Recombinant vector-based smallpox birds, recombinant vector for vaccination of poultry

 

(57) Abstract:

The invention relates to biotechnology and can be used to produce vaccines. The recombinant vector construct on the basis of attenuated smallpox birds by integrating a nucleotide sequence that determines the synthesis of the heterologous protein of the microorganism pathogenic to poultry in non-coding intergenic region located in the terminal inverted curves (TIR). Intergenic region1and2located between nucleotides 1675 and 2165 TIR and 2672 and 3605 TIR, respectively. Cells transformed designed vector that can Express the heterologous protein. The protein may be an antigen of a pathogenic for birds agents: a virus disease of Gumboro, virus, infectious bronchitis, Marek's disease virus, parasitic simplest, causing coccidiosis. The invention allows to create new recombinant vaccines against diseases of birds. 2 S. and 6 C.p. f-crystals, 13 ill., 9 table.

The invention relates to recombinant viruses derived from Avipox virus and, in particular, the virus responsible for smallpox in birds, commonly called Fowlpox. The invention relates also to method of obtaining these virus is them these viruses. The invention relates also to transfer sequences for inclusion in the genome of this recombinant virus, as well as plasmids that carry these sequences.

The virus responsible for smallpox in birds, or Fowlpox virus (FPV), belongs to the genus Avipox from the poxviridae family.

The Fowlpox virus has characteristics typical Pox. These viruses are of large size, whose genome is composed of DNA in the form of a linear double helix value of 300 to which an end part of a simple circuit connected covalent manner. About 20 to this genome were collected in a sequence, in particular a sequence of inverse terminal repeats (for TIR, Terminal Inverted Repeats) (Campbell et al. 1989; Tomley et al. 1988). The opposite terminal of repetition are two long identical sequences located at each end of the genome, with the opposite orientation.

Vaccinia virus or Vaccinia was the first of Pox, which was launched as a live recombinant virus capable of expressing foreign proteins. The list of expressed proteins is very long. In particular, expression of multiple foreign antigens allowed to obtain immunization against various bol is ora expression of heterologous proteins with the purpose of receiving the vaccine, i.e. the application of the recombinant virus, which has integrated in non-coding vnutrennuu region of its genome a DNA sequence encoding a heterologous protein; this area is a short sequence of 32 nucleotides located between the open phases for reading (ORF Open Reading Frame ORF 7 and ORF 9, and this area has been transformed by genetic non-core site inclusion. In such short vnutrennih areas of high risk that the inclusion of separate signal transcription from the gene and thereby introduce a mutation that inactivates the gene. It is confirmed: indeed, this sequence of 32 nucleotides is an essential area because she had to grow by 55 nucleotides, in order to avoid a possible overlap between the promoter phase ORF 9 and the coding sequence 3' of the phase ORF 7. In case the area that separates the ORF 7 of ORF 9, the inclusion in the natural sequence is also unstable (Spehner et al., 1990), and his conversion is necessary. Moreover, the proposed method of converting short vnutrivennoi region is not shared.

On the other hand, in the application for international is audacia the incorporation of foreign DNA into the gene timedancing. This inclusion creates a mutation in the gene, which could reduce infecting the ability of the virus. This is the risk of too much strain weakening and reduced immunogenic ability of the vaccine, derived from the recombinant virus.

In addition, the application for the European patent 0 353 851 been proposed the use of recombinant Fowlpox virus as a vector for the expression of heterologous protein. The inclusion of a foreign gene into the viral genome is carried out in open phases for reading (ORF) located in the opposite terminal repeats (TIR). The inclusion of one or more foreign genes into the inner portion of the open phases for reading has the risk of producing a mutation of a gene whose function is not known.

The present invention provides other areas to enable one or more foreign genes into the viral genome, which no longer have the disadvantages inherent in the above areas inclusions.

In this regard, the present invention relates to a recombinant Avipox virus, derived from a weakened strain of the virus and Avipox containing non-core parts of its genome at least one DNA sequence encoding a full and who possess this protein in the cell, infected with the indicated recombinant virus, and a minor portion of the genome is formed vnutrivennoi non-coding region of the Avipox virus, i.e., the area located between the two open phases for reading, including the signals of the expression.

Under the non-core part of the genome of the virus, Avipox understand the area that can be modified without affecting the functions involved in the transformation of the virus in vitro and in vivo. As a non-core part of the select non-coding vnutrennuu region of the genome, i.e., located between two ORFS, including their expression signals.

Nutrigenie region according to the invention are wide, so that the risk that the inclusion of separate signals of transcription of their respective genes, was minimal, and that their conversion would not be necessary. On the other hand, the sequence of these vnutrennih regions are non-coding, and therefore, the risk of mutations in the ORF is zero. Under a wide region see region having a sequence of more than 60 nucleotides. Usually choose a region having a sequence of more than 100 nucleotides. Preferably choose a region having a sequence of more than 200 nuclearization.

On the other hand, usually choose vnutrennuu region, which is cloned at least one DNA sequence that is located in one of the two regions TIR Avipox virus. Usually at least one DNA sequence is cloned in at least one of the two regions TIR Avipox virus. Preferably at least one DNA sequence is cloned in both areas TIR virus. In an even more preferred method is chosen vnutrennuu region, called region1and/or vnutrennuu region, called region2and nutregena region, called region1located between nucleotides 1675 and 2165, and nutregena region, called region2located between nucleotides 2672 and 3605, taking as a starting nucleotide of the restriction site BamHI, located in the TIR. This restriction site BamHI chosen here for reference vnutrennih areas, represents the first nucleotide sequence, published in Tomley et al. 1988. You can also define these areas according to the invention is as follows: nutregena region, called region1located between the open phases for reading ORF 1 and ORF 2, and open FASA phase for reading ORF 2 is located between nucleotides 2166 (A nucleotide of the ATG) and 2671 (3rd nucleotide of the stop codon), taking as a starting nucleotide of the restriction site BamHI and TIR, and nutregena region, called region2located between the open phases for reading ORF 2 and ORF 3, and the open phase for reading ORF 2 is located between nucleotides 2166 (A nucleotide of the ATG) and 2671 (3rd nucleotide of the stop codon), and the open phase for reading ORF 3 is located between nucleotides 3606 (3rd nucleotide of the stop codon) and 4055 (A nucleotide of the ATG), taking as a starting nucleotide of the restriction site BamHI in TIR. A more preferred method in the1choose the part located between nucleotides 1775 and 2065, and in2- part located between nucleotides 2772 and 3505. Good results were obtained by cloning the nucleotide level 1842, called site include B1, which is located in the1and/or at the level of nucleotide 3062, called site enable B2, which is located in the2.

Good results were obtained when at least one DNA sequence is cloned in the field2in one of the two regions TIR Avipox virus. Good results were also obtained when one DNA sequence is cloned in the field1in each of the two areas TIR is Telenesti DNA on one gene.

Under recombinant Avipox virus, derived from a weakened strain of the virus, Avipox, understand the weakened virus belonging to the genus Avipox, the genome of which contains the heterologous sequence is accompanied by a signal expression. Usually the virus using a virus of the genus Avipox, such as Fowlpox, Pigeonpox or Canarypox. Usually a virus use Fowlpox. Preferably as Fowlpox virus use vaccination strain of the virus of chicken pox or such as Fowlpox vaccine, manufactured and supplied to the trade by the company SOLVAY under the name POXINE or vaccine, manufactured and supplied to the trade by the company SOLVAY under the brands CHICK-N-POX, POULVAC CHICK-N-POX, CHICK-N-POX TC, POULVAC CHICK-N-POX TC, POULVAC POXINE, POULVAC P. In the preferred method as Fowlpox virus use the vaccine supply trade company SOLVAY under the brands CHICK-N-POX, POULVAC CHICK-N-POX, CHICK-N-POX TC, POULVAC CHICK-N-POX Tc, POULVAC POXINE, POULVAC P, abbreviated CNP.

Weakening is obtained by successive passages through the embryo or, if the virus was adapted to cultured cells, as a result of passing through the cell culture.

Under elements, capable of expression of a gene which encodes a heterologous protein in the cell, sargenti promoter and terminator, i.e. the elements known to the specialist, such as, in particular, are described, for example, Moss, 1990. In General the use of the promoter for Pox. Commonly used promoter for Vaccinia or Fowlpox. Preferably use the P11 promoter and promoter P7.5 for Vaccinia, such as described by Venkatesan et al., 1981; Cochran et al., 1985 and Bertholet et al, 1985.

Under at least one DNA sequence that encodes all or part of the protein that is heterologous in relation to the Avipox virus, understand any fragment of DNA extracted from the genome, or a copy DNCC or even synthesized DNA accompanied by signals of expression, a sequence which encodes all or part of the protein, alien Avipox.

Fig. 1-11 were prepared to allow a better understanding of the invention.

Fig. 1 depicts the construction of recombinant Avipox virus, modified according Mackett et al. 1984. Cells, infected (I : infection) virus Fowplpox (FPV), transfections (T : "transfection") plasmid transfer. This plasmid (P) is oriented promoter (Pr), to ensure the expression of adjacent heterologous gene (q), with the and with the other hand which are the sequences of viral DNA. Homologous recombination (R) is akademiese viral genome. This leads to the inclusion of a heterologous gene into the viral genome. This genome can be packaged and produce recombinant virus particles (VP). N indicates the nucleus of the cell (C).

Fig. 2 schematically depicts an end part of the genome of Fowlpox with TIR to the right (TIR-R) and left (TIR-L), denoted by rectangles, and the only sequence indicated below. The dotted line indicates the Central region of the genome. The fragment EcoRI restriction from TIR-L has a value of 6.2 to, whereas a similar fragment from TIR-R has a value of 9.0 to. This diagram shows that these two fragments have a common sequence, and only the sequence. Shows the BamHI site located below EcoRI TIR.

Fig. 3 depicts a restriction map of plasmid pTIRBI (PTIRBI) for some individual sites and the position of the ORF. A single BamHI site used for klonirovanie heterologous genes.

ori (ORI): start replication of plasmids in Escherichia coli (E. coli); Ap: gene for resistance to ampicillin; ORF 1 - ORF 6; open phase for reading in Fowlpox.

Fig. 4 is a drawing similar to Fig. 3, but for plasmids pTIRB2 (RTIRB2).

Fig. 5 depicts a restriction map of plasmid pIILac (PIILAC). This plasmid carries the LacZ gene of the microorganism E. coli pkaii in E. coli; Brin + : chain, Packed in phage M13 : AP : gene for resistance to ampicillin.

Fig. 6 depicts an example of a vector transfer for gene LacZ in the TIP. This vector is a plasmid pTIRBIP75Lac (PTIRBIP75LAC). Cassette P7.5-LacZ cloned in the BamHI site of plasmid pTIRBI (in clockwise direction). P7.5 : the P7 promoter.5 for Vaccinia.

Fig. 7 is a schematic depiction of inclusions LacZ gene (represented by dashed rectangle) in the genome of the CNP. Cassette P7.5 LacZ and PII-LacZ included in the terminal region (TIR) in one direction or another. The TIR region on the right is not represented. The arrows indicate the direction of transcription of the genes depicted as rectangles. The scale is not respected.

Fig. 8 depicts a segment for IBDV, the position of the primers and various fragments obtained by reverse transcription and amplification ARN segment of A strain Edgar.

A : the structure of the ORF

p : "primers" that are used to implement the amplification of segment A.

> < : the orientation of the primers relative to the coding sequence of segment A.

fgt PCR fragments generated by amplification ARN segment A.

pb : base pairs.

Fig. 9 (9a to 9e) depicts acids phases ORF 1, 2 and 3.

Primers 0,1, 1b, 2, 3, 4, 5, and 6 : primers used to generate (by reverse transcription and amplification of DNA fragments corresponding to segment A strain Edgar.

Fig. 10 depicts a scheme for the reconstruction of the DNA segment corresponding to a segment of A strain Edgar, on the basis of the fragments obtained by reverse transcription and amplification.

* : directed mutagenesis.

Fig. 11 depicts a restriction map of plasmid pTIR75EI LAC (PTIR75EILAC).

Part plasmids indicated by a solid line, corresponds to the homologous sequences of the genome of FPV. ori (ORI) : begin replication of the plasmid in E. coli; Ar : the gene for resistance to ampicillin; P7.5 : the P7 promoter.5 for vaccinia; ORF 1 - IBDV : ORF of the first segment of A strain Edgar; P11 : P11 promoter for vaccinia; LACZ : gene, encoding-galactosidase; pb : base pairs; OFR 1 - ORF 6 : open phase for reading in Fowlpox.

Fig. 12 depicts the description using ELISA test IBDV proteins expressed in recombinant FPV (IBDV). The description was carried out as follows: 50 μl of cell suspensions and serial twofold dilutions of these suspensions were placed in a Cup. After incubation of the second antibody anti-IBDV (monoclonal anti-VP3 or anti-VP2) was done by the STN with alkaline phosphatase.

Fig. 12a corresponds to the detection using monoclonal antibodies anti-VP3. Fig. 12B corresponds to the detection by means of monoclonal antibodies anti-VP2.

These figures abcissa depicts serial dilution in 2 times of cell extracts in logarithmic units, and the ordinate represents the ratio of the absorptions measured at 690 mm and 405 mm

Fig. 13 depicts the description using Western blot IBDV proteins expressed in recombinant FPV (IBDV). This study was performed as follows: cell suspensions centrifugeuse with acceleration 3000 g, and the resulting emergent products are subjected to ultracentrifugation with acceleration 185 000 g; residue obtained after two centrifugation processes are combined and again converted into a slurry by using buffer solution PBS (Phosphate Buffered Saline, Gibco).

Sucks protein in an amount of from 20 to 40 mcg for recombinant FPV/IBDV and 5 μg for cells infected with the help of IBDV (positive control) is applied per well on the gel type acrylamide-SDS. After transfer to the membrane IBDV proteins are detected using a polyclonal serum that recognizes all IBDV proteins, Fig. 13A, or by using monoclonius and mating system of streptavidin-alkaline phosphatase. AM (mW): proteins with known molecular masses.

The method used for carrying out the invention is schematically shown in Fig. 1 (modified according Mackett et al., 1984). Cells infected with the help of Avipox or Fowlpox, transfections the plasmid transfer. This plasmid carries the promoter is oriented to allow the expression of adjacent heterologous gene, bordered on both sides by sequences of viral DNA. Homologous recombination occurs in the cytoplasm of cells between sequences that fringe gene, and sequences located on the viral genome. Thus, there is the inclusion of a heterologous gene into the viral genome. This genome can be packaged and produce recombinant virus particles. This method of inclusion by infecting cells with the help of Pox with subsequent transfer of the plasmid was designed to Vaccinia and is commonly used in many laboratories for constructing recombinant Pox (Mackett et al., 1985).

The method according to the invention usually includes the following stages:

the first stage is the construction of plasmid transfer, permitting screening,

the second stage consists in cloning genes that code is combinant Avipox viruses, and

- the fourth stage is to conduct tests on vaccination with recombinant viruses.

Preferably the method includes the following stages:

a. Construction of plasmid transfer, permitting screening:

- cloning of fragments carrying TIR,

- the inclusion of a site cloning in TIR,

- cloning of promoters P11 and P7.5 of Vaccinia;

- construction of the cassette carrying LacZ gene, and cloning it below P11 or P7.5,

- cloning elements P11 LacZ or P. 7.5 LacZ in plasmid transfer TIR.

b. Construction of recombinant viruses CNP (LacZ gene to perform pre-test):

- transfer QT35 cells virus CNP and plasmid transfer,

- screening with subsequent purification of the recombinant thanks to the LacZ gene expression,

analysis of recombinant genomes and gene expression of LacZ

c. Test vaccination:

- vaccination of chickens with recombinant viruses and protection from avian pox made using recombinant LacZ virus.

d. Cloning of genes encoding heterologous antigens (heterologous protein) plasmid transfer:

- cloning of genes encoding heterologous antigens below P11 or P7.5,

- cloning cassettes P11-ant the BR> - transfer QT35 cells virus CNT and plasmid transfer,

- screening with subsequent purification of the recombinant viruses due to the LacZ gene expression,

analysis of the expression of the antigen on the cell culture and analysis of the genome of the recombinant virus.

f. Vaccination:

vaccination with recombinant viruses CNP,

- assessment of the degree of protection due to the expression of the antigen that is heterologous with respect to CNP.

The invention relates also to sequences of transfer, which allow you to enable heterologous DNA into the genome of a virus, Avipox, as defined above, and, in particular, at sites B1 or B2. The invention relates to also to plasmids that carry these sequences. Under the plasmid transfer understand plasmid, which contains a heterologous gene under the control of a promoter recognized by the Avipox virus, which is bordered on both sides by sequences located on either side of site B1 or from the site B2. The length of these sequences homologous to FPV that fringe the heterologous gene, should be sufficient to allow recombination of the left and right of the heterologous gene. Usually choose the sequence value is not less than 1 ceverha and 4321 nucleotides from the bottom, and for B2 - about 3070 nucleotides at the top and 3100 nucleotides from the bottom. Plasmid transfer involved in the process to isolate recombinant Avipox in a way that enforces the migration of cells quail QT35 previously infected with a virus, Avipox.

The invention relates also to the cultivation of eukaryotic cells infected with recombinant Avipox virus, derived from a weakened strain of the virus and Avipox containing non-core parts of its genome at least one DNA sequence encoding all or part of the protein that is heterologous to the virus, Avipox, as well as elements capable of expression of this protein in cells infected with the indicated recombinant virus, and non-core part is formed of non-coding vnutrivennoi region of the Avipox virus, which is located between two OFR, including their expression signals.

Preferably used cell cultivation poultry. Good results were obtained with cell culture quail. Excellent results were obtained with cell culture quail QT35 such that the described Cho (1981).

Recombinant Avipox virus according to the invention develops as vectors that can Express floor is where the virus according to the invention as a vector, able to Express fully or partially heterologous protein, such as, in particular, the antigen.

The invention relates also to a vaccine containing recombinant Avipox virus, derived from a weakened strain of the virus and Avipox containing non-core parts of its genome at least one DNA sequence encoding all or part of the protein that is heterologous to the virus, Avipox, as well as elements capable of expression of this protein in cells infected with the indicated recombinant virus, and a minor portion of the genome formed by non-coding vnutrivennoi region of the Avipox virus, which is located between the two ORFS, including their expression signals.

Under the heterologous protein is understood as any protein or part protein that alien in relation to the Avipox virus, the protein may be a natural or modified. In the General case, this protein may be an antigen, allowing you to develop a vaccine against pathogenic organism. Usually antigens are, in particular, proteins of agents that infect birds: viruses, bacteria, mantles, mycoplasmas, protozoa single-celled organisms, fungi and worms.

This, in particular, Adenoviridae, such as a virus,and Gumboro (IBDV), Coronaviridae such as infectious bronchitis virus (IBY), Herpesviridae (such as a virus disease of Makara (MDV) and the virus responsible for infectious laryngotracheitis (ILTV), Orthomyxoviridae, such as the virus responsible for influenza (Influeusa), Paramyxoviridae, such as Newcastle disease virus (NDV), Picornaviridae, such as the virus responsible for encephalomyelitis (AE), Roeviridae responsible for tenosynovitis, Retrovirus, such as the virus responsible for lymphoid leukosis (LL), the virus responsible for anemia in chickens (CAV).

This, in particular, mantles, such as Chlamydia psittaci.

This, in particular, Mycoplasma, such as Mycoplasma gallisepticum and M. synoviae.

This, in particular, bacteria, such as Escherichia coli, Haemophilus, including H. paragallinarum, which is responsible for the common cold, Pasteurella, including P. multocida, which is responsible for cholera, Salmonella, including S. gallinarium, responsible for typhoid fever, and S. pullorum, Clostridium, including C. botulinum, which produces the toxin responsible for botulism, C. perfringens and C. septicum responsible for dermatitis, C. colinum, Campilobacter jejuni, Streptococcus aures.

This, in particular, the simplest single-celled organisms, such as Eimeria responsible for coccidiosis and Cryptosporidiosis baileyi responsible for cryptosporidia.

This, in particular, worms, gillus fumigatis.

Preferred antigens are antigens of the virus diseases of Gumboro (IBDV), infectious bronchitis virus (IBV), the virus responsible for anemia in chickens (CAV), the simplest single-celled organism Eimeria responsible for coccidiosis, Newcastle disease virus (NDY) and Marek's disease virus (MDV).

Particularly preferred antigens are antigens of the virus diseases of Gumboro (IBDV), infectious bronchitis virus (IBV), the virus responsible for anemia in Kus (CAV) and the simplest unicellular organism Eimeria responsible for coccidia. Good results were obtained under the following conditions:

for IBDV : fully or partially open phase for reading, including polyprotein and part of polyprotein. Such proteins are, in particular, VP2, VP3, VP4. It also includes all combinations or modifications. These modifications represent, for example, the inclusion of a site cleavage,

for IBV : antigen E2,

for Eimeria : natural surface TA4 antigen and surface antigen whose sequence site of proteolytic cleavage has been modified,

for CAV : protein P50.

Excellent results were obtained with IBDV, and heterologous protein is one which this part included protein VP2, as is shown in Fig. 9.

The DNA sequence may encode protein completely or fragments of the protein and can cause the animal corresponding to the response of the immune system. Other molecules that are different from the antigens can also be taken into account, including growth factors, and immune modulators, such as interleukins. The aim of the present invention is also a production in vivo (in poultry) factor that is involved in the metabolism of the animal, such as, for example, growth hormone or factor, inducing the latter. The present invention relates also to obtain protein or peptide by culturing cells in vitro or in vivo in animals. Depending on the introduced heterologous protein it can act, in particular, as an enzyme, as a component of food or health of humans or animals as a pharmaceutical product for medical or veterinary use.

On the other hand, can be generated multiplet recombinant viruses carrying several heterologous genes, i.e., by cloning of several genes in the same genome, in the same site or in different regions or sites. Polyfunkci The

Target animal for Avipox virus according to the invention are birds, particularly poultry. The recombinant virus can also be applied to objects other than birds.

The vaccine according to the invention may be introduced in the form of various forms known to the specialist. Typically, the vaccine is given orally with food or water to drink, the intranasal route, the subcutaneous injection, in the form of an aerosol spray in the intramuscular injection or by perforation of the wing in accordance with the method, called "wing web. Preferably the vaccine is given by piercing the membrane of the wing in accordance with the method, called "wing web, intramuscular injection or as a result of subcutaneous injection.

The vaccine formulation according to the invention is made known to the specialist way. Typically vaccination composition contains stabilizers, the appropriate method of vaccine administration. The vaccine may be stored in lyophilized form.

The present invention is illustrated in the subsequent examples.

Example 1. Purification of virus CNP and its DNA.

As Fowlpox virus use vaccination strain supply trade company Solvay under the occurs from the soil strain, missed several times through eggs and adapted to cells in vitro in the 2 passing through CEF (chicken embryo fibroblasts) and 5 pass through the cell progeny quail QT35 described Chno (1981). Cell progeny quail QT35 obtained from Dr.Moscovici (6816, Northwest, 18th Avenue, Gainesville, Florida 32605, United States), which it has allocated and distributes it.

CNR cultivated at the cellular progeny QT35, environment for growth which consists of E199 (Gibco 500 ml), F12 (Gibco 500 ml), LAH (Gibco, hydrolyzed lactalbumin, 25 ml), FCS (Gibco, Foetal Calf Serum (25 ml) and fructose (5 ml of solution with a concentration of 200 g/l), incubation at a temperature of 38oC, 3% CO2. Normal multipletness of infection (MOI) is 0.01 of virus per cell, and cells at 80% are in the mail merge.

Viral colony was purified as follows: cells infected with Fowlpox virus CNP and extracted from phosphate buffer solution PBS (Phosphate Buffered Saline, Gibco) centrifugeuse with acceleration 6000 g (15 minutes). The precipitate after centrifugation again translated in suspension in a buffer solution of Tris (pH 9.1 mm), treated with trypsin (final concentration of trypsin 0.25 mg/ml; Gibco) and ultrasound. This product is placed in an environment with a 36% sucrose (weight/ogvernor the Tris solution with a concentration of 1 mm, pH = 9,0. Viruses centrifugeuse last for 30 minutes with acceleration 40000 g. The precipitate after centrifugation is extracted in a buffer solution for the decomposition of lysine : 10 mm Tris-HCl, pH = 8.0, 10mm EDTA, 1% SDS (sodium dodecyl sulphate) and 500 ág/ml proteinase K (Boehringer) 0.1 mg/ml RN ASE (Boehringer) and incubated for 2 hours at a temperature of 37oC. Extracted in phenol DNA precipitated in ethanol. It was cleaned about 20 μg DNA from 40 infected cups with an area of 150 cm2.

Example 2. Construction of genomic Bank EcoRI and genomic Bank BamHI for CNP

Used genetic methods describe in Maniatis et al., 1982. Restriction enzymes, polymerase, ligase and phosphatase were supplied by firms Pharmacia, Boehringer et Biolabs. Synthetic DNA were raised by the company Eurogentec.

The restriction fragments of viral DNA by EcoRI or BamHI were linked in the vector PUC18, described by Messing (1983), and is introduced into the bacterium E. coli MC1061 (araD139, (ara, Leu) 7697, LacX74, galU, galK, hsdR, strA, sold by Pharmacia). Both genomic Bank stored at -70oC in glycerol in the form of two transformed cell suspensions. Eighty percent of plasmids contain one on. Seventeen fragments BamHI and 45 EcoRI fragments were differencer the amount described thus inclusions BamHI is 82 and 210 to to to EcoRI fragments. The genome size Pox birds close to 300 TB (Coupar et al. , 1990). The selected fragments are thus 25% for BamHI and 70% for EcoRI from the whole genome.

Example 3. Cloning of fragments carrying TIR

The opposite terminal repeat (TIR) represent two identical sequences located at the ends of the genome of viruses Pox and having the opposite direction. Sequence size over 17 to one end of the genome of strain HP Fowlpox virus was published (Campbell et al., 1989 and Tomley et al., 1988). It contains about 10 to repeat the sequence and 7.0 to its adjacent sequence.

Carry out the cloning of the EcoRI fragments for TIR. Repeat the sequence contains an EcoRI site. As shown in Fig. 2, two fragments are cloned EcoRI: everyone has one part TIR and one single contiguous sequence. A single sequence is limited to the closest EcoRI site of TIR. These two fragments were selected by the oligonucleotide, complementary to a sequence located in the repeat part. The first fragment has a size of 6.2 to and will be called (by agreement) TIR-L. the Second is the size of 9.0 to and will be called (by agreement) TIR-R. Pic is>The connection between TIR and single sequence identified by comparing sequences TIR-L and TIR-R, located on the nucleotide 4007, and the BamHI site sequence, published in Tomley et al., (1988), is the nucleotide number 1. The connection is in the open phase for reading, called ORF 3 these authors.

Example 4. The creation of the site-directed mutagenesis sites BamHI TIR

Open phase for reading (ORF) were identified in the analysis of nucleotide sequences TIR. In particular, OFR 1 is located between nucleotides 416 and 1674, OFR 2 is located between nucleotides 2166 and 2671 and, finally, ORF 3 is located between nucleotides 3606 and 4055 and is encoded on the complementary chain. We accept as nucleotide N 1 is the first nucleotide G BamHI site of TIR. These ORF limit two large non-coding vnutrennih sequence, called the area1between ORF 1 and ORF 2, on the one hand, and region2between ORF 2 and ORF 3, on the other hand. These two nutrigenie region are selected as areas for inclusion.

A single cloning site was introduced by directed mutagenesis (equipment for mutagenesis film with other restriction enzymes cut sites, including Bg11 and Bg12. The position of the cloning site was chosen approximately in the middle of each vnutrivennoi area to avoid any risk to separate the signals of transcription of their respective genes, i.e.:

- in the1: site B1 is a sequence 5' GATC 3' to the nucleotide 1842, transformed into BamHI GGATCC by including G 5' C 3';

- in the2: site B2 is a sequence 5' GGATT 3' to the nucleotide 3062, transformed into BamHI by mutation of the second T in CC.

Two BamHI site located in the plasmid, which carries a fragment of TIR-L (example 3). One site of the vector pUC18. The second site is from TIR and is located at 71 below the nucleotide EcoRI site selected for cloning (Campbell et al., 1989, Fig. 2). These two BamHI site were subjected to deletions of the plasmid carrying TIR-L, by restriction with BamHI followed by filling the Klenow polymerase and binding. Plasmid, which in this case turns out, is a pTIRI.

To generate a BamHI site in the B1 fragment Hind P value of 0.9 to plasmids pTIRI was cloned in the vector pBSPIus supply trade firm Stratagen. Primer mutagenesis were:

5' TTTCGAAAGACTTTGGAT

CCGTAGTATAATATTATA 3'

Emphasize the work BamHI site in B2 Xbal fragment size of 1.7 to plasmids pTIRI was cloned in PBSRI us. Primer were:

5' TATATCACGGGATCCA

AAAGGTTATTAGTAGTC 3'

The underlined nucleotides, i.e., GGATCC, represent a sequence recognizable by a BamHI site.

Re-enabling each mutant fragment in pTIRI is the result bindings:

- B1: Hind 3 - Scal plasmids pTIRI (1491) + Scal-Aat 2 of pBSPIus after mutagenesis (485) + Aat 2 - EcoRI from pTIRI (4220) + EcoRI-Hind 3 of plasmids pTIRI (2635)

for B2: Xhol-SpII from plasmids pTIRI (8512) + SpII-Xhol of pBSPIus after mutagenesis (318).

These two binding generated vectors pTIRBI and PTIRB2. Their maps of the restriction shown in Fig. 3 and 4.

On the basis of plasmids pTIRI was generated plasmid pTIRBID in the deletion of your fragment EcoRI-Hpal value 2657.

Example 5. Cloning of the promoter P11 and P7.5 of Vaccinia.

P11 and P7.5 are known promoters for Vaccinia. P11 is a promoter of the gene encoding the protein P11, which undergoes transcription during the late phase of viral infection. P7.5 is a promoter of the gene encoding the protein P7.5, which undergoes transcription simultaneously during the immediate and late phase.

Construction of vectors with individual sites Bg12 and Bc11.

Two vectors for the cloning cassette, compatible with BamHI. The sequence of sites Bg12 and Bc11 were introduced into the BamHI site of pBSPIus by cloning the following synthetic DNA:

< / BR>
Vectors are pBSLK1 and pBSLK2. The linkers are:

pBSLK1... Ava/Smal-BamHI-Bc11-Bg12-Xbal...

pBSLK2... Ava/Smal-Bg12-Bc11-BamHI-Xbal...

Cloning of promoter P7.5

Fragment Bc11-BamHI length 143 on plasmids pGS20 contains the promoting sequence p7.5 (Mackett et al. , 1984). This sequence is shown below (Venkatesan et al., 1981, Cochran et al., 1985). The trailing and the local promoters are underlined. The last base at the BamHI separated on 10 from the ATG initiating gene.

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Fragment Bc11-BamHI was cloned in sites Bc11 and BamHI plasmid pBSLK1 and pBSLK2, generating plasmids, respectively p1P65 and p2P75. To allow digestion Bc11, plasmids pBSLK1 and pBSLK2 extracted from strain JM110, which is available in ATS (American Type Culture Collection) under the code of ATSS 47013.

Cloning of promoter P11

The sequence of the P11 promoter was cloned in the form of synthetic DNA based on sequence data Bertholet et al., (1985), Hanggi et al. , (1986) showed that a fragment of 30 nucleotides above the first nucleotide ATNm contains the promoter.

The synthesized DNA is St. pBSLK2, to generate p2P11. Bg12 fragment-BamHI from p2P11 was then cloned in the sites Xho2-BamHI from pACYC184. pACYC184 is a vector, described by Chang et Cohen (1978). Linking between Xho2 (GGATCT) and Bg12 (AGATCT) restores Bg12.

Sequence synthetic DNA carrier p11, is:

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Example 6. The design of the cassette carrying LacZ gene that is compatible with the restriction site BamHI.

Expression of the LacZ gene will provide an opportunity for screening recombinant viruses.

Cassette Bg12-BamHI carrying LacZ gene, was transformed by directed mutagenesis. This cassette was cloned in the plasmid pBSPIus (stratagen), subject to the deletion of the LacZ fragment, the latter in order to avoid recombination between two homologous sequences: one of the LacZ and the other on LacZ. This plasmid is a pASMut LacZ1. The sequence at the ends of the LacZ gene after mutagenesis are described below. Numbers correspond to the numbers of amino acids of the natural protein.

At the 5'end:

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On a horse 3':

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Example 7. Cloning of the gene LacZ promoters behind P7.5 or P11.

The LacZ cassette, Bg12-BamHI from pBSMut 1 LacZ was cloned in sites Bg12-BamHI from p2P75 and p2P11 to generate sootvetstvenno colonies have blue color on the environment, containing X-Gal, a chromogenic substrate: 5-bromo-4-chloro-3-indolyl--D-galactopyranoside), which causes a blue color under the action of a-galactosidase. This test proves that the gene is functional. Cassette P11 Lac in the form of Bg12 fragment-Hind 3 plasmid P11 Lac was also cloned in the sites Xho2 and Hind3 plasmids pACYC184, generating pACP11 Lac. Map p11 Lac is shown in Fig. 5.

Example 8. Cloning cassettes p75 Lac and P11 Lac in plasmid pTIRB1 and pTIRB2

Bg12 fragment-BamHI from p11 Lac (or pACP11 Lac) and Bg12 fragment-BamHI from p75 Lac carry the cassette promoter P7.5 or P11, followed by the LacZ gene (example 7). These fragments were cloned into the BamHI sites of plasmid pTIRB1 and plasmids pTIRB2 (example 4) in the two possible directions with the help of pathotype LacZ for sifting. Were allocated to six plasmids of the eight possible recombinant. Their numbers below. We assume that the orientation is " + "is the one that places the LacZ gene transcription in the direction from ORF 1 ORF 2 or ORF 2 to ORF 3, and the orientation is" - " is the one that corresponds to the opposite orientation. The names of the plasmids "P75 Lac" and "P11 Lac" mean that these tapes have orientation "+", whereas "LacP75" and "LacP11" means that these cartridges have the orientation is "-". As an example in Fig. 6 provides a map of plasmid pTIRBIP75 Lac (t Site B1 or B2 website cloning BamHI allocated in TIR.

The PR - orientation. The orientation is "+", when the direction of transcription of the LacZ coincides with the direction from ORF 1 ORF 2 or ORF 2 to ORF 3. The orientation is "-" is the opposite.

Name - the name of the plasmid transfer.

Example 9. Clarifying the terms of the transfer and screening and selection of recombinant viruses CNP/LacZ

The principle of constructing recombinant virus CNP shown in Fig. 1. The Protocol of the experience of migration is the following:

In day D = 1 : in terms of a Cup with an area of 25 cm2inoculant 2,5 106QT35 cells in the growth medium (composition described in example 1).

In day D = 2 transfer.

Viruses: 1 ml of freeze-dried vaccine CNP in vitro regidratirtee using 3 ml of sterile water, Milli-Q and stored at -70oC. This colony viruses thawed and subjected to mild ultrasonic treatment for 1 minute. Then diluted in the environment of the cultivation, denoted E119-F12, which corresponds to the environment E119 described in example 1, but without'lah, serum and fructose, in order to achieve a multiplicity of infection value of 0.05 viruses per cell in 2 ml of medium. Then replace the environment for smidi: mix 20 μg of plasmids with 400 μl of water and 100 μl of 1.25 M CaCl2, add one drop of 500 μl of buffer solution BBS /BES Buffer Saline/ double concentration /50 mm BES (Sigma), 280 mm NaCl, 1.5 mm Na2HPO4, pH = 7.0/ and incubated in the range from 15 to 30 minutes at a temperature of 25oC. Then 1 ml of the preparation of plasmids is applied to the cells after removal of the medium. Then incubated for 30 minutes at a temperature of 38oC. Then was added 4 ml of medium without'lah, but with the addition of 5% FCS and 15 mm solution of HEPES (Sigma) at pH = 7,2. Again incubated for 4 hours at a temperature of 38oC. Then replace the medium in 5 ml of medium growth, denoted E199 (composition described in example 1).

In day D = 0: gather grown viruses as described in example 1.

Screening is based on the getting-galactosidase activity detected in the result of experience on the Petri dish "plaque assay". Protocol experience for one of the Petri dishes of 6 cm is as follows.

In day D = 1 : inoculant 2,5 106cells in 5 ml of medium for growth.

In day D = 2 : infect the culture with 1 ml of virus dilutions 1: 10 and 1:100, incubated for 4 hours at a temperature of 38oC, then add 4 ml of medium for growth (composition described in example 1).

In day D = 3 : replace Wednesday at 5 ml layer of agarose, abrasco), 0.5 ml of LAH (Gibco) and 0.1 ml of HEPES (pH = 7,2, the final concentration 15 mm) and 1.0 ml of FCS. The melted agarose and the mixture is cured at a temperature of 38oC before mixing. Allow the agarose to form a jelly for 30 minutes at ambient temperature, then incubated at a temperature of 38oC.

In the D-day = 6 : cover the culture with 2 ml of 1% agarose in PBS (Gibco) containing 0.3 ml/ml X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside, Boehringer). X-Gal dissolved with a concentration of 30 mg/ml in DMSO (dimethylsulfoxide, Fluka).

In the D-day = 7 : calculate the number and proportion of blue areas in relation to the number of unpainted areas. Then individually selected 5 areas using a Pasteur pipette. Keep the viruses in 500 μl of medium for growth at -70oC.

The recombination frequency for each plasmid is shown in the table. 2. Recombinant viruses denoted by the symbol "V" followed by the number plasmid transfer (as defined in example 8). The percentage of recombinant varies from 0.1% to 0.5%, which corresponds to literature data for Pox. The image of recombinant genomes is given schematically in Fig. 7.

Note to the table. 2.

Blue/total - number chistka recombinant viruses CNP/LacZ

The clone is obtained by sequential purification one blue region on the Petri dish (as described in example 9). In principle, when all areas are blue, all viruses expressing LacZ gene and is no longer contaminated by wild virus. Three to four transitions necessary to achieve such uniformity. For example, the evolution of the share of blue areas during transitions was as follows (table. 3).

Note to the table. 3.

Blue/total - number of blue areas/total number of fields.

% - percentage of blue areas.

In pictures obtained by the method of electron microscopy, slice QT35 cells infected with recombinant virus V8, clearly illustrate typical patterns of Pox viruses, in particular internal structure, which has the shape of a dog bone.

Example 11. Production of large quantities of each recombinants

Six recombinant were increased as a result of three consecutive transitions on the cups. The title of the second transition varies from 4105to 1,110 land only7TCID50 (Tissue Culture Infections Dose) per ml Only virus V10 has a titer of less than 104. Other recombinants breed is almost as good as the wild type virus, for which the titles on LacZ, embedded in TIR

Gene LacZ embedded in the viral genome, can be stable or unstable depending on the type of recombination that has occurred (this is due to the Shuman et al., (1989). So, if there are two recombination on both sides of the inclusion, the introduced gene LacZ is stable. For example, a simple recombination leads to the inclusion of the entire plasmid transfer and, consequently, gene LacZ. This virus gives the blue areas. However, the viral genome carries homologous sequences straight direction, the recombination of which may lead to loss of inclusion and to the appearance of unpainted areas.

On the other hand, viruses, Pox change in the sequence of one TIR can be transferred to another TIR during cycles of infection and virus replication. Double recombination can implement LacZ in TIR-L, then the area of this virus becomes blue. During subsequent infections recombination between this recombinant TIR-L and wild TIR-R generates a mixed viral population TIR-L/LacZ, TIR-L/LacZ-TIR-R/LacZ and WT /wild/. Other recombination may also generate the last possible type, which is a TIR-R/LacZ. Therefore, the blue region can contain three types of viruses: viruses, and LacZ.

Check the homogeneity of viral drug "plaque assay" after several consecutive infections in liquid medium (example 9). "Plague assay" 3-year transition viruses V3, V4, V5, V8, V9 and V10 have the following percentages of cyan areas (PL. 4).

Note to the table. 4.

P3 and P6 - 3-th and 6-th transition of the virus.

Blue/total - number of blue areas/total number of fields.

% - percentage of blue areas.

- was not carried out

Thus, the recombinant viruses V8 and V9 are homogeneous. Recombinant V8 is stable up to 6-th transition. For example, preparations of viruses V3, V4 and V5 are not homogeneous. They contain a proportion of from 5 to 20% of wild-type virus. This share is even more important for virus V10.

Example 13. Analysis of the genomes of CNP/LacZ method Southern blot.

Check the homogeneity of the viral preparation by the method of Southern blot (Maniatis et al., 1982). The restriction fragments of the genomes of recombinant viruses and non-recombinant viruses vary by size.

Protocol experience to produce viral DNA is as follows:

In day D = 1: in the Cup area of 25 cm2containing 5 ml of culture medium, inoculant 2 106QT35 cells.

In describe (Costar). Centrifuged for 10 minutes with acceleration 6000 g. To the residue after centrifugation add 500 μl of buffer solution for the decomposition of lysine, which consists of 10 mm Tris-HCl, pH = 8.0, 10mm EDTA, 0,1% SDS, 0.1 mg/ml RN ASE, 0.1 mg/ml proteinase K. Transfer of cells into Eppendorf tube. Then mix the resulting suspension and incubated for 1 hour at a temperature of 50oC. Then the aqueous phase is extracted with 3 times the phenol with chloroform. Precipitated DNA using 0.3 M sodium acetate and 2 volumes of ethanol at -20oC for 15 minutes. Centrifuged for 10 minutes with acceleration 18300 g. The precipitate after centrifugation was transferred to suspension by 500 ál of water. About 5 μl of this suspension full DNA is sufficient for the method.

For site B1

The genomes of EcoRI digested. The probe is a plasmid pTIRB1 LacP75, digested by EcoRI. This probe distinguishes between recombinant and related fragments TIR-L and TIR-R Fragments have a size of 9.3 and 6.2 to wild virus, 7,4, 5,1 (doublet) and 4.4 for virus V3, 7,4, a 4.9 and 4.4 to to virus V8 and, finally, for virus V9 - 10,4, to 7.7 and 2.0 (doublet) to. Viruses V8 and V9 bought two copies of the LacZ gene (one in each TIR) and do not contain a fragment of the TIR related type. On the contrary, in Sulmona to conclude, that site enable B1 is not essential to the development of the virus and its growth on the cells during culturing. On the other hand, because the LacZ gene has a size of approximately 3 to, the allocation of these double recombinant proves that it is possible to implement 6 to on the virus genome CNP.

For site B2

Results according to the method of Southern show the presence of recombinant fragments of TIR-L and TIR-R, but also of related fragments in the preparations of viruses V4, V5 and V10. These three drugs, therefore, are not homogeneous.

Example 14. Alternative for selection of double recombinants in the website include B1

The evolution of recombinations in TIR predicts the emergence of a stable double recombinants in the progeny virus carrying the wild and recombinant TIR TIR. Viral region re-allocated from the blue region in a Petri dish "plaque assay" or as a result of new experience "plaque assay", or on plates for titration with 96 wells. You can analyze the genome of each new region or by the method of Southern or PCR. These various possibilities have been considered.

The method of Southern, carried out on the genomes of viruses from 10 different blue areas obtained on a "plaque assay" for the 3rd transition virus V3, showed, Celestine titration dilution is such that zero or one viral region accounts for one hole. This is the optimal dilution is estimated using preliminary titration. Good results are obtained when infection plastics for titration (200 µl per well, 20 ml per plate) using 50 µl of viral suspension, taken from the Petri dishes "plaque assay after extraction in 1 ml When the area is well visible, that there are wells that contain one region. After freezing and subsequent thawing take samples 100 ál of medium per well.

In the case of PCR total DNA quickly extracted the following conditions: centrifuged for 10 minutes with an acceleration of 1000 g, extract the residue after centrifugation at 200 μl of buffer solution for the decomposition of lysine (10 mm Tris-HCl, pH = 8.0, 10mm EDTA, 0.1 percent SDS) with the addition of RN-basics (0.1 mg/ml) and proteinase K (0.1 mg/ml) and incubated thus obtained mixture for 1 hour at a temperature of 50oC. Then the aqueous phase is extracted with three times in a mixture of phenol/chloroform. From this aqueous phase precipitated DNA by ethanol. Remove the precipitate in 50 ál of water. Use 10 µl of this suspension method for PCR. Conditions used for PCR are those which are recommended by the company Per the 168 and 5169 hybridize with the two sides of the site include B1, respectively above and below this site. The sequence is as follows:

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Fragment size 220 increases by wild TIR, and the fragment size over 3 Ko for recombinant TIR containing the LacZ gene. To identify the gene LacZ second amplification uses primer 5168 below site B1, and primer 3254, located in the LacZ gene. Increasing fragment size 674. This is a reasonable selection of primers allows, therefore, to distinguish recombinant TIR and non-recombinant TIR. The viral suspension is homogeneous, double recombinant, if a fragment of WT (wild) is not increased. Control is virus genome WT and plasmids pTIRB1 and pTIRB1 Lac (PL. 5).

Note to the table. 5.

- - no enlarged.

Example 15. The expression of LacZ gene in the tissue cultivation of recombinant viruses CNR/LacZ

The activity of-galactosidase (- gal) was measured using o-nicotinyl--galactopyranoside (ONPG) as a substrate. Enzyme changes ONPG in galactose and o-NITROPHENOL (BIP) is yellow, the amount of which is measured in the absorption at 420 nm. This absorption is transferred in units-gal using the calibration curve in accordance with ol CLASS="ptx2">

This experience "Gal" is, as will be seen in the Cup with the surface 25 cm2. For Pox viruses distinguish between immediate and delayed phases of infection. The nearest phase is the shortest; it lasts about 6 hours. It ends when the replication of the genome. Delayed phase begins with replication and ends at the end of the cycle of infection by the release of viral particles, i.e., three days later. In order to have sufficient LacZ gene expression during the short phase, prodlevajut artificially next phase using cytosine-D-arabinofuranoside (AGAS), which is an inhibitor of replication.

a. Cells.

In day D = 1: on the Cup inoculant 3 106QT35 cells in 5 ml of culture medium.

In day D = X2: spend infection with multipletness infection 3 of the virus to the cell. Incubated for 1 hour at a temperature of 38oC. Then remove the viral inoculum with a pipette. Then add (one Cup) 5 ml of medium to save with addition or without addition of AGAS (Sigma) with a concentration of 40 μg/ml Incubated in culture for 16 hours at a temperature of 38oC.

b. Picking up in the D-day = 3

Soskrebajut cells with cups.

Centrifuged culture in t is CL PBS, mix the suspension and transfer it into the Eppendorf tube with a volume of 1.4 ml

Then cells are lysed by adding 50 μl of CHCl3and 5 μl of 10% SDS. Stirred suspension of cells in a short time. Then this suspension centrifuged for 5 minutes with acceleration 9000 g.

C. the Enzymatic reaction

The substrate ONPG for 25 samples are prepared as follows: mix of 27.7 mg ONPG (Sigma) and 50 ml of diluent, consisting of 1 ml of 0.1 M MgSO4and 1 ml of 2-mercaptoethanol (Merck) in a final volume of 100 ml buffer solution (90 ml of 0.1 M NaHPO4with pH = 7.0, 0.1 M MgSO4, 1 ml of mercaptoethanol.

Mix of 1.95 ml of ONPG solution and 50 μl of the cell suspension, which is incubated for 1 hour at a temperature of 28oC. Then added to this suspension 2 ml of 1 M Na2CO3and measure the absorption of this suspension at 420 nm.

Comparison of activity-gal for recombinant viruses is given below in units of gal (PL. 6).

Note to the table. 6.

WT - wild type, non-recombinant.

The nearest (AGAS) is the closest phase of growth, artificially extended to 16 hours due to AGAS.

Nearest + Delayed (AGAS) is the nearest natural and the s.

1:5 and 1:10 - preliminary dilution of the extracts in 5 and 10 times.

The P7 Promoter.5 for Vaccinia acts during the immediate and delayed phases of infection (V3, V4 and V5), the promoter P11 is extremely delayed activity (V8 and V9), the P11 promoter is stronger than the P7 promoter.5, as Vaccinia. Temporary regulation, as well as the relative strength of the two promoters, therefore, are stored in the virus CNP, whether the inclusion of B1 or B2 and whatever the orientation of LacZ.

Example 16. Vaccination of chickens with recombinant virus CNP/LacZ. Protection from avian pox.

Immune response against-gal.

Feel Vaccinium the ability of recombinant CNP/LacZ. Two ordering criteria are protection against avian virus and the immune response against-galactosidase.

a. Introduction by piercing the wing

Suspension of recombinant viruses V4, V8 or suspension vaccination strain CNP was administered by injection chickens at the age of one day, examined in the fact that they are free from pathogens (SPF), resulting in perforation of the membrane of the wing (method, called "wing web (WW).

The tests are distributed into three groups of 28 to 30 chickens, negative control.

Each vaccinated chicken received 10 μl of the suspension containing viruses 5 105TSD (Tissue Culture Infections Dose)/ml, which is equivalent to 5 to 103TCID50 per bird. 29 day old Chicks were brought into contact with virulent virus (a strain of Fowlpox Challenge Virus obtained from Afica, USA).

The lack of health disorders assessed ten days later, refers to the protection of all vaccinated chickens against avian pox. In contrast, half of the unvaccinated chickens had health problems. Analysis of antibody titers against-galactosidase direct ELISA method showed that 21 of 29 (72%) of chickens vaccinated with virus V4, and 20 out of 38 /71%/ chickens vaccinated with virus V8, have serological reaction, i.e. they are seropositive.

Because the ELISA titer is the last dilution giving an optical density greater than 100, the average titer ELISA anti-galactosidase activity in the serum of these Chicks is 1:800.

b. Introduction intramuscular and subcutaneous by

Day old Chicks vaccinated on the first day by 104,4TSD virus V8 by intramuscular (VM, 27 chickens) or by subcutaneous (SC, 34 chicken). They tested health after 27 days. They semisavannah VM by, and in 27 of 34 (79%) of chickens vaccinated with PC by.

Thus, the results of immunization show

that recombinant viruses that contain the LacZ gene in the areas of TIR their genome in a site B1 or B2, retains its immunogenic capacity,

what the promoters P7.5 and P11 are in animals,

- what is the response of antibodies directed against a-galactosidase, considered here as a heterologous protein expressed recombinant CNP,

that intramuscular (IM) route, subcutaneous (SC) way by piercing the membrane of the wing (WW) are equivalent at the same time in relation to the protection from avian pox and to share serological reactions against-galactosidase.

Example 17. The inclusion of genes that encode glycoprotein E2 of avian bronchitis, transfer vectors

The virus responsible for avian infectious bronchitis (IBV) is a coronavirus. The most important surface antigen of the virus is the E2 protein consisting of two subunits, S1 and S2 (Cavanagh, 1983, Cavanagh et al., 1988). There are numerous serotypes, including Massachusetts, called M41, and the serotypes, in particular D and D (Kusters et al., 1987).

Develop retwitting for avian infectious bronchitis (IBV).

Gene protein E2 serotype M41 was received by Dr. Custerson from Utrecht University (the Netherlands), as well as large fragments of E2 genes of serotypes D1466, D207 and D274.

The BamHI cassette E2 gene for M41 was constructed by directed mutagenesis. Full gene of strain D1466 and full hybrid gene D207/D274 were designed on the basis of the fragments, and on tapes that are compatible with BamHI. These three cassettes were cloned following the P7 promoter.5, generating three plasmids p75M41, p75D1466 and p75D207.

Cassette P7.5-E2 cloned in the BamHI site of the vector transfer pTIRBI, then the cassette P11Lac below is cloned E2 in the BamHI site. These plasmids transfer are pTIRBIP75M41Lac, pTIRBIP75D1446Lac and pTIRBIP75D207Lac. Recombinant viruses constructed by transferring and purified as described in examples 9, 10 and 11. Genomes are analyzed by the method of Southern blot as described in example 13. The expression of the E2 antigen detected using immunological methods: ELISA, Western blot or immunofluorescence using specific antibodies. Get a colony of recombinant viruses. Poultry vaccinated dose of these recombinant. Vaccine efficacy is measured after infection with pathogenic viruses in accordance with the classical methods for infectious bside transfer - construction of recombinant CNP/TA4

Genus Eimeria includes parasites of poultry, responsible for coccidiosis. Were described surface antigens, in particular, for microorganisms E. tenella, E. necatrix, E. maxima (a European patent application 0 164 176 and 0 231 537).

Develop effective recombinant vaccine against coccidiosis.

Antigen for E. tenella, denoted TA4 or A4, is a protein with molecular weight of 25 kDa, consisting of two subunits with 17 kDa and 8 kDa, linked by a disulfide bridge. Gene was isolated from a genomic Bank, as well as on the basis of mARN. A full description of the gene was published in the above mentioned patent applications.

The BamHI cassette gene TA4 was constructed as the result of sublimirovanny. Plasmid pTA406 contains the coding sequence of the TA4. Plasmid pTA410 carries the gene TA4 with a modification in proteolytic sequence that is shared by the two subunits. By directed mutagenesis natural sequence Arg-Arg-Leu was replaced by the sequence Arg-Glu-Lys-Arg (described Kieny et al., 1988). These two cassettes were cloned BamHI below the P7 promoter.5 plasmids pIP7.5 in a direction that places the gene TA4 under the control of the P7.5.

Cassette P7.5-TA406 and P7.5-T is DAMI transfer are pTIRTA406Lac and pTIRA410Lac.

Recombinant viruses were obtained by transfer as described in examples 9, 10, 11. Get the blue areas. Recombinant carrying the gene TA406, is a virus V20, recombinant carrying the gene TA410, is a virus V21.

Double recombinant virus V21, i.e., the carrier single copy TA410 and one copy of LacZ in each area TIR, cleared on vinyl for titration, and its genome is analyzed by PCR as described in example 14. The used primers and the size of the enlarged fragments are given in the table below. Primer 1871 is complementary to the LacZ gene (table. 7).

The expression of the TA4 antigen detected using immunological methods: ELISA, Western blot or immunofluorescence using specific antibodies.

Get a colony of recombinant viruses. Poultry vaccinated dose of these recombinant. Vaccine efficacy is measured after the test ("Challenge") virulent Eimeria organisms in accordance with the classical methods for coccidiosis, such as the analysis of serum, the rate of increment of weight and clinical signs.

Example 19. The inclusion of gene polyprotein Gumboro in the transfer vectors

The agent responsible for the disease Gumboro, disease Gumboro), which affects young chickens and characterized by destruction of lymphoid cells handbags fabritsiusa. The virus has a genome consisting of two segments of double chains of RNA, called A segment (about 3400 base pairs;) and segment B (approximately 2900). Viral particles are non swirling and have the shape of a regular icosahedron with a diameter of about 60 nm. Four viral proteins were clearly identified: VP1 molecular weight (MV) 90 kDa (kDa: kilo Dalton), VP2 with MW of 37 kDa to 40 kDa; VP3 with MV from 32 kDa to 35 kDa and VP4 with MV from 24 kDa to 29 kDa (Dobos, 1979, Fahey et al., 1985). The VP2 protein is derived predecessor VPX with MV from 41 kDa and 54 kDa.

Segment B encodes VP1, which in reality is a viral polymerase. Segment A encodes 3 other protein. The latter are generated as a result of proteolytic cleavage, based on its predecessor with a MW of approximately 110 kDa, which corresponds to the large open phase for reading the segment A. this proteolytic cleavage involved in protein VP4 (Jagadish et al., 1988). Proteins VP2 and VP3 form the viral capsid. Protein VP2 contains antigenic determinants capable of inducing the synthesis of antibodies that neutralize the virus (Becht et al., 1988, Fahey et al., 1988).

Developed p the
1. The selection of genetic material from a selected strain of IBDV, namely from strain EDGAR. The virus strain EDGAR can get in the agricultural Department of the United States (USDA, APHIS 6505 Belcrest Road, Hyattsville, MD 20782, United States).

2. Synthesis, cloning and sequencing of the DNA corresponding to the segment A.

3. Introduction DNCC, and sequences necessary for the expression of this genetic material in the cells of the animal is infected with a virus, Fowlpox, and sequences allowing sift recombinant Fowlpox virus, plasmid transfer pTIRBI described in example 4.

4. Isolation, purification of recombinant viruses.

5. Genetic analysis of recombinant viruses.

6. Analysis of gene expression in vitro (in culture cells) gene of IBDV, wear these recombinant viruses.

7. Vaccination of chickens with recombinant viruses and analysis of protection from disease, Gumboro.

Stage 1.

Viral RNA strain EDGAR was selected on the basis of bags of chickens infected with a virus.

About 40 g of bags collected after 7 days after virus infection, are crushed in 40 ml of buffer rastvorovoda phase is applied to pre-formed sucrose gradient, consisting of 2 layers with a sucrose content of 40% and 60% (% as the ratio of weight/volume, in TNE buffer solution). The gradient is centrifuged with acceleration 134000 g for 2 hours and 30 minutes. Collect the phase interface surface 40 to 60% sucrose gradient, which contains the partially purified virus. Add 5 ml of this phase to 5 ml of the buffer solution containing 10 mm Tris-HCl, 100 mm NaCl, 0,5% SDS, 10 mm EDTA, 2 mg/ml Proteinase K at pH = 7,5. The mixture is incubated for 1 hour at a temperature of 37oC. Then the aqueous phase is extracted in a mixture of phenol/chloroform. Nucleic acid aqueous phase are precipitated in ethanol in the presence of 0.8 M LiCl and extracted in 500 μl of water.

Stage 2.

Synthesis and amplification DNCC corresponding to the segment A, were carried out by the method described in the "GENEAMP RNA PCR KIT, PERKIN ELMER CETUS", using synthetic oligonucleotides or primers", complementary to the sequence of segment A, as a seed for the synthesis of the first DNA chain under the action of reverse transcriptase. The choice of these synthetic nucleotides was determined by analysis of published sequences of segments A and other IBDV strains: Australian strain 002-73 (Hudson et al. , 1986), German strain CU-1 (Spies et al., 1989) Amma EDGAR shown in Fig. 8 and 9.

Analysis of published sequences of strains of IBDV shows that in addition to a large open-phase reading (ORF 1), encoding proteins VP2, VP4, VP3, there are two other open phase for a read on the same circuit, the coding segment A. One, ORF 2, starting at 34 on higher ATG phase ORF 1, is higher than the last and has a length of 435. Another, ORF 3, starts at 32 above the ORF 2 and has a length of 31. The possible role of these ORFS in the biology of the virus is not currently defined.

Were generated by four pieces of double chains DNCC limited respectively in pairs "primers": 0-1b/585/ 1-2 (1129), 3-4 (670), 5-6 (1301), and covering phase ORF 1 and ORF 2 segment A (see Fig. 8).

These fragments were cloned in the plasmid pBSPIus or pBSLKI, six plasmids were constructed in this way (see Fig. 8 and 10) (table. 8).

The nucleotide sequences of fragments of IBDV these plasmids were identified and aligned so as to restore the sequence of the enlarged segment of A strain EDGAR PCR (see Fig. 9). The original sequence of strain EDGAR, which were replaced in the result of the use of "primers" that are defined on the basis of published posledovatelnuju", located outside the latter.

The reconstruction of A segment on the basis of the obtained clones, schematically depicted in Fig. 10.

The stages are as follows:

1. Design pEDGAR12:

pBSIA2 (EcoRI + T4 DNA polymerase, SacI) + pBSIB (SphI) + T4 DNA polymerase, SacI)

2. Construction of plasmids pEADGARM12 by directed mutagenesis on pEDGAR12:

a) deletion of the SphI site of pEDGAR12 located above the IBDV sequence:

- initial sequence:

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5' GTCTGATCTCTACGG

TTCCCTTTAGTGAGGG 3'

b) the deletion of the EcoRI site of the plasmid pEDGAR12 and the introduction of the SphI site below IBDV:

- initial sequence:

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3. Design pACEDGARM12:

The inclusion of the fragment Bc11-SphI plasmids pEDGARM12 in sites Bc11-SphI plasmid pACYC184.

4. Design pEDGARM34i by site-directed mutagenesis on pEDGAR34i

Replacing the SphI site at the NsiI site:

- initial sequence:

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5. Design pEDGAR45:

pBS3A (Hinc2 + Pst1) + fragment Hinc2-Pst1 plasmids pEDGAR34i

6. Design pACEDGAR14:

pACEDGARM12 (SphI + T4 DNA polymerase, BamHI) + pEDGARM34i (NsiI + T4 DNA polymerase, BamHI)

7. Design pACEDGAR15:

pACEDGAR14 (BamHI + T4 DNA polymerase, SaII) + pEDGAR45 (PVu2-SaII)

8. Constr phase ORF 1 segment A on the BcII fragment-BamHI.

9. Construction of plasmids pBSIBDVOb by directed mutagenesis pBSIBDV0:

Introduction phase ORF 3 in plasmid pBSIBDV0, which is the beginning phases of ORF 1 and ORF 2:

- initial sequence:

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10. Design pACEDGAR2 and pACEDGAR3:

Plasmids pACEDGAR2 and pACEDGAR3 were obtained by replacing the field of BcI-I-Rsr2 plasmids pACEDGAR1 on the BcII fragment-Rsr2 plasmids pBSIBDVO and pBSIBDVOb respectively.

3 plasmids pACEDGAR1, pACEDGAR2 and pACEDGAR3 allow, therefore, to allocate on the BcII fragment-BamHI respectively ORF 1, ORF 1 - ORF 2 and ORF 1 - ORF 2 - ORF 3 segment A strain EDGAR.

The sequence of these 3 plasmids from ATG phases ORF below:

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Stage 3.

Then these fragments BcII-BamHI was cloned in the BamHI site of plasmid p2P75, which generates plasmid p75EDGAR1, p75EDGAR2 and p75EDGAR3 and places one or more coding sequences of A segment under the control of a promoter P7.5.

These coding sequences were placed under the control of the promoter P11. The BcII fragment-BamHI plasmid pACP11 carrying the p11 promoter, was included in the BamHI site of the plasmid pACEDGAR1, pACEDGAR2 and pACEDGAR3 to generate plasmids, respectively pIIEDGAR1, pIIEDGAR2 and pIIEDGAR3.

Cassette BcII-BamHI P75-EDGAR or P11-EDGAR were isolated from these different and pTIR11EDGAR3. The orientation of the cassettes BcII-BamHI plasmid pTIRBI that has been stored, is such that the direction of transcription initiated from promoters P7.5 and P11, would coincide with the direction of phase ORF 1 and ORF 2 in the plasmid pTIRBI.

Bg12 fragment-BamHI plasmid pACP11LAC bearing cartridge P11-LacZ, was included in the BamHI site of the plasmid pTIR75EDGAR1 and pTIR75EDGAR2 to generate plasmids, respectively pTIR75E1LAC and pTIR75E2LAC. Orientation P11 - LacZ in these plasmids is such that the direction of transcription of the LacZ gene would coincide with the direction of the open phases of the ORF 1 and ORF 2.

Similarly plasmids pTIR11E1LAC, pTIR11E2LAC and pTIR11E3LAC were generated by adding a BamHI site, respectively, plasmids pTIR11EDGAR1, pTIR11EDGAR2 and pTIR11EDGAR3 of the cassette P75-LacZ allocated in the form of Bg12 fragment-BamHI plasmid p75LAC.

Finally, plasmid pTIR11VP2LAC was generated by including in the PpuMI sites (treated DNA polymerase, T4) and Bg12 plasmids pTIR11E1LAC XhoI fragment (treated DNA polymerase, T4) - Bg12 plasmids p11EDGAR1. Plasmid transfer pTIR11VP2LAC contains under the control of the promoter P11 complete sequence of the VP2 protein, and amino-terminal part of the protein VP4; for design are combined in the phase of the last three amino acids (Asp, Leu, Glu; carboxy-terminally protein match therefore, one part of polyprotein containing amino acids 1 to 493, followed by amino acids with 1010 in 1012, which includes protein VP2.

As an example in Fig. 11 depicts a plasmid pTIR75E1LAC.

Stage 4 and stage 5.

Recombinant Fowlpox viruses, which have been integrated open phase ORF strain Edgar, were isolated, purified and characterized in accordance with the methods described in examples 9, 10, 11, 12, 13 and 14.

Designation of recombinant viruses is as follows:

V11 = P7.5E1; V12 = P7.5E2;

V14 = P11E1; V15 = P11E2;

V17 = P11VP2; V16 = P11E3.

The primers used for breeding double recombinant TIR PCR method, there are TIR WT, TIR containing LacZ, E1, E2, E3 or VP2. Combinations of primers and the size of the enlarged fragments are given in table. 9.

Primers IBDV16, 3, 1b and 22 hybridize with IBDV. Primers IBDV16, 1b and 3 hybridize with the coding part of the VP2 protein. Primer IBDV22's hybrid c coding part of the protein VP3 and is, therefore, 's hybrid with genome V17. Primers 5168 and 5169 hybridize with FPV. Primer 1871's hybrid gene-galactosidase. The sequence of these primers are given in table List and sequence of primers". CLASS="ptx2">

Used antibodies that are configured against IBDV strain C-1 were kindly provided by Professor G. müller, Institute of Virology, Justus-Liebig-University, Giessen, Germany.

Namely:

1 - polyclonal serum (N B22) hyperimmunizing rabbit against strain C-1,

2 - monoclonal antibody mouse anti-VP3 (N 1/A10), raspoznavaya protein VP3 in natural and denatured form,

3 - monoclonal antibody mouse anti-VP2 (N B1) that recognizes a conformational epitope of the VP2 protein; antibody B1 is an antibody capable of neutralizing viral strain C-1.

a. Experimental conditions infection

In day D = 1: per one Cup size 25 cm2inoculant 2,5 106QT35 cells in 5 ml of culture medium (composition described in example 1). Prepare eight cups.

In day D = 2 : infect cells with multipletness of infection of 0.01 viruses per cell, with each recombinant virus, V11, V12, V14, V15, V16, V17, V8 (negative control) and IBDV (strain Bursine 2, positive control).

In the D-day = 5 : collect infected cells and Wednesday after freezing and subsequent thawing.

b. Analysis of products expression using ELISA method t the lo B22, and as the second antibody or monoclonal antibody anti-VP3 (N 1/A10), or a monoclonal antibody, a neutralizing anti-VP2 (N B1). Curves ELISA methods is shown in Fig. 12A for anti-VP3 and 12B for anti-VP2.

The results of these analyses show that:

1 - Fig. 12A: protein VP3 is expressed in all recombinants (V11, V12, V14, V15 and V16), which contain at least a sequence encoding polyprotein (ORF 1, proteins VP2-VP4-VP3), but not expressed in recombinant V17, because it only contains the region encoding VP2, and amino-terminal part of the protein VP4, and is not expressed in the recombinant V8, because it does not contain any sequence of IBDV.

The expression level of the protein VP3 is weaker in the recombinants V11 and V12 than recombinants V14, V15 and V16, which is in fact correlated with the strength of the promoters present in various recombinants : the activity of promoter p7.5 (recombinants V11 and V12) is less strong than the activity of promoter p11 (recombinants V14, V15, V16) (see example 15).

Not observe differences in the level of expression of the protein VP3 depending on the phases of the reading segment A, introduced in various recombinants: ORF 1 for recombinant V11 and V14, ORF 1 + for ORF2 recombinant V12 and V15 and ORF 1 + OR the which does not contain sequences of IBDV.

The expression level is highest for recombinants V17; in this case it is close to the level of expression of the VP2 protein expressed in cells infected with the virus IBDV.

Again see the correlation between the strength of the promoters present in different recombinants, and the level of expression of the VP2 protein.

These results show, on the one hand that the epitope of the VP2 protein recognized by the antibody, neutralizing B1, which is inclined against strain C-1, is also present in the protein VP2 American strain Edgar, and, on the other hand, the initial conformation of the protein VP2 stored in all recombinants at least in this area.

c. Analysis of the products of expression by Western blot method.

Analysis using the Western blot of proteins produced by different recombinants was carried out, on the one hand, using polyclonal serum (B22), raspoznavaniya set of IBDV proteins, and, on the other hand, using monoclonal antibodies anti-VP3 (1/A10).

The results, illustrated in Fig. 13A shows that the polyclonal serum recognizes in extracts of cells infected with the help of IBDV, mostly 3 protein corresponding to VP2 ( 40 kDa), VP3 (the ü a predecessor (VPX, 48-49 kDa) protein VP2.

For all recombinant (V11, V12, V14, V15 and V16), which contain at least a sequence encoding polyprotein (ORF1, VP2-VP3-VP4), detected using polyclonal serum anti-IBDV (figure 13A) proteins with molecular weight (MW) coincident respectively with MV for VP3, VP4 and probably VPX. For recombinants V17 detected only one protein, the molecular weight of which is close to the molecular weight of the fusion protein VP2 + aminoterminal region of the protein VP4 ( 52 kDa).

Monoclonal antibody anti-VP3 (Fig. 13B) recognizes mainly two proteins: VP3 and probably a product of the decomposition of VP3 in cells infected with the help of IBDV, and all recombinants expressing polyprotein. No protein of IBDV is not recognized recombinant V17, expressing the fusion protein VP2-VP4.

The combination of these results indicates that the phase for reading the coding polyprotein (ORF 1), translated in all recombinants regardless of the presence of additional phases for reading ORF 2 (recombinants V12, V15) or ORF 2 and ORF 3 (recombinant V16). Cleavage of the precursor of polyprotein is correct, but is incomplete in regard to VP2, it was suggested that full maturation et al., 1982).

Stage 7.

Feel Vaccinium the ability of recombinant FPV/IBDV.

a. Serological reaction under the action VII

The first company of vaccination with recombinant FPV/IBDV compared the serological response against-galactosidase and against IBDV virus for three different routes of administration: by intramuscular (IM), subcutaneous route (SC) and by piercing the wing (WW or wing web).

Day old Chicks vaccinated with the help of 104,4recombinant viruses V11 in accordance with the three ways of introducing VMS, PC, or WW. They wordplays 11 days later.

Analysis of sera direct ELISA method showed that 2/14 (14%) chickens vaccinated by intramuscular, 3/6 (50%) of chickens vaccinated by subcutaneous, and 8/15 (53%) of chickens vaccinated by piercing the wing, have antibodies against a-galactosidase, whereas 5/14 (36%) of chickens vaccinated by intramuscular, 1/6 (17%) of chickens vaccinated by subcutaneous and 1/15 (7%) chickens vaccinated by piercing the wing, have antibodies against the virus IBDV.

Thus, the IBDV proteins are expressed recombinant FPV/IBDV11 vaccinated animal and cause a reaction of antibodies in some chickens. From tre is NNAA protection under the action of viruses V11, V15 and V16.

Day-old Chicks received a dose to a value of 104,4viruses V8 (negative control) V11, V15 or V16 by intramuscular and were subjected to injection. 10 days later under the action of 100 LD50(a hundred-fold lethal dose) of the virus 849 VB entered through the eye. Strain 849 VB described by Van Den Berg et al., 1991.

Chickens wordplays 210 days later.

Multiplied by 100 the ratio between the weight of the bag and the total weight is the protection factor.

For the control group vaccinated with virus V8 and not subjected to the test IBDV, this ratio is 0.57 (average for 10 chickens).

For groups V8 subjected to the test, this ratio comes to 0.11 (9 chickens).

For chickens vaccinated with virus V11, V15 and V16, this ratio is respectively 0,1 (20 Chicks), 0,12 (18 chickens) and 0.12 (18 chickens).

The use of this criterion shows that there is no early protection against IBDV virus under the action of recombinant V11, V15 and V16.

c. Late protection under the action of V11, V15 and V16.

Chickens vaccinated with viruses V8, V11, V15 and V16, as described in the previous paragraph, were tested virus IBDV after 42 days after injection.

Selected DL the b-galactosidase and virus IBDV.

Response against-galactosidase shows that 100% of serological reaction is obtained in all vaccinated chickens, because only 70 chickens. Titers ranged from 1:200 to 1:51200. There is no significant difference between the titers obtained for V11, in which the LacZ gene is under the control of P7.5, and V8, V15 and V16, which LacZ genes are under the control of the promoter P11.

Response against IBDV 10 chickens vaccinated with virus V8 is zero, it is positive in all chickens vaccinated with virus V11, V15 and V16. Details: title responses from 20 chickens vaccinated with virus V11, range from 1:800 to 1:51200, with an average of 1:6400. They range from 1:6400 and 1:102400, with an average 1:25600 for 20 chickens vaccinated with virus V15. They are between 1:6400 and 1: 204800 with an average 1: 25600 20 chickens vaccinated with virus V16. The content of antibodies is clearly higher for the groups vaccinated with viruses V15 or V16 than V11.

The titles of seroneutralization vaccination strain PBG68 adapted to cultured cells is 1:10 to V11, as a negative control, 4 serum from 20 to V15 and 1 serum from 20 to V16 are the titles of seroneutralization up to 1:20. Followed is the overall weight) 100 average of 0.66 in the control group, V8, not subjected to infusion (10 birds). It comes up to 0.1 in the group V8 subjected to the test "Challenge" (1 survived). It is 0.1, 0,11 0,11 for the three groups V11 (3 chicken), V15 (11 chickens) and V16 (8 Chicks), respectively. There is no protection against atrophy bags.

Mortality in four days that follow challenge "Challenge" was almost full for the control group (9 chickens out of 10). In the group V11 survived 3 chicken out of 20, whereas survived 11 chickens of 20 vaccinated with virus V15, and 8 chickens of 20 vaccinated with virus V16. Thus, see protection of about 50% of the animals vaccinated with the virus V15 or V16.

d. Protection under the action of viruses V14, V15 and V17.

Four groups of poultry from the age of three weeks, we received 0.2 ml of recombinant viruses V8 (negative control), V14, V15 or V17. The number of viruses per chicken was 106,4TSD. The route of administration is intramuscular.

After twenty-one days taken for analysis of serum from each animal to assess the titer of the antibodies of the type of anti-IBDV. Five lethal dose of IBDV is introduced animals from groups V14, V15 or V17 and parts of animals from groups V8 (i.e., from a group of poultry, received recombinant wheelie test, wordplays, and is determined by the ratio (weight bags/weight poultry) 100.

The results are as follows:

- Mortality: number of poultry, failed investments, are as follows:

for V8: 12 birds of 14 (85%), V14: 21/24 (87%),

for V15 : 17/25 (68%) and for V17 : 0/25 (0%).

In other words, all poultry vaccinated recombinantly V17-resistant infusion. In this group, the protection is complete. In addition, none of the poultry without showing any clinical sign of disease. Protect the value of 32% obtained for recombinants V15, close to the protection of the value of 50%, obtained in the previous experiment.

- ELISA method: estimated average titres against virus are: for V8 titers less than 1: 100 and is seen as negative, for V14 titers up to 1: 12800 (1:6400 and 1:51200), V15 titers up to 1:6400 (from 1: 800 to 1: 25600) for V17 titers less than 1:100, with the exception of 3 poultry (1:800, 1:6400 and 1:51200).

Therefore, the response of antibodies against IBDV-induced expression of polyprotein recombinant FPV/IBDV is very high. Response against a full virus caused by V17, is, on the contrary, weak.

- Seroneutralization: the title is otricatelniy; for V17 5 poultry out of 20 have a titer greater than 1:20, i.e. in particular 1:20 (twice), 1:40, 1:80 and 1:320.

Therefore, seroneutralization observed exclusively in the serum of poultry vaccinated with virus V17.

- Bags: average ratio (weight bags/total weight) 100 is as follows: for a V8, not subjected to infusion, the ratio is 0.68 ( 0,13) for V8 subjected to the test "Challenge", the ratio is 0.09 ( 0.01), and for V14 ratio is 0.10 ( 0,02), V15 ratio is 0.13 ( 0.04), and for V17 ratio is about 0.34 to 0.23).

Therefore, partial protection bags fabritsiusa can be observed only in the group V17. Indeed, 8 poultry from 24 have a relation similar to that which have witnesses are not subjected to infusion. Sixteen other relevant higher than the witnesses subjected to the test "Challenge".

Conclusions.

Key findings from these companies on vaccination are as follows

- Recombinants that Express polyprotein of IBDV, cause in poultry a strong backlash ELISA with high titers against the virus.

- Recombinant that expresses part of VP2 polyprotein, fully protects home FR the successive primers following claims.

1. Recombinant vector-based smallpox birds originating from the attenuated strain of the smallpox virus of birds, which determines the synthesis of the heterologous protein of the microorganism pathogenic for poultry, characterized by the following features: contains at least one sequence of a heterologous protein of the microorganism pathogenic for poultry, integrated in the portion of the genome is not essential for the activity of the virus, which represent non-coding intergenic region is not more than 60 nucleotides, located in the terminal inverted repeats (TIR) between the two open reading frames (ORFS) and enclosing the expression signals, as well as structural elements, capable of expression of the specified heterologous protein in cells transformed data with the recombinant vector, and the intergenic region is a1and/or2and1located between nucleotides 1675 and 2165 TIR, provided that the point of reference adopted by the restriction site for WAMN nucleases in TIR, and between the two open reading frames ORF1 and ORF2, located between nucleotides 416-1674 and 2166-2671 respectively, and2located between nucleotides 2672 and 3605 TIR at usloviia ORF2 and ORF3, located between nucleotides 2166-2671 and 3606 and 4055, respectively.

2. Vector under item 1, characterized in that obtained using attenuated strain of the smallpox virus of chickens.

3. Vector for p. 2, characterized in that the sequence of the heterologous protein is integrated into the region1in each of the two TIR.

4. Vector for p. 2, characterized in that the sequence of the heterologous protein is integrated into the region2one of the two TIR.

5. Vector under item 3, characterized in that the sequence of the heterologous protein is a sequence of the antigenic protein of a virus disease of Gumboro (MSF), or infectious bronchitis virus (SHAKING), or chicken anemia virus (VAC), or Newcastle disease virus, or virus, Marek's disease, or the sequence of the antigenic protein of parasitic simplest Eimeria tenella, causing coccidiosis.

6. Vector for p. 5, characterized in that the sequence of the antigenic protein in the case of MSF is a sequence palibelo or part thereof, in the case of SHAKING the sequence of the E2 protein, or part thereof, in the case of VAC - sequence P50 protein or part thereof in the case of parasitic simplest Eimeria tenella-the action scene, what part of the sequence palibelo MSF contains amino acids 1-493 and further amino acids 1010-1012.

8. Vector-based smallpox birds described in any of paragraphs. 1-7 formula used for vaccination of poultry.

 

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