CN104962527B - Attenuated vaccine strain of VII type NDV L gene mutations and preparation method thereof - Google Patents

Abstract

The present invention relates to a kind of using attenuated vaccine strain of VII type NDV L gene mutations of reverse genetics manipulation technology production and preparation method thereof, belong to biological products and biological technical field, the attenuated vaccine strain virulence of VII type NDV L gene mutations substantially weakens, have high titre, heritability stable, high with the antigen matching of popular virus in chicken embryo simultaneously, it is suitable for the large-scale production of vaccine, available for vaccine is manufactured, with conventional vaccine（Gene I and gene II type）Compare, the attenuated vaccine strain of VII type NDV L gene mutations will show wide application prospect in terms of the morbidity of control ewcastle disease and prevalence.

Description

Attenuated vaccine strain of VII type NDV L gene mutations and preparation method thereof

Technical field

The present invention relates to a kind of subtracting for VII type NDV L gene mutations using reverse genetics manipulation technology production Toxic vaccine strain and preparation method thereof, belong to biological products and biological technical field.

Background technology

Ewcastle disease is by NDV（Newcastle disease virus, NDV）Cause, encroach on chicken and turkey, also A kind of acute, highly contagious disease of other birds, birds can be infected, is that the big birds of universally acknowledged most important two are infected One of disease（Another is highly pathogenic bird flu）.NDV belongs to Paramyxoviridae Rubulavirus, has cyst membrane structure, base Because organizing the RNA virus for sub-thread minus strand non-segmented negative, it is made up of 6 kinds of structural proteins, respectively NP, P, M, F, HN and L albumen：M、 F and HN albumen is related to the formation of virus envelope；NP albumen is nucleocapsid protein, P albumen be viral RNA synthesis it is required because Son；L albumen is the maximum albumen of virus, and it is the most conservative in 6 kinds of structural proteins of NDV, is repaiied after having RNA polymerase, transcription The biological activities such as decorations, can collectively form viral replication complex, the complex participates in virus genomic turn with NP and P albumen Record and duplication, L albumen is in addition to participating in virus genomic duplication, also with the 5 ' methylated transferases that end caps, cap relies on The function such as activity and 3 ' end addition polyadenylic acid tails.

Virulence Difference between different NDV separation strains is very big, can will according to different with the pathogenic and virulence of chicken embryo to chicken NDV is divided into three classes：I.e. strong malicious type（Anaphylactic type）, medium virulence type（Middle hair style）With weak malicious type（Delayed type）.

Research in the past thinks that NDV infectivity and virulence are determined by F genes, and velogen strain F protein cracking site is RRQKRF, and low virulent strain LaSota F protein cracking site is GRQGRL.But with reverse Genetics Technique continuous development and Deep application in NDV researchs, NDV mechanism of causing a disease have new progress again.Peeters, De Leeuw and Panda Deng having saved out new recombinant virus after the F gene cracking sites of low virulent strain are sported into the cracking site consistent with velogen strain, Although experiment shows that its virulence significantly improves, the level consistent with velogen strain virulence is not reached, this is indicated that in NDV In genome in addition to the cracking site of F genes, it is also possible to other genes that can influence virus virulence be present.There is research to refer to recently Go out, also with the virulence of virus and pathogenic relevant, L albumen can be strengthened by improving the levels of replication of virus for L, HN and V protein NDV virulence.Rout etc. replaces with the L genes of NDV BC strains LaSota L genes, and virulence strengthens on the contrary after virus is rescued.Separately Outside, we, which successfully rescue, obtains the NDV L mutant strains of 3 pnca gene VII -97（K1756A, K1917A and E1954Q amino acids are mutated）With 1 The common mutant strains of the NDV F of pnca gene VII -97 and L（E1954Q+F）, found by virus virulence index determining, this 4 plants of VII types are new The attenuated vaccine strain of city epidemic disease poison L gene mutations can weaken virus virulence, we it is reasonable to conclude that NDV L albumen to virus Virulence play vital effect.These experiments absolutely prove that the virulence presence of L albumen and virus contacts.

The main vaccine strains of prevention and control NDV are two kinds of strains of LaSota and B1 on our times, and LaSota and the low virulent strains of B1 two are all Belong to gene II types.But domestic scholar Yan Weiwei etc. reported genotype VII NDV in 2000 in China's Mainland first In the presence of；2001, the representative strains of the domestic different regions to having been chosen since 1994 such as Liu Hualei carried out genetic analysis confirmation, institute 8 plants of strains of separation are similar to NDV velogen strain in the amino acid sequence of cracking site.Pass through Phylogenetic tree analytical table Bright, it is genotype Ⅶ NDV to have 7 plants in 8 plants of separation strains, and these data fully show the current Major Epidemic genotype Ⅶ new city in China Epidemic disease poison.Two kinds of attenuated vaccines of LaSota and B1 have applied decades as vaccine strain, and vaccine is although can provide certain Immunoprotection, but can not prevent poultry toxin expelling and suppress virus transmission capacity, therefore, ewcastle disease be still threat support fowl One of most important epidemic disease of industry development, China thereby results in huge economic loss every year.And the poison that China is popular Strain is mainly genotype Ⅶ, and with the continuous variation of virus, the research and development for the homogenic type vaccine of genotype Ⅶ turn into current Main task, the newcastle disease vaccine development of the type of China VII has been achieved with good progress, after industrialization, will turn into scale Change a necessary ring in plant's immune programme for children.The present invention establishes to carry out the development of NDV attenuated vaccines and NDV related basic researches Solid foundation is determined.

The content of the invention

The VII type NDV L gene mutations invented it is an object of the invention to overcome the deficiencies in the prior art Attenuated vaccine strain and preparation method thereof, so as to solve NDV vaccine strains used in current China market（Gene I and gene II type） With current NDV epidemic strains（Genotype Ⅶ）Genotype it is inconsistent caused by protective efficacy it is incomplete, it is impossible to effectively prevent disease The practical problem that poison is propagated.

The present invention is achieved by following technical solution：

A kind of attenuated vaccine strain of VII types NDV L gene mutations, it is characterised in that：VII type NDVs L The attenuated vaccine strain virulence of gene mutation substantially weakens, while has high titre, heritability stabilization and prevalence in chicken embryo The antigen matching of virus is high, is suitable for the large-scale production of vaccine, available for manufacturing vaccine, the VII type NDV L bases Because the antigen nucleotides sequence of the attenuated vaccine strain of mutation is classified as SEQ ID NO:1 to SEQ ID NO:Shown in 4.

A kind of preparation method of the attenuated vaccine strain of described VII type NDV L gene mutations, it is characterised in that：

Five cDNA clone fragments of structure covering whole gene group, using the restriction enzyme site of each intersegmental lap, The whole cDNA clone for obtaining 15192nt is assembled in transcription vector pBR322 plasmid.At 5 ' ends of full-length cDNA fragment Prefix t7 rna polymerase promoter is held, the fourth hepatovirus ribozyme with self catalysis is added after cDNA fragments（RiB）With T7 transcription stop signalses, the plasmid built are named as prNDV-97；By pcr gene group by the L albumen in genome cDNA The bit base of code area the 14056th is G by A same sense mutations, eliminates the restriction enzyme sites of Hind III at 14056bp, and as rescue disease The molecular labeling of poison, the plasmid for building completion are named as pmNDV-97；In T7 polymerase promoters in 5 ' ends of genome cDNA End introduces two unnecessary G, contributes to the virus rescue of paramyxovirus reverse genetic manipulation.

5 pairs of primers of design are used to expand the pnca gene group SEQ ID NO of NDV VII -97:5, primer sequence is as follows：

F1：5'ATGCGGCCGCTAATACGACTCACTATAGGGACCAAACAGAGAATCTG

TGAGGTACGATAA3'

R1：5'TAGGTACCCTTGCAGTGGCAGACTTAATCAATTCAGAG3'

F2：5'GGGTGAAATGACGCTCAATAAACTCTCACA3'

R2：5'ATGGTACCCTCCCTGTTGCAGATGTCCGAAGCACACCA3'

F3：5'TGCAGAGATCACTCACACTCACATCAATAC3'

R3：5'ACGGTACCAGGTGACCAGCTTCTGTTTCGGGCATAATC3'

F4：5'ATTAGTTTCTTCCAATATGTGCTCGCTGAC3'

R4：5'GTAGTAATGATACAGACCTGTAGGGTACCA3'

F5：5'GGAGAGAGAATGCAGAAGAAAGTGACCTGACCTCAGATAAAGCAG3'

R5：5'AGCCGGCGCCAGCGAGGAGGCTGGGACCATGCCGGCCACCAAACAG

AGATTTGGTGAATGACATC3'

2 pairs of design is used to be mutated at 14056bpHind III digestion site primer, eliminates this restriction enzyme site, primer sequence It is as follows：

AFa：5' CATGTTTTAAAGTTGTCAAG 3'

F6：5' GGGTCCAATCAGAGCTTATTAGATC 3'

R6：5' GATCTAATAAGCTCTGATTGGACCC 3'

ARa：5' GCG GTCGAC ACCGCTTTCAGGTATTTAG3'

On the basis of the full-length genome plasmids of pmNDV- 97, NDV-97 L gene mutation attenuated strains, concrete operations are built For：

By following primer, respectively through Overlapping PCR methods, by NDV parent poison L protein structure domains VI 1756th, 1917 and 1954 amino acids difference simple point mutation, the purpose fragment after amplification and pmNDV-97 carriers are passed throughHind Ⅲ WithXbaI restriction enzyme distinguishes double digestion, carries out nucleic acid electrophoresis, is carried out after glue reclaim purpose fragment and carrier pmNDV-97 Connection, the mutant plasmid obtained are respectively designated as pK1756A, pK1917A and pE1954Q.

4 pairs of primers of design are used for the L Argine Monohydrochloride rite-directed mutagenesises of NDV VII -97, and primer sequence is as follows：

AF1：5' CATGTTTTAAAGTTGTCAAG 3'

AF2：5' CTTCTTGGTATGCGGCATCTCATCTTC 3'

AF3：5' GTAGTGATCATCGCAGTACTGTATGC3'

AF4：5' GAGGGGATATGCAATGTTACCTGATAT3'

AF5：5' GCG GTCGAC ACCGCTTTCAGGTATTTAG3'

AF6：5'GAAGATGAGATGCCGCATACCAAGAAG3'

AF7：5'GCATACAGTACTGCGATGATCACTA3'

AF8：5'ATATCAGGTAACATTGCATATCCCCT3'

Wherein, AF1+AF6, AF1+AF7 and AF1+AF8 primer expand purpose band respectively using PCR method, name respectively For A1, B1 and C1；AF5+AF2, AF5+AF3 and AF5+AF4 primer expand purpose band respectively using PCR method, name respectively For A2, B2 and C2.On this basis, using AF1+AF5 primers respectively through Overlapping PCR methods, with A1+A2, B1+B2 It is respectively template with C1+C2, expands about 6000bp（Containing mutational site）Purpose fragment, obtain respectively L mutant fragments L1756, L1917 and L1954.

A kind of L gene mutations of NDV VII -97 and F are mutated the construction method of weakening strain with L genes altogether, are specially：With separation Strain NDV VII -97 genome L genes carry out rite-directed mutagenesis, to carrying out single-point amino acid mutation in L protein structure domains VI, obtain Highly cause weak NDV VII -97 L attenuated strains K1756A, K1917A and E1954Q.On the basis of E1954Q, F protein is cracked Site is mutated, by the F protein cracking site of velogen strain（RRQKRF）Sport the F protein cracking site of LaSota low virulent strains （GRQGRL）, the attenuated strain obtained is E1954Q+F.

Primer for being mutated F protein cracking site is as follows：

AF9：CTCCGACCAAAACCCTCCCACACTCCCTGACCC

AR9：GCAGAGTAGAGAAGAATACCCTCCCTGTTGCAGAT

AF10：TCTGTGTCCACGTCTGGAGGAGGGAGACAGGGGCGCCTTATAGGTGCTGT

TATTGGCAG

AR10：CTGCCAATAACAGCACCTATAAGGCGCCCCTGTCTCCCTCCTCCAGACGT

GGACACAGA

Wherein, AF9+AR10 and AF10+AR9 2 expands purpose band respectively to primer using PCR method, after glue reclaim again Using glue reclaim product as template, performing PCR amplification is entered using Overlapping methods with this 1 pair of primer of AF9+AR9, contained The fragment of F protein mutation is about 5458bp, using the mutant that this is obtained as E1954Q+F.

A kind of attenuated vaccine strain K1756A, K1917A, E1954Q and E1954Q of the L gene mutations of VII types NDV + F construction method, it is specially：

1）The structure of the pnca gene group full-length cDNAs of NDV VII -97；

2）The connection of Amino Acid-Induced Site-Directed Mutation and mutant full-length cDNA in the L protein structure domains VI of NDV VII -97；

3）On the basis of E1954Q mutation, the mutation of the F protein cracking sites of NDV VII -97 and mutant full-length cDNA Connection；

4）Attenuated vaccine strain K1756A, K1917A, E1954Q of VII type NDV L gene mutations

With E1954Q+F rescue；

By plasmid pK1756A, pK1917A, pE1954Q and pE1954Q+F respectively with pBSK-NP, pBSK-P and pBSK-L Three eukaryon expression plasmid cotransfection BHK-21 cells（Hamster kidney cell）, transfect 72h after by cell conditioned medium be inoculated with 9-11 ages in days SPF chicken embryos, every part of 3 pieces of sample inoculation, 0.2mL/ pieces, successfully rescue the attenuated vaccine strain for obtaining VII type NDV L gene mutations K1756A, K1917A and E1954Q and F protein and L albumen are mutated attenuated vaccine strain E1954Q+F altogether.

The present invention has following good effect：

The present invention, by transforming related gene, constructs viral drop using the NDV reverse genetic manipulation platforms established Degree is high, heritability is stable, antigen matching is high and the attenuated vaccine strain low to the birds pathogenicity such as host chicken, and this will be to grind Efficient vaccine kind poison processed lays the foundation.With conventional vaccine（Gene I and gene II type）Compare, VII type NDV L genes are dashed forward The attenuated vaccine strain of change will show wide application prospect in terms of the morbidity of control ewcastle disease and prevalence.

Brief description of the drawings

Fig. 1 is to assemble total length NDV cDNA using subgenome overlapping cDNA fragments caused by high-fidelity RT-PCR.Will CDNA fragments connect in shared restriction enzyme site, and are assembled in pBR322 plasmid is transcribed, in transcription plasmid PBZ and T7 terminator sequences are cloned wherein in advance in pBR322.Fig. 1 represents the ideograph of connection full-length cDNA.

Fig. 2 is that the nucleotides change for introducing modification enzyme site is produced by RT-PCR method, and is surveyed by automatic sequencer Sequence verifies that add frame is after being mutated by PCR at 14056bpHind III restriction enzyme site（G is changed into from A）.

Fig. 3 is the RT-PCR identifications and the sequencing of mutated site of the attenuated vaccine strain of VII type NDV L gene mutations As a result.Wherein, A figures are RT-PCR qualification results；B figures are K1756A sequencing results；C figures are K1917A sequencing results；D figures are E1954Q sequencing results；E figures are E1954Q+F sequencing results.

Fig. 4 is genetic stability analysis and the sequencing result of the attenuated vaccine strain of VII type NDV L gene mutations. Wherein, A figures are K1756A sequencing results；B figures are K1917A sequencing results；C figures are E1954Q sequencing results；D figures are E1954Q+ F sequencing results.

Fig. 5 is that attenuated vaccine strain K1756A, K1917A, E1954Q, L and F of VII type NDV L gene mutations are total to It is mutated attenuated strain E1954Q+F and rNDV indirect immunofluorescence analysis.With K1756A, K1917A, E1954Q, E1954Q+F and RNDV infects BHK-21 cells respectively（Negative control is set simultaneously）.Infective dose is 1 MOI, and 24h consolidates cell after infection It is fixed, permeable membrane, detected with the anti-NDV antiserums of chicken, then the rabbit-anti chicken igg antibody with FITC- couplings is incubated.Show in Leica fluorescence Micro- Microscopic observation fluorescence.

Embodiment

The present invention combines newcastle disease virus strain accumulation in recent years and the analysis and research to its whole genome sequence, by China VII type ewcastle disease strain L genes carry out pinpoint amino acid mutation and the amino acid of F and L genes is mutated altogether, and to L gene mutations Saved with the total length NDV genome cDNAs that L genes and F genes are mutated altogether, height of being rescued causes weak VII type NDVs L Attenuated vaccine strain K1756A, K1917A, E1954Q and E1954Q+F of gene mutation, this 4 plants of VII type NDV L genes The attenuated vaccine strain virulence of mutation substantially weakens, while has high titre, heritability stable, with popular virus in chicken embryo Antigen matching is high, is suitable for the large-scale production of vaccine, available for the production of vaccine, the VII type NDV L genes are dashed forward The antigen nucleotides sequence of the attenuated vaccine strain of change is classified as SEQ ID NO:1 to SEQ ID NO:Shown in 4.

The present invention is further described through with reference to specific implementation example.

The structure of the transcription vector of embodiment 1.：

BHK-21 cells used in the present inventor are hamster kidney cell；Culture medium is containing 10% hyclone（Gibco）；NDV VII -97 plant is preserved by poultry diease research department of Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences；SPF chicken embryos and SPF chick buy in Beijing Cimmeria Wei Tong Bioisystech Co., Ltd.

The reverse genetic manipulation platform that VII -97 plant of NDV is to transform to form on the basis of pBR322 plasmids, respectively in NDV T7 promoters and T7 ribozyme transcription terminators are contained in the both ends of full-length genome respectively, while insert with self splicing function Fourth hepatovirus ribozyme sequence.Being cloned in the DNA fragmentation between T7 promoters and ribozyme can obtain in the presence of t7 rna polymerase To transcription, and due to the autocatalysis function of fourth hepatovirus ribozyme, it is ensured that 3 ' ends of transcription product and the DNA of clone Fragment is accurately consistent.

The structure of embodiment 2.NDV Genomic full_length cDNAs：

5 cDNA clone fragments of structure covering whole gene group, using the restriction enzyme site of each intersegmental lap, Transcription vector pBR322 plasmid assembles the whole cDNA clone for obtaining 15192nt.In 5 ' ends of full-length cDNA fragment Prefix t7 rna polymerase promoter, the fourth hepatovirus ribozyme with self catalysis and T7 transcriptions are added after cDNA fragments Termination signal.The plasmid that structure is completed is named as prNDV.To eliminate at 14056bpHind III restriction enzyme site, passes through pcr gene The bit base of L protein-coding regions the 14056th in genome cDNA is G by A same sense mutations by group, and as the molecule of Revive virus Mark, we introduce two unnecessary G in T7 polymerase promoters in 5 ' ends of genome cDNA simultaneously, and this is potentially contributed to The virus rescue of paramyxovirus reverse genetic manipulation.

5 pairs of primers of design are used to expand the pnca gene group SEQ ID NO of NDV VII -97:5, primer sequence is as follows：

F1：5'ATGCGGCCGCTAATACGACTCACTATAGGGACCAAACAGAGAATCTG

TGAGGTACGATAA3'

R1：5'TAGGTACCCTTGCAGTGGCAGACTTAATCAATTCAGAG3'

F2：5'GGGTGAAATGACGCTCAATAAACTCTCACA3'

R2：5'ATGGTACCCTCCCTGTTGCAGATGTCCGAAGCACACCA3'

F3：5'TGCAGAGATCACTCACACTCACATCAATAC3'

R3：5'ACGGTACCAGGTGACCAGCTTCTGTTTCGGGCATAATC3'

F4：5'ATTAGTTTCTTCCAATATGTGCTCGCTGAC3'

R4：5'GTAGTAATGATACAGACCTGTAGGGTACCA3'

F5：5'GGAGAGAGAATGCAGAAGAAAGTGACCTGACCTCAGATAAAGCAG3'

R5：5'AGCCGGCGCCAGCGAGGAGGCTGGGACCATGCCGGCCACCAAACAG

AGATTTGGTGAATGACATC3'

2 pairs of design is used to be mutated at 14056bpHind III digestion site primer, eliminates this restriction enzyme site, primer sequence It is as follows：

AFa：5' CATGTTTTAAAGTTGTCAAG 3'

F6：5' GGGTCCAATCAGAGCTTATTAGATC 3'

R6：5' GATCTAATAAGCTCTGATTGGACCC 3'

ARa：5' GCG GTCGAC ACCGCTTTCAGGTATTTAG3'；

The geneome RNA in the egg inoculation allantoic fluids of NDV VII -97 is extracted using Trizol methods, detailed step is as follows：

1. taking chick embryo allantoic liquid 250uL, 750 uL Trizol are added（Invitrogen, USA）, concussion mixing, room temperature Place 5 minutes；

2. often pipe adds chloroform 200uL, centrifuge tube is covered tightly, acutely sways centrifuge tube 15 seconds；Room temperature places 10min, 12000rpm centrifuges 15min；

3. taking upper strata aqueous phase to be positioned in new centrifuge tube, add 700uL isopropanols, 4 degree are placed 10 minutes, 12000rpm centrifugations 10 minutes；

4. abandoning supernatant, the ratio that at least 1ml is added in every milliliter of Trizol liquid adds 75% ethanol, mixes, at 4 DEG C 7500g is centrifuged 5 minutes；

5. careful abandoning supernatant, then drying at room temperature 5-10 minutes, it is careful not to dry too, otherwise can reduces RNA Solubility；It is finally that RNA is soluble in water.By RNA reverse transcriptions into after cDNA, each genetic fragment is expanded using PCR method, cuts glue Purpose fragment is reclaimed, it is standby.

Whole gene component is 5 overlapping fragments of end section（F1-F5）Enter performing PCR amplification, cDNA fragments are cloned into On pBR322 carrier for expression of eukaryon；Nucleocapsid protein（NP）, phosphoprotein（P）With big polymerase protein（L）The open reading of gene Frame（ORF）CDNA is cloned in pBluescript II respectively（+/-）Plasmid T7 promoters downstream, respectively constitutes transcription helper plasmid PBSK-NP, pBSK-P and pBSK-L.

The full-length cDNA of the corresponding gene group of clone is the basis for establishing NDV reverse genetic operating systems, as genome Strand RNA transcription templates.

VII -97 plant of L gene of embodiment 3.NDV carries out pinpoint amino acid mutation and the connection of L mutant full-length cDNAs

By primer respectively through Overlapping PCR methods, by 1756 in VII -97 plant of L protein structure domain VI of NDV, 1917 and 1954 amino acids difference simple point mutation (table 1), the purpose fragment after amplification and prNDV-97 carriers are passed throughHind Ⅲ WithXbaI restriction enzyme distinguishes double digestion, carries out nucleic acid electrophoresis, is carried out after glue reclaim purpose fragment and carrier prNDV-97 Connection, the mutant plasmid obtained are respectively designated as pK1756A, pK1917A and pE1954Q.

On the basis of pE1954Q, F protein cracking site is mutated, by the F protein cracking site of velogen strain （RRQKRF）Sport the F protein cracking site of LaSota low virulent strains（GRQGRL）.F protein mutated site is distinguished by primer Expanded through Overlapping PCR methods, purpose fragment and pE1954Q carriers warp after amplificationSalI HeAfeI restriction enzyme Enzyme distinguishes double digestion, carries out nucleic acid electrophoresis, is attached after glue reclaim purpose fragment and carrier pE1954Q, the mutation obtained Plasmid is respectively designated as pE1954Q+F.Primer for being mutated F protein cracking site is as follows：

AF9：CTCCGACCAAAACCCTCCCACACTCCCTGACCC

AR9：GCAGAGTAGAGAAGAATACCCTCCCTGTTGCAGAT

AF10：TCTGTGTCCACGTCTGGAGGAGGGAGACAGGGGCGCCTTATAGGTGCTGT

TATTGGCAG

AR10：CTGCCAATAACAGCACCTATAAGGCGCCCCTGTCTCCCTCCTCCAGACGT

GGACACAGA

Wherein, AF9+AR10 and AF10+AR9 2 expands purpose band respectively to primer using PCR method, after glue reclaim again Using glue reclaim product as template, performing PCR amplification is entered using Overlapping methods with this 1 pair of primer of AF9+AR9, contained The fragment of F protein mutation is about 5458bp, using the mutant that this is obtained as E1954Q+F.

The L gene mutations position of table 1.

Attenuated vaccine strain K1756A, K1917A, E1954Q of embodiment 4.VII type NDV L gene mutations and E1954Q+F rescue

Poxvirus infection BHK-21 cells are first used before transfection, cell is washed 2-3 times after 1h, then transfects Revive virus.Will Plasmid pK1756A, pK1917A, pE1954Q and pE1954Q+F respectively with tri- eucaryon tables of pBSK-NP, pBSK-P and pBSK-L Up to plasmid co-transfection BHK-21 cells, cell is gently scraped after 72h, and 9-11 is inoculated with after being sufficiently mixed filtering with cell conditioned medium Age in days SPF chicken embryos, every part of 3 pieces of sample inoculation, 0.2mL/ pieces, the SPF embryos after inoculation continue culture 3 days, take chick embryo allantoic liquid 50uL routinely carries out NDV blood clotting（HA）Suppress with blood clotting（HI）Experiment, obtain VII type NDV L gene mutations Attenuated vaccine strain K1756A, K1917A, E1954Q and E1954Q+F.

Attenuated vaccine strain K1756A, K1917A, E1954Q of embodiment 5.VII type NDV L gene mutations and E1954Q+F identification

5.1 genetic stabilities are analyzed

Experimental method：By the attenuated vaccine strain of VII type NDV L gene mutations（K1756A、 K1917A、E1954Q With E1954Q+F）It was passaged to for the 10th generation using 9 age in days SPF chicken embryos respectively, extracts the 10th generation viral RNA, reverse transcription is into after cDNA Expand purpose fragment, and gel extraction purpose fragment, sample presentation sequencing.

Experimental result：Each mutant strain mutational site is still present, and genetic stability is good（Fig. 4）.

Indirect immunofluorescence assay

To detect attenuated strain（K1756A, K1917A, E1954Q and E1954Q+F）In the intracellular duplications of BHK-21, allantois Poison infects about 70%-80% individual layer BHK-21 cells using MOI as 1 virus quantity respectively, and 24h, exempts from SPF with NDV height after infection Chicken positive serum carries out indirect immunofluorescence assay for detection antibody, and sets negative control.Sent out by indirect immunofluorescence assay Existing, rescued virus can breed on BHK-21 cells.

Detailed step is as follows：

Attenuated strain chick embryo allantois poison is diluted with DMEM suitable multiples respectively, is that the infection of 1,300uL volumes is grown on according to MOI The BHK-21 cells of six orifice plates, 37 DEG C be incubated 1h after washed three times with DMEM, then add complete DMEM and continue to cultivate, after 24h Cell 10min is fixed with 4% paraformaldehyde, PBS is washed after cell and carried out closing 30min with 5% BSA, then exempted from NDV height SPF chickens positive serum is primary antibody, 1:500 times dilute, and are washed 5 times with PBS after 37 DEG C of effect 1h, add 1:The fluorescein of 800 dilutions （FITC）The secondary antibody of mark（Sigma）, 45min is acted on, PBS washes rear fluorescence microscope result（Fig. 5）.

The growth characteristics and pathogenic property of the attenuated strain of embodiment 6.

Experiment purpose：The attenuated vaccine of the VII type NDV L gene mutations obtained is rescued in detection using Reverse Genetics Growth characteristics of the strain in chicken embryo and its to the pathogenic of SPF chickens.

Experimental method：K1756A, K1917A, E1954Q and E1954Q+F attenuated strain F4 are pressed respectively for allantoic fluid and rNDV 10⁶EID₅₀The age in days SPF chick embryo allantoic cavities of dose inoculation 9, without dead germ in 96 hours；Chicken embryo is freezed to death after 96 hours, collects allantois Liquid, HA titres are respectively 2⁵、2⁶、2⁶ 、2⁸With 2⁸, and every milliliter of allantoic fluid EID₅₀It is respectively then 2.15 × 10⁹、3.16×10⁹、 1.78×10⁹、1.78×10⁹With 1 × 10¹⁰（Table 2）.According to OIE standard tests rNDV and its chicken embryo median lethal of attenuated strain Amount（MDT）, intracranial inoculation pathogenic index（ICPI）With intravenous inoculation pathogenic index（IVPI）Toxicity index measure.It the results are shown in Table 3（OIE write [Animal Husbandry and Veterinary Bureau of the Ministry of Agriculture is translated] mammals, birds and honeybee A classes and B classes disease diagnostic tests and Vaccine standards handbook Beijing：Chinese agriculture Science Press, 1996:140-146）.

Experimental result：Show to rescue the VII type NDV L bases obtained using reverse genetics manipulation technology by data above Because the attenuated vaccine strain virulence of mutation is substantially less than parent's strain, there is higher growth titre in chicken embryo and to SPF chickens poison The biological characteristics that power substantially weakens.

The attenuated vaccine strain HA and EID of the VII type NDV L gene mutations of table 2.₅₀Detection

The virus virulence assessment of indices of the attenuated vaccine strain of the VII type NDV L gene mutations of table 3.

* virus is inoculated with 9 age in days SPF chicken embryos after proper proportion dilutes（100uL/ is only）, all chicken embryos can be made lethal Viral maximum dilution multiple be virus minimum lethal dose（MLD）, MDT is minimum lethal dose（MLD）Cause chicken embryo death Average time, utilize the method measure MDT.

The age in days SPF chick of intracranial inoculation 1 after * virus kind 10 times of dilutions of poisons（50uL/ is only）, determine ICPI.

It is injected intravenously under * * wings（1×10⁶EID₅₀/ only）, determine IVPI.

The immune effect of the induced animal protection antibody of embodiment 7.

Experiment purpose：Measure rescues the attenuation of the VII type NDV L gene mutations obtained using reverse genetic operating system Immune protective of the vaccine strain to 2 week old SPF chick.

Experimental method：The virus expanded respectively using 9 age in days SPF chicken embryos, 1 × 10⁶EID₅₀Dosage adds some points eye on the way through collunarium Footpath 10 whites of difference artificial immunity carry out prosperous SPF chick, separately set nonimmune control group 10, immune group and nonimmune group are raised respectively Support and filtered in air negative pressure in isolator.Hind wing venous blood collection separation serum routinely detects the special HI antibody of NDV within 21 days.Separately Outside, latter 21 days are being immunized with strong malicious NDV VII -74（This room is preserved with kind of a poison）With 1 × 10⁶EID₅₀Dosage is added some points an approach through collunarium Carry out attacking poison, observe two weeks, statistics morbidity and death condition.

Experimental result：In allantois virus liquid F4 generations, are with 1 × 10⁶EID₅₀Dosage comes through collunarium approach artificial immunity white of adding some points Prosperous SPF chick, observed after immunization 3 weeks, 4 groups of all chick of immune group of period without any exception, food consumption and grow with Nonimmune control group no significant difference.3 weeks detection special HI antibody levels of serum N DV after immune（Table 4）.4 groups of immune groups attack poison Do not occur any morbidity or death afterwards, completely protection（10/10）, with the nonimmune control group of age in days, then all morbidity is dead（0/10） （Table 5）.As a result show, mutation attenuated strain primary immune response chick can induced high levels protection antibody reaction, have good Protecting effect.

The NDV L of table 4. are mutated the immunogenicity of low virulent strain

* dosage 1 × 10⁶EID₅₀/ only.

Venous blood collection under 3 weeks hind wings is immunized in *, determines HI titres, calculates average value.

The NDV L of table 5. mutation low virulent strains attack malicious protectiveness experiment

* 2 week old SPF chick, K1756A, K1917A, E1954Q and E1954Q+F are inoculated with through collunarium eye droppings respectively（1× 10⁶EID₅₀/ only）.

Venous blood collection under * wings, HI titres are determined, calculate average value.

After * * are immune 21 days, carry out attacking poison by collunarium and eyedripping way with the strong poison of NDV VII -74（1×10⁶EID₅₀/ Only）.

Attenuated strain K1756A, K1917A, E1954Q and E1954Q+F the toxin expelling situation analysis of embodiment 8.

Experiment purpose：Measure rescues the L of 3 plants of NDV VII -97 mutation attenuated strains obtained and 1 plant of L using reverse genetic operating system It is mutated the toxin expelling situation after attenuated strain is immunized altogether with F.

Experimental method：The virus that this experiment is expanded respectively using 9 age in days SPF chicken embryos, 1 × 10⁶EID₅₀Dosage adds through collunarium Eye droppings approach difference artificial immunity 10 2 week old carry out prosperous SPF chick, separately set LaSota control groups 10, immune group and nonimmune Group is raised respectively to be filtered in isolator in air negative pressure.Latter 21 days are being immunized with strong malicious NDV VII -74（This room is preserved with kind of a poison）With 1×10⁶EID₅₀Dosage carries out attacking poison through a collunarium approach of adding some points, and the 3d and 6d attacked after poison gathers throat swab and nephrodinic respectively Chamber swab, titrated using SPF chicken embryos, detect the toxin expelling situation of virus.

Experimental result：Poison, the equal adaptive immune protection of chicken used are attacked after immune group and control group are immune（Except blank chicken）, but Toxin expelling situation is detected after Immunization, the shedding virus of LaSota throat swabs is higher than other 4 groups of L mutation attenuated strains（Table 6）.

The NDV L of table 6. are mutated and F and L is mutated the detection of attenuated strain toxin expelling altogether

Note："/" represents and does not detect virus

The present invention infects BHK-21 cells using the recombinant poxvirus of stable expression T7 RNA polymerase systems, thin in transfection Full-length genome strand RNA is transcribed in born of the same parents, while expresses NP, P and L albumen needed for startup virus replication, is achieved very high Virus rescue efficiency.

The L of 3 plants of NDV VII -97 mutation attenuated strain K1756A, K1917A for being rescued using reverse genetics manipulation technology and E1954Q and 1 plant of F protein cracking site is mutated attenuated strain E1954Q+F to L altogether can reach similar to parent rNDV in chicken embryo Growth titre, and rescued virus is with 10⁶EID₅₀9 age in days SPF chicken embryos of inoculation in dosage allantoic cavity, not lethal chicken embryo in 96h, HA is respectively 2⁵、2⁶、2⁶With 2⁸；Every milliliter of allantoic fluid EID₅₀Respectively 2.15 × 10⁹、3.16×10⁹、1.78×10⁹、1.78 ×10⁹, 14 age in days SPF chick, which are immunized, can induce high-caliber protection antibody reaction, 21d antibody titers titre point after being immunized Wei 2⁵、2⁴、2⁴With 2⁴, reverse genetic operating system 4 plants of VII type NDV L genes of being rescued of the fully display present invention dash forward The attenuated vaccine strain of change can be used as vaccine strain.The attenuated vaccine strain has good chicken embryo growth characteristics and higher viral growth Titre, low production cost and easy to use.NDV attenuated vaccines be immunized be almost all newborn chick in China's aviculture must not The immune programme for children that can lack, and genotype is consistent with the epidemic strain in current China, 4 plants of VII type Newcastle Diseases of being naturally rescued The attenuated vaccine strain of malicious L gene mutations turns into the vaccine candidate of most attraction.

Sequence table

<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences

<120>Attenuated vaccine strain of VII type NDV L gene mutations and preparation method thereof

<160> 5

<170> PatentIn Version 3.3

<210> 1

<211> 15192

<212> DNA

<213>Artificial sequence (SEQ ID NO：1)

<400> 1

accaaacaga gaatctgtga ggtacgataa aaggcgaaga agcaatcgag atcgtacggg 60

tagaaggtgt gaatcccgag tgcgaggccg aagctcgaac ctgaggggac cttctaccga 120

tatgtcgtct gttttcgacg agtacgagca gctcctcgct gctcagaccc gccctaacgg 180

aactcatgga gggggagaga aagggagcac tttaaaagtt gaggtcccag tattcaccct 240

aaacagtgat gatccagagg atagatggaa ttttgcggta ttctgtcttc ggattgctgt 300

tagcgaggat gccaacaaac cactcaggca aggtgctctt atatccctct tatgctccca 360

ttctcaggtg atgagaaacc atgttgccct tgcagggaaa caaaatgagg ccacactggc 420

tgttcttgag atcgatggtt ttgctaacag tgtgccccag ttcaacaata ggagtggagt 480

gtctgaggaa agagcacaga gattcatggt aatcgcagga tctctccctc gggcgtgcag 540

caacggtact ccgtttgtca cggctggggt tgaagatgat gcaccagaag atatcactga 600

cactctggaa agaatcctat ctatccaagt tcaggtatgg gtcacagtag caaaggccat 660

gactgcatat gagacagcag atgagtcaga aacaagaaga ataaataagt atatgcagca 720

aggtcgagtt cagaagaagt acatccttca tcctgtatgc aggagtgcaa ttcaactcac 780

aatcagacat tctctggcag tccgtatctt cctagttagt gagctcaaga ggggccgcaa 840

tacagcaggt gggagctcta catattacaa cttggtcggg gatgtagact catacatcag 900

gaacaccggg cttactgcat ttttcctaac actcaaatat ggaatcaata ccaagacgtc 960

agccctcgca ctcagcagcc tcacaggtga tatccaaaaa atgaaacagc tcatgcgttt 1020

atatcggatg aaaggtgaaa atgcaccata catgacattg ttaggtgaca gtgaccagat 1080

gagctttgca cctgctgagt atgcacaact ttattctttt gccatgggca tggcatcagt 1140

cttagataag ggaactggca agtaccaatt cgccagggac tttatgagca catcattctg 1200

gagacttgga gtagagtatg ctcaggccca gggaagtagc attaatgagg acatggctgc 1260

tgagctaaaa ctaaccccgg cagcaaggag aggcctggca gctgctgccc aacgagtatc 1320

cgaagaaatc ggcagcatgg acattcccac tcaacaggcg ggagtcctca ccgggctcag 1380

tgatgaaggc ccccgaactc cacagggcag atcaaacaag ccgcaagggc aaccagatgc 1440

tggggatggg gagacccaat tcttggattt tatgagagca gtggcgaacg gcatgcggga 1500

agcgccaaat cctgtacaga gcaccaccca tccagagcct cccccaaccc ctggggcatc 1560

ccaagacaac gacactgact gggggtactg atcgacaaca cccagcctgc cttcacagga 1620

tcacaccaaa ccctccgacc aaaaccctcc cacactccct gacccacaac cccgcacgac 1680

cacaccaaca aaagctcccc cccaccctct cccccactcc cagccacacg atcccgccca 1740

cccgggacaa cacaggcaca gctcagttcg tcgacaatcc gcccagagcc caaggtatta 1800

gaaaaaaata cgggtagaag agagacatcc agagaccagg acgagtcacc aagttctctg 1860

ttctcccttc tacccagtga attagggtga agatggccac ttttacagat gcggagatag 1920

atgacatatt tgagaccagt gggactgtca ttgacagcat aattacggcc cagggcaaat 1980

cagctgagac cgttggaaga agtgcgatcc cgcagggcaa gaccaaagct ctaagcacag 2040

catgggagaa gcacgggagt atccagccac acgccagtca ggacgcccct gaccaacaag 2100

acagaataga aaaacagcca tccacacctg agcaggcgac tctacacaac aatccgccga 2160

tcgcatccac tgaaccgcct cccactcagg ccgcaagcga gaccagcgac acacagctca 2220

agactggggc aagcaactcc cttctgtcca tgctcgacaa actgagtaat aaatcgtcca 2280

atgctaaaaa gggcccatgg tcgggatccc aagaagggca tcaccaacct ccggcccaac 2340

aacatgggaa ccagccgagc tatggaagca accagggaag accgcagcat caggccaagg 2400

ccgtccctgg aaaccggggc acagacgaga acacagcata tcatggacag cggaaggagt 2460

cacaaccatc agctggtgca acccctcatg cgccccagtc agggcagagc caagacaata 2520

ctcctgcacc tgtggatcgt gtccagctac ctgccgactt tgtgcaggcg atgatgtcta 2580

tgatggaggc attatcacag aaggtaagta aagttgatca tcagctggac ctagtcttga 2640

aacagacatc ctccattcct atgatgcgat ctgaaatcca acagctcaag acatctgttg 2700

cgatcatgga agctaactta ggcatgatga aaattctgga ccctggttgt gctaatgttt 2760

catccttaag tgatctccgg gcagtagccc gatcccatcc agtcctagtt tcaggccctg 2820

gagacccatc tccttacgtg acacaagggg gtgaaatgac gctcaataaa ctctcacaac 2880

cggtgcagca cccctctgaa ttgattaagt ctgccactgc aagcgggcct gacatgggag 2940

tggagaagga cactgtccgc gcattaatca cctcgcgccc gatgcatcca agctcctcgg 3000

ctaagctcct gagcaagcta gatgcagcca ggtcaattga agagatcagg aagatcaaac 3060

gccttgcgct gaatggttga tggccatcac aactcataac aggctcccgt cactttagcg 3120

tcacacggaa tccctcgggg gccctccctc gcaaatctat gcttcaacac ccaaaacaac 3180

agccctctct cacccccccc aatccctcga atgaccgcac aactgcaacc aatccagcag 3240

cattagaaat taagaaaaaa tacgggtaga atcaaagtgc cttgattgca ccaaaatgga 3300

ctcatccagg acaattgggc tgtactttga ttctgccctc ccttccagca gcctgttagc 3360

atttccgatt gtcttacaag acacgggaga cgggaagaag caaatcaccc cacaatacag 3420

gatccagcgt cttgattcgt ggacagacag taaggaagac tcggtattca tcaccaccta 3480

cgggttcatc tttcaaattg ggaatgaaga aaccaccgtc ggtgtgatca atgacaatcc 3540

caggcacgag ctactctctt ccgcaatgct ctgcttaggg agtgtcccga acgacggaga 3600

tcttgttgag ctggcgagag cctgcctcac catggtggta acttgcaaaa agagtgcaac 3660

taacactgag agaatagtct tttcagtagt gcaggctcct cgggtgctgc aaagctgtat 3720

ggttgtggca aataggtact catcagtgaa tgcagtgaag catgtgaagg cgccagaaaa 3780

gatccctggg agcggaaccc tagagtataa agtgaatttt gtctctttga ccgtggtgcc 3840

aagaagggat gtctacagga tcccaactgc agtattgaaa gtgtctggct caagcctgta 3900

caatcttgcg ctcaatgtca ctattgatgt ggacgtggat ccgaagagcc cgttagtcaa 3960

atccctttcc aagtccgata gcggatacta tgcgaatctt tttctgcata tcgggcttat 4020

gtccactgta gataagaagg gaaagaaagt gacatttgac aagatagagg aaaagataag 4080

gagactcaat ctatccgtcg ggctcagtga tgtgctcgga ccctctgtgc ttgtgaaggc 4140

gagaggtgca cggactaagc tacttgctcc ttttttctct agcagtggga cagcctgcta 4200

tcctatagca aatgcctctc cccaggttgc caagatactc tggagccaaa ctgcgcacct 4260

gcggagtgtg aaagtcatca ttcaagccgg cactcagcgt gctgtcgcag tgaccgctga 4320

tcatgaggta acctccacta agatagagag gaggcatgcc attgctaaat acaatccttt 4380

caggaaataa gttgcatccc taagactgca gttcacctgc tttcccgaat caccattaca 4440

ccagacaatg atccatctcg actgcttata gttagttcac ctgtctagca aattagaaaa 4500

aacacgggta gaagagtctg gatcccgacc ggcacactca ggacgcaaca tgggctccaa 4560

accttctacc aggatcccag cacctctgat gctggtcacc cggattatgc tgatattggg 4620

ctgtattcgt tcgacaagct ctcttgacgg caggcctctt gcagctgcag gaattgtagt 4680

aacaggagat aaggcagtca atgtatacac ctcgtctcag acagggtcaa tcatagtcaa 4740

gttgctcccg aatatgccca gggataagga ggcgtgtgcg aaagccccat tagaggcata 4800

taacagaaca ctgactactt tgctcactcc tcttggcgac tccatccgca agattcaagg 4860

gtctgtgtcc acgtctggag gaaggaggca aaaacgcttt ataggtgctg ttattggcag 4920

tgtagctctt ggggttgcaa cagcggcaca gataacagca gctgcggccc taatacaagc 4980

caacaagaat gctgccaaca tccttcggct taaggagagc attgctgcaa ccaatgaagc 5040

tgtgcatgaa gtcaccgacg gattatcaca actatcagtg gcagttggga agatgcagca 5100

gtttgtcaat gaccagttta ataatacggc gcgagaattg gactgtataa aaatcacaca 5160

acaggttggt gtagaactca acctatacct aactgaattg actacagtat tcgggccaca 5220

gatcacctct cctgcattaa ctcagctgac catccaggca ctttataatt tagctggtgg 5280

caatatggat tacttattaa ctaagttagg tatagggaac aatcaactca gctcattaat 5340

tggtagcggc ctgatcactg gttaccctat actgtatgac tcacagactc aactcttggg 5400

catacaagtg aatttgccct cagtcgggaa cttaaataat atgcgtgcca cctatttgga 5460

gaccttatct gtaagtacaa ccaaaggata tgcctcagca cttgtcccga aagtagtgac 5520

acaagtcggt tctgtgatag aagagcttga cacctcatac tgtatagagt ccgatctgga 5580

tttatattgt actagaatag tgacattccc catgtcccca ggtatttatt cctgtttgag 5640

cggcaacaca tcagcttgca tgtattcaaa gactgaaggc gcactcacta cgccgtatat 5700

ggcccttaaa ggctcagtta ttgccaattg taagataaca acatgtagat gtacagaccc 5760

tcctggtatc atatcgcaaa attatggaga agctgtatcc ctgatagata gacattcgtg 5820

caatgtctta tcattagacg ggataactct gaggctcagc ggagaatttg atgcaactta 5880

tcaaaagaac atctcaatac tagattctca agtcatcgtg acaggcaatc ttgatatatc 5940

aactgaactt ggaaacgtca acaattcaat cagcaatgcc ttggataagt tggcagaaag 6000

caacagtaag atagaaaaag tcaatgtcag attaaccagc acatctgctc tcattaccta 6060

tattgttcta actgtcattt ctctattttt cggtgcactt agtctgggtt tagcgtgtta 6120

cctgatgtac aaacagaagg cacaacaaaa gaccttgcta tggcttggga ataatgccct 6180

tgatcagatg agagccacta caagagcatg aatgcagata agaggtggat atatacccaa 6240

cagcagcctg tgtgtcaatt ccgataatct gtcaagtaga agacttaaga aaaaactact 6300

gggaacaagc aaccaaagag caatacacgg gtagaacggt cagaggagcc acccttcaat 6360

cggaaattag gcttcacaac atccgttcta ccacatcacc atcaacaaga gtcaatcatg 6420

gaccgcgcgg ttaacagagt cgtgctggag aatgaggaaa gagaagcaaa gaacacatgg 6480

cgcctggttt tccggatcgc agtcttactt ttaatggtaa tgactctagc tatctccgca 6540

gctgccctgg catacagtat ggaggccagt acgccgcacg accttgcagg catatcgact 6600

gtgatctcta agacagaaga taaggttacg tctttactca gttcgagtca agatgtgata 6660

gataggatat acaagcaggt ggctcttgaa tccccgctgg cgctactaaa cactgaatct 6720

acaattatga atgcaataac ctctctttct tatcaaatta acggggctgc gaacaatagc 6780

ggatgtgggg cgcctgttca tgacccagat tatatcgggg ggataggcaa agaactcata 6840

gtggacgaca tcagtgatgt cacatcattt tatccttctg catatcaaga acacctgaat 6900

ttcatcccgg cgcctactac aggatccggt tgcactcgga taccctcatt tgacatgagc 6960

accacccatt attgttatac tcacaatgta atactatccg gttgcagaga tcactcacac 7020

tcacatcaat acttagcgct tggtgtgctt cggacatctg caacagggag ggtattcttc 7080

tctactctgc gctccatcaa tttagatgac acccaaaatc ggaagtcctg cagtgtgagt 7140

gcaacccctt taggttgtga tatgctgtgc tctaaggtca cagggactga agaggaggat 7200

tacaagtcag ctgccccaac atcaatggtg cacggaaggc tagggtttga cggtcaatac 7260

catgagaagg acttagacac cacggtctta tttaaggatt gggtggcaaa ttacccagga 7320

gtgggaggag ggtcttttat tgacgaccgt gtatggttcc cagtttacgg agggctcaaa 7380

cccaattcac ccagtgacac tgcacaagaa gggaaatatg taatatacaa gcgccataac 7440

aacacatgcc ccgatggaca agattaccaa attcggatgg ctaaatcttc atataaaccc 7500

gggcgatttg gtggaaagcg cgtacagcaa gccatattat ccatcaaagt gtcaacatcc 7560

ttgggtaagg acccggtgct gactattcca cctaatacaa tcacactcat gggagccgaa 7620

ggcagaatcc tcacagtagg gacatctcac ttcttgtacc aacgagggtc ttcatatttc 7680

tcccctgcct tattatatcc catgacagta aataacaaaa cggctacact ccatagtcct 7740

tatacgttta atgctttcac tcggccaggt agcgtccctt gccaggcatc agcaagatgc 7800

cccaactcat gcatcactgg ggtctatact gatccatatc ccttaatctt ccataggaat 7860

catactctac gaggggtctt tgggacgatg cttgatgatg aacaagcgag acttaacccc 7920

gtatctgcag tattcgacaa catatcccgc agtcgtgtca cccgggtgag ttcaagcagc 7980

accaaggcag catacacgac atcgacatgt tttaaagttg tcaagaccaa taaagcttat 8040

tgtcttagta ttgcagagat atccaatacc ctattcgggg aatttaggat cgttccctta 8100

ttagttgaga tcctcaagga tgatagagtt taagaagcta gacttggccg attgagccaa 8160

tcataggatg gttgggaaga cgacaccaca tcaatcatct cccacaatgc ttagagtcaa 8220

tctgaatatt aacataagcc aggatcccat gttgttgggc agccacaatc agacaatact 8280

gacatgatca ttctgagtcc tgcccactat caccttatta agaaaaaata caaaaagcat 8340

tgagatataa ggggaaacaa ccaacaagag ggaacacggg taggacatgg cgggctccgg 8400

tcccgaaagg gcagagcacc agatcatcct accagagtca catctatcct ctccattggt 8460

caagcacaaa ttgctatact actggaaatt gactgggcta ccgcttcctg atgaatgcga 8520

ctttgatcat ctcattatca gcaggcaatg gaagagaata ctggagtcgg ccactcctga 8580

cacagagaga atgataaaac tcgggcgggc agtgcaccag actctcaacc acaactccaa 8640

gataaccgga gtgctccatc ccaggtgttt agaagaactg gctagtattg aggtcccaga 8700

ttcaactaac aaattccgga agattgaaaa gaagatccag attcacaaca caaggtatgg 8760

agacctgttc acaaagctgt gcacgcatgt tgagaagaaa ttgctaggat catcccggtc 8820

taataatgtc ccacgatcag aggaattcag tagcatccgt acagatccgg cattctggtt 8880

tcactcaaaa tggtccagag ctaagttcgc gtggctccat ataaaacaag tccaaaggca 8940

tctgattgta gcagcaagga caaggtctgc agtcaacaag ttagtaacat taagtcataa 9000

gataggccac gtctttgtta ctcctgagct tgtcattgtg acacatacag acgagaacaa 9060

gttcacatgc ctcacccagg aacttgtatt gatgtatgcg gatatgatgg aaggcaggga 9120

catggtcaat ataatatctt ctacagcagc acatctcaga aacctatccg agaaaattga 9180

cgatattctg cggttagtag atgccctggc aaaggactta ggtaatcaag tctatgacgt 9240

tgtagcatta atggagggat tcgcatacgg tgctgttcag ctgcttgagc catcaggtac 9300

atttgcagga gatttctttg catttaacct acaggagctc aaagacactt taatcgaact 9360

tctcccaaat aatatagcgg aatcagtaac tcacgctatt gccgctgtat tctccggttt 9420

agaacagaat caagcagctg agatgttgtg cttgctacgt ttgtggggtc atcctttgct 9480

tgagtctcgt agtgcagcaa gagcagtcag gagccagatg tgtgcaccga agatggtaga 9540

cttcgatatg atcctccagg tactatcttt ctttaaagga acaatcatca atggatacag 9600

aaagaagaat tcaggtgtgt ggccgcgtgt caaagtagat acaatatacg ggaatatcat 9660

tgggcagctg tatgctgatt cagcagagat ctcacatgat gtcatgttga aggagtacaa 9720

gagtttatct gcttttgaat ttgagccatg tatagactat gaccctgtta ccaatctaag 9780

catgttccta aaagacaagg caatcgcaca tcctagtgat aattggctcg cctcatttag 9840

gcggaaccta ctctctgagg accagaagaa acagataaaa gaggcaactt caactaaccg 9900

cctcttgata gagttcttag aatcaaatga ttttgatcca tataaagaaa tggaatacct 9960

gacaaccctc gagtacctaa gaggtgacag tgtggcagta tcgtactcac tcaaagagaa 10020

agaggtaaaa gtgaatgggc ggatttttgc taagttaaca aagaaactaa ggaactgcca 10080

ggtaatggca gaaggaattc tagctgacca gattgcacct ttctttcagg gaaatggggt 10140

cattcaagat agcatatcct tgacaaagag tatgttagcg atgagtcaac tgtcctttaa 10200

cagcaataag aaacgtatca ctgactgcaa agagagggtt tcctcgaacc gcaatcatga 10260

tcagaagagc aagaatcgta gaagagttgc cacttttatc acgactgacc tacaaaagta 10320

ttgtcttaac tggagatatc agacagtcaa actattcgcc catgctatca atcagctgat 10380

gggcctacct catttcttcg agtggattca tcttaggctg atggacacta caatgtttgt 10440

aggggatcct ttcaatcctc caagtgaccc gactgactgt gatctatcaa gagtcccaaa 10500

cgatgacata tatattgtca gtgctagagg gggcattgag ggactctgcc agaagctatg 10560

gacgatgatc tcaattgctg caatccaact tgctgcagca agatctcatt gtcgagttgc 10620

ctgcatggta caaggtgaca atcaagtaat ggctgtaacg agagaggtaa gatcagatga 10680

ttccccggat atggtgttga cgcagttgca tcaagctagt gataatttct tcaaggaatt 10740

gattcatgtc aatcatttga ttggccataa tctgaaggat cgtgaaacca ttagatcaga 10800

cacattcttc atatacagca aacgaatatt caaggatgga gcaatactca gtcaggtcct 10860

caaaaattca tctaaattgg tgctaatatc aggcgacctt agcgaaaaca ctgtaatgtc 10920

ctgtgccaac attgcatcca ctgtagcacg actatgtgag aatgggcttc caaaggattt 10980

ctgttactat ttgaactacc taatgagttg tgtgcagaca tactttgatt cggagttctc 11040

tattacccac agctcgcaat cagattccaa ccagtcctgg atcgaggata tctctttcgt 11100

acactcatac gtgttaaccc ctgcccagct ggggggactg agcaaccttc aatactcaag 11160

gctctacaca aggaatattg gtgacccagg gaccactgct ttcgcagagg tcaagcgact 11220

ggaagcagtg gggttgctga gtcccagcat catgactaac atcttaacca ggccacctgg 11280

caatggagac tgggccagcc tatgcaacga cccatactct tttaattttg aaactgttgc 11340

aagcccaaat attgtcctca agaaacatac acagaaagtc ctatttgaga catgttcaaa 11400

ccccttatta tccggggtac atacagagga caatgaggca gaggagaaag cattggctga 11460

attcttactc aatcaagaag tgattcaccc acgtgtcgca catgctatca tggaagcaag 11520

ctctgtgggt aggagaaagc aaattcaagg gcttgttgac acaacgaaca ctgtgattaa 11580

gattgcactg actaggaggc ccctcggtat caaaaggctg atgcggataa tcaattactc 11640

gagcatgcat gcaatgttgt tcagagatga tattttctta tccaatagat ccaaccaccc 11700

attagtttct tccaatatgt gctcgctgac gctagcagat tatgcccgaa acagaagctg 11760

gtcacctctg acagggggca ggaaaatact gggtgtatcc aaccctgata ccatagaact 11820

tgtggaggga gagattctca gcgtcagtgg agggtgcaca aaatgtgaca gcggagatga 11880

gcagtttact tggttccatc ttccaagcaa tatagagctg actgatgaca ccagaaaaaa 11940

tcccccgatg agagtgccat atcttgggtc gaagactcaa gagaggagag ctgcctcgct 12000

tgcgaaaata gcccacatgt caccacatgt gaaagcagca ctaagggcat catccgtgtt 12060

aatctgggct tatggggaca acgaagtgaa ctggactgct gctcttaata ttgcaaggtc 12120

tcgatgcaac ataagctcag agtaccttcg gctattgtca cccctgccca cggctgggaa 12180

tctccaacat agattggatg atggcataac ccagatgaca tttacccctg catctctcta 12240

cagagtgtcg ccttacgttc acatatccaa tgattctcaa aggctattca ccgaagaagg 12300

ggtcaaagag ggaaacgtgg tttaccaaca aattatgctc ttgggtttat ccctaattga 12360

atcactcttc ccaatgacag caaccagaac atatgatgag atcacattac acctccacag 12420

taaatttagc tgctgtatcc gagaagcgcc tgttgcggtt cccttcgagc tcctcgggct 12480

ggcaccggaa ttaagaatgg taacctcaaa taagttcatg tatgatccta gccctatatc 12540

agagagagat ttcgcgagac ttgacttagc tatcttcaag agttacgagc ttaatttgga 12600

atcatattcc acgctggagc taatgaacat tctttcaata tctagcggga agttgattgg 12660

ccaatctgtg gtttcttatg atgaagacac ctctataaag aatgatgcta taatagtgta 12720

tgacaacaca cgaaattgga ttagtgaggc gcagaactca gatgtggtcc gcttgtttga 12780

gtatgcagca ctcgaagtgc ttcttgactg tgcttatcaa ctctactatc tgagggtaag 12840

gggtctaaac aacatcgtcc tatacatgaa tgacttatat aagaacatgc cagggatcct 12900

actctccaat attgcggcca cgatatccca ccccatcatt cactcaaggt tgaatgcagt 12960

aggcctaatt aaccatgacg ggtcacacca gctcgcagat atagacttcg tcgaggtgtc 13020

tgcaaaattg ttagtctctt gcactcgacg cgtggtctca ggcttatatg cagggaataa 13080

gtacgatctg ctgttcccat ctgtcttaga tgataacctg aatgagaaga tgctccaact 13140

gatttcccgg ttatgctgtc tgtacacagt gctctttgct acaacaagag aaatcccaaa 13200

aataaggggc ctatcagcag aagagaaatg ctcaatactc actgagtatc tactgtcaga 13260

tgctgtaaaa ccattgctta ggcccgaaca agtgaattct atcatgtctc ccaacataat 13320

cacgttccca gccaatctat attacatgtc taggaagagc cttaatttga tcagagaacg 13380

agaggacaga gatactatct tgtcattgtt gttccctcag gaaccactgc ttgagcttcg 13440

cccagtacgg gacattggtg ctcgagtgaa agacccgttt acccggcaac ccgcatcatt 13500

catacaagag ctagatctga gtgccccagc aaggtacgac gcatttacac tgagtaaggc 13560

ctgcttcgag catacattac cgaacccaaa ggaggattac ctagtacggt acttgttcag 13620

aggaataggg actgcttcat cttcttggta tgcggcatct catcttctat ccgtacctga 13680

ggtcaggtgt gcaagacatg ggaactcctt atacttagcg gaaggaagcg gagccatcat 13740

gagtcttctt gaattgcata taccacatga gactatctat tacaatacac ttttctcgaa 13800

tgagatgaac cctccacagc gacatttcgg acctacacca acacagtttc taaactcggt 13860

cgtttatagg aatctacaag cggaagtgcc atgtaaagat ggatatgtcc aggagttctg 13920

cccattatgg agagagaatg cagaagaaag tgacctgacc tcagataaag cagttggata 13980

tatcacatcc gtggtaccct acaggtctgt atcattacta cattgtgaca ttgagattcc 14040

tccagggtcc aatcaaagct tattagatca actggctact aatttatccc tgattgccat 14100

gcattctgtg agggagggcg gggtagtgat catcaaagta ctgtatgcaa tggggtacta 14160

cttccattta ctcatgaatt tattcactcc atgttccaca aaaggatata tactctccaa 14220

tggctatgcc tgtagagggg atatggagtg ttacctgata ttcgttatgg gctacttggg 14280

cgggcccacc ttcgtgcacg aagtggtaag gatggcaaaa actctaatac aacgacacgg 14340

tacactccta tctaaatcag atgaaattac attgactaag ctatttacct cacagcagcg 14400

tcatgtaaca gatatcctat ccagtccttt accgaagcta atgaagctct tgagagaaaa 14460

tattgatgcc gcactaattg aagctggggg acagcccgtc cgtccattct gtgcggaaag 14520

tttggtgagc acactaacag atatgactca gacagctcag atcattgcca gccacattga 14580

cacagtcatt cggtctgtaa tttacatgga ggctgagggt gacctcgccg acacagtgtt 14640

cttatttact ccttacaatc tatccacaga cggtaaaagg agaacatcac ttaagcagtg 14700

caccaaacag atcttggaag tcacaatact gggtctcaga gccaaagata tcaataaagt 14760

aggtgatgta atcagtttag tactcagagg tgcggtttct ctagaggacc tcatcccatt 14820

aaggacatac ctgaagcgca gtacctgcac taaatacctg aaagcggtcc taggtattac 14880

taaactcaaa gaaatgttca cagatacctc attactgtac ttgactcgtg ctcaacaaaa 14940

attctacatg aaaaccatag gtaatgctgc caagggatat tactgtaata atgactctta 15000

aaggcaatcg tacgccaatc agttatcttc ctaactgatg actccctcat tgactcaatt 15060

ataccagatt agaaaaaagt taaattccga ctctttggaa ctcgtattcg gattcagtta 15120

gttaacttta agcaagagtg cgcaaagtca tccctaatta tagtgatgtc attcaccaaa 15180

tctctgtttg gt 15192

<210> 2

<211> 15192

<212> DNA

<213>Artificial sequence（SEQ ID NO：2）

<400> 2

accaaacaga gaatctgtga ggtacgataa aaggcgaaga agcaatcgag atcgtacggg 60

tagaaggtgt gaatcccgag tgcgaggccg aagctcgaac ctgaggggac cttctaccga 120

tatgtcgtct gttttcgacg agtacgagca gctcctcgct gctcagaccc gccctaacgg 180

aactcatgga gggggagaga aagggagcac tttaaaagtt gaggtcccag tattcaccct 240

aaacagtgat gatccagagg atagatggaa ttttgcggta ttctgtcttc ggattgctgt 300

tagcgaggat gccaacaaac cactcaggca aggtgctctt atatccctct tatgctccca 360

ttctcaggtg atgagaaacc atgttgccct tgcagggaaa caaaatgagg ccacactggc 420

tgttcttgag atcgatggtt ttgctaacag tgtgccccag ttcaacaata ggagtggagt 480

gtctgaggaa agagcacaga gattcatggt aatcgcagga tctctccctc gggcgtgcag 540

caacggtact ccgtttgtca cggctggggt tgaagatgat gcaccagaag atatcactga 600

cactctggaa agaatcctat ctatccaagt tcaggtatgg gtcacagtag caaaggccat 660

gactgcatat gagacagcag atgagtcaga aacaagaaga ataaataagt atatgcagca 720

aggtcgagtt cagaagaagt acatccttca tcctgtatgc aggagtgcaa ttcaactcac 780

aatcagacat tctctggcag tccgtatctt cctagttagt gagctcaaga ggggccgcaa 840

tacagcaggt gggagctcta catattacaa cttggtcggg gatgtagact catacatcag 900

gaacaccggg cttactgcat ttttcctaac actcaaatat ggaatcaata ccaagacgtc 960

agccctcgca ctcagcagcc tcacaggtga tatccaaaaa atgaaacagc tcatgcgttt 1020

atatcggatg aaaggtgaaa atgcaccata catgacattg ttaggtgaca gtgaccagat 1080

gagctttgca cctgctgagt atgcacaact ttattctttt gccatgggca tggcatcagt 1140

cttagataag ggaactggca agtaccaatt cgccagggac tttatgagca catcattctg 1200

gagacttgga gtagagtatg ctcaggccca gggaagtagc attaatgagg acatggctgc 1260

tgagctaaaa ctaaccccgg cagcaaggag aggcctggca gctgctgccc aacgagtatc 1320

cgaagaaatc ggcagcatgg acattcccac tcaacaggcg ggagtcctca ccgggctcag 1380

tgatgaaggc ccccgaactc cacagggcag atcaaacaag ccgcaagggc aaccagatgc 1440

tggggatggg gagacccaat tcttggattt tatgagagca gtggcgaacg gcatgcggga 1500

agcgccaaat cctgtacaga gcaccaccca tccagagcct cccccaaccc ctggggcatc 1560

ccaagacaac gacactgact gggggtactg atcgacaaca cccagcctgc cttcacagga 1620

tcacaccaaa ccctccgacc aaaaccctcc cacactccct gacccacaac cccgcacgac 1680

cacaccaaca aaagctcccc cccaccctct cccccactcc cagccacacg atcccgccca 1740

cccgggacaa cacaggcaca gctcagttcg tcgacaatcc gcccagagcc caaggtatta 1800

gaaaaaaata cgggtagaag agagacatcc agagaccagg acgagtcacc aagttctctg 1860

ttctcccttc tacccagtga attagggtga agatggccac ttttacagat gcggagatag 1920

atgacatatt tgagaccagt gggactgtca ttgacagcat aattacggcc cagggcaaat 1980

cagctgagac cgttggaaga agtgcgatcc cgcagggcaa gaccaaagct ctaagcacag 2040

catgggagaa gcacgggagt atccagccac acgccagtca ggacgcccct gaccaacaag 2100

acagaataga aaaacagcca tccacacctg agcaggcgac tctacacaac aatccgccga 2160

tcgcatccac tgaaccgcct cccactcagg ccgcaagcga gaccagcgac acacagctca 2220

agactggggc aagcaactcc cttctgtcca tgctcgacaa actgagtaat aaatcgtcca 2280

atgctaaaaa gggcccatgg tcgggatccc aagaagggca tcaccaacct ccggcccaac 2340

aacatgggaa ccagccgagc tatggaagca accagggaag accgcagcat caggccaagg 2400

ccgtccctgg aaaccggggc acagacgaga acacagcata tcatggacag cggaaggagt 2460

cacaaccatc agctggtgca acccctcatg cgccccagtc agggcagagc caagacaata 2520

ctcctgcacc tgtggatcgt gtccagctac ctgccgactt tgtgcaggcg atgatgtcta 2580

tgatggaggc attatcacag aaggtaagta aagttgatca tcagctggac ctagtcttga 2640

aacagacatc ctccattcct atgatgcgat ctgaaatcca acagctcaag acatctgttg 2700

cgatcatgga agctaactta ggcatgatga aaattctgga ccctggttgt gctaatgttt 2760

catccttaag tgatctccgg gcagtagccc gatcccatcc agtcctagtt tcaggccctg 2820

gagacccatc tccttacgtg acacaagggg gtgaaatgac gctcaataaa ctctcacaac 2880

cggtgcagca cccctctgaa ttgattaagt ctgccactgc aagcgggcct gacatgggag 2940

tggagaagga cactgtccgc gcattaatca cctcgcgccc gatgcatcca agctcctcgg 3000

ctaagctcct gagcaagcta gatgcagcca ggtcaattga agagatcagg aagatcaaac 3060

gccttgcgct gaatggttga tggccatcac aactcataac aggctcccgt cactttagcg 3120

tcacacggaa tccctcgggg gccctccctc gcaaatctat gcttcaacac ccaaaacaac 3180

agccctctct cacccccccc aatccctcga atgaccgcac aactgcaacc aatccagcag 3240

cattagaaat taagaaaaaa tacgggtaga atcaaagtgc cttgattgca ccaaaatgga 3300

ctcatccagg acaattgggc tgtactttga ttctgccctc ccttccagca gcctgttagc 3360

atttccgatt gtcttacaag acacgggaga cgggaagaag caaatcaccc cacaatacag 3420

gatccagcgt cttgattcgt ggacagacag taaggaagac tcggtattca tcaccaccta 3480

cgggttcatc tttcaaattg ggaatgaaga aaccaccgtc ggtgtgatca atgacaatcc 3540

caggcacgag ctactctctt ccgcaatgct ctgcttaggg agtgtcccga acgacggaga 3600

tcttgttgag ctggcgagag cctgcctcac catggtggta acttgcaaaa agagtgcaac 3660

taacactgag agaatagtct tttcagtagt gcaggctcct cgggtgctgc aaagctgtat 3720

ggttgtggca aataggtact catcagtgaa tgcagtgaag catgtgaagg cgccagaaaa 3780

gatccctggg agcggaaccc tagagtataa agtgaatttt gtctctttga ccgtggtgcc 3840

aagaagggat gtctacagga tcccaactgc agtattgaaa gtgtctggct caagcctgta 3900

caatcttgcg ctcaatgtca ctattgatgt ggacgtggat ccgaagagcc cgttagtcaa 3960

atccctttcc aagtccgata gcggatacta tgcgaatctt tttctgcata tcgggcttat 4020

gtccactgta gataagaagg gaaagaaagt gacatttgac aagatagagg aaaagataag 4080

gagactcaat ctatccgtcg ggctcagtga tgtgctcgga ccctctgtgc ttgtgaaggc 4140

gagaggtgca cggactaagc tacttgctcc ttttttctct agcagtggga cagcctgcta 4200

tcctatagca aatgcctctc cccaggttgc caagatactc tggagccaaa ctgcgcacct 4260

gcggagtgtg aaagtcatca ttcaagccgg cactcagcgt gctgtcgcag tgaccgctga 4320

tcatgaggta acctccacta agatagagag gaggcatgcc attgctaaat acaatccttt 4380

caggaaataa gttgcatccc taagactgca gttcacctgc tttcccgaat caccattaca 4440

ccagacaatg atccatctcg actgcttata gttagttcac ctgtctagca aattagaaaa 4500

aacacgggta gaagagtctg gatcccgacc ggcacactca ggacgcaaca tgggctccaa 4560

accttctacc aggatcccag cacctctgat gctggtcacc cggattatgc tgatattggg 4620

ctgtattcgt tcgacaagct ctcttgacgg caggcctctt gcagctgcag gaattgtagt 4680

aacaggagat aaggcagtca atgtatacac ctcgtctcag acagggtcaa tcatagtcaa 4740

gttgctcccg aatatgccca gggataagga ggcgtgtgcg aaagccccat tagaggcata 4800

taacagaaca ctgactactt tgctcactcc tcttggcgac tccatccgca agattcaagg 4860

gtctgtgtcc acgtctggag gaaggaggca aaaacgcttt ataggtgctg ttattggcag 4920

tgtagctctt ggggttgcaa cagcggcaca gataacagca gctgcggccc taatacaagc 4980

caacaagaat gctgccaaca tccttcggct taaggagagc attgctgcaa ccaatgaagc 5040

tgtgcatgaa gtcaccgacg gattatcaca actatcagtg gcagttggga agatgcagca 5100

gtttgtcaat gaccagttta ataatacggc gcgagaattg gactgtataa aaatcacaca 5160

acaggttggt gtagaactca acctatacct aactgaattg actacagtat tcgggccaca 5220

gatcacctct cctgcattaa ctcagctgac catccaggca ctttataatt tagctggtgg 5280

caatatggat tacttattaa ctaagttagg tatagggaac aatcaactca gctcattaat 5340

tggtagcggc ctgatcactg gttaccctat actgtatgac tcacagactc aactcttggg 5400

catacaagtg aatttgccct cagtcgggaa cttaaataat atgcgtgcca cctatttgga 5460

gaccttatct gtaagtacaa ccaaaggata tgcctcagca cttgtcccga aagtagtgac 5520

acaagtcggt tctgtgatag aagagcttga cacctcatac tgtatagagt ccgatctgga 5580

tttatattgt actagaatag tgacattccc catgtcccca ggtatttatt cctgtttgag 5640

cggcaacaca tcagcttgca tgtattcaaa gactgaaggc gcactcacta cgccgtatat 5700

ggcccttaaa ggctcagtta ttgccaattg taagataaca acatgtagat gtacagaccc 5760

tcctggtatc atatcgcaaa attatggaga agctgtatcc ctgatagata gacattcgtg 5820

caatgtctta tcattagacg ggataactct gaggctcagc ggagaatttg atgcaactta 5880

tcaaaagaac atctcaatac tagattctca agtcatcgtg acaggcaatc ttgatatatc 5940

aactgaactt ggaaacgtca acaattcaat cagcaatgcc ttggataagt tggcagaaag 6000

caacagtaag atagaaaaag tcaatgtcag attaaccagc acatctgctc tcattaccta 6060

tattgttcta actgtcattt ctctattttt cggtgcactt agtctgggtt tagcgtgtta 6120

cctgatgtac aaacagaagg cacaacaaaa gaccttgcta tggcttggga ataatgccct 6180

tgatcagatg agagccacta caagagcatg aatgcagata agaggtggat atatacccaa 6240

cagcagcctg tgtgtcaatt ccgataatct gtcaagtaga agacttaaga aaaaactact 6300

gggaacaagc aaccaaagag caatacacgg gtagaacggt cagaggagcc acccttcaat 6360

cggaaattag gcttcacaac atccgttcta ccacatcacc atcaacaaga gtcaatcatg 6420

gaccgcgcgg ttaacagagt cgtgctggag aatgaggaaa gagaagcaaa gaacacatgg 6480

cgcctggttt tccggatcgc agtcttactt ttaatggtaa tgactctagc tatctccgca 6540

gctgccctgg catacagtat ggaggccagt acgccgcacg accttgcagg catatcgact 6600

gtgatctcta agacagaaga taaggttacg tctttactca gttcgagtca agatgtgata 6660

gataggatat acaagcaggt ggctcttgaa tccccgctgg cgctactaaa cactgaatct 6720

acaattatga atgcaataac ctctctttct tatcaaatta acggggctgc gaacaatagc 6780

ggatgtgggg cgcctgttca tgacccagat tatatcgggg ggataggcaa agaactcata 6840

gtggacgaca tcagtgatgt cacatcattt tatccttctg catatcaaga acacctgaat 6900

ttcatcccgg cgcctactac aggatccggt tgcactcgga taccctcatt tgacatgagc 6960

accacccatt attgttatac tcacaatgta atactatccg gttgcagaga tcactcacac 7020

tcacatcaat acttagcgct tggtgtgctt cggacatctg caacagggag ggtattcttc 7080

tctactctgc gctccatcaa tttagatgac acccaaaatc ggaagtcctg cagtgtgagt 7140

gcaacccctt taggttgtga tatgctgtgc tctaaggtca cagggactga agaggaggat 7200

tacaagtcag ctgccccaac atcaatggtg cacggaaggc tagggtttga cggtcaatac 7260

catgagaagg acttagacac cacggtctta tttaaggatt gggtggcaaa ttacccagga 7320

gtgggaggag ggtcttttat tgacgaccgt gtatggttcc cagtttacgg agggctcaaa 7380

cccaattcac ccagtgacac tgcacaagaa gggaaatatg taatatacaa gcgccataac 7440

aacacatgcc ccgatggaca agattaccaa attcggatgg ctaaatcttc atataaaccc 7500

gggcgatttg gtggaaagcg cgtacagcaa gccatattat ccatcaaagt gtcaacatcc 7560

ttgggtaagg acccggtgct gactattcca cctaatacaa tcacactcat gggagccgaa 7620

ggcagaatcc tcacagtagg gacatctcac ttcttgtacc aacgagggtc ttcatatttc 7680

tcccctgcct tattatatcc catgacagta aataacaaaa cggctacact ccatagtcct 7740

tatacgttta atgctttcac tcggccaggt agcgtccctt gccaggcatc agcaagatgc 7800

cccaactcat gcatcactgg ggtctatact gatccatatc ccttaatctt ccataggaat 7860

catactctac gaggggtctt tgggacgatg cttgatgatg aacaagcgag acttaacccc 7920

gtatctgcag tattcgacaa catatcccgc agtcgtgtca cccgggtgag ttcaagcagc 7980

accaaggcag catacacgac atcgacatgt tttaaagttg tcaagaccaa taaagcttat 8040

tgtcttagta ttgcagagat atccaatacc ctattcgggg aatttaggat cgttccctta 8100

ttagttgaga tcctcaagga tgatagagtt taagaagcta gacttggccg attgagccaa 8160

tcataggatg gttgggaaga cgacaccaca tcaatcatct cccacaatgc ttagagtcaa 8220

tctgaatatt aacataagcc aggatcccat gttgttgggc agccacaatc agacaatact 8280

gacatgatca ttctgagtcc tgcccactat caccttatta agaaaaaata caaaaagcat 8340

tgagatataa ggggaaacaa ccaacaagag ggaacacggg taggacatgg cgggctccgg 8400

tcccgaaagg gcagagcacc agatcatcct accagagtca catctatcct ctccattggt 8460

caagcacaaa ttgctatact actggaaatt gactgggcta ccgcttcctg atgaatgcga 8520

ctttgatcat ctcattatca gcaggcaatg gaagagaata ctggagtcgg ccactcctga 8580

cacagagaga atgataaaac tcgggcgggc agtgcaccag actctcaacc acaactccaa 8640

gataaccgga gtgctccatc ccaggtgttt agaagaactg gctagtattg aggtcccaga 8700

ttcaactaac aaattccgga agattgaaaa gaagatccag attcacaaca caaggtatgg 8760

agacctgttc acaaagctgt gcacgcatgt tgagaagaaa ttgctaggat catcccggtc 8820

taataatgtc ccacgatcag aggaattcag tagcatccgt acagatccgg cattctggtt 8880

tcactcaaaa tggtccagag ctaagttcgc gtggctccat ataaaacaag tccaaaggca 8940

tctgattgta gcagcaagga caaggtctgc agtcaacaag ttagtaacat taagtcataa 9000

gataggccac gtctttgtta ctcctgagct tgtcattgtg acacatacag acgagaacaa 9060

gttcacatgc ctcacccagg aacttgtatt gatgtatgcg gatatgatgg aaggcaggga 9120

catggtcaat ataatatctt ctacagcagc acatctcaga aacctatccg agaaaattga 9180

cgatattctg cggttagtag atgccctggc aaaggactta ggtaatcaag tctatgacgt 9240

tgtagcatta atggagggat tcgcatacgg tgctgttcag ctgcttgagc catcaggtac 9300

atttgcagga gatttctttg catttaacct acaggagctc aaagacactt taatcgaact 9360

tctcccaaat aatatagcgg aatcagtaac tcacgctatt gccgctgtat tctccggttt 9420

agaacagaat caagcagctg agatgttgtg cttgctacgt ttgtggggtc atcctttgct 9480

tgagtctcgt agtgcagcaa gagcagtcag gagccagatg tgtgcaccga agatggtaga 9540

cttcgatatg atcctccagg tactatcttt ctttaaagga acaatcatca atggatacag 9600

aaagaagaat tcaggtgtgt ggccgcgtgt caaagtagat acaatatacg ggaatatcat 9660

tgggcagctg tatgctgatt cagcagagat ctcacatgat gtcatgttga aggagtacaa 9720

gagtttatct gcttttgaat ttgagccatg tatagactat gaccctgtta ccaatctaag 9780

catgttccta aaagacaagg caatcgcaca tcctagtgat aattggctcg cctcatttag 9840

gcggaaccta ctctctgagg accagaagaa acagataaaa gaggcaactt caactaaccg 9900

cctcttgata gagttcttag aatcaaatga ttttgatcca tataaagaaa tggaatacct 9960

gacaaccctc gagtacctaa gaggtgacag tgtggcagta tcgtactcac tcaaagagaa 10020

agaggtaaaa gtgaatgggc ggatttttgc taagttaaca aagaaactaa ggaactgcca 10080

ggtaatggca gaaggaattc tagctgacca gattgcacct ttctttcagg gaaatggggt 10140

cattcaagat agcatatcct tgacaaagag tatgttagcg atgagtcaac tgtcctttaa 10200

cagcaataag aaacgtatca ctgactgcaa agagagggtt tcctcgaacc gcaatcatga 10260

tcagaagagc aagaatcgta gaagagttgc cacttttatc acgactgacc tacaaaagta 10320

ttgtcttaac tggagatatc agacagtcaa actattcgcc catgctatca atcagctgat 10380

gggcctacct catttcttcg agtggattca tcttaggctg atggacacta caatgtttgt 10440

aggggatcct ttcaatcctc caagtgaccc gactgactgt gatctatcaa gagtcccaaa 10500

cgatgacata tatattgtca gtgctagagg gggcattgag ggactctgcc agaagctatg 10560

gacgatgatc tcaattgctg caatccaact tgctgcagca agatctcatt gtcgagttgc 10620

ctgcatggta caaggtgaca atcaagtaat ggctgtaacg agagaggtaa gatcagatga 10680

ttccccggat atggtgttga cgcagttgca tcaagctagt gataatttct tcaaggaatt 10740

gattcatgtc aatcatttga ttggccataa tctgaaggat cgtgaaacca ttagatcaga 10800

cacattcttc atatacagca aacgaatatt caaggatgga gcaatactca gtcaggtcct 10860

caaaaattca tctaaattgg tgctaatatc aggcgacctt agcgaaaaca ctgtaatgtc 10920

ctgtgccaac attgcatcca ctgtagcacg actatgtgag aatgggcttc caaaggattt 10980

ctgttactat ttgaactacc taatgagttg tgtgcagaca tactttgatt cggagttctc 11040

tattacccac agctcgcaat cagattccaa ccagtcctgg atcgaggata tctctttcgt 11100

acactcatac gtgttaaccc ctgcccagct ggggggactg agcaaccttc aatactcaag 11160

gctctacaca aggaatattg gtgacccagg gaccactgct ttcgcagagg tcaagcgact 11220

ggaagcagtg gggttgctga gtcccagcat catgactaac atcttaacca ggccacctgg 11280

caatggagac tgggccagcc tatgcaacga cccatactct tttaattttg aaactgttgc 11340

aagcccaaat attgtcctca agaaacatac acagaaagtc ctatttgaga catgttcaaa 11400

ccccttatta tccggggtac atacagagga caatgaggca gaggagaaag cattggctga 11460

attcttactc aatcaagaag tgattcaccc acgtgtcgca catgctatca tggaagcaag 11520

ctctgtgggt aggagaaagc aaattcaagg gcttgttgac acaacgaaca ctgtgattaa 11580

gattgcactg actaggaggc ccctcggtat caaaaggctg atgcggataa tcaattactc 11640

gagcatgcat gcaatgttgt tcagagatga tattttctta tccaatagat ccaaccaccc 11700

attagtttct tccaatatgt gctcgctgac gctagcagat tatgcccgaa acagaagctg 11760

gtcacctctg acagggggca ggaaaatact gggtgtatcc aaccctgata ccatagaact 11820

tgtggaggga gagattctca gcgtcagtgg agggtgcaca aaatgtgaca gcggagatga 11880

gcagtttact tggttccatc ttccaagcaa tatagagctg actgatgaca ccagaaaaaa 11940

tcccccgatg agagtgccat atcttgggtc gaagactcaa gagaggagag ctgcctcgct 12000

tgcgaaaata gcccacatgt caccacatgt gaaagcagca ctaagggcat catccgtgtt 12060

aatctgggct tatggggaca acgaagtgaa ctggactgct gctcttaata ttgcaaggtc 12120

tcgatgcaac ataagctcag agtaccttcg gctattgtca cccctgccca cggctgggaa 12180

tctccaacat agattggatg atggcataac ccagatgaca tttacccctg catctctcta 12240

cagagtgtcg ccttacgttc acatatccaa tgattctcaa aggctattca ccgaagaagg 12300

ggtcaaagag ggaaacgtgg tttaccaaca aattatgctc ttgggtttat ccctaattga 12360

atcactcttc ccaatgacag caaccagaac atatgatgag atcacattac acctccacag 12420

taaatttagc tgctgtatcc gagaagcgcc tgttgcggtt cccttcgagc tcctcgggct 12480

ggcaccggaa ttaagaatgg taacctcaaa taagttcatg tatgatccta gccctatatc 12540

agagagagat ttcgcgagac ttgacttagc tatcttcaag agttacgagc ttaatttgga 12600

atcatattcc acgctggagc taatgaacat tctttcaata tctagcggga agttgattgg 12660

ccaatctgtg gtttcttatg atgaagacac ctctataaag aatgatgcta taatagtgta 12720

tgacaacaca cgaaattgga ttagtgaggc gcagaactca gatgtggtcc gcttgtttga 12780

gtatgcagca ctcgaagtgc ttcttgactg tgcttatcaa ctctactatc tgagggtaag 12840

gggtctaaac aacatcgtcc tatacatgaa tgacttatat aagaacatgc cagggatcct 12900

actctccaat attgcggcca cgatatccca ccccatcatt cactcaaggt tgaatgcagt 12960

aggcctaatt aaccatgacg ggtcacacca gctcgcagat atagacttcg tcgaggtgtc 13020

tgcaaaattg ttagtctctt gcactcgacg cgtggtctca ggcttatatg cagggaataa 13080

gtacgatctg ctgttcccat ctgtcttaga tgataacctg aatgagaaga tgctccaact 13140

gatttcccgg ttatgctgtc tgtacacagt gctctttgct acaacaagag aaatcccaaa 13200

aataaggggc ctatcagcag aagagaaatg ctcaatactc actgagtatc tactgtcaga 13260

tgctgtaaaa ccattgctta ggcccgaaca agtgaattct atcatgtctc ccaacataat 13320

cacgttccca gccaatctat attacatgtc taggaagagc cttaatttga tcagagaacg 13380

agaggacaga gatactatct tgtcattgtt gttccctcag gaaccactgc ttgagcttcg 13440

cccagtacgg gacattggtg ctcgagtgaa agacccgttt acccggcaac ccgcatcatt 13500

catacaagag ctagatctga gtgccccagc aaggtacgac gcatttacac tgagtaaggc 13560

ctgcttcgag catacattac cgaacccaaa ggaggattac ctagtacggt acttgttcag 13620

aggaataggg actgcttcat cttcttggta taaggcatct catcttctat ccgtacctga 13680

ggtcaggtgt gcaagacatg ggaactcctt atacttagcg gaaggaagcg gagccatcat 13740

gagtcttctt gaattgcata taccacatga gactatctat tacaatacac ttttctcgaa 13800

tgagatgaac cctccacagc gacatttcgg acctacacca acacagtttc taaactcggt 13860

cgtttatagg aatctacaag cggaagtgcc atgtaaagat ggatatgtcc aggagttctg 13920

cccattatgg agagagaatg cagaagaaag tgacctgacc tcagataaag cagttggata 13980

tatcacatcc gtggtaccct acaggtctgt atcattacta cattgtgaca ttgagattcc 14040

tccagggtcc aatcaaagct tattagatca actggctact aatttatccc tgattgccat 14100

gcattctgtg agggagggcg gggtagtgat catcgcagta ctgtatgcaa tggggtacta 14160

cttccattta ctcatgaatt tattcactcc atgttccaca aaaggatata tactctccaa 14220

tggctatgcc tgtagagggg atatggagtg ttacctgata ttcgttatgg gctacttggg 14280

cgggcccacc ttcgtgcacg aagtggtaag gatggcaaaa actctaatac aacgacacgg 14340

tacactccta tctaaatcag atgaaattac attgactaag ctatttacct cacagcagcg 14400

tcatgtaaca gatatcctat ccagtccttt accgaagcta atgaagctct tgagagaaaa 14460

tattgatgcc gcactaattg aagctggggg acagcccgtc cgtccattct gtgcggaaag 14520

tttggtgagc acactaacag atatgactca gacagctcag atcattgcca gccacattga 14580

cacagtcatt cggtctgtaa tttacatgga ggctgagggt gacctcgccg acacagtgtt 14640

cttatttact ccttacaatc tatccacaga cggtaaaagg agaacatcac ttaagcagtg 14700

caccaaacag atcttggaag tcacaatact gggtctcaga gccaaagata tcaataaagt 14760

aggtgatgta atcagtttag tactcagagg tgcggtttct ctagaggacc tcatcccatt 14820

aaggacatac ctgaagcgca gtacctgcac taaatacctg aaagcggtcc taggtattac 14880

taaactcaaa gaaatgttca cagatacctc attactgtac ttgactcgtg ctcaacaaaa 14940

attctacatg aaaaccatag gtaatgctgc caagggatat tactgtaata atgactctta 15000

aaggcaatcg tacgccaatc agttatcttc ctaactgatg actccctcat tgactcaatt 15060

ataccagatt agaaaaaagt taaattccga ctctttggaa ctcgtattcg gattcagtta 15120

gttaacttta agcaagagtg cgcaaagtca tccctaatta tagtgatgtc attcaccaaa 15180

tctctgtttg gt 15192

<210> 3

<211> 15192

<212> DNA

<213>Artificial sequence（SEQ ID NO：3）

<400> 3

accaaacaga gaatctgtga ggtacgataa aaggcgaaga agcaatcgag atcgtacggg 60

tagaaggtgt gaatcccgag tgcgaggccg aagctcgaac ctgaggggac cttctaccga 120

tatgtcgtct gttttcgacg agtacgagca gctcctcgct gctcagaccc gccctaacgg 180

aactcatgga gggggagaga aagggagcac tttaaaagtt gaggtcccag tattcaccct 240

aaacagtgat gatccagagg atagatggaa ttttgcggta ttctgtcttc ggattgctgt 300

tagcgaggat gccaacaaac cactcaggca aggtgctctt atatccctct tatgctccca 360

ttctcaggtg atgagaaacc atgttgccct tgcagggaaa caaaatgagg ccacactggc 420

tgttcttgag atcgatggtt ttgctaacag tgtgccccag ttcaacaata ggagtggagt 480

gtctgaggaa agagcacaga gattcatggt aatcgcagga tctctccctc gggcgtgcag 540

caacggtact ccgtttgtca cggctggggt tgaagatgat gcaccagaag atatcactga 600

cactctggaa agaatcctat ctatccaagt tcaggtatgg gtcacagtag caaaggccat 660

gactgcatat gagacagcag atgagtcaga aacaagaaga ataaataagt atatgcagca 720

aggtcgagtt cagaagaagt acatccttca tcctgtatgc aggagtgcaa ttcaactcac 780

aatcagacat tctctggcag tccgtatctt cctagttagt gagctcaaga ggggccgcaa 840

tacagcaggt gggagctcta catattacaa cttggtcggg gatgtagact catacatcag 900

gaacaccggg cttactgcat ttttcctaac actcaaatat ggaatcaata ccaagacgtc 960

agccctcgca ctcagcagcc tcacaggtga tatccaaaaa atgaaacagc tcatgcgttt 1020

atatcggatg aaaggtgaaa atgcaccata catgacattg ttaggtgaca gtgaccagat 1080

gagctttgca cctgctgagt atgcacaact ttattctttt gccatgggca tggcatcagt 1140

cttagataag ggaactggca agtaccaatt cgccagggac tttatgagca catcattctg 1200

gagacttgga gtagagtatg ctcaggccca gggaagtagc attaatgagg acatggctgc 1260

tgagctaaaa ctaaccccgg cagcaaggag aggcctggca gctgctgccc aacgagtatc 1320

cgaagaaatc ggcagcatgg acattcccac tcaacaggcg ggagtcctca ccgggctcag 1380

tgatgaaggc ccccgaactc cacagggcag atcaaacaag ccgcaagggc aaccagatgc 1440

tggggatggg gagacccaat tcttggattt tatgagagca gtggcgaacg gcatgcggga 1500

agcgccaaat cctgtacaga gcaccaccca tccagagcct cccccaaccc ctggggcatc 1560

ccaagacaac gacactgact gggggtactg atcgacaaca cccagcctgc cttcacagga 1620

tcacaccaaa ccctccgacc aaaaccctcc cacactccct gacccacaac cccgcacgac 1680

cacaccaaca aaagctcccc cccaccctct cccccactcc cagccacacg atcccgccca 1740

cccgggacaa cacaggcaca gctcagttcg tcgacaatcc gcccagagcc caaggtatta 1800

gaaaaaaata cgggtagaag agagacatcc agagaccagg acgagtcacc aagttctctg 1860

ttctcccttc tacccagtga attagggtga agatggccac ttttacagat gcggagatag 1920

atgacatatt tgagaccagt gggactgtca ttgacagcat aattacggcc cagggcaaat 1980

cagctgagac cgttggaaga agtgcgatcc cgcagggcaa gaccaaagct ctaagcacag 2040

catgggagaa gcacgggagt atccagccac acgccagtca ggacgcccct gaccaacaag 2100

acagaataga aaaacagcca tccacacctg agcaggcgac tctacacaac aatccgccga 2160

tcgcatccac tgaaccgcct cccactcagg ccgcaagcga gaccagcgac acacagctca 2220

agactggggc aagcaactcc cttctgtcca tgctcgacaa actgagtaat aaatcgtcca 2280

atgctaaaaa gggcccatgg tcgggatccc aagaagggca tcaccaacct ccggcccaac 2340

aacatgggaa ccagccgagc tatggaagca accagggaag accgcagcat caggccaagg 2400

ccgtccctgg aaaccggggc acagacgaga acacagcata tcatggacag cggaaggagt 2460

cacaaccatc agctggtgca acccctcatg cgccccagtc agggcagagc caagacaata 2520

ctcctgcacc tgtggatcgt gtccagctac ctgccgactt tgtgcaggcg atgatgtcta 2580

tgatggaggc attatcacag aaggtaagta aagttgatca tcagctggac ctagtcttga 2640

aacagacatc ctccattcct atgatgcgat ctgaaatcca acagctcaag acatctgttg 2700

cgatcatgga agctaactta ggcatgatga aaattctgga ccctggttgt gctaatgttt 2760

catccttaag tgatctccgg gcagtagccc gatcccatcc agtcctagtt tcaggccctg 2820

gagacccatc tccttacgtg acacaagggg gtgaaatgac gctcaataaa ctctcacaac 2880

cggtgcagca cccctctgaa ttgattaagt ctgccactgc aagcgggcct gacatgggag 2940

tggagaagga cactgtccgc gcattaatca cctcgcgccc gatgcatcca agctcctcgg 3000

ctaagctcct gagcaagcta gatgcagcca ggtcaattga agagatcagg aagatcaaac 3060

gccttgcgct gaatggttga tggccatcac aactcataac aggctcccgt cactttagcg 3120

tcacacggaa tccctcgggg gccctccctc gcaaatctat gcttcaacac ccaaaacaac 3180

agccctctct cacccccccc aatccctcga atgaccgcac aactgcaacc aatccagcag 3240

cattagaaat taagaaaaaa tacgggtaga atcaaagtgc cttgattgca ccaaaatgga 3300

ctcatccagg acaattgggc tgtactttga ttctgccctc ccttccagca gcctgttagc 3360

atttccgatt gtcttacaag acacgggaga cgggaagaag caaatcaccc cacaatacag 3420

gatccagcgt cttgattcgt ggacagacag taaggaagac tcggtattca tcaccaccta 3480

cgggttcatc tttcaaattg ggaatgaaga aaccaccgtc ggtgtgatca atgacaatcc 3540

caggcacgag ctactctctt ccgcaatgct ctgcttaggg agtgtcccga acgacggaga 3600

tcttgttgag ctggcgagag cctgcctcac catggtggta acttgcaaaa agagtgcaac 3660

taacactgag agaatagtct tttcagtagt gcaggctcct cgggtgctgc aaagctgtat 3720

ggttgtggca aataggtact catcagtgaa tgcagtgaag catgtgaagg cgccagaaaa 3780

gatccctggg agcggaaccc tagagtataa agtgaatttt gtctctttga ccgtggtgcc 3840

aagaagggat gtctacagga tcccaactgc agtattgaaa gtgtctggct caagcctgta 3900

caatcttgcg ctcaatgtca ctattgatgt ggacgtggat ccgaagagcc cgttagtcaa 3960

atccctttcc aagtccgata gcggatacta tgcgaatctt tttctgcata tcgggcttat 4020

gtccactgta gataagaagg gaaagaaagt gacatttgac aagatagagg aaaagataag 4080

gagactcaat ctatccgtcg ggctcagtga tgtgctcgga ccctctgtgc ttgtgaaggc 4140

gagaggtgca cggactaagc tacttgctcc ttttttctct agcagtggga cagcctgcta 4200

tcctatagca aatgcctctc cccaggttgc caagatactc tggagccaaa ctgcgcacct 4260

gcggagtgtg aaagtcatca ttcaagccgg cactcagcgt gctgtcgcag tgaccgctga 4320

tcatgaggta acctccacta agatagagag gaggcatgcc attgctaaat acaatccttt 4380

caggaaataa gttgcatccc taagactgca gttcacctgc tttcccgaat caccattaca 4440

ccagacaatg atccatctcg actgcttata gttagttcac ctgtctagca aattagaaaa 4500

aacacgggta gaagagtctg gatcccgacc ggcacactca ggacgcaaca tgggctccaa 4560

accttctacc aggatcccag cacctctgat gctggtcacc cggattatgc tgatattggg 4620

ctgtattcgt tcgacaagct ctcttgacgg caggcctctt gcagctgcag gaattgtagt 4680

aacaggagat aaggcagtca atgtatacac ctcgtctcag acagggtcaa tcatagtcaa 4740

gttgctcccg aatatgccca gggataagga ggcgtgtgcg aaagccccat tagaggcata 4800

taacagaaca ctgactactt tgctcactcc tcttggcgac tccatccgca agattcaagg 4860

gtctgtgtcc acgtctggag gaaggaggca aaaacgcttt ataggtgctg ttattggcag 4920

tgtagctctt ggggttgcaa cagcggcaca gataacagca gctgcggccc taatacaagc 4980

caacaagaat gctgccaaca tccttcggct taaggagagc attgctgcaa ccaatgaagc 5040

tgtgcatgaa gtcaccgacg gattatcaca actatcagtg gcagttggga agatgcagca 5100

gtttgtcaat gaccagttta ataatacggc gcgagaattg gactgtataa aaatcacaca 5160

acaggttggt gtagaactca acctatacct aactgaattg actacagtat tcgggccaca 5220

gatcacctct cctgcattaa ctcagctgac catccaggca ctttataatt tagctggtgg 5280

caatatggat tacttattaa ctaagttagg tatagggaac aatcaactca gctcattaat 5340

tggtagcggc ctgatcactg gttaccctat actgtatgac tcacagactc aactcttggg 5400

catacaagtg aatttgccct cagtcgggaa cttaaataat atgcgtgcca cctatttgga 5460

gaccttatct gtaagtacaa ccaaaggata tgcctcagca cttgtcccga aagtagtgac 5520

acaagtcggt tctgtgatag aagagcttga cacctcatac tgtatagagt ccgatctgga 5580

tttatattgt actagaatag tgacattccc catgtcccca ggtatttatt cctgtttgag 5640

cggcaacaca tcagcttgca tgtattcaaa gactgaaggc gcactcacta cgccgtatat 5700

ggcccttaaa ggctcagtta ttgccaattg taagataaca acatgtagat gtacagaccc 5760

tcctggtatc atatcgcaaa attatggaga agctgtatcc ctgatagata gacattcgtg 5820

caatgtctta tcattagacg ggataactct gaggctcagc ggagaatttg atgcaactta 5880

tcaaaagaac atctcaatac tagattctca agtcatcgtg acaggcaatc ttgatatatc 5940

aactgaactt ggaaacgtca acaattcaat cagcaatgcc ttggataagt tggcagaaag 6000

caacagtaag atagaaaaag tcaatgtcag attaaccagc acatctgctc tcattaccta 6060

tattgttcta actgtcattt ctctattttt cggtgcactt agtctgggtt tagcgtgtta 6120

cctgatgtac aaacagaagg cacaacaaaa gaccttgcta tggcttggga ataatgccct 6180

tgatcagatg agagccacta caagagcatg aatgcagata agaggtggat atatacccaa 6240

cagcagcctg tgtgtcaatt ccgataatct gtcaagtaga agacttaaga aaaaactact 6300

gggaacaagc aaccaaagag caatacacgg gtagaacggt cagaggagcc acccttcaat 6360

cggaaattag gcttcacaac atccgttcta ccacatcacc atcaacaaga gtcaatcatg 6420

gaccgcgcgg ttaacagagt cgtgctggag aatgaggaaa gagaagcaaa gaacacatgg 6480

cgcctggttt tccggatcgc agtcttactt ttaatggtaa tgactctagc tatctccgca 6540

gctgccctgg catacagtat ggaggccagt acgccgcacg accttgcagg catatcgact 6600

gtgatctcta agacagaaga taaggttacg tctttactca gttcgagtca agatgtgata 6660

gataggatat acaagcaggt ggctcttgaa tccccgctgg cgctactaaa cactgaatct 6720

acaattatga atgcaataac ctctctttct tatcaaatta acggggctgc gaacaatagc 6780

ggatgtgggg cgcctgttca tgacccagat tatatcgggg ggataggcaa agaactcata 6840

gtggacgaca tcagtgatgt cacatcattt tatccttctg catatcaaga acacctgaat 6900

ttcatcccgg cgcctactac aggatccggt tgcactcgga taccctcatt tgacatgagc 6960

accacccatt attgttatac tcacaatgta atactatccg gttgcagaga tcactcacac 7020

tcacatcaat acttagcgct tggtgtgctt cggacatctg caacagggag ggtattcttc 7080

tctactctgc gctccatcaa tttagatgac acccaaaatc ggaagtcctg cagtgtgagt 7140

gcaacccctt taggttgtga tatgctgtgc tctaaggtca cagggactga agaggaggat 7200

tacaagtcag ctgccccaac atcaatggtg cacggaaggc tagggtttga cggtcaatac 7260

catgagaagg acttagacac cacggtctta tttaaggatt gggtggcaaa ttacccagga 7320

gtgggaggag ggtcttttat tgacgaccgt gtatggttcc cagtttacgg agggctcaaa 7380

cccaattcac ccagtgacac tgcacaagaa gggaaatatg taatatacaa gcgccataac 7440

aacacatgcc ccgatggaca agattaccaa attcggatgg ctaaatcttc atataaaccc 7500

gggcgatttg gtggaaagcg cgtacagcaa gccatattat ccatcaaagt gtcaacatcc 7560

ttgggtaagg acccggtgct gactattcca cctaatacaa tcacactcat gggagccgaa 7620

ggcagaatcc tcacagtagg gacatctcac ttcttgtacc aacgagggtc ttcatatttc 7680

tcccctgcct tattatatcc catgacagta aataacaaaa cggctacact ccatagtcct 7740

tatacgttta atgctttcac tcggccaggt agcgtccctt gccaggcatc agcaagatgc 7800

cccaactcat gcatcactgg ggtctatact gatccatatc ccttaatctt ccataggaat 7860

catactctac gaggggtctt tgggacgatg cttgatgatg aacaagcgag acttaacccc 7920

gtatctgcag tattcgacaa catatcccgc agtcgtgtca cccgggtgag ttcaagcagc 7980

accaaggcag catacacgac atcgacatgt tttaaagttg tcaagaccaa taaagcttat 8040

tgtcttagta ttgcagagat atccaatacc ctattcgggg aatttaggat cgttccctta 8100

ttagttgaga tcctcaagga tgatagagtt taagaagcta gacttggccg attgagccaa 8160

tcataggatg gttgggaaga cgacaccaca tcaatcatct cccacaatgc ttagagtcaa 8220

tctgaatatt aacataagcc aggatcccat gttgttgggc agccacaatc agacaatact 8280

gacatgatca ttctgagtcc tgcccactat caccttatta agaaaaaata caaaaagcat 8340

tgagatataa ggggaaacaa ccaacaagag ggaacacggg taggacatgg cgggctccgg 8400

tcccgaaagg gcagagcacc agatcatcct accagagtca catctatcct ctccattggt 8460

caagcacaaa ttgctatact actggaaatt gactgggcta ccgcttcctg atgaatgcga 8520

ctttgatcat ctcattatca gcaggcaatg gaagagaata ctggagtcgg ccactcctga 8580

cacagagaga atgataaaac tcgggcgggc agtgcaccag actctcaacc acaactccaa 8640

gataaccgga gtgctccatc ccaggtgttt agaagaactg gctagtattg aggtcccaga 8700

ttcaactaac aaattccgga agattgaaaa gaagatccag attcacaaca caaggtatgg 8760

agacctgttc acaaagctgt gcacgcatgt tgagaagaaa ttgctaggat catcccggtc 8820

taataatgtc ccacgatcag aggaattcag tagcatccgt acagatccgg cattctggtt 8880

tcactcaaaa tggtccagag ctaagttcgc gtggctccat ataaaacaag tccaaaggca 8940

tctgattgta gcagcaagga caaggtctgc agtcaacaag ttagtaacat taagtcataa 9000

gataggccac gtctttgtta ctcctgagct tgtcattgtg acacatacag acgagaacaa 9060

gttcacatgc ctcacccagg aacttgtatt gatgtatgcg gatatgatgg aaggcaggga 9120

catggtcaat ataatatctt ctacagcagc acatctcaga aacctatccg agaaaattga 9180

cgatattctg cggttagtag atgccctggc aaaggactta ggtaatcaag tctatgacgt 9240

tgtagcatta atggagggat tcgcatacgg tgctgttcag ctgcttgagc catcaggtac 9300

atttgcagga gatttctttg catttaacct acaggagctc aaagacactt taatcgaact 9360

tctcccaaat aatatagcgg aatcagtaac tcacgctatt gccgctgtat tctccggttt 9420

agaacagaat caagcagctg agatgttgtg cttgctacgt ttgtggggtc atcctttgct 9480

tgagtctcgt agtgcagcaa gagcagtcag gagccagatg tgtgcaccga agatggtaga 9540

cttcgatatg atcctccagg tactatcttt ctttaaagga acaatcatca atggatacag 9600

aaagaagaat tcaggtgtgt ggccgcgtgt caaagtagat acaatatacg ggaatatcat 9660

tgggcagctg tatgctgatt cagcagagat ctcacatgat gtcatgttga aggagtacaa 9720

gagtttatct gcttttgaat ttgagccatg tatagactat gaccctgtta ccaatctaag 9780

catgttccta aaagacaagg caatcgcaca tcctagtgat aattggctcg cctcatttag 9840

gcggaaccta ctctctgagg accagaagaa acagataaaa gaggcaactt caactaaccg 9900

cctcttgata gagttcttag aatcaaatga ttttgatcca tataaagaaa tggaatacct 9960

gacaaccctc gagtacctaa gaggtgacag tgtggcagta tcgtactcac tcaaagagaa 10020

agaggtaaaa gtgaatgggc ggatttttgc taagttaaca aagaaactaa ggaactgcca 10080

ggtaatggca gaaggaattc tagctgacca gattgcacct ttctttcagg gaaatggggt 10140

cattcaagat agcatatcct tgacaaagag tatgttagcg atgagtcaac tgtcctttaa 10200

cagcaataag aaacgtatca ctgactgcaa agagagggtt tcctcgaacc gcaatcatga 10260

tcagaagagc aagaatcgta gaagagttgc cacttttatc acgactgacc tacaaaagta 10320

ttgtcttaac tggagatatc agacagtcaa actattcgcc catgctatca atcagctgat 10380

gggcctacct catttcttcg agtggattca tcttaggctg atggacacta caatgtttgt 10440

aggggatcct ttcaatcctc caagtgaccc gactgactgt gatctatcaa gagtcccaaa 10500

cgatgacata tatattgtca gtgctagagg gggcattgag ggactctgcc agaagctatg 10560

gacgatgatc tcaattgctg caatccaact tgctgcagca agatctcatt gtcgagttgc 10620

ctgcatggta caaggtgaca atcaagtaat ggctgtaacg agagaggtaa gatcagatga 10680

ttccccggat atggtgttga cgcagttgca tcaagctagt gataatttct tcaaggaatt 10740

gattcatgtc aatcatttga ttggccataa tctgaaggat cgtgaaacca ttagatcaga 10800

cacattcttc atatacagca aacgaatatt caaggatgga gcaatactca gtcaggtcct 10860

caaaaattca tctaaattgg tgctaatatc aggcgacctt agcgaaaaca ctgtaatgtc 10920

ctgtgccaac attgcatcca ctgtagcacg actatgtgag aatgggcttc caaaggattt 10980

ctgttactat ttgaactacc taatgagttg tgtgcagaca tactttgatt cggagttctc 11040

tattacccac agctcgcaat cagattccaa ccagtcctgg atcgaggata tctctttcgt 11100

acactcatac gtgttaaccc ctgcccagct ggggggactg agcaaccttc aatactcaag 11160

gctctacaca aggaatattg gtgacccagg gaccactgct ttcgcagagg tcaagcgact 11220

ggaagcagtg gggttgctga gtcccagcat catgactaac atcttaacca ggccacctgg 11280

caatggagac tgggccagcc tatgcaacga cccatactct tttaattttg aaactgttgc 11340

aagcccaaat attgtcctca agaaacatac acagaaagtc ctatttgaga catgttcaaa 11400

ccccttatta tccggggtac atacagagga caatgaggca gaggagaaag cattggctga 11460

attcttactc aatcaagaag tgattcaccc acgtgtcgca catgctatca tggaagcaag 11520

ctctgtgggt aggagaaagc aaattcaagg gcttgttgac acaacgaaca ctgtgattaa 11580

gattgcactg actaggaggc ccctcggtat caaaaggctg atgcggataa tcaattactc 11640

gagcatgcat gcaatgttgt tcagagatga tattttctta tccaatagat ccaaccaccc 11700

attagtttct tccaatatgt gctcgctgac gctagcagat tatgcccgaa acagaagctg 11760

gtcacctctg acagggggca ggaaaatact gggtgtatcc aaccctgata ccatagaact 11820

tgtggaggga gagattctca gcgtcagtgg agggtgcaca aaatgtgaca gcggagatga 11880

gcagtttact tggttccatc ttccaagcaa tatagagctg actgatgaca ccagaaaaaa 11940

tcccccgatg agagtgccat atcttgggtc gaagactcaa gagaggagag ctgcctcgct 12000

tgcgaaaata gcccacatgt caccacatgt gaaagcagca ctaagggcat catccgtgtt 12060

aatctgggct tatggggaca acgaagtgaa ctggactgct gctcttaata ttgcaaggtc 12120

tcgatgcaac ataagctcag agtaccttcg gctattgtca cccctgccca cggctgggaa 12180

tctccaacat agattggatg atggcataac ccagatgaca tttacccctg catctctcta 12240

cagagtgtcg ccttacgttc acatatccaa tgattctcaa aggctattca ccgaagaagg 12300

ggtcaaagag ggaaacgtgg tttaccaaca aattatgctc ttgggtttat ccctaattga 12360

atcactcttc ccaatgacag caaccagaac atatgatgag atcacattac acctccacag 12420

taaatttagc tgctgtatcc gagaagcgcc tgttgcggtt cccttcgagc tcctcgggct 12480

ggcaccggaa ttaagaatgg taacctcaaa taagttcatg tatgatccta gccctatatc 12540

agagagagat ttcgcgagac ttgacttagc tatcttcaag agttacgagc ttaatttgga 12600

atcatattcc acgctggagc taatgaacat tctttcaata tctagcggga agttgattgg 12660

ccaatctgtg gtttcttatg atgaagacac ctctataaag aatgatgcta taatagtgta 12720

tgacaacaca cgaaattgga ttagtgaggc gcagaactca gatgtggtcc gcttgtttga 12780

gtatgcagca ctcgaagtgc ttcttgactg tgcttatcaa ctctactatc tgagggtaag 12840

gggtctaaac aacatcgtcc tatacatgaa tgacttatat aagaacatgc cagggatcct 12900

actctccaat attgcggcca cgatatccca ccccatcatt cactcaaggt tgaatgcagt 12960

aggcctaatt aaccatgacg ggtcacacca gctcgcagat atagacttcg tcgaggtgtc 13020

tgcaaaattg ttagtctctt gcactcgacg cgtggtctca ggcttatatg cagggaataa 13080

gtacgatctg ctgttcccat ctgtcttaga tgataacctg aatgagaaga tgctccaact 13140

gatttcccgg ttatgctgtc tgtacacagt gctctttgct acaacaagag aaatcccaaa 13200

aataaggggc ctatcagcag aagagaaatg ctcaatactc actgagtatc tactgtcaga 13260

tgctgtaaaa ccattgctta ggcccgaaca agtgaattct atcatgtctc ccaacataat 13320

cacgttccca gccaatctat attacatgtc taggaagagc cttaatttga tcagagaacg 13380

agaggacaga gatactatct tgtcattgtt gttccctcag gaaccactgc ttgagcttcg 13440

cccagtacgg gacattggtg ctcgagtgaa agacccgttt acccggcaac ccgcatcatt 13500

catacaagag ctagatctga gtgccccagc aaggtacgac gcatttacac tgagtaaggc 13560

ctgcttcgag catacattac cgaacccaaa ggaggattac ctagtacggt acttgttcag 13620

aggaataggg actgcttcat cttcttggta taaggcatct catcttctat ccgtacctga 13680

ggtcaggtgt gcaagacatg ggaactcctt atacttagcg gaaggaagcg gagccatcat 13740

gagtcttctt gaattgcata taccacatga gactatctat tacaatacac ttttctcgaa 13800

tgagatgaac cctccacagc gacatttcgg acctacacca acacagtttc taaactcggt 13860

cgtttatagg aatctacaag cggaagtgcc atgtaaagat ggatatgtcc aggagttctg 13920

cccattatgg agagagaatg cagaagaaag tgacctgacc tcagataaag cagttggata 13980

tatcacatcc gtggtaccct acaggtctgt atcattacta cattgtgaca ttgagattcc 14040

tccagggtcc aatcaaagct tattagatca actggctact aatttatccc tgattgccat 14100

gcattctgtg agggagggcg gggtagtgat catcaaagta ctgtatgcaa tggggtacta 14160

cttccattta ctcatgaatt tattcactcc atgttccaca aaaggatata tactctccaa 14220

tggctatgcc tgtagagggg atatgcaatg ttacctgata ttcgttatgg gctacttggg 14280

cgggcccacc ttcgtgcacg aagtggtaag gatggcaaaa actctaatac aacgacacgg 14340

tacactccta tctaaatcag atgaaattac attgactaag ctatttacct cacagcagcg 14400

tcatgtaaca gatatcctat ccagtccttt accgaagcta atgaagctct tgagagaaaa 14460

tattgatgcc gcactaattg aagctggggg acagcccgtc cgtccattct gtgcggaaag 14520

tttggtgagc acactaacag atatgactca gacagctcag atcattgcca gccacattga 14580

cacagtcatt cggtctgtaa tttacatgga ggctgagggt gacctcgccg acacagtgtt 14640

cttatttact ccttacaatc tatccacaga cggtaaaagg agaacatcac ttaagcagtg 14700

caccaaacag atcttggaag tcacaatact gggtctcaga gccaaagata tcaataaagt 14760

aggtgatgta atcagtttag tactcagagg tgcggtttct ctagaggacc tcatcccatt 14820

aaggacatac ctgaagcgca gtacctgcac taaatacctg aaagcggtcc taggtattac 14880

taaactcaaa gaaatgttca cagatacctc attactgtac ttgactcgtg ctcaacaaaa 14940

attctacatg aaaaccatag gtaatgctgc caagggatat tactgtaata atgactctta 15000

aaggcaatcg tacgccaatc agttatcttc ctaactgatg actccctcat tgactcaatt 15060

ataccagatt agaaaaaagt taaattccga ctctttggaa ctcgtattcg gattcagtta 15120

gttaacttta agcaagagtg cgcaaagtca tccctaatta tagtgatgtc attcaccaaa 15180

tctctgtttg gt 15192

<210> 4

<211> 15192

<212> DNA

<213>Artificial sequence（SEQ ID NO：4）

<400> 4

accaaacaga gaatctgtga ggtacgataa aaggcgaaga agcaatcgag atcgtacggg 60

tagaaggtgt gaatcccgag tgcgaggccg aagctcgaac ctgaggggac cttctaccga 120

tatgtcgtct gttttcgacg agtacgagca gctcctcgct gctcagaccc gccctaacgg 180

aactcatgga gggggagaga aagggagcac tttaaaagtt gaggtcccag tattcaccct 240

aaacagtgat gatccagagg atagatggaa ttttgcggta ttctgtcttc ggattgctgt 300

tagcgaggat gccaacaaac cactcaggca aggtgctctt atatccctct tatgctccca 360

ttctcaggtg atgagaaacc atgttgccct tgcagggaaa caaaatgagg ccacactggc 420

tgttcttgag atcgatggtt ttgctaacag tgtgccccag ttcaacaata ggagtggagt 480

gtctgaggaa agagcacaga gattcatggt aatcgcagga tctctccctc gggcgtgcag 540

caacggtact ccgtttgtca cggctggggt tgaagatgat gcaccagaag atatcactga 600

cactctggaa agaatcctat ctatccaagt tcaggtatgg gtcacagtag caaaggccat 660

gactgcatat gagacagcag atgagtcaga aacaagaaga ataaataagt atatgcagca 720

aggtcgagtt cagaagaagt acatccttca tcctgtatgc aggagtgcaa ttcaactcac 780

aatcagacat tctctggcag tccgtatctt cctagttagt gagctcaaga ggggccgcaa 840

tacagcaggt gggagctcta catattacaa cttggtcggg gatgtagact catacatcag 900

gaacaccggg cttactgcat ttttcctaac actcaaatat ggaatcaata ccaagacgtc 960

agccctcgca ctcagcagcc tcacaggtga tatccaaaaa atgaaacagc tcatgcgttt 1020

atatcggatg aaaggtgaaa atgcaccata catgacattg ttaggtgaca gtgaccagat 1080

gagctttgca cctgctgagt atgcacaact ttattctttt gccatgggca tggcatcagt 1140

cttagataag ggaactggca agtaccaatt cgccagggac tttatgagca catcattctg 1200

gagacttgga gtagagtatg ctcaggccca gggaagtagc attaatgagg acatggctgc 1260

tgagctaaaa ctaaccccgg cagcaaggag aggcctggca gctgctgccc aacgagtatc 1320

cgaagaaatc ggcagcatgg acattcccac tcaacaggcg ggagtcctca ccgggctcag 1380

tgatgaaggc ccccgaactc cacagggcag atcaaacaag ccgcaagggc aaccagatgc 1440

tggggatggg gagacccaat tcttggattt tatgagagca gtggcgaacg gcatgcggga 1500

agcgccaaat cctgtacaga gcaccaccca tccagagcct cccccaaccc ctggggcatc 1560

ccaagacaac gacactgact gggggtactg atcgacaaca cccagcctgc cttcacagga 1620

tcacaccaaa ccctccgacc aaaaccctcc cacactccct gacccacaac cccgcacgac 1680

cacaccaaca aaagctcccc cccaccctct cccccactcc cagccacacg atcccgccca 1740

cccgggacaa cacaggcaca gctcagttcg tcgacaatcc gcccagagcc caaggtatta 1800

gaaaaaaata cgggtagaag agagacatcc agagaccagg acgagtcacc aagttctctg 1860

ttctcccttc tacccagtga attagggtga agatggccac ttttacagat gcggagatag 1920

atgacatatt tgagaccagt gggactgtca ttgacagcat aattacggcc cagggcaaat 1980

cagctgagac cgttggaaga agtgcgatcc cgcagggcaa gaccaaagct ctaagcacag 2040

catgggagaa gcacgggagt atccagccac acgccagtca ggacgcccct gaccaacaag 2100

acagaataga aaaacagcca tccacacctg agcaggcgac tctacacaac aatccgccga 2160

tcgcatccac tgaaccgcct cccactcagg ccgcaagcga gaccagcgac acacagctca 2220

agactggggc aagcaactcc cttctgtcca tgctcgacaa actgagtaat aaatcgtcca 2280

atgctaaaaa gggcccatgg tcgggatccc aagaagggca tcaccaacct ccggcccaac 2340

aacatgggaa ccagccgagc tatggaagca accagggaag accgcagcat caggccaagg 2400

ccgtccctgg aaaccggggc acagacgaga acacagcata tcatggacag cggaaggagt 2460

cacaaccatc agctggtgca acccctcatg cgccccagtc agggcagagc caagacaata 2520

ctcctgcacc tgtggatcgt gtccagctac ctgccgactt tgtgcaggcg atgatgtcta 2580

tgatggaggc attatcacag aaggtaagta aagttgatca tcagctggac ctagtcttga 2640

aacagacatc ctccattcct atgatgcgat ctgaaatcca acagctcaag acatctgttg 2700

cgatcatgga agctaactta ggcatgatga aaattctgga ccctggttgt gctaatgttt 2760

catccttaag tgatctccgg gcagtagccc gatcccatcc agtcctagtt tcaggccctg 2820

gagacccatc tccttacgtg acacaagggg gtgaaatgac gctcaataaa ctctcacaac 2880

cggtgcagca cccctctgaa ttgattaagt ctgccactgc aagcgggcct gacatgggag 2940

tggagaagga cactgtccgc gcattaatca cctcgcgccc gatgcatcca agctcctcgg 3000

ctaagctcct gagcaagcta gatgcagcca ggtcaattga agagatcagg aagatcaaac 3060

gccttgcgct gaatggttga tggccatcac aactcataac aggctcccgt cactttagcg 3120

tcacacggaa tccctcgggg gccctccctc gcaaatctat gcttcaacac ccaaaacaac 3180

agccctctct cacccccccc aatccctcga atgaccgcac aactgcaacc aatccagcag 3240

cattagaaat taagaaaaaa tacgggtaga atcaaagtgc cttgattgca ccaaaatgga 3300

ctcatccagg acaattgggc tgtactttga ttctgccctc ccttccagca gcctgttagc 3360

atttccgatt gtcttacaag acacgggaga cgggaagaag caaatcaccc cacaatacag 3420

gatccagcgt cttgattcgt ggacagacag taaggaagac tcggtattca tcaccaccta 3480

cgggttcatc tttcaaattg ggaatgaaga aaccaccgtc ggtgtgatca atgacaatcc 3540

caggcacgag ctactctctt ccgcaatgct ctgcttaggg agtgtcccga acgacggaga 3600

tcttgttgag ctggcgagag cctgcctcac catggtggta acttgcaaaa agagtgcaac 3660

taacactgag agaatagtct tttcagtagt gcaggctcct cgggtgctgc aaagctgtat 3720

ggttgtggca aataggtact catcagtgaa tgcagtgaag catgtgaagg cgccagaaaa 3780

gatccctggg agcggaaccc tagagtataa agtgaatttt gtctctttga ccgtggtgcc 3840

aagaagggat gtctacagga tcccaactgc agtattgaaa gtgtctggct caagcctgta 3900

caatcttgcg ctcaatgtca ctattgatgt ggacgtggat ccgaagagcc cgttagtcaa 3960

atccctttcc aagtccgata gcggatacta tgcgaatctt tttctgcata tcgggcttat 4020

gtccactgta gataagaagg gaaagaaagt gacatttgac aagatagagg aaaagataag 4080

gagactcaat ctatccgtcg ggctcagtga tgtgctcgga ccctctgtgc ttgtgaaggc 4140

gagaggtgca cggactaagc tacttgctcc ttttttctct agcagtggga cagcctgcta 4200

tcctatagca aatgcctctc cccaggttgc caagatactc tggagccaaa ctgcgcacct 4260

gcggagtgtg aaagtcatca ttcaagccgg cactcagcgt gctgtcgcag tgaccgctga 4320

tcatgaggta acctccacta agatagagag gaggcatgcc attgctaaat acaatccttt 4380

caggaaataa gttgcatccc taagactgca gttcacctgc tttcccgaat caccattaca 4440

ccagacaatg atccatctcg actgcttata gttagttcac ctgtctagca aattagaaaa 4500

aacacgggta gaagagtctg gatcccgacc ggcacactca ggacgcaaca tgggctccaa 4560

accttctacc aggatcccag cacctctgat gctggtcacc cggattatgc tgatattggg 4620

ctgtattcgt tcgacaagct ctcttgacgg caggcctctt gcagctgcag gaattgtagt 4680

aacaggagat aaggcagtca atgtatacac ctcgtctcag acagggtcaa tcatagtcaa 4740

gttgctcccg aatatgccca gggataagga ggcgtgtgcg aaagccccat tagaggcata 4800

taacagaaca ctgactactt tgctcactcc tcttggcgac tccatccgca agattcaagg 4860

gtctgtgtcc acgtctggag gagggagaca ggggcgcctt ataggtgctg ttattggcag 4920

tgtagctctt ggggttgcaa cagcggcaca gataacagca gctgcggccc taatacaagc 4980

caacaagaat gctgccaaca tccttcggct taaggagagc attgctgcaa ccaatgaagc 5040

tgtgcatgaa gtcaccgacg gattatcaca actatcagtg gcagttggga agatgcagca 5100

gtttgtcaat gaccagttta ataatacggc gcgagaattg gactgtataa aaatcacaca 5160

acaggttggt gtagaactca acctatacct aactgaattg actacagtat tcgggccaca 5220

gatcacctct cctgcattaa ctcagctgac catccaggca ctttataatt tagctggtgg 5280

caatatggat tacttattaa ctaagttagg tatagggaac aatcaactca gctcattaat 5340

tggtagcggc ctgatcactg gttaccctat actgtatgac tcacagactc aactcttggg 5400

catacaagtg aatttgccct cagtcgggaa cttaaataat atgcgtgcca cctatttgga 5460

gaccttatct gtaagtacaa ccaaaggata tgcctcagca cttgtcccga aagtagtgac 5520

acaagtcggt tctgtgatag aagagcttga cacctcatac tgtatagagt ccgatctgga 5580

tttatattgt actagaatag tgacattccc catgtcccca ggtatttatt cctgtttgag 5640

cggcaacaca tcagcttgca tgtattcaaa gactgaaggc gcactcacta cgccgtatat 5700

ggcccttaaa ggctcagtta ttgccaattg taagataaca acatgtagat gtacagaccc 5760

tcctggtatc atatcgcaaa attatggaga agctgtatcc ctgatagata gacattcgtg 5820

caatgtctta tcattagacg ggataactct gaggctcagc ggagaatttg atgcaactta 5880

tcaaaagaac atctcaatac tagattctca agtcatcgtg acaggcaatc ttgatatatc 5940

aactgaactt ggaaacgtca acaattcaat cagcaatgcc ttggataagt tggcagaaag 6000

caacagtaag atagaaaaag tcaatgtcag attaaccagc acatctgctc tcattaccta 6060

tattgttcta actgtcattt ctctattttt cggtgcactt agtctgggtt tagcgtgtta 6120

cctgatgtac aaacagaagg cacaacaaaa gaccttgcta tggcttggga ataatgccct 6180

tgatcagatg agagccacta caagagcatg aatgcagata agaggtggat atatacccaa 6240

cagcagcctg tgtgtcaatt ccgataatct gtcaagtaga agacttaaga aaaaactact 6300

gggaacaagc aaccaaagag caatacacgg gtagaacggt cagaggagcc acccttcaat 6360

cggaaattag gcttcacaac atccgttcta ccacatcacc atcaacaaga gtcaatcatg 6420

gaccgcgcgg ttaacagagt cgtgctggag aatgaggaaa gagaagcaaa gaacacatgg 6480

cgcctggttt tccggatcgc agtcttactt ttaatggtaa tgactctagc tatctccgca 6540

gctgccctgg catacagtat ggaggccagt acgccgcacg accttgcagg catatcgact 6600

gtgatctcta agacagaaga taaggttacg tctttactca gttcgagtca agatgtgata 6660

gataggatat acaagcaggt ggctcttgaa tccccgctgg cgctactaaa cactgaatct 6720

acaattatga atgcaataac ctctctttct tatcaaatta acggggctgc gaacaatagc 6780

ggatgtgggg cgcctgttca tgacccagat tatatcgggg ggataggcaa agaactcata 6840

gtggacgaca tcagtgatgt cacatcattt tatccttctg catatcaaga acacctgaat 6900

ttcatcccgg cgcctactac aggatccggt tgcactcgga taccctcatt tgacatgagc 6960

accacccatt attgttatac tcacaatgta atactatccg gttgcagaga tcactcacac 7020

tcacatcaat acttagcgct tggtgtgctt cggacatctg caacagggag ggtattcttc 7080

tctactctgc gctccatcaa tttagatgac acccaaaatc ggaagtcctg cagtgtgagt 7140

gcaacccctt taggttgtga tatgctgtgc tctaaggtca cagggactga agaggaggat 7200

tacaagtcag ctgccccaac atcaatggtg cacggaaggc tagggtttga cggtcaatac 7260

catgagaagg acttagacac cacggtctta tttaaggatt gggtggcaaa ttacccagga 7320

gtgggaggag ggtcttttat tgacgaccgt gtatggttcc cagtttacgg agggctcaaa 7380

cccaattcac ccagtgacac tgcacaagaa gggaaatatg taatatacaa gcgccataac 7440

aacacatgcc ccgatggaca agattaccaa attcggatgg ctaaatcttc atataaaccc 7500

gggcgatttg gtggaaagcg cgtacagcaa gccatattat ccatcaaagt gtcaacatcc 7560

ttgggtaagg acccggtgct gactattcca cctaatacaa tcacactcat gggagccgaa 7620

ggcagaatcc tcacagtagg gacatctcac ttcttgtacc aacgagggtc ttcatatttc 7680

tcccctgcct tattatatcc catgacagta aataacaaaa cggctacact ccatagtcct 7740

tatacgttta atgctttcac tcggccaggt agcgtccctt gccaggcatc agcaagatgc 7800

cccaactcat gcatcactgg ggtctatact gatccatatc ccttaatctt ccataggaat 7860

catactctac gaggggtctt tgggacgatg cttgatgatg aacaagcgag acttaacccc 7920

gtatctgcag tattcgacaa catatcccgc agtcgtgtca cccgggtgag ttcaagcagc 7980

accaaggcag catacacgac atcgacatgt tttaaagttg tcaagaccaa taaagcttat 8040

tgtcttagta ttgcagagat atccaatacc ctattcgggg aatttaggat cgttccctta 8100

ttagttgaga tcctcaagga tgatagagtt taagaagcta gacttggccg attgagccaa 8160

tcataggatg gttgggaaga cgacaccaca tcaatcatct cccacaatgc ttagagtcaa 8220

tctgaatatt aacataagcc aggatcccat gttgttgggc agccacaatc agacaatact 8280

gacatgatca ttctgagtcc tgcccactat caccttatta agaaaaaata caaaaagcat 8340

tgagatataa ggggaaacaa ccaacaagag ggaacacggg taggacatgg cgggctccgg 8400

tcccgaaagg gcagagcacc agatcatcct accagagtca catctatcct ctccattggt 8460

caagcacaaa ttgctatact actggaaatt gactgggcta ccgcttcctg atgaatgcga 8520

ctttgatcat ctcattatca gcaggcaatg gaagagaata ctggagtcgg ccactcctga 8580

cacagagaga atgataaaac tcgggcgggc agtgcaccag actctcaacc acaactccaa 8640

gataaccgga gtgctccatc ccaggtgttt agaagaactg gctagtattg aggtcccaga 8700

ttcaactaac aaattccgga agattgaaaa gaagatccag attcacaaca caaggtatgg 8760

agacctgttc acaaagctgt gcacgcatgt tgagaagaaa ttgctaggat catcccggtc 8820

taataatgtc ccacgatcag aggaattcag tagcatccgt acagatccgg cattctggtt 8880

tcactcaaaa tggtccagag ctaagttcgc gtggctccat ataaaacaag tccaaaggca 8940

tctgattgta gcagcaagga caaggtctgc agtcaacaag ttagtaacat taagtcataa 9000

gataggccac gtctttgtta ctcctgagct tgtcattgtg acacatacag acgagaacaa 9060

gttcacatgc ctcacccagg aacttgtatt gatgtatgcg gatatgatgg aaggcaggga 9120

catggtcaat ataatatctt ctacagcagc acatctcaga aacctatccg agaaaattga 9180

cgatattctg cggttagtag atgccctggc aaaggactta ggtaatcaag tctatgacgt 9240

tgtagcatta atggagggat tcgcatacgg tgctgttcag ctgcttgagc catcaggtac 9300

atttgcagga gatttctttg catttaacct acaggagctc aaagacactt taatcgaact 9360

tctcccaaat aatatagcgg aatcagtaac tcacgctatt gccgctgtat tctccggttt 9420

agaacagaat caagcagctg agatgttgtg cttgctacgt ttgtggggtc atcctttgct 9480

tgagtctcgt agtgcagcaa gagcagtcag gagccagatg tgtgcaccga agatggtaga 9540

cttcgatatg atcctccagg tactatcttt ctttaaagga acaatcatca atggatacag 9600

aaagaagaat tcaggtgtgt ggccgcgtgt caaagtagat acaatatacg ggaatatcat 9660

tgggcagctg tatgctgatt cagcagagat ctcacatgat gtcatgttga aggagtacaa 9720

gagtttatct gcttttgaat ttgagccatg tatagactat gaccctgtta ccaatctaag 9780

catgttccta aaagacaagg caatcgcaca tcctagtgat aattggctcg cctcatttag 9840

gcggaaccta ctctctgagg accagaagaa acagataaaa gaggcaactt caactaaccg 9900

cctcttgata gagttcttag aatcaaatga ttttgatcca tataaagaaa tggaatacct 9960

gacaaccctc gagtacctaa gaggtgacag tgtggcagta tcgtactcac tcaaagagaa 10020

agaggtaaaa gtgaatgggc ggatttttgc taagttaaca aagaaactaa ggaactgcca 10080

ggtaatggca gaaggaattc tagctgacca gattgcacct ttctttcagg gaaatggggt 10140

cattcaagat agcatatcct tgacaaagag tatgttagcg atgagtcaac tgtcctttaa 10200

cagcaataag aaacgtatca ctgactgcaa agagagggtt tcctcgaacc gcaatcatga 10260

tcagaagagc aagaatcgta gaagagttgc cacttttatc acgactgacc tacaaaagta 10320

ttgtcttaac tggagatatc agacagtcaa actattcgcc catgctatca atcagctgat 10380

gggcctacct catttcttcg agtggattca tcttaggctg atggacacta caatgtttgt 10440

aggggatcct ttcaatcctc caagtgaccc gactgactgt gatctatcaa gagtcccaaa 10500

cgatgacata tatattgtca gtgctagagg gggcattgag ggactctgcc agaagctatg 10560

gacgatgatc tcaattgctg caatccaact tgctgcagca agatctcatt gtcgagttgc 10620

ctgcatggta caaggtgaca atcaagtaat ggctgtaacg agagaggtaa gatcagatga 10680

ttccccggat atggtgttga cgcagttgca tcaagctagt gataatttct tcaaggaatt 10740

gattcatgtc aatcatttga ttggccataa tctgaaggat cgtgaaacca ttagatcaga 10800

cacattcttc atatacagca aacgaatatt caaggatgga gcaatactca gtcaggtcct 10860

caaaaattca tctaaattgg tgctaatatc aggcgacctt agcgaaaaca ctgtaatgtc 10920

ctgtgccaac attgcatcca ctgtagcacg actatgtgag aatgggcttc caaaggattt 10980

ctgttactat ttgaactacc taatgagttg tgtgcagaca tactttgatt cggagttctc 11040

tattacccac agctcgcaat cagattccaa ccagtcctgg atcgaggata tctctttcgt 11100

acactcatac gtgttaaccc ctgcccagct ggggggactg agcaaccttc aatactcaag 11160

gctctacaca aggaatattg gtgacccagg gaccactgct ttcgcagagg tcaagcgact 11220

ggaagcagtg gggttgctga gtcccagcat catgactaac atcttaacca ggccacctgg 11280

caatggagac tgggccagcc tatgcaacga cccatactct tttaattttg aaactgttgc 11340

aagcccaaat attgtcctca agaaacatac acagaaagtc ctatttgaga catgttcaaa 11400

ccccttatta tccggggtac atacagagga caatgaggca gaggagaaag cattggctga 11460

attcttactc aatcaagaag tgattcaccc acgtgtcgca catgctatca tggaagcaag 11520

ctctgtgggt aggagaaagc aaattcaagg gcttgttgac acaacgaaca ctgtgattaa 11580

gattgcactg actaggaggc ccctcggtat caaaaggctg atgcggataa tcaattactc 11640

gagcatgcat gcaatgttgt tcagagatga tattttctta tccaatagat ccaaccaccc 11700

attagtttct tccaatatgt gctcgctgac gctagcagat tatgcccgaa acagaagctg 11760

gtcacctctg acagggggca ggaaaatact gggtgtatcc aaccctgata ccatagaact 11820

tgtggaggga gagattctca gcgtcagtgg agggtgcaca aaatgtgaca gcggagatga 11880

gcagtttact tggttccatc ttccaagcaa tatagagctg actgatgaca ccagaaaaaa 11940

tcccccgatg agagtgccat atcttgggtc gaagactcaa gagaggagag ctgcctcgct 12000

tgcgaaaata gcccacatgt caccacatgt gaaagcagca ctaagggcat catccgtgtt 12060

aatctgggct tatggggaca acgaagtgaa ctggactgct gctcttaata ttgcaaggtc 12120

tcgatgcaac ataagctcag agtaccttcg gctattgtca cccctgccca cggctgggaa 12180

tctccaacat agattggatg atggcataac ccagatgaca tttacccctg catctctcta 12240

cagagtgtcg ccttacgttc acatatccaa tgattctcaa aggctattca ccgaagaagg 12300

ggtcaaagag ggaaacgtgg tttaccaaca aattatgctc ttgggtttat ccctaattga 12360

atcactcttc ccaatgacag caaccagaac atatgatgag atcacattac acctccacag 12420

taaatttagc tgctgtatcc gagaagcgcc tgttgcggtt cccttcgagc tcctcgggct 12480

ggcaccggaa ttaagaatgg taacctcaaa taagttcatg tatgatccta gccctatatc 12540

agagagagat ttcgcgagac ttgacttagc tatcttcaag agttacgagc ttaatttgga 12600

atcatattcc acgctggagc taatgaacat tctttcaata tctagcggga agttgattgg 12660

ccaatctgtg gtttcttatg atgaagacac ctctataaag aatgatgcta taatagtgta 12720

tgacaacaca cgaaattgga ttagtgaggc gcagaactca gatgtggtcc gcttgtttga 12780

gtatgcagca ctcgaagtgc ttcttgactg tgcttatcaa ctctactatc tgagggtaag 12840

gggtctaaac aacatcgtcc tatacatgaa tgacttatat aagaacatgc cagggatcct 12900

actctccaat attgcggcca cgatatccca ccccatcatt cactcaaggt tgaatgcagt 12960

aggcctaatt aaccatgacg ggtcacacca gctcgcagat atagacttcg tcgaggtgtc 13020

tgcaaaattg ttagtctctt gcactcgacg cgtggtctca ggcttatatg cagggaataa 13080

gtacgatctg ctgttcccat ctgtcttaga tgataacctg aatgagaaga tgctccaact 13140

gatttcccgg ttatgctgtc tgtacacagt gctctttgct acaacaagag aaatcccaaa 13200

aataaggggc ctatcagcag aagagaaatg ctcaatactc actgagtatc tactgtcaga 13260

tgctgtaaaa ccattgctta ggcccgaaca agtgaattct atcatgtctc ccaacataat 13320

cacgttccca gccaatctat attacatgtc taggaagagc cttaatttga tcagagaacg 13380

agaggacaga gatactatct tgtcattgtt gttccctcag gaaccactgc ttgagcttcg 13440

cccagtacgg gacattggtg ctcgagtgaa agacccgttt acccggcaac ccgcatcatt 13500

catacaagag ctagatctga gtgccccagc aaggtacgac gcatttacac tgagtaaggc 13560

ctgcttcgag catacattac cgaacccaaa ggaggattac ctagtacggt acttgttcag 13620

aggaataggg actgcttcat cttcttggta taaggcatct catcttctat ccgtacctga 13680

ggtcaggtgt gcaagacatg ggaactcctt atacttagcg gaaggaagcg gagccatcat 13740

gagtcttctt gaattgcata taccacatga gactatctat tacaatacac ttttctcgaa 13800

tgagatgaac cctccacagc gacatttcgg acctacacca acacagtttc taaactcggt 13860

cgtttatagg aatctacaag cggaagtgcc atgtaaagat ggatatgtcc aggagttctg 13920

cccattatgg agagagaatg cagaagaaag tgacctgacc tcagataaag cagttggata 13980

tatcacatcc gtggtaccct acaggtctgt atcattacta cattgtgaca ttgagattcc 14040

tccagggtcc aatcaaagct tattagatca actggctact aatttatccc tgattgccat 14100

gcattctgtg agggagggcg gggtagtgat catcaaagta ctgtatgcaa tggggtacta 14160

cttccattta ctcatgaatt tattcactcc atgttccaca aaaggatata tactctccaa 14220

tggctatgcc tgtagagggg atatgcaatg ttacctgata ttcgttatgg gctacttggg 14280

cgggcccacc ttcgtgcacg aagtggtaag gatggcaaaa actctaatac aacgacacgg 14340

tacactccta tctaaatcag atgaaattac attgactaag ctatttacct cacagcagcg 14400

tcatgtaaca gatatcctat ccagtccttt accgaagcta atgaagctct tgagagaaaa 14460

tattgatgcc gcactaattg aagctggggg acagcccgtc cgtccattct gtgcggaaag 14520

tttggtgagc acactaacag atatgactca gacagctcag atcattgcca gccacattga 14580

cacagtcatt cggtctgtaa tttacatgga ggctgagggt gacctcgccg acacagtgtt 14640

cttatttact ccttacaatc tatccacaga cggtaaaagg agaacatcac ttaagcagtg 14700

caccaaacag atcttggaag tcacaatact gggtctcaga gccaaagata tcaataaagt 14760

aggtgatgta atcagtttag tactcagagg tgcggtttct ctagaggacc tcatcccatt 14820

aaggacatac ctgaagcgca gtacctgcac taaatacctg aaagcggtcc taggtattac 14880

taaactcaaa gaaatgttca cagatacctc attactgtac ttgactcgtg ctcaacaaaa 14940

attctacatg aaaaccatag gtaatgctgc caagggatat tactgtaata atgactctta 15000

aaggcaatcg tacgccaatc agttatcttc ctaactgatg actccctcat tgactcaatt 15060

ataccagatt agaaaaaagt taaattccga ctctttggaa ctcgtattcg gattcagtta 15120

gttaacttta agcaagagtg cgcaaagtca tccctaatta tagtgatgtc attcaccaaa 15180

tctctgtttg gt 15192

<210> 5

<211> 15192

<212> DNA

<213>NDV（SEQ ID NO：5）

<400> 5

accaaacaga gaatctgtga ggtacgataa aaggcgaaga agcaatcgag atcgtacggg 60

tagaaggtgt gaatcccgag tgcgaggccg aagctcgaac ctgaggggac cttctaccga 120

tatgtcgtct gttttcgacg agtacgagca gctcctcgct gctcagaccc gccctaacgg 180

aactcatgga gggggagaga aagggagcac tttaaaagtt gaggtcccag tattcaccct 240

aaacagtgat gatccagagg atagatggaa ttttgcggta ttctgtcttc ggattgctgt 300

tagcgaggat gccaacaaac cactcaggca aggtgctctt atatccctct tatgctccca 360

ttctcaggtg atgagaaacc atgttgccct tgcagggaaa caaaatgagg ccacactggc 420

tgttcttgag atcgatggtt ttgctaacag tgtgccccag ttcaacaata ggagtggagt 480

gtctgaggaa agagcacaga gattcatggt aatcgcagga tctctccctc gggcgtgcag 540

caacggtact ccgtttgtca cggctggggt tgaagatgat gcaccagaag atatcactga 600

cactctggaa agaatcctat ctatccaagt tcaggtatgg gtcacagtag caaaggccat 660

gactgcatat gagacagcag atgagtcaga aacaagaaga ataaataagt atatgcagca 720

aggtcgagtt cagaagaagt acatccttca tcctgtatgc aggagtgcaa ttcaactcac 780

aatcagacat tctctggcag tccgtatctt cctagttagt gagctcaaga ggggccgcaa 840

tacagcaggt gggagctcta catattacaa cttggtcggg gatgtagact catacatcag 900

gaacaccggg cttactgcat ttttcctaac actcaaatat ggaatcaata ccaagacgtc 960

agccctcgca ctcagcagcc tcacaggtga tatccaaaaa atgaaacagc tcatgcgttt 1020

atatcggatg aaaggtgaaa atgcaccata catgacattg ttaggtgaca gtgaccagat 1080

gagctttgca cctgctgagt atgcacaact ttattctttt gccatgggca tggcatcagt 1140

cttagataag ggaactggca agtaccaatt cgccagggac tttatgagca catcattctg 1200

gagacttgga gtagagtatg ctcaggccca gggaagtagc attaatgagg acatggctgc 1260

tgagctaaaa ctaaccccgg cagcaaggag aggcctggca gctgctgccc aacgagtatc 1320

cgaagaaatc ggcagcatgg acattcccac tcaacaggcg ggagtcctca ccgggctcag 1380

tgatgaaggc ccccgaactc cacagggcag atcaaacaag ccgcaagggc aaccagatgc 1440

tggggatggg gagacccaat tcttggattt tatgagagca gtggcgaacg gcatgcggga 1500

agcgccaaat cctgtacaga gcaccaccca tccagagcct cccccaaccc ctggggcatc 1560

ccaagacaac gacactgact gggggtactg atcgacaaca cccagcctgc cttcacagga 1620

tcacaccaaa ccctccgacc aaaaccctcc cacactccct gacccacaac cccgcacgac 1680

cacaccaaca aaagctcccc cccaccctct cccccactcc cagccacacg atcccgccca 1740

cccgggacaa cacaggcaca gctcagttcg tcgacaatcc gcccagagcc caaggtatta 1800

gaaaaaaata cgggtagaag agagacatcc agagaccagg acgagtcacc aagttctctg 1860

ttctcccttc tacccagtga attagggtga agatggccac ttttacagat gcggagatag 1920

atgacatatt tgagaccagt gggactgtca ttgacagcat aattacggcc cagggcaaat 1980

cagctgagac cgttggaaga agtgcgatcc cgcagggcaa gaccaaagct ctaagcacag 2040

catgggagaa gcacgggagt atccagccac acgccagtca ggacgcccct gaccaacaag 2100

acagaataga aaaacagcca tccacacctg agcaggcgac tctacacaac aatccgccga 2160

tcgcatccac tgaaccgcct cccactcagg ccgcaagcga gaccagcgac acacagctca 2220

agactggggc aagcaactcc cttctgtcca tgctcgacaa actgagtaat aaatcgtcca 2280

atgctaaaaa gggcccatgg tcgggatccc aagaagggca tcaccaacct ccggcccaac 2340

aacatgggaa ccagccgagc tatggaagca accagggaag accgcagcat caggccaagg 2400

ccgtccctgg aaaccggggc acagacgaga acacagcata tcatggacag cggaaggagt 2460

cacaaccatc agctggtgca acccctcatg cgccccagtc agggcagagc caagacaata 2520

ctcctgcacc tgtggatcgt gtccagctac ctgccgactt tgtgcaggcg atgatgtcta 2580

tgatggaggc attatcacag aaggtaagta aagttgatca tcagctggac ctagtcttga 2640

aacagacatc ctccattcct atgatgcgat ctgaaatcca acagctcaag acatctgttg 2700

cgatcatgga agctaactta ggcatgatga aaattctgga ccctggttgt gctaatgttt 2760

catccttaag tgatctccgg gcagtagccc gatcccatcc agtcctagtt tcaggccctg 2820

gagacccatc tccttacgtg acacaagggg gtgaaatgac gctcaataaa ctctcacaac 2880

cggtgcagca cccctctgaa ttgattaagt ctgccactgc aagcgggcct gacatgggag 2940

tggagaagga cactgtccgc gcattaatca cctcgcgccc gatgcatcca agctcctcgg 3000

ctaagctcct gagcaagcta gatgcagcca ggtcaattga agagatcagg aagatcaaac 3060

gccttgcgct gaatggttga tggccatcac aactcataac aggctcccgt cactttagcg 3120

tcacacggaa tccctcgggg gccctccctc gcaaatctat gcttcaacac ccaaaacaac 3180

agccctctct cacccccccc aatccctcga atgaccgcac aactgcaacc aatccagcag 3240

cattagaaat taagaaaaaa tacgggtaga atcaaagtgc cttgattgca ccaaaatgga 3300

ctcatccagg acaattgggc tgtactttga ttctgccctc ccttccagca gcctgttagc 3360

atttccgatt gtcttacaag acacgggaga cgggaagaag caaatcaccc cacaatacag 3420

gatccagcgt cttgattcgt ggacagacag taaggaagac tcggtattca tcaccaccta 3480

cgggttcatc tttcaaattg ggaatgaaga aaccaccgtc ggtgtgatca atgacaatcc 3540

caggcacgag ctactctctt ccgcaatgct ctgcttaggg agtgtcccga acgacggaga 3600

tcttgttgag ctggcgagag cctgcctcac catggtggta acttgcaaaa agagtgcaac 3660

taacactgag agaatagtct tttcagtagt gcaggctcct cgggtgctgc aaagctgtat 3720

ggttgtggca aataggtact catcagtgaa tgcagtgaag catgtgaagg cgccagaaaa 3780

gatccctggg agcggaaccc tagagtataa agtgaatttt gtctctttga ccgtggtgcc 3840

aagaagggat gtctacagga tcccaactgc agtattgaaa gtgtctggct caagcctgta 3900

caatcttgcg ctcaatgtca ctattgatgt ggacgtggat ccgaagagcc cgttagtcaa 3960

atccctttcc aagtccgata gcggatacta tgcgaatctt tttctgcata tcgggcttat 4020

gtccactgta gataagaagg gaaagaaagt gacatttgac aagatagagg aaaagataag 4080

gagactcaat ctatccgtcg ggctcagtga tgtgctcgga ccctctgtgc ttgtgaaggc 4140

gagaggtgca cggactaagc tacttgctcc ttttttctct agcagtggga cagcctgcta 4200

tcctatagca aatgcctctc cccaggttgc caagatactc tggagccaaa ctgcgcacct 4260

gcggagtgtg aaagtcatca ttcaagccgg cactcagcgt gctgtcgcag tgaccgctga 4320

tcatgaggta acctccacta agatagagag gaggcatgcc attgctaaat acaatccttt 4380

caggaaataa gttgcatccc taagactgca gttcacctgc tttcccgaat caccattaca 4440

ccagacaatg atccatctcg actgcttata gttagttcac ctgtctagca aattagaaaa 4500

aacacgggta gaagagtctg gatcccgacc ggcacactca ggacgcaaca tgggctccaa 4560

accttctacc aggatcccag cacctctgat gctggtcacc cggattatgc tgatattggg 4620

ctgtattcgt tcgacaagct ctcttgacgg caggcctctt gcagctgcag gaattgtagt 4680

aacaggagat aaggcagtca atgtatacac ctcgtctcag acagggtcaa tcatagtcaa 4740

gttgctcccg aatatgccca gggataagga ggcgtgtgcg aaagccccat tagaggcata 4800

taacagaaca ctgactactt tgctcactcc tcttggcgac tccatccgca agattcaagg 4860

gtctgtgtcc acgtctggag gaaggaggca aaaacgcttt ataggtgctg ttattggcag 4920

tgtagctctt ggggttgcaa cagcggcaca gataacagca gctgcggccc taatacaagc 4980

caacaagaat gctgccaaca tccttcggct taaggagagc attgctgcaa ccaatgaagc 5040

tgtgcatgaa gtcaccgacg gattatcaca actatcagtg gcagttggga agatgcagca 5100

gtttgtcaat gaccagttta ataatacggc gcgagaattg gactgtataa aaatcacaca 5160

acaggttggt gtagaactca acctatacct aactgaattg actacagtat tcgggccaca 5220

gatcacctct cctgcattaa ctcagctgac catccaggca ctttataatt tagctggtgg 5280

caatatggat tacttattaa ctaagttagg tatagggaac aatcaactca gctcattaat 5340

tggtagcggc ctgatcactg gttaccctat actgtatgac tcacagactc aactcttggg 5400

catacaagtg aatttgccct cagtcgggaa cttaaataat atgcgtgcca cctatttgga 5460

gaccttatct gtaagtacaa ccaaaggata tgcctcagca cttgtcccga aagtagtgac 5520

acaagtcggt tctgtgatag aagagcttga cacctcatac tgtatagagt ccgatctgga 5580

tttatattgt actagaatag tgacattccc catgtcccca ggtatttatt cctgtttgag 5640

cggcaacaca tcagcttgca tgtattcaaa gactgaaggc gcactcacta cgccgtatat 5700

ggcccttaaa ggctcagtta ttgccaattg taagataaca acatgtagat gtacagaccc 5760

tcctggtatc atatcgcaaa attatggaga agctgtatcc ctgatagata gacattcgtg 5820

caatgtctta tcattagacg ggataactct gaggctcagc ggagaatttg atgcaactta 5880

tcaaaagaac atctcaatac tagattctca agtcatcgtg acaggcaatc ttgatatatc 5940

aactgaactt ggaaacgtca acaattcaat cagcaatgcc ttggataagt tggcagaaag 6000

caacagtaag atagaaaaag tcaatgtcag attaaccagc acatctgctc tcattaccta 6060

tattgttcta actgtcattt ctctattttt cggtgcactt agtctgggtt tagcgtgtta 6120

cctgatgtac aaacagaagg cacaacaaaa gaccttgcta tggcttggga ataatgccct 6180

tgatcagatg agagccacta caagagcatg aatgcagata agaggtggat atatacccaa 6240

cagcagcctg tgtgtcaatt ccgataatct gtcaagtaga agacttaaga aaaaactact 6300

gggaacaagc aaccaaagag caatacacgg gtagaacggt cagaggagcc acccttcaat 6360

cggaaattag gcttcacaac atccgttcta ccacatcacc atcaacaaga gtcaatcatg 6420

gaccgcgcgg ttaacagagt cgtgctggag aatgaggaaa gagaagcaaa gaacacatgg 6480

cgcctggttt tccggatcgc agtcttactt ttaatggtaa tgactctagc tatctccgca 6540

gctgccctgg catacagtat ggaggccagt acgccgcacg accttgcagg catatcgact 6600

gtgatctcta agacagaaga taaggttacg tctttactca gttcgagtca agatgtgata 6660

gataggatat acaagcaggt ggctcttgaa tccccgctgg cgctactaaa cactgaatct 6720

acaattatga atgcaataac ctctctttct tatcaaatta acggggctgc gaacaatagc 6780

ggatgtgggg cgcctgttca tgacccagat tatatcgggg ggataggcaa agaactcata 6840

gtggacgaca tcagtgatgt cacatcattt tatccttctg catatcaaga acacctgaat 6900

ttcatcccgg cgcctactac aggatccggt tgcactcgga taccctcatt tgacatgagc 6960

accacccatt attgttatac tcacaatgta atactatccg gttgcagaga tcactcacac 7020

tcacatcaat acttagcgct tggtgtgctt cggacatctg caacagggag ggtattcttc 7080

tctactctgc gctccatcaa tttagatgac acccaaaatc ggaagtcctg cagtgtgagt 7140

gcaacccctt taggttgtga tatgctgtgc tctaaggtca cagggactga agaggaggat 7200

tacaagtcag ctgccccaac atcaatggtg cacggaaggc tagggtttga cggtcaatac 7260

catgagaagg acttagacac cacggtctta tttaaggatt gggtggcaaa ttacccagga 7320

gtgggaggag ggtcttttat tgacgaccgt gtatggttcc cagtttacgg agggctcaaa 7380

cccaattcac ccagtgacac tgcacaagaa gggaaatatg taatatacaa gcgccataac 7440

aacacatgcc ccgatggaca agattaccaa attcggatgg ctaaatcttc atataaaccc 7500

gggcgatttg gtggaaagcg cgtacagcaa gccatattat ccatcaaagt gtcaacatcc 7560

ttgggtaagg acccggtgct gactattcca cctaatacaa tcacactcat gggagccgaa 7620

ggcagaatcc tcacagtagg gacatctcac ttcttgtacc aacgagggtc ttcatatttc 7680

tcccctgcct tattatatcc catgacagta aataacaaaa cggctacact ccatagtcct 7740

tatacgttta atgctttcac tcggccaggt agcgtccctt gccaggcatc agcaagatgc 7800

cccaactcat gcatcactgg ggtctatact gatccatatc ccttaatctt ccataggaat 7860

catactctac gaggggtctt tgggacgatg cttgatgatg aacaagcgag acttaacccc 7920

gtatctgcag tattcgacaa catatcccgc agtcgtgtca cccgggtgag ttcaagcagc 7980

accaaggcag catacacgac atcgacatgt tttaaagttg tcaagaccaa taaagcttat 8040

tgtcttagta ttgcagagat atccaatacc ctattcgggg aatttaggat cgttccctta 8100

ttagttgaga tcctcaagga tgatagagtt taagaagcta gacttggccg attgagccaa 8160

tcataggatg gttgggaaga cgacaccaca tcaatcatct cccacaatgc ttagagtcaa 8220

tctgaatatt aacataagcc aggatcccat gttgttgggc agccacaatc agacaatact 8280

gacatgatca ttctgagtcc tgcccactat caccttatta agaaaaaata caaaaagcat 8340

tgagatataa ggggaaacaa ccaacaagag ggaacacggg taggacatgg cgggctccgg 8400

tcccgaaagg gcagagcacc agatcatcct accagagtca catctatcct ctccattggt 8460

caagcacaaa ttgctatact actggaaatt gactgggcta ccgcttcctg atgaatgcga 8520

ctttgatcat ctcattatca gcaggcaatg gaagagaata ctggagtcgg ccactcctga 8580

cacagagaga atgataaaac tcgggcgggc agtgcaccag actctcaacc acaactccaa 8640

gataaccgga gtgctccatc ccaggtgttt agaagaactg gctagtattg aggtcccaga 8700

ttcaactaac aaattccgga agattgaaaa gaagatccag attcacaaca caaggtatgg 8760

agacctgttc acaaagctgt gcacgcatgt tgagaagaaa ttgctaggat catcccggtc 8820

taataatgtc ccacgatcag aggaattcag tagcatccgt acagatccgg cattctggtt 8880

tcactcaaaa tggtccagag ctaagttcgc gtggctccat ataaaacaag tccaaaggca 8940

tctgattgta gcagcaagga caaggtctgc agtcaacaag ttagtaacat taagtcataa 9000

gataggccac gtctttgtta ctcctgagct tgtcattgtg acacatacag acgagaacaa 9060

gttcacatgc ctcacccagg aacttgtatt gatgtatgcg gatatgatgg aaggcaggga 9120

catggtcaat ataatatctt ctacagcagc acatctcaga aacctatccg agaaaattga 9180

cgatattctg cggttagtag atgccctggc aaaggactta ggtaatcaag tctatgacgt 9240

tgtagcatta atggagggat tcgcatacgg tgctgttcag ctgcttgagc catcaggtac 9300

atttgcagga gatttctttg catttaacct acaggagctc aaagacactt taatcgaact 9360

tctcccaaat aatatagcgg aatcagtaac tcacgctatt gccgctgtat tctccggttt 9420

agaacagaat caagcagctg agatgttgtg cttgctacgt ttgtggggtc atcctttgct 9480

tgagtctcgt agtgcagcaa gagcagtcag gagccagatg tgtgcaccga agatggtaga 9540

cttcgatatg atcctccagg tactatcttt ctttaaagga acaatcatca atggatacag 9600

aaagaagaat tcaggtgtgt ggccgcgtgt caaagtagat acaatatacg ggaatatcat 9660

tgggcagctg tatgctgatt cagcagagat ctcacatgat gtcatgttga aggagtacaa 9720

gagtttatct gcttttgaat ttgagccatg tatagactat gaccctgtta ccaatctaag 9780

catgttccta aaagacaagg caatcgcaca tcctagtgat aattggctcg cctcatttag 9840

gcggaaccta ctctctgagg accagaagaa acagataaaa gaggcaactt caactaaccg 9900

cctcttgata gagttcttag aatcaaatga ttttgatcca tataaagaaa tggaatacct 9960

gacaaccctc gagtacctaa gaggtgacag tgtggcagta tcgtactcac tcaaagagaa 10020

agaggtaaaa gtgaatgggc ggatttttgc taagttaaca aagaaactaa ggaactgcca 10080

ggtaatggca gaaggaattc tagctgacca gattgcacct ttctttcagg gaaatggggt 10140

cattcaagat agcatatcct tgacaaagag tatgttagcg atgagtcaac tgtcctttaa 10200

cagcaataag aaacgtatca ctgactgcaa agagagggtt tcctcgaacc gcaatcatga 10260

tcagaagagc aagaatcgta gaagagttgc cacttttatc acgactgacc tacaaaagta 10320

ttgtcttaac tggagatatc agacagtcaa actattcgcc catgctatca atcagctgat 10380

gggcctacct catttcttcg agtggattca tcttaggctg atggacacta caatgtttgt 10440

aggggatcct ttcaatcctc caagtgaccc gactgactgt gatctatcaa gagtcccaaa 10500

cgatgacata tatattgtca gtgctagagg gggcattgag ggactctgcc agaagctatg 10560

gacgatgatc tcaattgctg caatccaact tgctgcagca agatctcatt gtcgagttgc 10620

ctgcatggta caaggtgaca atcaagtaat ggctgtaacg agagaggtaa gatcagatga 10680

ttccccggat atggtgttga cgcagttgca tcaagctagt gataatttct tcaaggaatt 10740

gattcatgtc aatcatttga ttggccataa tctgaaggat cgtgaaacca ttagatcaga 10800

cacattcttc atatacagca aacgaatatt caaggatgga gcaatactca gtcaggtcct 10860

caaaaattca tctaaattgg tgctaatatc aggcgacctt agcgaaaaca ctgtaatgtc 10920

ctgtgccaac attgcatcca ctgtagcacg actatgtgag aatgggcttc caaaggattt 10980

ctgttactat ttgaactacc taatgagttg tgtgcagaca tactttgatt cggagttctc 11040

tattacccac agctcgcaat cagattccaa ccagtcctgg atcgaggata tctctttcgt 11100

acactcatac gtgttaaccc ctgcccagct ggggggactg agcaaccttc aatactcaag 11160

gctctacaca aggaatattg gtgacccagg gaccactgct ttcgcagagg tcaagcgact 11220

ggaagcagtg gggttgctga gtcccagcat catgactaac atcttaacca ggccacctgg 11280

caatggagac tgggccagcc tatgcaacga cccatactct tttaattttg aaactgttgc 11340

aagcccaaat attgtcctca agaaacatac acagaaagtc ctatttgaga catgttcaaa 11400

ccccttatta tccggggtac atacagagga caatgaggca gaggagaaag cattggctga 11460

attcttactc aatcaagaag tgattcaccc acgtgtcgca catgctatca tggaagcaag 11520

ctctgtgggt aggagaaagc aaattcaagg gcttgttgac acaacgaaca ctgtgattaa 11580

gattgcactg actaggaggc ccctcggtat caaaaggctg atgcggataa tcaattactc 11640

gagcatgcat gcaatgttgt tcagagatga tattttctta tccaatagat ccaaccaccc 11700

attagtttct tccaatatgt gctcgctgac gctagcagat tatgcccgaa acagaagctg 11760

gtcacctctg acagggggca ggaaaatact gggtgtatcc aaccctgata ccatagaact 11820

tgtggaggga gagattctca gcgtcagtgg agggtgcaca aaatgtgaca gcggagatga 11880

gcagtttact tggttccatc ttccaagcaa tatagagctg actgatgaca ccagaaaaaa 11940

tcccccgatg agagtgccat atcttgggtc gaagactcaa gagaggagag ctgcctcgct 12000

tgcgaaaata gcccacatgt caccacatgt gaaagcagca ctaagggcat catccgtgtt 12060

aatctgggct tatggggaca acgaagtgaa ctggactgct gctcttaata ttgcaaggtc 12120

tcgatgcaac ataagctcag agtaccttcg gctattgtca cccctgccca cggctgggaa 12180

tctccaacat agattggatg atggcataac ccagatgaca tttacccctg catctctcta 12240

cagagtgtcg ccttacgttc acatatccaa tgattctcaa aggctattca ccgaagaagg 12300

ggtcaaagag ggaaacgtgg tttaccaaca aattatgctc ttgggtttat ccctaattga 12360

atcactcttc ccaatgacag caaccagaac atatgatgag atcacattac acctccacag 12420

taaatttagc tgctgtatcc gagaagcgcc tgttgcggtt cccttcgagc tcctcgggct 12480

ggcaccggaa ttaagaatgg taacctcaaa taagttcatg tatgatccta gccctatatc 12540

agagagagat ttcgcgagac ttgacttagc tatcttcaag agttacgagc ttaatttgga 12600

atcatattcc acgctggagc taatgaacat tctttcaata tctagcggga agttgattgg 12660

ccaatctgtg gtttcttatg atgaagacac ctctataaag aatgatgcta taatagtgta 12720

tgacaacaca cgaaattgga ttagtgaggc gcagaactca gatgtggtcc gcttgtttga 12780

gtatgcagca ctcgaagtgc ttcttgactg tgcttatcaa ctctactatc tgagggtaag 12840

gggtctaaac aacatcgtcc tatacatgaa tgacttatat aagaacatgc cagggatcct 12900

actctccaat attgcggcca cgatatccca ccccatcatt cactcaaggt tgaatgcagt 12960

aggcctaatt aaccatgacg ggtcacacca gctcgcagat atagacttcg tcgaggtgtc 13020

tgcaaaattg ttagtctctt gcactcgacg cgtggtctca ggcttatatg cagggaataa 13080

gtacgatctg ctgttcccat ctgtcttaga tgataacctg aatgagaaga tgctccaact 13140

gatttcccgg ttatgctgtc tgtacacagt gctctttgct acaacaagag aaatcccaaa 13200

aataaggggc ctatcagcag aagagaaatg ctcaatactc actgagtatc tactgtcaga 13260

tgctgtaaaa ccattgctta ggcccgaaca agtgaattct atcatgtctc ccaacataat 13320

cacgttccca gccaatctat attacatgtc taggaagagc cttaatttga tcagagaacg 13380

agaggacaga gatactatct tgtcattgtt gttccctcag gaaccactgc ttgagcttcg 13440

cccagtacgg gacattggtg ctcgagtgaa agacccgttt acccggcaac ccgcatcatt 13500

catacaagag ctagatctga gtgccccagc aaggtacgac gcatttacac tgagtaaggc 13560

ctgcttcgag catacattac cgaacccaaa ggaggattac ctagtacggt acttgttcag 13620

aggaataggg actgcttcat cttcttggta taaggcatct catcttctat ccgtacctga 13680

ggtcaggtgt gcaagacatg ggaactcctt atacttagcg gaaggaagcg gagccatcat 13740

gagtcttctt gaattgcata taccacatga gactatctat tacaatacac ttttctcgaa 13800

tgagatgaac cctccacagc gacatttcgg acctacacca acacagtttc taaactcggt 13860

cgtttatagg aatctacaag cggaagtgcc atgtaaagat ggatatgtcc aggagttctg 13920

cccattatgg agagagaatg cagaagaaag tgacctgacc tcagataaag cagttggata 13980

tatcacatcc gtggtaccct acaggtctgt atcattacta cattgtgaca ttgagattcc 14040

tccagggtcc aatcaaagct tattagatca actggctact aatttatccc tgattgccat 14100

gcattctgtg agggagggcg gggtagtgat catcaaagta ctgtatgcaa tggggtacta 14160

cttccattta ctcatgaatt tattcactcc atgttccaca aaaggatata tactctccaa 14220

tggctatgcc tgtagagggg atatggagtg ttacctgata ttcgttatgg gctacttggg 14280

cgggcccacc ttcgtgcacg aagtggtaag gatggcaaaa actctaatac aacgacacgg 14340

tacactccta tctaaatcag atgaaattac attgactaag ctatttacct cacagcagcg 14400

tcatgtaaca gatatcctat ccagtccttt accgaagcta atgaagctct tgagagaaaa 14460

tattgatgcc gcactaattg aagctggggg acagcccgtc cgtccattct gtgcggaaag 14520

tttggtgagc acactaacag atatgactca gacagctcag atcattgcca gccacattga 14580

cacagtcatt cggtctgtaa tttacatgga ggctgagggt gacctcgccg acacagtgtt 14640

cttatttact ccttacaatc tatccacaga cggtaaaagg agaacatcac ttaagcagtg 14700

caccaaacag atcttggaag tcacaatact gggtctcaga gccaaagata tcaataaagt 14760

aggtgatgta atcagtttag tactcagagg tgcggtttct ctagaggacc tcatcccatt 14820

aaggacatac ctgaagcgca gtacctgcac taaatacctg aaagcggtcc taggtattac 14880

taaactcaaa gaaatgttca cagatacctc attactgtac ttgactcgtg ctcaacaaaa 14940

attctacatg aaaaccatag gtaatgctgc caagggatat tactgtaata atgactctta 15000

aaggcaatcg tacgccaatc agttatcttc ctaactgatg actccctcat tgactcaatt 15060

ataccagatt agaaaaaagt taaattccga ctctttggaa ctcgtattcg gattcagtta 15120

gttaacttta agcaagagtg cgcaaagtca tccctaatta tagtgatgtc attcaccaaa 15180

tctctgtttg gt 15192

Claims (4)

1. A kind of 1. attenuated vaccine strain of VII types NDV L gene mutations, it is characterised in that：The VII type ewcastle diseases The attenuated vaccine strain virulence of viral L gene mutations substantially weakens, while in chicken embryo there is high titre, heritability to stablize, It is high with the antigen matching of popular virus, it is suitable for the large-scale production of vaccine, for manufacturing vaccine, the VII type ewcastle diseases The antigen nucleotides sequence of the attenuated vaccine strain of viral L gene mutations is classified as SEQ ID NO:Shown in 2.
2. A kind of 2. preparation method of the attenuated vaccine strain of VII types NDV L gene mutations, it is characterised in that：Construct Five cDNA cloned sequences of whole gene group are covered, using the restriction enzyme site of each intersegmental lap, in transcription vector matter Grain pBR322 assembles the global cDNA clone for obtaining 15192nt；Added before 5 ' ends of full-length cDNA fragment T7 rna polymerase promoter, the fourth hepatovirus ribozyme with self catalysis is added after full-length cDNA fragment（RiB）Sequence With T7 transcription stop signalses, the plasmid built is named as prNDV-97；By PCR method by the L in Full_length cDNA The bit base of protein-coding region the 14056th is G by A same sense mutations, eliminates the restriction enzyme sites of Hind III at 14056bp, and be used as and save The molecular labeling of virus is rescued, the plasmid for building completion is named as pmNDV-97；Simultaneously in t7 rna polymerase promoter and total length base Because introducing two unnecessary G between group cDNA 5 ' ends, this contributes to the virus rescue of paramyxovirus reverse genetic manipulation；

   The specific method of virus rescue is：Poxvirus infection BHK-21 cells are first used before transfection, cell is washed 2-3 times after 1h, with After transfect Revive virus, by full-length cDNA plasmid respectively with tri- eukaryon expression plasmid corotation of pBSK-NP, pBSK-P and pBSK-L BHK-21 cells are contaminated, gently scrape cell after 72h, and 9-11 age in days SPF chickens are inoculated with after being sufficiently mixed filtering with cell conditioned medium Embryo, every part of 3 pieces of sample inoculation, 0.2mL/ pieces, the SPF embryos after inoculation continue culture 3 days, take chick embryo allantoic liquid 50uL routinely to enter Row NDV blood clotting（HA）Suppress with blood clotting（HI）Experiment is detected；

   5 pairs of primers of design are used to expand the pnca gene group SEQ ID NO of NDV VII -97:5, primer sequence is as follows：

   F1 ：5'ATGCGGCCGCTAATACGACTCACTATAGGGACCAAACAGAGAATCTG

   TGAGGTACGATAA3'

   R1 ：5'TAGGTACCCTTGCAGTGGCAGACTTAATCAATTCAGAG3'

   F2 ：5'GGGTGAAATGACGCTCAATAAACTCTCACA3'

   R2 ：5'ATGGTACCCTCCCTGTTGCAGATGTCCGAAGCACACCA3'

   F3 ：5'TGCAGAGATCACTCACACTCACATCAATAC3'

   R3 ：5'ACGGTACCAGGTGACCAGCTTCTGTTTCGGGCATAATC3'

   F4 ：5'ATTAGTTTCTTCCAATATGTGCTCGCTGAC3'

   R4 ：5'GTAGTAATGATACAGACCTGTAGGGTACCA3'

   F5 ：5'GGAGAGAGAATGCAGAAGAAAGTGACCTGACCTCAGATAAAGCAG3'

   R5 ：5'AGCCGGCGCCAGCGAGGAGGCTGGGACCATGCCGGCCACCAAACAG

   AGATTTGGTGAATGACATC3'

   2 pairs of design is used to be mutated Hind III restriction enzyme site primers at 14056bp, eliminates this restriction enzyme site, primer sequence is such as Under：

   AFa ：5' CATGTTTTAAAGTTGTCAAG 3'

   F6 ：5' GGGTCCAATCAGAGCTTATTAGATC 3'

   R6 ：5' GATCTAATAAGCTCTGATTGGACCC 3'

   ARa ：5' GCG GTCGAC ACCGCTTTCAGGTATTTAG3' ；

   On the basis of pmNDV-97 full-length genome plasmids, NDV-97 L gene mutation attenuated strains are built, concrete operations are：It is logical Following primer is crossed, through Overlapping PCR methods, 1917 amino acids single-points in NDV L protein structure domains VI are dashed forward Become, the purpose fragment after amplification and pmNDV-97 carriers are distinguished into double digestion through Hind III and the restriction enzymes of Xba I, entered It is attached after row nucleic acid electrophoresis, glue reclaim purpose fragment and carrier pmNDV-97, the mutant plasmid obtained is named as pK1917A；

   4 pairs of primers of design are used for the L Argine Monohydrochloride rite-directed mutagenesises of NDV VII -97, and primer sequence is as follows：

   AF1 ：5' CATGTTTTAAAGTTGTCAAG 3'

   AF2 ：5' CTTCTTGGTATGCGGCATCTCATCTTC 3'

   AF3 ：5' GTAGTGATCATCGCAGTACTGTATGC3'

   AF4 ：5' GAGGGGATATGCAATGTTACCTGATAT3'

   AF5 ：5' GCG GTCGAC ACCGCTTTCAGGTATTTAG3'

   AF6 ：5'GAAGATGAGATGCCGCATACCAAGAAG3'

   AF7 ：5'GCATACAGTACTGCGATGATCACTA3'

   AF8 ：5'ATATCAGGTAACATTGCATATCCCCT3'

   Wherein, AF1+AF6, AF1+AF7 and AF1+AF8 primer expand purpose band respectively using PCR methods, name respectively For A1, B1 and C1；AF5+AF2, AF5+AF3 and AF5+AF4 primer expand purpose band respectively using PCR methods, respectively It is named as A2, B2 and C2；On this basis, using AF1+AF5 primers through Overlapping PCR methods, using B1+B2 as Template, the purpose fragment containing mutational site is expanded, obtain L mutant fragments L1917.
3. 3. preparation method as claimed in claim 2, it is characterised in that：Entered with the L genes of the full-length genomes of separation strains NDV VII -97 Row rite-directed mutagenesis, to carrying out single-point amino acid mutation in L protein structure domains VI, obtain height and cause the weak L of NDV VII -97 to subtract Toxic vaccine strain K1917A.
4. 4. the preparation method according to claim 3, it is characterised in that：

   1）The structure of the pnca gene group full-length cDNAs of NDV VII -97；

   2）The connection of Amino Acid-Induced Site-Directed Mutation and mutant full-length cDNA in the L protein structure domains VI of NDV VII -97；

   3）The attenuated vaccine strain K1917A of VII type NDV L gene mutations rescue；

   By plasmid pK1917A and tri- eukaryon expression plasmid cotransfection BHK-21 cells of pBSK-NP, pBSK-P and pBSK-L, Cell conditioned medium is inoculated with 9-11 age in days SPF chicken embryos after transfection 72h, every part of 3 pieces of sample inoculation, 0.2mL/ pieces, successfully rescues and obtains The L gene mutation attenuated strains K1917A of NDV VII -97.