CN109321534A - A kind of recombination VIII type newcastle disease virus low virulent strain - Google Patents

Abstract

The present invention relates to Veterinarian virus Protocols in Molecular Biology and immunological technique fields, disclose one plant of recombination, VIII type newcastle disease virus low virulent strain NDV-rHR09-LaSota-HN.The deposit number of the low virulent strain NDV-rHR09-LaSota-HN is CCTCC NO:V201830.Research shows that; NDV-rHR09-LaSota-HN strain virulence is weak; MDT is greater than 120h; ICPI value is 0.026; immunity test proof has good immune protective effect to Virulent Newcastle Disease Virus strain; through compareing with common vaccine strain LaSota, with NDV-rHR09-LaSota-HN plants of 1 age in days SPF chickens of inoculation, the protecting effect etc. that the antibody level of chicken, right pop velogen strain attack poison after 3 weeks is better than or not less than control group.

Description

A kind of recombination VIII type newcastle disease virus low virulent strain

Technical field

The present invention relates to Veterinarian virus Protocols in Molecular Biology and immunological technique fields, utilize reverse genetic manipulation platform The replacement for carrying out HN genetic fragment again on the basis of F gene mutation to the VIII type strain of gene that screening obtains, obtains one plant The gene VIII type newcastle disease virus low virulent strain of recombination.

Background technique

Newcastle disease virus (Newcastle disease virus, NDV) is huge to the harm of World Poultry aquaculture, it can To infect a variety of birds, often results in aviculture and generate huge economic loss [Alexander, D.J.2001.Gordon Memorial Lecture.Newcastle disease.Br Poult Sci 42:5-22.].The virus belongs on taxology In Paramyxoviridae, paramyxovirus subfamily, fowl Rubulavirus (Avulavirus) member [Mayo, M.A.2002.Virus taxonomy-Houston 2002.Arch Virol 147:1071-1076.].Newcastle disease virus has Cyst membrane, genome are sub-thread, minus strand, the RNA virus of non-segmented negative, genome structure mode are as follows: 3 '-NP-P-M-F-HN-L- 5 ', successively encode 6 kinds of structural proteins: nucleocapsid protein (NP), phosphoprotein (P), stromatin (M), fusion protein (F), blood clotting Element-neuraminidase protein (HN) and high molecular weight protein (L) i.e. nucleoprotein (NP), P, M, F, HN and L albumen.According to the poison to chicken Power, NDV strain can be virulent (Velogenic), medium virulence (Mesogenic) and weak malicious (Lentogen) three types. Existing a large amount of research illustrated pathogenicity molecule determinant [Huang Z, Krishnamurthy S, Panda A,Samal SK.Newcastle disease virus V protein is associated with viral pathogenesis and functions as an alpha interferon antagonist.Journal of virology 2003Aug；77 (16): 8676-85.], wherein most is the effect about F and HN albumen to virus virulence, Amino acid sequence especially at F protein cracking site is main determining factor [the Seal BS.Nucleotide of virus virulence and predicted amino acid sequence analysis of the fusion protein and hemagglutinin-neuraminidase protein genes among Newcastle disease virus isolates.Phylogenetic relationships among the Paramyxovirinae based on attachment glycoprotein sequences.Funct IntegrGenomics 2004Oct；4(4):246-57.]. The molecular basis of NDV Virulence Difference depends mainly on the amino acid sequence of the protease cracking site of F0 albumen, virulent cracking There is pairs of basic amine group acid sequence at site both ends, and mode motif is¹¹²R/K-R-Q-K/R-R-F¹¹⁷；The cracking site of weak poison Then lack polybases acidic amino acid sequence, mode motif is¹¹²-E/G-R/K-Q-E/G-R-L¹¹⁷.HN albumen is also NDV virulence Important determinant.Existing research has shown that the length of HN protein amino acid sequence is related with virulence, utilizes reverse genetic manipulation Technology, Roemer-Oberdoerfer etc. have proven to the different length of HN albumen and the virulence of NDV and it is pathogenic have it is very heavy Relationship (Roemer-Oberdoerfer A, Werner O, Veits J, the et al.Contribution of the wanted length of the HN protein and the sequence of the F protein cleavage site to Newcastle disease virus pathogenicity.J Gen Virol.2003,84(11):3121-3129.)。HN The length of gene coding region there are many type, encoded HN length protein has 616,585,581,580,578,577,572, 571 amino acid etc. is a variety of.HN precursor protein containing 616 amino acid exists only in avirulent strain, as D26, The strains such as Ulster and Queensland, do not find in velogen strain, and the HN albumen containing 571 amino acid exists only in by force In strain, the HN albumen of other length exists in highly-wetting liquid.

Since newcastle disease (ND) nineteen twenty-six is found, had occurred repeatedly big prevalence so far, and it is popular each time all There is the strain of different genotype to occur.The 4th for newcastle disease occur after the nineties is very popular, and gives China and world's aviculture Cause huge economic loss, current pandemic newcastle disease virus based on genotype VII, but V, VI and VIII etc. other Genotype virus also has presence.There is expert's proposition in recent years, in the emerging gene VIIh of the multiple countries in Asia and Middle East It may result in global the 5th newcastle disease with VIIi and VIIIa and VIIIb hypotype newcastle disease virus to be very popular (Miller PJ, Haddas R, Simanov L, et al.Identificationof new sub-genotypes of virulent Newcastle disease virus withpotential panzootic features.Infect Genet Evol.2015,29:216-29.).It the use of vaccine is most effective most economical method, newcastle disease during controlling epidemic disease Vaccine used decades, due to being widely used for ND vaccine, this disease has substantially obtained effective control, but clinical The generation of upper atypia ND is still commonplace, and ND is still one of the principal disease for threatening aviculture, the reason of immuning failure Except immune programme is unreasonable, in addition to the presence of immunosuppressive disease, the genotype of vaccine strain and popular strain and it is antigenic not With may be one of the main reasons, this illustrates that the conventional vaccine attack virulent for NDV can not provide ideal protection.From me State is in recent years from the point of view of ND epidemic characteristic, clinically it is separated to most of NDV strain belong to genotype VII [Liu, X.F., H.Q.Wan,X.X.Ni,Y.T.Wu,and W.B.Liu.Pathotypical and genotypical characterization of strain of Newcastle disease isolated from outbreaks in chicken and goose flocks in some regions of China during 1985-2001.Archives Of Virology, 2003,148 (7): 1387-1403.], and the genotype of conventional ND vaccine strain be mainly I and II type (such as V4, LaSota etc.), routine ND vaccine strain differs farther out with NDV epidemic strain on genetic distance, this makes poultry when virulent infection exist There are a large amount of toxin expelling phenomenons in early stage, leads to the generation of atypia ND.Currently, gene VIII type strain is in Africa, South Asia and the southeast Still there are a prevalence in many countries and regions such as Asia, and China has also had separation to find since 80 years, and the vaccine used is from existing report See to be still gene I, II low virulent strain, there is no the vaccine strains to match with VIII type, therefore formulate the weak poison of NDV gene VIII type Vaccine strain is worth with Important Economic.

Summary of the invention

It is an object of the invention to invent a kind of VIII type newcastle disease virus low virulent strain of gene of less toxic power, one will be provided for future Kind gene VIII type attenuated live vaccines, namely a kind of vaccine candidate strain to match with popular NDV gene VIII type is provided.

VIII type newcastle disease virus low virulent strain of recombination provided by the invention, is newcastle disease virus NDV-rHR09- LaSota-HN, its deposit number are CCTCC NO:V201830.

The invention also discloses the complete of the VIII type newcastle disease virus low virulent strain NDV-rHR09-LaSota-HN of recombination Genome sequence, as shown in SEQ ID No.1.

Attenuated vaccine Candidate Strain NDV-rHR09-LaSota-HN in the present invention is passed through from heat-resisting velogen strain NDV/HR09 Reverse genetic operating system carries out mutation transformation to virus F gene, on the basis of acquisition causes weak NDV/rAHR09 strain, into one The HN full length gene segment of vaccine strain LaSota is replaced NDV/rAHR09 respective segments and obtained by step.NDV/HR09 plants are inventions Laboratory where people separates from inspection pathological material of disease identification and is obtained.The systematic evolution tree that the strain is constructed through F genetic fragment proves Belong to VIII type of gene.The present invention has initially set up HR09 plants of reverse genetic operating system, to virus F gene cracking site base Sequence is mutated, make the amino acid of virus F gene corresponding encoded by¹¹²R-R-Q-K¹¹⁵-R-F¹¹⁷Sporting has typical weak poison The amino acid sequence of strain feature¹¹²G-R-Q-G¹¹⁵-R-L¹¹⁷, rescue obtains the NDB/rAHR09 strain of weak poison, and the MDT value of the strain > 120h, ICPI value are 0.057.In order to weaken the virulence of virus further, LaSota plants of HN gene is introduced, building obtains Retain NDV gene VIII type F gene antigen characteristic, the recombinant virus NDV-rHR09-LaSota- with LaSota plants of HN genes HN.Strain MDT > 120h, ICPI value is 0.026, Immunoprotection test, every group 12 1 age in days SPF chicken muscle injection inoculations 10⁶EID₅₀Dosage, with 10 after 3 weeks⁵EID₅₀The genotype VII ZJ1 strain of dosage carries out eye droppings, collunarium attacks poison, the results showed that, exempt from For epidemic disease chicken antibody HI potency with this strain group highest, when 3 week old, averagely reaches 6.35 ± 0.6, is significantly higher than LaSota plants, attacks malicious guarantor Shield rate is 100%.

The construction method of NDV-rHR09-LaSota-HN of the present invention is as follows:

(1) rescue of VIII type HR09 strain reverse genetic operating system of gene building and low virulent strain

Construct the full-length clone of HR09 pnca gene group cDNA and 3 eukaryotic expressions containing strain NP, P and L gene Carrier, through cotransfection BSR-T7/5 cell, rescue obtains infection clones, verifying reverse genetic platform construction success.Design two To primer, the method through Overlap PCR dashes forward HR09 plants F protein cracking site the 112nd, 115 and 117 basic amino acid Become non-alkaline amino acid relevant to low virulent strain, full-length cDNA transcription vector prNDV/HR09 is constructed, by transcription vector PrNDV/HR09 and 3 auxiliary expression plasmid co-transfection BSR-T7/5 cell, saves out virus N DV/rAHR09.Viral biology CHARACTERISTICS IDENTIFICATION result is MDT value > 120h, and ICPI value is 0.057, and it is weak to show that the NDV/rAHR09 of rescue has been caused.

(2) LaSota plants of HN gene recombined virus NDV-rHR09-LaSota-HN rescues are replaced

It is expanded using RT-PCR and obtains LaSota plants of HN gene ORF sequences, in the above-mentioned reverse genetic manipulation having built up The methods of on platform base, coupled using Overlap PCR and digestion, LaSota plants of HN gene ORF sequences are replaced into HR09 Corresponding segment in pnca gene group, constructs recombinant virus genomes full length cDNA clone, then with 3 auxiliary matter having had been built up Grain cotransfection BSR-T7/5 cell, rescue obtain recombinant virus.

(3) identification of recombination NDV-rHR09-LaSota-HN low virulent strain virulence and Immunoprotection test

The recombination low virulent strain that rescue is obtained is grown in chicken embryo to pass on, and it is special to carry out biology after chicken embryo growth is stablized after virus Property identification, measure MDT, ICPI value of virus, determine that the virulence of virus is strong and weak.

By strain of the present invention and the LaSota plants of progress immunological comparison tests of traditional vaccine strain.Test includes blank control group Totally 3 groups, every group of 12 SPF chickens, 1 age in days carries out 10⁶EID₅₀Dose immunization intramuscular injection inoculation, the HI for measuring each group chicken respectively weekly are anti- Body potency presses 10 after being immunized 3 weeks with popular genotype VII ZJ1 strain⁵EID₅₀Dosage carries out attacking poison, after finally statistics attacks poison As a result it is higher than control group level to prove that strain antibody of the present invention is generated, and has complete immunoprotection for the case fatality rate of each group chicken (protective rate 100%).

The NDV-rHR09-LaSota-HN low virulent strain that the present invention obtains remains VIII type NDV/HR09 strain F of NDV gene The antigenicity of albumen, after replacing low virulent strain LaSota plants of HN gene ORF, the virulence of recombinant virus is caused than independent F gene mutation The strain virulence of weak acquisition also wants weak, while also having preferable immunogenicity, and the attack that chicken right pop velogen strain is immunized obtains 100% protection, the level that antibody generates are high.

Detailed description of the invention

Fig. 1 is the building schematic diagram of HR09 plants of full-length genome cDNA transcription vectors.

Fig. 2 is 6 fragment electrophoretic figures of full-length genome of RT-PCR segmentation amplification.M:200bp；1-6 is respectively V1, R1, R2, R3, L1, V2 segment.

Fig. 3 is NDV/HR09 plants of full-length genome cDNA transcription vector digestion qualification result figures.1. plasmid of M:DL15000 pNDV/HR09；2,3 pNDV/HR09 after HpaI digestion.

Fig. 4 is GFP expression effect figure after helper plasmid cotransfection.

Fig. 5 is virus infection clones PCR qualification result figure.M:200bp Marker 1,2: 9 amplified fragments of primer

Fig. 6 is R2 and R3 segment building schematic diagram.

Fig. 7 is recombinant virus overall length plasmid construction ideograph.

NDV-rHR09-LaSota-HN strain of the invention is preserved in China typical culture collection on June 19th, 2018 Center, depositary institution address: Wuhan, China university；Classification naming are as follows: newcastle disease virus NDV-rHR09-LaSota-HN；Preservation Number is CCTCC NO:V201830.

NDV/rAHR09 involved in the present invention plants was preserved in China typical culture collection on September 20th, 2017 The heart, depositary institution address: Wuhan, China university；Classification naming are as follows: VIII type NDV/rAHR09 of newcastle disease virus gene；Deposit number For CCTCC NO:V201739.

Specific embodiment

Biomaterial according to the present invention:

LaSota plants are purchased from China Veterinery Drug Inspection Office (CVCCNo:AV615).

ZJ1 plants of (Liu X F, Wan H Q, Ni X X, et al.Pathotypical and genotypical characterization of strain of Newcastle disease virusisolated from out breaks in chicken and goose flocks in some regions of China during 1985-2001.Arch Virol,2003,148(7):1387-1403.)。

NDV/HR09 plants of (Cao Y, Liu Q, Zhang X, Hu H, Wu Y.2017.Complete genome sequence of heatresistantNewcastle disease virus strain HR09.Genome Announc 5:e01149-17.).GenBank accession number: MF285077.

One, the foundation of VIII type NDV/HR09 strain reverse genetic operating system of gene

It is as follows to test the main experimental materials and methods being related to:

Cell and chicken embryo: the BSR-T7/5 cell and minigenome TVT-LGT that can stablize expression t7 rna polymerase are by China Academy of Agricultural Sciences's Shanghai veterinary institute give, and TVT7R (0.0) transcription vector is saved by this laboratory, and SPF chicken embryo is by Beijing plum Li Yaweitong experimental animal Technology Co., Ltd. provides.

Experiment reagent: cell culture medium DMEM is purchased from Shanghai Yuan Pei Biotechnology Co., Ltd, and new fetal calf serum is purchased from GIMINI company, Easytaq archaeal dna polymerase, pEASYTM-Blunt cloning vector, pCR2.1 cloning vector, Trans-T1 sense Beijing Quanshijin Biotechnology Co., Ltd is purchased from by state cell；TRIzol Reagent, M-MLV reverse transcription system, high-fidelity Archaeal dna polymerase, dNTP are purchased from Beijing Quanshijin Biotechnology Co., Ltd, and DNA gel QIAquick Gel Extraction Kit, which is purchased from, likes biology of pursuing progress Technology (Hangzhou) Co., Ltd, DNA Marker are purchased from precious bioengineering (Dalian) Co., Ltd, Q5, restriction enzyme (Apa I, BsmB I, Mlu I, Bbs I, Spe I, Not I, Xba I) etc. be purchased from NEB company, T4DNA Ligase, restriction enzyme FspA I Purchased from Thermo Fisher company.It is the limited public affairs of century biotechnology that EndoFree Plasmid Midi Kit, which is purchased from Beijing health, Department.Other common biochemical reagents are domestic analytical reagents.

Design of primers and synthesis: it according to the complete genome sequence (GenBank accession number: MF285077) of NDV/HR09, uses Primer Premier5.0 software design 6 carries out segmentation amplification to its full genome of primer pair, and building schematic diagram is shown in Fig. 1.By gene Molecular labeling of 13048 bit bases of group as Revive virus.Primer sequence is shown in Table 1, and helper plasmid building primer is shown in Table 2, primer By Nanjing, Jin Sirui Bioisystech Co., Ltd is synthesized.

1.NDV/HR09 plants of full-length cDNAs of table are segmented amplimer

Table 2. constructs NDV/HR09 helper plasmid the primer

The clone of 1.HR09 plants of each segments of full-length genome cDNA and sequencing

Viral RNA is extracted according to the method for document and carries out reverse transcription.Using the cDNA of acquisition as template, target fragment L1 is used High-fidelity DNA polymerase amplification, remaining target fragment are expanded with Q5 enzyme.

PCR after reaction, using 1% agarose gel electrophoresis detects all PCR products, cuts under ultraviolet light Ago-Gel containing target fragment, chopping, and referring to DNA gel QIAquick Gel Extraction Kit specification, to the target fragment of amplification It is recycled, is carried out according to pEASYTM-BluntCloning Kit specification, by the product of recycling and pEASY TM-Blunt Cloning vector is placed in connection (the glue recovery product of segment L1 is connect with pCR2.1 carrier) in the dactylethrae of 1.5mL, and every pipe adds Enter 30 μ L Trans-T1 competent cells, mixes gently, be immediately placed in ice, ice bath 30min；Heat shock in 42 DEG C of water-baths 30s places cooled on ice 5min；It is added 600 μ L LB culture mediums, according to the speed oscillation 1h of 200r/min on 37 DEG C of shaking tables, takes 5000r/min is centrifuged 5min after out；Remaining about 100 μ L supernatants, are coated on LB plate with ampicillin after precipitating is resuspended On, 37 DEG C of incubators are inverted 12~16h of culture；Next day selects 5 single colonies and is inoculated with LB Liquid Culture with ampicillin respectively Base overnight incubation.According to the method that pEASY TM-Blunt Cloning Kit specification provides, with universal primer M13F and M13R is identified by PCR method.The positive clone of PCR identification, which send to Anhui General Corporation, to be sequenced.Sequencing is correctly cloned It is respectively designated as T-R1, T-R2, T-R3, T-V1, T-V2, T-NP, T-P, T-L1, T-L2, T-L3.

Segmentation amplification carried out to NDV/HR09 full-length genome, obtains totally 6 DNA fragmentations, identified, clip size and pre- Phase is consistent (such as Fig. 2).

The building of 2.NDV/HR09 plants of full-length genome cDNA transcription vectors

The extraction of each segment recombinant plasmid of NDV/HR09 full-length genome: the mother liquor containing each target fragment clone is connect respectively Kind LB culture medium with ampicillin is cultivated, using EndoFree Plasmid Midi Kit, according to operation instructions Plasmid is extracted, and carries out the measurement of concentration and purity.

The connection at the end of NDV/HR09 genome 5 ' and 3 ' ends: plasmid T-V1 and T-V2 is through I double digestion of Not I and Mlu, digestion Product is detected with agarose gel electrophoresis, and the purpose piece of 2300bp and 6100bp or so are separately recovered under ultraviolet irradiation Section, after the connection of T4 ligase, transformed competence colibacillus cell identifies that positive clone designation is T-V.Plasmid T-V BsmBI digestion, Digestion products are detected with agarose gel electrophoresis, and 4500bp segment, transcription vector TVT7R are recycled under ultraviolet irradiation (0.0) it with 3100bp segment is recycled after BbsI restriction enzyme digestion and electrophoresis, is connected using T4 ligase, transformed competence colibacillus cell, identification is positive Clone designation be TVT-V.

The connection of NDV/HR09 genome TM (2296nt~13048nt): using plasmid T-R1 and T-R2 as template, R1-F/ R2-R is primer, Overlap PCR amplification R12 segment, it is contemplated that clip size 4519bp.Amplified fragments connect pCR2.1 clone Carrier, transformed competence colibacillus cell, I site picking carrier S pe are located at the cloning and sequencing in R12 segment downstream.Correct clone designation For T-R12.

Double digestion, digestion products fine jade are carried out to plasmid T-R12 and T-R3 respectively with restriction enzyme BsmB I and Spe I The target fragment of 8500bp and 2800bp or so, T4 connection is separately recovered in sepharose electrophoresis detection under ultraviolet irradiation After enzyme connection, transformed competence colibacillus cell identifies that positive clone designation is TR123.

Plasmid TR123 and TL2 is through I double digestion of Spe I and FspA, and digestion products are detected with agarose gel electrophoresis, ultraviolet The segment of 12300bp and 3500bp or so is recycled under the irradiation of line, T4 ligase is attached, transformed competence colibacillus cell, bacterium solution Positive colony is identified in PCR and digestion, is named as TM.

The acquisition of transcription vector containing NDV/HR09 plants of full-length genome cDNA: plasmid TM and TVT-V are respectively through Apa I and Mlu I double digestion, digestion products are detected with agarose gel electrophoresis, and the band of 10kb and 9kb or so is recycled under ultraviolet irradiation, After the connection of T4 ligase, transformed competence colibacillus cell.To construct the transcription vector pNDV/ containing NDV genome cDNA overall length HR09。

The identification of NDV/HR09 plants of full-length genome cDNA transcription vectors: transcription vector is identified by digestion.Turn through analysis Record carrier on restriction enzyme site distribution, using I digestion of restriction enzyme Hpa identify, electrophoresis detection digestion band whether in advance Phase stripe size is consistent.Digestion system are as follows: sterilizing 12 μ L of ultrapure water, 1 μ L of Hpa I, cutsmart2 μ L, 5 μ L of Plasmid DNA (1 μ g).

The NDV/HR09 full-length genome cDNA transcription vector built is identified through digestion, and 2 bands are obtained after I digestion of Hpa, That is 4949bp and 13348bp, electrophoresis result and expection consistent (Fig. 3).

3. the building and identification of helper plasmid

Helper plasmid pCI-NP building: in 103nt-1643nt design primers of genome, NP gene, the segment 5 ' are expanded End addition I restriction enzyme site of Xba, connects pEASYTM-Blunt cloning vector.Sequencing result is correctly cloned through Xba I and Not I couple After digestion, digestion products are detected with agarose gel electrophoresis, and the band of 1500bp or so is recycled under ultraviolet irradiation, uses T4 Ligase is connected in the pCI-neo carrier equally through I double digestion of Xba I and Not, transformed competence colibacillus cell, PCR and digestion It identifies positive colony, is named as pCI-NP.

Helper plasmid pCI-P building: in 1874nt-3096nt design primers of genome, P gene, the segment 5 ' are expanded End addition I restriction enzyme site of Xba, connects pEASYTM-Blunt cloning vector.Sequencing result is correctly cloned through Xba I and Not I couple After digestion, digestion products are detected with agarose gel electrophoresis, and the band of 1200bp or so is recycled under ultraviolet irradiation, uses T4 Ligase is connected in the pCI-neo carrier equally through I double digestion of Xba I and Not, transformed competence colibacillus cell, PCR and digestion Method identifies positive colony, is named as pCI-P.

Helper plasmid pCI-L building: plasmid T-L2 is through I digestion of FspAI and Not, digestion products agarose gel electrophoresis Detection is recycled the band of 3500bp or so under ultraviolet irradiation, is connected to equally with T4 ligase through I enzyme of FspAI and Not After cutting in linear T-L1, after transformed competence colibacillus cell, the positive colony identified is named as T-L12.

Plasmid T-L12 and T-L3 is through I double digestion of Mlu I and Not, and digestion products are detected with agarose gel electrophoresis, ultraviolet The segment of 8500bp and 2000bp or so is recycled under the irradiation of line, T4 ligase is attached, transformed competence colibacillus cell, PCR with And enzymatic cleavage methods identify positive colony, are named as T-L123.

Plasmid T-L123 is after I double digestion of Spe I and Not, and digestion products are detected with agarose gel electrophoresis, in ultraviolet light Irradiation under recycle the band of 6600bp or so, be connected in the pCI-neo carrier equally through I double digestion of Xba I and Not, T4 connects It connects enzyme to be attached, transformed competence colibacillus cell, PCR and enzymatic cleavage methods identify positive colony, to be built into helper plasmid pCI-L。

The identification of helper plasmid: tri- eukaryon expression plasmids of pCI-NP, pCI-P and pCI-L and micro genome TVT-LGT After cotransfection BSR-T7/5 cell 48h, the expression that can see GFP gene is seen whether under inverted fluorescence microscope.Cell turns Dyeing method refers to X-treme GENE HPDNA Transfection Reagent specification.

Tri- eukaryon expression plasmids of pCI-NP, pCI-P and pCI-L and micro genome TVT-LGT cotransfection BSR-T7/5 After cell 48h, the expression (Fig. 4) of GFP gene can be seen under inverted fluorescence microscope, illustrates pCI-NP, pCI-P and pCI-L It can be expressed in BSR-T7/5 cell, RNPs structure can be formed with micro genome, to start micro genome Duplication and transcription.

4 virus rescues and reverse genetic platform are established

The preparation of cell and transfection plasmid: the BSR-T7/5 cell of transfection carries out prescreening training with the G418 of 1mg/mL It supports, in the day before transfection by 5 × 10⁵Cell inoculation in 35mm dish, about 60%~80% when being paved with for transfecting.Turn All plasmids (TVT/LGT, pNDV/HR09, pCI-NP, pCI-P, pCI-L) used when dye use EndoFree Plasmid Midi Kit is extracted, and has been extracted and has been measured its concentration and purity, concentration is more than 0.1 μ g/ μ L and purity is 1.8~2.0 (OD260/280), the plasmid of > 2 (OD260/230) is for transfecting, and plasmid extracting method is according to EndoFree Plasmid Midi Kit specification carries out.

Cotransfection: by the transcription vector pNDV/HR09 of NDV genome cDNA overall length and three helper plasmids (pCI-NP, PCI-P, pCI-L) the ready BSR-T7/5 cell of cotransfection, transfection sample is sealed with sealing film after transfecting 60h, is shifted To -70 degree refrigerator multigelation 3 times, it then will transfect under cell scraper, 9~11 ages in days are inoculated with after being sufficiently mixed with cell conditioned medium SPF chicken embryo, 0.4mL/ pieces, 37 DEG C of cultures.Timing observation chicken embryo state, discards the chicken embryo of unusual death in for 24 hours.It will after 96h Chicken embryo is placed in 4 DEG C of refrigerators, after chicken embryo vessel retraction, collects chick embryo allantoic liquid.

Viruses indentification: the measurement of HA: collection connect embryo allantoic liquid, with freshly prepared 1% chicken red blood cell according to OIE standard into Row HA measurement.The positive chick embryo allantoic liquid of HA detection is continued to connect in SPF chicken embryo and passed for 2 generations, allantoic fluid is collected and extracts virus RNA, after configuration reverse transcription system carries out reverse transcription and inactivates, using it as template, with the specific primer 9 (ND-9) of sequencing, It expands the part NDV L gene region length about 1892bp (12362nt~14253nt), according to the MluI (13048nt) mutated Base variation, to determine whether to save out virus.Heat resistance, the biological nature of rescued virus are carried out referring to method above-mentioned.

After transfection sample connects embryo, for first generation chicken embryo in 80h or so death, carrying out hemagglutination test has coagulation, rescued virus It is named as NDV/rHR09, and after resuming for 2 generations in SPF chicken embryo.The chick embryo allantoic liquid for collecting for the 3rd generation, using the method for RT-PCR It is detected, according to the mark molecule of genomic marker, is finally determined.Sequencing result shows genome the 13048th Base becomes A (Fig. 5) by G, it was demonstrated that viral genome is derived from transcription vector NDV/rHR09.As a result gene VIII is also turned out The success of type NDV/HR09 strain reverse genetic platform construction.

Two, the rescue of F gene mutation attenuated virus

1. design of primers and synthesis

By primer through Overlap PCR method, by the nucleosides at the F gene cracking site of recombinant virus pNDV/HR09 Acid is mutated, to make the amino acid of corresponding encoded by R¹¹²-R-Q-K¹¹⁵-R-F¹¹⁷Become the amino acid sequence of weak malicious feature: G¹¹²-R-Q-G¹¹⁵-R-L¹¹⁷, the plasmid after mutation is named as prNDV/HR09, and primer is limited by Nanjing Jin Sirui biotechnology Company's synthesis.Primer sequence is as follows:

Rr1-R:5’-AAGGCGCCCCTGTCCCCCCCCACCAGATGTAG-3’(SEQ ID No.24)

Rr2-F:5’-GGGGGACAGGGGCGCCTTATAGGTGCCGTTATTG-3’(SEQ ID No.25)

2. the clone and sequencing of segment Rr1 and segment Rr2

Using the recombinant plasmid TR-12 of above-mentioned acquisition as template, with R1-F (being shown in Table 6)/Rr1-R, Rr2-F/R2-R (is shown in Table 6) For primer, amplification obtains segment Rr1 and segment Rr2.25 μ L PCR reaction systems are as follows: sterilizing ultrapure water 15.75 μ L, 5 × Q5Buffer5 μ L, dNTPs (2.5mmol/L) 2 μ L, upstream primer (10 μm of ol/ μ L) 0.5 μ L, downstream primer (10 μm of ol/ μ L) 0.5 μ L, 1 0.25 μ L of μ L, Q5 of template.

PCR reaction cycle parameter are as follows: 98 DEG C of initial denaturations 30s, 98 DEG C of denaturation 10s, 55 DEG C of annealing 10s, 72 DEG C of extension 1min, Carry out 30 circulations；72 DEG C of extension 2min.

PCR after reaction, with 1% agarose gel electrophoresis detects all PCR products, cuts and contains under ultraviolet light Have the Ago-Gel of purpose segment, shred, and referring to DNA gel QIAquick Gel Extraction Kit specification, to the target fragment of amplification into Row recycling, puts recovery product and pEASY TM-Blunt cloning vector according to pEASYTM-BluntCloning Kit specification It is placed in connection (the glue recovery product of segment L1 is connect with pCR2.1 carrier) in the dactylethrae of 1.5mL, 30 μ L are added in every pipe Trans-T1 competent cell, mixes gently, and is immediately placed in ice, ice bath 30min；Heat shock 30s in 42 DEG C of water-baths places ice Upper cooling 5min；600 μ L LB culture mediums are added, according to the speed oscillation 1h, 5000r/ after taking-up of 200r/min on 37 DEG C of shaking tables Min is centrifuged 5min；Remaining about 100 μ L supernatants, are coated on LB plate with ampicillin, 37 DEG C of incubators after precipitating is resuspended It is inverted 12~16h of culture；Next day selects 5 single colonies and is inoculated with LB liquid medium overnight incubation with ampicillin respectively. According to pEASY TM-Blunt Cloning Kit specification method, identified with primer M13F and M13R by PCR method. The positive clone of PCR identification send company to be sequenced.Correctly clone is respectively designated as T-Rr1 and T-Rr2 for sequencing.

3. the building of attenuated virus full-length genome cDNA transcription vector

Using T-Rr1 and T-Rr2 amplified fragments as template, R1-F/Rr1-R is used respectively, Rr2-F/R2-R is primer, Overlap PCR amplification Rr12 segment, it is contemplated that clip size 4519bp.Reaction system is 50 μ L Overlap PCR systems: Sterilize ultrapure water 36.5 μ L, 10 × TransTaqHiFi Buffer5 μ L, dNTPs (2.5mmol/L) 4 μ L, 1 μ L of upstream primer, 1 μ L, T-Rr1 amplified fragments of downstream primer, 1 μ L, T-Rr2 amplified fragments, 1 μ L, TransTaq HiFi DNA polymerase (5U/μL)0.5μL.PCR reaction cycle parameter: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend 5min, 30 circulations；72 DEG C of extension 10min.

Amplified fragments connect pCR2.1 cloning vector, convert DH5 α competent cell, I site picking carrier S pe is located at R12 The cloning and sequencing in segment downstream.Correct clone designation is T-Rr12.

The same first part of remaining building process, final building obtain full-length cDNA overall length transcription vector, are named as pNDV/rAHR09。

4. the rescue of attenuated IBDVs and virulence identification

By the transcription vector pNDV/rAHR09 of above-mentioned acquisition and three helper plasmid (pCI-NP, pCI-P, pCI-L) corotation Contaminate ready BSR-T7/5 cell, the same first part of transfection method.

10% sterile allantoic fluid is added after transfecting 12h, in each dish.By transfection sample sealed membrane after transfection 60h It seals, is transferred to -70 degree refrigerator multigelation 3 times, then will transfect under cell scraper, and be inoculated with 9 after being sufficiently mixed with cell conditioned medium ~11 age in days SPF chicken embryos, 0.4mL/ pieces, 37 DEG C of cultures.The state of chicken embryo is observed in timing, discards the chicken of unusual death in for 24 hours Embryo.Embryo is placed in 4 DEG C of refrigerators after 96h, after chicken embryo vessel retraction, detection blood clotting (HA) potency simultaneously collects allantoic fluid, after resuming Generation.

Continue the positive chick embryo allantoic liquid of HA detection to upload for 2 generations in SPF chicken embryo, collects allantoic fluid and extract virus RNA after reverse transcription, using it as template, with the specific primer 4 (ND-4) of sequencing, expands virus F gene region partial sequence Length about 1755bp (4098nt~5872nt), according to the variation of F gene cracking region base, to determine whether to save out disease Poison.

The attenuated IBDVs of acquisition are named as NDV/rAHR09, carry out biological characteristics (MDT, ICPI) identification.By toxic urine Cyst fluid titre is diluted to 10^-3Afterwards, it is inoculated with CEF cell, observes cytopathy situation under the microscope after 48h, while setting maternal poison Strain is as control.

Rescued virus detects F gene cracking site sequence variation situation using the method for RT-PCR.Show F base through sequencing Because cracking region base has occurred and the consistent change of design scheme.

After rescued virus NDV/rAHR09 dilution, MDT and ICPI is measured respectively, the results show that MDT value > 120h；ICPI value It is 0.057.The virulence of comprehensive judgement standard declaration rescued virus meets the standard of low virulent strain.

Observation cytopathy is sent out after female parent virus HR09 and attenuated virus NDV/rAHR09 are infected CEF cell 48h simultaneously Existing, the CEF cell for being inoculated with attenuated virus does not occur cytopathy, and drawing in the net and falling off now then occur in the cell for being inoculated with maternal virus As forming apparent plaque.Illustrate that the F protein for the virus saved out cannot be cracked effectively on CEF, to illustrate its virulence Obviously weakened, virus weakening success.

Three, the rescue of LaSota plants of HN gene recombined virus is replaced

The amplification of 1.LaSota plants of HN gene

According to the position (see Fig. 1) of HN gene in the above-mentioned reverse genetic platform having built up, by LaSota plants of HN genes It is divided into two segments of b and c piece, separately designs 2 pairs of primers and expand, primer sequence is as follows:

R2-b-F primer: CTAATAGCAGACTCCAGTCATGGACCGCGCCGTTAG (SEQ ID No.26)

R2-b-R primer: CTCAGTATTTAACAATGCCAACGG (SEQ ID No.27)

R3-c-F primer: GTGACCTTGGCTATATCTGTAG (SEQ ID No.28)

R3-c-R primer: CTAGCCAGACCTGGCTTCTCTAACC (SEQ ID No.29)

50ul pcr amplification reaction system are as follows: Buffer10 μ l；dNTP(2.5)4μl；2 μ l of F/R primer (10 μM)； cDNA1.5μl；0.5 μ l of Q5 enzyme；H2O to 50 μ l.Reaction condition are as follows: 98 DEG C of initial denaturation 30s；98 DEG C of denaturation 8s, 52 DEG C of annealing 30s, 72 DEG C of extensions carry out 30 circulations；72 DEG C of extension 2min.

PCR after reaction, is detected using HN fragment PCR products of 1.2% agarose gel electrophoresis to LaSota, The Ago-Gel containing target fragment (about 0.3kb and 1.6kb respectively) is cut under ultraviolet light, is shredded, and referring to AxyPrep The method of DNA Gel Extraction Kit specification recycles the target fragment of amplification respectively.

2. the building of plasmid R2 and R3 segment

R2 the and R3 plasmid in above-mentioned reverse genetic platform is rebuild, forming types are shown in Fig. 6.

(1) building of R2 segment

Go out a segment using above-mentioned T-R2 plasmid as template amplification, primer sequence is as follows:

R2-a-F:AAGCTGTTGGCACCTTTCTTCT(SEQ ID No.30)

R2-a-R:GACTGGAGTCTGCTATTAGTGATG(SEQ ID No.31)

Reaction system is same as above with reaction condition.

PCR after reaction, detects PCR product using 1.2% agarose gel electrophoresis, cuts and contains under ultraviolet light There is the Ago-Gel of purpose segment, shreds, and referring to the method for AxyPrep DNA Gel Extraction Kit specification The target fragment about 1.4kb of amplification is recycled.

A the and b segment of OverLap PCR connection R2: respectively using a and b segment as template, upstream primer is the R2- in table 1 F, downstream primer are above-mentioned R2-b-R, pass through PCR connection a and b segment.

Reaction system is same as above with reaction condition.

PCR after reaction, detects PCR product using 1.2% agarose gel electrophoresis, cuts and contains under ultraviolet light There is the Ago-Gel of purpose segment, shreds, and referring to the method for AxyPrep DNA Gel Extraction Kit specification The target fragment about 2.6kb of amplification is recycled.

PCR product is according to pEASY^TMBlunt Cloning Kit specification method is attached reaction.Connection product is normal Rule method converts Trans-T1 competent cell, is coated on LB plate with ampicillin, and 37 DEG C of incubators are inverted culture 14h selects monoclonal colonies and carries out PCR identification respectively, and by identification, correctly positive bacterium solution send sequencing further verifying.It will verifying Correct R2 segment bacterium solution is inoculated with LB liquid medium overnight incubation with ampicillin.Next day extracts the plasmid of R2 segment ,- 20 DEG C of preservations.

(2) building of R3 segment

Go out d segment as template amplification using the above-mentioned T-R3 plasmid constructed, primer sequence is as follows:

R3-d-F primer: GAAGCCAGGTCTGGCTAGGAAATTAGATCTGGCTGGC (SEQ ID No.32)

R3-d-R primer: CATCTTTGGTGCGCACATCTG (SEQ ID No.33)

Reaction system and reaction condition is with reference to above-mentioned.

PCR after reaction, detects PCR product using 1.2% agarose gel electrophoresis, cuts and contains under ultraviolet light There is the Ago-Gel of purpose segment (size about 1.4kb), shreds, and referring to AxyPrep DNA Gel Extraction Kit The method of specification recycles the target fragment of amplification.

Pass through c the and d segment of OverLap PCR connection R3

PCR reaction condition is with reference to above-mentioned.

PCR after reaction, detects PCR product using 1.2% agarose gel electrophoresis, cuts and contains under ultraviolet light There is the Ago-Gel of purpose segment (about 2.8kb), shreds, and referring to AxyPrep DNA Gel Extraction Kit explanation The method of book recycles the target fragment of amplification.

PCR product conventional method is attached reaction.Connection product converts Trans-T1 competent cell, is coated on containing ammonia On the LB plate of parasiticin, 37 DEG C of incubators are inverted culture 14h, select monoclonal colonies and carry out PCR identification respectively, will be accredited as Correctly positive bacterium solution send sequencing further verifying.Correct R3 segment bacterium solution will be verified and be inoculated with LB liquid with ampicillin Culture medium overnight incubation.Next day extracts the plasmid of R3 segment, -20 DEG C of preservations.

3. the building of recombinant virus overall length plasmid

Recombinant virus overall length plasmid construction mode such as Fig. 7.

With reference to the method for aforementioned building overall length plasmid, it connect R2, R3 segment built to form R123 segment with R1, then It connect to form R123L2 (TM) segment (plasmid) with L2 segment, by plasmid TM and the TVT-V plasmid of aforementioned building respectively through Apa I With I double digestion of Mlu, digestion products are detected with agarose gel electrophoresis, cut size about 10kb respectively under ultraviolet irradiation Target fragment and recycling with 9kb, overnight with 14 DEG C of T4DNA ligase connections by product after the recovery, reaction condition is with reference to upper It states, converts 5 α competent cell of Trans, be coated on LB plate with ampicillin, 37 DEG C of incubators are inverted culture 14h, choose Selected monoclonal bacterium colony carries out PCR identification respectively, and by identification size, correctly positive bacterium solution send sequencing further verifying, will verify just True bacterium solution is inoculated with LB liquid medium overnight incubation with ampicillin, extracts plasmid and is named as pNDV-rHR09- LaSota-HN, -20 DEG C save backup.

4. the rescue and identification of recombinant virus

Step and method are the same as aforementioned.

By the allantoic fluid of HA test positive after SPF chicken embryo uploaded for two generations, the RNA of allantoic fluid extracting virus is collected, is led to It crosses RT-PCR and amplifies HN gene, connect T3 carrier, picking positive bacterium solution sends to sequencing identification.

Identified, acquisition recombinant virus sequence is correct, is named as NDV-rHR09-LaSota-HN, its full-length genome base Sequence is as shown in SEQ ID No.1.

Four, NDV-rHR09-LaSota-HN plants of virulence identifications and Immunoprotection test

By NDV-rHR09-LaSota-HN plants of recombinant virus by SPF chicken embryo is inoculated with after standard method dilution, measure respectively MDT, ICPI and IVPI.

The measurement of MDT: it is diluted to 10 again with the continuous normal saline 10 of sterilizing^-6~10^-9, the virus liquid of each dilution SPF chicken embryo through allantoic cavity 5 piece of 9~10 age in days of inoculation, 0.1mL/ pieces, 37 DEG C of culture 7d.It is primary at interval of 12h observation, continuously 7d is observed, observation all records the time of chicken embryo death every time.The value of MDT is calculated according to the following formula.

The measurement of ICPI: fresh viral allantoic fluid of the HA potency greater than 24 is taken to do 10 using not antibiotic sterilizing PBS After dilution, using 10 1 age in days SPF chick of intracranial inoculation, 50 μ L/ are only.Negative control group is set up simultaneously, and negative control group uses PBS carries out intracranial inoculation.Observation chicken mass-sending disease and death condition daily, are observed continuously 8d.Normal 0 point of meter, 1 point of morbidity meter, extremely 2 points of meter is died, the value of ICPI is calculated according to the following formula.

The results show that NDV-rHR09-LaSota-HN plants of MDT values of recombinant virus are greater than 120h, ICPI value is 0.026.With Aforementioned independent F gene causes weak acquisition strain to compare, and ICPI value reduces, and illustrates that the virulence of virus reduces.

NDV-rHR09-LaSota-HN plants are immunized with conventional vaccine strain LaSota to comparative test, and is compareed Group sets 4 groups, every group of 12 SPF chickens altogether together, and 1 age in days carries out 10⁶EID₅₀Dosage intramuscular immunity inoculation, be immunized 3 weeks after with Popular genotype VII ZJ1 strain presses 10⁵EID₅₀Dosage carries out attacking poison, measures the HI antibody titer of each group chicken, system respectively weekly Meter each group attacks the case fatality rate of chicken after poison, and it is higher than control group level as a result to prove that strain antibody of the present invention is generated, and has complete Immunoprotection (protective rate 100%).Specific test grouping inoculation is shown in Table 2, table 3 with HI detection case.

2 immunity test scheme of table

3 test group HI antibody titer of table and attack malicious survival rate situation

SEQUENCE LISTING

<110>Yangzhou University

<120>a kind of recombination VIII type newcastle disease virus low virulent strain

<130>

<160> 33

<170> PatentIn version 3.3

<210> 1

<211> 15210

<212> DNA

<213>newcastle disease virus NDV-rHR09-LaSota-HN

<400> 1

accaaacaga gaatctgtga ggtacgataa aaggcgaaga agcaatcgga atcgtacggg 60

tagaaggtgt gaacctcgag tgcgaggccg aagctcaaac tagagggagc cttctaccaa 120

catgtcgtcc gtattcgatg aatacgagca gctcctcgct gctcagactc gtcctaatgg 180

agctcacgga gggggagaga gagggagcac tttaaaagtt gaggtcccag tattcactct 240

taacagtgac gatccagaag atagatggaa ttttgcggta ttctgtcttc ggattgctgt 300

tagcgaggat gccaacaaac cgctcaggca aggtgctctc atatcccttt tatgctccca 360

ttcccaagtg atgaggaacc atgttgccct tgcaggaaaa cagaatgagg ccacactggc 420

tgttcttgaa atcgatggtt ttaccaacag cgtgccccag ttcaacaaca ggagtggagt 480

gtctgaggag agagcacaga gattcatggt aatagcaggg tccctccctc gggcatgcag 540

taacggtact ccattcgtca cggctggggt tgaagatgat gcaccagaag atatcactga 600

tactctggaa agaatcttat ctatccaggc tcaagtatgg gtcacagtag caaaggctat 660

gaccgcgtat gagacagcag atgagtcgga aacaagaaga atcaataagt atatgcagca 720

aggcagggtc cagaagaagt gcatcctcca ccccgtatgt aggagtgcga ttcaactcac 780

aatcagacat tctctggcag tccgtatttt cttagtcagc gaacttaaga ggggccgcaa 840

tacggcaggt gggagctcca catattacaa cttagtaggg gatgtagact catacatcag 900

gaacactggg cttactgcat tcttcctgac acttaaatat ggaattaaca ctaagacatc 960

agccctagca ctcagcagcc tcacaggcga tatccaaaaa atgaagcagc tcatgcgttt 1020

atatcggatg aagggagaaa atgcgccgta catgacattg ctaggtgaca gtgatcagat 1080

gagctttgca ccagctgagt atgcacagct ttactctttt gccatgggca tggcgtcagt 1140

cttggataaa ggaactggta aataccaatt tgccagagat tttatgagca catcattctg 1200

gagactcgga gtggagtatg ctcaggcgca ggggagtagc atcaatgagg acatggctgc 1260

tgagctaaaa ttaaccccgg cagcaaggag gggcctggca gctgctgccc aacgagtgtc 1320

tgaggaagct ggcagcgtgg atattcctac tcaacaagcc agagtcctca ctgggctcag 1380

cgacggaagc ccccgagcct cacaaggcag atcgaacgag ccgcaagggc aaccagacgt 1440

cggagatgga gagacccaat tcttggactt gatgagagca gtgacgaata gcatgcgaga 1500

agcaccaaac tcctcacaga gcaccaccca tccggagcct cccccaactc ctgggccatc 1560

acaagacaac gacactgact gggggtactg atcgatcaca cccagtccac ctccacaggg 1620

ctatcccaaa tcctccgtct gacccccccc cccaccccct gacccacagc cccgcacggc 1680

cgaaccaacg aaagcactcc tccatcctcc ttcccacccc cagccgcacg atccaaccca 1740

cccgagacaa cacgggcaca actcaatcca ccaataatcc atacggagcc aaagacatta 1800

gaaaaaaata cgggtagaag agagacaccc ggagatcaag gccaatcact aaggtctccg 1860

tcctcccttc tacccagtgg attagggcga agatggccac ctttacagat gcagagatag 1920

atgatatatt tgagaccagt ggaactgtca ttgacagcat aattacggcc cagggcaaat 1980

cagcagagac tgtcggaagg agcgcaatcc cacaaggcaa gaccaaagcg ctgagcacag 2040

catgggagaa gcacgggagc atccaaccat ccgccagtca ggacaacccc gaccggcagg 2100

atggaccaga caaacagcca tccacgcctg atcaggcaac cccgcacgac ggctcgccga 2160

tcacatccgc cgaaccgctc tctgcccagg ccgcaggcgc agccggcgat acacagctca 2220

aaactggagc aagcaactct cttttgtcca tgctcgacaa gctgagcaat aagtcatcca 2280

atgctaaaaa gggcccacgg tcgagcccac aggaaggata tcatcaacct ccgacccaac 2340

aacaggggaa tcagctgagc cgcggaagca accaggaaag atcgcagtac caagccaagg 2400

ccgtccctgg aagccggggc acagacgcga acacagcata tcatggacaa cggaaggagt 2460

cacaaccatc agctggtgca acccctcatg tgctccaatc aggacggagc ccagacaata 2520

ctcctgtacc tgtggatcat gtccagccac ctgtcgactt tgtgcaggcg atgatgacta 2580

tgatggaggc gttatcacag aaagtaagta aagttgacta tcagctagac ttagtcttaa 2640

agcagacatc ctccatccct atgatgcggt ctgaaatcca acagcttaag acatctgtcg 2700

cagtaatgga agctaattta ggcatgatga aaattctgga ccctggttgt gctaacattt 2760

catctttaag tgacctgcgg gcagttgccc gatcccaccc ggttttagtt tcaggccccg 2820

gagatccgtc cccctacgtg acacaagggg gtgagatgac actcaataaa ctctcgcaac 2880

caatacaaca cccttctgag ttgattaaat ctgccacggc gggtgggcct gatatgggag 2940

tggagaagga cactgtccgt gcattgatca cctcgcgccc gatgcatcca agttcctcag 3000

ctaagctcct gagtaagctg gatgcagccg ggtcgattga cgagatcaga aagattaaac 3060

gccttgcgct gaatggttga tcatcaccgc aacccacaac aggttcctgc cccctgagtg 3120

ccaaaaagaa tccaccccga gccccccttc cccgacccgt gctccaacac tctaagcaat 3180

agccctctcc cacccctctt gcccccttga atgattgcac aaccacggtt aatctagcag 3240

cttcaaaaat taagaaaaaa tacgggtaga atcgaagtgc cccgactgtg ccaaaatgga 3300

ctcatccagg acaatcgggc tgtattttga ttctgccctc ccctccagca gcctactagc 3360

attcccgatc gtcctacaag acacagggga cgggaagaag caaatcaccc cacaatacag 3420

gatccagcgt cttgactcgt ggacagacag taaagaagat tcagtattca ttaccactta 3480

tggattcatc ttccaagttg ggaatgaaga agtcactgtt ggcatgatca atggtaatcc 3540

caggcacgaa ttactctctt ctgcaatgct ttgcctaggg agtgtcccga acgacagtga 3600

tcttgttgag ctggcgaggg cctgcctcac tatggtgata acatgcaaga agagtgcaac 3660

taatactgag agaatagtct tctcagtagt gcaggcaccc cgtgtgctgc aaagctgtat 3720

ggttgtggca aacaggtact cgtcagtgaa tgcagtgaag catgtgaaag caccagagaa 3780

gatccctggg tgcggaaccc tagagtataa ggtgaacttt gtctccttga ctgtggtgcc 3840

aaaaaaggat gtctacagga tcccagcggc agctttgaaa gtatctggct cgagcctgta 3900

taatcttgca cttaacgtca ctattgatgt ggaggtggac ccgaagagcc cattggtcaa 3960

atccctttct aggtccgata atggatacta tgctaatctt ttcttgcata tcgggcttat 4020

gtccactgta gataagcggg gaaagaaagt gacatttgac aagctagagg agaagataag 4080

gagactcaat ctatctgtcg ggctcagtga tgtgctcgga ccctccgtgc ttgtgaaggc 4140

gagaggtgca cggactaagc tgttggcacc tttcttctct agcagtggga cagcctgcta 4200

tcctatagca aatgcctctc cccaggttgc taaaatactc tggagtcaaa ctgcacatct 4260

gcggagtgtg aaagtcgtca tccaagcagg cacccaacgt gctgtcgcag tgaccgccga 4320

tcatgaggtt acttctacca agatagagaa gaagtatatc attgctaaat acaatccttt 4380

caaaaaatag gttgcatttc tgagactgtg atctgcctgc tttcttgaat catcacaaca 4440

ctatataatg atccgtcttg attgcttaca gttagttcac ctgtttatct agttagaaaa 4500

aacacgggta gaagagtccg gatcctagtt ggcacatcca aggcacaata tgggttccaa 4560

atcttctacc aggttcttaa caccctcgat gttgatcacc cggattatgc tgatactgag 4620

ttgtatctgt ccgacaggct ctttagacgg caggcctctt gcagctgcag ggattgtggt 4680

aacaggggat aaagcagtca acatatacac ctcatctcag acggggtcaa tcatagtcaa 4740

gttgctccca aatatgccca aggataaaga ggcgtgtgca agaactccgt tggaggcata 4800

caacagaaca ctgactactt tactcacccc ccttggtgat tccatccgca ggatacaagg 4860

gtctgtgact acatctggtg gggggagaca ggggcgcctt ataggtgccg ttattggtag 4920

tgtagccctt ggagttgcaa cagctgcaca gataacagca gccgcggctc tgatacaggc 4980

caaccagaat gccgccaaca tcctccggct taaggagagc attgctgcaa ccaatgaagc 5040

tgtgcacgag gtcactgacg gattatcaca actagcagtg gcagttggga agatgcagca 5100

gtttgttaac gaccaattta ataataccgc acgagaattg gactgtataa agattgcgca 5160

gcaggttggt gtagaactca acttgtacct aactgaattg actacagtat tcgggccaca 5220

aatcacttcc cctgccttaa ctcagctgac tgtccaggca ctttataatt tagctggtgg 5280

caacatggat tacttgttga ctaaattagg tgtagggaac aatcagctca gctcattaat 5340

tggtagtggc ttgatcaccg gcaaccctat actatatgac tcacagactc aacttttagg 5400

catacaggta actttaccct cagtcgggaa cctaaataat atgcgtgcca cctacctgga 5460

gaccttatct gtaagcacaa ccaaagggtt tgcctcagca cttgtcccga aggtagtgac 5520

acaagtcggt tccgtgatag aagaacttga cacctcatac tgtatagagt ctgatctgga 5580

tttatactgt acaaggatag tgacattccc catgtctcca ggtatttatt cctgtctgag 5640

tggtaataca tcagcttgca tgtattcaaa gactgaaggt gcactcacca ctccatatat 5700

ggccctcaaa ggctcagtca tcgccaactg caagatgaca acatgtagat gtgcagaccc 5760

tccgggtatc atctcgcaaa actatggaga agctgtatct ctaatagata aacattcatg 5820

caatgtcttg tctttagacg ggataactct gaggctcagt ggggaatttg atgcaactta 5880

tcaaaagaat atctcaatcc tagactctca agtcatcgtg acaggcaatc tcgatatatc 5940

aacagagctt gggaatgtca acaactcaat aagcaatgcc ctggataagt tatcagaaag 6000

taacagcaaa ctagacaaag tcaatgtcaa gctaactagc acatctgctc tcattatcta 6060

tattgtctta attgtcatat ctcttgtttc cggtgtactt agcctggttc taacatgtta 6120

tctgatgtac aaacaaaagg cacagcaaaa gaccttatta tggcttggga ataataccct 6180

cgatcagatg agagccacca caagaacatg aatacggacg agaggtggat atgtccttaa 6240

tagcaactca tgtgtcaatt ctgacagcct gttaattaga aggattaaga aaaaactgtt 6300

ggatataagt gccaaaaggg caatacacgg gtagaacggt cggagaaacc ccctttcaac 6360

cgggaaccag gcctcacaac gtccgttcta ccgcatcact aatagcagac tccagtcatg 6420

gaccgcgccg ttagccaagt tgcgttagag aatgatgaaa gagaggcaaa aaatacatgg 6480

cgcttgatat tccggattgc aatcttattc ttaacagtag tgaccttggc tatatctgta 6540

gcctcccttt tatatagcat gggggctagc acacctagcg atcttgtagg cataccgact 6600

aggatttcca gggcagaaga aaagattaca tctacacttg gttccaatca agatgtagta 6660

gataggatat ataagcaagt ggcccttgag tctccgttgg cattgttaaa aactgagacc 6720

acaattatga acgcaataac atctctctct tatcagatta atggagctgc aaacaacagt 6780

gggtgggggg cacctatcca tgacccagat tatatagggg ggataggcaa agaactcatt 6840

gtagatgatg ctagtgatgt cacatcattc tatccctctg catttcaaga acatctgaat 6900

tttatcccgg cgcctactac aggatcaggt tgcactcgaa taccctcatt tgacatgagt 6960

gctacccatt actgctacac ccataatgta atattgtctg gatgcagaga tcactcacat 7020

tcatatcagt atttagcact tggtgtgctc cggacatctg caacagggag ggtattcttt 7080

tctactctgc gttccatcaa cctggacgac acccaaaatc ggaagtcttg cagtgtgagt 7140

gcaactcccc tgggttgtga tatgctgtgc tcgaaagtca cggagacaga ggaagaagat 7200

tataactcag ctgtccctac gcggatggta catgggaggt tagggttcga cggccagtac 7260

cacgaaaagg acctagatgt cacaacatta ttcggggact gggtggccaa ctacccagga 7320

gtagggggtg gatcttttat tgacagccgc gtatggttct cagtctacgg agggttaaaa 7380

cccaattcac ccagtgacac tgtacaggaa gggaaatatg tgatatacaa gcgatacaat 7440

gacacatgcc cagatgagca agactaccag attcgaatgg ccaagtcttc gtataagcct 7500

ggacggtttg gtgggaaacg catacagcag gctatcttat ctatcaaggt gtcaacatcc 7560

ttaggcgaag acccggtact gactgtaccg cccaacacag tcacactcat gggggccgaa 7620

ggcagaattc tcacagtagg gacatctcat ttcttgtatc aacgagggtc atcatacttc 7680

tctcccgcgt tattatatcc tatgacagtc agcaacaaaa cagccactct tcatagtcct 7740

tatacattca atgccttcac tcggccaggt agtatccctt gccaggcttc agcaagatgc 7800

cccaacccgt gtgttactgg agtctataca gatccatatc ccctaatctt ctatagaaac 7860

cacaccttgc gaggggtatt cgggacaatg cttgatggtg tacaagcaag acttaaccct 7920

gcgtctgcag tattcgatag cacatcccgc agtcgcatta ctcgagtgag ttcaagcagt 7980

accaaagcag catacacaac atcaacttgt tttaaagtgg tcaagactaa taagacctat 8040

tgtctcagca ttgctgaaat atctaatact ctcttcggag aattcagaat cgtcccgtta 8100

ctagttgaga tcctcaaaga tgacggggtt agagaagcca ggtctggcta ggaaattaga 8160

tctggctggc tgagtcaccc atgggaggtt tgagaagacg atattgtacc acctatcttc 8220

tgcaatgcta aggatcgagc cgaataccga tgcatgctcg aatcctacgc tgccggtcag 8280

ctataaccgg ataatgctga cgtgatcagt ctgaatcttg tcgatagtca ctttatttag 8340

aaaaaatatg aaaggtagtg aggtataaga aaaaacaacc caaagaggat aatacgggta 8400

ggacatggcg agctccggtc ccgaaagggc agagcatcag atcatcctac cagagtcaca 8460

tctatcttct ccattggtca agcacaaatt gctctattac tggaaattaa ctgggctacc 8520

acttcctgat gaatgtgact ttgaccatct tattatcagc agacaatgga agaaaatact 8580

tgagtcagcc accccggaca ctgagaggat gataaaactc ggacgagcag tacaccagac 8640

tctcaaccac aacttcaaga taaccggagt actccatccc aggtgtctcg aagaactggc 8700

tagtattgag gtccctgact caaccaacaa atttcggaag atcgaaaaga aaatccagat 8760

ccacaacaca aggtacggag aactgttcac aaggttgtgc acgaacgttg aaagtaaatt 8820

gctagggtca tcttggtcta gcaatgtccc acggtcagag gaatttaaca gcatccgtac 8880

agatccggca ttttggtttc actcaaaatg gtctagagcc aagtttgcat ggctccatat 8940

aaaacaggtc caaaggcatt tgatcgtggc agcaagaacg aggtctgcag tcaacaaatt 9000

ggtgacgctg actcataagg caggccaagt ctttgttact cctgagcttg tcattgtgac 9060

acatacagat gagaacaggt tcacatgcct cacccaggag cttgttttga tgtatgcgga 9120

tatgatggag ggcagggata tggtcaacat aatatcctcc actgcaacac atctcagaag 9180

cttatcagag aaaattgatg atattctacg gttagtagat gctctggcaa aagatttggg 9240

caatcaagtc tatgatgttg tagcactgat ggaagggttc gcatacggtg ccgttcagct 9300

gcttgagccg tcgggtacgt ttgcaggaga tttctttgca ttcaacctac aggagctcaa 9360

agatactcta atcgaacttc tcccaaatga tatcgcagaa ttagtgactc atacaatcgc 9420

tatcatattc tctggcttag agcagaatca agcagctgag atgttatgcc tgcttcgttt 9480

gtggggtcac ccactgcttg agtcccgtat cgcagcaaaa gcagtcagga gccagatgtg 9540

cgcaccaaag atggtagact tcgatatgat cctccaggta ttatccttct ttaagggaac 9600

aatcataaat ggatatagga agaagaactc gggtgtgtgg ccacgtgtca aaatggatac 9660

gatatacggg aaggtcatag ggcagctaca cgctgattcg gcagagattt cacatgatgt 9720

catgttgagg gagtacaaga gtctatctgc acttgaattc gagccatgta tagaatatga 9780

ccctgtcacc aatctaagca tgttcttaaa agacaaagca atcgcacatc cgagggataa 9840

ctggctcgcc tcatttaggc gaaaccttct ctctgaggaa cagaaaagac acataaagga 9900

ggcgacctca actaaccgcc tcctgataga gttcttagaa tcaaacgatt ttgatccgta 9960

taaggagatg gaatatttga ctacccttga gtacctaaga gatgacaatg tggcagtgtc 10020

atactcactc aaagaaaagg aagtgaaagt taatggacga attttcgcta agttaacaaa 10080

gaaattaagg aactgccagg taatggcaga gggaattcta gctgaccaga tcgcaccttt 10140

tttccaggga aatggagtca ttcaagatag catatctttg actaagagta tgttggcgat 10200

gagtcaactg tccttcaaca gcaataagaa acgtatcact gactgcaaag aaagggtttc 10260

ctcaacccgc aatcacgatc caaaaagcaa gaatcgccga agagttgcca cttttatcac 10320

gacggatctg caaaagtatt gtcttaactg gagatatcag acagtcaagc tatttgccca 10380

tgccatcaat cagctgatgg gcctacctca cttcttcgag tggattcatc ttagactaat 10440

ggacactacg atgtttgtag gagatccttt caatcctccg agtgacccta ccgattgtga 10500

tctatcaaga gtcccaaatg atgacatata cattgtcagt gctagggggg gcattgaggg 10560

attatgccag aagctatgga caatgatctc aattgccgca atccaacttg ctgcggcaag 10620

atcgcactgt cgagttgcct gcatggtaca aggtgacaat caagtaatag ctgtaacgag 10680

agaggtaaga tcagatgact ctccggatat ggtgttggca cagttgcatc aagccagtga 10740

taatttcttc aaagagttga ttcacgtcaa tcatctgatt ggccacaacc tgaaagatcg 10800

tgaaaccatc aggtcagaca cattcttcat atacagtaag cgaatattta aagatggagc 10860

aatactcagt caggtcctca aaaactcatc caaattggtg ctaatatcag gtgatctcag 10920

tgaaaacact gtaatgtctt gtgccaacat tgcatccact atagcacggc tgtgtgagaa 10980

cgggcttcct aaggatttct gttattattt aaactaccta atgagttgcg tgcagacata 11040

ctttgattct gagttttcta ttactcacaa ctcacaatca gactccaacc agtcttggat 11100

tgaggatatc tctttcgtac actcatacgt cttaacccct gcccagctag ggggactgag 11160

taaccttcaa tactcaaggc tctacacaag gaacatcggt gacccgggga ctactgcttt 11220

tgcagaggtc aagcgactag aagcagtggg gttgttgagt cctagcatca tgactaacat 11280

tctaactagg ccacctggca atggagattg ggccagtctg tgcaatgatc catactcctt 11340

taattttgag actgtcgcaa gcccaaatat tgtccttaag aaacacacac agaaagtctt 11400

atttgaaact tgctcaaacc ccttattatc cggagtacat acagaggata atgaggcaga 11460

agagaaggca ttggctgaat tcttactcaa tcaggaagtg gttcacccgc gtgtcgcgca 11520

tgctatcatg gaatcaagct ctgtaggtag gagaaaacaa attcaaggac ttgttgacac 11580

aacaaacact gtgatcaaga ttgcgttgac tagaaggccc ctcggtatca agaggctgat 11640

gcggataatc aattattcga gcatgcatgc aatgttattc agagatgatg tcttcttatc 11700

caacaggccc aaccacccct tagtctcttc tagtatgtgc tcgctaacgc tagcagatta 11760

cgcacggaac agaagctggt cacctctgac aggaggcaga aaaatactgg gtgtatctaa 11820

tcccgatacc atagaacttg tcgagggtga gattcttagt gtcagcggag ggtgcacaaa 11880

gtgtgatagc ggagatgagc agttcacttg gtttcatctt ccaagcaata tagagttgac 11940

tgatgacacc agcaagaatc ccccgatgag agtgccatat cttgggtcaa agactcagga 12000

gaggagggcc gcttcgcttg cgaaaatagc ccatatgtca ccacatgtga aggcagcact 12060

aagggcatca tctgtgttaa tctgggctta tggagacaac gaaataaact ggactgctgc 12120

tcttaatatt gcaaggtctc gatgcaacat aagctcagag tacctccgac tattgtcacc 12180

cctgcctaca gctgggaatc tccaacatag actggatgat ggcataaccc agatgacatt 12240

tacccctgcg tctctttaca gggtgtcacc ttacattcac atatctaatg attctcaaag 12300

gctatttacc gaggaaggaa taaaggaggg gaatgtggtt tatcagcaga ttatgctctt 12360

gggcttatca ctaattgaat cacttttccc aatgacaaca actaagacat atgatgaaat 12420

cacattacac ctccacagta aatttagctg ctgtatcagg gaagcacctg ttgcggttcc 12480

tttcgagcta ttagggttgg caccagaatt aaggacagta acctcaaata agttcatgta 12540

tgatcctagc cctgtatcag agagagactt tgcgagactt gacctagcta tcttcaaaag 12600

ttacgagctc aatttagagt catattccac aatggagcta atgaacattc tctcaatatc 12660

tagtgggaag ttgattggcc agtccatggt ttcttacgat gaagatacct ctataaagaa 12720

tgacgctata atagtgtatg acaacacacg aaattggatc agtgaagccc agaattcaga 12780

tgtggtccgc ttattcgagt atgcagcact cgaagtgctt ctcgactgtt cgtatcaact 12840

ctactatctg agagtgaggg gcttaaataa catcgtcctg tacatgagtg atttatacaa 12900

gaatatgcca ggaattctac tctctaatat tgcggcaaca atatctcacc ccatcattca 12960

ttcaaggtta aatgcagtag gtctagtcaa ccatgacggg tcacaccagc ttgcagacac 13020

agatttcatc gaaatgtctg caaaactatt agtctcttgc actcggcgcg tggtttcagg 13080

tttatatgca gggaataagt acgatctatt gttcccgtct gtcttggatg ataacctgag 13140

tgaaaagatg cttcagctaa tttcccgatt atgttgtctg tacacagtgc tctttgcgac 13200

aacaagagaa attcctaaaa taagaggcct atcagcggaa gaaaaatgct cagtactcac 13260

tgagtaccta ctgtcagatg ttgtgaaacc attgcttaag tctgagcaag tgagctctat 13320

catgtctccc aacataatca cgttcccagc caatttatat tacatgtcta ggaagagcct 13380

taatttgatc agggaaagag aggacaggga taccatctta gcattgttgt tccctcagga 13440

accactgctt gagttttgtc cagtgcagga tattggtgct cgagtgaaag atccatttac 13500

tcgacaacct gcagcgttca tacaagagtt agatttgagt gctccagcga ggtatgacgc 13560

atttacactt agtgaggttc actccgagca tacattgcca aattcagagg aagattattt 13620

agtacgatac atgtttagag gaatagggac tgcgtcatct tcttggtata aggcatctca 13680

tcttctttct gtacctgagg tcaggtgtgc aagacatggg aactccctat atttggcgga 13740

gggaagtgga gccattatga gtcttcttga attgcatata ccacacgaga ccatctacta 13800

taatacgctt ttctcgaatg agatgaaccc cccacagcga cacttcggac cgaccccaac 13860

acagtttcta aattcagtag tttataggaa tctacaggca gaagtgccgt gtaaagatgg 13920

atttgtccag gaattccgcc cattgtggag agagaatgca gaagagagtg acctgacctc 13980

agataaagcg gtgggataca tcacatctgc agtgccctac agatctgtat cattactaca 14040

ttgcgacatc gaaattccac cgggatctaa tcaaagctta ctagatcaac tggctaccaa 14100

tttatcgctg attgccatgc attctgtaag ggagggcgga gttgtgatca tcaaagtact 14160

gtatgcaatg ggatattact tccatctact catgaattta ttcactccat gttccacaaa 14220

aggatacatt ctctctaatg gctatgcttg cagaggggat atggagtgtt acctgatatt 14280

tgtcatgggc tatttaggcg ggcctacatt cgtgcacgag gtggtgagga tggcaaaaac 14340

tttagtgcag cggcatggca cacttctgtc taaatcagac gaaattacat tgactaggtt 14400

atttacctca cagcggcatc atgtaacaga catcctatcc agccctttgc cgagactaat 14460

gaaattcttg agagagaaca ttgatgctgc actgattgaa gctgggggac agcccgtccg 14520

tccgttctgt gcagagagtt tagtgagcac actaacagat gtgactcaga tgacccagat 14580

catcgccagc cacattgaca cagtcattcg atccgtaatc tacatggaag ccgagggtga 14640

ccttgctgat acagtgttct tattcactcc ttacaatctc tctacagacg gtaagaagag 14700

aacatcactt aaacagtgca caagacagat cttagaggtc acaatactag gtctcagagt 14760

caaagatctc aataaagtag gtgatgtaat tggcttaata ctcaaaggta tggtttctct 14820

agaggacctc atcccgctga ggacatactt gaagcgtagt acctgcccta aatacttgaa 14880

ggcagtccta ggtattacta agctcaaaga aatgttcaca gacacctctt tattatactt 14940

gactcgtgct caacaaaaat tctatatgaa aaccataggc aatgcagcca agggatatta 15000

cagtaacaat gactcttgaa ggcaatcaca tatcaataga ctatcttctt agctgattgt 15060

actctcatta acctggttat gccatattag aaaaaagttg aattccgacc ctttgaaact 15120

cgtattcgga ttcgaataat tatctcaaaa caggagtgcg cgtagttatc cttgattgca 15180

gccctgtcat tcaccaaatc tttgtttggt 15210

<210> 2

<211> 22

<212> DNA

<213>artificial sequence

<400> 2

catgctcgac aagctgagca at 22

<210> 3

<211> 23

<212> DNA

<213>artificial sequence

<400> 3

gaaaggtgcc aacagcttag tcc 23

<210> 4

<211> 22

<212> DNA

<213>artificial sequence

<400> 4

aagctgttgg cacctttctt ct 22

<210> 5

<211> 23

<212> DNA

<213>artificial sequence

<400> 5

tcaggtgttc ctggtatgca gat 23

<210> 6

<211> 25

<212> DNA

<213>artificial sequence

<400> 6

gctagcgttg cttaacactg aatct 25

<210> 7

<211> 21

<212> DNA

<213>artificial sequence

<400> 7

catctttggt gcgcacatct g 21

<210> 8

<211> 24

<212> DNA

<213>artificial sequence

<400> 8

atgtgcgcac caaagatggt agac 24

<210> 9

<211> 29

<212> DNA

<213>artificial sequence

<400> 9

cgacgcgtcg agtgcaagag actaatagt 29

<210> 10

<211> 39

<212> DNA

<213>artificial sequence

<400> 10

cgtctcgtat agggaccaaa cagagaatct gtgaggtac 39

<210> 11

<211> 29

<212> DNA

<213>artificial sequence

<400> 11

agacgcgtcc gttgtccatg atatgctgt 29

<210> 12

<211> 26

<212> DNA

<213>artificial sequence

<400> 12

tcgacgcgtg gtttcaggtt tatatg 26

<210> 13

<211> 36

<212> DNA

<213>artificial sequence

<400> 13

cgtctctacc caccaaacaa agatttggtg aatgac 36

<210> 14

<211> 32

<212> DNA

<213>artificial sequence

<400> 14

agactagtag gataatacgg gtaggacatg gc 32

<210> 15

<211> 19

<212> DNA

<213>artificial sequence

<400> 15

ttggtgcgca catctggct 19

<210> 16

<211> 24

<212> DNA

<213>artificial sequence

<400> 16

atgtgcgcac caaagatggt agac 24

<210> 17

<211> 29

<212> DNA

<213>artificial sequence

<400> 17

cgacgcgtcg agtgcaagag actaatagt 29

<210> 18

<211> 22

<212> DNA

<213>artificial sequence

<400> 18

cgacgcgtgg tttcaggttt at 22

<210> 19

<211> 31

<212> DNA

<213>artificial sequence

<400> 19

ttaatgagag tacaatcagc taagaagata g 31

<210> 20

<211> 29

<212> DNA

<213>artificial sequence

<400> 20

tatctagaga gggagccttc taccaacat 29

<210> 21

<211> 21

<212> DNA

<213>artificial sequence

<400> 21

gtcagacgga ggatttggga t 21

<210> 22

<211> 29

<212> DNA

<213>artificial sequence

<400> 22

agtctagacc agtggattag ggcgaagat 29

<210> 23

<211> 19

<212> DNA

<213>artificial sequence

<400> 23

gggggcagga acctgttgt 19

<210> 24

<211> 32

<212> DNA

<213>artificial sequence

<400> 24

aaggcgcccc tgtccccccc caccagatgt ag 32

<210> 25

<211> 34

<212> DNA

<213>artificial sequence

<400> 25

gggggacagg ggcgccttat aggtgccgtt attg 34

<210> 26

<211> 36

<212> DNA

<213>artificial sequence

<400> 26

ctaatagcag actccagtca tggaccgcgc cgttag 36

<210> 27

<211> 24

<212> DNA

<213>artificial sequence

<400> 27

ctcagtattt aacaatgcca acgg 24

<210> 28

<211> 22

<212> DNA

<213>artificial sequence

<400> 28

gtgaccttgg ctatatctgt ag 22

<210> 29

<211> 25

<212> DNA

<213>artificial sequence

<400> 29

ctagccagac ctggcttctc taacc 25

<210> 30

<211> 22

<212> DNA

<213>artificial sequence

<400> 30

aagctgttgg cacctttctt ct 22

<210> 31

<211> 24

<212> DNA

<213>artificial sequence

<400> 31

gactggagtc tgctattagt gatg 24

<210> 32

<211> 37

<212> DNA

<213>artificial sequence

<400> 32

gaagccaggt ctggctagga aattagatct ggctggc 37

<210> 33

<211> 21

<212> DNA

<213>artificial sequence

<400> 33

catctttggt gcgcacatct g 21

Claims (2)

1. a kind of VIII type newcastle disease virus low virulent strain of recombination, is newcastle disease virus NDV-rHR09-LaSota-HN, its guarantor Hiding number is CCTCC NO:V201830.

2. the full genome of VIII type newcastle disease virus low virulent strain NDV-rHR09-LaSota-HN of recombination described in claim 1 Group sequence, as shown in SEQ ID No.1.