CN109321535A

CN109321535A - A kind of heat-staple newcastle disease virus attenuated vaccine Candidate Strain

Info

Publication number: CN109321535A
Application number: CN201811244135.7A
Authority: CN
Inventors: 曹永忠; 吴艳涛; 张小荣; 刘倩
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2018-10-24
Filing date: 2018-10-24
Publication date: 2019-02-12

Abstract

The present invention relates to Veterinarian virus Protocols in Molecular Biology and immunological technique, novel (VIII type of gene) the attenuated vaccine Candidate Strain NDV/rAHR09 of one plant of heat-staple newcastle disease virus and its construction method are disclosed.The deposit number of the heat-staple newcastle disease virus attenuated vaccine Candidate Strain is CCTCC NO:V201739.Research shows that; NDV/rAHR09 plants have good thermal stability; 56 DEG C, 60min still keep hemagglutination activity and infection ability; immunity test proof has good immune protective effect to Virulent Newcastle Disease Virus strain; it is inoculated with 1 age in days SPF chicken respectively with common vaccine strain LaSota and heat-resisting V4 strain, detects the antibody titer of chicken, experiment results proved weekly; the antibody titer that chicken is immunized in strain of the present invention is significantly higher than LaSota plants and V4 plants, and it is 100% that right pop velogen strain, which attacks malicious protective rate,.

Description

A kind of heat-staple newcastle disease virus attenuated vaccine Candidate Strain

Technical field

The present invention relates to Veterinarian virus Protocols in Molecular Biology and field of immunology, and in particular to one plant of new gene type, resistance to The newcastle disease virus attenuated vaccine Candidate Strain of heat.

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, Especially unanimously think that the amino acid sequence at F protein cracking site is the main determining factor [Seal of virus virulence BS.Nucleotide 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.FunctIntegr Genomics 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。

Vaccine is needed to carry out the infection and propagation of immunization campaign control NDV in production.Conventional attenuated live vaccines are to temperature-sensitive Sense, needs cold chain to maintain in storage and transportation process.Australian early stage separation identifies some natural heat-resisting low virulent strains, such as V4, I-2 etc., this kind of strain belong to Genotype I on genotypic categorization, once make in the tropic countries such as Africa, Southeast Asia and area For vaccine strain use, reduce the dependence of cold chain, produce good economic and social benefit (Spradbrow P B, Copland J W.Production of thermostable Newcastle disease virus in developing countries.Preventive Veterinary Medicine[J],1996,29:157–159.).In generation China's nineteen ninety, was once The strains such as V4 have been introduced, due to being external patent protection strain, scientific research has been only used for, not can be carried out commercialized development use, Therefore South China Tropical rural area (lacking good cold chain system) lacks the autonomous heat-resisting vaccine strain that can be used.Separately Outside, the popularity of China's newcastle disease is complicated, traditional Genotype I vaccine strain popular strain immunoprotection current to China The effect is unsatisfactory, and there is autonomous property right, heat-resisting novel Newcastle disease attenuated vaccine strain to be of great significance for screening, exploitation.

NDV to heat tolerance it is not high, 50~55 DEG C, 30min can cause it to lose its activity (lose infection host energy Power).The result of study of early stage is shown, in the strain of NDV different virulence type, there is to the higher poison of temperature tolerance Strain, being mainly shown as after 56 DEG C, 30min processing still has the ability of infection host, although or lose infectious, but still have There is HA active.[Lomniczi B.Thermostability of Newcastle disease virus strains of Different virulence.Arch of Virol.1975, (47): 249-255.] typically now think, by 56 DEG C, The still NDV strain with infection ability is heat resistance strain after 30min heat treatment.

Summary of the invention

The object of the present invention is to provide a kind of heat-resisting new gene type (VIII type) newcastle disease virus attenuated vaccine Candidate Strains NDV/rAHR09 and its reverse genetic construct system, the heat resistance having by strain itself, solve attenuated live vaccines storage, It is that torrid areas and remote grass roots (being generally deficient of cold chain system) provide reliably to the dependence of cold chain in transportational process Prevention and control candidate vaccine.

A kind of heat-staple newcastle disease virus attenuated vaccine Candidate Strain provided by the invention, is VIII type of newcastle disease virus gene NDV/rAHR09, its deposit number are CCTCC NO:V201739.

The invention also discloses the heat-staple newcastle disease virus attenuated vaccine Candidate Strain NDV/rAHR09 genome sequences Column, as shown in SEQ ID No.47.

The invention also discloses the bases for containing the heat-staple newcastle disease virus attenuated vaccine Candidate Strain NDV/rAHR09 Because of the overall length transcription vector of group cDNA.

Heat-resisting attenuated vaccine Candidate Strain NDV/rAHR09 in the present invention is from heat-resisting velogen strain NDV/HR09 by reversed Genetic operating system carries out mutation transformation to virus F gene.NDV/HR09 plants are that laboratory is sick from inspection where inventor It separates in material and is obtained through Heat tolerance identification, which still has the energy of hemagglutination activity and infection cell through 56 DEG C, 60min Power, minimum lethal dose chicken embryo median lethal time (MDT) are 57.6h, and 1 age in days chicken intracranial inoculation pathogenic index (ICPI) is 1.8, 6 week old chicken intravenous inoculation pathogenic index (IVPI) are that the systematic evolution tree of 1.56, F genetic fragment building proves that strain belongs to gene VIII type has higher with the heat-resisting velogen strain NDV/AF2240 (No. GenBank: JX012096) that Malaysian early stage separates identification Homology, homology 94.0%.The present invention has initially set up HR09 plants of reverse genetic operating system, and successfully rescue obtains Retain the infection clones of the original biological characteristics of virus.Using the operating system, to virus F gene cracking site base sequence It is mutated, designs 1 pair of primer through Overlap PCR method, the nucleotide at the F gene cracking site of viral HR09 is carried out Mutation, make the amino acid of corresponding encoded by¹¹²R-R-Q-K¹¹⁵-R-F¹¹⁷Sport the amino acid sequence with typical low virulent strain feature Column¹¹²G-R-Q-G¹¹⁵-R-L¹¹⁷, the NDV/rAHR09 strain for saving acquisition remains the heat resistance of former maternal velogen strain, 56 DEG C of processing 60min still has HA activity, but MDT value > 120h, and ICPI value is 0.057, meets the standard of low virulent strain.Through with traditional vaccine LaSota plants of strain, the external heat-resisting strain V4 plants of progress Immunoprotection test introduced, every group 12 1 age in days SPF chicken muscle injections connect Kind 10⁶EID₅₀Dosage, with 10 after 3 weeks⁵EID₅₀Dosage genotype VII ZJ1 strain carries out eye droppings, collunarium attacks poison, the results showed that, exempt from For epidemic disease chicken antibody HI potency with strain group highest of the invention, when 3 week old, averagely reaches 6.0 ± 0.6, be significantly higher than LaSota plants and V4 plants, attacking malicious protective rate is 100%.

The construction method of NDV/rAHR09 of the present invention is as follows:

(1) identification of heat-resisting velogen strain HR09

The Reference strains such as the dozens of NDV separation strains save to this room and V4, LaSota carry out heat-resistance test jointly, with Still have HA activity as screening criteria when 56 DEG C of resistance to heat treatment 30min, obtains heat-resisting strain HR09 and given birth to after plaque purification Object CHARACTERISTICS IDENTIFICATION, chicken embryo average death time (MDT), 1 age in days chicken intracranial inoculation pathogenic index (ICPI), 6 including strain Week old chicken intravenous inoculation index (IVPI) etc..The whole genome sequence for measuring strain, identifies the molecular biological characteristic of strain.

(2) rescue of heat-resisting strain HR09 reverse genetic operating system 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 NDV/rHR09 plants, the Heat-tolerance Determination of rescued virus The results show that 56 DEG C of processing 60min still have hemagglutination activity, it was demonstrated that the NDV/rHR09 plants of heat resistances for remaining parent plant.Measurement NDV/rHR09 plants of MDT value, ICPI value, it was demonstrated that it is still NDV velogen strain.Design two pairs of primers, the side through Overlap PCR NDV/rHR09 strain F protein cracking site the 112nd, 115 and 117 basic amino acid is mutated into related to low virulent strain by method Non-alkaline amino acid, construct full-length cDNA transcription vector prNDV/HR09, by transcription vector prNDV/HR09 and 3 supplementary tables Up to plasmid co-transfection BSR-T7/5 cell, virus N DV/rAHR09 is saved out.Through 56 DEG C, 45min processing, NDV/rAHR09 plants still With coagulation, MDT value > 120h, ICPI value is 0.057.The result shows that NDV/rAHR09 plants have heat resistance, and virulence is It obviously causes weak, can be used as heat-resisting NDV attenuated vaccine candidate's strain.

(3) heat-resisting weak NDV/rAHR09 plants of continuous passage tests of poison and Immunoprotection test

The heat-resisting low virulent strain of acquisition is carried out continuous heat-resisting passage by chicken embryo to test, every 5 generations measurement virus MDT value and Heat resistance, and measure F gene order cracking site attachment sequence fragment, examine viral genetic stability, heat-resisting stability and Toxicity variation.It is passed on by continuous 15 generation chicken embryo, viral MDT value is always more than 120h, and heat resistance does not decline, viral F The base sequence segment mutation that there is no causing protein amino acid sequence to change near gene cracking site, it was demonstrated that virus is lost Transfer performance is stablized.

Strain of the present invention and traditional vaccine strain LaSota, V4 are immunized, 4 are set altogether to comparative test, including control group Group, every group of 12 SPF chickens, 1 age in days carry out 10⁶EID₅₀Dose immunization intramuscular injection inoculation, with popular genotype VII after being immunized 3 weeks ZJ1 strain presses 10⁵EID₅₀Dosage carries out attacking poison, measures the HI antibody titer of each group chicken respectively weekly, and statistics each group attacks chicken after poison Case fatality rate, it is higher than control group level as a result to prove that strain antibody of the present invention is generated, and with complete immunoprotection (protective rate 100%).

The VIII type strain of heat-resisting NDV/rAHR09 gene that the present invention obtains has stable heat resistance, by continuous passage Test proves that chicken embryo continuously passed for 15 generations, and heat resistance is stablized, and F gene sequencing does not find the mutation of virulence enhancing.

With heat-resisting V4 strain, heat labile LaSota plants of tradition common progress immunoprotection comparative test, poison of the invention Strain immunogenicity is good, and test chicken all obtains the protection to strong virus attack, and the level that antibody generates is high.

Detailed description of the invention

Fig. 1 is NDV/HR09 plants of full-length genome primer distributions.

Fig. 2 is RT-PCR segmentation amplification 10 fragment electrophoretic figures of full-length genome.M:200bp；1~10 is sequentially table 1 respectively In 10 pairs of primer amplification segments.

Fig. 3 is the NDV/HR09 phylogenetic tree based on the building of F genetic fragment.

Fig. 4 is the building schematic diagram of HR09 plants of full-length genome cDNA transcription vectors.

Fig. 5 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. 6 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. 7 is GFP expression effect figure after helper plasmid cotransfection.

Fig. 8 is virus infection clones PCR qualification result figure.M:200bp Marker；1,2: 9 amplified fragments of primer.

The heat-resisting vaccine candidate strain of NDV/rAHR09 of the invention was preserved in Chinese Typical Representative culture guarantor on September 20th, 2017 Hiding center, depositary institution address: Wuhan, China university；Classification naming are as follows: VIII type NDV/rAHR09 of newcastle disease virus gene；Preservation Number is 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.)。

Step 1: the identification of heat-resisting strain NDV/HR09

To livestock and poultry pestology key lab of the Ministry of Agriculture of Yangzhou University early stage separation save strain with LaSota plants into Row Evaluation of Heat Tolerance (table 1) still has HA activity as criterion using after 56 DEG C of processing 30min, obtains 1 heat-resisting strain, name It is NDV/HR09 plants.

1.LaSota plants, ZJ1 plants and HR09 plants heat-resisting hemagglutination activities of table

NDV/HR09 plants carry out Identification of Biological Characteristics after plaque purification.

The measurement of minimum lethal dose chicken embryo average death time (MDT), 1 age in days chicken intracranial inoculation pathogenic index (ICPI) are surveyed The method of fixed, 6 week old chicken intravenous inoculation pathogenic index (IVPI) the equal reference literature of measurement carries out.The results show that NDV/HR09 The MDT of strain is 57.6h, ICPI 1.8, IVPI 1.56, meets NDV velogen strain feature.

Devise 10 pairs of primers (Fig. 1, table 2), NDV/HR09 plants of whole genome sequences are expanded, measure and biology letter Cease credit analysis.Amplification is shown in Fig. 2.Sequencing results (GenBank accession number: MF285077) display, the strain genome Overall length is 15192nt, and the F protein cracking site amino acid sequence of derivation is¹¹²RRQKRF¹¹⁷, HN length protein is 571 amino Sour residue meets NDV velogen strain feature；Genome G/C content is 46.27%, with heat-resisting V4 plants, I-2 plants of low virulent strain and heat-resisting AF2240 plants of full-length genomes of velogen strain (GenBank accession number: V4 plants be JX524203, I-2 AY935499, AF2240 plant It is 86.7%, 85.4%, 94.0% for JX012096) nucleotide sequence homology, corresponding 6 structural protein coding genes Nucleotide sequence homology between 83.1%~95.6%, and the amino acid sequence homology of 6 structural proteins between Between 81.6%~98.0% (table 3,4)；It is shown based on chadogram constructed by F full length gene, NDV/HR09 plants belong to gene VIII type strain (Fig. 3).Illustrating HR09 plants, there are different in molecular biological characteristic from the strain delivered.

Table 2. expands NDV/HR09 plants of full-length genome primers

3 NDV/HR09 plants of table and different strain full-length genomes and 6 gene nucleic acid sequence homologies (%)

4 NDV/HR09 plants of table and different strain structural proteins amino acid sequence homologies (%)

Step 2: the building of heat-resisting strain NDV/HR09 reverse genetic operating system

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: according to the complete genome sequence of the NDV/HR09 of aforementioned acquisition, with 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. 4.By genome 13048 Molecular labeling of the bit base as Revive virus.Primer sequence is shown in Table 5, and helper plasmid building primer is shown in Table 6, and primer is by Nanjing The synthesis of Jin Sirui Bioisystech Co., Ltd.

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

Table 6 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-Blunt Cloning 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 pEASY TM-Blunt Cloning Kit (being purchased from Beijing Quanshijin Biotechnology Co., Ltd) explanation The method that book provides, is identified with universal primer M13F and M13R by PCR method.The positive clone of PCR identification send to peace The sequencing of emblem General Corporation.Correctly clone is respectively designated as T-R1, T-R2, T-R3, T-V1, T-V2, T-NP, T-P, T- for sequencing 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. 5).

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. 6).

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, connection pEASYTM-Blunt cloning vector (are purchased from Beijing Quanshijin Biotechnology Co., Ltd). Sequencing result is correctly cloned after I double digestion of Xba I and Not, and digestion products are detected with agarose gel electrophoresis, in ultraviolet light Irradiation under recycle the band of 1500bp or so, the pCI-neo equally through I double digestion of Xba I and Not is connected to T4 ligase In carrier (being purchased from Beijing Quanshijin Biotechnology Co., Ltd), transformed competence colibacillus cell, PCR and digestion identify positive gram It is grand, it 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 HP DNA 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. 7) 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.

The acquisition and identification of 4 virus infection clones

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.

The identification of rescued virus: the measurement of HA: collection connects embryo allantoic liquid, with freshly prepared 1% chicken red blood cell according to OIE Standard carries out 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 The RNA of virus, after configuration reverse transcription system carries out reverse transcription and inactivates, using it as template, with the specific primer 9 of sequencing (ND-9), the part NDV L gene region length about 1892bp (12362nt~14253nt) is expanded, according to the MluI mutated The base of (13048nt) changes, to determine whether to save out virus.The heat resistance of rescued virus, biological nature are referring to above-mentioned Method carries out.

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 sequence and NDV/ HR09 plants of sequences (GenBank accession number: MF285077) are identical；13048th base becomes A (Fig. 8) by G, it was demonstrated that disease The genome of poison is derived from transcription vector NDV/rHR09.

Point carries out 56 DEG C of heat resistant tests between taking rescued virus to be inoculated with the allantoic fluid timesharing being collected into after instar chicken embryo on the 9th~11, It still has hemagglutination activity in 56 DEG C of test 60min as the result is shown, identical as the heat-resisting duration of wild-type virus.

By rescued virus NDV/rHR09 according to OIE standard dilution after, respectively measure MDT and ICPI, the results show that MDT value For 60h, ICPI value is 1.625.(MDT value is 57.6h to the virulence and wild-type virus for showing rescued virus, 1.8) ICPI value is Compared to the apparent variation of not generation.

Step 3: the rescue and identification of F gene mutation and heat-resisting low virulent strain NDV/rAHR09

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.45)

Rr2-F:5’-GGGGGACAGGGGCGCCTTATAGGTGCCGTTATTG-3’(SEQ ID No.46)

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

The recombinant plasmid TR-12 obtained using step 2 is template, and with R1-F (being shown in Table 6)/Rr1-R, Rr2-F/R2-R (is shown in Table It 6) is 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, according to pEASYTM-Blunt Cloning Kit specification by recovery product and pEASY TM-Blunt cloning vector 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 full-length genome cDNA transcription vector pNDV/rAHR09

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 × TransTaq HiFi 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, TransTaqHiFi DNA polymerase (5U/ μL)0.5μL.PCR reaction cycle parameter: 94 DEG C of initial denaturations 5min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 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.

Remaining building process refers to step 2, and final building obtains full-length cDNA overall length transcription vector pNDV/ rAHR09。

4. the rescue and identification of heat-resisting low virulent strain NDV/rAHR09

By transcription vector pNDV/rAHR09 (the genome cDNA full length sequence such as SEQ ID containing genome cDNA overall length Shown in No.47) and three ready BSR-T7/5 cells of helper plasmid (pCI-NP, pCI-P, pCI-L) cotransfection, transfection side The same step 2 of 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 L 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, and (method is same for progress Evaluation of Heat Tolerance and Identification of Biological Characteristics Step 2).And toxic allantoic fluid titre is diluted to 10^-3Afterwards, it is inoculated with CEF cell, observes cytopathy feelings under the microscope after 48h Condition, while maternal strain is set 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 is changed, genome cDNA full length sequence is as shown in SEQ ID No.47, it was demonstrated that viral base Because group is derived from transcript pNDV/rAHR09.

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 Obvious weaken is occurred.

Step 4: NDV/rAHR09 strain Immunoprotection test

48 1 age in days SPF chickens are divided into 4 groups, and every group of 12 raisings are in 3 grades of isolators of bio-safety, by NDV/rAHR09 Strain and conventional vaccine strain LaSota, V4 carry out immune comparative test, control group PBS, and every chicken presses 10⁶EID₅₀/0.1mL The dosage intramuscular immunity of (PBS dilution) is inoculated with, and presses 10 after being immunized 3 weeks with popular genotype VII ZJ1 strain⁶EID₅₀/0.1mL Dosage carries out attacking poison, measures the HI antibody titer of each group chicken respectively weekly, and statistics each group is attacked the case fatality rate of chicken after poison, as a result proved The antibody titer that strain of the present invention generates test chicken is significantly higher than other control strains, and the attack of right pop velogen strain has complete Immune protective effect (protective rate 100%).Specific test grouping is shown in Table 7, table 8 with HI detection, strong protective rate situation.

7 immunity test scheme of table

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

。

SEQUENCE LISTING

<110>Yangzhou University

<120>a kind of heat-staple newcastle disease virus attenuated vaccine Candidate Strain

<130>

<160> 47

<170> PatentIn version 3.3

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accaaacaga gaatctgtga g 21

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ccttcatccg atayaarcgc at 22

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caatacgaca ggtgggagct c 21

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atcataggga tggaggatgt ctg 23

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agggcagagc caaracarta c 21

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cgcrgtttgr ctccagagta t 21

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tcgggctcag tgaygtgctc 20

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atcraattcc ccactgagcc t 21

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tgtcyccwgg yatttattcc tg 22

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gatrgatarg atrgcytgct g 21

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tcggaagtcy tgcagtgtga gt 22

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tgcrgacctt gtyctwgctg 20

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tggtttcact caaaatggtc c 21

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gagtattgct ccrtcyttra a 21

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ggattmtgyc agaagctatg 20

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ataggcgrac cacatctga 19

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cactcttycc aatgacraca ac 22

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taacactcca tatcccctct aca 23

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<212> DNA

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aacttgtcyc tgattgccat g 21

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<212> DNA

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ccaaacaaag atttggtgaa tgac 24

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<212> DNA

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gattacgcca agctttcctc agatactcgt tgggcagcag ct 42

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<212> DNA

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gattacgcca agcttcgaat gagatgaacc ccccacagcg 40

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catgctcgac aagctgagca at 22

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gaaaggtgcc aacagcttag tcc 23

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aagctgttgg cacctttctt ct 22

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tcaggtgttc ctggtatgca gat 23

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gctagcgttg cttaacactg aatct 25

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catctttggt gcgcacatct g 21

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atgtgcgcac caaagatggt agac 24

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cgacgcgtcg agtgcaagag actaatagt 29

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cgtctcgtat agggaccaaa cagagaatct gtgaggtac 39

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agacgcgtcc gttgtccatg atatgctgt 29

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tcgacgcgtg gtttcaggtt tatatg 26

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<212> DNA

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cgtctctacc caccaaacaa agatttggtg aatgac 36

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<212> DNA

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agactagtag gataatacgg gtaggacatg gc 32

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ttggtgcgca catctggct 19

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atgtgcgcac caaagatggt agac 24

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cgacgcgtcg agtgcaagag actaatagt 29

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cgacgcgtgg tttcaggttt at 22

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ttaatgagag tacaatcagc taagaagata g 31

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tatctagaga gggagccttc taccaacat 29

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gtcagacgga ggatttggga t 21

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agtctagacc agtggattag ggcgaagat 29

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gggggcagga acctgttgt 19

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aaggcgcccc tgtccccccc caccagatgt ag 32

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gggggacagg ggcgccttat aggtgccgtt attg 34

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<213>artificial sequence

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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

gaccgcgcag tcaacagagt tgtgctagag aatgaagaaa gagaagcaaa gaatacgtgg 6480

cgcttagttt tccggatcgc agttttattc ttaatagtaa cgactttagc catctctgca 6540

gctgccctga tatatagcat gggggctagc acaccgagca accttgcagg catacctact 6600

gcaatctcta aggcagagga tcggattacg tccttactca gctcaaatca agatgtcgta 6660

gataggatat ataagcaggt ggcccttgag tctccgctag cgttgcttaa cactgaatct 6720

ataattatga atgcaataac gtctctctct tatcaaatta atggggctgc aaataatagt 6780

gggtgtgggg cacctgttca tgatccagat tatatcgggg ggataggcaa agaacttata 6840

gtagacgacg ttagtgacgt cacatcattc tatccatctg cataccagga acacctgaat 6900

tttatcccag cacctactac aggatcaggt tgcacacgga taccctcatt cgacatgagt 6960

gctacccact actgttatac ccacaacgta atattgtctg gttgcagaga tcactcacac 7020

tcatatcagt acttagcact cggcgtgctt cggacatctg caacggggag ggtattcttt 7080

tctactctgc gttccatcaa tttggatgat actcaaaatc ggaagtcctg cagtgtgagt 7140

gccactcctt taggttgtga tatgctatgc tctaaggtca cggagactga ggaagaggat 7200

tataagtcag tcaccccaac gtcaatggta catggaaggt tagggtttga cggtcaatat 7260

catgaggagg atttagacat cacagtctta ttcaaggatt gggtggcaaa ttacccagga 7320

gtgggaggtg ggtccttcat tgacgaccgt gtatggttcc cagtttatgg agggctcgac 7380

cccaactcac ctagcgacac tgcacaagaa gggagatacg taatctacaa gcgctataat 7440

aacacatgcc ccgatgaaca ggattaccag attcgaatgg ctaaatcttc gtataagcct 7500

aggcgattcg gtggaaagcg tgtacagcaa gctatcttgt ctatcaaagt gtcaacatct 7560

ctaggtgagg acccagtgct gactgtaccg cccaatacag ttacactcat gggggccgaa 7620

ggcagagtcc tcaccgtagg gacatctcat ttcctgtacc aacgagggtc ttcatacttc 7680

tctcctgcct tattataccc tatgacagta aacaataaaa cagctactct tcatagtcct 7740

tatacattta atgctttcac ccggccaggt agtgtccctt gtcaggcatc agcaagatgt 7800

cctaactcat gtatcactgg agtctatact gatccgtatc ccttaatctt ccataggaac 7860

cacaccttgc gaggggtgtt cgggacaatg cttgatgatg aacaagcaag actcaacccc 7920

gtatctgcag tatttgataa catatctcgc agtcgcatga ctcgggtaag ttcaagcagc 7980

actaaggcag catacacgac gtcgacatgc tttaaggtcg tcaagaccaa taaagtttat 8040

tgtcttagca ttgccgaaat atccaacacc ttattcgggg aattcagaat tgtcccttta 8100

ctggttgaga ttctcaaaga tgatagggtt taggaaatta gatctggctg gctgagtcac 8160

ccatgggagg tttgagaaga cgatattgta ccacctatct tctgcaatgc taaggatcga 8220

gccgaatacc gatgcatgct cgaatcctac gctgccggtc agctataacc ggataatgct 8280

gacgtgatca gtctgaatct tgtcgatagt cactttattt agaaaaaata tgaaaggtag 8340

tgaggtataa gaaaaaacaa cccaaagagg ataatacggg taggacatgg cgagctccgg 8400

tcccgaaagg gcagagcatc agatcatcct accagagtca catctatctt ctccattggt 8460

caagcacaaa ttgctctatt actggaaatt aactgggcta ccacttcctg atgaatgtga 8520

ctttgaccat cttattatca gcagacaatg gaagaaaata cttgagtcag ccaccccgga 8580

cactgagagg atgataaaac tcggacgagc agtacaccag actctcaacc acaacttcaa 8640

gataaccgga gtactccatc ccaggtgtct cgaagaactg gctagtattg aggtccctga 8700

ctcaaccaac aaatttcgga agatcgaaaa gaaaatccag atccacaaca caaggtacgg 8760

agaactgttc acaaggttgt gcacgaacgt tgaaagtaaa ttgctagggt catcttggtc 8820

tagcaatgtc ccacggtcag aggaatttaa cagcatccgt acagatccgg cattttggtt 8880

tcactcaaaa tggtctagag ccaagtttgc atggctccat ataaaacagg tccaaaggca 8940

tttgatcgtg gcagcaagaa cgaggtctgc agtcaacaaa ttggtgacgc tgactcataa 9000

ggcaggccaa gtctttgtta ctcctgagct tgtcattgtg acacatacag atgagaacag 9060

gttcacatgc ctcacccagg agcttgtttt gatgtatgcg gatatgatgg agggcaggga 9120

tatggtcaac ataatatcct ccactgcaac acatctcaga agcttatcag agaaaattga 9180

tgatattcta cggttagtag atgctctggc aaaagatttg ggcaatcaag tctatgatgt 9240

tgtagcactg atggaagggt tcgcatacgg tgccgttcag ctgcttgagc cgtcgggtac 9300

gtttgcagga gatttctttg cattcaacct acaggagctc aaagatactc taatcgaact 9360

tctcccaaat gatatcgcag aattagtgac tcatacaatc gctatcatat tctctggctt 9420

agagcagaat caagcagctg agatgttatg cctgcttcgt ttgtggggtc acccactgct 9480

tgagtcccgt atcgcagcaa aagcagtcag gagccagatg tgcgcaccaa agatggtaga 9540

cttcgatatg atcctccagg tattatcctt ctttaaggga acaatcataa atggatatag 9600

gaagaagaac tcgggtgtgt ggccacgtgt caaaatggat acgatatacg ggaaggtcat 9660

agggcagcta cacgctgatt cggcagagat ttcacatgat gtcatgttga gggagtacaa 9720

gagtctatct gcacttgaat tcgagccatg tatagaatat gaccctgtca ccaatctaag 9780

catgttctta aaagacaaag caatcgcaca tccgagggat aactggctcg cctcatttag 9840

gcgaaacctt ctctctgagg aacagaaaag acacataaag gaggcgacct caactaaccg 9900

cctcctgata gagttcttag aatcaaacga ttttgatccg tataaggaga tggaatattt 9960

gactaccctt gagtacctaa gagatgacaa tgtggcagtg tcatactcac tcaaagaaaa 10020

ggaagtgaaa gttaatggac gaattttcgc taagttaaca aagaaattaa ggaactgcca 10080

ggtaatggca gagggaattc tagctgacca gatcgcacct tttttccagg gaaatggagt 10140

cattcaagat agcatatctt tgactaagag tatgttggcg atgagtcaac tgtccttcaa 10200

cagcaataag aaacgtatca ctgactgcaa agaaagggtt tcctcaaccc gcaatcacga 10260

tccaaaaagc aagaatcgcc gaagagttgc cacttttatc acgacggatc tgcaaaagta 10320

ttgtcttaac tggagatatc agacagtcaa gctatttgcc catgccatca atcagctgat 10380

gggcctacct cacttcttcg agtggattca tcttagacta atggacacta cgatgtttgt 10440

aggagatcct ttcaatcctc cgagtgaccc taccgattgt gatctatcaa gagtcccaaa 10500

tgatgacata tacattgtca gtgctagggg gggcattgag ggattatgcc agaagctatg 10560

gacaatgatc tcaattgccg caatccaact tgctgcggca agatcgcact gtcgagttgc 10620

ctgcatggta caaggtgaca atcaagtaat agctgtaacg agagaggtaa gatcagatga 10680

ctctccggat atggtgttgg cacagttgca tcaagccagt gataatttct tcaaagagtt 10740

gattcacgtc aatcatctga ttggccacaa cctgaaagat cgtgaaacca tcaggtcaga 10800

cacattcttc atatacagta agcgaatatt taaagatgga gcaatactca gtcaggtcct 10860

caaaaactca tccaaattgg tgctaatatc aggtgatctc agtgaaaaca ctgtaatgtc 10920

ttgtgccaac attgcatcca ctatagcacg gctgtgtgag aacgggcttc ctaaggattt 10980

ctgttattat ttaaactacc taatgagttg cgtgcagaca tactttgatt ctgagttttc 11040

tattactcac aactcacaat cagactccaa ccagtcttgg attgaggata tctctttcgt 11100

acactcatac gtcttaaccc ctgcccagct agggggactg agtaaccttc aatactcaag 11160

gctctacaca aggaacatcg gtgacccggg gactactgct tttgcagagg tcaagcgact 11220

agaagcagtg gggttgttga gtcctagcat catgactaac attctaacta ggccacctgg 11280

caatggagat tgggccagtc tgtgcaatga tccatactcc tttaattttg agactgtcgc 11340

aagcccaaat attgtcctta agaaacacac acagaaagtc ttatttgaaa cttgctcaaa 11400

ccccttatta tccggagtac atacagagga taatgaggca gaagagaagg cattggctga 11460

attcttactc aatcaggaag tggttcaccc gcgtgtcgcg catgctatca tggaatcaag 11520

ctctgtaggt aggagaaaac aaattcaagg acttgttgac acaacaaaca ctgtgatcaa 11580

gattgcgttg actagaaggc ccctcggtat caagaggctg atgcggataa tcaattattc 11640

gagcatgcat gcaatgttat tcagagatga tgtcttctta tccaacaggc ccaaccaccc 11700

cttagtctct tctagtatgt gctcgctaac gctagcagat tacgcacgga acagaagctg 11760

gtcacctctg acaggaggca gaaaaatact gggtgtatct aatcccgata ccatagaact 11820

tgtcgagggt gagattctta gtgtcagcgg agggtgcaca aagtgtgata gcggagatga 11880

gcagttcact tggtttcatc ttccaagcaa tatagagttg actgatgaca ccagcaagaa 11940

tcccccgatg agagtgccat atcttgggtc aaagactcag gagaggaggg ccgcttcgct 12000

tgcgaaaata gcccatatgt caccacatgt gaaggcagca ctaagggcat catctgtgtt 12060

aatctgggct tatggagaca acgaaataaa ctggactgct gctcttaata ttgcaaggtc 12120

tcgatgcaac ataagctcag agtacctccg actattgtca cccctgccta cagctgggaa 12180

tctccaacat agactggatg atggcataac ccagatgaca tttacccctg cgtctcttta 12240

cagggtgtca ccttacattc acatatctaa tgattctcaa aggctattta ccgaggaagg 12300

aataaaggag gggaatgtgg tttatcagca gattatgctc ttgggcttat cactaattga 12360

atcacttttc ccaatgacaa caactaagac atatgatgaa atcacattac acctccacag 12420

taaatttagc tgctgtatca gggaagcacc tgttgcggtt cctttcgagc tattagggtt 12480

ggcaccagaa ttaaggacag taacctcaaa taagttcatg tatgatccta gccctgtatc 12540

agagagagac tttgcgagac ttgacctagc tatcttcaaa agttacgagc tcaatttaga 12600

gtcatattcc acaatggagc taatgaacat tctctcaata tctagtggga agttgattgg 12660

ccagtccatg gtttcttacg atgaagatac ctctataaag aatgacgcta taatagtgta 12720

tgacaacaca cgaaattgga tcagtgaagc ccagaattca gatgtggtcc gcttattcga 12780

gtatgcagca ctcgaagtgc ttctcgactg ttcgtatcaa ctctactatc tgagagtgag 12840

gggcttaaat aacatcgtcc tgtacatgag tgatttatac aagaatatgc caggaattct 12900

actctctaat attgcggcaa caatatctca ccccatcatt cattcaaggt taaatgcagt 12960

aggtctagtc aaccatgacg ggtcacacca gcttgcagac acagatttca tcgaaatgtc 13020

tgcaaaacta ttagtctctt gcactcgacg cgtggtttca ggtttatatg cagggaataa 13080

gtacgatcta ttgttcccgt ctgtcttgga tgataacctg agtgaaaaga tgcttcagct 13140

aatttcccga ttatgttgtc tgtacacagt gctctttgcg acaacaagag aaattcctaa 13200

aataagaggc ctatcagcgg aagaaaaatg ctcagtactc actgagtacc tactgtcaga 13260

tgttgtgaaa ccattgctta agtctgagca agtgagctct atcatgtctc ccaacataat 13320

cacgttccca gccaatttat attacatgtc taggaagagc cttaatttga tcagggaaag 13380

agaggacagg gataccatct tagcattgtt gttccctcag gaaccactgc ttgagttttg 13440

tccagtgcag gatattggtg ctcgagtgaa agatccattt actcgacaac ctgcagcgtt 13500

catacaagag ttagatttga gtgctccagc gaggtatgac gcatttacac ttagtgaggt 13560

tcactccgag catacattgc caaattcaga ggaagattat ttagtacgat acatgtttag 13620

aggaataggg actgcgtcat cttcttggta taaggcatct catcttcttt ctgtacctga 13680

ggtcaggtgt gcaagacatg ggaactccct atatttggcg gagggaagtg gagccattat 13740

gagtcttctt gaattgcata taccacacga gaccatctac tataatacgc ttttctcgaa 13800

tgagatgaac cccccacagc gacacttcgg accgacccca acacagtttc taaattcagt 13860

agtttatagg aatctacagg cagaagtgcc gtgtaaagat ggatttgtcc aggaattccg 13920

cccattgtgg agagagaatg cagaagagag tgacctgacc tcagataaag cggtgggata 13980

catcacatct gcagtgccct acagatctgt atcattacta cattgcgaca tcgaaattcc 14040

accgggatct aatcaaagct tactagatca actggctacc aatttatcgc tgattgccat 14100

gcattctgta agggagggcg gagttgtgat catcaaagta ctgtatgcaa tgggatatta 14160

cttccatcta ctcatgaatt tattcactcc atgttccaca aaaggataca ttctctctaa 14220

tggctatgct tgcagagggg atatggagtg ttacctgata tttgtcatgg gctatttagg 14280

cgggcctaca ttcgtgcacg aggtggtgag gatggcaaaa actttagtgc agcggcatgg 14340

cacacttctg tctaaatcag acgaaattac attgactagg ttatttacct cacagcggca 14400

tcatgtaaca gacatcctat ccagcccttt gccgagacta atgaaattct tgagagagaa 14460

cattgatgct gcactgattg aagctggggg acagcccgtc cgtccgttct gtgcagagag 14520

tttagtgagc acactaacag atgtgactca gatgacccag atcatcgcca gccacattga 14580

cacagtcatt cgatccgtaa tctacatgga agccgagggt gaccttgctg atacagtgtt 14640

cttattcact ccttacaatc tctctacaga cggtaagaag agaacatcac ttaaacagtg 14700

cacaagacag atcttagagg tcacaatact aggtctcaga gtcaaagatc tcaataaagt 14760

aggtgatgta attggcttaa tactcaaagg tatggtttct ctagaggacc tcatcccgct 14820

gaggacatac ttgaagcgta gtacctgccc taaatacttg aaggcagtcc taggtattac 14880

taagctcaaa gaaatgttca cagacacctc tttattatac ttgactcgtg ctcaacaaaa 14940

attctatatg aaaaccatag gcaatgcagc caagggatat tacagtaaca atgactcttg 15000

aaggcaatca catatcaata gactatcttc ttagctgatt gtactctcat taacctggtt 15060

atgccatatt agaaaaaagt tgaattccga ccctttgaaa ctcgtattcg gattcgaata 15120

attatctcaa aacaggagtg cgcgtagtta tccttgattg cagccctgtc attcaccaaa 15180

tctttgtttg gt 15192

Claims

1. a kind of heat-staple newcastle disease virus attenuated vaccine Candidate Strain is VIII type NDV/rAHR09 of newcastle disease virus gene (Newcastle DiseaseVirus, NDV/rAHR09), its deposit number are CCTCC NO:V201739.

2. heat-staple newcastle disease virus attenuated vaccine Candidate Strain NDV/rAHR09 genome sequence, such as SEQ described in claim 1 Shown in ID No.47.

3. the genome cDNA containing heat-staple newcastle disease virus attenuated vaccine Candidate Strain NDV/rAHR09 described in claim 1 Overall length transcription vector.