CN107287168B - Newcastle disease virus rescue method and application thereof - Google Patents
Newcastle disease virus rescue method and application thereof Download PDFInfo
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- CN107287168B CN107287168B CN201610200554.5A CN201610200554A CN107287168B CN 107287168 B CN107287168 B CN 107287168B CN 201610200554 A CN201610200554 A CN 201610200554A CN 107287168 B CN107287168 B CN 107287168B
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Abstract
The invention relates to a Newcastle disease virus rescue method, which introduces a hammerhead ribozyme DNA sequence at the 5 'end of cDNA of a Newcastle disease virus genome and introduces a hepatitis D ribozyme DNA sequence at the 3' end, effectively ensures the accuracy of the 5 'end and the 3' end, simultaneously uses a CMV promoter on a transcription vector pCMVTNT plasmid to improve the transcription efficiency and the rescue efficiency, and rescues the Newcastle disease virus on BHK-21 cells. The invention also relates to gene VII Newcastle disease virus attenuated strain N7a rescued by the method. The strain has high titer and stable genetic performance on chick embryos, is suitable for large-scale production of vaccines, and can be used for preparing vaccines.
Description
Technical Field
The invention relates to a method for rescuing newcastle disease virus by applying a reverse genetic manipulation technology, a newcastle disease virus attenuated strain prepared by using the method and a vaccine composition prepared by the attenuated strain, belonging to the technical field of biology.
Background
Newcastle Disease (ND) is an acute highly-contagious infectious disease of various birds such as chickens and turkeys caused by serotype I avian paramyxovirus (NDV) among 9 serotypes of avian paramyxovirus, and often presents as a sepsis symptom. Newcastle disease is a serious highly contagious viral disease that is a major threat to the poultry industry due to its widespread distribution and possible significant economic losses in many countries around the world.
In recent years, newcastle disease vaccines are widely used in the world, but newcastle disease virulent virus infection is still common in immune chicken flocks, and meanwhile, the phenomenon that newcastle disease viruses and other respiratory pathogens are co-pathogenic in clinic is also common, so that newcastle disease is still one of important epidemic diseases which plague vast farmers at present. From the epidemic characteristics of newcastle disease in the last decade, most of clinically isolated newcastle disease virulent strains belong to gene type VII, while the genotypes of conventional newcastle disease vaccine strains are mainly genotype type II (such as LaSota, clone 30, V4 and the like), and the genetic distance between the newcastle disease epidemic strains and the conventional vaccine strains is far from each other, so that the conventional vaccine cannot provide comprehensive immune protection efficacy for the current newcastle disease virulent strains.
In the process of preparing new newcastle disease vaccines, newcastle disease virus rescue methods are often used to weaken newcastle disease virus strains, and therefore, the efficiency of the newcastle disease virus rescue method is the key to successful weakening of the strains.
Therefore, if a new epidemic strain can be weakened by using a high-efficiency newcastle disease virus rescue method, a vaccine composition which is matched with a clinical epidemic strain and has good immunogenicity is developed, and the epidemic of newcastle disease in China can be effectively prevented.
Disclosure of Invention
The invention relates to a Newcastle disease virus rescue method, which comprises the following steps: reverse transcription of the newcastle disease virus genome RNA to prepare cDNA of the newcastle disease virus genome; step (2), constructing seven cDNA cloning fragments covering the whole genome by using the cDNA of the Newcastle disease virus genome obtained in the step (1), connecting and assembling a transcription vector pCMVTNT plasmid by utilizing enzyme cutting sites of overlapped parts among the fragments to obtain a complete cDNA clone, introducing a hammerhead ribozyme DNA sequence into the 5 'end of the full-length cDNA fragment, and introducing a hepatitis D ribozyme DNA sequence into the 3' end of the full-length cDNA fragment to obtain a pCMV-FL plasmid; respectively constructing three auxiliary plasmids for cloning NP, P and L proteins; and (4) co-transfecting the full-length cDNA transcription vector plasmid constructed in the step (2) and the three helper plasmids obtained in the step (3) into BHK-21 cells to rescue the Newcastle disease virus strain.
The invention also relates to a method for attenuating the Newcastle disease virus, in which method the F group of the Newcastle disease virus is rescuedCharacteristic of attenuated strain due to mutation of cleavage site112G-R-Q-G-R-L117。
The invention also relates to a gene VII type Newcastle Disease Virus low virulent strain, wherein the gene VII type Newcastle Disease Virus low virulent strain is Newcastle Disease Virus (gene VII type), N7a strain (Newcastle Disease Virus (genotyp VII), strain N7a), which is preserved in China center for type culture Collection with the preservation address of Wuhan university in China, and the preservation number is as follows: CCTCC NO: v201545, preservation time 2015, 10 months and 19 days. The vaccine composition prepared from the gene VII Newcastle disease virus attenuated strain can provide effective immune protection for the epidemic gene VII Newcastle disease wild strain and show good immunogenicity.
The invention also relates to a vaccine composition, wherein the vaccine composition contains an immunizing dose of the antigen of the gene VII type Newcastle disease virus low virulent strain N7a or the culture thereof and a pharmaceutical carrier.
The invention has the following positive effects:
the reverse genetic technology used by the invention introduces a hammerhead ribozyme DNA sequence at the 5 'end of the cDNA of the Newcastle disease virus, and the sequence ensures that the transcribed RNA has an accurate 5' end and promotes transcription. Meanwhile, the sequence does not reduce the replication and proliferation capacity of the virus on cells, and high rescue efficiency is ensured. The method of the invention relies on RNA polymerase II under the control of cytomegalovirus promoter (CMV promoter), and takes eukaryotic expression vector pCMVTNT with CMV promoter as a skeleton to construct the full length, and under the drive of RNA polymerase II, the full length cRNA of the minus-strand virus can be stably expressed, and the virus rescue efficiency is effectively improved.
The present invention utilizes reverse genetic technology to save gene VII Newcastle disease virus attenuated strain successfully. The strain has high titer and stable genetic performance on chick embryos, is suitable for large-scale production of vaccines, and can be used for preparing vaccines. The strain is consistent with the genotype of the dominant epidemic strain of the newcastle disease virus in China, and shows wide application prospect in the aspect of controlling the morbidity and the epidemic of the current newcastle disease.
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FIG. 1 is a schematic diagram of construction of plasmid pCMV-FL containing full-length cDNA, FIG. 1A is a schematic diagram of construction of expression plasmid containing segments 1, 2 and 3 of Newcastle disease virus cDNA, FIG. 1B is a schematic diagram of construction of expression plasmid containing segments 4, 5, 6 and 7 of Newcastle disease virus cDNA, and FIG. 1C is a schematic diagram of construction of expression plasmid containing full-length cDNA of Newcastle disease virus;
FIG. 2 is a schematic diagram showing the F gene cleavage site mutation;
FIG. 3 is the restriction enzyme identification map of plasmid pCMV-FL-N7a of full-length cDNA, lane 1 is the electrophoresis map after XhoI restriction enzyme, lane 2 is the electrophoresis map after BglII restriction enzyme, lane M is Marker DL 15000;
FIG. 4 is a diagram showing the result of RT-PCR identification of gene VII Newcastle disease virus attenuated strain N7 a.
Detailed Description
Hereinafter, embodiments of the present invention will be described.
The invention relates to a Newcastle disease virus rescue method, which comprises the following steps: reverse transcription of the newcastle disease virus genome RNA to prepare cDNA of the newcastle disease virus genome; step (2), constructing seven cDNA cloning fragments covering the whole genome by using the cDNA of the Newcastle disease virus genome obtained in the step (1), connecting and assembling a transcription vector pCMVTNT plasmid by utilizing enzyme cutting sites of overlapped parts among the fragments to obtain a complete cDNA clone, introducing a hammerhead ribozyme DNA sequence into the 5 'end of the full-length cDNA fragment, and introducing a hepatitis D ribozyme DNA sequence into the 3' end of the full-length cDNA fragment to obtain a pCMV-FL plasmid; respectively constructing three auxiliary plasmids for cloning NP, P and L proteins; and (4) co-transfecting the full-length cDNA transcription vector plasmid constructed in the step (2) and the three helper plasmids obtained in the step (3) into BHK-21 cells to rescue the Newcastle disease virus strain.
As an embodiment of the present invention, in the step (1) of the rescue method, the newcastle disease virus is gene VII newcastle disease virus strain; the helper plasmid in the rescue method step (3) is a pCI-neo plasmid.
As an embodiment of the present invention, the step (2) includes:
according to the sequencing result of the viral genome and the analysis of the enzyme cutting sites in the transcription vector pCMVTNT, the genome is divided into 7 segments, the full length of the viral genome is amplified, and the primer sequences are 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’
PV 7-R: 5'-TGCAATTGGCGTACGATTGCCT-3', the pCMV-FL plasmid containing the complete cDNA clone is obtained by connecting and assembling the amplified 7 cDNA fragments in a transcription vector pCMVTNT plasmid;
the step (3) comprises the following steps: according to enzyme cutting site analysis of eukaryotic expression vector pCI-neo and NP, P and L genes, five pairs of primers are designed to amplify three genes, the L gene is divided into three segments to be amplified, wherein a kozak sequence of GCCACC is added to each upstream primer enzyme cutting site. The primer sequences are as follows:
NP-F：5’-TATTGAATTCGCCACCATGTCGTCTGTTTTCGACGAAT-3’
NP-R：5’-TATAGTCGACTCAGTACCCCCAGTCAGTGTCGTT-3’
P-F：5’-TATTGAATTCGCCACCATGGCTACTTTTACAGATGCGGA-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', plasmids pCI-NP and pCI-P, pCI-L were obtained by cloning the amplified cDNA of NP, P, and L genes into pCI-neo plasmid.
As an embodiment of the present invention, in the step (2), the F gene cleavage site of the Newcastle disease virus is mutated into the characteristics of a low virulent strain by using the Overlap PCR method112G-R-Q-G-R-L117，
The mutant primer sequences were as follows:
PV2-F：5’-CCGGAGCTCATCTCCTTACGTGACACA-3’
P1：5’-AAGGCGTCCTTGTCTCCCTCCTCCGGACGTGGACACA-3’
P2：5’-GAGGGAGACAAGGACGCCTTATAGGTGCTGTTATT-3’
PV3-R：5’-CGGGGTACCAGATCACAGTCGATAT-3’，
and the pCMV-FL-N7a plasmid containing the complete cDNA clone is obtained by connecting and assembling the amplified 6 cDNA fragments and the fragment containing the mutant F gene cleavage site in a transcription vector pCMVTNT plasmid;
in the step (4), the plasmid pCMV-FL-N7a and three auxiliary plasmids pCI-NP and pCI-P, pCI-L are co-transfected into BHK-21 cells, TPCK-trypsin with the final concentration of 1 mu g/ml is added 6 hours after transfection, culture solution and cells are harvested after 3-4 days, freeze thawing is carried out for 3 times repeatedly, centrifugation is carried out, and the supernatant is inoculated into SPF chick embryos of 10 days old, so that the gene VII Newcastle disease virus attenuated strain N7a is obtained.
As an embodiment of the invention, the preparation method of the gene VII Newcastle disease virus attenuated strain comprises the following specific steps:
1. sequencing of the whole genome of the newcastle disease virus PLK-N-06 strain:
by referring to the submitted NDV sequence (JN631747.1) in GenBank, 14 pairs of primers are designed, the amplified fragments are partially overlapped with each other and can be connected to cover the whole genome, and the primer sequences 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’
2. construction of full-Length infectious clone pCMV-FL of Newcastle disease Virus PLK-N-06 Strain:
according to the sequencing result of the viral genome and the analysis of the enzyme cutting sites in the transcription vector pCMVTNT, the genome is divided into 7 segments, the full length of the viral genome is amplified, and the primer sequences are 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’
3. construction of three auxiliary plasmids of Newcastle disease virus PLK-N-06 strain
According to enzyme cutting site analysis of eukaryotic expression vector pCI-neo and NP, P and L genes, five pairs of primers are designed to amplify three genes, the L gene is divided into three segments to be amplified, wherein a kozak sequence (GCCACC) is added to each upstream primer enzyme cutting site. The primer sequences are as follows:
NP-F：5’-TATTGAATTCGCCACCATGTCGTCTGTTTTCGACGAAT-3’
NP-R：5’-TATAGTCGACTCAGTACCCCCAGTCAGTGTCGTT-3’
P-F：5’-TATTGAATTCGCCACCATGGCTACTTTTACAGATGCGGA-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’
4. rescue of newcastle disease virus attenuated strain N7a
The F gene cleavage site of the Newcastle disease virus PLK-N-06 is mutated into the characteristics of a low virulent strain by using an Overlap PCR method112G-R-Q-G-R-L117The mutant primer sequences are as follows:
PV2-F：5’-CCGGAGCTCATCTCCTTACGTGACACA-3’
P1：5’-AAGGCGTCCTTGTCTCCCTCCTCCGGACGTGGACACA-3’
P2：5’-GAGGGAGACAAGGACGCCTTATAGGTGCTGTTATT-3’
PV3-R：5’-CGGGGTACCAGATCACAGTCGATAT-3’
the plasmid pCMV-FL-N7a and three auxiliary plasmids pCI-NP and pCI-P, pCI-L are co-transfected into BHK-21 cells, TPCK-trypsin with the final concentration of 1 mu g/ml is added 6 hours after transfection, culture solution and cells are harvested 3-4 days later, repeated freeze thawing is carried out for 3 times, centrifugation is carried out, and the supernatant is inoculated into SPF chick embryos of 10 days old, so that the gene VII Newcastle disease virus attenuated strain N7a is obtained.
The invention relates to a Newcastle Disease Virus attenuated strain, which is a gene VII type Newcastle Disease Virus attenuated strain prepared by the rescue method, wherein the gene VII type Newcastle Disease Virus attenuated strain is Newcastle Disease Virus (gene VII), N7a strain (Newcastle Disease Virus (genotype VII), strain N7a) and is preserved in China center for type culture Collection, the preservation address is Wuhan-Wuhan university, and the preservation number is as follows: CCTCC NO. V201545, and preservation time is 2015, 10 months and 19 days. The invention utilizes reverse genetic operation technology to carry out attenuated mutation on F gene of gene VII Newcastle disease virus PLK-N-06 strain separated, identified and stored by Pocke bioengineering GmbH, and then saves the attenuated gene VII Newcastle disease virus strain N7 a. The strain has high titer and stable genetic performance on chick embryos, is suitable for large-scale production of vaccines, and can be used for preparing vaccines.
The invention relates to a vaccine composition, wherein the vaccine composition contains an immunizing dose of antigen of the gene VII type Newcastle disease virus low virulent strain N7a or a culture thereof and a pharmaceutical carrier.
"cultures" are subcultures of different generations of the virus, and those skilled in the art know that only minor variations in the gene sequence are possible between generations.
As an embodiment of the invention, the antigen of the gene VII type Newcastle disease virus low virulent strain N7a or the culture thereof is inactivated whole virus antigen, subunit antigen or split virus antigen.
The term "vaccine composition" as used herein refers to a pharmaceutical composition comprising the immunogenicity of a gene type VII Newcastle disease virus, which pharmaceutical composition induces, stimulates or enhances the immune response in a chicken against the gene type VII Newcastle disease virus. The vaccine composition comprises an immunizing amount of attenuated live vaccine, inactivated vaccine, subunit vaccine or synthetic peptide vaccine of the gene VII type Newcastle disease virus strain.
Preferably, the vaccine composition comprises an immunizing amount of an inactivated vaccine of the gene type vii newcastle disease attenuated vaccine strain N7a strain or a culture thereof.
The term "inactivated vaccine", also known as inactivated vaccine, as used herein, refers to a suspension of inactivated virus used as an antigen to generate immunity. Examples of inactivated vaccines include whole virus vaccines and split vaccines. Inactivated vaccines can be readily produced using known methods. For example, whole virus inactivated vaccines can be obtained by treating the virus with formaldehyde solution. Split vaccines can be prepared from the viral envelope after treatment with ether. For example, the attenuated vaccine strain N7a of the present invention can be used to prepare inactivated vaccines by inactivation.
Preferably, the gene type vii newcastle disease virus antigen in said vaccine composition is an inactivated whole virus antigen of said gene type vii newcastle disease virus N7a strain; the vaccine composition further comprises an adjuvant.
As an embodiment of the invention, the culture of the gene VII type Newcastle disease virus low virulent strain N7a is a culture of 1-50 generations; preferably, the culture of the gene VII type Newcastle disease virus low virulent strain N7a is a culture of 1-40 generations; more preferably, the culture of the gene VII type Newcastle disease virus low virulent strain N7a is a culture of 1-35 generations; further preferably, the culture of the gene VII type Newcastle disease virus low virulent strain N7a is a culture of 1-30 generations; further preferably, the culture of the gene VII type Newcastle disease virus low virulent strain N7a is a culture of 1-25 generations; further preferably, the culture of the gene VII type Newcastle disease virus low virulent strain N7a is a culture of 1-20 generations; most preferably, the culture of the gene VII type Newcastle disease virus low virulent strain N7a is a culture of 1-16 generations. The amount of an ingredient or component of the composition of the present invention is preferably a therapeutically effective amount. The therapeutically effective amount refers to the amount necessary to exert their immunological effects in the host to which the composition is administered without causing undue side effects. The precise amounts of the ingredients used and the composition to be administered will vary depending on factors such as the type of disease being treated, the type and age of the animal being treated, the mode of administration, and the other ingredients in the composition.
As an embodiment of the invention, the content of the inactivated whole virus antigen of the gene VII type Newcastle disease virus attenuated strain is more than or equal to 10 before inactivation8.0EID50/0.1ml。
When the gene VII type Newcastle disease virus is less than 10 before inactivation8.0EID50When used in an amount of 0.1ml, the vaccine was not effective in stimulating antibody production. On the other hand, the excess amount may be uneconomical.
As a preferred embodiment of the present invention, the aboveThe inactivated whole virus antigen content of the gene VII type Newcastle disease virus attenuated strain is 10 before inactivation8.0EID50/0.1ml～109.0EID50/0.1ml。
As an embodiment of the invention, the pharmaceutically acceptable carrier comprises an adjuvant comprising (1) aluminium hydroxide, saponin, avridine, DDA, (2) a polymer of acrylic or methacrylic acid, maleic anhydride and an alkenyl derivative, or (3) an oil-in-water emulsion, a water-in-oil emulsion or a water-in-oil-in-water emulsion, the concentration of the adjuvant ranging from 10% to 70% V/V, preferably from 30% to 60% V/V, more preferably 60% V/V.
As a preferred embodiment of the present invention, the adjuvant comprises (1) the saponin QuilA; (2) carbomers which are the cross-linked products of a polymer of acrylic or methacrylic acid and a polyalkenyl ether of a sugar or polyol; or (3) the adjuvant comprises an emulsion based on light liquid paraffin oil, isoprenoid oil, such as squalane or squalene; oils resulting from the oligomerization of olefins, in particular isobutene or decene, esters of acids or alcohols with linear alkyl groups, more in particular vegetable oils, ethyl oleate, propylene glycol di (caprylate/caprate), glycerol tri (caprylate/caprate), propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters, oils and emulsifiers, preferably nonionic surfactants, are used together to form emulsions; in particular polyoxyethylated fatty acids (e.g. oleic acid), sorbitan, mannitol (e.g. anhydromannitol oleate), glycerol, polyglycerol, esters of propylene glycol and optionally ethoxylated oleic acid, isostearic acid, ricinoleic acid, hydroxystearic acid, ethers of fatty alcohols and polyols (e.g. oleyl alcohol), polyoxypropylene-polyoxyethylene block copolymers, in particular pluronic r, in particular L121.
The term "adjuvant" refers to a substance added to the composition of the present invention to increase the immunogenicity of the composition. Known adjuvants include, but are not limited to: (1) aluminium hydroxide, saponins (saponin) (e.g. QuilA), alfuzidine, DDA, (2) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivatives, or (3) vaccines can be made in the form of oil-in-water, water-in-oil or water-in-oil-in-water emulsions.
In particular, the emulsion may be based on light liquid paraffin oil, isoprenoid oil, such as squalane or squalene; oils resulting from the oligomerization of olefins, in particular isobutene or decene, esters of acids or alcohols with linear alkyl groups, more in particular vegetable oils, ethyl oleate, propylene glycol di (caprylate/caprate), glycerol tri (caprylate/caprate), propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used with an emulsifier to form an emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of polyoxyethylated fatty acids (e.g.oleic acid), sorbitan, mannitol (e.g.anhydromannitol oleate), glycerol, polyglycerol, propylene glycol and optionally ethoxylated oleic acid, isostearic acid, ricinoleic acid, hydroxystearic acid, ethers of fatty alcohols and polyols (e.g.oleyl alcohol), polyoxypropylene-polyoxyethylene block copolymers, in particular Pluronic R, especially L121 (see Hunter et al, 1995, "The Theory and Practical applications of Advances" (Steward-Tull, D.E.S. eds.) John Wiley and sons, NY, 51-94; Todd et al, Vaccine, 1997, 15, 564 + 570).
In particular, the acrylic or methacrylic acid polymers are crosslinked by polyalkenyl ethers of sugars or polyols. These compounds are known as carbomers.
Preferably, the adjuvant is a white oil adjuvant, and the water-in-oil emulsion is prepared.
The concentration of the adjuvant in the final vaccine composition ranges from 10% to 70% V/V, preferably from 30% to 60% V/V, more preferably 60% V/V.
The vaccine composition of the present invention may further comprise other agents added to the composition of the present invention. For example, the compositions of the present invention may also comprise agents such as: drugs, immunostimulants (e.g., alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 2(IL2)), antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives. To prepare such compositions, methods well known in the art may be used.
The invention relates to application of the vaccine composition in preparing a medicament for preventing and treating diseases related to Newcastle disease.
The term "prevention" means that the symptoms of the infection or disease by which the gene type vii newcastle disease virus is associated are blocked or delayed; the term "treatment" refers to a process by which the symptoms of an infection or disease associated with the gene type VII Newcastle disease virus are alleviated or eliminated altogether.
The invention has the following outstanding advantages:
the invention uses PCMVTNT plasmid to clone the full-length cDNA of the Newcastle disease genome, and adds a hammerhead ribozyme DNA sequence at the 5' end, thereby successfully rescuing the Newcastle disease virus in BHK-21 cells.
NDV has only one serotype, but the vaccine strain and the epidemic strain have larger difference in genotype and antigenic site; domestic and foreign researches show that when the genotypes of the vaccine strain and the epidemic strain are consistent, the vaccine strain not only can provide ideal clinical protection, but also can obviously reduce the virulent infection rate and the toxin expelling amount of the immunized chicken, and can effectively control the occurrence of atypical newcastle disease in the immunized chicken flock clinically; the gene VII type Newcastle disease attenuated vaccine strain provided by the invention has a good immune effect, and is favorable for preventing the infection of Newcastle disease virus.
The invention is further described below with reference to specific embodiments.
Example 1: sequencing of the whole genome of the newcastle disease virus PLK-N-06 strain:
the total RNA of NDV PLK-N-06 strain virus liquid is extracted according to the instruction of an RNA extraction kit, reverse transcription reaction is carried out by respectively using downstream primers of P1-P14 to prepare cDNA of the virus, and the whole genome of the virus is amplified by using the obtained cDNA as a template and using 14 designed pairs of primers. After the size of the PCR product is identified to be consistent with the expected size through 1% agarose gel electrophoresis, the PCR product is recovered and purified according to the instructions of the gel recovery kit. Cloning the recovered DNA fragment to pEASY-Blunt vector, transforming into E.coli DH5 alpha competent cell, screening positive clone, and sequencing analysis.
Example 2: construction of full-Length infectious clone pCMV-FL of Newcastle disease Virus PLK-N-06 Strain:
according to analysis of a genome sequence of a Newcastle disease virus PLK-N-06 strain and an enzyme cutting site in a transcription vector pCMVTNT, a genome is divided into 7 fragments, a target fragment is obtained by amplification from allantoic fluid infected by the PLK-N-06 strain through RT-PCR, and the target fragments are respectively connected into a cloning vector pEasy-blast. Firstly, hammer-head ribozyme (HamRz) is introduced into the 5 'end of the full-length cDNA fragment, hepatitis D ribozyme sequence (HdvRz) is introduced into the 3' end, the 3 'end sequence and the 5' end sequence of the genome are connected into a transcription vector, then seven fragments are sequentially connected into the transcription vector through enzyme digestion and connection, and finally the transcription vector pCMV-FL containing the virus full-length cDNA is obtained. FIG. 1A shows the process of connecting genomic cDNA fragments 1, 2 and 3 of the Newcastle disease virus PLK-N-06 strain to a transcription vector pCMVTNT, the prepared plasmid is pCMVTNT-PV123, FIG. 1B shows the process of connecting genomic cDNA fragments 4, 5, 6 and 7 of the Newcastle disease virus PLK-N-06 strain to a transcription vector pCMVTNT, the prepared plasmid is pCMVTNT-PV4567, FIG. 1C shows the process of preparing an expression vector containing the full-length cDNA of the Newcastle disease virus genome from the plasmid pCMVTNT-PV123 and the plasmid pCMVTNT-PV4567, and the prepared plasmid is pCMVTNT-FL.
The sequences of the primers used were 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’。
example 3: construction of three helper plasmids of the Newcastle disease virus PLK-N-06 strain:
the cDNA of the Newcastle disease virus PLK-N-06 is taken as a template, and two pairs of primers of the designed NP and P genes are used for amplifying the NP and P genes of the virus. After the size of the PCR product is identified to be consistent with the expected size through 1% agarose gel electrophoresis, the PCR product is recovered and purified according to the instructions of the gel recovery kit. Cloning the recovered DNA fragment to pCI-neo vector, transforming into Escherichia coli DH5 alpha competent cell, screening positive clone, enzyme cutting to identify correct plasmid named pCI-NP and pCI-P, and sequencing.
The cDNA of the Newcastle disease virus PLK-N-06 is used as a template, and three pairs of primers of the designed L gene are used for amplifying virus L1, L2 and L3 fragments. After the size of the PCR product is identified to be consistent with the expected size through 1% agarose gel electrophoresis, the PCR product is recovered and purified according to the instructions of the gel recovery kit. The recovered L1 fragment was digested with NheI and SalI, and the recovered L1 fragment was ligated with the pCI-neo vector digested with NheI and SalI, followed by transformation to obtain plasmid pCI-L1. The recovered L2 fragment was digested with Bsu36I and SalI, and then the recovered L2 fragment was ligated with the pCI-L1 vector digested with the same restriction enzymes, followed by transformation to obtain plasmid pCI-L12. And (3) carrying out double digestion on the recovered L3 fragment by MluI and SalI, recovering the L3 fragment, connecting the recovered L3 fragment with a pCI-L12 vector subjected to the same double digestion, transforming to finally obtain a plasmid pCI-L123, namely pCI-L, and carrying out sequencing analysis.
The sequences of the primers used were as follows:
NP-F：5’-TATTGAATTCGCCACCATGTCGTCTGTTTTCGACGAAT-3’
NP-R：5’-TATAGTCGACTCAGTACCCCCAGTCAGTGTCGTT-3’
P-F：5’-TATTGAATTCGCCACCATGGCTACTTTTACAGATGCGGA-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’。
example 4: rescue of newcastle disease virus attenuated strain N7 a:
utilizing two pairs of primers of PV2-F, P1, P2 and PV3-R, and mutating F gene cleavage site of Newcastle disease virus PLK-N-06 into low virulent strain characteristic by using Overlap PCR method112G-R-Q-G-R-L117. FIG. 2 shows the nucleotide sequence changes before and after mutating the F gene cleavage site and the corresponding amino acid sequence changes.
The mutant primer sequences were as follows:
PV2-F：5’-CCGGAGCTCATCTCCTTACGTGACACA-3’
P1：5’-AAGGCGTCCTTGTCTCCCTCCTCCGGACGTGGACACA-3’
P2：5’-GAGGGAGACAAGGACGCCTTATAGGTGCTGTTATT-3’
PV3-R：5’-CGGGGTACCAGATCACAGTCGATAT-3’。
the expression vector pCMV-FL-N7a containing the mutated full-length cDNA of the Newcastle disease virus PLK-N-06 is subjected to enzyme digestion identification by using restriction enzyme, and an electrophoresis diagram of a product after enzyme digestion is shown in figure 3, wherein a Lane 1 is a product after enzyme digestion by using XhoI, a Lane M is a molecular Marker, and a Lane 2 is a product after enzyme digestion by BglII.
BHK-21 cells were plated in a six-well plate to grow to 80% -90% monolayers, cells were gently washed 3 times with PBS before transfection, DMEM medium was changed to Opti-MEM medium, and a total of 6. mu.g of full-length plasmid, pCI-NP, pCI-P, and pCI-L were transfected with transfection reagents
2000 Co-transfection of B in the ratio 2: 1And (3) slightly washing the HK-21 cells for 6 hours by using PBS (phosphate buffer solution) for 3 times, changing the solution, adding TPCK-trypsin (1 mu g/ml) into an Opti-MEM culture medium, uniformly mixing, and continuously incubating for 3-4 days. The culture solution and cells were harvested, frozen and thawed repeatedly for 3 times, centrifuged, and the supernatant was inoculated to 10-day-old SPF chick embryos, 0.2ml per embryo. And (3) continuously incubating at 37 ℃, checking for 1-2 times per day by taking eggs, selecting dead chick embryos after inoculation for 72-120 hours and alive chick embryos after inoculation for 120 hours, and harvesting chick embryo liquid which is named as N7a strain.
Example 5: RT-PCR identification of the attenuated strain N7a of Newcastle disease virus:
extracting RNA of virus liquid of a Newcastle disease virus attenuated strain N7a, carrying out reverse transcription, amplifying by using PV3-F and PV3-R in the construction of a full-length infectious clone pCMV-FL, and sequencing, wherein the full-length of the F gene is contained, the sequencing result of an amplified fragment shows that the cleavage site of the F gene is mutated successfully, and the sequencing result is shown in figure 4. The primer sequence for amplifying the F gene in the attenuated strain N7a of the Newcastle disease virus is as follows:
PV2-F：5’-CCGGAGCTCATCTCCTTACGTGACACA-3’
PV3-R：5’-CGGGGTACCAGATCACAGTCGATAT-3’
example 6: determination of erythrocyte agglutination value and virus content of Newcastle disease virus attenuated strain N7a
The HA titer of the N7a strain virus strain is determined according to the appendix of the current Chinese veterinary pharmacopoeia. Diluting the N7a strain with sterilized normal saline solution by 10 times, and collecting 10-7、10-8、10-9And 10-104 dilutions, inoculating 5 SPF (specific pathogen free) chick embryos of 10 days old through allantoic cavities, placing each embryo with 0.1ml at 36-37 ℃ for continuous incubation, discarding the chick embryos dead before 48 hours, taking out the chick embryos dead after 48-120 hours at any time, placing at 2-8 ℃ for 120 hours, taking out all live embryos, determining the agglutination value of red blood cells one by one, judging the chick embryos dead after 48-120 hours and alive after 120 hours are infected, calculating EID (EID), and determining the number of red blood cells by one, wherein the agglutination value is not less than 1: 128 (micro method)50. The results are shown in Table 1.
TABLE 1 measurement results of HA potency and virus content of Newcastle disease virus N7a strain
| Viral strains | HA potency | Viral content (lgEID)50/0.1ml) |
| |
1∶512 | 9.5 |
The result shows that the N7a Newcastle disease virus attenuated vaccine strain has high growth titer and good biological property.
Example 7: determination of Newcastle disease Virus-attenuated strain N7a on the intracisternal pathogenicity index (ICPI) and on the mean death time (MDT/MLD) of the minimum lethal dose of chicken embryos:
the ICPI of the N7a strain virus strain was measured according to the appendix of veterinary biological product protocol (good year version). The average death time (MDT/MLD) of the minimum lethal dose of chicken embryos was measured in accordance with the appendix of veterinary biological products of the people's republic of China (good quality year edition) in the two-good quality, respectively, for the MDT/MLD of the N7a strain of virus.
The results show that the ICPI value of the Newcastle disease virus attenuated strain N7a strain is 0.25, and the MDT/MLD is 112 respectively, which accords with the characteristics of an attenuated strain.
Example 8: specificity of newcastle disease virus attenuated strain N7 a:
(1) HI test
Respectively preparing 4 units of antigen from N7a strains of virus seeds according to HA titer, performing HI test on NDV positive serum, EDSV positive serum, AIV (subtype H9) positive serum, AIV (subtype H7) positive serum and AIV (subtype H5) positive serum according to the appendix of the current Chinese veterinary pharmacopoeia, and determining HI titer of each serum.
The hemagglutination activity of the N7a strain virus strain can be only inhibited by positive serum of the newcastle disease virus by HI test determination, which shows that the rescued N7a strain is the newcastle disease virus and has no other hemagglutination virus. The results are shown in Table 2.
TABLE 2 HI test results of recombinant Newcastle disease Virus N7a strain
(2) Chick embryo neutralization test
Diluting the N7a strain seed virus to 10% with sterile normal saline5.0EID500.1ml, mixing with equal amount of anti-newcastle disease virus positive serum, neutralizing at room temperature for 1 hour, inoculating 10 SPF embryos of 10 days old through allantoic cavities, each embryo is 0.2ml, incubating at 37 ℃ for 120 hours, observing and recording the result.
The recombinant Newcastle disease virus N7a strain virus seeds are respectively neutralized with anti-Newcastle disease virus positive serum, all chicken embryos survive in 10/10 hours after being inoculated with chicken embryos, and chicken embryo solution has no hemagglutination. Indicating that the virus seeds can be completely neutralized by positive serum of the newcastle disease. The results are shown in Table 3.
TABLE 3 chick embryo neutralization test results for testing the specificity of the recombinant Newcastle disease Virus N7a strain
| Test items | N7a strain |
| Survival rate of chick embryo | 10/10 |
| Hemagglutination of chick embryo fluid | 10/10(－) |
Example 9 preparation of N7a Strain Newcastle disease Virus inactivated vaccine
The NDV N7a strain was diluted 10,000 times with sterile physiological saline, and 20 SPF embryos of 10 days old were inoculated, 0.1ml of each embryo was inoculated, and incubation was continued at 37 ℃. Discarding the dead embryos within 24h after inoculation, immediately placing the dead embryos for 24 h-120 h at 4 ℃, collecting the mixed sample for 120h, and measuring HA and EID of the prepared vaccine virus509.6 and 10, respectively9.5EID500.1 ml. Introducing the newcastle disease virus liquid with the determined titer into an inactivation tank, metering and adding 10% formaldehyde solution, starting a stirrer to stir so as to fully mix the solution, wherein the final concentration of formaldehyde is 0.1%, and inactivating the solution for 16 hours at 37 ℃.
According to the component ratio shown in the table 4, the virus liquid which is completely inactivated is slowly added into the white oil adjuvant, a motor is started at the same time, the mixture is stirred for 30-40 minutes at 4000r/min, and a 1% thimerosal solution is added before the stirring is stopped, so that the final concentration of the thimerosal solution is 0.01%.
TABLE 4 inactivated vaccine ratio of N7a strain Newcastle disease virus
| | Vaccine | 1 | |
| Strain N7a antigen (EID)50/0.1ml) | 108.0 | 109.0 | |
| White oil adjuvant (V/V) | 60％ | 60％ |
Example 10 efficacy test of N7a Strain Newcastle disease Virus inactivated vaccine
50 SPF chickens of 30 days old were divided into 5 groups of 10, groups 1 and 2 were each administered by breast intramuscular injection of vaccine 1 prepared in EXAMPLE 9, groups 3 and 4 were each administered by breast intramuscular injection of vaccine 2 prepared in EXAMPLE 9, at an immunization dose of 20. mu.l, and group 5 was not immunized as a control. All test chickens were kept separately, immunized for 21 days, and serum was isolated and measured for HI antibody titer along with the control group blood, while HN1101 strain 10 was used in group 1, group 3 and group 55EID50Dose intramuscular challenge, PLK-N-06 strains 10 in groups 2, 4 and 65EID50Intramuscular challenge/dose, observed for 14 days, and the number of morbidity, mortality, and protection recorded. The results are shown in Table 5.
TABLE 5 test results of efficacy of N7a inactivated Newcastle disease Virus vaccine
Note: HI antibodies were determined as geometric means of the immunized chicken antibodies, indicated as X ± SD, with X representing the mean and SD representing the standard deviation.
The results show that vaccine 1 and vaccine 2 both produced higher antibodies 21 days after immunization and that both groups were fully protective against virulent challenge compared to the control.
Proves that the vaccine 1 and the vaccine 2 can protect the virulent attack of lethal dose, and the content of the vaccine is not less than 108.0EID500.1ml provides complete protection for the chicken flocks.
Example 11 preparation of La Sota Strain Newcastle disease Virus inactivated vaccine
La Sota strain virus seeds are taken to prepare the La Sota strain Newcastle disease virus inactivated vaccine according to the preparation method of the example 9, and the content of the vaccine components is shown in the table 6.
TABLE 6 La Sota strain Newcastle disease inactivated vaccine ratio
| Components | Vaccine 3 |
| La Sota(EID50/0.1ml) | 108.0 |
| White oil adjuvant (V/V) | 60％ |
Example 12 comparative experiment on immunogenicity of inactivated vaccine against N7a Newcastle disease Virus
21-28 day-old SPF chickens were immunized with vaccine 1 prepared in example 9 and vaccine 3 prepared in example 11, 20 chickens were immunized with vaccine 1, 20 chickens were immunized with vaccine 3, 20. mu.l/chicken, 40 chickens were used in total, 10 chickens were used in the control group, and serum was isolated by intramuscular injection into the breast, and all chickens were bled 21 days before and after immunization and 28 days after immunization, and HI antibody titer measurement (autoantigen) was performed. 28 days after immunization, HN1101 strain and F48E9 were used for challenge, and the challenge dose was 105ELD50Dropping nose and eyes to attack virus, observing every day after attacking virus, observing for 14 days, recording appetite, excrement and disease symptoms and death of chickens in an immunization group and an attack control group, respectively collecting a larynx and a cloaca swab of each chicken in the immunization group on the 5 th day after attacking virus, respectively connecting all the larynx and cloaca swabs with embryos, respectively connecting 3 embryos of each swab with the embryos to perform virus separation, wherein the chick embryo fluid HA is more than 1: and 16, the virus is judged to be positive. The negative samples need to be judged after blind generation. The experimental groupings are shown in Table 7.
TABLE 7 grouping of the immunogenicity control experiments for the N7a inactivated Newcastle disease Virus vaccine
The HI antibody average value of the vaccine 1 immunization group from 21 days to 28 days after immunization reaches more than 1: 64, and the ratio of the control group chicken is not higher than 1: 4. The result of serological method test of the vaccine reaches the quality standard of the similar vaccine, and the HI antibody generated by the vaccine 1 to the self-antigen 28 days after immunization is higher than that of the vaccine 3. The specific results are shown in Table 8.
TABLE 8 comparison of HI antibody detection 7, 14, 21 and 28 days after immunization
After each immune group is observed for 14 days after virus challenge, each immune group has no morbidity or mortality and reaches the quality standard of the current NDV vaccine. Collecting laryngeal cotton swabs and cloaca cotton swabs on the 5 th day after the detoxification, respectively carrying out virus separation detection and detoxification, and carrying out blind transmission on negative samples for one generation. As a result, no detoxification was observed in each of the N7a strain immunized groups. The two swab samples of the control group live chickens are positive in detoxification. The results are shown in tables 9 and 10.
TABLE 9 comparative test toxicity challenge protection results
TABLE 10 comparative experiment for toxin expelling of larynx and cloaca on day 5 of challenge
Note: "/" indicates that the item is nothing; "+ A" indicates positive detoxification of larynx and cloaca; "-" indicates negative expulsion of both laryngeal and cloaca.
And (4) conclusion: the antibody can be detected 7 days after immunization, the antibody titer is higher 21 days after immunization, NDV N7a is higher than that of a La Sota antibody of a classical vaccine strain, the attack of gene VII type wild virus and standard virulent F48E9 can be completely protected after NDV N7a immunization, the vaccine composition can not only prevent the infection of gene VII type NDV but also effectively immunize classical NDV, and has higher protection rate than that of a La Sota immune group, compared with the attack of the La Sota immune group, the toxin expelling can not be ensured to be detected, and the vaccine composition has an important effect on the prevention of the outbreak of atypical newcastle disease. The vaccine composition can protect chicken flocks more comprehensively and is more beneficial to biological safety.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (15)
1. A vaccine composition comprising an immunizing amount of an antigen of genotype VII Newcastle disease virus attenuated strain N7a or a culture thereof and a pharmaceutical carrier; the antigen of the gene VII type Newcastle disease virus low virulent strain N7a or the culture thereof is inactivated whole virus antigen, and the preservation number of the Newcastle disease virus low virulent strain N7a is CCTCC NO. V201545; the culture of the gene VII type Newcastle disease virus low virulent strain N7a is a culture of 1-50 generations.
2. The vaccine composition according to claim 1, wherein the genotype VII Newcastle disease virus low virulent strain N7a is cultured for 1-40 generations.
3. The vaccine composition according to claim 1, wherein the culture of the genotype VII Newcastle disease virus low virulent strain N7a is a culture of 1-35 generations.
4. The vaccine composition according to claim 1, wherein the genotype VII Newcastle disease virus low virulent strain N7a is cultured for 1-30 generations.
5. The vaccine composition according to claim 1, wherein the genotype VII Newcastle disease virus low virulent strain N7a is cultured for 1-25 generations.
6. The vaccine composition according to claim 1, wherein the genotype VII Newcastle disease virus low virulent strain N7a is cultured in 1-20 generations.
7. The vaccine composition according to claim 1, wherein the culture of the genotype VII Newcastle disease virus low virulent strain N7a is a culture of 1-16 generations.
8. The vaccine composition according to claim 1, wherein the inactivated whole virus antigen content of the gene VII type Newcastle disease virus attenuated strain is not less than 10 before inactivation8.0EID50/0.1ml。
9. The vaccine composition according to claim 1, wherein the inactivated whole virus antigen content of the gene VII type Newcastle disease virus attenuated strain is 10 before inactivation8.0EID50/0.1ml～109.0EID50/0.1ml。
10. The vaccine composition of claim 1, wherein the pharmaceutically acceptable carrier comprises an adjuvant comprising (1) aluminum hydroxide, saponin, avridine, DDA, (2) polymers of acrylic or methacrylic acid, maleic anhydride, and alkenyl derivatives, or (3) oil-in-water, water-in-oil, or water-in-oil-in-water emulsions, the adjuvant being in a concentration ranging from 10% to 70% V/V.
11. The vaccine composition of claim 10, wherein the adjuvant comprises (1) the saponin QuilA; (2) carbomers which are the cross-linked products of a polymer of acrylic or methacrylic acid and a polyalkenyl ether of a sugar or polyol; or (3) the adjuvant comprises an emulsion based on light liquid paraffin oil, isoprenoid oil; oils resulting from the oligomerization of olefins, esters of acids or alcohols with linear alkyl groups; esters of branched fatty acid esters or alcohols, oils and emulsifiers are used together to form emulsions; ethers of fatty alcohols and polyhydric alcohols, polyoxypropylene-polyoxyethylene block copolymers.
12. The vaccine composition of claim 11, wherein the isoprenoid oil is squalane or squalene; the olefin is isobutene or decene; the acid or alcohol forming ester with a linear alkyl group is a vegetable oil, ethyl oleate, propylene glycol di (caprylate/caprate), glycerol tri (caprylate/caprate), or propylene glycol dioleate; the ester of branched fatty acid or alcohol is isostearic acid ester, and the emulsifier is nonionic surfactant; the branched fatty acid ester or ester of an alcohol is an ester of a polyoxyethylated fatty acid, sorbitan, mannitol, glycerol, polyglycerol, propylene glycol and ethoxylated oleic, isostearic, ricinoleic, hydroxystearic acid; the fatty alcohol and the polyhydric alcohol are oleyl alcohol; the polyoxypropylene-polyoxyethylene block copolymer is Pluronic R.
13. The vaccine composition of claim 12, wherein the polyoxyethylated fatty acid is oleic acid; the ester formed from mannitol is anhydromannitol oleate; the polyoxypropylene-polyoxyethylene block copolymer is L121.
14. The vaccine composition according to claim 10, wherein the concentration of the adjuvant ranges from 30% to 60% V/V.
15. The vaccine composition of claim 10, wherein the adjuvant is at a concentration of 60% V/V.
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| CN101182494B (en) * | 2007-09-05 | 2010-09-08 | 扬州大学 | Gene VII type new castle disease virus weakening strain A-NDV-VII and construction method thereof |
| CN103525777B (en) * | 2013-07-01 | 2016-05-18 | 中国农业科学院兰州兽医研究所 | Attenuated vaccine strain of VII type NDV L gene mutation and preparation method thereof |
| CN104962526B (en) * | 2013-09-30 | 2018-01-02 | 中国农业科学院兰州兽医研究所 | Attenuated vaccine strain of VII type NDV L gene mutations and preparation method thereof |
| CN104195116B (en) * | 2014-08-13 | 2018-03-06 | 吉林大学 | A kind of recombinant Newcastle disease virus and its construction method for expressing goose parvovirus VP3 genes |
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