JP3675569B2 - Recombinant virus and vaccine comprising the same - Google Patents

Description

【００３８】
この結果によれば、未接種、親株ＦＰＶ、ｆＮＺ２９ＭＤ−ｇＥ接種群の鶏は、強毒ＮＤＶの攻撃試験に対し、ほとんどすべてニューカッスル病で死亡したが、ＮＤＶのＦ遺伝子を挿入した組み換えＦＰＶであるｆＮＺ５９２９を接種した鶏では、２４％が生存し、ｆＮＺ５９２９にｇＥ遺伝子を挿入したｆＮＺ５９２９−ｇＥ、ｇＥとｇＩの両遺伝子を挿入したｆＮＺ５９２９−ｇＩ−ｇＥの本発明の組み換えＦＰＶを接種した鶏の生存率は、それぞれ３８％、４７％であった。特に、ｆＮＺ５９２９−７．５ｇＩ−ｇＥのワクチン効果はｆＮＺ５９２９のそれと比較して約２倍であったことから、ヘルペス属ウイルスのｇＩ遺伝子とｇＥ遺伝子とを抗原遺伝子と共に組み込んだ組み換えウイルスは、移行抗体存在条件下でも有効なワクチンとして機能することが判った。
【００３９】
（実施例１０）ＦＰＶ組み換え用プラスミドｐＮＺ９９２９ＶＰ２Ｓ−７．５ｇＩ−ｇＥの構築と組み換えＦＰＶの作製
大腸菌ＮＺ−９１０１（微工研寄託番号：微工研菌寄第１２４２２号）から常法により抽出できる約６．０ｋｂのプラスミドｐＩＢＤＶをＰｖｕＩで消化後、接着末端をＫｌｅｎｏｗで平滑末端にし、さらにＫｐｎＩで消化して約３．２ｋｂｐのＩＢＤＶ ＳｅｇＡ遺伝子を含むＤＮＡ断片を回収した。実施例３で構築したプラスミドｐＮＺ１８２９ＲをＢａｍＨＩで消化後、接着末端をＫｌｅｎｏｗで平滑末端にし、さらにＫｐｎＩで消化して開裂した部位に上記の約３．２ｋｂｐのＳｅｇＡ ＤＮＡ断片を挿入して、プラスミドｐＮＺ２９ＲＳｅｇＡを構築した。次に実施例７で得たプラスミドｐＧＴＰ７．５−ｇＩをＢｇｌＩで消化後、約１．４ｋｂｐのＤＮＡ断片を回収した。このＤＮＡ断片を上記プラスミドｐＮＺ２９ＲＳｅｇＡのＳｆｉＩ部位に挿入し、プラスミドｐＮＺ２９ＲＳｅｇＡ−７．５ｇＩを構築した．さらに実施例５で得たプラスミドｐＧＴＰｓ−ｇＥをＢｇｌＩで消化後、約１．７ｋｂｐのＤＮＡ断片を回収した。このＤＮＡ断片をプラスミドｐＮＺ２９ＲＳｅｇＡ−７．５ｇＩのＳｆｉＩ部位に挿入し、目的のＦＰＶ組み換え用プラスミドｐＮ２９ＲＳｅｇＡ−７．５ｇＩｇＥを構築した。
【００４０】
このプラスミドを用いて実施例８と同様の手法で組み換えＦＰＶを作製し、純化した。得られた組み換えＦＰＶは、実施例９で示されたのと同様、組み換え生ワクチンとして有効であると期待される。
【００４１】
【発明の効果】
かくして本発明によれば、親ウイルスの増殖に非必須なゲノム領域にニューカッスル病の感染防御抗原をコードした遺伝子とともにＭＤＶのｇＩ、ｇＥ遺伝子を組み込まれた組み換えウイルスが得られ、この組み換えウイルスは、ニューカッスル病に対する移行抗体を保有している鶏に接種すると、この移行抗体の影響を低減し、ワクチンとしての高い効果を有する。さらに本発明のワクチンは、マレック病、伝染性ファブリキウス嚢病、伝染性喉頭気管炎等のニューカッスル病以外の病原体の感染防御抗原をコードした遺伝子とともにＭＤＶのｇＩ遺伝子とｇＥ遺伝子とを組み換えウイルスに挿入した場合にも同様のワクチン効果が期待できる。
【００４２】
【配列表】
配列番号：1
配列の長さ：31
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列

【００４３】
【配列表】
配列番号：2
配列の長さ：22
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列

【００４４】
【配列表】
配列番号：3
配列の長さ：22
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列

【００４５】
【配列表】
配列番号：4
配列の長さ：14４
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列

【００４６】
【配列表】
配列番号：5
配列の長さ：24
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列

【００４７】
【配列表】
配列番号：6
配列の長さ：17
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列

【００４８】
【配列表】
配列番号：7
配列の長さ：22
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列

【００４９】
【配列表】
配列番号：8
配列の長さ：32
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列

【００５０】
【配列表】
配列番号：9
配列の長さ：28
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列

【００５１】
【配列表】
配列番号：10
配列の長さ：14
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列

【００５２】
【配列表】
配列番号：11
配列の長さ：2760
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：genomic DNA
起源
生物名：マレック病ウィルス Ｉ型
株名：ＧＡ株
配列

【００５３】
【配列表】
配列番号：12
配列の長さ：355
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：タンパク質
起源
生物名：マレック病ウィルス Ｉ型
株名：ＧＡ株
配列

【００５４】
【配列表】
配列番号：13
配列の長さ：493
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：タンパク質
起源
生物名：マレック病ウィルス Ｉ型
株名：ＧＡ株
配列

【００５５】
【図面の簡単な説明】
【図１】 組み換え用プラスミドｐＮＺ５９２９の構築手順を示した説明図である。
【図２】 組み換え用プラスミドＰＧＰＴｓ−ｇＥの構築手順を示した説明図である。
【図３】 組み換え用プラスミドｐＮＺ２９ＲＭＤｇＥの構築手順を示した説明図である。
【図４】 組み換え用プラスミドｐＮＺ５９２９−ｇＥの構築手順を示した説明図である。
【図５】 組み換え用プラスミドｐＮＺ５９２９−７．５ｇＩの構築手順を示した説明図である。
【図６】 組み換え用プラスミドｐＮＺ５９２９／７．５ｇＥｇＩの構築手順を示した説明図である。
【図７】 組み換え用プラスミドｐＮＺ２９ＲＳｅｇＡ−７．５ｇＩの構築手順を示した説明図である。
【図８】 組み換え用プラスミドｐＮＺ２９ＲＳｅｇＡ−７．５ｇＩｇＥの構築手順を示した説明図である。[0001]
[Industrial application fields]
  The present inventionFoul poxMore specifically, a virus and a vaccine comprising the same, a DNA encoding the glycoprotein gE of a virus belonging to the genus Herpes, or a DNA encoding the glycoprotein gE and a DNA encoding gI together with a DNA encoding a heterologous foreign antigen protein. , Recombinant foulpox virus inserted into a genomic region not essential for its growth, and the recombinationFoul poxThe present invention relates to a vaccine containing a virus as an active ingredient.
[0002]
[Prior art]
  In the current poultry farming field, disease prevention by vaccination is a pillar of hygiene management, regardless of whether it is a breeding chicken, a laying hen or a meat chicken. However, the vaccination program is really overcrowded, so the manpower, time and cost required for this is a major problem.
[0003]
  In recent years, as a solution to this, it has become possible to extract only a gene encoding a protein necessary for immunity induction from a certain pathogen by a recombinant DNA technique and construct a recombinant virus containing the gene. By inoculating such a recombinant virus as a recombinant live vaccine, it became possible to induce immunity against the antigen protein encoded by the inserted gene. Avipoxviruses belonging to poxviruses are suitable viruses for use in the construction of such recombinant viruses. These viruses typified by foulpox virus (hereinafter referred to as FPV) have large genomic DNA, many of which are non-essential for virus growth, and a plurality of antigen genes in these non-essential regions of the same virus. Can be inserted. Such a recombinant virus vaccine is presumed to be able to induce humoral immunity and cellular immunity of the vaccinated host. This method has been applied as a recombinant FPV in which a foreign gene has been inserted into an attenuated FPV that has been conventionally used as a live vaccine against fowlpox using genetic engineering techniques. It is a chicken disease virus as a foreign geneNewcastleRecombinant FPV inserted with a gene encoding a disease virus antigen (hereinafter referred to as an antigen gene), a Marek's disease virus antigen gene, and a chicken influenza virus antigen gene has been constructed and experimentally inoculated into SPF chickens, and vaccine effect (Boursnell et al., Virology, 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 FPV can insert various foreign antigen genes according to the purpose, and can simultaneously insert a plurality of antigen genes. Therefore, a multivalent vaccine effective against a plurality of pathogens in a single inoculation. It is promising that it will be possible, and as a result, a solution to the above-mentioned overcrowded vaccine program problem.
[0004]
  On the other hand, one of the important matters that must be considered in the process of putting a new vaccine into practical use is a transition antibody. Newborn individuals are at risk of pathogen infection due to immature immune mechanisms. It is born by receiving antibodies against various pathogens from the mother as a defense function on the living body side. Such maternal antibodies are called transitional antibodies and protect individuals from pathogen infection for several weeks after birth. However, on the other hand, the attenuated virus vaccinated at this time is also eliminated by the transfer antibody. As a result, the priming effect of the vaccine cannot be obtained, and the risk of infection with a pathogen increases when the transfer antibody disappears. Furthermore, since the components and titers of this transfer antibody vary from individual to individual, it is desirable to inoculate a collective vaccination in time for the disappearance of the transfer antibody, but this is almost impossible. For example, inoculate chickensNewcastleIn the case of a disease virus (hereinafter referred to as NDV) live vaccine, efforts are made to minimize the reduction of the vaccine effect due to variations in the transfer antibody titer of individuals by inoculating a few times at the time of the initial chick. In the case of an infectious bursal disease virus (hereinafter referred to as IBDV) vaccine, an inactivated oil-emulsion was given to breeding hens before the start of the spawning season at approximately 22 weeks of age.SexThe aim is to protect the chickens for several weeks after emergence by inoculating the virus and containing high levels of migrating antibodies in the fertilized eggs. However, when the recombinant FPV reported in the literature that the above-mentioned vaccine effect was observed was inoculated into a chicken that possessed a commercially available migrating antibody instead of an SPF chicken raised at the laboratory level, the effect of the migrating antibody In response, the vaccine effect is expected to decrease. Furthermore, when a multivalent recombinant FPV in which a plurality of antigen genes are inserted is inoculated, the transfer antibody against each antigen quickly eliminates this recombinant FPV, so that the effect of the transfer antibody is further increased. It is expected not to be possible.
[0005]
  By the way, recent herpes simplex virus (HSV) studies have identified two glycoproteins gI and gE that have the property of binding to host immunoglobulin (hereinafter referred to as IgG). These two proteins formed a complex and exhibited a function as an Fc receptor that binds to the Fc region of IgG (Johnson et al., J. Virol., 62, 1347-1354 (1988)). GE or gI gene deletion mutant HSV produced using recombinant DNA technology was significantly inhibited in vitro in the presence of anti-HSV antibodies (Johnson et al., Virology, 177, 437-444 (1990); Friedman Et al., J. Virol., 65, 7046-7050 (1991)). From these research results, it is inferred that HSV gI and gE play an important role to escape from immune responses involving viral antibodies. Although the functions of gI and gE in vivo are not yet elucidated, it has been clarified that only gI and gE are not essential for virus growth.
[0006]
  Also in MDV, the full length of the gI gene and the partial base sequence of the gE gene have recently been clarified (Velicer et al., US Pat. No. 5,252,716). However, their functional aspects have not been clarified at all, and it has been difficult to predict what phenomenon will occur when these genes are expressed in recombinant viruses.
[0007]
[Problems to be solved by the invention]
  The present inventors have developed a new recombination that is not easily affected by the transfer antibody.Foul poxAs a result of earnest examination to obtain a vaccine,Foul poxWhen a DNA encoding gE, a glycoprotein of a virus belonging to the herpes genus, is inserted together with a pathogen antigen gene in a region that is not essential for virus growth, it functions as a new type of vaccine that is not easily affected by the transfer antibody. RecombinationFoul poxThe inventors have found that a virus can be obtained and have completed the present invention.
[0008]
[Means for Solving the Problems]
  Thus, according to the present invention,The genomic region that is not essential for foulpox virus growth contains (1) DNA encoding the Marek's disease virus glycoprotein gE, or DNA encoding the Marek's disease virus glycoprotein gE and DNA encoding the glycoprotein gI. And (2) containing a DNA encoding a foreign foreign antigen proteinRecombinationFoul poxA virus is provided and the recombinantFoul poxA vaccine comprising a virus as an active ingredient is provided.
[0009]
  Recombination of the present inventionFoul poxFor example, the virus is prepared as follows. Pre-parent virusFoul pox virusofNonThe gE genetic death struggled so as to be under the control of a promoter that functions in the parent virus is incorporated into the non-essential region of the plasmid into which the essential region has been incorporated to obtain a plasmid vector. Next, by introducing this plasmid vector into cells infected with the parent virus, homologous recombination is caused, and the resulting virus is selected and purified to produce the desired recombination.Foul poxGet a virus.
The foul pox virus (FPV)Specific examples include ATCC VR-251, ATCC VR-250, ATCC VR-229, ATCC VR-249, ATCC VR-288, Nishigahara strain, Suisui strain, CEVA strain, and CEVA vaccine strain-derived viruses. A narrowly-defined FPV such as one that forms large plaques when infected with fibroblasts (CEF), or a closely related virus such as an NP strain (chicken-bearing pigeon Nakano strain), Examples thereof include viruses used as a pigeon live vaccine strain. These can be easily obtained commercially or by distribution.
[0010]
(Non-essential area)
  The non-essential region used in the present invention is a DNA region that is non-essential for the growth of the parent virus., TThe K gene region and the region described in JP-A-1-168279 can be used. Specific examples thereof are described in, for example, the above publication.NEcoRI fragment (about 7.3 kb), EcoRI-HindIII fragment (about 5.0 kb), BamHI fragment (about 4.0 kb), HindIII fragment (about 5.2 kb) of P strain DNACan be mentioned.
[0011]
(Vector containing non-essential regions)
  For the construction of the recombination vector of the present invention described later, a vector for cloning a non-essential region of the virus is used. Such vectors include, for example, plasmids such as pBR322, pBR325, pUC7, pUC8, and pUC18, phages such as λ phage and M13 phage, cosmids such as pHC79, and the like, and treatment with an appropriate restriction enzyme. It can be obtained by incorporating a DNA fragment of a virus non-essential region as described above.
[0012]
(Recombination vector)
  The recombination vector used in the present invention contains a non-essential region into which an antigen gene and a promoter governing it are inserted. What is necessary is just to insert the promoter which controls each gE gene or gE gene and gI gene, and an antigen gene which are mentioned later into the virus non-essential region of the vector containing the above-mentioned non-essential region, and such a vector. Of the derived antigen gene and the promoter controlling itFoul poxA fragment containing a non-essential region of the virus may be incorporated into another vector. In addition, recombinationFoul poxFor efficiency such as virus purification, a marker gene such as the lacZ gene of E. coli and a promoter described below may be incorporated.
[0013]
(GE gene, gI gene)
  The gE gene and gI gene used in the present invention may be those derived from the genome of a virus belonging to the genus Herpes. Herpes virus infects mammals such as herpes simplex virus that infects humans, bovine herpes virus that infects cattle, equine herpes virus that infects horses, ooeskey disease virus that infects pigs, and cat rhinotracheitis virus that infects cats And herpes viruses that infect birds such as Marek's disease virus and infectious pharyngeal tracheitis virus. It is preferable to use gE and gI genes derived from viruses that infect animal species to be vaccinated. That is, it is preferable to use a gene derived from Marek's disease virus for use as a chicken vaccine.
[0014]
  The gE gene and gI gene used in the present invention are usually about 80% to about 120% due to natural or artificial mutation such as deletion, insertion or addition of a part of the base when the total length is 100%. Preferably, the total length (base length) varies in the range of about 90% to 110%.RemainsA part of the base of gE gene or gI gene by gene or natural or artificial mutation (the part mentioned here is usually 20% or less of the whole, preferably 10% or less, more preferably 5% or less ) Is replaced and changed to a base encoding another amino acidRemainsBeing an ancestorEven the remainsA protein encoded by a gene usually has a function substantially equivalent to that of natural gE or gI.That's fine. ThisThe function of natural gE and gI mentioned here is that a complex of gI and gE (gI-gE) or gE alone has a physiological activity to bind to the Fc site of IgG, and as a result, a carrier or an antibody Recombination in which an antigen gene, gE gene, and, if necessary, a gI gene are incorporated into an organism that still has about 50% of the transition antibody immediately after birth, which is a function that inhibits the immune response due to cell-dependent damage The virus is inoculated as a vaccine, the virulent strain is inoculated when the transfer antibody becomes about 10%, and the infection protection rate when the transfer antibody is substantially lost is the same in non-expression of gE and gI A function substantially equivalent to that of natural gE or gI is judged to be about 1.5 times or more, preferably about 2 times or more of the group vaccinated with the recombinant virus incorporating the antigen gene.
[0015]
  In the present invention, the method for causing an artificial mutation is not particularly limited, and can be performed according to a conventional method. As a specific example, after treating a natural gE gene with an appropriate restriction enzyme, an appropriate DNA fragment is inserted (or dropped) so as not to shift the reading frame for translation into amino acids, and then ligated again. And an in vitro mutation method (Ncleic Acid Research 10, 6, 6487-6497 (1982)) by Fritz Eckstein et al.
[0016]
  GE inheritance from such herpes virusesWith childExamples thereof include DNA encoding the amino acid sequence of SEQ ID NO: 13 derived from Marek's disease virus type I GA strain, for example, the 1207th to 2697th sequences of SEQ ID NO: 11. Also, FGI inheritance from the genus RupesWith childExamples thereof include DNA encoding the amino acid sequence described in SEQ ID NO: 12 derived from Marek's disease virus type I GA strain, for example, the first to 1065th sequences described in SEQ ID NO: 11.
[0017]
  In the present invention, the gE gene alone can provide an effect as a vaccine that is not easily affected by the transferred antibody, but in order to obtain a higher effect, it is desirable to incorporate it with the gI gene. The order of linking the gE gene, gI gene and the antigen gene described below in the recombinant virus is not particularly limited as long as each gene is linked so that it can substantially express gE, gI and the antigen protein. That is, from the 5 ′ upstream side, the order of gE gene-gI gene-antigen gene, the order of gI gene-gE gene-antigen gene, the order of gE gene-antigen gene-gI gene, the order of gI gene-antigen gene-gE gene The order of antigen gene-gE gene-gI gene and the order of antigen gene-gI gene-gE gene may be any of these, but for the sake of convenience of operation, gE is placed behind (3 'side) gI gene. It is desirable that the genes are linked and the antigen gene is linked to the 5 ′ side or 3 ′ side of these genes. In addition, when a marker gene useful for selection of a recombinant virus is incorporated, the linking site is not particularly limited. The method for linking these genes is not particularly limited, and a method for preparing genes to be inserted in advance by linking each gene using an appropriate linker or the like, or a direct preparation of a recombinant vector by homologous recombination using a vector having each gene. Can be linked according to conventional methods such as
[0018]
(Heterologous foreign antigen protein, antigen gene)
  In the present invention, the foreign foreign antigen protein is a protein derived from a virus or bacteria different from the parent virus, and may be any protein that is transcribed and translated in the parent virus and expressed as an antigen protein. Specific examples of DNA (antigen gene) encoding a heterologous antigen protein when obtaining a chicken vaccine include a gene encoding MDV glycoprotein (Ross et al., J. Gen. Virol., 70, 1789-1804 (1988). )), A gene encoding HN of NDV (Miller et al., J. Gen. Virol., 67, 1917-1927 (1986)), a gene encoding F protein (McGinnes et al., Virus Res., 5, 343-356). (1986)), which encodes an antigen involved in defense against infection such as the gene encoding the structural protein VP2 of infectious bursal disease virus (Bayliss et al., J. Gen. Virol., 71, 1303-1212 (1990)). Genes are preferred.
[0019]
(promoter)
  The promoter used in the present invention is a recombinantFoul poxThere is no particular limitation as long as it functions as a promoter in a virus-infected host. For example, 7. Examples include a vaccinia virus gene promoter encoding 5K polypeptide, a vaccinia virus gene promoter encoding 11K polypeptide, a vaccinia virus gene promoter encoding thymidine kinase, and the like, as long as it functions as a promoter. May be modified by deleting a part thereof, or may be synthesized. In the present invention, synthetic promoters having sequences of both early and late promoters (AJ Davidson et al., J. Mol. Biol., 215, 749-769, and p771-781 (1989)) A part of the sequence is deleted so long as the promoter activity is not lost, or the base is changed (for example, the base sequence is preferably 5′-TTTTTTTTTTTTTTTTTTTTGGCATATAAAATAATAATATACCGCGTAAAAATTGAAAAAACTATTCTA CTTTATTC ′). In this synthetic promoter or a modified product thereof, a large number of T (thymidine) is continuous at the 5 ′ end of the base sequence. From the viewpoint of the high promoter activity and the high expression level of the antigen gene. It is preferable that 15-40 T is continuous, and it is more preferable that 18-30 T are continuous.
[0020]
  Of course, promoters can be linked so as to control the gE gene, gI gene, antigen gene, and marker gene, respectively, but the promoters linked to each gene are not necessarily the same promoter.
[0021]
(RecombinationFoul poxVirus production method)
  RecombinationFoul poxThe method for producing the virus is not particularly limited, and may be performed according to a conventional method. That is, by introducing a recombinant vector having a gE gene, a gI gene, an antigen gene, etc. into a cell previously infected with the parent virus by, for example, calcium phosphate coprecipitation method, the vector and the viral genomic DNA in the infected cell Homologous recombination occurs betweenFoul poxVirus is constructed. The obtained recombinant virus infects host cells cultured in a medium such as Eagle MEM, and a hybridization method using an antigen gene incorporating a growing plaque as a probe, or expression of a marker gene incorporated together with the antigen gene The candidate strain is purified by a method such as that described above, and an antibody against the polypeptide encoded by the incorporated antigen gene is used.Foul poxWhat is necessary is just to confirm that it is a virus. For example, recombination in which the lacZ gene is incorporated as a marker geneFoul pox virusIn the case of β-galactosidase. Therefore, blue plaques are formed in the presence of Bluo-gal (GIBCO-BRL), which is one of the temperaments, and can be selected and purified using its properties. The host cell is not particularly limited as long as the virus to be used can be infected and propagated.,chickenExamples thereof include fibroblast (CEF) cells and embryonated chicken egg allantoic cells.
[0022]
(vaccine)
  The vaccine of the present invention comprises one or more recombinants of the present invention.Foul poxIt is a vaccine consisting of a virus. That is, the MDV-derived gE gene disclosed by the present invention or a recombinant virus having a gE gene and a gI gene, an antigen gene and the like may be used alone, or other two or three types of recombinant viruses may be combined. Also recombinationFoul poxIn addition to viruses, pharmacologically problematic carriers such as physiological saline and stabilizers may be included. The method for preparing the vaccine of the present invention is not particularly limited. For example, recombination in the present inventionFoul poxA cell in which a virus can grow is infected with the recombinant virus of the present invention,Foul poxIncubate until virus grows. Thereafter, the cells are collected and disrupted. This cell debris is recombined with the precipitate in a centrifuge tube by a centrifuge.Foul poxSeparate into high-titer supernatant containing virus. Essentially free of host cells and recombined with cell culture mediaFoul poxThis centrifugal supernatant containing the virus can be used as the vaccine of the present invention. Further, it may be diluted with a pharmacologically acceptable physiological saline or the like. It can also be used as a freeze-dried vaccine by freeze-drying the centrifugal supernatant.
[0023]
  When the vaccine of the present invention is a chicken vaccine, it may be administered to poultry by any method as long as the recombinant virus in the vaccine infects poultry and causes protective immunity. For example, wing puncture, scratching the skin and inoculating the vaccine, or vaccination subcutaneously in poultry with an injection needle or other device. It is also possible to suspend the vaccine in poultry drinking water or mix it with the solids of the feed and inoculate it orally. Furthermore, a method of inhaling a vaccine by aerosol or spray, an intravenous inoculation method, an intramuscular inoculation method, an intraperitoneal inoculation method, or the like can also be used.
[0024]
  For example, in the case of chickens, the inoculation amount is usually 10 to 10 per bird.⁶Plaque forming unit (PFU), preferably 10²-10⁴PFU. In the case of injection, this amount may be diluted to about 0.1 ml with a pharmacologically acceptable liquid such as physiological saline. The vaccine of the present invention can be stored and used under the same conditions as ordinary vaccines. For example, if the recombinant virus of the present invention is freeze-dried, it can be stored in a refrigerator (0 to 4 ° C.), and can be stored at room temperature (20 to 22 ° C.) for a shorter period. In addition, the virus suspension can be frozen at -20 to -70 ° C and stored.
[0025]
【Example】
  The present invention will be specifically described below with reference to examples.
[0026]
(Example 1) Extraction of MDV GA strain genomic DNA
  CEF cells infected with MDV GA strain were collected by trypsinization using a petri dish, washed twice with PBS, and then washed with proteinase K buffer (10 mM Tris-HCl (pH 7.8), 5 mM EDTA, 0.5% SDS. The proteinase K (manufactured by Boehringer Mannheim) was added to a concentration of 50 μg / ml. After leaving at 55 ° C. for 2 hours, the protein was removed twice with phenol / chloroform, and 2 times the amount of ethanol was added and left at −20 ° C. for 20 minutes, followed by centrifugation and genomic DNA of MDV GA strain. Got.
[0027]
Example 2 Cloning of MDV gI and gE Genes
  Nucleotide sequence of unique short region of MDV GA strain published by Velicer et al. for cloning gI gene and gE gene from genomic DNA of MDV GA strain (Velicer et al., US Pat. No. 5,252,716) Based on the above, two kinds of DNA primers (5′-CGGGAGATCTGCGATGTATGGTACTACATATA-3 ′ (SEQ ID NO: 1), 5′-GGATCCCGCATCGACAATAAATT-3 ′ (SEQ ID NO: 2)) described in SEQ ID NOS: 1 and 2 were used. The primer of SEQ ID NO: 1 includes the nucleotide sequence described in the first to 18th of the gI gene represented by SEQ ID NO: 11, and the primer of SEQ ID NO: 2 is the 1496th of the gE gene represented by SEQ ID NO: 11. The base sequence described in -1515th is included. By using these primers, the polymerase chain reaction (hereinafter referred to as PCR) using the MDV GA strain genomic DNA obtained in Example 1 as a template, the DNA fragment having the full length of gI gene and a part of gE gene. Amplification was performed. The reaction composition was 64.5 μl of an aqueous solution containing 20 picograms of MDV genomic DNA, 8 μl of 10-fold concentrated PCR reaction buffer (200 mM Tris-HCl (pH 8.4), 500 mM potassium chloride, 25 mM magnesium chloride), 4 μl of 2.5 mM dNTPs, 5 U. 0.5 μl of tack polymerase / μl concentration and 1 μl of each 20 μm primer were added. The reaction conditions were denaturation at 95 ° C. for 1 minute, annealing at 55 ° C. for 2 minutes, polymerase reaction at 72 ° C. for 2 minutes, and 30 cycles with a thermal cycler 480 manufactured by Perkin Elmer Citas. As a result, a DNA fragment of about 1.5 kb containing the full length of the gI gene and a partial base sequence at the 5 'end of the gE gene was obtained.
[0028]
  The MDV GA strain obtained in Example 1 was cleaved with various restriction enzymes, then electrophoresed on a 0.8% agarose gel, the DNA fragment in the gel was transferred to a nylon membrane, and the above PCR product was used as a probe. Southern hybridization was performed. As a result, it was confirmed that an about 1.1 kb BamHI-KpnI DNA fragment containing the gI gene and an about 6.0 kb BglII-HindIII DNA fragment containing the gE gene were hybridized. These DNA fragments were cloned into the BamHI-KpnI and BamHI-HindIII sites of pUC18 to obtain pUC-gI-1 and pUC-gE-1, respectively. The approximately 6.0 kb BglII-HindIII DNA fragment cloned into pUC-gE-1 was cleaved with various restriction enzymes to prepare a restriction enzyme map. Using this, an about 2.2 kb KpnI-HpaI DNA fragment containing the gE gene was subcloned into the KpnI-HincII site of pUC18 to obtain pUC-gE-2. The entire base sequence of the gE gene (base sequence Nos. 1207 to 2700 in SEQ ID NO: 11) is the Sanger dideoxy chain termination method (Sanger et al., Proc. Natl. Acad. Sci. USA, 74, 5463-5467). 1977)) by examining the sequence of a series of deletion mutants of pUC-gE-2.
[0029]
(Example 3) FPV recombination plasmid pNZ5929 (see FIG. 1)
  Plasmid pNZ98 (described in JP-A-3-2784) was partially digested with the restriction enzyme SacI, and further partially digested with the restriction enzyme BamHI to recover a DNA fragment of about 1.9 kb. On the other hand, plasmid pNZ1729R (Yanagida et al., J. Virol., 66, 1402-1408 (1992)) was digested with EcoRI and SacI, and two kinds of synthetic DNAs described in SEQ ID NO: 3 and SEQ ID NO: 4 (5 A synthetic adapter containing an SfiI site annealed with '-AATTCGGCCGGGGGGGGCCAGCT-3' (SEQ ID NO: 3) and 5'-GGCCCCCCGCGCCG-3 '(SEQ ID NO: 4)) was inserted to construct plasmid pNZ1829R. This plasmid pNZ1829R was digested with the restriction enzyme BamHI, further partially digested with the restriction enzyme SacI, and the above-mentioned approximately 1.9 kb BamHI-SacI DNA fragment was inserted to construct the desired plasmid pNZ5929.
[0030]
(Example 4) Construction of antigen gene insertion plasmids pGPTs and pGTP7.5
  Plasmid pGPTs is a DNA fragment of about 140 bp obtained by digesting plasmid pNZ1719R (Yanagida et al., J. Virol., 66, 1402-1408 (1992)) with restriction enzymes HindIII and SalI at the HindIII-SalI site of pUC18. Further, the synthetic DNA (5′-AGCTGCCCCCCCGGCAAGCTTGCA-3 ′) described in SEQ ID NO: 5 was inserted into the HindIII-PstI site, and then the synthetic DNA (5′-TCGACATTTTTATGTAC-3 described in SEQ ID NO: 6 was inserted into the SalI-EcoRI site. ') Was inserted, and finally the synthetic DNA (5'-AATTCGGCCGGGGGGGCCAGCT-3') described in SEQ ID NO: 7 was inserted into the SacI-EcoRI site. Plasmid pGTP7.5 is a DNA fragment of about 270 bp obtained by digesting plasmid pNZ1037 (Ogawa et al., Vaccine, 8, 486-490 (1990)) with restriction enzymes HindIII and SalI at the HindIII-SalI site of pUC18. Further, the synthetic DNA (5′-AGCTGCCCCCCCGGCAAGCTTGCA-3 ′) described in SEQ ID NO: 5 was inserted into the HindIII-PstI site, and then the synthetic DNA (5′-TCGACATTTTTATGTAC-3 described in SEQ ID NO: 6 was inserted into the SalI-EcoRI site. ') Was inserted, and finally the synthetic DNA (5'-AATTCGGCCGGGGGGGCCAGCT-3') described in SEQ ID NO: 7 was inserted into the SacI-EcoRI site.
[0031]
(Example 5) Construction of FPV recombination plasmid pNZ29MD-gE (see FIGS. 2 and 3)
  PCR was performed using pUC-gE-1 obtained in Example 2 as a template and the primers shown in SEQ ID NOs: 8 and 9. The reaction composition and conditions were the same as in Example 2. After digesting the obtained PCR product with restriction enzymes BglII and SalI, a DNA fragment of about 1.5 kb is recovered, and the synthesis described in SEQ ID NO: 10 for creating a BglII site between the HindIII site and the PstI site of plasmid pUC18 The plasmid pUC-gE-PCR was constructed by inserting it into the BglII-SalI site of the plasmid pUC-XG prepared by incorporating DNA (5′-AGCTAGATCTTGCA-3 ′). Although the nucleotide sequence of the inserted DNA fragment was confirmed, no mutation was found during PCR. After digesting this plasmid pUC-18-gE-PCR with restriction enzymes BglII and SalI, a DNA fragment of about 1.5 kb was recovered and inserted into the BamHI-SalI site of pGTPs to construct plasmid pGPTs-gE. After digesting this plasmid pGPTS-gE with the restriction enzyme BglI, a DNA fragment of about 1.7 kb was recovered. This DNA fragment was inserted into the SfiI site of the FPV recombination vector pNZ1829-Sfi to construct the desired recombination plasmid pNZ29MD-gE.
[0032]
(Example 6) Construction of FPV recombination plasmid pNZ5929-gE (see FIG. 4)
  After digesting the plasmid pGPTs-gE obtained in Example 5 with the restriction enzyme BglI, a DNA fragment of about 1.7 kb was recovered. This DNA fragment was inserted into the SfiI site of the FPV recombination vector pNZ5929 obtained in Example 3 to construct the desired recombination plasmid pNZ5929-gE.
[0033]
(Example 7) Construction of FPV recombination plasmid pNZ5929-7.5 gI-gE (FIGS. 5 and 6)
  The about 1.5 kb DNA fragment obtained by PCR in Example 2 was digested with the restriction enzyme KpnI, and further partially digested with the restriction enzyme BglII to recover an about 1.1 kb DNA fragment. This DNA fragment was inserted into the BanHI-KpnI site of pGTP7.5 obtained in Example 4 to construct plasmid pGTP7.5-gI. Although the nucleotide sequence of the inserted DNA fragment was confirmed, no mutation was found during PCR. After digesting this plasmid pGTP7.5-gI with the restriction enzyme BglI, a DNA fragment of about 1.4 kb was recovered. This DNA fragment was inserted into the SfiI site of the FPV recombination vector pNZ5929 obtained in Example 3 to construct plasmid pNZ5929-gI. Next, the plasmid pGTPs-gE obtained in Example 5 was digested with the restriction enzyme BglI, and then a DNA fragment of about 1.7 kb was recovered. This DNA fragment was inserted into the SfiI site of plasmid pNZ5929-7.5 gI to construct the desired plasmid pNZ5929-7.5 gIgE for FPV recombination.
[0034]
(Example 8) Production and purification of recombinant FPV
  Monolayer CEF was infected with a virus with a large plaque formation phenotype isolated from the FPV vaccine (Nazerian et al., Avian Dis. 33, 458-465 (1989)) at a multiplicity of infection of 0.1 did. Three hours later, these cells were detached by trypsin treatment to obtain a cell suspension. 2 x 10 from this suspension⁷A mixture of the cells and 10 μg of the recombination plasmid was added to Saline G (0.14 M sodium chloride, 0.5 mM potassium chloride, 1.1 mM disodium monohydrogen phosphate, 0.5 mM magnesium chloride hexahydrate, 0.011 % Of glucose) and electroporation at room temperature under conditions of Gene Pulser (Bio-Rad) 3.0 KV / cm, 0.4 msec. Cells into which the plasmid was introduced were then cultured at 37 ° C. for 72 hours, and the cells were lysed by lyophilization three times. The released recombinant virus was selected as follows.
[0035]
  A 10-fold serial dilution of the lysate containing the progeny virus released from the lysed cells was infected with the subcultured CEF, and 10 ml of agar solution containing the growth medium was overlaid. After setting the agar at room temperature, it was incubated at 37 ° C. until typical FPV plaques appeared. Further, another agar containing 250 μg / ml of Bluo-gal was layered on each culture plate, and further cultured at 37 ° C. for 24 hours. Blue plaques appeared at a rate of approximately 1% for all progeny viruses. These blue plaques were isolated, the contained virus was recovered, and the recombinant virus was further purified in the same manner until all plaques that formed formed stained blue with Bluo-gal. This process usually ends in 3-4 times. This purified virus was named fNZ5929. This fNZ5929 was analyzed by Southern hybridization, and it was confirmed that the NDV F gene and the lacZ gene were in the expected positions. The recombination plasmids pNZ29MD-gE, pNZ5929-gE, and pNZ5929-7.5gI-gE shown in Examples 4, 5, and 6 were introduced into FPV-infected cells in the same manner as described above. They were named fNZ29MD-gE, fNZ5929-gE, and fNZ5929-7.5 gI-gE, respectively.
[0036]
(Example 9) Infection protective effect against virulent NDV of chicken inoculated with recombinant virus
  Recombinant FPV is puncture needle 10⁴PFU was inoculated into the right wing membrane of a chicken with a transfer antibody against Newcastle disease at 3 days of age.⁴PFU highly toxic DNV Sato strain was inoculated into peach muscles. Thereafter, deaths due to Newcastle disease were recorded until age 45 days. The results are shown in Table 1.
[0037]
[Table 1]

[0038]
  According to this result, the chickens of the uninoculated, parental strain FPV, fNZ29MD-gE inoculated group almost all died of Newcastle disease against the aggressive NDV challenge test, but were recombinant FPV in which the NDV F gene was inserted. Among the chickens inoculated with fNZ5929, 24% survived, and the survival of chickens inoculated with the recombinant FPV of the present invention of fNZ5929-gE in which the gE gene was inserted into fNZ5929, and fNZ5929-gI-gE in which both the genes of gE and gI were inserted. The rates were 38% and 47%, respectively. In particular, since the vaccine effect of fNZ5929-7.5gI-gE was about twice that of fNZ5929, the recombinant virus incorporating the herpes virus gI gene and gE gene together with the antigen gene is a transfer antibody. It was found that it functions as an effective vaccine even under existing conditions.
[0039]
(Example 10) Construction of FPV recombination plasmid pNZ9929VP2S-7.5gI-gE and preparation of recombinant FPV
  After digesting approximately 6.0 kb of plasmid pIBDV, which can be extracted from E. coli NZ-9101 (Maikoken Deposit No .: Microtechnical Laboratories No. 12422) by a conventional method, with PvuI, the cohesive ends are made blunt with Klenow, and further KpnI And a DNA fragment containing about 3.2 kbp of the IBDV SegA gene was recovered. The plasmid pNZ1829R constructed in Example 3 was digested with BamHI, the cohesive end was made blunt with Klenow, and further digested with KpnI, and the above-mentioned approximately 3.2 kbp SegA DNA fragment was inserted into the plasmid pNZ29RSegA. Built. Next, the plasmid pGTP7.5-gI obtained in Example 7 was digested with BglI, and then a DNA fragment of about 1.4 kbp was recovered. This DNA fragment was inserted into the SfiI site of the plasmid pNZ29RSegA to construct plasmid pNZ29RSegA-7.5gI. Further, after digesting the plasmid pGTPs-gE obtained in Example 5 with BglI, a DNA fragment of about 1.7 kbp was recovered. This DNA fragment was inserted into the SfiI site of the plasmid pNZ29RSegA-7.5gI to construct the desired FPV recombination plasmid pN29RSegA-7.5gIgE.
[0040]
  Using this plasmid, recombinant FPV was prepared and purified in the same manner as in Example 8. The obtained recombinant FPV is expected to be effective as a live recombinant vaccine, as shown in Example 9.
[0041]
【The invention's effect】
  Thus, according to the present invention, a recombinant virus in which MDV gI and gE genes are incorporated into a genomic region that is not essential for the growth of the parent virus together with a gene encoding a Newcastle disease infection protective antigen is obtained. When inoculated into chickens carrying a transfer antibody against Newcastle disease, the effect of this transfer antibody is reduced and the vaccine is highly effective. Furthermore, the vaccine of the present invention inserts MDV gI gene and gE gene into a recombinant virus together with a gene encoding a protective antigen of a pathogen other than Newcastle disease such as Marek's disease, infectious bursal disease, and infectious laryngotracheitis. In this case, the same vaccine effect can be expected.
[0042]
[Sequence Listing]
SEQ ID NO: 1
Sequence length: 31
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids Synthetic DNA
Array

[0043]
[Sequence Listing]
SEQ ID NO: 2
Sequence length: 22
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids Synthetic DNA
Array

[0044]
[Sequence Listing]
SEQ ID NO: 3
Sequence length: 22
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids Synthetic DNA
Array

[0045]
[Sequence Listing]
SEQ ID NO: 4
Sequence length: 144
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids Synthetic DNA
Array

[0046]
[Sequence Listing]
SEQ ID NO: 5
Sequence length: 24
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids Synthetic DNA
Array

[0047]
[Sequence Listing]
SEQ ID NO: 6
Sequence length: 17
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids Synthetic DNA
Array

[0048]
[Sequence Listing]
SEQ ID NO: 7
Sequence length: 22
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids Synthetic DNA
Array

[0049]
[Sequence Listing]
SEQ ID NO: 8
Array length: 32
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids Synthetic DNA
Array

[0050]
[Sequence Listing]
SEQ ID NO: 9
Sequence length: 28
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids Synthetic DNA
Array

[0051]
[Sequence Listing]
SEQ ID NO: 10
Sequence length: 14
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids Synthetic DNA
Array

[0052]
[Sequence Listing]
SEQ ID NO: 11
Array length: 2760
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: genomic DNA
origin
Name of organism: Marek's disease virus type I
Stock name: GA stock
Array

[0053]
[Sequence Listing]
SEQ ID NO: 12
Sequence length: 355
Sequence type: amino acid
Topology: Linear
Sequence type: Protein
origin
Name of organism: Marek's disease virus type I
Stock name: GA stock
Array

[0054]
[Sequence Listing]
SEQ ID NO: 13
Sequence length: 493
Sequence type: amino acid
Topology: Linear
Sequence type: Protein
origin
Name of organism: Marek's disease virus type I
Stock name: GA stock
Array

[0055]
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is an explanatory diagram showing the construction procedure of a recombination plasmid pNZ5929.
FIG. 2 is an explanatory diagram showing a procedure for constructing a recombination plasmid PGPTs-gE.
FIG. 3 is an explanatory diagram showing a procedure for constructing a recombination plasmid pNZ29RMDgE.
FIG. 4 is an explanatory diagram showing a procedure for constructing a recombination plasmid pNZ5929-gE.
FIG. 5 is an explanatory diagram showing a procedure for constructing a recombination plasmid pNZ5929-7.5gI.
FIG. 6 is an explanatory diagram showing a procedure for constructing a recombination plasmid pNZ5929 / 7.5 gEgI.
FIG. 7 is an explanatory diagram showing a procedure for constructing a recombination plasmid pNZ29RSegA-7.5gI.
FIG. 8 is an explanatory diagram showing a procedure for constructing a recombination plasmid pNZ29RSegA-7.5gIgE.

Claims (4)

  The genomic region that is not essential for foulpox virus growth contains (1) DNA encoding the Marek's disease virus glycoprotein gE, or DNA encoding the Marek's disease virus glycoprotein gE and DNA encoding the glycoprotein gI. And (2) a recombinant foulpox virus containing DNA encoding a heterologous foreign antigen protein.   The recombinant foulpox virus according to claim 1, wherein the heterologous foreign antigen protein is a virus-derived antigenic protein selected from the group consisting of Newcastle disease virus, Marek's disease virus, infectious bursal disease virus, and infectious laryngotracheitis virus. .   The heterologous foreign antigen protein comprises the group consisting of Newcastle disease virus F antigen protein or HN antigen protein, Marek disease virus glycoprotein gE, infectious bursal disease virus VP2 protein, and infectious laryngotracheitis virus glycoprotein gB. The recombinant foulpox virus according to claim 2, which is an antigenic protein to be selected.   A vaccine comprising the recombinant foulpox virus according to any one of claims 1 to 3 as an active ingredient.

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