JP2003530076A - Gene cloning and expression vectors based on recombinant eggs and avian adenovirus - Google Patents

Abstract

  (57) [Summary] We describe a new technique for producing eggs containing recombinant proteins based on a novel use of avian adenovirus (AAV). Adenovirus-infected embryogenic eggs are used as host organisms. The synthesized protein is isolated in large quantities from each egg and purified without the time-consuming purification steps. As far as is known, the mechanisms of protein translation and post-translational modification in avian species are completely similar in the case of other higher eukaryotes such as humans, domestic animals and plants. Unlike bacterial, yeast, and plant host systems for producing recombinant proteins, avian embryos allow the isolation of proteins in endotoxin-free, biologically active forms. The combination of high productivity and the possibility of obtaining the protein in its native form make it the only biotechnological choice that is much better than other hosts currently used for this purpose.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to the field of molecular biology. In particular, the present invention relates to eggs containing recombinant proteins and / or recombinant DNA, as well as vectors and genes useful for producing recombinant proteins in eggs.

BACKGROUND OF THE INVENTION It is a norm in the pharmaceutical industry to transform genetic information into products that can be provided directly to the market. By 2000, the market for biological products is expected to be close to 150 billion yen, which is largely driven by the discovery of genomes and drugs. This growth reflects the enormous demand for characterizing the molecular characteristics of newly discovered genes whose function would provide disease cure. Once such genes have been identified, large scale manufacturing is often required for research and clinical trials to bring the product to market. There are very few recombinant protein production techniques that can be used to produce clinically usable quality products. It is still difficult and unpredictable to base bacteria, yeasts or insect cells on the manufacturing method,
Expensive. Therefore, there is a need to develop highly productive recombinant protein expression systems that are easy to use and inexpensive to scale up to industrial production levels.

DISCLOSURE OF THE INVENTION In order to meet these needs, the present invention aims at a recombinant protein expression vector based on the hen egg adenovirus expression vector (AdCEV), poultry adenovirus type 1 (Ad FAV1). It is a thing. The Ad FAV1 host is a fertilized chicken egg that has, for many years, provided a simple method for the growth of various viruses that can be used to immunize humans or animals. For example, human and animal influenza viruses, along with many poultry viruses, are still produced in fertilized eggs. The present invention is
It extends to useful and valuable products that can produce any protein that can be stably expressed by the AdCEV system of the present invention in fertilized eggs. Transfection of AdCEV converts each egg into a mini factory that produces large amounts of recombinant protein in egg yolk. Our method does not require a screening step and produces high rates of recombinant cells after transfection. The AdCEV / egg system of the present invention provides the following benefits. Ie 1. simple and low cost;
2. High productivity-mg protein / egg; 3. Short production time-72 hours incubation; 4. Accurate processing of eukaryotic proteins; 5. Simple purification procedure from egg solution; 6. For mass production Easy scale-up; and 7. High level of biosafety.

The present invention is further directed to eggs comprising the recombinant protein. The egg can be a poultry egg. The egg may be an egg of any poultry species susceptible to adenovirus infection.

The present invention is further directed to eggs comprised of recombinant DNA. The present invention is further directed to eggs containing recombinant RNA.

The present invention also provides a method of preparing a recombinant avian adenovirus vector containing a heterologous gene, comprising: (a) (i) at least one transcriptional regulatory sequence flanking at least one restriction endonuclease site; and (ii) A plasmid, cosmid or phage containing avian adenovirus DNA containing DNA from a nonessential region of the Ad FAV1 genome flanking the transcriptional regulatory sequence and the restriction endonuclease site is prepared, and (b) at least one from a heterologous gene is prepared. It is directed to a method comprising the steps of inserting a seed protein coding sequence into the restriction endonuclease site adjacent to the transcriptional regulatory sequence.

The invention further provides a method of genetically modifying an avian adenovirus, comprising the steps of (a
) (I) at least 1 adjacent to at least one restriction endonuclease site
Individual transcriptional regulatory sequences and (ii) a plasmid, cosmid or phage containing avian adenovirus consisting of DNA from the non-essential region of avian adenovirus flanking the transcriptional regulatory sequence and the restriction endonuclease site, ( b) inserting at least one protein coding sequence from a heterologous gene into the restriction endonuclease site flanking the transcriptional and translational regulatory sequences to provide (c) at least one cell infected with recombinant avian adenovirus. Then, (d) a recombinant avian adenovirus capable of expressing the above protein coding sequence is isolated from the above cells.

The invention further provides in a method of using a recombinant avian adenovirus as a vaccine, comprising (a) at least one avian adenovirus transcriptional and translational regulatory sequence and at least one protein coding sequence from a heterologous gene. An infectious avian adenovirus containing a chimeric gene is prepared, wherein the chimeric gene is flanked by DNA from a nonessential region of the avian adenovirus genome, the infectious avian adenovirus encoding the protein code upon infection of the cell. The method is intended to express the sequence. In the second step, the animal or human is inoculated with an inoculum containing the recombinant protein in a concentration sufficient to induce an immune response in the animal or human.

The invention further comprises (a) a plasmid, a cosmid or a phage, (b) at least one avian adenovirus transcriptional and translational regulatory sequence from a heterologous gene, and (c
) DN from a nonessential region of the avian adenovirus genome adjacent to the above chimeric gene
The purpose is a vector consisting of A.

The present invention is further an infectious avian adenovirus containing a chimeric gene consisting of at least one avian adenovirus transcriptional and translational regulatory sequence and at least one protein coding sequence from a heterologous gene, wherein: The chimeric gene is flanked by DNA from a nonessential region of the avian adenovirus genome, and the infectious avian adenovirus is intended for avian adenovirus capable of expressing the protein coding sequence upon infection of cells.

In another embodiment, the invention provides the following steps: (a) (i) at least one transcription and translation regulatory sequence adjacent to at least one restriction endonuclease site; and (ii) the above transcription. Prepare a vector consisting of a bacterial or yeast plasmid, cosmid, or phage containing avian adenovirus DNA consisting of DNA from a non-essential region of avian adenovirus flanking the regulatory sequence and the restriction endonuclease site, and (b) a heterologous gene. For the infectious avian adenovirus recombinants produced by the method comprising the step of inserting at least one protein coding sequence from Escherichia coli into the restriction endonuclease site flanking the transcription and translation regulatory sequences. is there.

In another embodiment, the present invention is directed to the production of large quantities of recombinant protein from genes expressed in egg fluid. The gene of interest may encode a protein that is poorly expressed. That is, a protein that is not produced in large amounts and / or transiently under natural physiological conditions. In other cases, the size of the "non-replaced" region of the virus may be exceeded by which the size encoding the desired gene (s) can be replaced without affecting the required infectivity of the recombinant virus. .

One approach to achieving increased protein production is the use of transient cell expression systems transfected with “minichromosomes” in which the cells are not expected to integrate into the host cell's genome. The minichromosomes used in the transient cell expression system can also be modified to further increase their copy number during replication in infected cells.

The present invention provides two cloned inverted adenovirus terminal sequences in a vector DNA molecule, as well as non-defective adenovirus DNA as both vector and helper.
Co-transfection of cells by (cotransfection). In these systems, the helper DNA provides in trans all of the protein functions required for the packaging of vectors containing only those cis-acting elements required for viral DNA replication and packaging, primarily in the reverse terminus. Repeat sequences (including the origin of viral replication) and packaging signal sequences.

In the present invention, the vector system comprises a transcribed transcriptional sequence (
Promoter)), a RNA-stabilizing sequence that enhances translation, at least one protein coding sequence from a heterologous gene, and a non-defective adenovirus DNA as a helper.

In another embodiment, the vector system comprises a DNA minichromosome consisting of a transcribed sequence (promoter), a translation-enhancing RNA stabilizing sequence,
It consists of at least one protein coding sequence from a heterologous gene and the inverted adenovirus ends required for replication of the vector, and a non-defective adenovirus particle as a helper.

Thus, based on this disclosure, those skilled in genetic engineering can construct transfectants that overcome the size limitations of expression vectors and the production problems associated with underexpressed genes. Those skilled in the art of recombinant DNA technology, in particular, will find suitable DNA encoding the protein of interest, origin of adenovirus DNA replication from both the right and left ends of the genome, transcriptional transactivators and translational stimulators, and helpers. Adenovirus DNA can be designed and then used for large-scale production of the desired protein by production of transgenic eggs using the transfectant production method disclosed by the present invention. Such proteins include their native forms or truncated analogs as well as fusion proteins or other engineered constructs that can mimic the biological activity of the protein of interest.

[0018]     (Brief description of drawings)   It is believed that the present invention can be better understood with reference to the accompanying drawings.

[0019]     (Detailed Description of the Invention)   Definition   To ensure a full understanding of the invention, the following definitions are provided.

Adenovirus: Any virus that belongs to the adenovirus family. The large adenoviridae (45 species) are adenoviruses (Mastaden) that infect natural hosts and mammals.
viridae) and adenoviruses that infect avian species (Aviadenoviridae).

Avian adenovirus: An adenovirus of the Aviadenoviridae family. There are at least 10 different serotypes of avian adenovirus, Fenner, F et al., Veterinary Vi
Commonly infect chickens and other avian species such as ducks, quails and turkeys as disclosed in Rology, Academic Press, Orlando, Florida, 1987, pp 329-337. The above description is incorporated herein by reference.

CELO: Chicken embryo lethal orphan virus. CELO is synonymous with poultry adenovirus serotype 1 or avian adenovirus serotype 1. CELO is an avian adenovirus of the adenoviridae family. The general organization of the CELO virus is an icosahedral capsid 70-80 nm in diameter made up of hexon and penton structures. The CELO virus genome is a linear, double-stranded DNA molecule, the DNA of which is concentrated in virions by a core protein encoded by the virus. CELO virus has a terminal protein by covalent bond and has an inverted terminal repeat structure.

Ad CEV: Recombinant adenovirus vector derived from chicken embryo lethal orphan virus

Promoter: A promoter is a minimal DNA sequence sufficient to direct transcription. Promoters allow control of transcription for cell type-specific, tissue-specific, organ-specific, or inducible expression. The promoter element is located in the 5'or 3'region of the native gene.

Polyadenylation site: A polyadenylation site is a nucleotide sequence that causes cleavage of mRNA at certain enzyme-specific sites and addition of an adenylate residue at the 3'end of the mRNA.

Polypeptide: Polypeptide refers to any chain of amino acids, regardless of length or post-translational modification (eg, glycosylation or phosphorylation), including proteins. Polypeptides useful in the present invention include, but are not limited to, HIV glycoprotein; rabies glycoprotein; hantavirus glycoprotein; Ebola virus glycoprotein; human papillomavirus glycoprotein; hepatitis B surface antigen; A Hepatitis C envelope protein; Hepatitis C envelope protein; Hepatitis E envelope protein; Human cytomegalosvirus major envelope glycoprotein; Pseudorabies virus glycoprotein; Vesicular stomatitis virus glycoprotein; RS virus envelope protein; Rubella virus glycoprotein; Measles Virus envelope protein; yellow fever virus envelope protein;
Influenza virus protein; tick-borne encephalitis virus envelope protein; parainfluenza virus envelope protein; osteocalcin; osteonectin; chymase; thyroid peroxidase; interleukin;
Caspase; Calpain; Apoptosis protein; Insulin; Tumor necrosis factor; Granulocyte macrophage colony stimulating factor; Epidermal growth factor; Erythropoietin; Interferon; Prostaglandin; Thrombolytic protein (plasminogen, urokinase, plasminogen tissue activator, etc.) ); Eosinophil-derived neurotoxin (major eosinophil ribonuclease); morphogenetic protein (human jagged, mouse jagged); integrin; fibronectin; vitronectin; osteopontin; cadherin; lavendustin A; receptor tyrosine kinase; pituitary protein; endoglin Β-2-macroglobulin antigen; human C
-Reactive protein; fatty acid binding protein; human chorionic gonadotropin; neuron-specific enolase; human growth hormone; cytomegalovirus envelope protein; Epstein-Barr virus protein; hepatitis core antigen; Diofil
aria immitis glycoprotein; bovine leukemia virus glycoprotein; mumps virus envelope protein; human parvovirus envelope protein; rotavirus glycoprotein; verotoxin; parathyroid hormone.

Substantially identical: Substantially identical to a polypeptide is one that exhibits at least 50%, preferably 70%, more preferably 90%, and most preferably 95% identity to a reference polypeptide. Means Substantially identical to a nucleic acid means exhibiting at least 85%, preferably 90%, more preferably 95%, and most preferably 97% identity to a reference nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids,
More preferably at least 25 amino acids, most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 30 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides. Sequence identity is typically determined by sequence analysis software (eg, Sequence Analysis Software Package of the Genetics Compu
ter Group (GCG), University of Wisconsin Biotechnology Center, 1710 Univ
ersity Avenue, Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions and other modifications. Conservative substitutions typically include the substitutions within the following groups: Glycine, alanine; valine,
Isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine and tyrosine.

Substantially pure polypeptide: By substantially pure polypeptide is meant a polypeptide that has been separated from components that naturally accompany it. Typically, a polypeptide will be at least 60% by weight from the protein with which it is associated and the organic molecules with which it is naturally associated.
Substantially pure when released. Preferably, the preparation contains at least 75%, more preferably at least 90%, most preferably at least 99% by weight of the polypeptide of interest. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis or HPLC analysis. Substantially pure DNA: Substantially pure DNA means DNA that does not contain genes flanking the gene of interest in the native genome of the organism from which the DNA of the present invention is derived. Thus, the term refers to, for example, recombinant DNA inserted into the vector of the invention.
Includes. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

Transformed Cell: A transformed cell is one (or its progeny) into which a DNA encoding a polypeptide (as used herein) has been introduced by recombinant DNA methods. Means a cell.

Transformed egg: A transformed egg is a recombinant DN
DNA encoding a polypeptide by method A (as used herein)
Means an introduced egg.

Arranged for expression: The term arranged for expression means a DNA sequence in which a DNA molecule directs the transcription and translation of the sequence (ie, promotes the production of, for example, a recombinant polypeptide or RNA molecule). It means that it is placed adjacent to.

Operably linked: The term operably linked refers to gene and regulatory sequences (
It is meant that the singular or plural is linked in such a way that the expression of the gene is possible when the appropriate molecule (eg transcription activator protein) is bound to the regulatory sequence (s).

Minichromosomes: DNA molecules consist of at least the 5'- and 3'-terminal repeat sequences of the adenovirus genome that contain origins of replication and packaging sequences.
Such DNA molecules will automatically replicate in cells infected with a helper virus that provides all necessary replication functions in trans.

DETAILED DESCRIPTION OF THE INVENTION In view of these definitions, the present invention is directed to a recombinant protein production system based on chicken eggs infected with avian adenovirus.

The large adenoviridae are adenoviruses that infect mammals (the Mastadenoviridae) and adenoviruses that infect birds (the Aviadeno vir) depending on the host range.
idae). Chicken embryo lethal orphan (CELO) virus is a vector for bovine skin disease (Van den Ende, M. et al., J. Gen. Microbiol. V. 3, 174-183, 1949)
Infection vector (Yates, VJ & D.
E. Fry, Am. J. Vet. Res.
tebrates, vol. 3. Viral infections of birds. Elsevier Scientific Publish
ers, Amsterdam, 1993; McFerran, JB & BM Adair, Avian Pathol. V. 6,
189-217, 1977). The CELO virus, classified as fowl adenovirus type 1 (FAV-1), has been a major subject of avian adenovirology for many years. FAV-1 adenovirus can be isolated from healthy chickens and reintroduced into chickens experimentally does not cause disease (Cowen, B. et al., Avian. Dis. V. 22, 459). -470, 1
978). The overall structural organization of the CELO virus is similar to that of mammalian adenoviruses, with icosahedral capsids 70-80 nm in diameter constructed from hexon and penton structures. The CELO virus genome is a linear double-stranded DNA molecule
Is condensed within the core protein encoded by the virus. The CELO virus genome is covalently linked to terminal proteins and has inverted terminal repeats (ITRs), which are shorter than mammalian ITRs. The CELO virus encodes a protease that has 61-69% homology with mammalian adenovirus proteases.

The DNA sequence and genomic organization of the CELO virus has been reported (Chiocca, S. et al.,
J. Virol. V. 70, N5, 2939-2949, 1996). Its sequence is 48.3
It is about 8 kb longer than the 35.9 kb of human subgenus C adenoviruses Ad2 and Ad5. Genes of major viral structural proteins (hexon, penton IIIa, fiber, pV
I, pVII and pVIII) are present and in expected positions in the genome. Initial area 2 (E
2) There are also genes (encoding DNA-binding proteins, DNA polymerase and terminal proteins). However, the CELO virus is a mammalian adenovirus E1,
It lacks the E3 and E4 regions. The CELO virus genome has a sequence of about 5 kb at the left end and a sequence of 15 kb at the right end, but there is limited homology or no homology with the mastadenovirus genome. These new sequences contain a large number of open reading frames (ORFs), which are missing E1, E2 and possibly E
It appears to encode a function that replaces four regions.

Deproteinized DN of AD CELO (herpes species) introduced into the allantoic cavity of a 9-day-old chicken embryo
A induces the infectious virus Ad CELO (Grabko, VI, Acta Virologia, 1987,
v. 31, No 1-2, 97-102). Using the method for heteroduplex analysis of virion DNA, we have placed a nonessential region in the EcoRI-B fragment of the Ad CELO genome. As a test of the feasibility of constructing a viral vector, the inventors inserted the plasmid pUC 19 into a nonessential region (2686 bp), assembled a CELO DNA fragment by in vitro ligation, and cultivated a 9-day-old chicken embryo with CELO. Transfected with overlapping fragments of DNA. As a result of viral DNA recombination in vivo, we obtained Ad CELO virions containing the genome of plasmid pUC19 in a nonessential region (Grabko VI,
Construction of recombinant adenovirus vector by method of insertion het
erologous DNA in genome of Adenovirus CELO, Russian patent N5061908 / 13, 1993
). In this way, a DNA fragment of up to 2700 bp can be inserted into the native genome of Ad CELO (in a nonessential region) without affecting the success or failure of viral replication. However, if the non-essential region (about 6% of the genome) is deleted, 2000-300
It is expected that the capacity of the genome can be enhanced up to 0 additional base pairs.

The main advantage of the prokaryotic system is that the genetic and phenotypic markers can easily be used as a tool to select the correct recombinant. In a eukaryotic system,
This process is usually difficult, and engineering an efficient system for recombinants is a major problem in vector design, especially in the chicken embryo system. In the present invention, the inventors have developed a powerful technique for assembling recombinant Ad CELO from two fragments in vitro. By using the assembly scheme of the present invention, manual and time-consuming selection and purification of recombinant clones using phenotypic markers is eliminated. In the scheme of the present invention, each fragment is not infectious. Recombinant infectious DNA is formed only after assembly in vitro, which induces regeneration of infectious recombinant virions upon transfection (Celis, E. et al., J. Immunol. V. 136, 629). -697, 1086).

Indeed, any polypeptide of interest can be expressed by use of the expression system of the invention. Examples of such polypeptides include vaccine species Vnukovo-
There are 32 rabies virus glycoproteins.

Vaccination against rabies remains the only effective means of preventing disease after rabies infection. Recombinant viruses expressing rabies glycoprotein (RG) used as vaccines have considerable potential to overcome some of these problems. RG can elicit protective virus neutralizing antibodies (Wiktor, TL et al., J. Am. Med. Assoc.
v. 224, 1170-1171, 1973), MHC class I- and class II-restricted cytotoxic T cells (Celis, E. et al., J. Immunol. v. 136, 629-697, 1086) and this antigen. The expressed vaccinia virus vector was evaluated as an oral vaccine (Kieny, MP
, Nature v. 312, 163-166, 1984). Adenovirus vector vaccine is a useful auxiliary vaccine for vaccines using vaccinia virus vector (Hruby,
DE, Clin. Microbiol. Rev. v. 3, 153-170, 1990), possibly considered as a suitable alternative vaccine (Kaplan, C., Arch. Virol. V. 106, 127-139,
1989). Cloning of rabies virus glycoprotein G in embryogenic eggs is described in the Examples section below.

The most thoroughly studied poxviridae vaccinia virus has been used as a live vaccine to kill smallpox. Medical interest in vaccinia virus will be diminished, but live vaccinia recombinants can express heterologous genes (Mackett, M. et al., Proc. Natl. Acad. Sci. USA v. 79, 7415-
7419, 1982) and rabies virus (Wiktor, TJ et al., Proc. Natl. Acad. Sci.
USA v. 81, 7194-7198, 1982) and many other viral infections have been shown to protect and immunize animals against re-stimulation.

Vaccinia virus can also be used as a cloning and expression vehicle (Grabko, VI et al., IV Conference of Russian Federation-New Direction.
Biotechnology, p. 102-103, 1994). Recombinant vaccinia virus expressing RG from strain Vnukovo-32 elicited protective antibodies against rabies virus in mice.

Rabies glycoprotein DNA coding sequences can also be traced by polyadenylation (poly (A)) sequences such as the SV40 early poly (A) region. The poly (A) region, a signal for polyadenylation of RNA transcripts, appears to play a role in stabilizing transcription. Similar poly (A) regions are derived from various naturally occurring genes.
This region may also be modified to alter its sequence, provided that polyadenylation and transcription stabilizing functions are not significantly adversely affected.

A recombinant DNA molecule consisting of the left end of the Ad CELO genome, the major late promoter, the bipartite leader, the rabies glycoprotein DNA coding sequence and poly (A) SV40 is ligated to the right large fragment of the Ad CELO genome. Along with this, these two ligated D
The NA strip contains the entire adenovirus genome and contains infectious virus information. The next step is the introduction of recombinant viral vector DNA into avian eggs or avian cell cultures.

Transcription and expression of the heterologous protein coding sequence in the system described above can be monitored. Southern blot analysis, for example, can be used to determine the copy number of the RG gene. Northern blot analysis can be used to determine the copy number of the RG gene. Northern blot analysis provides information on the size of transcribed gene sequences (see, eg, Maniatis et al., Supra). The level of transcription can also be quantified. Expression of the selected RG protein in avian cells or avian amniotic fluid can be further confirmed by Western blot analysis and activity test of the resulting glycoprotein.

Another example is an expression system using a vector, an avian egg composed of pKSII-DC5′-MLP-2BLD-RG-p (A) -DC3 ′ and a DNA Ad CELO as a helper. The vector, pKSII-DC5'-MLP-2BLD-RG-p (A) -DC3 ', is the Ad CELO genome, major late promoter, bipartite leader, rabies glycoprotein DNA coding sequence, poly (A) SV40 and Ad CELO genomes. Composed of the right end of. Embryogenic eggs can be obtained using this vector and purified Ad
Cotransfection with CELO DNA results in the expression of recombinant rabies glycoprotein that accumulates in allantoic fluid.

It is believed that the following examples will make these and other embodiments of the present invention apparent to those skilled in the art. Within the examples, poultry adenovirus type 1 (FAV1) is often mentioned, but for the purpose of illustration, the same characteristics are noted for other types of poultry adenovirus, in particular 1, 2, 3, 4, 5, 6 It also applies to types 7, 7, 8, 9, 10, 11 and 12, and the invention described herein is intended to cover all of these avian adenoviruses. Further, for the purposes of the present invention, it should be understood that any of these various adenoviruses can be used in eggs to produce any protein.

Example 1 Using the results of a Swedish researcher (J. Virology, 1982, v. 42, N1, 306-310) describing the homology region between the hexon genes of human Ad2 and Ad CELO, the present inventor Et al. Synthesized several primers that uniquely identify the hexon gene of human Ad2. The specificity of the primers was determined by sequencing the fragments cloned from Ad CELO and human Ad5. One of the primers was used to synthesize Ad CELO which hybridizes to the human Ad2 hexon gene. Reverse primer 5'AGGAACCAGTCTTTGGTCATGT-3 'from human Ad2 genomic position: 21164-21185 bp SEQ ID NO: 27

This primer was used for the synthesis of the Ad CELO hexon gene. First chain cDN
The synthesis of A was performed by AMV reverse transcriptase. For the synthesis of the second strand, RNAse H,
DNA-pol1 and T4 DNA-pol were used. Double-stranded cDNA (approximately 2500-3500 nucleotide base pairs) was eluted from the agarose gel and cloned into the pBluescript II SK (+) vector. Clones were selected by molecular weight and ³² P-labelled CELO DNA. The most likely size clone was sequenced from each end of the cloned fragment. All clones have a primer sequence at their 3'end, while the 5'of the cloned fragment.
It has a homologous sequence in the test clone at the end. Hybridization of ^{3 2} P- labeled fragments of these clones and Ad CELO (XbaI-B fragment and EcoRI-A fragment) is, CELO hexon genes they were shown to contain part and bisection leader sequence hexon gene ( MLP) is useful in the vectors of the invention for the expression of heterologous proteins.

Example 2 This example describes the generation of the plasmid pKSII-DC5'-MLP-2bLd described in Figure 1. Plasmid pKSII-DC5'-MLP-2bld was prepared by directional cloning in 3 consecutive steps.

1. Cloning the 5'end of the FAV1 genome First, using the oligonucleotide primer and polymerase chain reaction (PCR), the left end of the Ad CELO genome up to 538 base pairs (bp) (Avian adenovirus CEL
The position between the 0 and 538 bp DNA sequences of the O genome) was amplified and isolated. 5'CAAGTGGTACCGGCCAAATTGGCCGATGATGTATAATAACCTCA-3 '[SEQ ID NO: 1] 5' CAACCAAGCTTCTCTTCCGAAGTCATCTG-3 '[SEQ ID NO: 2] The amplified sequence of 538 bp is shown in Table 1 [SEQ ID NO: 21].

The amplified DNA contained the essential origin (ori) and the packaging sequence (pkg). This kPCR fragment (KpnI-ori / pkg-HindIII) was digested with HindIII alone, inserted into pSKII vector, and digested with HindIII and SmaI. Using this construct, DC5 '
EcoRI was deleted from the fragment (KpnI-ori / pkg-HindIII). This plasmid (pSKII
-DC5 ') was digested with EcoRI and the site was filled in by using T4 DNA polymerase. The vector was then ligated again. The deleted pSKII-DC5 'EcoRI [-] was replaced with KpnI-
Digested with HindIII. DC5 'EcoRI [-] was isolated and KpnI-HindIII cut pSKII
Ligated to vector. At this time, the region located between the 538 bp and 1988 bp genomes of Ad CELO was deleted.

2. Cloning of the major late promoter MLP Secondly, the region of the major late promoter was found near 7000 bp, with a 7488 bp T
An ATA box (Chiocca, S. et al., J. Virol. V. 70, N5, 2939-2949, 1996) was generated by PCR using the following primers. 5'-CAACTAAGCTTGAGCTGTACGTGTCACTTCC-3 '[SEQ ID NO: 3] 5'-CAACAGAATTCCTGGAAGTCGAGGCGACC-3' [SEQ ID NO: 3] The amplified DNA contained a major late promoter (MLP). Then, the PSKII vector was opened with HindIII-EcoRI, and the PCR fragment HindIII-MLP-EcoRI was inserted therein.
This sequence is shown in Table II [SEQ ID NO: 22].

3. Isolation and cloning of the bipartite leader sequence Nine day old chicken embryos were inoculated with 0.1 mL of CELO virus at a concentration of 10 ⁸ virions / mL per embryo. The inoculation procedure was as described in Rev. Roum. Med. Virol. 1985, 36, 4, 235-240. Two eggs were chilled on ice 18-20 hours post infection. Chorioallantoic membranes were isolated as described, rinsed with cold phosphate buffered saline, washed membranes were then washed with buffer A (10
mM Hepes pH 7.5; 25 mM NaCl; 5 mM MgCl ₂ ) in a Potter-Elevehjem homogenizer. The homogenate was dissolved by adding 5% Triton X-100 in buffer A.
Nuclei were pelleted by centrifugation at 3000 rpm for 30 minutes. The supernatant was added to 3% SDS, followed by phenol extraction (2x), chloroform extraction (2x) and ethanol precipitation. All cytoplasmic RNA was dissolved in 4M guanidinium isocyanate in 40mM Tris-HCl containing 20mM NaCl and RNA was passed through a 5.7M CsCl cushion to 150,000 xg.
Centrifuge at 20 ° C. (+ 4 ° C.) for sedimentation. The pellet was resuspended in 6M guanidine hydrochloride. 0.025 volumes of 1 M acetic acid and 0.5 volumes of ethanol are then added,
RNA was precipitated as above. Dissolve the RNA pellet in a minimum volume of DEP-treated water,
Used to generate double standard cDNA by RT / PCR as described by Grabko (Grabko, VI FEBS Lett. V. 387, pp. 189-192, 1996).

The reverse transcriptase (RT) mixture was 67 mM Tris-Cl (pH 8.8), 16.6 mM (NH ₄ ) ₂ SO ₄ , 0.25.
4 mM deoxynucleoside triphosphates (dATP, dCTP, dGTP, dTTP), 2 mM
MnCl ₂ or 1.5 mM MgCl ₂ , 20 pM XabI primer (reverse), 5 units Taq or Tth
It contained DNA polymerase and various amounts of virion RNA (0.2 μg to 2 pg). RNA
The template was added to the RT mixture heated to 60 ° C, covered with 40 mL of mineral oil, incubated at 60 ° C for 3 minutes, and the method was continued at 70 ° C for 15 minutes. After RT reaction, 67 mM Tris-HCl pH
8.8, 16.6 mM (NH ₄ ) ₂ SO ₄ , 0.01% Tween-20, 0.75 mM EDTA, 0.25 mM each of 4 deoxynucleoside triphosphates, 2 mM MgCl ₂ , and finally 20 mM primer (direct) EcoRI was added. Then add the standard incubation mixture (1000 μL)
Amplification was performed in a DNA thermocycler (Perkin-Elmer Cetus Instruments) as follows. That is, 35 cycles at 94 ° C for 1 minute, 56 ° C for 1 minute, and 72 ° C for 1.5 minutes. Aliquots (5 μL) were analyzed by electrophoresis on a 1% agarose gel. The RT / PCR incubation mixture containing the CDNA amplified fragment was extracted with chloroform and precipitated with 3 volumes of ethanol. Amplified fragment and plasmid pBluescript II S
K (+) was hydrolyzed with restriction enzymes XabI and EcoRI, ligated with T4 DNA ligase, transformed into Escherichia coli DH5 cells, amplification of insert by recombinant clone PCR, followed by electrophoresis on 1% agarose gel. Screened. The primer used was 5'-CATGGAATTCCAGGTCTACGCCGACGAGAGGATCG-3 '[SEQ ID NO: 5] 5'-CAACTCTAGAGCCTGAATTTGTTTTTCAAGTC-3' [SEQ ID NO: 6]

The amplified DNA contained the dichotomous leader sequence (2bld) and the sequence of hexon mRNA. The pSKII vector was then cleaved with EcoRI-XbaI and the PCR fragment EcoRI-2bld-5'hex-
I inserted XbaI there. Only bisected leaders were obtained by PCR with the following primers. 5'-CATGGAATTCGACTTCCAGGTCTACGGCGACGAGAGG-3 '[SEQ ID NO: 7] 5'-CCTGGATCCGATGTGTTCCTTGAACCAAAC-3' [SEQ ID NO: 8]

The amplified DNA contained only the bipartite leader sequence (2bld). The pSKII vector was then cleaved with EcoRI-BamHI and the PCR fragment EcoRI-2bld-BamHI was inserted therein. PS
The KII vector contains an essential origin (ori) and contains the packaging sequence (pkg) (KpnI-or
i / pkg-HindIII) with HindIII-EcoRI and the major late promoter (MPL) is ori
And a pkg element. Then (KpnI-ori / pkg-HindIII-MLP-EcoRI
) Was cleaved with EcoRI-BamHI to ligate the bipartite leader to the ori / pkg and MLP elements.

Example 3 This example describes the generation of the plasmid pKSII-DC5'-MLP-2BLD-RG-p (A) shown in Figure 2. The plasmid pKSII-DC5'-MLP-2BLD-RG-p (A) was prepared by directional cloning in 3 consecutive steps. First, the glycoprotein gene of rabies virus vaccine strain Vnukovo-32 is extracted from viral RNA using oligonucleotide primers, reverse transcriptase (Ame
rsham) and 1640 b of the rabies virus glycoprotein gene prepared using PCR.
Up to p DNA sequence was amplified and isolated. The primers used were: 5'-GGATCCAGGAAAGATGGTTCCTCAGGCTCTCCTGTTTG-3 '[SEQ ID NO: 9] 5'-GCTGCAGCAAGGGGAGGTGATCTTCAGACTTGGATCGT-3' [SEQ ID NO: 10]

The amplified fragment contained the glycoprotein gene of the rabies virus vaccine strain Vnukovo-32 (RG). The pSKII vector is then cleaved with BamHI-PstI and the PCR fragment BamH
I-RG-PstI was inserted there. Second, using the oligonucleotide primers and polymerase chain reaction (PCR), the genomic SV40 (simian virus 40
Up to 240 base pairs (bp) of 0-2770 base pairs (bp) located in the DNA sequence were amplified and isolated. The primers used were: 5'-CAATCTGCAGATCATAATCAGCCATACCAC-3 '[SEQ ID NO: 11] 5'-CAACTCTAGATCCAGACATGATAAGATACATTG-3' [SEQ ID NO: 12]

The amplified DNA contained the poly (A) site of SV40. pSKII vector followed by BamHI
It was cleaved with -XbaI and the fragment BamHI-RG-PstI and the PCR fragment PstI-p (A) -XbaI were inserted therein. Thirdly, the pSKII vector containing (KpnI-ori / pkg-HindIII-MLP-EcoRI-2bld-BamHI) is cleaved with BamHI-XbaI and the fragment BamHI-RG-PstI-p (A) -XbaI is inserted therein. did. The sequence of poly (A) SV40 is shown in Table V [SEQ ID NO: 25].

Example 4 This example uses the plasmid pKSII-DC5′-MLP-2bLD-RG-p (A) -DC (XbaI-No shown in FIG.
State the occurrence of tI). Plasmid pKSII-DC5'-MLP-2bld-RG-p (A) -DC (XbaI-No
tI) was prepared by directional cloning in one step. (KpnI-ori / pkg- HindIII-M
The pSKII vector containing LP-EcoRI-2bld-BamHI-RG-PstI-p (A) -XbaI) was cleaved with XbaI-NotI and the fragment Ad CELO genome XbaI- (present between 2 and 17.4 kb) -NotI was placed therein. Inserted. A collection of recombinant Ad CELO genomes was prepared by in vitro ligation in one step. This is shown in Figure 4. (KpnI-SfiI-ori / pkg-HindIII- MLP-EcoRI-2bld-BamHI-R
The pSKII vector containing G-PstI-p (A) -XbaI-Ad CELO-NotI) was cleaved with SfiI-NotI and the fragment Ad CELO genome NotI- (located at 17.4-48.3 kb) -3 'end Ad CELO It was ligated to the genome. This ligation mixture (about 1 μg per embryo) was injected into the allantoic cavity of 9-day-old chicken embryos. After 72 hours of incubation at 37 ° C. in a humidified incubator, allantoic water was harvested and assayed for rabies glycoprotein recombinant protein.

Example 5 Construction of CRP-expressing clones We removed the N-terminal leader peptide and expressed the subunit in the mature form-described in the structural data entry PDB: 1 GNH of the Protein Database Bank. A clone was constructed that mimics the type found in the crystallized molecule. Our recombinant construct thus encodes a protein beginning with the sequence (-NH-GLN THR ASP MET SER ARG ...) [SEQ ID NO: 23]. The present inventors considered the fact that the lung-forming egg system, in principle, correctly cleaves the precursor form of CRP, which does not result in the same arrangement in CELO-infected cells. Viral proteins were specifically transported to allantoic fluid by CELO-infected cells and appeared to be similar to those occurring in CRP subunits or pentamers.

In order to make the correct protein, we therefore
Methionine was artificially added by introducing methionine in the correct reading frame at the N-terminal end (-NH-MET GLN THR ASP MET SER ARG ...) [
SEQ ID NO: 13]. Since most proteins in eukaryotic cells are non-specifically processed by cellular enzymes to remove the N-terminal methionine, we believe that addition can actually increase the chance of the correct protein. Thought.

PCR Primers The construction of the restriction sites and recombinant CRP (rCRP) molecule described above was performed using Genbank (Acc. No. X56692
) Performed using the sequence as a guide. The primer introduces the restriction sites used to make the construct (Figure 5). 5'primer 5'-CGTTA GGATCC ATGCAGACAGACATGTCGAGGAAGGC-3 '[SEQ ID NO: 13] 3'primer 3'-CCAA GCGGCCGC TGCAGTCATCAGGGCCACAGCTGGGGTTTGGT-3' [SEQ ID NO: 14] I primer-For (37mer) 5'-CGTTAGGATCCATGTCGGTGAAGCCCCTT '[SEQ ID NO: 15] II primer-Rev (38 mer) 5'-CCAATCTGCAGTCATCAGGGCCACAGCTGGGGTTTGGT-3' [SEQ ID NO: 16]

Plasmid DNA was isolated from overnight cultures of the ATCC 324978 clone, EcoRI and Xho
Digested with I restriction enzyme. The expected fragment size of approximately 1.8 kb was observed on the gel. The plasmid DNA was used as a template for the PCR reaction together with the primers described in the Materials and Methods section. A fragment of approximately 650 bp was observed, consistent with published sequence data. The photograph of the agarose gel in FIG. 6 shows the products of PCR amplification with CRP primers along with lateral size markers. The sequence of our recombinant human CRP protein encoded by the PCR insert is shown in Table III [SEQ ID NO: 23].

The PCR fragment was introduced into the construct pKSII-DC5'-MLP-2bld-hCRP-p (A) -DC3 '. Selection of recombinants at each step was done by restriction digestion and fragment size determination on agarose gels. A viral terminal repeat sequence (TR) essential for viral genome replication was also included in the construct.

The cloned human C-reactive protein in eggs yielded a recombinant identical to the normal protein in its ability to cross-react with either pentamer and CRP-specific antisera. The results are shown in Fig. 7. In other experiments, recombinant CRP was assayed for its ability to bind to O-phosphorylethanolamine sepharose, a matrix commonly used for biospecific adsorption of CRP. The results suggested that the recombinant CRP formed a pentamer. This is because it is required to make the bond to the matrix (FIG. 8).

Example 6 Cloning of Rabies Virus Glycoprotein The nucleotide and deduced amino acid sequences of the glycoprotein gene of rabies virus vaccine strain Vnukovo-32 are known (Fodor, I, Grabko, VI et al. Arch. Virol. V.
135, N 3-4, 451-459, 1994). Rabies virus vaccine strain Vnukovo-32 was grown on primary hamster kidney cells or monkey kidney cell line 4647. After infection, the supernatant was clarified by ultrafiltration and virus particles were concentrated by ultracentrifugation. The pellet was resuspended in 4M guanidine thiocyanate and viral RNA was isolated by phenol extraction, chloroform extraction and ethanol precipitation. cDNA is RNA using 3'end specific primers
Created from. 5'-GGATCCAGGAAAGATGGTTCCTCAGGCTCTCCTGTTTG-3 '[SEQ ID NO: 17] This was made to overlap the translation initiation codon (ATG) using reverse transcriptase (Amersham). cDNA was amplified using PCR (GeneAmp, Perkin Elmer Cetus), and 3'- and 5'-
It is a specific primer.

The 5'-specific primer had the following sequence: 5'-GCTGCAGCAAGGGGAGGTGATCTTCAGACTTGGATCGT-3 '[SEQ ID NO: 18] The 5'-primer contained a stop codon. The PCR product was electrophoresed on a 0.1% agarose gel and the expected size band of double-stranded DNA was excised from the gel and then cut with BamHI and PstI. The cleaved product was ligated with similarly treated pUC 19 vector. Clones were obtained after transformation of E. coli DH5 bacterial cells on X-gel treated plates. Plasmid DNA containing the insert was identified by electrophoresis on agarose gel after digestion with BamHI and PstI.

Both strands of DNA were sequenced by the dideoxynucleotide termination method using the SEquence kit (USB, USA). DNA sequences were computer analyzed using the "GCG" package version 7.1. The nucleotide and deduced amino acid sequence of the glycoprotein was submitted to the EMBL data library and assigned accession number X71879.
The deduced sequence of the 524 amino acid polypeptide was identical in size and organization to the most recently characterized rabies glycoprotein. Comparing the nucleotide sequences of 8 gp G genes, Vnukovo-32 strain showed ERA (99.4%) and SAD B19 (99%).
.1%), showing the largest homology. Similar results were obtained by analysis of deduced amino acids. Functional activity of glycoprotein G gene was examined in bacterial cells (Grabko,
VI, Fragment of DNA coding glycoprotein G of Rabies virus. Russian patent N2
008355, 1994). The gene glycoprotein G was inserted into the pUC19 vector under the LacZ promoter. The recombinant plasmid DNA was transformed into E. coli DH5.
Clone PVG18-1 showed high immunogenic activity of rabies glycoprotein G.

Example 7 This example describes plasmid-DC5'-GRV-DC3 '(pKSII-DC5'-MLP-2BL described in FIG.
The occurrence of D-GRV-p (A) SV40-DC3 ') is described. Plasmid pKSII-DC5'-MLP- 2BLD-
GRV-p (A) SV40-DC3'-pSKII was prepared by directional cloning in two consecutive steps.

1. Cloning of the 3'end of the FAV1 genome First, using the oligonucleotide primer, DNA Ad CELO and polymerase chain reaction (PCR), up to 830 base pairs (bp) at the right end of the Ad CELO genome (42995 on avian adenovirus CELO genome). bp to 43804 bp (present in the DNA sequence) was amplified and isolated. The primers used were as follows. 5'-CAACCTCTAGACATCACCATAGCAATCATTGG-3 '[SEQ ID NO: 19] XbaI 42995-43016 bp Ad CELO 5'-CATTCGCGGCCGCGATGATGTATAATAACCTCAAAAACTAACG-3' NotI 43804-43774 bp Ad CELO [SEQ ID NO: 20] 830 bp The amplified sequence is shown in Table VI.

In the second step, the amplified DNA contained the essential ori. PSKII vector (Stratagen Cloning Systems, La Jolla, CA) was digested with XbaI-NotI and
The PCR fragment DC3 '(XbaI-ori-NotI) was inserted.

The vector pSKII-DC3 ′ (XbaI-ori-NotI) was cut with XbaI to give NotI site and FAV1.
An XbaI-NotI fragment containing the 3'end of the genome was isolated. The fragment containing the 3'end of this FAV1 genome (XbaI-NotI) was digested with the XbaI-NotI vector pSKII-DC5'-MLP-2bld-GRV-p (
A) Inserted into SV40. The ligated DNA was transformed into E. coli and the correct plasmid was identified by restriction with the enzymes XbaI, HindIII, KpnI, EcoRI, BamHI, NotI. All restriction conditions were as recommended by the manufacturer (New England BioLabs).

Example 8 Expression of rabies G protein in embryonated eggs Rabies glycoproteins have also been cloned into the vectors of the invention. This sequence is shown in SEQ ID NO: 24. The complete CELO (FAV1) viral genome sequence is shown in Table VIII [SEQ ID NO: 27].

The expression of rabies glycoprotein G in allantoic water of infected (R) and control (C) eggs is shown in FIG. Based on these results, the system of the present invention produced sufficient material for multiple doses of vaccine in each egg.

Example 9 This example describes transfection of avian eggs performed with recombinant adenovirus vector DNA (DC5'-GRV-DC3 ') and DNA Ad CELO as a helper.

Transfection was performed using Grabko VI (Acta Virologica, 1987, v. 31, N 1-2,
pp. 92-102). Deproteinized virion DNA Ad CEL
O preparation was diluted with sterile saline (1 to 5 μg / embryo) and vector DNA was first cut with SfiI-NotI, DC5'-GRV-DC3 '(pSKII-DC5'- MLP-2bld-RG- p (A) SV40-DC3'-
pSKII) was also diluted with the same sterile saline (5-10 μg / embryo). This mixture was introduced into the allantoic cavity of 9-day-old chicken embryos using individual glass capillaries. 72-96 embryos
Incubated at 37 ° C for hours. After cooling the embryos, allantoic water was collected and assayed for protein production.

Bradford's ability to easily and accurately measure the concentration of soluble proteins
The Bio-Rad protein assay based on the method of (Anal. Biochem. 1976, 72: 248-) was used. This is done by adding an acid dye to the protein solution and then measuring with a spectrophotometer at 595 nm. Comparison with a standard curve provides a relatively measure of protein concentration.

Protein levels are also measured by Western blot (immunoblot) using anti-RGP mouse monoclonal antibody. This antibody is Caprion Products, Inc (U
SA) and purified anti-mouse IgG (H +) with blotting grade affinity.
L) Alkaline phosphatase conjugate was provided by Bio-Ra. Sample purity is S
Stained by DS-PAGE (4-20% gel) followed by Gradipure electrophoretic gel staining (Figure 11). Table 7 summarizes the actual experimental transfections carried out to determine the effect of the above vector (alone and in combination with DNA Ad CELO) on the production of rabies glycoprotein G.

All references, including publications and patents, cited in this specification are indicated to be individually incorporated by reference, each reference in its entirety, and are hereby incorporated by reference in their entirety. Similarly, it is incorporated herein by reference. Although the present invention has been described with particular emphasis on preferred embodiments, it will be appreciated by those of ordinary skill in the art that the preferred embodiments may vary. In particular, it should be noted that any protein can be expressed in the galvanic transgenic egg system of the present invention. Similarly, it is contemplated that the present invention may be practiced other than as specifically described herein. Therefore, the present invention is intended to embrace all modifications without departing from the spirit and scope of the appended claims.

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[Brief description of drawings]

[Figure 1]   Schematic diagram showing the plasmid pSKII-DC5'-MLP-2bLD.

[Fig. 2]   Schematic diagram showing the plasmid pSKII-DC5'-MLP-2bLD-RGSV40-p (A).

FIG. 3 is a schematic diagram showing the plasmid pSKII-DC5′-MLP-2bLD-RG-p (A) -DC (XbaI-NotI).

[Figure 4]   Schematic diagram showing the steps of constructing a recombinant Ad CELO (Ad CEV) genome.

[Figure 5]   Schematic diagram of the rCRP construct.

[Figure 6]   The 660 base pair CRP PCR product used to construct the CRP clone (Figure 5).

FIG. 7 shows expression of human CRP in eggs. 7 μL allantoic fluid from eggs infected with CRP constructs
Was loaded on a 4-20% gradient SDS-PAGE gel. One half of the gel was stained with Coomassie blue dye and the other half was transferred to nitrocellulose and reacted with anti-CRP antiserum. The contents of the lane are as follows. 1. rCRP clone; 2. CR isolated from human body fluids
P control protein; 3. N / A; 4. FAV1 infected allantoic water; 5. Non-infected allantoic water; molecular weight marker before staining is indicated by MW.

FIG. 8 shows the expression of recombinant CRP in allantoic water of recombinant adenovirus (Ad CEV) infected chick embryos. Allantoic Water and Control Samples with O-Phosphorylethanolamine Sepharos
Incubated according to manufacturer's protocol with e (Sigma, USA). Samples were boiled and eluted with PAGE loading buffer containing 1% SDS and loaded on the gel. 1.
FAV1; 2. Non-infected allantoic water; 3. Recombinant CRP virus infection; 4. Recombinant CRP infection; 5
CRP purified from human body fluid; 6. Non-infected allantoic water; 7. Molecular weight 8. CRP purified from human body fluid.

FIG. 9 is a schematic diagram showing the preparation of plasmid pSKII-DC5′-MLP-2BLD-GRV-pA-DC3 ′.

FIG. 10 shows the expression of rabies glycoprotein G in allantoic fluid of infected (R) and control (C) eggs. In controls, wild-type virus was used to infect eggs and one of the components of adenovirus was found (A). A 68 kd protein corresponding to the expected size of the rabies construct was found in the rabies clone. MW is a molecular weight marker.

Figure 11. Expression of rabies glycoprotein G in allantoic water of chick embryos transfected with recombinant adenovirus vector pKSII-DC5'-MLP-2BLD-GRV-p (A) -DC3 'and helper CELO virus DNA. Indicates. 1. MW standard; 2. pKSII-DC5'- MLP-2BLD-G
Allantoic water from embryos transfected with RV-p (A) -DC3 'and helper CELO virus DNA (protein sample boiled before loading); 3. pKSII-DC5'-MLP-2BLD
-GRV-p (A) -DC3 'and allantoic fluid from embryos transfected with helper CELO virus DNA (protein sample boiled before loading); 4. Urine from embryos transfected with CELO virus DNA Membrane water; 5. Allantoic water from uninfected embryos.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Breiden, Elmuno, Earl             United States Maine, Portland,             Bolton Street 40 F term (reference) 4B024 AA01 AA10 AA20 BA32 BA61                       BA80 CA02 CA12 DA02 EA02                       EA03 EA06 FA01 GA11 HA17                 4B065 AA90X AA95X AA95Y AB01                       AC14 CA24 CA43 CA44 CA45                 4C085 AA03 BA77 CC04 DD61

Claims (43)

[Claims] 1. An egg containing a recombinant protein. 2. The egg according to claim 1, wherein the egg is a poultry egg. 3. The egg according to claim 1, wherein the egg is any egg of avian species. 4. An egg containing recombinant DNA. 5. The egg according to claim 4, wherein the egg is a poultry egg. 6. The egg according to claim 4, which is any egg of avian species. 7. A method of preparing a recombinant avian adenovirus vector containing a heterologous gene, comprising the steps of: (a) (i) at least one transcriptional regulatory sequence flanking at least one restriction endonuclease site; and (Ii) Ad CEL adjacent to the transcription regulatory sequence and the restriction endonuclease site
Preparing a plasmid, cosmid or phage containing avian adenovirus DNA containing DNA from a nonessential region in the O genome; then (b) flanking at least one protein coding sequence from a heterologous gene with the transcriptional regulatory sequence Inserting the restriction endonuclease site. 8. The method of claim 7, wherein the Ad CELO DNA further comprises DNA preceding and including the site where RNA synthesis begins. 9. The method of claim 7, wherein the transcriptional regulatory sequence regulates all viral mRNAs processed from the major late transcript. 10. The avian adenovirus DNA is Ad CELO DNA.
Method described in 7 ”. 11. The method according to claim 7, wherein the protein coding sequence from a heterologous gene comprises DNA extending beyond the translation initiation site of the heterologous gene and the translation stop site of the heterologous gene. 12. The method according to claim 11, wherein the protein coding sequence is obtained from a DNA copy of a DNA gene or a DNA copy of an RNA gene. 13. The non-essential region is Ad CEL from 1% to 8% map of Ad CELO.
The method of claim 7 including an O region. 14. The formed plasmid is KpnI-DC5′-HindIII, HindIII-ML.
P- EcoRI, EcoRI-2bld-BamHI, BamHI-GRV-PstI, PstI-p (A) SV40-XbaI, XbaI-DC-
The method according to claim 7, which is one of the group consisting of NotI. 15. A method of genetically modifying an avian adenovirus, comprising the steps of: (a) (i) at least one flanking at least one restriction endonuclease site.
And (ii) a plasmid, cosmid or phage containing avian adenovirus DNA containing DNA from a non-essential region of the avian adenovirus flanking the transcription control sequence and the restriction endonuclease site. And (b) at least one protein coding region from a heterologous gene is inserted into the restriction endonuclease site adjacent to the transcriptional and translational regulatory sequences, and (c) at least one cell infected with a recombinant avian adenovirus. Provide (d
) A method comprising the step of isolating a recombinant avian adenovirus capable of expressing the protein coding sequence from the cell. 16. A method of using a recombinant avian adenovirus as a vaccine, comprising the steps of: (a) a chimera comprising at least one avian adenovirus transcriptional and translational regulatory sequence and at least one protein coding sequence from a heterologous gene. Preparing an infectious avian adenovirus containing a gene, wherein said chimeric gene is flanked by DNA from a nonessential region of the avian adenovirus genome, said infectious avian adenovirus is said protein coding sequence upon infecting a cell. And (b) inoculating the animal or human with an inoculum containing the recombinant protein at a concentration sufficient to induce an immune response in the animal or human. 17. The method of claim 16, wherein the immune response comprises the production of antibodies against at least an antigenic portion encoded by the protein coding sequence. 18. The above chimera of (a) a plasmid, cosmid or phage; (b) a chimeric gene containing at least one avian adenovirus transcriptional and translational regulatory sequence from a heterologous gene, and (c) an avian adenovirus genome. A vector containing DNA from a nonessential region of DNA adjacent to a gene. 19. The vector according to claim 18, wherein the protein coding sequence is a sequence from a heterologous gene selected from the group consisting of rabies virus. 20. The vector according to claim 18, wherein the protein coding sequence encodes an immunogenic protein. 21. The vector of claim 20, wherein the protein coding sequence encodes at least an antigenic portion of the immunogenic protein. 22. The vector is KpnI-DC5'-HindIII, HindIII-MLP-EcoRI,
EcoRI-2bld-BamHI, BamHI-GRV-PstI, PstI-p (A) SV40-XbaI, DC5'-MLP-2bld- G
The vector according to claim 21, which is selected from the group consisting of RV-p (A) -DC (XbaI-NotI). 23. A chimeric gene comprising therein at least one avian adenovirus transcriptional and translational regulatory sequence and at least one protein coding sequence from a heterologous gene, wherein said chimeric gene is a non-essential avian adenovirus. An infectious adenovirus, flanked by DNA from a region, capable of expressing the protein coding sequence when the infectious avian adenovirus infects a cell. 24. The chimeric gene is further preceded by an initiation site of RNA synthesis,
The infectious avian adenovirus according to claim 23, which comprises avian adenovirus DNA and DNA containing the same. 25. The infectious avian adenovirus according to claim 24, wherein the protein coding sequence encodes an immunogenic protein. 26. The infectious avian adenovirus according to claim 25, wherein the protein coding sequence encodes at least an antigenic portion of the immunogenic protein. 27. The infectious avian adenovirus according to claim 26, wherein the immunogenic protein is rabies glycoprotein G antigen. 28. The infectious avian adenovirus is KpnI-DC5'-HindIII, Hi.
ndIII-MLP-EcoRI, EcoRI-2bld-BamHI, BamHI-GRV-PstI, PstI-p (A) SV40- XbaI,
The claim selected from the group consisting of DC5'-MLP-2bld-GRV-p (A) -DC (XbaI-NotI)
27 ”, the infectious avian adenovirus. 29. The following steps: (a) (i) at least one flanking at least one restriction endonuclease site.
A transcriptional and translational regulatory sequence; and (ii) a plasmid containing avian adenovirus DNA consisting of DNA from a non-essential region of the avian adenovirus genome flanking the transcriptional regulatory sequence and the restriction site,
Prepared by a method comprising a step of preparing a vector comprising a cosmid or a phage; (b) inserting at least one protein coding sequence from a heterologous gene into the restriction endonuclease site adjacent to the translation and transcription regulatory sequences. Infectious avian adenovirus recombinant. 30. An avian adenovirus recombinant vector obtainable by in vitro and in vivo manipulation of avian adenovirus DNA. 31. (a) both left and right inverted terminal repeat sequences and at least one packaging signal sequence; (b) at least one transcription regulator sequence and at least one translation stimulator sequence; (c) at least One protein coding sequence; (d) insertions and / or deletions and / or
Or the avian adenovirus recombinant vector of claim 30, characterized by containing at least one other region of the avian adenovirus DNA containing as much of a modification as a mutation. 32. The formed plasmid is KpnI-DC5'-HindIII, HindIII-.
MLP-EcoRI, EcoRI-2bld-BamHI, BamHI-GRV-PstI, PstI-p (A) SV40-XbaI, XbaI-DC
The avian adenovirus recombinant vector according to claim 30, which is one of the group consisting of 3'-NotI. 33. In a bacterium or yeast capable of producing recombinant protein after introduction into avian eggs or avian cells with avian adenovirus particles as helpers capable of producing avian adenovirus DNA or recombinant protein as much as possible. 33. The avian adenovirus recombinant vector according to claim 30, 31, or 32, which is present in a plasmid capable of replicating. 34. The avian adenovirus recombinant vector according to claim 30, 31, 32, 33, which contains a heterologous DNA. 35. The avian adenovirus recombinant vector according to claim 34, which comprises a heterologous DNA encoding a therapeutic protein. 36. The avian adenovirus recombinant vector according to claim 34, which comprises a heterologous DNA encoding an immunostimulatory protein. 37. The avian adenovirus recombinant vector according to claim 34, which contains a heterologous DNA encoding a protein derived from a human virus or a bacterial or protozoan pathogen. 38. The avian adenovirus recombinant vector according to claim 34, which contains a heterologous DNA encoding a protein derived from an animal virus or a bacterial or protozoan pathogen. 39. The avian adenovirus recombinant vector according to claim 34, which contains heterologous DNA encoding a protein derived from an avian virus or a bacterial or protozoan pathogen. 40. The avian adenovirus recombinant vector according to claim 34, which comprises a heterologous DNA encoding a cytokine protein. 41. A method according to claim 3, which contains a heterologous DNA encoding a plant protein.
The avian adenovirus recombinant vector described in 4). 42. The avian adenovirus recombinant vector according to claim 34, which contains a heterologous DNA encoding a tumor antigen protein or a fragment thereof. 43. A method for producing a recombinant protein in an avian egg according to any one of claims 30 to 40, comprising the steps of: (a) (i) left and right inverted terminal repeats. And (ii) at least one transcriptional regulator sequence and translational stimulator sequence; and (iii) at least one heterologous gene at the restriction endonuclease site flanking the transcriptional and translational regulatory sequences. A protein-encoding sequence comprising two protein coding sequences; a vector comprising a plasmid, cosmid or phage containing avian adenovirus; (b) preparing a mixture of the vector DNA and purified adenovirus or whole adenovirus particles; ) Introducing the mixture into an embryogenic avian egg or avian cell culture, (d) Incubating the avian egg treated in step 1 for a period of time, (e) after this period, harvesting liquid from the egg containing the specific recombinant protein molecule encoded by the vector DNA .