JPH05508540A - Recombinant pigeonpox virus vaccine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Recombinant fowlpox virus vaccine field of invention The present invention relates to the field of treatment of Newcastle disease in birds. More specifically, the present invention relates to a recombinant virus vaccine treatment for Newcastle disease.

Background of the invention Newcastle disease is the most important viral disease of poultry. newcastle disease attacks chickens, turkeys, pigeons and many species of wild and domestic birds. It is a highly contagious and devastating infectious disease. The mortality rate from Newcastle disease is It depends on both the immune status of the bird and the virulence of the Newcastle disease virus.

The mortality rate due to Newcastle disease has reached 100%. Chickens are the most susceptible It seems to be some kind of.

Newcastle disease virus, avian paramyxovirus type 1 causes Newcastle disease It is a pathogen. Newcastle disease virus strains have three pathotypes: fast-acting and subpathogenic. and long-latency. Fast-acting and subpathogenic Newcastle disease virus The long-incubation period strain is pathogenic, whereas the long-incubation period strain is lethal to birds at any time of the year. Currently, a live vaccine strain against Newcastle disease virus is available. It is used as Commonly used live vaccine strains include Hitchner, Lack and and Ulster (Hitchner, La5ota and U1ster) strains. include.

However, commonly used live vaccines have some drawbacks, such as heat sensitivity, residual virulence, potential for reversion to virulence and neutralization by corresponding antibodies, etc. It has its drawbacks. In addition, disease eradication plans should include serologically determined vaccination and susceptible chickens. This is hampered by the inability to distinguish between infected chickens.

Genetically engineered vaccines are expected to solve these problems. Recombinant type vaccines have no possibility of reversion to virulence of attenuated strains (and are effective against pathogenic bacteria). It has the advantage of conferring immunity. Baoretch dated July 29, 1986 and rice fed to P. aoletti and P. anicali National Patent No. 4.603゜112 covers non-essential regions of the vaccinia genome. ssential region), which does not exist naturally in vaccinia virus. Discloses a recombinant vaccinia virus that has been synthetically modified by the presence of ugly DNA. are doing. Vaccinia virus is modified at the vaccinia thymidine kinase gene locus. It is decorated with genes from herpesviruses and influenza viruses. It was expressed.

Meulemans et al. (1988) Avian Pathology -(AvianPathology) 17:821 At 827, live virus Generated from a recombinant vaccinia virus vector that protected chickens against virus challenge. We report the F glycoprotein of Newcastle disease virus. The reason Tinia virus vaccine protects chickens from infection with Newcastle disease virus. However, researchers are testing the recombinant virus vaccine in inoculated chickens. No seroconversion could be demonstrated for the components. Vaccinia virus is chicken It has been reported that the virus does not propagate in Vaccinia virus to chickens Although adaptation is possible, vaccinia nevertheless infects humans and their Vaccine use in chickens is prohibited because its use as a cutin may be harmful to human health. The use of T. tinia recombinant virus is not justified.

There have been several reports of the use of fowlpox virus in recombinant vaccines. ing.

PCT Application Publication No. WO38102022 is a recombinant fowlpox virus or A related virus modified by inserting sexual DNA into the non-essential region of the fowlpox virus. Avian pox virus is disclosed. EPO publication number 031 In No. 4569, fowlpox virus is modified in the non-coding region between genes, resulting in a heterologous structure. discloses a recombinant fowlpox virus containing DNA encoding the protein of Ru.

Fowlpox virus has a more limited range than vaccinia virus and is less likely to infect humans. However, some birds, especially the two commercially important turkeys, Generally more toxic in leopards and chickens.

Therefore, it is an object of the present invention to develop recombinant fowlpox in chickens and turkeys. A set of inoculations for birds against Newcastle disease infection that is less pathogenic than vaccines. To provide pigeon pox vaccines and pharmaceutical preparations. It's there.

Another object of the invention is to modify and select for use as a vaccine. The object of the present invention is to provide a fowlpox vector capable of expressing the gene. The features of this invention A further object is to provide a vaccine for inoculating chickens against pathogenic bacteria.

Summary of the invention The present invention is directed to a non-essential region of the fowlpox virus genome that is naturally occurring in the fowlpox virus. Provides a recombinant fowlpox virus that is synthetically modified by the presence of non-DNA. . In a preferred embodiment of the invention, fowlpox virus is isolated from the thymidine kinase gene range. modified to contain DNA that is not naturally present in fowlpox virus.

Preferred DNA sequences inserted within the thymidine kinase gene are This is the DNA that encodes the F protein of the Ssul disease virus.

The invention is pointed out in more detail in the appended claims and as set forth below. are described in preferred specific examples.

Brief description of the drawing Figure 1 shows the mRNA sense strand encoding the F gene of Newcastle disease. , clone Ⅰ, shows the nucleotide sequence of the insert of 14.

Detailed description of the invention The present invention is directed to a non-essential region of the fowlpox virus genome that is naturally occurring in the fowlpox virus. Provides a recombinant fowlpox virus that is synthetically modified by the presence of non-DNA. It is something. When using the recombinant virus of the present invention, at least two different It becomes possible to simultaneously vaccinate birds against diseases. Recombinant cormorant of the present invention Viruses include fowlpox virus and other viruses, such as Newcastle disease virus. or other microorganisms. In a preferred embodiment of the present invention The recombinant fowlpox virus contains the F gene of Newcastle disease virus. Ru.

If a bird is inoculated with live attenuated Newcastle disease virus, the bird is vaccinated. , when subsequent infections occur, and whether cranes are the only ones infected with the virus. It is up to you to decide whether it is possible. Recombination containing Newcastle disease F gene Birds inoculated with fowlpox virus will not be able to feed unless challenged with the virus itself. Because they do not produce antibodies against all antigenic epitopes of Castle disease virus , the recombinant fowlpox virus generates antibodies due to infection and responds to the recombinant fowlpox virus. Newcastle disease virus antibodies produced by

Fowlpox virus vaccine causes severe reactions if used incorrectly With the exception of chickenpox, fowlpox virus has little pathogenicity in chickens and turkeys. do not have. Thus, the opportunity for an adverse reaction against the viral vector is This can be reduced by using fowlpox virus as a virus.

Recombinant fowlpox virus can be used in chickens, including chickens, turkeys and pigeons. Species capable of infecting with smallpox virus? , and its characteristic DNA as fowlpox virus. It is useful for inoculation of species capable of infecting with microorganisms inserted therein.

The recombinant fowlpox virus of the present invention is compatible with established protocols for administering vaccines to birds. methods, such as the wing web method and the feather web method. Trivacy and foliicle methods, including the feather follicle method Tripathy and Cunningham Box in Diseases of Poultry (Avian Pox i) Diseases of Poultry), 8th edition, Iowa State ・University Press (I ova 5tate University Press, Awes, Iowa, USA, 1984, Chapter 23 Disclosed in Avian Pox (Chapter 23 Avian Pox) fowl pox or fowl pox vaccine that is commercially available. It can be administered as a pharmaceutical preparation according to the methods used in those cases. pharmaceutical preparation As a product, the recombinant virus of the invention may be added to a solution such as water, saline or other buffer. The compound is administered in combination with a pharmaceutically acceptable carrier or diluent. The pharmaceutical preparation is Additionally, additional ingredients such as adjuvants or stabilizers may be included. individual The route of administration, timing of administration to the bird and dose will depend on the species, age and weight of the bird. It depends on such factors. In addition, the route of administration, the time of the bird and the recombinant virus, The dosage of cutin or pharmaceutical preparation is determined by It can vary according to the type of DNA that does not occur naturally in the smallpox virus.

Using a recombinant fowlpox virus that expresses the F gene of Newcastle disease virus In a preliminary test, chicken embryos with a virus titer of 10"TCI Dse/ml Vaccination with a virus produced in skin cells prevents Newcastle disease virus. Strain Italian (Newcastle disease virus 5tra SPF is effective in protecting chickens when attacked by intalians. It turned out to be effective.

Commercially available fowlpox viruses useful as vaccines or research strains of fowlpox virus Viruses are suitable for use as viral vectors in the present invention. Preferences of the present invention The fowlpox viruses used in the new specific examples are Rit SA (Rit SA), Available from Genval, Belgium. , with registration number 6100.

The recombinant fowlpox virus of the present invention contains DNA that does not normally exist in fowlpox viruses. It is produced by synthetic insertion into the genome of a virus. Preferred of the present invention In a specific example, DNA that is not normally present in fowlpox virus, i.e., foreign D NA or DNA of a foreign structure is added to a non-essential part of the viral genome, i.e. Insert within the gin kinase gene. In other embodiments of the invention, exogenous The DNA may be inserted into a genetically open region of fowlpox virus.

A foreign D encoding the desired gene producer inserted into a fowlpox virus vector. NAs are selected according to the type of microorganism or other purposes against which protection is desired. It will be done. Preferably, the DNA not normally present in fowlpox virus is characteristic; Alternatively, it may cause the formation of neutralizing antibodies in the inoculated host, resulting in the formation of neutralizing antibodies against the scales. It encodes proteins involved in the fusion of viruses to host cells. Like The new DNA sequence is the Newcastle disease virus F gene. Contains this gene The recombinant fowlpox virus that has the ability to inoculate inoculated birds with fowlpox virus and new Protects against infection by both Sul disease virus. other viruses or other microorganisms Replace the DNA encoding the antigenic protein from the Newcastle disease F gene with and can confer protection against viruses or other microorganisms. foreignness The foreign gene is inserted into a fowlpox vector operably linked to a transcriptional regulatory factor. It is preferable to transfer the gene appropriately. In a preferred embodiment of the invention, the The foreign DNA is operably linked to the vaccinia pHK promoter. That reason The promoter in T. tinia p11 is the later 11,000-dalton polypeptide ( derived from the vaccinia virus gene encoding IIK): even though herein Bertolette, which is hereby incorporated by reference even if fully described. Bertholet, C.R. et al. (1985) Proceedings Inges of National Academy of Sciences Proe, Natl.

Acad, Sci, USA) 82:2096-2100; and Cooper ・B, E.

Coupar, B, E, H, et al. (1986) European Journal of Immunology (Eur, J, lm1uno1.) 1st +1479-1487 reference. In addition, fowlpox viruses such as the fowlpox virus promoter Other transcriptional regulators that are compatible with viruses may also be used.

To facilitate the insertion of foreign genes into fowlpox, a given vaccinia virus protein motor, restriction site for inserting foreign coding sequences and the furan of the fowlpox TK gene. Plasmid vectors containing King sequences may also be constructed. Preferred of the present invention In specific examples, the Newcastle disease F gene is functionally linked to the vaccinia promoter. The Newcastle disease F gene and vaccinia promoter are linked upstream. upstream and downstream with the flanking sequences of the fowlpox thymidine kinase gene ( Thymidine kinase gene sequence, promoter, Newcastle disease F gene, thymidine kinase gin kinase gene sequence).

The plasmid DNA is then transformed into wild-type or modified forms, i.e., combinations of heterologous structures. transfect cells infected with fowlpox virus that has undergone recombination.

Transfection can be performed by calcium phosphate coprecipitation or electroporation. This can be done by standard methods such as electroporation. Ru. The host cells are then exposed to the presence of a recombinant virus expressing the foreign gene. and test it. Insert foreign DNA into the thymidine kinase gene to create a recombinant virus. Build Luss TK-.

The TK-recombinant contains promothioxyuri, a nucleoside similar to thymidine. It may be isolated by plaque assay in the presence of BUdR. If present in the DNA contained in the It is mutagenic and toxic. Other methods for selecting recombinant viruses , Boyle and Coupar (1988) Virus Research 10:343-356 It is shown.

The recombinant virus of the present invention is cultured in chicken cells, but it is different from the fowlpox virus. Other cell types with infectivity are also suitable for culturing longitudinally recombinant viruses. .

In a preferred embodiment of the invention, two recombinant viruses expressing the F gene was isolated. High passage of recombinant PPV/F22 virus expressing the F gene Derived from pigeonpox virus. A recombinant PPV/F26 horse expressing the F gene The virus was derived from low-passage fowlpox virus.

Experimental example The virus strain developed as a vector virus is Rit. Fowlpox virus available from SAX Genval, Belgium) under registration number 6100. for vaccinating chickens against fowlpox by the wing-web method. It's a special thing.

All cell cultures used for fowlpox virus adaptation and propagation were performed using SPF chicken embryos. (LohUn, Fuchshalfen) (Cuxhaven, West Germany). -Next chicken embryo skin cells are all 12 days old. Obtained by trypsinization of embryos, The cells were grown in Opti-MEM (Gibco) containing % fetal bovine serum. I set it. For maintenance of the cells, fetal bovine serum was omitted from the medium. -Next Chicken embryo fibroblasts are obtained from finely chopped 9- to 10-day-old chicken embryos. prepared by psin treatment and grown in Opti-MEM. the cell For maintenance, fetal bovine serum was omitted from the medium. -Next chicken liver cells of 14-day-old cells in Opti-MEM containing 10% fetal bovine serum as growth medium. Prepared from embryonic liver. Maintenance medium is EL supplemented with 12% fetal bovine serum. It was Y (Gibco). All cell cultures were cultured at 1,38% in the presence of 5% carbon dioxide. Incubated at 5°C.

One vial containing 1,000 doses of fowlpox virus (10γ!EID50) Rehydrated in Opti-MEMIOml and inoculated into chicken hepatocytes. inoculation Six days later, the progeny virus was collected by repeating three cycles of freezing and thawing, and then Continuously in Watori hepatocytes, chicken embryo skin cells and chicken embryo fibroblasts Adapted to cell culture by passage. Two emerging adaptive viruses: low-passage fowlpox virus Rus and high passage pigeonpox viruses were obtained. Low-passage fowlpox virus is transmitted to chicken hepatocytes. 2 passages in chick embryo skin cells, followed by 2 passages in chicken embryo skin cells, then 2 passages in chicken embryo skin cells, followed by 2 passages in chicken embryo skin cells It was prepared by passage twice in embryonic fibroblast cells (six passages in total). last (6th passage) was titrated in chicken embryo fibroblast cells and 10'" TCI DB. It had a titer of /+*1. High-passage cells grow low-passage fowlpox virus were prepared by an additional 25 passages in embryonic fibroblasts (total 31 1st succession). High-passage pigeonpox virus was titrated in chicken embryo fibroblast cells and It had a titer of CI Dsa.

EID! . and TCIDs. measurement of EIDso is the dose of virus that infects 50% of the embryos in the test group.

TCIDso is the dose of virus that infects 50% of the cell cultures in the test group.

The EID5° and TCIDs. standard techniques, e.g. B.Iological S. Uspensions) J, 2nd edition, The. American Association of Avian Bathologists (The A+ 5erican As5ocation of AvianP atholo gists), Creative Printing Company (Creative ePrinting Company), 2011 East Main Street Villegas and Birthiyas, Endwell, New York 13760; Calculated in accordance with the 'J.i.l.legas and purchase) method. Can be done.

Construction of recombinant plasmid Unless otherwise specified, recombinant DNA manipulations were performed using Sambrook. ), Molecular Cloning, A Laboratory Manual, 2nd Edition ( Molecular Cloning, A Laboratory Manu al, 5th edition), Cold Spring + Harbor Laboratory Press (Cold Spring Harbor Laborat Ory Press), Cold Spring Harbor, Massachusetts, It was carried out according to the standard method described in 1989.

Cloning of the Newcastle disease F gene to construct a recombinant fowlpox virus Even if fully disclosed herein, it is considered a part of the disclosure of this specification, Co-pending U.S. Patent Application No. 07/441,18 filed November 22, 1989 The Newcastle disease gene disclosed in No. 2 was used. cloning of this gene The method is shown below.

A. Materials and Methods Virus, Newcastle disease virus, strain Italian, international ・Reference Laboratory Weybridge (I internationala) l Reference Laboratory Weybridge), UK Obtained from. It was propagated in 9 to 11 day old chicken embryos and It was used as a russe source. The virus was described by Le Long et al. (19 86) Journal of Biogy (J, Virol, ) Danyu 119 Purified according to the disclosure of G-1202.

Extraction of poly(A)''-+aRNA from NDV-infected cells, BHK-21 cells Infections were carried out at a multiplicity of 5-10 PFU (plaque forming units)/cell. 30 minutes of sucking After the harvest period, the cells were incubated in Eagle's minimal essential medium (MEM) at 37°C. I started.

At 230 hours post-infection, actinomycin D was added at a final concentration of 2 μg/ml; After 4 hours, the cells were soaked in TNE buffer (0.01 M Tris, pH 8, 3, 0, 15 M Washed twice with NaC1,0, OOIM EDTA), 400 tt g/m TNE-SDS buffer (0.01M Tris pH 8, Dissolved in 3,0,15M NaCl1,0゜001M EDTA, 0.5% 5DS) I let it happen. - volume of phenol:chloroform:in-amyl alcohol (25:2 4:1), destroy the DNA with a mixer, and mix the mixture with phenol. Extracted twice with :chloroform:isoamyl alcohol (-volume). ethanol After precipitation, the nucleic acids were pelleted and added to an elution buffer (0°1M Tris pH 7.5, O, O OIM EDTA, 0.5% 5DS) and the mixture was diluted to 2M in LiCl. and incubated at 4°C for 12 hours. After centrifugation, the pellet is Hybridization buffer (0.1M Tris, pH 7, 5, 0, 35M NaC 110, OOLM EDTA, 0.5% 5DS) Dissolve in 2111, 1°5ml of oligo(dT)-cellulose column. Elute poly(A)-containing NSs Eluted with buffer.

Before use, the poly(A)'-II RNA was incubated with protein in rabbit reticulocyte lysate. In order to test the stimulation of compound synthesis, Palha+ s, H, R, B, et al., European Journal of Biochemistry Lee (Eur, J, B1ochei, ) 67:247-256 (1 976)), its synthetic viral protein L6 Long et al. (1986) Journal of Bioscience (J, ViroL, ) 57:1 Immunoprecipitation reactions using specific anti-NDV antibodies according to the disclosure of No. 198-1202. Detected by.

Synthesis and cloning of cDNA, using poly(A)- from infected cells as a template -a+RNA and oligo(dT)+. -1, Reverse transposition using primers Single-stranded cDNA copies were synthesized with ribtase. mRNA-cDNA hive After subjecting the lid to heat/cool denaturation, the 3' end loop from the single-stranded cDNA was , used as a primer by DNA polymerase I to synthesize double strands. . Next, the hairpin loop of the obtained two-terminal cDNA was digested with Sl nuclease. digest and add oligo(dC) residues to the 3′ end of the cDNA, creating an oligo(dC) tail. A section was formed. Then, the cDNA was subjected to (dG)n (dC)n hybridization. Pst1 of pBR322 plasmid DNA in the oligo(dG) tail by site and the ligation mixture was used to create E. coli strain MM2. 94 (hsdR17, i.e. r-h1-k) to tetracycline resistance Transformed.

Detection of viral clones, clones containing viral cDNA as inserts (1 199 of 277 tetracycline-resistant and ampicillin-sensitive colonies ) and 5xp-labeled 50S viral RNA (sucrose gradient) as a probe. (isolated at 15-30% vt/vt)). Although fully disclosed in the Grunstein, M. and H. ogness, D., S.) (1975) Proceedings of Pear. National Academy of Sciences atl, Acad, Sci, U.S.A.) 2:3961. Therefore, by colony hybridization of specific part B (in 5 itu) Detected.

The viral clones were then used as probes to insert randomly selected cDNAs. 32p-labeled plasmid by nick translation containing Colony hybridization experiments between other specific regions using DNA classified into.

cDNA identification, using the known coding assignment of +lRNA of NDV Collins, P. L., et al., Journal ・Of Pyrology” Dan: 1024-1031 (1982)), mRNA The separation was performed in 0.025 M sodium citrate containing 6 M urea, pH 3.5. For electrophoresis of poly(A)''-mRNA from infected cells using a % agarose gel. RNA transfer was performed on nitrocellulose. Then, that second Cut the trocellulose sheet into several bands and nick-translate each of them. Hybridization was performed with the 32p-labeled cDNA of 1 by reaction.

Capture identification of cDNA was performed using the enzymatic method of Riezman, H. et al. Embo Journal (Embo J.) 12:2161 2168 (1983). The hybridization-selection method described was performed. This method selects immunization of the resulting protein with anti-NDV antibody. (Le Long et al., cited above).

DNA sequence analysis, DNA sequence analysis by Maxima & Gilbert (Maxam & G11be) rt) and Sanger dideoxy sequence method. It was measured by the method.

(2) Recombination of the nucleotide sequence of the fusion gene and the amino acid sequence of protein F type cDNA clone (It, 14) has a difference between the clones containing the F-specific sequence. It was chosen for DNA sequence analysis because it had the largest insert. F More than 94% of the open chain coding region of the protein was sequenced. mRNA sense The complete nucleotide sequence of the insert of clone n14 in the strand is shown in Figure 1. vinegar. It consists of 1764, excluding the poly(A) tract and G-C tail. It has base pairs. Start at ATG (nucleotides 68-70) and end at the nuclear stop codon ( One long open reading frame ending at nucleotides 1646-1648) It just exists. This terminal codon has four additional in-frame codons (in-fr ame) Following the terminator.

Its coding region consists of a 526 amino acid protein (Mr 56.464, (without any carbohydrate composition), which has three characteristic hydrophobic regions. do. F2 probably extends from residue 19 to residue 98, with 890 a Consisting of amino acids (Mr 8.304, without any carbohydrate composition): Fl is It is a polypeptide of 423 amino acids (residues 104-526; Mr 45, 541, if it is not a glycoprotein (unglycoslyatecl). F is Four potential glycoslyation sites on two F2 One, F, contains three.

High-passage fowlpox virus was purified by sucrose gradient ultracentrifugation, and virus D NA was extracted using a phenol/chloroform procedure. Precipitate DNA with ethanol Lambda gtlQ vector (prong (Pros+eda) EcoRI before connecting to Madison, WI (Madison, MO) It was digested by A clone containing the fowlpox virus thymidine kinase gene was Boyle and Coupar (1988) Disclosed in Virus Research 10:343-356 s! was produced using the thymidine kinase gene sequence of the fowlpox virus. P ori Identification was made by hybridization using oligonucleotides. Oligo used The sequence of the nucleotide probe is 5' GAT AAATACATA GCCG TG TGT AGG 3'. Positive clones were expanded and inserts were digested with restriction enzymes. 2,540 bases containing the entire pigeon virus thymidine kinase gene A paired C1aI-BamHI fragment was obtained. This fragment was added to plasmid pSP65 (pro (Madison, Wilcon, MO) into the AccI-SmaI site, Plasmid pSP657K was obtained.

Next, the EcoR1 site of pSP65TK was cloned with DNA polymerase I Klenow ( Klenow) fragment and the EcoRI site was deleted using the fowlpox virus thymidine fragment. was introduced into the unique NcoI site of the kinase gene.

383 containing p7.5 and pHK promoters from vaccinia virus An EcoRI fragment of 5 base pairs was obtained by enzymatically digesting plasmid psc11. Even if fully disclosed herein, it shall not be considered a part of the disclosure herein. Chakrabarti et al. (1985) Molecular ・Molecular and Cellular Biology lar B iology) 12:3403-3409゜ The F gene was extracted by enzymatic digestion using BaIIIHl and FspI restriction enzymes. Obtained from pBR322 clone (I[, 14). The BamH1 end of the F gene It was blunted using the Klenow fragment of DNA polymerase I. Then, the blunt end F The gene was cloned into the PvuII site of plasmid YEP36 (Bu (1984) Proceedings of the National Academy of Sciences Proc, Natl, Acad, Sci , USA) Dan 1: 3332-3336), it is genetically encoded by galactokinase. It was modified by deletion of the offspring to obtain plasmid YEP36/F. newcastle sick The EωRI fragment containing the virus F gene was subjected to enzymatic digestion of plasmid YEP36/F. and then convert it to pSP64 (Prong, Madison, Wilcon Mono State) ligated into the EcoRI site of.

The F gene from Newcastle disease virus was extracted into a plasmid by EcoR1 digestion. Extracted from SP64. The EcoRI fragment contains most of the F coding sequence (1721 nucleotide) and further contains an ATG codon, another one of the F sequences. do.

p7.5-pHK and F fragments together into pSP657 at the unique EcoRI site (ERI). The recombinant plasmid was inserted into the pHK protein in the correct orientation. The presence of F sequences downstream of the motor was characterized by restriction analysis. The program determined the sequence at the junction of the lomotor and Newcastle disease F genes and Passed by: Transfection and isolation of recombinant fowlpox virus - subchicken embryo fibroblasts Cells were incubated with either low-passage pigeonpox virus or high-passage fowlpox virus at 0.05PF. U/cells were infected and treated with lipofectin reagent (L ipofectin Reage). nt) (Bethesda Research Laboratory Lease (Bethesda Re5ea) rchL laboratories), Gaithersberg (Gaithers) burg), Maryland) and 5 μg of the plasmid according to the creator's instructions. was transfected with. Recombination between the plasmid and the viral genome is We generated a midine kinase-negative fowlpox virus recombinant and applied it to a concentration of 25 μg/a+1. In the presence of bromodeoxyuridine (BUdR) at a concentration of isolated by plaque assay. Two recombinant viruses expressing the F gene isolated. A recombinant PPV/F22 virus expressing the F gene was used as a high-passage fowlpox virus. Induced from a virus. Recombinant virus of PPV/F26 expressing F gene were induced from low-passage pigeonpox virus.

After repeated cloning four times in chicken embryo skin cells, virus D The presence of the F sequence in NA is confirmed by the F sequence in the thymidine kinase flanking region of the F gene. Using primers manufactured by Sambrook et al., supra. Confirmed by polymerase chain reaction according to the method. The sequence of the primer used was , 5°CTA TGT TTT CCG GTA AAA CAT CGG3' and 3'CTA TTT ATG TAT CGG CACACA TCC5 ’ is.

Expression of the F gene in infected cells, even if fully disclosed herein. However, Lee Long et al. (1986) Journal of Pyrology (J, Virol, ) Dan Uni 1198-1202, and Espion et al. (1987) Archives of Pyrology (Arch, Virol, ) 95 + According to the method of 7995, anti-Newcastle disease virus polyclonal anti- Demonstrated by immunofluorescence using both F and F protein monoclonal antibodies did.

The purity of the recombinant fowlpox virus was determined by a third clonage test. ), even if fully disclosed herein, any part of the disclosure herein Kousoulas et al. (1984) 135:379 -394 method, the biologe ( Using a biotinylated monoclonal antibody, we used a biotinylated monoclonal antibody to The plaques obtained in the cells were checked by staining them as they were.

All plaques were shown to express Newcastle disease virus F protein. It was made clear.

The stability of the recombinant viruses was determined by the fourth cluster of PPV/F22 and PPV/F26. After four consecutive expansions in chicken embryonic skin cells, Li Long et al. F protein or new protein according to the methods of Ibid. and Nispion et al., Id. Immunofluorescence and radioimmunology using polyclonal antiserum against Sul disease virus. It was evaluated by testing the progeny virus by epidemiological precipitation reaction experiment. both exams was positive.

Immunization experiment Preventing SPF chickens using PPV/F26 fourth chicken embryo skin cell passage Inoculated. The virus titer of the virus is 10""TCI D50/ml. there were.

Eighteen 6-week-old sPF chickens were vaccinated by the feather-polycle method.

After 20 days, fast-acting Newcastle disease virus strain Italian with 10'EID5Q The attack was carried out by intramuscular injection of Challenge 10 4 week old SPF chickens It was used as a control. All birds were observed for one month post-challenge. all contrasts of the chickens died within 6 days after challenge. Only one chicken was vaccinated died during the entire observation period. Serology tests, even if not fully disclosed herein. Meulema ns) et al. (1988) Avian Van Loong-CAvitarr Path,) 17:821-827+7) ELISA and hemagglutinin inhibition This was done due to adverse reactions. Serology tests showed that 17 out of 18 cats were tested 20 days after vaccination. The presence of F protein antibodies and hemagglutinin antibodies in vaccinated chickens proved the absence of One month after challenge, hemagglutinin and F protein antibodies are all present in all living organisms.

The foregoing description and illustrations fully disclose the invention, including preferred embodiments thereof. Ru. Modifications to the described method that are obvious to those skilled in molecular genetics and related fields are within the scope of the following claims.

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Claims (15)

[Claims] 1. Among the non-essential regions of the pigeonpox virus genome, D, which is not naturally present in pigeonpox virus. Recombinant pigeonpox virus synthetically modified by the presence of NA. 2. The pigeonpox virus according to claim 1, wherein the non-essential region is within the thymidine kinase gene. ilus. 3. The non-naturally occurring DNA in the pigeonpox virus is the Newcastle disease virus F gene. The pigeonpox virus according to claim 1, which is at least a part of the gene. 4. The non-naturally occurring DNA in the pigeonpox virus is the Newcastle disease virus F gene. The pigeonpox virus according to claim 2, which is at least a part of the gene. 5. Among the non-essential regions of the pigeonpox virus genome, D, which is not naturally present in pigeonpox virus. It is characterized by consisting of a recombinant pigeonpox virus that has been synthetically modified by the presence of NA. A vaccine with a sign. 6. Claim 5: The non-essential region of the pigeonpox genome is within the thymidine kinase gene. Vaccines listed. 7. The non-naturally occurring DNA in the pigeonpox virus is the Newcastle disease virus F gene. The vaccine according to claim 5, which is at least a part of the gene. 8. (a) Within the blanking sequence of the pigeonpox virus thymidine kinase gene. a desired gene product under the control of at least one vaccinia virus promoter Obtain exogenous DNA encoding; (b) Introducing the DNA from step (a) into the thymidine kinase locus of pigeonpox virus. A method for producing a vaccine for preventing diseases in birds, characterized by: 9. the foreign DNA is at least a portion of the Newcastle disease virus F gene The method according to claim 8. 10. It consists of the pigeonpox virus according to claim 1 and a pharmaceutically acceptable carrier. Characteristic pharmaceutical preparations. 11. A vaccine comprising the vaccine according to claim 4 and a pharmaceutically acceptable carrier. Pharmaceutical preparations. 12. An amount of the supplement effective to suppress infection of birds by Newcastle disease virus. The vaccine described in claim 4 is administered to birds susceptible to infection by Newcastle disease virus. A method for inoculating said birds, comprising administering to said bird. 13. A eukaryotic cell culture infected with the virus of claim 1. 14. 14. The eukaryotic cell culture according to claim 13, wherein the cell culture is a chicken cell culture. 15. The eukaryotic cell according to claim 14, wherein the cell culture is an Ewatori embryonic skin cell culture. culture.