PL201132B1 - Polypeptide, sequence, bacillary virus, method of manufacture of glycoprotein, application of sequence, application of bacillary virus, application of polypeptide - Google Patents

Abstract

A polypeptide consisting of a signal sequence and an amino acid sequence of a glycoprotein E2 or a fragment thereof, wherein the signal sequence is selected from the signal sequence baculovirus gp67, pseudo rabies virus gG or gD signal sequence.

Description

(22) Date of notification: April 29, 2003

The Patent Office of the Republic of Poland (19) PL (11) 201132 (13) B1 (51) Int.Cl.

C07K 14/005 (2006.01) C07K 14/185 (2006.01) C12N 15/40 (2006.01) C12N 15/866 (2006.01)

Polypeptide, sequence, baculovirus, glycoprotein production method, sequence use, baculovirus use, polypeptide use (43) Application announced:

(73) The right holder of the patent:

University of Gdańsk, Gdańsk, PL

Institute of Bioorganic Chemistry of the Polish Academy of Sciences in Poznań,

Poznan, PL

Institute of Biotechnology and Antibiotics, Warsaw, PL

Kucharczyk Krzysztof Techniki Elektroforetyczne Sp. z o.o., Warsaw, PL

02.11.2004 BUP 22/04 (45) The following was announced about the grant of the patent:

31.03.2009 WUP 03/09 (72) Inventor (s):

Bogusław Szewczyk, Gdańsk, PL Beata Adamiak, Gdynia, PL Krystyna Bieńkowska-Szewczyk, Gdańsk, PL Jolanta Ficińska, Gdańsk, PL Andrzej Płucienniczak, Warsaw, PL Anna Porębska, Warsaw, PL Andrzej B. Legocki, Poznań, PL Katarzyna Miedzińska, Rawicz , PL Krzysztof Kucharczyk, Warsaw, PL (74) Plenipotentiary:

Aleksandra Twardowska,

JAN WIERZCHOŃ & PARTNERZY, Patent and Trademark Office (57) 1. A polypeptide consisting of a signal sequence and an amino acid sequence of the E2 glycoprotein or a fragment thereof, the signal sequence being selected from the baculovirus gp67 signal sequence, the gG or gD signal sequence of pseudorabies virus.

PL 201 132 B1

Description of the invention

The subject of the invention is a polypeptide, sequence, baculovirus, a method of obtaining a glycoprotein, the use of the sequence, the use of a baculovirus, the use of a polypeptide for the production of a vaccine against CSFV. In general, the invention relates to the preparation of E2 glycoprotein, the major antigen of Classical Swine Fever Virus (CSFV), from the growth medium of insect cells infected with recombinant viruses belonging to the Baculoviridae family having recombinant protein evacuation sequences. Objects of the invention serve to use such a medium as an immunizing agent.

Some animal viruses can be used as vectors to express foreign genes. Baculoviruses are one of the most widely used groups of viruses used for this purpose. The most commonly used species of baculovirus for expression of foreign genes is Autographa californica nuclear polyhedrosis virus (AcNPV). The double-stranded circular DNA molecule of this virus is approximately 128,000 base pairs in size. Viral DNA is bound to a small basic protein with a molecular weight of approximately 7 kDa. The genome is surrounded by a protein capsid with envelope proteins on its surface. In the occlusive form of the virus, the virus particles are surrounded by a thick layer of polyhedrin protein.

There are two forms of virions in the life cycle of AcNPV: the occlusive form (OV) and the budding form (BV also called EV). These forms differ in morphology and their role in cell infection. The OV form forms occlusive bodies in the nuclei of infected cells. After the host dies, this form is used to infect other caterpillars. The main protein of the OV form is polyhedrin, which protects the virus against the adverse effects of the external environment. In the alkaline environment of the caterpillars' gut, viruses are released from the polyhedrin shell. They infect intestinal cells where primary viral growth and the formation of BV progeny takes place. These forms do not contain polyhedrin. They can infect other cells throughout the caterpillar's body (or further cells in tissue culture).

The expression of the AcNPV baculovirus genes takes place in several successive phases: early (often divided into very early and delayed early phases), late and very late. The late phase (6 - 18 hours after infection) is characterized by the expression of genes whose products are involved in the formation of the BV form. In this phase, the structural proteins of the capsid and core are synthesized. A glycoprotein with a molecular weight of 67 kDa is also synthesized, which plays an important role in secondary, intercellular infection by the BV form. The very late (occlusive) phase lasts from about 18 hours to about 80 hours post infection. In this phase, intensive synthesis of polyhedrin (molecular weight approx. 30 kDa) takes place, which can constitute up to 50% of the mass of all proteins in the cell. The AcNPV baculovirus produces approximately 70 OV particles in one cell nucleus and each OV particle contains numerous viral capsids. The second very late protein is the p10 protein. His role is much less understood. The p10 protein creates cytoskeletal structures in the cell and participates in the release of OV particles from the cells.

The second species of baculovirus frequently used to express foreign genes is Bombyx mori nuclear polyhedrosis virus, abbreviated as BmNPV. It is approximately 90% identical in nucleotide sequence to AcNPV. The similarity of the nucleotide sequence makes the protein composition of both viruses very similar, but they do not share any host. The development of both viruses is almost the same; the only notable difference is the amount of packed viral particles in the polyhedrin occlusions. AcNPV belongs to the group labeled MNPV (multiple nuclear polyhedrosis viruses) because many viral particles are packed into one polyhedrin occlusion, while BmNPV belongs to the group labeled SNPV (single nuclear polyhedrosis virus) because it is believed that viral particles are packed individually in the sheath polyhedrine. BmNPV is most often used as an expression system when it is planned to obtain foreign proteins in caterpillars rather than in tissue culture of insect cells. This is due, among other things, to the size of silkworm caterpillars (about 10 times greater in weight than those of AcNPV hosts), the simplicity of breeding and the lack of cannibalistic tendencies of silkworm caterpillars.

The baculovirus genome is very large, and therefore the direct insertion of a foreign gene into a specific place is not easy. Most often, appropriate transfer vectors are used to insert a gene into the baculovirus genome. They contain a replicon of a bacterial multicopy plasmid, a selection marker, a promoter and terminator region derived from the baculovirus genome with sequences flanking these regions, and a multiple cloning site (or a single restriction site) between the promoter and terminator where the foreign gene is inserted. , insect cells are most commonly infected with a mixture of viral DNA

And a transfer vector containing a foreign gene. In insect cells, recombination occurs between the homologous sequences of the plasmid and the viral genome, which leads to the incorporation of the foreign gene into the viral genome while deleting the corresponding baculovirus gene. Most often, foreign genes are inserted in place of the polyhedrin or p10 gene. Both of these genes have very strong promoters and therefore transcription is very efficient. Both proteins are synthesized in the occlusive phase, so they are not needed for the formation of BV particles. Baculovirus recombinants lacking the polyhedrin gene can be grown in tissue culture where infection is carried out by the BV form. Reproduction of the recombinant in caterpillars is possible by microinjection into the caterpillar body or by infection with a recombinant coated with polyhedrin previously obtained by mixed infection with a wild-type strain and a recombinant in defined proportions. In the case of recombinants that do not contain the p10 gene, multiplication is even easier. They can be multiplied both in tissue culture and by feeding the caterpillars with contaminated food.

Apart from the very high level of expression of foreign genes in the baculovirus system (the mass of the foreign protein can be up to 50% of the mass of all cellular proteins) there are several other factors that make this system extremely attractive. A very large piece of foreign DNA can be inserted into the baculovirus genome. Among other things, the expression of a cDNA which was a full copy of the RNA genome of an animal virus with a size of several thousand bases was obtained. Post-translational modification is very similar in insect and mammalian cells. The exception is N-glycosylation, which is more primitive in insect cells, resulting in baculovirus-derived vertebrate glycoproteins having a molecular weight slightly lower than their natural counterparts. Insect cells are particularly easy to cultivate in vitro. Most cell lines can be grown in both monolayer and suspension cultures. The optimum temperature for cultivation is 27 ° C, which in practice means that cultivation at room temperature is possible. The culture also does not require an atmosphere of carbon dioxide, so the cultivation of insect cells can be carried out in air or water shakers identical to those used for the cultivation of bacteria in a standard microbiological laboratory (Bieńkowska-Szewczyk K., Szewczyk B., Acta Bioch. Polon., 1999,46, 325-339).

In the classic Summers and Smith (Summers and Smith, Texas Agricult. Exp. Station Bull., 1987; 1555, 1-57) a foreign gene was inserted in place of a polyhedrin gene and recombinants were distinguished by plaque morphology. Recombinant viruses do not produce polyhedrin and therefore do not form occlusive bodies in the nuclei of the infected cells. This is noticeable under the microscope and even directly visually. However, this is a tedious and unreliable method. The gradual simplification of the individual stages of recombinant preparation led to the reduction of the time of their preparation to several weeks.

The first improvement was the increase in transfection efficiency after adding plasmid and genomic DNA to the insect cell suspension. Methods based on the precipitation of DNA in the presence of calcium phosphate have been replaced by methods where transfection occurs in the presence of cationic lipids. Another facilitation in the isolation of recombinants was the use of transfer vectors allowing the introduction of reporter genes together with the cloned gene into the baculovirus genome. The reporter gene, most often lacZ, was in the opposite orientation to that of the cloned gene and was under the control of a non-polyhedrin (mostly weak) baculovirus promoter. Since the reporter gene was introduced together with the foreign gene, the addition of an X-gal type substrate for β-galactosidase to the growth medium stained the plaques of the recombinant (but not the wild-type strain) blue. The isolation of such recombinants was much easier and the time needed to obtain baculovirus recombinants was significantly reduced. Another method was to replace the polyhedrin gene with a linker containing a sequence for a restriction enzyme that has no other natural cleavage site in the genome. This makes it possible to obtain linear genomic DNA cut in the polyhedrin gene. Transfection with such a linear genome and transfer vector significantly reduces the efficiency of transfection, but about 30% of the plaques are recombinant plaques. Further design changes in this linear baculovirus resulted in virtually 100% of the viral plaques being recombinant.

Homologous recombination in insect cells in vivo between the transfer vector and the baculovirus genome is an inefficient process. Consequently, several methods have been developed in which the introduction of a foreign gene into the baculovirus genome takes place outside the insect cells. The most common method is the site-specific transposition of an expression cassette into a shuttle vector containing a baculovirus (so-called bacmid) genome that can replicate in Escherichia coli bacteria (Luckow et al., J. Virol., 1993; 67, 4566-4579). Bakmid contains a low-copy mini-F replicon, a kanamycin resistance marker and a cod4 DNA segment

The lacZ peptide was derived from a plasmid of the pUC series. At the N-terminus of the lacZ gene there is a short segment containing the attachment site for the Tn7 transposon (mini-attTn7). The shuttle vector exists in Escherichia coli DH10B as a large plasmid that codes for kanamycin resistance and is responsible for the complementation of the lacZ deletion on the bacterial chromosome (shown by blue colony formation in the presence of X-gal and IPTG substrate). A shuttle vector can adopt a foreign gene from the transfer vector by transposing the mini-Tn7 into the mini-attTn7 site provided the helper plasmid encoded transposition proteins are present which also has a tetracycline resistance marker. Mini-Tn7 contains the gentamicin resistance marker, baculovirus promoter, foreign gene and transcription termination signal from SV40 virus. Transposition of mini-Tn7 into bacmid disrupts the expression of lacZ, making bacterial colonies containing the modified bacmid white instead of blue on an appropriate selection medium. This allows the modified bacmids to be easily isolated. Purified modified bacmid can be used for transfection; thus there is no selection of recombinants in insect cells. The method, although having a complicated theoretical background, is simple to apply and allows for quick obtaining of baculovirus recombinants. The time needed to obtain a recombinant is very short, usually about two weeks.

Despite the undoubted advantages, such as low production cost and high efficiency of recombinant protein synthesis, caterpillar cultures for obtaining foreign gene products in the baculovirus system are rarely used. In vitro cultures are used much more frequently (at least 90% of baculovirus system uses), possibly due to greater reproducibility and easier control of the experimental conditions. More than 400 insect cell lines are known, however only a few lines are very widely used as hosts for baculoviruses. AcNPV is most often propagated on cell lines derived from Spodoptera frugiperda or Trichoplusia ni. Several basic media for in vitro cultivation of insect cells in which baculovirus are propagated are known. These include Grace Medium, IPL-41 and TC-100, which are available in solid or liquid form. Before use, fetal bovine serum is added to them to a concentration of 10%. A few years ago, there appeared substrates that do not require the addition of bovine serum, which are irreplaceable in many cases, such as the isolation of proteins exported to the growth substrate. These include: ExCell 400 and ExCell 401 (J.R.H. Biosciences), HyQ-CCM-3 (HyClone), Sf900 and Sf900-II (GIBCO / BRL), SFM1 and SFM2 (Sigma).

Classical swine fever virus (CSFV), also often referred to as porcine cholera virus (HCV), for which the E2 gene was cloned into baculovirus, belongs to the genus Pestivirus, family Flaviviridae. This virus causes one of the most dangerous diseases of farm animals - classical swine fever, which very often results in the death of infected animals. Symptoms of the disease are primarily very high fever, leukopenia and bruising. In the past, the CSF virus caused many epidemics on the European continent. The most recent epidemic in 1997 killed several million pigs in the Netherlands and Germany. Virus-infected herds were slaughtered to prevent spread of the virus. However, the killing of animals is difficult to justify from both an economic and a humanitarian point of view. Mass vaccination is therefore probably the most rational method of eliminating the virus from the animal population in a given country.

The swine fever virus virion is spherical in shape; it consists of a hexagonal capsid containing a genome of approximately 12.5 kb. The viral genome is made up of a single, positive-stranded RNA. The cDNAs of two CSFV strains: Brescia (Moormann et al., Virology, 1990; 177: 184-198) and Alfort (Meyers et al., Virology, 1989; 171: 555-567) have been fully sequenced. A single, open reading frame (ORF) encoding a protein of about 4,000 amino acids was found. The ORF is flanked by two non-coding regions that are involved in genome replication and translation. The 5 'noncoding region of approximately 400 nucleotides contains the AUG codon as the ribosome binding site. This region is important in the initiation of translation of the CSFV genome. It is known, however, that there is no "cap" structure characteristic of eukaryotic mRNA. The 3 'non-coding region, approx. 200 nucleotides long, does not contain the poly-A sequence at the end, which is also characteristic of eukaryotic mRNA. The 5 'non-coding end has a complex secondary structure resembling that of the analogous region in picornaviruses (Brown et al., Nucleic Acids Res. 1992; 20: 5041-5045; Deng and Brock, Nucleic Acids Res. 1993; 21: 1949-1957). The presence of multiple AUG triplets upstream of the start codon suggests that translation of the CSFV genome may begin with internal ribosome binding independent of the "cap" structure (Poole et al., Virology, 1995; 206: 750-754).

PL 201 132 B1

The approximately 4,000 amino acid swine fever virus precursor protein undergoes a series of strictly controlled cotranslational and post-translational modifications. Cellular and viral proteases are involved in these modifications (Stark et al., J. Virol. 1993; 67: 7088-7095). The first transformations of the precursor protein resulted in the release from the N-terminal side of N ^pro protease (p23), capsid protein C (p14) and three structural proteins included in the viral envelope. These are the proteins:

1. E0 (also known as E ^ms ) - the former name is E2 or gp44-48 characterizing the azmolecular mass;

2. E1 - former name E3 or gp31;

3. E2 - former name E1or gp51-54;

All three of these proteins are glycosylated. Their arrangement in the precursor protein is as follows: E0, E1, E2. The glycoproteins form higher-order structures among themselves, connected with each other by disulfide bridges. E0 (gp44-48) and E2 (gp51-54) are exposed on the cell surface, while E1 (gp31) is directed inside the virion (Thiel et al., J. Virol. 1991; 65: 4705-4712; van Rijn et al. m., J. Virol. 1994; 68: 3934-3942).

A further segment of the genome codes for non-structural proteins. These are the proteins NS2, NS3, NS4A, NS4B, NS5A, NS5B (Meyers et al., Virology 1992; 191: 368-386). The NS3 analogs in other flaviruses exhibit serine protease, NTPase and helicase activities. NS5B is possibly a viral RNA-dependent RNA polymerase.

Swine fever virus infected animals develop antibodies against the E0, E2 and NS3 proteins (Wensvoort et al., Vet. Microbiol. 1988; 17: 129-140). However, only anti-E2 antibodies have a strong neutralizing effect against the fever virus. In contrast, anti-E0 and anti-NS3 antibodies have very little neutralizing activity (Wensvoort, J. Gen. Virol. 1989; 70: 352-358; Weiland et al., J. Virol. 1990; 64: 3563-3569).

Until recently, one of the widely used vaccines against CSF was a vaccine based on a live, attenuated strain of the fever virus. The vaccine strain C (Chinese strain) successfully protected animals against CSF, but it was not possible to serologically distinguish vaccinated animals from wild-type infected animals. A number of CSF vaccines are currently under development and are still being developed. Currently used vaccines have to meet several requirements: they should be genetically safe, they should immunize animals effectively, and they should distinguish vaccinated animals from animals infected with the wild-type virus using relatively simple tests. CSF vaccines developed in recent years can be divided into four groups:

1. Subunit vaccines constructed on the basis of baculoviral vectors;

2. subunit vaccines based on herpesviral vectors;

3. live attenuated porcine virus vaccines, which have introduced appropriate genetic markers by site-specific mutagenesis methods;

4. DNA vaccines.

A candidate subunit vaccine obtained in the baculovirus system relied on expression of the E2 gene in insect cells (Hulst et al., J. Virol. 1993; 67: 5435-5442). Doses of 20 to 100 µg of the purified E2 gene product (gp51-54) were sufficient to obtain a high level of neutralizing antibodies. In the same laboratory, a baculoviral recombinant was constructed which contained the E2 gene with a deletion responsible for the lack of one of the antigenic determinants. This recombinant produced the E2 glycoprotein, which continued to induce the formation of neutralizing antibodies (van Rijn et al., J. Gen. Virol. 1996; 77: 2737-2745). Potentially, a vaccine and a diagnostic test to differentiate between infected and vaccinated animals in this system can only be constructed based on the E2 glycoprotein if the missing antigenic determinant is used in the diagnostic test.

The herpesviral vector used to introduce the CSFV genes is an attenuated pseudorabies virus. Also in this case, the E2 gene was introduced (van Zijl et al., J. Virol. 1991; 65: 2761-2765). Animals vaccinated with such a recombinant herpesvirus developed resistance to CSFV and pseudorabies virus. In order to reduce the risk of recombinant transmission from vaccinated to unvaccinated animals, Peeters et al. (J. Gen. Virol. 1997; 78: 3311-3314) constructed a recombinant that could only spread through intercellular junctions, while the mature viral particles were non-infectious.

Marker vaccines based on the C strain of the virus are constructed in a rather complicated way. First, a complete DNA copy of the viral genome is obtained (Moormann et al., J. Vro /. 1996; 70: 763-770). To such

Mutations, such as deletions or insertions, are introduced into the copies, which will form the basis of a diagnostic test. This is followed by in vitro transcription and introduction of infectious RNA into the cell.

Patent description US4745051 (published 1988.05.17) describes a solution for a method for obtaining a recombinant expression baculovirus vector for expression of a selected gene in a host cell. Preferably, a baculovirus DNA cleavage method is used to generate a DNA fragment containing the polyhedrin gene, or a portion thereof including the polyhedrin promoter. A recombinant shuttle vector is obtained by introducing said DNA fragment into a cloning system, and then introducing the selected gene into such a modified vector under the transcriptional control of the polyhedrin promoter. The recombinant vector interacts with the baculovirus DNA in such a way that recombination occurs and the selected gene is incorporated into the baculovirus genome.

Patent descriptions US5925360 (published 1999.07.20), EP1087014 (published 2001.03.28), US5935582 (published 1999.08.10), EP0389034 (published 1999.07.14), US5811103 (published 1998.09.22), EP0614979 (published 1996.06.05), EP0713915 (published 1996/05/29), EP0389034 (published 1994/09/14), describe Classical Swine Fever Virus CSFV, formerly Hog cholera virus, solutions. A vaccine containing a classical swine fever polypeptide is provided. In addition, vector vaccines are provided that are capable of expressing a nucleotide sequence encoding such a polypeptide. The discussed polypeptide and nucleotide sequences can also be used for the detection of classical swine fever.

WO0220048 (published 2002.03.14) describes a classical swine fever epitope vaccine containing at least one epitope peptide conjugated to a conjugate transport protein, each conjugate having at least one mono-epitope or multi-epitope repeat. at least once. The epitope (s) in question is (are) selected from a neutralizing epitope of the classical swine fever virus E2 envelope protein and the mutant epitope. The method of preparing the vaccine comprises the following steps: (1) synthesizing at least one peptide, each having a neutralizing mono-epitope or multi-epitopes or a mutated epitope, the epitope obtained from classical swine fever virus E2 envelope protein repeated at least once; (2) attaching to an appropriately conjugated carrier; (3) forming said conjugates with the vaccine to be formed.

Patent description EP0924298 (published 1999.06.23) describes a solution for gene expression in baculovirus systems. This invention relates to a method for increasing protein synthesis in baculovirus expression systems. The invention provides a method of producing a recombinant protein in the culture of insect cells infected with a baculovirus recombinant carrying the gene encoding said protein. In culture, the insect cells are in a growth medium of at least 2 liters and are infected with an inoculum containing at least one baculovirus under conditions where the cell density is 1x10 ⁵ to 5x10 ⁶ cells / ml and the moi is <0.01. Furthermore, the invention describes a method for obtaining E2 or Erns pestiviral proteins or fragments thereof in an insect cell culture characterized in that the final concentration of said protein (s) in the growth medium at harvest is at least 100 µg / ml. Furthermore, the invention relates to methods of producing follicle hormone, α and / or β units, and complexes or fragments thereof, with a concentration in the medium of at least 15 µg / ml.

EP1203813 (published 2002.05.08) and EP0003642 (published 1999.12.16) and EP0965639 (published 1999.12.22) describe the construction of attenuated pestiviruses. The described solutions relate to attenuated pestiviruses characterized by the fact that their enzymatic activity present in the E ^ms glycoprotein is removed, and methods for the production, use and detection of attenuated pestiviruses are presented.

Patent description US5750383 (published 1998.05.12) describes a solution for cloning in baculovirus. The cloning system is based on the recovery of the marker using the necessary gp64 gene for development. In this system, a gene necessary for virus replication, growth and reproduction in cell culture is deleted or inactivated in the viral genome. After the production of a baculovirus lacking this gene, the virus is multiplied in the host cells, which express the protein necessary for development or its functional homologue. To clone a foreign gene into a baculovirus containing the mutation, the virus infects wild host cells

The same cells are transfected with a plasmid containing the necessary gene for development, or a functional homologue thereof, attached to a foreign gene, under the control of a selected promoter. The baculovirus is "recovered" thanks to the essential gene attached to the foreign gene and is capable of normal multiplication and expression of the foreign gene. Recombinant "recovered" baculovirus can be used for gene expression, biological control, or the presentation of a foreign protein on the surface of the virus for the production of vaccines and antibodies.

Patent description US6461863 (published 2002.10.08) relates to pathways for modifying glycosylation of insect cells using baculovirus vectors. In this embodiment, various recombinant baculovirus vectors and insect host cells are provided that contain at least one gene encoding an enzyme involved in oligosaccharide conversion processes. Vectors and cells may contain other heterologous structural genes, including protein transforming enzymes. Methods of producing and using recombinant baculoviruses and vectors are presented, including their use in the production of recombinant proteins and insecticides.

Patent description US6355240 (published 2002.03.12) describes a solution to enhance the insecticidal properties of an insect virus by expressing heterologous proteins with early promoters. The invention relates to recombinant insect viruses such as baculoviruses or granulosis viruses used as insecticides, and vectors used in conjunction with viruses used to express heterologous genes or fragments thereof (preferably encoding an insect controlling or modifying substance such as an insect toxin). attached to an early promoter.

Patent description US5965134 (published 1999.10.12) and EP0772632 (published 2001.10.04) describe solutions for an immunogenic composition against pestiviruses, especially CSFV, especially non-structural p10 protein, more specifically T-cell epitopes from this protein and nucleic acid molecules encoding these polypeptides, vaccines, and the use of these polypeptides and nucleic acid molecules in diagnostics.

The research described above has significantly expanded the knowledge about CSFV and methods of preventing diseases caused by this virus. However, there is still no preparation that could be used for immunization against CSFV, and it would be characterized by a very good ability to generate a protective immune response and at the same time a low price allowing for mass vaccination.

The object of the present invention is to provide means that could be used for the efficient production of the E2 glycoprotein for the production of a CSFV vaccine, and in particular for the production of the growth medium of insect cells infected with a baculovirus recombinant expressing classical swine fever E2 glycoprotein.

Unexpectedly, the realization of such a defined goal and the solution of the problems described in the prior art related to proteins generated in cells on the basis of exogenous DNA have been achieved in the present invention.

The invention relates to a polypeptide consisting of a signal sequence and an amino acid sequence of an E2 glycoprotein or a fragment thereof, characterized in that the signal sequence is selected from a baculovirus gp67 signal sequence, a pseudorabies virus gG or gD signal sequence.

Another object of the invention is a nucleotide sequence encoding a polypeptide consisting of a signal sequence and an amino acid sequence of the E2 glycoprotein or a fragment thereof, characterized in that the signal sequence is selected from the baculovirus gp67 signal sequence, the gG or gD signal sequence of pseudorabies virus.

Preferably, the sequence according to the invention comprises a sequence selected from among the sequences shown in Fig. 4 - Fig. 9.

Another object of the invention is a baculovirus containing a nucleotide sequence encoding a polypeptide consisting of the signal sequence and the amino acid sequence of the E2 glycoprotein or a fragment thereof, characterized in that the signal sequence is selected from the baculovirus gp67 signal sequence, the pseudorabies virus gG or gD signal sequence.

Another object of the invention is the use of a nucleotide sequence encoding a polypeptide consisting of the signal sequence and the amino acid sequence of the E2 glycoprotein or its fragment, characterized in that the signal sequence is selected from among the

The baculovirus gp67 signal sequence, the pseudo rabies virus gG or gD signal sequence, for the production of an E2 glycoprotein or a fragment thereof.

Another object of the invention is a method of producing E2 glycoprotein, the major antigen of the classical swine fever virus, characterized in that the insect cells are infected with AcNPV recombinant baculovirus containing the classical swine fever virus gene under the control of the polyhedrin promoter, the infected insect cells are grown, the cells are removed by centrifugation. and E2 glycoprotein is precipitated from the growth medium with glycoprotein precipitation reagents.

Preferably, the method according to the invention is characterized in that the reagent for precipitating glycoproteins is ammonium sulfate.

Preferably, the method according to the invention is characterized in that the cultivation of the insect cells is carried out for a period of up to 10 days.

Preferably, the gene encoding the E2 glycoprotein comprises baculovirus gp67, pseudorabies virus gG or gD signal sequences allowing the export of the glycoprotein into the growth medium.

Preferably, the gene encoding the E2 glycoprotein has been devoid of a glycoprotein anchor sequence in cell membranes.

Preferably, the insect cells are cultured in a synthetic medium devoid of fetal calf serum.

Another object of the invention is the use of a baculovirus containing a nucleotide sequence encoding a polypeptide consisting of the signal sequence and the amino acid sequence of the E2 glycoprotein or a fragment thereof, characterized in that the signal sequence is selected from the baculovirus gp67 signal sequence, the gG or gD signal sequence of pseudorabies virus to produce the glycoprotein E2 or a fragment of it.

A further object of the invention is the use of a polypeptide consisting of the signal sequence and the amino acid sequence of the E2 glycoprotein or a fragment thereof, characterized in that the signal sequence is selected from a baculovirus gp67 signal sequence, a pseudo rabies virus gG or gD signal sequence for the production of a CSFV vaccine.

Another object of the invention is the use of a polypeptide obtained by the method of producing glycoprotein E2, the major antigen of classical swine fever virus, wherein the insect cells are infected with recombinant AcNPV containing the classical swine fever virus gene under the control of the polyhedrin promoter, the infected insect cells are cultured, the cells are removed by centrifugation and E2 glycoprotein is precipitated from the growth medium with glycoprotein precipitation reagents for the preparation of a CSFV vaccine.

Preferably, the use according to the invention is characterized in that the glycoprotein precipitation reagent is ammonium sulfate.

The attached figures allow a better explanation of the essence of the invention.

Figure 1 shows a schematic of a transfer plasmid for cloning the E2 gene.

Figure 2 shows Western blotting analysis of E2 glycoprotein production in insect cell culture. The photo shows a nitrocellulose membrane which is a replica of a 10% polyacrylamide gel with applied:

1. whole-cell carotid infected with a different type of baculovim carrying a truncated gene encoding the E2 glycoprotein;

2. cytoplasmic fraction of the same cells;

3. Growth medium on which insect cells infected with a different type of baculovirus carrying a truncated gene encoding the E2 glycoprotein were cultured;

4. whole insect cells infected with wild-type baculovirus.

Western blotting was performed using monoclonal antibodies reactive with the linear E2 epitope.

On the right side there are the molecular weights of the color standards used to determine the molecular weight of the E2 glycoprotein.

Figure 3 shows the reaction of the E2 antigen with polyclonal sera obtained by inoculating animals with purified preparations of the E2 glycoprotein with the transmembrane sequence cleaved off.

The picture shows a nitrocellulose membrane which is a replica of a 10% polyacrylamide gel on which insect cells infected with a recombinant baculovirus bearing a truncated gene encoding the E2 glycoprotein collected as shown in the top of figure 2, 3, 4, 5 days after recombinant infection. Left panel - recombinant with gG signal sequence, right panel - without signal sequence. In the outer right lane, the preparation was used to immunize the animals. Western blotting was performed using monospecific polyclonal serum.

Figure 4 shows the nucleotide sequence of the E2 gene with a lead sequence and the corresponding amino acid sequence, designated GP67E2TM ^- includes the truncated form of E2 with the sequence gp67.

Figure 5 shows the nucleotide sequence of the E2 gene with the lead sequence and the corresponding amino acid sequence, designated GP67E2, includes the complete E2 form with the gp67 sequence.

Figure 6 shows the nucleotide sequence of the E2 gene with a lead-out sequence and the corresponding amino acid sequence, designated GGE2TM ^- includes a truncated form of E2 with a gG sequence.

Figure 7 shows the nucleotide sequence of the E2 gene with the lead sequence and the corresponding amino acid sequence, designated GGE2, includes the complete form of E2 with the gG sequence.

Figure 8 shows the nucleotide sequence of the E2 gene with a lead-out sequence and the corresponding amino acid sequence, designated GDE2TM ^- includes the truncated form of E2 with the gD sequence.

Figure 9 shows the nucleotide sequence of the E2 gene with the lead sequence and the corresponding amino acid sequence, designated GDE2, includes the complete form of E2 with the gD sequence.

Exemplary embodiments of the above-defined invention are presented below.

P r z k ł a d 1

Production of recombinant E2 glycoprotein.

a) construction of transfer vectors with signal sequences

In order to increase the amount of E2 glycoprotein exported outside the cell to the growth medium, special transfer vectors were constructed, derivatives of the pFastBac plasmid, where special signal sequences were introduced from the 5 'end of the multiple cloning site. These sequences, designated gG, gD, and gp67, have been selected on the basis of their role in leading some viral proteins outside the natural host cell. The nucleotide sequences of the signal sequences and their DNA base composition are given below.

GD signal sequence:

CTC AGA TCT AAA ATG CTG CTC GCA GCG CTA TTG GCG GCG CTG GTC GCC CGG ACG ACG CTC GGT GCG GAC GTG GAG GAT CCG TG

Number of rules - 83; composition - 13A; 24C; 30G; 16T

GG signal sequence:

GGC AGA TCT CAA CAA TGA AGT GGG CAA CGT GGA TTC TCG CCC TCG GGC TCC TCG TGG TCC GCA CCG TCG TGG CCG GAT CCG AGG

Number of bases - 84; from where - 14A; 26C; 28G; 16T

Gp67 signal sequence:

CGC AGA TCT CAA TGC TAC TAG TAA ATC AGT CAC ACC AAG GCT TCA ATA AGG AAC ACA CAA GCA AGA TGG TAA GCG CTA TTG TTT TAT ATG TGC TTT TGG CGG CGG CGG CGC ATT CTG CCT TTG CGG AA CG ACGA CG ACTC GCA TG AGC ACTC AGA CGG ATC CTG C

Number of bases - 163; thence - 44A; 40C; 42G; 37T

These sequences were obtained by PCR using the appropriate complementary primers (12 complementary bases each from the 5 'and 3' end) and the DNA of an organism containing genes with signal sequences. The primers, in addition to the complementary sequences, contained sequences recognized by the selected restriction enzymes: sequences for BglII on the 5 'primer and sequences for BamHI on the 3' primer. After the PCR reaction was performed, the reaction products were analyzed by agarose electrophoresis, the bands after staining with ethidium bromide were illuminated with ultraviolet radiation in the range of about 360 nm and visually analyzed on a transilluminator. Bands corresponding to the appropriate molecular weight were isolated from the agarose gel and digested with restriction enzymes that recognized the nucleotide sequences inserted at the 3 'and 5' ends. The restriction enzyme digested fragments were purified again to remove reagents. The purified fragments were added to the ligation mixture containing, in addition to bacteriophage T4 ligase and the appropriate ATP reaction buffer, also the pFastBac transfer vector cut at the multiple cloning site with the same restriction enzymes as the cloned fragments. After ligation overnight, the reaction mixture was used to transform Escherichia coli bacterial cells, preferably Escherichia coli DH5. Ampicillin was used as a selection agent. Ampicillin-resistant bacterial colonies were grown in liquid culture supplemented with ampicillin and DNA was isolated

Plasmid from a culture derived from each ampicillin resistant colony. Each of the plasmids was subjected to careful restriction analysis. The plasmids containing the cloned fragments and the unaltered starting plasmid were finally sequenced to confirm that the transfer plasmid with the signal sequence had a nucleotide sequence that was completely theoretical as predicted. The schematic diagram of the resulting transfer plasmids with signal sequences is shown in Figure 1, while the nucleotide sequence diagrams of the E2 gene with lead sequences and the corresponding amino acid sequences are shown in Figures 4-9.

b) construction of a baculovirus recombinant carrying the E2 gene of classical swine fever virus

The full-length E2 gene (gp51-54) was obtained by RT-PCR using the CSF virus genome as template. It was then cloned into the pUC19 multicopy plasmid. To obtain the transfer plasmid with the cloned E2 gene, the multicopy plasmid containing the complete 1500 bp gene was first cleaved with the SalI enzyme at the multiple cloning site and the sticky ends were filled in using the Klenow fragment of the Escherichia coli DNA polymerase. The fragment encoding the E2 gene was excised from the plasmid vector by cleaving the plasmid with the KpnI enzyme at the multiple cloning site on the opposite side of the E2 insert. The E2 gene was then cloned into a transfer plasmid.

A 1083 bp fragment of the E2 gene lacking the coding sequence for the C-terminal part of the glycoprotein was excised from the multicopy plasmid with the enzymes KpnI and Scal. and cloned into the transfer plasmid in the same way as the full-length gene. After ligation, the pFastBac1 plasmid with the cloned truncated E2 gene was used for the construction of a baculovirus recombinant using the method used in the Bac-to-Bac system.

For this purpose, 5 µL of purified transfer plasmid DNA with the cloned foreign gene was added to 100 µ 100 of Escherichia coli DH10Bac competent cells. After incubation on ice for 30 min, heat shock was performed, 1 ml of fresh growth medium was added and the bacteria were grown for 2 h. The transformed cells were then plated on a selection medium containing kanamycin, gentamicin, tetracycline, and a substrate for galactosidase X-gal and IPTG. After overnight incubation, white colonies were isolated and the bacteria grown on kanamycin and gentamicin medium. Bacmid DNA containing the E2 gene after transposition was isolated from the bacteria and used to transfect Spodoptera frugiperda Sf21 insect cells with lipofectin. The transfected insect cells were grown for 72 h, and then the recombinant was isolated.

In order to determine the production of E2 glycoprotein in insect cells in the first stages after transfection, the IPMA (immunoperoxidase monolayer assay) reaction was performed. This reaction is similar to the immunoblotting reaction, however, the subsequent steps of the IPMA reaction are carried out in situ within the infected cells. Monolayer cultures infected with the appropriate recombinant were fixed with cold methanol. After removal of methanol, cells were washed with PBS buffer and then incubated with horseradish peroxidase conjugated anti-E2 monoclonal antibodies. Excess antibodies were washed away and peroxidase substrates were added: hydrogen peroxide and 3-amino-9-ethylcarbazole. Cells in which the antigen was synthesized stained red.

In the next step, the localization of the E2 glycoprotein was checked during the cultivation of insect cells infected with baculovirus recombinants. Spodoptera frugiperda insect cells of the Sf21 line were cultured in medium supplemented with fetal bovine serum or in fully synthetic medium. Insect cells were infected with the appropriate recombinant at a multiplicity of infection of approximately 10. After 3-5 days after infection, the insect cells were centrifuged and fractionated into membrane and cytoplasmic fractions, and E2 glycoprotein was also isolated from the synthetic growth medium.

Proteins of the membrane and cytoplasmic fractions and the growth medium obtained after infection with baculovirus recombinants were analyzed on 10% polyacrylamide gels in the presence of dodecyl sulfate. Electrophoresis was performed according to the Laemmli method (Laemmli, U.K., Nature; 1970; 227; 680-685) and stained with Coomassie blue. Western blotting antigen analysis was performed according to Towbin et al. Proc.Natl.Acad.Sci.USA, 1979; 76, 4350-4354. Protein transfer was performed on nitrocellulose membranes or polyvinylidene fluoride membranes. Antibodies bound to alkaline phosphatase were used for detection.

The obtained glycoproteins on polyacrylamide gels gave quite blurred bands, which is characteristic of glycoproteins present in more than one glycoform. The molecular weight of the main band of the full-length glycoprotein synthesized in insect cells is over 50 kD and is close to the molecular weight of the native glycoprotein synthesized in the cells

PL 201 132 B1 in mammals (51-54 kD). The full-length E2 glycoprotein accumulates in insect cells mainly in the cell's membrane structures and is not transported out to the growth medium. The full-length protein synthesized by recombinant BacE2 (TM ⁺ ) was used to obtain a monospecific, polyclonal anti-E2 serum. The method of elution from a replica of polyacrylamide gel on a PVDF membrane was used to obtain a highly purified protein required for immunization of the rabbit (Szewczyk, B., Summers, DF, Anal. Biochem., 1988; 168; 48-53). An anti-E2 polyclonal serum was obtained which reacted well with the baculovirus product of the E2 gene and with the native E2 glycoprotein.

The second recombinant BacE2 (TM) contains a truncated form of the gene (1100 bp). The deleted region corresponds to the C-terminal part of the E2 glycoprotein which has no antigenic determinant. The aim of the modification by adding signal sequences and removing the C-terminal part was to obtain a protein product efficiently transported from the cell to the medium. Transport to the growing medium makes the product more accessible for possible insulation. The amount of truncated E2 protein synthesized was nearly three times the amount of full-length E2 protein expressed. The truncated E2 glycoprotein forms homodimers, which proves the proper folding of glycoproteins. The molecular weight of the truncated form of the E2 protein is 45-47 kD. It is efficiently transported to the growth medium (Fig. 2).

P r z k ł a d 2

Immunogenic properties of the truncated E2 glycoprotein.

Full-length E2 protein synthesized after infection of insect cells with recombinant BacE2 (TM +) and truncated E2 protein synthesized after infection with recombinant BacE2 (TM) were used to obtain monospecific polyclonal sera against E2 glycoprotein. The method of elution from a replica of a polyacrylamide gel on a polyvinylidene fluoride-PVDF membrane was used to obtain a highly purified protein required for immunization of the rabbit (Szewczyk, B., Summers, DF, Anal. Biochem., 1988; 168; 48-53). For this purpose, Spodoptera frugiperda insect cells of the Sf21 lineage were cultured in a completely synthetic medium. They were infected with the appropriate recombinant at a multiplicity of infection of approximately 10. After 4 days after infection, the insect cells were centrifuged and fractionated into membrane and cytoplasmic fractions, and E2 glycoprotein was also isolated from the synthetic growth medium. The membrane fraction in the case of full-length recombinant BacE2 (TM ⁺ ) or the growth medium of insect cells infected with recombinant BacE2 (TM) was loaded onto a 10% polyacrylamide gel. After the electrophoretic separation was completed, a gel replica was made on a polyvinylidene fluoride (PVDF) membrane using a wet transfer apparatus and transferring overnight at room temperature at 30V. After transfer, the membrane was reversibly stained with Ponceau red and the position of the full or truncated E2 glycoprotein was marked with a pencil. The E2 glycoprotein bands were cut precisely with small scissors not allowing the membrane to dry out. The excised bands were placed in an Eppendorf tube and flooded with a small amount of elution solution (2% dodecyl sulfate / 1% Triton X-100 in pH 7.0 buffer). After several minutes of shaking, the membrane was centrifuged and the elution solution was collected. The elution procedure was repeated once more and 4 volumes of acetone were added to the combined eluates. After 12 hours at -20 ° C, the pellet was centrifuged. The amount of pure protein in the pellet was assessed against a protein standard (bovine blood albumin) by applying a small portion of the pellet taken from an homogeneous suspension in water to a 10% polyacrylamide gel and electrophoresing in the presence of dodecyl sulfate. The gel was stained with Coomassie blue. The remainder of the pellet was used as antigen to vaccinate rabbits.

Rabbits were immunized by vaccinating twice with approximately 100 µg of antigen. The first vaccination was performed intradermally with an emulsion of antigen and Freund's complete adjuvant. The emulsion was prepared by brief 30-second sonication of a mixture of antigen (0.5 ml in water) and adjuvant (0.5 ml). After 4 weeks, a revaccination was performed with an emulsion prepared from the antigen suspension (100 µg in 0.5 ml water) and incomplete Freund's adjuvant. This vaccination was performed subcutaneously. After another 4 weeks, the animal's blood was collected and the serum was isolated. The thus obtained anti-E2 polyclonal sera reacted well with the baculovirus product of the E2 gene and with the native E2 glycoprotein in both IPMA and Western blotting (Fig. 3). No difference in serum reactivity was found whether the complete E2 antigen was used or a truncated product that contained sequences leading the E2 glycoprotein into the growth medium.

PL 201 132 B1

Claims (15)

Patent claims A polypeptide consisting of a signal sequence and an amino acid sequence of an E2 glycoprotein or a fragment thereof, wherein the signal sequence is selected from a baculovirus gp67 signal sequence, a pseudoragas virus gG or gD signal sequence. 2. A nucleotide sequence encoding a polypeptide consisting of a signal sequence and an amino acid sequence of E2 giicoprotein or a fragment thereof, wherein the signal sequence is selected from a bacuiovirus gp67 signal sequence, a pseudoraginous virus gG or gD signal sequence. 3. The sequence of claim 1 The sequencer of the sequences shown in Fig. 4 - Fig. 9. 4. Baculovirus containing the - ^ <c ^ nuWeotide sequence encode a polypeptide consisting of a signal sequence and an amino acid sequence of the E2 giicoprotein or a fragment thereof, the signal sequence being selected from the bacuiovirus gp67 signal sequence, the gG and or gD signal sequence of pseudoviral virus. 5. The use of a nucleotide sequence encoding a polypeptide consisting of the signal sequence and the amino acid sequence of the E2 giicoprotein or a fragment thereof, the signal sequence being selected from the bacuiovirus gp67 signal sequence, the pseudo-rabies virus gG or gD signal sequence, for the production of giikoprotein E2 and glycuboprotein E2 and a fragment thereof. its fragment. 6. A method of producing glycoprotein E2, a major virnsaclassical swine pomoou antigen, characterized in that the insect cells are infected with a recombinant AcNPV bacuioviral virus containing the kiassic swine fever virus gene as defined in claim 1. 2 under the control of the poiihedrin promoter, infected insect cells are grown, cells are removed by centrifugation, and giicoprotein E2 is precipitated from the growth medium with giicoprotein precipitation reagents. 7. The method according to principles. 6 by the fact that the reagent to extract glycoprophein ammonium sulfate. 8. The method according to p. 6. The method of claim 6, wherein the culturing of the insect cells is performed for up to 10 days. 9. The method according to p. 6, incl. that the gene encoding the E2 glycoprotein contains the bakuiovirus gp67, gG, or gD signal sequences of the pseudo rabies virus to export the giicoprotein to the growth medium. 10. The method according to Zasł. 6, in that the gene encoding the E2 glycoprotein was: devoid of a glycoprotein anchor sequence in cell membranes. 11. The method according to p. 6. The method of claim 6, wherein the insect cells are cultured in a synthetic medium devoid of fetal liquid serum. 12. Use of a bacuiovirus containing a nucleotide sequence encoding a polypeptide consisting of the signal sequence and the amino acid sequence of E2 giicoprotein or a fragment thereof, the signal sequence being selected from the bacuiovirus gp67 signal sequence, the gG and or gD signal sequence of the pseudo-rabies virus E2 and a giikuboprotein fragment thereof. 13. A polypeptide consisting of the sequence and the amino acid sequence of E2 glycoprotein, or a fragment thereof, was used, wherein the signal sequence was selected from the bakuiovirus gp67 signal sequence, the pseudo rabies virus gG or gD signal sequence, for the production of a CSFV vaccine. 14. The polypeptide obtained by the method of producing giicoprotein E2, the major antigen of the kiasic swine fever virus as defined in claim 14, is used. 2, where the insect cells are infected with the AcNPV recombinant bacuioviral virus containing the viral swine fever virus gene under the control of the poiihedrin promoter, the infected insect cells are grown, the cells are removed by centrifugation, and the E2 giicoprotein is precipitated from the growth medium with CSFein giicoprotein precipitation reagents. 15. Applications according to merit. 14 that the reagent for w '^ tr ^^ (^^ rΉ ^ glycoproteins is ammonium sulfate.