LV15007B - Production of potato pvm virus-like particles - Google Patents

Description

DESCRIPTION OF THE INVENTION

The invention relates to biotechnology, molecular biology as well as to the production of vaccines, diagnostic tools and new nanomaterials. It is based on regular, organized nanometer-sized filamentous structures that contain artificially attached protein sequences created by viral polypeptide self-assembly processes.

Analysis of the state of the art

Virus-like particles (VLPs) are multisubunit protein structures that are identical or structurally analogous to the viruses in question and are not infectious. Virus-like particles are widely known as candidates for vaccines [1]. Hepatitis B and human papillomavirus-like particles are the most popular and are used in commercial medical practice as vaccines against hepatitis B and cervical malignancy, the papillomavirus. The effectiveness of virus-like particles in stimulating the immune response is well-known: structurally tight and multiple repetitive amino acid sequence motifs on the surface of viral proteins are the determining signals for B-cell activation of the immune system and serve as a signal to the immune system. In addition, virus-like particle sizes (predominantly less than 50 nm in diameter) are suitable for entry into dendritic cells, where VLP is cleaved by subsequent immune system T-cell activation [3]. Thus, VLP elicits an effective humoral and cellular immune response, presence [4],

Plant viruses do not cause mammalian disease. However, plant viruses and similar particles artificially derived from their structural proteins (virus-like particles - VLPs) can be used as epitope carriers that exhibit pathogen-specific epitopes on the surface of their structural proteins. The term "epitope" is defined as the shortest amino acid sequence capable of binding its specific antibody molecules; epitopes can be either linear or conformational [1], Linear epitopes are short amino acid sequences (4 to 10 in length) whose activity is not affected by their spatial structure. Conformational epitopes are formed from distant regions of the polypeptide chain, forming a distinct spatial structure.

The idea of using peptides (epitopes) specific for pathogens in immunization has been known for more than 20 years. Low molecular weight substances, including short amino acid sequences (peptides), are often known to cause poor immune responses. Therefore, epitopes are attracted to protein carrier molecules that elicit increased immune responses in mammals [5]. With the development of genetic engineering and plant virology, the idea emerged that plant viruses could also be used as carriers of alien epitopes. Thus, by cloning the DNA sequence cDNA of the tobacco mosaic virus (TMV) surface protein encoding the DNA sequence of the peptide-specific peptide and expressing these recombinant genes in Escerichia

-2coli, a vaccine candidate was obtained. Localized poliovirus epitopes on the TMV surface produced poliovirus neutralizing antibodies following injection in laboratory rats [6]. A similar strategy has been demonstrated for several other plant viruses such as Cowpea mosaic virus, Tomato bushy stunt virus, Cucumber mosaic virus, Potato virus X (PVX), Johnsongrass mosaic virus, Papaya mosaic virus [7]. Various host organisms have been used to obtain the relevant vaccine candidates, e.g., plants infected with recombinant viruses, insect cells infected with modified baculoviruses, yeasts, and also E. coli platforms.

Plant viruses or VLPs derived from them can also be used to create new nanomaterials. Thus, a novel immunoadsorption based on tobamovirus has been developed which is capable of binding up to 2 g of monoclonal antibody per g of carrier. This is accomplished by adding the functional fragment of protein A of Staphylococcus aureus to the C-terminus of the tobamovirus surface protein and creating an appropriate expression system for obtaining VLP from tobacco leaves. As a result, nanoparticulate material (18x200 nm in size) was obtained, with 2100 copies of protein A derivative on each particle surface; it has been proposed by the authors to be used as a cost-effective, single-use immunosorbent for antibody purification [8].

Potato virus PVM belongs to the group of Carlavirus viruses, which includes 42 different species of plant viruses, including other potato viruses PVP, PVS and PVX [9]. The Genbank nucleotide sequence database contains sequences of more than 800 different Carlavirus representatives. The morphological PVM virions are lightly concave, 650 x 12 nm filamentous structures (filaments). Their single-stranded RNA consists of approximately 8500 nucleotides encoding a polyprotein consisting of methyltransferase, helicase, and polymerase domains. The remaining proteins are synthesized from 2 subgenomic RNAs (sgRNAs), including the surface protein (CP), which is translated from the smallest, -1.5 kb sgRNA [10]. Recently, the PVM surface protein has been expressed in the E.coli system in a coupled form with the carrier protein GST at + 30 ° C, no virus-like particle formation has been shown [11].

Like many filamentous viruses, the crystal structure of Carlavirus, including PVM, is unknown. This makes it difficult to use them as carriers of foreign amino acid sequences because it is not possible to determine localized regions on the viral surface and to predict whether the inserted sequences will be on the VLP surface and how their insertion into the viral surface protein structure will influence VLP formation. However, for filamentous viruses, it is possible to construct structural models based on the results of immunological and biochemical experiments [12]. It should be noted that in filamentous viruses, the N- and / or C-terminals of surface proteins are often localized to the surface of the particles, allowing epitopes or protein domains to be inserted at the N- or C-terminus of the surface protein. In such cases, however, it is necessary to experimentally test for VLP formation;

There are several examples of filamentous plant VLPs from E.coli expression systems used as carriers of medically important epitopes with high immunological activity. Papaya mosaic virus (PapMV - PVM-related Potexvirus group) VLPs with the surface protein C-terminal hepatitis C E2 epitopes obtained from the E. coli expression system induced a sustained humoral immune response in experimental animals after use in immunization. Interestingly, an immune response was observed only when E2-CP filamentous particles were used for immunization. The E2-CP monomer was found to be weakly immunogenic [13]. Thus, these experiments demonstrate the importance of epitope multimerization in inducing an efficient immune response through the placement of epitopes on the surface of VLPs. It should be noted that filamentous plant virus particles can also be used as adjuvants to stimulate antibody formation [14]. The C-terminal sequence of other filamentous plant viral surface proteins has also been used to represent foreign epitopes on the surface of VLPs, for example: Potyvirus group JGMV (Johnsongrass mosaic virus) [15] and PVA [16], and Tobamovirus group TMV [17] and CGMMV [18]. It should be noted that recent examples relate to the production of recombinant VLPs from highly complex plant expression systems, in which case the VLPs contain infectious viral nucleic acids which complicate their further use in production and medical practice.

Less known cases of foreign polypeptide sequences are located at the N-terminus of filamentous viruses CP. The infectious virus and plant host system already mentioned has been used to insert short epitopes (6 amino acids) at the N-terminus of Potexvirus PVX CP and to achieve relatively high immune responses to the epitope in laboratory animals [19], in the case of JGMV to be replaced by foreign protein sequences without interfering with VLP formation in recombinant E.coli cells [20]. However, the present invention does not provide data on the immunological properties of such VLPs, although the authors have indicated the potential use of such VLPs as vaccines. The authors also argued that it is possible to similarly partially or completely substitute both the N- and C-terminal amino acids in the structure of all Potyvirus surface proteins without interfering with VLP formation. Contrary to these data, the role of the N- and C-terminal amino acids in the formation of Potyvirus VLP is characterized by another mechanism for the formation of the PVVV (Pepper vein banding virus) viruses of the Potyvirus group. According to this model, virion formation begins with the interaction of the N- and C-terminus of the CP with positively and negatively charged amino acids, creating ring structures that further form viral filaments. The formation of PVBV CP N- or C-terminal truncated clones has been shown to interfere with VLP formation in E.coli cells [21]. Thus, the assertion that all N-terminal and C-terminal amino acids can be substituted for the surface proteins of all Potyvirus viruses is incorrect. This is also illustrated by our recent data on PVY virus-like particles, where up to 71 amino acid lengths could be inserted at the N-terminus of the surface protein, while C-terminal inserts disrupted VLP formation. In addition, PVY carrier VLP

In the formation process, partial proteolytic cleavage of the N-terminal amino acids of the surface protein occurs, leading to a reduced amount of inserted epitopes on the surface of VLPs, which in turn may reduce antibody formation activity against these epitopes [22].

Thus, it is clear that, despite known examples, there is still a technological need for alternative VLP-based polypeptide carriers, which can be derived from simple bacterial expression systems and can be used as novel nanomaterials for a wide variety of applications, including immunologically active materials and novel adsorbents that can be used for antibody production, purification processes of biologically active substances and development of new biochemical analysis methods.

Image descriptions

Fig. 1 Expression plasmid containing the described PVM surface protein gene. The PVM-CP gene was obtained by RT-PCR from infected potato meristem plants, cloned in the E. coli expression vector pET-28a + (Novagen). The localization of the PVY-CP gene, the kanamycin resistance gene (KmR), as well as some of the major restriction cleavage links are indicated.

Fig. 2 a plasmid fragment of pET-28a + E.coli expression containing the described PVM surface protein gene with a glycine-serine repeat at the 5 'end of the cDNA gene. The amino acid sequence of the PVM-CP protein is depicted below the fragment, the amino acids added at the N-terminus of the protein are underlined.

Fig. 3 a plasmid fragment of pET-28a + E.coli expression containing the described PVM surface protein gene at the 5 'end of the hepatitis B preSl epitope and the glycine-serine repeat cDNA gene. Below the fragment is the amino acid sequence of the PVM-CP protein, the amino acids attached to the N-terminus of the protein are underlined, the preSl amino acids are shown in italics in bold.

Fig. 4 A plasmid fragment of pET-28a + E.coli expression containing the described PVM surface protein gene with a glycine-serine repeat and a doubled protein ΑZ domain at the 5 'end of the cDNA gene. Below the fragment is the amino acid sequence of the PVM-CP protein, the amino acids added at the N-terminus of the protein are underlined, and the amino acids of both Z domains are shown in bold italics.

Fig. 5 Analysis of PVM-CP acquisition and purification on SDS-polyacrylamide gel.

M - protein marker (Fermentas, Lithuania), T - total protein, S - soluble protein, P insoluble protein, 1-6 sucrose gradient fractions.

Fig. 6 Potato virus PVM-like particles (VLPs) derived from recombinant E.coli pETPVM-CP cells. The scaled line at the bottom is 200 nm.

Fig. 7 Potato virus PVM-like particles (VLPs) derived from recombinant E.coli pETPVM-CP-NpreSl cells. The scaled line at the bottom is 100 nm.

-58. fig. Potato virus PVM-like particles (VLPs) derived from recombinant E.coli pETPVM-CP-Nzz cells. The scale line in the lower right corner corresponds to 100 nm.

Description of the Invention

The present invention demonstrates that a cDNA copy of the PVM RNA surface protein (CP) gene obtained from infected potato plants (SEQ ID NO: 2), when inserted into an E. coli expression plasmid (vector), is capable of inducing high yield surface protein synthesis. in bacterial cultures. As the expression vector, the pET-28a + vector mentioned herein containing the T7 promoter sequence, the ribosome binding site, the antibiotic (kanamycin) resistance gene and the expression regulating bear gene may be used. In this case, recombinant E. coli strains containing the T7 polymerase gene, such as BL21 (DE3), C2566, C3010 or others, which provide for the active synthesis of the target protein from the T7 promoter at various culture temperatures, should be used. In principle, other vectors and bacterial strains can also be used. They can be tested in a similar way as described in Example 1.

In bacterial cells, the CP molecule (SEQ ID NO: 1) synthesizes spontaneously virus-like particles (VLPs) that are morphologically similar to native PVM virions, as shown by electro-microscopic analysis. The invention demonstrates techniques for obtaining PVM virus-like particles using appropriate E.coli host cells and expression vectors. Genetic engineering of these VLPs can introduce polypeptide sequences without interfering with the autopolymerization process of VLPs in bacterial cells as described in Example 2.

These polypeptides can be of a wide variety of amino acid sequences, including antigens specific to various disease-causing agents, such as the hepatitis B preSl epitope (SEQ ID NO: 10), protein domains, e.g., the double A domain of protein A (SEQ ID NO: 16), also artificial sequences depending on their intended use. Our experimental data suggest that additional amino acid sequences may be added to the N-terminus of the potato virus PVM surface protein so as not to interfere with virus-like particle formation in heterologous host cells, particularly recombinant E.coli cells.

The fact that the resulting recombinant foreign polypeptide-containing VLPs show high morphological similarity to native viruses suggests the possibility that these polypeptides are arranged in a regularly organized manner within the structure of the VLP. In the case of PVM VIT, it provides a smooth, regular placement of the polypeptide on the surface of the filamentous VLP. Such regular epitopes or protein domains on the surface of the carrier are required for the production of high quality antibodies during the immunization process. In addition, the multiple placement of a particular amino acid sequence on the surface of a VLP can also serve as an essential component of various analytical techniques to increase the signal intensity per unit area, which is important, for example, for efficient microchipping.

Furthermore, the invention also demonstrates an effective method for purifying recombinant PVM VLPs by multiple fractionation by ultracentrifugation.

In the case of potato virus PVM, an effective technique for obtaining virus-like particles from recombinant E.coli cells, as well as PVM VLPs with heterologous protein sequences (up to 137 additional amino acids) in the surface protein structure, has not been found in the patent and other literature.

The VLPs of the invention may be used as immune response stimulating agents when administered to mammals. Antibodies derived from recombinant VLPs containing epitopes and protein domains can be used in the treatment and prevention of diseases as well as in the diagnosis of the respective diseases. Antibodies thus obtained can also be used as a key component in diagnostic reagent kits (eg ELISA kits) for detection of PVA infections in potatoes and other PVA sensitive crops. In addition, such VLPs can be used to create new nanomaterials. PVM VLPs containing protein A Z-domains can be used for purification of monoclonal antibodies.

As will be understood by those skilled in the art, the present invention is based on recombinant nucleic acid technologies such as purification of DNA and RNA from cells of various organisms, polymerase chain and other enzymatic reactions, cloning, expression and purification of recombinant proteins in prokaryotic cells and other technologies. These techniques are well known to those skilled in the art and their detailed descriptions can be found in popular collections of methods [24-27]. For a better understanding of the invention, the examples below illustrate the generation of PVM VLPs and the insertion of polypeptide sequences into the structure of VLPs.

Example 1: Cloning of PVM surface protein gene, expression in E.coli cells, production of virus-like particles

Total RNA from infected potato meristem plants was isolated with TRI reagent (Sigma, USA) according to the manufacturer's instructions. A PVM cDNA was obtained by reaction with M-MuLV H (-) reverse transcriptase (Fermantas, Lithuania) with 'random' oligohexamers.

The primer sequences for cDNA cloning were selected by analyzing those published in the Genbank database

PVM sequences in surface protein gene regions. For cloning the surface protein (CP) gene, primers were selected from regions of the genome that contain the CP gene with the highest possible sequence identity between the known isolates PVM_CP1F (SEQ ID NO: 3) and PVM_CP1R (SEQ ID NO: 4). Further, PCR reactions with these selected primers yielded a cDNA 1 copy of the CP gene.

The resulting viral surface protein cDNA PCR product was isolated from an agarose gel and directly ligated into the auxiliary vector for cloning pTZ57R / T PCR products (Fermentas, Lithuania). containing cDNA inserts

The -7 clones were sequenced with the universal primers M13 / pUC direct (-46) and M13 / pUC reverse (-46) to confirm the gene match to the required sequence according to the Applied Biosystems protocol. To avoid possible errors in the RT-PCR reactions, at least 4 different clones were sequenced and compared to the sequences found in the Genbank database at both nucleotide and amino acid levels.

The nucleotide sequence of our isolate PVM CP gene (SEQ ID NO: 2) is different from all previously known - it is 24 nucleotides away from the nearest published PVM isolate (Genbank KC866622), the gene encodes a protein (SEQ ID NO: 1) two different amino acids (Arg44 / Lys and Tyr278 / Phe).

From the cDNA sequences obtained, a primer sequence was derived for subcloning the respective CP genes in the expression vector pET-28a + (Novagen, USA) PVM_CP_Nco_F (SEQ ID NO:

5) and PVM_CP_Hind_R (SEQ ID NO: 6) by adding Ncol and HindIII restriction cleavage sites, respectively. The PCR products obtained with these primers and the E. coli expression vector pET28a (+) were cleaved at the restriction sites Ncol and HindIII, and the resulting vector and PVM-CP fragments were ligated with T4 ligase (Fermentas, Lithuania). Competent E.coli XLI cells were transformed with the resulting ligation mixture. Transformation mixtures were seeded on LB plates with kanamycin. Isolated plasmid DNA was screened by restriction analysis; sequences were prepared for clones with the appropriate restriction picture. The potato virus PVM CP expression plasmid surface protein from E.coli in preparative amounts was selected for analysis of sequencing results.

The corresponding plasmid map (pET-PVM-CP) is shown in Fig. 1. The resulting expression vector pETPVM-CP was transformed into competent E. coli C2566 (NEB, USA) expression cells. To select the highest expression clones, transformed cells were cultured in analytical (20 ml) volumes in 2xTY medium with kanamycin (Km; 25 mg / l) on a shaker (200 rpm) at + 30 ° C to OD (600 nm) - 0.8-1.0. Cells were induced with 0.2 mM IPTG and 2mM CaClo and 5mM MgCE were added. After induction, cultures were cultured for another 18 h on a shaker at + 20 ° C. Expression levels were determined by analysis of total cell lysates in SDS / polyacrylamide gels. The highest expression clone was cultured in preparative amounts in 200 ml flasks under the same conditions as in the clone selection experiments. The biomass obtained after fermentation was frozen (-20 ° C). Soluble fraction proteins after cell disruption in PBS buffer by ultrasonic disintegration and separation of insoluble proteins and membrane fragments were fractionated in an ultracentrifuge (Beckman, USA; SW28 rotor, 25000 rpm, 6h, + 5 ° C), sucrose gradient (20-60%). The gradient was divided into six fractions starting from the bottom of the gradient, which were further analyzed by SDS-polyacrylamide (SDS / PAA) gel (Fig. 5). As can be seen from SDS / PAA gel analysis, the PVM surface protein was synthesized in significant amounts and found predominantly

-8 soluble protein fractions. The PVM-CP protein was also found in the lower fractions I and 2 (50/60% sucrose) after graduation, indicating their possible presence in the form of high molecular weight aggregates. Fractions 1 and 2 of PVM-CP were pooled and dialyzed against 200 volumes of 1x PBS to remove sucrose. After dialysis, CP preparations were concentrated using an Amicon Ultra 15 centrifugation filter (Millipore, USA). If necessary, the ultracentrifugation procedure may be repeated to obtain clean preparations. Concentrated viral surface protein preparations were analyzed by electron microscopy. As can be seen from the image obtained by electron microscopy analysis (Fig. 6), the PVM surface protein gene of the cloned potato virus expresses E.coli-like particles. The PVM-CP VLP isolated from E.coli in E.coli cells forms filamentous virus-like particles that morphologically resemble native PVM virions and reach sizes up to 600 nm.

Example 2: Preparation of PVM VLPs containing heterologous polypeptides

In order to construct a PVM CP construct that could serve as a carrier for foreign epitopes or protein domains, it was first necessary to construct intermediate constructs in which the inserted epitopes or protein domains would not interfere with CP amino acids in the VLP assembly process. It was decided to add glycine and serine repeat sequences (SEQ ID NO: 7) at the N-terminus of PVM-CP, which would serve as a flexible amino acid linker (linker) between the CP amino acids and the inserted foreign protein sequence and ensure smooth VLP formation. Following the protocol already described, a (G4S) ₃ coding sequence and an additional BamHI restriction site epitope was introduced at the 5 'end of the PVM-CP gene with the appropriate primers for introduction of the linker at the N-terminus (SEQ ID NO: 8 / SEQ ID NO: 9). protein cDNA cloning (Fig. 2). The expression vector pET-PVM-CP and the PCR product were cleaved at the restriction sites Ncol and EcoR1 (intimal site of the PVM CP gene). Fragments were isolated from 0.8% agarose gel and ligated with T4 ligase; competent cells of E.coli XLI were transformed with ligation mixtures. After plasmid isolation from single colony cultures, clones corresponding to the respective gene sequences were selected by restriction analysis and sequencing. Next, E.coli C2566 expression strain cells were transformed with the selected pET-PVM-CP-NG4S plasmid.

The PVM-CP-NG4S protein was purified according to the protocol described above (Example 1).

Similar to PVM-CP, much of the protein under investigation was found in 50/60% sucrose gradient fractions (fractions 1 and 2), suggesting that they form high molecular weight VLP structures. Thus, it was shown that foreign peptide sequences can be added to the N-terminus of PVM-CP.

The hepatitis B virus preS 1 epitope (SEQ ID NO: 10), whose cDNA is in the collection of the Latvian Biomedical Center [27], was selected for PVM VLP as an epitope carrier.

The preSl epitope cDNA for cloning was obtained by PCR from complement-specific primers PVM-NpreSlF (SEQ ID NO: 11) and PVM-NpreSlR (SEQ ID NO: 12) without the use of reference DNA ("template DNA"). The PCR product was cut with NcoI and BamHI and ligated into pET-PVM-CP-NG4S, which was cut by NcoI and partial BamHI digestion. XL1 cells were transformed with the ligation mixture, plasmid DNA was isolated. By sequencing, a clone with the sequence planned was selected, thereby obtaining the expression vector pET-PVM-CP-NpreS1 (plasmid fragment map and target protein amino acid sequence - Fig. 3), which was introduced into C2566 expression cells by transformation. After selection of expression clones, the culture with the highest expression was cultured in preparative amounts, and cell lysate was purified in a sucrose gradient by ultracentrifugation according to the protocol described above. Similar to PVM-CP, the target protein was found in large amounts in 50/60% sucrose fractions, suggesting that it may form multimeric structures. To verify the formation of VLPs from this recombinant protein, these fractions were pooled, dialyzed and, after concentration with an Amicon Ultra-15 (100kDa) ultrafiltration device, prepared for electron microscopy analysis. As shown in Figure 7, the resulting protein construct, in which the HBV preSl 22 amino acids and 17 glycine-serine linker are added to the PVM surface protein at the N-terminus, is capable of forming virus-like particles that do not morphologically differ from the PVM-CP VLPs already described. This indicates that foreign epitope sequences can be "incorporated" into the PVM VLP structure and do not interfere with the VLP formation process.

To test the effect of longer, N-terminal polypeptides on VLP formation by PVM-CP autopolymerization, a double Z domain from Staphylococcus aureus, which is known to be capable of binding various immunoglobulins and can be used in antibody purification processes, was selected as model protein . The Z domain has been successfully expressed in E.coli cells [29]. A duplicate Z domain cDNA copy (SEQ ID NO: 13) for PCR amplification is found in the commercial vector pEZZ18 (Pharmacia, USA). To clone the Z domain cDNA at the 5 'end of the PVM CP gene, the corresponding pEZZ18 fragment was amplified in a PCR reaction using specific primers (SEQ ID NO: 14 / (SEQ ID NO: 15). Similarly, the resulting PCR product was Ncol and BamHI). directly cloned pET-PVM-CPNG4S A clone without PCR errors was selected by restriction analysis and sequencing to obtain the expression vector pET-PVM-CP-Nzz (Fig. 4). This plasmid was introduced into C2566 expression cells by transformation. The CP-Nzz protein was obtained by an analogous protocol as the other PVM-CP derivatives, again in the sucrose gradient the purified protein was autopolymerized in the form of virus-like particles according to electron microscopy (Fig. 8) with a morphology not significantly different from PVM-CP VLP. , although shorter filaments are predominant than in the case of VAT-CP, so it is shown that VAT can be added at the N-terminus

A polypeptide sequence of at least 137 amino acids long comprising 116 amino acids of the Z domain (SEQ ID NO: 16) without interfering with the process of VLP formation.

References

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SEQUENCE LISTING

SEQ ID NO: 1

ΤΥΡΕ: protein

LENGTH: 304 amino acids

ORGANISM: Potato virus M (PVM) TOPOLOGY: linear

MOLECULAR ΤΥΡΕ: coat protein (CP)

SEQUENCE: 1

Met Gly Asp Ser Thr 5 Lys Lys Ala Glu Ala Ala Lys 10 Asp Or Gly 15th Thr Ser Gln Glu Lys Arg Glu Ala Arg Pro Leu Pro Thr Ala Ala Asp Phe Glu Gly Arg Asp Thr Ser 20th 25th 30th 35 Glu Asn Thr Asp Gly Arg Ala Ala Asp Ala Asp Gly Glu Met Ser Leu Glu Arg Arg 40 45 50 55 Leu Asp Ser Leu Arg Glu Phe Leu Arg Glu Arg Arg Gly Ala Ile Arg Or Thr Asn 60 65 70 75 Pro Gly Leu Glu Thr Gly Arg Pro Arg Leu Gln Leu Ala Glu Asn Met Arg Pro Asp 80 85 90 95 Pro Thr Asn Pro Tyr Asn Arg Pro Ser Ile Glu Ala Leu Ser Arg Ile Lys Pro Ile 100 105 110 Ala Ile Ser Asn Asn Met Ala Thr Ser Glu Asp Met Met Arg Ile Tyr Or Asn Leu 115 120 125 130 Glu Gly Leu Gly Or Pro Thr Glu His Or Gln Gln Or Or Ile Gln Ala Or Leu 135 140 145 150 Phe Cys Lys Asp Ala Ser Ser Ser Or Phe Leu Asp Pro Arg Gly Ser Phe Glu Trp 155 160 165 170 Pro Arg Gly Ala Ile Thr Ala Asp Ala Or Leu Ala Or Leu Lys Lys Asp Ala Glu 175 180 185 190 Thr Leu Arg Arg Or Cys Arg Leu Tyr Ala Pro Or Thr Trp Asn His Met Leu Thr 195 200 205 His Asn Ala Pro Pro Ala Asp Trp Ala Ala Met Gly Phe Gln Tyr Glu Asp Arg Phe 210 215 220 225 Ala Ala Phe Asp Cys Phe Asp Tyr Or Glu Asn Thr Ala Ala Or Gln Pro Leu Glu 230 235 240 245 Gly Leu Ile Arg Arg Pro Thr Pro Arg Glu Lys Ile Ala His Asn Thr His Lys Asp 250 255 260 265 Ile Ala Leu Arg Gly Ala Asn Arg Asn Gln Or Tyr Ser Ser Leu Asn Ala Glu Or 270 275 280 285 Thr Gly Gly Met Asn Gly Pro Glu Leu Thr Arg Asp Tyr Gly Lys Ser Asn Arg Lys 290 295 300

SEQ ID NO: 2

ΤΥΡΕ: nucleic acid

LENGTH: 912 bases

ORGANISM: Potato virus M

TOPOLOGY: linear

MOLECULAR ΤΥΡΕ: coat protein cDNA

SEQUENCE: 2 atgggagattcaacgaagaaagctgaagctgccaaagatgtgggcacttcgcaagaaaag

-13agagaagcgcgaccactgccgactgctgctgactttgaggggagggacacatcggagaac 120 actgatgggcgtgctgcagatgctgatggggagatgtcattggagcggaggcttgacagc 180 ctccgagaattcctgcgagagcggaggggcgctattcgggtgacaaacccggggttagag 240 actggcaggccaaggttgcagctagctgaaaatatgcgccctgatcccacgaatccgtac 300 aacaggccgtccatagaagctctcagccggatcaaaccaatcgcgatctcaaacaatatg 360 gccacatctgaggatatgatgcgcatatatgtgaacctagaggggctaggggtgccgact 420 gagcacgtgcagcaggtggtgattcaggctgtgctattttgcaaggatgcgagcagctcc 480 gtattcctggatccgcgaggctcgtttgagtggccaaggggtgctataaccgctgatgcc 540 gtcttggctgtgctgaagaaggacgcagaaacactacgaagggtgtgtaggctgtatgcc 600 ccggtgacatggaatcatatgctgacgcacaacgcgcctccggccgactgggctgccatg 660 gggtttcagtatgaggatcgcttcgctgctttcgactgctttgattacgttgagaatact 720 gctgcagtccaacccctagagggattgatcaggcgacctaccccaagggaaaagatagct 780 cacaatacgcacaaagacatcgcactgcgtggagcaaatcgcaatcaggtgtacagctct 840 ctcaatgccgaggtcactggtggcatgaatggtccggagctcactagggattatgggaag 900 tcgaatagaaaa

SEQ ID NO: 3

ΤΥΡΕ: nucleic acid

LENGTH: 25 bases

ORGANISM: artificial sequence

TOPOLOGY: linear

MOLECULAR ΤΥΡΕ: synthetic oligonucleotide

OTHER INFORMATION: PVM_CP1F primer

SEQUENCE: 3 cgtttgggtgtggttcctttaggtc 25

SEQ ID NO: 4

ΤΥΡΕ: nucleic acid

LENGTH: 35 bases

ORGANISM: artificial sequence

TOPOLOGY: linear

MOLECULAR ΤΥΡΕ: synthetic oligonucleotide

OTHER INFORMATION: PVM_CPlR primer

SEQUENCE: 4 gcacactcagcaatactaaagcaatttcaaacaca 35

SEQ ID NO: 5

ΤΥΡΕ: nucleic acid

LENGTH: 34 bases

ORGANISM: artificial sequence

TOPOLOGY: linear

MOLECULAR ΤΥΡΕ: synthetic oligonucleotide

OTHER INFORMATION: PVM_CP_Nco_F primer

SEQUENCE: 5 caccatgggagattcaacgaagaaagctgaagct 34

SEQ ID NO: 6

ΤΥΡΕ: nucleic acid

-14LENGTH: 34 bases

ORGANISM: artificial sequence

TOPOLOGY: linear

MOLECULAR ΤΥΡΕ: synthetic oligonucleotide

OTHER INFORMATION: PVM_CP_Hind_R primer

SEQUENCE: 6 gcaagctttaaagccaccttggttacgtgcttca 34

SEQ ID NO: 7

ΤΥΡΕ: protein

LENGTH: 17 amino acids

ORGANISM: artificial sequence

TOPOLOGY: linear

MOLECULAR ΤΥΡΕ: amino acid linker

OTHER INFORMATION: VAT-CP N-terminal

SEQUENCE: 7

Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser 15 10 15

SEQ ID NO: 8

ΤΥΡΕ: nucleic acid

LENGTH: 93 bases

ORGANISM: artificial sequence

TOPOLOGY: linear

MOLECULAR ΤΥΡΕ: synthetic oligonucleotide

OTHER INFORMATION: PVM_Ng4sF primer

SEQUENCE: 8 taccatgggaaatgacggatccggaggaggtggaagcggaggaggtggatctggaggagg 60 tggttctatgggagattcaacgaagaaagctga 93

SEQ ID NO: 9

ΤΥΡΕ: nucleic acid

LENGTH: 22 bases

ORGANISM: artificial sequence

TOPOLOGY: linear

MOLECULAR ΤΥΡΕ: synthetic oligonucleotide OTHER INFORMATION: PVM_EcoR primer

SEQUENCE: 9 cgctctcgcaggaattctcgga

SEQ ID NO: 10

ΤΥΡΕ: protein

LENGTH: 21 amino acids

-15ORGANISM: Hepatitis B virus

TOPOLOGY: linear

MOLECULAR ΤΥΡΕ: preSl protein

OTHER INFORMATION: N-terminal fragment

SEQUENCE: 10

Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Lower Phe Arg Lower Asn Thr 15 10 15

Area Asn 20

SEQ ID NO: 11

ΤΥΡΕ: nucleic acid

LENGTH: 55 bases

ORGANISM: artificial sequence

TOPOLOGY: linear

MOLECULAR ΤΥΡΕ: synthetic oligonucleotide

OTHER INFORMATION: PVM-NpreSlF primer

SEQUENCE: 15 taccatgggaaatgacaatcctctgggattctttcccgaccaccagttggaccca 55

SEQ ID NO: 12

ΤΥΡΕ: nucleic acid

LENGTH: 58 bases

ORGANISM: artificial sequence

TOPOLOGY: linear

MOLECULAR ΤΥΡΕ: synthetic oligonucleotide

OTHER INFORMATION: PVM-NpreSlRprimer

SEQUENCE: 12 tccggatcctggatttgcggtgtttgctctgaaggctgggtccaactggtggtcggga 58

SEQ ID NO: 13

ΤΥΡΕ: nucleic acid

LENGTH: 348 bases

ORGANISM: Staphylococcus aureus

TOPOLOGY: linear

MOLECULAR ΤΥΡΕ: duplicated antibody binding domain Z cDNA

SEQUENCE: 13 gtagacaacaaattcaacaaagaacaacaaaacgcgttctatgagatcttacatttacct

-16aacttaaacgaagaacaacgaaacgccttcatccaaagtttaaaagatgacccaagccaa 120 agcgctaaccttttagcagaagctaaaaagctaaatgatgctcaggcgccgaaagtagac 180 aacaaattcaacaaagaacaacaaaacgcgttctatgagatcttacatttacctaactta 240 aacgaagaacaacgaaacgccttcatccaaagtttaaaagatgacccaagccaaagcgct 300 348 aaccttttagcagaagctaaaaagctaaatgatgctcaggcgccgaaa

SEQ ID NO: 14

ΤΥΡΕ: nucleic acid

LENGTH: 51 bases

ORGANISM: artificial sequence

TOPOLOGY: linear

MOLECULAR ΤΥΡΕ: synthetic oligonucleotide

OTHER INFORMATION: PVM-NzzF primer

SEQUENCE: 14 taccatgggaaatgacgtagacaacaaattcaacaaagaacaacaaaacgc 51

SEQ ID NO: 15

ΤΥΡΕ: nucleic acid

LENGTH: 34 bases

ORGANISM: artificial sequence

TOPOLOGY: linear

MOLECULAR ΤΥΡΕ: synthetic oligonucleotide

OTHER INFORMATION: PVM-NzzF primer

SEQUENCE: 15 ccggatcctttcggcgcctgagcatcatttagct 34

SEQ ID NO: 16

ΤΥΡΕ: protein

LENGTH: 116 amino acids

ORGANISM: Staphylococcus aureus

TOPOLOGY: linear

MOLECULAR ΤΥΡΕ: duplicated antibody binding domain Z OTHER INFORMATION: Sequence from pEZZ18

SEQUENCE: 16

Or 1 Asp Asn Lys Phe Asn Lys 5 Glu Gln Gln Asn 10th Ala Phe Tyr Glu Ile 15th Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro 20th 25th 30th 35 Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro 40 45 50 55 Lys Or Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His 60 65 70 75 Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp 80 85 90 Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 95 100 105 110 Pro Lys 115

Claims (4)

Patent Claims A nucleic acid molecule encoding a potato virus PVM surface protein according to SEQ ID NO: 2. A method for obtaining filamentous virus-like particles comprising (a) preparing a potato virus PVM nucleic acid molecule according to SEQ ID NO: 2; (b) administering said nucleic acid to the host cells; (c) culturing the host cells under conditions which yield a protein capable of forming virus-like particles. A method for obtaining filamentous virus-like particles according to claim 2, wherein said host cells are Escherichia coli cultured at + 20 ° C. 4. Filamentous virus-like particles containing at least one protein of the following group: (a) a protein comprising amino acids 1-304 of SEQ ID NO: 1; (b) a protein having an amino acid sequence showing at least 90% identity to the PVM surface protein amino acids of SEQ ID NO: 1; (c) a protein comprising amino acids 1-304 of SEQ ID NO: 1 to which a polypeptide sequence of up to 137 amino acids has been added in Ngal.