FI102835B - Western subtype peptides and polypeptides of FSME virus and diagnostic agents containing them - Google Patents

Description

1,102835 Western FSME Virus Peptides and Polypeptides and Diagnostic Agents Containing Them

Divided by application 881294 5

The present invention relates to peptides and polypeptides of the Western subtype of FSME virus and diagnostic agents containing them.

The western subtype of early summer meningoencephalitis (FSME) virus belongs to the genus Flaviviridae, which are spherical, lipid-surrounded RNAviruses (Westaway et al., Intervirology 24, 183-192, 1985). The prototype of these viruses is the yellow fever virus. By definition, all Flavi viruses are serologically related to each other, as shown by the haemagglutination inhibition test. However, by cross-neutralization, the genus can be subdivided into several serocomplexes (DeMadrid and Porterfield, J. Gen. Virol. 23, 91-96, 1974), which comprise viruses that are more closely related to each other or to ungrouped serocomplexes.

With Flavi viruses. FSME virus is a so-called. Member of the "Tick-Borne" serogroup, which also includes Loupung-111 virus, Langat virus, Omsker's hemorrhagic fever virus, Kyasanur Forest-Disease virus and Negishi virus. 25 FSME virus strains could be further subdivided into the western (European) subtype, which predominantly transmits via Ixodes ricinus, and the Far East subtype, whose primary agent is Ixodes persulcatus (Clarke, 1964; Bull. WHO 31, 45-56).

This subtype differentiation was demonstrated by both «competitive radioimmunoassay and peptide mapping using the limited proteolysis of the corresponding structural glycoproteins (Heinz and Kunz, J. Gen. Virol. 57, 263-274, 1981) and antigen analysis using? (Heinz et al., Virology 126, 525-537 (1983)).

The mature viral particles contain only three building proteins, designated E, C, and M, with approximate molecular weights of 50,000-60,000, 15,000, and 7,000.

The genome of Flavi viruses consists of single-stranded RNA of about 11,000 bases with mRNA direction and a molecular weight of 4 x 10 6 daltons. This RNA form-10, together with protein C, produces a spherical nucleocapid surrounded by a lipid membrane fused to proteins E and M. Experiments using purified preparations of E protein obtained with virus detergents after solubilization have shown that E exhibits viral haemagglutinin (i.e., it induces agglutination of certain erythrocytes under appropriate conditions) which, after immunization, induces haemagglutination inhibitors, protective antibodies as well as immunity against infection with live virulen-20 virus (Heinz et al., Infect. Immun. 33, 250-257, 1981).

In addition to these structural proteins, several virus-specific non-structural proteins can be found in the cells infected by Flavi viruses.

The genomic sequence of Flavi viruses has recently been determined using cDNA cloning and sequencing of yellow fever, West Nil and Murray Valley encephalitis viruses (Rice et al., Science 229, 726-733, 1985; Dalgarno et al., J. Mol. Biol. 187, 309-332, 1986; Castle et al., 30 Virology 147, 227-236, 1985; Castle et al., Virology 149, 10-26, 1986; Wengler et al., Virology 147, 264. 274 (1985). According to these analyzes, the Flavi viral genome RNA contains a single, long, open reading frame of approximately 11,000 nucleotides encoding all structural proteins and non-structural proteins.

The genes of the structural proteins are localized to the 5 'part of the RNA; they comprise about a quarter of the genome 5 in the order C-prM (M) -E. prM is a precursor of M present in immature forms of Flavi viruses (Shapiro et al., Virology 56, 88-94, 1973). Its proteolytic degradation, likely due to the presence of protease 10 in the cellular endoplasmic network system, results in the formation of M and is apparently a late event in the course of virus maturation.

It is assumed that translation of viral proteins is initiated and continues from the first or possibly second AUG at the 5 'end of the RNA until it encounters the termination 15 codon at the 3' end of the RNA. It is believed that the formation of individual proteins occurs through a series of specific proteolytic degradation events involving both cellular and viral specific proteases.

Pletnev et al. (FEBS 3660, 200, No. 2, 317-321, 20 1986) have cloned certain fragments of the Far East subtype of the FSME virus and demonstrated that the genome sequence of this subtype of FSME virus corresponded to the genomes of the above viruses but were unable to clone and sequence FSME. the entire sequence of the 25 structural genes of this subtype of the virus. In addition, the published protein E sequence derived from the clone Sofjin 2 has been shown to be incorrect because the reading frame shift resulted in the wrong sequence in the carboxy terminal region.

30 To date, more than 60 different Flavi viruses w · are known, and about two-thirds of them are transmitted through the infection of an infected Arthropod (Arthropode) and are thus known as Arthropode-Borne (ARBO) viruses. Several Flavi viruses are known human pathogens, including yellow fever virus, Dengue virus, Japanese encephalitis virus, and FSME virus (Shope; "The Togaviruses", pp. 47-82, Academic Press, New York).

The FSME virus is endemic in many European countries, Russia and 5 China. The disease is well documented in some Central European countries, such as Austria, Czechoslovakia or Hungary, and every year hundreds of cases of hospitalization are registered, which is a major public health problem.

The disease can be effectively prevented by vaccination with a highly purified, formalin-inactivated whole virus vaccine (Kunz et al., J. Med. Virol. 6, 103-109, 1980), which induces an immune response against the structural proteins of the virus. The major disadvantage of this vaccine is that large quantities of infectious and potentially dangerous viral suspensions must be handled during manufacture. Therefore, large and expensive security measures are necessary.

The present invention relates to a peptide or polypeptide characterized in that it comprises a portion of a protein selected from the group consisting of protein C, rpm, M or E of the western subtype of FSME virus and the amino acid sequence contained in Figure 3. .. amino acid sequence.

The present invention further relates to a diagnostic agent, characterized in that it contains the above peptide or polypeptide.

Within the scope of the present invention, there is provided a DNA molecule comprising a FSME virus from the west. 30 subspecies DNA encoding at least one portion of at least one protein selected from the group consisting of the Western subtype of FSME virus proteins C, M or E.

The described DNA molecule corresponds to the single stranded RNA of the FSME Western subtype 35 and is suitable for the generation of genetic information for expression of a polypeptide which may be the Western FSME virus subtype C, prM, M or E alone or in combination as described above. or portions of one or more of said 5 proteins that can be usefully used in diagnostics.

The DNA molecule may be used as a whole molecule having the sequence encoding structural proteins of the western subtype of the FSME virus, preferably as shown in Figure 1. The DNA sequence shown in Figure 1 represents the cloned genome of the western subtype of the FSME virus, corresponding to the RNA molecule contained in the naturally occurring western subtypes of the FSME virus. The DNA sequence shown in Figure 1 comprises a region of structural proteins and an untranslated 5 'region. A carefully studied DNA sequence shows only an open reading frame; the amino terminal origin of proteins C, prM, M and E, as well as the boundaries of clones A5 and P1-1 are indicated by arrows.

Preferably, the DNA molecules encode one of the structural proteins of the western subtype of the FSME virus, i.e., the entire C, prM, M or E protein. Individual portions of the DNA molecule in question can be used directly in expression systems to produce one of the desired structural proteins.

Preferably, a portion of a DNA molecule encoding at least the region of an antigenic determinant of a structural protein of at least the western subtype of the FSME virus is used for the production of diagnostic agents. It is known that regions containing antigenic determinants of a protein induce antibody induction. Therefore, it is not necessary to use whole proteins known to have antigenic properties, but only the region of the anti-β2282835 genetic determinant of these proteins can be used. It is known that in some cases the sequence of only a few amino acids may act as an antigen and may elicit an antibody response. A DNA sequence encoding the regions of the antigenic determinants of the FSME virus subtype 5 structural protein E is preferred.

There are two ways to make a DNA molecule. One way to obtain the DNA molecule is to extract viral RNA from the western FSME virus subtype and purify 10 RNA molecules, followed by reverse transcription of this RNA matrix into a DNA molecule using the reverse transcriptase enzyme. Another possibility is to chemically synthesize DNA molecules as soon as the DNA sequence is known.

In a preferred embodiment, the DNA molecules used in the context of the present invention can be linked to additional DNA sequences.

These additional sequences may allow replication and expression of the DNA molecule in cell culture. For this purpose, the most important DNA sequences are the DNA sequences of enhancers (promoters), promoters, polyadenylation signals, and splice signals. These additional DNA sequences may be combined with the DNA molecules used in the invention according to standard methods known to those skilled in the art.

The above for DNA molecules also applies to RNA molecules. RNA molecules useful in the invention may be obtained by isolation and purification from Western FSME virus subtype RNA or by recombinant RNA / DNA technology. Further, not only are the RNA molecules obtained by purification of the western subtype of FSME virus useful, but also the RNA molecules obtained by reverse transcription into isolated and purified viral RNA DNA and subsequently reverse transcribed-35 miles obtained DNA again into RNA.

•.

DNA v102835 DNA and RNA molecules can be inserted into vectors selected from the group consisting of plasmids, viruses and cosmids. One skilled in the art will recognize that the vectors used may comprise DNA sequences that control replication and expression of inserted RNA or DNA sequences, such as promoters, enhancers, etc.

A plasmid 10 pBR322, which is well described in the literature and suitable for cloning said DNA sequences.

A preferred plasmid prepared in this manner is plasmid A5 shown in Figure 5. Plasmid A5 derived from pBR322 contains a DNA sequence encoding protein E. Another preferred plasmid is 15 P1-1. Said preferred plasmids have been deposited with the Deutsche Sammlung von Mikroorganismen under a contract with Budapest and have been assigned the following deposit numbers: a) plasmid A5 in E. coli HB101 20 DSM 4382 b) plasmid P1-1 in E. coli HB101 DSM 4383 Starting from the described plasmid A5, insertion plasmids containing portions of the FSME virus DNA sequence cloned in plasmid A5 can be advantageously prepared, wherein these insertion plasmids contain foreign genetic information such that it can be combined with Vaccinia virus HindIII J by homologous recombination. The sub-fragments of plasmid A5 are cleaved by various restriction endonucleases and cloned into insertion plasmids, for example to obtain forms of protein E, which may or may not contain hydrophobic amino acid sequences, namely signal and membrane anchor sequences.

• «102835

Recombinant viruses with integrated protein-E DNA may be isolated from plaque. The integration of the FSME DNA sequences encoding the desired portions of protein E is confirmed by digestion with appropriate restriction endonucleases and hybridization with 32 P-labeled FSME cRNA, and it is noted that a characteristic Vaccinia HindIII J fragment can be detected.

Three examples of plasmids suitable for recombination in Vaccinia viruses include OVdt 10 pSCll-H1 containing the Haell fragment of cloned FSME DNA, plasmid pSCll-F41 containing the Fnu DII fragment of cloned FSME DNA, and plasmid pSC1-P6 containing the PvuII fragment of cloned FSME DNA. The plasmids described are shown in Figure 7. The starting plasmid pSC11 in E. coli is deposited with the depository at the Deutsche Sammlung von Mikroorganismen Budapest under the accession number DMS 4381.

The described insertion plasmids are preferably used to insert the desired sequences into Vaccinia expression vectors.

Bacteria can also be used as sequence transfer vehicles. A preferred bacterium i is Salmonella typhi.

Thus, the vectors or means used are preferably contained in cell cultures in which expression of the polypeptides of the invention encoded by RNA or DNA sequences occurs under the most natural conditions possible, preferably the cell culture is a mammalian cell culture.

Particularly suitable cell cultures for expression of the desired peptides or polypeptides are Vero cell cultures or chicken fetal fibroblast cell cultures containing the Vaccinia expression vector described above, expressing the proteins corresponding to the Vaccinia expression vectors.

5, 102835

Recombinant Vaccinia viruses can be analyzed using Southern Blot hybridization to detect the presence of inserted FSME DNA.

Thus, peptides or polypeptides comprising amino acid sequences encoded by any of the above nucleotide sequences are provided according to the invention. The advantages of preparing these peptides or polypeptides are in agreement with the advantages presented above for DNA and RNA molecules.

The amino acid sequences of the peptides or poly peptides obtainable according to the invention correspond to the DNA and RNA sequences described above and preferably have the amino acid sequence shown in Figure 3.

The plasmids pSC11-H1, pSC11-F41 and pSC11-P6 described above can be used to carry out the expression of the proteins they define, preferably by combining with the corresponding Vaccinia expression vectors.

While it is preferable to prepare peptides or polypeptides via expression of any of the 20 nucleotide sequences provided within the scope of this invention, it is also possible to synthesize the peptides or polypeptides of the invention chemically.

Preferably, the peptides or poly-v peptides of the invention are included in a composition that can be used in the diagnostic field.

The invention will be illustrated by the following detailed description.

A preferred embodiment of the invention is illustrated in the figures, as shown in the following description of the figures: Figure 1 shows the DNA sequence of the cloned genome of the western subtype of FSME virus comprising a structural region and a 5 'non-reverse transcript region. The amino terminal origin of proteins C, prM (M) and E, as well as the boundaries of clones A5 and P1-1 are indicated by arrows.

• «! O 102835

Figure 2 shows the RNA sequence of the western subtype of the FSME virus encoding structural proteins. The amino terminal origin of proteins C, prM (M) and E are indicated by arrows.

Figure 3 shows the amino acid sequence of the structural subunits of the Western subtype of FSME virus derived from the DNA sequence shown in Figure 1. The NH2 ends of the various proteins are indicated by arrows.

Figure 4 shows a photograph of agarose gel electrophoresis of plasmids containing inserts specific for the western subtype of the FSME virus after digestion with the restriction enzyme SmaI (lines 2-6). The upper bands represent plasmid DNA (4363ep), the lower bands represent specific insert DNA (in the range 15000 to 3500 bp). The DNA markers in lines 1 and 7 correspond to base pairs 23,130, 9,416, 6,557, 4,361, 2,322, 2,027, 564 and 125 (top to bottom).

Figure 5 shows plasmid A5 derived from pBR322, showing restriction endonuclease cleavage sites of selected sub-fragments 20, with corresponding regions encoding FSME protein E indicated. The arrow indicates the direction of transcription of the genomic RNA.

FIG. include a single SmaI insertion site, the Vaccinia promoter, the prokaryotic Lac Z gene, and Vaccinia-TK recombination sequences.

Figure 7 shows three specific, exemplary FSME insertion plasmids for in vivo recombination in a Vaccinia expression vector. The only SmaI site in each of the plasmids presented is nucleotide 6,500.

In each case, the first base of the FSME virus specific sequences in each of the plasmids presented is nucleotide 6,503.

Figure 8 shows the characteristic features of the subcloned FSME virus-specific sequences. Translation codon initiation codons and hydrophilic amino acid regions derived from the hydropathyplot have been described. These areas were identified using the IBI-Pustell computer program.

Hydrophobic sequences are indicated □, hydrophilic 5 sequences are I.

Figure 9 shows the physical mapping of FSME insertion plasmids and the identification of plasmid DNA fragments containing FSME-specific sequences. A shows an ethidium bromide-stained agarose gel showing DNA-10 fragments, and B shows the result of Southern Blot hybridization with a "FSMERi-boprobe" probe.

Section A) shows

Signal 1 HindIII digested lambda DNA as size 15 standard

Signal 2 Pvul DNA cleaved with Pvul Signal 3 Pvll DNA cleaved with Pvul Signal 4 BamHI digested pSC22-P6 DNA Signal 5 BamHI digested pSC11 DNA

Signal 6 BamHI digested pSCll-F41 DNA

Signal 7 EcoRI digested pSCll-F41 DNA

Signal 8 EcoRI digested pSC11 DNA

In section B), fragments containing FSME sequences indicated after hybridization are shown in 2.585 kb signal 3 0.919 kb signal 4 1.019 kb signal 6 1.879 kb signal 7 These fragments are expected from cloning 30 (see Figure 7). The DNA in signals 1, 2, 5 and 8 does not hybridize because it is only lambda DNA or pure pSC11 plasmid DNA.

Figure 10 shows Slot Blot hybridization for detection of FSME-Vaccinia virus combinations. Uninfected cells, cells infected with vSC8 (Dr. Moss), or cells infected with four P6 isolates, were transferred to nitrocellulose filters and hybridized with 32 P-labeled pSC115 DNA (nick-repair). or with FSME-specific "Ri-boprobe" in B. No signals are detected for non-infected cells. The recombinant virus vSC8 hybridizes with pSC11 for TK sequences. However, FSME-specific "Riboprobe" did not produce a positive signal with vSC8 as expected. Cells infected with the single isoforms (1-4) of the viral combination P6 showed hybridization with both "Riboprobe" (due to the FSME sequence) and pSC11 DNA (due to the TK sequence).

Figure 11 shows DNA fragment patterns obtained after wild-type virion DNA digestion with HindIII or Vaccinia virus / FSME combinations. Thereby, it becomes clear that the HindIII J fragment is lost by recombination events in 20 viral recombinant DNA cleavage models. Original

The HindIII J fragment of about 5 kb is only visible in signal 6 on the agarose gel. No other DNA preparations contain the HindIII J fragment.

. Signal 1 BstEII as a cut length lambda DNA standard

Signal 2 DNA of FSME combination F41 (insertion plasmid pSC11-F4)

Signal 3 FSME combination P6 DNA (insertion plasmid pSCll-P6) 30 Signal 4 viral combination P1 DNA (insertion plasmid pSC11)

Signal 5 DNA of vSC8 virus combination

Signal 6 DNA from wild type WR virions

▼ · • «13 102835

Figure 12 shows the detection of FSME-specific sequences in HindIII DNA fragments of virus combinations using hybridization with 32P-labeled FSME "Ribopro-be" (A). As a control, the same filter was hybridized a second time with 32 P (nick-repair) -labeled pSC22 plasmid DNA (B).

A) The FSME-specific "Riboprobe" hybridizes exclusively with DNA from viral combination F41 at signals 1, 2 and 3 and with 10 from P6 at signal 7. Hybridization is not detectable with P1 at signals 4, 5 and 6 and with DNA from vSC8 in signal 8 and from DNA from uninfected cells in signal 9.

B) The radiolabeled pSC11 DNA hybridizes with all DNA preparations that could be detected with FSME-specific "Riboprobe". This is due to the presence of Vaccinia TK sequences necessary for recombination in pSC11 DNA. Therefore, this plasmid pSC1120 DNA also hybridizes with the combination of P1 DNA at signals 4, 5 and 6 and with the combination of vSC8 DNA at signal 8. DNA from uninfected cells does not hybridize (signal 9).

Figure 13 shows FSME-specific protein Western

Blot analysis on extracts from Vero cells infected with P6 virus combination. The proteins were separated on a 15% polyacrylamide gel and prepared using standard methods for Western Blot 30. As a control, proteins derived from these infected cells were incubated with negative human serum (signals 1 and 3). A positive human serum with antibodies against FSME virus could detect the expected FSME-specific anti-? 102835 gene of size 38,000 at signals 2 and 4. Signals 1 and 3 obtained with negative serum , appear to be nonspecific.

The DNA useful in the present invention is prepared and sequenced using the following general methods.

First, viral RNA is extracted from Western FSME virus subtype or cells infected with Western FSME virus subtype. Using this RNA as mat-10 rice, double-stranded cDNA is synthesized, for example, using the reverse transcriptase enzyme. Using recombinant DNA techniques, this cDNA vector DNA, such as Escherichia coli plasmid DNA, is inserted to obtain the recombinant plasmid. Recombinant plasmids are used to transform suitable host cells, such as E. coli HB101, to amplify plasmids, or to express the corresponding proteins. Single colonies containing insert-containing plasmids are identified by the Plasmid-Mini-Preparation method in Birnboim and Dolyn muc-20 (Nucleic Acids Research 7, 1195-1204, 1979). The basic DNA sequence of the western subspecies of the virus present in the recombinant plasmid is determined using a rapid DNA sequencing method such as the dideoxy chain termination method.

* A detailed description of the method of making cDNA, recombinant plasmids, and cells transformed with these plasmids, and determining the nucleotide sequence is as follows: first, the viral RNA of the western subtype of FSME can be obtained from purified virus. For this purpose, the Western FSME virus subtype can be cultured in primary fetal cells of the fetus, concentrated by ultracentrifugation, and purified using duplicate sucrose density gradient centrifugation. After digestion of the proteins with SDS in the presence of proteinase K 10 · 102835 and an RNAase inhibitor, for example by incubation for one hour at 37 ° C, the RNA is extracted, for example, with phenol and ethanol precipitated. Using this RNA as a matrix, complementar-5 DNA of viral RNA is then synthesized in vitro using reverse transcriptase enzymes (e.g., derived from "Avian Myeloblastosis" virus) according to Gubler and Hofmänn (1983) 263-269. . Using primers, such as hex-sucleotide primers derived from calf thymus DNA, viral RNA is incubated under appropriate conditions, for example, as described in the Amersham (English) Handbook of "cDNA Synthesis System", with reverse transcription and using enzymes. phosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyguanidine triphosphate (dGTP) and deoxycytidine triphosphate (dCTP) as substrates. The cDNA.RNA hybrid thus obtained is then treated with RNAase H under defined conditions where nickels are formed in the RNA of the hybrid molecule. E. coli DNA-20 polymerase I can then be used to efficiently replace the RNA strand, where the nicked RNA acts as a primer. The double-stranded DNA thus obtained is purified from proteins using phenol-chloroform extraction, ethanol precipitated, and remaining RNA fragments are removed using RNAase treatment (e.g., in TE buffer, 30 min at 37 ° C).

For example, cloning of dsDNA is performed using synthetic adapters. For this purpose, dsDNA is treated with the Klenow fragment of E. coli DNA polymerase I under appropriate conditions (Maniatis et al., Molecular 30 Cloning, CSH 1982) to ensure maximum number of smooth ends suitable for cloning, followed by phenol extraction to remove proteins. The dsDNA with flat ends is phosphorylated at the 5 'end with polynucleotide kinase according to the standard procedure (Maniatis 1 102835 et al., Molecular Cloning, 1982). The phosphorylated dsDNA is incubated under appropriate conditions with BamHI-SmaI adapters in the presence of DNA ligase. The reaction mixture is applied to a 1% agarose gel, separated, and various size cDNAs bound to the adapters are excised from the gel and purified, for example, by electroelution. The DNA thus obtained is again phosphorylated with polynucleotide kinase as described above. On the other hand, plasmid DNA used as vector DNA, for example, pBR322 DNA of E. coli, is digested with restriction endonuclease BamHI and dephosphorylated using bacterial alkaline phosphatase. Synthetic dsDNA with BamHI adapters at both ends and BamHI digested vector DNA is incubated under suitable conditions to allow hybridization of complementary sequences to both ends of the DNA molecules and ligated using T4 DNA ligase. The recombinant DNA molecule is then used to transform a competent E. coli strain (e.g., E. coli HB101) as described by Maniatis et al. (Molecular Cloning 20 CSH, 1982). The vector DNA used contains two genes that confer resistance to ampicillin or tetracycline. The cloning method used deletes the tetracycline resistance gene. To select those combinations that are likely to contain virus-specific inserts, transformed bacteria are cultured in the presence of ampicillin and then sensitivity to tetracycline is analyzed. Plasmids from tetracycline-sensitive colonies are isolated by Plasmid-Mini-Preparation (Birnboim and Doly, Nucleic Acids Research 7, 30, 1195-1204, 1979) and insert sizes are analyzed by restriction enzyme analysis using SmaI and separation of the resulting fragments by 1%. agarose gel.

The base sequence of these inserts was determined by the di-deoxy chain termination method of Sanger et al. according to «17 102835 (Prot. Natl. Acad. Sci. U.S.A. 74, 5463-5467, 1977).

The resulting sequence is examined for homology with other sequences of Flavi viruses already known (Rice et al., Science 229, 726-733, 1985; Dalgarno et al., J.

5 Mol. Biol. 187, 309-323, 1986; Castle et al., Virology 147, 227-236, 1985; Castle et al., Virology 149, 10-26, 1986; Wengler et al., Virology 147, 264 274, 1985;

Pletnev et al., FEBS 3660 200, No. 2, 317 321, 1986) to locate the sequence studied by computer programs in the total sequence of genomic RNA. Figure 1 shows the complete nucleotide sequence of the western subtype of the FSME viral genome and Figure 3 the corresponding amino acid sequence.

The exact amino ends of E and M are determined using N-terminal amino acid sequence analyzes, for example, using the manual method of Chang et al. (FEBS Letters 93, 205-214, 1978). For this purpose, E protein is isolated, for example, using preparative SDS-PAGE, electroeluted from the gel, and N-terminal sequence analysis is performed. The resulting sequence is Ser-Arg-Cys, thus the amino terminus of E is in exactly the same position as the other Flavi viruses, i.e., two amino acids before the first and highly conserved cysteine moiety (Figure 3).

The following procedure may be proposed for the determination of the amino terminus of M protein. Purified virus is solubilized with a nonionic detergent (e.g., Triton X 100) and protein complexes containing E and M are obtained using density gradient centrifugation in a detergent si-30 in an unsaturated sucrose gradient (Heinz and Kunz J. Gen. Virol. 49, 125 1980). Sequence analysis of these protein complexes results in the following double sequence:

Ser-Arg-Cys 35 Ser-Val-Leu is 102835 wherein Ser-Arg-Cys corresponds to the sequence of E and the amino terminal end of Ser-Val-Leu M protein. These ends are indicated in the sequences shown in Figures 1 and 3.

Thus, for the first time, within the scope of the present invention, the nucleotide sequence of the 5 'region of the western subtype of the FSME viral genome is provided, including sequences encoding structural proteins, and consequently, the amino acid sequences of these structural proteins.

The base sequence of Figure 1 is a preferred example of DNA encoding polypeptides having the structural proteins of the western subtype of the FSME virus. As a result of the degeneracy of the genetic code, the same amino acid sequence may be encoded by bases other than those shown in the figure.

Thus, sequences that have been altered by mutation, transposition, and degradation, which equally encode an amino acid sequence that still has the essential antigenic properties of structural proteins, are also useful in this invention.

Furthermore, the present invention is not limited to exactly the same amino acid sequence as that shown in Figure 3, which only shows an exemplary sequence of natural isolation of the western subtype of FSME virus. It is a well-known fact that RNA viral genomes are alt-

V

Tin for a higher mutation frequency than those found with DNA viruses or cellular genes as a result of high RNA polymerase error rate and repair mechanism error (Holland et al., Science 215, 1577-1585, 1982; Reanney, Ann. Rev. Microbiol . 36, 47-73, 1982). Regardless of the characteristics of the virus, these mutations can lead to the rapid development of a new type of virus as a result of antigen transfer, as is the case with the influenza virus (Both et al: "The Origin of Pandemic

Influenza Viruses ", W.G. Laver (ed.), Elsevier, 1983).

35 On the other hand, RNA viruses may be more stable in natural

• I

19 102835 under conditions relative to the antigen structure, presumably as a result of functional constraints that do not permit extensive structural changes in antigen-active proteins. Equally, a certain number of variants 5 must be considered and this can be demonstrated using sequence comparison of different natural isolates.

This can be accomplished, for example, by sequencing the RNA of different isolates using the dideoxy chain termination method as primers synthetic oligonucleotides prepared according to the prototype isolate shown in Figure 1.

Analysis of the nucleotide sequences corresponding to the E protein of such different isolates has revealed different nucleotides. The following nucleotide changes were observed in natural RNA isolate (ZZ-9) compared to Neudörfl: • · 5 20 102835

Nucleotide F S ME ZZ-9 Amino Acid Site Change 2375 A U Thr - Ser

2347 U C

2323 hp

2317 hp

10

2281 CU

2266 U C

2263 hp

2021 C U

1876 CU

1868 A G Met - Vai 1773 C U Ala - Vai 2Q 1668 A G Glu - Gly

1663 CU

1661 A G Asn - Aso

1660 CU

25

': 1472 U C

1451 A G Ile - Vai

1343 CU

30 1114 U C -

1111 U C

1090 C U

1048 U C

35,994 U C

2i 102835

The observed changes are an expression of the natural variation level of the FSME virus which does not lead to a new serotype. The sequence homologies between Flavi viruses that do not belong to the same serocomplex are 40-50%, for example, 5,4 homology between YF and MVE viruses, and 70-80% between members of the same Flavi virus serogroup, and e.g. 77.1% homology between WN and MVE viruses.

Comparison between the published nucleotide sequences of the Far East subtype (Pletnev et al., FEBS 3660 200, No. 2, 10 317-321, 1986) and the nucleotide sequences of the Western subtype showed about 85% homology.

All sequences showing minor variations compared to prototype sequences, such as those found in other natural isolates of the western FSME virus subtype, are encompassed by the present invention. Moreover, such variations can be obtained using in vitro nucleic acid modification. Thus, all sequences that hybridize under stringent conditions, for example at least 90% nucleotide sequence sequence homology with the disclosed sequences or portions of sequences that preferably encode proteins or peptides having the Western blots of the FSME viral subtype -, femininity.

The person skilled in the art is well able to insert the described DNA or portions thereof into suitable vectors and to use recombinant vectors to transform suitable cells, either for amplification of the DNA or for expression of the corresponding proteins. Suitable hosts, vectors, and conditions for these operations are familiar to those skilled in the art.

There are many publications describing the synthesis of foreign proteins using recombinant DNA technology (for example, see "Maximizing Gene Expression," by Reznikoff and Gold, Ed., Butterworths, 1986; 35 Harris, Gen. Eng. 4, 127- 183; Wetzel and Goeddel, The Pep- • «22 102835 * Tides 5, 1-64, 1983). Using these methods, proteins encoded from cloned DNA can be expressed either in bacteria, in yeast or in mammalian cells. It is still known in the art to use such expressed proteins as vaccines for immunization purposes (Valenzuela et al., Nature 307, 178-180, 1984; McAleer et al., Nature 307, 178-180, 1984) or as diagnostic antigens.

Thus, all sequence combinations of the described sequences, such as, for example, genes encoding other proteins, or sequences that contribute to expression, such as promoters, enhancers, polyadenylation or signaling signals, are useful according to the invention.

One of ordinary skill in the art is well aware that it is not necessary to use the whole natural protein for diagnostic purposes (Lerner, Nature 299, 592-596, 1982; Arnon, TIBS 11, 521-524, 1986). For example, in the case of FSMEvirus Western subtype E protein, it was shown that hemagglutination-inhibiting and neutralizing antibodies can be induced by immunization with a 9,000 dalton proteolytic fragment, which also reacts with the polyclonal immune sera obtained by immunization with a full-length immunol. , J. Gen. Vi-: Rol. 65, 1921-1929, 1984). Other proteolytic fragments may also be suitable for diagnostic purposes.

Proteins, or portions of the proteins used as diagnostic agents in accordance with the invention, may be prepared not only using recombinant DNA technologies as described above, but the sequence information provided may likewise serve as a basis for the chemical synthesis of oligopeptides. There is an extensive literature in this field: synthesized peptides are made corresponding to DNA sequences encoding many different proteins and have been used for many purposes such as molecular biology and immunological studies (Lerner et al., Cell 23, 23 102835 309 310, 1981; Lerner, Nature 299, 592-596, 1982) or for vaccination (Shinnick et al., Ann. Rev. Microbiol. 37, 425-446, 1983; DiMarchi et al., Science 232, 639-83). 641, 1986).

The preparation and use of peptides or peptide combinations corresponding to the sequences disclosed in the present invention illustrate the state of the art.

The following examples illustrate the invention.

Example 1 Generation and Purification of FSME Virus A 10% suspension of the brain of neonatal mice infected with 10 Western subtypes of FSME virus was used to infect monolayer cultures of chicken fetal primary cells maintained with 15 mM HEPES and 15 mM EPPS pH 7. in buffered minimal media 15 (MEM). After incubation for 40 hours at 37 ° C, the supernatant solution was clarified at 4 ° C at 10,000 g and the virus was pelleted using 3 hours ultracentrifugation at 4 ° C and 50,000 g. The virus was then resuspended in an appropriate volume of 20 TAN buffer (0.05 M triethanolamine, 0.1 M NaCl, pH 8.0) and subjected to a Zonal (rate-Zonal) centrifugation for 110 minutes in a 5-20% sucrose density gradient at 170,000 g at 4 ° C. The viral peak was localized by scanning the gradient at 254 nm and performing a non-density gradient centrifugation in a 20-50% (w / w) sucrose density gradient for 18 hours at 4 ° C and 150,000 g / min. The viral peak was dialyzed against TAN buffer to remove excess sucrose.

Example 2.! Preparation of Virus RNA 100 μg of purified virus was diluted in 400 μΙ-ΆΆη proteinase K reaction buffer (10 mM Tris pH 7.8, 5 mM EDTA, 0.5% w / v SDS). Proteinase K was added at a final concentration of 200 µg / ml and the mixture was incubated for one hour for 35 hours at 37 ° C. The protein was then removed from the solution by extraction twice with the same volume (400 μΐ) of phenol and once with chloroform: isoamyl alcohol (24: 1). Then 26 μΐ of a 3 M sodium acetate solution was added and the RNA was precipitated 2.5 times with 5 volumes of ethanol. Prior to further use, a portion of the RNA was denatured with glyoxal and separated on a 1% agarose gel to test the yield and quality of the preparation.

Example 3

Synthesis of double-stranded cDNA 10 5 μg of ethanol precipitated RNA was resuspended in 40 μg of premium synthesis buffer (Amersham, UK) containing 5 μg of a random "oligonucleotide" dialect, heated for one minute at 70 ° C and was allowed to cool slowly to room temperature. All four deoxynucleotide triphosphates were added to a final concentration of 1 mM. Further, five units of human placental ribonuclease inhibitor and 10 µCi of α-32P-dCTP were added to the mixture. Synthesis of the first strand was initiated by addition of 100 units of reverse transcriptase (Amersham) and allowed to react for two hours at 42 ° C. The reaction mixture was then placed on ice and the reagents for double stranded synthesis were added in the following order: 93.5 μΐ double stranded synthesis buffer (Amersham, UK), four units of E. coli DNA polymerase; : and water to a final volume of 250 μΐ. Double-strand synthesis was performed using successive incubations at 12 ° C and 22 ° C (2 hours each) and stopped by heating the solution to 70 ° C for 20 minutes. The double-stranded cDNA was incubated with 10 units of T4 DNA polymerase for 30 minutes at 37 ° C to produce flat ends. Sen.! After 30, the cDNA was purified using phenol and chloroform extracts and precipitated with two volumes of ethanol.

Example 4

Adapter ligation 35 cDNA was treated with polynucleotide kinase in the presence of 2 mM 25 102835 ATP (Maniatis et al., Molecular Cloning, CSH 1982) to ensure that all 5 'ends were phosphorylated and could thus be ligated. BamHI-Smal adapters were obtained from Pharmacia. These adapters have a 5 'cohesive, BamHI-compatible cohesive end and a flat end and contain a complete SmaI recognition site in their sequence. At first, only the flat end had the 5 'phosphate group and could be attached, whereas the cohesive end was dephosphorylated. 5 µg of this adapter 10 was mixed with cDNA in 20 µl of ligation buffer (50 mM Tris, pH 7.5, 5 mM MgCl 2, 5 mM DTT, 1 mM ATP). The reaction was catalyzed by T4 DNA ligase (New England Biolabs) in an overnight reaction at 14 ° C.

Example 5 Size Separation of cDNA The cDNA was separated on a 1% low melting agarose gel. Different sizes were cut off from the gel and DNA was extracted from agarose with hot phenol according to the standard procedure. Despite the small amount of present DNA, which could hardly be detected by ethidium bromide staining, the extraction steps could be easily followed by the radioactive label incorporated into the cDNA. The size separation step allowed for large; Selective cloning of cDNA fragments and served in addition to separating cDNA from completely unrelated adapter molecules.

Example 6

Cloning into the bacterial plasmid The ends of the cDNA that bind to various sizes of BamHI were phosphorylated with polynucleotide kinase and protein removal was performed using phenol and chloroform extractions. The DNA was then precipitated with two volumes of ethanol and resuspended in a small volume of water. 20-30 ng of cDNA was mixed with 100 ng of Es-35 cherichia coli plasmid pBR322, linearized 26102835 using BamHI digestion and dephosphorylated to prevent self-ligation. Both of these components were ligated with T4 DNA ligase in 5 μΐ of ligation buffer (see above) in an overnight reaction at 16 ° C. The next day, the dilution-5 was ligated with water to a 5-fold volume and then used directly to transform E. coli HB101 cells. Useful bacteria were obtained from BRL (Maryland, USA) and transformed with 5 µl of diluted ligation mixture using exactly the manufacturer's instructions. Typically, hundreds of transformed cells were obtained after plating on amplicillin-containing agar plates.

Example 7

Identification of Insert Containing Plasmids Individual ampicillin-resistant colonies were plated on tetracycline-containing agar plates. About 5% of the colonies tested were tetracycline sensitive. These were used to inoculate 2 ml of ampicillin-containing LB media and incubated overnight at 37 ° C in a shaker at 20 rpm. The next day, plasmid rapid preparation was performed according to the standard procedure (Birnboim and Doly, Nucleic Acid Research 7, 1195-1204, 1979). Plasmids were digested with the restriction enzyme SmaI and analyzed on a 1% agarose gel (Figure 4). In this manner, 21 plasmids containing inserts ranging in size from 2,000 to 3,500 bp were identified.

Example 8

Initial characterization of inserts

Insert-containing plasmids were prepared on a large scale, according to the standard method (Maniatis et al., Molecular Cloning CSH, 1982) and purified by ethidium bromide density gradient centrifugation. About 500 ng of plasmid DNA was digested with a set of restriction enzymes and analyzed on a 1% agarose gel. Thus, a useful 35-region band model was obtained for each plasmid, making it possible to first predict which inserts may contain compatible or different sequence regions.

Example 9

Sequence analysis of clone A5 insert One of the insert containing plasmids, designated plasmid A5, was digested with Smal and separated on a 0.7% agarose gel. The band comprising the entire insert was excised from the gel and the DNA was purified from agarose by electroelution. Isolated fragment 10 was cleaved in various reactions with frequently cleaving restriction enzymes producing flat ends such as RsaI, AluI and Haelll. The double-stranded form of bacteriophage M13mp8 was digested with SmaI and dephosphorylated. Mixtures of DNA fragments were de-proteinized, precipitated, and ligated with 15 M13 vectors as described above. Transformation of E. coli JM105 cells, identification of recombinant phages using the color selection method, and production of single-stranded DNA according to standard methods were performed. Individual clones were selected and sequenced according to the dideoxy sequence-20 chimeric method, such as Sanger et al. (Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467, 1977). The commercially available M13 universal primer was generally used as the primer. Thus, the sequence was determined for a large number: fragments of clone A5, which eventually resulted in the total DNA sequence of this 25 clone. By comparing this sequence with published genome sequences of various Flavi viruses, it was found that A5 contains the region of the genome encoding the structural proteins C, M, and E, with the exception of the amino terminal half of the C protein. The exact localization of A5 is pre-s; 30 shown in Figure 1.

Example 10 cDNA Synthesis Using FSME Virus Specific Primer A FSME virus specific primer was used to construct a 35 clone containing the entire genome of the viral gene, including the entire sequence of the C protein. information as well as most of the untranslated 51 leader regions. This oligonucleotide primer consisted of 14 nucleotides complementary to the FSME-ge-5 nomenclature encoding the C-terminal amino acids of the E protein. Preparation of viral RNA and synthesis of double-stranded cDNA was performed essentially as described above, except that this time only 1 µg of RNA and 0.5 µg of synthetic oligonucleotide primer were used. Subsequently, incubation times of double-strand synthesis and T4 DNA polymerase treatment were reduced from one hour to three minutes.

Example 11

Cloning into a Blue Script Vector Using 15 EcoRI Linkers From the information available at this stage of the study, it could be assumed that the entire structural region does not contain the EcoRI recognition site. Thus, synthetic EcoRI linkers (New England Biolabs) could be used for the 20 cloning method. About 1 μg of cDNA was ligated with 1 μg of linker DNA for six hours at 14 ° C using T4 DNA ligase and buffer system as described above. After purification of the mixture using phenol and chloroform extracts, and after ethanol precipitation, 25 DNA were resuspended in 40 μ1: ΆΆη "medium sit" restriction enzyme buffer (CSH Handbook) and disrupted for 3 hours using excess EcoRI (80 units) at 37 ° C. :in. The mixture was then separated on a 1% agarose gel and a cDNA of 1500 to 2500 bp was again electro-eluted from the gel. 30 thio.

Simultaneously, a new multi-use vector Bluescript KS M13 + (Stratagene) was digested with EcoRI. 100 ng of this linearized plasmid was mixed with the entire cDNA preparation and the DNAs were co-precipitated with ethanol. Ligation of plasmidicDNA was performed as described above. Ligation-29 102835 mixtures were gradually diluted from two to one hundred times and used with transformants of E. coli XL-1 blue. Transformation and identification of insert containing plasmids were performed using color selection exactly according to the manufacturer's instructions (Stratagene). Thus, four plasmids were obtained containing inserts of approximately 2500 bp.

Example 12

Clone P1-1 Sequence Assay An insert-containing plasmid, designated Plasmid P1-1, was used to prepare single-stranded template DNA. This was done using the coupling of helper phage R408 to an exponentially growing bacterial culture of Pl-1 in M.O.I 20: 1. After incubation for 8 hours at 37 ° C on a shaker at 240 rpm, the supernatant containing phage particles was collected and prepared by single stranded P1-DNA using the standard method. Unlike the classical M13 system, the Bluescript system allows the production of yk-20 internal-stranded template DNA that contains the entire cDNA sequence, thus eliminating the time-consuming subcloning method. Sequencing of P1-1 was performed by standard deoxy method using primers as well as': general M13 and synthetic oligonucleotide primers, which correspond to different viral sequences. Thus, it was found that P1-1 covers the entire coding region for structural proteins and extends 120 bp beyond the start codon of the C protein. The exact location of P1-1 is likewise shown in Figure 1.

Example 13 Sequencing of Different Natural Isolates Using Direct RNA Sequencing The coding sequences for the structural proteins of various natural isolates of the western subtype of the FSME virus were determined using direct sequencing of genomic RNA from the S. Natl. Acad. Sci. USA 75: 4257-4261 (1978). Various synthetic oligonucleotides corresponding to different sites in the genome were used as primers. Approximately 1 μg of genomic RNA and 0.5 μg of each region-5 were mixed in a volume of 10 μ1, heated for three minutes at 65 ° C and then slowly cooled to 42 ° C. Sequencing reactions were catalyzed using reverse transcriptase (Amersham) for 15 minutes at 42 ° C. As a control, the pro-10 Neudörfl prototype of the FSME virus was simultaneously sequenced and analyzed on the same polyacrylamide gel. This allowed for simple and reliable identification of sequence variations of the various natural isolates. Sequence anomalies were found at the locations described in the general section of the specification.

Example 14

Amino-terminal sequence analysis of viral proteins on 1 mg of purified FSME virus western subtype was performed by SDS-PAGE according to Laemmli and Favre (J. Mol. 20 Biol. 80, 575-599, 1973) on 10% acrylamide gels. The E protein was localized by staining the gel filter paper blot with amide black, the protein was excised from the gel and electroeluted using an ISCOelution apparatus for 5 hours at 3 W. N-terminal sequence analysis was performed by Chang et al. (FEBS Letters 93, 205-214, 1978). The resulting sequence was

Ser-Arg-Cys and thus enabled the exact location of the E protein in the sequence shown in Figure 3 to be determined.

Alternatively, protein complexes containing both E and M were prepared by solubilization of Western subtype purified FSME viruses with Triton X 100 followed by density gradient centrifugation in a detergent-free mass gradient such as Heinz and Kun. Gen. Virol. 49, 125-132, 1980). As expected, 31102835 amino-terminal sequence analysis yielded a double-quency and

Ser-Arg-Cys 5 Ser-Val-Leu

Since it was already known that Ser-Arg-Cys was derived from E protein, the Ser-Val-Leu sequence was thus identified as the amino terminus of M (Figure 3).

Example 15

Construction of insertion plasmids containing foreign genes obtained by cloning the 5 'end sequences of the FSME virus by cloning the FSME virus using homologous recombination in Vaccinia Hind III J Figure 5 shows plasmid A5 prepared as described in stai by p. As shown in Figure 5, plasmid A5 contains the DNA sequence encoding the FSME virus E protein. Figure 5 further depicts restriction endonuclease cleavage sites of selected sub-fragments, wherein the FSME protein E coding regions are depicted. The transcript direction of the trans in genomic RNA is depicted by an arrow.

Other viral and plasmid material and methods used to prepare plasmids containing specific DNA fragments encoding the E protein are described below: 15.1. Virus cell culture

Vaccinia wild type WR can be obtained from ATCC under accession number VR-119. 30 Human TK-143 cells that do not produce thymidine kinase are commonly available and deposited at the Pasteur Institute (Accession No. 1-732). Vaccinia viruses are allowed to multiply in Vero cells between about 47 and 55 cycles. Cells are cultured in MEM (minimal essential medium) containing 32 102835 * 5% FCS (fetal calf serum), antibiotic and glutamine. The species of virus to be used for recombination is allowed to grow once in HAT (hypoxanthine, aminopterin, thymidine) medium containing 5% FCS to select TK + wild-type virus. Recombinant TK virus is assayed on human TK-143 cells grown in MEM with 5% FCS containing 25 μg / ml BUdR. Combinations can be detected by coating the cell monolayer with low melting point agarose containing MEM, 5% 10 FCS, 25 μg / ml BUdR, and 250 μg / ml X-gal. The cell culture is maintained for 48-72 hours in an incubator at 37 ° C. The amount of spontaneous TK virus mutants is determined by infecting a TK ~ cell monolayer with or without 25 μg / ml BUdR. One mutant 15 can usually be found among 104 viruses (Mackett et al., "DNA Cloning Vol. II, A Practical Approach", IRL Press, D.M. Glover (ed.), 1985).

15.2. Plasmids and cloning methods

Plasmid A5, a plasmid derived from pBR322, shown in Figure 5, shows a restriction endonucleation map.

Figure 6 shows the plasmid pSC11 containing the E. coli Lac Z gene for the "blue color" assay, TK protecting sequences and the Vaccinia promoter. Plasmid pSC11 is well known and generally available, however, in particular from the 25 National Institute of Health.

All restriction endonucleases used to modify the DNA were used according to the manufacturer's instructions. Southern blot analysis and plasmid DNA isolation were performed according to a standard method (Mani-30 atis et al., Molecular Cloning, CSH, 1982).

Radiolabeled samples were obtained using either the "nick-repair" method or the pSP6-"Riboprobe" method (Green et al., Cell 32, 681-694, 1983).

The "riboprobe" probe used in the present 33,102,835 inventions was prepared by inserting an approximately 1.0 kb internal FSME nucleotide sequence into pSP64. Before RNA synthesis was initiated, the plasmid was linearized with EcoRI. A typical analysis was:

Tris-HCl 7.5 40 mM

MgCl2 6 mM

Spermidine 2 mM

NaCl 20 mM

DTT 10 mM

ATP, CTP, GTP 0.5 mM each

20 U of human placental RNAase inhibitor

pSP64-P5 X EcoRI 2 μg a32P-UTP 400 Ci / mmol 100 uCi

SP6 RNA Polymerase 4 U

15 Total volume 20 μΐ

Incubation for 1 hour at 40 ° C

Non-linked nucleotides were separated from the radiolabeled cRNA using column chromatography.

20 15.3. In vivo recombination assays

Infection and transfection:

Recombinations were performed mainly as described by Mackett et al. (see 15.1.) have described. Human TK 'cells; in addition, Vero cells or 25 chicken fetal fibroblast cells could be used as host cells. Approximately 1-3 hours prior to transfection, cells were infected with an infection factor (m.o.i.) of 0.01 to 0.05 pfu / cell in wild-type WR. HEPES-buffered saline for DNA transfection was prepared as follows: 10 x HBS containing 8.18% NaCl (w / v); 5.94% HEPES (w / v) and 0.2% Na 2 HPO 4 (w / v) were diluted 2 x HBS before use. The pH was adjusted to 7.05 with 1 N NaOH. The buffer was diluted 1x with HBS and sterilized by filtration. To 1 ml of this 1x HBS was added a solution of 1-5 µg of plasmid DNA and 10-20 µg of carrier DNA 34 102835 (either wild type Vaccinia virus or salmon sperm DNA) . In particular, the addition of carrier DNA appeared to be essential. The described mixture was thoroughly stirred for one minute on a Vortex mixer. Subsequently, li-5 was slowly adjusted with 2 M CaCl 2 until a final concentration of 0.125 M. The DNA precipitate appeared at 25 ° C after 20-30 minutes. The medium was separated from the infected cells, the monolayer was washed once with cell culture medium, and the remaining solution was completely removed, followed by slow addition of DNA precipitate. 30 minutes later, fresh medium was added at 25 ° C and cells were incubated at 37 ° C. After 3-4 hours, the medium was again removed and the fresh cell culture medium replaced. Cells were usually harvested after 24 or 48 hours with a scraper-15 club, sedimented at low speed, and the cell pellet was harvested in media at a total volume of 500 μΐ.

Plaque analysis and purification of recombinant viruses The virus suspension was frozen and thawed again three times; 60 μΐ of the suspension was incubated with 1/5 volume of 1.25% trypsin for 30 minutes at 37 ° C. A series of three dilutions (1:10) was added to a single TK-143sso-lu monolayer grown on 10 cm 2 plates. After incubation for 30 to 60 minutes at 37 ° C, 25 medium was removed, then 3 ml of medium containing 25 μΐ / ml bromideoxyuridine was added. After 24 or 48 hours at 37 ° C, the medium was again removed and replaced with an agarose layer which containing 25 μg / ml bromideoxyuridine and 125 μ9 / τη1 X-Gala After an additional incubation for 24 to 48 hours, blue plaques could be observed 30 Individual plaques were detached and suspended in 500 μl of medium This virus-containing suspension was treated with trypsin as above Plaque cleaning was performed at least twice.

35 102835 15.4. Preparation and Characterization of Wild Type and Recombinant Vaccinia DNA From 175 cm 2 flasks, infected cells were scraped and washed twice with PBS-Ca 2+ / Mg 2+ at pH 7.4.

After centrifugation, the cells were resuspended in 250 μΐ PBS. 250 μ1: η volume of 2 x cell lysis buffer (1% Triton X 100; 70 mM β-mercaptoethanol; 40 mM EDTA) was added and cells lysed for 5 minutes on ice. The nuclei were separated from the cytoplasm for one minute at 10 16,000 x g. The supernatant solution containing the aggregated virus particles was transferred to a new tube and centrifuged for 30 minutes at 16,000 x g to pellet the virus. A viral lysis buffer (10 mM Tris-HCl pH 8; 1 mM EDTA; 5 mM β-mercaptoeta-15 nol; 200 mM NaCl; 1% SDS; 150 μg / ml proteinase K) was then added to the pellet in an amount of 100 μ1: η and incubated for 30 minutes at 56 ° C. The nucleic acids were then extracted 3x with Tris-HCl-saturated phenol, then re-extracted with chloroform: isoamyl alcohol (24: 1). Care was taken that DNA 20 was not exposed to shear forces. Nucleic acid was precipitated with 0.1 M NaCl and 2.5 times volume of ethanol at -20 ° C overnight. About 10 to 20 μg of DNA could be isolated from 10 cells. After centrifugation (30 min at 17,000 x; g), the DNA was dissolved in a small volume of 20-40 μ1: η H2.

The DNA was digested with restriction endonuclease HindIII and the fragments were separated on a 0.6% agarose gel in TA buffer (Rice et al., J. Virol. 56, 227-239, 1985).

15.5. Detection of Nucleic Acids by Hybridization After transferring 30 Southern Blot DNA from a gel (30 ', 0.5 N NaOH and 2 x 10', 10 x SSC / 0.5 M Tris-HCl pH 7.5) to nitrocellulose filters, At 4 ° C for four hours. The filters were pre-hybridized for 6 hours at 65 ° C. · 36 102835 in 5x Denhardt solution (1x Denhardt = 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% BSA) . The wet filters were sealed in plastic bags and incubated with hybridization solution and radioactive probe for 18 5 hours at 43 ° C. The filters were washed at 65 ° C three times each 30 'each with 2 x SSC, 1 x SSC and 0.1 x SSC with 0.1% SDS and 20 mM NaPO 4. After air drying the filters, these were placed on Kodak X-Own AR X-ray films.

10 "Riboprobe" vector (pSP6) containing the FSME fragment from plasmid A5 shown in Figure 5 was used to make a "Riboprobe" specific for FSME virus. The radiolabeled cRNA was mixed with the hybridization solution (50% formamide, Amberli-15 Telia purified; 5 x SSC, 1 x Denhardt's solution, 0.1% SDS; 100 µg / ml herring sperm DNA). The total volume of the hybridization solution was determined because 50 μΐ for 100 µl / cm2 filter granulate was required for optimal hybridization conditions. The amount of radioactive cDNA was about 10 cpm / ml on a 20 or about 10 cpm / cm filter. The solution was heated at 5 'to 65 ° C.

Spot Blot These experiments were performed by Mackett et al. (see.

*: 15.1.) With minor modifications. About 1/4 of 25 isolated plaques were used to infect a 35 mm Petri dish. After 48 hours of infection, the cells were harvested and washed in buffer containing 50 mM Tris-HCl pH 7.5 and 100 mM NaCl. Finally, the cells were resuspended in 500 μ tätä of this buffer. 100 μ1 · .η aliquots were aspirated under vacuum for nitrocellulose. After drying, the filters, which had been softened for 3 'with 0.5 N NaOH, were applied to chromatography paper. Filters on paper filters immersed in 2 x SSC / L M Tris-HCl, pH 7.5, were then neutralized for 3 'each. The filters were then heated for at least 37 102835 hours for two hours and further processed as described above for Southern Blot hybridization.

15.6. protein Analysis

Vero cells or KT-143 cells were infected with m.o.i.15 pfu / cell with a total volume of about 1-2 ml / 25 cm2 flask. In a few experiments, cells were labeled with 100 μ0: 1 → 35 S-methionine / ml. When CPE was high, cells were harvested, washed twice in PBS, and finally suspended in 100 μl RIPA buffer (150 mM NaCl; 10 mM Tris-HCl pH 7.2; 10 L sodium deoxycholate; 1% Triton X 100; 0.1% SDS; 100 µg / ml PMSF) (50 150 μΐ for 5 x 10 5 to 1.5 x 10 6 cells) After 10 minutes on ice, the samples were sonicated for 20 seconds. To 8 μΐ of this protein mixture was added 12 μΐ 2 x cooking buffer (0.2 M DDT; 4% SDS; 0.16 M Tris-HCl pH 6.8; 20% glycerin; 1/1 000 parts by volume of 5% BPB in ethanol); proteins were denatured for 10 minutes at 100 ° C and electrophoresed on a 15% SDS polyacrylamide gel (Bodemer et al., Virology 103, 340-349, 1980). Proteins were transferred from the gel to nitrosel-20 lulose filters in buffer containing 0.192 M Glycine, 0.025 M Tris pH 8.3 and 20% methanol at a current of about 1.5 A for 2.5 hours. The nitric cellulose filters were then incubated in a "blocking" solution in "MET" (0.15 M NaCl, 5 mM EDTA, 50 mM Tris pH 7.4, 0.05% Triton X 100, 2% BSA). Human serum and another antibody directed against human immunoglobulins were added to NET at a 1: 500 dilution and incubated for two hours each. The strips were then washed with NET for 3 x 10 minutes and then softened with TBS (20 mM Tris 30 pH 7.5, 0.9% NaCl, 2% BSA) for 3 x 10 minutes. The bands were developed in 50 ml of TBS and the chromogenic substrate DAB and H 2 O 2.

15.7. Preparation of insertion plasmids

Using the materials and methods described above, 38102835 plasmids containing the DNA sequences of E protein were now prepared. First, plasmid A5 (Figure 5) was digested with restriction endonucleases PvuII, FnuDII and Haell. The fragments were inserted into the sole SmaI-5 site of plasmid pSC11 (Figure 6). Three FSME fragments were isolated from agarose gels by electroelution and ligated into plasmid pSC11, whereby the latter was also linearized to a single SmaI site. The physical map of these plasmids is shown in Figure 7. The three fragments are 1022 bp (Pvu-10 II), 1485 bp (FnuDII) and 1553 bp (Haell). Said three restriction endonucleases were used because the nucleotide sequence encoding the interior of the E protein with and without the hydrophobic signal or membrane anchor sequence had been found using a hydropa-15 thiaplot performed using the IBI Pustell computer program (Figure 8). Orientation of the inserted protein E sequences was determined using Southern Blot analysis with respect to the 7.5 U / L Vaccinia promoter. Cleavage of the pSCII plasmid with PvuII or BamHI resulted in 20 distinct fragments of 2.585 kb or 0.919 kb (Figures 9A, lanes 3 and 4), which in each case hybridized with the P-labeled "Riboprobe" probe (Fig. 9B, lanes 3 and 4). Plasmid pSC11-F41 could be digested with BamHI or EcoRI fragment; ments of; The 1.019 kb or 1.879 kb fragments were hybridized with the "Riboprobe" probe, respectively (Figure 9B, lanes 6 and 7).

15.8. In vivo recombination with FSME insertion plasmid diene and wild type Vaccinia virus Vero cell monolayer infected with Vaccinia I. 30 species in WR, were transfected with plasmid DNA. After 48-72 hours, a strong cytopathogenic effect was observed and cells were harvested. The virus was released from the cells using repeated freezing and thawing and trypsin treatment as described. The TK cell monolayer was then infected with this 39

--1. A

102835 with virus suspension. Cells grew without BUdR. The developing plaques were visualized under a chromogenic substrate, an X-gal containing agarose layer. Virus dilutions were performed to give 10-30 plaques per 35 5 mm plate. Individual blue plaques were excised and used to further infect TK cells for further purification of infection selection or for infecting Vero cells for analysis of viral macromolecules.

15.9. Snot Blot analysis to generate recombinant viruses

Small samples (approximately 100 μΐ) of infected cell monolayer from 35 mm plates or 25 cm 2 culture flasks were harvested and hybridized with nick-repair-labeled pSCll plasmid (containing no FSME sequences) 15 or FSME. Riboprobe (Figure 10). False infected cells did not signal with either probe.

Virus pSC8 containing the Lac Z gene and TK-Vaccinia sequences but not the FSME sequences gave a negative result with the FSME "Riboprobe". However, all cells containing 20 viruses from four different single plaque isolates were positive as expected with the FSME and pSC11 probe.

15.10. DNA analysis of recombinant viruses ·: Vero cell monolayer was infected with m.o.i of 1 to 25 10 pfu / cell of plaque-purified, added Vaccinia compounds. Virus DNA was isolated from virus particles released from infected cells and analyzed using restriction endonuclease digestion and Southern Blot hybridization. 5 kb HindiII J fragment of wild-type DNA. 30 (as shown in Figure 6, lane 6) was replaced by a larger HindIII fragment consisting of the original HindIII J fragment and those DNA sequences inserted into the TK gene locus using in vivo recombination. The vSC8 virus contained the 3.5 kb Lac Z gene, the Hin 40102835 dill J fragment was thus altered to about 8.5 kb (Figure 11, lane 5). Since larger fragments could not be easily resolved, even in low-percentage gels, a final assay for recombinant viral DNA was performed using Southern Blot hybridization (Figure 12). 32P-labeled probes were able to clearly detect fragments that were the size of the prediction of the cloning strategy. The recombinant virus formed by recombination of pSC11-P6 had a negligible deviation (about 500 bp), a smaller Hind 10 fragment of HindIII as the "pSC11-F41" combination (Figures 12A and B). DNAs from uninfected cells did not show hybridization with the pSC11 "nickre-pair" labeled probe or "Riboprobe" (Figures 12A and B, lane 9). DNA hybri-15 from vSC8 virus was only disrupted into the pSC11 "nick-repair" labeled probe due to the common TK sequence and the Lac Z gene (Fig. 12B, lane 8). The FSME "Riboprobe" did not show, as expected, any hybridization. However, both the "Riboprobe" and the "nick-repair" labeled probe identified a "combination" -Hindlll J fragment of different sizes (Figures 12A and B, lanes 1-3 and 7). Vaccinia virus DNA generated by recombination with the insertion plasmid pSC11 and wild-type virus is shown in Figures 12A and B, ri-; vit 4-6. No hybridization was observed with "Riboprobe" (Fig. 12A), although plasmid with pSC11 (Fig. 12B).

15.11. protein Analysis

Cells were infected, wild-type or recombinant, with a m.o.i value of 1-10 pfu / cell. Cells were labeled with 35S-methionine at different times after infection, harvested and. 30 were analyzed sequentially on denaturing SDS polyacrylamide gels. Protein E conversion molecules were determined using Western Blot analysis with human antiserum. Cells infected with recombinant virus P6 were able to detect FSME virus-specific protein with a molecular weight of 10283535 at about 38,000 daltons. This protein had a size calculated from the primary sequence and consisted of 335 amino acids derived from FSME protein E and 87 amino acids from the Vaccinia TK gene. Since the FSME sequence 5 does not contain internal stop codons, this fusion was expected. Another recombinant virus comprising the FnuDIII fragment of the FSME protein E sequence is expected to have a protein of about 42 kd.

Synthesis time of FSME proteins in cells infected with Vaccinia virus combinations was determined by labeling the cells at various times after infection for a short time (4 hours) with 35S-methionine. Because the p7,5 promoter is active in both the early and late phase of the infection cycle, cells were expected to produce FSME-pro-15 proteins at an earlier and later time point after infection. In this regard, the late p11 promoter regulates the synthesis of the β-gal protein of 116 kd. This protein thus appeared relatively late after infection. The promoter early and late ac-20 densities described affected the maturation of cells, especially when larger amounts of virus had to be used to purify and prepare the desired proteins.

• · «

Claims (5)

42 102835 A peptide or polypeptide, characterized in that it comprises a portion of a protein selected from the group consisting of protein C, rpm, M or E of the western subtype of the FSME virus and the amino acid sequence contained in the amino acid sequence shown in Figure 3. A peptide or polypeptide according to claim 1, characterized in that it comprises a protein selected from the group consisting of protein C, rpm, M or E of the western subtype of the FSME virus. A peptide or polypeptide according to claim 1 or 2, characterized in that it comprises at least an antigenic determinant region of a protein selected from the group consisting of protein C, rpm, M or E of the western subtype of FSME virus, preferably protein E. . A peptide or polypeptide according to any one of claims 1 to 3, characterized in that it is produced by expression of a nucleotide sequence encoding it in a suitable expression system. Diagnostic agent, characterized in that it contains a peptide or polypeptide according to any one of claims 1 to 3. 25 3 * «102835