CN108676092A - A kind of Yolk antibody and preparation method thereof of prevention novel duck reovirus - Google Patents
A kind of Yolk antibody and preparation method thereof of prevention novel duck reovirus Download PDFInfo
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- CN108676092A CN108676092A CN201810494056.5A CN201810494056A CN108676092A CN 108676092 A CN108676092 A CN 108676092A CN 201810494056 A CN201810494056 A CN 201810494056A CN 108676092 A CN108676092 A CN 108676092A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
Abstract
The invention discloses a kind of Yolk antibody and preparation method thereof of prevention novel duck reovirus, the Yolk antibody contains anti-duck reovirus strain antibody；The deposit number of the duck reovirus strain is CCTCC NO：V201818.The safety of Yolk antibody prepared by the present invention is good, does not occur any locally or systemically adverse reaction caused by Yolk antibody；And can effectively prevent and/or treat the infection of novel duck reovirus, there is good commercialized development foreground.
Description
Technical field
The present invention relates to veterinary biologics technical fields, and in particular to a kind of yolk of prevention novel duck reovirus
Antibody and preparation method thereof.
Background technology
The genome of reovirus is bifilar segmented RNA, and the international virus purification committee (ICTV) will within 1978
It is set to Reoviridae (Reoviridae), including reovirus category, Orbivirus and rotavirus.Exhale intestines lonely
Viral (Avian reovirus) is isolated in the respiratory tract of chicken for the first time, after infecting susceptible chicken, can behave as myotenositis, cunning
The symptoms such as film inflammation and breathing problem.ARV can the birds such as infected chicken, turkey, duck, goose, parrot, pigeon, the virus of chicken can be caused
Property arthritis, tenosynovitis, while being also causes of disease of other diseases, including myocarditis, pericarditis, hydropericardium, hepatitis, thymus gland withers
Contracting, respiratory diseases and growth retardation etc..
From 2016, the outburst on a large scale of the ground such as China Shandong, Hebei, Henan and Jiangsu duck is with gambrel enlargement for main disease
The communicable disease of shape, the disease mainly cause each age level duck gambrel swelling, gambrel often to contain a small amount of yellow or blood
Sample exudate, several cases have cheesy exudate in articular cavity, suffer from duck and different degrees of limping occur.The disease mainly results in
The decline that kind duck is laid eggs with meat duck weight, feedstuff-meat ratio increase, and meat duck delivers qualification rate significant decrease for sale, to meat duck and kind duck cultivation
Industry causes serious financial consequences.
It has been investigated that the outburst of the disease is caused by a kind of novel duck reovirus infection, vaccine there is no at present
It can be prevented, conventional antiviral and antibacterial therapy method is invalid.
Yolk antibody refers to the antibody for specific antigen extracted from immune eggs, due to only having IgG in yolk
Class antibody, therefore it is called Yolk immunoglobulin IgG (egg yolkimmunoglobulins), referred to as IgY.It is anti-with serum
Body is compared, and Yolk antibody has the advantages that many uniquenesses：(1) it is not necessarily to blood sampling, immune height need to be only acquired and exempt from egg yolk purifying i.e.
It can purify to obtain Yolk antibody；(2) there is good stability, it is heat-resisting acidproof, and remain to keep certain activity under room temperature；(3)
Therapeutic effect is apparent, high specificity, and is suitable for mass producing；(4) safe efficient, noresidue, mild environmental protection.But due to
The disease belongs in China to be reported for the first time, be there is no vaccine and the antibody listing of commercialization at present, is used for the novel duck reovirus
Urgent prevent and the Yolk antibody of early infection treatment is even more to have not been reported；And due to containing a large amount of lipid in yolk,
Many inconvenience are brought to using, this also constrains the development and application for the Yolk antibody for preventing the novel duck reovirus.
Invention content
For the above-mentioned prior art, the object of the present invention is to provide a kind of yolk of prevention novel duck reovirus is anti-
Body, the novel reovirus infection for preventing and treating duck group's outburst, serious commercial caused by make up the disease damage
It loses.
It is a further object of the present invention to provide the preparation methods of the Yolk antibody of the prevention novel duck reovirus.The system
Preparation Method is easily operated, is suitble to large-scale production；And Yolk antibody property stabilization, purity height, the potency prepared is high, specific
By force, drug residue free, mild environmental protection, the prevention to novel duck reovirus infection and the great application value for the treatment of.
To achieve the above object, the present invention adopts the following technical scheme that：
The present invention be based on from serious gambrel enlargement symptom to suffer from isolated in duck tendon tissue one plant new
Type duck reovirus, deposit number are CCTCC NO：V201818, on the basis of the separation strains, this can be prevented by having developed
The inactivated vaccine of novel duck reovirus, using the inactivated vaccine high intensity immune health laying hen, then chicken after separating immune
Egg yolk, extraction prepare Yolk antibody.Specifically：
The first aspect of the present invention provides a kind of Yolk antibody of prevention novel duck reovirus, the Yolk antibody
Contain anti-duck reovirus strain antibody；The deposit number of the duck reovirus strain is CCTCC NO：V201818.
The second aspect of the present invention provides the preparation method of above-mentioned Yolk antibody, includes the following steps：
(1) it is CCTCC NO with deposit number：The duck reovirus of V201818 is made and goes out as production of vaccine strain
Live vaccine；
(2) with the inactivated vaccine injecting immune laying hen prepared, high-immunity egg is prepared；
(3) yolk is collected by high-immunity egg, through once inactivating, acidizing extraction, secondary inactivation, coarse filtration, aseptic filtration, concentration and
After inactivating three times, it is prepared into the Yolk antibody of prevention novel duck reovirus.
Preferably, in step (1), the inactivated vaccine is prepared by the following method：
It is CCTCC NO by deposit number：The duck reovirus of V201818 is inoculated with SPF chicken embryos, collects dead in 24-120h
Chick embryo allantoic liquid is died, virus liquid is obtained；Virus liquid is concentrated into viral level >=105.0ELD50, the virus liquid after concentration is through formaldehyde
Inactivation is added Tween-80 mixing and is used as water phase, mixed using white oil, aluminum stearate and Span-80 as oil phase, by water phase and oil
Mutually by volume 1:Inactivated vaccine is made in 1 mixing, emulsification.
Preferably, in step (2), divided 4 times with the inactivated vaccine of preparation and laying hen is immunized, each immunization interval 2
Week.Immunization route is that neck subcutaneously or intramuscularly injects inactivated vaccine.
Preferably, in step (3), the primary inactivation is specially：By yolk liquid and water by volume 1:(0.5~1.5)
Mixing, yolk diluent is obtained after stirring evenly, and 25~35min is kept the temperature under the conditions of 60~65 DEG C.
Preferably, in step (3), the acidizing extraction is specially：The yolk diluent is added into yolk diluent
Filtrate is collected by filtration after stirring in the acetate buffer that 2.5~3.5 times of volume.It is further preferred that the acetate buffer
PH value be 4.8-5.2；More preferably 5.0.
Preferably, in step (3), the secondary inactivation is specially：Octanoic acid is added into the filtrate after acidizing extraction to end
Concentration 3.5~4.5% stirs 30-120min, is placed 4~8 hours in 2~8 DEG C after stirring.
Preferably, in step (3), the coarse filtration is specially：It is then clear to filtrate with membrane filtration again first with filter-cloth filtering
Clearly.It is further preferred that the filter cloth is polypropylene fibre 750B filter clothes.
Preferably, in step (3), the aseptic filtration is specially：With 0.22 μm of membrane filtration degerming.
Preferably, in step (3), the concentration is specially：Using the PES hollow fiber ultrafiltration membranes of 30~50KD 2~8
It is concentrated under the conditions of DEG C, until antibody titer is not less than 1:512.
Preferably, in step (3), the inactivation three times is specially：Formalin is added, makes the final concentration of of formalin
0.1%, 37 DEG C of inactivation 16h.
The third aspect of the present invention provides above-mentioned Yolk antibody and is preparing for preventing and/or treating duck reovirus
Application in the product of infection.The deposit number of the duck reovirus is CCTCC NO：V201818.
Beneficial effects of the present invention：
(1) present invention is exhaled using isolated one plant of novel duck reovirus out of morbidity duckling body, the novel duck
There is intestines orphan's Strain excellent immunogenicity, the passage that can stablize in chicken embryo to be closed with instep for currently endemy
Saving the novel duck reovirus infection that enlargement is cardinal symptom has better protectiveness.
(2) it is CCTCC NO with deposit number：The duck reovirus of V201818 is prepared as production of vaccine strain and is inactivated
Vaccine carrys out Immune Laying Hens, can efficiently obtain Yolk antibody, and the safety of the Yolk antibody is good, does not occur being drawn by Yolk antibody
Any locally or systemically adverse reaction risen；And the infection of novel duck reovirus can be effectively prevented and treated, have
There is good commercialized development foreground.
(3) preparation process of Yolk antibody is optimized in the present invention, collects after height exempts from egg yolk and first carries out tentatively
Inactivation then carries out acidification with acetate buffer and extracts, then as inactivator and extracts the secondary inactivation of agent progress using octanoic acid,
It is filtered clarification, filtration sterilization on the basis of this, is most inactivated three times with formaldehyde after ultrafiltration concentration afterwards, yolk is finally obtained
Antibody-solutions.The content that lipid in final product is effectively reduced above by acidifying water-octanoic acid method, reduces adverse reaction
Risk, while step is concentrated by ultrafiltration and helps to ensure that antibody titer in solution, improve protective rate；Higher antibody titer will be in future
Higher extension rate is needed when clinical application, to reduce impurity content indirectly, and then promotes safety.
Description of the drawings
Fig. 1：Cytopathy of the virus isolated strain of the present invention on LMH cells；Wherein, A：Under normal growth state
LMH cells；B：The CPE that N-DAV-SD16 occurs on LMH cells.
Fig. 2：PCR qualification results；Wherein, M Marker；Swimming lane 1 is sample to be tested；Swimming lane 2 is blank control.
Specific implementation mode
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
As background technology is introduced, from 2016, the ground such as China Shandong, Hebei, Henan and Jiangsu duck is quick-fried on a large scale
Hair is using gambrel enlargement as the communicable disease of cardinal symptom.The symptom of disease duck reovirus infection different from the past,
There is the symptom of gambrel enlargement in duck group for the first time, thus deduce, which may be to exhale the lonely disease of intestines by novel
Caused by poison.It there is no the drug and method that can effectively control the novel duck reovirus infection at present.Intestines are exhaled using duck
Lonely viral inactivation vaccine inoculation is to prevent the effective measures of the reovirus infection and outburst, but obtain on condition that needing to detach
The excellent duck reovirus strains of immunogenicity.
Since reovirus is RNA virus, there are multiple segmentations, trained in antigenic structure, pathogenic, cell between different strains
It supports and is had a certain difference on characteristic and host specificity, in genetic evolution, be easy to happen variation, lead to traditional strain system
Standby vaccine cannot prevention and control be current popular well is infected by the Avianreovirus of cardinal symptom of gambrel enlargement.
Present inventor isolates one plant of duck reovirus N-DAV-SD16 from the tendon tissue of morbidity duck swelling,
Birds reovirus gene by segmented 10 genetic fragments (including：L1-L3, M1-M3, S1-S4) composition, the present invention couple
The duck reovirus N-DAV-SD16 of above-mentioned new separation has carried out genome sequencing, and respectively by 10 genetic fragments and now
The Avianreovirus having been reported that has carried out sequence alignment and homology analysis, as a result, it has been found that, new isolated strain N-DAV-SD16
10 genetic fragments in L1, L2, L3, M1, M2, S3, S4 be respectively positioned in a relatively independent branch, and at present on
Biography reovirus-originated duck homology in Genbank is relatively low, illustrates that the strain N-DAV-SD16 newly detached is different from others
Avianreovirus, it can be assumed that duck reovirus found in the present invention with it is existing it has been reported that duck reovirus
In the presence of compared with Big mutation rate, being 1 independent kind of Orthoreovirus.By further comparing discovery, which exhales the lonely disease of intestines
Part has been recombinated in malicious N-DAV-SD16 genomes leads to the S1 gene pieces of broiler chicken source reovirus of broiler chicken gambrel swelling
Section.And the novel duck reovirus is preserved in China typical culture collection center, deposit number is CCTCC NO：
V201818。
It is long the time required to generating antibody after being immunized since vaccine immunity is to aim at prevention, be difficult to when duck mass-sends disease into
Row in time, is quickly treated.Based on this, the present invention prepares vaccine immunity laying hen by the novel duck reovirus of separation, adopts
Collection height exempts from egg and prepares Yolk antibody, the urgent prevention for novel duck reovirus and early infection treatment.
In one embodiment of the invention, the Yolk antibody of the prevention novel duck reovirus provided is by such as lower section
Method is prepared：
(1) it takes novel duck reovirus height to exempt from egg yolk, is uniformly mixing to obtain yolk liquid；
(2) step (1) the yolk liquid and water are pressed 1:(0.5~1.5) (V/V) ratio mixes, and is obtained after stirring evenly
Yolk diluent keeps the temperature 25~35min under the conditions of 60~65 DEG C；
(3) acetate buffer that the yolk liquid accumulates 2.5~3.5 times is added into yolk diluent, is filtered after stirring
Filtrate is collected by filtration in cloth；
(4) octanoic acid is added into step (3) described filtrate to final concentration 3.5~4.5% (V/V), in 2~8 DEG C after stirring
It places 4~8 hours；
(5) solution after taking step (4) to place is filtered to filtrate and is clarified, and collects clear filtrate；
(6) step (5) the clear filtrate filtration sterilization is taken, formaldehyde is then added to final concentration 0.1% (V/V), in 35
~37 DEG C inactivate 12~20 hours.
Preferably, the novel reovirus height described in step (1), which exempts from egg, to be inactivated using novel reovirus
Produced egg after 4 immune hens of vaccine point, each immunization interval 2 weeks；Further preferably, the immunization route is that neck is subcutaneous
Or the novel reovirus inactivated vaccine of intramuscular injection.
Preferably, in step (1) the novel reovirus high immunity egg yolk, novel reovirus antibody titer
Not less than 1:1024.
Preferably, first exempting from egg to the height in step (1) carries out eggshell disinfection, yolk is then collected；It is further excellent
Choosing, the sterilization method is first to spray 75% alcohol again after eggshell drying with 1% bromogeramine solution soaking disinfection 15min
To eggshell surface.
Preferably, the water described in step (1), is prior to 80 DEG C holding 30min, is then cooled to 65 DEG C of notes below
Jetting.
Preferably, acetate buffer pH value described in step (2) is 4.8~5.2, more optimizedly 5.0.
Preferably, the stirring described in step (3), is persistently 30~120min, more optimizedly 90min.
Preferably, the filter cloth described in step (3) is polypropylene fibre 750B filter clothes.
Preferably, the stirring described in step (4), duration are 30~120min, more optimizedly 90min.
Preferably, the filtering described in step (5) is first with filter-cloth filtering, then clarified again with membrane filtration to filtrate；
It is further preferred that the filter cloth is polypropylene fibre 750B filter clothes.
Preferably, the filter sizes for being used for filtration sterilization in step (6) are 0.22 μm.
Preferably, novel reovirus antibody titer is not less than 1 in clear filtrate after step (6) degerming:512.
Preferably, being first concentrated by ultrafiltration after degerming in step (6), formalin-inactivated is then added；Further preferably
, the ultrafiltration concentration is realized using the PES hollow fiber ultrafiltration membranes of 30~50KD；It is further preferred that the concentration
It is carried out under the conditions of 2~8 DEG C.
In above technical scheme, collects after height exempts from egg yolk and first tentatively inactivated, then with acetate buffer
It carries out acidification to extract, then the secondary inactivation of agent progress as inactivator and is extracted using octanoic acid, be filtered clarification, mistake on this basis
Bacterium is filtered out, is most inactivated three times with formaldehyde after ultrafiltration concentration afterwards, Yolk antibody solution is finally obtained.Above by acidification
Water-octanoic acid method effectively reduces the content of lipid in final product, reduces the risk of adverse reaction, while step is concentrated by ultrafiltration
It helps to ensure that antibody titer in solution, improves protective rate；Higher antibody titer needs higher when future clinical is applied
Extension rate to reduce impurity content indirectly, and then promotes safety.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
The test material that test material is this field routine is not specifically described used in the embodiment of the present invention,
It can be commercially available by commercial channel.Specific experiment condition and method are not specified in the embodiment of the present invention, usually according to routine
Condition, such as J. Pehanorm Brooker chief editors, Science Press, 2002, Molecular Cloning:A Laboratory guide (third edition)；D.L. this peck
Top grade is edited, Science Press, and 2001, cell experiment guide；Or the condition suggested according to manufacturer.The embodiment of the present invention
In quantitative test, be respectively provided with three repeated experiments, results are averaged；% in the embodiment of the present invention, unless otherwise instructed,
It is mass percentage.
Embodiment 1：The discovery and identification of novel duck reovirus
1. virus purification：
(1) it clinically is accredited as positive arthritic duck through PCR reactions, the tendon tissue that dissect acquires its swelling is set
In 15mL centrifuge tubes, be added 5 times of volumes serum-free DMEM culture mediums, multigelation 3 times after homogenate, every time thaw after
Freeze thawing next time is being carried out after shaking 1-2min on oscillator；After freeze thawing by the 15mL centrifuge tubes equipped with sample in centrifuge
4000rpm centrifuges 5min；Take supernatant, it is spare after 0.22 μm of filtering with microporous membrane；
(2) according to the multiplication characteristic for exhaling the lonely disease of intestines, chicken gizzard cancer cell (LMH) is selected to carry out virus purification.According to conventional thin
Born of the same parents' cultural method, waits for 25cm2When cell confluent monolayers in cell bottle, suction, which is abandoned in bottle culture medium and swung with PBS, washes cell 2 times,
The sample freeze thawing supernatant that 0.5 milliliter of step (1) has filtered is added, cell is placed in 37 DEG C, 5%CO2Feel in the incubator of concentration
Make 30min, sense is added the DMEM culture mediums containing 2% fetal calf serum after making, observes and records the time for cytopathy occur.
If the acellular lesion of 1st generation occurs, then the culture harvested with 1st generation after freeze thawing continues to detach according to step (1) method, until
It obtains and stablizes strain；The 5th generation still acellular lesion is such as reached, then is considered as virus purification feminine gender.Finally obtain one plant of stable poison
Strain, occurs more apparent cytopathy for 3 days after being inoculated with LMH cells, that is, shows as cell rounding, fusion, the group of presentation
Block falls off lesion (Fig. 1), normal with batch blank control cell, which is named as N-DAV-SD16.
2. viruses indentification：
(1) PCR is identified
1) RNA is extracted：
By the virus liquid of strain N-DAV-SD16 according to the requirement of RNA extracts kit specifications, viral RNA is extracted, be placed in-
20 DEG C save backup；
2) reverse transcription obtains cDNA：
Reverse transcription reagent box used uses the article No. from precious biological (Dalian) Co., Ltd for RR036A's
PrimeScriptTMRT Master Mix sequentially add 5 × PrimeScript RT Master in 200 μ LPCR reaction tubes
The μ L of Mix × 2, the middle sample to be tested Total μ L of RNA~2 extracted of step 1), use RNase Free dH2O is supplemented to 10 μ l bodies
System.It is placed in PCR instrument and is reacted, reaction condition is 37 DEG C of 15min；4 DEG C of preservations after 85 DEG C of 5s.
3) PCR amplification：
It is expanded using 20 μ L systems：The μ L of template cDNA × 2, upstream and downstream primer is each × 1 μ L, 2 × Es
The μ L of TaqMasterMix × 10, using ddH2O is supplemented to 20 μ L systems.Mixing is simultaneously reacted in wink from PCR instrument is placed on, reaction
Condition is 95 DEG C, 5min, and 95 DEG C later, 45s, 56 DEG C, 30s, 72 DEG C, 30s carries out 30 and recycles, 72 DEG C, 4 DEG C after 10min
It saves backup.
Sense primer：5′-TGA GAC GCC TGA CTA CGA TT-3′(SEQ ID NO.1)；
Downstream primer：5′-ATG CTT GGA GTG AGA CGA CT-3′(SEQ ID NO.2).
4) PCR qualification results：
Product after carrying out PCR amplification with primer occurs big with desired value 513bp in 1% Ago-Gel after electrophoresis
The small specific band (Fig. 2) being consistent.Show that there are novel reoviruses in the cell culture of each separation strains.In addition to dividing
From the conventional duck source viral diagnosis that strain carries out, and other virus pollutions are not detected.
(2) sequencing：
Whole genome sequence measurement carried out to strain N-DAV-SD16 using two generation sequencing technologies, in whole genome sequence
The sequence of this 10 genetic fragments of L1, L2, L3, M1, M2, M3, S1, S2, S3, S4 is respectively such as SEQ ID NO.3-SEQ ID
Shown in NO.12.Then sequence analysis is carried out with the Avianreovirus sequence announced in the prior art, as a result, it has been found that, poison
L1, L2, L3, M1, M2, S3, S4 are respectively positioned in a relatively independent branch in 10 genetic fragments of strain N-DAV-SD16, and
With uploaded in Genbank that reovirus-originated duck homology is relatively low at present, illustrate the strain N-DAV-SD16 newly detached
Different from other Avianreovirus, it can be assumed that duck reovirus found in the present invention with it is existing it has been reported that
Duck reovirus exists compared with Big mutation rate, is 1 independent kind of Orthoreovirus.
The virus is subjected to preservation, deposit number is CCTCC NO：V201818.
Embodiment 2：The preparation of inactivated vaccine
(1) virus multiplication program：The novel duck reovirus strain that embodiment 1 detaches is connect according to 0.2ml/ pieces of dosage
Kind of 9 age in days health SPF chicken embryos discard dead germ in for 24 hours, collect dead chick embryo allantoic liquid in 24~120h, obtain virus liquid, and -20
DEG C preserve.
(2) virus liquid concentrates：By the virus liquid of harvest under the conditions of 2~8 DEG C, original volume is concentrated into ultrafiltration concentration machine
1/3, by existing《Chinese veterinary pharmacopoeia》Annex carries out steriling test, asepsis growth, and the malicious valence of standby survey that keeps sample.Virus liquid is per 0.1ml
Viral level answers >=105.0ELD50, the allantoic fluid after concentration inactivated immediately.
(3) it inactivates：Virus liquid after concentration is imported in inactivation tank, 10% formalin, which is added, keeps its final concentration of
0.1%.It is imported in another inactivation tank after adding formalin, fails to contact inactivator to avoid the virus that tank mouth nearby adheres to.37
DEG C sealing inactivation 24 hours (by temperature in tank reach 37 DEG C start in terms of), it is primary every stirring in 4~6 hours therebetween, after inactivation, set
It is saved backup in 2~8 DEG C.
(4) prepared by inactivated vaccine：
1) prepared by oil phase：94 parts of white oil for animals, 2 parts of aluminum stearate is taken to be placed in oil phase preparation tank after being heated to 80 DEG C, then
Add 6 parts of Span-80, until when temperature reaches 115 DEG C, maintains 30min, it is spare after cooling.
2) prepared by water phase：It will examine 96 parts of qualified virus liquid that 4 parts of sterilizing Tween-80 are added after inactivation, start refiner
20~30min is stirred, Tween-80 is made to be completely dissolved.
3) it emulsifies：Example is 1 to oil phase compared with water:1 (V/V), first imports colloid mill by oil phase, and 2500 revs/min of stirrings are delayed
It is slow that water phase is added, it is emulsified 5 minutes for 10000 revs/min after adding.Sulphur willow is added by vaccine total amount 0.01% before terminating emulsification
Mercury fully vibrates mixing.After emulsification, 10ml is taken, is centrifuged 15 minutes with 3000r/min, tube bottom is precipitated without water phase.
After aseptic subpackaged, roll lid and be stored in 2~8 DEG C.
Inactivated vaccine to preparation includes：Dosage form, centrifugal stability, viscosity and sterile quality inspection, specific side
Method refers to《Republic of China Veterinary Pharmacopoeia》(2015 editions).
As a result it is：The dosage form of inactivated vaccine prepared by the present invention is water-in-oil type (W/O)；Centrifugal stability, viscosity and nothing
Bacterium, which checks, to be met《Republic of China Veterinary Pharmacopoeia》The regulation of (2015 editions).
Embodiment 3：The preparation of novel duck reovirus Yolk antibody
1. using inactivated vaccine immunization laying hen：
Novel reovirus inactivated vaccine Immune Laying Hens prepared by embodiment 2, every chicken neck of first immunisation are subcutaneous
2.0ml inactivated vaccines are injected, second of immune, every chicken neck hypodermic injection 2.0ml inactivated vaccine, after two exempt from is carried out after 14 days
It is immune to carry out within 14 days third time, 2.0ml inactivated vaccines are subcutaneously injected in every chicken neck, and three exempt to carry out booster immunization in latter 14 days, often
Chicken neck is subcutaneously injected 2.0ml inactivated vaccines, and 14 days after booster immunization, acquisition yolk measures novel reovirus and neutralizes
Antibody titer answers >=1:1024.
2. Yolk antibody manufactures：
(1) eggshell sterilizes：Collection height exempts from egg and is soaked in 15min in 1% bromogeramine solution.After taking-up naturally dry or
After drying, spray 75% alcohol eggshell surface is sterilized it is spare.
(2) yolk detaches：It takes machinery to beat eggs mode, egg white, blastodisc and frenulum should be fully removed when beating eggs, collect ovum
It is yellow.
(3) I is inactivated：The yolk of collection is sufficiently stirred, it is in uniform paste to make yolk, starts peristaltic pump, by yolk liquid pump
Enter in interlayer retort, (water for injection is first sterilized through 80 DEG C of 30min, and is cooled to the isometric water for injection of yolk for addition
65 DEG C or less), after stirring and evenly mixing, 60~65 DEG C of heat preservation (inactivation) 30min.
(4) acidification extracts：The acetic acid that the pH value 5.0 for being equivalent to 3 times of volumes of former yolk is first added in retort is isolated is slow
Then yolk liquid is added in fliud flushing, open blender, abundant agitating 30 minutes.
(5) II is inactivated：The octanoic acid that final concentration (V/V) is 4% is added in yolk liquid liquid to make inactivator and extract agent, acutely stirs
90min is mixed, 2~8 DEG C are placed 4~8 hours.
(6) coarse filtration：It is filtered until clear after polypropylene fibre 750B filter-cloth filterings, then with column core filter.
(7) aseptic filtration：With 0.22 μm of membrane filtration degerming.2~8 DEG C of storages are set, should be no more than 14.Sample inspection simultaneously
Survey the neutralize antibody titers of novel reovirus.
(8) it concentrates：As the neutralize antibody titers of the novel reovirus of detection are less than 1:512, it should will be after aseptic filtration
Yolk antibody under the conditions of 2~8 DEG C, the ultrafiltration concentration of suitable multiple is carried out with the concentration film packet of 30~50KD, after concentration
Novel reovirus neutralize antibody titers should be not less than 1:512.
(9) III is inactivated：Solution after concentration is imported in inactivation tank, metered 10% formalin opens stirring
Machine stirs, it is made to be sufficiently mixed, and the ultimate density (V/V) of formalin is 0.1%, and 37 DEG C inactivate 16 hours.
(10) packing storage：Aseptically the filtrate inactivated is sub-packed in sterile glass vials, covers rubber plug, is rolled
Aluminium lid, it is labelled, it saves backup, storage temperature is -20 DEG C.
Embodiment 4：Yolk antibody quality inspection
1. safety examination：
With the susceptible duckling of 1-3 ages in days (the equal ＜ of novel duck reovirus maternal antibody 1:4) 10, each muscle branch injection
Yolk antibody 2.0ml prepared by the embodiment of the present invention 3 is observed 14, and all health survivals of susceptible duckling illustrate the ovum of the present invention
The safety of yellow antibody is good.
2. steriling test：
According to《Republic of China Veterinary Pharmacopoeia》(2015 editions) progress, result are：Without bacterium, mycoplasma and exogenous virus
Pollution.
3. efficacy test (Immunization method)：
The susceptible duckling of 1 age in days (the equal ＜ of novel duck reovirus maternal antibody 1:4) 20, it is randomly divided into tetra- groups of A, B, often
Group 10.Wherein, A is immune group, and neck subcutaneously or intramuscularly injects the Yolk antibody of the preparation of embodiment 3, every 0.5ml；B is to attack
Malicious control group, subcutaneously or intramuscularly injecting normal saline, every 0.5ml.Isolated rearing.
After 16 hours, two groups of ducklings of A, B respectively do 10 times of diluted novel ducks with physiological saline and exhale the lonely disease of intestines by intramuscular injection
(deposit number is CCTCC NO to poison：V201818), every 0.5ml.It is observed 10 after attacking poison, records every group of duckling morbidity, death
Situation.
As a result：A groups attack 10/10 protection after poison；B groups attack poison and start to fall ill after 12 hours, dead 9 in 10 days.
Embodiment 5：The application of Yolk antibody
1. the application in preventing novel duck reovirus
With the susceptible duckling of 1 age in days (the equal ＜ of novel duck reovirus maternal antibody 1:4) 30 are subjects, random point
It is three groups, every group 10.Wherein：
Test group：Neck subcutaneously or intramuscularly injects the Yolk antibody of the preparation of embodiment 3, every 0.5ml；
Control group：Laying hen is immunized with presently commercially available Avianreovirus inactivated vaccine, and by embodiment 3
Yolk antibody is prepared in method, and neck subcutaneously or intramuscularly injects the Yolk antibody, every 0.5ml；
Blank control group：Subcutaneously or intramuscularly injecting normal saline, every 0.5ml.
By above-mentioned three groups of duckling isolated rearings, after 16 hours, respectively intramuscular injection with physiological saline do 10 times it is diluted new
(deposit number is CCTCC NO to type duck reovirus：V201818), every 0.5ml.It is observed 10 after attacking poison, records every group
Duckling morbidity, death condition.It the results are shown in Table 1.
Table 1：
| Group | Attack poison strain | Prevention ＆ protection |
| Test group | Novel duck reovirus | 10/10 |
| Control group | Novel duck reovirus | 5/10 |
| Blank control group | Novel duck reovirus | 1/10 |
Note：Prevention ＆ protection is health survival duckling number/duckling sum after attacking poison.
As can be seen from Table 1, Yolk antibody of the invention has splendid prevention ＆ protection effect, and intestines are exhaled for the novel duck
Lonely virus is capable of providing 100% prevention ＆ protection, and Vaccine effectiveness is better than the Yolk antibody prepared using existing inactivated vaccine.
2. to the application in novel duck reovirus therapy
In novel duck reovirus infection region of disease, symptom is gambrel enlargement, is with 30 ducklings similar in the course of disease
Subjects, are randomly divided into 3 groups, every group 10, whole isolated rearings, wherein：
Test group：Neck subcutaneously or intramuscularly injects the Yolk antibody of the preparation of embodiment 3, every 1.0ml；
Control group：Laying hen is immunized with presently commercially available Avianreovirus inactivated vaccine, and by embodiment 3
Yolk antibody is prepared in method, and neck subcutaneously or intramuscularly injects the Yolk antibody, every 1.0ml；
Blank control group：Subcutaneously or intramuscularly injecting normal saline, every 1.0ml.
Observation 10 days, records every group of the duckling state of an illness, death condition.
As a result it is：Test group injected Yolk antibody after 2 days, and the feed intake of illness duckling starts to increase, and the state of mind is apparent
It improves, does not have duckling dead in 10 days；Wherein 1/2 duckling is slaughtered and dissected after 10 days, as a result, it has been found that illness duckling instep closes
Section enlargement symptom disappears substantially.After control group injects Yolk antibody, the feed intake and the state of mind of illness duckling do not have significantly
It improves, begins within 5th illness duckling death from injection Yolk antibody, the death rate of illness duckling is 60% in 10 days；10 days
It slaughters the 1/2 of the duckling of survival and dissects afterwards, as a result, it has been found that illness duckling gambrel enlargement symptom exists on a small quantity.Blank control
The death rate in the illness duckling 10 days of group is up to 70%.
The above test results show that the Yolk antibody safety of the present invention is good, preventive effect is good, cure rate is high, Ke Yiyong
In the prevention of novel duck reovirus infection, there is great economic and social benefit.
The foregoing is merely the preferred embodiments of the application, are not intended to limit this application, for the skill of this field
For art personnel, the application can have various modifications and variations.Within the spirit and principles of this application, any made by repair
Change, equivalent replacement, improvement etc., should be included within the protection domain of the application.
SEQUENCE LISTING
<110>Shandong Agricultural University
<120>A kind of Yolk antibody and preparation method thereof of prevention novel duck reovirus
<130> 2018
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
tgagacgcct gactacgatt 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
atgcttggag tgagacgact 20
<210> 3
<211> 3928
<212> DNA
<213>L1 genetic fragments
<400> 3
ctttttctcc gatctccgaa atgagttcgc gcaaagtggc tagacgtcgt cataaggatg 60
ctactgaatt taaatacact aagaacgtta acaagtctaa gccatcctct actgacgtta 120
aagaacctgc ggatagtgcc acagataaga aagtcactgt tccatcacca gacaatccag 180
ccgcttctac tccctcttcc actgatggag cttctcaaac atccgttgct aagcagacga 240
atgataatga cgcctcagtt aaggaatcag ctcccaagcc taccgtatct agcgatggga 300
aagacggaat gcatggtgct gtgaagttgc aagacgctaa ggccactgca gctgtggata 360
gtaataagga tagagatgtg gtatttggtg gcgcaggctc tggtgacaaa aatgctatta 420
cgaagactgg ttccgttgac aatgatgggg gcgttaaagt cgttccagcc aaggatgcta 480
cgatatcttc ggccaaagcc atgatggaac agaagcagtt agttgctggt cttccgaagc 540
aaccgaagtc tgctaatcat ttgtgcaccg tttgtatggc acaattcgcg tctgctgacg 600
ccctaactat ccatcagact acgcactcca ttggctccaa tgctgctctg acgagcttct 660
cgatttctac tgctgttgaa gaattcattc aatcatgggc tgctgctacg tccactgcca 720
ataccaagac ggccttaact gtgtctgacg tggactcgct gatgatgact gaaggaatac 780
gtctcataac ttgggattct gggttatgta catcttttga gcttgtcccg attgtccatt 840
caaacactgt tcaagatgta atttcgtact catggttcac atcaagttat aatatcacaa 900
ctcccttccc acaggcgcct gtcgtgcgaa ttgttttacg tactaattgg gctgccaaat 960
tggactctcc ctcgtcgtcg cgtgaatgtg atcttcgcct cgccccacct acagagagta 1020
atgctcgatc attctcaatg ctgctcaata ctggtgcgac tccagaaggt actttcaacc 1080
ccaacaccct tcgtatgaat gtgctgcaga tgtgtcttca gtatgttctg gctaacctac 1140
acttgaatcg tagcactcag ttcaccatgg atttgactgc cgcagctccc aatctgtcgg 1200
cgtctcaact ccgtatcgtt ccagatgata aagatggtaa atggttccct gtcatgtatc 1260
catctcgcgt gaacatccca ctgttcaata agacagctga ttttgttaat cagtgcattc 1320
gtgacagaat tggccgatat gaccgcgccc agactttcgc tggcgcaccc tctgaatggg 1380
ctgacatgtg ggaaacagcg gacgcgttaa ctctctccgt ccgtgaaatg tggatgtcac 1440
gtatttccca aatgaatatc tctcctgctg atatcgctga cgctatctct cgatgttctc 1500
agtccctgct cactgttgcc gcgccaacag ctccttctgt agctcgtctg ttaccttggc 1560
gggttagttc cgatgagagg cagctcctcc aattgttgat gtacttgaac gttgggacta 1620
gtgccgacta cgtccaacct attctgtctg cgttcgctcg aactctatct cgcgtgtcac 1680
cattgcgtat taatcccact ttaatcgcta atgccatgtc gacaattgtc gagagcacta 1740
ctaataccca gagtcctgcg gcagctattt tgtcgaagct taaacctgtt gcctcggact 1800
tctccgattt taggttggcg tgtgccgctt ggttgtataa tggttgcgtc cagacatact 1860
tgtctgagga ttcatatcca agcagtggtg gatctgttac tagcattgat acattggttg 1920
atatgtttgt gtgcttgttg gctttgcctc ttgttactga tcctaatgct ccttgccaag 1980
cctttatggt tgtcgctaat gccatggttg gttacgagaa tctaccgatg gacgacccta 2040
attttactca gcaaagattg gctgcagcgt tcaacaatcc cacgacctgg cctcaatgtt 2100
tcctccaccc tcaaaacatc gatcgacgcc aatgtccgat tctctcatgg tgggctcagc 2160
agattcatcg taattggccc acaccgtctc aaattactta tggggcgcct gacatcattg 2220
ggtccgctaa cttgtttact cctcctgacg tgctgttgct tccattacag cataggccca 2280
tccgtattac taatcccacc ctgaacttcg ataatgagtt gacgacttgg cgtaacaccg 2340
tggttgatct ggttctgcgc atcatcgaca gtggtcggta ccagcccaat tggaatcagt 2400
ccatccgtgc ctctatgcgg aatgcgatga caaatttcag aattattaag tcctatactc 2460
ctgcttacat agcagaactg ctacctgtgg aactggcagc tatcgctcca actctaccct 2520
tccagccttt tcaggtgccg tttgcccgct tagatcgcga cgctatcgtc actcacgtca 2580
atgtatctag acaagctccc aacaatcttg ctcaacctgc attgaacatg tccatgacgt 2640
accagcgcac aggggttcca atctctctta gtgcccgtcc cttggcagtc gctcttctgt 2700
caggccagta ccctactgat ccccctcttc agactaatgt ttggtacgta aacactctca 2760
cgcctctata ttccaatgat ggtctcttta ataacgtcca gcacgctatg gttgcttctg 2820
aagcttacgc taccttgatc accatgctgg ctcagtgcac tgacatgcag taccctgtgg 2880
atcggccatt gaattggctt cgtcagatta atttggctgc taatgaggcg acgattttcg 2940
gtcgatcaat taattcactt ttccagactg ctttcgacct ttcaccttcc actgtattgc 3000
ttcaaccgtt tttggaatct gatccacgtg cgacacagct agccatttct tacgttcgtt 3060
ataatggtga cagtgaaacc ttcgtgccaa cagtgcgtcc atctatgatt tcagaagcga 3120
cattgctcgt tgagcgtact ctctcgcacg aatacaacct cttcggttta tgccgtggtg 3180
acatcatcct gggacagcac atgactccaa ctgcgttcaa tcctttggct ccgcctcctt 3240
ccgtcgtttt taacaggggt gatactgatg tctatgagtt tggctctcgt agcttcgcca 3300
acttcggcat gaatggggag gagatcttgg tcatggatgc gaacggcgtg cgtcgtccat 3360
tactcggccg gtgggttatg ccactgcagc ttttgatggt taatattggc gtatttccta 3420
agctgttgct ggatcgtatc ctgaaggggc gcttgtatat ccgacttgaa gttggcgcgt 3480
atccatatac ggtgcagtat taccagggac gtgagtttac ggatggcttc actctgcttg 3540
agcaatggat gtctaaggtg tcgcccatgg gtatccctcc cgtccctttc ctcatgccgc 3600
agtccgaagg acacaacatc acttcaggca tggttactca ttacatctgg tccactgaat 3660
ataatgatgg gtccctcttc gccacgaaca ccgacctacc agttactgtg ttcggccctg 3720
accgcaccat cccaatcgag cgttatcggg cactcgtgga tccaggtgct cttcccgcta 3780
ctaaccaact gccgcacacc attgatcttt actgctcact gagacggtac tacttggaga 3840
cacctcccat cactgcgacc gtcaccactt atggcgatgg actccccgcg ctgaaccatt 3900
agagcggcga ggctagacgc gagctgat 3928
<210> 4
<211> 3830
<212> DNA
<213>L2 genetic fragments
<400> 4
gctttttcct caccatgcat gtcaacgggt ttgatgatgc tactctctcc tacgcacagt 60
ccatatcggg ggttatccca atgacaaata agttatttga gcaagcatct acctcaatac 120
gtgccctacc acgctcacat gtttatgcta tattagataa tgttaatttt tcagtttcat 180
gtgtcatccc gaatcgtatt ttccatcatc ctgatcactc tgagtacttt tacattgatg 240
cggttaacag agttagacgg aaacaagtta tcgatcctga tgatgtattt gtgccaaact 300
gcaatttgca gggtcttatt actccaatgg agagattacc aaattatggt caattgtctg 360
agactatttc gtcgaacgct cgggatggct tgccatctgc acgcgtagca gctacttttt 420
ataacacttc ggtgtcacag gctcgccaag ttaaagctcc acttgaatcc tttttgttgc 480
ctttgctatt atctgagact tgctcattat cagacgatcc ttgcggactt gacaccgcag 540
cttctcctcc gatccatacc aatctggcgt tatgggtgtt acgcgaaatt agtcgaacta 600
tttgtggatc ttcgaatgac cgttcacctt ggttactgct cgattcaggg gttgcgtggt 660
tcatgtctcc gttaatgtca tcggccattc cacctcttat ggctgactta acgaatctag 720
cgatctacaa acaaatttgt tctgtgtctg atgagcttca ttcccttgca gttcaagtgg 780
ttttgcaggc cgcggcgtca caatcgtatg gacactacat attgcagacg aagtcagtat 840
tcccccaaaa tacgttacat aacatgtttc gtacgctcac tgatggtatt gttccggtca 900
tagattggtt agaaccgcgt tcaaactatc gcttcatgct tcaaggtgcg cgtaaagtga 960
cttcagatga tgcgaatcag gctccggata acacggaagc cgcggagcaa ctcggtcgca 1020
agatggggtg cctcgatgtc gtacgctcct tacgtaagat gtcctcgtcc atcactgtcc 1080
attcacacga tgccatgacc ttcgtacgtg atgctatgtc gtgcactagt ggcatcttta 1140
tcacgcgtca acctactgag accgttttaa aagagtacac tcaagcgccg accattgaag 1200
tccctattcc tcaatcggac tggtcaccgc ctattggatc tctgcgatat ctctctgatg 1260
cctgttccct ccccgctgtg tatctggcta gggcttggcg cagagctgcc tctgctgtag 1320
tagataatcc gcacacttgg gaccctttat atcaggccat ccttcgctct caatatgtga 1380
cgtcacgcgg tgggtccggt gccgcgttaa gagatgcttt gaaggctgcg gaagttgagc 1440
ttcctcagta tcctggggtc agtgttaaag tggcgaccaa gatttatcaa gcggctcaga 1500
ctgctgacgt gcctttcgat aaattatctc gtgctgttct agctccattg tcgatgggct 1560
tacgtaacca agttcagcga cgtccaagaa ccatcatgcc catgaatgtg gttcaacagc 1620
agatttcagc ggctcatact ctctccgctg actacattaa ttatcacatg aacttgtcga 1680
cgacctcggg tagcgctgtg attgagaagg tggttccatt aggtatgtat gcatcctgtc 1740
ctcccgctca agcggttaat attgatatta aggcttgtga cgcgtccatc acgtaccagt 1800
attttctttc tgttatcgtc ggtgccattc atgagggtgc agcaggccgt cgtgtctcgt 1860
cttcattcat gggcgttcca ccaagcgtgc tgtccgttgt cgatgctagc ggagtgactt 1920
catccatgcc catctcaggt tttcaagtca tgtgtcaatg gttggctaaa ctttaccagc 1980
gaggttttga gtatcaagtg acggatacat tctcacctgg caatattttc acgcatcaca 2040
ctactacttt tccctctggt tcaacagcga cgtctacaga acatactgcc aataatagca 2100
cgatgatgga tggcttcctg cgcgcttgga ttccttcctc cggtgcgtcc gacgtactga 2160
agaagttctg caaatccatc tcaatacaac ggaattacgt ttgtcagggt gacgatggtc 2220
taatggtcgt tgatgggcta tcgacaggta aattatcagg cgagataatc gatgaatttg 2280
ttaaggaatt gagagcctat ggtaaatcat ttgggtggaa ctatgatata gagtttaccg 2340
gaaatgcgga gtatctaaag ttgtatttcc taaacggttg ccgtatacct aatgtttctc 2400
gacatccgat ctgtggcaaa gagcgcgctt caggggacaa gttagaaatg tggccgtcca 2460
ccattgacat cttcaatggc atatttgtga atggtgtgca tgatggtttg ccgtggcgca 2520
gatggttacg ttattgttgg gctcttgctc ttatgtattc tggaaagacc gtgcgtcatg 2580
atgattctga ggtgttgatc caatatccta tgtggtcttt tgtgtattgg ggtttgcctc 2640
ccgtgagcgc gtttgggtct gatccatgga tcttttctcc atatatgccc actggtgatc 2700
atggtttcta ttcaatgttg actttagtgc gccctctgat cactaacttg tccccatcgt 2760
cagacgcttc gggattattt ggtcaatgcg atcacaacgt tttgttcaat tctgagctag 2820
tttatcaggg ctattacatg gctcaatgcc cacggcaacc ttctcgctcg aaccgtagag 2880
atgatcccga ctctgtacag cgcttcgtca aggccttgga gtcttacctt tacatttccc 2940
ctgagctaaa atcgcgagtg cgccttggtc gtgaccgctg gcagaagttg gttgggtata 3000
cggaaaaatc tcccccgtcg cttgatgacg tggcgttcaa atggttccgt agtgcacagg 3060
aagctgatct cccaaccgct acagagattc aaagcatgga tctggccttg ctggcagcca 3120
gacgtaggac atatcagggc ttctccaagt tgttgaatac ctatttaagg gtaacctggg 3180
atttatctga tcctgttgaa cacgctgtag atccccgcgt acccttgtgt gccggtgtct 3240
ctccatcaaa tagcgagccg ttcctcaaat tgtactccgt aggtcctatg atgcaatcca 3300
cgcgtaaata ctttagcaat acgctattta tccatcggac tgtgtctggt cttgacgtcg 3360
atgtcgtcga tcgtgcgctc cttaggttgc gtgcccttaa tgcgcctgat gacgtggttg 3420
tagctcaact tttgatggta gggttgtctg aagccgaagc cgctacatta gcagcgaaaa 3480
tacggacgat ggatatcaat gccgtgcaat tggccagagt tgtcaactta tccatccctg 3540
actcatggat gaccatggat tttgatcgct tgatacgaga tatcgtgtct gtcactcctc 3600
tgaccgttcg atccctaacc accgatctac cctctggcgt accgtgggct cgcgcgatct 3660
tacagttctt aggtgcgggt gtcgccatga cggccgtcgg acccttgcgt cgtccctact 3720
tacactcagt tgccggaggc atgtcttcat tcattaagca gttccgccgg tggatgcgtg 3780
ccgaaacgag gtagcgtccg tgcccggcat ggctcgagga attactcatc 3830
<210> 5
<211> 3876
<212> DNA
<213>L3 genetic fragments
<400> 5
gcttttccac ccatggctca gattcgaggc cttcggttgt ctacgacgct ctcagctcca 60
cctccacgca agattataac atctcacact tatgatgagc tgatctccgc tctgaagtta 120
gcaaccaagc cttggcgccc tttgaagtcg cgaaataatg attctgtcac ggcagtgcag 180
ctcctttttc cccttaatgg ttatattgaa cccatgttca tgttggaaaa ggatatgacc 240
cttagtgatt ttgaggcctg gttgacgcct cttctatctg cactcgctga ccagttgctt 300
agacactacc ctatcgccgc ctatcacggg cgtttgatta atccgctgct atctaatgca 360
attgttgccg ctttcttgtc taacgtgcct tatgcgcatg cattggatca tctcttcctt 420
gttagaggaa acgtcgagga tattatggat gcggggatcg caattcagaa tcacttgtgg 480
ttcgatcgcg gtgcactagt gacccctgct ggacagaaat ttgttcagct gactggctat 540
aacttctcct ctaatgatcc gtgtctattt tctaagcaat tgcgttgtta tggcctcgtt 600
tactactttc tcgacatggc cgaatgtctg gcgtattgtt ggcgtcatct atccaattca 660
actccactga tacactttga ccgtccgtcc aatggagttc attgtttggt gccctctgaa 720
tccacgacgc ctatcgctgg ttcgttacca gtgtcagcac tcagctctat tttgttggaa 780
tcctgtcttc agcaatctac aattaatgcc cttactccca ctggttcgcc cgtcattaga 840
caaatagaag cattgttgcc tatatcatca ccgtttttcg aacgacggaa cactctggaa 900
tattctctct tcgcactgtc aaatgctctg gtaaatggtt atcagcttgt agacttgcgt 960
tccggccacc ctgattgcgc tactgttgct gccatcctag ctagattgat tgatttctct 1020
aaggatatca ccgttattca accgcgccct gctctctttg ctatcaacag cgacagtccg 1080
cttacgtata gtggagaaaa tgctaatttt atttcgcgct tgacttgtgc gtccggtaga 1140
cctattggtc cggtcgttgt cggtaaatcc gttgatcatg ccgttggttg gatgccccag 1200
tttgaccccg ccacgtccta caaccctgat ctctcgatgg actcacttgc tcgtgccacg 1260
acactgcctc tccgtgctaa gtattcgact ttctggtcag gcccagcatt gttttctttc 1320
gcttcatgta ataggcacaa tggtgtatat gacatacagt tcatggctca atttcctcct 1380
acttacttta gtgatgatga cgccttttct agatcacgat tctcttctta tcgtgcagtt 1440
agggaccggt cattgttgaa ggataccgct aatttgatgt acatctcgaa tttgtccagc 1500
tctcacgacc atcgtcttgt cccagattct aaaactatga tttatgtggg ttcctctggt 1560
actcatgtag ataatcaacc ttctatcatt aaacctctct tagctggaac tcttccgggt 1620
gtttttcgcc ccctttctat aaaacaggtt ggttgggagg tcactaatgg aacgatttgt 1680
gatgttgagc ttcctttagc cactggtacg ttcttcttcg tgtacagtga tgtggatcaa 1740
gtgcaatcag gtgattctga tttggacgcc tcctcgcgtc gcttttgctc ccaattggac 1800
atgctaatga agttgacgtt tactggtgga tcgcttgtcg ttaaatgcaa ttttccgact 1860
agtctagtgt ggcgtcacat cttctccact gtttctcctt atttctcggc tattcattta 1920
atgaagcctc tcgtgtcaaa taatttagaa ctgtatctat tgtttgcgga gcgtttgccc 1980
gttcctgacg tcgcgttccg tccttcagcg gacgttgtcg ttttctggcg atcacagcta 2040
caacgctatc gagtgttgcg tgattccttt tctaatgtgc cctccatcgg gtccactctc 2100
actttagatg aacctttgac tgtctctatt ctcaattttg ttgacgtcac ctccctttct 2160
tctcttgagg atcaacgagc cttatctgct ttttcagttt taacttctct agggtcacag 2220
aaactctcgc ttcatcctta ctttgatagc taccgcacgc agctcactgg aataattact 2280
ccacattcac gtaatcttct agatagactg gcgtacgtcc cgcgcgtttt tccttcgacg 2340
attgatgtgc aacatcgtgt catggcctct tcagatccag aaatttttgg ttttcgttct 2400
aattcatgga ctcaactgtc cttcttctac gacgcgacgt taacttctat tgattttact 2460
gatgtaaagc actggttaga tttagggacc gggcctgagg cgcgaccgtt gtcttttctg 2520
ccttccgatc ttcccgtcac gttatgtgac actcgtccat tcatcttccc ttccggctgc 2580
tgggctactt tcactgattt cttaagttat gactaccttg tcacgaatgt cgtgctctca 2640
actggtgccg atgtcgtatc ctgtgttctt tctctgggtg cggcctgtgc tgatgccaac 2700
ataactttac atgaaggcgt gcggcagctg atttcccaat gcgtggatgc caatgtcaag 2760
acattgttcc tgcagcttaa ttgtcctctc ccatctgcgg gtgatgtatc tcgggagatt 2820
cttgagatgg ttcagactaa ttcaacttac gtgtttcata ccttgggtcg tattgaaccg 2880
ttcattccat actccgctct ttcagagata gttgaggact tgtgtcccgg catcgtcatt 2940
gaaattaaga ctatggatcc ctctctctca tggcttgatt acgctgttca atccaatgcg 3000
tcagtgacgt cggatgatat cgtattggca atgcgtctgt ctcacttctg tccacttttc 3060
gtgtttcatt ttgaccgtca gtctgctcaa tttccggatg atgcgcgtgt tgggactcct 3120
tttactgtca cgctgttaga ttatgaagat actcgttcat acgaggtgac gttagataat 3180
gtcactatcg ccaccgttac cgcaggtgct ttggtgggtt tctcatctgg tgtcagcgtc 3240
agttcaacca acaatcagct tattttaact atcgattccg caagtccagg aattctctcg 3300
gtcattcaga ttcttcccgc tcgtatctct ttaggcagtt gtgtgataga agcaccggat 3360
ccatctctct ccttgatctt ccccgccacg ttagatacct ctttgtcggg aaccgatttg 3420
gagctgtatt tgtctgactg gtacgatgtt gcattatttt acgtcgatga aatccactcc 3480
cgcttgctgc cagtgtccga taccaagtat gaaatatatc gcaaggatca ggcgccgaac 3540
agccgggtga tcaactatat tttcgatcgg tctgatgtgt ttttcaagct agtgttatgt 3600
gacgtatctc cctcaggagt aggccgtttc atctaccgtg agttgccaga attaagttca 3660
cctgtttggc cagacaacgc gcgcactttc ttgtccatac cgttcgagtc acccatggtg 3720
attgtctcgc cggacggacc tgttaattac gatggcgcaa actttactcc tccaaactca 3780
tggttgacgg ttgatggcag tacctgcgtt gtagatggcc gtccttcgtt ctacgtgccc 3840
cctggccgat atggtctggt gagagtctaa acgacc 3876
<210> 6
<211> 2199
<212> DNA
<213>M1 genetic fragments
<400> 6
atggcctatc tagccacacc tgtgctagga gtcggttctc gcattaccgc tttagatcgt 60
actattgatg ctatcacgtt gaaacctcga atcgacttac aagatgtata cacaattgat 120
cccactttga ctctgcgtca gatagagtta atctcttcgg gaacttcaat ggacgatatc 180
gctcgtggac tgttgcaccg agactggcgt cgtcaatcca tcatcgtttt gcttccctcg 240
cgtcgctctc tccttgagta tctattgtct aacccttctg tctgtccaga cggtttagat 300
cgttctcgac ttaaaggatt tcagaagcgt ccaaatgatt ttcgtgttca agatttcttc 360
tctccactga tcacggactc gacgtcaatt gctacatact ctcgatggct taatgcccac 420
cctgttgtgt actcaactac tcataaggtc gctggtgctc gggtgcgtct ctttggacct 480
gccaaattat acattctgtc acctgacgtt cttcgcgaat tatccatttt gagatccacg 540
gatcgtgtcc tcgttgtacc tacagcacgt gtatatgttg gttgctttcc tagcgcttcc 600
actagtaatt gtgtgctcac tgcacgcgac cgctggaatg ctcctgacgt tcatcccgtt 660
gtcaaggcaa tccaattagc atatgaccat caatatcgtg tcaccgctcg ctatctttcg 720
gatccccttg tctccgccct tcttgttggg aatcggtcgg tgaagacctt gaaggtacag 780
ccagtagagg ccagagcagc acgatcagtc ggcattcgcg ttcaagcgat gacgccccct 840
cgtggtatca acacctctat catccaagtc gttgatctca ggctgcaatg tcgacattct 900
ctcattccca ccgaaaggcc cttcccgctg acatttatcg gcctcccatc ctgtttgctc 960
cagcatttgg atttgacgct atctgacgat tgggtgccta ttcgtgatcc cacgggcatg 1020
tttgaaatgt ggttcatgat tcttacgctc acttgtgata agattcttga tggacggggc 1080
aacgctgttt ttctcatccc cagttctact aatgcattgt cgattaatta tgtacagctt 1140
acatcgaccg cgtctcaacg ccctcagtca ttagcggcaa atgcatctgg acggatagat 1200
tctatcggac tatgtatgcc taaggggtct tttaagtcaa ctatgattaa atttctcact 1260
ggcttggaga tttgcggcac acgagtgatg tactcggacg tcgtgatgga cagtgatgac 1320
gtgggtgacg ctttggatcc tacttttgaa acggctttgt atgatgcact ggtagcactt 1380
gatccgcctt ttgacgttga taagttggct agccccactg atctagttaa tcaggagtac 1440
gttgcgtctc atatgtaccc gacattctta cggcttgtca atgagctgct gactcctaag 1500
gcttcagagt tgtactctga gcgtagcgtt gaattccgat ctcttactta cgcgcacgct 1560
gattctgaat ttcttaactc atgctggacc gctcgcttaa tgcgttgctt tatcaactat 1620
catgaagagc agaatatctt acttcgtcct ggacgcgttg gtggggtgtt atttcaagtc 1680
gcgttgagcc gttgctataa gatgttcgct acttccactc ctgcttcccc tctgtcattg 1740
ttcctcaagt cgttgttcgt tccttggatt gagtctgccc cactgttagc gaatctaacg 1800
ccaaatgagt cttctcgtgt gttagcatgg tatattcctt cctcgtactg gagcgacaat 1860
ggttggtgcg tttgtgacac tcatcgtcac gtcaccttct ccttcatccg cggtcttccc 1920
gccgacctgt cggtgttaga tctgtttgat tggtctcgat tccgcgcgac tataaacgtg 1980
gacacgtctc tcgtggagct aggcgcagac attcgtgcgg tcaaagtatc agtccattgg 2040
acatctcaga agcccactgt ggacgttttt gacaatcgtg cgcttttcac ccccttccag 2100
cactaccatt tgagtctcca ctgtaattgc gcacctggtc gacctttctt cgcgaagaac 2160
atgaagctat atttgtcgac ggtaggaggc gagcactga 2199
<210> 7
<211> 2208
<212> DNA
<213>M2 genetic fragments
<400> 7
accaccgaga tctacactct ttccctacac gacgctcttc cgatctcttt ctcacaagat 60
gggtaacgcg acgtctgttg tgcagaactt caatatccaa ggtgatggta atcattttgc 120
tccatctgct gagactactt catccgccgt accgtcatta tctttgaatc ccggactgtt 180
aaatccaggt ggtaaggcgt gggtcctgat tgatccatct ctaaatgctt ccgatccttc 240
atcactacgt ctgatgactt cggctgatct atcaacactt cctcgatctg ctactagtaa 300
ctctaccggg tttctcccca cttctggcat gtatgccatt gctactaagg agacgttgag 360
tgtaattact gagcacgcga tttcccagtt tgataagtta cagatggctt gtgagttgga 420
ccgcgattat ctggatgcta gaggtgtttc tcctgagtct gtgaatattc atagttatat 480
agcctacgtt gattgcttcg tgggtgtatc tgcaaggcag gctgcgtcaa attttaagcg 540
gcatgtgcca gttatcacca aatctcgtat gacacaattt atgacatccg cgcagaatat 600
gttgcaagtg cttgggccct gggaacgtga tgttcgtgag ttactcacta ttcttcctac 660
ttccactacc gctggtaaaa ttacgtgcga catgaagtct gttgtcgctt tcattgatga 720
tcagctctct gataccagtt tgtgtcgtct gtaccccgac tgtgctgctg cggcggtggc 780
tagacgtaat ggtggcattc gatggaagac acctgatact gacgaggctc cttcacttgc 840
aactaacgat attgctgctt caactatggg tacgcttgcg aatactacac cactggctga 900
gaagtcgaac tcgggcgagg agtcgatgcg cttggttagt gatgttggcg tggacatcgt 960
ttgttctcgt ggccccatca gttcttcagt ttggtcccgc acggttgaac ccaaatcgta 1020
caatattaga acccttcgtg tagaagaagc gctttggcta cgcgagtgcc aagcgactac 1080
tggttttgat gtacagtaca cgctgcccga ccagactaca cataagcatt tctggcttca 1140
gaaggggtca gtcgtcataa atcttgagca aacgggtagt atgatgttcg atgtgaacat 1200
agcgggtaaa gattacaaga agggcacctt taatcctgat aatcataaat tggtcctctt 1260
ggttatgcag tcaaagatcc ctttcgagtc ttggaccgtc gcttctcaaa ttactggtat 1320
cgctcaagtg gctgaggtca ctgtgcatgc tgctgatagt tcgactccta accaaaagat 1380
aatcggtgaa acttcgctgt cttatttatt tgagagggag acggtgacca catccaatac 1440
tgaagtcaat acatatctgt tgtgcacttg gcagcttgac gacgcgcaga gcaatgacgc 1500
aaacgcctgg ccagatgctt gggacgggat cacaacattg accccactta cgtccggtac 1560
tgtaaccatc aaggggactt cggtggattc tgtcgtaccg tctgatttag ttggtgctta 1620
tacacctgag gctttggctg ccgcgcttcc taacgacgct gggttaattt tggctaataa 1680
ggcaactaaa ttggctgacg ccatcaataa ggaggatgat tctgtgattg atgagtcttc 1740
tccctttagc acccccattc aaggagttct ggctgttcaa caacttgata ccgtggggac 1800
acgcggtaca cgtgcactcc agcctccatc cattctgaaa cgcatcgcct cacgagctct 1860
tcacatgttt cttggtgatc caaagtctat tctaaaacag gcgacgcccg tattgaggga 1920
ccctgacgtt tggaccggct ttgttcaagg tgttagagac ggcatccgga ctaagtcgct 1980
atccgctgga gtacggtccg tgtataataa cgttaccgcc acacagtctg tacaaacgtg 2040
gaaacagggg ttcctgacga aaatacagac gttgttcaag ccatcgtgag gtgctaaggc 2100
ctctctctgc ggcgggtcgg tgggcacgtc gtagtgacgc tgaatgcacg gggaggtgac 2160
gctccctgga ttggcaagat cggaagagcg tcgtgtaggg aaagagtg 2208
<210> 8
<211> 1996
<212> DNA
<213>M3 genetic fragments
<400> 8
gctttttgag tcctagcgtg gatcatggcg tcaaccaagt ggggagacaa gccgatgtcg 60
ctctcaatgt ctcacgatgg atcatctatc cgcagtgccg cctcacagtt tctgtcggtt 120
cccctgtctc actcaacgcc aatcccacct caacggaaga ccgtattgct gaaattcatg 180
atcggtgatg acctggttac cgtgcagggc gcgctcgctc cttttgatga atactggtac 240
gataaccaac ctctattgtc tcaagctgtt gagctgctcg cctctgagga tcgtctgcgt 300
caattcgagc attatgagaa gtttctactt aagaagggcc accaaattgc tgagatcatg 360
aataggctac gtcttttctt caccgacgtt ctcaaagtga agatggaagc tgatgctctt 420
ccttctctag ctcaatacct gatggctggt actttggatg ctgtctccac cgttcacgaa 480
cctgatgctt gtgttccagt cacttcaaaa atcatagcta agcagcagac tgtgtccaag 540
tcccctggac gtcttgctga agaggagtat aatgttatta gatcacgttt cctcactcat 600
gagatctttg acttaacgtc tgacttgccc ggtgttcaac cattcatgga tatgtactat 660
gccaccgttc ctcgtgccga ttccaccggt tggtgtgtct accgcagaaa aggtctattg 720
attcatgccc ctgatgagca atactcggat ctgactattt tcaccacccg tcttacggca 780
gcgcgtgagt tacagcttgt ggctggggag gtcgttgtgg cttgcttcga tcttatggat 840
atctctgata ttgctccatc tcatcatgca tcggttcaag aggaacgtac tctcggcact 900
agcaagtatt ccaacgttac agctaatgag catccgttgg tattcttttc acccaatgca 960
ttacgctggg caatagatca tgcctgtact gattccttga tttccactag gaatattcgg 1020
gtctgcgtcg gtattgaccc cttagtgacc agatggactc gcgatggcgt gcaggaggct 1080
gcaattctta tggatgacaa gctaccctca gcaggacgtg ctcggatggc tctacgaacc 1140
ttgcttctag cgcgtcgctc accaatgacg tccttcttac taggtgctct caagcagtcc 1200
ggtggtcagc taatggaaca ttatcgatgt gatgcggcta ataggtatgg atctcccacc 1260
attccagttt ctcaccctcc accgtgtcca aaatgtcctg agctgaaaga acagatcacc 1320
aaactttcgt cagctcctgc gcctaaagtt gactcgtccg ctggtcctgc cgtgctgttg 1380
tcgaagatcg ctgagctcca acgtgctaac cgagaactgt ccttgaagtt agtggacgtt 1440
cagccagccc gagaagacca ccttctagct tacctcaacg agcacgtatg tgttaacgct 1500
aaagatcacg agaaaggtct actagcccgt tgtaatgtct cgagtgattc aatcgccgct 1560
atccttggtc aacgtttgaa aaatcgagaa cggtttgaaa cgagactacg gcacgaggct 1620
ggtgcggagt gggagccacg agtggaagcg ttgaatcaag agttggctaa ggcgcgtatt 1680
gagcaacaag atatgatgac tcagtcctta cagtacctga atgaacgtga tgaactgctg 1740
cgcgaggtgg atgagctcaa acgcgaactg gctaccctac ggtttgctaa tgtgagacta 1800
aatgccgata accaccgcat gagtcgtgcg acccgtgttg gagatgtatt cgtcagtgat 1860
gttgatccct taccacccgg tcttcctggt gaatcgaaac catccattga agaactggta 1920
gatgatctgt gagctttgcc ttgtgactcg acttctctct gattccatgt acccacggcg 1980
gactcggcta ttcatc 1996
<210> 9
<211> 1644
<212> DNA
<213>S1 genetic fragments
<400> 9
gctttttcag tctcttgtat cgatgttccg tatgtcttcc ggttcatgca acggcgcgac 60
gtcaatattt ggtaacgtgc attgtcaagc ggcccaaaat accgcaggcg gagatcttca 120
agctacctcg tcattaattg cttattggcc ttatctcgct gctggtggtg gtctcattat 180
aattttaatt attgttatag gtataatctg ttgttgtaag gccaaagtta aggctgacgc 240
taccagaagt gtgttctatc gggagttgct tgctctgaac tcgggcaagt gtaatgcagc 300
acctccgtca tacgacgttt gatgtgcggc ggtttgagtt ttctccgacg gtgtttgaag 360
agtgtttgac tccatctttt accgctgtga ctgacactga ccctgtgagg tactttaata 420
ttgagcttcc gtcaactcac cgtctcctcc cttggcttcc cgttcttctg ttccaatcct 480
gtaaagtgca tgtttcttta gtacgtagat tctctttatg ctcgacttta tctgatattt 540
gtgagtacga ttgcaaattg cttccgtcta ttaacgctat tgtgtcgaat ccagtgtcga 600
gcgcggtttc atctatcgtc gttcactggg atggacggat taactcaaca gcagcgaaga 660
gaagtcgtgg ggttgatact gtcgttgact tcgagcgtga gtataagttc tggcgatttg 720
acgcaaattc gtgagcgtct ttccgctttg gaatctgcga ctgcgtcgct gaacgaatcc 780
gttaatacag ctttgtctag gttagtggat ttgtctgcat cgcttgataa cgtggcggcc 840
tcgttagcgg agacgaaagt ggaaatgaac tcactagttt ctgacgttca gggtttgcga 900
gcttcccttg actcttctgc ttcagagctg gcttctctat cttcattggt gcgtgatcac 960
ggctcttcga ttgctggcct acagagagaa gtaagtgcct tatcgagtga ggtaggcaac 1020
cttaaaacct cggtatcatc gcagggcctt cctatcacta gccttgagaa acgagtgcaa 1080
gctttagaag gtggttctag tacgactctg tcatttgctg atcctcttaa gttagaggct 1140
gggaccgtgt cactcgaggt agatccgtat ttctgctctg tgaatcgtaa tctgacgtcg 1200
tattctgctg atgctcagtt gatgcaattt cagtggtctg tgaaagggga agatggcgcg 1260
gccaactcta ttgatatgga cgtgaacgct cactctcatg gttcacgcac tgattatctg 1320
atgtcaacca agcaatcatt gactgttaca acgtctcccg ctactcttgt ctttgaactg 1380
gataggatta ttgctcttcc ctccgacctt tctcgcctaa ttccatgtta tggttttcag 1440
caagccactt ttcccgttga tatctccttc cagcgagatg gcgtttcgca tacgtatcaa 1500
gtctatggga agtacacatc ttctcgcgtc ttcaagacta cgttctcgcc tggctcctca 1560
ggtcccgcag tgattaagtt tttgaccgtg cgtacgggca tcgataccta aggtgtggcg 1620
ccgtacgggg gctggttatt catc 1644
<210> 10
<211> 1322
<212> DNA
<213>S2 genetic fragments
<400> 10
gggcgcgtgc cgtgtacgac ttcttttcta cgcctttcgg gaatcgtggt ctagcgacga 60
atcgtactca actatcatca ctactatcaa gctcgaattc cccatggcaa cgttttctat 120
catcaatgac tccattgaca gcgccgggca tcgtttcgac acctgaagca ccctatccag 180
gttcgttaat gtatcaagag tctatgctcc acagtgctac cgtccctgga gtacttggca 240
atcgcgacgc ttggcgtacg ttcaatgtct tcggactttc atggactgac gaaggactgt 300
caggactagt ggctacccaa gatcctcctc ccgccgcccc gtatcagcca gcctctgctc 360
agtggtcgga tcttctcaac taccccagat gggcaaacag acgtcgtgag ctgcaatcta 420
agtacccgct tctgcttcgc tccactctgc tctctgccat gcgagctggt cctgttctat 480
atgttgagac gtggccgaat atgatttctg gacgattagc tgattggttt atgtcccaat 540
atggcaataa tttcgttgac atgtgtgcta ggttgaccca gtcttgttcg aacatgcctg 600
ttgaacctga tgggaattat gatcaacaga tgcgtgcttt aattagtttg tggcttctgt 660
catacattgg ggtaatcaac caaaccaaca ccatcagcgg tttctacttc tcctcaaaga 720
ctcggggtca agcgttggac agttggactt tgttctatac cacgaatact aatcgtgtcc 780
aaattacgca gagacatttt gcttatgtgt gcgcccgatc tcctgattgg aacgtggaca 840
aatcatggat cgctgctgcg aacttaaccg ccattgttat ggcttgccgt caaccgccga 900
tgtttgctaa tcaaggcgtc attaatcagg cgcagaaccg acccggattc tccatgaatg 960
gggggacgcc cgtccacgag ctcaacttac ttactactgc gcaagagtgc atcaggcagt 1020
gggtggtagc aggcttggtg tcggcagcaa aggggcaagc actaacgcag gaagctaatg 1080
acttctcaaa cctcatccag gcggatctag gccagatcaa ggcgcaggac gacgctttgt 1140
acaatcagca gccgggatac gcgaggagaa taaaaccttt cgttaatggt gactggacac 1200
caggtatgac cgctcaagct ctggccgttc tagccacttt taccgcctag gcgtagggtc 1260
gtacgctgcc cgagtccagc cctccggcag cccgtggaga tcggacgagc gtcgtgtagg 1320
ga 1322
<210> 11
<211> 1202
<212> DNA
<213>S3 genetic fragments
<400> 11
gctttttgag tccttagcgt gcaagccgca atggaggtac gtgtgccaaa ctttcactcg 60
ttcgttgaag gaataacatc tagctacttg aagactcctg cttgctggaa tgcacaaaca 120
gcttgggata ctgtgacctt tcacgtccct gatgtaatta gagttggtaa cgcctactgt 180
tgttctcaat gttgtggtgt actctactac gggactctgc cctcggacgg taattatttc 240
cctcatcaca agtgtcatca gcaacagttt aggactgata ctccactgct tcgatacgtg 300
cgcattggta gaaccactga gcatctgatg gatcaatatg ctgtcgctct ggagtccatt 360
gctgaacact atgacgagat tagtcaacgt atggtcgatg agccagagaa tgacgaggtt 420
acacctcttg atatcgttac gcgtaccgaa tctatcagga gtgacaaggc agtcgaccca 480
gacttttgga catacccact tgagcggcgt tctgatgatt ctcgtagaga catcgcctca 540
gcatgctgga aaatgattga cgcgtcggcg cgtagtctca ctcttccaaa ttgcctcgtc 600
tccccctctt tgcactctcg ctccgtcttt ggtcagatgc aaacgaccac cactatatac 660
gatgttgcgg catcgggaaa ggccgttaag ttttcaccaa tggttgctac actagcgcaa 720
cgtgatgctg gccctgtaat gcttgcgaat gctgacccgg cggaaggcgt gtactctttc 780
tggacgtcgc acttcgcttt ctcaccgctc atcggcggag ttggaattac gggacagtac 840
gctcgtgagt cgtaccatca agtgggtcat ccagtgattg ggagtggtaa gaaggcatcg 900
cattacagga atctgttcat ggaagcgtgg cgcgggtggt cgaagtcagc tttcgcatgt 960
gctactggaa tggagccagc tgaatgtgaa tctcgtctga gaggacacgc tcgtactatg 1020
ctcggacgct ctctgccgcc cgtttgtgac gatgatgttg ctcagcagtc tggcgcggta 1080
ctgacttcgc tgcagaaaac gaacaagttc accgttgtgg agtgtggttg gtaagtacct 1140
ccgggtcaaa atgcacatag gctcccacct atgtgacggt tagcgggact cgcctattca 1200
tc 1202
<210> 12
<211> 1192
<212> DNA
<213>S4 genetic fragments
<400> 12
gctttttgag tccttgcgca gccatggaca acaccgtgcg tgttggagtt tcccgcaaca 60
catccggcgc agctggtcag actgttttta gaaactttta cttactacga tgcaatatct 120
cagctgacgg tcgtaatgca acaaaagctg tgcaatccca ttttccattc ctttctcgtg 180
ctgtccgttg cctatctcct ctagccgctc attgtgctga taggactctt cgtcgtgaca 240
atgtgaaaca aattcttact cgtgagctgc catttccatc ggatttaatc aattacgcac 300
atcatgtgaa ctcatcctcc cttactactt ctcagggtgt cgaggcggca cgtctagtgg 360
cccaagtcta tggagaacag ctatcgtttg atcacattta tcccactggt tccgcaactt 420
actgccctgg agcgattgct aatgcgattt cccgtatcat ggctggtttt gtgccccacg 480
aaggtgacaa ctttaccccg gacggttcta ttgactatct cgccgccgac ctggtcgcgt 540
ataagttcgt gctcccttac atgctagata ttgtggacgg acgtccgcag attgttcttc 600
catcacacac tgttgaggag atgctgtcca acacgagttt gcttaattcg attgacgctt 660
catttggtat tgaatcgaag agcgatcaac gcatgacccg tgacgcggct gaaatgagtt 720
ctcgctcact taatgagctt gaggatcatg agcagagggg tcgaatgcct tggaaaatca 780
tgacggcaat gttcgcggcg caattgaagg tggagttgga cgccctagct gatgagcgcg 840
ttgaatctca ggctaacgct catgtgacat cttttgggtc tcgtctgttc aaccaaatgt 900
ctgcttttgt cccaattgat cgtgagttga tggagctggc tctactcatc aaagagcagg 960
gtttcgcaat gaatccaggg caagtcgcat ctaaatggtc gctgatacga cgatctggcc 1020
ccactcgccc gctatcaggc gcacgccttg agatcaggaa tggcaactgg acaattcgtg 1080
aaggtgacca gacgcttctg tctgtctccc cagctaggat ggcgtaaacg ggacccatgg 1140
tgcgggtgag gggccgccac accctctgcc gcgacctgga ctcttattca tc 1192
Claims (10)
1. a kind of Yolk antibody of prevention novel duck reovirus, which is characterized in that the Yolk antibody contains anti-duck and exhales intestines
Lonely virus strain antibody；The deposit number of the duck reovirus strain is CCTCC NO：V201818.
2. the preparation method of Yolk antibody described in claim 1, which is characterized in that include the following steps：
(1) it is CCTCC NO with deposit number：Inactivation epidemic disease is made as production of vaccine strain in the duck reovirus of V201818
Seedling；
(2) with the inactivated vaccine injecting immune laying hen prepared, high-immunity egg is prepared；
(3) yolk is collected by high-immunity egg, through once inactivating, acidizing extraction, secondary inactivation, coarse filtration, aseptic filtration, concentration and three times
The Yolk antibody of prevention novel duck reovirus is prepared into after inactivation.
3. preparation method according to claim 2, which is characterized in that in step (1), the inactivated vaccine is by the following method
It is prepared：
It is CCTCC NO by deposit number：The duck reovirus of V201818 is inoculated with SPF chicken embryos, collects dead chicken in 24-120h
Embryo allantoic liquid obtains virus liquid；Virus liquid is concentrated into viral level >=105.0ELD50, the virus liquid after concentration goes out through formaldehyde
It is living, Tween-80 mixing is added and is used as water phase, is mixed using white oil, aluminum stearate and Span-80 as oil phase, by water phase and oil phase
By volume 1:Inactivated vaccine is made in 1 mixing, emulsification.
4. preparation method according to claim 2, which is characterized in that in step (2), divided 4 times with the inactivated vaccine of preparation
Laying hen is immunized, each immunization interval 2 weeks.
5. preparation method according to claim 2, which is characterized in that in step (3), the primary inactivation is specially：It will
Yolk liquid and water by volume 1:(0.5~1.5) it mixes, yolk diluent is obtained after stirring evenly, under the conditions of 60~65 DEG C
Keep the temperature 25~35min.
6. preparation method according to claim 2, which is characterized in that in step (3), the acidizing extraction is specially：To
The acetate buffer of 2.5~3.5 times of the yolk diluent volume is added in yolk diluent, filter is collected by filtration after stirring
Liquid；Preferably, the pH value of the acetate buffer is 4.8-5.2；More preferably 5.0.
7. preparation method according to claim 2, which is characterized in that in step (3), the secondary inactivation is specially：To
Octanoic acid is added in filtrate after acidizing extraction to final concentration 3.5~4.5%, stirs 30-120min, in 2~8 DEG C of placements after stirring
4~8 hours；
Preferably, in step (3), the aseptic filtration is specially：With 0.22 μm of membrane filtration degerming.
8. preparation method according to claim 2, which is characterized in that in step (3), the concentration is specially：Utilize 30-
The PES hollow fiber ultrafiltration membranes of 50KD are concentrated under the conditions of 2-8 DEG C, until antibody titer is not less than 1:512.
9. Yolk antibody described in claim 1 is preparing the system for preventing and/or treating novel duck reovirus infection
Application in product；The deposit number of the novel duck reovirus strain is CCTCC NO：V201818.
10. deposit number is CCTCC NO：The duck reovirus of V201818 is preparing treatment caused by duck reovirus
Application in the Yolk antibody of gambrel swelling.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110590946A (en) * | 2019-08-29 | 2019-12-20 | 广东渔跃生物技术有限公司 | Preparation method of duck reovirus egg yolk antibody |
| CN111440815A (en) * | 2020-03-12 | 2020-07-24 | 潍坊华英生物科技有限公司 | Novel duck reovirus composite vaccine and preparation method of yolk antibody |
| CN113493507A (en) * | 2020-12-31 | 2021-10-12 | 哈药集团生物疫苗有限公司 | Novel duck reovirus egg yolk antibody and preparation method and application thereof |
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| DE29904221U1 (en) * | 1999-02-26 | 1999-06-10 | Kobilke Hartmut Dr Med Vet | Specific IgY egg yolk antibodies to be obtained permanently |
| CN105153303A (en) * | 2015-09-21 | 2015-12-16 | 山东农业大学 | Egg yolk antibody for preventing and controlling duck parvovirus and preparation method thereof |
| CN105367654A (en) * | 2015-12-08 | 2016-03-02 | 天津瑞普生物技术股份有限公司 | Preparation method of I-type duck hepatitis refined yolk antibodies |
| CN106540250A (en) * | 2016-11-30 | 2017-03-29 | 山东农业大学 | The preparation method of goose source reovirus inactivated vaccine |
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Patent Citations (4)
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|---|---|---|---|---|
| DE29904221U1 (en) * | 1999-02-26 | 1999-06-10 | Kobilke Hartmut Dr Med Vet | Specific IgY egg yolk antibodies to be obtained permanently |
| CN105153303A (en) * | 2015-09-21 | 2015-12-16 | 山东农业大学 | Egg yolk antibody for preventing and controlling duck parvovirus and preparation method thereof |
| CN105367654A (en) * | 2015-12-08 | 2016-03-02 | 天津瑞普生物技术股份有限公司 | Preparation method of I-type duck hepatitis refined yolk antibodies |
| CN106540250A (en) * | 2016-11-30 | 2017-03-29 | 山东农业大学 | The preparation method of goose source reovirus inactivated vaccine |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110590946A (en) * | 2019-08-29 | 2019-12-20 | 广东渔跃生物技术有限公司 | Preparation method of duck reovirus egg yolk antibody |
| CN111440815A (en) * | 2020-03-12 | 2020-07-24 | 潍坊华英生物科技有限公司 | Novel duck reovirus composite vaccine and preparation method of yolk antibody |
| CN111440815B (en) * | 2020-03-12 | 2022-12-16 | 潍坊华英生物科技有限公司 | Novel duck reovirus composite vaccine and yolk antibody preparation method |
| CN113493507A (en) * | 2020-12-31 | 2021-10-12 | 哈药集团生物疫苗有限公司 | Novel duck reovirus egg yolk antibody and preparation method and application thereof |
| CN113493507B (en) * | 2020-12-31 | 2023-04-21 | 哈药集团生物疫苗有限公司 | Novel duck reovirus yolk antibody and preparation method and application thereof |
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|---|---|
| CN108676092B (en) | 2020-10-23 |
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