WO1998003635A1

WO1998003635A1 - Regions nonessential for the proliferation of avipoxviruses, and recombinant avipoxviruses and vaccines prepared with the use of the same

Info

Publication number: WO1998003635A1
Application number: PCT/JP1997/002508
Authority: WO
Inventors: Takanori Sato; Takashi Okuda
Original assignee: Nippon Zeon Co., Ltd.
Priority date: 1996-07-18
Filing date: 1997-07-18
Publication date: 1998-01-29
Also published as: AU3462797A; JPH1028586A

Abstract

Genomic DNA regions of avipoxviruses (hereinafter referred to as APVs) having at least two foreign genes in the genomic regions nonessential for the proliferation of the APVs: recombinant APVs carrying the above-mentionned genomic DNA regions integrated thereinto and having substantially the same proliferation properties as those of the parent strains; vaccines comprising these APVs; and a method for producing the above-mentionned recombinant APVs and a method for screening the same. Various foreign genes can be integrated into the genomic DNA regions as described above, which makes these regions usable as tools for integrating foreign genes over a considerably wide range into viruses usable in the construction of recombinant viruses via homologous integration.

Description

Description Non-essential region for propagation of avipox virus, recombinant avipox virus and vaccine using the same

The present invention relates to a recombinant of an avibox virus (hereinafter, referred to as APV) and a vaccine comprising the same, and more particularly, an APV recombinant having a foreign gene inserted therein but having substantially the same growth potential as a parent strain. And vaccines consisting thereof. Background art

In the current poultry farming field, prevention of disease by cultivation is a pillar of hygiene management, regardless of whether it is a breeder, a laying hen, or a meat hen. However, the construction program is overcrowded, and the manpower, time and costs involved are a major concern. As a solution, in recent years, using recombinant DNA technology, it has become possible to extract only arrested genes encoding proteins necessary for immunity induction from pathogens and construct a recombinant virus containing the same. By inoculating such a recombinant virus as a live recombinant vaccine, it is possible to induce immunity against the antigen protein encoded by the introduced gene. APV belonging to the box virus is a suitable virus for use in constructing such a recombinant virus. These viruses, represented by foul box virus (hereinafter sometimes abbreviated as FPV) and pigeon pox virus (hereinafter sometimes abbreviated as PPV), have large genomic DNA, and many regions of the virus Non-essential, multiple antigen genes can be inserted into these non-essential regions of the same virus. When chickens are inoculated with such a recombinant virus vaccine, humoral immunity and cellular immunity can be induced. This method has been applied as a recombinant APV in which a foreign gene has been inserted into attenuated FPV or PPV, which has been used as a live vaccine against fowlpox, using genetic engineering techniques. Use attenuated vaccine strain as parent strain The reason is that it occurs only with a negligible probability, but we take care not to become a virulent strain even if the gene inserted in the chicken drops out.

As foreign genes, arrested genes encoding antigenic determinants of fowl disease virus, Newcastle disease virus (hereinafter abbreviated as NDV), and antigens of Marek's disease virus Recombinant APV inserted with the gene and the antigen gene of the chicken influenza virus has been constructed, and the vaccine effect has been confirmed experimentally by inoculating SPF chickens (Boursnell et al., Viroloogy, 178). , 297-300, 1990; Nazerian et al., J. Virol., 66, 1409-1413, 1992; Taylor et al., Vaccine, 6, 504-508, 1988). In this way, recombinant APV can introduce various foreign antigen genes depending on the purpose, and can simultaneously introduce multiple antigen genes, so that a single inoculation can protect against multiple pathogens. It is promising that an effective multivalent vaccine will be possible and, as a result, a solution to the overcrowded Pectin program problem.

However, recombinant APV virus using an attenuated virus (such as the virus strain of vaccine) as the parent virus had weaknesses. Originally, recombinants in which an exogenous gene has been introduced have a lower growth rate in chickens than the parental virus. On the other hand, the attenuated parent virus is very low in its own right. Therefore, if an exogenous gene is inserted into the attenuated parent virus, the resulting recombinant APV naturally has a reduced proliferative potential. For this reason, vaccines using an attenuated virus as the parent virus often did not actually achieve sufficient effects in vivo.

This attenuation caused by the insertion of a foreign gene is a phenomenon that is also known in other recombinant viruses. Vaccinia virus belonging to the same box virus family as APV (hereinafter abbreviated as VV) has also been reported to be attenuated by the introduction of an exogenous telegene (Journal of Viology. 66: 2617-2630). (1992)). In addition, as shown in the examples of the present application, the present inventors have also experienced that incorporation of a foreign telegene attenuates APV in most cases. Disclosure of the invention

The present inventors have conducted intensive studies to develop a method for producing a recombinant so that the growth in chickens does not decrease even when an exogenous gene is inserted. Succeeded in identifying an insertion site at which proliferation was not substantially reduced as compared with the proliferation of, and found that a recombinant that did not attenuate could be made by inserting a foreign gene into the insertion site, thus completing the present invention. Was.

That is, the first aspect of the present invention is a recombinant APV having at least two or more foreign genes in a genomic DNA region that is not essential for the growth of APV, and having substantially the same growth potential as the parent strain. . Here, the region that is not essential for the growth of APV is characterized by having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing. The genomic DNA region that is not essential for APV growth also contains at least 300 bases 5 ′ to the most upstream gene and 3 ′ to the most downstream gene among the foreign genes. Characterized in that the base sequence has homology to a continuous part of the base sequence set forth in SEQ ID NO: 1.

Further, the foreign gene is selected from the group consisting of a gene encoding a promoter, a gene encoding a promoter, and a gene encoding an antigenic determinant of a bird pathogen.

The transgenes encoding the antigenic determinants of the avian pathogens include infectious bursal disease virus, pigeon infectious bronchitis virus, chicken meningitis virus, Newcastle disease virus, Marek's disease virus, and chicken flu virus. It is characterized by being a gene encoding a viral determinant selected from the group consisting of viruses. Furthermore, the foreign gene may be a gene encoding an antigenic determinant of Newcastle disease virus, and the gene encoding the antigenic determinant of Newcastle disease virus is a gene encoding F antigen or HN antigen. It is characterized by the following.

The APV is a fowlpox vaccine strain. In addition, the above-mentioned APV is characterized by a pigeonpox Nakano strain.

A second aspect of the present invention is a recombinant plasmid containing a genomic DNA region that is non-essential for the growth of APV from the pigeonpox Nakano strain. Furthermore, the genomic DNA region not essential for APV growth has at least the nucleotide sequence of SEQ ID NO: 1. The genomic DNA region of the recombinant plasmid that is not essential for the growth of APV may further contain at least two or more foreign genes. It is selected from the group consisting of a marker gene, a gene encoding a promoter, and a gene encoding an antigenic determinant of a chicken pathogen. The marker-gene is characterized in that the gene encoding the promoter and the gene encoding the antigenic determinant of the chicken pathogen are selected from the same group as described above for recombinant APV. Further, the exogenous gene may be an arresting gene encoding an antigenic determinant of Newcastle disease virus, in which case the distant gene encoding the antigenic determinant of Newcastle disease virus may be used. , F, or HN antigen.

A third aspect of the present invention is a vaccine containing the above-mentioned recombinant APV as an active ingredient. An anti-Newcastle disease vaccine comprising the above-mentioned recombinant virus as an active ingredient. A fourth aspect of the present invention provides: (l) a step of arbitrarily cleaving APV genomic DNA to obtain a DNA fragment; (2) a step of introducing two or more foreign genes into the DNA fragment; 3) a step of preparing a plasmid for recombination by introducing the DNA fragment into which the foreign gene has been introduced into a plasmid, and (4) a step of preparing recombinant APV by recombining APV with the plasmid for recombination. A method for producing a recombinant APV having substantially the same growth ability in a chicken as a parent strain, characterized by comprising:

The DNA fragment comprises a DNA region not essential for APV growth. In addition, the exogenous arrestor is selected from the group consisting of a marker 子 gene, a promoter-encoding gene and an avian pathogen antigenic determinant-encoding element.

In the production method of the present invention, the genes encoding the antigenic determinants of the avian pathogen are infectious bursal disease transmitting gene, pigeon infectious bronchitis virus, chicken meningitis virus, Newcastle disease virus, It is an arrestor encoding an antigenic determinant of a virus selected from the group consisting of Marek's disease virus and chicken influenza virus. In addition, the foreign gene is particularly preferably an arrestor encoding an NDV antigenic determinant, and the gene encoding the NDV antigenic determinant is a transgene encoding an F antigen or an HN antigen. It is characterized by being a child. A fifth aspect of the present invention is a method of (l) selecting a virus that forms a blue plaque when a cell line derived from a bird infected with recombinant APV is cultured on agar in vitro. And (2) measuring the box size after infecting chickens with the virus that forms the blue plaque in vivo, wherein the growth in chickens is substantially reduced. Not a screening method for recombinant APV.

A sixth embodiment of the present invention relates to a genomic DNA region that is not essential for APV growth, wherein an Escherichia coli-derived 3-galactosidase arrestor (lacZ gene) has been inserted into the region together with the VV7.5K promoter. It is possible to obtain a recombinant APV in which the growth of recombinant APV in chickens is not substantially reduced as compared to the growth of the virus in chickens before the introduction of the above-mentioned galactosidase enzyme. Genomic DNA region.

Further, the present invention is a genomic DNA region that is non-essential for APV growth and contains the nucleotide sequence of SEQ ID NO: 1 in which a foreign gene has been incorporated into a specific site. The specific site is a Clal site existing at nucleotides 962 to 967 of the nucleotide sequence described in SEQ ID NO: 1 and an Hpal site existing at Z or nucleotides 4, 267 to 4,272 of the nucleotide sequence. Features.

A seventh aspect of the present invention is the use of the above-mentioned vaccine in birds. Here, the above-mentioned birds are characterized in that they are selected from the group consisting of pigeons, duck, duck, duck, surprising bird, turkey, quail, chicken, pheasant, and slime.

An eighth aspect of the present invention is a method for preventing avian diseases using the above-mentioned vaccine. Here, the birds are characterized in that they are selected from the group consisting of pigeons, duck, duck, duck, eagle bird, turkey, quail, chicken, pheasant, and squirrel. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the change in pox size in SPF chickens inoculated with various recombinants expressing the lacZZ gene. Here, the horizontal axis shows the number of days (days) after inoculation of each recombinant, and the vertical axis shows the size of the varicella (wakefulness ³ ).

FIG. 2 shows a restriction map (a), a subclone (b), and a deletion mutant clone (c) of a non-essential region of PNP35, respectively. As shown in the figure, 2 kbp in (a) indicates the distance from Clal. Each number in (b) indicates the name of the subclone. (C) shows the deletion clones, 35dl1, 35dl2, 35dl3, 35dl4 and 35dl5 in order from the top.

Figure 3 shows the development of chickens carrying a transfer antibody against Newcastle disease immunized with each recombinant. It is a graph which showed transition of pox size. Here, the horizontal axis shows the number of days (days) after inoculation of each recombinant, and the vertical axis shows the size of the varicella (删³ ).

FIG. 4 is a diagram showing the construction of plasmid pNZ76. pNZ76 is a vector containing the 7.5V promoter of VV and ^ -galactosidase.

FIG. 5 is a diagram showing the construction of plasmid PNZ98. pNZ98 is a vector having the 7.5V promoter of VV and the F gene of NDV.

FIG. 6 is a diagram showing the construction of plasmid PNZ87. pNZ87 is a vector having the 7.5K promoter of VV and the HN gene of NDV.

FIG. 7 is a view showing a genomic DNA sequence of a region not essential for the growth of the APV of the present invention. Restriction enzyme cleavage sites in this genomic DNA region are underlined. The Cl site (base No. 962) or the Hpal site (base Nos. 3, 691 to 3, 696. 4, 267 to 4, 272) were indicated. BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, the region that is not essential for APV growth refers to a region that is not required for APV growth, and specifically refers to a region represented by the nucleotide sequence of SEQ ID NO: 1. Here, APV is a collective term for viruses belonging to the genus APV, a subfamily of the box virus family, which is an animal DNA virus. When the virus infects birds, it forms a bock, and the size of the bock formed can determine the degree of infectivity. Specific viruses include foul box virus, pigeon box virus, canary box virus, and quell box virus.

The region that is not essential for APV growth includes the nucleotide sequence of SEQ ID NO: 1. The nucleotide sequence set forth in SEQ ID NO: 1 is preferably derived from a fowlpox vaccine strain. Examples of the fowlpox vaccine strain include the fowlpox Nakano strain (fetal fowlpox poison toxin Nakano strain) and the CEVA Pectin strain. The nucleotide sequence set forth in SEQ ID NO: 1 is derived from the fowlpox Nakano strain among these fowlpox vaccine strains.

An exogenous gene is a gene not originally possessed by APV and may be of any animal species. Specific examples of such outpatients are Examples include the gene encoding the promoter, the gene encoding the various promoters, the gene encoding the antigenic determinant of the avian pathogen, and other genes.

Examples of various marker genes include a lacZ gene and a luciferase gene derived from Escherichia coli.

Genes encoding various promoters include those encoding the VV 7.5K polypeptide, those encoding the VV 11K polypeptide, and those encoding the VV 19K polypeptide. Examples include a gene, a gene encoding a 42K polypeptide of VV, a gene encoding a thymidine kinase, a gene encoding a 28K polypeptide of VV, and the like.

Genes encoding antigenic determinants of avian pathogens include antigens such as infectious bursal disease virus, pigeon infectious bronchitis virus, chicken meningitis virus, Newcastle disease virus, Marek's disease virus, and chicken flu virus Mention may be made of genes encoding determinants. If a gene encoding an antigenic determinant of the Castle Castle virus is selected as the foreign gene, an antibody against NDV is produced when a virus incorporating this gene infects chickens. If the gene encoding the antigenic determinant of the avian pathogen is the gene encoding the antigenic determinant of Newcastle disease virus, and if it is the gene encoding the F or HN antigen, the NDV It is particularly preferred because it produces antibodies that can prevent onset.

In the present invention, it is preferable that at least two or more foreign genes are integrated into a region that is not essential for APV growth. These exogenous transgenes may be introduced in any combination as long as the transgenes are expressed, and there is no limitation on the number of introduced transgenes. When introduced in combination with a gene encoding the antigenic determinant of the pathogen, a rapid gene encoding this antigenic determinant is expressed under the control of the introduced promoter, and the antigenic determinant of the bird pathogen is converted into a protein. Produced. The above two or more arrested children may be linked via a phosphorus sequence as necessary and introduced, or they may be linked and introduced.

Non-essential regions for APV growth due to the introduction of at least two foreign genes Is separated into a region that binds to the most upstream 5 ′ side of the foreign gene and a region that binds to the most downstream 3 ′ side.

At this time, the region that is not essential for the growth of the APV has a base sequence of at least 300 bp (hereinafter referred to as an upstream base sequence) on the 5 'side of the gene located at the most upstream position of the foreign gene, It is advantageous for homologous recombination to have a nucleotide sequence of at least 300 bp (hereinafter, referred to as a downstream nucleotide sequence) also at the 3 ′ side of the gene located at the most downstream gene. The upstream base sequence and the downstream base sequence are preferably about 600 bp or more, more preferably about 800 bp or more, and particularly preferably about l. OOObp. These nucleotide sequences have homology with a continuous part of the nucleotide sequence shown in SEQ ID NO: 1.

The continuous part of the nucleotide sequence of SEQ ID NO: 1 refers to a sequence in which at least 300 bp of the above-mentioned sequence of at least 300 bp start from a base having the nucleotide sequence of SEQ ID NO: 1.

Further, having homology means that it is only required that the upstream base sequence and the downstream base sequence are homologous to the base sequence described in SEQ ID NO: 1 by a certain ratio or more. A homology of at least about 90% or more, preferably about 95% or more, more preferably about 99% or more. The homology referred to in the present invention is a DNA sequence input analysis system.

The value was measured by "DNASYS" (Takara Shuzo).

The non-essential region of the APV of the present invention can be obtained, for example, as follows. That is, fetal chicken cells are inoculated with fowl fowl pox poison venom Nakano strain (manufactured by Nippon Pharmacy) at a predetermined multiplicity of infection (mo i), allowed to adsorb for a certain period of time, added with a medium, and counted at 37 ° C. After culturing for a day, collect the supernatant, collect virus particles from it, and obtain virus DNA. This viral DNA is digested with restriction enzymes to obtain a DNA fragment. An appropriate plasmid is digested with a restriction enzyme, and this plasmid and a DNA fragment digested with the restriction enzyme are ligated to prepare a transformed positive plasmid. The plasmid is used to transform competent Escherichia coli, and the concentration of the transformant is increased using a selection medium. The selected transformant Escherichia coli is further cultured to extract a plasmid, digested with restriction enzymes, and subjected to electrophoresis to detect whether there is the same restriction enzyme digest fragment of the original viral DNA. Can be obtained by a method such as the gun cloning method (JP-A-1-168279).

By inserting, for example, the β-galactosidase gene together with the gene encoding the 7.5 V promoter of VV into the genomic DNA region thus obtained, A genomic DNA region not essential for the growth of APV can be created. Furthermore, when a recombinant APV is produced by homologous recombination of an APV with a genomic DNA region that is not essential for the growth of an APV having an exogenous gene, the genomic DNA region affects the growth of the recombinant APV. Whether it can be confirmed by the growth of recombinant APV in chickens, as described below. This makes it possible to determine the insertion site of the foreign gene where the growth of the recombinant APV does not substantially decrease compared to that of the parent strain.

More specifically, a genomic DNA region that is non-essential for the growth of APV that has the ^ -galactosidase gene as a foreign gene together with the gene encoding the 7.5V promoter of VV can be obtained, for example, by the following steps. it can. That is, (a) a step of cleaving APV genomic DNA with a restriction enzyme and cloning into a predetermined vector to obtain a plurality of clones, and (b) a 7.5 KV of VV within the APV genomic DNA fragment of each clone. (C) transfection of each vector prepared in (b) into cells infected with APV, and (d) purification of each recombinant. (E) inoculating chickens with the same amount of each of the purified recombinants and the parent strain of the recombinants and measuring the magnitude of variability over 3 weeks after inoculation; and (f) variability And selecting a recombinant having substantially the same size as the parent strain. By using this method, it is confirmed that a genomic DNA region that is not essential for the growth of APV of the present invention has been cloned into the vector (a) used to prepare the recombinant.

Specific examples of the genomic DNA region that is not essential for the growth of APV of the present invention include a 5 Kb fragment (SEQ ID NO: 1) obtained by digesting genomic DNA of chicken embryonated pigeon pox Nakano strain (NP strain) with EcoRI. ). When the ^ -galactosidase gene is inserted into this fragment at a predetermined position together with an arresting gene encoding the 7.5V promoter of VV, the growth of APV is not affected and the growth is substantially equivalent to that of the parent strain. A recombinant virus having the following formula: Here, the predetermined position refers to the nucleotide sequence of SEQ ID NO: 1. Are the CI al site at base numbers 962-967, the Hpal site at 3,691-3,696 and the Hpal site at 4,267-4,272. The above-mentioned foreign gene can be inserted into any or a plurality of these sites.

The recombinant APV of the present invention contains a genomic DNA region that is non-essential for the growth of APV as described above, and its growth is substantially equivalent to that of the parent strain.

The parent strain refers to an APV that has not been recombined with a genomic DNA region that is not essential for the growth of APV, and a virus that can be recombined using a genomic DNA region that is not essential for the growth of APV described above. I do. Such a virus can be any virus as long as it infects birds, but propagates in cells of poultry, such as pigeons, ducks, duck, duck, eagle, turkey, quail, chicken, pheasant, and sarcophagi. Preferably, it is possible.

Examples of such viruses include fowl box virus (FPV), pigeon pox virus (PPV), canari box virus, turkey box virus, and various viruses belonging to the subgenus quell box virus. Specific examples of these include viruses derived from ATCC VR-251, ATCC VR-250, ATCC VR-229, ATCC VR-249, ATCC VR-288, Nishigahara strain, Sashimi strain, CEVA strain, and CEVA vaccine strain. A group of viruses called NPV in the narrow sense, such as those that form large plaques when infected with chicken embryo fibroblasts (CEF), and a narrow group of FPV, such as the fetal fowlpox Nakano strain (NP strain) Examples include closely related viruses used as live fowlpox vaccine strains. These can be obtained from depositaries, commercial vaccines, or from institutions such as the American 'Type' Culture Collection (ATCC).

Among these, it is preferable to use a smallpox vaccine strain and to be safe as a vaccine.

A recombinant APV having substantially the same growth potential as the parent strain refers to an APV having a growth potential defined as follows when the growth potential of the parent strain is set to 100. Proliferation in chickens is calculated from the box size formed as APV proliferates. It is considered that the smaller the bock size formed after infection with the virus, the lower its proliferation. Specifically, the parental strain or the recombinant APV prepared as described above is Inoculate the membrane, measure the height-width-height of the box of each individual using digital calipers every 3 days after the inoculation, and calculate the box size as the product of these. The box size formed by the parent strain is (A), the box size formed by the recombinant APV is (B), and BZAX 100 (%), which is the ratio of A to B, is used as an index of proliferation. This ratio is usually 90% or more and 110% or less, preferably 95% or more and 105% or less, and is preferably a genomic DNA region where APV growth is not essential. If this value is Ru der less 105% 95% or more, have preventive effects of disease when used as a vaccine which will be described later this virus _β

In order to produce a recombinant APV substantially equivalent to the parent strain, the above-described foreign gene is inserted into a predetermined site in a region not essential for the growth of the APV. For example, if the region not essential for the growth of APV is the nucleotide sequence of SEQ ID NO: 1, the ClaI site

Such a recombinant virus can be obtained by incorporating a foreign gene into one or both of the base (base No. 962) or the Hpal site (base Nos. 4, 267). For example, when the E. coli-derived / 3-galactosidase gene and the gene encoding the 7.5K promoter of the VV virus are integrated into the Cla site, a virus having substantially the same growth potential as the parent strain is obtained. Can be obtained. The / 3-galactosidase used herein has a sequence known by accession number V00296 of Gene Bank.

The method for producing the recombinant APV of the present invention is not particularly limited, and may be performed according to a conventional method. That is, by introducing a recombinant vector into cells infected with APV in advance by, for example, the calcium phosphate coprecipitation method, homologous recombination between the vector and the viral genomic DNA in the infected cells is caused, and the recombinant APV is infected. Can be constructed. The recombinant APV thus obtained is used to infect host cells cultured in a medium such as Eagle MEM. Purification of candidate strains from plaques growing on the culture medium by means such as hybridization using the incorporated antigen-gene as a probe or expressing the marker gene integrated with the antigen-gene Then, immunoassay using an antibody against the polypeptide encoded by the incorporated antigen fast gene may be performed to confirm that the desired recombinant virus has been obtained. For example, recombination in which the lacZ gene is In the case of APV, the virus expresses / 3-galactosidase. Therefore, such recombinant APV forms a blue plaque in the presence of Bluo-gal (GIBCO-BRL), one of the substrates for galactosidase, and is selected and purified using its properties. do it.

The host cell is not particularly limited as long as it can infect and proliferate the APV used. For example, when FPV is used, chicken-derived cells such as CEF cells (chick embryo fibroblasts) are used. Subcultured cells, embryonated chicken egg allantois cells, and the like.

The recombinant plasmid of the present invention contains a genomic DNA region consisting of the nucleotide sequence of SEQ ID NO: 1 that is not essential for APV propagation. It is preferable that the nucleotide sequence described in SEQ ID NO: 1 further have at least two foreign telegenes at the above-mentioned Cla! And Z or Hpal sites. Suitable exotic speed transducers are as described above. For example, when a gene encoding the 7.5K promoter of the VV virus and an antigenic determinant of the NDV F antigen or HN antigen are incorporated into the Clal site, when this plasmid is used to recombine APV, It is possible to obtain a recombinant virus which is highly effective in preventing bird diseases and can be used as a vaccine.

The vector for cloning the non-essential region of the virus used for construction of the above-mentioned recombinant plasmid is not particularly limited as long as it can be generally used. Specifically, plasmids such as pBR322, pBR325, pUC7, pUC8, and pUC18, phagemids such as λ phage and Μ13 phage, and cosmids such as pHC79 and pKT264 can be used. These plasmids contain the ampicillin resistance gene, the 3-galactosidase gene, the luciferase gene (Science, 234: 856-859 (1986), and Anal. Biochem., 188: 245-). 254 (1990)).

The recombinant plasmid of the present invention can be obtained by treating the above vector with an appropriate restriction enzyme and incorporating the above-mentioned genomic DNA region not essential for the growth of the APV of the present invention.

In the recombination vector used in the present invention, a gene encoding an antigenic determinant and a promoter gene that controls the gene as described below are inserted into a predetermined position in a genomic DNA region that is not essential for APV growth. It was done. By inserting these arrested children into predetermined positions, the living body can be exposed to the recombinant APV containing the foreign gene described above. When stained, it is possible to obtain an effect that the growth of the recombinant APV in the living body does not decrease. In addition, the above-mentioned marker gene, such as the Escherichia coli-derived ^ -galactosidase gene, may be incorporated together with the promoter for efficiency such as purification of recombinant APV.

In the present invention, a transgene encoding an antigenic determinant of a pathogen that integrates into a region of genomic DNA that is not essential for APV growth is transcribed in host cells when infection of these pathogens is established in the body of the bird. It is not particularly limited as long as it can be translated and expressed as an antigen protein and can produce an antibody as an antigen in the body of an infected bird. Such antigenic determinants include infectious bursal disease virus, pigeon infectious bronchitis virus, chicken meningitis virus, Newcastle disease virus, Marek's disease virus, chicken influenza virus and other chicken pathogen antigens. Determinants can be mentioned. Specifically, a gene encoding the NDV HN protein (Miller et al., 丄 Gen. Virol., 67: 1917-1927 (1986)), a gene encoding the F protein (McGinnes et al., Virus Res. 5: 343-356 (1986)), a gene encoding the glycoprotein gB of Marek's disease virus (Ross et al., Supra Gen. Virol., 70: 1789-1804 (1988)), infectious Fabrychus disease Genes encoding the VP2 structural protein of the virus (Bay) iss et al., J. Gen. Virol., 71: 1303-1312 (1990)). Encoded genes are preferred for their high immunogenicity.

The vaccine of the present invention contains the above-mentioned recombinant APV as an active ingredient. The peptide of the present invention may be one in which one kind of the above-mentioned recombinant APV is produced and contained alone. Further, a mixture of a plurality of recombinant APVs may be used, or a combination of these may be used. Furthermore, they may be combined with other vaccines. Here, other vaccines that can be combined include, for example, recombinant APV (described in JP-A-157381 and JP-A-3-27284) as an anti-NDV vaccine, or a recombinant as an anti-MDV vaccine. Examples include recombinant pectin such as APV (Japanese Patent Application Laid-Open No. 6-78764 and US Patent Application No. 08 / 499,474), and a turkey virus virus vaccine used as an anti-MDV vaccine. The vaccine of the present invention may be inoculated together with carrier.

The method for producing the vaccine of the present invention is not particularly limited. For example, the recombinant of the present invention Cells capable of growing APV are infected with the recombinant APV of the present invention, and cultured until the recombinant APV has sufficiently grown. Thereafter, the cells are collected and crushed by an appropriate means to obtain a crushed cell. The cell lysate is centrifuged and separated in a centrifuge tube into a precipitate and a supernatant containing high-titer recombinant APV.

Essentially free of host cells, this medium containing cell culture medium and recombinant APV can be used directly as a vaccine. It may be appropriately diluted with physiological saline, phosphate buffered saline (PBS) or other pharmacologically acceptable buffers. The vaccine of the present invention can be frozen and stored in a cell culture medium as it is, or the centrifuged supernatant can be lyophilized and stored as a lyophilized vaccine.

The vaccine of the present invention may be administered to poultry by any method as long as the recombinant APV in the vaccine infects poultry and elicits protective immunity. For example, the vaccine may be vaccinated by wing puncture, scratching the skin, or subcutaneously vaccinating poultry with a needle or other device. It is also possible to administer the vaccine in such a way that it is suspended in poultry drinking water for ingestion, or mixed with solid feed for oral inoculation. Further, various methods such as a method of inhaling a vaccine by aerosol spray, an intravenous inoculation method, an intramuscular inoculation method, and an intraperitoneal inoculation method can be used.

Inoculation amount of the vaccine of the present invention, for example, in the case of chickens, a per bird usually 10 to 10 ⁶ plaques forming units (Pfu), and preferably 10 ² ~10 ⁴ pfu. For inoculation by injection, dilute this volume to approximately 0.1 mL with a pharmaceutically acceptable carrier such as saline or sterile water, such as glycerin. Just do it. The above Kiyarya one, for example, be suspended in an amount of about 5 mg to about 40mg and Kiyarya foremost sterile water LOmL, about 10 ² to about 10 ⁶ pfu / mL of recombinant virus with inoculation when Wakuchineshiyon effect of Is high.

Since the recombinant APV of the present invention has substantially the same growth in the chicken as the parent strain, it can continuously exert an extremely excellent vaccine effect even on chickens having a transfer antibody.

For example, a recombinant APV in which a gene encoding an NDV antigenic determinant, preferably an HN endogenous gene and an F 遗 gene, is inserted as a foreign gene into a non-essential region of APV. Can produce antibodies against Newcastle disease virus. This recombinant APV was added to a flock of three-day-old hens whose Newcastle disease virus hemagglutination-inhibiting antibody titer was at least 4 or more and whose average value was 16 or more and 32 or less. 1 X l to 0 ⁴ pfu vaccination as times eyes. After 3 weeks from the first round of vaccination, challenged inoculated with 3 X l 0 ⁴ pfu virulent Newcastle disease virus Sato strain Niwatorimomo muscle, examine the survival rate of chickens that were challenged from the challenge after two weeks. Using the vaccine of the present invention, the survival rate of these chickens is 80%, preferably 90% or more.

In particular, using the EcoRI-digested DNA fragment (SEQ ID NO: 1) of the fetal fowler pox pox Nakano strain (NP strain), the 964th restriction enzyme Clal cleavage site in a region not essential for APV growth, or The 3rd, 694th and the 4th, 270th restriction enzymes at the Hpal cleavage site, a gene derived from NDV, preferably a gene encoding an antigenic determinant of NDV, preferably an HN gene and Recombinant APV incorporating the Z or F gene exhibits excellent protection against NDV infection. APV power In the case of chicken embryonated pigeon pox Nakano strain (Ν Ρ strain), excellent effects can be obtained.

Here, the hemagglutination-inhibiting antibody (NDV-HI antibody) titer is measured by an ordinary method. Specifically, the method described in the section “Hemagglutinating antigen for diagnosis of Newcastle disease” on page 282, published by Kinka Shobo Co., Ltd., 1996, “Chicken Vaccine”, 2nd edition.

In other words, the ND Ishii strain is inoculated into the urinary cavity of 9-11 day old embryonated chicken eggs, and cultured at 37 ° C for 3-5 days to collect urine fluid. Add an equal volume of ethyl ether to this urinary fluid and inactivate sensitization at 37 ° C for 60 minutes, with occasional shaking. Then except Echiruete Le, this 1/25 M KI0 ₄ was added an equal amount of, after shaking mixing, an additional 10% of the Budo © sugar solution is added at a ratio of 50%. Mix by shaking, dispense into aliquot containers, and freeze-dry to obtain Α Α antigen. Serum is collected from highly immunized chickens that are frequently inoculated with NDV, dispensed into small aliquots and lyophilized. Make serial dilutions of the antigen in a tube using saline (0.4 mL each), and add an equal volume of 0.5% chicken red blood cell suspension. After mixing by shaking, allow to stand at room temperature (around 25 ° C). Erythrocytes sedimented in front of the tube bottom and showed a film form (a positive image was formed). The titer is determined by comparison with the control.

Vaccines such as the above, pigeon, duck, duck, duck, amazing bird, turkey, quail, chicken, When inoculated into poultry such as pheasants and scabs, in these birds, the genes encoding the antigenic determinants of the pathogens of the birds integrated as described above into the recombinant APV used as vaccines are expressed in the birds. Thus, an antigenic determinant protein is produced. The antigenic determinants thus produced allow antibodies to be produced in the body of the vaccinated bird, thereby preventing the development of diseases having such antigenic determinants.

The recombinant APV used as the vaccine of the present invention can contain at least two or more exogenous genes in a region not essential for the growth of the APV incorporated therein. Thus, by incorporating a transgene encoding the antigenic determinant of multiple pathogens, one vaccine can be used to complete vaccines against multiple pathogens. Therefore, there is no need to administer vaccines for each type of pathogen as in the past, and a simple vaccine program can reduce manpower, time, and money.

The recombinant virus used as the vaccine of the present invention has substantially the same growth ability as the parent strain, unlike the conventionally used vaccine. For this reason, a sufficient effect can be obtained by the vaccine using the vaccine of the present invention.

Furthermore, taking the case of fowlpox as an example, in the past, a highly virulent strain was attenuated into a vaccine strain, and a foreign vaccine was incorporated into the vaccine strain and used as a live vaccine. However, it was not unusual for the integrated arrested child to be lost or for the attenuated vaccine strain to revert to the virulent strain. In the vaccine of the present invention, a virulent strain was not used, and a gene encoding an antigenic determinant of a pathogen was incorporated as a foreign gene by homologous recombination. If it is lost, it does not revert to the virulent strain.

As the subcultured cells (cell lines) derived from chickens used in carrying out the screening method of the present invention, the above-mentioned CEF cells are the most common, but are not particularly limited as long as they are cells capable of proliferating APV.

After infecting these subcultured cells with APV, a recombinant plasmid is introduced by an appropriate means, and agar is added to an appropriate liquid medium and a growth medium containing serum, amino acids, etc. as appropriate. And form plaque. With such a liquid medium Examples include LB, MEM. DMEM, 199 medium, and MacCoy5A. Avian-derived cells as described above are cultured in a monolayer and infected with APV at 0.05-0.2moi. 0. Power to infect at about lmo i It is preferable because the efficiency of obtaining the recombinant is high. After infection, the monolayer cultured cells are treated with trypsin to prepare a cell suspension, and the recombinant plasmid is introduced. The introduction of the recombinant plasmid can be carried out by various known means such as electrolysis and calcium phosphate method, but is preferably carried out by election-portion polish.

Recombinant virus was obtained from the cells into which the recombinant plasmid had been introduced, and the cells were again infected with bird-derived cells, and a growth medium containing agar was overlaid to form plaques. By the way, when another agar medium is overlaid, the integrated marker 遴 gene is expressed and plaque force is formed which can be easily selected. For example, when the recombinant APV contains the acZ gene, a blue plaque can be formed by adding Bluo-gal to an agar medium to be overlaid later. Bluo-gal is preferably added at a concentration of 300 to 900 μg gL, and when added at a concentration of 600 g / ml, virus selection can be performed easily. Select viruses that form blue plaques. This procedure is usually repeated 4 to 6 times, if necessary, to purify the recombinant APV. After the purified recombinant APV is propagated in an appropriate medium as described above, the chicken is infected as described above. By measuring the bock size formed as the virus grows in infected chickens as described above, it is possible to screen for recombinant APV that has substantially the same growth potential in chickens as the parent strain. it can.

Example

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited thereto. Example 1 Preparation of plasmid into which APV genomic DNA fragment was inserted

(1) Acquisition of genomic DNA of NP strain

CEF cells were monolayer cultured in a 75 cm ² culture flask. Here, APV chicken embryo poultry pox poison The Nakano strain (NP strain, manufactured by Nippon Pharmacy) was inoculated at 1 pfu / mL. After culturing at 37 ° C. for 2 hours in a 5% CO ₂ incubator, 10% tributose phosphate broth (manufactured by Difco) and single MEM containing 0.03% L-glutamine were added. Thereafter, the cells were cultured at 37 ° C in an incubator at 5 o / oCO for 4 days, and the culture supernatant was recovered. The culture supernatant was centrifuged at 3,000 rpm (1,500 × g) for 4 minutes at 4 t to obtain a centrifuged supernatant. The supernatant was centrifuged at about 98,000 xg for 1 hour at 4'C, and the precipitate was collected. The precipitate is suspended in 10 volumes of a DNase reaction buffer (50 mM Tris-HCl (pH 7.5), lmM MgCl ₂ ) of the culture supernatant, and DNasel (Boehringer Mannheim) is adjusted to 9 Zg / mL. The mixture was added and reacted at 37'C for 30 minutes. After completion of the reaction, proteinase K (Boehringer Mannheim) was added at 500 g / mL and SDS (sodium dodecyl sulfate) at 1%, and 37. Reaction was carried out overnight at C. Thereafter, the cells were gently treated with phenol-chloroform, and genomic DNA l Atg of NP strain, a live smallpox vaccine strain, was obtained by ethanol precipitation.

(2) Preparation of plasmid with APV genomic DNA fragment inserted

This was divided into two portions of 0.5 g each and digested with a restriction enzyme EcoRI or Hindi, respectively, in a buffer for restriction enzyme, and about 0.05 g of a 2 Kbp to 7 Kb DNA fragment was recovered by 0.8% agarose gel electrophoresis.

0.5; g of pl) C18 (Pharmacia) is also digested with the restriction enzymes EcoRI or Hindi II, and treated with alkaline phosphatase (10 units) to remove the phosphoric acid at the 5, -terminal, and then phenol: Extracted with (1: 1) and precipitated with ethanol, 0.4 μg of cleaved pUC18 was recovered. Hereinafter, PUC18 cleaved by EcoRI is called pUC18 (EcoRI), and pUC18 cleaved by Hindi 11 is called pUC18.

(Hindlll).

The recovered PUC18 (EcoRI) or pUC18 (Hindi II) and the previously prepared 0.05 g each of EcoRI or Hindlll digested NP strain genomic DNA fragments were added to 10 units of DNA ligase (T4 DNA ligase), respectively. The ligation was performed by incubating at ° C for 1 hour.

Combi competent E. coli TG 1 were grown in L-broth, 10 ⁹ were transformed with each recombinant plasmid 0.45 g of the above. The transformed E. coli TG1 was placed on an LB agar medium containing X-Ga] (100 μg / mL) and ampicillin (50 g / mL) for The cells were cultured at 37 ° C to form colonies.

Sixty white colonies were selected and cultured in LB liquid medium containing ampicillin (SO ^ g / mL). Thereafter, plasmid was extracted from each colony by the method of Birnboim and Dory [Birnboim, H. C. & Doly, J., Nucleic Acid Research, Vol. 7, pp. 1513- (1979)]. That is, a transformant of E. coli was solubilized with an alkali, treated with phenol, and then precipitated with ethanol to extract a plasmid (1 to 100 g).

Each of the obtained plasmids was cut with EcoRI or Hindlll to confirm the insertion of the APV genomic DNA fragment, and these plasmids were named pNP01 to pNP60. Example 2 Preparation of recombinant plasmid

The lacZ gene was inserted together with the promoter into the APV genomic DNA fragment of the plasmid obtained in Example 1 as follows to prepare a plasmid for recombination.

(1) Preparation of a cleavage plasmid having one restriction enzyme cleavage site in the inserted APV genomic DNA fragment

Since PUC18 does not have Clal, EcoRV, and Hpal restriction enzyme cleavage sites, pNP01 to pNP60 obtained in Example 1 were cleaved with the above restriction enzymes. By this restriction enzyme treatment, it was possible to easily select a brassmid in which one of the APV genomic DNA fragments cloned into the above-mentioned brasmid had been cut.

The selected plasmids were pNP03, pNP04, pNP25, pNP26, pNP29, pNP32, pNP35, pNP36, and pNP38.

PNP28, pNP29, and pNP35 were digested with Clal, pNP03, pP04, and pNP26 were digested with EcoKV, and pNP32, pNP36, and pNP38 were digested with Hpa]. The cohesive end of the above plasmid digested with Clal was blunt-ended by using MA polymerase.

Then, the phosphate group at the 5, -terminal of each cleaved plasmid was removed by treating with 10 units of alkaline phosphatase and 1 M Tris-HCl (pH 8.0) at 56 ° C for 30 minutes.

Then, the mixture was extracted with an equal volume of a mixture of phenol and black form (1: 1), and precipitated with cold ethanol to obtain a cleavage plasmid (0.05 to 0.5 / g).

(2) Incorporation of lacZ fast gene and promoter into the inserted APV genomic DNA fragment (2-a) Preparation of pNZ76

An approximately 0.26 Kbp Sa! L-RsaI fragment (Cell, 125: 805-813 (1983)) containing the 7.5 M dalton peptide coding DNA of the vaccinia virus WR strain (Cell, 125: 805-813 (1983)) and a PUC9 SalI-SmaI The plasmid pUWP-1 was constructed.

10 μg of pMAOOKShirakawa et al., Gene, 28: 127-, (1984)) was digested with BamHl. 2 g of pUC18 (Pharmacia) was digested with BamHI, extracted with phenol-chloroform (1: 1), and cleaved pUC18 was recovered by ethanol precipitation. The 5'-terminal phosphate was removed by treatment with alkaline phosphatase, and the DNA was again extracted with phenol-chloroform (1: 1) and recovered by precipitation with ethanol.

1 g of purified APV (NP strain) DNA digested with cleaved 0.2 ^ §11 (18, 8 ^ 1 and Hindi II) was ligated with ligase, thereby transforming a competent E. coli JM103. to obtain a transformant the transformant, 0.03% of 5-bromo-one ₄ -. chloro - 3 Indorinore - ^-D-galactopyranoside door Bruno Sid, isoproterenol building 0.03RaM --D-Garakutobira Nosids were cultured on LB agar medium containing 40 g / mL ampicillin for 15 hours at 37. Among the traits grown on the agar medium, white colonies were removed and 40 g / mL ampicillin was contained. The cells were cultured in LB liquid medium for 15 hours at 37. The transformants were collected, the plasmid was extracted according to the method of Birnboim and Dawley (Nucleic Acid Research, 7: 1513-, (1979)), and EcoRI and Hindi were extracted. The double digested fragment was subjected to 0.6% agarose electrophoresis to obtain 0- Galactosidase arrested children (about 3.3 Kbp) were recovered.

On the other hand, 0.3 μg of pUC19 was digested with BamHI, extracted with phenol-chloroform (1: 1), and recovered by ethanol precipitation. The recovered pUC19 and the 9-galactosidase arrested gene were ligated with ligase to produce a recombinant plasmid pNZ66.

On the other hand, 40 β g of pUWP_l was double digested with Hpall and EcoRI and subjected to 15% low melting point agarose electrophoresis (70 V, 6 hours) to separate a fragment of about 0.26 Kbp containing the 7.5K promoter. Then, the agarose gel was crushed into small pieces and the DNA was recovered with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Since this DNA fragment had cohesive ends, it was blunt-ended with DNA polymerase to obtain a 7.5K promoter single speed gene.

Digest 0.3 μg of pNZ66 with HincII and use phenol monoclonal form (1: 1) Extracted and recovered by ethanol precipitation. This Hincil digested pNZ66 (containing the 9 / -galactosidase gene) and the 7.5 K promoter gene obtained as described above were ligated using ligase to obtain plasmid pNZ76 (FIG. 4). .

(2-b) Preparation of plasmid for transformation

Using ligase (10 units), these telegenes were ligated to each of the cleaved plasmids recovered in (1) above to prepare a transformation plasmid (0.1 lzg). 10 ³ E. coli TG 1, were transformed with these transformants plasmid (0.1 At g).

The transformed E. coli TG1 was cultured on an LB agar medium containing 50 g / mL ambicilin at 37 ° C for 15 hours.

The colonies that appeared on the LB agar medium containing ampicillin were cultured in an LB liquid medium containing ampicillin at 37 ° C for 5 hours, and then the plasmid was extracted using the above-mentioned method of Birnboim and Dolly, and the lacZ gene transfer ability was determined. The inserted brasmid was selected. In this way, nine types of plasmids for recombination were obtained, in which the lacZ gene was inserted into each of the above nine types of plasmids. did. Example 3 Preparation and Purification of Recombinant APV Expressing 1 acZ Gene

Previously were monolayer cultured CEF cells in 5% C0 ₂ incubator one at 35 ° C. The confluent CEF cells (1 × 10 ⁷ ) were infected with the APV NP strain at a multiplicity of infection of 0.1 and cultured at 37 ° C. for 3 hours. Three hours later, these cells were treated with a 0.1% trypsin solution and detached to obtain a cell suspension. The cell suspension was centrifuged at 1,500 × g at 4 ° C. for 5 minutes to separate cells. Phosphate buffered saline (Saline G, (0.14M NaCl, 0.5mM KCl, 1. lraM Na 2 HP0 4, 0.5mM MgCl 2 -6H 2 0, 0.01 ^ glucose) 2 X 10 ⁷ cells / mL using a The suspension was re-suspended at a concentration of 1 and divided into nine equal parts in accordance with the number of recombinant plasmides prepared in Example 2.

To each of these suspensions (0.7 mL), 10 g of the above-mentioned plasmid for recombination is added one by one, and the mixture is added at room temperature to 3.0 KV / cm using Gene Pulser (Bio-Rad). The plasmid was introduced under an election port ratio of 0.4 msec. The cells transfected with the plasmid obtained as described above are then cultured at 37 ° C for 72 hours, and freeze-thaw three times using dry ice-ethanol to lyse the cells, and the recombinant virus is recovered. Was released from inside the cells.

Released recombinant viruses were screened as follows. Infection as described above in dissolved lysate containing the released progeny virus from the cells dilution was 10-fold serially diluted in CEF cells were seeded into each of the two plates (1 X 10 ⁷ cells / mL) Then, 10 mL of agar medium (0.8%) containing a growth medium (MEM containing 5% serum) was overlaid. After solidifying the agar at room temperature, the cells were cultured at 37'C until typical APV plaques appeared. Approximately one week after the plaques grew, another 10 mL of agar medium (0.8%) containing 600 ng / mL of Bluo-gal was overlaid on each culture plate, and further cultured for 24 hours at 37 ° C.

Colorless plaques and blue plaques stained with Blu-gai are formed on the plate. A blue plaque was extracted from these, and the virus contained therein was recovered according to a conventional method. This operation was repeated until all plaques formed were stained blue with Blu-gal, and the recombinant virus was further purified. Normally, this process is completed in 4-6 times.

The recombinant viruses purified in this way were named fNP1003, fNP1004, fNP1025, fNP1026, fNP1029, fNP1032, fNP1035, fNP1036, fNP1038, respectively. Example 4 Immunization experiments on chickens using recombinants and selection of non-attenuated recombinants Chicken was immunized using the recombinant APV prepared in Example 3 as follows, and non-attenuated recombinant APV was Selected.

The 10 ⁴ pfu of NP strain (parent virus) as a recombinant APV and control, were inoculated with the puncture needle on each wing membrane SPF chickens 4 day old ten chickens per group. On days 3, 7, 9, 11, 14, 17, and 21 after inoculation, the varicella (bock) size of each individual was measured, and the average for each group was calculated. The box size was calculated from the three measurements by measuring the height, width, and height using a digital caliper. Figure 1 shows the change in the mean value of varicella in each group.

As is clear from FIG. 1, only fNP1035 showed the same change in varicella as the parent strain, indicating that it was not attenuated. On the other hand, the box size of other recombinant APV was smaller than that of the parent strain, indicating that these recombinants were attenuated. Example 5 Analysis of insertion site of recombinant APV

The recombinant APV insertion site selected in Example 4 was analyzed for fNPI035.

(1) Creating a restriction map

The plasmid for recombination used in the preparation of the recombinant fNP1035 selected in Example 4 uses a non-essential region of PNP35 as an insertion site. Therefore, a detailed restriction map of pNP35 was prepared as follows.

fNP35 was digested with six different restriction enzymes (Hindi II, Bglll. Hpal, Sacl, EcoRI, Clal) alone or with two restriction enzymes. Each fragment obtained by this digestion was subjected to 0.8% agarose gel electrophoresis, and the number and number of bands appearing after digestion were examined. The restriction enzymes used for double digestion are as shown in Figure 2 (a). By changing the combination of the restriction enzymes shown here, it was confirmed that the cleavage site of the restriction enzyme was at the position shown in FIG. 2 (column (a) of FIG. 2).

(2) Base sequence analysis

The subclones in the EcoRI-5Kbp fragment shown in FIG. 2 (column (b) of FIG. 2) were each cloned into PUC18 according to a conventional method. Further, a deletion mutant clone of pNP35 was obtained according to the method of Henikoff (Gene, vol. 28, p. 357- (1984)). The deletion mutant clone thus obtained is shown in FIG. 2 (column (c) of FIG. 2).

The nucleotide sequence of the clone shown in column (b) of FIG. 2 was read using a nucleotide sequence analysis kit (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit) and an ABI fully automatic sequencer. The analysis data is described in SEQ ID NO: 1. Reference Example 1 Production of recombinant fNZ6927RL

(1) Construction of plasmid pNZ6927RL for fNZ6927RL production

Brasmid to generate recombinant fNZ6927RL to be used as control

PNZ6927RL is based on pNZ1729R (Yanagida et al., J. Virol., 66, 1402-1408 (1992)), a plasmid in which a promoter from pigeonpox P17 is connected to a lacZ arrested child from Escherichia coli. Figure 6 shows the NDV F gene linked to the 7.5K bromoester of PNZ98 (JP-A-3-27284) and pNZ87 (JP-A-1-157381) shown in Figure 5. Publication), the NDV HN telegene linked to the VV 7.5K promoter was inserted.

(1-a) Preparation of plasmid pNZ87

As described above, PNZ76 was digested with BamHI, and electrophoresed on a 0.8% agarose gel to recover an approximately 2.9 kb fragment not containing the -galactosidase gene.

On the other hand, the hybrid phage mplO-HN180 was double-digested with Bglll and BamHI, and electrophoresed on a 0.8% agarose gel to recover an approximately 1.8 kb NDV HN 遗 gene fragment.

Both were ligated by ligase to transform a competent E. coli TG-1 strain. This transgenic plant contains 0.03% of 5-bromo-4-chloro-3-indolyl-; 9-galactopyranoside, 0.03 mM of isopropyl- / 9-D-galactopyranoside, and 40 tg / mL of ampicillin. The cells were cultured on an LB agar medium for 15 hours at 37 ° C. Among the traits grown on the agar medium, white colonies were collected and cultured in LB liquid medium containing 40 μg / mL ampicillin at 37 for 15 hours. The transformant was recovered, and plasmid was extracted according to the method of Birnboim and Doley (Nucleic Acid Research, 7: 1513-, (1979)). Thus, a brasmid pNZ87 containing the HN fast gene was obtained.

(1-b) Preparation of pNZ98

Plasmid XL IΠ-1 OH (Virus Research, 7: 24-255 (1987)) containing NDV F and HN transgenes was used.

4 Atg of plasmid XLIII-10H was digested with Xbal, the cohesive ends generated were made blunt with DNA polymerase, extracted with phenol-chloroform (1: 1), and recovered by ethanol precipitation. The recovered DNA was digested with BamHI, and electrophoresed on a 0.8% agarose gel to recover a fragment completely containing about 2.1 kb of the F gene.

On the other hand, pNZ76 prepared as described above was double-digested with BamHI and Smal, and about 3. Okbp and BamHI-Sraai fragments excluding the lacZ gene were recovered. The recovered fragment was ligated with a fragment containing about 2.1 kb of the F gene completely by ligase to transform competent E. coli TG1 strain.

From the colonies grown on LB agar medium containing 50 μg / mL ampicillin, a plasmid was prepared by the same operation as above. This plasmid was digested with restriction enzymes (BamHI and Smal), and the desired clone was identified and named PNZ98 '. this PNZ98 'contains the full length of the F gene and about 300 bp of the 5'-end of the gene. To remove this part, ρΝΖ98 'was double-digested with Smal and Kpnl, and a Smal-Kpnl fragment of about 4,150 bp was recovered by 0.8% agarose gel electrophoresis. Further, ρΝΖ98 ′ was similarly double-digested with Smal and Avail, and about 650 bp of Smal Aval I was recovered by 1.5% agarose gel electrophoresis.

These two fragments were mixed, the cohesive end was made blunt by DNA polymerase, and then Escherichia coli TG1 which was concomitant was transformed to obtain a transformant. This transformant was grown on LB agar medium containing 50 β g / mL of ampicillin, and a plasmid was obtained from the formed colony as described above. This plasmid was digested again with Smal, and the cleaved one was selected to obtain a plasmid pNZ98.

(2) Production and purification of fNZ6927RL

A recombinant fNZ6927RL was prepared and purified in the same manner as described in Example 3, except that PNZ6927RL was used as a recombinant vector. Example 6 Production of recombinant fNP6935

(1) pNP35 the construction of recombinant for plasmid _P NP6935 was揷入the HN and F genes of NDV

PNP6935 was prepared by cleaving pNP35 (5 g) with Clal, blunting the cleaved end with DNA polymerase (Takara Shuzo), and removing the 5'-end phosphate by treatment with alkaline phosphatase. After that, it was extracted with phenol-cloth form (1: 1) and precipitated with ethanol to recover the cleavage plasmid. PNZ6927RL (5 ^ g) prepared in Reference Example 1 (1) was cut with Hindlll, the cut ends were made blunt with DNA polymerase, and the lacZ fast gene of about 7.5 Kbp and HN and F telegrams of NDV from agarose gel. The DNA fragment (1 _μδ ) containing the offspring was recovered. The cleavage plasmid (0.5 g) previously collected and this DNA fragment (0.5 g) were ligated with ligase (T4 DNA ligase) to obtain plasmid PNP6935 according to a conventional method. This plasmid and the above-mentioned pNZ6927RL have the same exogenous repressed gene, and differ only in the region and region that are indispensable for APV growth.

(2) Preparation and purification of recombinant fNP6935

A recombinant fNP6935 was produced and purified in the same manner as in Example 3 except that PNP6935 was used as a recombinant vector. Both recombinants of fNP6935 and fNZ6927RL are Each was analyzed by the Southern hybridization method, and it was confirmed that the F, HN 遗 and 1 acZ NDV genes of NDV were in the expected positions. Example 7 Pox size and toxicity of chickens immunized with the recombinants obtained in Example 6 and Comparative Example 1

Fifty three-day-old chicks were prepared having a transferable antibody having a hemagglutination-inhibiting antibody titer of Newcastle disease virus of at least 4 or more. The average hemagglutination-inhibiting antibody titer in these chicks was 24.3.

These chickens were divided into four groups of 14, 14, 13, and 9, respectively.

Two of the groups of 14 birds (inoculation groups 1 and 2) were inoculated with the recombinant APV fNZ6934 and fNZ6927RL (1 × 10 ⁴ pfu in each case) obtained in the above example into the wing membrane of a puncture needle.

Chicks of 13 groups (blood collection group) were whole-blood-collected from the heart, and the serum was stored at 4 ° C.

The group of 9 birds (non-vaccinated group) received no vaccination and was bred together with the vaccinated group in a chicken isolator for 3 weeks. During breeding for 3 weeks, the pock size of the chicks in the inoculated group and the non-inoculated group was measured in the same manner as in Example 4, and 3 x 10 ⁴ pfu of poison was given at 24 days of age after 3 weeks. Newcastle disease virus Sato strain was inoculated (challenge) into the muscles of the thighs, and observed for 2 weeks.

One week, two weeks after immunization, and immediately before challenge, blood was partially collected from chickens, and the NDV-HI titer for NDV present in the serum was constantly determined along with the blood collected from the blood collection group at the time of immunization. The measurement was carried out according to the method described in “Vaccines of chickens”, 2nd edition, published by Kiko Shobo Co., Ltd., published in 1992, page 282, “Hemagglutinating antigen for diagnosis of Newcastle disease”. . Figure 3 shows the changes in the average size of chicken pox in each group. Table 1 shows the transition of the average NDV-H1 antibody titer and the results of the NDV challenge test.

As is clear from FIG. 3 and Table 1, fNP6935, which is the recombinant of the present invention, has a larger pox size than fNZ6927RL, and has better NDV challenge test results and HI antibody induction than fNZ6927RL. Proven to be a vaccine. Particularly, in the group inoculated with the recombinant of the present invention, 90% or more of the challenged chickens survive, and the recombinant of the present invention is an effective and practical vaccine showing an extremely high survival rate. It was shown,

NDV-H1 antibody titer at the age of age NDV challenge 'test Inoculated virus 3 10 17 24 (day) Survival / test feather protection rate (%) fNP6935 24.3 8.0 18.0 7.3 13/14 92.9 fNZ6927RL 24.3 8.4 7.6 4.0 9/14 64.3 I (Non-inoculated) 24.3 11.3 2.8 2.0 2/9 22.2

Example 8 Preparation of a recombinant in which a foreign gene has been inserted into the Hpal region of pNP35

Cleavage of PNP35 (2 μg) with Hpal to see if a recombinant with the foreign gene inserted into the Hpal cleavage site at the two restriction enzymes in the nonessential region of pNP35 in Figure 1 can be obtained Then, the phosphoric acid at the 5, 1 terminal was removed by alkaline phosphatase treatment. Thereafter, extraction with phenol: cloth form (1: 1) was performed, followed by precipitation with ethanol, and cleaved pNP35 (lg) was recovered. As described in Example 2, lacZ gene (0.2 g) was ligated to the recovered pNZ35 (0.2 g) with T4 DNA ligase, E. coli TG1 was transformed, and the resulting transformant was Desired plasmid

1 βg of PNP1035-H was obtained. Using this plasmid, a recombinant fNP1035-H was produced and purified in the same manner as in Example 3. SPF chickens were inoculated in the same manner as in Example 4, and their pox size was compared with that of the parent virus, NP strain. As with fNP1035, the change in varicella was similar to that of the NP strain. This indicates that Hpal, in addition to Clal, can be used as an insertion site for the non-essential region of pNP35, and the non-essential region shown in SEQ ID NO: 1 can be used in vivo even when a foreign gene is inserted. It has been reconfirmed that it is useful as a genomic region where the growth of the DNA does not decrease.

Thus, according to the present invention, a recombinant APV can be obtained which has substantially the same in vivo proliferative properties as the parent strain even when a foreign gene is introduced into the genomic DNA region. Industrial applicability

The recombinant APV of the present invention can be used as a vaccine against various bird pathogens. Furthermore, the recombinant APV of the present invention can be used as an effective vaccine against chickens carrying a transfer antibody.

In addition, the genomic DNA region that is not essential for the growth of the APV of the present invention can incorporate various exogenous genes into the region. Therefore, it can be used as a tool for incorporating a wide variety of foreign genes into viruses capable of producing recombinant viruses by homologous recombination.

Sequence Listing SEQ ID NO: 1

Sequence length: 5055

Sequence type: nucleic acid

Number of chains: 1 strand

Topology: linear

Sequence type: genomic DNA

Origin

Organism name: Pigeonpox virus

Strain name: NP strain (fowlpox live vaccine strain)

Array

GAATTCTAGA TATTTCAGTA TTGCCATTTT CTATAGCGCA GTGCAAAAGA GAACACCCAT 60 ATTCATTAAT CATATTCGGG TCCGAGTCAT TGTCTTTTAA AGCTTTAATA ACATCAGAAA 120 CACAGCCAGA TTCGATAGCC TCAAATACCT CCATGTTGTG TAATATTAGT AGTATCCGTG 180 TATGTACACC GGATGTACCA AGCGATATAA AAATGATTAA AATTTCAATT TTTATTTAAC 240 ATTAGGATGT CTATTTCATA ATCCCGTTAT TAGTAGTTAG TGGTTAGTAA GTACCATATT 300 ACTACTAATA AGATAAAAAA TAAAACTGCT GATGTACAGT TTATACCCAT AATGAAATTA 360 TTTATAAAAT CCTCAGCCAT GGAAATAATG AAATGATACA ACCAATTCAT ATACAATGTT 420 TATTTATAGG ATCTATTATG AATTAATATT TTTTTACTTT TTAACTAAAC GCGATTTAAT 480 AAATCTACTA TAGTATCAAT ATTATTTCAA ATAATAAAAA AATATTATAC GGTTAGTAAT 540 GTACTTTAAT GTATACCAAA ATAAAATCTA ATAGAGTAAT GTCTTTTTAA TTTCATATAT 600 ATCATTAGGA TTTATACATT TAATTGGGTA TTCAGTTCCA TAACCTCTAT AATTATCATC 660 AGTAGCCTTA AACATTTCTT CTTTATTAAC ATACTTACAT TTATTGGTAT CGTTCATGTT 720 AGATTTCAAA TCGTCAGTAT ATACTAAAGG TGCCATTGCC AAACAGAACT CTTCTTGGGA 780 TAGTTCATCT ACATAAAATT TACCATCGCT GTCTACCATA CCAAGAAATT CATTTCTTGT 840 AGTATATTGG ATACATCCAG AAACATACTC TATATCAGAA TTTAAACATC CTATGAATAT 900 ATKATTATA ATACATGATG TCATAAACTT ACATTTAGTG TCGTAATCGA ACACCTTTAA 960 TATCGATTCC GGAGTAGTTT TAGTTCCATT TACCTGCATG AATACCATGC TATCAGTAAC 1020 TGGAGTTACT GTATTAAAGC ATGATTTGAT TTCACTACTT ATTTCACGTA GTATAACCAT 1080

TATTGTAAAG TGTAGTAGTT ATATATTATT CTGTAATAAG GAATTAATTT GCTAGTTGGG 1140

TTATAAAACG TTCTAGATAA ATCTATTAAT AATTCATTTT TATATATGTT ACTCGGAAGA 1200

AATTACTATT TACTAGTTAA TTATAGAATA GATAAGTCTT AATAATTTAC TTTTAGTATT 1260

ACTCGGAAGA AATTACTATA GTTGACTGTA TAAACATTAT AAGAAAGAAT AAATTCAAAA 1320

ATATTTATAA AATAAATATT CTACCATCTT CCGTAAACAT CTTTATATTC TGTTACTAGG 1380

TATTCTTTTC CTACATATCT TACAGGAAAT TCTCTGGCGT GTGTATCATT AAGCCAATAT 1440

CTACAATATG GAATTTCGAC ATGGCCGATG ACACTTTCGT TTACCTTTAT ACTTGTGTAT 1500

CTGTAATCCA CCACATTTTG TGTGAAGCCC TGCGGTCTTT CGTATGACAT TACACCTCCT 1560

GTTACTATAC CGGTTACTCT CCATCTAGGA TTGCCGTAAT TACTTCCTAC GAAGCAAGTT 1620

ATCTTGGTTT CGTTTTCATG TTTTTCTAGT AGATAGGTTA TACCTTCTTT TTTAATTTCG 1680

ATACATCTTT GATACGTTTC CTCTTTCCCA GAAGCAGTAA GTAGTACACA GGTAAAGCAC 1740

AAACTTTGAG GACCTGTTTT TTTAACTGTA AAAGTAGAAG TATCTCCACT AACAGTTACT 1800

TCATATATAT CTCTATAAGG ACTAGGAACA TAATTGAAAG CGTTAAGTCC GTTACCTCCT 1860

ACCTGCTCTT GGGCTACCAT AATCCCATCG CTGCCTCTCC AAGTAGCTCT TGATATTCCT 1920

TCTCCGTATT CGGTTCTGCA TTGTAATTTT ACAGGGCTAC CCGGTACACC TTCTTCTCCA 1980

TAGATATTTT TCAAGCTAAA TGCTATTATA GTAATTGCTA GAAATAAATA ATTAGCCTTC 2040

ATCTTGATTG CTTTATATTA AACACTGGAT AATGTACGAG GATCACATAT AGTAGTTAAT 2100

ATACTTATTC ACTTTTTAAT TAAAAATGTA TTAATCTTAA AAAAATATCA AAAATTAAAC 2160

CAACCACCTC TTATAACGAG ATTTCTGTCC AGGTTCCATC AAAGATGATC CAGAGATCAG 2220

AACCACAGAA AGGTCCTGTA ATTTTTCATC GTAAGAAGTC ATAGATGCTA CATAATCTCT 2280

ACTTAGACCC AAGTAACTTA CCGTAAATAT TACTGTAGTA GTACAATCAG TGTAATTTGT 2340

ATTTGCTACT ATTTTTTTAC ATTCATTATC TTTCGAATAT TCTACCAATA CTTGAATATC 2400

TTCGTTCAAC TTCACACCAC CTGCTCTAAC ATCTACTTTT ACTTCGTGGT CTTCTATAGT 2460

TCTAGTCCTA TTTATTATAA AATCAATCAA ATCTTTATCT ACATTATCTA TAACATACGC 2520

ATCTACGTGA GGCATTACTC TGAGTTCTAT ACCGTGTCTG TAACTCTCTG TTCCGTTGTA 2580

ATAGAAGATA CATCTATAGC GACCTTCGTC ATTTCTGGAT GACCTTTTAG GAAGTTCAAC 2640

ATTATATTCT TTAACTGGAT CGTTACAATA GCATATACTG TCTTCTCCGG CATCATGTAT 2700

GTCAATAGTT GCCTTAAACG CGTTATTTTT CATGTTTCCT TTTACTACAA TCAGTTTAGT 2760

AGCGTGTCTG TCAGGTGGTA GAAGACAAGT CAAATTAACC ATTACATCGT TATGTACTAC 2820 CATAGTATAA GATCGACATT GTAGGAAGGT AGCCAATAAG AATAAAGAAA CTACGTACAC 2880

TTTTCCAGCC ATTATTTTTT TTACCAACTA CTAATAATGC TACACTAGTG TTAGTGTTAT 2940

ATTTATGTTT TTTCCTAATA ATATCTGGAA ATCGTTTTAA GATCTTCCAT AGATAAATTT 3000

GACAATATTA CTCTATGTAT CTCAGGAGAT AGTAGACACC ATCTGCTGCT ATGATCTTTA 3060

CTAGCGTATT CATTGATGAT CGATAACGTC AAGTCTATAG CTGTCTTCCT TTTTTCCATG 3120

TATTTTATAT GCTTCTTAAT AAAACAACGA AATATCCTTA TATTTTTAAG ATCTATCCTT 3180

CGTATACGTT TTATAGAATT AGTTAGACCA TCCAGTTTAT CTAATATCAG AACATCGTAC 3240

AAGGATATTT TCTTATCACC CGTATAAAAT ACATTGTCTT TCATAATGGA CAGTTCGTGT 3300 pcs CTATTTCAT CCTTTAATGC TTTGGTTTCT TTATAGTTGT TTACAAATCT CATATTACGT 3360

ATAAAACCTT GTTTCTTCTT TATAGAAGAG TCGTTTATCA CAGAAAAGTA TAAATATATT 3420

ACAGCAATGA ACACAGCAGG ATTATTACGG GCTTTATGAA TAGTGATGGG AGTATGAAAT 3480

CTATTCATAA AACACATATC CGCTCCGTGT TCTAACAGTA CGCGAGTACA TTCTTCACAC 3540 cho TATTACGTA TAGCGTAAGT TAGTGCTGTA TTTTTATCAT AATCTGTTTG ATTAACGTCA 3600

GCTCCATTAG CTAATAAGTA TTTCATATTA CTTACTTTAC TTTCTCTAGA ACAAATCATT 3660

AAAGGTGTTA TCCCGTAATT ATCTAGTTTC GTTAACGTTA GCTCCGTTTT TTAGTAATAT 3720

CTTTATATTA CTAATCCTAG AGTACATACA GGCTAAATGT ATAGGTGTTT TACCGTATCT 3780

ATTTCTTACA TTAACATCAG CACCTTTATT AACAAGTAGT CTAGTAAGCC TAGAAGTATT 3840

TATAGAAACA GCCGCGTGTA TAGGGTACAT ATTACAAGCA TCGCATGATA CGTTTATATC 3900

CTTTATCTTC TTTAGCAGTT TTCTTGTAAT AGGTAAATTC CTTAATTCGT ATATACACAT 3960

GCAGAGTATA GAAATAGGTA GATAATGAAT AGGTAAACTA AGTATGTATG AGATAATACT 4020

ATAATACCTC TTACATATCG CTTCCATCAG TATATCGTTA CAACATCTTA TTTGAGCTCC 4080

GTGTTTAACT AGAATATTGA ATAATCTTAC ACCGTGTTTA CTAGACATTA TAGCGTATTT 4140

TAACATTGTA TATACATCTC CTATATCTGG ATCTGCGCCG TGGTTTAATA ATAAATTTAC 4200

CAGAGTAACA TTTTCTTGTT CTACAGCATG ATATAATGCA GTATGCGTGT TTTCGCATAT 4260

ACCCGTGTTA ACATCAACTC CTGCATCAAT GAATAACTTT ACTATTTTAG GACTATTAGT 4320

TTTAACGGCT TCTAAGAAAT AATCTTCTAA CATTGTATCT GTATCTACTA ATAAATTATT 4380

AGTTATAAAA TATTCTACTA GTTCTGTATT TCTAGCTCTA ATTGCGCACT TAATAGTAAT 4440

GTTTCGCATA TAATATAAAC TTGGATGTTT AGTTTCTTCC CATAGAATTT GACATAGGGG 4500

TATAGTACTA AAATATGAAT ATCCTGTAGC ATAACCGTTT TTAACACAAT ATGAACTACC 4560

CTTGTAAGAA TCTAATACGT AAGATCTACA CGCCATTTTC AAATCAGCGC CATGTTTTAT 4620 —I 2

—9

G TTAATAAT AATCTT CGC¾0 AG¾¾ CCA 504TTTG TTTTT TTT AT ACTATTA, ACA¾ TT.A TT

0 ACGTG98AGC CTGC TTCTT. GTATAT AAGCCGCG 4A TTACATTATTTTATTG AATTTATAAA

0G92 AGCGG CGCAATATTCT TACATC 4AA AT CTACTTAT TATTATCTTCT CTAATTTGTATT

0CG86 ACST CC TATTAACTTTAA TG 4TAT AAAACATTTTT TAATTCTAAG CATATTTTA

TCGGGAAC ATA GCA TGATGAGTATAT TCC800ATATGTTAA TTTTACCSAAACG 4ACT TAT

T CCGTTCT TATCTTTAATTAATTAAT0TT TTTT ATCACCCCCCGC 474TTC TAT

C¾ATTTTT iG TATATTCTT G680AMATATTCTTTT TATACAAGACGCC 4TT JTA GTT.

Claims

The scope of the claims

1. A recombinant avipox virus having at least two or more foreign genes in a genomic DNA region that is not essential for the growth of avibox virus, and having substantially the same growth potential as the parent strain.

2. The recombinant avibox virus according to claim 1, wherein the region that is not essential for propagation of the avibox virus has the nucleotide sequence of SEQ ID NO: 1 in the sequence listing.

3.A nucleotide sequence comprising at least 300 nucleotides on the 5 'side of the most upstream gene and the 3' side of the most downstream gene among the foreign genes, wherein the nucleotide sequence is 3. The recombinant avipoxvirus according to claim 1, which has homology to a continuous part of the nucleotide sequence described in No. 1.

4. The exogenous gene is selected from the group consisting of a marker, a gene encoding a promoter, and a gene encoding an antigenic determinant of a chicken pathogen. The recombinant avibox virus according to any one of the above.

5. The rapid gene encoding the antigenic determinant of the chicken pathogen is infectious bursal disease virus, pigeon infectious bronchitis virus, chicken meningitis virus, Newcastle disease virus, Marek's disease virus, and drone influenza. The recombinant avibox virus according to any one of claims 1 to 4, which is a gene encoding an antigenic determinant of a virus selected from the group consisting of viruses.

6. The recombinant transpoxvirus according to claim 5, wherein the foreign gene is a gene encoding an antigenic determinant of Newcastle disease virus.

7. The recombinant Avibock according to claim 6, wherein the arresting gene encoding the antigenic determinant of the Newcastle disease virus is a gene encoding an F antigen or an HN antigen. Swirls.

8. The recombinant avibox virus according to any one of claims 1 to 7, wherein the avibox virus is a fowlpox vaccine strain.

9. The recombinant avipoxvirus according to any one of claims 1 to 8, wherein the avibox virus is a pigeonpox Nakano strain.

10. Recombinant plasmid in which a genomic DNA region that is not essential for the growth of Avibox virus contains at least the nucleotide sequence of SEQ ID NO: 1.

1 1. Among the foreign genes, a nucleotide sequence consisting of at least 300 bases is located at the 5 'side of the farthest gene located at the most upstream position and at the 3 side of the downstream gene located at the most downstream position. A recombinant plasmid comprising a DNA sequence having homology to a continuous part of the nucleotide sequence of No. 1.

1 2. A vaccine comprising the recombinant avibox virus according to any one of claims 1 to 9 as an active ingredient.

1 3. An anti-Newcastle disease vaccine comprising the recombinant virus according to any one of claims 1 to 9 as an active ingredient.

14. (1) arbitrarily cleaving avibox virus genomic DNA to obtain a DNA fragment;

(2) introducing two or more exogenous speed genes into the DNA fragment;

(3) a step of preparing a plasmid for recombination by introducing the DNA fragment into which the foreign hesitant has been introduced into plasmid,

(4) a step of recombining the avibox virus with the recombinant plasmid to produce a recombinant avibox virus; A method for producing a recombinant avibox virus, which is substantially equivalent to a parent strain in terms of growth in a bird, comprising:

15. The method for producing a recombinant avipoxvirus according to claim 14, wherein the DNA fragment comprises a DNA region not essential for the growth of avibox virus.

16. The foreign gene is selected from the group consisting of a marker gene, a gene encoding a promoter, and a gene encoding an antigenic determinant of a bird pathogen. 3. The method for producing a recombinant avibox virus according to item 1.

1 7. The transgenes encoding the antigenic determinants of the avian pathogens are the infectious bursal disease gene, the pigeon infectious bronchitis virus, the chicken meningitis virus, the Newcastle disease virus, the Marek's disease virus, and 17. The method for producing a recombinant avibox virus according to any one of claims 14 to 16, wherein the gene encodes an antigenic determinant of a virus selected from the group consisting of chicken influenza viruses.

18. The method for producing a recombinant Avibox virus according to claim 17, wherein the foreign gene is a gene encoding an antigenic determinant of Newcastle disease virus.

19. The method according to any one of claims 14 to 18, wherein the gene encoding the antigenic determinant of the Newcastle disease virus is a gene encoding an F antigen or an HN antigen.

20. (I) selecting in vitro a virus that forms blue plaques when avian cell lines infected with the recombinant avibox virus are cultured on agar, (2) a step of measuring the size of the pock after infecting the chicken with the virus that forms the blue plaque in vivo,

A method for screening for a recombinant avipox virus, which does not substantially reduce the growth in the body.

21. A genomic DNA region that is non-essential for the growth of avibox virus, in which a / 9-galactosidase gene from Escherichia coli has been inserted into the region together with a vaccinia virus 7.5K motor. A recombinant avipox virus, wherein the growth of the avibox virus in chicken is not substantially reduced as compared with the growth of the virus in the chicken before the introduction of the ^ -galactosidase gene. The genomic DNA region from which is obtained.

22. A genomic DNA region which is non-essential for the growth of avipoxviruses and which comprises the nucleotide sequence of SEQ ID NO: 1 incorporating a foreign gene at a specific site.

23. The specific site is a C1al site existing at nucleotide numbers 962 to 967 and an HpaI site existing at nucleotide numbers 4,267 to 272 in the nucleotide sequence of SEQ ID NO: 1. A genomic DNA region that is non-essential for propagation of the avibox virus according to claim 22.