WO2019069720A1 - Paramyxovirus aviaire recombinant - Google Patents

Paramyxovirus aviaire recombinant Download PDF

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WO2019069720A1
WO2019069720A1 PCT/JP2018/035085 JP2018035085W WO2019069720A1 WO 2019069720 A1 WO2019069720 A1 WO 2019069720A1 JP 2018035085 W JP2018035085 W JP 2018035085W WO 2019069720 A1 WO2019069720 A1 WO 2019069720A1
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avian paramyxovirus
nucleotide
sequence
recombinant
paramyxovirus
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PCT/JP2018/035085
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English (en)
Japanese (ja)
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良太 常國
岳彦 西藤
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国立研究開発法人農業・食品産業技術総合研究機構
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Priority to JP2019510375A priority Critical patent/JP6573746B1/ja
Publication of WO2019069720A1 publication Critical patent/WO2019069720A1/fr

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    • 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
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses

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  • the present invention relates to a recombinant avian paramyxovirus and a composition for a vaccine comprising the virus as an active ingredient.
  • the present invention also relates to a nucleotide construct for producing the virus, a kit for producing the virus comprising the construct, and a method for producing the virus.
  • Influenza is an acute infection accompanied by symptoms of upper respiratory tract inflammation, respiratory disease and the like caused by influenza virus, and if the infectivity is strong, the symptoms of the infected person may be severe and may lead to death.
  • influenza virus The natural host of influenza virus is mainly wild birds such as waterfowl, and while the epidemic spreads among birds including chicken which is poultry, the virus series evolves and becomes acclimated to various hosts (pig, human) And interspecies transmission will occur.
  • HPAI highly pathogenic avian influenza virus
  • HPAIV highly pathogenic avian influenza virus
  • HPAIV highly pathogenic avian influenza virus
  • a recombinant vaccine having a Newcastle disease (ND) virus (NDV) as a vector As a vaccine against HPAIV, a recombinant vaccine having a Newcastle disease (ND) virus (NDV) as a vector has been developed.
  • ND Newcastle disease
  • a recombinant vaccine (recombinant virus) in which an antigen gene such as the hemagglutinin (HA) gene of HPAIV has been inserted into the virus enables labor-saving vaccination such as drinking water and spray administration, and, like HPAIV, to respiratory mucous membranes. There is an advantage such as induction of local immunity can be expected because of infection.
  • the recombinant virus based on NDV is a transcription comprising the gene initiation sequence and gene termination sequence of NDV endogenous and the antigen gene in the 3 'untranslated region in any transcription unit in NDV. It is produced by inserting a unit (non-patent documents 1 to 5). Furthermore, in these recombinant viruses, a Kozak sequence known as a sequence that improves translation efficiency in eukaryotic cells is inserted immediately before the start codon of the antigen gene (Non-patent Documents 1 and 3), which is an antigen. It is assumed that the translation efficiency of gene mRNA is enhanced.
  • the present inventors recombine using as a vector another virus (avian paramyxovirus, APMV) belonging to the same genus as the virus (paramyxoviridae Abravirus genus).
  • APMV avian paramyxovirus
  • APMV-2, APMV-6 and APMV-10 APMV-2, APMV-6 and APMV-10
  • a recombinant vaccine APMV- in which APMV-10 is a vector, and the HA gene of HPAIV is inserted into the untranslated region between P and M genes of its genome (between the ORF of P gene and the gene termination sequence).
  • 10 / HA was prepared, as a result of the vaccine was inoculated into chickens immunized with ND vaccine, although certain protective effect against HPAIV infection was observed et al., the protective effect (infectious units: 10 6 EID 50 APMV-10 / After HA inoculation, the survival rate of chickens when challenged with HPAIV is as low as 25%, and it can not be said that a sufficient vaccine effect was obtained (Non-patent Document 7).
  • the present invention has been made in view of the problems of the prior art, and it is a recombinant bird para that can increase the expression effect of foreign proteins such as pathogen antigens in host cells and enhance the vaccine effect against pathogens.
  • the purpose is to provide a myxovirus.
  • the present inventors have recombinant APMV-10 (recombinant avian paramyxovirus) into which a minus strand RNA encoding a foreign transcription unit has been inserted, having the following characteristics: It came to produce.
  • the foreign transcription unit comprises a nucleotide encoding the HA protein (foreign protein) of HPAIV, which is operably linked to the gene initiation sequence and gene termination sequence of avian paramyxovirus.
  • the 5 'untranslated region of the avian paramyxovirus is inserted between the gene initiation sequence and the initiation codon of the nucleotide.
  • the 3 'untranslated region of the avian paramyxovirus is inserted between the stop codon of the nucleotide and the gene termination sequence.
  • the minus strand RNA encoding the foreign transcription unit is inserted into the minus strand RNA encoding an intergenic region between the endogenous transcription units of avian paramyxovirus.
  • the recombinant avian paramyxovirus described in Non-Patent Document 7 that is, the recombinant virus in which the foreign transcription unit is inserted into the untranslated region in the endogenous transcription unit of the bird paramyxovirus, is foreign in infected cells. Almost no protein expression was observed.
  • a recombinant virus in which an exogenous transcription unit that does not contain the untranslated region of avian paramyxovirus is inserted into the intergenic region between the endogenous transcriptional units of avian paramyxovirus, Almost no expression was observed.
  • the survival rate of chicken after HPAIV infection is 25% or 50. %Met.
  • excretion of HPAIV was observed from all individuals of the chicken.
  • antibody titers to HPAIV at challenge were below the detection limit in most chickens.
  • Antibody titers to HPAIV were below the detection limit in most chickens.
  • the survival rate of the chicken after HPAIV infection was 100%.
  • almost no excretion of HPAIV was observed in the chicken, and even if it was, its virus titer was low.
  • antibody titers to HPAIV at the time of challenge were detected in many individuals immunized with the recombinant virus.
  • the present invention relates to a recombinant avian paramyxovirus and a composition for a vaccine comprising the active ingredient of the virus. Furthermore, the present invention relates to a nucleotide construct for producing the virus, a kit for producing the virus containing the construct, and a method for producing the virus using them, more specifically, I will provide a.
  • a recombinant avian paramyxovirus wherein a negative strand RNA encoding a foreign transcription unit is inserted into a negative strand RNA encoding avian paramyxovirus,
  • the translation region is linked to the gene termination sequence of avian paramyxovirus
  • a recombinant avian paramyxovirus wherein the minus strand RNA encoding the foreign transcription unit is inserted into the minus strand RNA encoding an intergenic region between endogenous transcription units of avian paramyxovirus.
  • ⁇ 2> The recombinant avian paramyxovirus according to ⁇ 1>, which is derived from avian paramyxovirus serotype 10.
  • the 5 'untranslated region is a region consisting of the nucleotide sequence of any one of SEQ ID NOs: 1 to 6, and the 3' untranslated region is described in any of SEQ ID NOs: 7 to 12
  • the recombinant avian paramyxovirus according to ⁇ 1> or ⁇ 2> which is a region consisting of the nucleotide sequence of ⁇ 4>
  • ⁇ 5> The recombinant avian paramyxovirus according to any one of ⁇ 1> to ⁇ 4>, wherein the foreign protein is a hemagglutinin protein of influenza virus.
  • ⁇ 6> A composition for vaccine comprising the recombinant avian paramyxovirus according to any one of ⁇ 1> to ⁇ 5> as an active ingredient.
  • the 5 'untranslated region is a region consisting of the nucleotide sequence of any one of SEQ ID NOs: 1 to 6, and the 3' untranslated region is described in any of SEQ ID NOs: 7 to 12
  • the nucleotide construct according to ⁇ 7> or ⁇ 8> which is a region consisting of the nucleotide sequence of ⁇ 10>
  • a promoter sequence recognized by a DNA-dependent RNA polymerase is linked to the 5 'end of the nucleotide encoding the avian paramyxovirus, and from the 5' end to the 3 'end of the nucleotide,
  • nucleotide construct according to ⁇ 10> wherein a nucleotide encoding a foreign protein is inserted into the site.
  • nucleotide construct according to ⁇ 11> wherein the foreign protein is an antigen protein derived from a pathogen.
  • nucleotide construct according to ⁇ 11>, wherein the foreign protein is a hemagglutinin protein of influenza virus.
  • ⁇ 14> A method for producing the recombinant avian paramyxovirus according to any one of ⁇ 1> to ⁇ 5>, Introducing the nucleotide construct according to any one of ⁇ 11> to ⁇ 13> into a transformed cell that expresses the DNA-dependent RNA polymerase and the core protein of the avian paramyxovirus; Recovering the recombinant avian paramyxovirus from the culture of the cells.
  • the recombinant bird paramyxo according to any one of ⁇ 1> to ⁇ 5>, which comprises at least one substance selected from the group consisting of the following (a) to (e) and instructions for use:
  • a kit for producing a virus (a) The nucleotide construct according to any one of ⁇ 10> to ⁇ 13> (b) the nucleotide construct capable of expressing the DNA-dependent RNA polymerase (c) the above Nucleotide constructs capable of expressing the core protein of avian paramyxovirus (d) Cells for introducing at least one nucleotide construct selected from the group consisting of (a) to (c) (e) said DNA dependence A transformed cell which expresses RNA polymerase and / or the core protein of the avian paramyxovirus.
  • the "avian paramyxovirus” which is the basis of the recombinant virus of the present invention is a virus belonging to the mononegavirus order, paramyxoviridae, Abravirus genus, avian paramyxovirus species, and a single strand of minus strand
  • the RNA is maintained as a genome.
  • the avian paramyxovirus RNA genome has a leader sequence located at the 3 'end, a trailer sequence located at the 5' end, and six major transcription units (NP, P, M, And a negative strand RNA encoding a transcription unit of F, HN and L proteins (note that in APMV-6, it further has a transcription unit of SH protein between F and HN).
  • proteins encoded by them three proteins (core proteins), phosphoprotein (P) that constitutes nucleoprotein (NP) and RNA-dependent RNA polymerase, and core protein, together with genomic RNA To form a complex (ribonucleoprotein complex, RNP complex) which is an autonomously replicable replicon.
  • the aforementioned RNP complex by an envelope composed of matrix (M) proteins and transmembrane glycoproteins (HN and F proteins) that play a role in virus assembly, budding and cell attachment and / or invasion of the virus.
  • M matrix
  • HN and F proteins transmembrane glycoproteins
  • Negative strand RNAs encoding each transcription unit are, in order from the 3 'end, negative strand RNA encoding a gene start sequence (GS: gene start), negative strand RNA encoding a 5' untranslated region, translation region ( A negative strand RNA encoding an ORF (open reading frame) and a negative strand RNA encoding a 3 'untranslated region are configured by being arranged in tandem. Furthermore, transcription units arranged in series are all separated by an intergenic region (IGR) that is not transcribed.
  • IGR intergenic region
  • the present invention it is possible to increase the expression level of foreign proteins such as pathogen antigens in the host and thus to enhance the vaccine effect against pathogens.
  • foreign proteins such as pathogen antigens
  • NDV Newcastle disease virus
  • FIG. 1 is a schematic view showing an outline of a vector for transcribing avian paramyxovirus (APMV-10) genomic RNA.
  • APMV-10 genome in which the foreign transcription unit is inserted between the transcription unit of P gene (gene termination sequence: GE) and the transcription unit of M gene (gene initiation sequence: GS), and the foreign transcription unit (in the figure, control)
  • FIG. 6 is a schematic view showing an outline of the embodiment to L-NCR).
  • FIG. 6 is a schematic view showing an outline of APMV-10 genome in which a foreign transcription unit is inserted into the non-translated region (P NCR) of P gene.
  • FIG. 2 is a schematic view showing an outline of a vector for expressing NPMV, P protein or L protein of APMV-10.
  • FIG. 5 is a schematic view showing a process of replicating recombinant avian paramyxovirus (such as pAPMV10 / HA) in cultured cells. It is a graph which shows the expression level (HA protein) of the foreign protein which each virus encodes in the host cell which infected the recombinant avian paramyxovirus.
  • the horizontal axis shows the recombinant avian paramyxovirus infected with host cells (see FIGS. 2 and 3).
  • the expression level of HA protein corrected by the expression level of actin protein in each host cell is further expressed as a relative value based on that in control.
  • the horizontal axis indicates the time after inoculation of each recombinant avian paramyxovirus into the chorioallantoic cavity of embryonated chicken eggs, and the vertical axis indicates the virus titer in the collected chorioallantoic fluid.
  • APMV-10 genome in which the foreign transcription unit is inserted between the transcription unit of F gene (gene termination sequence: GE) and the transcription unit of HN gene (gene initiation sequence: GS), and the foreign transcription unit (FHN in the figure) -HA to FHN-M-NCR) is a schematic view showing an outline. It is a graph which shows the expression level (HA protein) of the foreign protein which each virus encodes in the host cell which infected the recombinant avian paramyxovirus. In the figure, the horizontal axis shows a recombinant avian paramyxovirus infected with host cells (see FIG. 8).
  • FIG. 5 is a schematic view showing an outline of a foreign transcription unit encoding a luciferase gene, each inserted into minigenome transcription plasmids (Luc-F-NCR, Luc-HN-NCR and Luc-blank) mimicking the APMV-10 genome.
  • FIG. 5 is a schematic view showing an outline of a foreign transcription unit encoding a luciferase gene, each inserted into minigenome transcription plasmids (Luc-F-NCR, Luc-HN-NCR and Luc-blank) mimicking the APMV-10 genome.
  • FIG. 16 is a graph showing the results of measuring the luciferase activity in the culture solution of cultured cells into which minigenome transcription plasmids (Luc-F-NCR, Luc-HN-NCR and Luc-blank) mimicking the APMV-10 genome were respectively introduced.
  • "Negative Control” indicates luciferase activity in the culture solution of cultured cells to which the minigenome has not been introduced.
  • the “luciferase activity” shown on the vertical axis indicates the ratio when the measured value in Luc-blank-introduced cells is 1.
  • the present invention is a recombinant avian paramyxovirus, wherein the negative strand RNA encoding a foreign transcription unit is inserted into the negative strand RNA encoding avian paramyxovirus, In the said foreign transcription unit, the gene start sequence of the avian paramyxovirus, the 5 'untranslated region of the avian paramyxovirus, the nucleotide encoding the foreign protein, and the 3' non-avian paramyxovirus, in order from the 5 'side The translation region is linked to the gene termination sequence of avian paramyxovirus, and The recombinant avian paramyxovirus is provided, wherein the minus strand RNA encoding the foreign transcription unit is inserted into the minus strand RNA encoding an intergenic region between the endogenous transcription units of avian paramyxovirus.
  • recombinant avian paramyxovirus is a gene engineered to be able to introduce a nucleotide encoding a foreign protein into a host cell, and further to allow the protein to be expressed in the cell.
  • the recombinant avian paramyxovirus may be a nucleocapsid consisting of genomic RNA of the virus (minus strand RNA encoding the virus) and an NP protein, and a ribonucleoprotein complex further comprising P protein and L protein ( It may be an RNP complex) and may be an enveloped virus particle.
  • avian paramyxovirus which is the basis (derived from) of the recombinant virus of the present invention is as described above, for example, as a virus belonging to avian paramyxovirus species, serotype 2 to avian paramyxovirus 15 (APMV-2 to APMV-15).
  • APMV-2, APMV-6 or APMV-10 is preferable, and APMV-10 is more preferable, from the viewpoint that the origin of the recombinant virus of the present invention infects chickens but has low pathogenicity.
  • genome sequences of avian paramyxoviruses necessary for designing and preparing the recombinant avian paramyxoviruses of the present invention are known in the art. Such information can be obtained, for example, through the website of the National Center for Biotechnology Information (NCBI), for each virus sequence information (NCBI GenBank sequence information). More specifically, representative genomic sequences of APMV-2 to APMV-15 include the following. It should be understood that the sequences provided by NCBI and the like are represented by the sequences of DNA complementary to the genomic RNA (minus strand RNA) encoding each virus. Also, since two different sequences with low homology with each other are currently registered as the genome sequence of APMV-15, they are shown together in Table 1.
  • NP transcription unit .P transcription unit M transcription unit, F transcription unit, HN transcription unit, L transcription unit, etc.
  • sequences constituting them gene initiation sequence, 5 'non The translational region, open reading frame, 3 'non-translational region, gene termination sequence
  • intergenic region separating each transcription unit are as described above.
  • These specific sequences are also known and can be obtained, for example, through the NCBI website.
  • the respective sequences of APMV-10 are shown below based on the NCBI GenBank sequence information exemplified in Table 1. In the table, the IGR in the NP transcription unit is not the sequence number but the sequence itself.
  • the sequence of the virus can be easily mutated in nature. Therefore, it is to be understood that the sequence is not specified in the representative sequences shown in Tables 1 and 2 but also includes naturally mutated sequences.
  • the recombinant avian paramyxovirus may consist of the entire genome of the avian paramyxovirus, and a part of the genome (for example, an NP transcription unit, a P transcription unit, an M transcription unit, It may be one in which at least one transcription unit selected from the group consisting of F transcription unit, SH transcription unit, HN transcription unit and L transcription unit is deleted (so-called minigenome).
  • the "foreign transcription unit” comprises, in order from the 5 'side, the gene initiation sequence of the avian paramyxovirus, the 5' untranslated region of the avian paramyxovirus, the nucleotide encoding the foreign protein, and the avian paramyxo It is a transcription unit in which the 3 'untranslated region of the virus and the gene termination sequence of avian paramyxovirus are linked.
  • Binding between these sequences and regions may be direct binding or indirect binding via other RNA sequences (usually 1 to 20 RNAs).
  • the "foreign protein” is usually a protein which is not originally present as a component of avian paramyxovirus, and is desired to be expressed in the cell when the recombinant avian paramyxovirus of the present invention is infected into a host cell. If it is a protein, there is no restriction
  • gene transcription sequences, 5 'untranslated regions, 3' untranslated regions, and gene termination sequences contained in the foreign transcription unit include endogenous transcription units (NP, P, M, F) of avian paramyxovirus. , HN, L, etc.), and typically includes each sequence described in NCBI GenBank information shown in Table 1 (refer to Table 2 for each sequence of APMV-10). ). Furthermore, the sequence of the virus can be easily mutated in nature. Therefore, it is to be understood that each of the above-mentioned sequences is not typically specified as the above sequence, but also includes naturally mutated sequences.
  • each of the sequences contained in the foreign transcription unit may be substituted, deleted, inserted, and / or added with one or more nucleotides in each sequence as long as the foreign protein can be expressed from the recombinant avian paramyxovirus of the present invention.
  • sequence having high homology with each sequence refers to the number of nucleotides substituted or the like in a range capable of expressing a foreign protein, preferably 30% or less of the number of the sequence to be substituted or the like, more preferably 20% or less of the sequence The number is more preferably 10% or less in the sequence.
  • “high homology” means, for example, 60% or more homology, preferably 70% or more homology, more preferably 80% or more homology, still more preferably 90% or more homology (eg, 95%) (97% or more, 99% or more homology).
  • Sequence homology can be determined using the BLASTN (nucleic acid level) program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The specific method of the analysis method using such a program is known and can be analyzed using default parameters.
  • the origin of each of the sequences contained in the foreign transcription unit may be different from the type of virus that is the basis of the recombinant avian paramyxovirus, as long as it is derived from the avian paramyxovirus (for example, the origin of the sequence is
  • the virus which is AMPV-2 and the base of the recombinant avian paramyxovirus may be AMPV-10), preferably it is derived from the same avian paramyxovirus, and both may be derived from AMPV-10 More preferable.
  • the avian paramyxoviruses from which the aforementioned sequences are derived may also differ from each other (for example, the gene initiation sequence is derived from AMPV-2 and the 5 'untranslated region is derived from AMPV-10) However, it is preferable that all are derived from the same avian paramyxovirus, and it is more preferable that all be derived from AMPV-10.
  • sequences contained in the foreign transcription unit may be different from each other as long as they are derived from the endogenous transcription unit of avian paramyxovirus (eg, the gene initiation sequence is derived from the NP transcription unit, The 5 'untranslated region may be derived from a P transcription unit), preferably all these sequences are from the same endogenous transcription unit.
  • each of the sequences when each of the sequences is derived from an endogenous transcription unit of AMPV-10, it contains at least the sequence described in any one of SEQ ID NOs: 1 to 6 as a 3 'untranslated region, and A foreign transcription unit comprising the sequence described in any of SEQ ID NOs: 7 to 12 as the 5 'non-translated region, and the sequence described in any of SEQ ID NOs: 13 to 15 as a gene initiation sequence
  • the 3 'untranslated region includes the sequence described in any of SEQ ID NOs: 1 to 6
  • the 5' untranslated region includes the sequence described in any of SEQ ID NOs: 7 to 12, and gene termination It is a foreign transcription unit comprising the sequence set forth in any of SEQ ID NOs: 16-18 as a sequence.
  • a foreign transcription unit (a foreign transcription unit comprising a non-translated region derived from an NP transcription unit of AMPV-10) comprising at least the sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 7, at least SEQ ID NO: 2 and A foreign transcription unit comprising the sequence set forth in SEQ ID NO: 8 (a foreign transcription unit comprising a non-translated region derived from the P transcription unit of AMPV-10), a foreign body comprising at least the sequence set forth in SEQ ID NO: 3 and SEQ ID NO: 9
  • a transcription unit foreign transcription unit comprising a non-translated region derived from the M transcription unit of AMPV-10
  • a foreign transcription unit comprising at least the sequence set forth in SEQ ID NO: 4 and SEQ ID NO: 10 (from the F transcription unit for AMPV-10)
  • a translational transcription unit comprising a non-translational region of SEQ ID NO: 5 and at least a sequence as set forth in SEQ ID NO: 5 and SEQ ID NO: 11 A
  • the foreign transcription unit comprising the non-translated region derived from the NP transcription unit of the AMPV-10 or the AMPV-10
  • a foreign transcription unit comprising a non-translational region derived from the M transcription unit is preferred, and a foreign transcription unit comprising each sequence derived from the NP transcription unit of the AMPV-10 or a foreign transcription comprising each sequence derived from the M transcription unit of the AMPV-10 Units are more preferred.
  • a foreign transcription unit including a non-translated region derived from the L transcription unit of AMPV-10 is preferable, and the L transcription unit of AMPV-10 is preferable.
  • Foreign transcription units comprising each sequence derived from are more preferred.
  • a foreign transcription unit containing a non-translated region derived from the HN transcription unit of AMPV-10 is preferable, and the HN transcription unit of AMPV-10 is preferable.
  • Foreign transcription units comprising each sequence derived from are more preferred.
  • the above-mentioned sequences contained in the endogenous transcription unit of avian paramyxovirus and the nucleotide encoding the foreign protein are operably linked so as to express the protein.
  • the genome sequence of paramyxovirus is a multiple of 6
  • the number of nucleotides constituting the exogenous transcription unit according to the present invention is 6 from the viewpoint that replication (RNA synthesis) is efficiently performed. It is preferable that the number is a multiple, and it is more preferable that the number of nucleotides constituting the recombinant avian paramyxovirus is also a multiple of 6 (for the rule of six of so-called paramyxovirus, see Kolakofsky D. et al., J. Virol., February 1998, 72 (2), pp. 891-899. Of it).
  • the minus strand RNA encoding the aforementioned foreign transcription unit is inserted into the minus strand RNA encoding an intergenic region between the endogenous transcription units of the avian paramyxovirus.
  • intergenic region into which the foreign transcription unit is inserted there is no particular limitation on the intergenic region into which the foreign transcription unit is inserted, and an intergenic region (NP-P intergenic region) between the gene termination sequence of the NP transcription unit and the gene initiation sequence of the P transcription unit, P The intergenic region (P-M intergenic region) between the gene termination sequence of transcription unit and the gene initiation sequence of M transcription unit, between the gene termination sequence of M transcription unit and the gene initiation sequence of F transcription unit
  • SH-HN intergenic region intergenic region between gene termination sequence of HN transcription unit and gene start sequence of L transcription unit
  • the P-M intergenic region is preferable from the viewpoint of better expression efficiency of the protein
  • the F-HN intergenic region is preferred from the viewpoint of better growth efficiency of the virus.
  • minus strand RNA encoding a foreign transcription unit may be inserted between the leader sequence and the trailer sequence.
  • Such a recombinant avian paramyxovirus virus can be produced, for example, by using the nucleotide construct described below, a kit containing the same, and a method using the same.
  • ⁇ Composition for vaccine> As shown in the following examples, according to the present invention, foreign proteins encoded by the virus can be highly expressed in host cells infected with the above-mentioned recombinant avian paramyxovirus. Therefore, the vaccine effect on the pathogen in the host cell can be enhanced by using the foreign protein as the antigen protein of the pathogen causing the infection.
  • the present invention provides a composition for vaccine containing the above-mentioned recombinant avian paramyxovirus as an active ingredient.
  • pathogen includes, for example, viruses, bacteria, fungi, protozoa, and more specifically, influenza virus, Marek's disease virus (MDV), infectious laryngo tracheitis virus (ILTV), infection Bronchitis Virus (IBV), Infectious Bursal Disease Virus (IBDV), Chicken Anemia Virus (CAV), Reovirus, Avian retrovirus, Poultry Adenovirus, Turkey rhinotracheitis virus (TRTV), Newcastle Disease Virus (NDV), E. coli, Mycoplasma, Salmonella, Campylobacter, Ornitobacterium, Pasteurella, Eimeria, Cryptosporidium protozoa.
  • MDV Marek's disease virus
  • ILTV infectious laryngo tracheitis virus
  • IBV Infectious Bursal Disease Virus
  • CAV Chicken Anemia Virus
  • TRTV Turkey rhinotracheitis virus
  • NDV Newcastle Disease Virus
  • E. coli Mycoplasma
  • Salmonella Campylobacter
  • the antigenic proteins of these pathogens are proteins that constitute pathogens, and there is no particular limitation as long as they are proteins that elicit the host's immune response when the pathogen is infected, for example, in influenza virus, hemagglutinin (HA precursor, H1, H2, H3, H4, H5, H7, H8, H9, H11, H12, H13, H14, H15, H16 etc.), matrix proteins (M1, M2 etc.), neuraminidase NA1, NA2, NA3, NA4, NA5, NA6, NA7, NA8, NA9 etc., non-structural proteins (NS1, NS2 etc.), nucleoprotein (NP), polymerases (PA polymerase, PB1 polymerase 1, PB2 polymerase 2 etc.) Can be mentioned.
  • HA precursor H1, H2, H3, H4, H5, H7, H8, H9, H11, H12, H13, H14, H15, H16 etc.
  • matrix proteins M1, M2 etc.
  • influenza virus according to the present invention for example, subtypes (H1 to H16, etc.) divided based on the difference in structure of hemagglutinin, subtypes further classified according to types of neuraminidase produced by the O virus N1H1H1N2H18N18H18N10H18N5H1N8H1H6N5H1N8H1H5N4H5N1; H5N8; H5N1; H5N1; H5N1; H5N1; H5N1; H5N1; H5N1; H5N1; H5N1; H5N1; H5N1; H5N1; H5N1; H5N1; H5N1; H5N1; H5N1; H9N8, H9N9 etc.) Strains classified according to separated place, separated order, separated year (isolate; A / Hong Kong / 156/1997, A / duck / Vietnam / 2/2007, A /
  • a pharmacologically acceptable carrier or vehicle may be included in addition to the above-mentioned recombinant avian paramyxovirus as the active ingredient.
  • Pharmaceutically acceptable carriers include, for example, stabilizers, excipients, preservatives, surfactants, chelating agents, binders and the like.
  • a pharmacologically acceptable medium for example, water, physiological saline, phosphate buffer can be mentioned.
  • the form of the vaccine composition is not particularly limited, and may be, for example, in the form of a suspension or in a lyophilised form.
  • the vaccine composition of the present invention may also contain an adjuvant.
  • Adjuvants include, for example, aluminum hydroxide, aluminum phosphate, aluminum oxide, oil-in-water or water-in-oil emulsions, saponins, alum, CpG DNA.
  • the titer of the recombinant avian paramyxovirus contained in the vaccine composition of the present invention is not particularly limited, but is usually 10 5 EID 50 / individual to 10 7 EID 50 / individual, preferably 10 5.5 EID. 50 / individual to 10 6.5 EID 50 / individual, more preferably 10 6 EID 50 / individual.
  • the survival rate of chicken after HPAIV infection is 50% It is a virus having the above (more preferably 60% or more, further preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, more preferably 80% or more, particularly preferably 100%).
  • EID 50 when inoculated into embryonated chicken eggs, means a viral load of 50% is infected.
  • the present invention also provides a method of inhibiting infection or growth of a pathogen in an animal. That is, the present invention also relates to a method for suppressing infection or growth of a pathogen in an animal, comprising the step of inoculating the animal a vaccine composition containing the recombinant avian paramyxovirus of the present invention or the virus as an active ingredient. provide.
  • the "animal" to be inoculated in the present invention is not particularly limited as long as it can be infected by the recombinant avian paramyxovirus, but it is usually a bird, and examples are chicken, duck, duck, turkey, quail, pheasant, parrot , Hawks, crows, ostrich.
  • the recombinant avian paramyxovirus etc. of the present invention can be particularly suitably used for birds (such as chickens) in which an antibody against NDV is produced.
  • the "inoculation" of animals such as recombinant avian paramyxovirus can be performed using methods known in the art. Such methods include, but are not limited to, intranasal, oral, oronasal and subcutaneous and inhalation, intraocular, intradermal, intramuscular, intraperitoneal, intravenous, parenteral, suppository or transdermal . Also, such inoculation method can be carried out by using a sprayer, a spray, a spray, a syringe, a needleless injection device or a fine particle gene gun.
  • the inoculation scheme for an animal is appropriately adjusted according to the dosage form, type of animal, age and body weight, etc., and is not particularly limited as long as an immunologically effective amount can be administered to the animal, single or single It may be multiple inoculations, and may be inoculated simultaneously or sequentially.
  • the present invention also provides the following nucleotide construct useful in the production of the above-mentioned recombinant avian paramyxovirus.
  • a nucleotide construct encoding a recombinant avian paramyxovirus wherein a cloning region containing a site for inserting a nucleotide encoding a foreign protein is inserted into the nucleotide encoding avian paramyxovirus, In the cloning region, the gene start sequence of the avian paramyxovirus, the 5 'untranslated region of the avian paramyxovirus, the aforementioned site, the 3' untranslated region of the avian paramyxovirus, and the avian paramyxovirus gene start sequence from the 5 'side Is linked to the gene termination sequence of paramyxovirus, and A nucleotide construct, wherein the cloning region is inserted into a nucleotide encoding an intergenic region between the endogenous transcription units of avian paramyxovirus.
  • the cloning region is inserted between the nucleotide encoding the leader sequence and the nucleotide encoding the trailer sequence. It is also good.
  • the nucleotides constituting the nucleotide construct may be DNA (single-stranded DNA, double-stranded DNA), or may be positive-strand RNA, but are chemically stable.
  • DNA is preferred from the viewpoint of easy modification of the sequence of paramyxovirus and easy generation of recombinant avian paramyxovirus described later.
  • the nucleotide construct may, for example, take the form of a plasmid vector, a phage vector, a viral vector.
  • the “nucleotide encoding avian paramyxovirus” can be obtained, for example, as a plus-strand RNA by performing a synthesis reaction using RNA-dependent RNA polymerase, using genomic RNA (minus strand RNA) extracted from the virus as a template be able to. Also, by performing a synthesis reaction (reverse transcription reaction) using an RNA-dependent DNA polymerase using the genomic RNA (minus strand RNA) as a template, it can be obtained as a plus strand DNA and thus a double strand DNA.
  • genomic RNA extraction from viruses and synthesis methods using RNA-dependent polymerase are known.
  • the “nucleotide encoding avian paramyxovirus” may encode the full-length genomic RNA of avian paramyxovirus, and a part of the genome (eg, NP transcription unit, P transcription)
  • a unit which is deficient in at least one transcription unit selected from the group consisting of the unit, M transcription unit, F transcription unit, SH transcription unit, HN transcription unit and L transcription unit May be
  • the "cloning region" inserted into the nucleotide encoding avian paramyxovirus is for inserting the gene initiation sequence of avian paramyxovirus, the 5 'untranslated region of avian paramyxovirus, and a nucleotide encoding a foreign protein
  • the person skilled in the art can use the commercially available automatic DNA synthesizer to synthesize chemically based on the sequence of the site of 3, the untranslated region of the avian paramyxovirus 3 'and the gene termination sequence of the avian paramyxovirus it can.
  • the nucleotide encoding the prepared avian paramyxovirus is used as a template, and those skilled in the art appropriately use known genetic recombination techniques (PCR, restriction enzyme treatment, site-directed mutagenesis, etc.) as appropriate. It can also be prepared.
  • the site is not particularly limited as long as it can insert a nucleotide encoding a foreign protein, but usually a restriction enzyme recognition sequence is used, and the site is a site having a plurality of restriction enzyme recognition sequences. It may be (a so-called multi cloning site).
  • insertion of the thus obtained cloning region into the nucleotide encoding avian paramyxovirus can also be carried out using known gene recombination techniques (PCR, restriction enzyme treatment, site-directed mutagenesis, etc.) by those skilled in the art. As appropriate.
  • the nucleotide construct described above further comprises a promoter sequence recognized by a DNA-dependent RNA polymerase linked to the 5 'end of the nucleotide encoding the avian paramyxovirus, and the 3' end of the nucleotide.
  • the ribozyme sequence and the terminator sequence recognized by the DNA-dependent RNA polymerase may be linked to the side sequentially from the 5 'side.
  • the DNA-dependent RNA polymerase recognizes the promoter sequence, and the minus-strand RNA encoding the recombinant avian paramyxovirus is transcribed. Ru. Furthermore, in order for the recombinant virus to initiate an infection cycle, an exact cleavage at the 3 'end of the minus strand RNA is required, but the cleavage is achieved by self-cleavage of the ribozyme. become.
  • the binding of the promoter sequence recognized by the DNA-dependent RNA polymerase to the 5 'end of the nucleotide encoding the avian paramyxovirus can be performed as long as transcription of the negative strand RNA can be performed under the control of the promoter sequence.
  • it may be a direct bond or an indirect bond via another nucleotide.
  • the DNA-dependent RNA polymerase is not particularly limited as long as it can transcribe RNA using DNA as a template, for example, T7 RNA polymerase, T3 RNA polymerase, Sp6 RNA polymerase, RNA polymerase I, II or eukaryote-derived RNA polymerase Although III may be mentioned, T7 RNA polymerase is preferred from the viewpoint of higher transcription efficiency.
  • the ribozyme sequence disposed between the terminator of the DNA-dependent RNA polymerase and the 3 'end of the nucleotide encoding the avian paramyxovirus is preferably a ribozyme sequence having a self-cleaving activity, more preferably These include ribozyme sequences derived from Hepatitis delta virus, hammerhead ribozyme sequences, and hairpin ribozyme sequences.
  • the nucleotide construct has, in addition to the above sequences, a drug resistance gene (ampicillin resistance gene etc.), an expression control site of the gene (for example, ampicillin resistance gene) for cloning using E. coli etc. It may contain an AmpR promoter etc.), an origin of replication (f1 origin of replication, pBR322 origin of replication etc).
  • the foreign protein is as described above, and the nucleotide encoding the protein is not particularly limited, but from the viewpoint of improving the expression efficiency of the protein in the host cell, according to the usage frequency of codons in the host It is desirable that the sequence is a modified (codon-optimized) nucleotide.
  • the recombinant avian paramyxovirus of the present invention can be produced as follows.
  • nucleotide construct as described above in which a nucleotide encoding a foreign protein is inserted at the site; Recovering the recombinant avian paramyxovirus from the culture of the cells.
  • the "cell” into which the nucleotide construct described above is introduced is not particularly limited as long as it can produce the recombinant avian paramyxovirus encoded by the construct, for example, Vero cell, DF1 cell, Chicken primary cultured cells (chicken fetal fibroblast (CEF) cells, chicken fetal (CEP) cells), 293T cells, MDCK cells, MDBK cells can be mentioned, but from the viewpoint of better gene transfer efficiency and virus growth efficiency, Vero cells are preferred.
  • RNA-dependent RNA polymerase in order to express the minus strand RNA encoded by the nucleotide construct (recombinant avian paramyxovirus genomic RNA) under the control of the above-mentioned promoter and further to transcribe the plus strand RNA from the minus strand RNA, It has to be transformed so that the above-mentioned DNA-dependent RNA polymerase and the avian paramyxovirus core protein can be expressed.
  • the “core protein” may be any protein capable of transcribing positive strand RNA from the negative strand RNA, and includes phosphoprotein (P) and RNA-dependent RNA polymerase constituting nucleoprotein (NP) and RNA-dependent RNA polymerase (L) is mentioned. Also, they may be derived from avian paramyxovirus, but are preferably derived from the same recombinant avian paramyxovirus encoded by the nucleotide construct.
  • nucleotide construct a plasmid vector, a phage vector, a virus vector, etc.
  • the cells are introduced into the cells using a known method. It can be done.
  • Such transformation may occur prior to, simultaneously with, or after the introduction of the nucleotide construct encoding the recombinant avian paramyxovirus.
  • nucleotide construct there is no particular limitation on known methods for introducing the nucleotide construct into cells, and calcium phosphate method, lipofection method, DEAE dextran method, electroporation method and microinjection method can be mentioned.
  • the plus strand RNA encoding the virus is transcribed, and the virus is further used as a template.
  • the encoded viral protein and foreign protein will be expressed.
  • the negative strand RNA encoding the recombinant avian paramyxovirus is linked to the virus protein, the recombinant avian paramyxovirus will be propagated in the culture of the cells.
  • the “culture” of the cells may be any as long as it can contain a recombinant avian paramyxovirus, and the cells infected with the virus, which are obtained by culturing transformed cells in a culture medium, the secretory product of the cells And culture media containing the metabolites of the cells, etc., including dilutions and concentrates thereof.
  • virus purification / separation methods such as filtration, centrifugation, adsorption and column purification. It can be carried out by using in combination.
  • the recombinant avian paramyxovirus thus obtained is proliferated by additionally seeding cells susceptible to infection with the virus, such as primary cultured chicken cells, as shown in the following examples. You may Alternatively, they can be proliferated by inoculating embryonated eggs (for example, chicken eggs or quail eggs) instead of or in combination with such cells.
  • embryonated eggs for example, chicken eggs or quail eggs
  • recombinant avian paramyxovirus can be produced by using the nucleotide construct of the present invention. Accordingly, the present invention also provides a kit for producing the following recombinant avian paramyxovirus.
  • a kit for producing a recombinant avian paramyxovirus of the present invention which comprises at least one substance selected from the group consisting of the following (a) to (e) and instructions for use: (a) a nucleotide construct of the present invention (b A nucleotide construct capable of expressing the DNA-dependent RNA polymerase (c) A nucleotide construct capable of expressing the core protein of the avian paramyxovirus (d) selected from the group consisting of (a) to (c) A cell for introducing at least one nucleotide construct (e) A transformed cell expressing the DNA-dependent RNA polymerase and / or the core protein of the avian paramyxovirus.
  • the APMV-10 genome was divided into four fragments by RT-PCR, amplified, and cloned into pCR-XL-TOPO vector (Thermo Fisher Scientific). The vector portion of the pNDV / B1 plasmid (see Nakaya, T. et al., J Virol 75: 11868-11873.
  • NDV Newcastle disease virus
  • pSL1180 manufactured by Amersham Pharmacia Biotech
  • APMV-10 genome was excised using the In-Fusion HD cloning kit (Clontech) to assemble the vector.
  • FIG. 1 A schematic diagram of the APMV-10 genomic RNA transcription plasmid pAPMV-10 constructed in this way is shown in FIG.
  • the sequences of transcription units constituting the APMV-10 genome and the region between the transcription units (intergenic region) are shown in Table 2 above.
  • pAPMV-10 is obtained by inserting the T7 promoter sequence, the full-length genome of APMV-10, the hepatitis D virus ribozyme sequence and the T7 terminator sequence into pSL1180.
  • the hepatitis D virus ribozyme sequence is for cleaving the 3 'end of the transcribed RNA, and is not included in the recombinant viral genome.
  • a restriction enzyme (RsrII) cleavage sequence is inserted into the untranslated region (between P gene ORF and termination sequence) located between P gene and M gene. There is.
  • a foreign gene (antigen gene) amplified by PT-PCR was inserted between the PM genes of pAPMV-10 to construct a full-length APMV-10 genomic RNA transcription plasmid pAPMV-10 / HA containing the antigen gene.
  • a highly pathogenic avian influenza virus (HPAIV) HA gene optimized for chicken codons is used, and the untranslated region (NCR) in each transcription unit of APMV-10 endogenously flanked on both sides, and gene initiation at both ends The one flanked by the sequence (GS) and the gene termination sequence (GE) was used (see FIG. 2).
  • recombinant viruses (10 / HA and 10 / opHA) were prepared in which the antigen gene was inserted into the RsrII cleavage sequence located in the NCR behind the P gene.
  • the antigen gene has a 2 bp NCR before its start codon and GS, and a 113 bp NCR between the stop codon and GE, in the transcription unit of the P gene. It will be inserted.
  • HA indicates that highly pathogenic avian influenza virus (HPAIV) HA gene itself was inserted in each recombinant virus, and "opHA” is most suitable for chicken codons It shows that the transformed HPA IV HA gene was inserted in each recombinant virus.
  • recombinant viruses incorporating NCR used GS and GE corresponding to each NCR (see Table 2). Those in the transcription unit of the M gene were used as GS and GE in 10 / HA, 10 / opHA and control.
  • the NPMV gene, AP gene and AP gene of APMV-10 are amplified by RT-PCR, inserted into the protein expression vector pCAGGS, and downstream of the CMV enhancer and chicken ⁇ -actin promoter, cloning site Plasmids for protein expression pCAGGS-NP, pCAGGS-P, and pCAGGS-L, each of which has each gene of APMV-10 inserted in and PolyA, were constructed respectively.
  • the pAPMV or pAPMV-10 / HA prepared as described above and pCAGGS-NP, pCAGGS-P and pACGGS-L were infected with a recombinant vaccinia virus expressing T7 polymerase.
  • the cultured cells (Vero cells) were cotransfected with a gene transfer reagent (Mirus Co., product name: TransIT (registered trademark) -LT1 reagent).
  • TransIT registered trademark
  • the collected supernatant and cell suspension were inoculated into the chorioallantoic cavity of 10-day-old embryonated chicken eggs, cultured at 37 ° C. for 48 hours, cooled, and the chorioallantoic fluid collected.
  • the collected allantoic fluid was used as a virus solution for various tests.
  • T7 RNA polymerase is expressed in cultured cells infected with the vaccinia virus MVA / T7 strain, and full-length APMV genomic RNA is synthesized from pAPMV or pAPMV / HA having a T7 promoter.
  • NPMV, P protein and L protein of APMV-10 are synthesized from pCAGGS-NP, pCAGGS-P and pCAGGS-L. Since NP, P and L proteins are proteins involved in transcriptional replication of viral RNA, mRNA transcription and RNA genomic replication of APMV-10 virus are initiated using the full-length APMV-10 genomic RNA as a template.
  • APMV or pAPMV / HA is produced in cultured cells. Furthermore, these recombinant APMV or APMV / HA infect and propagate in cell lines and primary chicken culture cells.
  • NP-NCR, P-NCR, M-NCR, F-NCR, HN-NCR, L-NCR in which NCR was added to the antigen gene were 10 / HA, 10 / opHA and NCR.
  • NCR was added to the antigen gene
  • 10 / HA 10 / opHA
  • NCR was found to express 11.6 to 31.8 times, 18.3 to 50.2 times and 199.3 to 545.3 times the antigen protein in infected cells, respectively, as compared to the control not containing .
  • the chorioallantoic cavity of 10-day-old embryonated chicken eggs was inoculated with 100 EID 50 (infective unit) of virus, and the allantoic fluid was collected after 12, 24, 36, 48 hours. The titer of the collected chorioallantoic fluid was measured using embryonated chicken eggs. The chorioallantoic cavity was serially diluted 10-fold with PBS, and 0.2 ml each was inoculated into the chorioallantoic cavity of five 10-day-old embryonated chicken eggs at each dilution. After culturing at 37 ° C.
  • the allantoic fluid was collected, and the presence or absence of virus growth was determined by a hemagglutination test using chicken erythrocytes.
  • the virus titer was calculated by the Reed and Muench calculation method. The obtained result is shown in FIG.
  • NDV efficacy test Immunization to NDV
  • attenuated NDV B1 was administered by 10 6 EID 50 instillation to 2-week-old chicken, and ⁇ -propiolactone-inactivated NDV B1 was administered intramuscularly 10000 HAU at 4 weeks two weeks later. did.
  • Vaccination with recombinant virus Next, at 7 weeks of age three weeks later, blood was collected from the wing vein, and recombinant APMV-10 / HA was administered with 10 6 EID 50 by eye drops.
  • HPAIV A / chicken / Yamaguchi / 7/2004 (H5N1) strain was intranasally inoculated at 10 6 EID 50 .
  • swabs were taken from the larynx and the general cavity. The symptoms were observed for 10 days and mortality was calculated.
  • the virus titer contained in the collected swab was calculated using embryonated chicken eggs.
  • the antibody titer in the collected blood was measured by the hemagglutination inhibition (HI) test. The obtained results are shown in Table 5.
  • the serum was heat treated at 55 ° C. for 30 minutes to inactivate complement.
  • 25 ⁇ L of serum diluted with PBS 25 ⁇ L of HPAIV A / chicken / Yamaguchi / 7/2004 (H5N1) strain adjusted to 8 HA was added and allowed to stand for 30 minutes.
  • the maximum dilution of serum in which red blood cell aggregation did not occur was taken as the HI antibody titer.
  • the insertion of the transcription unit containing the antigen gene (foreign gene) flanked by the untranslated region of APMV is not particularly limited as long as it is an intergenic region between the endogenous transcription units of APMV. It was confirmed that the expression level of the protein encoded by the gene can be improved.
  • minigenome transcription system In order to confirm the improvement effect of the APMV-10 untranslated region on the protein expression amount encoded by the luciferase gene, a minigenome transcription system (minigenome transcription plasmid) that mimics the genome of APMV-10 was used.
  • the minigenome transcription plasmid of APMV-10 is a hepatitis D ribozyme sequence, a leader sequence of APMV-10, an APMV between the polyA sequence of pCMV-GLuc2 (manufactured by NEW ENGLAND BioLabs) and the CMV promoter. It was constructed by inserting a gene start sequence of -10, a luciferase gene, a gene termination sequence of APMV-10, a trailer sequence of APMV-10 and a hammerhead ribozyme. In addition, the sequence of the said luciferase gene used the Gaussia luciferase gene optimized to the human codon.
  • RNA transcribed by the minigenome transcription plasmid is cleaved by the ribozyme, and the leader sequence of APMV-10, the gene start sequence of APMV-10, the gene termination sequence of the luciferase gene, the gene termination sequence of APMV-10, APMV-10 in order from the 3 'end It becomes RNA in which the Trailer sequence is arranged.
  • APMV-10 at both ends of the luciferase gene was added to generate two transcription plasmids (Luc-F-NCR and Luc-HN-NCR).
  • a transcription plasmid (Luc-blank) to which a non-translated region was not added was also prepared.
  • each mini-genome transcription plasmid described above, and a polymerase expression plasmid of APMV-10 (pCAGGS-NP, pCAGGS-P and pCAGGS-L) in a 12-well cell culture plate at 2.8 ⁇ 10 5 / well
  • the Transfected Vero cells were transfected with TransIT (registered trademark) -LT1 Reagent (manufactured by TAKARA), and after 48 hours, the culture fluid was recovered.
  • the luciferase activity of the collected culture solution was measured using Dual-Luciferase (registered trademark) Reporter Assay System (manufactured by Promega). The obtained result is shown in FIG.
  • Luc-HN-NCR in which the untranslated region of HN gene of APMV-10 is imparted to the luciferase gene Showed 20 times higher activity than Luc-blank which was not given.
  • Luc-F-NCR in which the untranslated region of the F gene is added to the luciferase gene, also showed about 3 times higher activity than Luc-blank.
  • HPAIV highly pathogenic avian influenza virus
  • the recombinant avian paramyxovirus of the present invention is extremely useful in the prevention of HPAIV and the like.

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Abstract

L'invention concerne un paramyxovirus aviaire recombinant, dans lequel de l'ARN à brin négatif codant pour une unité de transcription exogène est inséré, l'unité de transcription exogène comprenant un nucléotide codant pour une protéine exogène, le nucléotide étant lié de manière fonctionnelle à une séquence d'initiation de gène et à une séquence de terminaison de gène du paramyxovirus aviaire, des régions non traduites du paramyxovirus aviaire étant insérées entre la séquence d'initiation de gène et le nucléotide et entre le nucléotide et la séquence de terminaison de gène, l'ARN à brin négatif codant pour l'unité de transcription exogène étant inséré dans un ARN à brin négatif codant pour une région intergénique entre des unités de transcription endogènes du paramyxovirus aviaire et le paramyxovirus aviaire recombinant permettant une expression élevée de la protéine exogène dans des cellules hôtes.
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CN111334464A (zh) * 2020-03-30 2020-06-26 重庆市畜牧科学院 鹅副粘病毒在加快鹅成纤维细胞增殖速度中的应用

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