CN104762271A - Preparation method and use of duck-derived coronavirus attenuated strain DCV35 - Google Patents
Preparation method and use of duck-derived coronavirus attenuated strain DCV35 Download PDFInfo
- Publication number
- CN104762271A CN104762271A CN201510037732.2A CN201510037732A CN104762271A CN 104762271 A CN104762271 A CN 104762271A CN 201510037732 A CN201510037732 A CN 201510037732A CN 104762271 A CN104762271 A CN 104762271A
- Authority
- CN
- China
- Prior art keywords
- generation
- dcv35
- chicken
- duck
- virulent strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to a preparation method and use of a duck-derived coronavirus attenuated strain DCV35. The preparation method is characterized in that a duck-derived coronavirus virulent strain IBV ZZ2004 (with microbial preservation number of CGMCC NO.3842) separated by the inventor is subjected to passage attenuation, the S1 gene sequence of the screened attenuated strain DCV35 has obvious characteristics, and the variant sequence has stable heredity in 22th-35th generations. In SPF chicken infection adopting an artificial infection test and a horizontal transmission test, the screened attenuated strain DCV35 does not produce virulence enhancement. An experiment proves that after inoculation of a chicken with the attenuated strain, the attenuated strain can effectively activate a chicken immunization system, has good effects of preventing chicken infectious bronchitis and can be used as a vaccine for preventing chicken infectious bronchitis.
Description
Technical field
The present invention relates to the preparations and applicatio of a strain duck-origin coronavirus (Duck Coronaviruse, DCV) low virulent strain DCV35, belong to biological technical field.
Background technology
Avian infectious bronchitis virus (Infectious bronchitis virus, IBV) aquaculture of serious harm China, the financial loss caused because of chicken infectious bronchitis is every year very serious, and prevent this disease main be attenuated vaccine, wherein the most widely used attenuated vaccine is H120 and H52.But because IBV serotype is numerous, IBV genome easily makes a variation again, new IB variant constantly occurs, makes currently available vaccines, existing vaccines can not produce effective provide protection to emerging variation epidemic isolates, causes the control of IBV to become difficulty.
IBV ZZ2004 is from causing immunosuppression and growth-inhibiting to be the one virus be separated to kind duck of cardinal symptom, this virus can cause broiler chicken that similar symptom occurs, sickness rate is 80%, mortality ratio is more than 10%, morbidity chicken group loses weight about 37%, immunity degradation, financial loss reaches more than 30%.Duck source property IBVZZ2004 exists significantly different from domestic reported IBV on pathogenic, causes significant immunosuppression and growth-inhibiting, and all can infect chicken and duck.IBVZZ2004 strain and domestic popular IBV affinity very low, also very low with the affinity of domestic main vaccine strain, the homology as M41 strain, H120, Beaudette, IBV4/91 is 85.2% ~ 87.9%.The correlation properties such as the separation andpreconcentration method of this virus isolated strain ZZ2004, pathogenic and genovariation have all been shown to some extent in patent of invention ZL201010225577.4; in production practice, currently available vaccines, existing vaccines can not produce effective protection to duck source property IBVZZ2004, makes this disease constantly popular.From obtaining research data, the immunogenicity of duck source property IBVZZ2004 strain is good, possesses the fundamental as vaccine virus.Therefore, be necessary to cause weak research to it, to preparing the good low virulent strain of a strain, for the research and development of IBVZZ2004 attenuated vaccine lay the foundation.
Summary of the invention
Technical problem to be solved by this invention is just to provide the preparation method of a kind of duck-origin coronavirus low virulent strain DCV35, and studies its gene expression characteristics and the application in infectious bronchitis prevention.
For solving above technical problem, content of the present invention is: the preparation of duck-origin coronavirus low virulent strain DCV35, it is characterized in that selected strong poison is IBV ZZ2004 the 6th generation kind poison, this virulent strain has delivered the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, and deposit number is: CGMCC NO.3842.Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center; Unit address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date on April 29th, 2010, Classification And Nomenclature: duck-origin coronavirus.
The application of duck-origin coronavirus low virulent strain DCV35 as above, the preparation method of duck-origin coronavirus low virulent strain, it is characterized in that: using this avian infectious bronchitis virus ZZ2004 the 6th generation as seed poison, after 56 DEG C of water-bath 20min, inoculate 9 age in days SPF chicken embryos, every embryo allantoic cavity inoculation 0.2mL, is placed in SPF hatching device 31-33 DEG C and hatches; To discard within 24 hours dead chicken embryo, after 72 hours, select that the allantoic fluid of typical cytopathic chicken embryo is aseptic collects virus; Do same process with preceding method again, go down to posterity, reached for 25 generations continuously, from the 26th generation, adopt 37 DEG C to hatch cultivate, then continuous passage is to the 35th generation, obtain DCV35.Above-mentioned viral S1 gene has obvious feature, and when virus reached for the 22nd generation, S1 gene has 12 bases to there occurs variation, and this variation still keeps stable reaching for the 35th generation.This described low virulent strain seedling can induce target animals generation for the specific immune response of avian infectious bronchitis virus, can prevent the generation of this disease.
First, first through 56 DEG C of water-bath 20min before seed virus inoculated into chick embryo, after inoculation SPF chicken embryo, carry out 31 ~ 33 DEG C of Low-temperature culture, then do same process with preceding method, reached for the 25th generation continuously, from the 26th generation, adopt 37 DEG C of cultivations, then continuous passage is to the 35th generation, obtains attenuated IBDVs DCV35.
Secondly, the variation situation of comparative analysis each generation virus S1 gene order in succeeding generations, when finding to reach for the 22nd generation, S1 gene has 12 bases to there occurs variation, and this variation still keeps stable reaching for the 35th generation.12 variant sites of S1 gene are respectively: 188ntT → C(Val → Ala), 191ntA → G (Asn → Ser), 228ntT → G (Asn → Lys), 232ntA → C (Ser → Arg), 347ntT → A (Phe → Try), 351ntG → T (Arg → Ser), 360nt T → G (Ser → Arg), 454ntG → A (Val r → Ile), 471ntT → G (Ser → Arg), 560ntC → A (Thr → Lys), 1477ntT → C (Ser → Pro), 1497ntA → C (Gln → His).
Finally, EID is passed through
50mensuration, the test of SPF chicken Virulence detection, cohabitation infection and virulence are returned strong test and are measured the pathogenic of DCV35; The immunogenicity of DCV35 is measured by this strain inoculation SPF chicken, deactivation strain inoculation rabbit and protest test.Test-results shows that DCV35 strain strengthens chicken embryo adaptability, does not have lethality to SPF chicken, and live together with only infecting and do not fall ill, virulence is not returned by force; After the target animals of DCV35 immunity simultaneously, body antibody horizontal raises, and the malicious protection ratio of attacking of chicken also raises.
Accordingly, low virulent strain DCV35 of the present invention is through and special causes that incompetent person's section obtains, and S1 gene has obvious genetic stability feature;
By described low virulent strain DCV35 direct immunization animal or to mix with acceptable carrier in immunity thus the Attenuate vaccine obtained or inactivated vaccine can both significantly improve the immunological competence of animal body.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed limitation of the present invention, to amendment or the replacement of the inventive method, step or condition, all belong to scope of the present invention.
Embodiment 1 Low-temperature culture induction low virulent strain
By ZZV6 seed poison (EID
50be 10
-4.88/ 0.2mL) first through 56 DEG C of water-bath 20min, after filtering with the bacterial filter of 0.22um, inoculate 9 age in days SPF chicken embryos, every pickup kind 5 pieces, every embryo 0.2mL, in 31 ~ 32 DEG C of hatchings.Every day observes, chicken embryo dead within 24 hours abandons it, dead chicken embryo puts 4 DEG C of refrigerators temporarily, 72 hours aseptic collects virus, observes the pathology situation of chicken embryo simultaneously, selects the allantoic fluid of typical cytopathic chicken embryo to go down to posterity, every Dai Jun does same process and cultivates, reached for the 25th generation continuously, from the 26th generation, adopt 37 DEG C of cultivations, then reach the 35th generation acquisition DCV35 continuously.
Embodiment 2 passaged virus S1 gene sequencing
Choose in continuous passage process the 7th generation, the 15th generation, 20th ~ 35 generations virus, RT-PCR clones S1 gene, measures its nucleotide sequence.S1 gene primer: upstream (P1): 5'-AGTTATTGGTTAGAGATGTTGGGGA-3', downstream (P2): 5'-C GTA TGG ACA GCT TGT GAC ATT TTC-3'.
Application DNASTAR analysis software by the 7th of order-checking the, 15,20, the base sequence of S1 gene of 22-35 generation poison and female parent poison and the difference of derivation aminoacid sequence, in table 1, respectively the results are shown in appendix A and Appendix B for base and amino acid comparative analysis.When result shows to reach for the 22nd generation, S1 gene has 12 bases to there occurs variation, and this variation still keeps stable reaching for the 35th generation.12 variant sites of S1 gene are respectively: 188ntT → C(Val → Ala), 191ntA → G (Asn → Ser), 228ntT → G (Asn → Lys), 232ntA → C (Ser → Arg), 347ntT → A (Phe → Try), 351ntG → T (Arg → Ser), 360nt T → G (Ser → Arg), 454ntG → A (Val r → Ile), 471ntT → G (Ser → Arg), 560ntC → A (Thr → Lys), 1477ntT → C (Ser → Pro), 1497ntA → C (Gln → His).
The former sequence of table 1 and the 7th, 15,20, the base of 22 ~ 35 generation S1 genovariations and amino acid
| Base/amino acid | 188/63 | 191/64 | 228/76 | 232/78 | 347/116 | 351/117 | 360/120 | 454/152 | 471/157 | 560/187 | 594/ | 610/204 | 1061/354 | 1329/ | 1477/493 | 1497/499 |
| Former sequence | T/V | A/N | T/N | A/S | T/F | G/R | T/S | G/V | T/S | C/T | T/ | A/M | A/Y | T/ | T/S | A/Q |
| V7 | A/ | C/H | ||||||||||||||
| V15 | C/ | G/V | G/C | C/ | ||||||||||||
| V20 | G/V | |||||||||||||||
| V22 | C/A | G/S | G/K | C/R | A/Y | T/S | G/R | A/I | G/R | A/K | C/P | C/H | ||||
| V35 | C/A | G/S | G/K | C/R | A/Y | T/S | G/R | A/I | G/R | A/K | C/P | C/H |
Embodiment 3 DCV35 is to the pathogenicity of SPF chick
(1) DCV35 EID
50after measuring the sterile filters filtration of getting DCV35 1mL 0.22um, do 10 times with physiological saline and increase progressively dilution, each extent of dilution inoculates the SPF chicken embryo of 5 piece of 9 age in days, the every embryo of 0.2mL/, 37 DEG C of cultivations, observational data every day dead germ number, chicken embryo dead within 24 hours abandons it, hatch to 19 ages in days dissection residue embryo alive, using death and dwarf embryo as the criterion of infection.The SPF chicken embryo of 9 ages in days is inoculated in contrast with same dosage physiological saline.EID is calculated with Reed-Muench Er Shi method
50.
Result shows: along with the increase of viral passages number of times, and virus is more and more stronger to the adaptability of SPF chicken embryo, EID
50also increase, calculate the EID of DCV35 through Reed-Muench method
50be 10
-6.75/ 0.2mL.
(2) DCV35 artificial challenge SPF chick 39 5 age in days SPF chick, adopt completely random method to be divided into 3 large groups, the 1st and 2 groups often organize 15 chickens, control group 9 chickens, by IBV ZZ2004 the 6th generation poison (10
4.88eID
50/ 0.2mL), 35 generation poison (10
6.75eID
50/ 0.2mL) after the sterile filter of 0.22 μm, inoculate the 1st group, the 2nd group respectively through eye droppings, collunarium approach, often group inoculation 2mL, the physiological saline of the 3rd group of chicken injection Isodose.From inoculation, every day, observed and recorded incidence, cutd open inspection to the chicken of dead chicken pathology, observed dead chicken pathology.
Result is as shown in table 2, and DCV35 strain inoculation SPF chicken does not fall ill, also not lethal SPF chicken.
The virulence of table 2 IBV ZZ2004 ZZV6 and DCV35 virus to SPF chick
| Group | Dosage | Sickness rate | Case fatality rate |
| ZZV6 | 2mL 10 4.88EID 50 | 71.43% (10/14) | 30% (3/10) |
| DCV35 | 2mL 10 6.75EID 50 | 0% (0/14) | 0% (0/14) |
| Control (3 groups) | The physiological saline of 2mL 1% | 0% (0/9) | 0% (0/9) |
(3) the viral allantoic fluid of DCV35 infected chicken cohabitation infection test DCV35 inoculates 5 age in days SPF chicken 4 through collunarium, eye droppings, and every dosage is not less than 10
6.75eID
50/ 0.2mL, only supports in same SPF shield retaining with group feeding with the healthy SPF chicken 4 of the non-virus inoculation of same age in days, observes 7 days, the clinical manifestation situation of record chicken; Put to death all chickens after 7 days, RT-PCR detection is carried out to the chicken internal organs not inoculating poison that DCV35 supports with group feeding.
Result shows: the SPF chick of 5 inoculated DCV35 and 5 support 7 days with group feeding with age in days SPF chicken, and none is morbidity only.RT-PCR detected result is carried out to the chicken internal organs of living together and is the positive.
(4) DCV35 virulence returns strong test DCV35 for passaged virus liquid (10
6.75eID
50/ 0.2mL) inoculate 5 age in days SPF chicken 5, every inoculate slaughter after 7 days only from the incidence observing chicken from inoculation, get its kidney and tracheae and add physiological saline and make 1:2 and mix.After grinding, centrifuging and taking supernatant liquor, inoculates SPF chick 5 after dual anti-process, and it is the s-generation, so passes for 5 generations continuously by susceptible chick.And the every virus in generation is measured it as RT-PCR whether be with poison.
Result shows: virus returns chicken body 5 generation, and per generation returns and all detects its M gene with RT-PCR is positive, and inoculation chick but has no inoculation chicken any clinical manifestation, illustrates that the virulence of this attenuated virus is stablized.
The immunogenicity determining of embodiment 4 DCV35
(1) the weak poison of DCV35 inoculates the antibody test of SPF chicken serum by SPF fowl raising in negative pressure isolator, free choice feeding and drinking-water, and carries out first immunisation when 15 age in days, and DCV35 dosage of inoculation is 10
6.75eID
50/ 0.2mL.After first immunisation 14 days, often organize the blood that chicken takes 4 chickens at random, the thermostat container that the blood sample of collection is placed in 37 DEG C is hatched 2h, then the centrifugal 10min of 1000r/min, collects serum, is sub-packed in the PCR pipe of 200uL,-20 DEG C of preservations, indirect ELISA method to be used detects the antibody in chicken group serum.
Result is as shown in table 3: inoculated DCV35 strain chicken serum antibody is when Dilution ratio is 1:800, and the value of P/N reaches 2.1, and nonvaccinated control group chicken serum sample antibody detects and is feminine gender.
Table 3 chicken group serum antibody produces level
| Extent of dilution | 1:50 | 1:100 | 1:200 | 1:400 | 1:800 | 1:1600 | 1:3200 | 1:6400 | 1:12800 | 1:25600 | Blank | Negative |
| 1 | 0.901 | 0.818 | 0.664 | 0.488 | 0.355 | 0.200 | 0.175 | 0.145 | 0.123 | 0.112 | 0.087 | 0.096 |
| P/N | 9.385 | 8.52 | 6.916 | 5.083 | 3.697 | 2.080 | 1.822 | 1.479 | 1.51 | 1.166 | ||
| 2 | 0.924 | 0.892 | 0.599 | 0.47 | 0.293 | 0.198 | 0.156 | 0.127 | 0.104 | 0.092 | 0.093 | 0.098 |
| P/N | 9.428 | 9.102 | 6.112 | 4.795 | 2.989 | 2.020 | 1.591 | 1.295 | 1.061 | 0.938 | ||
| 3 | 1.067 | 0.851 | 0.553 | 0.418 | 0.285 | 0.190 | 0.183 | 0.129 | 0.111 | 0.107 | 0.087 | 0.093 |
| P/N | 11.473 | 9.15 | 5.946 | 4.494 | 3.064 | 2.04 | 1.967 | 1.387 | 1.193 | 1.15 | ||
| 4 | 0.809 | 0.595 | 0.372 | 0.294 | 0.205 | 0.157 | 0.133 | 0.108 | 0.114 | 0.107 | 0.098 | 0.093 |
| P/N | 8.698 | 6.397 | 4 | 3.161 | 2.204 | 1.688 | 1.43 | 1.161 | 1.225 | 1.15 |
(2) after the weak malicious immunize rabbit of DCV35, Serum Antibody Detection selects health, body weight at the White Rabbit 2 of about 2.0kg, (inactivation antigen concentration is 2.4mg/mL to the emulsion made of the virus antigen-Freund's complete adjuvant (FAC) of first immunisation deactivation, antigen: FAC volume is 1:1), neck dorsal sc multi-point injection, the each position of 0.2mL/, every rabbit injection 1mL; The emulsion two that the full adjuvant (FIC) that cannots be used up after two weeks is made is exempted from, and dosage is 1mL; After two weeks directly with the 35th generation virus carry out booster immunization.Three exempt from latter 7 days ear edge vein exploitating bloods, detect it tire with indirect ELISA.
Result shows: the albumen wrapper sheet using this laboratory to preserve DCV35 weak malicious 3 is exempted from after rabbit blood sampling carry out ELISA detection, the rabbit of DCV35 immunity can produce good antibody, tires and reaches 1:3200, in table 4.
Table 4 rabbit anteserum antibody horizontal
| Extent of dilution | 1:50 | 1:100 | 1:200 | 1:400 | 1:800 | 1:1600 | 1:3200 | 1:6400 | 1:12800 | 1:25600 | Blank | Negative |
| 1 | 2.038 | 1.508 | 1.378 | 0.977 | 0.805 | 0.506 | 0.479 | 0.305 | 0.234 | 0.215 | 0.104 | 0.152 |
| P/N | 15.18 | 9.921 | 9.065 | 6.427 | 5.625 | 3.328 | 3.151 | 2.006 | 1.539 | 1.414 | ||
| 2 | 2.029 | 1.522 | 1.355 | 1.066 | 0.804 | 0.564 | 0.439 | 0.241 | 0.234 | 0.18 | 0.109 | 0.165 |
| P/N | 12.296 | 9.224 | 8.212 | 6.46 | 5.09 | 3.418 | 2.66 | 1.46 | 1.418 | 1.09 |
(3) protest test 14 SPF chick, at 5 ages in days and 19 ages in days immune DCV35 respectively, dosage of inoculation is every 10
6.75eID
50/ 0.2mL.Two exempt to use ZZV6 to carry out attacking poison after two weeks, and every chicken is through collunarium, eye droppings inoculation 2mL (10
4.88eID
50), establish simultaneously and nonimmunely attack malicious control group.From self tapping poison, every day observes, record morbidity, the death condition of chicken group, cuts open inspection, observe pathological change, Continuous Observation 7 days to dead chicken.
Result shows: DCV35 strain group chicken does not fall ill, and case fatality rate is 0, but not the morbidity of immune group chicken, and occur dead.Its result is as table 5.
Table 5 DCV35 is to the immune protective efficiency of SPF chick
| Group | Dosage | Sickness rate | Case fatality rate |
| DCV35 group | 2mL 10 4.88EID 50 | 0% (0/14) | 0% (0/0) |
| Nonimmune control group | 2mL10 4.88EID 50 | 22.2% (2/9) | 50% (1/2) |
In annex be former sequence, 7,15,20,22 ~ 35 generation base sequence, and former sequence, 7,15,20,22 ~ 35 generation aminoacid sequence.
Organization Applicant
----------------------
Street :
City :
State :
Country :
PostalCode :
PhoneNumber :
FaxNumber :
EmailAddress :
<110> OrganizationName: Henan Science and Technology College
Application Project
-------------------
<120> Title: the preparations and applicatio of duck-origin coronavirus low virulent strain DCV35
<130> AppFileReference : 1
<140> CurrentAppNumber :
<141> CurrentFilingDate : ____-__-__
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
atgttgggga agtcactgtt tttagtgacc attttgtgtg cactatgcag tgcaaacttg 60
tttgattttg ataataatta tgtgtactac taccaaagtg catttaggcc tccaagtggg 120
tggcatttgc aagggggtgc ttatgcagta gtgaattcta ctaattatac taataatgca 180
ggtcaggtaa atgagtgcac tgttggtgtt attaaggacg tctataatca aagtgcggct 240
gctatagcta tgacagcacc tcttcagggt atggcttggt ctaagtcaca attttgtagt 300
gcacactgta acttttctga aattacagtt tttgtcacac attgttttag gagcggtagt 360
gggtcttgtc ctataacagg catgattcca cagaaccata ttcgtatttc tgcaatgaaa 420
aatggcactt tattttataa tttaacagtt agcgtatcta aataccctag ttttaaatct 480
tttcaatgtg ttaacaactt cacatctgtt tatttaaatg gtgatcttgt ttttacttct 540
aacacaacta ctgatgtcac gtcagcaggt gtgtatttta aagcaggtgg acctgtaaat 600
tataatatta tgaaagaatt taatgttctg gcttattttg tcaatggtac tgtgcaagat 660
gtaattctgt gtgatgacac accgagaggc ttgcttgcat gtcaatataa tactggtaat 720
ttttcagatg ggttttaccc ttttactaat tctagtttag ttaaggaaaa gtttattgtt 780
tatcgtgaga atagtgttaa tactactctt actttaacta actatacttt ttataatgtg 840
actaatgccg cgcctaatca aggtggtgtt cattccattt taacttatca aacacaaaca 900
gctcagagtg gctattataa ttttaattta tcatttctga gtagttttgt gtataaagag 960
tctaattaca tgtatgggtc ttaccaccct gcatgtaatt ttagattaga aactattaat 1020
aatggcttgt ggtttaattc actggcagtt tcacttgcat acggaccact tcaaggtggc 1080
tgtaagcaat cagtttttag tagtagagct acatgttgtt atgcttattc gtataacggt 1140
cctcgcgctt gtaaaggcgt ttacagcggt gagttgacac aaagttttga atgtggttta 1200
ttagtttatg ttactaagag cgatggctct cgtatacaaa cagccactga accaccaatt 1260
ataactcaac acaattataa taatattact ttaaatactt gtgttgatta taatatatat 1320
ggcagaactg gccaagggtt tattactaat gtaactgact cagctgctag ctataattat 1380
ttagctaatg cagggttggc tattttagac acttctggtg ccatagacgt ctttgttgta 1440
caaggcgaat atggtcttaa tttttataag gttaattcct gtgaggacat taaccaacag 1500
tttgtagttt ctggtggtaa tatagttggt attctcactt ctcgtaatga aactggttct 1560
cagcttattg agaatcaatt ttatgttaaa ctcactaatg taagtcgtcg tcacagacgt 1620
<212> Type : DNA
<211> Length : 1620
SequenceName: former sequence
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
atgttgggga agtcactgtt tttagtgacc attttgtgtg cactatgcag tgcaaacttg 60
tttgattttg ataataatta tgtgtactac taccaaagtg catttaggcc tccaagtggg 120
tggcatttgc aagggggtgc ttatgcagta gtgaattcta ctaattatac taataatgca 180
ggtcaggtaa atgagtgcac tgttggtgtt attaaggacg tctataatca aagtgcggct 240
gctatagcta tgacagcacc tcttcagggt atggcttggt ctaagtcaca attttgtagt 300
gcacactgta acttttctga aattacagtt tttgtcacac attgttatag gagcggtagt 360
gggtcttgtc ctataacagg catgattcca cagaaccata ttcgtatttc tgcaatgaaa 420
aatggcactt tattttataa tttaacagtt agcgtatcta aataccctag ttttaaatct 480
tttcaatgtg ttaacaactt cacatctgtt tatttaaatg gtgatcttgt ttttacttct 540
aacacaacta ctgatgtcac gtcagcaggt gtgtatttta aagcaggtgg acctgtaaat 600
tataatatta tgaaagaatt taatgttctg gcttattttg tcaatggtac tgtgcaagat 660
gtaattctgt gtgatgacac accgagaggc ttgcttgcat gtcaatataa tactggtaat 720
ttttcagatg ggttttaccc ttttactaat tctagtttag ttaaggaaaa gtttattgtt 780
tatcgtgaga atagtgttaa tactactctt actttaacta actatacttt ttataatgtg 840
actaatgccg cgcctaatca aggtggtgtt cattccattt taacttatca aacacaaaca 900
gctcagagtg gctattataa ttttaattta tcatttctga gtagttttgt gtataaagag 960
tctaattaca tgtatgggtc ttaccaccct gcatgtaatt ttagattaga aactattaat 1020
aatggcttgt ggtttaattc actggcagtt tcacttgcat acggaccact tcaaggtggc 1080
tgtaagcaat cagtttttag tagtagagct acatgttgtt atgcttattc gtataacggt 1140
cctcgcgctt gtaaaggcgt ttacagcggt gagttgacac aaagttttga atgtggttta 1200
ttagtttatg ttactaagag cgatggctct cgtatacaaa cagccactga accaccaatt 1260
ataactcaac acaattataa taatattact ttaaatactt gtgttgatta taatatatat 1320
ggcagaactg gccaagggtt tattactaat gtaactgact cagctgctag ctataattat 1380
ttagctaatg cagggttggc tattttagac acttctggtg ccatagacgt ctttgttgta 1440
caaggcgaat atggtcttaa tttttataag gttaattcct gtgaggacat taaccaccag 1500
tttgtagttt ctggtggtaa tatagttggt attctcactt ctcgtaatga aactggttct 1560
cagcttattg agaatcaatt ttatgttaaa ctcactaatg taagtcgtcg tcacagacgt 1620
<212> Type : DNA
<211> Length : 1620
SequenceName: the 7 generation sequence
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
atgttgggga agtcactgtt tttagtgacc attttgtgtg cactatgcag tgcaaacttg 60
tttgattttg ataataatta tgtgtactac taccaaagtg catttaggcc tccaagtggg 120
tggcatttgc aagggggtgc ttatgcagta gtgaattcta ctaattatac taataatgca 180
ggtcaggtaa atgagtgcac tgttggtgtt attaaggacg tctataatca aagtgcggct 240
gctatagcta tgacagcacc tcttcagggt atggcttggt ctaagtcaca attttgtagt 300
gcacactgta acttttctga aattacagtt tttgtcacac attgttttag gagcggtagt 360
gggtcttgtc ctataacagg catgattcca cagaaccata ttcgtatttc tgcaatgaaa 420
aatggcactt tattttataa tttaacagtt agcgtatcta aataccctag ttttaaatct 480
tttcaatgtg ttaacaactt cacatctgtt tatttaaatg gtgatcttgt ttttacttct 540
aacacaacta ctgatgtcac gtcagcaggt gtgtatttta aagcaggtgg acccgtaaat 600
tataatattg tgaaagaatt taatgttctg gcttattttg tcaatggtac tgtgcaagat 660
gtaattctgt gtgatgacac accgagaggc ttgcttgcat gtcaatataa tactggtaat 720
ttttcagatg ggttttaccc ttttactaat tctagtttag ttaaggaaaa gtttattgtt 780
tatcgtgaga atagtgttaa tactactctt actttaacta actatacttt ttataatgtg 840
actaatgccg cgcctaatca aggtggtgtt cattccattt taacttatca aacacaaaca 900
gctcagagtg gctattataa ttttaattta tcatttctga gtagttttgt gtataaagag 960
tctaattaca tgtatgggtc ttaccaccct gcatgtaatt ttagattaga aactattaat 1020
aatggcttgt ggtttaattc actggcagtt tcacttgcat gcggaccact tcaaggtggc 1080
tgtaagcaat cagtttttag tagtagagct acatgttgtt atgcttattc gtataacggt 1140
cctcgcgctt gtaaaggcgt ttacagcggt gagttgacac aaagttttga atgtggttta 1200
ttagtttatg ttactaagag cgatggctct cgtatacaaa cagccactga accaccaatt 1260
ataactcaac acaattataa taatattact ttaaatactt gtgttgatta taatatatat 1320
ggcagaaccg gccaagggtt tattactaat gtaactgact cagctgctag ctataattat 1380
ttagctaatg cagggttggc tattttagac acttctggtg ccatagacgt ctttgttgta 1440
caaggcgaat atggtcttaa tttttataag gttaattcct gtgaggacat taaccaacag 1500
tttgtagttt ctggtggtaa tatagttggt attctcactt ctcgtaatga aactggttct 1560
cagcttattg agaatcaatt ttatgttaaa ctcactaatg taagtcgtcg tcacagacgt 1620
<212> Type : DNA
<211> Length : 1620
SequenceName: the 15 generation sequence
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
atgttgggga agtcactgtt tttagtgacc attttgtgtg cactatgcag tgcaaacttg 60
tttgattttg ataataatta tgtgtactac taccaaagtg catttaggcc tccaagtggg 120
tggcatttgc aagggggtgc ttatgcagta gtgaattcta ctaattatac taataatgca 180
ggtcaggtaa atgagtgcac tgttggtgtt attaaggacg tctataatca aagtgcggct 240
gctatagcta tgacagcacc tcttcagggt atggcttggt ctaagtcaca attttgtagt 300
gcacactgta acttttctga aattacagtt tttgtcacac attgttttag gagcggtagt 360
gggtcttgtc ctataacagg catgattcca cagaaccata ttcgtatttc tgcaatgaaa 420
aatggcactt tattttataa tttaacagtt agcgtatcta aataccctag ttttaaatct 480
tttcaatgtg ttaacaactt cacatctgtt tatttaaatg gtgatcttgt ttttacttct 540
aacacaacta ctgatgtcac gtcagcaggt gtgtatttta aagcaggtgg acctgtaaat 600
tataatattg tgaaagaatt taatgttctg gcttattttg tcaatggtac tgtgcaagat 660
gtaattctgt gtgatgacac accgagaggc ttgcttgcat gtcaatataa tactggtaat 720
ttttcagatg ggttttaccc ttttactaat tctagtttag ttaaggaaaa gtttattgtt 780
tatcgtgaga atagtgttaa tactactctt actttaacta actatacttt ttataatgtg 840
actaatgccg cgcctaatca aggtggtgtt cattccattt taacttatca aacacaaaca 900
gctcagagtg gctattataa ttttaattta tcatttctga gtagttttgt gtataaagag 960
tctaattaca tgtatgggtc ttaccaccct gcatgtaatt ttagattaga aactattaat 1020
aatggcttgt ggtttaattc actggcagtt tcacttgcat acggaccact tcaaggtggc 1080
tgtaagcaat cagtttttag tagtagagct acatgttgtt atgcttattc gtataacggt 1140
cctcgcgctt gtaaaggcgt ttacagcggt gagttgacac aaagttttga atgtggttta 1200
ttagtttatg ttactaagag cgatggctct cgtatacaaa cagccactga accaccaatt 1260
ataactcaac acaattataa taatattact ttaaatactt gtgttgatta taatatatat 1320
ggcagaactg gccaagggtt tattactaat gtaactgact cagctgctag ctataattat 1380
ttagctaatg cagggttggc tattttagac acttctggtg ccatagacgt ctttgttgta 1440
caaggcgaat atggtcttaa tttttataag gttaattcct gtgaggacat taaccaacag 1500
tttgtagttt ctggtggtaa tatagttggt attctcactt ctcgtaatga aactggttct 1560
cagcttattg agaatcaatt ttatgttaaa ctcactaatg taagtcgtcg tcacagacgt 1620
<212> Type : DNA
<211> Length : 1620
SequenceName: the 20 generation sequence
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
atgttgggga agtcactgtt tttagtgacc attttgtgtg cactatgcag tgcaaacttg 60
tttgattttg ataataatta tgtgtactac taccaaagtg catttaggcc tccaagtggg 120
tggcatttgc aagggggtgc ttatgcagta gtgaattcta ctaattatac taataatgca 180
ggtcaggtaa atgagtgcac tgttggtgtt attaaggacg tctataatca aagtgcggct 240
gctatagcta tgacagcacc tcttcagggt atggcttggt ctaagtcaca attttgtagt 300
gcacactgta acttttctga aattacagtt tttgtcacac attgttatag tagcggtagg 360
gggtcttgtc ctataacagg catgattcca cagaaccata ttcgtatttc tgcaatgaaa 420
aatggcactt tattttataa tttaacagtt agcatatcta aataccctag gtttaaatct 480
tttcaatgtg ttaacaactt cacatctgtt tatttaaatg gtgatcttgt ttttacttct 540
aacacaacta ctgatgtcac gtcagcaggt gtgtatttta aagcaggtgg acctgtaaat 600
tataatatta tgaaagaatt taatgttctg gcttattttg tcaatggtac tgtgcaagat 660
gtaattctgt gtgatgacac accgagaggc ttgcttgcat gtcaatataa tactggtaat 720
ttttcagatg ggttttaccc ttttactaat tctagtttag ttaaggaaaa gtttattgtt 780
tatcgtgaga atagtgttaa tactactctt actttaacta actatacttt ttataatgtg 840
actaatgccg cgcctaatca aggtggtgtt cattccattt taacttatca aacacaaaca 900
gctcagagtg gctattataa ttttaattta tcatttctga gtagttttgt gtataaagag 960
tctaattaca tgtatgggtc ttaccaccct gcatgtaatt ttagattaga aactattaat 1020
aatggcttgt ggtttaattc actggcagtt tcacttgcat acggaccact tcaaggtggc 1080
tgtaagcaat cagtttttag tagtagagct acatgttgtt atgcttattc gtataacggt 1140
cctcgcgctt gtaaaggcgt ttacagcggt gagttgacac aaagttttga atgtggttta 1200
ttagtttatg ttactaagag cgatggctct cgtatacaaa cagccactga accaccaatt 1260
ataactcaac acaattataa taatattact ttaaatactt gtgttgatta taatatatat 1320
ggcagaactg gccaagggtt tattactaat gtaactgact cagctgctag ctataattat 1380
ttagctaatg cagggttggc tattttagac acttctggtg ccatagacgt ctttgttgta 1440
caaggcgaat atggtcttaa tttttataag gttaatccct gtgaggacat taaccaacag 1500
tttgtagttt ctggtggtaa tatagttggt attctcactt ctcgtaatga aactggttct 1560
cagcttattg agaatcaatt ttatgttaaa ctcactaatg taagtcgtcg tcacagacgt 1620
<212> Type : DNA
<211> Length : 1620
SequenceName: the 22 generation sequence
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
atgttgggga agtcactgtt tttagtgacc attttgtgtg cactatgcag tgcaaacttg 60
tttgattttg ataataatta tgtgtactac taccaaagtg catttaggcc tccaagtggg 120
tggcatttgc aagggggtgc ttatgcagta gtgaattcta ctaattatac taataatgca 180
ggtcaggtaa atgagtgcac tgttggtgtt attaaggacg tctataatca aagtgcggct 240
gctatagcta tgacagcacc tcttcagggt atggcttggt ctaagtcaca attttgtagt 300
gcacactgta acttttctga aattacagtt tttgtcacac attgttatag tagcggtagg 360
gggtcttgtc ctataacagg catgattcca cagaaccata ttcgtatttc tgcaatgaaa 420
aatggcactt tattttataa tttaacagtt agcatatcta aataccctag gtttaaatct 480
tttcaatgtg ttaacaactt cacatctgtt tatttaaatg gtgatcttgt ttttacttct 540
aacacaacta ctgatgtcac gtcagcaggt gtgtatttta aagcaggtgg acctgtaaat 600
tataatatta tgaaagaatt taatgttctg gcttattttg tcaatggtac tgtgcaagat 660
gtaattctgt gtgatgacac accgagaggc ttgcttgcat gtcaatataa tactggtaat 720
ttttcagatg ggttttaccc ttttactaat tctagtttag ttaaggaaaa gtttattgtt 780
tatcgtgaga atagtgttaa tactactctt actttaacta actatacttt ttataatgtg 840
actaatgccg cgcctaatca aggtggtgtt cattccattt taacttatca aacacaaaca 900
gctcagagtg gctattataa ttttaattta tcatttctga gtagttttgt gtataaagag 960
tctaattaca tgtatgggtc ttaccaccct gcatgtaatt ttagattaga aactattaat 1020
aatggcttgt ggtttaattc actggcagtt tcacttgcat acggaccact tcaaggtggc 1080
tgtaagcaat cagtttttag tagtagagct acatgttgtt atgcttattc gtataacggt 1140
cctcgcgctt gtaaaggcgt ttacagcggt gagttgacac aaagttttga atgtggttta 1200
ttagtttatg ttactaagag cgatggctct cgtatacaaa cagccactga accaccaatt 1260
ataactcaac acaattataa taatattact ttaaatactt gtgttgatta taatatatat 1320
ggcagaactg gccaagggtt tattactaat gtaactgact cagctgctag ctataattat 1380
ttagctaatg cagggttggc tattttagac acttctggtg ccatagacgt ctttgttgta 1440
caaggcgaat atggtcttaa tttttataag gttaatccct gtgaggacat taaccaacag 1500
tttgtagttt ctggtggtaa tatagttggt attctcactt ctcgtaatga aactggttct 1560
cagcttattg agaatcaatt ttatgttaaa ctcactaatg taagtcgtcg tcacagacgt 1620
<212> Type : DNA
<211> Length : 1620
SequenceName: the 35 generation sequence
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
MLGKSLFLVTILCALCSANLFDFDNNYVYYYQSAFRPPSGWHLQGGAYAVVNSTNYTNNAGQVNECTVGVIKDVYNQSAAAIAMTAPLQGMAWSKSQFCSAHCNFSEITVFVTHCFRSGSGSCPITGMIPQNHIRISAMKNGTLFYNLTVSVSKYPSFKSFQCVNNFTSVYLNGDLVFTSNTTTDVTSAGVYFKAGGPVNYNIMKEFNVLAYFVNGTVQDVILCDDTPRGLLACQYNTGNFSDGFYPFTNSSLVKEKFIVYRENSVNTTLTLTNYTFYNVTNAAPNQGGVHSILTYQTQTAQSGYYNFNLSFLSSFVYKESNYMYGSYHPACNFRLETINNGLWFNSLAVSLAYGPLQGGCKQSVFSSRATCCYAYSYNGPRACKGVYSGELTQSFECGLLVYVTKSDGSRIQTATEPPIITQHNYNNITLNTCVDYNIYGRTGQGFITNVTDSAASYNYLANAGLAILDTSGAIDVFVVQGEYGLNFYKVNSCEDINQQFVVSGGNIVGILTSRNETGSQLIENQFYVKLTNVSRRHRR
<212> Type : PRT
<211> Length : 540
SequenceName: former sequence amino acid
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
MLGKSLFLVT ILCALCSANL FDFDNNYVYY YQSAFRPPSG WHLQGGAYAV VNSTNYTNNA 60
GQVNECTVGV IKDVYNQSAA AIAMTAPLQG MAWSKSQFCS AHCNFSEITV FVTHCFRSGS 120
GSCPITGMIP QNHIRISAMK NGTLFYNLTV SVSKYPSFKS FQCVNNFTSV YLNGDLVFTS 180
NTTTDVTSAG VYFKAGGPVN YNIMKEFNVL AYFVNGTVQD VILCDDTPRG LLACQYNTGN 240
FSDGFYPFTN SSLVKEKFIV YRENSVNTTL TLTNYTFYNV TNAAPNQGGV HSILTYQTQT 300
AQSGYYNFNL SFLSSFVYKE SNYMYGSYHP ACNFRLETIN NGLWFNSLAV SLAYGPLQGG 360
CKQSVFSSRA TCCYAYSYNG PRACKGVYSG ELTQSFECGL LVYVTKSDGS RIQTATEPPI 420
ITQHNYNNIT LNTCVDYNIY GRTGQGFITN VTDSAASYNY LANAGLAILD TSGAIDVFVV 480
QGEYGLNFYK VNSCEDINQQ FVVSGGNIVG ILTSRNETGS QLIENQFYVK LTNVSRRHRR 540
<212> Type : PRT
<211> Length : 540
SequenceName: for amino acid the 7th
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
MLGKSLFLVT ILCALCSANL FDFDNNYVYY YQSAFRPPSG WHLQGGAYAV VNSTNYTNNA 60
GQVNECTVGV IKDVYNQSAA AIAMTAPLQG MAWSKSQFCS AHCNFSEITV FVTHCFRSGS 120
GSCPITGMIP QNHIRISAMK NGTLFYNLTV SVSKYPSFKS FQCVNNFTSV YLNGDLVFTS 180
NTTTDVTSAG VYFKAGGPVN YNIMKEFNVL AYFVNGTVQD VILCDDTPRG LLACQYNTGN 240
FSDGFYPFTN SSLVKEKFIV YRENSVNTTL TLTNYTFYNV TNAAPNQGGV HSILTYQTQT 300
AQSGYYNFNL SFLSSFVYKE SNYMYGSYHP ACNFRLETIN NGLWFNSLAV SLAYGPLQGG 360
CKQSVFSSRA TCCYAYSYNG PRACKGVYSG ELTQSFECGL LVYVTKSDGS RIQTATEPPI 420
ITQHNYNNIT LNTCVDYNIY GRTGQGFITN VTDSAASYNY LANAGLAILD TSGAIDVFVV 480
QGEYGLNFYK VNSCEDINQQ FVVSGGNIVG ILTSRNETGS QLIENQFYVK LTNVSRRHRR 540
<212> Type : PRT
<211> Length : 540
SequenceName: the 15 generation amino acid
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
MLGKSLFLVT ILCALCSANL FDFDNNYVYY YQSAFRPPSG WHLQGGAYAV VNSTNYTNNA 60
GQVNECTVGV IKDVYNQSAA AIAMTAPLQG MAWSKSQFCS AHCNFSEITV FVTHCFRSGS 120
GSCPITGMIP QNHIRISAMK NGTLFYNLTV SVSKYPSFKS FQCVNNFTSV YLNGDLVFTS 180
NTTTDVTSAG VYFKAGGPVN YNIMKEFNVL AYFVNGTVQD VILCDDTPRG LLACQYNTGN 240
FSDGFYPFTN SSLVKEKFIV YRENSVNTTL TLTNYTFYNV TNAAPNQGGV HSILTYQTQT 300
AQSGYYNFNL SFLSSFVYKE SNYMYGSYHP ACNFRLETIN NGLWFNSLAV SLAYGPLQGG 360
CKQSVFSSRA TCCYAYSYNG PRACKGVYSG ELTQSFECGL LVYVTKSDGS RIQTATEPPI 420
ITQHNYNNIT LNTCVDYNIY GRTGQGFITN VTDSAASYNY LANAGLAILD TSGAIDVFVV 480
QGEYGLNFYK VNSCEDINQQ FVVSGGNIVG ILTSRNETGS QLIENQFYVK LTNVSRRHRR 540
<212> Type : PRT
<211> Length : 540
SequenceName: the 20 generation amino acid
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
MLGKSLFLVT ILCALCSANL FDFDNNYVYY YQSAFRPPSG WHLQGGAYAV VNSTNYTNNA 60
GQVNECTVGV IKDVYNQSAA AIAMTAPLQG MAWSKSQFCS AHCNFSEITV FVTHCFRSGS 120
GSCPITGMIP QNHIRISAMK NGTLFYNLTV SVSKYPSFKS FQCVNNFTSV YLNGDLVFTS 180
NTTTDVTSAG VYFKAGGPVN YNIMKEFNVL AYFVNGTVQD VILCDDTPRG LLACQYNTGN 240
FSDGFYPFTN SSLVKEKFIV YRENSVNTTL TLTNYTFYNV TNAAPNQGGV HSILTYQTQT 300
AQSGYYNFNL SFLSSFVYKE SNYMYGSYHP ACNFRLETIN NGLWFNSLAV SLAYGPLQGG 360
CKQSVFSSRA TCCYAYSYNG PRACKGVYSG ELTQSFECGL LVYVTKSDGS RIQTATEPPI 420
ITQHNYNNIT LNTCVDYNIY GRTGQGFITN VTDSAASYNY LANAGLAILD TSGAIDVFVV 480
QGEYGLNFYK VNSCEDINQQ FVVSGGNIVG ILTSRNETGS QLIENQFYVK LTNVSRRHRR 540
<212> Type : PRT
<211> Length : 540
SequenceName: the 22 generation amino acid
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
MLGKSLFLVTILCALCSANLFDFDNNYVYYYQSAFRPPSGWHLQGGAYAVVNSTNYTNNAGQVNECTVGVIKDVYNQSAAAIAMTAPLQGMAWSKSQFCSAHCNFSEITVFVTHCFRSGSGSCPITGMIPQNHIRISAMKNGTLFYNLTVSVSKYPSFKSFQCVNNFTSVYLNGDLVFTSNTTTDVTSAGVYFKAGGPVNYNIMKEFNVLAYFVNGTVQDVILCDDTPRGLLACQYNTGNFSDGFYPFTNSSLVKEKFIVYRENSVNTTLTLTNYTFYNVTNAAPNQGGVHSILTYQTQTAQSGYYNFNLSFLSSFVYKESNYMYGSYHPACNFRLETINNGLWFNSLAVSLAYGPLQGGCKQSVFSSRATCCYAYSYNGPRACKGVYSGELTQSFECGLLVYVTKSDGSRIQTATEPPIITQHNYNNITLNTCVDYNIYGRTGQGFITNVTDSAASYNYLANAGLAILDTSGAIDVFVVQGEYGLNFYKVNSCEDINQQFVVSGGNIVGILTSRNETGSQLIENQFYVKLTNVSRRHRR
<212> Type : PRT
<211> Length : 540
SequenceName: the 35 generation amino acid
SequenceDescription :
Organization Applicant
----------------------
Street :
City :
State :
Country :
PostalCode :
PhoneNumber :
FaxNumber :
EmailAddress :
<110> OrganizationName: Henan Science and Technology College
Application Project
-------------------
<120> Title: the preparations and applicatio of duck-origin coronavirus low virulent strain DCV35
<130> AppFileReference : 1
<140> CurrentAppNumber :
<141> CurrentFilingDate : ____-__-__
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
atgttgggga agtcactgtt tttagtgacc attttgtgtg cactatgcag tgcaaacttg 60
tttgattttg ataataatta tgtgtactac taccaaagtg catttaggcc tccaagtggg 120
tggcatttgc aagggggtgc ttatgcagta gtgaattcta ctaattatac taataatgca 180
ggtcaggtaa atgagtgcac tgttggtgtt attaaggacg tctataatca aagtgcggct 240
gctatagcta tgacagcacc tcttcagggt atggcttggt ctaagtcaca attttgtagt 300
gcacactgta acttttctga aattacagtt tttgtcacac attgttttag gagcggtagt 360
gggtcttgtc ctataacagg catgattcca cagaaccata ttcgtatttc tgcaatgaaa 420
aatggcactt tattttataa tttaacagtt agcgtatcta aataccctag ttttaaatct 480
tttcaatgtg ttaacaactt cacatctgtt tatttaaatg gtgatcttgt ttttacttct 540
aacacaacta ctgatgtcac gtcagcaggt gtgtatttta aagcaggtgg acctgtaaat 600
tataatatta tgaaagaatt taatgttctg gcttattttg tcaatggtac tgtgcaagat 660
gtaattctgt gtgatgacac accgagaggc ttgcttgcat gtcaatataa tactggtaat 720
ttttcagatg ggttttaccc ttttactaat tctagtttag ttaaggaaaa gtttattgtt 780
tatcgtgaga atagtgttaa tactactctt actttaacta actatacttt ttataatgtg 840
actaatgccg cgcctaatca aggtggtgtt cattccattt taacttatca aacacaaaca 900
gctcagagtg gctattataa ttttaattta tcatttctga gtagttttgt gtataaagag 960
tctaattaca tgtatgggtc ttaccaccct gcatgtaatt ttagattaga aactattaat 1020
aatggcttgt ggtttaattc actggcagtt tcacttgcat acggaccact tcaaggtggc 1080
tgtaagcaat cagtttttag tagtagagct acatgttgtt atgcttattc gtataacggt 1140
cctcgcgctt gtaaaggcgt ttacagcggt gagttgacac aaagttttga atgtggttta 1200
ttagtttatg ttactaagag cgatggctct cgtatacaaa cagccactga accaccaatt 1260
ataactcaac acaattataa taatattact ttaaatactt gtgttgatta taatatatat 1320
ggcagaactg gccaagggtt tattactaat gtaactgact cagctgctag ctataattat 1380
ttagctaatg cagggttggc tattttagac acttctggtg ccatagacgt ctttgttgta 1440
caaggcgaat atggtcttaa tttttataag gttaattcct gtgaggacat taaccaacag 1500
tttgtagttt ctggtggtaa tatagttggt attctcactt ctcgtaatga aactggttct 1560
cagcttattg agaatcaatt ttatgttaaa ctcactaatg taagtcgtcg tcacagacgt 1620
<212> Type : DNA
<211> Length : 1620
SequenceName: former sequence
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
atgttgggga agtcactgtt tttagtgacc attttgtgtg cactatgcag tgcaaacttg 60
tttgattttg ataataatta tgtgtactac taccaaagtg catttaggcc tccaagtggg 120
tggcatttgc aagggggtgc ttatgcagta gtgaattcta ctaattatac taataatgca 180
ggtcaggtaa atgagtgcac tgttggtgtt attaaggacg tctataatca aagtgcggct 240
gctatagcta tgacagcacc tcttcagggt atggcttggt ctaagtcaca attttgtagt 300
gcacactgta acttttctga aattacagtt tttgtcacac attgttatag gagcggtagt 360
gggtcttgtc ctataacagg catgattcca cagaaccata ttcgtatttc tgcaatgaaa 420
aatggcactt tattttataa tttaacagtt agcgtatcta aataccctag ttttaaatct 480
tttcaatgtg ttaacaactt cacatctgtt tatttaaatg gtgatcttgt ttttacttct 540
aacacaacta ctgatgtcac gtcagcaggt gtgtatttta aagcaggtgg acctgtaaat 600
tataatatta tgaaagaatt taatgttctg gcttattttg tcaatggtac tgtgcaagat 660
gtaattctgt gtgatgacac accgagaggc ttgcttgcat gtcaatataa tactggtaat 720
ttttcagatg ggttttaccc ttttactaat tctagtttag ttaaggaaaa gtttattgtt 780
tatcgtgaga atagtgttaa tactactctt actttaacta actatacttt ttataatgtg 840
actaatgccg cgcctaatca aggtggtgtt cattccattt taacttatca aacacaaaca 900
gctcagagtg gctattataa ttttaattta tcatttctga gtagttttgt gtataaagag 960
tctaattaca tgtatgggtc ttaccaccct gcatgtaatt ttagattaga aactattaat 1020
aatggcttgt ggtttaattc actggcagtt tcacttgcat acggaccact tcaaggtggc 1080
tgtaagcaat cagtttttag tagtagagct acatgttgtt atgcttattc gtataacggt 1140
cctcgcgctt gtaaaggcgt ttacagcggt gagttgacac aaagttttga atgtggttta 1200
ttagtttatg ttactaagag cgatggctct cgtatacaaa cagccactga accaccaatt 1260
ataactcaac acaattataa taatattact ttaaatactt gtgttgatta taatatatat 1320
ggcagaactg gccaagggtt tattactaat gtaactgact cagctgctag ctataattat 1380
ttagctaatg cagggttggc tattttagac acttctggtg ccatagacgt ctttgttgta 1440
caaggcgaat atggtcttaa tttttataag gttaattcct gtgaggacat taaccaccag 1500
tttgtagttt ctggtggtaa tatagttggt attctcactt ctcgtaatga aactggttct 1560
cagcttattg agaatcaatt ttatgttaaa ctcactaatg taagtcgtcg tcacagacgt 1620
<212> Type : DNA
<211> Length : 1620
SequenceName: the 7 generation sequence
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
atgttgggga agtcactgtt tttagtgacc attttgtgtg cactatgcag tgcaaacttg 60
tttgattttg ataataatta tgtgtactac taccaaagtg catttaggcc tccaagtggg 120
tggcatttgc aagggggtgc ttatgcagta gtgaattcta ctaattatac taataatgca 180
ggtcaggtaa atgagtgcac tgttggtgtt attaaggacg tctataatca aagtgcggct 240
gctatagcta tgacagcacc tcttcagggt atggcttggt ctaagtcaca attttgtagt 300
gcacactgta acttttctga aattacagtt tttgtcacac attgttttag gagcggtagt 360
gggtcttgtc ctataacagg catgattcca cagaaccata ttcgtatttc tgcaatgaaa 420
aatggcactt tattttataa tttaacagtt agcgtatcta aataccctag ttttaaatct 480
tttcaatgtg ttaacaactt cacatctgtt tatttaaatg gtgatcttgt ttttacttct 540
aacacaacta ctgatgtcac gtcagcaggt gtgtatttta aagcaggtgg acccgtaaat 600
tataatattg tgaaagaatt taatgttctg gcttattttg tcaatggtac tgtgcaagat 660
gtaattctgt gtgatgacac accgagaggc ttgcttgcat gtcaatataa tactggtaat 720
ttttcagatg ggttttaccc ttttactaat tctagtttag ttaaggaaaa gtttattgtt 780
tatcgtgaga atagtgttaa tactactctt actttaacta actatacttt ttataatgtg 840
actaatgccg cgcctaatca aggtggtgtt cattccattt taacttatca aacacaaaca 900
gctcagagtg gctattataa ttttaattta tcatttctga gtagttttgt gtataaagag 960
tctaattaca tgtatgggtc ttaccaccct gcatgtaatt ttagattaga aactattaat 1020
aatggcttgt ggtttaattc actggcagtt tcacttgcat gcggaccact tcaaggtggc 1080
tgtaagcaat cagtttttag tagtagagct acatgttgtt atgcttattc gtataacggt 1140
cctcgcgctt gtaaaggcgt ttacagcggt gagttgacac aaagttttga atgtggttta 1200
ttagtttatg ttactaagag cgatggctct cgtatacaaa cagccactga accaccaatt 1260
ataactcaac acaattataa taatattact ttaaatactt gtgttgatta taatatatat 1320
ggcagaaccg gccaagggtt tattactaat gtaactgact cagctgctag ctataattat 1380
ttagctaatg cagggttggc tattttagac acttctggtg ccatagacgt ctttgttgta 1440
caaggcgaat atggtcttaa tttttataag gttaattcct gtgaggacat taaccaacag 1500
tttgtagttt ctggtggtaa tatagttggt attctcactt ctcgtaatga aactggttct 1560
cagcttattg agaatcaatt ttatgttaaa ctcactaatg taagtcgtcg tcacagacgt 1620
<212> Type : DNA
<211> Length : 1620
SequenceName: the 15 generation sequence
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
atgttgggga agtcactgtt tttagtgacc attttgtgtg cactatgcag tgcaaacttg 60
tttgattttg ataataatta tgtgtactac taccaaagtg catttaggcc tccaagtggg 120
tggcatttgc aagggggtgc ttatgcagta gtgaattcta ctaattatac taataatgca 180
ggtcaggtaa atgagtgcac tgttggtgtt attaaggacg tctataatca aagtgcggct 240
gctatagcta tgacagcacc tcttcagggt atggcttggt ctaagtcaca attttgtagt 300
gcacactgta acttttctga aattacagtt tttgtcacac attgttttag gagcggtagt 360
gggtcttgtc ctataacagg catgattcca cagaaccata ttcgtatttc tgcaatgaaa 420
aatggcactt tattttataa tttaacagtt agcgtatcta aataccctag ttttaaatct 480
tttcaatgtg ttaacaactt cacatctgtt tatttaaatg gtgatcttgt ttttacttct 540
aacacaacta ctgatgtcac gtcagcaggt gtgtatttta aagcaggtgg acctgtaaat 600
tataatattg tgaaagaatt taatgttctg gcttattttg tcaatggtac tgtgcaagat 660
gtaattctgt gtgatgacac accgagaggc ttgcttgcat gtcaatataa tactggtaat 720
ttttcagatg ggttttaccc ttttactaat tctagtttag ttaaggaaaa gtttattgtt 780
tatcgtgaga atagtgttaa tactactctt actttaacta actatacttt ttataatgtg 840
actaatgccg cgcctaatca aggtggtgtt cattccattt taacttatca aacacaaaca 900
gctcagagtg gctattataa ttttaattta tcatttctga gtagttttgt gtataaagag 960
tctaattaca tgtatgggtc ttaccaccct gcatgtaatt ttagattaga aactattaat 1020
aatggcttgt ggtttaattc actggcagtt tcacttgcat acggaccact tcaaggtggc 1080
tgtaagcaat cagtttttag tagtagagct acatgttgtt atgcttattc gtataacggt 1140
cctcgcgctt gtaaaggcgt ttacagcggt gagttgacac aaagttttga atgtggttta 1200
ttagtttatg ttactaagag cgatggctct cgtatacaaa cagccactga accaccaatt 1260
ataactcaac acaattataa taatattact ttaaatactt gtgttgatta taatatatat 1320
ggcagaactg gccaagggtt tattactaat gtaactgact cagctgctag ctataattat 1380
ttagctaatg cagggttggc tattttagac acttctggtg ccatagacgt ctttgttgta 1440
caaggcgaat atggtcttaa tttttataag gttaattcct gtgaggacat taaccaacag 1500
tttgtagttt ctggtggtaa tatagttggt attctcactt ctcgtaatga aactggttct 1560
cagcttattg agaatcaatt ttatgttaaa ctcactaatg taagtcgtcg tcacagacgt 1620
<212> Type : DNA
<211> Length : 1620
SequenceName: the 20 generation sequence
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
atgttgggga agtcactgtt tttagtgacc attttgtgtg cactatgcag tgcaaacttg 60
tttgattttg ataataatta tgtgtactac taccaaagtg catttaggcc tccaagtggg 120
tggcatttgc aagggggtgc ttatgcagta gtgaattcta ctaattatac taataatgca 180
ggtcaggtaa atgagtgcac tgttggtgtt attaaggacg tctataatca aagtgcggct 240
gctatagcta tgacagcacc tcttcagggt atggcttggt ctaagtcaca attttgtagt 300
gcacactgta acttttctga aattacagtt tttgtcacac attgttatag tagcggtagg 360
gggtcttgtc ctataacagg catgattcca cagaaccata ttcgtatttc tgcaatgaaa 420
aatggcactt tattttataa tttaacagtt agcatatcta aataccctag gtttaaatct 480
tttcaatgtg ttaacaactt cacatctgtt tatttaaatg gtgatcttgt ttttacttct 540
aacacaacta ctgatgtcac gtcagcaggt gtgtatttta aagcaggtgg acctgtaaat 600
tataatatta tgaaagaatt taatgttctg gcttattttg tcaatggtac tgtgcaagat 660
gtaattctgt gtgatgacac accgagaggc ttgcttgcat gtcaatataa tactggtaat 720
ttttcagatg ggttttaccc ttttactaat tctagtttag ttaaggaaaa gtttattgtt 780
tatcgtgaga atagtgttaa tactactctt actttaacta actatacttt ttataatgtg 840
actaatgccg cgcctaatca aggtggtgtt cattccattt taacttatca aacacaaaca 900
gctcagagtg gctattataa ttttaattta tcatttctga gtagttttgt gtataaagag 960
tctaattaca tgtatgggtc ttaccaccct gcatgtaatt ttagattaga aactattaat 1020
aatggcttgt ggtttaattc actggcagtt tcacttgcat acggaccact tcaaggtggc 1080
tgtaagcaat cagtttttag tagtagagct acatgttgtt atgcttattc gtataacggt 1140
cctcgcgctt gtaaaggcgt ttacagcggt gagttgacac aaagttttga atgtggttta 1200
ttagtttatg ttactaagag cgatggctct cgtatacaaa cagccactga accaccaatt 1260
ataactcaac acaattataa taatattact ttaaatactt gtgttgatta taatatatat 1320
ggcagaactg gccaagggtt tattactaat gtaactgact cagctgctag ctataattat 1380
ttagctaatg cagggttggc tattttagac acttctggtg ccatagacgt ctttgttgta 1440
caaggcgaat atggtcttaa tttttataag gttaatccct gtgaggacat taaccaacag 1500
tttgtagttt ctggtggtaa tatagttggt attctcactt ctcgtaatga aactggttct 1560
cagcttattg agaatcaatt ttatgttaaa ctcactaatg taagtcgtcg tcacagacgt 1620
<212> Type : DNA
<211> Length : 1620
SequenceName: the 22 generation sequence
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
atgttgggga agtcactgtt tttagtgacc attttgtgtg cactatgcag tgcaaacttg 60
tttgattttg ataataatta tgtgtactac taccaaagtg catttaggcc tccaagtggg 120
tggcatttgc aagggggtgc ttatgcagta gtgaattcta ctaattatac taataatgca 180
ggtcaggtaa atgagtgcac tgttggtgtt attaaggacg tctataatca aagtgcggct 240
gctatagcta tgacagcacc tcttcagggt atggcttggt ctaagtcaca attttgtagt 300
gcacactgta acttttctga aattacagtt tttgtcacac attgttatag tagcggtagg 360
gggtcttgtc ctataacagg catgattcca cagaaccata ttcgtatttc tgcaatgaaa 420
aatggcactt tattttataa tttaacagtt agcatatcta aataccctag gtttaaatct 480
tttcaatgtg ttaacaactt cacatctgtt tatttaaatg gtgatcttgt ttttacttct 540
aacacaacta ctgatgtcac gtcagcaggt gtgtatttta aagcaggtgg acctgtaaat 600
tataatatta tgaaagaatt taatgttctg gcttattttg tcaatggtac tgtgcaagat 660
gtaattctgt gtgatgacac accgagaggc ttgcttgcat gtcaatataa tactggtaat 720
ttttcagatg ggttttaccc ttttactaat tctagtttag ttaaggaaaa gtttattgtt 780
tatcgtgaga atagtgttaa tactactctt actttaacta actatacttt ttataatgtg 840
actaatgccg cgcctaatca aggtggtgtt cattccattt taacttatca aacacaaaca 900
gctcagagtg gctattataa ttttaattta tcatttctga gtagttttgt gtataaagag 960
tctaattaca tgtatgggtc ttaccaccct gcatgtaatt ttagattaga aactattaat 1020
aatggcttgt ggtttaattc actggcagtt tcacttgcat acggaccact tcaaggtggc 1080
tgtaagcaat cagtttttag tagtagagct acatgttgtt atgcttattc gtataacggt 1140
cctcgcgctt gtaaaggcgt ttacagcggt gagttgacac aaagttttga atgtggttta 1200
ttagtttatg ttactaagag cgatggctct cgtatacaaa cagccactga accaccaatt 1260
ataactcaac acaattataa taatattact ttaaatactt gtgttgatta taatatatat 1320
ggcagaactg gccaagggtt tattactaat gtaactgact cagctgctag ctataattat 1380
ttagctaatg cagggttggc tattttagac acttctggtg ccatagacgt ctttgttgta 1440
caaggcgaat atggtcttaa tttttataag gttaatccct gtgaggacat taaccaacag 1500
tttgtagttt ctggtggtaa tatagttggt attctcactt ctcgtaatga aactggttct 1560
cagcttattg agaatcaatt ttatgttaaa ctcactaatg taagtcgtcg tcacagacgt 1620
<212> Type : DNA
<211> Length : 1620
SequenceName: the 35 generation sequence
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
MLGKSLFLVTILCALCSANLFDFDNNYVYYYQSAFRPPSGWHLQGGAYAVVNSTNYTNNAGQVNECTVGVIKDVYNQSAAAIAMTAPLQGMAWSKSQFCSAHCNFSEITVFVTHCFRSGSGSCPITGMIPQNHIRISAMKNGTLFYNLTVSVSKYPSFKSFQCVNNFTSVYLNGDLVFTSNTTTDVTSAGVYFKAGGPVNYNIMKEFNVLAYFVNGTVQDVILCDDTPRGLLACQYNTGNFSDGFYPFTNSSLVKEKFIVYRENSVNTTLTLTNYTFYNVTNAAPNQGGVHSILTYQTQTAQSGYYNFNLSFLSSFVYKESNYMYGSYHPACNFRLETINNGLWFNSLAVSLAYGPLQGGCKQSVFSSRATCCYAYSYNGPRACKGVYSGELTQSFECGLLVYVTKSDGSRIQTATEPPIITQHNYNNITLNTCVDYNIYGRTGQGFITNVTDSAASYNYLANAGLAILDTSGAIDVFVVQGEYGLNFYKVNSCEDINQQFVVSGGNIVGILTSRNETGSQLIENQFYVKLTNVSRRHRR
<212> Type : PRT
<211> Length : 540
SequenceName: former sequence amino acid
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
MLGKSLFLVT ILCALCSANL FDFDNNYVYY YQSAFRPPSG WHLQGGAYAV VNSTNYTNNA 60
GQVNECTVGV IKDVYNQSAA AIAMTAPLQG MAWSKSQFCS AHCNFSEITV FVTHCFRSGS 120
GSCPITGMIP QNHIRISAMK NGTLFYNLTV SVSKYPSFKS FQCVNNFTSV YLNGDLVFTS 180
NTTTDVTSAG VYFKAGGPVN YNIMKEFNVL AYFVNGTVQD VILCDDTPRG LLACQYNTGN 240
FSDGFYPFTN SSLVKEKFIV YRENSVNTTL TLTNYTFYNV TNAAPNQGGV HSILTYQTQT 300
AQSGYYNFNL SFLSSFVYKE SNYMYGSYHP ACNFRLETIN NGLWFNSLAV SLAYGPLQGG 360
CKQSVFSSRA TCCYAYSYNG PRACKGVYSG ELTQSFECGL LVYVTKSDGS RIQTATEPPI 420
ITQHNYNNIT LNTCVDYNIY GRTGQGFITN VTDSAASYNY LANAGLAILD TSGAIDVFVV 480
QGEYGLNFYK VNSCEDINQQ FVVSGGNIVG ILTSRNETGS QLIENQFYVK LTNVSRRHRR 540
<212> Type : PRT
<211> Length : 540
SequenceName: for amino acid the 7th
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
MLGKSLFLVT ILCALCSANL FDFDNNYVYY YQSAFRPPSG WHLQGGAYAV VNSTNYTNNA 60
GQVNECTVGV IKDVYNQSAA AIAMTAPLQG MAWSKSQFCS AHCNFSEITV FVTHCFRSGS 120
GSCPITGMIP QNHIRISAMK NGTLFYNLTV SVSKYPSFKS FQCVNNFTSV YLNGDLVFTS 180
NTTTDVTSAG VYFKAGGPVN YNIMKEFNVL AYFVNGTVQD VILCDDTPRG LLACQYNTGN 240
FSDGFYPFTN SSLVKEKFIV YRENSVNTTL TLTNYTFYNV TNAAPNQGGV HSILTYQTQT 300
AQSGYYNFNL SFLSSFVYKE SNYMYGSYHP ACNFRLETIN NGLWFNSLAV SLAYGPLQGG 360
CKQSVFSSRA TCCYAYSYNG PRACKGVYSG ELTQSFECGL LVYVTKSDGS RIQTATEPPI 420
ITQHNYNNIT LNTCVDYNIY GRTGQGFITN VTDSAASYNY LANAGLAILD TSGAIDVFVV 480
QGEYGLNFYK VNSCEDINQQ FVVSGGNIVG ILTSRNETGS QLIENQFYVK LTNVSRRHRR 540
<212> Type : PRT
<211> Length : 540
SequenceName: the 15 generation amino acid
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
MLGKSLFLVT ILCALCSANL FDFDNNYVYY YQSAFRPPSG WHLQGGAYAV VNSTNYTNNA 60
GQVNECTVGV IKDVYNQSAA AIAMTAPLQG MAWSKSQFCS AHCNFSEITV FVTHCFRSGS 120
GSCPITGMIP QNHIRISAMK NGTLFYNLTV SVSKYPSFKS FQCVNNFTSV YLNGDLVFTS 180
NTTTDVTSAG VYFKAGGPVN YNIMKEFNVL AYFVNGTVQD VILCDDTPRG LLACQYNTGN 240
FSDGFYPFTN SSLVKEKFIV YRENSVNTTL TLTNYTFYNV TNAAPNQGGV HSILTYQTQT 300
AQSGYYNFNL SFLSSFVYKE SNYMYGSYHP ACNFRLETIN NGLWFNSLAV SLAYGPLQGG 360
CKQSVFSSRA TCCYAYSYNG PRACKGVYSG ELTQSFECGL LVYVTKSDGS RIQTATEPPI 420
ITQHNYNNIT LNTCVDYNIY GRTGQGFITN VTDSAASYNY LANAGLAILD TSGAIDVFVV 480
QGEYGLNFYK VNSCEDINQQ FVVSGGNIVG ILTSRNETGS QLIENQFYVK LTNVSRRHRR 540
<212> Type : PRT
<211> Length : 540
SequenceName: the 20 generation amino acid
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
MLGKSLFLVT ILCALCSANL FDFDNNYVYY YQSAFRPPSG WHLQGGAYAV VNSTNYTNNA 60
GQVNECTVGV IKDVYNQSAA AIAMTAPLQG MAWSKSQFCS AHCNFSEITV FVTHCFRSGS 120
GSCPITGMIP QNHIRISAMK NGTLFYNLTV SVSKYPSFKS FQCVNNFTSV YLNGDLVFTS 180
NTTTDVTSAG VYFKAGGPVN YNIMKEFNVL AYFVNGTVQD VILCDDTPRG LLACQYNTGN 240
FSDGFYPFTN SSLVKEKFIV YRENSVNTTL TLTNYTFYNV TNAAPNQGGV HSILTYQTQT 300
AQSGYYNFNL SFLSSFVYKE SNYMYGSYHP ACNFRLETIN NGLWFNSLAV SLAYGPLQGG 360
CKQSVFSSRA TCCYAYSYNG PRACKGVYSG ELTQSFECGL LVYVTKSDGS RIQTATEPPI 420
ITQHNYNNIT LNTCVDYNIY GRTGQGFITN VTDSAASYNY LANAGLAILD TSGAIDVFVV 480
QGEYGLNFYK VNSCEDINQQ FVVSGGNIVG ILTSRNETGS QLIENQFYVK LTNVSRRHRR 540
<212> Type : PRT
<211> Length : 540
SequenceName: the 22 generation amino acid
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
MLGKSLFLVTILCALCSANLFDFDNNYVYYYQSAFRPPSGWHLQGGAYAVVNSTNYTNNAGQVNECTVGVIKDVYNQSAAAIAMTAPLQGMAWSKSQFCSAHCNFSEITVFVTHCFRSGSGSCPITGMIPQNHIRISAMKNGTLFYNLTVSVSKYPSFKSFQCVNNFTSVYLNGDLVFTSNTTTDVTSAGVYFKAGGPVNYNIMKEFNVLAYFVNGTVQDVILCDDTPRGLLACQYNTGNFSDGFYPFTNSSLVKEKFIVYRENSVNTTLTLTNYTFYNVTNAAPNQGGVHSILTYQTQTAQSGYYNFNLSFLSSFVYKESNYMYGSYHPACNFRLETINNGLWFNSLAVSLAYGPLQGGCKQSVFSSRATCCYAYSYNGPRACKGVYSGELTQSFECGLLVYVTKSDGSRIQTATEPPIITQHNYNNITLNTCVDYNIYGRTGQGFITNVTDSAASYNYLANAGLAILDTSGAIDVFVVQGEYGLNFYKVNSCEDINQQFVVSGGNIVGILTSRNETGSQLIENQFYVKLTNVSRRHRR
<212> Type : PRT
<211> Length : 540
SequenceName: the 35 generation amino acid
SequenceDescription :
Claims (4)
1. the preparation of duck-origin coronavirus low virulent strain DCV35, it is characterized in that selected strong poison is IBV ZZ2004 the 6th generation kind poison, this virulent strain has delivered the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, and deposit number is: CGMCC NO.3842.
2. apply as claimed in claim 1, the preparation method of duck-origin coronavirus low virulent strain, it is characterized in that this avian infectious bronchitis virus ZZ2004 the 6th generation as seed poison, after 56 DEG C of water-bath 20min, inoculate 9 age in days SPF chicken embryos, every embryo allantoic cavity inoculation 0.2mL, is placed in SPF hatching device 31-33 DEG C and hatches; To discard within 24 hours dead chicken embryo, after 72 hours, select that the allantoic fluid of typical cytopathic chicken embryo is aseptic collects virus; Do same process with preceding method again, go down to posterity, reached for 25 generations continuously, from the 26th generation, adopt 37 DEG C to hatch cultivate, then continuous passage is to the 35th generation, obtain DCV35.
3. the low virulent strain DCV35 obtained as claimed in claim 2, its viral S1 gene has obvious feature, and when virus reached for the 22nd generation, S1 gene has 12 bases to there occurs variation, and this variation still keeps stable reaching for the 35th generation.
4. the low virulent strain DCV35 obtained as described in claim 1-3, is characterized in that this low virulent strain seedling can induce target animals generation for the specific immune response of avian infectious bronchitis virus, can prevent the generation of this disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510037732.2A CN104762271A (en) | 2015-01-26 | 2015-01-26 | Preparation method and use of duck-derived coronavirus attenuated strain DCV35 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510037732.2A CN104762271A (en) | 2015-01-26 | 2015-01-26 | Preparation method and use of duck-derived coronavirus attenuated strain DCV35 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104762271A true CN104762271A (en) | 2015-07-08 |
Family
ID=53644364
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510037732.2A Pending CN104762271A (en) | 2015-01-26 | 2015-01-26 | Preparation method and use of duck-derived coronavirus attenuated strain DCV35 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104762271A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107653231A (en) * | 2017-09-28 | 2018-02-02 | 河南科技学院 | Duck-origin coronavirus low virulent strain IBVDCV35 and its application |
| CN110699331A (en) * | 2019-10-25 | 2020-01-17 | 天津瑞普生物技术股份有限公司 | Preparation method and application of goose astrovirus attenuated virus |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0073856A1 (en) * | 1981-08-28 | 1983-03-16 | Gist-Brocades N.V. | Infectious-bronchitis vaccines for poultry, combined infectious-bronchitis vaccines, process for preparing such vaccines, process for preventing infectious bronchitis and infectious-bronchitis virus strain |
| EP0221609A1 (en) * | 1985-10-31 | 1987-05-13 | Duphar International Research B.V | New antigenically active proteins and peptides, and infectious bronchitis virus (IBV) vaccines |
| CN103497934A (en) * | 2013-10-08 | 2014-01-08 | 南京天邦生物科技有限公司 | Avian infectious bronchitis virus vaccine strain (HF2 strain) and application thereof |
| CN103849632A (en) * | 2012-12-06 | 2014-06-11 | 普莱柯生物工程股份有限公司 | S1 gene of low virulent strain of infectious bronchitis and low virulent strain and application thereof |
-
2015
- 2015-01-26 CN CN201510037732.2A patent/CN104762271A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0073856A1 (en) * | 1981-08-28 | 1983-03-16 | Gist-Brocades N.V. | Infectious-bronchitis vaccines for poultry, combined infectious-bronchitis vaccines, process for preparing such vaccines, process for preventing infectious bronchitis and infectious-bronchitis virus strain |
| EP0221609A1 (en) * | 1985-10-31 | 1987-05-13 | Duphar International Research B.V | New antigenically active proteins and peptides, and infectious bronchitis virus (IBV) vaccines |
| CN103849632A (en) * | 2012-12-06 | 2014-06-11 | 普莱柯生物工程股份有限公司 | S1 gene of low virulent strain of infectious bronchitis and low virulent strain and application thereof |
| CN103497934A (en) * | 2013-10-08 | 2014-01-08 | 南京天邦生物科技有限公司 | Avian infectious bronchitis virus vaccine strain (HF2 strain) and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| JI-YONG ZHOU等: "Generation of the transgenic potato expressing full-length spike protein of infectious bronchitis virus", 《JOURNAL OF BIOTECHNOLOGY》 * |
| SHENGWANG LIU等: "A new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and non-vaccinated flocks in China", 《AVIAN PATHOLOGY》 * |
| 刘瑞生等: "鸡传染性支气管炎研究进展", 《科学养禽》 * |
| 王文秀等: "鸡传染性支气管炎病毒变异机理及疫苗研究现状", 《动物医学进展》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107653231A (en) * | 2017-09-28 | 2018-02-02 | 河南科技学院 | Duck-origin coronavirus low virulent strain IBVDCV35 and its application |
| CN110699331A (en) * | 2019-10-25 | 2020-01-17 | 天津瑞普生物技术股份有限公司 | Preparation method and application of goose astrovirus attenuated virus |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102066407A (en) | Novel avian astrovirus | |
| CN102220287B (en) | Avian infectious bronchitis cold adaptation attenuated vaccine strain and application thereof | |
| CN102851257A (en) | Attenuated vaccine strain for avian infectious bronchitis virus and application thereof | |
| CN105671003A (en) | Infectious bronchitis low-virulent live vaccine YX10 D90 strain | |
| CN106282130A (en) | A kind of I group 4 type aviadenovirus, inactivated vaccine and preparation method thereof | |
| CN103497934B (en) | Avian infectious bronchitis virus vaccine strain (HF2 strain) and application thereof | |
| CN105441393B (en) | Very virulent infectious bursal disease virus DF-1 cell adapted strain and its application | |
| CN105802920B (en) | A11 plants of infectious bursal disease virus and its application | |
| CN103710314A (en) | Avian Encephalomyelitis virus attenuated vaccine strain | |
| CN105770881A (en) | Mycoplasma gallisepticum (MG) and mycoplasma synoviae (MS) combined inactivate vaccine | |
| CN101843900B (en) | Bird flu inactivated vaccine and preparation method thereof | |
| CN111057682B (en) | Avian H9N2 subtype avian influenza strain separation identification and application | |
| CN104762271A (en) | Preparation method and use of duck-derived coronavirus attenuated strain DCV35 | |
| CN109097340A (en) | A kind of aviadenovirus, a kind of quadruple vaccine and preparation method thereof | |
| CN107653231B (en) | Duck-origin coronavirus low-virulent strain IBVDCV35 and application thereof | |
| CN104130981A (en) | Application of avian infectious bronchitis virus vaccine strain in preparation of inactivated vaccine | |
| KR101102271B1 (en) | Attenuated Avian Infectious Bronchitis Virus and Vaccine for Avian Infectious Bronchitis Comprising the Same | |
| CN108588034B (en) | Variant rabbit viral hemorrhagic disease virus and application thereof in preparation of inactivated vaccine | |
| CN103721253B (en) | Live avian encephalomylitis and henpox combined vaccine | |
| CN109207437B (en) | Group I8 avian adenovirus strain and application thereof | |
| CN113832113B (en) | Novel duck reovirus attenuated strain and application thereof | |
| CN104195114A (en) | Avian pneumovirus and application thereof | |
| CN102533673B (en) | Chicken infectious bursal disease very virulent cell adapted strain and application thereof | |
| KR101092977B1 (en) | Avian Infectious Bronchitis Virus and Vaccine for Avian Infectious Bronchitis Comprising the Same | |
| CN105727275B (en) | A kind of duck hepatitis bivalent vaccine and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150708 |