WO1996014409A1 - Production of recombinant peptides as natural hydrophobic peptide analogues - Google Patents

Production of recombinant peptides as natural hydrophobic peptide analogues Download PDF

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Publication number
WO1996014409A1
WO1996014409A1 PCT/FR1995/001464 FR9501464W WO9614409A1 WO 1996014409 A1 WO1996014409 A1 WO 1996014409A1 FR 9501464 W FR9501464 W FR 9501464W WO 9614409 A1 WO9614409 A1 WO 9614409A1
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Prior art keywords
peptide
sequence
recombinant
aaa
acc
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PCT/FR1995/001464
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French (fr)
Inventor
Thien Nguyen Ngoc
Hans Binz
Mathias Uhlen
Stefan Stahl
Per Ake Nygren
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Pierre Fabre Medicament
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Application filed by Pierre Fabre Medicament filed Critical Pierre Fabre Medicament
Priority to NZ296562A priority Critical patent/NZ296562A/en
Priority to JP8515108A priority patent/JPH10508479A/en
Priority to EP95939336A priority patent/EP0791059A1/en
Priority to AU41200/96A priority patent/AU704594B2/en
Publication of WO1996014409A1 publication Critical patent/WO1996014409A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to recombinant peptides, analogues of natural peptides, and having conserved the biological activity of these natural peptides. These peptides can be expressed by different types of cells and their production is improved compared to that of the natural peptide.
  • peptide any substance composed of a chain of amino acids, that is to say oligopeptide, polypeptide and protein.
  • Peptides have a wide variety of biological properties, and to avoid the problems of contamination, in particular, linked to purification techniques from biological products, we have been led to produce them by genetic engineering.
  • peptides are excreted through the cell membrane, in order to obtain them in the extracellular medium or, in the case of Gram-negative bacteria, in the periplasmic space.
  • a system can be introduced into a cell, in particular a bacterium, allowing expression of the peptide in a form fused with a membrane anchoring sequence, so as to obtain the fusion product covalently linked to the membrane surface.
  • RSV respiratory syncitial virus
  • the respiratory syncytial virus (RSV) is the most common cause of respiratory diseases in newborns: bronchopneumopathies (bronchiolitis). WHO estimates 5 ⁇ million cases of RSV each year, including 160,000 deaths worldwide. There are two subgroups of the virus (subgroups ⁇ and B).
  • RSV is classified in the family of Paramyxoviridae, genus pneumovirus comprising a non-segmented ⁇ RN genome, of negative polarity, coding for 10 specific proteins.
  • RSV structural proteins for a vaccine, such as the envelope proteins called protein F (fusion protein) or protein G, a 22 Kd glycoprotein, a protein of 9.5 Kd, or the major capsid protein
  • Application WO 89/02935 describes the protective properties of the entire F protein of RSV, optionally modified in monomeric or deacetylated form. A series of fragments of the F protein have been cloned in order to search for their neutralizing properties.
  • RSV infections of the upper airways treatment is essentially based on symptomatic medications identical to those of other viral infections.
  • RSV infections of the lower airways treatment in infants is based on maintaining proper hydration, aspirating secretions and administering oxygen if necessary.
  • a positive effect has been observed with ribavirin, a nucleotide active in vitro against RSV.
  • ribavirin a nucleotide active in vitro against RSV.
  • the applicants have built an original vector system, also called a shuttle vector, which is functional in Esche chia coli and Staphylococcus xylosus: the vector contains a secretory sequence signal S, and an XM region of membrane anchoring of protein A origin. Staphylococcus aureus, with a cloning site between S and XM allowing the insertion of one or more genes.
  • the hydrophobicity of the molecule must in certain cases be modified. However, these modifications must not alter the biological, in particular immunogenic, properties of the product.
  • the subject of the present invention is a method for producing a recombinant biologically active peptide, analogous to a natural peptide having at least one hydrophobic region, characterized in that it comprises a step in which a cell a DNA construct, coding for a peptide whose amino acid sequence differs from the sequence of the natural peptide by at least one modification in said hydrophobic region, and comprising elements ensuring expression and secretion of said peptide by cell, and in that, after culturing the cells, the peptide and / or the cells carrying said recombinant peptide are recovered.
  • the amino acid sequence is modified at least in a region different from the transmembrane region of the natural peptide.
  • the modification should preferably take place in a region which is not essential for the biological activity of interest of the peptide, which must be maintained.
  • This process involves a recombinant DNA construct in which a functional secretory signal sequence is linked to a structural gene which has been altered in order to modify the structure which allows the recombinant product to cross the membrane of the host cell, then that the recombinant product of the original structural gene is not when linked to the same secretory signal sequence; and the structural modifications of the recombinant product should be carried out by genetic engineering by altering the localization of the recombinant product expressed in a host cell.
  • the modifications aim to modify the hydrophobicity of the recombinant product.
  • the subject of the invention is therefore a method for producing a recombinant peptide in which the structural modifications of the gene lead to a peptide in which at least one hydrophobic amino acid of the sequence of the natural peptide is replaced by a non-hydrophobic amino acid.
  • at least one hydrophobic amino acid is deleted from the sequence of the natural peptide.
  • the hydrophobic amino acid is chosen from the following group: Tryptophan, Phenylalanine, Proline, Valine, Alanine, Isoleucine, Leucine and Methionine.
  • Structural modifications of the gene can be made by insertion of nucleotides, or by deletion of nucleotides.
  • the constructions in which the structural modifications of the gene are made by substitution of nucleotides by site-directed mutagenesis are also included in the invention.
  • the structural modifications of the gene could change the localization in such a way that the recombinant product is exposed to the membrane surface of the cell by a covalent bond to the membrane anchoring part.
  • the structural modifications of the gene can change the location so that the recombinant product is secreted into the culture medium.
  • the invention therefore also relates to the construction of DNA which comprises a secretion signal sequence operably linked to the DNA sequence coding for the recombinant peptide, and ensuring the translocation of said peptide and its extracellular secretion.
  • the invention relates to a method characterized in that the DNA construction comprises a signal sequence operably linked to the DNA sequence coding for said peptide and allowing the translocation of the peptide across the membrane of the host cell and its membrane anchoring.
  • Another object of the invention is recombinant peptides capable of being obtained by the process, characterized in that they differ from the natural peptide by at least one modification in the hydrophobic region of the natural peptide. They may appear anchored to the surface of the host cell.
  • peptides will in particular be chosen from analogs of a protein of the RSV structure or of a fragment of such a protein; more particularly, the recombinant peptide comprises a sequence analogous to protein G of RSV, in group A or B, in particular comprised between residues 130 and 230 of protein G of RSV, having at least 80% homology.
  • Protein G is an RSV envelope glycoprotein, with a molecular weight between 84 and 90 Kd, poor in methionine.
  • the sequence of protein G differs for subgroups A and B of RSV; the terms "protein G sequence" when used in the present application, should be understood as referring to both the sequence of subgroup A or of subgroup B, when this is not specified differently.
  • the Applicant has demonstrated that the sequence between amino acids 130 and 230 of the natural protein G is particularly suitable for inducing effective protection against infection by RSV, subgroups A and B, without inducing the pathologies observed. with vaccines based on the whole virus inactivated by formalin, or observed with whole F and G proteins.
  • the means allowing the expression of the polypeptide are known to those skilled in the art and are adapted according to the bacteria used.
  • the DNA sequence is introduced in the form of a plasmid, such as a shuttle plasmid.
  • the RSV proteins have to date been expressed in different expression systems such as vaccinia virus, baculoviruses or adenoviruses. However, potential problems are associated with the presence of residual virus particles.
  • the method according to the present invention uses a commensal bacterium from humans, apathogenic and edible.
  • the bacteria can belong to the genus Staphylococcus.
  • Staphylococcus xylosus which is a bacterium used in the food industry for many years, and can be administered, alive, orally.
  • Expression systems of heterologous epitopes on the surface of S. xylosus have been described in particular by N'guyen et al in Gene, 1993, 128: 89-94.
  • the heterologous polypeptide is expressed on the surface of the membrane of the bacterium, in a conformation essentially identical to that of the corresponding epitope of the natural protein G.
  • the presentation of the recombinant protein on the membrane surface of the bacteria depends on its chemical nature and its peptide sequence.
  • the amino acid cysteine at positions 173 and / or 186 has been replaced by an amino acid which does not form of disulfide bridge, in particular serine.
  • Such a mutation promotes the formation of the disulfide bridge between the cysteine residues remaining in positions 176 and 182, which is critical for the immunogenicity of the sequence; it prevents the formation of disordered disulfide bridges.
  • Peptides useful for the implementation of such a process are those comprising in particular one of the sequences ID No. 3 or ID No. 4.
  • the amino acids phenylalanine corresponding to positions 163, 165, 168 and / or 170 of the sequence of protein G are replaced by an acid polar amine, especially serine.
  • This modification can be associated with the mutations mentioned above.
  • a polypeptide can in particular have the sequence ID No. 5.
  • the suppression of the hydrophobic region situated upstream of the critical loop formed by the disulfide bridge between the amino acids cysteine in positions 176 and 182 allows the recombinant protein to better cross the bacterial membrane and to correctly expose the immunodominant part on the membrane surface.
  • the sequence between the amino acids numbered 162 and 170 is deleted. More particularly, the sequence of the heterologous peptide expressed in the bacterium can include the sequence ID No. 6.
  • the invention also comprises a bacterium expressing a peptide or a protein, capable of being obtained by the method described in the present application. Said bacteria can be used alive or killed.
  • Polypeptides or bacteria having one or more of the above characteristics are useful as medicaments.
  • the invention comprises pharmaceutical compositions, characterized in that they comprise a polypeptide or a bacterium according to the invention in admixture with pharmaceutically acceptable adjuvants.
  • the live vector oral vaccine must include the modified protein which has the optimal conformation to induce the best protection against RSV. This is why the present invention also relates to an application of such a pharmaceutical composition to the preparation of an oral vaccine intended to prevent infections caused by the respiratory syncytial virus.
  • the subject of the invention is the nucleotide sequences coding for a recombinant peptide analogous to a natural peptide as described above; these sequences may also contain elements ensuring expression of the peptide in one or more specific host cells. These elements make it possible to target the cells in which the construction will be expressed during its administration to a mammal, human or animal.
  • These can be DNA or RNA constructs, which will preferably be incorporated into a vector.
  • Suitable vectors are in particular plasmids or viruses of the Adenovirus type, which can be formulated in pharmaceutical compositions with acceptable excipients.
  • the subject of the invention is nucleotide sequences coding for a polypeptide carried by a peptide sequence comprised between the amino acid residues 130 and 230 of the protein G of the respiratory virus or for a polypeptide having at least 80% of homology with said peptide sequence, and further comprising the means allowing the expression of the polypeptide on the surface of the membrane of a non-pathogenic bacterium of the genus Staphylococcus.
  • a DNA sequence coding for a recombinant peptide analogous to a natural peptide, the recombinant peptide sequence exhibiting at least one modification in the non-transmembrane hydrophobic region of the natural peptide, forms part of the invention.
  • the method according to the invention comprises the following steps: a. transforming the host cells with a recombinant DNA construct containing a signal sequence which is operably linked to a structural gene, and the latter is modified so that the recombinant product can be translocated across the membrane of the host cell ; b. fermenting said host cell to express the recombinant product; vs. recover the extracellular proteins secreted by the cells transformed by the constructs.
  • the invention also comprises a recombinant cell which contains a DNA sequence or a construct as defined above.
  • This host cell can be a bacterium, Gram + or Gram-, a yeast cell or a mammalian cell.
  • Particularly suitable bacteria are chosen from the group comprising Escherichia coli, Staphylococcus xylosus, Staphylococcus carnosus.
  • the DNA sequence can be integrated into the chromosome of the bacteria, Gram positive or Gram negative.
  • This recombinant DNA construct may contain the gene coding for protein G of amino acid 130 to 230 of human RSV subgroup A fused upstream and / or downstream of that of subgroup B.
  • a type of construction can be carried out with the gene coding for the amino acid protein 130 to 230 of bovine RSV belonging to subgroup A, or subgroup B.
  • FIG. 3 Schematic of shuttle vector constructions, above pSE'mlpl ⁇ BBXM, in (A) vector pSE'G2BBXM; (B) vector pSE'G2subBBXM; and (C) vector pSE * G2delBBXM;
  • FIG. 6 Diagram of the principle of construction of the secretion vectors from the respective shuttle vectors pSE'G2subBBXM and pSE'G2delBBXM whose descriptions are detailed in Example 4.
  • the products secreted from the culture medium of S are illustrated xylosus carrying vectors whose stop codon (T) has been inserted upstream of the XM membrane anchoring region;
  • Figure 7 Analysis by SDS-Page and immunoblot of the fusion proteins secreted by S. xylosus;
  • G2 Construction of G2 by assembly of synthetic genes: The gene coding for the amino acid region 130-230 of the glycoprotein G of the RS virus, named G2, where we have in addition changed two Cys residues at position 173 and 186 into Ser with respect to the original sequence, is obtained by techniques of assembly of genes in solid phase (Stahl S. et al., 1993. BioTechniques, .L4.424-434). The oligonucleotide sequence has been optimized by combining the usual codons of bacteria such as Ecoli and Staphylococcus.
  • the oligonucleotides were synthesized by phosphoramidite chemistry on the automatic DNA synthesizer (Gene Assembler Plus, Pharmacia Biotech) according to the manufacturer's recommendations.
  • the oligonucleotides to be fixed in the solid phase are biotinylated at the 5 ′ end by the reagent Biotin-on phosphoramidite (Clontech).
  • the other oligonucleotides are phosphorylated in 5 ′ by the phosphate-on amidite reagent (Clontech) according to the Clontech protocol.
  • the oligonucleotides are deprotected and are purified according to the recommendations of Pharmacia.
  • the biotinylated oligonucleotides are purified by reverse phase liquid chromatography (PEP RPC column, Pharmacia).
  • the gene is assembled in two parts: G2am (upstream) from AA 130 to 177 with two restriction sites BamHI and PstI in 5 'and 3' of the gene, G2av (downstream) from AA 177 to 230 with two sites of restriction PstI and Ba HI in 5 'and 3' of the gene.
  • the G2 gene is reconstituted by ligating the two fragments G2am and G2av via the PstI site.
  • the immobilized double strand comprises a BamHI restriction site and the strand complementary to that which is biotinylated has 6 to 15 nucleotides protruding from its 5 ′ side (phosphorylated) allowing the next oligonucleotide to come to hybridize.
  • Hybridization of the latter oligonucleotide is carried out by raising the temperature of the medium to 70 ° C. to avoid the formation of secondary structures. Ligation is done by adding T4 DNA ligase (Gibco BRL). Thus, the gene is constructed successively, taking the precaution at each cycle of rinsing the solid support in order to eliminate the excess of unbound oligonucleotides before adding the following oligonucleotide. The last ligated double strand contains a PstI restriction site.
  • the double strand is then released from its solid support by digesting it with the restriction enzymes BamHI and PstI and then ligated into the cloning and sequencing vector prit28 (Hultman et al., 1988, Nucleosides Nucleotides 7 629-638) digested by same enzymes: the resulting vector is pR! T28G2am of size 3067bp.
  • the nucleic acid sequence of G2am is determined by DNA sequencing on the ABI automatic sequencer, according to the manufacturer's recommendations (Applied Biosystem).
  • the double strand is released from the solid support by enzymatic digestion successively with PstI and HindIII, cloned in pRIT28: the resulting vector is named pRIT28G2av of size 3091 bp.
  • the nucleic acid sequence of G2av is determined by DNA sequencing on the ABI automatic sequencer, according to the manufacturer's recommendations (Applied Biosystem).
  • the two fragments G2am and G2av are ligated by the restriction site PstI and the gene formed is cloned into pRIT28 by the BamHI sites in 5 ′ and HindIII in 3 ′: the resulting vector is named pRIT28G2 of size 3220 bp.
  • the nucleic sequence of G2 is determined by DNA sequencing on the ABI automatic sequencer, according to the manufacturer's recommendations (Applied Biosystem).
  • the G2 fragment is digested with BamHI and HindIII and cloned into the shuttle vector: pSE'G2BBXM (7666 bp) (FIGURE 3). List of oligonucleotides required for gene assembly
  • HindlII TH12 (33mer): 5'-GCCGACCACC AAACCGGTCG ACTAAGCrTC ACA-3 ' HindlII TH13B (19mer): 5'-Biotin-CCCTGTGAAG CTTGGTTTG-3 'TH14 (32mer): 5'-CATAAACCGC AGACCACCAA ACCGAAAGAA GT-3' TH15 (32mer): 5'-GTGGTCGGCA CTTCTTTCGG TTTGGTGGTC TG 3 ' : 5'-AAAACCGACC TTCAAAACCA CCAAAAAAGA T-3 "TH17 (3 lmer): 5'-CGGTTTATGA TCTTTTTTGG TGGTTTTGAA C-3 'TH18 (30mer): 5'-GGGCAAAAA ⁇ ACCACGACCA AACCGACCAA-3' TH19 (3 lmer): 5 ' GTCCrGTITrrTGGTCGGTTTGGTCGTGGTTT-3 'TH20 (34mer): 5
  • the gene fragment where the four phenylalanine residues at positions 163, 165, 168 and 170 are replaced by Serines is generated by gene amplification (PCR), from the G2 gene inserted in the vector pRIT28, using as pairs of primers RIT27 / TNG73 and RIT28 / TNG72 (see FIGURE 2. (1)).
  • the primers TNG72 and TNG73 are complementary to a region of 19 nucleotides containing three of the four phenylalanines: Rit27: 5 , -GCnCCGGCr CGTATGlTGTGTG-3 'Rit28: 5'-AAAGGGGGAT GTGCTGCAAG GCC-3'
  • the primer TNG73 introduces the first three mutations (TTC in TCC) on the upstream fragment amplified with RIT27.
  • the primer TNG72 introduces the last three mutations (TTC in TCC) on the downstream fragment amplified with RIT28.
  • Five temperature cycles (9 'c, 15 sec; 50 ' c, 1 min; 72 " c, 1 min) followed by twenty cycles (9 " c, 1 5 sec; 60 "c, 15 sec; 72 'c, 15 sec)
  • the two amplified fragments are mixed in a single tube diluted in PCR buffer without primers and the extension reaction is carried out in five temperature cycles (96 'c, 15 sec; 54 * c, 30 sec; 72 * c, 1 min).
  • the extension product is diluted 1/100 in PCR buffer containing the primers RIT27 and RIT28 and the gene amplification is carried out in 30 temperature cycles (96 * c, 15 sec; 54 'c, 15 sec; 72 ' c, 30 sec).
  • the fragment is then digested with the restriction enzymes BamHI / HindIII and cloned into the vector prit28 digested with the same enzymes: pRIT28G2sub.
  • the nucleic sequence of G2Sub is determined by DNA sequencing on the ABI automatic sequencer device, according to the manufacturer's recommendations (Applied Biosystem).
  • the G2sub fragment is digested with BamHI and HindIII and cloned into the shuttle vector: pSE'G2subBBXM (7666 bp) (FIGURE 3).
  • G2del (see FIGURE 2. (2)) In the same way, the G2 gene fragment deleted from the part containing the 4 phenylalanine residues from aa 162 to aa 170 is generated by PCR in two fragments: upstream with the primers RIT27 / TH48 on the one hand and downstream with RIT28 / TH 1 1 on the other hand.
  • the primers TH1 1 and TH48 are complementary to 13 nucleotides.
  • TH 1 1 5'-GTGCCGAGCA GCATCTGCAG CA-3 '
  • the fragment is then digested with the restriction enzymes I.amlll / Hindlll cl cloned in the vector prit28 digested with the same enzymes: pRIT28G2del.
  • the nucleic sequence of G2del is determined by DNA sequencing on the ABI automatic sequencer device, according to the manufacturer's recommendations (Applied Biosystem).
  • the G2dcl fragment is digested with BamHI and HindIII and cloned into the shuttle vector: pSE'G2delBBXM (7639 bp) (FIGURE 3).
  • oligonucleotide linker (5'-AGCTTGGCTG TTCCGCCATG GCTCGAG-3 ', with the complementary strand) is inserted into the HindIII site of the plasmid pSZZmpl ⁇ XM (Hansson et al, 1992, J. Bacteriol 174: 4239-4245), thus creating two additional restriction sites Ncol and Xhol downstream of the HindIII site of the resulting vector pSZZmpl 8 (XhoI) XM.
  • BB gene fragment coding for 198 amino acids, named from the binding region of the serum albumin of the streptococcal protein G (Nygren et al, 1988. J. Mol.
  • TSB 12.5 g of Yeast extract
  • Chloramphenicol (20 ⁇ g / ml)
  • preheating overnight
  • S. xylosus transformed by the shuttle vector pSE'G2BBXM or pSE'G2subBBXM or pSE'G2delBBXM Gôtz et al. protocol, 1 81, J Bacteriol., 145: 74-81.
  • the proteins are purified by affinity: the supernatant is passed through an HSA-Sepharose affinity column (Human Albumin Serum). After rinsing the column, the proteins are eluted with a pH 2.7 acid buffer and lyophilized.
  • the proteins are separated on two identical SDS-PAGE gels (12%) with the precolored standard molecular size markers (Gibco BRL). A gel is colored with Coomassie Blue. The second is transferred to the ProblotTM membrane (Applied Biosystem) for the immunoblot with the specific anti-G l antibody (obtained from rabbit serum immunized with the G l peptide (aal 74-1 87) according to current protocols. 'immunization). See Figure 5.
  • the cultures of the recombinant S. xylosus bacteria are made as described above.
  • the bacteria are resuspended in a PBS solution at 0.1% Sodium Azide (w / v) at the final concentration estimated by optical density (600 nm) equal to the unit.
  • 30 ⁇ l of stock solution are aliquoted in each conical well of a microtiter plate, centrifuged at 550 g for 10 minutes at 4 ° C.
  • the bacterial pellet is resuspended in a volume of 150 ⁇ l of PBS solution containing rabbit serum polyclonal anti-G2 (titre 1/1 280 000) diluted at l / 200th, incubation for 30 minutes.
  • the bacterial cells are rinsed twice with PBS and incubated in 150 ⁇ l of a PBS solution containing anti-rabbit FITC (Sigma) diluted to 1/100 for a period of 30 minutes. After rinsing the cells twice with PBS buffer, it is resuspended in a Falcon tube containing 1 ml of PBS-Paraformaldehyde buffer 1% (w / v). The prepared samples are analyzed on the FACScan TM device (Becton Dickinson). The fluorescence distribution of each cell suspension is analyzed by LYSIS II TM software and is represented by fluorescence histograms. See Figure 4.
  • Termination codons upstream of the region coding for the XM membrane anchoring, as shown in FIG. 6.
  • a unique Xhol restriction site between BB and XM was used to insert a double strand of oligonucleotides coding for three termination codons (Ter) in both directions of orientation, with the introduction of an Aat II restriction site:
  • pSE'G2subBBXM and pSE'G2delBBXM are thus digested with Xho I and are ligated with the double strand of oligonucleotides previously phosphorylated at 5 '.
  • the resulting vectors are pSE'G2subBB [Ter] XM (7693 bp) and pSE'G2delBB [Tcr] XM (7666 bp) respectively.
  • FIG. 7 shows: A) the SDS-PAGE gel, the separation of the secreted proteins from S. xylosus, under reduced conditions, in 1 and in 2 respectively represent the proteins G2subBB and G2delBB at the expected size: 35.23 Kda and 34.28 Kda; in B) the immunoblot of the proteins shows that the antibody specific to the G region (aal 74-187) or G l of the RSV clearly recognizes the two secreted proteins. Very few proteolytic degradations have been observed.
  • FIG. 8 shows that the spectra of S. xylosus carrying shuttle vectors pSE'mpl ⁇ BBXM, pSE'G2subBBXM and pSE'G2delBBXM are displaced in the axis of the fluorescence intensity towards the right, that is to say towards the presence of heterologous antigens on the surface of the bacteria. While the spectra of S.
  • xylosus carrying shuttle vectors pSE'G2subBBTerXM and pSE'G2delBBTerXM are not displaced there, this indicates that the heterologous antigens are absent on the surface of the bacteria and that they have been found and purified from the culture medium.
  • Comassie blue staining SDS-Page gel of the fusion proteins extracted from the bacterial membrane and purified by affinity on the Albumin column of the different constructions:

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Abstract

A method for secreting a biologically active recombinant peptide as an analogue of a natural peptide having at least one hydrophobic region, by culturing cells transformed by a nucleic acid construct comprising elements for the expression and secretion of said peptide by the cell, and a sequence coding for a peptide with an amino acid sequence that differs from the natural peptide sequence in that it has at least one modification in a non-transmembrane hydrophobic region of the peptide; and by recovering the peptide and/or cells carrying said recombinant peptide. The resulting recombinant peptide, a corresponding DNA sequence, and a bacterium containing same, are also disclosed.

Description

PRODUCTION DE PEPTIDES RECOMBINANTS ANALOGUES DE PEPTIDES NATURELS HYDROPHOBES PRODUCTION OF RECOMBINANT PEPTIDES SIMILAR TO HYDROPHOBIC NATURAL PEPTIDES
La présente invention se rapporte à des peptides recombinants, analogues de peptides naturels, et ayant conservé l'activité biologique de ces peptides naturels. Ces peptides peuvent être exprimés par différents types de cellules et leur production est améliorée par rapport à celle du peptide naturel.The present invention relates to recombinant peptides, analogues of natural peptides, and having conserved the biological activity of these natural peptides. These peptides can be expressed by different types of cells and their production is improved compared to that of the natural peptide.
Par peptide on entend toute substance composée d'un enchaînement d'acides aminés, c'est-à-dire oligopeptide, polypeptide et protéine. Les peptides possèdent une grande variété de propriétés biologiques, et pour éviter les problèmes notamment de contamination liés aux techniques de purification à partir de produits biologiques, on a été amené à les produire par génie génétique.By peptide is meant any substance composed of a chain of amino acids, that is to say oligopeptide, polypeptide and protein. Peptides have a wide variety of biological properties, and to avoid the problems of contamination, in particular, linked to purification techniques from biological products, we have been led to produce them by genetic engineering.
Toutefois, la production de peptides par voie recombinante se heurte à des difficultés liées au système d'expression et à la cellule hôte. En effet, pour faciliter leur récupération, il est souhaitable que les peptides soient excrétés à travers la membrane cellulaire, afin de les obtenir dans le milieu extracellulaire ou, dans le cas de bactérie- Gram négatif, dans l'espace périplasmique. On peut introduire dans une cellule, en particulier une bactérie, un système permettant l'expression du peptide sous forme fusionnée avec une séquence d'ancrage membranaire, de manière à obtenir le produit de fusion lié de manière covalente, à la surface membranaire.However, the production of peptides by the recombinant route encounters difficulties linked to the expression system and to the host cell. Indeed, to facilitate their recovery, it is desirable that the peptides are excreted through the cell membrane, in order to obtain them in the extracellular medium or, in the case of Gram-negative bacteria, in the periplasmic space. A system can be introduced into a cell, in particular a bacterium, allowing expression of the peptide in a form fused with a membrane anchoring sequence, so as to obtain the fusion product covalently linked to the membrane surface.
C'est ainsi que dans le cadre de la préparation de vaccin contre le VRS, les demandeurs ont mis au point un procédé de technique d'ADN recombinant permettant de modifier ponctuellement par mutagénèse dirigée, des nucléotides dans un gène codant pour une séquence polypeptidique, utile notamment pour l'obtention de vaccins par voie orale contre le virus respiratoire syncitial (VRS) . Le virus respiratoire syncytial (VRS) est la cause la plus fréquente de maladies respiratoires chez le nouveau-nc : bronchopneumopathies (bronchiolites). L'OMS estime chaque année 5ϋ millions de cas atteints du VRS, dont 160 000 décès dans le monde entier. Il existe deux sous groupes du virus (sous groupes Λ et B). Le VRS est classé dans la famille des Paramyxoviridae, genre pneumovirus comportant un génome ΛRN non segmenté, de polarité négative, codant pour 10 protéines spécifiques. Il n'existe pas actuellement de vaccin disponible, contre le VRS. Les vaccins à virus inactivé se sont montrés inefficaces et ont même parfois aggravé les infections des nourrissons. Dans les années 60, les tentatives de vaccination avec le VRS inactivé à la formaline ont conduit à l'échec : au lieu de conférer une protection lors de la réinfection due au VRS, le vaccin a eu pour effet d'aggraver la maladie chez l'enfant.Thus in the context of the preparation of a vaccine against RSV, the applicants have developed a method of recombinant DNA technique making it possible to modify punctually by site-directed mutagenesis, nucleotides in a gene coding for a polypeptide sequence, useful especially for obtaining oral vaccines against respiratory syncitial virus (RSV). The respiratory syncytial virus (RSV) is the most common cause of respiratory diseases in newborns: bronchopneumopathies (bronchiolitis). WHO estimates 5ϋ million cases of RSV each year, including 160,000 deaths worldwide. There are two subgroups of the virus (subgroups Λ and B). RSV is classified in the family of Paramyxoviridae, genus pneumovirus comprising a non-segmented ΛRN genome, of negative polarity, coding for 10 specific proteins. There is currently no vaccine available for RSV. Inactivated virus vaccines have been shown to be ineffective and have sometimes even aggravated infections in infants. In the 1960s, attempts to vaccinate with RSV inactivated with formalin led to failure: instead of conferring protection during reinfection due to RSV, the vaccine had the effect of worsening the disease in 'child.
La demande WO 87/04185 a proposé d'utiliser des protéines structurales du VRS en vue d'un vaccin, comme les protéines d'enveloppe appelées protéine F (protéine de fusion) ou protéine G, une glycoprotéine de 22 Kd, une protéine de 9,5 Kd, ou la protéine majeure de capsideApplication WO 87/04185 proposed using RSV structural proteins for a vaccine, such as the envelope proteins called protein F (fusion protein) or protein G, a 22 Kd glycoprotein, a protein of 9.5 Kd, or the major capsid protein
(protéine N).(protein N).
La demande WO 89/02935 décrit les propriétés de protection de la protéine F entière du VRS, éventuellement modifiée sous forme monomérique ou désacétylée. Une série de fragments de la protéine F a été clonée en vue de rechercher leurs propriétés neutralisantes.Application WO 89/02935 describes the protective properties of the entire F protein of RSV, optionally modified in monomeric or deacetylated form. A series of fragments of the F protein have been cloned in order to search for their neutralizing properties.
Toutefois les vaccins immunitaires testés à ce jour se sont montrés inefficaces ou ont induit une pathologie pulmonaire (bronchiolite ou péribronchite). A l'heure actuelle il n'existe pas de traitement de fond des infections dues au VRS.However, the immune vaccines tested to date have been shown to be ineffective or have induced pulmonary pathology (bronchiolitis or peribronchitis). There is currently no basic treatment for RSV infections.
Les infections au VRS des voies aériennes supérieures : le traitement repose essentiellement sur les médications symptomatiques identiques à celles des autres infections virales. Les infections au VRS des voies aériennes inférieures : le traitement chez les nourrissons repose sur le maintien d'une hydratation correcte, l'aspiration des sécrétions et l'administration d'oxygène si besoin. Un effet positif a été observé avec la ribavirine, nucléotide actif in vitro contre le VRS. Afin de faciliter l'administration du vaccin, il serait souhaitable de disposer d'un produit actif par voie orale, générant une bonne immunité, avec des effets secondaires réduits. Les demandeurs ont construit un système vecteur original, appelé encore vecteur navette, qui est fonctionnel dans Esche chia coli et Staphylococcus xylosus : le vecteur renferme un signal séquence de sécrétion S, et une région XM d'ancrage membranaire d'origine de la protéine A du Staphylococcus aureus, avec un site de clonage entre S et XM permettant d'insérer un ou plusieurs gènes.RSV infections of the upper airways: treatment is essentially based on symptomatic medications identical to those of other viral infections. RSV infections of the lower airways: treatment in infants is based on maintaining proper hydration, aspirating secretions and administering oxygen if necessary. A positive effect has been observed with ribavirin, a nucleotide active in vitro against RSV. In order to facilitate the administration of the vaccine, it would be desirable to have an active product orally, generating good immunity, with reduced side effects. The applicants have built an original vector system, also called a shuttle vector, which is functional in Esche chia coli and Staphylococcus xylosus: the vector contains a secretory sequence signal S, and an XM region of membrane anchoring of protein A origin. Staphylococcus aureus, with a cloning site between S and XM allowing the insertion of one or more genes.
Afin de faciliter le passage transmembranaire du peptide, l'hydrophobicité de la molécule doit dans certains cas être modifiée. Cependant, ces modifications ne doivent pas altérer les propriétés biologiques, notamment immunogènes du produit.In order to facilitate the transmembrane passage of the peptide, the hydrophobicity of the molecule must in certain cases be modified. However, these modifications must not alter the biological, in particular immunogenic, properties of the product.
C'est pourquoi la présente invention a pour objet un procédé de production d'un peptide recombinant biologiquement actif, analogue d'un peptide naturel présentant au moins une région hydrophobe, caractérisé en ce qu'il comprend une étape dans laquelle on introduit dans une cellule une construction d'ADN, codant pour un peptide dont la séquence d'acides aminés diffère de la séquence du peptide naturel par au moins une modification dans ladite région hydrophobe, et comportant des éléments assurant l'expression et la sécrétion dudit peptide par la cellule, et en ce que, après culture des cellules, on récupère le peptide et/ou les cellules portant ledit peptide recombinant.This is why the subject of the present invention is a method for producing a recombinant biologically active peptide, analogous to a natural peptide having at least one hydrophobic region, characterized in that it comprises a step in which a cell a DNA construct, coding for a peptide whose amino acid sequence differs from the sequence of the natural peptide by at least one modification in said hydrophobic region, and comprising elements ensuring expression and secretion of said peptide by cell, and in that, after culturing the cells, the peptide and / or the cells carrying said recombinant peptide are recovered.
De préférence, la séquence d'acides aminés est modifiée au moins dans une région différente de la région transmembranaire du peptide naturel.Preferably, the amino acid sequence is modified at least in a region different from the transmembrane region of the natural peptide.
La modification devra de préférence intervenir dans une région non essentielle pour l'activité biologique d'intérêt du peptide, qui doit être maintenue.The modification should preferably take place in a region which is not essential for the biological activity of interest of the peptide, which must be maintained.
Ce procédé met en jeu une construction d'ADN recombinant dans laquelle une séquence signal de sécrétion fonctionnelle est liée à un gène de structure qui a été altéré afin de modifier la structure qui permette au produit recombinant de traverser la membrane de la cellule hôte, alors que le produit recombinant du gène de structure d'origine ne l'est pas quant il est lié à la même séquence signal de sécrétion ; et les modifications structurelles du produit recombinant devraient être réalisées par génie génétique en altérant la localisation du produit recombinant exprimé dans une cellule hôte. Les modifications, selon un aspect de l'invention, visent à modifier l'hydrophobicité du produit recombinant.This process involves a recombinant DNA construct in which a functional secretory signal sequence is linked to a structural gene which has been altered in order to modify the structure which allows the recombinant product to cross the membrane of the host cell, then that the recombinant product of the original structural gene is not when linked to the same secretory signal sequence; and the structural modifications of the recombinant product should be carried out by genetic engineering by altering the localization of the recombinant product expressed in a host cell. The modifications, according to one aspect of the invention, aim to modify the hydrophobicity of the recombinant product.
L'invention a donc pour objet un procédé de production de peptide recombinant dans lequel les modifications structurelles du gène conduisent à un peptide dans lequel au moins un acide aminé hydrophobe de la séquence du peptide naturel est remplacé par un acide aminé non hydrophobe. Dans un autre mode de réalisation dans le peptide recombinant, au moins un acide aminé hydrophobe est délété de la séquence du peptide naturel. Avantageusement, l'acide aminé hydrophobe est choisi dans le groupe suivant : Tryptophane, Phénylalanine, Proline, Valine, Alanine, Isoleucine, Leucine et Méthionine.The subject of the invention is therefore a method for producing a recombinant peptide in which the structural modifications of the gene lead to a peptide in which at least one hydrophobic amino acid of the sequence of the natural peptide is replaced by a non-hydrophobic amino acid. In another embodiment in the recombinant peptide, at least one hydrophobic amino acid is deleted from the sequence of the natural peptide. Advantageously, the hydrophobic amino acid is chosen from the following group: Tryptophan, Phenylalanine, Proline, Valine, Alanine, Isoleucine, Leucine and Methionine.
Les modifications structurelles du gène peuvent être faites par insertion de nucléotides, ou par délétion de nucléotides. Les constructions dans lesquelles les modifications structurelles du gène sont faites par substitution de nucléotides par mutagénèse dirigée sont également comprises dans l'invention.Structural modifications of the gene can be made by insertion of nucleotides, or by deletion of nucleotides. The constructions in which the structural modifications of the gene are made by substitution of nucleotides by site-directed mutagenesis are also included in the invention.
Les modifications structurelles du gène pourront changer la localisation de telle manière que le produit recombinant soit exposé à la surface membranaire de la cellule par une liaison covalente à la partie d'ancrage membranaire.The structural modifications of the gene could change the localization in such a way that the recombinant product is exposed to the membrane surface of the cell by a covalent bond to the membrane anchoring part.
Dans un autre mode de mise en oeuvre, les modifications structurelles du gène peuvent changer la localisation de telle manière que le produit recombinant soit sécrété dans le milieu de culture. L'invention a donc également pour objet la construction d'ADN qui comprend une séquence signal de sécrétion liée de manière opérationnelle à la séquence d'ADN codant pour le peptide recombinant, et assurant la translocation dudit peptide et sa sécrétion extracellulaire.In another embodiment, the structural modifications of the gene can change the location so that the recombinant product is secreted into the culture medium. The invention therefore also relates to the construction of DNA which comprises a secretion signal sequence operably linked to the DNA sequence coding for the recombinant peptide, and ensuring the translocation of said peptide and its extracellular secretion.
Selon un autre aspect, l'invention se rapporte à un procédé caractérisé en ce que la construction d'ADN comprend une séquence signal liée de manière opérationnelle à la séquence d'ADN codant pour ledit peptide et permettant la translocation du peptide à travers la membrane de la cellule hôte et son ancrage membranaire. Des peptides recombinants susceptibles d'être obtenus par le procédé caractérisés en ce qu'ils diffèrent du peptide naturel par au moins une modification dans la région hydrophobe du peptide naturel sont un autre des objets de l'invention. Ils peuvent se présenter ancrés à la surface de la cellule hôte. Ces peptides seront notamment choisis parmi les analogues d'une protéine de structure du VRS ou d'un fragment d'une telle protéine ; plus particulièrement, le peptide recombinant comprend une séquence analogue de la protéine G du VRS, sous groupe A ou B, notamment comprise entre les résidus 130 et 230 de la protéine G du VRS en présentant au moins 80 % d'homologie.According to another aspect, the invention relates to a method characterized in that the DNA construction comprises a signal sequence operably linked to the DNA sequence coding for said peptide and allowing the translocation of the peptide across the membrane of the host cell and its membrane anchoring. Another object of the invention is recombinant peptides capable of being obtained by the process, characterized in that they differ from the natural peptide by at least one modification in the hydrophobic region of the natural peptide. They may appear anchored to the surface of the host cell. These peptides will in particular be chosen from analogs of a protein of the RSV structure or of a fragment of such a protein; more particularly, the recombinant peptide comprises a sequence analogous to protein G of RSV, in group A or B, in particular comprised between residues 130 and 230 of protein G of RSV, having at least 80% homology.
La protéine G est une glycoprotéine d'enveloppe du VRS, de poids moléculaire compris entre 84 et 90 Kd, pauvre en méthionine. La séquence de la protéine G diffère pour les sous-groupes A et B du VRS ; les termes "séquence de la protéine G" lorsqu'ils sont employés dans la présente demande, doivent s'entendre comme se référant à la fois à la séquence du sous-groupe A ou du sous-groupe B, lorsque cela n'est pas précisé différemment.Protein G is an RSV envelope glycoprotein, with a molecular weight between 84 and 90 Kd, poor in methionine. The sequence of protein G differs for subgroups A and B of RSV; the terms "protein G sequence" when used in the present application, should be understood as referring to both the sequence of subgroup A or of subgroup B, when this is not specified differently.
La Demanderesse a mis en évidence que la séquence comprise entre les acides aminés 130 et 230 de la protéine G naturelle est particulièrement appropriée pour induire une protection efficace contre l'infection par le VRS, sous-groupes A et B, sans induire les pathologies observées avec les vaccins basés sur le virus entier inactivé par le formol, ou observées avec les protéines F et G entières.The Applicant has demonstrated that the sequence between amino acids 130 and 230 of the natural protein G is particularly suitable for inducing effective protection against infection by RSV, subgroups A and B, without inducing the pathologies observed. with vaccines based on the whole virus inactivated by formalin, or observed with whole F and G proteins.
Les moyens permettant l'expression du polypeptide sont connus de l'homme du métier et sont adaptés en fonction de la bactérie utilisée.The means allowing the expression of the polypeptide are known to those skilled in the art and are adapted according to the bacteria used.
De préférence, la séquence d'ADN est introduite sous forme d'un plasmide, tel qu'un plasmide navette.Preferably, the DNA sequence is introduced in the form of a plasmid, such as a shuttle plasmid.
Les protéines du VRS ont été à ce jour exprimées dans différents systèmes d'expression comme le virus de la vaccine, les baculovirus ou les adénovirus. Cependant, des problèmes potentiels sont associés à la présence de particules virales résiduelles. Dans un de ses modes de mise en oeuvre, le procédé selon la présente invention utilise une bactérie commensale de l'homme, apathogène et comestible. En particulier, la bactérie peut appartenir au genre Staphylococcus. Staphylococcus xylosus qui est une bactérie utilisée dans l'industrie alimentaire depuis de nombreuses années, et peut être administrée, vivante, par voie orale. Des systèmes d'expression d'épitopes hétérologues à la surface de S. xylosus ont été notamment décrits par N'guyen et al dans Gène, 1993, 128 : 89-94.The RSV proteins have to date been expressed in different expression systems such as vaccinia virus, baculoviruses or adenoviruses. However, potential problems are associated with the presence of residual virus particles. In one of its embodiments, the method according to the present invention uses a commensal bacterium from humans, apathogenic and edible. In particular, the bacteria can belong to the genus Staphylococcus. Staphylococcus xylosus which is a bacterium used in the food industry for many years, and can be administered, alive, orally. Expression systems of heterologous epitopes on the surface of S. xylosus have been described in particular by N'guyen et al in Gene, 1993, 128: 89-94.
De préférence, le polypeptide hétérologue est exprimé à la surface de la membrane de la bactérie, sous une conformation essentiellement identique à celle de l'épitope correspondant de la protéine G naturelle.Preferably, the heterologous polypeptide is expressed on the surface of the membrane of the bacterium, in a conformation essentially identical to that of the corresponding epitope of the natural protein G.
La présentation de la protéine recombinante à la surface membranaire de la bactérie dépend de sa nature chimique et de son enchaînement peptidique. On peut utiliser la séquence naturelle de la protéine G du VRS et introduire une séquence d'ADN codant pour un peptide comportant la séquence ID n° 1 ou la séquence ID n° 2.The presentation of the recombinant protein on the membrane surface of the bacteria depends on its chemical nature and its peptide sequence. One can use the natural sequence of the G protein of RSV and introduce a DNA sequence coding for a peptide comprising the sequence ID No. 1 or the sequence ID No. 2.
Selon un aspect de l'invention, dans la séquence correspondant à la séquence comprise entre les acides aminés 130 et 230 de la protéine G, l'acide aminé cystéine en positions 173 et/ou 186 a été remplacé par un acide aminé ne formant pas de pont disulfure, en particulier la serine.According to one aspect of the invention, in the sequence corresponding to the sequence between amino acids 130 and 230 of protein G, the amino acid cysteine at positions 173 and / or 186 has been replaced by an amino acid which does not form of disulfide bridge, in particular serine.
Une telle mutation favorise la formation du pont disulfure entre les résidus cystéine restant en positions 176 et 182, qui est critique pour l'immunogénicité de la séquence ; elle évite la formation de ponts disulfures désordonnés.Such a mutation promotes the formation of the disulfide bridge between the cysteine residues remaining in positions 176 and 182, which is critical for the immunogenicity of the sequence; it prevents the formation of disordered disulfide bridges.
Des peptides utiles pour la mise en oeuvre d'un tel procédé sont ceux comportant notamment l'une des séquences ID n° 3 ou ID n° 4.Peptides useful for the implementation of such a process are those comprising in particular one of the sequences ID No. 3 or ID No. 4.
Selon un autre aspect de l'invention dans la séquence du polypeptide hétérologue correspondant à la protéine G du VRS, les acides aminés phénylalanine correspondant aux positions 163, 165, 168 et/ou 170 de la séquence de la protéine G sont remplacés par un acide aminé polaire, en particulier la serine.According to another aspect of the invention in the sequence of the heterologous polypeptide corresponding to protein G of RSV, the amino acids phenylalanine corresponding to positions 163, 165, 168 and / or 170 of the sequence of protein G are replaced by an acid polar amine, especially serine.
Cette modification peut être associée aux mutations précédemment citées. Un tel polypeptide peut notamment présenter la séquence ID n° 5. Lors de la mise en oeuvre du procédé, la suppression de la région hydrophobique située en amont de la boucle critique formée par le pont disulfure entre les acides aminés cystéine en positions 176 et 182, permet à la protéine recombinante de mieux traverser la membrane bactérienne et d'exposer correctement la partie immunodominante à la surface membranaire.This modification can be associated with the mutations mentioned above. Such a polypeptide can in particular have the sequence ID No. 5. During the implementation of the method, the suppression of the hydrophobic region situated upstream of the critical loop formed by the disulfide bridge between the amino acids cysteine in positions 176 and 182, allows the recombinant protein to better cross the bacterial membrane and to correctly expose the immunodominant part on the membrane surface.
Selon encore un autre aspect de l'invention, dans la séquence peptidique correspondant à la protéine G du VRS, la séquence comprise entre les acides aminés numérotés 162 et 170 est délétée. Plus particulièrement la séquence du peptide hétérologue exprimée dans la bactérie peut comporter la séquence ID n° 6.According to yet another aspect of the invention, in the peptide sequence corresponding to protein G of the RSV, the sequence between the amino acids numbered 162 and 170 is deleted. More particularly, the sequence of the heterologous peptide expressed in the bacterium can include the sequence ID No. 6.
L'invention comprend également une bactérie exprimant un peptide ou une protéine, susceptibles d'être obtenus par le procédé décrit dans la présente demande. Ladite bactérie peut être utilisée vivante ou tuée.The invention also comprises a bacterium expressing a peptide or a protein, capable of being obtained by the method described in the present application. Said bacteria can be used alive or killed.
Les polypeptides ou les bactéries présentant une ou plusieurs des caractéristiques ci-dessus sont utiles à titre de médicament.Polypeptides or bacteria having one or more of the above characteristics are useful as medicaments.
L'invention comprend des compositions pharmaceutiques, caractérisées en ce qu'elles comprennent un polypeptide ou une bactérie selon l'invention en mélange avec des adjuvants pharmaceutiquement acceptables.The invention comprises pharmaceutical compositions, characterized in that they comprise a polypeptide or a bacterium according to the invention in admixture with pharmaceutically acceptable adjuvants.
Le vaccin oral à base de vecteur vivant doit comprendre la protéine modifiée qui présente la conformation optimale pour induire la meilleure protection contre le VRS. C'est pourquoi la présente invention à également pour objet une application d'une telle composition pharmaceutique à la préparation d'un vaccin par voie orale destiné à prévenir les infections provoquées par le virus respiratoire syncytial.The live vector oral vaccine must include the modified protein which has the optimal conformation to induce the best protection against RSV. This is why the present invention also relates to an application of such a pharmaceutical composition to the preparation of an oral vaccine intended to prevent infections caused by the respiratory syncytial virus.
Enfin, l'invention a pour objet les séquences nucléotidiques codant pour un peptide recom binant analogue d'un peptide naturel tel que décrit précédemment ; ces séquences peuvent comporter en outre des éléments assurant l'expression du peptide dans une ou plusieurs cellules hôtes spécifiques. Ces éléments permettent de cibler les cellules dans lesquelles la construction va s'exprimer lors de son administration à un mammifère, humain ou animal. Il peut s'agir de constructions d'ADN ou d'ARN, qui seront de préférence incorporées dans un vecteur. Des vecteurs appropriés sont notamment des plasmides ou des virus du type Adénovirus, qui pourront être formulés dans des compositions pharmaceutiques avec des excipients acceptables.Finally, the subject of the invention is the nucleotide sequences coding for a recombinant peptide analogous to a natural peptide as described above; these sequences may also contain elements ensuring expression of the peptide in one or more specific host cells. These elements make it possible to target the cells in which the construction will be expressed during its administration to a mammal, human or animal. These can be DNA or RNA constructs, which will preferably be incorporated into a vector. Suitable vectors are in particular plasmids or viruses of the Adenovirus type, which can be formulated in pharmaceutical compositions with acceptable excipients.
Selon l'un de ses aspects, l'invention a pour objet des séquences nucléotidiques codant pour un polypeptide porté par une séquence peptidique comprise entre les résidus aminoacides 130 et 230 de la protéine G du virus respiratoire ou pour un polypeptide présentant au moins 80 % d'homologie avec ladite séquence peptidique, et comportant en outre les moyens permettant l'expression du polypeptide à la surface de la membrane d'une bactérie non pathogène du genre Staphylococcus. Une séquence d'ADN qui comprend - une séquence signal de sécrétion fonctionnelle,According to one of its aspects, the subject of the invention is nucleotide sequences coding for a polypeptide carried by a peptide sequence comprised between the amino acid residues 130 and 230 of the protein G of the respiratory virus or for a polypeptide having at least 80% of homology with said peptide sequence, and further comprising the means allowing the expression of the polypeptide on the surface of the membrane of a non-pathogenic bacterium of the genus Staphylococcus. A DNA sequence that includes - a functional secretory signal sequence,
- une séquence d'ADN codant pour un peptide recombinant analogue d'un peptide naturel, la séquence de peptide recombinant présentant au moins une modification dans la région hydrophobe non transmembranaire du peptide naturel, fait partie de l'invention.a DNA sequence coding for a recombinant peptide analogous to a natural peptide, the recombinant peptide sequence exhibiting at least one modification in the non-transmembrane hydrophobic region of the natural peptide, forms part of the invention.
Le procédé selon l'invention comprend les étapes suivantes : a. transformer les cellules hôtes avec une construction d'ADN recombinant renfermant une séquence signal qui est liée de manière opérationnelle à un gène structurel, et que ce dernier soit modifié de telle sorte que le produit recombinant peut être translocaté à travers la membrane de la cellule hôte ; b. fermenter ladite cellule hôte pour exprimer le produit recombinant ; c. récupérer les protéines extracellulaires, sécrétées par les cellules transformées par les constructions. L'invention comprend également une cellule recombinante qui contient une séquence d'ADN ou une construction telle que définie précédemment.The method according to the invention comprises the following steps: a. transforming the host cells with a recombinant DNA construct containing a signal sequence which is operably linked to a structural gene, and the latter is modified so that the recombinant product can be translocated across the membrane of the host cell ; b. fermenting said host cell to express the recombinant product; vs. recover the extracellular proteins secreted by the cells transformed by the constructs. The invention also comprises a recombinant cell which contains a DNA sequence or a construct as defined above.
Cette cellule hôte peut être une bactérie, Gram + ou Gram-, une cellule de levure ou une cellule de mammifère.This host cell can be a bacterium, Gram + or Gram-, a yeast cell or a mammalian cell.
Des bactéries particulièrement adaptées sont choisies dans le groupe comprenant Escherichia coli, Staphylococcus xylosus, Staphylococcus carnosus.Particularly suitable bacteria are chosen from the group comprising Escherichia coli, Staphylococcus xylosus, Staphylococcus carnosus.
La séquence d'ADN peut être intégrée dans le chromosome de la bactérie, Gram positif ou Gram négatif.The DNA sequence can be integrated into the chromosome of the bacteria, Gram positive or Gram negative.
Cette construction d'ADN recombinant peut renfermer le gène codant pour la protéine G de l'amino-acide 130 à 230 du VRS humain sous- groupe A fusionnée en amont et/ou en aval de celle du sous-groupe B.This recombinant DNA construct may contain the gene coding for protein G of amino acid 130 to 230 of human RSV subgroup A fused upstream and / or downstream of that of subgroup B.
Un type de construction peut être réalisé avec le gène codant pour la protéine de l'amino-acide 130 à 230 du VRS bovin appartenant au sous- groupe A, ou sous sous-groupe B.A type of construction can be carried out with the gene coding for the amino acid protein 130 to 230 of bovine RSV belonging to subgroup A, or subgroup B.
Les exemples qui suivent sont destinés à illustrer l'invention sans aucunement en limiter la portée.The examples which follow are intended to illustrate the invention without in any way limiting its scope.
Dans ces exemples, on se référera aux figures suivantes :In these examples, reference is made to the following figures:
- Figure 1 : Construction par assemblage de gènes du plasmide pRIT28G2av ;- Figure 1: Construction by assembly of genes from the plasmid pRIT28G2av;
- Figure 2 : 1 ) Construction du gène G2 substitué par des résidus serine ;- Figure 2: 1) Construction of the G2 gene substituted by serine residues;
2) Construction du gène G2 délété des résidus 162 à 170 ;2) Construction of the G2 gene deleted from residues 162 to 170;
- Figure 3 : Schéma des constructions de vecteurs navettes, en haut pSE'mlplδBBXM, en (A) vecteur pSE'G2BBXM ; (B) vecteur pSE'G2subBBXM ; et (C) vecteur pSE*G2delBBXM ;- Figure 3: Schematic of shuttle vector constructions, above pSE'mlplδBBXM, in (A) vector pSE'G2BBXM; (B) vector pSE'G2subBBXM; and (C) vector pSE * G2delBBXM;
- Figure 4 .Analyse par cytométrie de flux des protéines recombinantes de surface ;- Figure 4. Analysis by flow cytometry of recombinant surface proteins;
- Figure 5 : Analyse par SDS-Page des protéines extraites de la membrane de S. Xylosus recombinant ;- Figure 5: Analysis by SDS-Page of proteins extracted from the membrane of recombinant S. Xylosus;
- Figure 6 : Schéma du principe de construction des vecteurs de sécrétion à partir des vecteurs navettes respectifs pSE'G2subBBXM et pSE'G2delBBXM dont les descriptions sont détaillées dans l'exemple 4. Sont illustrés les produits sécrétés à partir du milieu de culture de S. xylosus portant des vecteurs dont le codon Stop (T) a été inséré en amont de la région d'ancrage membranaire XM ; - Figure 7 : Analyse par SDS-Page et immunoblot des protéines de fusion sécrétées par S. xylosus ;- Figure 6: Diagram of the principle of construction of the secretion vectors from the respective shuttle vectors pSE'G2subBBXM and pSE'G2delBBXM whose descriptions are detailed in Example 4. The products secreted from the culture medium of S are illustrated xylosus carrying vectors whose stop codon (T) has been inserted upstream of the XM membrane anchoring region; - Figure 7: Analysis by SDS-Page and immunoblot of the fusion proteins secreted by S. xylosus;
- Figure 8 : Analyse par cytométrie de flux des protéines sécrétées par S. xylosus portant différents vecteurs navettes.- Figure 8: Analysis by flow cytometry of proteins secreted by S. xylosus carrying different shuttle vectors.
EXEMPLESEXAMPLES
EXEMPLE 1 :EXAMPLE 1:
I) Construction de G2 par assemblage de gènes synthétiques : Le gène codant pour la région en amino-acides 130-230 de la glycoprotéine G du virus RS, nommé G2, où nous avons en plus changé deux résidus Cys en position 173 et 186 en Ser par rapport à la séquence d'origine, est obtenu par les techniques d'assemblage de gènes en phase solide (Stahl S. et col., 1993. BioTechniques, .L4.424-434). La séquence des oligonucléotides a été optimisée en combinant les codons usuels de bactéries telles que Ecoli et Staphylococcus.I) Construction of G2 by assembly of synthetic genes: The gene coding for the amino acid region 130-230 of the glycoprotein G of the RS virus, named G2, where we have in addition changed two Cys residues at position 173 and 186 into Ser with respect to the original sequence, is obtained by techniques of assembly of genes in solid phase (Stahl S. et al., 1993. BioTechniques, .L4.424-434). The oligonucleotide sequence has been optimized by combining the usual codons of bacteria such as Ecoli and Staphylococcus.
Les oligonucléotides ont été synthétisés par la chimie des phosphoramidites sur le synthétiseur d'ADN automatique (Gène Assembler Plus, Pharmacia Biotech) selon les recommandations du constructeur. Les oligonucléotides à fixer en phase solide sont biotinylés en extrémité 5' par le réactif Biotin-on phosphoramidite (Clontech). Les autres oligonucléotides sont phosphorylés en 5' par le réactif Phosphate-on amidite (Clontech) selon le protocole de Clontech. Les oligonucléotides sont déprotégés et sont purifiés selon les recommandations de Pharmacia. Les oligonucléotides biotinylés sont purifiés par chromatographie liquide sur phase réverse (colonne PEP RPC, Pharmacia).The oligonucleotides were synthesized by phosphoramidite chemistry on the automatic DNA synthesizer (Gene Assembler Plus, Pharmacia Biotech) according to the manufacturer's recommendations. The oligonucleotides to be fixed in the solid phase are biotinylated at the 5 ′ end by the reagent Biotin-on phosphoramidite (Clontech). The other oligonucleotides are phosphorylated in 5 ′ by the phosphate-on amidite reagent (Clontech) according to the Clontech protocol. The oligonucleotides are deprotected and are purified according to the recommendations of Pharmacia. The biotinylated oligonucleotides are purified by reverse phase liquid chromatography (PEP RPC column, Pharmacia).
Le gène est assemblé en deux parties : G2am (amont) de l'aa 130 à 177 avec deux sites de restriction BamHI et PstI en 5' et 3' du gène, G2av (aval) de l'aa 177 à 230 avec deux sites de restriction PstI et Ba HI en 5' et en 3' du gène. Le gène G2 est reconstitué en ligaturant les deux fragments G2am et G2av par le site PstI.The gene is assembled in two parts: G2am (upstream) from AA 130 to 177 with two restriction sites BamHI and PstI in 5 'and 3' of the gene, G2av (downstream) from AA 177 to 230 with two sites of restriction PstI and Ba HI in 5 'and 3' of the gene. The G2 gene is reconstituted by ligating the two fragments G2am and G2av via the PstI site.
a) Assemblage de gène de G2am ( FIGURE 1 ) :a) G2am gene assembly (FIGURE 1):
Dans un micro tube, deux oligonucléotides complémentaires dont l'un est biotinylé en 5' : TH1 B = 5'-Biotin-CCGGATCCT ATGACCGTGA A-3' et TH2 = 5 '-GTΠTΓGGTT TTCACGGTCA TAGGATCCGG-3' sont hybrides et immobilisés sur des billes magnétiques couplées à la streptavidine (Dynal, Oslo, Norway). Le double brin immobilisé comprend un site de restriction BamHI et le brin complémentaire à celui qui est biotinylé possède 6 à 15 nucléotides de dépassement de son côté 5' (phosphorylé) permettant à l'oligonucléotide suivant de venir s'hybrider. L'hybridation de ce dernier oligonucléotide est effectuée en élevant la température du milieu à 70 'C pour éviter la formation de structures secondaires. La ligature est faite en ajoutant de la T4 DNA ligase (Gibco BRL). Ainsi le gène est construit successivement en prenant la précaution à chaque cycle de rincer le support solide afin d'éliminer l'excès d'oligonucléotides non liés avant d'ajouter l'oligonucléotide suivant. Le dernier double brin ligaturé contient un site de restriction PstI.In a micro tube, two complementary oligonucleotides, one of which is biotinylated in 5 ': TH1 B = 5'-Biotin-CCGGATCCT ATGACCGTGA A-3' and TH2 = 5 '-GTΠTΓGGTT TTCACGGTCA TAGGATCCGG-3' are hybridized and immobilized on magnetic beads coupled to streptavidin (Dynal, Oslo, Norway). The immobilized double strand comprises a BamHI restriction site and the strand complementary to that which is biotinylated has 6 to 15 nucleotides protruding from its 5 ′ side (phosphorylated) allowing the next oligonucleotide to come to hybridize. Hybridization of the latter oligonucleotide is carried out by raising the temperature of the medium to 70 ° C. to avoid the formation of secondary structures. Ligation is done by adding T4 DNA ligase (Gibco BRL). Thus, the gene is constructed successively, taking the precaution at each cycle of rinsing the solid support in order to eliminate the excess of unbound oligonucleotides before adding the following oligonucleotide. The last ligated double strand contains a PstI restriction site.
Le double brin est ensuite libéré de son support solide en le digérant avec les enzymes de restrictions BamHI et PstI puis ligaturé dans le vecteur de clonage et de séquençage prit28 (Hultman et col., 1988, Nucleosides Nucléotides 7 629-638) digéré par les mêmes enzymes : le vecteur résultant est pR!T28G2am de taille 3067pb. La séquence nucléotique de G2am est déterminée par séquençage d'ADN sur le séquenceur automatique ABI, selon les recomandations du constructeur (Applied Biosystem).The double strand is then released from its solid support by digesting it with the restriction enzymes BamHI and PstI and then ligated into the cloning and sequencing vector prit28 (Hultman et al., 1988, Nucleosides Nucleotides 7 629-638) digested by same enzymes: the resulting vector is pR! T28G2am of size 3067bp. The nucleic acid sequence of G2am is determined by DNA sequencing on the ABI automatic sequencer, according to the manufacturer's recommendations (Applied Biosystem).
b) Assemblage de gène de G2av (FIGURE 1 ) :b) G2av gene assembly (FIGURE 1):
De la même manière le gène G2av est assemblé en phase solide sur billes magnétiques par les deux premiers oligonucléotides complémentaires don t l'un est biotinylé en 5 ': TH 13 B (5'-Biotin-TGTGAAGCTT AGTCGACCGG TTTG-3') et TH12 = (5'-GCCGACCACC AAACCGGTCG ACTAAGCTTC ACA-3'), ainsi de suite. Le double brin est libéré du support solide par digestion enzymatique successivement par PstI et HindlII, cloné dans pRIT28 : le vecteur résultant est nommé pRIT28G2av de taille 3091 pb. La séquence nucléotique de G2av est déterminée par séquençage d'ADN sur le séquenceur automatique ABI, selon les recomandations du constructeur (Applied Biosystem). c) Construction du gène G2 ≈ G2am + G2av :In the same way the G2av gene is assembled in solid phase on magnetic beads by the first two complementary oligonucleotides don t one is biotinylated in 5 ': TH 13 B (5'-Biotin-TGTGAAGCTT AGTCGACCGG TTTG-3') and TH12 = (5'-GCCGACCACC AAACCGGTCG ACTAAGCTTC ACA-3 '), and so on. The double strand is released from the solid support by enzymatic digestion successively with PstI and HindIII, cloned in pRIT28: the resulting vector is named pRIT28G2av of size 3091 bp. The nucleic acid sequence of G2av is determined by DNA sequencing on the ABI automatic sequencer, according to the manufacturer's recommendations (Applied Biosystem). c) Construction of the G2 ≈ G2am + G2av gene:
Les deux fragments G2am et G2av sont ligaturés par le site de restriction PstI et le gène constitué est cloné dans pRIT28 par les sites BamHI en 5' et HindlII en 3': le vecteur résultant est nommé pRIT28G2 de taille 3220pb. La séquence nucléotique de G2 est déterminée par séquençage d'ADN sur le séquenceur automatique ABI, selon les recomandations du constructeur (Applied Biosystem). Le fragment G2 est digéré par BamHI et HindlII et cloné dans le vecteur navette : pSE'G2BBXM (7666 pb) (FIGURE 3). Liste des oligonucléotides nécessaires pour l'assemblage de gènesThe two fragments G2am and G2av are ligated by the restriction site PstI and the gene formed is cloned into pRIT28 by the BamHI sites in 5 ′ and HindIII in 3 ′: the resulting vector is named pRIT28G2 of size 3220 bp. The nucleic sequence of G2 is determined by DNA sequencing on the ABI automatic sequencer, according to the manufacturer's recommendations (Applied Biosystem). The G2 fragment is digested with BamHI and HindIII and cloned into the shuttle vector: pSE'G2BBXM (7666 bp) (FIGURE 3). List of oligonucleotides required for gene assembly
G2:G2:
BamHI TH1B (20mer) : 5'-Biotin-CCGGATCCT ATGACCGTGA A-3* BamHI TH1B (20mer): 5'-Biotin-CCGGATCCT ATGACCGTGA A-3 *
BamHI TH2 (30mer) : 5 '-Gl'l l T1GGTT TTCACGGTCA TAGGATÇCGG-3 ' TH3 (28mer) : 5'-AACCAAAAAC ACCACGACCA CCCAGACC-3' TH4 (3 lmer) : 5'-GTTTGCTCGG CIT GTCTGG GTGGTCGTGG T-3' TΗ5 (3 lmer) : 5'-CAGCCGAGCA AACCGACCAC CAAACAGCGTC-3* TH6 (29mer) : 5'-CC -TTTGTTC TGACGCTGTTTC-GTGGTCG-3, TH7 (29mer) : S'-AGAACAAACC GCCGAACAAΛ CCGAACAAC-3'BamHI TH2 (30mer): 5 '-Gl ' ll T1GGTT TTCACGGTCA TAGGATÇCGG-3 'TH3 (28mer): 5'-AACCAAAAAC ACCACGACCA CCCAGACC-3' TH4 (3 lmer): 5'-GTTTGCTCGG CIT GTCTGG GTGGTCGTG5 T-3 (3 lmer): 5'-CAGCCGAGCA AACCGACCAC CAAACAGCGTC-3 * TH6 (29mer): 5'-CC -TTTGTTC TGACGCTGTTTC-GTGGTCG-3 , TH7 (29mer): S'-AGAACAAACC GCCGAACAAΛ CCGAACAAC-3 '
TH8 (34mer) : 5'-CTTCGAAATG GΛAATCGTTG TTCGGTTTGTTCGG-3' TH9 (27mer) : 5'-GAτiTCCATTTCGAAGTGTTCAACrrC-3'TH8 (34mer): 5'-CTTCGAAATG GΛAATCGTTG TTCGGTTTGTTCGG-3 'TH9 (27mer): 5'-GAτiTCCATTTCGAAGTGTTCAACrrC-3'
Pst I TH10 (33mer) : 5'-TGCTGÇAGAT GCTGCTCGGC ACGAΛGTTGA ACΛ-3' Pst IPst I TH10 (33mer): 5'-TGCTGÇAGAT GCTGCTCGGC ACGAΛGTTGA ACΛ-3 'Pst I
TH11 (22mer) : 5'-GTGCCGAGCA GCATCTGCAG CA-3'TH11 (22mer): 5'-GTGCCGAGCA GCATCTGCAG CA-3 '
HindlII TH12 (33mer) : 5'-GCCGACCACC AAACCGGTCG ACTAAGCrTC ACA-3' HindlII TH13B ( 19mer) : 5'-Biotin-CCCTGTGAAG CTTGGTTTG-3' TH14 (32mer) : 5'-CATAAACCGC AGACCACCAA ACCGAAAGAA GT-3' TH15 (32mer) : 5'-GTGGTCGGCA CTTCTTTCGG TTTGGTGGTC TG-3' TH16 (31mer) : 5'-AAAACCGACC TTCAAAACCA CCAAAAAAGA T-3" TH17 (3 lmer) : 5'-CGGTTTATGA TCTTTTTTGG TGGTTTTGAA C-3' TH18(30mer) : 5'-GGGCAAAAAΛ ACCACGACCA AACCGACCAA-3' TH19 (3 lmer) : 5'-GTCCrGTITrrTGGTCGGTTTGGTCGTGGTTT-3' TH20 (34mer) : 5'-GGGCGATCAG CAAACGTATC CCGAACAAAA AACC-3' TH21 (33mer) : 5'-TTTTGCCCGG TT1TTTGTTC GGGATACGTT TGC-3'HindlII TH12 (33mer): 5'-GCCGACCACC AAACCGGTCG ACTAAGCrTC ACA-3 ' HindlII TH13B (19mer): 5'-Biotin-CCCTGTGAAG CTTGGTTTG-3 'TH14 (32mer): 5'-CATAAACCGC AGACCACCAA ACCGAAAGAA GT-3' TH15 (32mer): 5'-GTGGTCGGCA CTTCTTTCGG TTTGGTGGTC TG 3 ' : 5'-AAAACCGACC TTCAAAACCA CCAAAAAAGA T-3 "TH17 (3 lmer): 5'-CGGTTTATGA TCTTTTTTGG TGGTTTTGAA C-3 'TH18 (30mer): 5'-GGGCAAAAAΛ ACCACGACCA AACCGACCAA-3' TH19 (3 lmer): 5 ' GTCCrGTITrrTGGTCGGTTTGGTCGTGGTTT-3 'TH20 (34mer): 5'-GGGCGATCAG CAAACGTATC CCGAACAAAA AACC-3' TH21 (33mer): 5'-TTTTGCCCGG TT1TTTGTTC GGGATACGTT TGC-3
Pst I TH22(25mer) : 5'-ATC TGCAGCAACA ACCCGACCTG CT-3'Pst I TH22 (25mer): 5'-ATC TGCAGCAACA ACCCGACCTG CT-3 '
Pst I TH23 (34mer) : 5'-TGATCGCCCA GCAGGTCGGG TTGTTGCTGC AGAT-3'Pst I TH23 (34mer): 5'-TGATCGCCCA GCAGGTCGGG TTGTTGCTGC AGAT-3 '
II) Construction de G2sub par mutaeénèse dirigée :II) Construction of G2sub by site-directed mutaeenesis:
Le fragment de gène où les quatre résidus Phénylalanine en position 163, 165, 168 et 170 sont remplacés par des Serines, est généré par amplification génique (PCR), à partir du gène G2 inséré dans le vecteur pRIT28, en utilisant comme couples d'amorces RIT27/TNG73 et RIT28/TNG72 (voir FIGURE 2.( 1 )). Les amorces TNG72 et TNG73 sont complémentaires d'une région de 19 nucléotides renfermant trois des quatres Phénylalanine : Rit27 : 5,-GCnCCGGCr CGTATGlTGTGTG-3' Rit28 : 5'-AAAGGGGGAT GTGCTGCAAG GCC-3'The gene fragment where the four phenylalanine residues at positions 163, 165, 168 and 170 are replaced by Serines, is generated by gene amplification (PCR), from the G2 gene inserted in the vector pRIT28, using as pairs of primers RIT27 / TNG73 and RIT28 / TNG72 (see FIGURE 2. (1)). The primers TNG72 and TNG73 are complementary to a region of 19 nucleotides containing three of the four phenylalanines: Rit27: 5 , -GCnCCGGCr CGTATGlTGTGTG-3 'Rit28: 5'-AAAGGGGGAT GTGCTGCAAG GCC-3'
TNG72 5 '- C CAT TCC GAA GTG TCC AAC 'ICC GTG CCG AGC AC-3 'TNG72 5 '- C CAT TCC GAA GTG TCC AAC' ICC GTG CCG AGC AC-3 '
TNG73 3 '- GC TTC CTA ΛGG GTA AGG CTT CAC ΛGG TTG-5 'TNG73 3 '- GC TTC CTA ΛGG GTA AGG CTT CAC ΛGG TTG-5'
D'une part, l'amorce TNG73 introduit les trois premières mutations (TTC en TCC) sur le fragment en amont amplifié avec RIT27. D'autre part l'amorce TNG72 introduit les trois dernières mutations (TTC en TCC) sur le fragment en aval amplifie avec RIT28. Cinq cycles de températures (9 ' c, 15 sec; 50 ' c, 1 min; 72 " c, 1 min) suivis de vingt cycles (9 " c, 1 5 sec; 60 " c, 15 sec; 72 ' c, 15 sec) Les deux fragments amplifiés sont mélangés dans un seul tube dilué dans du tampon de PCR sans amorces et la réaction d'extension est faite en cinq cycles de températures (96 ' c, 15 sec; 54 * c, 30 sec; 72 * c, 1 min). Le produit d'extension est dilué à 1/100 dans du tampon PCR contenant les amorces RIT27 et RIT28 et l'amplification génique est faite en 30 cycles de températures (96 *c, 15 sec; 54 ' c, 15 sec; 72 ' c, 30 sec). Le fragment est ensuite digéré avec les enzymes de restrictions BamHI/HindIII et cloné dans le vecteur prit28 digéré par les mêmes enzymes: pRIT28G2sub. La séquence nucléotique de G2Sub est déterminée par séquençage d'ADN sur l'appareil de séquenceur automatique ABI, selon les recomandations du constructeur (Applied Biosystem). Le fragment G2sub est digéré par BamHI et HindlII et cloné dans le vecteur navette : pSE'G2subBBXM (7666 pb)(FIGURE 3).On the one hand, the primer TNG73 introduces the first three mutations (TTC in TCC) on the upstream fragment amplified with RIT27. On the other hand, the primer TNG72 introduces the last three mutations (TTC in TCC) on the downstream fragment amplified with RIT28. Five temperature cycles (9 'c, 15 sec; 50 ' c, 1 min; 72 " c, 1 min) followed by twenty cycles (9 " c, 1 5 sec; 60 "c, 15 sec; 72 'c, 15 sec) The two amplified fragments are mixed in a single tube diluted in PCR buffer without primers and the extension reaction is carried out in five temperature cycles (96 'c, 15 sec; 54 * c, 30 sec; 72 * c, 1 min). The extension product is diluted 1/100 in PCR buffer containing the primers RIT27 and RIT28 and the gene amplification is carried out in 30 temperature cycles (96 * c, 15 sec; 54 'c, 15 sec; 72 ' c, 30 sec). The fragment is then digested with the restriction enzymes BamHI / HindIII and cloned into the vector prit28 digested with the same enzymes: pRIT28G2sub. The nucleic sequence of G2Sub is determined by DNA sequencing on the ABI automatic sequencer device, according to the manufacturer's recommendations (Applied Biosystem). The G2sub fragment is digested with BamHI and HindIII and cloned into the shuttle vector: pSE'G2subBBXM (7666 bp) (FIGURE 3).
III) Construction de G2del : (voir FIGURE 2.(2)) De la même manière, le fragment de gène G2 délété de la partie renfermant les 4 résidus Phénylalanine de l'aa 162 à l'aa 170 est généré par PCR en deux fragments : en amont avec les amorces RIT27/TH48 d'une part et en aval avec RIT28/TH 1 1 d'autre part. Les amorces TH1 1 et TH48 sont complémentaires de 13 nucléotides. TH 1 1 5'-GTGCCGAGCA GCATCTGCAG CA-3 'III) Construction of G2del: (see FIGURE 2. (2)) In the same way, the G2 gene fragment deleted from the part containing the 4 phenylalanine residues from aa 162 to aa 170 is generated by PCR in two fragments: upstream with the primers RIT27 / TH48 on the one hand and downstream with RIT28 / TH 1 1 on the other hand. The primers TH1 1 and TH48 are complementary to 13 nucleotides. TH 1 1 5'-GTGCCGAGCA GCATCTGCAG CA-3 '
3'-GGC TGTTT GGCTlC.'lTG
Figure imgf000016_0001
TH483'-GGC TGTTT GGCTlC. ' lTG
Figure imgf000016_0001
TH48
Cinq cycles de températures (96 * c, 15 sec; 52 ' c, 1 min; 72 ' c, 1 min) suivis de vingt cycles (96 ' c, 15 sec; 60' c, 15 sec; 72 ' c, 15 sec) Les deux fragments amplifiés sont mélangés dans un seul tube dilué dans du tampon de PCR sans amorce et la réaction d'extension est faite en cinq cycles de températures (96 ' c, 15 sec; 42 ' c, 30 sec; 72 ' c, 1 min). Le produit d'extension est dilué à 1/100 dans du tampon PCR contenant les amorces RIT27 et RIT28 et l'amplification génique est faite en 30 cycles de températures (96 ' c, 1 5 sec; 60 * c, 15 sec; 72 ' c, 30 sec). Le fragment est ensuite digéré avec les enzymes de restrictions I.amlll/Hindlll cl cloné dans le vecteur prit28 digéré par les mêmes enzymes: pRIT28G2del. La séquence nucléotique de G2del est déterminée par séquençage d'ADN sur l'appareil de séquenceur automatique ABI, selon les recomandations du constructeur (Applied Biosystem). Le fragment G2dcl est digéré par BamHI et HindlII et cloné dans le vecteur navette : pSE'G2delBBXM (7639 pb)(FIGURE 3).Five temperature cycles (96 * c, 15 sec; 52 ' c, 1 min; 72 ' c, 1 min) followed by twenty cycles (96 ' c, 15 sec; 60 ' c, 15 sec; 72 ' c, 15 sec) The two amplified fragments are mixed in a single tube diluted in PCR buffer without primer and the extension reaction is carried out in five temperature cycles (96 'c, 15 sec; 42' c, 30 sec; 72 ' c, 1 min). The extension product is diluted 1/100 in PCR buffer containing the primers RIT27 and RIT28 and the gene amplification is carried out in 30 temperature cycles (96 'c, 15 sec; 60 * c, 15 sec; 72 'c, 30 sec). The fragment is then digested with the restriction enzymes I.amlll / Hindlll cl cloned in the vector prit28 digested with the same enzymes: pRIT28G2del. The nucleic sequence of G2del is determined by DNA sequencing on the ABI automatic sequencer device, according to the manufacturer's recommendations (Applied Biosystem). The G2dcl fragment is digested with BamHI and HindIII and cloned into the shuttle vector: pSE'G2delBBXM (7639 bp) (FIGURE 3).
IV) Construction du vecteur navette :IV) Construction of the shuttle vector:
Un linker d'oligonucléotides (5'-AGCTTGGCTG TTCCGCCATG GCTCGAG-3', avec le brin complémentaire) est inséré dans le site HindlII du plasmide pSZZmpl δXM (Hansson et col, 1992, J.Bacteriol 174 : 4239-4245), créant ainsi deux sites de restriction supplémentaires Ncol et Xhol en aval du site HindlII du vecteur résultant pSZZmpl 8(XhoI)XM. Un fragment de gène codant 198 acides aminés, nommé BB, de la région de fixation du sérum albumine de la protéine G streptococcique ( Nygren et col, 1988. J.Mol.Reconig., 1 :69-74), est généré par PCR avec les amorces ( 1 = S'-CCGAATTCAA GCTTAGATGC TCTAGCAAAA GCCAAG-3" et 2 = 5'-CCCCTGCAGT TAGGATCCCT CGAGAGGTAA TGCAGCTΛAA ATTTCATC-3') sur le template plasmidique pSPG l (Guss et col, 1986.EMBO J., 5 : 1567-1575 ). Le fragment est digéré avec HindlII et Xhol et cloné en aval du site multiple de clonage mp l 8 du vecteur pSZZmp l 8(XhoI)XM; le vecteur résultant pSZZmp l 8(XhoI) BBXM est digéré par Notl et HindlII. Le fragment renfermant ZZ est remplacé par un autre fragment digéré par les mêmes enzymes de restriction du vecteur pE'mpl δ (Sophia 1 lober, non publié). Le vecteur navette résultant est nommé pSE'mpl δBBXM (FIGURE 3).An oligonucleotide linker (5'-AGCTTGGCTG TTCCGCCATG GCTCGAG-3 ', with the complementary strand) is inserted into the HindIII site of the plasmid pSZZmpl δXM (Hansson et al, 1992, J. Bacteriol 174: 4239-4245), thus creating two additional restriction sites Ncol and Xhol downstream of the HindIII site of the resulting vector pSZZmpl 8 (XhoI) XM. A gene fragment coding for 198 amino acids, named BB, from the binding region of the serum albumin of the streptococcal protein G (Nygren et al, 1988. J. Mol. Reconig., 1: 69-74), is generated by PCR with the primers (1 = S'-CCGAATTCAA GCTTAGATGC TCTAGCAAAA GCCAAG-3 "and 2 = 5'-CCCCTGCAGT TAGGATCCCT CGAGAGGTAA TGCAGCTΛAA ATTTCATC-3 ') on the plasmid template pSPG l (Guss et al, 1986.EMBO J., 5: The fragment is digested with HindIII and Xhol and cloned downstream of the multiple cloning site mp l 8 of the vector pSZZmp l 8 (XhoI) XM; the resulting vector pSZZmp l 8 (XhoI) BBXM is digested with Notl and The fragment containing ZZ is replaced by another fragment digested with the same restriction enzymes of the vector pE'mpl δ (Sophia 1 lober, unpublished) The resulting shuttle vector is named pSE'mpl δBBXM (FIGURE 3).
EXEMPLE 2 :EXAMPLE 2:
Extraction et analyse de protéines membranaires des S. xylosus recombinants : Dans un erlenmeycr de 1 litre, on inocule 250 ml de milieu (7,5 g deExtraction and analysis of membrane proteins from recombinant S. xylosus: In a 1 liter Erlenmeyer flask, 250 ml of medium (7.5 g of
TSB, 12,5 g de Yeast extract) contenant du Chloramphénicol (20 μg/ml) avec 5 ml de préeuhure (overnight) de S. xylosus transformé par le vecteur navette pSE'G2BBXM ou pSE'G2subBBXM ou pSE'G2delBBXM selon le protocole de Gôtz et col, 1 81 , J Bacteriol., 145:74-81. Incuber sous agitation à température = 32° C pendant une durée de 6 heures. Centrifuger le milieu à 5000 rpm, 12 min et à température •= 4° C. Le culot bactérien est resuspendu dans 40 ml de TST, on rajoute 200 μl de solution contenant de la lysostaphine ( 1 mg/ml) et 200 μl de lysozyme (50 mg/ml). Incuber pendant une heure à température = 37° C sous agitation modérée. Soniquer la solution pendant 2 min, avec l'appareil Vibra cell équipé d'une sonde dont la puissance est réglée à 7. Centrifuger à 13 500 rpm, pendant 20 min, à température = 4° C. Les protéines sont purifiées par affinité : le surnageant est passé sur colonne d'affinité HSA-Sépharose (Sérum Albumine Humaine). Après avoir rincé la colonne, les protéines sont éluées avec un tampon acide pH 2,7 et lyophilisées.TSB, 12.5 g of Yeast extract) containing Chloramphenicol (20 μg / ml) with 5 ml of preheating (overnight) of S. xylosus transformed by the shuttle vector pSE'G2BBXM or pSE'G2subBBXM or pSE'G2delBBXM Gôtz et al. protocol, 1 81, J Bacteriol., 145: 74-81. Incubate with shaking at temperature = 32 ° C for a period of 6 hours. Centrifuge the medium at 5000 rpm, 12 min and at temperature • = 4 ° C. The bacterial pellet is resuspended in 40 ml of TST, 200 μl of solution containing lysostaphin (1 mg / ml) and 200 μl of lysozyme are added (50 mg / ml). Incubate for one hour at temperature = 37 ° C with moderate shaking. Sonicate the solution for 2 min, with the Vibra cell device equipped with a probe whose power is set to 7. Centrifuge at 13,500 rpm, for 20 min, at temperature = 4 ° C. The proteins are purified by affinity: the supernatant is passed through an HSA-Sepharose affinity column (Human Albumin Serum). After rinsing the column, the proteins are eluted with a pH 2.7 acid buffer and lyophilized.
Les protéines sont séparées sur deux gels SDS-PAGE ( 12%) identiques avec les marqueurs de tailles moléculaires standard (Gibco BRL) précolorés. Un gel est coloré au Bleu de Coomassie. Le deuxième est transféré sur membrane ProblotTM (Applied Biosystem) pour l'immunoblot avec l'anticorps spécifique anti-G l (obtenu à partir du sérum de lapin immunisé avec le peptide G l (aal 74- 1 87) selon les protocoles courants d'immunisation). Voir Figure 5.The proteins are separated on two identical SDS-PAGE gels (12%) with the precolored standard molecular size markers (Gibco BRL). A gel is colored with Coomassie Blue. The second is transferred to the ProblotTM membrane (Applied Biosystem) for the immunoblot with the specific anti-G l antibody (obtained from rabbit serum immunized with the G l peptide (aal 74-1 87) according to current protocols. 'immunization). See Figure 5.
EXEMPLE 3 :EXAMPLE 3:
Analyse par Cytométrie de flux ( FACScanl ) des protéines recombinantes à la surface de S. xylosus :Analysis by flow cytometry (FACScanl) of the recombinant proteins on the surface of S. xylosus:
Les cultures des bactéries recombinantes S. xylosus sont faites comme décrit précédemment. Pour constituer une solution stock, on resuspend la bactérie dans une solution de PBS à 0, 1% de Sodium Azide (p/v) à la concentration finale estimée par densité optique (600nm) égale à l'unité. On aliquote 30 μl de solution stock dans chaque puits conique d'une plaque de microtitres, on centrifuge à 550 g pendant 10 minutes à 4° C. On resuspend le culot bactérien dans un volume de 150 μl de solution de PBS contenant du sérum lapin polyclonal anti-G2 (titre 1/ 1 280 000) dilué au l/200ème, incubation pendant 30 minutes. On rince deux fois les cellules bactériennes avec du PBS et on incube dans 150 μl d'une solution de PBS renfermant de l'anti-lapin FITC (Sigma) dilué au l/100ème pendant une durée de 30 minutes. Après avoir rincé les cellules deux fois avec du tampon PBS, on resuspend dans un tube Falcon contenant 1 ml de tampon PBS-Paraformaldéhyde 1% (p/v). Les échantillons préparés sont analysés sur l'appareil FACScan™ (Becton Dickinson). La distribution de fluorescence de chaque suspension cellulaire est analysée par le logiciel LYSIS II™ et est représentée par des histogrammes de fluorescence. Voir Figure 4.The cultures of the recombinant S. xylosus bacteria are made as described above. To constitute a stock solution, the bacteria are resuspended in a PBS solution at 0.1% Sodium Azide (w / v) at the final concentration estimated by optical density (600 nm) equal to the unit. 30 μl of stock solution are aliquoted in each conical well of a microtiter plate, centrifuged at 550 g for 10 minutes at 4 ° C. The bacterial pellet is resuspended in a volume of 150 μl of PBS solution containing rabbit serum polyclonal anti-G2 (titre 1/1 280 000) diluted at l / 200th, incubation for 30 minutes. The bacterial cells are rinsed twice with PBS and incubated in 150 μl of a PBS solution containing anti-rabbit FITC (Sigma) diluted to 1/100 for a period of 30 minutes. After rinsing the cells twice with PBS buffer, it is resuspended in a Falcon tube containing 1 ml of PBS-Paraformaldehyde buffer 1% (w / v). The prepared samples are analyzed on the FACScan ™ device (Becton Dickinson). The fluorescence distribution of each cell suspension is analyzed by LYSIS II ™ software and is represented by fluorescence histograms. See Figure 4.
EXEMPLE 4 :EXAMPLE 4:
Modulation sécrétion-insertion en sécrétion :Secretion-insertion modulation in secretion:
A partir des différents vecteurs navettes, il est possible d'insérer des codons de terminaison en amont de la région codant pour l'ancrage membranaire XM, comme le montre la figure 6. Un site unique de restriction Xhol entre BB et XM a été utilisé pour insérer un double brin d'oligonucléotides codant pour trois codons terminaison (Ter) dans les deux sens d'orientation, avec introduction d'un site de restriction Aat II :From the various shuttle vectors, it is possible to insert termination codons upstream of the region coding for the XM membrane anchoring, as shown in FIG. 6. A unique Xhol restriction site between BB and XM was used to insert a double strand of oligonucleotides coding for three termination codons (Ter) in both directions of orientation, with the introduction of an Aat II restriction site:
Aat II Ter Ter Ter --> 5'-TC GAC GTC TAΛ TGA TAΛ TTA TCA TTA C-3' 3'-G CAG ATT ACT ATT AΛT AGT AAT CΛG CT-5' <— Ter Ter Ter Les vecteurs pSE'G2subBBXM et pSE'G2delBBXM sont ainsi digérés par Xho I et sont ligaturés avec le double brin d'oligonucléotides préalablement phosphorylés en 5 '. Les vecteurs résultants sont respectivement pSE'G2subBB[Ter]XM (7693 pb) et pSE'G2delBB [Tcr]XM (7666 pb). La culture des E.coli ou des S. ylosus transformées respectivement avec ces deux vecteurs suivie d'une purification sur colonne d'affinité ( USA Sépharose) permet d'obtenir des protéines G2subBB et G2delBB. I.a figure 7 montre: en A) le gel SDS-PAGE, la séparation des protéines sécrétées à partir des S. xylosus, dans des conditions réduites, en 1 et en 2 représentent respectivement les protéines G2subBB et G2delBB à la taille attendue: 35,23 Kda et 34,28 Kda; en B) l'immunoblot des protéines montre que l'anticorps spécifique à la région G(aal 74-187) ou G l du VRS reconnaît bien les deux protéines sécrétées. Très peu de dégradations protéolytiques ont été observées. De plus, l'analyse FACSCAN avec l'anticorps polyclonal anti¬ lapin, anti-BB a été réalisée sur les différents S.xylosus. La figure 8 montre que les spectres de S.xylosus portant des vecteurs navettes pSE'mplδBBXM, pSE'G2subBBXM et pSE'G2delBBXM sont déplacés dans l'axe de l'intensité de fluorescence vers la droite, c'est-à-dire vers la présence des antigènes hétérologues à la surface de la bactérie. Alors que les spectres de S.xylosus portant des vecteurs navettes pSE'G2subBBTerXM et pSE'G2delBBTerXM n'y sont pas déplacés, ceci indique que les antigènes hétérologues sont absents à la surface de la bactérie et que l'on les a retrouvés et purifiés à partir du milieu de culture. Aat II Ter Ter Ter ->5'-TC GAC GTC TAΛ TGA TAΛ TTA TCA TTA C-3 '3'-G CAG ATT ACT ATT AΛT AGT AAT CΛG CT-5'<- Ter Ter Ter Vectors pSE'G2subBBXM and pSE'G2delBBXM are thus digested with Xho I and are ligated with the double strand of oligonucleotides previously phosphorylated at 5 '. The resulting vectors are pSE'G2subBB [Ter] XM (7693 bp) and pSE'G2delBB [Tcr] XM (7666 bp) respectively. The culture of E.coli or S. ylosus transformed respectively with these two vectors followed by purification on an affinity column (USA Sepharose) makes it possible to obtain proteins G2subBB and G2delBB. Figure 7 shows: A) the SDS-PAGE gel, the separation of the secreted proteins from S. xylosus, under reduced conditions, in 1 and in 2 respectively represent the proteins G2subBB and G2delBB at the expected size: 35.23 Kda and 34.28 Kda; in B) the immunoblot of the proteins shows that the antibody specific to the G region (aal 74-187) or G l of the RSV clearly recognizes the two secreted proteins. Very few proteolytic degradations have been observed. In addition, the FACSCAN analysis with the anti-rabbit, anti-BB polyclonal antibody was carried out on the various S. xylosus. FIG. 8 shows that the spectra of S. xylosus carrying shuttle vectors pSE'mplδBBXM, pSE'G2subBBXM and pSE'G2delBBXM are displaced in the axis of the fluorescence intensity towards the right, that is to say towards the presence of heterologous antigens on the surface of the bacteria. While the spectra of S. xylosus carrying shuttle vectors pSE'G2subBBTerXM and pSE'G2delBBTerXM are not displaced there, this indicates that the heterologous antigens are absent on the surface of the bacteria and that they have been found and purified from the culture medium.
LISTE DE SEQUENCESLIST OF SEQUENCES
(1) INFORMATIONS GENERALES :(1) GENERAL INFORMATION:
(i) DEPOSANT:(i) DEPOSITOR:
(A) NOM: PIERRE FABRE MEDICAMENT(A) NAME: PIERRE FABRE MEDICAMENT
(B) RUE: 45 PLACE ABEL GANCE(B) STREET: 45 PLACE ABEL GANCE
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(ii) TITRE DE L' INVENTION: PRODUCTION DE PEPTIDES ANALOGUES DE PEPTIDES HYDROPHOBES, PEPTIDE RECOMBINANT, SEQUENCE D'ADN CORRESPONDANTE(ii) TITLE OF THE INVENTION: PRODUCTION OF HYDROPHOBIC PEPTIDE-LIKE PEPTIDES, RECOMBINANT PEPTIDE, CORRESPONDING DNA SEQUENCE
(iii) NOMBRE DE SEQUENCES: 6(iii) NUMBER OF SEQUENCES: 6
(iv) FORME DECHIFFRABLE PAR ORDINATEUR:(iv) COMPUTER-DETACHABLE FORM:
(A) TYPE DE SUPPORT: Floppy disk(A) TYPE OF SUPPORT: Floppy disk
(B) ORDINATEUR: IBM PC compatible(B) COMPUTER: IBM PC compatible
(C) SYSTEME D' EXPLOITATION: PC-DOS/MS-DOS(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) LOGICIEL: Patentln Release #1.0, Version #1.30 (OEB)(D) SOFTWARE: Patentln Release # 1.0, Version # 1.30 (EPO)
(vi) DONNEES DE LA DEMANDE ANTERIEURE:(vi) DATA FROM THE PREVIOUS APPLICATION:
(A) NUMERO DE LA DEMANDE: FR 9413307(A) REQUEST NUMBER: FR 9413307
(B) DATE DE DEPOT: 07-NOV-1994(B) DEPOSIT DATE: 07-NOV-1994
(2) INFORMATIONS POUR LA SEQ ID NO: 1:(2) INFORMATION FOR SEQ ID NO: 1:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 303 paires de bases(A) LENGTH: 303 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADN(ii) TYPE OF MOLECULE: DNA
(ix) CARACTERISTIQUE:(ix) CHARACTERISTIC:
(A) NOM/CLE: CDS(A) NAME / KEY: CDS
(B) EMPLACEMENT:!..303 (xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 1:(B) LOCATION:! .. 303 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:
ACC GTG AAA ACC AAA AAC ACC ACG ACC ACC CAG ACC CAG CCG AGC AAA 48 Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gin Thr Gin Pro Ser Lys 1 5 10 15ACC GTG AAA ACC AAA AAC ACC ACG ACC ACC CAG ACC CAG CCG AGC AAA 48 Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gin Thr Gin Pro Ser Lys 1 5 10 15
CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG CCG AAC AAA CCG AAC AAC 96 Pro Thr Thr Lys Gin Arg Gin Asn Lys Pro Pro Asn Lys Pro Asn Asn 20 25 30CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG CCG AAC AAA CCG AAC AAC 96 Pro Thr Thr Lys Gin Arg Gin Asn Lys Pro Pro Asn Lys Pro Asn Asn 20 25 30
GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG CCG TGC AGC ATC TGC AGC 144 Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Cys Ser Ile Cys Ser 35 40 45GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG CCG TGC AGC ATC TGC AGC 144 Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Cys Ser Ile Cys Ser 35 40 45
AAC AAC CCG ACC TGC TGG GCG ATC TGC AAA CGT ATC CCG AAC AAA AAA 192 Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro Asn Lys Lys 50 55 60AAC AAC CCG ACC TGC TGG GCG ATC TGC AAA CGT ATC CCG AAC AAA AAA 192 Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro Asn Lys Lys 50 55 60
CCG GGC AAA AAA ACC ACG ACC AAA CCG ACC AAA AAA CCG ACC TTC AAA 240 Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys 65 70 75 80CCG GGC AAA AAA ACC ACG ACC AAA CCG ACC AAA AAA CCG ACC TTC AAA 240 Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys 65 70 75 80
ACC ACC AAA AAA GAT CAT AAA CCG CAG ACC ACC AAA CCG AAA GAA GTG 288 Thr Thr Lys Lys Asp His Lys Pro Gin Thr Thr Lys Pro Lys Glu Val 85 90 95ACC ACC AAA AAA GAT CAT AAA CCG CAG ACC ACC AAA CCG AAA GAA GTG 288 Thr Thr Lys Lys Asp His Lys Pro Gin Thr Thr Lys Pro Lys Glu Val 85 90 95
CCG ACC ACC AAA CCA 303CCG ACC ACC AAA CCA 303
Pro Thr Thr Lys Pro 100Pro Thr Thr Lys Pro 100
(2) INFORMATIONS POUR LA SEQ ID NO: 2:(2) INFORMATION FOR SEQ ID NO: 2:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 303 paires de bases(A) LENGTH: 303 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADN(ii) TYPE OF MOLECULE: DNA
(ix) CARACTERISTIQUE:(ix) CHARACTERISTIC:
(A) NOM/CLE: CDS(A) NAME / KEY: CDS
(B) EMPLACEMENT:!..303 (xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 2:(B) LOCATION:! .. 303 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
ACC GCG CAG ACC AAA GGC CGT ATC ACC ACC AGC ACC CAG ACC AAC AAA 48 Thr Alα Gin Thr Lys Gly Arg Ile Thr Thr Ser Thr Gin Thr Asn Lys 1 5 10 15ACC GCG CAG ACC AAA GGC CGT ATC ACC ACC AGC ACC CAG ACC AAC AAA 48 Thr Alα Gin Thr Lys Gly Arg Ile Thr Thr Ser Thr Gin Thr Asn Lys 1 5 10 15
CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG CCG AAA AAA CCG AAA GAT 96 Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro Pro Lys Lys Pro Lys Asp 20 25 30CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG CCG AAA AAA CCG AAA GAT 96 Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro Pro Lys Lys Pro Lys Asp 20 25 30
GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG CCC TGC AGC ATC TGC GGC 144 Asp Tyr His Phe Glu Val Phe Asn Phe Val Pro Cys Ser Ile Cys Gly 35 40 45GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG CCC TGC AGC ATC TGC GGC 144 Asp Tyr His Phe Glu Val Phe Asn Phe Val Pro Cys Ser Ile Cys Gly 35 40 45
AAC AAC CAG CTG TGC AAA AGC ATC TGC AAA ACC ATC CCG AGC AAC AAA 192 Asn Asn Gin Leu Cys Lys Ser Ile Cys Lys Thr Ile Pro Ser Asn Lys 50 55 60AAC AAC CAG CTG TGC AAA AGC ATC TGC AAA ACC ATC CCG AGC AAC AAA 192 Asn Asn Gin Leu Cys Lys Ser Ile Cys Lys Thr Ile Pro Ser Asn Lys 50 55 60
CCG AAA AAG AAA CCG ACC ATC AAA CCG ACC AAC AAA CCG ACC ACC AAA 240 Pro Lys Lys Lys Pro Thr Ile Lys Pro Thr Asn Lys Pro Thr Thr Lys 65 70 75 80CCG AAA AAG AAA CCG ACC ATC AAA CCG ACC AAC AAA CCG ACC ACC AAA 240 Pro Lys Lys Lys Pro Thr Ile Lys Pro Thr Asn Lys Pro Thr Thr Lys 65 70 75 80
ACC ACC AAC AAA CGT GAT CCG AAA ACC CCG GCG AAA ATG CCG AAG AAG 288 Thr Thr Asn Lys Arg Asp Pro Lys Thr Pro Ala Lys Met Pro Lys Lys 85 90 95ACC ACC AAC AAA CGT GAT CCG AAA ACC CCG GCG AAA ATG CCG AAG AAG 288 Thr Thr Asn Lys Arg Asp Pro Lys Thr Pro Ala Lys Met Pro Lys Lys 85 90 95
GAA ATC ATC ACC AAC 303GAA ATC ATC ACC AAC 303
Glu Ile Ile Thr Asn 100Glu Ile Ile Thr Asn 100
(2) INFORMATIONS POUR LA SEQ ID NO: 3:(2) INFORMATION FOR SEQ ID NO: 3:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 303 paires de bases(A) LENGTH: 303 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADN(ii) TYPE OF MOLECULE: DNA
(ix) CARACTERISTIQUE:(ix) CHARACTERISTIC:
(A) NOM/CLE: CDS(A) NAME / KEY: CDS
(B) EMPLACEMENT:!..303 (xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 3:(B) LOCATION:! .. 303 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:
ACC GTG AAA ACC AAA AAC ACC ACG ACC ACC CAG ACC CAG CCG AGC AAA 48ACC GTG AAA ACC AAA AAC ACC ACG ACC ACC CAG ACC CAG CCG AGC AAA 48
Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gin Thr Gin Pro Ser Lys 1 5 10 15Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gin Thr Gin Pro Ser Lys 1 5 10 15
CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG CCG AAC AAA CCG AAC AAC 96CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG CCG AAC AAA CCG AAC AAC 96
Pro Thr Thr Lys Gin Arg Gin Asn Lys Pro Pro Asn Lys Pro Asn Asn 20 25 30Pro Thr Thr Lys Gin Arg Gin Asn Lys Pro Pro Asn Lys Pro Asn Asn 20 25 30
GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG CCG AGC AGC ATC TGC AGC 144GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG CCG AGC AGC ATC TGC AGC 144
Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Ser Ser Ile Cys Ser 35 40 45Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Ser Ser Ile Cys Ser 35 40 45
AAC AAC CCG ACC TGC TGG GCG ATC AGC AAA CGT ATC CCG AAC AAA AAA 192AAC AAC CCG ACC TGC TGG GCG ATC AGC AAA CGT ATC CCG AAC AAA AAA 192
Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys Arg Ile Pro Asn Lys Lys 50 55 60Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys Arg Ile Pro Asn Lys Lys 50 55 60
CCG GGC AAA AAA ACC ACG ACC AAA CCG ACC AAA AAA CCG ACC TTC AAA 240CCG GGC AAA AAA ACC ACG ACC AAA CCG ACC AAA AAA CCG ACC TTC AAA 240
Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Phe LysPro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys
65 70 75 8065 70 75 80
ACC ACC AAA AAA GAT CAT AAA CCG CAG ACC ACC AAA CCG AAA GAA GTG 288ACC ACC AAA AAA GAT CAT AAA CCG CAG ACC ACC AAA CCG AAA GAA GTG 288
Thr Thr Lys Lys Asp His Lys Pro Gin Thr Thr Lys Pro Lys Glu Val 85 90 95Thr Thr Lys Lys Asp His Lys Pro Gin Thr Thr Lys Pro Lys Glu Val 85 90 95
CCG ACC ACC AAA CCA 303CCG ACC ACC AAA CCA 303
Pro Thr Thr Lys Pro 100Pro Thr Thr Lys Pro 100
(2) INFORMATIONS POUR LA SEQ ID NO: 4:(2) INFORMATION FOR SEQ ID NO: 4:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 303 paires de bases(A) LENGTH: 303 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADN(ii) TYPE OF MOLECULE: DNA
(ix) CARACTERISTIQUE:(ix) CHARACTERISTIC:
(A) NOM/CLE: CDS(A) NAME / KEY: CDS
(B) EMPLACEMENT:!..303 (xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 4:(B) LOCATION:! .. 303 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
ACC GCG CAG ACC AAA GGC CGT ATC ACC ACC AGC ACC CAG ACC AAC AAA 48 Thr Alα Gin Thr Lys Gly Arg Ile Thr Thr Ser Thr Gin Thr Asn Lys 1 5 10 15ACC GCG CAG ACC AAA GGC CGT ATC ACC ACC AGC ACC CAG ACC AAC AAA 48 Thr Alα Gin Thr Lys Gly Arg Ile Thr Thr Ser Thr Gin Thr Asn Lys 1 5 10 15
CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG CCG AAA AAA CCG AAA GAT 96 Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro Pro Lys Lys Pro Lys Asp 20 25 30CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG CCG AAA AAA CCG AAA GAT 96 Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro Pro Lys Lys Pro Lys Asp 20 25 30
GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG CCC AGC AGC ATC TGC GGC 144 Asp Tyr His Phe Glu Val Phe Asn Phe Val Pro Ser Ser Ile Cys Gly 35 40 45GAT TAC CAC TTC GAA GTG TTC AAC TTC GTG CCC AGC AGC ATC TGC GGC 144 Asp Tyr His Phe Glu Val Phe Asn Phe Val Pro Ser Ser Ile Cys Gly 35 40 45
AAC AAC CAG CTG TGC AAA AGC ATC AGC AAA ACC ATC CCG AGC AAC AAA 192 Asn Asn Gin Leu Cys Lys Ser Ile Ser Lys Thr Ile Pro Ser Asn Lys 50 55 60AAC AAC CAG CTG TGC AAA AGC ATC AGC AAA ACC ATC CCG AGC AAC AAA 192 Asn Asn Gin Leu Cys Lys Ser Ile Ser Lys Thr Ile Pro Ser Asn Lys 50 55 60
CCG AAA AAG AAA CCG ACC ATC AAA CCG ACC AAC AAA CCG ACC ACC AAA 240 Pro Lys Lys Lys Pro Thr Ile Lys Pro Thr Asn Lys Pro Thr Thr Lys 65 70 75 80CCG AAA AAG AAA CCG ACC ATC AAA CCG ACC AAC AAA CCG ACC ACC AAA 240 Pro Lys Lys Lys Pro Thr Ile Lys Pro Thr Asn Lys Pro Thr Thr Lys 65 70 75 80
ACC ACC AAC AAA CGT GAT CCG AAA ACC CCG GCG AAA ATG CCG AAG AAG 288 Thr Thr Asn Lys Arg Asp Pro Lys Thr Pro Ala Lys Met Pro Lys Lys 85 90 95ACC ACC AAC AAA CGT GAT CCG AAA ACC CCG GCG AAA ATG CCG AAG AAG 288 Thr Thr Asn Lys Arg Asp Pro Lys Thr Pro Ala Lys Met Pro Lys Lys 85 90 95
GAA ATC ATC ACC AAC 303GAA ATC ATC ACC AAC 303
Glu Ile Ile Thr Asn 100Glu Ile Ile Thr Asn 100
(2) INFORMATIONS POUR LA SEQ ID NO: 5:(2) INFORMATION FOR SEQ ID NO: 5:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 303 paires de bases(A) LENGTH: 303 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADN(ii) TYPE OF MOLECULE: DNA
(ix) CARACTERISTIQUE:(ix) CHARACTERISTIC:
(A) NOM/CLE: CDS(A) NAME / KEY: CDS
(B) EMPLACEMENT:!..303 (xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 5:(B) LOCATION:! .. 303 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:
ACC GTG AAA ACC AAA AAC ACC ACG ACC ACC CAG ACC CAG CCG AGC AAA 48 Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gin Thr Gin Pro Ser Lys 1 5 10 15ACC GTG AAA ACC AAA AAC ACC ACG ACC ACC CAG ACC CAG CCG AGC AAA 48 Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gin Thr Gin Pro Ser Lys 1 5 10 15
CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG CCG AAC AAA CCG AAC AAC 96 Pro Thr Thr Lys Gin Arg Gin Asn Lys Pro Pro Asn Lys Pro Asn Asn 20 25 30CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG CCG AAC AAA CCG AAC AAC 96 Pro Thr Thr Lys Gin Arg Gin Asn Lys Pro Pro Asn Lys Pro Asn Asn 20 25 30
GAT TCC CAT TCC GAA GTG TCC AAC TCC GTG CCG AGC AGC ATC TGC AGC 144 Asp Ser His Ser Glu Val Ser Asn Ser Val Pro Ser Ser Ile Cys Ser 35 40 45GAT TCC CAT TCC GAA GTG TCC AAC TCC GTG CCG AGC AGC ATC TGC AGC 144 Asp Ser His Ser Glu Val Ser Asn Ser Val Pro Ser Ser Ile Cys Ser 35 40 45
AAC AAC CCG ACC TGC TGG GCG ATC AGC AAA CGT ATC CCG AAC AAA AAA 192 Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys Arg Ile Pro Asn Lys Lys 50 55 60AAC AAC CCG ACC TGC TGG GCG ATC AGC AAA CGT ATC CCG AAC AAA AAA 192 Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys Arg Ile Pro Asn Lys Lys 50 55 60
CCG GGC AAA AAA ACC ACG ACC AAA CCG ACC AAA AAA CCG ACC TTC AAA 240 Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys 65 70 75 80CCG GGC AAA AAA ACC ACG ACC AAA CCG ACC AAA AAA CCG ACC TTC AAA 240 Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys 65 70 75 80
ACC ACC AAA AAA GAT CAT AAA CCG CAG ACC ACC AAA CCG AAA GAA GTG 288 Thr Thr Lys Lys Asp His Lys Pro Gin Thr Thr Lys Pro Lys Glu Val 85 90 95ACC ACC AAA AAA GAT CAT AAA CCG CAG ACC ACC AAA CCG AAA GAA GTG 288 Thr Thr Lys Lys Asp His Lys Pro Gin Thr Thr Lys Pro Lys Glu Val 85 90 95
CCG ACC ACC AAA CCA 303CCG ACC ACC AAA CCA 303
Pro Thr Thr Lys Pro 100Pro Thr Thr Lys Pro 100
(2) INFORMATIONS POUR LA SEQ ID NO: 6:(2) INFORMATION FOR SEQ ID NO: 6:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 276 paires de bases(A) LENGTH: 276 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADN(ii) TYPE OF MOLECULE: DNA
(ix) CARACTERISTIQUE:(ix) CHARACTERISTIC:
(A) NOM/CLE: CDS(A) NAME / KEY: CDS
(B) EMPLACEMENT:!..276 (xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 6:(B) LOCATION:! .. 276 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:
ACC GTG AAA ACC AAA AAC ACC ACG ACC ACC CAG ACC CAG CCG AGC AAA 48 Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gin Thr Gin Pro Ser Lys 1 5 10 15ACC GTG AAA ACC AAA AAC ACC ACG ACC ACC CAG ACC CAG CCG AGC AAA 48 Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gin Thr Gin Pro Ser Lys 1 5 10 15
CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG CCG AAC AAA CCG AAC AAC 96 Pro Thr Thr Lys Gin Arg Gin Asn Lys Pro Pro Asn Lys Pro Asn Asn 20 25 30CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG CCG AAC AAA CCG AAC AAC 96 Pro Thr Thr Lys Gin Arg Gin Asn Lys Pro Pro Asn Lys Pro Asn Asn 20 25 30
GTG CCG AGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC AGC 144 Val Pro Ser Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Ser 35 40 45GTG CCG AGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC AGC 144 Val Pro Ser Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Ser 35 40 45
AAA CGT ATC CCG AAC AAA AAA CCG GGC AAA AAA ACC ACG ACC AAA CCG 192 Lys Arg Ile Pro Asn Lys Lys Pro Gly Lys Lys Thr Thr Thr Lys Pro 50 55 60AAA CGT ATC CCG AAC AAA AAA CCG GGC AAA AAA ACC ACG ACC AAA CCG 192 Lys Arg Ile Pro Asn Lys Lys Pro Gly Lys Lys Thr Thr Thr Lys Pro 50 55 60
ACC AAA AAA CCG ACC TTC AAA ACC ACC AAA AAA GAT CAT AAA CCG CAG 240 Thr Lys Lys Pro Thr Phe Lys Thr Thr Lys Lys Asp His Lys Pro Gin 65 70 75 80ACC AAA AAA CCG ACC TTC AAA ACC ACC AAA AAA GAT CAT AAA CCG CAG 240 Thr Lys Lys Pro Thr Phe Lys Thr Thr Lys Lys Asp His Lys Pro Gin 65 70 75 80
ACC ACC AAA CCG AAA GAA GTG CCG ACC ACC AAA CCA 276ACC ACC AAA CCG AAA GAA GTG CCG ACC ACC AAA CCA 276
Thr Thr Lys Pro Lys Glu Val Pro Thr Thr Lys Pro 85 90 Thr Thr Lys Pro Lys Glu Val Pro Thr Thr Lys Pro 85 90
i FΓ.FNΠF DES FIGURESi FΓ.FNΠF OF FIGURES
Figure 5 :Figure 5:
A) Coloration au bleu de Comassie : Gel SDS-Page des protéines de fusion extraites de membrane bactérienne et purifiées par affinité sur colonne Albumine des différents constructions :A) Comassie blue staining: SDS-Page gel of the fusion proteins extracted from the bacterial membrane and purified by affinity on the Albumin column of the different constructions:
- Puits HW : Marqueurs de taille moléculaire (en Kda).- HW well: Molecular size markers (in Kda).
- puits 1 : S.xylosus (ρSE,G2BBXM].- well 1: S. xylosus (ρSE , G2BBXM].
- puits 2 : S.xylosus [pSE'G2 subBBXM].- well 2: S. xylosus [pSE'G2 subBBXM].
- puits 3 : S.xylosus [pSE"G2delBBXM].- well 3: S. xylosus [pSE "G2delBBXM].
B ) Immunoblo t des protéines de fusion extraites de membrane bactérienne et purifiées par affinité sur colonne Albumine des différents constructions avec anticorps polyclonal apin anti-G l .B) Immunoblo t of the fusion proteins extracted from the bacterial membrane and purified by affinity on the Albumin column of the various constructions with anti-G l polyclonal antibody.
- Puits HW : Marqueurs de taille moléculaire précoiorés (en Kda).- HW well: Precolored molecular size markers (in Kda).
- puits 1 : S.xylosus [ρSE'G2BBXM].- well 1: S.xylosus [ρSE'G2BBXM].
- puits 2 : S.xylosus (pSE'G2subBBXM).- well 2: S. xylosus (pSE'G2subBBXM).
- puits 3 : S.xylosus [pSE'G2delBBXM].- well 3: S. xylosus [pSE'G2delBBXM].
Figure 7 :Figure 7:
A) Coloration au bleu de Comassie : Gel SDS-Page des protéines de fusion sécrétées et purifiées par affinité sur colonne Albumine des différentes constructions:A) Comassie blue staining: SDS-Page gel of secreted and purified affinity fusion proteins on albumin column of the different constructions:
- puits 1 : S.xylosus [pSE'G2delBB] (34,28 Kda).- well 1: S. xylosus [pSE'G2delBB] (34.28 Kda).
- puits 2 : S.xylosus [pSE'G2subBB] (35,23 Kda).- well 2: S. xylosus [pSE'G2subBB] (35.23 Kda).
- puits HW ; Marqueurs de taille moléculaire (en Kda).- HW well; Molecular size markers (in Kda).
B) Immunoblot des protéines de fusion sécrétées et purifiées par affinité sur colonne Albumine des différentes constructions avec anticorps polyclonal lapin anti-G l (VRS).B) Immunoblot of the fusion proteins secreted and purified by affinity on an albumin column of the various constructions with rabbit anti-G l polyclonal antibody (RSV).
- puits 1 : S.xylosus [pSE'G2delBB] (34,28 Kda).- well 1: S. xylosus [pSE'G2delBB] (34.28 Kda).
- puits 2 : S.xylosus [pSE'G2subBB] (35,23 Kda).- well 2: S. xylosus [pSE'G2subBB] (35.23 Kda).
- puits HW : Marqueurs de taille moléculaire précolorés (en Kda). - HW well: Precolored molecular size markers (in Kda).

Claims

REVENDICATIONS
1. Procédé de sécrétion d'un peptide recombinant biologiquement actif, analogue d'un peptide naturel présentant au moins une région hydrophobe, caractérisé en ce qu'on cultive des cellules transformées par une construction d'acides nucléiques comportant - des éléments assurant l'expression et la sécrétion dudit peptide par la cellule, et1. Method for secreting a biologically active recombinant peptide, analogous to a natural peptide having at least one hydrophobic region, characterized in that cells transformed by a nucleic acid construction comprising - elements ensuring the expression and secretion of said peptide by the cell, and
- une séquence codant pour un peptide dont la séquence en acides aminés diffère de la séquence du peptide naturel par au moins une modification dans une région hydrophobe non transmembranaire du peptide, et en ce que l'on récupère le peptide et/ou les cellules portant ledit peptide recombinant.a sequence coding for a peptide whose amino acid sequence differs from the sequence of the natural peptide by at least one modification in a non-transmembrane hydrophobic region of the peptide, and in that the peptide and / or the cells carrying are recovered said recombinant peptide.
2. Procédé selon la revendication 1, caractérisé en ce qu'au moins un acide aminé hydrophobe de la séquence du peptide naturel est remplacé par un acide aminé non hydrophobe.2. Method according to claim 1, characterized in that at least one hydrophobic amino acid of the natural peptide sequence is replaced by a non-hydrophobic amino acid.
3. Procédé selon l'une des revendications 1 ou 2, caractérisé en ce qu'au moins un acide aminé hydrophobe est délélé de la séquence du peptide naturel. 3. Method according to one of claims 1 or 2, characterized in that at least one hydrophobic amino acid is loosened from the sequence of the natural peptide.
4. Procédé selon l'une des revendications 1 à 3, caractérisé en ce que l'acide aminé hydrophobe est choisi dans le groupe suivant : Tryptophane, Phénylalanine, Proline, Valine, Alanine, Isolcuci e, Leucine et Méthionine.4. Method according to one of claims 1 to 3, characterized in that the hydrophobic amino acid is chosen from the following group: Tryptophan, Phenylalanine, Proline, Valine, Alanine, Isolcuci e, Leucine and Methionine.
5. Procédé selon l'une des revendications 1 à 4, caractérisé en ce que la construction d'ADN comprend une séquence signal de sécrétion liée de manière opérationnelle à la séquence d'ADN codant pour le peptide recombinant, et assurant la translocation dudit peptide et sa sécrétion extracellulaire.5. Method according to one of claims 1 to 4, characterized in that the DNA construction comprises a secretion signal sequence operably linked to the DNA sequence coding for the recombinant peptide, and ensuring the translocation of said peptide and its extracellular secretion.
6. Procédé selon l'une des revendications 1 à 4, caractérisé en ce que la construction d'ADN comprend une séquence signal liée de manière opérationnelle à la séquence d'ADN codant pour ledit peptide et permettant la translocation du peptide à travers la membrane de la cellule hôte et son ancrage membranaire. 6. Method according to one of claims 1 to 4, characterized in that the DNA construction comprises a signal sequence operably linked to the DNA sequence coding for said peptide and allowing the translocation of the peptide through the membrane of the host cell and its membrane anchoring.
7. Peptide recombinant susceptible d'être obtenu par le procédé selon l'une des revendications 1 à 6, caractérisé en ce qu'il diffère du peptide naturel par au moins une modification dans la région hydrophobe du peptide naturel. 7. Recombinant peptide capable of being obtained by the method according to one of claims 1 to 6, characterized in that it differs from the natural peptide by at least one modification in the hydrophobic region of the natural peptide.
8. Peptide recombinant selon la revendication 7, caractérisé en ce qu'il est ancré à la surface de la cellule hôte.8. Recombinant peptide according to claim 7, characterized in that it is anchored to the surface of the host cell.
9. Peptide recombinant selon l'une des revendications 7 ou 8, caractérisé en ce qu'il s'agit d'un analogue d'une protéine de structure du VRS ou d'un fragment d'une telle protéine. 9. Recombinant peptide according to one of claims 7 or 8, characterized in that it is an analogue of a protein of RSV structure or a fragment of such a protein.
10. Peptide recombinant selon l'une des revendications 7 à 9, caractérisé en ce qu'il comprend une séquence analogue de la protéine G du VRS, sous groupe A ou B.10. Recombinant peptide according to one of claims 7 to 9, characterized in that it comprises an analogous sequence of the protein G of RSV, in group A or B.
1 1. Peptide recombinant selon l'une des revendications 7 à 10, caractérisé en ce qu'il comprend une séquence analogue de la séquence comprise entre les résidus 130 et 230 de la protéine G du VRS.1 1. Recombinant peptide according to one of claims 7 to 10, characterized in that it comprises a sequence analogous to the sequence between residues 130 and 230 of the G protein of RSV.
12. Peptide recombinant selon l'une des revendications 7 à 1 1 , caractérisé en ce qu'il présente l'une des séquences ID n° 1 , n° 2, n° 3, n° 4, n° 5 ou n° 6.12. Recombinant peptide according to one of claims 7 to 1 1, characterized in that it has one of the sequences ID No. 1, No. 2, No. 3, No. 4, No. 5 or No. 6.
13. Séquence nucléotidique codant pour un peptide selon l'une des revendications 7 à 12.13. Nucleotide sequence coding for a peptide according to one of claims 7 to 12.
14. Séquence nucléotidique selon la revendication 13, caractérisée en ce qu'elle comporte en outre des éléments assurant l'expression du peptide dans une ou plusieurs cellules hôtes spécifiques.14. Nucleotide sequence according to claim 13, characterized in that it further comprises elements ensuring the expression of the peptide in one or more specific host cells.
15. Séquence nucléotidique selon l'une des rev endications 13 ou 14, caractérisée en ce qu'il s'agit d'ADN.15. Nucleotide sequence according to one of claims 13 or 14, characterized in that it is DNA.
16. Séquence nucléotidique selon l'une des revendications 13 ou 14, caractérisée en ce qu'il s'agit d'ARN.16. Nucleotide sequence according to one of claims 13 or 14, characterized in that it is RNA.
17. Vecteur d'expression, caractérisé en ce qu'il comprend une séquence nucléotidique selon l'une des revendications 13 à 16. 17. Expression vector, characterized in that it comprises a nucleotide sequence according to one of claims 13 to 16.
18. Composition pharmaceutique destinée à être administrée à un mammifère pour provoquer la production in situ d'un peptide, caractérisée en ce qu'elle contient un vecteur d'expression selon la revendication 17. 18. Pharmaceutical composition intended to be administered to a mammal to cause the production in situ of a peptide, characterized in that it contains an expression vector according to claim 17.
19. Séquence d'ADN susceptible d'être utilisée dans le procédé selon l'une des revendications 1 à 6, caractérisée en ce qu'elle comprend :19. DNA sequence capable of being used in the method according to one of claims 1 to 6, characterized in that it comprises:
- une séquence signal de sécrétion fonctionnelle,- a signal sequence of functional secretion,
- une séquence d'ADN codant pour un peptide recombinant analogue d'un peptide naturel, la séquence de peptide recombinant présentant au moins une modification dans une région hydrophobe non transmembranaire du peptide naturel.a DNA sequence coding for a recombinant peptide analogous to a natural peptide, the recombinant peptide sequence exhibiting at least one modification in a non-transmembrane hydrophobic region of the natural peptide.
20. Cellule recombinante, caractérisée en ce qu'elle contient une séquence d'ADN selon l'une des revendications 15 à 16 ou 19. 20. Recombinant cell, characterized in that it contains a DNA sequence according to one of claims 15 to 16 or 19.
21. Cellule selon la revendication 20, caractérisée en ce qu'il s'agit d'une bactérie Gram-négatif.21. Cell according to claim 20, characterized in that it is a Gram-negative bacterium.
22. Cellule selon la revendication 20, caractérisée en ce qu'il s'agit d'une bactérie Gram-positif.22. Cell according to claim 20, characterized in that it is a Gram-positive bacterium.
23. Cellule selon la revendication 20, caractérisée en ce qu'il s'agit d'une cellule de levure.23. Cell according to claim 20, characterized in that it is a yeast cell.
24. Cellule selon la revendication 20, caractérisée en ce qu'il s'agit d'une cellule de mammifère.24. Cell according to claim 20, characterized in that it is a mammalian cell.
25. Bactérie selon l'une des revendications 22 ou 23, caractérisée en ce qu'elle est choisie parmi Escherichia coli, Staphylococcus xylosus, Staphylococcus carnosus.25. Bacterium according to one of claims 22 or 23, characterized in that it is chosen from Escherichia coli, Staphylococcus xylosus, Staphylococcus carnosus.
26. Bactérie Gram-négatif selon la revendication 21 , caractérisée en ce que la séquence d'ADN recombinant, s'est intégrée dans le chromosome de l'hôte.26. Gram-negative bacterium according to claim 21, characterized in that the recombinant DNA sequence has integrated into the chromosome of the host.
27. Bactérie Gram-positif selon la revendication 22, caractérisée en ce qu'elle contient une séquence d'ADN recombinant, qui s'est intégrée dans le chromosome de l'hôte.27. Gram-positive bacterium according to claim 22, characterized in that it contains a recombinant DNA sequence, which has integrated into the chromosome of the host.
28. Composition pharmaceutique, caractérisée en ce qu'elle contient une cellule selon l'une des revendications 20 à 27. 28. Pharmaceutical composition, characterized in that it contains a cell according to one of claims 20 to 27.
PCT/FR1995/001464 1994-11-07 1995-11-07 Production of recombinant peptides as natural hydrophobic peptide analogues WO1996014409A1 (en)

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AU673650B2 (en) * 1995-01-10 1996-11-14 University Of Edinburgh, The Novel vectors and use thereof for capturing target genes
EP0970115A1 (en) * 1996-06-05 2000-01-12 Biomolecular Research Institute Ltd. Viral peptides with structural homology to protein g of respiratory syncytial virus
EP0970115A4 (en) * 1996-06-05 2002-02-13 Biomolecular Res Inst Ltd Viral peptides with structural homology to protein g of respiratory syncytial virus
WO1999003987A2 (en) * 1997-07-17 1999-01-28 Pierre Fabre Medicament Syncytial respiratory virus epitopes and antibodies comprising them, useful in diagnosis and therapy
WO1999003987A3 (en) * 1997-07-17 1999-04-08 Pf Medicament Syncytial respiratory virus epitopes and antibodies comprising them, useful in diagnosis and therapy
FR2819810A1 (en) * 2001-01-23 2002-07-26 Pf Medicament NON-GLYCOSYL PEPTIDES DERIVED FROM RSV PROTEIN G AND THEIR USE IN A VACCINE
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US8993517B2 (en) 2001-12-21 2015-03-31 Human Genome Sciences, Inc. Albumin fusion proteins
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