing, by bioinformatics means analysis
Go out the specific sequence region of its mutation, design specific primer, obtain a kind of detection of PCR-based Canine Parvovirus and/or
The diagnostic reagent of canine parvovirus.More specifically, conventional reagent (for example, dNTP, archaeal dna polymerase, buffer can be detected with PCR
Deng), electrophoresis detection conventional reagent (e.g., agarose, buffer, EB or) together combination formed PCR-based detection for diagnosing
The test kit of Canine Parvovirus and/or canine parvovirus.
The vaccine combination of the present invention of embodiment 5.
The present embodiment provides a kind of vaccine combination for preventing and/or treating Canine Parvovirus associated diseases, its bag
Include attenuation, and/or weak poison, and/or inactivation the arbitrary described canine parvovirus poison strain CPV- of embodiment 1- embodiment 4
YH。
Further, the vaccine combination also includes the medicinal load that can mix with the canine parvovirus poison strain CPV-YH
Body.
Preferably, the vaccine combination also includes pharmaceutically acceptable adjuvant.
The protective agent refers to that substantially nontoxic to laboratory animal and to including in vaccine combination CPV-YH strains are lived
Property substantially without the reagent for affecting, it can be solid, liquid or gel form, and usually aseptic.
What the adjuvant was well known to those skilled in the art, for example Freund adjuvant, aluminium hydroxide, aluminum phosphate, aluminium oxide,
Saponin, glucosan such as DEAE- glucosans, vegetable oil (such as Oleum Arachidis hypogaeae semen, olive oil and/or Vitamin E acetate), mineral oil,
Lecithin, bacteria lipopolysaccharide, Peptidoglycan and Dan Baiduotang proteoglycan PG etc..Can also have polyhydric alcohol such as Propylene Glycol, Polyethylene Glycol, glycerol etc. and
Antioxidant.To ensure protectant effect, above-mentioned protective agent can change with the different of vaccine dosage, can be above-mentioned one
The combination of kind or several adjuvants (protective agent).
The checkout and diagnosis reagent of the present invention of embodiment 6.
The present embodiment provides a kind of reagent for detecting Canine Parvovirus virulent strain and/or diagnosis canine parvovirus, and it is special
Levy and be, including the antibody obtained as immunogen with the arbitrary described canine parvovirus poison strain CPV-YH of embodiment 1- embodiment 4.
The checkout and diagnosis reagent that the present embodiment is provided can be specially a kind of Test paper, and detection line and right is provided with reagent paper
According to line；The above-mentioned antibody obtained as immunogen with CPV-YH strains is coated with detection line；And detection is coated with control line and is examined
The conventional antibody in disconnected field, e.g., sheep anti-mouse igg antibody.Meanwhile, the Test paper also includes, uses in coating, preparation
Other conventional reagents in checkout and diagnosis field, for example, BSA, CBS or PBS etc..
Embodiment 7：The sequencing detection of Canine Parvovirus virulent strain (CPV-YH) full genome
1 materials and methods
1.1 material
Virus：CPV-YH strains, are separated by Anheal Laboratories Co., Ltd, are preserved.
1.2 method
1.2.1 the design of primer
With reference to the CPV full genomes included on Genbank conserved sequence application DNAMAN software designs 5 to primer, by English
The synthesis of Weihe River victory base (Shanghai) trade Co., Ltd.
The CPV full genome amplimers of table 2
1.2.2 the amplification of CPV-YH full-length genomes
Virus genom DNA with extraction is expanded as template with 5 pairs of primers of design.
PCR reaction system volumes are：
Response procedures：95 DEG C of 4min, 95 DEG C of 30s, 50～58 DEG C of 45s, 72 DEG C of 90s, 35 circulations, 72 DEG C of 10min.
1.2.3 PCR primer clone and sequencing
Amplified production is identified and reclaimed with 1.5% agarose gel electrophoresiies.
Recovery product is connected with PMD19-T carriers, and linked system is：
Take 5ul connection products to convert to the 50ulDH5 α competent cells for preparing, be applied to the LB containing ampicillin and put down
Plate, 37 DEG C of 12～16h of culture.Picking single bacterium colony is inoculated in LB culture fluid of the 3ml containing ampicillin, 37 DEG C of vibrations from flat board
16～20h of culture.Select bacterium solution PCR method identification recombiant plasmid.Positive strain will be accredited as and send Invitrogen (Shanghai) trade
Company limited is sequenced.
1.2.4 sequence analysis
The correct each sequence of sequencing is spliced with DNAMAN, is carried out with the CPV sequences delivered both at home and abroad on GenBank
Compare, sequencing result is analyzed with softwares such as DNAMAN and MEGA4.0 is compared.
2 results
It is analyzed after sequencing result splicing, as a result shows：The Canine Parvovirus CPV-YH pnca gene group total lengths of the present invention
4921bp, sequence is as shown in SEQ ID NO.1, including 2 open reading frame (ORF), is separately encoded the regulation egg of early transcription
Viral capsid proteins (VP1 and VP2) of (NS1 and NS2) and late transcription in vain.Non-structural protein NS 1 and the common long 2007bp of NS2,
Between 273～2279nt of genome, wherein NS1 and NS2 genes overlap each other.Structural protein VP1 gene is located at gene
Between the 2286-2363nt of group, Viral structural protein VP2 full length gene 1755bp, between the 2787-4541nt of genome.5’-
There is the hairpin structure that palindrome is formed at end, and there is the repetitive sequence for being about 60bp of a copy at 3 ' ends.According to VP2 the 426th
Amino acid variation situation, the separation strains belong to CPV-2a.
VP2 evolution of amino acid trees show that CPV-YH separation strains with 2013-BJ-P27 (China) separation strains and UY306 (draw by crow
Gui) separation strains are in same little branch (see Fig. 3).Whole genome sequence comparison result shows, CPV-YH strains and 2011
The detached CPV-LZ2 separation strains homology highest 99% in year Lanzhou.The aminoacid sequence that NS1 genes are derived is separated with CPV-LZ2
Strain NS1 homologys are 99.55%, have at 3 and morph.Meanwhile, VP1 gene amino acid sequences also occur in that 3 variations.
Wherein first variation is present in 25, and this position is in introne position, at present with regard in Canine Parvovirus VP1
Correlational study containing son is still weak, and cut mode and particular location not yet determine, therefore the variation of this strain this position aminoacid
The change of virus virulence may be directly resulted in.
Found by the gene analysiss to its major antigen albumen, on major antigenic sites, the ammonia of CPV-YH Strain
Base acid sequence with it is domestic and international, especially it has been reported that domestic separation strains have larger difference, as shown in table 4, with 2013-BJ-P27
Compare, morph at 406 (MS/2014 is S, and 2013-BJ-P27 is T)；Compared with UY306, have 5 to go out and there occurs variation,
Be respectively the 7th (MS/2014 is Q, and UY306 is H) 87 (MS/2014 is L, and UY306 is M) 101 (MS/2014 is T,
UY306 is I) 305 (MS/2014 is Y, and UY306 is D) 406 (MS/2014 is S, and UY306 is T)；With CPV-N point of the U.S.
Compare from strain, have there occurs variation at 10 points, be respectively that 87 M become L, 101 I become Y, and 267 F become Y, and 297 S become
A, 300 become G by A, and 305 D become Y, and 324 Y become I, and 367 Y become D, and 406 T become S, and 340 T become A；
In addition compared with other separation strains, CPV-YH separation strains there occurs special variation at 406, and by T S is become, it is shown that virus
Evolution, relative specific variation it is less with strains of pathogenic Journal of Sex Research, however not excluded that its dependency, be shown in Table 3.
Table 3.CPV-YH strains and CPV-LZ2 strain NS1 and VP1 gene amino acid sequences difference
The amino acid residue of non-synonymous displacement in table 4.CPV-YH strain VP2
It can be seen from analysis above, CPV-YH Strain is the new epidemic strain of one plant of Chinese Canine Parvovirus, and pole has can
The newest trend of evolution of CPV can be represented, can be used as the new seed culture of viruses for preparing canine parvovirus vaccine.CPV is extremely strong as a kind of variability
Animal viruss, especially major antigen albumen VP2 has king-sized mutability, in view of the variation of CPV-YH Strain is big, pathogenic
The characteristics of power is strong, mortality rate is high and reacts virus evolution trend, can apply to the development of currently a popular CPV vaccines, be conducive to
The prevention and control of current canine parvovirus disease.
SEQUENCE LISTING
<110>Anheal Laboratories Co., Ltd
<120>One plant of canine parvovirus poison strain CPV-YH and its application
<130> P160461/SJY
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 4921
<212> DNA
<213> artificial sequence
<220>
<223>Canine Parvovirus（Canine parvovirus）Strain CPV-YH whole genome sequences
<400> 1
attctttaga accaactgac caagttcacg tacgtatgac gtgatgacgc gcgctgcgcg 60
cgctgcctac ggcagtcaca cgtcatacgt acgctccttg gtcagttggt tctaaagaat 120
gataggcggt ttgtgtgttt aaacttgggc gggaaaaggt ggcgggctaa ttgtgggcgt 180
ggttaaaggt ataaaagaca aaccatagac cgttactgac attcgcttct tgtctttgac 240
agagtgaacc tctcttactt tgactaacca tgtctggcaa ccagtatact gaggaagtta 300
tggagggagt gaattggttg aagaaacatg cagaaaatga agcattttcg tttgttttta 360
aatgtgacaa cgtccaacta aatggaaagg atgttcgctg gaacaactat accaaaccaa 420
ttcaaaatga agagctaaca tctttaatta gaggagcaca aacagcaatg gatcaaaccg 480
aagaagaaga aatggactgg gaatcggaag ttgatagtct cgccaaaaag caagtacaaa 540
cttttgatgc attaattaaa aaatgtcttt ttgaagtctt tgtttctaaa aatatagaac 600
caaatgaatg tgtttggttt attcaacatg aatggggaaa agatcaaggc tggcattgtc 660
atgttttact tcatagtaaa aacttacaac aagcaactgg taaatggcta cgcagacaaa 720
tgaatatgta ttggagtaga tggttggtga ctctttgttc ggtaaactta acaccaactg 780
aaaagattaa gctcagagaa attgcagaag atagtgaatg ggtgactata ttaacataca 840
gacataagca aacaaaaaaa gactatgtta aaatggttca ttttggaaat atgatagcat 900
attacttttt aacaaaaaaa aaaattgtcc acatgacaaa agaaagtggc tattttttaa 960
gtactgattc tggttggaaa tttaacttta tgaagtatca agacagacaa attgtcagta 1020
cactttacac tgaacaaatg aaaccagaaa ccgttgaaac cacagtgacg acagcacagg 1080
aaacaaagcg cgggagaatt caaactaaaa aggaagtgtc aatcaaatgt actttgcggg 1140
acttggttag taaaagagta acatcacctg aagactggat gatgttacaa ccagatagtt 1200
atattgaaat gatggcacaa ccaggaggtg aaaatctttt aaaaaataca cttgaaattt 1260
gtactttgac tttagcaagg acaaaaacag catttgaatt aatacttgaa aaagcagata 1320
atactaaact aactaacttt gatcttgcaa attctagaac atgtcaaatt tttagaatgc 1380
atggatggaa ttggattaaa gtttgtcacg ctatagcatg tgttttaaat agacaaggtg 1440
gtaaaagaaa tacagttctt tttcatggac cagcaagtac aggaaaatct atcattgctc 1500
aagccatagc acaagctgtg ggtaatgttg gttgttataa tgcagcaaat gtgaattttc 1560
catttaatga ctgtaccaat aaaaatttaa tttggattga agaagctggt aactttggtc 1620
aacaagttaa tcaatttaaa gcaatctgtt ctggacaaac aattagaatt gatcaaaaag 1680
gtaaaggaag taagcaaatt gaaccaactc cagtaattat gacaactaat gaaaatataa 1740
caattgtgag aattggatgc gaagaaagac ctgaacatac acaaccaata agagacagaa 1800
tgttgaacat taagttagta tgtaagcttc caggagactt tggtttggtt gataaagaag 1860
aatggccttt aatatgtgca tggttggtta aacatggttt tgtatcaacc atggctaact 1920
atacacatca ttggggaaaa gtgccagaat gggatgaaaa ctgggcggag cctaaaatac 1980
aagaaggtat aaattcacca ggttgcaaag acttaaagac acaagcggca agcaatcctc 2040
agagtcaaga ccaagttcta actcctctga ctccggacgt agtggacctt gcactggaac 2100
cgtggagtac tccagatacg cctattgcag aaactgcaaa tcaacaatca aaccaacttg 2160
gcgttactca caaagacgtg caagcgagtc caacgtggtc cgaaatagag gcagacctga 2220
gagccatctt tacttctgaa caattagaag aagattttcg agacgacttg gattaaggta 2280
cgatggcacc tccggcaaag agagccagga gaggtaaggg tgtgttagtg aagtgggggg 2340
aggggaagaa tttaatgact taactaagta tgtgtttttt tataggactt gtgcctccag 2400
gttataaata tcttgggcct gggaacagtc ttgaccaagg agaaccaact aacccttctg 2460
acgccgctgc aaaagaacac gacgaagctt acgctgctta tcttcgctct ggtaaaaacc 2520
catacttata tttctcgcca gcagatcaac gctttataga tcaaactaag gacgctaaag 2580
attggggggg gaaaatagga cattattttt ttagagctaa aaaggcaatt gctccagtat 2640
taactgatac accagatcat ccatcaacat caagaccaac aaaaccaact aaaagaagta 2700
gaccaccacc tcatattttc atcaatcttg caaaaaaaaa aaaagccggt gcaggacaag 2760
taaaaagaga caatcttgca ccaatgagtg atggagcagt tcaaccagac ggtggtcagc 2820
ctgctgtcag aaatgaaaga gctacaggat ctgggaacgg gtctggaggc gggggtggtg 2880
gtggttctgg gggtgtgggg atttctacgg gtactttcaa taatcagacg gaatttaaat 2940
ttttggaaaa cggatgggtg gaaatcacag caaactcaag cagacttgta catttaaata 3000
tgccagaaag tgaaaattat agaagagtgg ttgtaaataa tttggataaa actgcagtta 3060
acggaaacat ggctttagat gatacccatg cacaaattgt aacaccttgg tcattggttg 3120
atgcaaatgc ttggggagtt tggtttaatc caggagattg gcaactaatt gttaatacta 3180
tgagtgagtt gcatttagtt agttttgaac aagaaatttt taatgttgtt ttaaagactg 3240
tttcagaatc tgctactcag ccaccaacta aagtttataa taatgattta actgcatcat 3300
tgatggttgc attagatagt aataatacta tgccatttac tccagcagct atgagatctg 3360
agacattggg tttttatcca tggaaaccaa ccataccaac tccatggaga tattattttc 3420
aatgggatag aacattaata ccatctcata ctggaactag tggcacacca acaaatatat 3480
accatggtac agatccagat gatgttcaat tttacactat tgaaaattct gtgccagtac 3540
acttactaag aacaggtgat gaatttgcta caggaacatt ttattttgat tgtaaaccat 3600
gtagactaac acacacatgg caaacaaata gagcattggg cttaccacca tttctaaatt 3660
ctttgcctca agctgaagga ggtactaact ttggttatat aggagttcaa caagataaaa 3720
gacgtggtgt aactcaaatg ggaaatacaa acattattac tgaagctact attatgagac 3780
cagctgaggt tggttatagt gcaccatatt attcttttga ggcgtctaca caagggccat 3840
ttaaaacacc tattgcagca ggacgggggg gagcgcaaac agatgaaaat caagcagcag 3900
atggtgatcc aagatatgca tttggtagac aacatggtca aaaaactacc acaacaggag 3960
aaacacctga gagatttaca tatatagcac atcaagatac aggaagatat ccagaaggag 4020
attggattca aaatattaac tttaaccttc ctgtaacaaa tgataatgta ttgctaccaa 4080
cagatccaat tggaggtaaa gcaggaatta actatactaa tatatttaat acttatggtc 4140
ctttaactgc attaaataat gtaccaccag tttatccaaa tggtcaaatt tgggataaag 4200
aatttgatac tgacttaaaa ccaagacttc atgtaaatgc accatttgtt tgtcaaaata 4260
attgtcctgg tcaattattt gtaaaagttg cgcctaattt aacaaatgaa tatgatcctg 4320
atgcatctgc taatatgtca agaattgtaa cttactcaga tttttggtgg aaaggtaaat 4380
tagtatttaa agctaaacta agagcctctc atacttggaa tccaattcaa caaatgagta 4440
ttaatgtaga taaccaattt aactatgtac caagtaatat tggaggtatg aaaattgtat 4500
atgaaaaatc tcaactagca cctagaaaat tatactaaca tacttactat gtttttatgt 4560
ttattacata ttattttaag attaattaaa ttacagcata gaaatattat acttgtattt 4620
gatataggat ttagaaggtt tgttatatgg tatacaataa ctgtaagaaa tagaagaaca 4680
tttagatcat agttagtagt ttgttttata aaatgtattg taaaccatta atgtatgttg 4740
ctatggtgtg ggtggttggt tggtttgccc ttagaatatg ttaaggacca aaaaaatcaa 4800
taaaagacat ttaaaactaa atggcctcgt atactgtcta taagcaacca accaggccag 4860
tcagtctact gatgttgtgc atctcccacc caccccccgc ttaaagacag attgatactt 4920
a 4921
<210> 2
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223>Primer PNS1
<400> 2
attctttaga accaactgac ca 22
<210> 3
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223>Primer PNS1 downstream sequences
<400> 3
gcaatttctc tgagcttaat c 21
<210> 4
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223>PNS2 forward primer
<400> 4
cggtaaactt aacaccaact g 21
<210> 5
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223>PNS2 downstream primers
<400> 5
agcttgtgct atggcttgag 20
<210> 6
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223>PNS3 forward primer
<400> 6
catggaccag caagtacagg a 21
<210> 7
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223>PNS3 downstream primers
<400> 7
tataacctgg aggcacaagt cc 22
<210> 8
<211> 17
<212> DNA
<213> artificial sequence
<220>
<223>PVP1 forward primer
<400> 8
atggcacctc cggcaaa 17
<210> 9
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223>PVP1 downstream primers
<400> 9
cactcattgg tgcaagattg tc 22
<210> 10
<211> 18
<212> DNA
<213> artificial sequence
<220>
<223>PVP2 forward primer
<400> 10
gccggtgcag gacaagta 18
<210> 11
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223>PVP2 downstream primers
<400> 11
taacatattc taagggcaaa cc 22
Claims (10)
1. one plant of Canine Parvovirus (Canine parvovirus) strain CPV-YH, its preserving number is CGMCC NO.12990, its
Whole genome sequence is as shown in SEQ ID No.1.
2. Canine Parvovirus (Canine parvovirus) strain CPV-YH described in pair claim 1 carries out passing on breeding
Method, it is characterised in that the strain described in claim 1 is inoculated in host cell, is cultivated and/or is passed on, preferably
Ground, the host cell adopts A-72 cells.
3. a kind of method for preparing canine parvovirus vaccine, it is characterised in that：To the Canine Parvovirus described in claim 1
(Canine parvovirus) strain CPV-YH is attenuated and/or inactivation becomes weak poison or without poison strain.
4. the method for preparing the antibody of anti-dog parvovirus, it is characterised in that comprise the following steps：
Using Canine Parvovirus (Canine parvovirus) strain described in claim 1 as immunogen immune animal.
5. what the method described in claim 4 was prepared has the antibody of identification and/or binding activity to Canine Parvovirus.
6. purposes of the canine parvovirus strain CPV-YH described in claim 1 in terms of Canine Parvovirus diagnostic reagent is prepared.
7. purposes according to claim 6, it is characterised in that include, with described canine parvovirus strain CPV-YH as
Immunogen immune animal obtains antibody；Or, designing specificity according to the whole genome sequence of the canine parvovirus strain CPV-YH
Primer.
8. a kind of bacterin preparation for preventing and/or treating Canine Parvovirus associated diseases, it is characterised in that：Its active component
Including the canine parvovirus strain CPV-YH described in attenuation, and/or weak poison, and/or inactivation claim 1.
9. bacterin preparation according to claim 8, it is characterised in that also including can be with the canine parvovirus poison strain CPV-
The pharmaceutical carrier of YH mixing；And/or, pharmaceutically acceptable adjuvant.
10. it is a kind of for detect Canine Parvovirus and/or diagnosis canine parvovirus reagent, it is characterised in that include with right will
Seek the antibody that described canine parvovirus strain CPV-YH is that immunogen is obtained；Or, based on the canine parvovirus strain CPV-YH's
The specific primer of whole genome sequence design.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109735505A (en) * | 2018-12-26 | 2019-05-10 | 河南农业大学 | The amplification and application of one plant of canine parvovirus poison strain and its gene order |
| CN110846284A (en) * | 2019-11-07 | 2020-02-28 | 衡阳师范学院 | Canine parvovirus CPV-HuN1703 strain and application thereof |
| CN111635890A (en) * | 2020-06-03 | 2020-09-08 | 中国农业科学院北京畜牧兽医研究所 | Dog parvovirus new CPV-2b strain and application thereof |
| CN111763766A (en) * | 2020-04-14 | 2020-10-13 | 南京农业大学 | Primer pair, TaqMan probe and method for detecting canine diarrhea virus by one-step method and application |
| CN112079916A (en) * | 2020-02-28 | 2020-12-15 | 衡阳师范学院 | Canine parvovirus egg yolk antibody and preparation method thereof |
| CN112195161A (en) * | 2020-10-20 | 2021-01-08 | 辽宁益康生物股份有限公司 | Canine parvovirus vaccine strain, vaccine and preparation method thereof |
| CN113073083A (en) * | 2020-01-03 | 2021-07-06 | 普莱柯生物工程股份有限公司 | Canine parvovirus low virulent strain, vaccine composition prepared from same and application of vaccine composition |
| CN116376981A (en) * | 2023-04-21 | 2023-07-04 | 西北农林科技大学 | Recombinant canine parvovirus pseudovirus |
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| CN109735505A (en) * | 2018-12-26 | 2019-05-10 | 河南农业大学 | The amplification and application of one plant of canine parvovirus poison strain and its gene order |
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| CN113073083A (en) * | 2020-01-03 | 2021-07-06 | 普莱柯生物工程股份有限公司 | Canine parvovirus low virulent strain, vaccine composition prepared from same and application of vaccine composition |
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| CN111763766B (en) * | 2020-04-14 | 2022-12-27 | 南京农业大学 | Primer pair, taqMan probe and method for detecting canine diarrhea virus by one-step method and application |
| CN111635890A (en) * | 2020-06-03 | 2020-09-08 | 中国农业科学院北京畜牧兽医研究所 | Dog parvovirus new CPV-2b strain and application thereof |
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| CN116376981A (en) * | 2023-04-21 | 2023-07-04 | 西北农林科技大学 | Recombinant canine parvovirus pseudovirus |
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