CN110128508A - A kind of aviadenovirus fiber protein subunit vaccine - Google Patents
A kind of aviadenovirus fiber protein subunit vaccine Download PDFInfo
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- CN110128508A CN110128508A CN201910442879.8A CN201910442879A CN110128508A CN 110128508 A CN110128508 A CN 110128508A CN 201910442879 A CN201910442879 A CN 201910442879A CN 110128508 A CN110128508 A CN 110128508A
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Abstract
The present invention provides a kind of aviadenovirus fiber protein subunit vaccine, it is the antigen fiber albumen novel using aviadenovirus, fiber albumen is set to obtain soluble expression in Escherichia coli after optimizing to sequence, expression product prepares subunit vaccine, wherein the amino acid sequence of aviadenovirus fiber antigen protein is SEQ ID NO:4, and the nucleotides sequence of encoding gene is classified as SEQ ID NO:3.The subunit vaccine of aviadenovirus fiber albumen preparation of the invention has Antigen Stability high, purity is high, high specificity do not generate other uncorrelated antibody, detection method facilitates accurate feature, has established solid foundation for industrialized production aviadenovirus subunit vaccine and diagnostic reagent.
Description
Technical field
The invention belongs to veterinary biologics technical fields, and in particular to a kind of aviadenovirus fiber protein subunit epidemic disease
Seedling.
Background technique
Aviadenovirus (Fowl Adenovirus, FAdV) belongs to Adenoviridae Aviadenovirus, is divided into 5 kinds (A~E),
12 serotypes；Aviadenovirus FAdV has been reported that all over the world.FAdV, which infects, generally causes subclinical situation, and acute sense
Dye mainly causes inclusion body hepatitis, hydropericardium and gizzard erosion etc..From 2013, domestic chicken group forgave as caused by FAdV
Body hepatitis, hydropericardium case gradually increase.By 2015, multiple province chicken groups were broken out at home for FAdV infection.FAdV at present
Outburst not only occurs on 3~4 week old broiler chicken, also betides the laying hen of 10~20 week old, causes serious warp to domestic poultry husbandry
Ji loss.
Aviadenovirus is nonencapsulated ball shape structure, and capsid is 20 face body cubic symmetries, is made of 252 capsomeres, wherein
240 are hexon (Hexon, 120KD), are main envelope protein；The capsomere of 12 apex angles be known as penton (Penton,
82KD)；There are also the fiber of trimer protein (62KD).Wherein penton substrate one end passes through non-covalent bond and tripolymer fiber phase
Even, the other end is adsorbed on cell membrane.Wherein virus fiber is main immune protein, can be effectively protected chicken from FAdV
Infection.
Existing aviadenovirus vaccine is to use complete pathogen as antigen for vaccine, but currently manufactured vaccine uses
Strain and the antigenicity of clinical popular strain there are biggish differences, attack malicious protecting effect to affect, restrict inactivation epidemic disease
The application of seedling.And the effect of Attenuate vaccine is easy to the interference by maternal antibody and antibiotic, and there are virulence to return for less-virulent strain
A possibility that strong and induction subclinical infection.Therefore, subunit vaccine is developed for aviadenovirus immune protective antigen, is fowl
The Main way of the novel sub- vaccine research of adenovirus.
Summary of the invention
It is an object of the present invention to provide a kind of aviadenovirus fiber protein subunit vaccines, are new using a kind of aviadenovirus
The antigen fiber albumen of type makes fiber albumen obtain soluble expression, table in Escherichia coli after optimizing to sequence
Subunit vaccine is prepared up to product, to make up the deficiencies in the prior art.
Present invention firstly provides a kind of aviadenovirus fiber antigen proteins, include:
1) amino acid sequence is the albumen of SEQ ID NO:2；
2) replace on albumen 1), lack, adding one or several amino acid, and there is the egg of protein function in 1)
It is white；
The gene of above-mentioned aviadenovirus fiber albumen is encoded, nucleotides sequence is classified as SEQ ID NO:1；
The present invention also provides the optimization body of above-mentioned fiber antigen protein, amino acid sequence is SEQ ID NO:4, is compiled
The nucleotides sequence of code gene is classified as SEQ ID NO:3；
Aviadenovirus fiber albumen of the invention can be used for preparing vaccine or diagnostic reagent.
Another aspect of the present invention provides a kind of subunit vaccine, is that above-mentioned fiber albumen is used to prepare as antigen
's.
The subunit vaccine of aviadenovirus fiber albumen preparation of the invention has Antigen Stability height, with high purity, specifically
Property it is strong, do not generate other uncorrelated antibody, detection method facilitates accurate feature, be industrialized production aviadenovirus subunit epidemic disease
Seedling and diagnostic reagent have established solid foundation.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Method applied by the present invention can use epidemic disease
Common method in seedling preparation field is not limited solely to the specific record of the embodiment of the present invention, those skilled in the art
The present invention can be realized with other conventional methods.
The amplification of embodiment 1:fiber gene and sequence analysis
Doubtful hydropericardium syndrome occurred in Shandong Yantai chicken farm by 2017, is examined through clinical investigation and laboratory
It surveys, tentative diagnosis is avian adenovirus infection.It is investigated, had injected aviadenovirus epidemic disease before firmly believing above-mentioned morbidity chicken group
Seedling.Suspection has aviadenovirus virus to be mutated under the selection pressure of vaccine, is successfully separated out from the liver of morbidity chicken
Virus, the template as amplification.
1, aviadenovirus fiber gene is expanded
According to the aviadenovirus fiber gene order delivered in NCBI, primer, the sequence information of primer have been designed and synthesized
It is as follows:
Primer1:5 '-ATGCTCCGAGCCCCTAAAAG-3 '；
Primer2:5 '-TTACGGGAGGGAGCCCGCTG-3 '.
Isolated aviadenovirus nucleic acid carries out PCR amplification target fragment as template, with primer primer1 and primer2,
Through sequencing, nucleotides sequence is classified as SEQ ID NO:1, and deriving its amino acid sequence according to sequencing result is SEQ ID NO:
2。
It is compared with the aviadenovirus fiber albumen (AOS50917.1) announced in NCBI, as a result amino acid
Homology is about 97%, has multiple sites to be made a variation, and the 40th (K becomes M), the 219th (D becomes E), the 272nd (T becomes S),
291st (P becomes A), the 295th (T becomes R), the 299th (T becomes M), the 300th (T becomes R), the 405th (S becomes R), the 406th
Position (I becomes A), the 421st (N becomes K), the 439th (E becomes Q), the 453rd (A becomes T), the 459th (N becomes K).The result shows that point
From strain be the aviadenovirus to morph, contain new fiber gene.
2, the shearing, optimization of fiber gene
The antigen site of the structural protein gene fiber of isolated strain is analyzed, its N-terminal 180aa is deleted, is stayed
The globular domain of lower fiber and a small amount of rod-shaped handle can completely express the immunization sites of fiber albumen and be formed
Good space structure.Codon is optimized simultaneously, improves the expression efficiency of albumen.Amino acid sequence after optimization modification
Respectively SEQ ID NO:4.Sequence after optimization modification is that can give expression to antigen site well.The gene of selection is carried out
Codon optimization, the nucleotides sequence of the gene after optimization are classified as SEQ ID NO:3.
Embodiment 2, the building for expressing fiber gene recombination plasmid
2.1 endonuclease reaction
2.1.1 label needs well the 1.5mL EP used to manage, and sample-adding is carried out according to the following table in 1.5mL EP pipe, mixes:
Reaction system is 50 μ L, and sample-adding is as follows:
2.1.2 the 1.5mL EP pipe in step 2.2.1 is placed in 37 DEG C of thermostat water baths, water-bath 2-3h.
2.1.3 double enzyme digestion product glue recycles
Above-mentioned double digestion system is taken out, carries out agarose gel electrophoresis to recycle DNA fragmentation therein.
(1) sample collection EP pipe, adsorption column and collecting pipe have been marked.
(2) the empty EP pipe weight marked is weighed, and records numerical value.
(3) single target DNA band is carefully cut from Ago-Gel with scalpel on bale cutting instrument be put into it is dry
In net 1.5mL centrifuge tube.
(4) 600 μ L PC buffer50 DEG C water-baths are added in the 1.5mL centrifuge tube in step (3) and place 5min or so,
Centrifuge tube is mildly constantly spun upside down therebetween, to ensure that blob of viscose sufficiently dissolves.
(5) column equilibration: into adsorption column CB2,500 μ L equilibrium liquid BL, centrifugation is added in (adsorption column is placed in advance in collecting pipe)
12,000rpm, 1min outwell the waste liquid in collecting pipe, adsorption column are placed back in collecting pipe.
(6) step (5) acquired solution is added in adsorption column CB2, stand 2min, 10,000rpm, be centrifuged 30s, outwell receipts
Waste liquid in collector, then adsorption column CB2 is put into collecting pipe.
(7) 600 μ L rinsing liquid PW buffer are added into adsorption column, stand 3min, is centrifuged 10,000rpm, 30s, outwells
Adsorption column CB2 is put into collecting pipe by the waste liquid in collecting pipe.
(8) step (7) are repeated.
(9) suction attached column is centrifuged, 12,000rpm, 2min, as far as possible removing rinsing liquid.Adsorption column is placed in and is placed at room temperature for
10min thoroughly dries.
(10) adsorption column CB2 is put into collecting pipe, 50 μ L is vacantly added dropwise to adsorbed film middle position
Elutionbuffer (65 DEG C of preheatings), stands 3min, is centrifuged 12,000rpm, 2min.
(11) from centrifuge tube in step (10) is taken out in centrifuge, intermediate adsorption column CB2 is abandoned, centrifuge tube lid is covered
Son retains the DNA sample in centrifuge tube.
(12) DNA sample in step 11 is placed in 4 DEG C of preservations, prepares agarose gel electrophoresis identification glue and recycles DNA piece
Section.
2.2 connection reactions
(1) label needs the 0.2mL centrifuge tube used.
(2) it is loaded in marking complete 0.2mL pipe according to 20 μ L reaction systems of following table:
(3) it after completing sample-adding, is gently blown and beaten with pipettor and mixes each component several times.
(4) 0.2mL centrifuge tube is placed in 37 DEG C of reaction 30min, to after the reaction was completed, reaction tube is placed in ice-water bath immediately
Middle cooling 5min.
(5) reaction product of step (4) can directly carry out transformation experiment, can also be stored in -20 DEG C, turn wait need to thaw
Change.
2.3 conversion reaction
(1) 10 μ L connection reaction solutions are rapidly joined in 100 μ L competent cells, and blows and beats mixing, ice bath 30min.
(2) after the completion of step (1), sample cell is taken out, is placed in 42 DEG C of water-bath 100s, immediately after ice bath 2min.
(3) it after the completion of step (2), takes out sample cell and 600 μ L liquid LB is added into sample cell in superclean bench
Culture medium, is then placed in 37 DEG C of constant-temperature tables for sample cell, and 220rpm cultivates 1h.
(4) preparation conversion plate, according to plasmid resistance preparation conversion LB resistant panel.
(5) coated plate: taking out sample cell in step (3), and room temperature is centrifuged 8,000rpm, 2min, removes 600 μ L supernatant fluids,
The thallus of bottom of the tube is resuspended in remaining supernatant, and the bacterium solution of resuspension is put into corresponding conversion plate center, will be converted with bacteria stick is applied
The bacterium solution of plate center is uniformly spread out.
(6) step (5) plate is just being placed in biochemical constant incubator, after 37 DEG C of culture 1h, will conversion plate be inverted into
Row culture 15h.
(7) conversion results are observed and recorded.
2.4 plasmid extractions and PCR are identified
2.4.1 plasmid extraction
(1) with 10 μ L liquid transfer gun heads from conversion plate in picking monoclonal to 5ml that resistance containing card LB liquid medium
In, 37 DEG C, 220rpm shakes bacterium and stays overnight.
(2) bacterium solution is drawn into 1.5mL EP pipe, and room temperature centrifugation, 12,000rpm, 2min abandon supernatant.
(3) 250 μ L plasmids are added in the EP pipe in step (2) and extract reagent P1buffer, thorough suspension thalline.
(4) 250 μ L P2buffer are added into step (3) solution, immediately mildly 5-10 mixing of reverse centrifuge tube.Room
Temperature stands 2-4min.
(5) 350 μ L P3buffer are added into step (4) solution, immediately mildly 5-10 mixing of reverse centrifuge tube.Room
Temperature stands 2-4min.
(6) by step (5) solution, room temperature centrifugation, 14,000rpm, 10min.
(7) supernatant solution in step (6) is moved into adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell collection
Liquid in pipe.
(8) 500 μ L Buffer DW1 are added to adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell collecting pipe
Middle liquid.
(9) 500 μ L wash solution are added to adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell receipts
Liquid in collector.It is repeated once.
(10) suction attached column, room temperature centrifugation, 12,000rpm, 2min.
(11) adsorption column is put into a clean 1.5ml centrifuge tube, 30 μ L is added to absorption center membrane
Elutionbuffer is stored at room temperature 5min, and room temperature is centrifuged, 12,000rpm, 2min, DNA solution in 4 DEG C of preservation pipes.
2.4.2 PCR is identified
(1) PCR pipe for needing to use well is marked, sample-adding is carried out according to the following table, mixes, reaction system is 25 μ L:
(2) PCR amplification program:
(3) it is sequenced: sending sequencing company to be sequenced the positive plasmid of pcr identification.
2.5 conversion
The positive plasmid of sequencing is converted into BL21, the same 2.3. of method
2.6 picking plasmids and PCR are identified
2.6.1 picking plasmid
(1) from the plate of 2.5 conversions, with 10 μ L liquid transfer gun heads, picking monoclonal colonies contain to 5ml from conversion plate
In the LB liquid medium of kalamycin resistance, 37 DEG C, 220rpm shakes bacterium and stays overnight.
(2) bacterium solution is drawn into 1.5mL EP pipe, and room temperature centrifugation, 12,000rpm, 2min abandon supernatant.
(3) 250 μ L plasmids are added in the EP pipe in step (2) and extract reagent P1buffer, thorough suspension thalline.
(4) 250 μ L P2buffer are added into step (3) solution, immediately mildly 5-10 mixing of reverse centrifuge tube.Room
Temperature stands 2-4min.
(5) 350 μ L P3buffer are added into step (4) solution, immediately mildly 5-10 mixing of reverse centrifuge tube.Room
Temperature stands 2-4min.
(6) by step (5) solution, room temperature centrifugation, 14,000rpm, 10min.
(7) supernatant solution in step (6) is moved into adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell collection
Liquid in pipe.
(8) 500 μ L Buffer DW1 are added to adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell collecting pipe
Middle liquid.
(9) 500 μ L wash solution are added to adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell receipts
Liquid in collector.It is repeated once.
(10) suction attached column, room temperature centrifugation, 12,000rpm, 2min.
(11) adsorption column is put into a clean 1.5ml centrifuge tube, 30 μ L is added to absorption center membrane
Elutionbuffer is stored at room temperature 5min, and room temperature is centrifuged, 12,000rpm, 2min, DNA solution in 4 DEG C of preservation pipes.
2.6.2 PCR identifies the DNA that will be extracted in 2.6.1, carries out pcr identification, identifies that positive plasmid serves marine growth
Engineering Co., Ltd is sequenced, and the positive corresponding strain of plasmid is sequenced for protein expression.
Expression, purifying, the identification of 3 albumen of embodiment
3.1 expression will be sequenced positive strain with identification and be inoculated on the LB plate containing kanamycin sulfate, 37 DEG C of cultures
Overnight.Picking colony is cultivated in the LB culture solution and escherichia coli bacterium culture medium containing kanamycin sulfate to bacterium solution
When OD600 is 0.6~0.8, the lactose of final concentration of 0.02M is added, carries out 5~7h of Fiber differentiation, thalline were collected by centrifugation.Bacterium mud
It suspended, be crushed with PBS.SDS-PAGE sample loading buffer [50mM Tris-HCl (pH6.8), 100mM is added
Dithiothreitol (DTT), 2%SDS, 0.05%Bromophemol blue, 10%Glycerol], 5min is boiled, is used
12% separation gel, 5% concentration glue carry out polyacrylamide gel electrophoresis, 100 volts of about 2.5h, coomassie brilliant blue R250 dyeing, hair
The fiber gene being now not optimised is not expressed in Escherichia coli, and the fiber gene optimized has carried out table in Escherichia coli
It reaches, and is solubility expression.
3.2 purifying suspend the thallus of collection with PBS, set ultrasonic disruption thalline.By the thallus of collection with PBS into
Row suspends, and sets ultrasonic disruption thalline, and 12000r/min is centrifuged 15min, and supernatant is taken to abandon precipitating.Supernatant is subjected to layer with Ni2+ column
Analysis purifying.
Albumen prepared by aviadenovirus totivirus and 3.2 is carried out SDS-PAGE simultaneously by 3.3 Western blot, is used
Destination protein band is transferred to pvdf membrane by 20 volts of transfer 30min of semidry method, and transfer film is closed overnight with confining liquid, and PBST is washed
It washs 3 times, 1: 500 37 DEG C of diluted aviadenovirus positive serum effect 1.5h, PBST is washed 3 times, with 1: 2000 diluted HRP label
37 DEG C of goat-anti chicken enzyme labelled antibody effect 1.5h, PBST washs 3 times, and substrate solution acts on 5min, develop the color in chemiDOC,
As a result totivirus band is very weak, and the nucleotides sequence after optimizing is classified as the gene of SEQ ID NO:3 in e. coli expression
Fiber protein band is very bright, illustrates that the antigen protein immunogenicity of expression is good.
3.4 AGP are detected carries out fine jade expansion detection for the totivirus that the supernatant of expression separates simultaneously.The supernatant of results expression
It is 1:500 that fine jade, which expands potency, and totivirus positive control only has 1:4.This illustrates that the immunogenicity of the albumen after present invention optimization is good.
The preparation of 4 vaccine of embodiment
1, subunit vaccine is prepared
(1) mutually preparation takes 95 parts of white oil for animals to oil, 1 part of aluminum stearate, is placed in oily phase preparation tank after being heated to 80 DEG C, then
5 parts of Jia Siben -80, until 30min is maintained when temperature reaches 115 DEG C, it is spare after cooling.
(2) by fiber albumen, using normal saline dilution, at 10 μ g/0.2ml, (fine jade expands potency as 1:10), matches for water phase preparation
At antigen for vaccine liquid.5 parts of Tween-80s after taking sterilizing are added in Agitation Tank, while being added 95 parts of antigen for vaccine liquid, stir
20~30min is mixed, Tween-80 is completely dissolved.
(3) emulsification takes 2 parts of oily phase to be put in high-speed shearing machine, starts the stirring of motor slow rotation, while water being added slowly
It 1 part of phase, with 10000r/min, emulsifies 5 minutes.After emulsification, 10ml is taken, with 3000r/min centrifugation 15 minutes, the water that tube bottom is precipitated
Mutually it is no more than 0.5ml.
2, the safety of vaccine and efficacy test
For 2.1 safety verifications with 7 age in days SPF chickens 10, subunit vaccine 2.0ml is subcutaneously injected in every neck, while being set pair
It according to 5, raises, is observed continuously 14 at identical conditions, the feeding of record test chicken, drinking-water and clinical setting.The result shows that
There is not any locally and systemically adverse reaction as caused by vaccine in the vaccine of preparation.
2.2 efficacy tests take SPF chicken 30 of 28 ages in days, are randomly divided into 3 groups, every group 10.One group of immune the present embodiment
The subunit vaccine of preparation, neck subcutaneous injection, dosage are 0.2ml/；One group of immune market vaccine；One group not immune to do pair
According to.21 days after inoculation, blood sampling carries out AGP detection.All immune chickens and control chicken simultaneously, 1 type fowl adenopathy of intramuscular injection embodiment
The virus liquid of malicious isolated viral is (containing about 105.5TCID50/0.1ml), every 0.2ml.Observation 14 days.As a result control chicken 10/10 sends out
Disease, subunit's immune group 10/10 are protected, and market vaccine 6/10 is protected.Illustrate that the subunit vaccine effect is certain, is highly resistant to
The attack (table 1) of aviadenovirus prevalence strain.
Table 1: efficacy test results
Note: 1, fall ill: 1. spiritual depressed, feather is slightly random, closes one's eyes, squats, and serious person is dead；2. dead or appearance is clinical
There are not hydropericardium, the pathological changes such as liver enlargement or kidney enlargement in dead morbidity chicken, dissect in 2~3 days after symptom.
The above results show that subunit vaccine can prevent the infection of currently a popular aviadenovirus, and subunit's epidemic disease well
The antibody that seedling generates is much higher than market vaccine group, so having a good application prospect.
To sum up, the present invention is by the fiber albumen being not optimised and the fiber albumen of optimization while building is used to express antigen
The recombinant vector of albumen, as a result not optimized fiber gene cannot in expression in escherichia coli, and optimize fiber gene
Soluble recombinant protein is successfully given expression in Escherichia coli.Its immune effect of the subunit vaccine of preparation can be pre- well
The attack of anti-popularity strain.
Sequence table
<110>YEBIO Bioengineering Co., Ltd of Qingdao
<120>a kind of aviadenovirus fiber protein subunit vaccine
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1440
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atgctccgag cccctaaaag aagacattcc gaaaacggga tgcccgagac cgaagcggga 60
ccttccccgg ctccaatcaa gcgcgccaaa cgcatggtga gagcatccca gcttgacctg 120
gtttatcctt tcgattacgt ggccgacccc gtcggagggc tcaacccgcc ttttttggga 180
ggctcaggac ccctagtgga ccagggcgga cagcttacgc tcaacgtcac cgatcccatc 240
atcatcaaga acagatcggt ggacttggcc cacgacccca gtctcgatgt caacgcccaa 300
ggtcaactgg cggtggccgt tgaccccgaa ggggccctgg acatcacccc cgatggactg 360
gacgtcaagg tcgacggagt gaccgtaatg gtcaacgatg actgggaact ggccgtaaaa 420
gtcgacccgt ccggcggatt ggattccacc gcgggtggac tgggggtcag cgtggacgac 480
accttgctcg tggatcaggg agaactgggc gtacacctca accaacaagg acccatcact 540
gccgatagca gtggtatcga cctcgagatc aatcctaaca tgttcacggt caacacctcg 600
accggaagcg gagtgctgga actcaaccta aaagcgcagg gaggcatcca agccgagagt 660
tcgggagtgg gcgtttccgt ggatgaaagc ctacagattg tcaacaacac tctggaagtg 720
aaaccggatc ccagcggacc gcttacggtc tccgccaatg gcctagggct gaagtacgac 780
actaataccc tagcggtgac cgcgggcgct ttatccgtgg tcggaggggg gagcgtctcc 840
acacccatcg ctacttttgt ctcgggaagt gccagcctca accgttacaa tgccatgcgt 900
gtcaattcca gcgcgaacgc cttctcttgc gcctactacc ttcaacagtg gaacatacag 960
gggctccttg ttacctccct ctacttgaaa ttggacagcg ccaccatggg gaatcgccct 1020
ggggacctca actccgccaa tgccaaatgg ttcacctttt gggtgtccgc ctatctccag 1080
caatgcaacc cctccgggat tcaagcggga acggtcagcc cctccaccgc caccctcacg 1140
gactttgaac ccatggccaa taggagcgtg accagcccat ggacgtactc ggccaatgga 1200
tactatgaac cacgcgctgg ggaattccaa gtgttcagcc cggtggtaac aggtgcctgg 1260
aagccgggaa acatagggat ccgcgtcctc cccgtgccgg tttcggcctc cggacagcga 1320
tacacccttc tatgctatag tctgcagtgc acgaacacca gcatttttaa tccaaagaac 1380
agcggaacca tgatcgtggg acccgtgctc tacagctgtc cagcgggctc cctcccgtaa 1440
<210> 2
<211> 479
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Met Leu Arg Ala Pro Lys Arg Arg His Ser Glu Asn Gly Met Pro Glu
1 5 10 15
Thr Glu Ala Gly Pro Ser Pro Ala Pro Ile Lys Arg Ala Lys Arg Met
20 25 30
Val Arg Ala Ser Gln Leu Asp Leu Val Tyr Pro Phe Asp Tyr Val Ala
35 40 45
Asp Pro Val Gly Gly Leu Asn Pro Pro Phe Leu Gly Gly Ser Gly Pro
50 55 60
Leu Val Asp Gln Gly Gly Gln Leu Thr Leu Asn Val Thr Asp Pro Ile
65 70 75 80
Ile Ile Lys Asn Arg Ser Val Asp Leu Ala His Asp Pro Ser Leu Asp
85 90 95
Val Asn Ala Gln Gly Gln Leu Ala Val Ala Val Asp Pro Glu Gly Ala
100 105 110
Leu Asp Ile Thr Pro Asp Gly Leu Asp Val Lys Val Asp Gly Val Thr
115 120 125
Val Met Val Asn Asp Asp Trp Glu Leu Ala Val Lys Val Asp Pro Ser
130 135 140
Gly Gly Leu Asp Ser Thr Ala Gly Gly Leu Gly Val Ser Val Asp Asp
145 150 155 160
Thr Leu Leu Val Asp Gln Gly Glu Leu Gly Val His Leu Asn Gln Gln
165 170 175
Gly Pro Ile Thr Ala Asp Ser Ser Gly Ile Asp Leu Glu Ile Asn Pro
180 185 190
Asn Met Phe Thr Val Asn Thr Ser Thr Gly Ser Gly Val Leu Glu Leu
195 200 205
Asn Leu Lys Ala Gln Gly Gly Ile Gln Ala Glu Ser Ser Gly Val Gly
210 215 220
Val Ser Val Asp Glu Ser Leu Gln Ile Val Asn Asn Thr Leu Glu Val
225 230 235 240
Lys Pro Asp Pro Ser Gly Pro Leu Thr Val Ser Ala Asn Gly Leu Gly
245 250 255
Leu Lys Tyr Asp Thr Asn Thr Leu Ala Val Thr Ala Gly Ala Leu Ser
260 265 270
Val Val Gly Gly Gly Ser Val Ser Thr Pro Ile Ala Thr Phe Val Ser
275 280 285
Gly Ser Ala Ser Leu Asn Arg Tyr Asn Ala Met Arg Val Asn Ser Ser
290 295 300
Ala Asn Ala Phe Ser Cys Ala Tyr Tyr Leu Gln Gln Trp Asn Ile Gln
305 310 315 320
Gly Leu Leu Val Thr Ser Leu Tyr Leu Lys Leu Asp Ser Ala Thr Met
325 330 335
Gly Asn Arg Pro Gly Asp Leu Asn Ser Ala Asn Ala Lys Trp Phe Thr
340 345 350
Phe Trp Val Ser Ala Tyr Leu Gln Gln Cys Asn Pro Ser Gly Ile Gln
355 360 365
Ala Gly Thr Val Ser Pro Ser Thr Ala Thr Leu Thr Asp Phe Glu Pro
370 375 380
Met Ala Asn Arg Ser Val Thr Ser Pro Trp Thr Tyr Ser Ala Asn Gly
385 390 395 400
Tyr Tyr Glu Pro Arg Ala Gly Glu Phe Gln Val Phe Ser Pro Val Val
405 410 415
Thr Gly Ala Trp Lys Pro Gly Asn Ile Gly Ile Arg Val Leu Pro Val
420 425 430
Pro Val Ser Ala Ser Gly Gln Arg Tyr Thr Leu Leu Cys Tyr Ser Leu
435 440 445
Gln Cys Thr Asn Thr Ser Ile Phe Asn Pro Lys Asn Ser Gly Thr Met
450 455 460
Ile Val Gly Pro Val Leu Tyr Ser Cys Pro Ala Gly Ser Leu Pro
465 470 475
<210> 3
<211> 900
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gccgacagta gtggtatcga cttggagata aaccccaata tgttcaccgt taacacgagt 60
accggtagtg gcgtactgga gttgaacttg aaagcccagg gcggtattca agccgagagt 120
agtggagtgg gcgtttcggt agacgaaagc ctgcaaattg tcaataatac tctcgaagtt 180
aagcccgatc ctagtggacc tttgacagta tcggcgaatg gactgggact caaatatgat 240
acgaatactc tggctgtcac ggcaggagcc ttgtcggtgg ttggtggtgg ctccgtcagc 300
actcccatcg ccacgtttgt tagcggaagt gcaagcttga atcgttataa cgccatgcgt 360
gtaaatagct ccgccaatgc gttcagttgc gcttattact tgcagcagtg gaatattcaa 420
ggtctgttgg ttacgtccct gtacttgaag ttggacagtg ccaccatggg caataggccg 480
ggtgacttga actccgctaa tgccaagtgg ttcacatttt gggtttcggc ttacctccaa 540
caatgcaatc catcgggaat tcaggccgga accgtatcgc ccagtaccgc aaccctgacc 600
gatttcgagc cgatggccaa ccgttcggtt acgagtcctt ggacatatag tgcgaatggt 660
tattatgaac ctagggctgg tgagtttcag gttttctccc ctgtcgttac tggcgcgtgg 720
aagccgggta acataggcat ccgagttctc cccgtcccgg tctccgcaag cggccaacgg 780
tacaccttgc tgtgttactc gctccaatgt actaatactt ccatattcaa cccgaaaaat 840
agcggaacaa tgatcgtcgg tcctgtcttg tactcgtgtc ctgctggctc gttgccataa 900
<210> 4
<211> 299
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Ala Asp Ser Ser Gly Ile Asp Leu Glu Ile Asn Pro Asn Met Phe Thr
1 5 10 15
Val Asn Thr Ser Thr Gly Ser Gly Val Leu Glu Leu Asn Leu Lys Ala
20 25 30
Gln Gly Gly Ile Gln Ala Glu Ser Ser Gly Val Gly Val Ser Val Asp
35 40 45
Glu Ser Leu Gln Ile Val Asn Asn Thr Leu Glu Val Lys Pro Asp Pro
50 55 60
Ser Gly Pro Leu Thr Val Ser Ala Asn Gly Leu Gly Leu Lys Tyr Asp
65 70 75 80
Thr Asn Thr Leu Ala Val Thr Ala Gly Ala Leu Ser Val Val Gly Gly
85 90 95
Gly Ser Val Ser Thr Pro Ile Ala Thr Phe Val Ser Gly Ser Ala Ser
100 105 110
Leu Asn Arg Tyr Asn Ala Met Arg Val Asn Ser Ser Ala Asn Ala Phe
115 120 125
Ser Cys Ala Tyr Tyr Leu Gln Gln Trp Asn Ile Gln Gly Leu Leu Val
130 135 140
Thr Ser Leu Tyr Leu Lys Leu Asp Ser Ala Thr Met Gly Asn Arg Pro
145 150 155 160
Gly Asp Leu Asn Ser Ala Asn Ala Lys Trp Phe Thr Phe Trp Val Ser
165 170 175
Ala Tyr Leu Gln Gln Cys Asn Pro Ser Gly Ile Gln Ala Gly Thr Val
180 185 190
Ser Pro Ser Thr Ala Thr Leu Thr Asp Phe Glu Pro Met Ala Asn Arg
195 200 205
Ser Val Thr Ser Pro Trp Thr Tyr Ser Ala Asn Gly Tyr Tyr Glu Pro
210 215 220
Arg Ala Gly Glu Phe Gln Val Phe Ser Pro Val Val Thr Gly Ala Trp
225 230 235 240
Lys Pro Gly Asn Ile Gly Ile Arg Val Leu Pro Val Pro Val Ser Ala
245 250 255
Ser Gly Gln Arg Tyr Thr Leu Leu Cys Tyr Ser Leu Gln Cys Thr Asn
260 265 270
Thr Ser Ile Phe Asn Pro Lys Asn Ser Gly Thr Met Ile Val Gly Pro
275 280 285
Val Leu Tyr Ser Cys Pro Ala Gly Ser Leu Pro
290 295
Claims (7)
1. a kind of aviadenovirus fiber antigen protein, which is characterized in that the fiber antigen protein includes:
1) amino acid sequence is the albumen of SEQ ID NO:2；
2) replace on albumen 1), lack, adding one or several amino acid, and there is the albumen of protein function in 1).
2. aviadenovirus fiber antigen protein as described in claim 1, which is characterized in that the fiber antigen protein
Amino acid sequence is SEQ ID NO:4.
3. a kind of gene, which is characterized in that the gene encodes aviadenovirus fiber antigen egg of any of claims 1 or 2
It is white.
4. gene as claimed in claim 3, which is characterized in that encode aviadenovirus fiber antigen egg described in claim 1
The nucleotides sequence of white gene is classified as SEQ ID NO:1.
5. gene as claimed in claim 3, which is characterized in that encode aviadenovirus fiber antigen egg as claimed in claim 2
The nucleotides sequence of white gene is classified as SEQ ID NO:3.
6. aviadenovirus fiber antigen protein of any of claims 1 or 2 is preparing the application in vaccine or diagnostic reagent.
7. a kind of subunit vaccine, which is characterized in that the subunit vaccine is using fowl adenopathy as claimed in claim 2
What malicious fiber antigen protein was prepared as antigen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910442879.8A CN110128508A (en) | 2019-05-25 | 2019-05-25 | A kind of aviadenovirus fiber protein subunit vaccine |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910442879.8A CN110128508A (en) | 2019-05-25 | 2019-05-25 | A kind of aviadenovirus fiber protein subunit vaccine |
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| Publication Number | Publication Date |
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| CN110128508A true CN110128508A (en) | 2019-08-16 |
Family
ID=67581654
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910442879.8A Withdrawn CN110128508A (en) | 2019-05-25 | 2019-05-25 | A kind of aviadenovirus fiber protein subunit vaccine |
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| CN (1) | CN110128508A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112142830A (en) * | 2020-09-18 | 2020-12-29 | 长江大学 | Recombinant avian adenovirus type 4fiber2 protein and preparation method and application thereof |
| CN112940084A (en) * | 2021-03-02 | 2021-06-11 | 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） | Serum type4 avian adenovirus subunit vaccine and application thereof |
| CN113817764A (en) * | 2021-10-28 | 2021-12-21 | 华南农业大学 | Preparation and application of group I4 avian adenovirus Fiber-2 protein |
| CN114085293A (en) * | 2021-11-14 | 2022-02-25 | 吉林大学 | Recombinant protein for preventing avian Ankara disease and construction method and application thereof |
| CN114470182A (en) * | 2022-02-11 | 2022-05-13 | 贵州大学 | Avian adenovirus serum 4-type mRNA vaccine and preparation method and application thereof |
| CN116875563A (en) * | 2023-09-05 | 2023-10-13 | 广东永顺生物制药股份有限公司 | Avian adenovirus, strain, fiber-2 protein, vaccine, subunit vaccine and application |
-
2019
- 2019-05-25 CN CN201910442879.8A patent/CN110128508A/en not_active Withdrawn
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112142830A (en) * | 2020-09-18 | 2020-12-29 | 长江大学 | Recombinant avian adenovirus type 4fiber2 protein and preparation method and application thereof |
| CN112142830B (en) * | 2020-09-18 | 2022-03-22 | 长江大学 | Recombinant avian adenovirus type 4fiber2 protein and preparation method and application thereof |
| CN112940084A (en) * | 2021-03-02 | 2021-06-11 | 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） | Serum type4 avian adenovirus subunit vaccine and application thereof |
| CN112940084B (en) * | 2021-03-02 | 2022-02-11 | 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） | Serum type4 avian adenovirus subunit vaccine and application thereof |
| CN113817764A (en) * | 2021-10-28 | 2021-12-21 | 华南农业大学 | Preparation and application of group I4 avian adenovirus Fiber-2 protein |
| CN114085293A (en) * | 2021-11-14 | 2022-02-25 | 吉林大学 | Recombinant protein for preventing avian Ankara disease and construction method and application thereof |
| CN114470182A (en) * | 2022-02-11 | 2022-05-13 | 贵州大学 | Avian adenovirus serum 4-type mRNA vaccine and preparation method and application thereof |
| CN116875563A (en) * | 2023-09-05 | 2023-10-13 | 广东永顺生物制药股份有限公司 | Avian adenovirus, strain, fiber-2 protein, vaccine, subunit vaccine and application |
| CN116875563B (en) * | 2023-09-05 | 2023-11-24 | 广东永顺生物制药股份有限公司 | Avian adenovirus, strain, fiber-2 protein, vaccine, subunit vaccine and application |
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Application publication date: 20190816 |