CN108330108A - CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus - Google Patents
CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus Download PDFInfo
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Abstract
The present invention provides a kind of 3 12 2014 plants of type inactivated vaccine CH GD of Ana 1 aviadenovirus, and China typical culture collection center is preserved in November 27 in 2017, and deposit number is CCTCC NO：V201764, preservation address are：China, Wuhan, Wuhan University；Complete genome sequence submits Genebank, Accession No：KR135164；The present invention also provides a kind of polypeptide, amino acid sequence such as SEQ ID NO：Shown in 2；The nucleotide sequence such as SEQ ID NO of coding polypeptide are provided：Shown in 1.The inactivation strain is only safe to various ducks, and without side reaction, the inactivation strain is made that vaccine safety is effective, and can protect same source strength poison attacks poison, have it is practical be widely applied value.
Description
Technical field
The present invention relates to a kind of inactivated vaccine, CH-GD-12-2014 plants of 3 type inactivated vaccine of specifically a kind of Ana 1 aviadenovirus,
Belong to biotechnology.
Background technology
Avian adenovirus infection mainly causes chicken egg-decreasing syndrome, chicken inclusion body hepatitis, broiler and the respiratory tract of turkey
Syndrome, bronchitis, poult hemorrhagic enteritis, turkey virus hepatitis, pheasant marble lienopathla etc..Aviadenovirus
Infection increases year by year, the incidence getting worse of especially nearly 2 years aviadenovirus characterized by inclusion body hepatitis.Fowl gland
Virus is divided into I, II, III group according to different group specific antigens.I group I fowl adenovirus includes traditional aviadenovirus and other birds
Isolate has common group antigen, totally 12 serotype, common are chicken embryo lethal orphan virus and inclusion body hepatitis disease
Poison etc..This group of viruses generally existing in goose, chicken, duck body makes chicken group be negative infection, and as secondary cause of disease collective effect man
Fowl, and energy vertical transmission, pollute chicken embryo.II group I fowl adenovirus includes hemorrhagic enteritis of turkeys virus and marble lienopathla virus
Deng these viral group antigens and I group I fowl adenovirus difference.III group I fowl adenovirus, i.e., a viroid related with egg-decreasing syndrome,
It can detach and obtain out of goose, chicken, duck body.Aviadenovirus is the common infectious agent of poultry, can directly or indirectly be caused a disease,
Most of aviadenovirus replicate in poultry body but do not generate apparent clinical symptom, work as undesirable element, especially other diseases
Immunity of poultry is set to decline, adenovirus then plays the effect of its chance pathogen quickly.Aviadenovirus can also inductive infection fowl
Class generates high-caliber neutrality circulating antibody：I.e. in the case where having infected virus, host is still again by virus
Subinfection again.Adenovirus is largely regulated and controled by host immune response in cell endoreduplication, duplication.
Currently, document report Ana 1 aviadenovirus has 2 type of Duck adenovirus type 1 and Ana 1 aviadenovirus.Ana 1 aviadenovirus l types (Duck
Adenovirus 1, DAdV1) virus is and a kind of relevant distrand DNA aviadenovirus of no cyst membrane of egg drop syndrome to belong to
In gland thymus gland Tobamovirus (Atdaneoviurs), there are the common antigens of very little with I group I fowl adenovirus.Duck laying rate fall ill substantially
Degree declines.Minority morbidity duck feed intake is reduced, and slightly depressed, output deformity egg, soft-shelled egg, the death rate are 1%~5% to spirit.
Early stage increases feed mineral, protein content and trace element, the state of an illness and does not improve.French the first explosion duck in 1977
Adenovirus 2 type disease, infects sick duck weight loss, is not steady on one's legs, continue 10d since 35 ages in days or so, there is 1-1.5%'s daily
The death rate.Nineteen eighty-two Northern Ireland scientist Bouquet etc. is compared by aviadenovirus group antigen, is confirmed that the virus is a kind of new
The aviadenovirus of type.2011, the scientists such as Harrach demonstrated again that the virus is a new virus and proposal is named as
DAdV-2, but due to lacking sequence, it is proposed that do not adopted by ICTV.Until 2014, Austrian scientist Ana Marek passed through height
Flux sequencing technologies just obtain the virus complete sequence, and analyze the sequence.The same year, In Guangdong Province kind duck occur similar
The case where the dying of dying of illness, dissect find that the symptoms such as major Liver enlargement, mottling bleeding, white spotty necrosis, duck occurs in sick duck
The death rate be more than 20%.Find that the virus is another novel Ana 1 aviadenovirus by identifying, the strain is in many places all tables
Reveal symptom similar with fowl inclusion body hepatitis and hydropericardium syndrome, fowl inclusion body hepatitis is by I group I fowl adenovirus (fowl
Adenovirus I, FAdV-I) certain some serotype caused by, to encroach on based on liver and be formed in core in liver cell
Infectious disease with the characteristics of occlusion body.Chicken infected, initial stage can not see apparent symptom, have individual chickens meeting dies by visitation of God, but more
For the good chicken of body condition.A small number of chickens show as losing the appetite after 2~3d of infection, spiritual depressed, the symptoms such as drowsiness, chicken face also
Face is pale, and skin is in yellow, and cockscomb fades, and visible subcutaneous hemorrhage.The characteristic pathology of the disease changes in liver, clinical condition
Shape shows as liver enlargement, and there are different degrees of blutpunkte and blood spots in surface, and it is in filbert to yellow that liver, which fades, and matter is crisp.
Liver also can see the necrosis region to differ in size, and necrosis region and bleeding friendship sometimes mixes, and liver surface shows recessed
The feeling of convex injustice.Liver hyperemia, bleeding, hepatic cell fattydegeneration, and occur together focal necrosis and courage are shown as in histology
Juice deposits, and basophilla or oxyphilous occlusion body is can be also found that in liver cell nuclear, edge is larger and clear, rounded or shape
Irregularly.In recent years, Ana 1 aviadenovirus prevalence was in outburst trend, and special novel variant strain occurs, and seriously endangers the hair of aviculture
Exhibition, in order to reduce production and national economy loss, the vaccine of development of new variation strain is extremely urgent, and the country does not have also at present
There are report and the production of 3 type vaccine of Ana 1 aviadenovirus.
The present invention is to be separated to one plant of doubtful new virus out of Guangdong duck morbidity kind duck body, and success is in duck embryos at fibre
It ties up after being bred on cell, it is special to the form and physics and chemistry of virion by transmission electron microscope, blood coagulation tests and physical and chemical experiment
Sign is studied, and is as a result proved that the virus belongs to New-type adenovirus, and be named as CH-GD-12-2014, is then passed through high throughput
Sequencing, obtains the whole genome sequence of the virus, complete genome sequencing shows CH-GD-12-2014 plants and Austria in 2014
GR plants of 2 type of Ana 1 aviadenovirus that land productivity scientist Ana Marek are delivered has highest homology, is 92%；Full-length genome is evolved
Tree also indicate that CH-GD-12-2014 plants, GR plants of 2 type of Ana 1 aviadenovirus and goose Adenovirus Type 4 (GAdV-4) belong to I group I fowl adenovirus
A branch on category, further analysis shows that CH-GD-12-2014 plant with GR plants and be not belonging to same type of Ana 1 aviadenovirus,
Fiber genes there are two CH-GD-12-2014 plants, and fiber genes there are one GR plants, CH-GD-12-2014 plants of hexon
Amino acid sequence and GR plants only 60% of similitude, the results showed that CH-GD-12-2014 plants are another novel duck adenopathies
Poison, is named as 3 type of Ana 1 aviadenovirus (DAdV-3), and CH-GD-12-2014 has apparent pathogenic, and infection rate 100% is felt
The liver, heart and kidney and other organs lesion for contaminating animal are apparent, and wherein liver is the most serious, show as yellow, enlargement hyperemia, matter
Ground becomes fragile, and surface has the symptoms such as different degrees of blutpunkte, blood spots and the necrosis region that differs in size, animal immune protection real
Test the result shows that, inactivated vaccine can stimulate animal body to generate a certain amount of antibody, be played a certain protective role to animal body.
Invention content
The purpose of the present invention is being directed to currently a popular strain to lack related attenuated vaccine or inactivated vaccine, a kind of duck gland is provided
Viral CH-GD-12-2014 plants of 3 type inactivated vaccine, complete genome sequence submit Genebank, Accession No：KR135164,
The generation of 3 type epidemic disease of Ana 1 aviadenovirus can not only be effectively controlled, and there is prodigious economic benefit and good social benefit.
Second object of the present invention is to provide the application of CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus；
Third object of the present invention is to provide the drugs containing CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus.
Purpose of the present invention is to what is be achieved by the following technical programs：
CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus was preserved in Chinese Typical Representative culture on November 27th, 2017
Object collection, deposit number are CCTCC NO：V201764, Classification And Nomenclature Duck Adenovirus Type III, DAdv
III CH-GD-12-2014, preservation address are：China, Wuhan, Wuhan University；Complete genome sequence submits Genebank,
Accession No：KR135164.
The present invention also provides a kind of polypeptide, amino acid sequence such as SEQ ID NO：Shown in 2.
The present invention also provides the nucleotide sequence of coding said polypeptide, the sequence such as SEQ ID NO：Shown in 1.
Through being sequenced and analyzing, the hexon gene codes of CH-GD-12-2014 plants of 3 type inactivated vaccine of the Ana 1 aviadenovirus
Polypeptide sequence such as SEQ ID NO：Shown in 2；The hexon genes of CH-GD-12-2014 plants of 3 type inactivated vaccine of the Ana 1 aviadenovirus
Nucleotides sequence is classified as SEQ ID NO：Shown in 1, or in SEQ ID NO：In 1 sequence basis through replacement, lack and/or add
Add one or more nucleotide and coding SEQ ID NO：The gene order of the 2 same functions of albumen.
Treatment Ana 1 aviadenovirus is being prepared the present invention also provides 3 type inactivated vaccine of the Ana 1 aviadenovirus for CH-GD-12-2014 plants
Drug in application.
The present invention also provides the drugs containing CH-GD-12-2014 plants of 3 type inactivated vaccine of the Ana 1 aviadenovirus, specifically can be with
It is vaccine, the vaccine includes CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus and pharmaceutically acceptable adjuvant.
Compared with prior art, the invention has the advantages that：
The present invention provides a kind of CH-GD-12-2014 plants of 3 type inactivated vaccines of Ana 1 aviadenovirus, in 27 preservation November in 2017
In China typical culture collection center, deposit number is CCTCC NO：V201764, preservation address are：China, Wuhan, Wuhan
University；The inactivation strain is only safe to various ducks, and without side reaction, it is effective that the inactivation strain is made vaccine safety, can protect
It protects and attacks poison with source strength poison, have practical and be widely applied value.
Description of the drawings
Attached drawing：For CH-GD-12-2014 plants of 3 type of Ana 1 aviadenovirus of the present invention chadogram, CH-GD-12- are built with Reference strains
2014 plants, GR plants of 2 type of Ana 1 aviadenovirus and goose Adenovirus Type 4 (GAdV-4) belong to the branch that I group I fowl adenovirus belongs to.
Specific implementation mode
Further specific descriptions, design philosophy of the invention or same are done to the present invention below by specific embodiment and attached drawing
The simple replacement of substance belongs to the scope of protection of the present invention, and is this if experimental method is without specified otherwise used in following
The existing conventional method of technical field, used dispensing or material are unless otherwise specified available by commercial sources
Dispensing or material.
Embodiment one：The CH-GD-12-2014 plants of separation identifications of 3 type of Ana 1 aviadenovirus
For CH-GD-12-2014 in 2014 from the collected sick duck liver separation identification of duck, detailed process is as follows：
A. viral to be separately cultured
A.1 the culture of duck embryo fibroblasts
The nonimmune fertilization duck embryos embryos of 11 Elderly of aseptic collection, remove head, wing, pawl and the internal organ of duck embryos, PBS solution is washed
It 3 times, is cut to be less than 3mm with sterilizing scissors3Tissue block after, add PBS solution jog, then add 0.25% trypsase, in 37
Water-bath digests in DEG C water bath, until serum, which can be uniformly added, in liquid change terminates digestion, this process generally requires 20-
30min；By suspension with 100 mesh cell sieves filter after, collect filtered fluid in 5mL centrifuge tubes, 8000 leave heart 5min, remove on
DMEM is added after clear, and is diluted to 5 × 105A/mL (serum for containing 10%), is seeded to 25ml Kolle flasks, 37 DEG C of merging, 50%
CO2, saturated humidity CO2Culture, as DEF cells, are digested when cell compactness reaches 80%~90% in incubator
It passes on, correlation test can be carried out after the second generation；
A.2CH-GD-12-2014 it is separately cultured
Aseptic collection duck liver of dying of illness is dirty, and by volume 1：Sterile phosphate buffer solution, grinding, freeze thawing 3 is added in 5 ratio
Secondary, 12000rpm centrifuges 10min, and supernatant is applied to list through 0.22 μm of filter filtration sterilization, by the 0.3ml pathological material of disease lapping liquids of acquisition
On layer DEF cells, control group then adds the DMEM of equivalent, same to operate；Inoculation is placed on 37 DEG C, CO2It is cultivated in incubator
After 3h, DMEM maintaining liquids (containing 2% serum) is added to cultivate, pays attention to the DEF cell growth status of observation and record, culture daily
After 4-5d, 2 lytic cells of freeze thawing carry out receiving poison, and so lasting passage, each of harvest is stored in -80 DEG C for virus, until the 6th generation opened
Begin, DEF cells start lesion occur, and cell starts to split after karyopycnosis occurs for 24 hours in infection, grape cluster lesion sample, 96h occurs in 48h
Solution, largely expands numerous 6th generation virus liquid, and with spare, it is 10 to measure viral level5.25TCID50/0.1ml；
B. viruses indentification
B.1 blood coagulation tests：1~6 withholds the virus liquid obtained carries out duck blood hemagglutination test and chicken blood hemagglutination test respectively, as a result
The chicken red blood cell and duck red blood cell of each generation virus liquid pair 1% are not aggregated, exclude to have for orthomyxovirus, paramyxovirus etc.
The possibility of coagulation avian viral；
B.2PCR it identifies：The sick duck liver lapping liquid for being stored in -80 DEG C is centrifuged after three three solutions of jelly, 10000rpm
It is 0.22 μm of filtering with microporous membrane that supernatant aperture is taken after centrifugation 10min, takes the virus provided according to OMEGA companies after filtrate
DNA extracts the extracting that kit operation instructions carry out DNA, is carried out according to the Ana 1 aviadenovirus primers announced on NCBI
PCR is detected；
Upstream:5’-CACTCACGGGAACTG-3’
Downstream:5’-GGGCACCACAAACG-3’
As a result PCR product is coincide, preliminary proof isolated viral is through 1.5% agarose gel electrophoresis with purposeful band
Ana 1 aviadenovirus；
B.3 full genome is sequenced
A.2 10000rpm centrifuges 10min after the virus liquid collected is thawed, the miillpore filter mistake for being 0.22 μm with aperture
Then filter takes filtered fluid to extract the extracting that kit operation instructions carry out DNA according to the viral DNA that OMEGA companies provide,
The concentration of DNA is more than 280 ≈ 1.8 of 100ng/ μ L, OD 260/OD, carries out high-flux sequence and shows：Viral complete genome sequence is
43842bp, is named as CH-GD-12-2014, and complete genome sequence submits Genebank, Accession No：KR135164, the sequence
Row G/C content is 47.14%, and inverted terminal repetitive sequence (ITRs) is 721bp, contains 37 genes, lacks ORF51,1C, 54,
Possess ORF55A and five unique gene of others (ORF63-67), containing there are two fiber genes.The sequence and GoAdV-4 bases
Quite similar because organizing, the sequence similarity with GR plants is 92.3%；
It is higher that analysis CH-GD-12-2014 coded amino acids show that GR plants of CH-GD-12-2014 and 2 type of Ana 1 aviadenovirus have
Homology, but there are prodigious differences for some key genes, for example, CH-GD-12-2014 has fiber-1 and fiber-2 two
A gene, and GR plants of 2 type of Ana 1 aviadenovirus only 1 fiber-1 gene；
B.4 morphological observation
CH-GD-12-2014 strain virus liquid visible diameter about 65-75nm under Electronic Speculum after centrifugation, phosphotungstic acid negative staining,
In icosahedral symmetry structure；
B.5 animal Orthogonal Rotational Regressive Tests
1 Elderly SPF Shaoxing pockmark ducks 50 are randomly divided into two groups, and test group leg muscle injects 0.3mL
(0.3x105.25TCID50) CH-GD-12-2014 virus liquids, control group injects the same dose of DMEM culture solutions, puts isolation respectively
Device is raised, and the congested brittle and aetiolation of liver enlargement is fairly obvious when infecting 7d, and Cardiac Manifestation is loose ponding, in heart
There are two phymatoid blood black neoplasms for upper end；Kidney oedema is congested；When 14d, 21d, liver can be observed and show as Huang
Change phenomenon, Cardiac Manifestation is hydropericardium phenomenon, and kidney oedema is congested；When 30d, there is inclusion body hepatitis phenomenon, liver surface
Many places are distributed blood black grumeleuse, and heart deformation, in heart upper end there are two phymatoid blood black parcel, kidney is shown as slightly
Hyperemia, by animal Orthogonal Rotational Regressive Tests confirm 3 type of Ana 1 aviadenovirus that is separated to infect susceptible duckling it is reproducible go out clinical case.
Embodiment two：3 type CH-GD-12-2014 inactivated vaccines of Ana 1 aviadenovirus are studied
A. viral Physico-chemical tests
Physicochemical test result show CH-GD-12-2014 it is acidproof not alkaline-resisting, it is not high to the sensibility of temperature, ether, acetone,
Influence of the chloroform to CH-GD-12-2014 is smaller, and ultraviolet irradiation and formaldehyde can be such that CH-GD-12-2014 inactivates；
B. viral level measures
Measure the TCID of CH-GD-12-2014 strain virus50, it is viral to use 10 times of doubling dilutions of sterile saline, it dilutes
Virus inoculation to 96 holes on single layer DEF cells in, each dilution is inoculated with a tandem totally 8 hole, per hole 0.1m1, and is arranged
Control group；The DMEM maintaining liquids that virus liquid contains 2% serum to every hole 100 μ L of addition, 37 DEG C, CO are sucked after being incubated 2h2Constant temperature
After incubator carries out culture 4-5d, the lesion situation of cell is observed and recorded, TCID is calculated by Reed-Muench methods50, measure knot
Fruit is shown in Table 1, and result of calculation shows the virus liquid TCID of 1mL50Number is 105.25；
1 TCID of table50Test result
C. sterile and detection of mycoplasma
By CH-GD-12-2014 strain virus according to existing《Republic of China Veterinary Pharmacopoeia》It is former that annex carries out sterile and branch
Physical examination is tested, and testing result is feminine gender；
D. exogenous virus detects
By CH-GD-12-2014 strain virus according to existing《Republic of China Veterinary Pharmacopoeia》Annex carries out exogenous virus inspection
It surveys, testing result is feminine gender.
It is prepared by three CH-GD-12-2014 inactivated vaccines of embodiment
The preparation of a.CH-GD-12-2014 virus liquids
6th is withheld collection DEF cell virus lyolysis freeze after 10000rpm centrifuge 10min, be 0.22 μm micro- with aperture
Hole membrane filtration is inoculated into 200 and is covered in the 75cm Tissue Culture Flasks of 80%-90% single layer DEF cells, every bottle of 1ml, inoculation
It is placed on 37 DEG C, CO2After carrying out culture 3h in incubator, DMEM maintaining liquids (containing 2% serum) is added to cultivate 4-5 days, virus
10000rpm centrifugations 10min takes supernatant to collect cell toxicant with the filtering with microporous membrane that pore size is 0.22um after liquid three freezes three solutions
Liquid, it is separately sampled to test, it is placed in -20 DEG C and saves backup；
The inspection of b.CH-GD-12-2014 venom
The virus liquid of harvest is by existing《Chinese veterinary pharmacopoeia》Annex method makees steriling test and hemagglutination test (HA test) by bottle,
And a measurement viral level (TCID that samples50), the virus liquid asepsis growth as a result harvested is red to 1% chicken red blood cell and duck thin
Born of the same parents' suspension agglutination test is feminine gender, is 10 per 0.1ml viral levels5.52TCID50More than；
C. match seedling and packing
C.1 the preparation of inactivation of viruses liquid：10000rpm is taken to centrifuge, 0.22 μm of aperture membrane filtration virus liquid 99.82mL adds
Adding 0.18mL formalins, (final concentration of 0.18%) of formaldehyde, then 37 DEG C of inactivations for 24 hours, are rocked once per 0.5h；
C.2 prepared by water phase：96mL inactivation of viruses liquid is taken, 4% Tween-80s of 4mL are added, oscillation keeps mixing abundant；
C.3 prepared by oil phase：94mL white oils are taken, 2% aluminum stearates of 2g and 6% Span-80s of 6mL is added, sets on electrothermal furnace
It dissolves by heating to transparent, is cooled to room temperature spare；
C.4 prepared by inactivated vaccine：Oil phase and water phase are pressed 2:1 proportional arrangement, when emulsification, first refuel phase, and then side is shaken
Water phase (antigen) is added in side, and after water phase adds, fully emulsified (being shaken with mulser) is carried out in clean environment or the workshops SPF,
It before emulsification, is added in water phase dual anti-(penicillin and streptomysin), 2000 units/mL, after emulsification, takes appropriate emulsification vaccine
10min is centrifuged in 3000rpm, whether there is or not layerings for observation, are such as layered, then should continue to emulsify, until it is not stratified, then take upper layer
Lotion removes the precipitated metal that following a little mulser leaves, and finally carries out packing label and be put into 4 DEG C to save backup；
D. the safety testing of vaccine
1 age in days and 10 Elderly Shaoxing pockmark ducks are immunized for the first time with large dosage of, it is immune for the first time after 10d distinguish Isodose into
Row is immune for the second time, examines the safety of vaccine, passes through the immune inactivated vaccine 20 prepared of leg muscle, every 1.5ml
(containing 5 plumage part vaccines), while 10 not immunized controls are set, negative pressure isolator raising, free water feeding observes and records chicken daily
Health condition only, observation to after exempting from 21 days for the second time, and as a result immune group is with control group chicken without morbidity, and increase weight indifference, table
Bright vaccine is safe；
E. the potency test of vaccine
43 day Elderly SPF, Shaoxing pockmark duck was put into isolator raising 1d, and when 1 age in days dissects 3 at random, and observation internal organs are
It is no normal, and internal organs is taken to do PCR detections, health condition is checked, after determining health status, by remaining 40 Shaoxing SPF
Sheldrake is randomly divided into two groups, and experimental group 20, control group 20, the progress of 10 ages in days is immune for the first time, and experimental group passes through leg
Intramuscular injection enters the inactivated vaccine 0.3ml prepared, and control group is injected into 0.3ml DMEM culture solutions by leg muscle；20 days
Age carries out second and is immunized, and experimental group is injected into the inactivated vaccine 0.3ml control groups prepared by leg muscle and passes through leg
Intramuscular injection enters 0.3mlDMEM culture solutions；2d carries out attacking poison to duck after exempting from for the second time, the method pair injected by leg muscle
Every SPF Shaoxing pockmark duck of experimental group and control group all injects 300 μ L CH-GD-12-2014 virus liquids (105.25TCID50), attack poison
Observe and record daily afterwards chicken group morbidity, death condition, timely dissect death chicken, and the 3rd after attacking poison, 7,14,21,28,35,
42,50d take at random experimental group and control group each 3 SPF Shaoxing pockmark ducks acquisition liver, heart, lungs, kidney, spleen, the bursa of farbricius,
The samples such as thymus gland, small intestine are placed in 10% formalin fixer fixed, progress HE dyeing making pathological sections, then aobvious
Tissues observed lesion under micro mirror.As a result it shows：Immune rear experimental group and control group chicken group are normal, attack rear dissection discovery after poison 7
Control group SPF Shaoxing pockmark ducks show as liver and a small number of necrosis regions occur；Simultaneously there is black tubercle in heart slight bleeding；Kidney shows
It is slight congested.And immune group is dead without morbidity, chicken health is without exception；Pathological section is the results show that be non-immune group when 42d
Bleeding and inflammatory cell aggregation occur for liver, and heart has inflammatory cell aggregation, and slight hyperemia occurs in kidney, and control group is without apparent
Disease states.Show that the vaccine safety prepared with CH-GD-12-2014 inactivations is effective, can protect same source strength poison attacks poison.
Gene order table
SEQ ID NO：1 (nucleotide sequence of the hexon genes of CH-GD-12-2014)
atggccgctctgacccctgacttgacgacggctacgccgaggctgtcctattttcacattgccggtccgagcacacg
ggagtatctgtcagaggaccttcagcagtttatgagtgcaacatccagctactttgagttgcgaaacaaattcaggc
aaacggttgctgccccaacgcgtaatgttactaccgagaaagctcaacgtctccagatacgctactatcccattcag
actgacgaaacatcaaacacacacagagtgagattttccatgaatgtcggtgacagttgggttcttgacatgggatc
aacttattttgacataaagggtgtgctagataggggttcttctttcaaaccatacagtggaacagcttacaatccac
tagctcctaaagaatcggtgtttaacttttggtacacgcacacagactctaagaactacattggtgcgcagctgtcg
actctgtacgaaaacacagaccctggcacaggtacaccaacacaaaatgtagtaaaagcaatgagtggagtcaatcc
tgatccaaaccaagggtcaagtatctcagttcctgaattgcttataggagacacaaatgacaataagtttagtggag
tggcgaaagtagctaaggctgaattgatgcttgctcatggtgcttacgttaagccagtggcacccactgggtctcag
tctctatcccaaactggatatgtgttgtcttcagatgggtccacaaagtataacggagctatttcggttgaggacta
tacatcatcacttcagtatccagatagcctgtacattccaccaaactcgactgctgtagacaactttggtgtgacaa
agggactcagacctaactacataggttttcgcgataatttcattaacattctttatcatgactcaggtgtctgctca
ggtacgcttaattctgagaggtcaggcatgaacgttgttgtagcattacaggacaggaatactgaactaagctatca
gtacatgttggctgacatgatgtcaaggcatcattattttgcactatggaatcaggcagtagaccagtatgatcatg
acgtaagagtcttcaacaatgacggatatgaagacgtatctagctcctatgcgttctaccctaattgcttaggcttt
caacctggtggagaactctatacgaaattgaaggtagtgaagacagactcttcttcaggcgctatgtctggcggtga
ggtggccaacacatcaagtgcatttggagtaggcaacattcctgcctatgaaattaatatcccggcttctatgaaac
gaattttcattatgagcaacattgctgattacctgccagacaaatacaaggtcagcatagactcaacagacggtgtg
gaccagaactcatacgagtatatgaacaagcgtgtacctcttaccaacattgtggatctttttacaaatataggtgc
cagatggtcagtggatcagatggataacgttaatccatttaatcaccacagaaattggggcctgaaatacaggtctc
agctcctaggaaacagccgttactgtcagtttcacattcaggtaccgcagaagtatttcgctataaaaaatctacta
cttctgccaggaacatacacctatgaatgggtactccgaaaggatccgaatatggtgctgcagtcaagcttggggaa
cgatttacggcttgacaaggcttccattacatttacagaggtgaatttgatggccagcttcatgcctatggaccata
ataccagtaaccaactagagctcatgatgcgcaatgctactaatgatcagacattcatggactacctcggtgcaaag
aacgcactttacagcattcctgcagggtcaaaccaagtaactatcaacattcctgctagaacctgggaaggcatgcg
aggatggtctttcactcgtctcaaaacaaaagaaacacctcaacaaggagcacaatatgacgtggcgtttaagtatt
ccggatcaattccatacttagacggtacattctatcttaaccacacatttaagaatatgagtgtgttgtttgataca
tcgataaattggccaggaaatgataggctcatgtcaccgaacatgttcgaaattaaaagagcaccagcagctgactc
tgaaggatttactatgagtcaatgcgacattacgaaagattggtggctaattcagatggccacaaactacaactttg
tgtataacgggtacaggttctggcctgacagacattacttccagtatgactttttgcgtaactttgacccaatgtcc
agacagtctcccaatttctctcagtctaatggattgtatgatctggtctctgtggataacacaccatccactggagc
acctaaacaggaatacgttcgtaacaactcggggttcgtggcacctagggccgaaccagtctcaaatgcgagacagg
gacacgcatggcccgctaattggccttatccactgattggtaaacactgcatagacagtgctaacattacccagtac
aagaaattcctgtgcgacaactacctgtggaccattccctttagctccgactttatgtatatgggtgagctgacgga
tctgggtcagaatcccatgtacaccaacaactcccacagcatggtgatcaactttgaggtagaccccatggacgagg
acacctatctctacatgctgtacggagtgtttgatgcggtcagggtgaaccagcctgagcgcaatgtgctggccatg
gcttacttccgtacgcctttcgctacaggtaacgcggtgtaa
SEQ ID NO：2(SEQ ID NO：The amino acid sequence of 1 coding)
MAALTPDLTTATPRLSYFHIAGPSTREYLSEDLQQFMSATSSYFELRNKFRQTVAAPTRNVTTEKAQRL
QIRYYPIQTDETSNTHRVRFSMNVGDSWVLDMGSTYFDIKGVLDRGSSFKPYSGTAYNPLAPKESVFNFWYTHTDSK
NYIGAQLSTLYENTDPGTGTPTQNVVKAMSGVNPDPNQGSSISVPELLIGDTNDNKFSGVAKVAKAELMLAHGAYVK
PVAPTGSQSLSQTGYVLSSDGSTKYNGAISVEDYTSSLQYPDSLYIPPNSTAVDNFGVTKGLRPNYIGFRDNFINIL
YHDSGVCSGTLNSERSGMNVVVALQDRNTELSYQYMLADMMSRHHYFALWNQAVDQYDHDVRVFNNDGYEDVSSSYA
FYPNCLGFQPGGELYTKLKVVKTDSSSGAMSGGEVANTSSAFGVGNIPAYEINIPASMKRIFIMSNIADYLPDKYKV
SIDSTDGVDQNSYEYMNKRVPLTNIVDLFTNIGARWSVDQMDNVNPFNHHRNWGLKYRSQLLGNSRYCQFHIQVPQK
YFAIKNLLLLPGTYTYEWVLRKDPNMVLQSSLGNDLRLDKASITFTEVNLMASFMPMDHNTSNQLELMMRNATNDQT
FMDYLGAKNALYSIPAGSNQVTINIPARTWEGMRGWSFTRLKTKETPQQGAQYDVAFKYSGSIPYLDGTFYLNHTFK
NMSVLFDTSINWPGNDRLMSPNMFEIKRAPAADSEGFTMSQCDITKDWWLIQMATNYNFVYNGYRFWPDRHYFQYDF
LRNFDPMSRQSPNFSQSNGLYDLVSVDNTPSTGAPKQEYVRNNSGFVAPRAEPVSNARQGHAWPANWPYPLIGKHCI
DSANITQYKKFLCDNYLWTIPFSSDFMYMGELTDLGQNPMYTNNSHSMVINFEVDPMDEDTYLYMLYGVFDAVRVNQ
PERNVLAMAYFRTPFATGNAV
Sequence table
<110>Agricultural University Of South China
<120>CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2814
<212> DNA
<213>CH-GD-12-2014 plants of 3 type of Ana 1 aviadenovirus (Duck Adenovirus Type III, DAdVIII CH-GD-
12-2014)
<400> 1
atggccgctc tgacccctga cttgacgacg gctacgccga ggctgtccta ttttcacatt 60
gccggtccga gcacacggga gtatctgtca gaggaccttc agcagtttat gagtgcaaca 120
tccagctact ttgagttgcg aaacaaattc aggcaaacgg ttgctgcccc aacgcgtaat 180
gttactaccg agaaagctca acgtctccag atacgctact atcccattca gactgacgaa 240
acatcaaaca cacacagagt gagattttcc atgaatgtcg gtgacagttg ggttcttgac 300
atgggatcaa cttattttga cataaagggt gtgctagata ggggttcttc tttcaaacca 360
tacagtggaa cagcttacaa tccactagct cctaaagaat cggtgtttaa cttttggtac 420
acgcacacag actctaagaa ctacattggt gcgcagctgt cgactctgta cgaaaacaca 480
gaccctggca caggtacacc aacacaaaat gtagtaaaag caatgagtgg agtcaatcct 540
gatccaaacc aagggtcaag tatctcagtt cctgaattgc ttataggaga cacaaatgac 600
aataagttta gtggagtggc gaaagtagct aaggctgaat tgatgcttgc tcatggtgct 660
tacgttaagc cagtggcacc cactgggtct cagtctctat cccaaactgg atatgtgttg 720
tcttcagatg ggtccacaaa gtataacgga gctatttcgg ttgaggacta tacatcatca 780
cttcagtatc cagatagcct gtacattcca ccaaactcga ctgctgtaga caactttggt 840
gtgacaaagg gactcagacc taactacata ggttttcgcg ataatttcat taacattctt 900
tatcatgact caggtgtctg ctcaggtacg cttaattctg agaggtcagg catgaacgtt 960
gttgtagcat tacaggacag gaatactgaa ctaagctatc agtacatgtt ggctgacatg 1020
atgtcaaggc atcattattt tgcactatgg aatcaggcag tagaccagta tgatcatgac 1080
gtaagagtct tcaacaatga cggatatgaa gacgtatcta gctcctatgc gttctaccct 1140
aattgcttag gctttcaacc tggtggagaa ctctatacga aattgaaggt agtgaagaca 1200
gactcttctt caggcgctat gtctggcggt gaggtggcca acacatcaag tgcatttgga 1260
gtaggcaaca ttcctgccta tgaaattaat atcccggctt ctatgaaacg aattttcatt 1320
atgagcaaca ttgctgatta cctgccagac aaatacaagg tcagcataga ctcaacagac 1380
ggtgtggacc agaactcata cgagtatatg aacaagcgtg tacctcttac caacattgtg 1440
gatcttttta caaatatagg tgccagatgg tcagtggatc agatggataa cgttaatcca 1500
tttaatcacc acagaaattg gggcctgaaa tacaggtctc agctcctagg aaacagccgt 1560
tactgtcagt ttcacattca ggtaccgcag aagtatttcg ctataaaaaa tctactactt 1620
ctgccaggaa catacaccta tgaatgggta ctccgaaagg atccgaatat ggtgctgcag 1680
tcaagcttgg ggaacgattt acggcttgac aaggcttcca ttacatttac agaggtgaat 1740
ttgatggcca gcttcatgcc tatggaccat aataccagta accaactaga gctcatgatg 1800
cgcaatgcta ctaatgatca gacattcatg gactacctcg gtgcaaagaa cgcactttac 1860
agcattcctg cagggtcaaa ccaagtaact atcaacattc ctgctagaac ctgggaaggc 1920
atgcgaggat ggtctttcac tcgtctcaaa acaaaagaaa cacctcaaca aggagcacaa 1980
tatgacgtgg cgtttaagta ttccggatca attccatact tagacggtac attctatctt 2040
aaccacacat ttaagaatat gagtgtgttg tttgatacat cgataaattg gccaggaaat 2100
gataggctca tgtcaccgaa catgttcgaa attaaaagag caccagcagc tgactctgaa 2160
ggatttacta tgagtcaatg cgacattacg aaagattggt ggctaattca gatggccaca 2220
aactacaact ttgtgtataa cgggtacagg ttctggcctg acagacatta cttccagtat 2280
gactttttgc gtaactttga cccaatgtcc agacagtctc ccaatttctc tcagtctaat 2340
ggattgtatg atctggtctc tgtggataac acaccatcca ctggagcacc taaacaggaa 2400
tacgttcgta acaactcggg gttcgtggca cctagggccg aaccagtctc aaatgcgaga 2460
cagggacacg catggcccgc taattggcct tatccactga ttggtaaaca ctgcatagac 2520
agtgctaaca ttacccagta caagaaattc ctgtgcgaca actacctgtg gaccattccc 2580
tttagctccg actttatgta tatgggtgag ctgacggatc tgggtcagaa tcccatgtac 2640
accaacaact cccacagcat ggtgatcaac tttgaggtag accccatgga cgaggacacc 2700
tatctctaca tgctgtacgg agtgtttgat gcggtcaggg tgaaccagcc tgagcgcaat 2760
gtgctggcca tggcttactt ccgtacgcct ttcgctacag gtaacgcggt gtaa 2814
Claims (7)
1. CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus, which is characterized in that 3 type inactivated vaccine CH-GD- of Ana 1 aviadenovirus
12-2014 plants, China typical culture collection center is preserved on November 27th, 2017, deposit number is CCTCC NO：
V201764, preservation address are：China, Wuhan, Wuhan University；Complete genome sequence submits Genebank, Accession No：
KR135164。
2. CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus according to claim 1, which is characterized in that also provide one
Kind polypeptide, amino acid sequence such as SEQ ID NO：Shown in 2.
3. CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus according to claim 1, which is characterized in that also provide volume
The nucleotide sequence of the code polypeptide, the sequence such as SEQ ID NO：Shown in 1.
4. according to CH-GD-12-2014 plants of claims 1 or 2 or 3 Ana 1 aviadenovirus, 3 type inactivated vaccine, which is characterized in that institute
State the polypeptide sequence such as SEQ ID NO of the hexon gene codes of CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus：2 institutes
Show；The nucleotides sequence of the hexon genes of CH-GD-12-2014 plants of 3 type inactivated vaccine of the Ana 1 aviadenovirus is classified as SEQ ID NO：1
It is shown, or in SEQ ID NO：Through replacing, lacking and oring add one or more nucleotide and volume in 1 sequence basis
Code SEQ ID NO：The gene order of the 2 same functions of albumen.
5. CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus according to claim 1, which is characterized in that also provide duck
Application of CH-GD-12-2014 plants of the adenovirus type III inactivated vaccine in the drug for preparing treatment Ana 1 aviadenovirus.
6. CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus according to claim 1, which is characterized in that also provide and contain
The drug for having CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus can be specifically vaccine.
7. CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus according to claim 1 or 6, which is characterized in that described
Vaccine includes CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus and pharmaceutically acceptable adjuvant.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711430436.4A CN108330108A (en) | 2017-12-26 | 2017-12-26 | CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus |
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| CN201711430436.4A CN108330108A (en) | 2017-12-26 | 2017-12-26 | CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108842004A (en) * | 2018-07-28 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | Distinguish real-time fluorescence quantitative PCR primer and its differentiating method and the application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
| CN108842005A (en) * | 2018-07-28 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | The MGB probe and its mating primer and method of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B are detected simultaneously |
| CN108950070A (en) * | 2018-07-28 | 2018-12-07 | 福建省农业科学院畜牧兽医研究所 | Identify the PCR primer and its discrimination method and application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
| CN110117576A (en) * | 2019-04-23 | 2019-08-13 | 广东迈科特生物科技有限公司 | A kind of 3 type Strain of Ana 1 aviadenovirus and its Yolk antibody preparation and application |
| CN110208518A (en) * | 2019-05-28 | 2019-09-06 | 福建省农业科学院畜牧兽医研究所 | Indirect ELISA detection method and its detection kit based on 3 type adenovirus antibody of Fiber1 Protein Detection duck |
| CN110564699A (en) * | 2019-09-30 | 2019-12-13 | 山东信得科技股份有限公司 | Novel Muscovy duck adenovirus strain, inactivated vaccine and preparation method thereof |
| CN113198011A (en) * | 2021-04-28 | 2021-08-03 | 重庆永健生物技术有限责任公司 | Duck adenovirus type-3 strain inactivated vaccine and application thereof |
| CN113278595A (en) * | 2021-04-28 | 2021-08-20 | 重庆永健生物技术有限责任公司 | Duck adenovirus type-3 strain, duck adenovirus egg yolk antibody and preparation method and application thereof |
| CN114561367A (en) * | 2022-02-25 | 2022-05-31 | 华南农业大学 | Recombinant duck adenovirus type 3, preparation method and application thereof |
| CN114668838A (en) * | 2021-12-30 | 2022-06-28 | 哈药集团生物疫苗有限公司 | Tetrad inactivated vaccine for preventing and treating duck circovirus disease, novel duck reovirus disease, duck viral hepatitis and duck adenovirus type 3 |
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|---|---|---|---|---|
| CN108842004A (en) * | 2018-07-28 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | Distinguish real-time fluorescence quantitative PCR primer and its differentiating method and the application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
| CN108842005A (en) * | 2018-07-28 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | The MGB probe and its mating primer and method of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B are detected simultaneously |
| CN108950070A (en) * | 2018-07-28 | 2018-12-07 | 福建省农业科学院畜牧兽医研究所 | Identify the PCR primer and its discrimination method and application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
| CN110117576A (en) * | 2019-04-23 | 2019-08-13 | 广东迈科特生物科技有限公司 | A kind of 3 type Strain of Ana 1 aviadenovirus and its Yolk antibody preparation and application |
| CN110117576B (en) * | 2019-04-23 | 2023-02-10 | 广东迈科特生物科技有限公司 | Duck adenovirus type-3 virus strain and preparation and application of yolk antibody thereof |
| CN110208518A (en) * | 2019-05-28 | 2019-09-06 | 福建省农业科学院畜牧兽医研究所 | Indirect ELISA detection method and its detection kit based on 3 type adenovirus antibody of Fiber1 Protein Detection duck |
| CN110564699A (en) * | 2019-09-30 | 2019-12-13 | 山东信得科技股份有限公司 | Novel Muscovy duck adenovirus strain, inactivated vaccine and preparation method thereof |
| CN110564699B (en) * | 2019-09-30 | 2021-08-03 | 山东信得科技股份有限公司 | Novel Muscovy duck adenovirus strain, inactivated vaccine and preparation method thereof |
| CN113198011A (en) * | 2021-04-28 | 2021-08-03 | 重庆永健生物技术有限责任公司 | Duck adenovirus type-3 strain inactivated vaccine and application thereof |
| CN113278595A (en) * | 2021-04-28 | 2021-08-20 | 重庆永健生物技术有限责任公司 | Duck adenovirus type-3 strain, duck adenovirus egg yolk antibody and preparation method and application thereof |
| CN114668838A (en) * | 2021-12-30 | 2022-06-28 | 哈药集团生物疫苗有限公司 | Tetrad inactivated vaccine for preventing and treating duck circovirus disease, novel duck reovirus disease, duck viral hepatitis and duck adenovirus type 3 |
| CN114561367A (en) * | 2022-02-25 | 2022-05-31 | 华南农业大学 | Recombinant duck adenovirus type 3, preparation method and application thereof |
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