WO2019069720A1

WO2019069720A1 - Recombinant avian paramyxovirus

Info

Publication number: WO2019069720A1
Application number: PCT/JP2018/035085
Authority: WO
Inventors: 良太常國; 岳彦西藤
Original assignee: 国立研究開発法人農業・食品産業技術総合研究機構
Priority date: 2017-10-02
Filing date: 2018-09-21
Publication date: 2019-04-11
Also published as: JP6573746B1; JPWO2019069720A1

Abstract

Provided is a recombinant avian paramyxovirus into which negative-strand RNA encoding an exogenous transcription unit is inserted, wherein the exogenous transcription unit includes a nucleotide encoding an exogenous protein, the nucleotide is operably linked to a gene initiation sequence and a gene termination sequence of the avian paramyxovirus, untranslated regions of the avian paramyxovirus are inserted between the gene initiation sequence and the nucleotide and between the nucleotide and the gene termination sequence, the negative-strand RNA encoding the exogenous transcription unit is inserted into negative-strand RNA encoding an intergenic region between endogenous transcription units of the avian paramyxovirus, and the recombinant avian paramyxovirus enables high expression of the exogenous protein in host cells.

Description

Recombinant bird paramyxovirus

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.

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.

For example, the highly pathogenic avian influenza (HPAI) virus (HPAIV) virus (HPAIV) H5N1 virus has killed millions of poultry in many countries in Asia, Europe and Africa, and in 1997, Since the epidemic, the number of HPAI H5N1 avian-to-human transmissions leading to clinically significant and lethal human infections has continued to grow.

In view of the sensitivity of animals including humans to HPAIV, means for preventing infection of the virus in birds and its spread are essential, and effective vaccines against HPAIV are sought.

As a vaccine against HPAIV, a recombinant vaccine having a Newcastle disease (ND) virus (NDV) as a vector has been developed. 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.

Moreover, 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.

However, there is a problem that the vaccine effect is diminished if the antibody against the underlying NDV is generated in the chicken or the like to be administered. In the poultry industry, ND vaccines are administered to chickens sequentially over time, so it is assumed that recombinant vaccines based on NDV are not effective as emergency vaccines at the time of HPAI outbreak. Therefore, it is desirable to develop a vaccine that can also be used in chickens immunized with the ND vaccine.

In order to solve this problem, instead of NDV, the present inventors recombine using as a vector another virus (avian paramyxovirus, APMV) belonging to the same genus as the virus (paramyxoviridae Abravirus genus). We are trying to develop a vaccine. Then, they are inoculated with APMV serotypes 2, 6 and 10 (APMV-2, APMV-6 and APMV-10), and these viruses grow efficiently in chickens regardless of the presence of antibodies against NDV. Have found that. Furthermore, the present inventors have reported that these APMV-based recombinant viruses are promising as a vaccine for imparting anti-influenza virus activity to chickens already having antibodies against NDV. (Non-patent document 6).

However, 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).

Thus, for practical use of recombinant viruses based on APMV, it is necessary to enhance the vaccine effect, for example, it is considered to increase the expression amount of an antigen such as HA. However, such a method has not been developed.

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.

As a result of intensive studies to achieve the above object, 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.
(1) 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.
(2) The 5 'untranslated region of the avian paramyxovirus is inserted between the gene initiation sequence and the initiation codon of the nucleotide.
(3) The 3 'untranslated region of the avian paramyxovirus is inserted between the stop codon of the nucleotide and the gene termination sequence.
(4) Furthermore, 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.

Then, as a result of infecting chicken primary culture cells with this recombinant avian paramyxovirus, high expression of foreign protein was observed in the cells.

On the other hand, 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. In addition, even if 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.

In addition, in order to test the vaccine effect of the recombinant avian paramyxovirus, chickens were previously infected with NDV, and then these recombinant viruses were respectively administered and further infected with HPAIV.

As a result, when using the recombinant avian paramyxovirus in which the foreign transcription unit is inserted in the intergenic region between the endogenous transcription units, the survival rate of chicken after HPAIV infection is 25% or 50. %Met. In addition, excretion of HPAIV was observed from all individuals of the chicken. Furthermore, antibody titers to HPAIV at challenge were below the detection limit in most chickens. In addition, even when using a recombinant virus in which an exogenous transcription unit that does not contain the untranslated region of the avian paramyxovirus is inserted into the intergenic region between the endogenous transcriptional units of the avian paramyxovirus, Antibody titers to HPAIV were below the detection limit in most chickens.

On the other hand, in the case of using the recombinant avian paramyxovirus in which the foreign transcription unit was inserted in the intergenic region between the endogenous transcription units, the survival rate of the chicken after HPAIV infection was 100%. In addition, almost no excretion of HPAIV was observed in the chicken, and even if it was, its virus titer was low. In addition, antibody titers to HPAIV at the time of challenge were detected in many individuals immunized with the recombinant virus.

In addition, it was confirmed that such a high vaccine effect was achieved even if the intergenic region in which the foreign transcription unit was inserted was changed in the avian paramyxovirus. Furthermore, even if the foreign protein is a protein other than HA protein, it is confirmed that the expression amount of the protein can be similarly increased when the above-mentioned HA protein is expressed using a recombinant avian paramyxovirus, and the present invention It came to complete.

That is, 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.
<1> A recombinant avian paramyxovirus, wherein a negative strand RNA encoding a foreign transcription unit is inserted into a 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
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.
<3> 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> The recombinant avian paramyxovirus according to any one of <1> to <3>, wherein the foreign protein is an antigen protein derived from a pathogen.
<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.
<7> A nucleotide construct encoding a recombinant avian paramyxovirus, wherein a cloning region including 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.
<8> The nucleotide construct according to claim 7, wherein the avian paramyxovirus is avian paramyxovirus serotype 10.
<9> 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> Furthermore, 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, The nucleotide construct according to any one of <7> to <9>, in which a ribozyme sequence and a terminator sequence recognized by the DNA-dependent RNA polymerase are linked in sequence.
<11> The nucleotide construct according to <10>, wherein a nucleotide encoding a foreign protein is inserted into the site.
<12> The nucleotide construct according to <11>, wherein the foreign protein is an antigen protein derived from a pathogen.
<13> The 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.
<15> 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). Among the 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. Also, 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. By being packaged, the basic structure (virus particle) of avian paramyxovirus is formed.

Moreover, in the genome of avian paramyxovirus, the order of minus strand RNA encoding a transcription unit is extremely highly conserved, and each transcription in the order of NP, P, M, F, HN and L from the 3 'side Negative strand RNAs encoding units are arranged in tandem (in APMV-6, SH is further arranged between F and HN).

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.

According to 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. In particular, even in chickens that retain antibodies against Newcastle disease virus (NDV), according to the present invention, it is possible to exert a vaccine effect against highly pathogenic avian influenza virus (HPAIV).

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. In the figure, the horizontal axis shows the recombinant avian paramyxovirus infected with host cells (see FIGS. 2 and 3). On the vertical axis, 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. It is a graph which shows the result of having measured the proliferation (titer) of the virus by the hemagglutination test using chicken erythrocytes. In the figure, 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). On the vertical axis, the expression level of HA protein corrected by the expression level of actin protein in each host cell is shown. A schematic diagram showing an outline of a minigenome transcription system imitating an APMV-10 genome for expressing a luciferase gene as a foreign gene and a process of measuring luciferase activity in a culture solution of cultured cells in which the minigenome transcription system is expressed. It is. 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. In the figure, "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.

<Recombinant bird paramyxovirus>
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.

In the present invention, “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. Means bird paramyxovirus. 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.

The “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). Among these, 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.

In addition, 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.

Avian paramyxovirus endogenous transcription units (NP transcription unit .P transcription unit, M transcription unit, F transcription unit, HN transcription unit, L transcription unit, etc.) and sequences constituting them (gene initiation sequence, 5 'non The translational region, open reading frame, 3 'non-translational region, gene termination sequence), and the 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.

Furthermore, 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. In the present invention, 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).

In the present invention, 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 | limiting in particular, For example, as mentioned later, when using a recombinant avian paramyxovirus as a vaccine, the antigenic protein of the pathogen which causes an infection is mentioned.

In the present invention, 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.

In addition, 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. Or a sequence having high homology with each sequence. Here, "plurality" 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. Also, “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.

The 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. More specifically, 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 And 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. Furthermore, preferably, 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 foreign transcription unit containing a translation region) or a foreign transcription unit containing at least the sequence set forth in SEQ ID NO: 6 and SEQ ID NO: 12 (a foreign transcription unit containing a non-translated region derived from the L transcription unit of AMPV-10) And more preferably, a foreign transcription unit comprising the sequence set forth in SEQ ID NO: 13, SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 16 (a foreign transcription comprising each sequence derived from an NP transcription unit of AMPV-10) Unit), SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 8 and SEQ ID NO: 17 as an exogenous transcription unit (foreign transcription unit comprising each sequence derived from P transcription unit of AMPV-10), A foreign transcription unit (a foreign transcription unit containing each sequence derived from the M transcription unit of AMPV-10) comprising the sequence set forth in SEQ ID NO: 15, SEQ ID NO: 3, SEQ ID NO: 9 and SEQ ID NO: 18, SEQ ID NO: 1 SEQ ID NO: 4, SEQ ID NO: 10 and SEQ ID NO: 16 (The foreign transcription unit containing the respective sequences derived from the F transcription unit of AMPV-10) SEQ ID NO: 14, SEQ ID NO: SEQ ID NO: 11, SEQ ID NO: 11 and SEQ ID NO: 16 (SEQ ID NO: 16), or exogenous transcription unit (foreign transcription unit comprising each sequence derived from HN transcription unit of AMPV-10) 6, a foreign transcription unit (a foreign transcription unit comprising each sequence derived from the L transcription unit of AMPV-10) comprising the sequence set forth in SEQ ID NO: 12 and SEQ ID NO: 16.

In addition, from the viewpoint that the expression level of the foreign protein expressed from the recombinant avian paramyxovirus tends to be higher, 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.

In addition, from the viewpoint that the titer of the recombinant avian paramyxovirus tends to be higher, 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.

In addition, from the viewpoint that the proliferation of the recombinant avian paramyxovirus tends to be higher, 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.

In the foreign transcription unit according to the present invention, 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. However, when 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).

In the recombinant avian paramyxovirus of the present invention, 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. 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 An intergenic region (M-F intergenic region), an intergenic region between the gene termination sequence of the F transcription unit and the gene start sequence of the HN transcription unit (F-HN intergenic region. In APMV-6) The intergenic region (F-SH intergenic region) between the gene termination sequence of the F transcription unit and the gene initiation sequence of the SH transcription unit, the gene termination sequence of the SH transcription unit and the gene initiation sequence of the HN transcription unit Intergenic region (SH-HN intergenic region), intergenic region (HN-L intergenic region) between gene termination sequence of HN transcription unit and gene start sequence of L transcription unit Although at least one of them may be used, the P-M intergenic region is preferable from the viewpoint of better expression efficiency of the protein, and the F-HN intergenic region is preferred from the viewpoint of better growth efficiency of the virus. In addition, when the recombinant avian paramyxovirus of the present invention takes the form of the above-mentioned minigenome, 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.

Therefore, the present invention provides a composition for vaccine containing the above-mentioned recombinant avian paramyxovirus as an active ingredient.

In the present invention, "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.

Furthermore, 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.

In addition, as the 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; 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 / chicken / I donesia / 7/2003, A / Indonesia / CDC596 / 2006, A / Indonesia / CDC940 / 2006, A / Egypt / 1394-NAMRU3 / 2007, A / duck / Hunan / 127/2005, A / whooper swan / Akita / 1 / 2008, A / chicken / Guiyang / 3055/2005, A / Viet Nam / HN 31242/2007, A / Chicken / Yunnan / 493/2005, A / chicken / Yamaguchi / 7/2004, A / Pheasant / Hong Kong / FY155 / 2001, A / goose / Guiyang / 337/2006, A / goose / Guangxi / 914/2004, A / duck / H bei / wg / 2002, A / Beijing / 01/2003, A / chicken / Hong Kong / 61.9 / 2002, A / Goose / Shantou / 1621/2005, etc.), but what is limited thereto Absent.

In the vaccine composition of the present invention, 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. Moreover, as a pharmacologically acceptable medium, for example, water, physiological saline, phosphate buffer can be mentioned. Those skilled in the art can appropriately select and use known carriers and media known in the art according to the type and method of use of the vaccine composition, as these carriers and vehicles.

In addition, 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. Furthermore, in terms of enhancing the vaccine effect, 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.

In addition, as the recombinant avian paramyxovirus of the present invention, more preferably, as shown in the examples below, when the virus is inoculated at 10 ⁶ EID ₅₀ , 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%).

Note that the "EID _50", when inoculated into embryonated chicken eggs, means a viral load of 50% is infected.

<Preventive method against infection of pathogens>
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. In addition, as shown in Examples described later, 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.

In addition, 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.

<A nucleotide construct for recombinant bird paramyxovirus production>
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.
In the nucleotide construct of the present invention, when the avian paramyxovirus takes the form of a minigenome, the cloning region is inserted between the nucleotide encoding the leader sequence and the nucleotide encoding the trailer sequence. It is also good.

In the present invention, 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. Also, when constituted by DNA, 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. In addition, genomic RNA extraction from viruses and synthesis methods using RNA-dependent polymerase are known. Furthermore, in the present invention, 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 (the so-called minigenome) 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

In addition, 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. In addition, as described above, 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).

In addition, 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.

In the present invention, 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.

When the nucleotide construct is introduced into a transformed cell described below by adopting such a configuration, 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.

Therefore, 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. There is no particular limitation, and 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.

Furthermore, in the present invention, 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).

In addition, as described below, in order to produce (produce) recombinant avian paramyxovirus, it is necessary to insert a nucleotide encoding a foreign protein, which is desired to be expressed in a host, at the above-mentioned site.

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.

<Method of producing recombinant bird paramyxovirus>
By using the above-described nucleotide construct, the recombinant avian paramyxovirus of the present invention can be produced as follows.

Introducing into the transformed cell expressing the DNA-dependent RNA polymerase and the core protein of the avian paramyxovirus a 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.

In the production method of the present invention, 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.

Moreover, in these cells, 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.

There is no particular limitation on the transformation, and by introducing a nucleotide construct (a plasmid vector, a phage vector, a virus vector, etc.) capable of expressing these proteins in the cells, 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.

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.

Thus, in the transformed cell into which the nucleotide construct encoding the recombinant avian paramyxovirus has been introduced, as described above, 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. Then, when 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.

There is no particular limitation on the method of “recovering” the recombinant avian paramyxovirus from such culture, and it is possible to appropriately use known virus purification / separation methods such as filtration, centrifugation, adsorption and column purification. It can be carried out by using in combination.

Furthermore, in the present invention, 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.

<Kit for producing recombinant bird paramyxovirus>
As described above, 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.

Hereinafter, the present invention will be more specifically described based on examples, but the present invention is not limited to the following examples.

(Preparation of recombinant virus)
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. 2001) cloning the Newcastle disease virus (NDV) genome (ie, pSL1180 (manufactured by Amersham Pharmacia Biotech)) Was excised at the restriction enzyme cleavage site, and the cloned APMV-10 genome was excised using the In-Fusion HD cloning kit (Clontech) to assemble the vector. 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.

As shown in FIG. 1, 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. In addition, in the genome of APMV-10 used in this example, 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.

Furthermore, 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. . For 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).

Also, as a recombinant virus for control experiments, 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. As shown in FIG. 3, in these recombinant viruses, 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.

The sequences of the foreign transcription unit inserted into each recombinant virus are shown in Table 3.

In the item of "foreign gene" in Table 3, "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. In addition, as shown in the table, 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.

In addition, as shown in FIG. 4, 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 β-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.

Then, as shown in FIG. 5, 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). One day later, primary chicken cultured cells (fetal fibroblast (CEF) cells) were replated, and one day later, the supernatant and cell suspension were collected. 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.

In the above steps, 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. In addition, 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. Then, synthesis of each viral protein takes place, and finally, recombinant 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.

(Comparison of antigen expression level in infected cells)
Chicken primary cultured cells (CEF cells) were inoculated with moi = 1 (the amount infected with 1 infectious unit of virus per cell), and after 24 hours, the cells were subjected to SDS-PAGE sample buffer (manufactured by ATTO, product name: EzApply) ) And recovered. The collected lysate was electrophoresed by a fully automatic capillary electrophoresis immunoassay system (product name: Wes, manufactured by Protein Simple Co., Ltd.), and an antigen protein was detected using an anti-HA2 chicken monoclonal antibody and an HRP-labeled anti-chicken IgY antibody. The amount of protein was measured using Compass software (manufactured by Protein Simple Co., Ltd.). The obtained results are shown in FIG. 6 and Table 4.

As shown in FIG. 6 and Table 4, 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. 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 .

(Proliferative in developing chicken eggs)
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. for 48 hours, 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.

As shown in FIG. 7, although virus growth was somewhat slow, virus titer after 48 hours was similar. Therefore, it became clear that it is possible to produce a vaccine using an embryonated chicken egg as well as NDV etc. by the recombinant virus which gave NCR. L-NCR has the highest virus titer after 48 hours and can be expected to reduce the cost of vaccine production. In addition, HN-NCR, 10 / opHA and control showed high proliferative activity 24 hours after inoculation. On the other hand, M-NCR and NP-NCR were slightly slower in growth than the other viruses 24 hours after inoculation.

(Vaccine efficacy test)
Immunization to NDV First, attenuated NDV B1 was administered by 10 ⁶ EID ₅₀ instillation to 2-week-old chicken, and β-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.

After challenge with HPAIV and at 9 weeks of age 2 weeks after vaccination, blood was collected from the wing vein, and then HPAIV A / chicken / Yamaguchi / 7/2004 (H5N1) strain was intranasally inoculated at 10 ⁶ EID ₅₀ . On

days

2 and 4, 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. Moreover, the antibody titer in the collected blood was measured by the hemagglutination inhibition (HI) test. The obtained results are shown in Table 5.

As shown in Table 5, chickens immunized with NCR-given recombinant virus survived 100% without any clinical symptoms when challenged with HPAIV. Virus excretion after HPAIV challenge was not detected by NP-NCR, F-NCR and HN-NCR, but was detected by P-NCR, M-NCR and L-NCR from 1 to 2 out of 10 birds. , Its virus titer was low. The HI antibody titer against HPAIV at the time of challenge was detected in many individuals immunized with the NCR-added recombinant virus, and HN-NCR in particular showed an increase in HI antibody titer in 8 out of 10 birds.

Chickens immunized with 10 / HA and 10 / opHA without NCR remained at 25% and 50% by virus challenge, and 100% virus elimination from infected chickens was confirmed. In addition, the HI titer at the time of HPAIV challenge was below the detection limit in most chickens.

From these facts, it was confirmed that the vaccine effect of the NCR-added recombinant virus was enhanced as compared to the non-added recombinant virus.

(Hemagglutination inhibition test)
The serum was heat treated at 55 ° C. for 30 minutes to inactivate complement. To 25 μL of serum diluted with PBS, 25 μL of HPAIV A / chicken / Yamaguchi / 7/2004 (H5N1) strain adjusted to 8 HA was added and allowed to stand for 30 minutes. 50 μL of a blood cell suspension obtained by adding chicken erythrocytes to PBS at a ratio of 0.55% was added, and after 45 minutes, it was determined whether or not there was aggregation of erythrocytes. The maximum dilution of serum in which red blood cell aggregation did not occur was taken as the HI antibody titer.

(Examination of antigen gene insertion site)
In order to investigate whether the expression level of the antigen is increased by NCR insertion even if the site to insert the antigen gene is not between P and M, recombinant viruses FHN-HA and FHN- with the antigen gene inserted between F-HN transcription units opHA and FHN-M-NCR were produced (see FIG. 8 and Table 6). In Table 6, the 3 'untranslated region and the 5' untranslated region of the recombinant viruses FHN-HA and FHN-opHA indicate not the sequence numbers but the sequences themselves.

And about the recombinant virus which inserted the antigen gene between these F-HN transcription units, the protein expression amount in an infected cell and the vaccine effect were investigated. The obtained results are shown in FIG. 9 and Table 7.

As is clear from the results shown in FIG. 9, the amount of protein expression in infected cells was significantly higher in those to which NCR of M gene was imparted than in the others. Furthermore, as shown in Table 7, vaccine effects are also obtained when the insertion position is between F-HN transcription units, as in the case where the insertion position of the antigen gene is between PM transcription units (see Table 5 etc.). It also became clear that it was expensive.

Therefore, 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.

(Examination of antigen gene)
As described above, it has been revealed that the present invention can improve the expression level of the protein encoded by the HA gene. Therefore, next, even when a gene other than the HA gene (luciferase gene) is used as the foreign gene, according to the present invention, the method shown below can improve the expression level of the protein encoded by the gene. It confirmed by.

(Construction of mini-genome 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, as shown in FIG. 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.

The 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.

In addition, as shown in FIG. 11 and Table 8, in order to confirm the improvement effect of the APMV-10 untranslated region on the protein expression amount encoded by the luciferase gene, in the minigenome transcription plasmid described above, APMV-10 at both ends of the luciferase gene The untranslated region of the F or HN gene was added to generate two transcription plasmids (Luc-F-NCR and Luc-HN-NCR). In addition, as a control, a transcription plasmid (Luc-blank) to which a non-translated region was not added was also prepared.

In addition, in Table 8, about "the adjustment | control arrangement | sequence to the multiple of 6", not sequence number but the arrangement | sequence itself is shown. In addition, the relevant sequences were provided in each transcription plasmid in order to satisfy the above-mentioned "6 rules of paramyxovirus".

(Measurement of luciferase activity)
As shown in FIG. 11, 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.

The above tests (transfection, recovery of culture solution and measurement of luciferase activity) were performed on cells of 2 wells of each sample as targets, and three tests were performed separately.

As a result of measuring the luciferase activity of the cell supernatant of cultured cells into which each minigenome transcription plasmid has been introduced, as shown in FIG. 12, 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. In addition, 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.

From the above, even when the protein to be expressed was luciferase, it was confirmed that the addition of the untranslated region of APMV-10 enhanced the expression as in the case of the HA gene of highly pathogenic avian influenza virus .

As described above, according to the present invention, it is possible to increase the expression level of foreign proteins such as pathogen antigens in a host, and thus to enhance the vaccine effect against pathogens. In particular, even in chickens that retain antibodies against Newcastle disease virus (NDV), according to the present invention, it is possible to exert a vaccine effect against highly pathogenic avian influenza virus (HPAIV).

Therefore, the recombinant avian paramyxovirus of the present invention is extremely useful in the prevention of HPAIV and the like.

SEQ ID NO: 25
<223> SEQ ID NO: 26 with codon optimization
<223> Synthetic construct SEQ ID NO: 29
<223> Luciferase gene synthetic construct SEQ ID NO: 30
<223> Synthetic Constructs

Claims

A recombinant avian paramyxovirus, wherein a negative strand RNA encoding a foreign transcription unit is inserted into a 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
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.
The recombinant avian paramyxovirus according to claim 1, which is derived from avian paramyxovirus serotype 10.
The 5 'untranslated region is a region consisting of the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 6, and the 3' untranslated region is a nucleotide sequence set forth in any of SEQ ID NOs: 7 to 12. The recombinant avian paramyxovirus according to claim 1 or 2, which is a region consisting of
The recombinant avian paramyxovirus according to any one of claims 1 to 3, wherein the foreign protein is an antigen protein derived from a pathogen.
The recombinant avian paramyxovirus according to any one of claims 1 to 4, wherein the foreign protein is a hemagglutinin protein of influenza virus.
A composition for a vaccine comprising the recombinant avian paramyxovirus according to any one of claims 1 to 5 as an active ingredient.
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.
8. The nucleotide construct of claim 7, wherein said avian paramyxovirus is avian paramyxovirus serotype 10.
The 5 'untranslated region is a region consisting of the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 6, and the 3' untranslated region is a nucleotide sequence set forth in any of SEQ ID NOs: 7 to 12. The nucleotide construct according to claim 7 or 8, which is a region consisting of
Furthermore, a promoter sequence recognized by a DNA-dependent RNA polymerase is linked to the 5 'end of the avian paramyxovirus-encoding nucleotide, and the 5' end of the nucleotide sequence is a ribozyme in this order from the 5 'end. The nucleotide construct according to any one of claims 7 to 9, wherein a sequence is linked to a terminator sequence recognized by said DNA dependent RNA polymerase.
11. The nucleotide construct according to claim 10, wherein a nucleotide encoding a foreign protein is inserted at said site.
The nucleotide construct according to claim 11, wherein the foreign protein is an antigenic protein derived from a pathogen.
The nucleotide construct according to claim 11, wherein the foreign protein is a hemagglutinin protein of influenza virus.
A method for producing the recombinant avian paramyxovirus according to any one of claims 1 to 5, comprising
Introducing the nucleotide construct according to any one of claims 11 to 13 into a transformed cell expressing 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 method for producing the recombinant avian paramyxovirus according to any one of claims 1 to 5, comprising at least one substance selected from the group consisting of the following (a) to (e) and instructions for use: Kit of (a) a nucleotide construct according to any one of claims 10 to 13 (b) a nucleotide construct capable of expressing said DNA-dependent RNA polymerase (c) a core protein of said avian paramyxovirus (D) a cell for introducing at least one nucleotide construct selected from the group consisting of (a) to (c) (e) said DNA-dependent RNA polymerase and / or said bird A transformed cell that expresses a core protein of paramyxovirus.