CN109402065B

CN109402065B - Muscovy duck subtype C avian metapneumovirus attenuated strain and preparation and application thereof

Info

Publication number: CN109402065B
Application number: CN201810897411.3A
Authority: CN
Inventors: 刘佳佳; 陈�峰; 王占新; 鲁俊鹏; 覃健萍; 操胜
Original assignee: Wens Foodstuff Group Co Ltd
Current assignee: Wens Foodstuff Group Co Ltd
Priority date: 2018-08-08
Filing date: 2018-08-08
Publication date: 2021-07-30
Anticipated expiration: 2038-08-08
Also published as: CN109402065A

Abstract

The invention belongs to the field of microbial animal viruses, and particularly relates to a Muscovy duck subtype C avian metapneumovirus attenuated strain as well as preparation and application thereof. The Muscovy duck subtype C avian metapneumovirus low virulent strain has the preservation number as follows: CCTCC NO of V201823, and the nucleotide sequence is SEQ ID NO: 1, amino acid sequence of SEQ ID NO: 2 to SEQ ID NO: 9. the Muscovy duck subtype C avian metapneumovirus attenuated strain is prepared by subculturing the subtype C avian metapneumovirus S-01 strain to 50 generations. The invention obtains the Muscovy duck subtype C avian metapneumovirus S-01 strain attenuated culture strain for the first time, and lays a foundation for the development of attenuated vaccine.

Description

Muscovy duck subtype C avian metapneumovirus attenuated strain and preparation and application thereof

Technical Field

The invention belongs to the field of microbial animal viruses, and particularly relates to a Muscovy duck subtype C avian metapneumovirus attenuated strain as well as preparation and application thereof.

Background

Avian metapneumopathy is one of the acute highly contagious diseases of upper respiratory infection caused by avian metapneumovirus (aMPV). Avian lung disease was first isolated in young turkeys, with clinical symptoms of sneezing, tracheal rales, adherent secretions in the nose and eyes, conjunctivitis, and the like. It is therefore called turkey rhinotracheitis. In south africa in 1978, aMPV was first isolated and was subsequently reported in europe, asia and south america. The separation of avian pneumovirus A subtype in the chicken flocks with club head syndrome is reported for the first time in 1998 in China, and the separation of B, C subtype in the chicken flocks is carried out in 2012 and subsequently. A C subtype avian metapneumovirus is separated from a duck group which is generated in a duck farm in Guangdong province in 7 months in 2010 and is characterized by cough of ducklings and laying ducks and abnormal reduction of laying rate of the laying ducks, and is named as an S-01 strain, and the duck source C subtype avian metapneumovirus is only reported in France and Canada.

The most effective method for aMPV prevention is currently the use of vaccines, and for already infected poultry, there is no more effective treatment except for the use of different antibiotics to reduce the degree of morbidity and secondary infections. The improvement of the biological safety and the enhancement of the management of the feeding environment are important for the occurrence of the disease, and the vaccination can greatly reduce the morbidity and the economic loss. The avian metapneumovirus is mainly based on cellular immunity, and at present, a plurality of vaccines (attenuated vaccine and inactivated vaccine) aiming at aMPV have been developed, but only aiming at the aMPV-A and aMPV-B subtype of the attenuated live vaccine, the attenuated live vaccine is widely applied in the world. Cross-protection exists between different subtypes of aMPV, especially between aMPV-A and aMPV-B. However, the cross-protection effect between the aMPV-A, aMPV-B strain and the aMPV-C strain is not ideal among different aMPV-C strains. Therefore, it is necessary to develop attenuated vaccines against the aMPV-C subtype strain.

Duck origin subtype C avian metapneumovirus is only reported in France and Canada, and attenuated vaccines aiming at Muscovy duck origin aMPV-C subtype are not reported at present.

Disclosure of Invention

In view of the above, a duck subtype C avian metapneumovirus attenuated strain and a preparation and application thereof are provided for solving the above problems.

The invention is realized by the following technical scheme:

a Muscovy duck subtype C avian metapneumovirus low virulent strain has a preservation number of: CCTCC NO: V201823; the preservation unit: china center for type culture Collection; the preservation date is as follows: year 2018, month 08, day 02; and (4) storage address: wuhan, Wuhan university; the nucleotide sequence is SEQ ID NO: 1 is shown.

A Muscovy duck subtype C avian metapneumovirus low virulent strain has a preservation number of: CCTCC NO: V201823; the preservation unit: china center for type culture Collection; the preservation date is as follows: year 2018, month 08, day 02; and (4) storage address: wuhan, Wuhan university; the reading frame of the attenuated strain comprises an N gene, a P gene, an M2 gene, an SH gene, an F gene, a G gene and an L gene, and the amino acid sequences of the reading frame respectively correspond to the amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO: 9.

a preparation method of Muscovy duck subtype C avian metapneumovirus low virulent strain comprises the step of carrying out subculture on the subtype C avian metapneumovirus S-01 strain for generations of 40-62.

Further, the preparation method of the Muscovy duck subtype C avian metapneumovirus attenuated strain comprises the step of subculturing the subtype C avian metapneumovirus S-01 strain to 50 generations.

Further, the preparation method of the muscovy duck subtype C avian metapneumovirus attenuated strain specifically comprises the following steps:

inoculating a full monolayer African green monkey kidney cell (Vero) T25 cell bottle with Muscovy duck subtype C avian metapneumovirus S-01 strain, and inoculating 500ul of virus solution diluted by 100 times into each bottle; after adsorbing for 2 hours, removing virus liquid, adding 5ml of DMEM culture solution containing 2% fetal calf serum, and culturing for 24-96 hours until cytopathic effect appears; repeatedly freezing and thawing for 3 times, and absorbing virus liquid to inoculate the next generation; in this way, the cells were subcultured continuously up to 40 generations, and then purified continuously 5 times by limiting dilution, followed by subculture.

A duck immune product containing the low virulent strain or the low virulent strain prepared by the method, wherein the content of the low virulent strain in each immune product (namely the dosage of each immunization) is not less than 10^2.5TCID₅₀(ii) a Preferably ≧ 10^4.5TCID₅₀。

The invention has the beneficial effects that:

at present, no report of duck source subtype C avian metapneumovirus attenuated vaccine exists temporarily, and the invention belongs to first separation, passage and weakening culture.

The attenuated strain is relatively stable, and S-01-42P, S-01-52P, S-01-62P is completely attenuated and does not have an unstable phenomenon.

In order to develop a vaccine capable of effectively preventing and controlling duck subtype C avian metapneumovirus, the research continuously subcultures Muscovy duck subtype C avian metapneumovirus S-01 strain on Vero cells to achieve the aim of weakening, and lays a foundation for the development of attenuated vaccines.

After the subtype C avian metapneumovirus S-01 strain is continuously passaged on Vero cells, through different generations of pathogenicity tests, the strain is already attenuated after 40 generations. The titer of the attenuated strain is obviously increased from the original 10 by the titer of the cell before attenuation^3.0TCID₅₀0.1ml to 10⁵TCID₅₀More than 0.1 ml. The attenuated progeny (S-01-50P) of the invention has the best immunogenicity, 10^2.5TCID₅₀0.1ml clinical protection can reach 100%, can not completely prevent detoxification, 10^4.5TCID₅₀0.1ml of the medicine can achieve 100 percent of clinical protection and can completely prevent detoxification. Therefore, S-01-50P is selected as a candidate strain of the attenuated vaccine (attenuated and stable, the first 10 generations and the next 10 generations are attenuated) according to the research result, and 10^4.5TCID₅₀0.1ml can achieve 100% of clinical protection rate and 0% of detoxification rate. Sequencing analysis of the whole gene sequence of the attenuated strain S-01-50P shows that the attenuated strain has 16 amino acid variations compared with the initial isolated strain S-01-5P, and the amino acid variations have great relation with the variation of the virulence of the strain.

Drawings

Fig. 1 shows the nasal cavity of a diseased duck.

Fig. 2 shows the nasal cavity of an unharmed duck.

Detailed Description

To better illustrate the problems addressed by the present invention, the technical solutions adopted and the effects achieved, reference will now be made to the following detailed description and related information. It should be noted that the present disclosure includes, but is not limited to, the following examples and combinations thereof.

The specific techniques or conditions not specified in the examples of the present invention are performed according to the techniques or conditions described in the literature in the art or according to the product specification. The reagents or instruments used are not indicated by manufacturers, and are all conventional products which can be obtained by commercial purchase and the like.

Example 1 subculture of Duck subtype C metapneumovirus

Inoculating a duck C subtype avian metapneumovirus S-01 strain which is separated in a laboratory and causes muscovy duck morbidity in nature, inoculating a full monolayer African green monkey kidney cell (Vero) T25 cell bottle, inoculating 100 times of diluted 500ul virus liquid in each bottle, adsorbing for 2 hours, discarding the virus liquid, adding 5ml of DMEM culture solution containing 2% fetal calf serum, culturing for 24-96 hours, repeating freeze thawing for 3 times until cytopathic effect appears, and absorbing the virus liquid to inoculate the next generation. Subcultured continuously to 40 generations, then purified continuously 5 times by limiting dilution, and then transferred to 62 generations.

Subculture results

Vero cells are inoculated with F3 generation duck embryo yolk liquid, no obvious lesion exists in F1-F3 generation, cytopathic effect, suspension cytocytosis, syncytium occurrence and the like appear in F4 generation on day 4. The F4 generation titer is lower to 10³TCID₅₀0.1 ml. With the increase of the passage number, the virus titer is increased until F60 reaches 10^5.5TCID₅₀0.1 mL. During the passage, the time of cytopathic effect of the cells is shortened along with the increase of the passage times, and the cytopathic effect is changed from the original few suspension dead cells to the original more syncytial formation to the suspension dead cells to the cells with more syncytial formation and less syncytial formation. The titer at each passage is shown in Table 1.

TABLE 1 measurement results of titer of S-01 strain at different generations

Example 2 subtype C avian metapneumovirus S-01 Strain passage sub-pathogenicity test

Selecting 9 groups of S-01 strain passage generation S-01-5P, S-01-17P, S-01-28P, S-01-32P, S-01-40P, S-01-50P, S-01-60P, Vero cell liquid control groups, and applying eye drops to the nose to infect the ducklings with the infection dosage of 10 weeks⁴TCID₅₀And/ml, observing clinical symptoms every day after immunization, recording the number and severity of diseased ducks in each group, collecting cloaca swabs and throat swabs in each group on the 5 th day after immunization, placing the swabs of 5 ducks in an EP (ethylene-propylene-diene) tube of 15ml, and carrying out RT-PCR (reverse transcription-polymerase chain reaction) detection, wherein 6 samples to be detected in each group are totally. The pathogenicity of ducks by different generations is analyzed by observing the clinical symptoms and pathological changes after the autopsy. (RT-PCR detection method refer to Ali A, Reynolds DL: A reverse transcription-polymerase chain reaction assay for the detection of avian pneumovirus(Colorado strain).Avian Dis1999,43:600–603.)

The experimental results are as follows: according to clinical observation, the diseases of F5, F17 and F32 occur, the disease does not occur in other generations, the clinical symptoms mainly include two symptoms of cough and tracheal rale, mucus accumulation in the nasal cavity of the duck with the typical caesarean section symptom can be only seen (as indicated by an arrow in figure 1), and the symptoms do not occur in a negative control (figure 2). F28 showed no morbidity, while F32 showed an unstable viral attenuation at this stage. No disease was observed in F42 and the subsequent generations, indicating that the disease was already weakened. The incidence rates of F5, F17 and F32 are reduced in sequence and are 73%, 33% and 13% respectively. The toxin expelling condition appears after each generation of immunity. The toxin expelling and the incidence rate of each generation are shown in the following table 2.

TABLE 2 toxin expelling and disease onset of each generation of S-01 strain

Example 3 subtype C avian metapneumovirus S-01 Strain attenuated surrogate vaccine efficacy experiment

The weakened generations in the above results are subjected to immunogenicity and different titer immune toxicity attack tests, F40, F50 and F60 are selected in the tests, and the specific implementation operation is that 200 1-day old Muscovy ducks (which are not immunized with aMPV and are detected as negative with aMPV) are randomly divided into 10 groups, and each group comprises 20. The 40, 50 and 60 generations are divided into three gradients (10)^2.5、10^3.5、10^4.5TCID₅₀0.1 ml). Vero cell sap 0 was a 10 th blank. The duckling is immunized in an eye-dropping nose-dropping manner at the time point of 2 w. Clinical symptoms were observed daily after immunization, cloaca swabs and throat swabs were collected on day 5, and 10 ducks in each group were collected randomly and placed in 2ml EP tubes, respectively. 300ul of saline containing 2000U of double antibody was added to each EP tube. Acquired swabRT-PCR detection is carried out. Performing F5 generation toxin counteracting in the manner of eye drop and nose drop in the experimental group at 3w after immunization, wherein the toxin counteracting dosage is 10^4.5TCID₅₀. Clinical symptoms were observed every day after challenge, cloaca swabs and throat swabs were collected 5 days after challenge, and 10 ducks in each group were randomly collected and placed in 2ml EP tubes, respectively. 300ul of physiological saline containing 2000U of the double antibody was added to each EP tube, and the collected swabs were subjected to RT-PCR detection.

The experimental results are as follows: the disease does not occur after each group of immunization, F5 passages are attacked after 3w of immunization, and the swab detection result collected in 5 days shows that only 10 passages are immunized^4.5TCID₅₀The ducks in the group have no toxin expelling condition, and the other ducks in each generation have 10 times^3.5And 10^2.5The situation of expelling toxin occurs. Indicating that 104.5 has the best immune effect. After the blank control group had attacked the toxin, clinical symptoms such as cough appeared, and the condition of expelling toxin was detected in 5 days, indicating that the control group was true. The toxicant elimination statistics are shown in table 3.

TABLE 3 toxin expelling statistics table for efficacy experiment

Example 4 determination and analysis of Gene sequence of attenuated Strain S-01-50P of subtype C avian Metapneumovirus

The C subtype avian metapneumovirus attenuated strain S-01-50P is subjected to whole gene sequence determination and analysis, is compared with the whole gene of the initial isolate S-01, and is subjected to nucleotide and amino acid site variation analysis.

The experimental results are as follows: the length of the weakening strain S-01-50P whole gene sequence is 14112bp (excluding polyA tail), an N protein sequence (395aa), a P protein sequence (295aa), an M protein sequence (255aa), an F protein sequence (538aa), an M2 protein sequence (185aa), an SH protein sequence (176aa), a G protein sequence (586aa) and an L protein sequence (2006 aa). Through the comparison and analysis of the weakening strain S-01-50P and the initial separation lesion generation S-01 whole gene sequence, the homology is relatively high, and 16 amino acids in total have variation. The N gene has no variation; the P gene has 2 amino acid site variations, namely 129Aa R-K and 233Aa M-I; the M gene has a 1-amino acid variation 136Aa V-D; the F gene has 6 amino acid variations, namely 60Aa C-R, 231AaV-I, 334Aa F-L, 453Aa E-K, 485Aa E-K and 528Aa N-S; the gene has 3 amino acid variations, namely 60Aa P-L, 65 Aa-V and 85Aa T-I; the L gene has 4 amino acid variations, namely 14Aa T-P, 30Aa I-L, 186Aa F-L and 1756Aa I-T. These amino acid changes may be associated with attenuated virulence of the strain and enhanced viral replication.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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cacagctaaa tggtccaatt gttttggcac atctctacct gtaagggtga attgcacagc 5820

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aaaggtctga ttgcaagaaa aaactttttg ccttgatttt agggctgatc agctttgaca 6240

tcactatgaa tataatgctg tctgtcatgt atgtggagtc aaatgaggcc ctgagctcat 6300

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cagacaaaca caaaacaaca agagcaacaa cagaggcaga gcaggagaaa caaagcaagc 6540

aaaccacaga gccaggcacc agcaccccga agcacatccc cgcaaggcca agcagcaaat 6600

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cagcacccaa gaacagacag acaacaacca aaaagaccga agcggacacc ccgacaacaa 6720

gcagagcaaa gcaaaccaac aaacccacag ggacagaaac aacacccccc agagcaacaa 6780

cagaaacaga caaggacaaa gaagggccaa cacagcacac aaccaaagag cagcccgaga 6840

caacggcagg agggacaaca actccacagc caagaagaac aaccagcagg ccagccccaa 6900

caacaaacac caaagagggg gcagaaacca ccgggaccag aacaaccaag agcacccaaa 6960

caagcgcaag cccaccaaga ccaacaagaa gcacacccag caaaacagca acaggaacca 7020

acaagagagc cacaacaaca aagggaccga acacagcaag cacagacaga agacaacaga 7080

ccaggacaac cccaaagcaa gaccaacaga cccaaaccaa ggcaaagaca accacaaaca 7140

aggcccacgc aaaagccgcc accacaccag agcacaacac agacacaaca gacagcatga 7200

aagaaaactc caaggaggac aaaaccacca gggaccccag cagcaaagca acaaccaagc 7260

aagaaaacac cagcaaagga acaaccgcaa caaacctcgg aaacaacacc gaggcaggcg 7320

cgagaacacc cccaacaacc accccgacaa ggcacaccac ggaaccagcc acaagcacag 7380

cggggggaca caccaaagca agaactacaa gatggaaaag cacagccgcc agacagccca 7440

caagaaacaa cacaacagca gataccaaaa cagcccagag caaacaaaca acaccggcgc 7500

aactaggcaa caacacaaca ccagagaaca caacaccacc ggacaacaaa agcaacagcc 7560

aaacaaacgt tgcaccaact gaagaaatag aaattggctc aagcctctgg cgaagaaggt 7620

atgtttatgg accctgcaga gagaatgctc tggagcaccc aatgaatccg tgtcttaagg 7680

acaacactac atggatttat ttggacaatg gaaggaattt gccagcaggt tattatgata 7740

gtaaaacaga taaaataatt tgctatggaa tctatagagg aaactcatat tgttacgggc 7800

gaatagagtg cacatgtaaa aacggcacag gactgctatc ctattgctgt aattcataca 7860

actggagtta gtccaagagc aaattagtta attaaaaaga aggaccaagt taaaaatgga 7920

tccactaaat gaaggggttg tgaatgtgta cttgcctgat tcttatttga aaggtgtaat 7980

atcttttagt gaaaccaatg cattaggatc ttgccttcta gggaaacact accttaaaaa 8040

ggataacaca tcaaaagtag ctatagagag ccctgttgta gaacacatca gattgagaaa 8100

tgctttttag actaggatca aggagaaaaa cctaagggta gtagaacccg tcaacatgca 8160

aagtgaagtc atgagaaatt cttacacctg tgaattgaat ttactcaaac aacttataac 8220

aagaagtaaa gacataagct cactcaaact tgacatgata tgtgactggt tgcaattaaa 8280

atcaacaccc gaaaatccat ctgtattaaa gtttgtggat gtaaggtgta ttcccgactg 8340

ggtaagcact tggtttagca gctggtacaa tttaaacaaa cttatattag agtttaggcg 8400

tgaagaggta gcctgcacgg ggtcaatcat atgtaaaaca atagggagca taatgtttat 8460

tatttcatcg ctcgggtgtg tcatcaaaag caacaagagc aaaagaatta gctttatgac 8520

atacaatcag gtgctgacat ggaaagatgt catgttaagc aggttcaatg caaatctatg 8580

tgtgtggatt agcaacagtc ttaacaaaaa tcaggaagga ttaggtctta gaagcaatct 8640

acaagggacc ctggttaaca aattatatga gattgttgac tctatgctca gtgtttgcag 8700

caatgagggg ttcacccttg ttaaagaatt tgaagggttc ataatgagtg agattctgaa 8760

aataacagag catgctcagt ttagtacaag gttcagaaac actttgttga atgggttagt 8820

agaccaactg gtaaagatga ggggcctaaa caggaaaagg gtttcaggca ctgtcttgga 8880

aggaaaccag tatccaatgt atgagacaac attagctaca ctaggggaag ctttaaaaac 8940

aataagactc ctggtaaaca agaacttaga caatgccgct gagctgtact acattttcag 9000

gatttttggc catccaatgg ttgaagagag agaagctatg gacgcagtca gacttaataa 9060

tgagatcaca aaaattttga aattagagag ccttacagag ttgagggggg ccttcatttt 9120

aagaatcata aaaggctttg ttgacacaaa taagagatgg cctaaaatca aaaatctcaa 9180

agtgttgagc agaaggtggg ttatgtactt taaagcaaag agttatccaa gccaattgga 9240

gctgagtagt caagatttcc ttgaattagc aggtgtgcag tttgagcagg aatttgcaat 9300

accagagaga acaaatcttg agatggtctt gaatgataag gccatctcac cacccaaaaa 9360

tttaatatgg tcagtgttcc ctaaaaatta ccttccaaca aacatcaggg aaaaattcac 9420

tgatgaaatg ttcaactcaa gtgaaaagtt gaagacaaga agggtgttgg agtactactt 9480

aaaagacaac aagtttgatc agaatgacct gaaaaaatat gttgttaggc aagagtactt 9540

gggtgataaa gaacatgtag tgtcactgac agggaaagaa cgtgagttaa gtgtgggaag 9600

gatgttcgcg atgcagccag ggaaacaaag gcaagttcag atcttagcag aaaagctact 9660

tgcagacaat atagtaccat tcttccctga gacattgaca aaatatggtg atctagaact 9720

acaaaggatt atggaaataa aatctgagtt gtcatcagta aagtcacgga gaaatgacag 9780

ttacaacaat tatattgcta gggcatcaat agtgactgat cttagtaaat tcaaccaagc 9840

ttttcggtac gagacatctt ctgtctgtgc agatgtagtt gatgaattgc atggcacaca 9900

aagtctcttc tgctggctac atttgactgt gccactgaca actatgattt gcacatatag 9960

acatgctcct ccagaaacag aaggagtgta tgacattgat aaaatcaagg aacaaagtgg 10020

gctttacagg tttcatatgg gggggattga agggtggtgc caaaagttgt ggactatgga 10080

ggctatttct ttattagatg tagtatcagt caaaaaccgt gtccaactaa catcattgtt 10140

aaatggagac aatcaatcta ttgatgttag caaacctgtt aggttgtcac aaggagttga 10200

tgaagttaaa gctgactata gcctggcagt gaaaatgtta aaagaaataa gaaatgctta 10260

caaagacata gggcacaagt taaaagaagg agagacctat atatccagag atctacaatt 10320

catgagcaag gtcattcaat cagagggtgt gatgcatccg tcaccaataa agaaaattct 10380

gagggtcgga ccatggatta atactatctt agatgacatc aagaccagtg cagagtcaat 10440

aggaagccta tgtcaagagt tagagtttag aggtgaaagc ctcttggtta gccttattct 10500

aaggaatttt tggttatacg agttatggat gcatgaatct aaaagtcatc cacttgcagg 10560

gaagcagcta tacagacagc tgagcaagac attagcaata actcaaaaat tctttggaat 10620

aacaaaagaa actgatgttg ttaatctgtg gatgaatgtt ccaatgcaat ttggtggggg 10680

tgaccctgta gtgttgtata ggtcatttta ccggaggaca ccagatttct tgactgaagc 10740

agtaagtcat atgagtgtac tactcaaggt gtatgggaaa gcaaaagagg gatcaaagaa 10800

agattttttc aaagcattat tatcagtaga caaaaataag agggccactc tgaccacatt 10860

aatgagagac cctcaagcag tgggatcaga gaggcaggca agagttacta gtgaaattaa 10920

tagagcagca gtcacaagtg tcttgagctt atcacctaac cagcttttct gtgatagtgc 10980

aatacactac agcagaaatg aagaagaagt tgggttaata gcccaaaaca ttacaccagt 11040

ctatccacac gggctgaggg tgttgtatga gtcattgcca ttccacaagg cggaaaaagt 11100

tgtaaacatg atatctggaa caaaatctat cacaaactta ctacaaagga catcagcaat 11160

caatggagaa gatattgata gagcagtctc aatgatgttg gagaatttag ggttactctc 11220

taggatactg tctgtgagcc aagatgatat aatcttgcct accaaggcaa atggtgatct 11280

gatatgttgt caagtctcca ggactttgag ggaaaggtcc tgggataaca tggaaattgt 11340

tggagtcacc tcaccgagca tagttacatg tatgaacata gtgtattcca gcagctctca 11400

actaaaagga ataacaatcg aaaagttcag cacagataaa actacaagag gtcaaagagg 11460

accaaaaagt ccttgggttg gatccagcac acaggagaaa aaattggttc ctgtttacaa 11520

tagacaaatc ctctcaaagc aacagaaaga acaactagag gcaatcggaa aactgagatg 11580

ggtttataag gggactcaag ggttaaggag gttgttggat aaaatctgca tagggagctt 11640

agggatcagt tacaagaatg ttaagccact cttgccaaga tttatgagtg ttaacttcct 11700

acacagactt tccgtcagta gtagaccaat ggagtttcca gcatctgttc ctgcttacag 11760

aacgaccaat taccatttcg acacaagtcc tgtaaaccag actctgagtg aaagatttgg 11820

aaatgaggac atcaatttgg tgtttcagaa cgctatcagc tgtgggataa gtgtgatgag 11880

tgtagtagag caacttacag gcagaagccc taaacagtta gtaatgattc cccaattaga 11940

agaaattgac ataatgcctc cgcctgtctt ttcaggaaaa tttgattata aactggtaga 12000

aaagatatca tcagaccaac acattttcag ccctgacaaa ttggacctag tgactctagg 12060

aaaaatgtta atgccttcaa caagtggagc aaagtctgat cagttttgga acaggaaaga 12120

aaacttcttc catggcaata atttggtaga gtccctatct gcagcattgg cttgtcattg 12180

gtgcgggatc ttaactgaac agtgtaatga gaacaatatt ttcagaagag aatggggtga 12240

tggttttgtt acagatcatg ccttcataga tttcaaaata tttatcggcg tatttaagac 12300

caaactattg tgtggatggg gatccagggg gggggacatt aaagatcgag acatgataga 12360

cgaatctatt gacaaattaa taagagttga caactcattc tggaggatgt ttagcaaagt 12420

gatacttgaa ccaaaagtaa ggaaaagagt aatgcttttt gatgtgaaga tcttatcact 12480

tgtaggctat gcaggattta aaaattggtt tattgaccac ctgagatcta gtgacttgtg 12540

tgaagtgcca tgggttgtca atgctgatag tgaaattgtt gaggtgtctg ctgtgaaaat 12600

atatttacag ctgctgaggg tgagttctcc cttgaggatt acagtgttga attattcaga 12660

catggcccat gctattacca ggctaataag aaggaaatca atgcatgaca atgtgccatc 12720

aatatctagg acactgtcac ctgcagagtt agctcctgta gttgaaccaa ccgtgcagat 12780

gaacttgttc cctaagatca catttgaaag attgaagaat tatgaaacta tatcagggtc 12840

tactagaggg aagttgacaa gaaattacat ggtgatgttg ccttggcagc atattaatag 12900

gttcaacttt gtattcagct ctaccggttg caaaataagt gtcaaatcat gtattgggaa 12960

gttgattcaa gacttaaatc cgacagtttt ttattttgtg ggagaaggag ccggcaattg 13020

gatggcaaga acagcttgtg agtacccaaa tacaaagttt gtttacagga gtcttaaaga 13080

tgatttagac caccatttcc ctttggaatt tcagagagtc ctagggaaca tgaatagagt 13140

tatagatggt ggagaaggtc tatccatgga cacaacagat gcaactcaga aaactcattg 13200

ggatctgaca catagaattt gtaaagatgc attactgatt actttgtgtg atgctgaatt 13260

taaagatagg gatgactttt tcaagatggt gacactatgg agaaagcatg ttttgtcatg 13320

taggatatgt acaacctatg gaacagatct ctacctgttc gcaaaatatc atgcaaaaga 13380

ggaaagcata aaacttccct attttgttag atcaattgcc acatatgtca tgcaaggcag 13440

taaattgtca ggatctgagt gctatgttct tctgacactt ggtcacccac aacaacctac 13500

catgtcacgg ggaagtacag agctccaaat taaaaatggc tgtgtgtaat gacttcagca 13560

ttcctaggaa agtagaagtg aaggctgttg aggcaaactg caaatcgctg ctttctggcc 13620

taaggacacc tataaacaga gcagaattgg atcgacaaaa gaagatgttg acactaagaa 13680

gttatcattc atcagtagca acagttggag gcagcagagt catcgaatca aaatggttga 13740

gcaagaaagc taccactata atcgagtggt tagaacatat cttgaattcc ccaaaagggg 13800

agttaaacta tgattttttt gaggcactag agaacaccta tccaaatatg gttaaattat 13860

tagataatct tggcagtgct gaactaaaaa aactgatcaa agtgacaggt tacatgctga 13920

tgagtaagaa ataataggag aaaattacct taagagatta cttataattg aggctaaaaa 13980

ctaacatatt catgctgaag gatattaaaa tgatagttgt ttaaatttag caatctgaat 14040

atgaagcaca caatcagcaa ttagttatta aaaaatagag aaactaaaat tggatgaata 14100

cggttttttt gc 14112

<210> 2

<211> 394

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ser Leu Gln Gly Ile Gln Leu Ser Asp Leu Ser Tyr Lys His Ala

1 5 10 15

Ile Leu Lys Glu Ser Gln Tyr Thr Ile Lys Arg Asp Val Gly Thr Thr

20 25 30

Thr Ala Val Thr Pro Ser Ser Leu Gln Arg Glu Val Ser Leu Leu Cys

35 40 45

Gly Glu Ile Leu Tyr Ala Lys His Thr Asp Tyr Ser His Ala Ala Glu

50 55 60

Val Gly Met Gln Tyr Val Ser Thr Thr Leu Gly Ala Glu Arg Thr Gln

65 70 75 80

Gln Ile Leu Lys Asn Ser Gly Ser Glu Val Gln Ala Val Leu Thr Lys

85 90 95

Thr Tyr Ser Leu Gly Lys Gly Lys Asn Ser Lys Gly Glu Asp Leu Gln

100 105 110

Met Leu Asp Ile His Gly Val Glu Lys Ser Trp Val Glu Glu Val Asp

115 120 125

Lys Glu Ala Arg Lys Thr Met Ala Ser Ala Thr Lys Asp Asn Ser Gly

130 135 140

Pro Ile Pro Gln Asn Gln Arg Pro Ser Ser Pro Asp Ala Pro Ile Ile

145 150 155 160

Leu Leu Cys Ile Gly Ala Leu Ile Phe Thr Lys Leu Ala Ser Thr Ile

165 170 175

Glu Val Gly Leu Glu Thr Ala Val Arg Arg Ala Asn Arg Val Leu Asn

180 185 190

Asp Ala Leu Lys Arg Phe Pro Arg Ile Asp Ile Pro Lys Ile Ala Arg

195 200 205

Ser Phe Tyr Asp Leu Phe Glu Gln Lys Val Tyr Tyr Arg Ser Leu Phe

210 215 220

Ile Glu Tyr Gly Lys Ala Leu Gly Ser Ser Ser Thr Gly Ser Lys Ala

225 230 235 240

Glu Ser Leu Phe Val Asn Ile Phe Met Gln Ala Tyr Gly Ala Gly Gln

245 250 255

Thr Met Leu Arg Trp Gly Val Ile Ala Arg Ser Ser Asn Asn Ile Met

260 265 270

Leu Gly His Val Ser Val Gln Ala Glu Leu Lys Gln Val Thr Glu Val

275 280 285

Tyr Asp Leu Val Arg Glu Met Gly Pro Glu Ser Gly Leu Leu His Leu

290 295 300

Arg Gln Ser Pro Lys Ala Gly Leu Leu Ser Leu Ala Asn Cys Pro Asn

305 310 315 320

Phe Ala Ser Val Val Leu Gly Asn Ala Ser Gly Leu Gly Ile Leu Gly

325 330 335

Met Tyr Arg Gly Arg Val Pro Asn Thr Glu Leu Phe Ala Ala Ala Glu

340 345 350

Ser Tyr Ala Arg Ser Leu Lys Glu Ser Asn Lys Ile Asn Phe Ser Ser

355 360 365

Leu Gly Leu Thr Glu Glu Glu Lys Glu Ala Ala Glu Asn Phe Leu Asn

370 375 380

Ile Asn Glu Glu Gly Gln Asn Asp Tyr Glu

385 390

<210> 3

<211> 294

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Ser Phe Pro Glu Gly Lys Asp Ile Leu Leu Met Gly Asn Glu Ala

1 5 10 15

Ala Lys Ala Ala Glu Ala Phe Gln Arg Ser Leu Lys Lys Val Gly His

20 25 30

Arg Arg Thr Gln Ser Ile Val Gly Asp Lys Ile Ile Thr Val Ser Glu

35 40 45

Ile Val Glu Lys Pro Thr Ile Ser Lys Ser Thr Lys Val Thr Thr Pro

50 55 60

Pro Glu Arg Lys Asn Ala Trp Gly Glu Lys Pro Asp Thr Thr Arg Ser

65 70 75 80

Pro Thr Glu Glu Ala Lys Asn Glu Ala Thr Leu Glu Asp Ala Ser Arg

85 90 95

Leu Tyr Glu Glu Val Phe Ala Pro Thr Ser Asp Gly Lys Thr Leu Ala

100 105 110

Glu Lys Gly Lys Glu Thr Thr Glu Lys Pro Lys Lys Lys Val Lys Phe

115 120 125

Lys Asn Asp Glu Ser Gly Lys Tyr Thr Lys Leu Glu Met Glu Ala Leu

130 135 140

Glu Leu Leu Ser Asp Asn Glu Asp Asp Asp Ala Glu Ser Ser Val Leu

145 150 155 160

Thr Phe Glu Glu Lys Asp Thr Ser Ala Leu Ser Leu Glu Ala Arg Leu

165 170 175

Glu Ser Ile Asp Glu Lys Leu Ser Met Ile Leu Gly Leu Leu Arg Thr

180 185 190

Leu Asn Val Ala Thr Ala Gly Pro Thr Ala Ala Arg Asp Gly Ile Arg

195 200 205

Asp Ala Met Val Gly Leu Arg Glu Glu Leu Ile Ala Asp Ile Ile Lys

210 215 220

Glu Ala Lys Gly Lys Ala Ala Glu Ile Met Lys Glu Glu Ala Lys Gln

225 230 235 240

Lys Ser Lys Ile Gly Asn Gly Ser Val Gly Leu Thr Glu Lys Ala Lys

245 250 255

Glu Leu Asn Lys Ile Val Glu Asp Glu Ser Thr Ser Gly Glu Ser Glu

260 265 270

Glu Glu Glu Glu Glu Glu Asp Glu Glu Glu Ser Asn Pro Asp Asp Asp

275 280 285

Leu Tyr Ser Leu Thr Met

290

<210> 4

<211> 254

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Glu Ser Tyr Leu Val Asp Thr Tyr Gln Gly Val Pro Tyr Thr Ala

1 5 10 15

Ala Val Gln Thr Asp Leu Val Glu Lys Asp Gln Leu Pro Ala Arg Leu

20 25 30

Thr Val Trp Phe Pro Leu Phe Gln Thr Asn Thr Pro Pro Thr Val Leu

35 40 45

Leu Glu Gln Leu Lys Thr Leu Thr Ile Thr Thr Leu Tyr Thr Ala Ser

50 55 60

Gln Asn Gly Pro Ile Leu Lys Val Asn Ala Ser Ala Gln Gly Ala Ala

65 70 75 80

Met Ser Ala Leu Pro Lys Ser Phe Asp Val Ser Ala Ser Val Ala Leu

85 90 95

Asp Asp Tyr Ser Lys Leu Glu Phe Asp Lys Leu Thr Val Cys Glu Leu

100 105 110

Lys Ala Val Tyr Leu Thr Thr Met Lys Pro Tyr Gly Met Val Ser Lys

115 120 125

Phe Val Asn Ser Ala Lys Ala Asp Gly Lys Lys Thr His Asp Leu Ile

130 135 140

Ala Leu Cys Asp Phe Leu Asp Leu Glu Lys Gly Val Pro Val Thr Ile

145 150 155 160

Pro Ala Tyr Ile Lys Ser Val Ser Ile Lys Glu Ser Glu Ser Ala Thr

165 170 175

Val Glu Ala Ala Ile Ser Gly Glu Ala Asp Gln Ala Ile Thr Gln Ala

180 185 190

Arg Ile Ala Pro Tyr Ala Gly Leu Ile Met Ile Met Thr Met Asn Asn

195 200 205

Pro Lys Gly Ile Phe Lys Lys Leu Gly Ala Gly Val Gln Val Ile Val

210 215 220

Glu Leu Gly Ala Tyr Val Gln Ala Glu Ser Ile Ser Arg Ile Cys Arg

225 230 235 240

Asn Trp Ser His Gln Gly Thr Arg Tyr Val Leu Lys Ser Arg

245 250

<210> 5

<211> 184

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Ser Arg Lys Ala Pro Cys Lys Tyr Glu Val Arg Gly Lys Cys Asn

1 5 10 15

Arg Gly Ser Glu Cys Lys Phe Asn His Asn Tyr Trp Ser Trp Pro Asp

20 25 30

Arg Tyr Leu Leu Leu Arg Ser Asn Tyr Leu Leu Asn Gln Leu Leu Arg

35 40 45

Asn Thr Asp Arg Ser Asp Gly Leu Ser Leu Ile Ser Gly Ala Gly Arg

50 55 60

Asp Asp Arg Thr Gln Asp Phe Val Leu Gly Ser Thr Asn Val Val Gln

65 70 75 80

Asn Tyr Ile Asp Asn Asn Glu Asn Ile Thr Lys Ala Ser Ala Cys Tyr

85 90 95

Ser Leu Tyr Asn Ile Ile Lys Gln Leu Gln Glu Thr Asp Val Arg Gln

100 105 110

Ala Arg Asp Asn Lys Val Asp Asp Ser Lys His Val Ala Leu His Asn

115 120 125

Leu Val Leu Ser Tyr Met Glu Met Ser Lys Thr Pro Ala Ser Leu Ile

130 135 140

Asn Asn Leu Lys Lys Leu Pro Lys Glu Lys Leu Lys Lys Leu Ala Lys

145 150 155 160

Leu Ile Ile Glu Leu Ser Ala Gly Val Glu Asn Asp Ser Thr Ala Thr

165 170 175

Met Gln Asp Ser Ala Asn Ser Asp

180

<210> 6

<211> 175

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Glu Pro Leu Lys Val Ser Gly Ser Gly Gly Met Pro Met Lys Thr

1 5 10 15

Arg Leu Asn Ile Ile Leu Glu Lys Ser Ile Asn Lys Ile Leu Ile Ile

20 25 30

Leu Gly Leu Leu Leu Thr Ala Ser Thr Val Ile Thr Ile Ile Leu Thr

35 40 45

Val Glu Tyr Ile Arg Val Glu Asn Glu Leu Gln Leu Cys Lys Met Gly

50 55 60

Ala Glu Val Ala Lys Thr Thr Gln Glu Thr Gln Thr Leu Pro Val Lys

65 70 75 80

Thr Thr Pro Met Leu Thr Ser Thr Arg Ser Thr Thr Ile Ser Val Lys

85 90 95

Thr Arg Ser Val Ser Arg Thr Thr His Pro Thr Gly Pro Ser Cys Trp

100 105 110

Arg Glu Glu Glu Lys Cys Gln Asn Ile Thr Ala Lys Trp Ser Asn Cys

115 120 125

Phe Gly Thr Ser Leu Pro Val Arg Val Asn Cys Thr Ala Leu Arg Glu

130 135 140

Leu Cys Asp Glu Gln Leu Gly Asn His Thr Thr Val Gln Ala Ser Arg

145 150 155 160

Lys Cys Thr Cys Ile Tyr Ala Leu Asn Trp Asp Cys Gly Tyr Ala

165 170 175

<210> 7

<211> 537

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Met Ser Trp Lys Val Val Leu Leu Leu Val Leu Leu Ala Thr Pro Thr

1 5 10 15

Gly Gly Leu Glu Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Val Thr

20 25 30

Arg Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe

35 40 45

Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Arg Thr Asp Gly Pro

50 55 60

Ser Leu Ile Arg Thr Glu Leu Glu Leu Thr Lys Asn Ala Leu Glu Glu

65 70 75 80

Leu Lys Thr Val Ser Ala Asp Gln Leu Ala Lys Glu Ala Arg Ile Met

85 90 95

Ser Pro Arg Lys Ala Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val

100 105 110

Ala Thr Ala Ala Ala Val Thr Ala Gly Val Ala Ile Ala Lys Thr Ile

115 120 125

Arg Leu Glu Gly Glu Val Ala Ala Ile Lys Gly Ala Leu Arg Lys Thr

130 135 140

Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val Leu Ala Thr

145 150 155 160

Ala Val Asn Asp Leu Lys Asp Phe Ile Ser Lys Lys Leu Thr Pro Ala

165 170 175

Ile Asn Lys Asn Lys Cys Asp Ile Ser Asp Leu Lys Met Ala Val Ser

180 185 190

Phe Gly Gln Tyr Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser

195 200 205

Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu Met Thr Asp

210 215 220

Ala Glu Leu Val Arg Ala Ile Ser Asn Met Pro Thr Ser Ser Gly Gln

225 230 235 240

Ile Asn Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe

245 250 255

Gly Ile Leu Ile Gly Val Tyr Gly Ser Ser Val Val Tyr Met Val Gln

260 265 270

Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Lys Val Lys Ala

275 280 285

Ala Pro Leu Cys Ser Gly Lys Asp Gly Ser Tyr Ala Cys Leu Leu Arg

290 295 300

Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr

305 310 315 320

Pro Asn Glu Glu Asp Cys Glu Val Arg Ser Asp His Val Leu Cys Asp

325 330 335

Thr Ala Ala Gly Ile Asn Val Ala Lys Glu Ser Glu Glu Cys Asn Arg

340 345 350

Asn Ile Ser Thr Thr Lys Tyr Pro Cys Lys Val Ser Thr Gly Arg His

355 360 365

Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys

370 375 380

Tyr Asp Gly Val Ser Cys Ser Ile Gly Ser Asn Lys Val Gly Ile Ile

385 390 395 400

Arg Pro Leu Gly Lys Gly Cys Ser Tyr Ile Ser Asn Gln Asp Ala Asp

405 410 415

Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly

420 425 430

Glu Gln His Thr Ile Lys Gly Lys Pro Val Ser Ser Asn Phe Asp Pro

435 440 445

Ile Glu Phe Pro Lys Asp Gln Phe Asn Ile Ala Leu Asp Gln Val Phe

450 455 460

Glu Ser Val Glu Lys Ser Gln Ser Leu Ile Asp Gln Ser Asn Lys Ile

465 470 475 480

Leu Asp Ser Thr Lys Lys Gly Asn Ala Gly Phe Val Ile Val Ile Val

485 490 495

Leu Ile Val Leu Leu Met Leu Ala Ala Val Gly Val Gly Ile Phe Phe

500 505 510

Val Val Lys Lys Arg Lys Thr Ala Pro Lys Phe Pro Met Glu Met Ser

515 520 525

Gly Val Asn Asn Lys Gly Phe Ile Pro

530 535

<210> 8

<211> 585

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Glu Val Lys Ile Glu Asn Val Gly Lys Ser Gln Glu Leu Arg Val

1 5 10 15

Lys Val Lys Asn Phe Ile Lys Arg Ser Asp Cys Lys Lys Lys Leu Phe

20 25 30

Ala Leu Ile Leu Gly Leu Ile Ser Phe Asp Ile Thr Met Asn Ile Met

35 40 45

Leu Ser Val Met Tyr Val Glu Ser Asn Glu Ala Leu Ser Ser Cys Arg

50 55 60

Val Gln Gly Thr Pro Ala Pro Arg Asp Asn Arg Thr Asn Thr Glu Asn

65 70 75 80

Thr Ala Lys Glu Thr Thr Leu His Thr Met Thr Thr Thr Arg Asn Thr

85 90 95

Glu Ala Gly Gly Thr Lys Thr Thr Lys Pro Gln Ala Asp Glu Arg Ala

100 105 110

Thr Ser Pro Ser Lys Asn Pro Thr Ile Gly Ala Asp Lys His Lys Thr

115 120 125

Thr Arg Ala Thr Thr Glu Ala Glu Gln Glu Lys Gln Ser Lys Gln Thr

130 135 140

Thr Glu Pro Gly Thr Ser Thr Pro Lys His Ile Pro Ala Arg Pro Ser

145 150 155 160

Ser Lys Ser Pro Ala Thr Thr Lys Thr Thr Thr Gln Pro Thr Thr Pro

165 170 175

Thr Val Ala Lys Gly Gly Thr Ala Pro Lys Asn Arg Gln Thr Thr Thr

180 185 190

Lys Lys Thr Glu Ala Asp Thr Pro Thr Thr Ser Arg Ala Lys Gln Thr

195 200 205

Asn Lys Pro Thr Gly Thr Glu Thr Thr Pro Pro Arg Ala Thr Thr Glu

210 215 220

Thr Asp Lys Asp Lys Glu Gly Pro Thr Gln His Thr Thr Lys Glu Gln

225 230 235 240

Pro Glu Thr Thr Ala Gly Gly Thr Thr Thr Pro Gln Pro Arg Arg Thr

245 250 255

Thr Ser Arg Pro Ala Pro Thr Thr Asn Thr Lys Glu Gly Ala Glu Thr

260 265 270

Thr Gly Thr Arg Thr Thr Lys Ser Thr Gln Thr Ser Ala Ser Pro Pro

275 280 285

Arg Pro Thr Arg Ser Thr Pro Ser Lys Thr Ala Thr Gly Thr Asn Lys

290 295 300

Arg Ala Thr Thr Thr Lys Gly Pro Asn Thr Ala Ser Thr Asp Arg Arg

305 310 315 320

Gln Gln Thr Arg Thr Thr Pro Lys Gln Asp Gln Gln Thr Gln Thr Lys

325 330 335

Ala Lys Thr Thr Thr Asn Lys Ala His Ala Lys Ala Ala Thr Thr Pro

340 345 350

Glu His Asn Thr Asp Thr Thr Asp Ser Met Lys Glu Asn Ser Lys Glu

355 360 365

Asp Lys Thr Thr Arg Asp Pro Ser Ser Lys Ala Thr Thr Lys Gln Glu

370 375 380

Asn Thr Ser Lys Gly Thr Thr Ala Thr Asn Leu Gly Asn Asn Thr Glu

385 390 395 400

Ala Gly Ala Arg Thr Pro Pro Thr Thr Thr Pro Thr Arg His Thr Thr

405 410 415

Glu Pro Ala Thr Ser Thr Ala Gly Gly His Thr Lys Ala Arg Thr Thr

420 425 430

Arg Trp Lys Ser Thr Ala Ala Arg Gln Pro Thr Arg Asn Asn Thr Thr

435 440 445

Ala Asp Thr Lys Thr Ala Gln Ser Lys Gln Thr Thr Pro Ala Gln Leu

450 455 460

Gly Asn Asn Thr Thr Pro Glu Asn Thr Thr Pro Pro Asp Asn Lys Ser

465 470 475 480

Asn Ser Gln Thr Asn Val Ala Pro Thr Glu Glu Ile Glu Ile Gly Ser

485 490 495

Ser Leu Trp Arg Arg Arg Tyr Val Tyr Gly Pro Cys Arg Glu Asn Ala

500 505 510

Leu Glu His Pro Met Asn Pro Cys Leu Lys Asp Asn Thr Thr Trp Ile

515 520 525

Tyr Leu Asp Asn Gly Arg Asn Leu Pro Ala Gly Tyr Tyr Asp Ser Lys

530 535 540

Thr Asp Lys Ile Ile Cys Tyr Gly Ile Tyr Arg Gly Asn Ser Tyr Cys

545 550 555 560

Tyr Gly Arg Ile Glu Cys Thr Cys Lys Asn Gly Thr Gly Leu Leu Ser

565 570 575

Tyr Cys Cys Asn Ser Tyr Asn Trp Ser

580 585

<210> 9

<211> 2005

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Asp Pro Leu Asn Glu Gly Val Val Asn Val Tyr Leu Pro Asp Ser

1 5 10 15

Tyr Leu Lys Gly Val Ile Ser Phe Ser Glu Thr Asn Ala Leu Gly Ser

20 25 30

Cys Leu Leu Gly Lys His Tyr Leu Lys Lys Asp Asn Thr Ser Lys Val

35 40 45

Ala Ile Glu Ser Pro Val Val Glu His Ile Arg Leu Arg Asn Ala Phe

50 55 60

Gln Thr Arg Ile Lys Glu Lys Asn Leu Arg Val Val Glu Pro Val Asn

65 70 75 80

Met Gln Ser Glu Val Met Arg Asn Ser Tyr Thr Cys Glu Leu Asn Leu

85 90 95

Leu Lys Gln Leu Ile Thr Arg Ser Lys Asp Ile Ser Ser Leu Lys Leu

100 105 110

Asp Met Ile Cys Asp Trp Leu Gln Leu Lys Ser Thr Pro Glu Asn Pro

115 120 125

Ser Val Leu Lys Phe Val Asp Val Arg Cys Ile Pro Asp Trp Val Ser

130 135 140

Thr Trp Phe Ser Ser Trp Tyr Asn Leu Asn Lys Leu Ile Leu Glu Phe

145 150 155 160

Arg Arg Glu Glu Val Ala Cys Thr Gly Ser Ile Ile Cys Lys Thr Ile

165 170 175

Gly Ser Ile Met Phe Ile Ile Ser Ser Leu Gly Cys Val Ile Lys Ser

180 185 190

Asn Lys Ser Lys Arg Ile Ser Phe Met Thr Tyr Asn Gln Val Leu Thr

195 200 205

Trp Lys Asp Val Met Leu Ser Arg Phe Asn Ala Asn Leu Cys Val Trp

210 215 220

Ile Ser Asn Ser Leu Asn Lys Asn Gln Glu Gly Leu Gly Leu Arg Ser

225 230 235 240

Asn Leu Gln Gly Thr Leu Val Asn Lys Leu Tyr Glu Ile Val Asp Ser

245 250 255

Met Leu Ser Val Cys Ser Asn Glu Gly Phe Thr Leu Val Lys Glu Phe

260 265 270

Glu Gly Phe Ile Met Ser Glu Ile Leu Lys Ile Thr Glu His Ala Gln

275 280 285

Phe Ser Thr Arg Phe Arg Asn Thr Leu Leu Asn Gly Leu Val Asp Gln

290 295 300

Leu Val Lys Met Arg Gly Leu Asn Arg Lys Arg Val Ser Gly Thr Val

305 310 315 320

Leu Glu Gly Asn Gln Tyr Pro Met Tyr Glu Thr Thr Leu Ala Thr Leu

325 330 335

Gly Glu Ala Leu Lys Thr Ile Arg Leu Leu Val Asn Lys Asn Leu Asp

340 345 350

Asn Ala Ala Glu Leu Tyr Tyr Ile Phe Arg Ile Phe Gly His Pro Met

355 360 365

Val Glu Glu Arg Glu Ala Met Asp Ala Val Arg Leu Asn Asn Glu Ile

370 375 380

Thr Lys Ile Leu Lys Leu Glu Ser Leu Thr Glu Leu Arg Gly Ala Phe

385 390 395 400

Ile Leu Arg Ile Ile Lys Gly Phe Val Asp Thr Asn Lys Arg Trp Pro

405 410 415

Lys Ile Lys Asn Leu Lys Val Leu Ser Arg Arg Trp Val Met Tyr Phe

420 425 430

Lys Ala Lys Ser Tyr Pro Ser Gln Leu Glu Leu Ser Ser Gln Asp Phe

435 440 445

Leu Glu Leu Ala Gly Val Gln Phe Glu Gln Glu Phe Ala Ile Pro Glu

450 455 460

Arg Thr Asn Leu Glu Met Val Leu Asn Asp Lys Ala Ile Ser Pro Pro

465 470 475 480

Lys Asn Leu Ile Trp Ser Val Phe Pro Lys Asn Tyr Leu Pro Thr Asn

485 490 495

Ile Arg Glu Lys Phe Thr Asp Glu Met Phe Asn Ser Ser Glu Lys Leu

500 505 510

Lys Thr Arg Arg Val Leu Glu Tyr Tyr Leu Lys Asp Asn Lys Phe Asp

515 520 525

Gln Asn Asp Leu Lys Lys Tyr Val Val Arg Gln Glu Tyr Leu Gly Asp

530 535 540

Lys Glu His Val Val Ser Leu Thr Gly Lys Glu Arg Glu Leu Ser Val

545 550 555 560

Gly Arg Met Phe Ala Met Gln Pro Gly Lys Gln Arg Gln Val Gln Ile

565 570 575

Leu Ala Glu Lys Leu Leu Ala Asp Asn Ile Val Pro Phe Phe Pro Glu

580 585 590

Thr Leu Thr Lys Tyr Gly Asp Leu Glu Leu Gln Arg Ile Met Glu Ile

595 600 605

Lys Ser Glu Leu Ser Ser Val Lys Ser Arg Arg Asn Asp Ser Tyr Asn

610 615 620

Asn Tyr Ile Ala Arg Ala Ser Ile Val Thr Asp Leu Ser Lys Phe Asn

625 630 635 640

Gln Ala Phe Arg Tyr Glu Thr Ser Ser Val Cys Ala Asp Val Val Asp

645 650 655

Glu Leu His Gly Thr Gln Ser Leu Phe Cys Trp Leu His Leu Thr Val

660 665 670

Pro Leu Thr Thr Met Ile Cys Thr Tyr Arg His Ala Pro Pro Glu Thr

675 680 685

Glu Gly Val Tyr Asp Ile Asp Lys Ile Lys Glu Gln Ser Gly Leu Tyr

690 695 700

Arg Phe His Met Gly Gly Ile Glu Gly Trp Cys Gln Lys Leu Trp Thr

705 710 715 720

Met Glu Ala Ile Ser Leu Leu Asp Val Val Ser Val Lys Asn Arg Val

725 730 735

Gln Leu Thr Ser Leu Leu Asn Gly Asp Asn Gln Ser Ile Asp Val Ser

740 745 750

Lys Pro Val Arg Leu Ser Gln Gly Val Asp Glu Val Lys Ala Asp Tyr

755 760 765

Ser Leu Ala Val Lys Met Leu Lys Glu Ile Arg Asn Ala Tyr Lys Asp

770 775 780

Ile Gly His Lys Leu Lys Glu Gly Glu Thr Tyr Ile Ser Arg Asp Leu

785 790 795 800

Gln Phe Met Ser Lys Val Ile Gln Ser Glu Gly Val Met His Pro Ser

805 810 815

Pro Ile Lys Lys Ile Leu Arg Val Gly Pro Trp Ile Asn Thr Ile Leu

820 825 830

Asp Asp Ile Lys Thr Ser Ala Glu Ser Ile Gly Ser Leu Cys Gln Glu

835 840 845

Leu Glu Phe Arg Gly Glu Ser Leu Leu Val Ser Leu Ile Leu Arg Asn

850 855 860

Phe Trp Leu Tyr Glu Leu Trp Met His Glu Ser Lys Ser His Pro Leu

865 870 875 880

Ala Gly Lys Gln Leu Tyr Arg Gln Leu Ser Lys Thr Leu Ala Ile Thr

885 890 895

Gln Lys Phe Phe Gly Ile Thr Lys Glu Thr Asp Val Val Asn Leu Trp

900 905 910

Met Asn Val Pro Met Gln Phe Gly Gly Gly Asp Pro Val Val Leu Tyr

915 920 925

Arg Ser Phe Tyr Arg Arg Thr Pro Asp Phe Leu Thr Glu Ala Val Ser

930 935 940

His Met Ser Val Leu Leu Lys Val Tyr Gly Lys Ala Lys Glu Gly Ser

945 950 955 960

Lys Lys Asp Phe Phe Lys Ala Leu Leu Ser Val Asp Lys Asn Lys Arg

965 970 975

Ala Thr Leu Thr Thr Leu Met Arg Asp Pro Gln Ala Val Gly Ser Glu

980 985 990

Arg Gln Ala Arg Val Thr Ser Glu Ile Asn Arg Ala Ala Val Thr Ser

995 1000 1005

Val Leu Ser Leu Ser Pro Asn Gln Leu Phe Cys Asp Ser Ala Ile His

1010 1015 1020

Tyr Ser Arg Asn Glu Glu Glu Val Gly Leu Ile Ala Gln Asn Ile Thr

1025 1030 1035 1040

Pro Val Tyr Pro His Gly Leu Arg Val Leu Tyr Glu Ser Leu Pro Phe

1045 1050 1055

His Lys Ala Glu Lys Val Val Asn Met Ile Ser Gly Thr Lys Ser Ile

1060 1065 1070

Thr Asn Leu Leu Gln Arg Thr Ser Ala Ile Asn Gly Glu Asp Ile Asp

1075 1080 1085

Arg Ala Val Ser Met Met Leu Glu Asn Leu Gly Leu Leu Ser Arg Ile

1090 1095 1100

Leu Ser Val Ser Gln Asp Asp Ile Ile Leu Pro Thr Lys Ala Asn Gly

1105 1110 1115 1120

Asp Leu Ile Cys Cys Gln Val Ser Arg Thr Leu Arg Glu Arg Ser Trp

1125 1130 1135

Asp Asn Met Glu Ile Val Gly Val Thr Ser Pro Ser Ile Val Thr Cys

1140 1145 1150

Met Asn Ile Val Tyr Ser Ser Ser Ser Gln Leu Lys Gly Ile Thr Ile

1155 1160 1165

Glu Lys Phe Ser Thr Asp Lys Thr Thr Arg Gly Gln Arg Gly Pro Lys

1170 1175 1180

Ser Pro Trp Val Gly Ser Ser Thr Gln Glu Lys Lys Leu Val Pro Val

1185 1190 1195 1200

Tyr Asn Arg Gln Ile Leu Ser Lys Gln Gln Lys Glu Gln Leu Glu Ala

1205 1210 1215

Ile Gly Lys Leu Arg Trp Val Tyr Lys Gly Thr Gln Gly Leu Arg Arg

1220 1225 1230

Leu Leu Asp Lys Ile Cys Ile Gly Ser Leu Gly Ile Ser Tyr Lys Asn

1235 1240 1245

Val Lys Pro Leu Leu Pro Arg Phe Met Ser Val Asn Phe Leu His Arg

1250 1255 1260

Leu Ser Val Ser Ser Arg Pro Met Glu Phe Pro Ala Ser Val Pro Ala

1265 1270 1275 1280

Tyr Arg Thr Thr Asn Tyr His Phe Asp Thr Ser Pro Val Asn Gln Thr

1285 1290 1295

Leu Ser Glu Arg Phe Gly Asn Glu Asp Ile Asn Leu Val Phe Gln Asn

1300 1305 1310

Ala Ile Ser Cys Gly Ile Ser Val Met Ser Val Val Glu Gln Leu Thr

1315 1320 1325

Gly Arg Ser Pro Lys Gln Leu Val Met Ile Pro Gln Leu Glu Glu Ile

1330 1335 1340

Asp Ile Met Pro Pro Pro Val Phe Ser Gly Lys Phe Asp Tyr Lys Leu

1345 1350 1355 1360

Val Glu Lys Ile Ser Ser Asp Gln His Ile Phe Ser Pro Asp Lys Leu

1365 1370 1375

Asp Leu Val Thr Leu Gly Lys Met Leu Met Pro Ser Thr Ser Gly Ala

1380 1385 1390

Lys Ser Asp Gln Phe Trp Asn Arg Lys Glu Asn Phe Phe His Gly Asn

1395 1400 1405

Asn Leu Val Glu Ser Leu Ser Ala Ala Leu Ala Cys His Trp Cys Gly

1410 1415 1420

Ile Leu Thr Glu Gln Cys Asn Glu Asn Asn Ile Phe Arg Arg Glu Trp

1425 1430 1435 1440

Gly Asp Gly Phe Val Thr Asp His Ala Phe Ile Asp Phe Lys Ile Phe

1445 1450 1455

Ile Gly Val Phe Lys Thr Lys Leu Leu Cys Gly Trp Gly Ser Arg Gly

1460 1465 1470

Gly Asp Ile Lys Asp Arg Asp Met Ile Asp Glu Ser Ile Asp Lys Leu

1475 1480 1485

Ile Arg Val Asp Asn Ser Phe Trp Arg Met Phe Ser Lys Val Ile Leu

1490 1495 1500

Glu Pro Lys Val Arg Lys Arg Val Met Leu Phe Asp Val Lys Ile Leu

1505 1510 1515 1520

Ser Leu Val Gly Tyr Ala Gly Phe Lys Asn Trp Phe Ile Asp His Leu

1525 1530 1535

Arg Ser Ser Asp Leu Cys Glu Val Pro Trp Val Val Asn Ala Asp Ser

1540 1545 1550

Glu Ile Val Glu Val Ser Ala Val Lys Ile Tyr Leu Gln Leu Leu Arg

1555 1560 1565

Val Ser Ser Pro Leu Arg Ile Thr Val Leu Asn Tyr Ser Asp Met Ala

1570 1575 1580

His Ala Ile Thr Arg Leu Ile Arg Arg Lys Ser Met His Asp Asn Val

1585 1590 1595 1600

Pro Ser Ile Ser Arg Thr Leu Ser Pro Ala Glu Leu Ala Pro Val Val

1605 1610 1615

Glu Pro Thr Val Gln Met Asn Leu Phe Pro Lys Ile Thr Phe Glu Arg

1620 1625 1630

Leu Lys Asn Tyr Glu Thr Ile Ser Gly Ser Thr Arg Gly Lys Leu Thr

1635 1640 1645

Arg Asn Tyr Met Val Met Leu Pro Trp Gln His Ile Asn Arg Phe Asn

1650 1655 1660

Phe Val Phe Ser Ser Thr Gly Cys Lys Ile Ser Val Lys Ser Cys Ile

1665 1670 1675 1680

Gly Lys Leu Ile Gln Asp Leu Asn Pro Thr Val Phe Tyr Phe Val Gly

1685 1690 1695

Glu Gly Ala Gly Asn Trp Met Ala Arg Thr Ala Cys Glu Tyr Pro Asn

1700 1705 1710

Thr Lys Phe Val Tyr Arg Ser Leu Lys Asp Asp Leu Asp His His Phe

1715 1720 1725

Pro Leu Glu Phe Gln Arg Val Leu Gly Asn Met Asn Arg Val Ile Asp

1730 1735 1740

Gly Gly Glu Gly Leu Ser Met Asp Thr Thr Asp Ala Thr Gln Lys Thr

1745 1750 1755 1760

His Trp Asp Leu Thr His Arg Ile Cys Lys Asp Ala Leu Leu Ile Thr

1765 1770 1775

Leu Cys Asp Ala Glu Phe Lys Asp Arg Asp Asp Phe Phe Lys Met Val

1780 1785 1790

Thr Leu Trp Arg Lys His Val Leu Ser Cys Arg Ile Cys Thr Thr Tyr

1795 1800 1805

Gly Thr Asp Leu Tyr Leu Phe Ala Lys Tyr His Ala Lys Glu Glu Ser

1810 1815 1820

Ile Lys Leu Pro Tyr Phe Val Arg Ser Ile Ala Thr Tyr Val Met Gln

1825 1830 1835 1840

Gly Ser Lys Leu Ser Gly Ser Glu Cys Tyr Val Leu Leu Thr Leu Gly

1845 1850 1855

His His Asn Asn Leu Pro Cys His Gly Glu Val Gln Ser Ser Lys Leu

1860 1865 1870

Lys Met Ala Val Cys Asn Asp Phe Ser Ile Pro Arg Lys Val Glu Val

1875 1880 1885

Lys Ala Val Glu Ala Asn Cys Lys Ser Leu Leu Ser Gly Leu Arg Thr

1890 1895 1900

Pro Ile Asn Arg Ala Glu Leu Asp Arg Gln Lys Lys Met Leu Thr Leu

1905 1910 1915 1920

Arg Ser Tyr His Ser Ser Val Ala Thr Val Gly Gly Ser Arg Val Ile

1925 1930 1935

Glu Ser Lys Trp Leu Ser Lys Lys Ala Thr Thr Ile Ile Glu Trp Leu

1940 1945 1950

Glu His Ile Leu Asn Ser Pro Lys Gly Glu Leu Asn Tyr Asp Phe Phe

1955 1960 1965

Glu Ala Leu Glu Asn Thr Tyr Pro Asn Met Val Lys Leu Leu Asp Asn

1970 1975 1980

Leu Gly Ser Ala Glu Leu Lys Lys Leu Ile Lys Val Thr Gly Tyr Met

1985 1990 1995 2000

Leu Met Ser Lys Lys

2005

Claims

1. A Muscovy duck subtype C avian metapneumovirus low virulent strain has a preservation number of: CCTCC NO: V201823; the nucleotide sequence is SEQ ID NO: 1.

2. the Muscovy duck subtype C avian metapneumovirus attenuated strain according to claim 1, wherein the reading frame amino acid sequence of the Muscovy duck subtype C avian metapneumovirus attenuated strain is SEQ ID NO: 2 to SEQ ID NO: 9.

3. the use of the low virulent strain of muscovy duck subtype C avian metapneumovirus according to any one of claims 1 to 2 for the preparation of muscovy duck immune products.

4. A muscovy duck immunization preparation comprising the attenuated strain of muscovy duck subtype C avian metapneumovirus according to any one of claims 1 to 2, wherein the content of the attenuated strain in each immunization preparation is ≧ 10^2.5TCID₅₀。

5. The muscovy duck immunizing product according to claim 4, wherein the content of the attenuated strain in each immunizing product is ≧ 10^4.5TCID₅₀。