CN107287168A - A kind of NDV saves method and its application - Google Patents

A kind of NDV saves method and its application Download PDF

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Publication number
CN107287168A
CN107287168A CN201610200554.5A CN201610200554A CN107287168A CN 107287168 A CN107287168 A CN 107287168A CN 201610200554 A CN201610200554 A CN 201610200554A CN 107287168 A CN107287168 A CN 107287168A
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ndv
culture
low virulent
gene
cdna
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CN107287168B (en
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田克恭
程艺
孙进忠
张许科
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LUOYANG HUIZHONG BIOTECH Co.,Ltd.
Pulaike Biological Engineering Co Ltd
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Pulaike Biological Engineering Co Ltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18161—Methods of inactivation or attenuation
    • C12N2760/18162—Methods of inactivation or attenuation by genetic engineering

Abstract

Method is saved the present invention relates to a kind of NDV, this method introduces hammerhead ribozyme DNA sequence dna at the cDNA of newcastle disease virus gene group 5 ' ends, 3 ' ends introduce fourth liver ribozymal DNA sequence, it is effectively guaranteed 5 ' and holds accurate with 3 ' ends, simultaneously using the CMV promoter on transcription vector pCMVTNT plasmids, transcriptional efficiency and rescue efficiency are improved, NDV has been saved on the cells of BHK 21.The invention further relates to the gene VII NDV low virulent strains N7a saved out using this method.The strain has high titre, heritability stable in chicken embryo, is suitable for the large-scale production of vaccine, available for manufacture vaccine.

Description

A kind of NDV saves method and its application
Technical field
The present invention relates to reverse genetics manipulation technology is applied, produce a kind of NDV rescue method, The new castle disease virus weakening strain prepared using this method, and vaccine combination prepared by the weakening strain, Belong to biological technical field.
Background technology
Ewcastle disease (Newcastle disease, ND) is the blood in 9 kinds of serotypes of avian paramyxovirus A variety of birds such as chicken and turkey caused by clear I type avian paramyxovirus (i.e. NDV, NDV) Acute highly contagious disease, is in often septicemia symptom.Ewcastle disease is that a serious height is passed Metachromia viral disease, because it in global many distribution of countries extensively and is likely to result in great warp Ji loss, is a chief threat of poultry industry.
In recent years, newcastle disease vaccine is worldwide widely used, but in vaccinated flock Virulent Newcastle Disease Virus infection is still common, meanwhile, clinically NDV and other respiratory pathogenses The pathogenic phenomenon of body collaboration is also relatively conventional, and therefore, ewcastle disease is still the vast cultivation of puzzle One of the important epidemic disease at family.It is clinically separated from the point of view of the epidemic characteristic of ewcastle disease nearly ten years To most of Virulent Newcastle Disease Virus belong to genotype Ⅶ, and the genotype of conventional newcastle disease vaccine strain Predominantly gene II type (such as LaSota, clone 30, V4), ewcastle disease epidemic strain and routine Vaccine strain is differed farther out on genetic distance, therefore, and conventional vaccine is malicious by force to current NDV The attack of strain can not provide comprehensive immune protection effectiveness.
During new newcastle disease vaccine is prepared, method often is saved to new city using NDV The progress cause of epidemic disease strain is weak, therefore, and the efficiency of NDV rescue method is that strain causes weak success Key.
Therefore, new epidemic strain is carried out if efficient NDV can be used to save method Cause is weak, develops and clinical popular strain the matches, vaccine combination that immunogenicity is good, The prevalence of China's ewcastle disease can effectively be prevented.
The content of the invention
Method is saved the present invention relates to a kind of NDV, wherein, methods described includes：Step (1), newcastle disease virus gene group RNA described in reverse transcription, prepares the newcastle disease virus gene The cDNA of group；Step (2), uses the NDV obtained in the step (1) The cDNA of genome, builds seven cDNA clone fragments of covering whole gene group, using each The restriction enzyme site of the intersegmental lap of piece, acquisition is assembled in transcription vector pCMVTNT plasmids Whole cDNA clone, hammerhead ribozyme DNA sequences are introduced at 5 ' ends of full-length cDNA fragment Row, 3 ' ends introduce fourth liver ribozymal DNA sequence, obtain pCMV-FL plasmids；Step (3), The helper plasmid of three clone's NP, P, L albumen is built respectively；And step (4), will be described Obtained in the cDNA total length transcription vector plasmids and the step (3) that are built in step (2) Three helper plasmid cotransfection BHK-21 cells, save out newcastle disease virus strain.
The invention further relates to a kind of cause weak method of NDV, by institute in the rescue method The F gene cracking sites for stating NDV sport low virulent strain feature112G-R-Q-G-R-L117。
The invention further relates to a kind of genotype VII NDV low virulent strain, the gene VII is new Epidemic disease malicious low virulent strain in city is NDV (genotype VII), N7a plants of (Newcastle Disease Virus (genotype VII), strain N7a), China typical culture collection center is preserved in, is protected Tibetan address is Wuhan, China Wuhan University, deposit number：CCTCC NO：V201545, preservation Time is on October 19th, 2015.Vaccine prepared by the gene VII NDVs low virulent strain Composition can provide effective immunoprotection with the strain of pop genotype Ⅶ newcastle disease virus, show Good immunogenicity.
The invention further relates to a kind of vaccine combination, wherein, the vaccine combination contains immune The described genotype VII NDV low virulent strain N7a of amount or the antigen and medicine of its culture Carrier on.
The present invention has following good effect：
The Reverse Genetics that the present invention is used introduce hammerhead shape at NDV cDNA 5 ' ends Ribozymal DNA sequence, the sequence causes the RNA transcribed out to have accurate 5 ' end, and promotes Transcription.The sequence does not reduce duplication multiplication capacity of the virus on cell simultaneously, it is ensured that high Save efficiency.The inventive method cytomegalovirus promoter (CMV promoter) control under, Dependenc RNA polymerase II, the carrier for expression of eukaryon of CMV promoter is carried with itself PCMVTNT carries out total length structure as skeleton, under the driving of rna plymerase ii, bears Chain virus full length cRNA, which can stablize, to be expressed, and virus rescue efficiency is effectively improved.
The present invention has successfully saved out the weak poison of gene VII NDVs using Reverse Genetics Strain.The strain has high titre, heritability stable in chicken embryo, is suitable for the extensive of vaccine Production, available for manufacture vaccine.The gene of the strain and the advantage epidemic strain of China's NDV Type is consistent, and wide application prospect is shown in terms of the morbidity of current ewcastle disease and prevalence is controlled.
Brief description of the drawings
Fig. 1 is the structure schematic diagram of the plasmid pCMV-FL comprising full-length cDNA, Tu1AWei The structure schematic diagram of the expression plasmid of 1,2,3 fragments comprising NDV cDNA, Figure 1B For 4 comprising NDV cDNA, the structure schematic diagram of the expression plasmid of 5,6,7 fragments, Fig. 1 C are the structure schematic diagram of the expression plasmid comprising NDV full-length cDNA；
Fig. 2 is that F genes cracking site is mutated schematic diagram；
Fig. 3 is the plasmid pCMV-FL-N7a of full-length cDNA digestion qualification figure, and swimming lane 1 is Electrophoretogram after XhoI digestions, swimming lane 2 is the electrophoretogram after BglII digestions, and swimming lane M is Marker DL15000；
Fig. 4 is gene VII new castle disease virus weakening strains N7a RT-PCR qualification result figures.
Embodiment
Hereinafter, embodiments of the present invention are illustrated.
Method is saved the present invention relates to a kind of NDV, wherein, methods described includes：Step (1), newcastle disease virus gene group RNA described in reverse transcription, prepares the newcastle disease virus gene The cDNA of group；Step (2), uses the NDV obtained in the step (1) The cDNA of genome, builds seven cDNA clone fragments of covering whole gene group, using each The restriction enzyme site of the intersegmental lap of piece, acquisition is assembled in transcription vector pCMVTNT plasmids Whole cDNA clone, hammerhead ribozyme DNA sequences are introduced at 5 ' ends of full-length cDNA fragment Row, 3 ' ends introduce fourth liver ribozymal DNA sequence, obtain pCMV-FL plasmids；Step (3), The helper plasmid of three clone's NP, P, L albumen is built respectively；And step (4), will be described Obtained in the cDNA total length transcription vector plasmids and the step (3) that are built in step (2) Three helper plasmid cotransfection BHK-21 cells, save out newcastle disease virus strain.
As one embodiment of the present invention, ewcastle disease described in the rescue method and step (1) Virus is gene VII newcastle disease virus strains；Helper plasmid described in the rescue method and step (3) For pCI-neo plasmids.
As one embodiment of the present invention, the step (2) includes：
According to point of restriction enzyme site in viral genome sequencing result and transcription vector pCMVTNT Analysis, is divided into 7 fragments, amplicon virus full-length genome, primer sequence is as follows by genome：
PV1-F：5’-CCGGAGCTCAATCGAATCGTACGGGTAG-3’
PV1-R：5’-CGGATCAATTCAGAAGGGTGCT-3’
PV2-F：5’-CCGGAGCTCATCTCCTTACGTGACACA-3’
PV2-R：5’-CGGGGTACCGTGTAGTTGCACTTCTT-3’
PV3-F：5’-CCGGAGCTCAATGCTCTGCTTAGGAGTG-3’
PV3-R：5’-CGGAGATCACAGTCGATAT-3’
PV4-F：5’-CCGGAGCTCATGGTAATGATCTACT-3’
PV4-R：5’-CGGGGTACCACTCTATCATCCTTGA-3’
PV5-F：5’-TGCGAGCTCATTGTCTTAGTATCCA-3’
PV5-R：5’-CGGGGTACCTCATTAGAGTTCAAAT-3’
PV6-F：5’-TGCTCTTGAATGCATCCACTTAGCA-3’
PV6-R：5’-CGGGGTACCAAGCAGATCGTACTT-3’
PV7-F：5’-CCGGAGCTCATAGACTTCTCGAGGT-3’
PV7-R：5 '-TGCAATTGGCGTACGATTGCCT-3 ', by by the 7 of amplification Individual cDNA fragments are assembled in transcription vector pCMVTNT plasmids and obtained comprising complete The pCMV-FL plasmids of cDNA clone；
The step (3) includes：According to carrier for expression of eukaryon pCI-neo and NP, P and L The restriction enzyme site analysis of gene, five pairs of primers of design expand three genes, and L genes are divided into three pieces Section amplification, wherein GCCACC kozak sequences are added to after each sense primer restriction enzyme site. Primer sequence is as follows：
NP-F：5’-TATTGAATTCGCCACCATGTCGTCTGTTTTCGACGA AT-3’
NP-R：5’-TATAGTCGACTCAGTACCCCCAGTCAGTGTCGTT-3’
P-F：5’-TATTGAATTCGCCACCATGGCTACTTTTACAGATGCG GA-3’
P-R：5’-TATAGTCGACTCAACCATTCAGCGCAAGGCGT-3’
L1-F：5’-TATTGCTAGCGCCACCATGGCGGGCTCCGGTCC-3’
L1-R：5’-TATAGTCGACCAGGATTGGTTGGAATCTGACTG-3’
L2-F：5’-AGATGGAACAATACTCAGTCAGGTC-3’
L2-R：5’-TATAGTCGACTCTTCTCACTCAGGTTA-3’
L3-F：5’-TCATTCACTCAAGGTTGAATGCAGT-3’
L3-R：5 '-TATAGTCGACTTAAGAGTCATTATTACTG-3 ', by inciting somebody to action NP, P and L gene cDNA clone of amplification to pCI-neo plasmids obtain pCI-NP, pCI-P, PCI-L plasmids.
As one embodiment of the present invention, in the step (2), Overlap PCR are utilized Method, low virulent strain feature is sported by the F gene cracking sites of the NDV112G-R-Q-G-R-L117,
Mutant primer sequence is as follows：
PV2-F：5’-CCGGAGCTCATCTCCTTACGTGACACA-3’
P1：5’-AAGGCGTCCTTGTCTCCCTCCTCCGGACGTGGACACA -3’
P2：5’-GAGGGAGACAAGGACGCCTTATAGGTGCTGTTATT-3’
PV3-R：5 '-CGGGGTACCAGATCACAGTCGATAT-3 ',
And by the way that 6 cDNA fragments of the amplification and the mutation F genes that include are cracked The fragment in site is assembled in transcription vector pCMVTNT plasmids and obtained comprising global cDNA The pCMV-FL-N7a plasmids of clone；
In the step (4), by plasmid pCMV-FL-N7a and three helper plasmid pCI-NP, PCI-P, pCI-L cotransfection BHK-21 cells, add final concentration of 1 μ g/ml in 6 hours after transfection TPCK-trypsin, harvest nutrient solution and cell after 3~4 days, centrifuged after multigelation 3 times, Supernatant is inoculated with 10 age in days SPF chicken embryos, obtains gene VII new castle disease virus weakening strains N7a.
It is used as one embodiment of the present invention, gene VII new castle disease virus weakening strains of the invention Preparation method includes step in detail below：
1st, the sequencing of NDV PLK-N-06 plants of full-length genomes：
With reference to the NDV sequences (JN631747.1) being had been filed in GenBank, 14 pairs of design is drawn Part is overlapped between thing, amplified fragments, and full-length genome can be covered by connecting, primer sequence Row are as follows：
P1-1：5’-ACCAAACAGAGAATCCGT-3’
P1-2：5’-CCGTTGGGCAGCAGCTGCCA-3’
P2-1：5’-AGCTCATGCGTTTATATCGGAT-3’
P2-2：5’-TTAGCATTGGACGATTTATT-3’
P3-1：5’-AGACCAGTGGGACTGTCATT-3’
P3-2：5’-GCACTTTGATTCTACCCGTAT-3’
P4-1：5’-ATTGAAGAGATCAGGAAGAT-3’
P4-2：5’-CAGGCTGTCCCACTGCTAGAG-3’
P5-1：5’-AATCTTGCGCTCAATGTCACT-3’
P5-2：5’-CCTCTAATGGGGCTTTCGC-3’
P6-1：5’-AATTGTAGTAACAGGAGA-3’
P6-2：5’-TGAGAATCTAGTATTGAG-3’
P7-1：5’-GAAGCTGTATCCCTGATAG-3’
P7-2：5’-TGGTGTCTAAGTCCTTCTCAT-3’
P8-1：5’-TCACAGGGACTGAAGAGGA-3’
P8-2：5’-TCATCAGGAAGCGGTAGCCCA-3’
P9-1：5’-TGACATGATTATTCTGAGT-3’
P9-2：5’-ACATCTCAGCTGCTTGATT-3’
P10-1：5’-TCAGGTACATTTGCAGGA-3’
P10-2：5’-AGCAAGTTGGATTGCAGCA-3’
P11-1：5’-TGTCTTAACTGGAGATATCA-3’
P11-2：5’-TTGCTTGGAAGATGGAACCA-3’
P12-1：5’-AGTCTCTTCTAATATGTGTT-3’
P12-2：5’-TCTCTTGTTGTAGCAAAGA-3’
P13-1：5’-ACGGATCACACCAACTTGCA-3’
P13-2：5’-GTGCACTGTTTAAGTGATGT-3’
P14-1：5’-GAGTGTTACCTGGTATTTGT-3’
P14-2：5’-CCAAACAAAGATTTGGTGAA-3’
2nd, NDV PLK-N-06 plants of full-length infectious clone pCMV-FL structure：
According to point of restriction enzyme site in viral genome sequencing result and transcription vector pCMVTNT Analysis, is divided into 7 fragments, amplicon virus full-length genome, primer sequence is as follows by genome：
PV1-F：5’-CCGGAGCTCAATCGAATCGTACGGGTAG-3’
PV1-R：5’-CGGATCAATTCAGAAGGGTGCT-3’
PV2-F：5’-CCGGAGCTCATCTCCTTACGTGACACA-3’
PV2-R：5’-CGGGGTACCGTGTAGTTGCACTTCTT-3’
PV3-F：5’-CCGGAGCTCAATGCTCTGCTTAGGAGTG-3’
PV3-R：5’-CGGAGATCACAGTCGATAT-3’
PV4-F：5’-CCGGAGCTCATGGTAATGATCTACT-3’
PV4-R：5’-CGGGGTACCACTCTATCATCCTTGA-3’
PV5-F：5’-TGCGAGCTCATTGTCTTAGTATCCA-3’
PV5-R：5’-CGGGGTACCTCATTAGAGTTCAAAT-3’
PV6-F：5’-TGCTCTTGAATGCATCCACTTAGCA-3’
PV6-R：5’-CGGGGTACCAAGCAGATCGTACTT-3’
PV7-F：5’-CCGGAGCTCATAGACTTCTCGAGGT-3’
PV7-R：5’-TGCAATTGGCGTACGATTGCCT-3’
3rd, the structure of three helper plasmids of NDV PLK-N-06 strains
According to the restriction enzyme site analysis to carrier for expression of eukaryon pCI-neo and NP, P and L genes, Five pairs of primers of design expand three genes, and L genes are divided into three fragment amplifications, wherein by kozak Sequence (GCCACC) is added to after each sense primer restriction enzyme site.Primer sequence is as follows：
NP-F：5’-TATTGAATTCGCCACCATGTCGTCTGTTTTCGACG AAT-3’
NP-R：5’-TATAGTCGACTCAGTACCCCCAGTCAGTGTCGTT-3’
P-F：5’-TATTGAATTCGCCACCATGGCTACTTTTACAGATGCG GA-3’
P-R：5’-TATAGTCGACTCAACCATTCAGCGCAAGGCGT-3’
L1-F：5’-TATTGCTAGCGCCACCATGGCGGGCTCCGGTCC-3’
L1-R：5’-TATAGTCGACCAGGATTGGTTGGAATCTGACTG-3’
L2-F：5’-AGATGGAACAATACTCAGTCAGGTC-3’
L2-R：5’-TATAGTCGACTCTTCTCACTCAGGTTA-3’
L3-F：5’-TCATTCACTCAAGGTTGAATGCAGT-3’
L3-R：5’-TATAGTCGACTTAAGAGTCATTATTACTG-3’
4th, new castle disease virus weakening strain N7a rescue
Using Overlap PCR methods, NDV PLK-N-06 F genes are cracked into position Point mutation is low virulent strain feature112G-R-Q-G-R-L117, mutant primer sequence is as follows：
PV2-F：5’-CCGGAGCTCATCTCCTTACGTGACACA-3’
P1：5’-AAGGCGTCCTTGTCTCCCTCCTCCGGACGTGGACACA -3’
P2：5’-GAGGGAGACAAGGACGCCTTATAGGTGCTGTTATT-3’
PV3-R：5’-CGGGGTACCAGATCACAGTCGATAT-3’
By plasmid pCMV-FL-N7a and three helper plasmid pCI-NP, pCI-P, pCI-L corotation BHK-21 cells are contaminated, the TPCK-trypsin that 6 hours add final concentration of 1 μ g/ml after transfection, Harvest nutrient solution and cell after 3~4 days, centrifuged after multigelation 3 times, supernatant is inoculated with 10 ages in days SPF chicken embryos, that is, obtain gene VII new castle disease virus weakening strains N7a.
The present invention relates to a kind of NDV low virulent strain, the NDV low virulent strain is described The genotype VII NDV low virulent strain that rescue method is prepared, the gene VII new cities Epidemic disease poison low virulent strain is NDV (genotype VII), N7a plants of (Newcastle Disease Virus (genotype VII), strain N7a), China typical culture collection center is preserved in, is protected Tibetan address is Wuhan, China Wuhan University, deposit number：CCTCC NO.V201545, during preservation Between be on October 19th, 2015.The present invention is using reverse genetics manipulation technology to the biological works of Pu Laike The separation of journey limited company, identification, PLK-N-06 plants of the gene VII NDVs preserved F genes carry out saving out the weak gene VII newcastle disease virus strains N7a of cause after causing weak mutation.Should Strain has high titre, heritability stable in chicken embryo, is suitable for the large-scale production of vaccine, Available for manufacture vaccine.
The present invention relates to a kind of vaccine combination, wherein, the vaccine combination contains immune amount Described genotype VII NDV low virulent strain N7a or its culture antigen and pharmacy On carrier.
" culture " is the different generation subcultures of virus, and those skilled in the art know difference Its gene order is only possible between generation can occur small variation.
As one embodiment of the present invention, the genotype VII NDV low virulent strain The antigen of N7a or its culture is inactivated whole virus antigen, subunit antigen or lytic virus Antigen.
Term used herein " vaccine combination " refers to containing genotype Ⅶ NDV immunogenicity Pharmaceutical composition, the pharmaceutical composition can induce, stimulate or strengthen chicken for genotype Ⅶ it is new The immune response of city epidemic disease poison.The vaccine combination includes the genotype Ⅶ Newcastle Disease of immune amount Attenuated live vaccine, inactivated vaccine, subunit vaccine or the synthetic peptide vaccine of strain.
Preferably, the vaccine combination includes the genotype Ⅶ Newcastle disease attenuated vaccine strain of immune amount The inactivated vaccine of N7a plants or its culture.
Term used herein " inactivated vaccine ", also referred to as inactivated vaccines, refer to being used as antigen With the suspension for the inactivation of viruses for producing immunity.The example of inactivated vaccine include whole virus vaccine and Cracking type vaccine.Inactivated vaccine can be easily produced using known method.For example, by using Formalin processing virus can obtain inactivated virus vaccine.Cracking type vaccine can be after being handled with ether Prepared by peplos.For example inactivation can be passed through with N7a plants of the attenuated vaccine strain of the present invention Method be prepared into inactivated vaccine.
Preferably, genotype Ⅶ NDV antigen is the described of inactivation in the vaccine combination N7a plants of totivirus antigens of genotype Ⅶ NDV；The vaccine combination is further comprising assistant Agent.
As one embodiment of the present invention, the genotype VII NDV low virulent strain N7a culture is the culture in 1~50 generation；Preferably, the genotype VII Newcastle Disease Malicious low virulent strain N7a culture is the culture in 1~40 generation；It is highly preferred that the gene VII Type NDV low virulent strain N7a culture is the culture in 1~35 generation；It is further preferred that The culture of the genotype VII NDV low virulent strain N7a is the culture in 1~30 generation； It is further preferred that the culture of the genotype VII NDV low virulent strain N7a is 1~25 The culture in generation；It is further preferred that the genotype VII NDV low virulent strain N7a Culture is the culture in 1~20 generation；Most preferably, the genotype VII NDV is weak Strain N7a culture is the culture in 1~16 generation.The composition or component of the composition of the present invention Amount be preferably therapeutically effective amount.The therapeutically effective amount refers in the host that composition is applied Their immunological role is played without causing excessive side effect institute necessary amounts.Composition used and treat The accurate amount of the composition of administration is by according to type of the factor such as the disease for the treatment of, and to be treated is dynamic The type of thing and age, the mode of administration, and other compositions in composition and change.
As one embodiment of the present invention, the genotype Ⅶ NDV low virulent strain goes out Totivirus antigenic content living for before inactivation >=108.0EID50/0.1ml。
It is preceding less than 10 to inactivate when genotype Ⅶ NDV8.0EID50/ 0.1ml amount in use, Vaccine is unable to the generation of effective stimulus antibody.On the other hand, the amount exceeded is probably uneconomic.
It is used as a kind of preferred embodiment of the present invention, the genotype Ⅶ NDV low virulent strain Inactivated whole virus antigenic content for inactivation preceding 108.0EID50/ 0.1ml~109.0EID50/0.1ml。
As one embodiment of the present invention, the pharmaceutically acceptable carrier includes adjuvant, The adjuvant include (1) aluminium hydroxide, saponin(e, Avridine, DDA, (2) acrylic acid or The polymer of the polymer of methacrylic acid, maleic anhydride and alkenyl derivative, or (3) Oil in water emulsion, water-in-oil emulsion or W/O/W emulsion, the concentration range of the adjuvant be from 10% arrives 70%V/V, preferably from 30% to 60%V/V, more preferably 60%V/V.
As a kind of preferred embodiment of the present invention, the adjuvant includes (1) saponin(e QuilA； (2) acrylic or methacrylic acid polymer and sugar or the poly alkenyl ether cross-linking products card of polyalcohol Ripple nurse；Or (3) described adjuvant includes the emulsion based on light liquid paraffin oil, isoprenoid oil, Such as saualane or squalene；The oil that alkene, particularly isobutene or decene oligomerizationization are produced, band The ester of acid or the alcohol formation of straight chained alkyl, more particularly vegetable oil, ethyl oleate, propane diols two (caprylate/decylate), glycerine three (caprylate/decylate), Rikemal PO 200； The ester of branched aliphatic acid esters or alcohol, particularly isostearate, oil are used together to be formed with emulsifying agent Emulsion, emulsifying agent preferred nonionic surfactants；Particularly polyoxyethylated fatty acid is (for example Oleic acid), it is sorbitan, mannitol (such as anhydromannitol oleate ester), glycerine, poly- Glycerine, propane diols and alternatively the oleic acid of ethoxylation, isostearic acid, castor oil acid, hydroxyl are hard The ether of the ester of resin acid formation, fatty alcohol and polyalcohol (such as oleyl alcohol), polyoxy propylene-polyoxy second Alkene block copolymer, particularly PluronicR, especially L121.
Term " adjuvant ", which refers to, to be added in the composition of the present invention to increase the immunogenicity of composition Material.Known adjuvant includes, but are not limited to：(1) aluminium hydroxide, saponin(e (Saponine) (such as QuilA), Avridine, DDA, the polymer of (2) acrylic or methacrylic acid, The polymer of maleic anhydride and alkenyl derivative, or (3) vaccine can be with oil-in-water, oil Bag water or W/O/W emulsion form are made.
Especially, emulsion can be based on light liquid paraffin oil, isoprenoid oil, such as saualane Or squalene；The oil that alkene, particularly isobutene or decene oligomerizationization are produced, with straight chained alkyl Acid or the ester of alcohol formation, more particularly vegetable oil, the ethyl oleate, the (caprylate/last of the ten Heavenly stems of propane diols two Acid esters), glycerine three (caprylate/decylate), Rikemal PO 200；Branched aliphatic acid esters Or the ester of alcohol, particularly isostearate.Oil is used together to form emulsion with emulsifying agent.Emulsifying agent Preferred nonionic surfactants, particularly polyoxyethylated fatty acid (such as oleic acid), dehydration D-sorbite, mannitol (such as anhydromannitol oleate ester), glycerine, polyglycereol, propane diols The ester that alternatively oleic acid of ethoxylation, isostearic acid, castor oil acid, hydroxy stearic acid are formed, The ether of fatty alcohol and polyalcohol (such as oleyl alcohol), polyoxypropylene polyoxyethylene block copolymer, Particularly PluronicR, especially L121 are (with reference to Hunter etc., 1995, " The Theory and Practical Application ofAdjuvants " (Steward-Tull, D.E.S are edited) John Wiley AndSons, NY, 51-94；Todd etc., Vaccine, 1997,15,564-570).
Especially, acrylic or methacrylic acid polymer passes through sugar or the poly alkenyl ether of polyalcohol Crosslinking.These compounds are referred to as carbomer.
Preferably, the present invention is white-oil adjuvant from adjuvant, prepares water-in-oil emulsion.
In final vaccine combination, the concentration range of adjuvant is from 10% to 70%V/V, preferably From 30% to 60%V/V, more preferably 60%V/V.
Other reagents can also further be added to the combination of the present invention by vaccine combination of the present invention Thing.For example, the composition of the present invention can also include reagent, such as：Medicine, immunostimulant is (such as： Alpha-interferon, beta-interferon, gamma interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulatory factor (M-CSF) and interleukin-22 (IL2)), Antioxidant, surfactant, colouring agent, ethereal oil, buffer, dispersant, propellant And preservative.In order to prepare such composition, method well known in the art can be used.
Prevention and treatment ewcastle disease relevant disease is being prepared the present invention relates to described vaccine combination Application in medicine.
Term " prevention " refers to the disease by its infection related to genotype Ⅶ NDV or disease Shape is blocked or postponed；Term " treatment " refers to by the infection related to genotype Ⅶ NDV Or the process that the symptom of disease is alleviated or is completely eliminated.
The present invention has the advantages that following prominent：
The present invention uses PCMVTNT plasmid cloning ewcastle disease Genomic full_length cDNAs, and 5 ' End addition hammerhead ribozyme DNA sequence dna, successfully saves out ewcastle disease in BHK-21 cells Virus.
Although NDV only one of which serotypes, vaccine strain and epidemic strain are in genotype and antigen position All differed greatly on point；Research shows both at home and abroad, when vaccine strain is consistent with the genotype of epidemic strain, It can not only provide preferable clinical protection, and can significantly reduce immune chicken strong malicious infection rate and Shedding virus, clinically can effectively control the generation of non typical newcastle disease in vaccinated flock；The present invention The genotype Ⅶ Newcastle disease attenuated vaccine strain of offer, immune effect is good, advantageously prevents ewcastle disease The infection of virus.
With reference to specific implementation example, the present invention will be further described.
Embodiment 1：The sequencing of PLK-N-06 plants of full-length genomes of NDV：
With reference to the specification of RNA extracts kits, NDV PLK-N-06 strain virus liquid is extracted Total serum IgE, carries out reverse transcription reaction using P1~P14 anti-sense primer respectively, prepares virus CDNA, the cDNA to obtain uses 14 pairs of primer amplicon virus full genomes of design as template Group.PCR primer identifies stripe size through 1% agarose gel electrophoresis after expected be consistent, by PCR Product carries out recovery purifying according to glue reclaim kit specification.By the DNA fragmentation gram after recovery Grand to be transformed into pEASY-Blunt carriers in E.coli DH5 α competent cells, screening is positive Clone, and carry out sequencing analysis.
Embodiment 2：The full-length infectious clone pCMV-FL of PLK-N-06 plants of NDV structure Build：
According to NDV PLK-N-06 pnca gene group sequences and transcription vector pCMVTNT The analysis of middle restriction enzyme site, is divided into 7 fragments by genome, through RT-PCR from PLK-N-06 Amplification obtains purpose fragment in the allantoic fluid of strain infection, and cloning vector pEasy-Blunt is connected into respectively In.Hammerhead ribozyme (HamRz) is introduced at 5 ' ends of full-length cDNA fragment first, 3 ' ends are drawn Enter fourth liver ribozyme sequence (HdvRz), the terminal sequence of genome 3 ' and 5 ' be connected into transcription vector, Seven fragments are connected into transcription vector successively by digestion, connection again, finally obtained containing virus The transcription vector pCMV-FL of full-length cDNA.Figure 1A illustrates NDV PLK-N-06 Pnca gene group cDNA fragments 1,2,3 are connected to transcription vector pCMVTNT process, prepare Plasmid be pCMVTNT-PV123, Figure 1B illustrates PLK-N-06 plants of bases of NDV Because group cDNA fragments 4,5,6,7 are connected to transcription vector pCMVTNT process, prepare Plasmid be pCMVTNT-PV4567, Fig. 1 C are illustrated by plasmid pCMVTNT-PV123 The table containing newcastle disease virus gene group full-length cDNA is prepared with plasmid pCMVTNT-PV4567 Up to the process of carrier, the plasmid of preparation is pCMVTNT-FL.
Using primer, its sequence is as follows：
PV1-F：5’-CCGGAGCTCAATCGAATCGTACGGGTAG-3’
PV1-R：5’-CGGATCAATTCAGAAGGGTGCT-3’
PV2-F：5’-CCGGAGCTCATCTCCTTACGTGACACA-3’
PV2-R：5’-CGGGGTACCGTGTAGTTGCACTTCTT-3’
PV3-F：5’-CCGGAGCTCAATGCTCTGCTTAGGAGTG-3’
PV3-R：5’-CGGAGATCACAGTCGATAT-3’
PV4-F：5’-CCGGAGCTCATGGTAATGATCTACT-3’
PV4-R：5’-CGGGGTACCACTCTATCATCCTTGA-3’
PV5-F：5’-TGCGAGCTCATTGTCTTAGTATCCA-3’
PV5-R：5’-CGGGGTACCTCATTAGAGTTCAAAT-3’
PV6-F：5’-TGCTCTTGAATGCATCCACTTAGCA-3’
PV6-R：5’-CGGGGTACCAAGCAGATCGTACTT-3’
PV7-F：5’-CCGGAGCTCATAGACTTCTCGAGGT-3’
PV7-R：5’-TGCAATTGGCGTACGATTGCCT-3’.
Embodiment 3：The structure of NDV PLK-N-06 three helper plasmids of strain：
Using NDV PLK-N-06 cDNA as template, NP, P gene of design are used Two pairs of primer amplicon virus NP, P genes.PCR primer is identified through 1% agarose gel electrophoresis Stripe size is with after expected be consistent, PCR primer is reclaimed according to glue reclaim kit specification Purifying.DNA fragmentation after recovery is cloned into pCI-neo carriers, bacillus coli DH 5 alpha is transformed into In competent cell, screening positive clone, digestion identify correct plasmid be named as pCI-NP, PCI-P, and carry out sequencing analysis.
Using NDV PLK-N-06 cDNA as template, the three of the L genes of design are used To primer amplicon virus L1, L2, L3 fragment.PCR primer is reflected through 1% agarose gel electrophoresis Stripe size is determined with after expected be consistent, PCR primer is returned according to glue reclaim kit specification Receive purifying.By the L1 fragments after recovery with after NheI and SalI double digestions, reclaim L1 fragments with It is attached, converts by the pCI-neo carriers of same double digestion, obtains plasmid pCI-L1.Will L2 fragments after recovery are same with passing through with L2 fragments after Bsu36I and SalI double digestions, are reclaimed The pCI-L1 carriers of double digestion are attached, converted, and obtain plasmid pCI-L12.After recovery L3 fragments are with after MluI and SalI double digestions, and recovery L3 fragments are with passing through same double digestion PCI-L12 carriers are attached, converted, and finally give plasmid pCI-L123, i.e. pCI-L, and Carry out sequencing analysis.
Using primer, its sequence is as follows：
NP-F：5’-TATTGAATTCGCCACCATGTCGTCTGTTTTCGACGA AT-3’
NP-R：5’-TATAGTCGACTCAGTACCCCCAGTCAGTGTCGTT-3’
P-F：5’-TATTGAATTCGCCACCATGGCTACTTTTACAGATGCG GA-3’
P-R：5’-TATAGTCGACTCAACCATTCAGCGCAAGGCGT-3’
L1-F：5’-TATTGCTAGCGCCACCATGGCGGGCTCCGGTCC-3’
L1-R：5’-TATAGTCGACCAGGATTGGTTGGAATCTGACTG-3’
L2-F：5’-AGATGGAACAATACTCAGTCAGGTC-3’
L2-R：5’-TATAGTCGACTCTTCTCACTCAGGTTA-3’
L3-F：5’-TCATTCACTCAAGGTTGAATGCAGT-3’
L3-R：5’-TATAGTCGACTTAAGAGTCATTATTACTG-3’.
Embodiment 4：New castle disease virus weakening strain N7a rescue：
Using PV2-F, P1 and P2, PV3-R two to primer, by Overlap PCR methods, NDV PLK-N-06 F gene cracking sites are sported into low virulent strain feature112G-R-Q-G-R-L117.Fig. 2 illustrates the nucleotides sequence before and after F genes cracking site is mutated Row change and the change of corresponding amino acid sequence.
Mutant primer sequence is as follows：
PV2-F：5’-CCGGAGCTCATCTCCTTACGTGACACA-3’
P1：5’-AAGGCGTCCTTGTCTCCCTCCTCCGGACGTGGACACA -3’
P2：5’-GAGGGAGACAAGGACGCCTTATAGGTGCTGTTATT-3’
PV3-R：5’-CGGGGTACCAGATCACAGTCGATAT-3’.
By the expression vector containing the NDV PLK-N-06 full-length cDNAs after mutation The electrophoretogram that pCMV-FL-N7a carries out product after digestion identification, digestion with restriction enzyme is shown in Fig. 3, wherein, swimming lane 1 is that, using product after XhoI digestions, swimming lane M is molecule Marker, Swimming lane 2 is product after BglII digestions.
BHK-21 cells are laid in six orifice plates and grow to 80%~90% individual layer, PBS is used before transfection Gently washing cell 3 times, Opti-MEM culture mediums are changed to by DMEM culture mediums, will 6 μ g altogether Total length plasmid, pCI-NP, pCI-P and pCI-L transfection reagent2000 According to 2: 2: 1: 1 ratio cotransfection BHK-21 cells, gently washed with PBS after 6 hours Changed after 3 times and TPCK-trypsin (1 μ g/ml) is added in liquid, Opti-MEM culture mediums, mixing is equal It is even to continue to be incubated 3~4.Harvest nutrient solution and cell, centrifuged after multigelation 3 times, supernatant connects 10 age in days SPF chicken embryos are planted, per embryo 0.2ml.37 DEG C are put to continue to be incubated, per sunshine egg inspection 1~ 2 times, 72~120 hours dead chicken embryos with survival in 120 hours after choosing inoculation harvest chicken embryo liquid, It is named as N7a plants.
Embodiment 5：New castle disease virus weakening strain N7a RT-PCR identifications：
The RNA of new castle disease virus weakening strain N7a strain virus liquid is extracted, total length sense is used after reverse transcription PV3-F and PV3-R in metachromia clone pCMV-FL structure are expanded and are sequenced, and it is wrapped The total length of the gene containing F, the sequencing result of amplified fragments shows that the cracking site of F genes has been mutated into Work(, sequencing result is shown in Fig. 4.For expanding drawing for F genes in N7a plants of new castle disease virus weakening strain Thing sequence is：
PV2-F：5’-CCGGAGCTCATCTCCTTACGTGACACA-3’
PV3-R：5’-CGGGGTACCAGATCACAGTCGATAT-3’
Embodiment 6：The measure of new castle disease virus weakening strain N7a erythrocyte agglutinations valency and viral level
By existing《Chinese veterinary pharmacopoeia》Annex is carried out, and determines the HA potency of N7a plants of seeds culture of viruses.Will N7a plants of seed culture of viruses sterile salines are made 10 times and are serially diluted, and take 10-7、10-8、10-9And 10-104 Individual dilution factor, is respectively inoculated with 10 5 pieces of age in days SPF chicken embryos through allantoic cavity, per embryo 0.1ml, put 36~ 37 DEG C are continued to be incubated, and dead chicken embryo, which is discarded, before 48 hours disregards, dead at 48~120 hours Chicken embryo is taken out at any time put 2~8 DEG C, to 120 hours, took out all embryos living, and 48~120 hours dead The chicken embryo with survival in 120 hours is died, erythrocyte agglutination valency is determined by embryo, agglutination titer is not less than 1: 128 (micromethod) person is judged to infection, calculates EID50.It the results are shown in Table 1.
The HA potency and viral level measurement result of table N7a plants of seeds culture of viruses of 1 newcastle disease virus
Strain HA potency Viral level (lgEID50/0.1ml)
N7a plants 1∶512 9.5
As a result show, N7a plants of NDV attenuated vaccine strain growth titres are high, with good Biological characteristics.
Embodiment 7：New castle disease virus weakening strain N7a is to chicken ICPI (ICPI) and right The measure of the average death time (MDT/MLD) of chicken embryo minimum lethal dose：
Press《Regulations》(two 〇 〇 〇 versions) annex is carried out, and N7a is determined respectively The ICPI of strain seed culture of viruses.Average death time (MDT/MLD) to chicken embryo minimum lethal dose is pressed《In Magnificent people's republic's regulations》(two 〇 〇 〇 versions) annex is carried out, and is surveyed respectively Determine the MDT/MLD of N7a plants of seeds culture of viruses.
As a result the ICPI values for showing N7a plants of seeds culture of viruses of new castle disease virus weakening strain are 0.25, MDT/MLD is respectively 112, meets the feature of low virulent strain.
Embodiment 8：New castle disease virus weakening strain N7a specificity：
(1) HI is tested
N7a plants of seeds culture of viruses are prepared into 4 unit antigens according to HA potency respectively, NDV positive bloods are used Clearly, EDSV positive serums, AIV (H9 hypotypes) positive serum, AIV (H7 hypotypes) are positive Serum and AIV (H5 hypotypes) positive serum are respectively by existing《Chinese veterinary pharmacopoeia》Annex is carried out HI is tested, and determines the HI potency of each serum.
Test and determine through HI, the hemagglutination activity of N7a plants of seeds culture of viruses only can be by newcastle disease virus positive blood Suppressed clearly, shown, the N7a strains of rescue are newcastle disease virus, are deposited without other coagulation viruses .It the results are shown in Table 2.
Table 2 recombinates the HI result of the tests of N7a plants of seeds culture of viruses of newcastle disease virus
(2) chicken embryo neutralization test
N7a plants of seeds culture of viruses are diluted to 10 with sterile saline5.0EID50/ 0.1ml, respectively with equivalent Anti-newcastle disease virus positive serum is mixed, in room temperature and after 1 hour, is respectively inoculated with 10 through allantoic cavity 10 pieces of age in days SPF chicken embryos, per embryo 0.2ml, are inoculated with rearmounted 37 DEG C and are incubated 120 hours, observation is simultaneously Record result.
Newcastle disease virus N7a plants of seed culture of viruses of restructuring is entered with anti-newcastle disease virus positive serum respectively Row is neutralized, 120 hours after inoculated into chick embryo, equal 10/10 survival of chicken embryo, and chicken embryo liquid is without blood clotting Property.Show that seed culture of viruses can completely be neutralized by newcastle disease positive serum.It the results are shown in Table 3.
The specific outcome of the chicken embryo neutralization test of table 3 detection restructuring N7a plants of seeds culture of viruses of newcastle disease virus
Pilot project N7a plants
Chicken embryo survival ratio 10/10
Chicken embryo liquid is coagulation 10/10 (-)
The preparation of 9N7a plants of NDV inactivated vaccines of embodiment
N7a plants of seeds culture of viruses of NDV dilute 10,000 times with sterile saline, are inoculated with 10 age in days SPF 20 pieces of chicken embryo, per embryonic breeding kind 0.1ml, puts 37 DEG C and continues to be incubated.Dead germ in 24h after inoculation is abandoned Go, 24h~120h dead germs put 4 DEG C in time, 120h receives aggregate sample, determine seedling poison HA and EID50Respectively 9.6 and 109.5EID50/0.1ml.The Newcastle Disease venom for measuring potency is imported Inactivate in tank, metered 10% formalin, open mixer stirring, be sufficiently mixed it, The ultimate density of formaldehyde is 0.1%, and 37 DEG C inactivate 16 hours.
According to the component proportion of table 4, the complete virus liquid of inactivation is blown slowly and is added in white-oil adjuvant, Motor is started simultaneously, is stirred 30~40 minutes with 4000r/min, 1% sulphur is added before stirring is terminated Willow mercury solution, it is 0.01% to make its ultimate density.
The N7a plants of NDV inactivated vaccine proportionings of table 4
Component Vaccine 1 Vaccine 2
N7a plants of antigen (EID50/0.1ml) 108.0 109.0
White-oil adjuvant (V/V) 60% 60%
10N7a plants of NDV inactivated vaccine potency tests of embodiment
The SPF chickens 50 of 30 ages in days are taken, are divided into 5 groups, every group 10, the 1st group and the 2nd The group vaccine 1 that respectively prepared by chest muscle injecting immune embodiment 9, the 3rd group and the 4th group of difference Vaccine 2 prepared by chest muscle injecting immune embodiment 9, immunizing dose is 20 μ l, and the 5th group is not It is immune to be used as control.All equal isolated rearings of test chicken, after being immunized 21, take a blood sample together with control group Serum is separated, HI antibody titers are determined, while the 1st group, the 3rd group and the 5th group is used HN1101 Strain 105EID50/ only dosage intramuscular injection attack, the 2nd group, the 4th group and the 6th group PLK-N-06 Strain 105EID50/ dosage intramuscular injection attack only, is observed 14, record morbidity, dead and protect Protect number.It the results are shown in Table 5.
The N7a plants of NDV inactivated vaccine efficacy test results of table 5
Note：HI TPPAs are the geometric mean of immune chicken antibody, are denoted as X ± SD, X generations Table average, SD represents standard deviation.
As a result show, 2 groups of vaccine 1 and vaccine can produce higher antibody in 21 days after immune, And two groups compared with control, the attack of strong poison can be protected completely.
The strong virus attack of lethal dose can be protected by demonstrating vaccine 1 and vaccine 2, and vaccine contg is not Less than 108.0EID50/ 0.1ml can provide the complete protection to chicken group.
The preparation of Sota plants of NDV inactivated vaccines of embodiment 11La
Sota plants of seeds culture of viruses of La are taken to prepare Sota plants of Newcastle Diseases of La according to the preparation method of embodiment 9 Malicious inactivated vaccine, vaccine component content is shown in Table 6.
Sota plants of ewcastle disease inactivated vaccine proportionings of 6 La of table
Component Vaccine 3
La Sota(EID50/0.1ml) 108.0
White-oil adjuvant (V/V) 60%
12N7a plants of NDV inactivated vaccine immunogenicity contrast tests of embodiment
Take the vaccine 3 for implementing 9 vaccines 1 prepared and the preparation of embodiment 11 that 21~28 are immunized respectively 20 chickens are immunized in age in days SPF chickens, vaccine 1, and 20 chickens are immunized in vaccine 3, and 20 μ l/ are only, common 40,10 chickens of control group, chest muscle injection before exempting from, exempts from latter 21 days and 28 days to complete Portion's chicken blood sampling separation serum, carries out HI antibody titers measure (autoantigen).28 days after exempting from, Poison is attacked with HN1101 plants and F48E9, toxic agent amount 10 is attacked5ELD50, collunarium eye droppings carry out attack poison, Attack after poison and observe daily, observe 14 days, record immune group and attack malicious control group chicken appetite, excrement, Disease symptom and whether there is death, after poison is attacked the 5th day respectively the larynx of collection every chicken of immune group and Cloacal swab, embryo is connect by all larynx and cloacal swab respectively, and each swab connects 3 pieces of embryo Carry out virus purification, chicken embryo liquid HA ＞ 1：16 are judged to the virus purification positive.Negative sample needs blind Judged again after passing a generation.Experiment packet situation is shown in Table 7.
The N7a plants of NDV inactivated vaccine immunogenicity contrast test packets of table 7
After the immune group of vaccine 1 is immune 21 to 28 HI antibody average values reached 1: 64 with On, and control group chicken is not higher than 1: 4.Illustrate that the serological method assay of vaccine reaches The quality standard of similar vaccine, and 28 days vaccines 1 are produced to autoantigen after immune HI antibody is high compared with vaccine 3.Concrete outcome is shown in Table 8.
7 days, 14 days, 21 days and 28 days HI antibody test situations after the contrast test of table 8 is immune
Each immune group is attacked after poison and observed 14 days, and each immune group is showed no morbidity or dead, reaches The quality standard of present NDV vaccines.The 5th day collection larynx and cloacal swabs after poison are attacked, Virus purification detection toxin expelling, a negative sample blind passage generation are carried out respectively.As a result N7a plants of each immune groups It is showed no toxin expelling.Control group survival two kinds of swab samples of chicken are that toxin expelling is positive.Concrete outcome is shown in Table 9、10。
The contrast test of table 9 attacks poison protection result
The contrast test of table 10 attacks the toxin expelling situation of malicious 5th day larynx and cloaca
Note："/" represents this without content；"+A " represents that larynx and cloaca toxin expelling are the positive；“—” Represent that larynx and cloaca toxin expelling are feminine gender.
Conclusion：Antibody can be detected after immune within 7th day, and antibody titer is higher after being immunized 21 days, , can be complete after NDV N7a are immune and the more classical vaccine strain La Sota antibody of NDV N7a is high The attack of the wild poison of the type of Protecting gene VII and the strong poison F48E9 of standard, vaccine combination of the invention is not only Genotype Ⅶ NDV infection can be prevented and classical NDV also can be effectively immunized, and compared with La Sota immune group protective rates are higher, compared with La Sota immune groups are attacked after poison, ensure that detection Less than toxin expelling, play the role of for the outburst for preventing atypical ewcastle disease important.Illustrate this hair Bright vaccine combination can more fully be protected to chicken group, be more beneficial for bio-safety.
Described above is only the preferred embodiments of the present invention, and any shape is not done to the present invention Limitation in formula, although the present invention is disclosed above with preferred embodiment, but and be not used to limit The fixed present invention, any those skilled in the art are not departing from technical solution of the present invention In the range of, when the technology contents using the disclosure above make a little change or are modified to equivalent change The equivalent embodiment of change, as long as being the content without departing from technical solution of the present invention, according to the present invention Technical spirit any simple modification, equivalent variations and the modification made to above example, In the range of still falling within technical solution of the present invention.

Claims (10)

1. a kind of NDV saves method, it is characterised in that methods described includes：
Step (1), newcastle disease virus gene group RNA described in reverse transcription, prepares the ewcastle disease Virus genomic cDNA；
Step (2), uses the newcastle disease virus gene group obtained in the step (1) CDNA, builds seven cDNA clone fragments of covering whole gene group, intersegmental heavy using each The restriction enzyme site of folded part, assembles in transcription vector pCMVTNT plasmids and obtains completely CDNA clone, hammerhead ribozyme DNA sequence dna, 3 ' are introduced at 5 ' ends of full-length cDNA fragment End introduces fourth liver ribozymal DNA sequence, obtains pCMV-FL plasmids；
Step (3), builds the helper plasmid of three clone's NP, P, L albumen respectively；And
Step (4), by the cDNA total length transcription vector plasmids built in the step (2) and The three helper plasmid cotransfection BHK-21 cells obtained in the step (3), save out new city Epidemic disease strain.
2. NDV according to claim 1 saves method, it is characterised in that described It is gene VII newcastle disease virus strains to save NDV described in method and step (1)；
Helper plasmid is pCI-neo plasmids described in the rescue method and step (3).
3. NDV according to claim 2 saves method, wherein, the step (2) Include：
According to point of restriction enzyme site in viral genome sequencing result and transcription vector pCMVTNT Analysis, is divided into 7 fragments, amplicon virus full-length genome, primer sequence is as follows by genome：
PV1-F：5’-CCGGAGCTCAATCGAATCGTACGGGTAG-3’
PV1-R：5’-CGGATCAATTCAGAAGGGTGCT-3’
PV2-F：5’-CCGGAGCTCATCTCCTTACGTGACACA-3’
PV2-R：5’-CGGGGTACCGTGTAGTTGCACTTCTT-3’
PV3-F：5’-CCGGAGCTCAATGCTCTGCTTAGGAGTG-3’
PV3-R：5’-CGGAGATCACAGTCGATAT-3’
PV4-F：5’-CCGGAGCTCATGGTAATGATCTACT-3’
PV4-R：5’-CGGGGTACCACTCTATCATCCTTGA-3’
PV5-F：5’-TGCGAGCTCATTGTCTTAGTATCCA-3’
PV5-R：5’-CGGGGTACCTCATTAGAGTTCAAAT-3’
PV6-F：5’-TGCTCTTGAATGCATCCACTTAGCA-3’
PV6-R：5’-CGGGGTACCAAGCAGATCGTACTT-3’
PV7-F：5’-CCGGAGCTCATAGACTTCTCGAGGT-3’
PV7-R：5 '-TGCAATTGGCGTACGATTGCCT-3 ', by by the 7 of amplification Individual cDNA fragments are assembled in transcription vector pCMVTNT plasmids and obtained comprising complete The pCMV-FL plasmids of cDNA clone；
The step (3) includes：According to carrier for expression of eukaryon pCI-neo and NP, P and L The restriction enzyme site analysis of gene, five pairs of primers of design expand three genes, and L genes are divided into three pieces Section amplification, wherein GCCACC kozak sequences are added to after each sense primer restriction enzyme site. Primer sequence is as follows：
NP-F：5’-TATTGAATTCGCCACCATGTCGTCTGTTTTCGACGA AT-3’
NP-R：5’-TATAGTCGACTCAGTACCCCCAGTCAGTGTCGTT-3’
P-F：5’-TATTGAATTCGCCACCATGGCTACTTTTACAGATGCG GA-3’
P-R：5’-TATAGTCGACTCAACCATTCAGCGCAAGGCGT-3’
L1-F：5’-TATTGCTAGCGCCACCATGGCGGGCTCCGGTCC-3’
L1-R：5’-TATAGTCGACCAGGATTGGTTGGAATCTGACTG-3’
L2-F：5’-AGATGGAACAATACTCAGTCAGGTC-3’
L2-R：5’-TATAGTCGACTCTTCTCACTCAGGTTA-3’
L3-F：5’-TCATTCACTCAAGGTTGAATGCAGT-3’
L3-R：5 '-TATAGTCGACTTAAGAGTCATTATTACTG-3 ', by inciting somebody to action NP, P and L gene cDNA clone of amplification to pCI-neo plasmids obtain pCI-NP, pCI-P, PCI-L plasmids.
4. NDV according to claim 3 saves method, wherein, in the step (2) in, using Overlap PCR methods, by the F gene cracking sites of the NDV Sport low virulent strain feature112G-R-Q-G-R-L117,
Mutant primer sequence is as follows：
PV2-F：5’-CCGGAGCTCATCTCCTTACGTGACACA-3’
P1：5’-AAGGCGTCCTTGTCTCCCTCCTCCGGACGTGGACACA -3’
P2：5’-GAGGGAGACAAGGACGCCTTATAGGTGCTGTTATT-3’
PV3-R：5 '-CGGGGTACCAGATCACAGTCGATAT-3 ',
And by the way that 6 cDNA fragments of the amplification and the mutation F genes that include are cracked The fragment in site is assembled in transcription vector pCMVTNT plasmids and obtained comprising global cDNA The pCMV-FL-N7a plasmids of clone；
In the step (4), by plasmid pCMV-FL-N7a and three helper plasmid pCI-NP, PCI-P, pCI-L cotransfection BHK-21 cells, add final concentration of 1 μ g/ml in 6 hours after transfection TPCK-trypsin, harvest nutrient solution and cell after 3~4 days, centrifuged after multigelation 3 times, Supernatant is inoculated with 10 age in days SPF chicken embryos, obtains gene VII new castle disease virus weakening strains N7a.
5. the genotype VII Newcastle Disease that rescue method is prepared according to claim 4 Malicious low virulent strain N7a, the gene VII NDV low virulent strains N7a are preserved in Chinese Typical Representative training Thing collection is supported, preservation address is Wuhan, China Wuhan University, preserving number：CCTCC NO.V201545, the preservation time is on October 19th, 2015.
6. a kind of vaccine combination, wherein, the vaccine combination contains the right of immune amount It is required that the antigen of genotype VII NDV low virulent strain N7a or its culture described in 5 and Carrier pharmaceutically.
7. vaccine combination according to claim 6, wherein, the genotype VII is new The antigen of city epidemic disease poison low virulent strain N7a or its culture be inactivated whole virus antigen, subunit antigen, Or the antigen of lytic virus.
8. vaccine combination according to claim 6, wherein, the genotype VII is new The malicious low virulent strain N7a of city epidemic disease culture is the culture in 1~50 generation；Preferably, the gene VII type NDV low virulent strains N7a culture is the culture in 1~40 generation；It is highly preferred that The culture of the genotype VII NDV low virulent strain N7a is the culture in 1~35 generation； It is further preferred that the culture of the genotype VII NDV low virulent strain N7a is 1~30 The culture in generation；It is further preferred that the genotype VII NDV low virulent strain N7a Culture is the culture in 1~25 generation；It is further preferred that the genotype VII Newcastle Disease Malicious low virulent strain N7a culture is the culture in 1~20 generation；Most preferably, the gene VII Type NDV low virulent strain N7a culture is the culture in 1~16 generation.
9. vaccine combination according to claim 6, wherein, the genotype Ⅶ new city Epidemic disease poison low virulent strain inactivated whole virus antigenic content for inactivation before >=108.0EID50/0.1ml；It is preferred that Ground, before the inactivated whole virus antigenic content of the genotype Ⅶ NDV low virulent strain is inactivation 108.0EID50/ 0.1ml~109.0EID50/0.1ml。
10. vaccine combination according to claim 6, wherein, it is described pharmaceutically acceptable Carrier include adjuvant, the adjuvant include (1) aluminium hydroxide, saponin(e, Avridine, DDA, (2) polymer of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative is poly- Compound, or (3) oil in water emulsion, water-in-oil emulsion or W/O/W emulsion, the adjuvant Concentration range be from 10% to 70%V/V, preferably from 30% to 60%V/V, more preferably 60% V/V；
Preferably, the adjuvant includes (1) saponin(e QuilA；(2) acrylic or methacrylic Acid polymer and sugar or the poly alkenyl ether cross-linking products carbomer of polyalcohol；Or (3) described adjuvant Including the emulsion based on light liquid paraffin oil, isoprenoid oil, such as saualane or squalene； The oil that alkene, particularly isobutene or decene oligomerizationization are produced, acid or alcohol with straight chained alkyl are formed Ester, more particularly vegetable oil, ethyl oleate, propane diols two (caprylate/decylate), Glycerine three (caprylate/decylate), Rikemal PO 200；The ester of branched aliphatic acid esters or alcohol, Particularly isostearate, oil are used together to form emulsion with emulsifying agent, the preferred nonionic of emulsifying agent Surfactant；Particularly polyoxyethylated fatty acid (such as oleic acid), sorbitan, Mannitol (such as anhydromannitol oleate ester), glycerine, polyglycereol, propane diols and alternatively second The oleic acid of epoxide, isostearic acid, castor oil acid, the ester of hydroxy stearic acid formation, fatty alcohol and The ether of polyalcohol (such as oleyl alcohol), polyoxypropylene polyoxyethylene block copolymer, particularly PluronicR, especially L121.
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