RU2326942C1 - Method of preparation of recombinant birds virus for vaccination and genetic therapy - Google Patents

Abstract

FIELD: technological processes; veterinary medicine.

SUBSTANCE: on the basis of genomic DNA of birds adenovirus CELO two plasmids are designed, one of which contains its left fragment (1-20028 bps), which includes expressing cassette under control of own main late promoter of adenovirus MLP, and the other contains the right fragment of genome CELO (17,390-43,804 bps) with zone that is homologous to nucleotide sequence of the first plasmid for performance of recombination, and also with deletion in 3,787 bps for increase of packing capacity of adenovirus CELO. Expressing cassette is cloned in the area of deletion, which consists of exogenic promoter that provides high level of expression in eukaryotic cells, polylinkers for cloning of one or more target genes and signal of polyadenylation. Homologous recombination between designed plasmids is performed in the culture of cells of leghorn rooster hepatoma (LMH), which provides preparation of recombinant birds adenovirus CELO. For cloning in expressing cassettes genes of cytokines, growth factors, antigens and oncosuppressors of human being, birds and microorganisms are used, both taken separately and in certain combinations for achievement of required genetic therapeutic effect or high level of immunisation.

EFFECT: creation of genetically engineered recombinant vaccines of new generation and preparations for genetic therapy.

32 cl, 4 dwg, 22 ex

Description

The invention relates to molecular biology, biotechnology, genetic engineering of viruses and relates to an original method of creating a recombinant CELO bird adenovirus containing a target gene or genes under the control of an exogenous promoter, at least one target gene under the control of its own promoter of the CELO virus, the expression of which in birds , mammals of animals and humans, immunity to a disease or diseases occurs, or the necessary gene-therapeutic effect is achieved, which can be used To create a genetically engineered recombinant new generation of vaccines and drugs for gene therapy.

To create immunity to any disease, vaccines are introduced into the body - preparations from microorganisms or viruses to stimulate the production of the corresponding own antibodies in the body. A more modern method of vaccination is the use of infectious agents created by genetic engineering methods with the necessary set of antigens. New types of vaccines for birds, mammals, and humans must be effective in their intended purpose, safe for the body and cheap.

Gene therapy is a modern direction in the treatment of many hereditary and acquired diseases by introducing a DNA fragment into the cells of a sick person in order to replace the functions of the mutant gene and treat hereditary diseases. For this purpose, as in the case of the creation of a new generation of vaccines, the genomes of viruses are used in which the necessary target genes are inserted. Based on the property of viruses to deliver genetic material to body cells, they are used as vectors. Thus, retroviruses, lentiviruses, adeno-associated viruses, herpes viruses, poxviruses and adenoviruses are currently successfully used.

Adenoviruses (adenovirus) make up a group of DNA viruses and cause acute respiratory diseases, pneumonia, epidemiological keratoconjunctivitis, and infectious diseases of the gastrointestinal tract and bladder. Adenoviral vectors are a widely used tool for delivering genes to mammalian cells. Compared with other vector systems, recombinant adenoviruses are highly efficient in expressing the target transgene in various types of cells, vector safety for humans and animals, the accumulation of recombinant viruses in producer cells in high titer, and the induction of both humoral and cellular immune responses to transgenic products in human and animal body, large packing capacity of the vector, etc. Currently known methods for producing recombinant vaccines based on human and animal adenoviruses (Hum Gene Ther. 2005, V.16, p. 157-168; Proc. Natl. Acad. Sci. USA 2004, V.101, p.14567- 14571).

It is known that the most commonly used in practice is human adenovirus of the 5th serotype. Thus, a recombinant human adenovirus of the 5th serotype (Ad5) was obtained containing the nucleotide sequence of the human influenza virus hemagglutinin for further use as a vaccine for humans and animals against a pathogenic strain of influenza virus (Vaccine. 2005, V 23, p. 1029-1036 ) The H5N1 avian influenza virus hemagglutinin gene was cloned into human adenovirus of the 5th serotype also to use the resulting recombinant virus as a vaccine for humans, mammals and birds (Vaccine 2006, J. of Virology, 2006, V. 80, No. 4, p. 1959-1964). Also, this adenovirus was used for gene therapy of tumors in the case when the p53 tumor suppressor gene was cloned into it (US 7033750). All of the above genes were cloned under the control of the CMV cytomegalovirus promoter.

However, adenoviruses have their own promoter - the major late promoter (MLP), which is more effective compared to exogenous promoters, for example, the aforementioned CMV. In this case, the MLP promoter is able to function only in the permissive (suitable for virus reproduction) system and only when it is part of a replicatively competent adenovirus. This is due to the fact that the transcription of genes under his control begins after undergoing the process of replication of the viral genome. Recently, vectors based on replicatively competent adenoviruses are finding more and more use, in connection with which the use of the main late promoter becomes relevant. It is known that in the genome of the human adenovirus of the 5th serotype, the dog papillomavirus capsid antigen gene was integrated under the control of the main late MLP promoter (PNAS 2005, V.102, No. 12, p. 4590-4595). For gene cloning, a site located in the late region of the L5 genome was used after the sequence encoding the fiber gene.

Thus, it is known to use recombinant adenoviruses with only one expression cassette carrying one or two target genes, the reading frames of which are small (up to 1000 nucleotides for each of the two genes), although the adenovirus system can potentially contain at least two cassettes, which can contain one or more target genes, the protein products of which can belong to different functional groups: cytokines, oncosuppressors, growth factors, antigens of pathogenic organisms.

Along with human adenoviruses, CELO chicken embryo lethal orphan (adenovirus of birds 1 serotype FAV-1) is used to create vector systems. The CELO virus is an infectious agent, but the infection it causes does not have significant economic consequences or a threat to the health of the bird. This virus can be isolated from healthy chickens and does not cause disease as a result of experimental infection of chickens. Recombinant CELO adenoviruses expressing the genes of protective antigens of various pathogens have a pronounced tropism in relation to the epithelial tissues of birds. This allows efficient and prolonged expression of the target gene in the body of immunized birds, which in turn leads to the formation of a powerful specific humoral and cytotoxic immune response against the pathogen. An additional advantage of CELO recombinant avian adenoviruses is the low cost of producing preparative amounts of the virus in the chicken embryo allantois, a safe and high-tech system traditionally used for vaccine production. In this regard, the recombinant CELO virus was used to clone the bird infectious bursitis virus (IBDV) VP2 gene (Vaccine 2004, V.22, p. 2351-2360) and the rabies virus glycoprotein G gene (Microbiology, 2006, v. 4, p. .69-71). The obtained recombinant CELO adenoviruses were used for vaccination of mammalian animals and birds, as a result of which their protection against pathogenic virus strains was obtained.

It is known that several methods are used to produce recombinant adenoviruses. The first involves targeted ligation of the target gene with fragments of the adenovirus genome (T. Maniatis et al. Molecular Cloning. M: Mir, 1984). Since the adenovirus genome has a large size of the order of 35000-45000 n.p. and contains a small number of unique restriction sites, this method is technically complicated and ineffective, especially in the case of the introduction of several target genes.

The second known method is based on the use of lambda phage, which also limits the possibilities for obtaining recombinant adenoviruses, since this technology requires expensive materials and the process for producing recombinant virus is multi-stage (J. Virology, 2001, V.75, N 11, p. 5288- 5301).

A third method is also known, based on the cloning of the target gene in a “shuttle” vector, which contains regions homologous to terminal fragments of the adenovirus genome, followed by homologous recombination, as a result of which the target gene is transferred to the adenovirus genomic DNA.

It is known that to obtain recombinant CELO virus using homologous recombination, use the full genomic DNA of the CELO virus or a plasmid containing the full genomic DNA of the CELO virus, as well as a plasmid construct that simultaneously contains the right and left portions of the CELO genome and one expression cassette from the target gene under control eukaryotic promoter and polyadenylation signal. The expression cassette is contained either in the left region of the CELO genome, or in its right region. Two types of deletions are carried out in the right part of the CELO genome from the plasmid construct, either in the region of 40065-43684 n.a. or in the region 41731-43684 n.a. due to the limitation of the packing capacity of the viral particle (US, 6841158, 2005). Moreover, in the region of deletions 41731-43684 n.a. clone an expression cassette with one target gene due to the limitation of the packaging capacity of the viral particle, allowing in this case to integrate a gene whose size does not exceed 2153 bp

Due to the homologous regions of the plasmid construct with the expression cassette and the full genomic DNA of the CELO virus, E. coli cells carry out homologous recombination, the result of which is to obtain a plasmid with the full CELO genome and the expression cassette turned on. The obtained plasmid is introduced by transfection into the CELO-permissive hepatoma LMH rooster hepatoma cell line (leggom male hepatoma), where the recombinant adenovirus is assembled and propagated (J. Virology 1999, V.73, No. 2, p.1399-1410).

This method has limitations in use, since due to the large size of the CELO genome (about 44000 bp), the target inserts must be of a limited size, that is, it is possible to clone only one target gene. The production of recombinant adenovirus through recombination steps in E. coli propagation cells in LMH cells leads to an increase in the cost of obtaining a vaccine or gene therapeutic drug. Also, the main late promoter of the adenovirus MLP, which is effective for the expression of target genes in avian cells, is not involved.

The objective of the invention is to create a new method for producing recombinant adenovirus CELO birds using two plasmid constructs created on the basis of the CELO bird adenovirus genome and containing its sections, in order to increase the capacity of recombinant adenovirus for cloning target genes under the control of an exogenous promoter and create an additional expression cassette under control own effective adenovirus promoter for cloning target genes, as well as for combining them with genes under control m exogenous promoter whose expression leads to immunize avian organism, mammals and man, as well as carrying out gene therapy.

The problem is solved in that in the method of creating recombinant avian adenovirus for vaccination and gene therapy, the genomic DNA of the adenovirus CELO is taken, which includes its own main late promoter, MLP adenovirus, and based on its left fragment (1-20028 bp) a plasmid containing an inserted splicing site and at least one target gene under the control of the MLP promoter. Also, based on the right fragment of the CELO genome (17390-43804), a second plasmid is constructed in which there is a region homologous to the zone of the first construct for recombination, while a 3787 bp deletion is made in the nucleotide sequence of the plasmid. to increase the packing capacity of the CELO adenovirus, in the area of which at least one expression cassette is then cloned, consisting of an exogenous promoter providing a high level of expression in eukaryotic cells, from at least one target gene and a polyadenylation signal. Homologous recombination is carried out in a Leghorn rooster hepatoma cell culture (LMH), providing a recombinant adenovirus consisting of CELO avian adenovirus genomic DNA with an expression cassette including a splicing site, at least one target gene under the control of the MLP adenovirus promoter and avian polyadenylation signal from the genome CELO, as well as with another expression cassette consisting of at least one target gene under the control of an exogenous promoter for expression in eukaryotic cells and a poly signal adenylation. In LMH cell culture, the recombinant CELO recombinant avian adenovirus is assembled and propagated for subsequent use as a vaccine or gene therapeutic agent. The following genes are used as target genes in expression cassettes: cytokines (bird γ-interferon, human α16-interferon, human α-interferon α-2a, human β-interferon, human γ-interferon, granulocyte-macrophage colony stimulating factor GMCSF human, interleukin - 2 people), growth factors (insulin-like growth factor IGF1 human, vascular endothelial growth factor human VEGF, neurotrophic factor glial cell GDNF human), cancer suppressors (thymidine kinase HSV1 human herpes virus type I person, p53 h Lovek) and antigens (protective antigens of mycobacteria M. tuberculosis Ag85A, Ag85B, ESAT6, hexon antigens of avian EDS-76 and appendix Penton (The Fiber) avian virus EDS-76). The bird genes are cloned under the control of the adenovirus promoter MLP, the genes of microorganisms and humans under the exogenous promoter. Cloning is carried out either by single genes or several genes in a specific combination: the bird cytokine gene under the control of the MLP promoter or the exogenous promoter, the bird cytokine gene under the control of the MLP promoter and the bird cytokine gene under the control of the exogenous promoter, the human cytokine gene under the control of the exogenous promoter, the factor gene human growth under the control of the exogenous promoter, the human oncosuppressor gene under the control of the exogenous promoter, the gene for mycobacterial protective antigen under the control of the exogenous promoter, two and the gene for protective antigens of mycobacteria under the control of the exogenous promoter, the bird antigen gene under the control of the MLP promoter or exogenous promoter, the bird antigen gene under the control of the MLP promoter and the bird antigen gene under the control of the exogenous promoter, the cytokine gene under the control of the MLP promoter in combination with the antigen gene under the control exogenous promoter.

The invention is illustrated by drawings, where Fig. 1 shows a plasmid construct bearing in its composition a fragment of the CELO virus genome from 1 n.p. 20028 bp in which the expression cassette is located, consisting of the own MLP promoter (I) of CELO adenovirus, splicing site (II), polylinker with a unique SpeI restriction site for cloning target (reporter) genes (III). Figure 2 shows a plasmid construct bearing in its composition a fragment of the genome of the CELO virus from 17390 N. p. 43804 n.p. with a deletion of 3787 bp, in the region of which there is an expression cassette consisting of the human cytomegalovirus CMV (IV) promoter for efficient expression of eukaryotic genes, a polylinker with a unique SpeI restriction site for cloning target (reporter) genes (V) and a polyadenylation signal (Vi). Figure 3 shows a diagram of homologous recombination in the region of 17882-20028 n.a. (VII) in cells of the LMH line between the linearized plasmid constructs depicted in figure 1 and figure 2. Figure 4 shows the full-length genome of recombinant CELO adenovirus obtained by homologous recombination containing an expression cassette from the own promoter of MLP (I) CELO adenovirus, splicing site (II), at least one target (reporter) gene (III) and polyadenylation signal adenovirus (VIII), as well as an expression cassette from the human cytomegalovirus promoter CMV (IV), at least one target (reporter) gene (V) and polyadenylation signal (VI), for example, bovine growth hormone gene BGH p (A).

The method for creating recombinant CELO avian adenovirus for vaccination and gene therapy is based on homologous recombination between two plasmid constructs specially created on the basis of the full-sized CELO avian adenovirus genome.

The first plasmid construct, which is shown in FIG. 1, contains a left fragment of the CELO virus genome from 1 n.p. up to 20028 bp and an expression cassette consisting of the CELO adenovirus MLP (I) promoter, which provides expression levels of reporter genes in avian cells sufficient for immunization, from a specially integrated splicing site (II), from the target gene (III), which is cloned using a unique SpeI restriction site . The main late MLP promoter controls transcription on a sufficiently large portion of the viral genome, for the CELO virus, from 12193 nucleotides to 39751 nucleotides (+) of the DNA chain. To carry out the cloning of the target genes under his control, a plot was selected (17501-17502 bp) located between the polyadenylation signal of the late reading frame L2 and ATG of the codon of the pVI protein, in which the reading frames of late and early genes do not overlap. When cloning a reporter gene at a given site, it will function as another reading frame as part of the late reading frame L1, provided that there is a splicing site at its 3'-end that needs to be embedded. The polyadenylation signal for the site-built reporter gene will be the intrinsic polyA of the adenovirus (VIII), which is shown in Fig. 4.

The second plasmid construct, which is shown in FIG. 2, contains the right fragment of the CELO genome from the NotI restriction enzyme recognition site (17390 bp) to the right inverted repeat (the last nucleotide of the CELO virus genome is 43804 bp). This fragment has a delegated area of 3787 pp to increase the packaging capacity of the CELO virus, in which at least one expression cassette is cloned, consisting of an exogenous CMV (IV) promoter providing a high level of expression in eukaryotic cells, from the target gene (s) (V) and from the bovine gene polyadenylation signal growth hormone BGH p (A) (VI).

Both created plasmid constructs have genes responsible for antibiotic resistance (R), as well as a homologous region for recombination of 17390-2002 8 n.p. (Vii) of 2638 n.a. the genome of the CELO virus. Homologous recombination, which is shown in FIG. 3, is carried out directly in CELO-permissive leghorn rooster hepatoma (LMH) cell culture.

As a result, a recombinant adenovirus (shown in Fig. 4) is obtained with an increased capacity, consisting of the full-sized genomic DNA of CELO bird adenovirus, including two expression cassettes, in which target genes are cloned for expression in cells of birds, mammals, animals and humans. In LMH cell culture, recombinant CELO adenovirus is assembled and propagated.

Using this method, it is possible to obtain recombinant CELO adenoviruses: - carrying the reporter gene (s) in the deletion region at the right end of the genome in the expression cassette under the control of the exogenous CMV promoter;

- carrying at least one reporter gene under the control of the own promoter of the adenovirus MLP,

- carrying two reporter genes, one of which is in the deletion region at the right end of the genome in the expression cassette under the control of the exogenous CMV promoter, and the second under the control of its own MLP promoter.

As target (reporter) genes in the expression cassettes, the genes necessary to achieve a high immune effect and the necessary gene therapeutic effect are used. These genes belong to various functional groups: cytokines, cancer suppressors, growth factors and antigens of pathogenic organisms.

Cytokines are proteins of intercellular interactions. These include interferons, interleukins, GM-CSF and other proteins.

Interferon α and interferon β, having a common cellular receptor, are called type I interferons. Interferon α encodes about 20 similar genes, while interferon β encodes two genes. Type I interferons have immunoregulatory and antiproliferative activity (Int J Oncol., 2006, V 29, No. 6, p. 1405-1412, Nat Clin Pract Oncol. 2006, V 3, No. 11, p.633-639). Recombinant interferon is approved for use in chronic myelogenous leukemia, hairy cell leukemia, the epidemic form of Kaposi’s sarcoma. Interferon y produced by lymphocytes is classified as type II interferon. It has other immunoregulatory properties, has less antitumor activity and is used to treat chronic granulomatous disease (Hematol Oncol., 2006, V 24, No. 3, p. 151-158).

GM-CSF is a granulocyte macrophage colony stimulating factor. GM-CSF has been shown to stimulate bone marrow stem cells to form mixed colonies of granulocytes and macrophages on agar (Nimmer S. et al., 1988, Int. J. Oncol. 2006, V.28, No. 4, p. 947-953).

Interleukins are cytokines that mediate interactions between white blood cells. At least 17 interleukins have been detected. Interleukin 2, previously known as the growth factor of T-lymphocytes, is considered the main regulator of cellular immunity. It stimulates the proliferation and cytotoxic activity of T and NK lymphocytes.

Neurotrophic growth factors (neurotrophins) play an important role in the processes of development and functioning of the nervous system, and also, which is especially important for practical medicine, in the regeneration of damaged neuronal structures (Lindsay ea 1996, Lindsay ea 1996, Cuello ea 1997, Ebadi ea 1997). Insufficient expression of neurotrophic growth factors is the cause of many diseases of the nervous system. For example, degeneration of axons of nerve cells is associated with a change in the expression of factors such as insulin-like growth factor - insulin like grouth factor (IGF1IGF2), glial derived neurotrophic factor (GDNF), etc.

VEGF - (Vascular endothelial growth factor) - a vascular endothelial growth factor glycoprotein that binds only to endothelial cells and stimulates their proliferation. This factor is intensely produced by the cells of some human tumors, in particular ovarian cancer, contributing to neovascularization of the tumor and, possibly, its early dissemination. In addition to the angiogenic effect, VEGF significantly enhances vascular permeability; this property is associated with local extravasation observed in a number of tumors, such as ascites formation in ovarian cancer (Karp, J.E., and Broder, S. 1995).

In gene therapy, cancer suppressor genes are used to treat malignant tumors. In particular, targeted introduction of “suicide genes” into the tumor tissues, the production of which has a cytotoxic effect, is used. The most promising in this regard are the so-called conditionally lethal genes encoding proteins, which in themselves are not toxic to cells, but when interacting with any substance can cause their destruction. These genes include the HSV1 herpes virus thymidine kinase gene. Its introduction into the patient’s tumor cells will lead to the destruction of the patient’s tumor cells genetically modified by this gene (Cancer Gene Ther., 2005, V.12, No. 7, p.617-626).

The p53 protein is a key element in controlling the cellular response to various types of stress: its activation in response to stress leads to a halt in proliferation or apoptosis (cell death). Inactivation of p53, observed in most tumors, testifies in favor of its antitumor activity (Cancer Gene Ther., 2004, V.11, No. 1, p. 79-84.).

Another relevant group of genes for cloning in recombinant CELO adenovirus are nucleotide sequences encoding antigens. The genes Ag85A, Ag85B, ESAT6 encode the main protective antigens of the tuberculosis pathogen Mycobacterium tuberculosis, which are used to create genetically engineered vaccines based on plasmid and viral vectors, in particular, on the basis of the human adenovirus genome 5th serotype (J. Immunol., 2004, Nov, 15, V.173, No. 10, p.6357-6365). Hexon and fiber are surface antigens of the SSN-76 virus (egg-laying syndrome-76), which causes laying hen disease, which is characterized by softening, absence or depigmentation of the eggshell and is accompanied by a significant decrease in egg production. In order to prevent SSN-76 disease, ineffective inactivated vaccines are currently used. Genetic vaccines containing the genes for hexon and fiber of the SSN-76 virus have not yet been developed. Therefore, the cloning in the recombinant CELO adenovirus of the genes of the hexon and the process of the penton (fiber) of the avian virus SSN-76 can be used to effectively combat the egg production syndrome in chickens.

The main characteristic that distinguishes the new method for producing recombinant avian adenovirus for vaccination and gene therapy from the described analogue is the use of two plasmid constructs that are based on the CELO genome to produce recombinant CELO adenovirus, one of which contains an end fragment of the CELO genome with an expression cassette in the field of deletion, and another initial fragment of the CELO genome with an expression cassette, which includes its own adenovirus promoter. The resulting recombinant CELO adenovirus contains two expression cassettes, each containing one or more target genes. Also, the resulting recombinant CELO adenovirus is capable of replication and packaging at an increased size (due to several inserts of foreign DNA in its genome) directly in the LMH cell culture, bypassing the stage using E. coli cells.

Obtaining a recombinant adenovirus that simultaneously contains several identical / different genes under the control of the exogenous and native MLP promoter is relevant, because:

1) allows for the expression of several different genes in the same cell, the probability of which is greatly reduced during infection by two separate recombinant adenoviruses carrying the same genes;

2) serves to increase the level of gene expression (at least twice) by increasing the number of copies of the gene in the dose of the virus (in the case of the same gene);

3) it is economically more profitable to obtain one recombinant virus than two;

4) can provide enhanced cellular immune response, the case when the cytokine is in combination with an antigen is especially effective.

The invention is illustrated by the following examples.

Example 1. The creation of the first plasmid construct containing the left fragment of the CELO virus genome with the cloning site of the target gene under the control of the own promoter of the adenovirus MLP to claim 1.

To obtain the construct, the following oligonucleotides are used: NotI-F 5'-cctatcattgcggccgctatcg-3'and Not-R 5'-cagcatcactttttgtcccc-g-3 'containing the NotI restriction endonuclease site; HindIII-F 5'-actggacgcactaaaaggccc-3 '; HindIII-R 5'-cccaaagctttacgccgcagc-3 ', comprising the HindIII restriction endonuclease site. Polymerase chain reaction (PCR) is carried out in 25 μl of the reaction mixture containing 2 μg of CELO virus DNA as a template, 10 pcM of each HindIII-R and HindIII-F primer, 67 mM Tris-HCl (pH 8.8 at 25 ° C) , 15 mM ammonium sulfate, 2.5 mM magnesium chloride, 0.01% Tween-20, a mixture of deoxynucleotide triphosphates (dATP, dCTP, dTTP and dGTP, 2.5 mM each) and 1 unit. Taq DNA polymerase under liquid paraffin under the following conditions: 94 ° C for 5 minutes; 5 cycles: 58 ° С - 2 min, 72 ° С - 20 s and 94 ° С - 30 s; 20 cycles: 58 ° C - 30 s, 72 ° C - 20 s and 94 ° C - 30 s; then 58 ° C for 5 minutes and 72 ° C for 10 minutes. The amplification product is treated with chloroform, reprecipitated with ethanol and dissolved in bidistilled water. Then, the resulting construct, consisting of a portion of the CELO genome 17502-17882 bp, and also containing a single SpeI site, is cloned into the pGEM-T Easy plasmid vector (Promega No. A1360). Cloning is carried out according to the protocol attached to the pGEM-T Easy PCR product cloning kit.

As a result of the following PCR on the matrix of the CELO genome with primers NotI-F and Not-R under conditions of 94 ° C - 5 min; 5 cycles: 58 ° С - 2 min, 72 ° С - 20 s and 94 ° С - 30 s; 20 cycles: 58 ° C - 30 s, 72 ° C - 20 s and 94 ° C - 30 s; then 58 ° C for 5 min and 72 ° C for 10 min, a construct consisting of a portion of the CELO genome 17390-17501 bp is obtained. The resulting intermediate structure for further preparation of the first structure is cloned into the commercial vector pGEM-T Easy ("Promega" No. A 1360) according to the attached protocol. Thus, pGEM-Hind and pGEM-Not plasmids are obtained.

To incorporate the obtained constructs into the CELO genome, it is necessary to create a plasmid vector where the above constructs would be flanked by DNA homologous to the viral regions, which would ensure the further production of the complete CELO recombinant adenovirus genome. For this purpose, a section of adenovirus DNA with 12657 bp (NdeI restriction enzyme site) at 19960 bp (BamHI restriction enzyme site) are cloned into the pShuttle-CMV plasmid (Invitrogen, USA), treated with restriction endonucleases NdeI and DraIII (Fermentas, Latvia) (T. Maniatis et al. Methods of genetic engineering. Molecular cloning. M .: World, 1984). In the resulting plasmid pCELO12-19, the first part of the construct from the plasmid pGEM-Hind is cloned at the NotI and HindIII sites, and then the second part of the construct from pGEM-Not at the two sites for NotI, and thus the plasmid pCELOHindNot is obtained.

To obtain the full-sized genome of the recombinant adenovirus, it is necessary to increase the left and right homologous shoulders to at least 2000 nucleotides. However, the left shoulder must be completely extended to 1 nucleotide of the CELO genome in order to avoid obtaining a product without foreign insertion during homologous recombination. To this end, an increase in the right homology region is carried out by cloning the sequence of the CELO genome from 19146 to 22653 (SphI restriction enzyme sites) into the pCELO-HindNot plasmid processed by SphI (Fermentas, Latvia). The result is the plasmid construct pCELOHNS.

Next, a fragment of the pCELOHNS construct containing the construct as part of the CELO 12657-22653 bp genome, after treatment with restriction enzymes Ndel and Bstll07I, was cloned in the plasmid vector pCDNA 3.1.Zeo + (Invitrogen, USA), processed with restriction enzymes NdeI and EcoRV (Fermentas ”, Latvia). The result is a design p31N.

To create a plasmid construct containing a unique AscI site flanking the first nucleotide of the CELO genome, oligonucleotides are synthesized, the first of which 5'-attggcgcgccgatgatgtat-3 'encodes at the 5'end of the AscI recognition site flanking the first CELO nucleotide. The reverse primer 5'-ctacagcacccatacctgtg-3 'is complementary to the CELO 1260-1280 bp genome sequence As a result, PCR on a CELO DNA matrix under conditions of 94 ° C for 5 min; 5 cycles: 58 ° С - 2 min, 72 ° С - 20 s and 94 ° С - 30 s; 20 cycles: 58 ° C - 30 s, 72 ° C - 20 s and 94 ° C - 30 s; then 58 ° C for 5 min and 72 ° C for 10 min, followed by cloning of the product into the pGEM-T Easy vector to construct pGEM1280, which contains a unique AscI site, flanking the CELO genome from 1 to 1280 bp

Next, in the plasmid pGEM1280, treated with the restriction enzyme MunI (736 bp of the CELO genome), a fragment of the CELO genome of MunI (736 bp) - MunI (14631 bp) was cloned. The result is a construct pGEM014 containing a unique AscI site flanking the CELO genome from 1 to 14631 bp Then, a fragment of the obtained plasmid obtained by its hydrolysis at sites for NotI and XhoI is subcloned into the commercial vector pBlueScript KS (II) + (Invitrogen, USA) processed at sites for the same restriction enzymes. Thus, a plasmid construct pBS014 is obtained containing a unique AscI site flanking the CELO genome from 1 to 14631 bp

At the last stage, the p31N construct fragment containing the construct as part of the CELO 12657-20028 bp genome site obtained after processing of this plasmid at two XhoI restriction enzyme sites was cloned into the pBS014 construct processed at the sites of the same restriction enzyme obtained at the previous stage . The result is a pCELO20HN construct containing a construct comprising a CELO genome region of 1-20028 bp, where the first nucleotide of the CELO genome is flanked by a unique site for recognition by AscI restriction enzyme, but without the insertion of a reporter gene at the unique SpeI site (shown in figure 1) .

Example 2. The creation of a second plasmid construct containing the right fragment of the CELO genome from the recognition site of the NotI restriction enzyme (17390 bp) to the right inverted repeat (the last nucleotide of the adenovirus genome - 43804 bp), in which the expression cassette was cloned at the site of deletion to claim 1 of the claims.

Fragment of the CELO adenovirus genome from 36278 bp up to 40065 bp obtained by hydrolysis of CELO virus DNA at ClaI and KpnI restriction enzyme sites (No. ER0141, ER0521 Fermentas, Latvia). Cloning of the obtained fragment is carried out in the plasmid vector pBluescript II KS (+) (Invitrogen, USA) at the sites for restriction enzymes ClaI and KpnI (No. ER0141, ER0521 Fermentas, Latvia) (T. Maniatis et al. Methods of genetic engineering. Molecular Cloning), Moscow: Mir, 1984). The presence of the insert is confirmed by restriction analysis using restriction enzymes XhoI and XbaI. From the obtained plasmid vector pBS-CELO-R, the indicated fragment of the adenovirus genome is subcloned into plasmid pCBEdlRV (Mol. Genetics, Microbiology and Virology, 2002, No. 2, pp. 30-35) carrying the fragment of the adenovirus CELO 43685-43804 n.p. on sites for restriction enzymes KpnI and Eco321. Restriction analysis is carried out on the sites XhoI, XbaI and Eco321. An expression cassette from pcDNA 3.1.Zeo + plasmid (Invitrogen, United States) hydrolyzed from the BglII and PvuII sites (No. ER0081, ER0631 “Fermentas”, Latvia) and processed Klenov fragment (Klenow fragment No. EP0051 “Fermentas”, Latvia). Restriction analysis of the obtained plasmid construct is carried out at EcoRV and XbaI sites. The new plasmid construct pCBEdl3kb contains an expression cassette with the E1 region promoter of the CMV human cytomegalovirus, a polylinker for cloning target genes (with unique SpeI restriction enzyme) and a signal for the polyadenylation of the bovine growth hormone gene BGH p (A), as well as parts of the CELO virus genome 36 HL-p24 (CEL 36). P. and 43685-43804 n.p., with an increased insertion volume of foreign DNA up to 3787 n.p.

Cells of the LMH line are transfected with this plasmid together with CELO virus DNA hydrolyzed by Swa I. As a result of homologous recombination, recombinant bird adenovirus CELO-d13 with a deletion of a 3787 bp genome fragment is obtained. (Thus, the capacity of the vector is increased to 5100 n.p.).

Plasmid pCELO, containing the full-length bird adenovirus CELO genome, flanked by Pacl restriction endonuclease sites, is hydrolyzed at the NotI site and partially hydrolyzed at the Pacl site, then ligation is performed. The result is the plasmid pCELO-Not-43804 containing the genome of the CELO virus from 17390 N. p. (site for NotI) up to 43804 n.p. By homologous recombination in E. coli between the plasmid pCELO-Not-43804 linearized at the site for ApaLI and the genomic DNA of the virus CELO-dl3 hydrolyzed at the site for Ndd, the plasmid construct pCELOdl3right was obtained. This design is depicted in figure 2, contains a fragment of the genome of the CELO virus from the NotI site to 43804 bp, with the right inverted repeat flanked by a single restriction enzyme Pacl. Also, this plasmid contains an expression cassette in the region of deletion of the CELO genome with unique Spel restriction enzyme. (shown in figure 2).

Example 3. Obtaining recombinant bird adenovirus CELO containing two expression cassettes - with the promoter of the adenovirus MLP and with the exogenous promoter to claim 1.

The plasmid construct pCELO20HN of Example 1, containing a CELO virus genome portion from 1 to 20,028 bp and a single site for AscI restriction endonuclease, is hydrolyzed with AscI restriction enzyme, and the plasmid construct pCELOdl3right of Example 2 is hydrolyzed with Pacl restriction enzyme ( , Latvia). Both constructs are then co-transfected into the hepatoma cells of Leghorn LMH rooster by calcium phosphate precipitation (J. Gen. Virol., 1977). As a result of homologous recombination, which is shown in Fig. 3, between the fragment of the viral genome CELO 17882-20028 bp from the first construct and a similar fragment from another construct in LMH cells, a complete recombinant CELO adenovirus is obtained (Fig. 4), the recombination of which is confirmed using the PCR method.

Example 4. Construction of recombinant adenoviruses CELON-chIFNγ / dl3 and CELONdl3-chIFN carrying the gene of γ-interferon of birds, and the study of antiviral activity in vitro to claims 2-3.

The γ-interferon gene of birds is obtained by PCR on the DNA matrix of the chicken genome. For cDNA synthesis, a pair of primers IFN-F 5'-catgaaggacagcactaagg-3 'and IFN-R 5'-aatagttagtcttctattag-3' selected at the ends of the gene according to the GENE-Bank sequence No. AN009942 (Gallus gallus interferon gamma, complete cds) are used. As a result, PCR under conditions of 94 ° C for 5 min; 5 cycles: 58 ° С - 2 min, 72 ° С - 20 s and 94 ° С - 30 s; 20 cycles: 58 ° C - 30 s, 72 ° C - 20 s and 94 ° C - 30 s; then 58 ° C for 5 min and 72 ° C for 10 min receive cDNA of the γ-interferon gene, which is then cloned into the pGEM-T Easy vector (Promega, Cat. No A1360). Thus, the plasmid pGEM-γIFN is obtained. The chemical synthesis of oligodeoxyribonucleotides is carried out by the solid-phase aminophosphite method using an AFM 100-2 synthesizer (Novosibirsk). Purification is carried out by electrophoresis in 12% PAAG.

At the next stage, from the obtained plasmid pGEM-γIFN treated with NotI restriction endonuclease (No. ER0592 Fermentas, Latvia) and the Klenov fragment (No. ER0052 Fermentas, Latvia), the γIFN gene was cloned in the plasmid construct pCELOdl3right according to Example 2, hydrolyzed according to Example 2 SpeI site (No. ER1251 “Fermentas”, Latvia) and processed Klenov fragment. The result is a plasmid construct pCELOdl3-chIFNγ carrying a fragment of the CELO avian virus genome, where an expression cassette containing the avian γ-interferon gene is contained in the deletion region of the genome. Further, this plasmid treated with Pacl restriction enzyme (No. K0547R "New England BioLabs", USA) and plasmid pCELO20HN treated with AscI restriction enzyme (No. R0558S "New England BioLabs", USA) co-transfect LMH cells. Thus, as a result of homologous recombination in LMH cells, recombinant avian adenovirus CELONdl3-chIFN is obtained that carries the γ-interferon gene of birds under the control of the CMV promoter.

Also from the obtained plasmid pGEM-γIFN treated with NotI restriction endonuclease (No. ER0592 Fermentas, Latvia) and the Klenov fragment (No. EP0052 Fermentas, Latvia), the γ-IFN γ gene was cloned into the plasmid construct pCELO20HN according to Example 1, hydrolyzed according to Example 1 SpeI site (No. ER1251 “Fermentas”, Latvia) and processed Klenov fragment. The result is a plasmid construct pCELO20HN-chIFNγ carrying a fragment of the CELO avian virus genome and the avian IFNγ gene under the control of the MLP promoter. Further, this plasmid treated with AscI restrictase and the pCELOdl3right plasmid treated with PacI restrictase co-transfected LMH cells. As a result of homologous recombination in cells of the LMH line, recombinant avian adenovirus CELON-chIFNγ / dl3 is obtained, carrying the γ-interferon gene of birds under the control of the MLP promoter.

The next step is the study of the antiviral activity of the preparations of the culture fluid containing alFNγ. For this, viruses CELON-chIFNγ / d13, CELONd13-chIFN and wild-type CELO (as a negative control) infect LMH line cells at a dose of 5 PFU / cell. 72 hours after infection, culture medium is taken. The study of antiviral activity of drugs is carried out according to the standard method of titration of interferon in a bird cell culture. Mouse encephalomyocarditis virus is used as an indicator test virus. The analysis revealed that the culture medium of cells infected with wild-type CELO virus does not have antiviral activity. The antiviral activity of the test samples, the culture medium of cells infected with the virus CELON-chIFNγ / dl3 and CELONdl3-chIFN, is 1 · 10 ⁴ IU / ml and 1.2 · 10 ⁴ IU / ml.

Example 5. Construction of a recombinant adenovirus carrying the genes of γ-interferon of birds simultaneously under the control of its own promoter of adenovirus and under the control of the exogenous promoter, and the study of antiviral activity to claims 4-5.

A plasmid, pCELOdl3-chIFNγ, carrying a fragment of the CELO avian virus genome, where the expression cassette containing the bird γ-interferon gene treated with PacI restriction enzyme (No. R0547S “New England BioLabs”, USA) and plasmid pCELO20HN-example NIFIF 4, carrying a fragment of the CELO avian virus genome and avian IFNγ gene under the control of the MLP promoter treated with AscI restrictase enzyme (No. R0558S "New England BioLabs", USA), cotransfects LMH cells. Thus, as a result of homologous recombination in LMH cells, the recombinant bird adenovirus CELON-chIFN / dl3-chIFN is obtained, which carries the bird γ-interferon gene under the control of the CMV promoter and under the control of the MLP promoter.

The next step is the study of the antiviral activity of the preparations of the culture fluid containing aIFNγ. For this, wild-type CELON-chIFN / d13-chIFN and CELO viruses (as a negative control) infect LMH line cells at a dose of 5 PFU / cell. 72 hours after infection, culture medium is taken. The study of antiviral activity of drugs is carried out according to the standard method of titration of interferon in a bird cell culture. Mouse encephalomyocarditis virus is used as an indicator test virus. The analysis revealed that the culture medium of cells infected with wild-type CELO virus does not have antiviral activity. The antiviral activity of the test sample, the culture medium of cells infected with the CELON-chIFN / dl3-chIFN virus, is 2.8 · 10 ⁴ IU / ml.

Example 6. Construction of a recombinant adenovirus CELONd13-IFNα16 carrying the gene of human α16-interferon under the control of an exogenous promoter to claims 6, 7 of the claims.

The human α16-interferon gene is obtained by PCR on the DNA matrix of the human genome. For the synthesis of cDNA using a pair of primers selected at the ends of the gene according to the sequence GENE-Bank No. NM002173 (Homo sapiens interferon, alpha 16 (IFNA16), mRNA). The resulting cDNA of the α16-interferon gene is cloned into the pGEM-T Easy plasmid vector (Promega, Cat. No. A1360). In the next step, from the obtained plasmid pGEM-α16IFN treated with the NotI restriction endonuclease (No. ER0592 Fermentas, Latvia) and the Klenov fragment (No. EP0052 Fermentas, Latvia), the α16IFN gene was cloned into the plasmid construct pCELOd13right according to Example 2, hydrolyzed from the site Spel (No. ER1251 “Fermentas”, Latvia) and processed Klenov fragment. The result is a plasmid construct pCELOd13-IFNα16 with a fragment of the CELO avian virus genome, where an expression cassette with the human IFNα16 gene is contained in the genome deletion region.

This plasmid treated with PacI restriction enzyme (No. R0547S "New England BioLabs", USA) and the plasmid pCELO20HN treated with AscI restriction enzyme (No. R0558S "New England BioLabs", USA) co-transfect LMH cells. Thus, as a result of homologous recombination in LMH cells, a recombinant avian adenovirus CELONdB-IFNα16 is obtained that carries the human α16-interferon gene under the control of the exogenous CMV promoter.

Example 7. Construction of a recombinant adenovirus CELONd13-IFNα-2α carrying the human α-interferon α-2a gene under the control of an exogenous promoter to claims 6, 8.

The α-interferon α-2a gene is cloned from the plasmid pORF-hIFN-α (Invivogen, Cat. Code porf-hifna), hydrolyzed at the sites for restriction endonucleases NcoI (No. ER0571 “Fermentas”, Latvia) and NheI (No. ER0971 “ Fermentas ”, Latvia), followed by treatment with a Klenov fragment (No. EP0052“ Fermentas ”, Latvia), in the plasmid construct pCELOdl3right according to Example 2, hydrolyzed at SpeI site (No. ER1251“ Fermentas ”, Latvia) and processed with a Klenov fragment. The result is a plasmid construct pCELOdl3-IFNα-2a with a fragment of the CELO avian virus genome, where an expression cassette containing the IFNα-2a gene is contained in the genome deletion region.

This plasmid treated with PacI restriction enzyme (No. R0547S "New England BioLabs", USA) and the plasmid pCELO20HN of Example 1 treated with AscI restriction enzyme (No. R0558S "New England BioLabs", USA) cotransfect LMH cells. As a result of homologous recombination in LMH cells, recombinant avian adenovirus CELONdl3-IFNα-2a is obtained, which carries the human α-interferon α-2a gene under the control of an exogenous promoter.

Example 8. Construction of the recombinant adenovirus CELONd13-IFNβ carrying the human β-interferon gene under the control of an exogenous promoter, to claims 6, 9 of the claims.

The β-interferon gene is cloned from the plasmid pORF-hINF-β ("Invivogen", Cat. Code porf-hifnb), hydrolyzed at the sites for restriction endonucleases AgeI (No. ER1461 "Fermentas", Latvia) and NheI (No. ER0971 "Fermentas", Latvia), followed by treatment with a Klenov fragment (No. EP0052 Fermentas, Latvia), in the plasmid construct pCELOdl3right according to Example 2, hydrolyzed at SpeI site (No. ER1251 Fermentas, Latvia) and processed with a Klenov fragment. The result is a plasmid construct pCELOdl3-IFNβ with a fragment of the CELO avian virus genome, where an expression cassette containing the IFN β gene is contained in the genome deletion region.

Further, this plasmid treated with the restriction enzyme PacI (No. R0547S "New England BioLabs", USA) and the plasmid pCELO20HN of Example 1 treated with the restriction enzyme AscI (No. R0558S "New England BioLabs", USA) co-transfect LMH cells. As a result of homologous recombination in LMH cells, recombinant avian adenovirus CELONdl3-IFNβ is obtained that carries the human β-interferon gene.

Example 9. Construction of a recombinant adenovirus CELONdl3-IFNγ carrying the human γ-interferon gene, to claims 6, 10 of the claims.

The human γ-interferon gene is cloned from the plasmid pORF-hINF-γ (Invivogen, Cat. Code porf-hifng), hydrolyzed at the sites for restriction endonucleases SgrAI (No. R0603S "New England BioLabs", USA) and NheI (No. ER0971 " Fermentas ”, Latvia), followed by processing with a Klenov fragment (No. EP0052“ Fermentas ”, Latvia), in the plasmid construct pCELOdl3right according to Example 2, hydrolyzed on the SpeI site (No. ER1251“ Fermentas ”, Latvia) and processed with a Klenov fragment. The result is the plasmid construct pCELOdl3-IFNγ carrying a fragment of the CELO avian virus genome, where an expression cassette containing the IFNγ gene is contained in the genome deletion region.

This plasmid treated with PacI restriction enzyme (No. R0547S "New England BioLabs", USA) and the plasmid pCELO20HN of Example 1 treated with AscI restriction enzyme (No. R0558S "New England BioLabs", USA) cotransfect LMH cells. As a result of homologous recombination in LMH cells, recombinant avian adenovirus CELONdl3-IFNγ is obtained that carries the human γ-interferon gene under the control of an exogenous promoter.

Example 10. The construction of the recombinant adenovirus CELONdl3-GMCSF, carrying the gene of granulocyte-macrophage colony stimulating factor GMCSF human under the control of an exogenous promoter, to PP.6, 11 of the claims.

The GMCSF gene is cloned from the plasmid pORF-hGMCSF (Invivogen, Cat. Code porf-hgmcsf), hydrolyzed at the sites for restriction endonucleases SgrAI (No. R0603S “New England BioLabs”, USA) and NheI (No. ER0971) Fermentas, Latvia , followed by treatment with a Klenov fragment (No. EP0052 Fermentas, Latvia), in the plasmid construct pCELOdl3right according to Example 2, hydrolyzed at the SpeI site (No. ER1251 Fermentas, Latvia) and processed with a Klenov fragment. The result is a plasmid construct pCELOd13-GMCSF carrying a fragment of the CELO avian virus genome, where an expression cassette with the GMCSF gene is contained in the genome deletion region.

This plasmid treated with PacI restriction enzyme (No. R0547S "New England BioLabs", USA) and the plasmid pCELO20HN treated with AscI restriction enzyme (No. R0558S "New England BioLabs", USA) co-transfect LMH cells. As a result of homologous recombination in LMH cells, the recombinant avian adenovirus CELONd13-GMCSF is obtained that carries the gene for granulocyte-macrophage colony stimulating factor GMCSF under the control of an exogenous promoter.

Example 11. Construction of a recombinant adenovirus CELONdl3-IL-2 carrying the human interleukin-2 gene under the control of an exogenous promoter, to claims 6, 12 of the claims.

The human interleukin-2 gene is cloned from the plasmid pORF-hIL-2 (Invivogen, Cat. Code porf-hiI2), hydrolyzed at the sites for restriction endonucleases SgrAI (No. R0603S "New England BioLabs", USA) and NheI (No. ER0971 " Fermentas ”, Latvia), followed by processing with a Klenov fragment (No. EP0052“ Fermentas ”, Latvia), in the plasmid construct pCELOd13right, hydrolyzed at SpeI site (No. ER1251“ Fermentas ”, Latvia) and processed with a Klenov fragment. The result is a plasmid construct pCELOdl3-IL-2 carrying a fragment of the CELO avian virus genome, where an expression cassette with the human IL-2 gene is contained in the genome deletion region.

This plasmid treated with PacI restriction enzyme (No. R05478 "New England BioLabs", USA) and the plasmid pCELO20HN treated with AscI restriction enzyme (No. R0558S "New England BioLabs", USA) co-transfect LMH cells. As a result of homologous recombination in LMH cells, recombinant avian adenovirus CELONdl3-IL-2 is obtained that carries the human interleukin-2 gene under the control of an exogenous promoter.

Example 12. The construction of the recombinant adenovirus CELONdl3-IGF1, carrying the gene of insulin-like growth factor human IGF1, to claims 13, 14 of the claims.

The gene for insulin-like growth factor 1, IGF1 is cloned from the plasmid pBLAST49-hIGF1A (Invivogen, Cat. Code pbla-higfa), hydrolyzed at the sites for restriction endonucleases NcoI (No. ER0571 “Fermentas”, Latvia) and NheI (No. ER0971 “Fermentas” , Latvia), followed by treatment with a Klenov fragment (No. EP0052 Fermentas, Latvia), in the plasmid construct pCELOdl3right, hydrolyzed at SpeI site (No. ER1251 Fermentas, Latvia) and processed with a Klenov fragment. The result is a plasmid construct pCELOdl3-IGF1 carrying a fragment of the CELO avian virus genome, where an expression cassette with the IGF1 gene is contained in the genome deletion region.

This plasmid treated with restriction enzyme PacI (No. K0547S "New England BioLabs", USA) and the plasmid pCELO20HN treated with restriction enzyme AscI (No. R0558S "New England BioLabs", USA) co-transfect LMH cells. As a result of homologous recombination in cells of the LMH line, recombinant avian adenovirus CELONd13-IGF1 carrying the gene of insulin-like growth factor 1, human IGF1, is obtained.

Example 13. Construction of a recombinant adenovirus CELONdl3-VEGF carrying the human vascular endothelial growth factor gene human VEGF under the control of an exogenous promoter, to claims 13, 15 of the claims.

The VEGF vascular endothelial growth factor gene is cloned from the plasmid pBLAST49-hVEGF (Invivogen, Cat. Code pbla-hvegf), digested at the sites for restriction endonucleases AgeI (No. ER1461 “Fermentas”, Latvia) and NheI (No. ER0971 “Fermentas”, Latvia), followed by treatment with a Klenov fragment (No. EP0052 Fermentas, Latvia), in the plasmid construct pCELOdl3right, hydrolyzed at SpeI site (No. ER1251 Fermentas, Latvia) and processed with a Klenov fragment. The result is a plasmid construct pCELOdl3-VEGF carrying a fragment of the CELO avian virus genome, where an expression cassette with the VEGF gene is contained in the genome deletion region.

This plasmid treated with PacI restriction enzyme (No. R0547S "New England BioLabs", USA) and the plasmid pCELO20HN treated with AscI restriction enzyme (No. R0558S "New England BioLabs", USA) co-transfect LMH cells. As a result of homologous recombination in LMH cells, recombinant avian adenovirus CELONdl3-VEGF is obtained that carries the human vascular endothelial growth factor gene VEGF under the control of an exogenous promoter.

Example 14. Construction of a recombinant adenovirus CELONdl3-GDNF carrying the gene for the neurotrophic factor of glial cells of human GDNF under the control of an exogenous promoter, to claims 13, 16 of the claims.

The gene for the neurotrophic factor of glial GDNF cells is cloned from the plasmid pBLAST49-hGDNFa (Invivogen, Cat. Code pbla-hgdfna), hydrolyzed at the sites for restriction endonucleases BspHI (No. ER1281 “Fermentas”, Latvia) and NheI (No. ER0971 “Fermentas”, Latvia), followed by treatment with a Klenov fragment (No. EP0052 Fermentas, Latvia), in the plasmid construct pCELOdl3right, hydrolyzed at the SpeI site (No. ER1251 Fermentas, Latvia) and processed with a Klenov fragment. The result is a plasmid construct pCELOd13-GDNF carrying a fragment of the CELO avian virus genome, where an expression cassette with the GDNF gene is contained in the genome deletion region.

This plasmid treated with PacI restriction enzyme (No. R0547S "New England BioLabs", USA) and the plasmid pCELO20HN treated with AscI restriction enzyme (No. R0558S "New England BioLabs", USA) co-transfect LMH cells. As a result of homologous recombination in LMH cells, recombinant avian adenovirus CELONdI3-GDNF is obtained that carries the human neurotrophic factor of glial GDNF cells under the control of an exogenous promoter.

Example 15. Construction of a recombinant adenovirus CELONd13-TK carrying the thymidine kinase gene of the HSV1 virus under the control of an exogenous promoter, to claims 17, 18 of the claims.

The HSV1 virus thymidine kinase gene is cloned from the plasmid pORF-HSVltk (Invivogen, Cat. Code pmod-tk) hydrolyzed at the NcoI restriction endonuclease sites (No. ER 0571 Fermentas, Latvia) and NheI (No. ER0971 Fermentas, Latvia ), followed by treatment with a Klenov fragment (No. EP0052 Fermentas, Latvia), in the plasmid construct pCELOd13 right, hydrolyzed at SpeI site (No. ER 1251 Fermentas, Latvia) and processed with a Klenov fragment. The result is a plasmid construct pCELOd13-TK carrying a fragment of the CELO avian virus genome, where an expression cassette with the HSV1 virus thymidine kinase gene is contained in the genome deletion region.

This plasmid treated with PacI restriction enzyme (No. R0547S "New England BioLabs", USA) and the plasmid pCELO20HN treated with AscI restriction enzyme (No. R0558S "New England BioLabs", USA) co-transfect LMH cells. As a result of homologous recombination in LMH cells, recombinant avian adenovirus CELONd13-TK is obtained that carries the HSV1 virus thymidine kinase gene under the control of an exogenous promoter.

Example 16. Construction of a recombinant adenovirus CELONdl3-p53 carrying the human p53 gene under the control of an exogenous promoter, and determination of its antitumor activity to claims 17, 19 of the claims.

The human p53 gene is cloned from the plasmid pORF-hp53 (Invivogen, Cat. Code porf-hp53), hydrolyzed at the sites for restriction endonucleases AgeI (No. ER1461 "Fermentas", Latvia) and NheI (No. ER0971 "Fermentas", Latvia), followed by treatment with a Klenov fragment, in the plasmid construct pCELOd13right, hydrolyzed at SpeI site (No. ER1251 Fermentas, Latvia) and processed with a Klenov fragment (No. EP0052 Fermentas, Latvia). The resulting plasmid construct pCELOdl3-p53 contains a fragment of the CELO avian virus genome, where an expression cassette with the human p53 gene is located in the region of the deletion of the genome.

This plasmid treated with PacI restriction enzyme (No. R0547S "New England BioLabs", USA) and the plasmid pCELO20HN treated with AscI restriction enzyme (No. R0558S "New England BioLabs", USA) co-transfect LMH cells. As a result of homologous recombination in LMH cells, recombinant avian adenovirus CELONdl3-p53 carrying the human p53 gene is obtained.

Then, two groups of mice (10 mice per group) are injected subcutaneously with A431 / waflConALacZ tumor cells. The first group, together with tumor cells, is introduced with recombinant avian adenovirus CELONdl3-p53 at a dose of 10 ⁹ PFU / mouse. The second group does not enter the virus (control group). 14 days after the injection, the tumor volume is measured. The tumor size in mice of the control group is 4 times larger compared to the tumor volume of mice that were injected with the CELONdl3-p53 virus.

Example 17. Construction of recombinant adenoviruses CELONdl3-Ag85A, CELONdl3-Ag85B, CELONdl3-ESAT6 carrying the genes of protective antigens of Mycobacterium tuberculosis under the control of an exogenous promoter, to claims 20, 21, 22, 23 of the claims. In vitro detection of Ag85B gene expression after infection with appropriate recombinant cell adenovirus.

Genes of antigens of M. tuberculosis Ag85A, Ag85B, ESAT6 obtained by PCR under the condition of 94 ° C for 5 min; 5 cycles: 58 ° С - 2 min, 72 ° С - 20 s and 94 ° С - 30 s; 20 cycles: 58 ° C - 30 s, 72 ° C - 20 s and 94 ° C - 30 s; then 58 ° C for 5 min and 72 ° C for 10 min, where M. tuberculosis genomic DNA is used as a matrix. For each antigen, a pair of tub-F 5'-atgacagacgtgagccgaaa-3 primers and tub-R 5'-tcagccggcgcctaacgaactct-3 primers selected at the ends of the gene according to the GENE-Bank BX842579 DNA sequence of M. tuberculosis strain H37Rv are used. Each of the PCR products encoding the genes of the above antigens is cloned into the pGEM-T Easy plasmid (Promega, Cat. No. A1360). From the obtained plasmids pGEM-Ag85A, pGEM-Ag85B and pGEM-ESAT6 treated with the NotI restriction endonuclease (No. ER0592 Fermentas, Latvia) and the Klenov fragment (No. EP0052 Fermentas, Latvia), the antigen genes were cloned into the plasmid construct pCELOdl3right according to example 2, hydrolyzed on the SpeI site (No. ER1251 Fermentas, Latvia) and processed with a Klenov fragment. As a result, plasmid constructs pCELOdl3-Ag85A, pCELOdl3-Ag85B and pCELOdl3-ESAT6 carrying the fragment of the CELO avian virus genome are obtained, where the expression cassette containing the gene for the protective antigen M tuberculosis Ag85A, Ag85B and ESAT6, respectively, is contained.

These plasmids treated with PacI restriction enzyme (No. R0547S "New England BioLabs", USA) and plasmid pCELO20HN treated with AscI restriction enzyme (No. R0558S "New England BioLabs", USA) cotransfect LMH cells. As a result of homologous recombination in LMH cells, recombinant avian adenoviruses CELONdl3-Ag85A, CELONdl3-Ag85B, CELONdl3-ESAT6 carrying the genes of protective antigens M. tuberculosis Ag85A, Ag85B and ESAT6, respectively, are obtained.

At the next stage, the expression of the gene for the protective antigen of M. tuberculosis Ag85B is detected during cell infection with the CELONdl3-Ag85B virus in vitro.

For this, the wild-type CELONdl3-Ag85B and CELO viruses (as a negative control) infect LMH line cells at a dose of 5 PFU / cell. 48 hours after infection, the cells are treated with lysis buffer (0.05 M Tris HCl ph 7.2; 0.15 M NaCl; 0.1% SDS; 0.1% Dezoxycholat Na; 0.1% Triton X100, 1 mM DTT , protease inhibitor) for detecting the expression of the gene for the protective antigen of M. tuberculosis Ag85B in the cytoplasmic fraction of cells using Western blotting using monoclonal antibodies obtained against the ag85B protein of M. tuberculosis. As a result, it was found that the recombinant ag85B protein in its antigenic properties and molecular weight corresponds to the ag85B protein of M. tuberculosis.

Example 18. Construction of a recombinant adenovirus CELONdl3-Ag85AB, carrying the genes of protective antigens of Mycobacterium tuberculosis under the control of an exogenous promoter, and the identification of its immune effect to claims 24-25.

From the resulting plasmid pGEM-Ag85A treated with EcoRI restriction endonuclease, the M. tuberculosis Ag85A gene was cloned into the pIRES plasmid construct (Clontech, No. 631605) treated with the same restriction endonuclease. The result is the plasmid construct pIRES-Ag85A, to which, after treatment with the NotI restriction endonuclease, the Ag85B gene is cloned from the previously obtained plasmid pGEM-Ag85B. Thus, the plasmid pIRES-Ag85AB carrying the IRES and the Ag85A, Ag85B M. tuberculosis genes is created. Next, the fragment of the plasmid pIRES-Ag85AB, consisting of two genes (Ag85A, Ag85B) and IRES, obtained by processing this plasmid with restriction endonucleases NheI and NotI (Fermentas, Latvia), as well as the Klenov fragment, is cloned into the plasmid pCELOdl3right, hydrolyzed by SpeI website (No. ER1251 “Fermentas”, Latvia) and processed by Klenov fragment.

The result is a plasmid construct pCELOd13-Ag85AB carrying a fragment of the CELO avian virus genome, where an expression cassette with the genes of protective antigens M. tuberculosis Ag85A and Ag85B is contained in the genome deletion region. The Pad restriction enzyme treated plasmid (No. R0547S “New England BioLabs”, USA) and the AscI restriction enzyme treated pCEL020HN plasmid (New England BioLabs No. R0558S, USA) cotransfect LMH cells. As a result of homologous recombination in LMH cells, recombinant avian adenovirus CELONd13-Ag85AB is obtained that carries the genes of protective antigens M. tuberculosis Ag85A and Ag85B under the control of the exogenous CMV promoter.

To increase the protective function of the BCG vaccine against tuberculosis, which is a non-pathogenic M. tuberculosis strain, 50,000 CFU / mouse of the BCG vaccine strain are introduced into two groups of mice (15 mice in each group), and after 8 weeks recombinant avian adenovirus CELONd13-Ag85AB is administered to one group of mice at a dose of 109 PFU / mouse. 12 weeks after BCG administration, both groups of mice were aerosolized with a pathogenic strain of M. tuberculosis at a dose of 10 ⁹ CFU / mouse. Four weeks after infection, M. tuberculosis is seeded from the spleen and lung of both groups of mice. The number of colonies formed by M. tuberculosis seeded from the group of mice injected with the CELONd13-Ag85AB virus is 4 times less than in the control group where the infection with the CELONd1l3-Ag85AB virus was not performed.

Example 19. The construction of the recombinant adenovirus CELONd13-hex carrying the gene for the bird virus virus SSN-76 under the control of an exogenous promoter, to claims 26, 27 of the claims.

The HSSA-76 bird virus hexon gene is obtained by PCR on the HSSA-76 virus genome DNA matrix (Gene bank No. Z86065, U63515) under conditions of 94 ° C. for 5 minutes; 5 cycles: 58 ° С - 2 min, 72 ° С - 20 s and 94 ° С - 30 s; 20 cycles: 58 ° C - 30 s, 72 ° C - 20 s and 94 ° C - 30 s; then 58 ° C for 5 min and 72 ° C for 10 min using hex-F 5'-tgttacagatgaagcgactacgg-3 and hex-R 5'-caaagtctactgtgctccaac-3 primers. The resulting cDNA of the hexon gene is cloned into the pGEM-T Easy plasmid vector (Promega, Cat. No A1360). In the next step, from the obtained plasmid pGEM-hex treated with NotI restriction endonuclease (No. ER0592 Fermentas, Latvia) and the Klenov fragment (No. EP0052 Fermentas, Latvia), the hexon gene hex was cloned into the plasmid construct pCELOd13right hydrolyzed at the SpeI site ( No. ER1251 “Fermentas”, Latvia) and processed Klenov fragment. The result is a plasmid construct pCELOd13-hex carrying a fragment of the CELO avian virus genome, where an expression cassette containing the CCJ-76 avian virus hex hex gene is contained in the genome deletion region.

This plasmid treated with PacI restriction enzyme (No. R0547S "New England BioLabs", USA) and plasmid pCEL020HN treated with AscI restriction enzyme (No. R0558S "New England BioLabs", USA) carry out cotransfection of LMH cells. As a result of homologous recombination in cells of the LMH line, recombinant avian adenovirus CELONd13-hex is obtained, which carries the gene for the bird virus virus SSN-76 under the control of the exogenous CMV promoter.

Example 20. Construction of recombinant adenoviruses CELOHNd13-fiber, CELOHN-fiber, carrying the gene of the process of the penton (fiber) of the bird virus SSYA-76 under the control of the CMV and MLP promoters, respectively to claims 26, 28 of the claims.

The gene of the process of the penton (fiber) virus of the hen egg production syndrome CCY-76 is obtained by PCR using the genome of the CCY-76 virus genome (Gene bank No. Z86065, U63515) under conditions of 94 ° C for 5 min; 5 cycles: 58 ° С - 2 min, 72 ° С - 20 s and 94 ° С - 30 s; 20 cycles: 58 ° C - 30 s, 72 ° C - 20 s and 94 ° C - 30 s; then 58 ° C for 5 min and 72 ° C for 10 min using primers: fib-F 5'-ggaggcattgggatcgtctcg-3, fib-R 5'-gattactcaatcagctttgggaa-3. The resulting cDNA of the fiber gene is cloned into the pGEM-T Easy plasmid vector (Promega, Cat. No. A1360).

From the obtained plasmid pGEM-fiber treated with NotI restriction endonuclease (No. ER0592 Fermentas, Latvia) and the Klenov fragment (No. EP0052 Fermentas, Latvia), the fiber gene was cloned into the plasmid construct pCELOd13right hydrolyzed at SpeI site (No. ER 1251 “ Fermentas ”, Latvia) and processed fragment of Klenov. The result is a plasmid construct pCELOd13-fiber carrying a fragment of the CELO avian virus genome, where an expression cassette containing the FIB fiber virus gene CCL-76 is contained in the genome deletion region.

This plasmid treated with PacI restriction enzyme (No. R0547S "New England BioLabs", USA) and plasmid pCELO20HN treated with AscI restriction enzyme (No. R0558S "New England BioLabs", USA) carry out cotransfection of LMH cells. As a result of homologous recombination in LMH cells, a recombinant bird adenovirus CELOHNdl3-fiber is obtained that carries the penton-fiber process gene of the bird virus CCYA-76 under the control of the CMV promoter.

Also from the obtained plasmid pGEM-fiber treated with NotI restriction endonuclease (No. ER0592 Fermentas, Latvia) and the Klenov fragment (No. EP0052 Fermentas, Latvia), the penton process gene (fiber) was cloned into the plasmid construct pCELO20HN, hydrolyzed at the site SpeI (No. ER1251 "Fermentas", Latvia) and processed fragment Klenova. As a result, the pCELO20HN-fiber plasmid construct was obtained, carrying the fragment of the CELO bird virus genome and the penton process (fiber) process gene of the BSS virus-76 under the control of the MLP promoter.

Further, LMH cells were cotransfected with this plasmid treated with AscI restrictase and plasmid pCELOd13right treated with PacI. Thus, as a result of homologous recombination in cells of the LMH line, recombinant bird adenovirus CELOHN-fiber was obtained, carrying the gene of the penton process sprout of the bird virus SSN-76 under the control of the MLP promoter.

Example 21. The construction of the recombinant adenovirus CELOHN-fib / dI3-fib carrying the genes of the penton process (fiber) virus of the CCN-76 virus under the control of the MLP and CMV promoters, and the detection of the level of immunization in ovo when used to claims 29, 30 of the claims . As a result of homologous recombination in LMH cells upon their co-transfection with plasmid constructs pCELO20HN-fiber treated with AscI restrictase and pCELOd13-fiber treated with PacI restrictase (Example 20), the CELOHN-fib / dl3-fib virus carrying the process gene was obtained (fiber) of the BWV-76 bird virus under the control of the MLP promoter and the penton process gene (fiber) of the BWW-76 bird virus under the control of the CMV promoter.

To determine the increase in the effect of the dose of the introduced target gene on the level of specific antibodies, chickens at the age of three weeks are infected with the CELOHNd13-fiber and CELOHN-fib / d13-fib viruses. The first group is administered the CELOHNd13-fiber virus (as in Example 20), the second group is CELOHN-fib / d13-fib, the third group is the wild-type CELO virus. All virus preparations are administered at a dose of 10 ⁸ PFU / individual. 3 weeks after infection in the blood serum of chickens of all three groups, a study of the level of specific antibodies to the SSJ-76 virus is carried out by ELISA ("A kit for determining antibodies to the egg-laying syndrome-76 virus (SSJ-76) by enzyme-linked immunosorbent assay", FSI ARRIAH). No antibodies were detected in the blood serum of chickens infected with wild-type CELO virus. In the blood serum of chickens infected with the CELOHNd13-fiber virus, the antibody titer is 1:64, and those infected with CELOHN-fib / d13-fib are 1: 128.

Example 22. The construction of the recombinant adenovirus CELOHN-aIFNγ / d13-fib, carrying the interferon genes in birds and the penton process (fiber) of the BSS virus-76 under the control of MLP and CMV promoters, respectively, and the determination of its antiviral activity to claims 31, 32 of the formula inventions.

The plasmid construct CELOHNd13-fiber, carrying the penton process gene of the bird virus CCYA-76 under the control of the CMV promoter, and pCELO20HN-chIFNγ, carrying the IFNγ gene of birds under the control of the MLP promoter, after treatment with PacI restriction enzymes (No. R0547S "New England BioLabs , USA) and AscI (No. R0558S "New England BioLabs", USA), respectively, carry out the cotransfection of LMH cell lines. As a result of homologous recombination, a recombinant avian adenovirus CELOHN-aIFNγ / d13-fib is obtained that carries the penton process gene (fiber) of the CCV-76 bird virus under the control of the CMV promoter and the bird IFNγ gene under the control of the MLP promoter.

At the next stage of the work, the expression of avian IFNγ genes and the penton process (fiber) of the CCV-76 virus is detected during cell infection with CELOHN-aIFNγ / d13-fib virus in vitro. For this, wild-type CELOHN-aIFNγ / d13-fib and CELO viruses (as a negative control) infect LMH line cells at a dose of 5 PFU / cell. 48 hours after infection, culture medium was selected to detect expression of the bird IFNγ gene by examining the biological activity of the protein, and the cells were treated with lysis buffer (0.05 M Tris HCl ph 7.2; 0.15 M NaCl; 0.1% SDS; 0 , 1% Dezoxycholat Na; 0.1% Triton X100, 1 mM DTT, protease inhibitor) for detection of expression of the penton process (fiber) process gene of the bird virus SSN-76 in the cytoplasmic fraction of cells by immunoblotting.

The study of antiviral activity of drugs is carried out according to the standard method of titration of interferon in a bird cell culture. Mouse encephalomyocarditis virus is used as an indicator test virus. The analysis revealed that the culture medium of cells infected with wild-type CELO virus does not have antiviral activity. The antiviral activity of the test samples, the culture medium of cells infected with the CELOHN-aIFNγ / d13-fib virus, is 1.2 · 10 ⁴ IU / ml.

The expression of the gene for the process of the fiber of the bird virus SSJ-76 in the cytoplasmic fraction of cells is detected by Western blotting using native purified fiber of the virus SSJ-76. As a result, it was revealed that the recombinant fiber protein, in terms of its antigenic properties and molecular weight, corresponds to the protein of the CCF-76 virus fiber monomer, there is expression of the fiber gene in the composition of the recombinant adenovirus CELOHN-aIFNγ / d13-fib, and the expressed protein contains unchanged antigenic determinants and biologically active.

Claims (32)

1. A method of creating a recombinant avian adenovirus for vaccination and gene therapy, which consists of using two plasmids with parts of the CELO adenovirus genome, one of which contains an expression cassette and a CELO genome for subsequent homologous recombination, characterized in that they take the genomic DNA of bird adenovirus CELO, which includes its own main late promoter of MLP adenovirus, and based on its left fragment (1-20028 bp) construct the first plasmid containing the built-in splicing site and as at least one target gene under the control of the MLP promoter, as well as on the basis of the right fragment of the CELO genome (17390-43804) construct a second plasmid in which there is a region homologous to the zone of the first construct for recombination, while in the nucleotide sequence of the plasmid they form a deletion of 3787 n .P. to increase the packing capacity of the CELO adenovirus, in the region of which at least one expression cassette is then cloned, consisting of an exogenous promoter providing a high level of expression in eukaryotic cells, from at least one target gene and a polyadenylation signal, after which homologous recombination is performed in a hepatoma cell culture Leghorn rooster (LMH), providing a recombinant adenovirus consisting of CELO avian adenovirus genomic DNA with an expression cassette including a splice site inga, at least one target gene under the control of the MLP adenovirus promoter and a polyadenylation signal from the CELO adenovirus genome, as well as with another expression cassette consisting of at least one target gene under the control of an exogenous promoter for expression in eukaryotic cells and a polyadenylation signal, As a result, the recombinant CELO recombinant avian adenovirus is assembled and propagated in LMH cell culture for subsequent use as a vaccine or gene therapeutic agent. 2. The method according to claim 1, characterized in that the avian cytokine gene is used as the target gene in the expression cassette under the control of the MLP adenovirus own promoter or under the control of the exogenous promoter. 3. The method according to claim 2, characterized in that the gene of avian interferon γ is used as the bird cytokine gene. 4. The method according to claim 1, characterized in that the avian cytokine gene is simultaneously used as the target gene in the expression cassette under the control of the MLP adenovirus own promoter and under the control of the exogenous promoter. 5. The method according to claim 4, characterized in that the gene of avian interferon γ is used as the bird cytokine gene. 6. The method according to claim 1, characterized in that the human cytokine gene is used as the target gene in the expression cassette under the control of an exogenous promoter. 7. The method according to claim 6, characterized in that the gene of human α16-interferon is used as the human cytokine gene. 8. The method according to claim 6, characterized in that the human α-interferon α-2a gene is used as the human cytokine gene. 9. The method according to claim 6, characterized in that the human interferon β gene is used as the human cytokine gene. 10. The method according to claim 6, characterized in that the human interferon γ gene is used as the human cytokine gene. 11. The method according to claim 6, characterized in that the gene of granulocyte-macrophage colony stimulating factor GMCSF is used as a human cytokine gene. 12. The method according to claim 6, characterized in that the human interleukin-2 gene is used as the human cytokine gene. 13. The method according to claim 1, characterized in that the human growth factor gene is used as the target gene in the expression cassette under the control of an exogenous promoter. 14. The method according to p. 13, characterized in that the gene of human insulin-like growth factor IGF1 is used as the human growth factor gene. 15. The method according to p. 13, characterized in that the gene for human vascular endothelial growth factor VEGF is used as a human growth factor gene. 16. The method according to p. 13, characterized in that the gene for the human growth factor gene is the gene for the neurotrophic factor of glial human GDNF cells. 17. The method according to claim 1, characterized in that the human oncosuppressor gene is used as the target gene in the expression cassette under the control of an exogenous promoter. 18. The method according to 17, characterized in that the human thymidine kinase gene of human HSV1 virus is used as the human oncosuppressor gene. 19. The method according to 17, characterized in that the human p53 gene is used as the human cancer suppressor gene. 20. The method according to claim 1, characterized in that the bacterial antigen gene is used as the target gene in the expression cassette under the control of the exogenous promoter. 21. The method according to claim 20, characterized in that the gene of a bacterial antigen Ag85A of the mycobacterium M. tuberculosis Ag85A is used as a bacterial antigen gene. 22. The method according to claim 20, characterized in that the gene of a bacterial antigen Ag85B of the mycobacterium M. tuberculosis Ag85B is used as a bacterial antigen gene. 23. The method according to claim 20, characterized in that the gene for the bacterial antigen ESAT6 of the mycobacterium M. tuberculosis is used as a bacterial antigen gene. 24. The method according to claim 1, characterized in that the genes of bacterial antigens are used as target genes in the expression cassette under the control of an exogenous promoter. 25. The method according to p. 24, characterized in that the genes of bacterial antigens are the genes of protective antigens Ag85A and Ag85B of mycobacterium M. tuberculosis. 26. The method according to claim 1, characterized in that the bird antigen gene is used as the target gene in the expression cassette under the control of the MLP adenovirus own promoter or under the control of the exogenous promoter. 27. The method according to p. 26, characterized in that the gene of the bird virus antigen gene SSJA-76 is used as a bird antigen gene. 28. The method according to p. 26, characterized in that the gene of the process of the penton (fiber) of the bird virus SSN-76 is used as a bird antigen gene. 29. The method according to claim 1, characterized in that the avian antigen gene is simultaneously used as the target gene in the expression cassette under the control of the MLP adenovirus own promoter and under the control of the exogenous promoter. 30. The method according to clause 29, characterized in that the gene of the process of the penton (fiber) of the bird virus SSN-76 is used as a bird antigen gene. 31. The method according to claim 1, characterized in that the cytokine gene is used as the target gene in the expression cassette under the control of the MLP adenovirus own promoter, and the antigen gene is used in the expression cassette under the control of the exogenous promoter. 32. The method according to p. 31, characterized in that the gene γ of the interferon of birds is used as the cytokine gene, and the gene of the process of the penton (fiber) of the bird virus SSN-76 is used as the gene of antigen.

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