JP2009195136A - New recombinant herpesvirus of turkey, and vaccine for domestic fowl utilizing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a recombinant HVT (herpesvirus of turkey) having vaccine effects on avian IB (infectious bronchitis). <P>SOLUTION: The recombinant HVT is obtained by inserting a gene encoding a spike protein of the avian IBV (infectious bronchitis virus), a gene encoding a membrane protein, and a gene encoding an envelope protein together with a promoter into a genome. A promoter selected from among the group consisting of an initial promoter of cytomegalovirus, a promoter of an avian β-actin gene, and a promoter of UL50 gene of Marek's disease virus is preferable. The gene encoding each of the proteins is preferably inserted between open leading frames of UL45 and UL46 of the HVT. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a chicken infectious bronchitis virus (hereinafter, sometimes referred to as IBV) spike protein, membrane protein, and envelope in a non-essential region of the turkey herpesvirus (hereinafter sometimes referred to as HVT) genome. The present invention relates to a novel recombinant turkey herpesvirus in which a gene encoding a protein is inserted together with a promoter, and a poultry vaccine using the same.

  Infectious bronchitis (hereinafter sometimes referred to as IB) is a highly contagious respiratory disease first reported in the United States in 1931. A chicken disease that occurs when infected with the infectious chicken infectious bronchitis virus (IBV), and there are strains that cause oviposition disorders such as respiratory symptoms and decreased egg production, and nephritis in an incubation period of 36 hours. It is not uncommon to end up with subclinical infection, but the mortality rate is higher at 6 weeks of age or younger. Histologically, epithelial cells such as trachea, kidneys and fallopian tubes are degenerated and destroyed. The disease occurs and is prevalent in most countries that run the poultry industry.

  IBV is a single-stranded RNA virus belonging to the Coronaviridae family and possesses an outer membrane. Viral particles are four structural proteins, three of which are spike (S) protein (glycoprotein), membrane (M) protein (glycoprotein), and envelope (E) protein that constitute the outer membrane, and the other is nucleocapsid ( N) It is a protein.

  IBV is a natural host for chickens and is a subject of surveillance epidemics. Many serotypes are believed to exist. As typical serotypes, there are Massachetts (commonly known as Mass) type, Arkansas (commonly known as Ark) type, and Connecticut (commonly known as Conn) type, but there is no clear relationship between serotype and disease type.

There are many live vaccines and inactivated vaccines for this disease, but the preventive effect is not complete due to differences in serotypes between viruses and vaccine viruses that are prevalent in the field. All of them use a virus obtained by propagating a chicken infectious bronchitis virus in a growing chicken egg as a raw material, and a manufacturing method that does not use the growing chicken egg has been desired in terms of manufacturing cost.

With the advancement of gene recombination technology, attempts have been made to produce IB vaccines by gene recombination. In the old days, there is a report (J. Gen. Virology (1987) 68: 2291-2298) in which a spike protein is expressed in vaccinia virus. However, in this report, the inoculated mouse was raised in the antibody titer against the S protein, but there has been no report that the chicken had a vaccine effect thereafter.
In addition, there are documents and patents in which spike proteins are expressed using fowlpox virus (FPV) and herpes virus as vectors (Avian Disease 46: 831-838 (2002), US Pat. No. 5,310,671, and JP-A 05-103667. However, there is no report that there was a sufficient vaccine effect. In addition, there is a report of a recombinant turkey herpesvirus (hereinafter sometimes referred to as “HVT”) in which genes encoding S and M proteins of IBV are inserted (US Pat. No. 5,596,1982), but there is no data on vaccine effect .

  Many reports of successful genetically engineered vaccines have been achieved by making virus-like particles. For example, virus-like particles of AIDS virus (HIV) (J. Virology (1990) 64: 2653-2659, J. Virology (1996) 70: 2643-2649) virus-like particles of rotavirus (Vaccine (1998) 16: 507). -516) and human papillomavirus (Infect. Immunity (1999) 67: 3674-3679). However, there are no reports of successful formation of such virus-like particles for recombinant IBV vaccines.

US Pat. No. 5,310,671 Japanese Patent Laid-Open No. 05-103667 US Patent No. 5961982

J. et al. Gen. Virology 68: 2291-2298 (1987) Avian Disease 46: 831-838 (2002) J. et al. Virology 64: 2653-2659 (1990) J. et al. Virology 70: 2643-2649 (1996) Vaccine 16: 507-516 (1998) Infect. Immunity 67: 3674-3679 (1999)

  The present invention has been made in view of the above-mentioned problems of the prior art, and aims to provide a recombinant turkey herpesvirus having a vaccine effect against chicken infectious bronchitis, and a poultry vaccine using the same. .

  As a result of intensive studies to solve the above problems, the present inventor has inserted a gene encoding an IBV spike protein, a gene encoding a membrane protein, and a gene encoding an envelope protein into the genome together with a promoter. A recombinant turkey herpesvirus was prepared, and it was found that the recombinant turkey herpesvirus can be used as a vaccine, thereby completing the present invention.

  Thus, according to the present invention, a gene encoding a chicken infectious bronchitis virus spike protein (hereinafter referred to as “S protein”), a gene encoding a membrane protein (hereinafter referred to as “M protein”), and an envelope protein ( Hereinafter, a recombinant turkey herpesvirus in which a gene encoding "E protein" is inserted into the genome together with a promoter is provided, which promoter is a cytomegalovirus early promoter, a chicken β-actin gene promoter, and Marek's disease Provided is the recombinant turkey herpesvirus selected from the group consisting of viral UL50 gene promoters, and a gene encoding the S protein, a gene encoding the M protein, and a gene encoding the E protein Turkey herpesvirus UL45 and UL46 the recombinant herpesvirus of turkeys is obtained is inserted between the open reading frame is provided, and further, a vaccine for poultry as an active ingredient the recombinant herpesvirus of turkeys is provided.

The present invention is described in detail below.
Chicken Infectious Bronchitis Virus In the present invention, the chicken infectious bronchitis virus (IBV) from which the gene encoding S protein, the gene encoding M protein, and the gene encoding E protein are derived from cells, microorganisms, The American Type Culture Collection (which can be obtained from the ATCC etc. for a fee or free of charge), or directly obtained from an IBV-infected chicken. The serotype is not particularly limited, but is widely distributed. Therefore, a Mass type or Ark type virus is desirable, and particularly desirable is a Mass type, and the virus strain is not particularly limited, and specific examples include Mass41 strain, MassH120 strain, and Ark DPI strain.

A gene encoding a protein derived from chicken infectious bronchitis virus A gene encoding S protein derived from chicken infectious bronchitis virus, a gene encoding M protein, and a gene encoding E protein are the above chicken infectious bronchitis The gene is not particularly limited as long as it is a gene corresponding to S protein, M protein, and E protein of cDNA prepared from RNA, which is the genome of flame virus.
For these genes, the cDNA sequence derived from the viral genome can be used as it is, but it may be used after modification such as adding a stop codon or a restriction enzyme site so that it can be easily subcloned into a homologous vector. Specifically, examples of the gene encoding the S protein, the gene encoding the M protein, and the gene encoding the E protein include the sequences of SEQ ID NOs: 1, 3, and 5, respectively. These are particularly preferred examples derived from the Mass 41 strain, but are not limited thereto, and one or more amino acids may have an amino acid sequence substituted, deleted, or added. . For example, the homology between the S protein of the Mass41 strain and the S protein of the ArkDPI strain is 86.6%. That is, if the homology with the protein of Mass 41 strain is 85% or more, it can be used in the present invention. Here, the homology is the homology search “The Basic Local Alignment Search Tool” of the Japan DNA Data Bank (DNA Data Bank of Japan; http://www.ddbj.nig.ac.jp/Welcome-j.html). BLAST) ”.

Turkey Herpesvirus (HVT)
The turkey herpesvirus used as a parent strain of the recombinant turkey herpesvirus of the present invention can be obtained from the American Type Culture Collection (ATCC), which is a cell, microorganism or gene bank, for a fee or free of charge, or directly obtained from an HVT-infected chicken. It may be what was made. Preferable examples of HVT include those belonging to the gammaherpesvirus subfamily, naturally non-pathogenic, and non-neoplastic viruses used as vaccines for poultry. Specific examples include FC126 (ATCC VR-584B), PB-THV1, H-2, YT-7, WTHV-1, HPRS-26, and the like. For example, the FC126 strain can be preferably used.

Promoter The promoter used in the present invention is not particularly limited as long as the recombinant HVT is a gene that functions as a promoter in an infected cell (ie, has the ability to bind RNA polymerase for mRNA synthesis and promote transcription). . Specifically, from 91927th to 92196th of the genome sequence of heterologous viral promoters and MDV (Genbank accession number: AF243438) such as Cytomegalovirus (CMV) promoter, Rose Sarcoma virus (RSV) promoter, SV40 early promoter Examples thereof include a base region or a chicken cell-derived β-actin promoter (chicken β-actin promoter). From the viewpoint of the stability of recombinant HVT, it is preferable to add three different promoters to each of the genes encoding S, M and E proteins derived from chicken infectious bronchitis virus and insert them into the HVT genome.

Non-essential region A gene region encoding a S protein derived from a chicken infectious bronchitis virus, a gene encoding an M protein, and a genomic region not essential for the growth of avian herpesvirus inserted with a gene encoding an E protein are known in the art. In addition to the region used for recombinant avian herpesvirus (a known non-essential region), a newly found non-essential region can also be used.
Specific examples of known non-essential regions include the UL43 gene used in WO89 / 01040, the US2 gene used in WO93 / 25665, and UL44 to UL46 shown in International Publication WO99 / 18215. A region between open reading frames (ORF) is exemplified, and a particularly preferable region is a region between the ORFs of UL45 and UL46.
A method for finding a new non-essential region is not particularly limited, and examples thereof include the following general method. That is, a genomic DNA fragment of avian herpesvirus is cloned, its restriction enzyme map is prepared, a recombinant plasmid in which a marker gene is incorporated into the appropriate restriction enzyme site together with a promoter is constructed. What is necessary is just to investigate whether a recombinant avian herpesvirus which is stable in vitro or in vivo can be obtained by a virus production method.

Method for Producing Recombinant Turkey Herpesvirus The method for producing a recombinant turkey herpesvirus may be a commonly known general method. One example is as follows. (1) First, a plasmid in which a genomic DNA fragment of turkey herpesvirus containing the above-mentioned non-essential region is cloned is obtained, and a gene encoding the above-mentioned IBV S protein in the non-essential region of the HVT genomic region in this plasmid A recombinant plasmid is constructed by inserting a gene encoding the M protein and a gene encoding the E protein (both usually cDNA) together with a promoter. (2) The constructed recombinant plasmid is introduced into turkey herpesvirus-infected CEF cells, or the recombinant plasmid is introduced into CEF cells together with infectious genomic DNA prepared from turkey herpesvirus. These introduction methods may be generally used methods such as electroporation. (3) CEF cells into which the recombinant plasmid has been introduced are seeded in culture dishes and cultured until the plaques become sufficiently large. (4) Since this plaque contains a mixture of recombinant virus and wild-type virus, the target recombinant virus is purified from here. The purification method is not particularly limited, however, for example, anti-M41 chicken serum prepared from chickens that have been appropriately diluted with plaque-expressing CEF cells, seeded in 96-well culture plates, and hyperimmunized with Mass41 strain of IBV. A method of detecting plaques of recombinant virus expressing S protein by antigen-antibody reaction using as a primary antibody, or if a marker protein gene such as GFP protein has also been inserted, fluorescence A method of detecting plaques of recombinant virus expressing GFP protein under a microscope is exemplified.

IBV vaccine Since the recombinant turkey herpesvirus of the present invention has a gene encoding the S protein of IBV, a causative virus of chicken infectious bronchitis, a gene encoding the M protein, and a gene encoding the E protein, When the FC126 strain, which is used as a chicken infectious bronchitis vaccine for chickens and widely used as an MDV vaccine as a turkey herpesvirus, is used, it is effective against Marek's disease as well as chicken infectious bronchitis. It becomes a vaccine.
In addition to the recombinant turkey herpesvirus of the present invention, the vaccine comprising the recombinant turkey herpesvirus of the present invention as a storage stabilizer, pH adjuster, adjuvant for immune response factories, cells, medium components, etc. It may contain ingredients that are not physically problematic. Furthermore, other recombinant or non-recombinant virus may be mixed as a component other than the recombinant virus. Specifically, the use of MDV-1 or MDV-2 vaccine strain mixed with the recombinant HVT of the present invention is an example.
Although the preparation method of the vaccine of this invention is not specifically limited, For example, it prepares by the following method. After the recombinant HVT of the present invention is infected with CEF cells capable of growing the virus and propagated, the infected cells are peeled off with a scraper or trypsin, and separated into cells and supernatant by centrifugation. The obtained cells are suspended in a culture medium containing 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen.

A method for administering the vaccine of the present invention to chickens is not particularly limited, and a general method used as a Marek's disease vaccine can be used. That is, 10-10 ⁵ PFU / dose, preferably 10 ² to 10 ⁴ PFU / dose is dissolved in an appropriate amount of phosphate buffer or the like, and is subcutaneously in the neck of the first day-old chicken or before hatching. Can be administered using injection or inoculation devices.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited thereto.
Unless otherwise specified, the plasmid construction of the present invention is described in Molecular Cloning: A Laboratory Manual. 2nd Edition. (Cold Spring Harbor Laboratory. Cold Spring Harbor. NY 1989). The restriction enzyme fragment was purified from agarose gel using QIAquick Gel Extraction Kit (manufactured by QIAGEN, catalog number # 28704).

1. Preparation of IBV-derived cDNA RNA was recovered from IBV Mass type field isolate Mass # 14 (manufactured by BioMune) as follows. First, the stored virus (1 tube) was transferred to an ultracentrifugation tube, and 28 ml of PBS was added, followed by ultracentrifugation at 432000 × g for 1 hour. After centrifugation, the supernatant was discarded, 0.6 ml of RLT buffer (RNeasy mini kit, QIAGEN, catalog #: 74104) was added to the precipitate, and the precipitate was homogenized with a 20 gauge needle syringe. Thereafter, 0.6 ml of 70% ethanol was added and mixed. They were placed on two RNeasy spin columns (provided with the above mini kit) and centrifuged at 10,000 rpm for 15 seconds in an Eppendorf centrifuge. The liquid that passed through the column was discarded, and 0.7 ml of RW1 buffer (included in the mini kit) was placed on the column and centrifuged again for 15 seconds. Further, the column was washed twice with 0.5 ml of RPE buffer (included in the above mini kit). Then, centrifuge at 14,000 rpm for 1 minute in an Eppendorf centrifuge to remove the liquid remaining on the column, place the column on a new tube, add 30 μl of sterilized water, and rotate at 10,000 rpm for 1 minute. The RNA was recovered by centrifugation.

  CDNA synthesis from the recovered viral RNA was performed as follows using Super Script First-strand Synthesis System for RT-PCR (RT-PCR kit manufactured by Invitrogen, catalog #: 11904-018). 1 μl of random hexamer and 1 μl of 10 mM dNTP were added to 8 μl of the collected RNA solution, and placed at 65 ° C. for 5 minutes, and then ice-cooled. To this was added 2 μl of 10 × RT buffer, 4 μl of 25 mM magnesium chloride, 2 μl of 0.1 mM DTT, and 1 μl of RNaseOut solution (200 mM Tris-HCl (pH 8.4), 500 mM potassium chloride), and placed at 25 ° C. for 2 minutes. After that, 1 μl of the enzyme Super Script II RT was added. After being placed at 25 ° C. for 10 minutes, it was allowed to stand at 42 ° C. for 50 minutes, then at 70 ° C. for 15 minutes, and then transferred to 4 ° C. Here E. 1 μl of ColiRNase H (2 units / μl) was added and the mixture was allowed to stand at 37 ° C. for 20 minutes. IBV virus cDNA was prepared by the above operation.

2. Cloning of Genes Encoding IBV Proteins Using the cDNA prepared in Example 1 as a template, PCR cloning of genes encoding S protein was performed. The PCR primers used were XBA-MASS-S (SEQ ID NO: 7) and MASS-SAL-S (SEQ ID NO: 8), and the enzyme was Taq polymerase (manufactured by Takara Bio Inc., product name “TAKARA Ex Taq”). It was. As a result of PCR, DNA of 3.5 kbp was amplified and probed into a TA cloning vector (Invitrogen, product name “TOPO TA PCR2.1 Miniprep”) according to the method recommended by the manufacturer.
Plasmid DNA was prepared from the resulting transformant by miniprep method, cleaved with restriction enzyme SalI, and the cleavage pattern was confirmed by agarose gel electrophoresis.
As a result, 2 clones (# 7 and # 8) were obtained as plasmid pCR2.1-S (mass) in which the gene encoding S protein was cloned.

  The two clones were sequenced. The sequence primer used is Mass-S-F2 (SEQ ID NO: 9), in addition to the commercially available T7 promoter primer (Toyobo, catalog number: PRM-102) and P8 primer (Toyobo, catalog number: PRM-008), Mass-S-R2 (SEQ ID NO: 10), Mass-S-F3 (SEQ ID NO: 11), Mass-S-R3 (SEQ ID NO: 12), Mass-S-F4 (SEQ ID NO: 13), Mass-S-F5 ( SEQ ID NO: 14), Mass-S-F6 (SEQ ID NO: 15), Mass-S-R4 (SEQ ID NO: 16), Mass-S-R5 (SEQ ID NO: 17), Mass-S-R6 (SEQ ID NO: 18) Primers were used.

  As a result of the base sequence analysis, clone # 8 has 3,489 bases, and compared with the gene encoding S protein of Mass41 strain (Accession #: AY851295) registered in GeneBank, 4 bases (C1896T, A2241G, A2679G, T3444C), which were all silent mutations in which the amino acids were not changed. The gene sequence encoding the cloned S protein is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

The primers used for PCR cloning of the gene encoding the M protein and the gene encoding the E protein are as follows.
PCR primers for cloning the gene encoding M protein were XbaMASS_M (SEQ ID NO: 19) and MASS_MSal (SEQ ID NO: 20), and the DNA amplified by PCR was ligated to a TOPO cloning vector, and three candidate clones (# 7, 11 and 12) were sequenced, only the clone # 11 had a silent base difference at two locations, and the clone # 11 had the same amino acid sequence as the Mass41 strain.
The ORF of the gene encoding the M protein of the # 11 clone is 678 bp (SEQ ID NO: 3), and the amino acid sequence is shown in SEQ ID NO: 4. This plasmid was named pCR2.1-M (mass).

  On the other hand, PCR primers XbaMASS_E (SEQ ID NO: 21) and MASS_ESal (SEQ ID NO: 22) were used for cloning of the gene encoding the E protein. Again, DNA amplified by PCR was ligated to a TOPO cloning vector to obtain two candidate clones. pCR2.1-E (mass) clones # 4 and # 5. These two clones had the same sequence in opposite directions, but differed from GeneBank's Mass41 strain (AY851295) by only one base (one amino acid) (T157G, L53V). The ORF of the gene encoding E protein is 330 bp, and its amino acid sequence is shown in SEQ ID NO: 6.

3. 3. Construction of plasmid DNA in which genes encoding IBV proteins are inserted together with a promoter that works in eukaryotic cells 3-1. Preparation of p46CoaM (mass) Turkey Herpesvirus FC126 strain (hereinafter simply referred to as “HVT”) DNA was prepared as described in Example 1 of US Pat. No. 6,632,664. In order to introduce an SfiI restriction enzyme site for inserting a foreign gene, PCR was performed using two PCR primer sets and HVT DNA as a template.
One set is HVT45Sph (SEQ ID NO: 23) and 45SfiIR (SEQ ID NO: 24), and the other set is a set of 46SfiF (SEQ ID NO: 25) and HVT46Xho (SEQ ID NO: 26). After confirming that each PCR product (0.4 kbp and 0.6 kbp) was amplified, the products were mixed at a ratio of 1: 1, and PCR was performed again with a primer set of HVT45Sph and HVT46Xho.
As a result, a 0.98 kbp fragment was amplified. This amplified fragment was double-cut with SphI and XhoI and ligated with pNZ45 / 46SfiI (Example 2 of US Pat. No. 6,632,664) that was also double-cut with SphI and XhoI to construct p46Sfi (5491 bp).

Synthetic oligonucleotides Sfi-polA-Sal-Pst-Sfi-U (SEQ ID NO: 27) and Sfi-polA-Sal-Pst-Sfi-L (SEQ ID NO: 28) were annealed and ligated to p46Sfi cleaved with restriction enzyme SfiI. P46Sfi-pA (5543 bp) was produced.
A chicken β-actin (Bac) promoter fragment (1.57 kbp) obtained by digesting plasmid pNZ45 / 46BacpA described in US Pat. No. 7,153,511 with PstI and SalI was recovered from a 1.2% agarose gel. The plasmid p46Sfi-pA was double-cut with PstI and SalI and ligated with the recovered Bac promoter fragment to prepare p46Bac (7101 bp).

The core region (about 300 bases) of the Bac promoter recovered from the agarose gel by double-cutting the plasmid pGICOA described in Example 1 of US Pat. No. 6,866,852 with PstI and XbaI, and p46Bac was cut with PstI and XbaI. Ligation with the fragment produced p46Coa (5822 bp).
Plasmid pCR2.1-M (mass) was cleaved with XbaI and SalI, and the gene encoding the M protein (678 bp) recovered from the agarose gel was ligated with plasmid p46Coa that was also double-cut with XbaI and SalI to obtain p46CoaM. (Mass) (6511 bp) (FIG. 1) was produced.

3-2. Construction of p46cmvS (mass) The cytomegalovirus IE promoter was obtained from the commercially available plasmid pBK-CMV (Stratagene; catalog #: 212209). Within this promoter, there are three recognition sites for restriction enzyme BglI. Since this site interferes with plasmid construction, in vitro mutation was performed using PCR using four primer sets to crush this site. The primer sets were (a) a set of PrCMV1 (SEQ ID NO: 29) and PrCMV3 (SEQ ID NO: 30), (a) a set of PrCMV4 (SEQ ID NO: 31) and PrCMV5 (SEQ ID NO: 32), and (c) PrCMV6 (SEQ ID NO: 33). ) And PrCMV2 (SEQ ID NO: 34), and (d) a set of PrCMVo1 (SEQ ID NO: 35) and PrCMVR1 (SEQ ID NO: 36). Using the plasmid pBK-CMV as a template, PCR was performed with each of these four primer sets. These PCR products were mixed, and the second PCR was performed with PrCMV1 and PrCMVR1 to change the nucleotide sequence at three locations. Of DNA fragment (604 bp). The sequence of this CMV promoter is SEQ ID NO: 37.

  This CMV promoter fragment was cleaved with PstI and XbaI and inserted into a plasmid pUC18polASfi (US Pat. No. 6,866,852) which was also cleaved with PstI and XbaI to prepare pGICMV (−) (3321 bp). The SV40 poly A signal sequence is obtained by PCR amplification with pBK-CMV as a template with a primer set of PolyA-SalKpn (SEQ ID NO: 38) and PolyA-SfiF2 (SEQ ID NO: 39), and recovered by double digestion with SalI and SfiI. The resulting fragment was ligated with pGICMV (−), which was also double-cut with SalI and SfiI, to produce plasmid pGICMVpA (3354 bp).

In order to modify the ends of the fragment from the CMV promoter to polyA, PCR was performed with a primer set of PrCMV1Bgl (SEQ ID NO: 40) and PolA-SfiR (SEQ ID NO: 41) using pGICMVpA as a template. The 955 bp amplification product was cleaved with BglI and inserted into p46Sfi cleaved with SfiI to prepare p46CMV.
A gene encoding an approximately 3.5 kbp S protein obtained by double digestion of plasmid pCR2.1-S (mass) with XbaI and SalI was ligated with p46CMV which was also double digested with XbaI and SalI. p46 cmvS (mass) (FIG. 2) was constructed.

3-3. Preparation of p46CoaMcmvS (mass) After cleaving p46cmvS (mass) with the restriction enzyme BglI, the approximately 4.4 kbp DNA fragment recovered from the agarose gel was ligated with p46CoaM (mass) cleaved with the restriction enzyme SfiI, and the plasmid p46CoaMcmvS (mass). ) (FIG. 3).

3-4. Preparation of pPmd50E (mass) md51GFP CVI988 strain commercially available as a Marek's disease vaccine was infected with CEF cells in a 15 cm diameter culture dish to increase the virus, washed with PBS, and then lysis buffer (0.5% SDS, 10 mM). Add 4 ml of Tris-HCl (pH 8), 100 mM NaCl, 1 mM EDTA, 200 μg / ml proteinase), incubate for 4 hours in a CO ₂ incubator (37 ° C.), transfer the cell lysate to a 50 ml falcon tube, and add an equal volume of phenol ( TE saturation) was added and rotated slowly for 15 minutes to mix. Next, after centrifugation at 1500 × g for 5 minutes, the aqueous layer was slowly sucked with a pipette and transferred to a new tube. An equal amount of chloroform: isoamyl alcohol = 24: 1 was added and mixed slowly for 15 minutes. Thereafter, the mixture was centrifuged at 1500 × g for 5 minutes, the aqueous layer was slowly sucked with a pipette, transferred to a new tube, and 100% ethanol was added twice as much as the aqueous layer, mixed well, and then centrifuged at 1500 × g for 5 minutes. The precipitate was rinsed with 70% ethanol and then air-dried to prepare a nucleic acid fraction containing MDV genomic DNA.
Using the MDV genomic DNA fraction prepared above as a template, PCR was performed with a primer set of MD50F (SEQ ID NO: 42) and MD51R (SEQ ID NO: 43), and the DNA region from 91709 to 92261 of the MDV genomic sequence Was amplified and ligated into a pPCR-Script Amp vector (Stratagene) to transform JM109. Thus, plasmids were prepared from the obtained three transformants, and the nucleotide sequence of the inserted DNA was analyzed. Of the three strains, the base sequences of the inserted DNAs of the two strains were completely identical. These plasmids were named pPCR-Pmd50 (3493 bp).

Two synthetic nucleotides, Ad_Kp-Bg-SaU (SEQ ID NO: 44) and Ad_Kp-Bg-SaL (SEQ ID NO: 45), were obtained by digesting pPCR-Pmd50F with the restriction enzyme KpnI and further recovering it by digesting with SalI. An annealed synthetic DNA adapter was inserted. The plasmid thus obtained was named pPmd50M1 (3516 bp).
Two synthetic nucleotides, Ad_No-Sf-SaU (SEQ ID NO: 46) and Ad_No-Sf-SaL (SEQ ID NO: 47), were annealed to the plasmids obtained by cleaving pPmd50M1 with the restriction enzyme SacI and then further cut with NotI. The synthesized synthetic DNA adapter was inserted. The plasmid thus obtained was named pPmd50M2 (3542 bp).

Next, two syntheses of Ad_Nc-Bg-Xh-NoU (SEQ ID NO: 48) and Ad_Nc-Bg-Xh-NoL (SEQ ID NO: 49) were obtained by recovering pPmd50M2 by double digestion with restriction enzymes NcoI and NotI. A synthetic DNA adapter annealed with nucleotides was inserted. The plasmid thus obtained was named pPmd50M3 (3060 bp).
Using pPCR-Pmd50 as a template, PCR was performed with PCR primers of Pmd50F (SEQ ID NO: 50) and Pmd51R (SEQ ID NO: 51). An approximately 380 bp DNA fragment obtained by double digestion of the amplified PCR product with BamHI and NcoI was ligated with pPmd50M3 double digested with BamHI and NcoI to construct pPmd50F (3409 bp). The base sequence from the KpnI site to the SacI site of this pPmd50F is SEQ ID NO: 52.
PCR was carried out using a primer set of GFPBspF (SEQ ID NO: 53) and GFP SalR (SEQ ID NO: 54) using a commercially available plasmid vector pGFP (Clontech, catalog number: 6097-1) having a green fluorescent protein GFP gene derived from Aequorea jellyfish as a template. Carried out. The amplified PCR product was double-cut with restriction enzymes BspHI and SalI, and ligated with pPmd50F double-cut with NcoI and XhoI to prepare pPmd51GFP.
Next, pCR2.1-E (mass) (clone # 5) was double-cut with the restriction enzymes BamHI and SalI and ligated to pPmd51GFP that was also double-cut with BamHI and SalI, and the plasmid pPmd50E (mass) md51GFP ( 4685 bp) (FIG. 4) was constructed.

3-5. Preparation of p46CoaMcmvSmd50Emd51GFP Plasmid pPmd50E (mass) md51GFP was cleaved with restriction enzyme BglI, and an approximately 1.8 kbp DNA fragment recovered from the agarose gel was ligated with p46CoaMcmvS (mass) c46mGFGFE (FIG. 5) was obtained.

4). Production and purification of recombinant HVT 4-1. Preparation and purification of rHVT / IB_S The DNA of the parent strain HVT (FC126 strain) was prepared by the method of Morgan et al. (Avian Diseases, 34: 345-351 (1990)), together with the plasmid p46cmvS (mass) constructed in 3.2 above It was introduced into CEF cells using electroporation, and purification of recombinant HVT was carried out using an antibody against S protein.

1 × 10 ⁷ chicken embryo fibroblasts (CEF) were suspended in 0.1 ml of MEF-1 buffer (Amaxa Biosystems, catalog No. VPD-1004), and 1 μg of DNA of recombinant plasmid p46 cmvS (mass) was added. 5 μg of HVT genomic DNA was added, and gene introduction was performed at room temperature using a program A-23 of a gene introduction apparatus (Amaxa Biosystems, product name “Nucleofector”). Immediately mixed with Leibovitz's L-15 containing 4% bovine serum and McCoy's 5A Medium (both manufactured by GIBCO BRL, catalog numbers # 41300-039, # 21500-061) at the same volume ratio (hereinafter, LM (+) medium) was added, and the cells were cultured in a cell culture dish having a diameter of 60 mm in a 37 ° C., 5% CO ₂ incubator for 6 days. The cells were appropriately diluted, mixed with CEF suspended in LM (+) medium, dispensed into 96-well culture plates, and cultured until plaques appeared. Cells in each well of the plate were trypsinized and fresh CEF cells were added and transferred to each well of two 96-well culture plates. The replica plate thus prepared was cultured until plaques appeared. After that, cells on the one plate are fixed with methanol, and whether or not recombinant HVT expressing S protein is present by antigen-antibody reaction using anti-M41 chicken antiserum (SPAFAS) as the primary antibody. Was confirmed. Cells were collected from a replica corresponding to a well confirmed recombinant HVT, diluted, mixed with fresh CEF cells suspended in LM (+) medium, dispensed into a 96-well plate, and cultured. This repetition of dilution, replica production, and confirmation of recombinant HVT was repeated until it was confirmed that all plaques derived from one well were almost 100% recombinant HVT. This recombinant HVT was propagated with CEF to about 10 ⁵ pfu, then subjected to ultrasonic disruption, and the centrifugal supernatant was infected with CEF cells in a 96-well plate. After culturing for one week, this operation was repeated again until the recombinant HVT was 100%. The purified recombinant HVT virus was named rHVT / IB_S.

4-2. Production and purification of rHVT / IB_S + M As described in 4-1, except that p46CoaMcmvS (mass) is used instead of p46cmvS (mass) for the recombinant plasmid. In the same manner as above, recombinant HVT was purified and named rHVT / IB_S + M.

4-3. Preparation and purification of rHVT / IB_S + M + E 1 × 10 ⁷ CEF cells were suspended in 0.1 ml of MEF-1 buffer, 1 μg of recombinant plasmid p46CoaMcmvSmd50Emd51GFP DNA and 5 μg of HVT genomic DNA were added, and the same gene introduction apparatus as described above was used. Used for gene transfer. Immediately after adding LM (+) medium, the cells were cultured in a cell culture dish having a diameter of 60 mm for 6 days in a 37 ° C., 5% CO ₂ incubator. The cells were appropriately diluted, mixed with CEF suspended in LM (+) medium, dispensed into a 96-well culture plate, and cultured until plaques appeared. The 96-well culture plate was observed under a fluorescence microscope to search for HVT plaques expressing GFP protein. Cells are collected from a well in which a recombinant HVT emitting green fluorescence indicating the expression of GFP protein is confirmed, diluted, mixed with fresh CEF cells suspended in LM (+) medium, and dispensed into a 96-well culture plate. And cultured. By repeating this series of operations, it was confirmed that it was almost 100% recombinant HVT. This recombinant HVT was propagated in CEF cells to about 10 ⁵ pfu, then subjected to ultrasonic disruption, and the centrifugal supernatant was infected with CEF cells in a 96-well culture plate. After culturing for one week, this operation was repeated again until the recombinant HVT was 100%. The purified recombinant HVT virus was named rHVT / IB_S + M + E.

5. Confirmation of recombinant HVT expressed protein (black plaque assay)
Recombinant HVT prepared in 4 above was mixed with CEF cells, cultured in 6-well culture plates, washed cells twice with PBS after 3 days, and then -20 ° C. methanol: acetone (2: 1 ) Was added at 500 μl / well and left at room temperature for 5 minutes. Thereafter, it was washed twice with PBS and reacted with the primary antibody. The primary antibodies used were (1) monoclonal antibody (MAb) 1588 against serotype Mass strain of IBV (distributed by Cornell University. Reference papers are Avian Disease (1992) 36: 903-915), (2) Anti-M41 chicken serum, (3) 97th to 225th region of M protein expressed in E. coli using plasmid pGEX-6P-3 (Amersham Biosciences, catalog No. 27-4599-01) Chicken serum (anti-M chicken serum) obtained by immunizing chickens with recombinant proteins, (4) Immunizing chickens with recombinant proteins from the 3rd to 108th region of E protein using the same E. coli expression vector Thus obtained chicken serum (anti-E chicken serum) was used. Biotinylated anti-mouse IgG (Vector Laboratories, catalog No. BA-9200) or biotinylated anti-chicken IgG antibody (vector), which was allowed to stand at 37 ° C. for 1 hour, washed twice with PBS, and diluted 5000 times as a secondary antibody. Reaction with Laboratory, BA-9010). After standing again for 1 hour, the plate was washed twice with PBS, and left for 30 minutes in a vector stain ABC-ALP (Vector Laboratories, AK-5000) solution. After washing with PBS, the color was developed with NBT / BCIP (Roche Diagnostics, catalog No. 11681451001). The progress of the reaction was confirmed under a microscope, and the color reaction was stopped by discarding the reagent and replacing it with water. The results are shown in Table 1, assuming that the plaque stained black was positive (+) and the plaque not stained was (-).

  As shown in Table 1, only rHVT / IB_S + M + E infected CEF cells reacted with a monoclonal antibody against S protein (1588) or anti-E chicken serum. Since 1588 is a serum-specific monoclonal antibody and does not react in Western blotting, it is considered that the three-dimensional structure of S protein is recognized (Avian Diseases (1992) 36: 903-915). The reaction to this monoclonal antibody suggests that the spike protein expressed in rHVT / IB_S + M + E-infected cells has a three-dimensional structure that is similar to the S protein of the IBV Mass strain. It was found that such a three-dimensional structure of the S protein cannot be obtained with the replacement HVT.

6). Vaccine Effect Animal Test of Recombinant Chicks born from SPF eggs purchased from Nissei Laboratories were inoculated subcutaneously with 3 × 10 ³ / wings of each recombinant HVT shown in Table 2 (10 birds per group). Four weeks later, blood was collected and the antibody titer was measured using an ELISA plate (manufactured by SPAFAS) ground with Mass41 strain as an antigen. HRPO labeled anti-chicken IgG sheep serum (BETHYL) diluted with 4000 times after reacting with serum collected from chicken diluted 500 times for 1 hour, washed well with PBS-T (PBS containing 0.1% Tween-20) After reacting for 1 hour and thoroughly washing again with PBS-T, the substrate (Bio-Rad, catalog number 172-1644) was added and allowed to react for 15 minutes. Those having an absorbance of 0.2 or more were regarded as positive, and the number of chickens that became positive was counted. The results are shown in Table 2.

The presence or absence of neutralizing antibodies (VN) against IBV was also measured. The Mass 41 strain of 10 ³ EID ₅₀ was mixed with chicken serum serially diluted 10-fold (the collected serum pool) and allowed to stand for 1 hour. It was inoculated into a 10-day embryo SPF fertilized egg, and 7 days later, the fetus was taken out from the egg to determine whether it was alive or dead. The 50% neutralizing antibody titer (NI) was calculated by the method of Reed et al. (Am. J. Hyg. (1938) 27: 493-497). The results are shown in Table 2.
As shown in Table 2, antibodies against IBV were detected only in rHVT / IB_S + M + E, and neutralizing antibodies were also observed.

FIG. 1 is a schematic diagram of the plasmid p46CoaM (mass). FIG. 2 is a schematic view of the plasmid p46cmvS (mass). FIG. 3 is a schematic diagram of the plasmid p46CoaMcmvS (mass). FIG. 4 is a schematic diagram of the plasmid pPmd50E (mass) md51GFP. FIG. 5 is a schematic diagram of the plasmid p46CoaMcmvSmd50Emd51GFP.

Claims (4)

A recombinant turkey herpesvirus in which a gene encoding a spike protein of chicken infectious bronchitis virus, a gene encoding a membrane protein, and a gene encoding an envelope protein are inserted into the genome together with a promoter. The recombinant turkey herpesvirus according to claim 1, wherein the promoter is selected from the group consisting of a cytomegalovirus early promoter, a chicken β-actin gene promoter, and a Marek's disease virus UL50 gene promoter. The gene encoding a spike protein, the gene encoding a membrane protein, and the gene encoding an envelope protein are inserted between the open reading frames of turkey herpesvirus UL45 and UL46. Recombinant turkey herpesvirus. A poultry vaccine comprising the recombinant turkey herpesvirus according to any one of claims 1 to 3 as an active ingredient.

Images