FR2721032A1 - New protein from Bombyx mori that binds bacterial lipopoly-saccharide - Google Patents

Abstract

Proteins (I) contg. at least part of the sequence (A) or (B) are new. Arg Ile Ser Val Gln Asp Val Pro Lys Met Thr Leu Phe Ala Phe Gln Gly Asn Leu (A) Phe His Thr Tyr Ser Val Gln Trp Thr Pro Asp Phe Ile Ala Leu Ser Val Asp Gly (B) Also claimed are: (1) fragments of (I);(2) nucleic acid (II) encoding (I) or its fragments, (3) eukaryotic, pref. plant, or prokaryotic cells that express (I) or its fragments, or contain (II).

Description

The subject of the present invention is a protein binding

  the lipopolysaccharide complex (LPS).

  It also relates to the applications of this protein, in particular in a transgenic plant expressing this protein. Mechanical attacks, for example of phytopathogenic insects, and infections by fungi, bacteria and viruses induce in the tissues of affected plants the synthesis of proteins called "Pathogenis

related protein ".

  P-1,3 glucanases are among the best known and best studied of these PR proteins. This observation and the fact that P-1,3 glucans constitute the majority components of the walls of fungal cells led to the hypothesis of an implication of P-1,3 glucanases

  in the molecular defenses of plants.

  Several authors have suggested that such enzymes constitute factors for resistance to disease and act by attacking the cell walls of fungi.

  pathogens thus inducing lysis or inhibitory effects.

  If the theoretical mechanism of some P-1,3 glucanases has been studied, no practical application of this type of enzyme

  has so far been proposed.

  Thus, to the knowledge of the applicant, no transgenic plant coding for / -1.3 exogenous glucanases has been

  described in the state of the art.

  Note that if the presence of P-1,3 glucanase has been

  noted in plants and bacteria, none 0-1.3-

  glucanase, ni / -1.3-1.4 glucanase, is not known to

  Applicant, synthesized in animals.

  In humans and some animals, infections with gram-negative bacteria can cause serious pathological changes related to septic shock. For example, in the United States septic shock is responsible for

About 100,000 deaths per year.

  To the applicants' knowledge, no effective medication is currently available against such ailments. Proteins from a marine arthropede (Xiphosma) commonly known as the Moluccan crab (Horseshoe crab)

  have been proposed as drugs.

  The hemocytes of these crustaceans were known to

  aggregate bacterial endotoxins.

  AKETAGAWA et al. have highlighted in 1986 (The Journal of Biological Chemistry, 261, n'16, 7357-7365), the structure of a protein of the Japanese species (Tachypleus

  tridentatus) with anti-activity

  lipopolysaccharide. The sequencing of a protein, having a sequence identity of 83% with the previous one, was carried out from the American species (Limulus Polyphemus) by Muta et al. (J. Biochem. 101, 1321-1330,

1987).

  In international application PCT / JP 88/00823, a protein having a strong affinity for the lipopolysaccharide complex is also described, as well as its use for eliminating bacterial endotoxins and

  as a therapeutic agent against bacterial infections.

  However, these proteins have the notable disadvantage of being from organisms far removed from

  mammals and in particular humans.

  It is therefore necessary to find new molecules making it possible to effectively prevent and combat septic shock due to infections, in particular by

gram-negative bacteria.

  The lipopolysaccharide complex (LPS) of gram-negative bacteria can also constitute a contaminant of various fluids, both biological and synthetic, and

  especially liquids injectable into the human body.

  The molecules known to bind the lipopolysaccharide complex (LPS), called LPSBP (in English LPS binding protein), and mentioned above for their therapeutic effect have already been proposed for the elimination of LPS

  preparations where they might be present.

  Nevertheless, it is necessary to find molecules having greater specificity and greater sensitivity for their bonds with respect to the lipopolysaccharide complex. The plaintiff therefore endeavored to find a means allowing on the one hand to combat septic shock in mammals and in particular in humans and on the other hand to

  fight phytopathogenic bacteria.

  The applicant has demonstrated that the Bombyx mori silkworm produces a protein meeting these two requirements. He thus demonstrated that this protein makes it possible to combat the septic shock induced by the presence of lipopolysaccharide complex (LPS) and on the other hand to eliminate this complex from the preparations intended to be injected in

peculiar to man.

  He also showed that the integration into the genome of plants of a gene coding for this protein allows

  fight phytopathogenic fungal infections.

  The subject of the present invention is a protein characterized in that it comprises in whole or in part the following sequence SEQ ID N'1: Arg Ile Ser Val Gln Asp Val Pro Lys Met Thr Leu Phe Ala Phe

Gln Gly Asn Leu.

  It also relates to a protein characterized in that it partly or wholly comprises the following sequence SEQ ID N'2: Phe His Thr Tyr Ser Val Gln Trp Thr Pro Asp Phe Ile Ala Leu

Ser Val Asp Gly.

  Such proteins advantageously have a molecular weight of the order of 50 kD. They preferably include the sequence SEQ ID N'3 or a part of the following sequence SEQ ID N'3: Lys Ile Ser Tyr Ala Gln Met Pro Asp Val Lys Ile Gln Ala Phe Arg Pro Lys Gly Leu Arg Ile Ser Val Gln Asp Val Pro Lys Met Thr Leu Phe Ala Phe Gln Gly Asn Leu Asn His Lys Leu Asp Ser Thr Ser Val Gly Thr Leu Ser Ala Glu Val Leu Asp Pro Val Asn Gly Arg Trp Val Tyr Glu Glu Pro Asp Leu Lys Leu Lys Val Lys Asp Val Val Tyr Tyr Asn Ala Val Phe Ser Ile Asn Lys Lys Ile Tyr Glu Lys Thr Asn Gln Gln Phe Thr Val Thr Glu Leu Glu Asp Pro Asn Ala Ser Thr Asp Ser Gln Lys Pro Glu Cys Lys Pro Thr Lys Thr Arg Val Arg Gly Gly Lys Ala Cys Ala Gly Gln Thr Ile Phe Glu Glu Gln Phe Asp Ser Leu Asp Glu Asn Val Trp Gln Ile Glu Gln Tyr Ile Pro Ile Tyr His Pro Glu Tyr Pro Phe Val Ser Tyr Gln Arg Asn Asn Leu Thr Val Ser Thr Ala Asp Gly Asn Leu His Ile Asn Ala Lys Leu Gln Gln His Met Pro Gly Phe Leu Asp Asp Ser Ile Tyr Ser Gly Thr Leu Asn Leu Phe Ser Gly Cys Thr Ser Ser Ala Glu Ala Cys Ile Lys Gln Ala Ser Gl y Ala Asp Ile Leu Pro Pro Ile Val Ser Gly Arg Ile Thr Ser Ile Gly Phe Ala Phe Thr Tyr Gly Thr Val Glu Ile Arg Ala Lys Leu Pro Gln Gly Asp Trp Leu Tyr Pro Glu Ile Leu Glu Pro Phe Leu Lys Lys Tyr Gly Ser Met Asn Tyr Ala Ser Gly Val Val Lys Ile Ala Cys Ala Arg Gly Asn Ala Glu Leu Tyr Ser Gly Pro Asn Asp Tyr Ser Asn Thr Val Leu Tyr Gly Gly Gly Pro Ile Met Asp Leu Glu Cys Arg Glu Asn Phe Leu Ser Thr Lys Arg Arg Arg Asp Gly Thr Ser Trp Gly Asp Ser Phe His Thr Tyr Ser Val Gln Trp Thr Pro Asp Phe Ile Ala Leu Ser Val Asp Gly Glu Glu Trp Ala Arg Val Glu Ala Pro Arg Asp Ala Leu Pro Ala Val Cys Ala His Ala Pro Arg His Leu Leu Gln Ala Gly Ser Gln Met Ala Pro Phe Asp Asp His Phe Tyr Ile Thr Leu Gly Val Ala Ala Gly Gly Ile Thr Glu Phe Arg Asp Gly Ser Ile Thr Ser Gly Gly Val Thr Lys Pro Trp Arg Asp Ser Ala Arg Lys Ala Ser Val His Phe Trp Arg His Met Ser Asp Trp Phe Pro Arg Trp Ser Gln Pro Ser Leu Ile Val Asp Phe Val Lys Val Ile Ala Leu Said proteins can also have the sequence ID N'5 or part of the following SEQ ID N'5: Met Gly Gly Arg Val Leu Cys Leu Ile Leu Phe Ile Lys Ile Ser Tyr Ala Gln Met Pro Asp Val Lys Ile Gln Ala Phe Arg Pro Lys Gly Leu Arg Ile Ser Val Gln Asp Val Pro Lys Met Thr Leu Phe Ala Phe Gln Gly Asn Leu Asn His Lys Leu Asp Ser Thr Ser Val Gly Thr Leu Ser Ala Glu Val Leu Asp Pro Val Asn Gly Arg Trp Val Tyr Glu Glu Pro Asp Leu Lys Leu Lys Val Lys Asp Val Val Tyr Tyr Asn Ala Val Phe Ser Ile Asn Lys Lys Ile Tyr Glu Lys Thr Asn Gln Gln Phe Thr Val Thr Glu Leu Glu Asp Pro Asn Ala Ser Thr Asp Ser Gln Lys Pro Glu Cys Lys Pro Thr Lys Thr Arg Val Arg Gly Gly Lys Ala Cys Ala Gly Gln Thr Ile Phe Glu Glu Gln Phe Asp Ser Leu Asp Glu Asn Val Trp Gln Ile Glu Gln Tyr Ile Pro Ile Tyr His Pro Glu Tyr Pro Phe Val Ser Tyr Gln Arg Asn Asn Leu Thr Val Ser Thr Ala Asp Gly Asn Leu His Ile Asn Ala Lys Leu Gln Gln His Met Pro Gly Phe Leu Asp Asp Ser Ile Tyr Ser Gly Thr Leu Asn Leu Phe Ser Gly Cys Thr Ser Ser Ala Glu Ala Cys Ile Lys Gln Ala Ser Gly Ala Asp Ile Leu Pro Pro Ile Val Ser Gly Arg Ile Thr Ser Ile Gly Phe Ala Phe Thr Tyr Gly Thr Val Glu Ile Arg Ala Lys Leu Pro Gln Gly Asp Trp Leu Tyr Pro Glu Ile Leu Leu Glu Pro Phe Leu Lys Lys Tyr Gly Ser Met Asn Tyr Ala Ser Gly Val Val Lys Ile Ala Cys Ala Arg Gly Asn Ala Glu Leu Tyr Ser Gly Pro Asn Asp Tyr Ser Asn Thr Val Leu Tyr Gly Gly Pro Ile Met Asp Leu Glu Cys Arg Glu Asn Phe Leu Ser Thr Lys Arg Arg Arg Asp Gly Thr Ser Trp Gly Asp Ser Phe His Thr Tyr Ser Val Gln Trp Thr Pro Asp Phe Ile Ala Leu Ser Val Asp Gly Glu Glu Trp Ala Arg Val Glu Ala Pro Arg Asp Ala Leu Pro Ala Val Cys Ala His Ala Pro Arg His Leu Leu Gln Ala Gly Ser Gln Met Ala Pro Phe Asp Asp His Phe Tyr Ile Thr Leu Gly Val Ala Ala Gly Gly Ile Thr Glu Phe Arg Asp Gly Ser Ile Thr Ser Gly Gly Val Thr Lys Pro Trp Arg Asp Ser Ala Arg Lys Ala Ser Val His Phe Trp Arg His Met Ser Asp Trp Phe Pro Arg Trp Ser Gln Pro Ser Leu Ile Val Asp Phe Val Lys Val Ile Ala Leu The latter protein advantageously has glycosylation, in particular in pos ition 182 of its sequence

amino acids.

  It will be noted that the present invention relates not

  only proteins whose sequence is shown above

  above but also their fragments as well as all the variants of these proteins which would consist in modifications in the structure not modifying the function of these proteins, that it is in particular a function of protection of plants against phytopathogens, of shock treatment

septic tank or LPS connection.

  In particular, such variants can consist of substitutions of an amino acid by another amino acid having a different formula but a function

substantially identical.

  Lehninger's book (Biochemistry, Molecular Bases of Cellular Structure and Functions, second edition 1979, Flammarion Médecine Science Ed.) Specifies that the twenty most frequently encountered amino acids can be subdivided into four groups, according to their structures chemicals. Note that according to the present invention, the

  substitution of an amino acid for one of the proteins described above

  above by another amino acid belonging to the same group does not introduce a functional change in the structure of the protein, and that proteins in which there is a simple substitution of this type, also called substitution

  conservative, fall within the scope of the present invention.

  This does not imply, however, that substitutions of an amino acid from a given group by another amino acid belonging to another group have the effect of

  such modified proteins within the scope of the present invention.

  Indeed, the present invention relates to any molecule having a similar structure and whose function as

  as defined above is retained.

  The present invention also relates to oligonucleotides, and in particular DNA molecules encoding at least one of the proteins or one of its fragments described above, and relates in particular to the molecule DNA comprising the following sequence SEQ ID N'4:

  GAGGATCCGG GTACC ATG GGT GGA CGC GTG TTG TGT CTG ATT TTA TTT

  ATA AAA ATA TCG TAC GCT CAA ATG CCC GAT GTG AAA ATA CAA GCT

  TTT CGC CCG AAA GGA CTT CGA ATA TCC GTT CAA GAT GTC CCC AAA

  ATG ACA CTG TTC GCG TTC CAA GGC AAC TTG AAC CAT AAA CTG GAC

  AGC ACC AGC GTC GGG ACA CTG AGT GCA GAG GTA CTG GAT CCA GTC

  AAC GGC CGA TGG GTG TAC GAA GAG CCC GAT CTT AAA CTA AAA GTC

  AAG GAC GTC GTC TAT TAT AAC GCG GTC TTC TCA ATC AAC AAG AAA

  ATA TAC GAG AAA ACA AAC CAA CAG TTC ACC GTA ACA GAG CTA GAA

  GAT CCT AAT GCA AGC ACA GAT TCT CAG AAA CCA GAA TGT AAG CCA

  ACA AAG ACG AGA GTG CGA GGC GGC AAA GCG TGT GCC GGA CAA ACA

  ATA TTC GAG GAG CAA TTT GAT TCC CTG GAC GAA AAC GTT TGG CAA

  ATC GAG CAG TAT ATA CCG ATT TAT CAC CCC GAA TAC CCC TTC GTG

  TCC TAC CAG CGT AAT AAT TTA ACA GTA TCT ACC GCA GAT GGA AAC

  CTA CAT ATT AAC GCC AAA CTT CAA CAA CAT ATG CCC GGC TTT CTG

  GAC GAC TCT ATA TAT TCT GGC ACA CTT AAT TTG TTC AGT GGG TGT

  ACT TCG TCA GCA GAG GCA TGC ATC AAA CAG GCT TCC GGT GCT GAT

  ATT CTA CCA CCA ATC GTC AGC GGC AGA ATC ACA TCA ATA GGA TTT

  GCA TTT ACG TAC GGA ACA GTC GAA ATC AGA GCG AAA TTA CCG CAA

  GGG GAC TGG CTG TAT CCG GAA ATT CTA TTG GAG CCG TTC TTA AAG

  AAG TAT GGA AGT ATG AAT TAT GCG TCC GGC GTA GTG AAG ATA GCG

  TGT GCG CGC GGT AAT GCA GAA CTC TAT TCC GGA CCT AAT GAC TAC

  AGC AAT ACG GTT TTG TAC GGA GGA CCG ATC ATG GAT CTG GAG TGC

  CGC GAG AAC TTT CTC TCC ACA AAA AGA CGC AGA GAC GGC ACG TCG

  TGG GGC GAC AGT TTT CAT ACA TAT TCC GTT CAA TGG ACA CCT GAT

  TTC ATA GCC CTG TCT GTG GAC GGC GAG GAG TGG GCG CGA GTG GAG

  GCG CCG CGG GAC GCC CTG CCG GCT GTC TGC GCG CAC GCC CCG CGG

  CAC CTG CTG CAG GCC GGC TCG CAG ATG GCG CCA TTC GAT GAC CAC

  TTC TAC ATA ACG CTA GGC GTG GCG GCA GGA GGC ATA ACG GAG TTC

  CGC GAC GGG TCT ATA ACC TCC GGG GGA GTC ACC AAG CCC TGG AGA

  GAC AGC GCT CGG AAG GCA TCC GTA CAT TTC TGG CGG CAC ATG TCC

  GAT TGG TTC CCA CGG TGG AGT CAG CCA TCT TTA ATC GTG GAC TTT

  GTC AAA GTT ATC GCC TTA TGATCTAGTC ATTTAGATTT TGTATAATTA

  AGTTATTAGT ATAGTTTCTG CAGGCAAAAT TAATTGCATT CTGAGTTTTT

  ATTGTCAAAC TACAATTTAA AATTTAATTC CTAAAAGATC TCTCTTCTTT

  CTAAATCTGT AATTTAGCTA ATTTTATTTG TATCATTTTC TTTTTTTAAA

  TTTTGATAGA GCGGCGAATG ACTAGAAGAT GTGATCTGCG AAAATAAAAC

  TAAGTGACAT GCATGCAAAG GCATACACAC AACAGAGTTT TGCATTATTA

  TTACCTTTCT TGAGCGAGAT GAGGACTACT GACGTGACCT GGAGTGATAT

  TTTGGAGCAA GCCTAAGACA GGGTCAAGTG GAGACAGCTC TAAGGCCCCT

  ATGTACCGGC GGGGTGTCTT AGGGCTGCAA TAACAACATT ACCTTTCTTA

  ACCAAACAGC GATATTATCT GTAATATTAT AGTTAGAAAG CCTGCATCTT

  ATACTCTTAT TGACACACGT ACCTGGATGA TTTTAACGAT GAAGAAATAA

  CATCGTGTAA AAAATTTAGC TGGAAAAGAT TTTGAGGAAT CAAGAAATAC

  CCTATGAACC AGCACAGGTA CGTGTCACCA CTCTGGCTAA TTCTGCCACG

  TTGCGGTTGG TTTGAAGTTT GAGACAACCT TTGCACGATA CAATTGAGAC

  TAAGACCTCA TGGCTCGGAG TGAGTGGAGT CATTCAAGTT GCGACTGGCT

  CCAGTAACCA CTTATAACCA GGTAGATCGT GAGCCTATCA GCTCATTTAG

  AGCAACAAAA AACAAAATAA TGTACTCTTG AAAAAATCAT TTTAATAAAA

GACCAGAGTG AAAAAAAAAAAA AAAAA

  The present invention further relates to eukaryotic or prokaryotic cells expressing one of the proteins described above or one of their fragments, or carrying at least one

  part of an oligonucleotide mentioned above.

  One of the cells is advantageously a bacterial cell or a plant cell, for example from a plant belonging to the family of the vine, tobacco,

tomato or potato.

  Another object of the present invention is a process for obtaining one of the proteins described above or one of their fragments, characterized in that an oligonucleotide, such as one of those mentioned above, is expressed in a

  adapted prokaryotic or eukaryotic cell.

  The proteins which are the subject of the present invention can nevertheless be obtained by any other method available to those skilled in the art, such as chemical synthesis, and in particular those listed in the general manual: "Molecular cloning: A Laboratory manual" (Sambrook et al, 1989, Cold Spring Harbor Lab., Cold Spring Harbor, NY,

  2nd edition) and in its recent re-editions.

  In general, those skilled in the art will also refer to this manual for the implementation of the present invention. In addition to the objects mentioned above, the present invention relates to a medicament containing one of the proteins described above or one of their fragments and the use of such a protein for the manufacture of a medicament for the treatment of affections linked to the lipopolysaccharide complex, and in particular to treat septic shock. Note that the genetic distance between mammals and the silkworm is smaller than that between mammals and Xiphosma. The proteins which are the subject of the present invention, which are derived from a silkworm protein, will therefore be better accepted by mammals, and in particular humans, than the proteins extracted from Limulus, or, from neighboring species, of which l is proposed in

PCT / JP88 / 00823.

  The proteins according to the invention can be administered by any suitable method, preferably by injection, in order to rapidly combat the effects of septic shock. Another object of the present invention is a process for removing the lipopolysaccharide complex from preparations containing the latter, said process consisting in binding said complex to one of the proteins described above or

to one of their fragments.

  Such a method can in particular be implemented in

  fixing on a support the protein binding said complex.

  The present invention also relates to a plant carrying at least one gene coding for an exogenous protein

  having an activity (-1.3 glucanase.

  Such a protein is advantageously one of the proteins defined above or one of its fragments. The gene carried by the plant can, for its part, comprise at least one

part of a DNA defined above.

  Such a plant preferably belongs to the family of the vine, tobacco, tomato or apple

earthen.

  Finally, the present invention relates to the use of fragments of the oligonucleotides described above for gene amplification. Such an amplification can be implemented according to the method described in the

request FR-92.13.562.

  The present invention is illustrated without however being limited by the examples which follow and with reference to the appended drawings in which: FIG. 1A represents an SDS-PAGE gel of extracts of gram-negative bacteria. Well 1 corresponds to a mixture of protein markers (phosphorylase b, 92 kD; bovine serum albumin 66.2 kD; ovalbumin 43 kD; carbonic anhydrase 30 kD). Well 2 is a witness to the binding test carried out without hemolymph (bacteria alone). Well 3 is a binding test performed with immune hemolymph (immunization by injection of E. cloacae). Well 4 corresponds to a binding test carried out with immune hemolymph, the immunization having been carried out by abrasion

  procuticular and topical application of E. cloacae.

  FIG. 1B is a peptide mapping carried out in CHLP following the in situ digestion of the protein P-50 by the endopeptidase Lys-C. The peptide peaks indicated by the black and white arrows were subjected to an analysis of

their amino acid sequence.

  Figure 2 illustrates a binding test performed using non-immune hemolymph (well 3) or immune hemolymph (well 1, 2, 4, 5 and 6). The incubation times for the binding test are one minute (well 1); 2.5 minutes (wells 2 and 3), 5 minutes (wells 4), 15 minutes (wells 5), and 30 minutes (wells 6). Well 7 corresponds to molecular weight markers whose

sizes are indicated in kD.

  FIG. 3A illustrates the position of the two pairs of degenerate primers (P1 and P3 for the sense strand and P2 and P4

for the anti-sense strand).

  FIG. 3B represents the analysis of the MOPAC products on a 2% agarose gel. Well 1 corresponds to a 10 μl aliquot of the MOPAC product obtained using P1 and P4. Well 2 corresponds to an aliquot of 10p1 of the MOPAC product obtained using P3 and P2. Well 3 corresponds to the molecular weight marker (100 bp Ladder Pharmacia). The

  arrow indicates the product position of 160 base pairs.

  FIG. 4 represents a restriction map used for the analysis of cDNA P-50 lambdaBP5001. The region

  of the coding protein is indicated in white.

  FIG. 5 illustrates the analysis of the hydropathy of the P-50 protein. The numbers of the amino acid residues are indicated on the horizontal line while the hydrophobicity and

  hydrophilicity are indicated above and respectively

below the horizontal line.

  Figure 6 illustrates similarities and identities

  of sequences between P-50 and various glucanases.

  The meanings of the abbreviations are as follows: Bci: 13-1.3 glucanase from Bacillus circulans, Fsu: f-1,3-1,4 glucanase from Fibrobacter succinogenes; Cth: 0-1.3-1.4 glucanase from Clostridium thermocellum; Bsu: P-1,3-1,4 glucanase from Bacillus subtilis; Bam: 3-1.3-1.4 glucanase from Bacillus amylofiquefaciens; Bma: P-1,3-1,4 glucanase from Bacillus macerans; Bli: 1-1.3-1.4 glucanase from Bacillus licheniformis. Figure 7 illustrates the similarities and sequence identities between P-50 and the LPS binding domains of human LBP and BPI proteins, as described by SCHUMANN et al. (1990, Science, 249, 1429) and HOESS et al.

  (1993, EMBO J., 12, N'9, 3351-3356).

  In these last two figures, the similarities and identities between the amino acids are indicated by boxes. Identical amino acids are indicated by dots. The numbers shown on the left refer to

  at the position of the residue in each protein.

  Figure 8 is a photograph of a fluorographed SDS-PAGE gel of messenger RNA translation products encoding the P-50 protein (well 1). Well 2 corresponds to a control without messenger RNA. Reference molecular weights

  are shown on the right of the figure.

  FIG. 9A is an autoradiogram of a transfer of messenger RNA hybridized with the Pst I / EcoRI fragment of

lambdaBP5001 (1.2 kb).

  Wells 1.3 and 5 correspond to total RNA extracted from fatty larvae and wells 2.4 and 6 correspond to total RNA extracted from epidermal cells, before procuticular abrasion (wells 1 and 2) and six hours after abrasion in the presence of bacteria (wells 3 and 4) or six hours after

  abrasion without bacteria (wells 5 and 6).

  The size of molecular weight marker is indicated

on the left of the autoradiogram.

  FIG. 9B represents the same dehybridized transfer then rehybridized with a probe corresponding to the DNA complementary to the α-tubulin (white arrow) and with a

  probe corresponding to the cecropin B2 gene (black arrow).

EXAMPLE 1:

  Determination of the P-50 protein sequence

A) MATERIALS AND METHODS

  1. Chemical reagents and instruments The reagents were supplied by Bio-Rad Labs

  (Richmond, CA) for acrylamide, N, N'-

  methylenebisacrylamide, ammonium persulfate, N, N, N ', N'-tetramethylethylenediamine, Tris, glycine and Bromophenol Blue, by Wako Pure Chemical Industries Ltd. (Tokyo, Japan) for 2-mercaptoethanol, by Scharz / Mann Biotech (Cleveland, OH) for tricin, by Fluka Biochemika (Buchs, Germany), for dodecyl sodium sulfate, by Perkin-Elmer Cetus for polymerase Thermus aquaticus, by Sigma (St, Louis MO) for Ponceau S, by Boehringer for endopeptidase Lys-C, by Amersham for the ECL system, by BRL for reverse transcriptase of MuLV, by Stratagene for the PCR cloning kit , by Amersham for the Mega Prime DNA labeling system, by Stratagene for the RNA transcription kit, by Pharmacia for the T7 sequencing kit, by Promega for the Taq Track sequencing kit, by Pharmacia-LKB (Uppsala , Sweden) for mixing molecular weight markers, and by Applied Biosystems (Foster City,

  CA), for the Milli-ProBlott PVDF high capacity membrane.

  All other chemicals are chemicals

high quality commercial.

  2. Organisms, cells, immunization and recovery of the hemolymph Bombyx mori silkworms (Asahi x Tokai) were

  supplied by Toyo Sangyo Consulting, Inc., Tokyo, Japan.

  They were grown on an artificial medium (powder

  mulberry) at 23 ° C with a 14 hour photoperiod.

  Larvae in the fifth molt stage are cultivated until the 5th day of this stage and immunized by injection of pl of Enterobacter cloacae (strain 57-9, Collection of the Pasteur Institute) at the end of the logarithmic growth phase. The larvae are then kept 24 hours in

  presence of food then the hemolymph is recovered.

  Immunization by cuticular abrasion and application of bacteria is carried out as described by Brey et al.

  (1993, Proc. Natl. Acad. Sci. USA.90. 6275-6279).

  For some experiments, silkworms are also cultivated under axenic conditions (Brey et al.,

1993, previously cited).

  The hemolymph is recovered by making an incision of the body of the larvae in tubes containing a few phenylthiourea crystals then is centrifuged at 4000 g for 10

minutes at 4'C.

  The supernatant, made up of hemolymph plasma, is

  used immediately or stored & - 80'C.

  3. Bacteria in vitro binding test The in vitro bacteria-plasma hemolymph binding test was carried out substantially as

  described by Sun et al. (1990, Science, 250: 1729-1732).

  ml of gram-negative E. cloacae or gram bacteria

  positive Bacillus licheniformis at the end of the logarithmic growth phase are centrifuged, washed and finally resuspended in 200 μl of Tris-HCl, 10 mM, pH 8 according to Sun and

al. (1990, previously cited).

  The bacterial suspension is added to 1 ml of immune hemolymph plasma and incubated at room temperature with a

  moderate agitation during given periods.

  The mixture is centrifuged for 2 minutes at 10,000 g, at 4 ° C, and the supernatant is removed before carrying out another

bond test.

  The pellet is washed successively with 200 μl of a NaCl solution in which the NaCl concentration is increased arithmetically after each washing (0.1; 0.2; 0.3; 0.4 and 0.5 M in NaCl) or washed twice in 500 p1 of a 0.5 M NaCl solution and finally eluted with 200 p1 of a solution comprising 0.5 M NaCl and 0.1 M acetate

ammonium, pH 4.0.

  The eluent is used to make an SDS / PAGE gel.

  4. Electrophoresis and immobilization of the proteins The electrophoresis on the SDS-Polyacrylamide gel is carried out by the method of Laemmli (1970, Nature 227: 680) using an apparatus for electrophoresis BioRad

miniprotein.

  The sequencing of the N-terminal end of the intact protein as well as of the peptide fragments obtained by enzymatic cleavage in situ was carried out using the gel.

  Tricin SDS-PAGE (Ploug, 1989, Anal. Biochem. 181: 33-39).

  The proteins separated by electrophoresis are transferred onto a mini-Problott PVDF membrane according to the

manufacturer's instructions.

  5. In situ enzymatic cleavage and peptide mapping of internal sequences After electrophoresis, the gel is fixed in a solution containing 50% methanol and 10% acetic acid and stained with

amide black for 2 minutes.

  The protein bands are cut and washed in Milli Q quality water, then put back in the same tube and dehydrated using a Speed-Vac.

  Digestion with Endopeptidase Lys-C (final concentration: 2 μg / ml) is carried out in 300 μl of Tris-HCl, 0.1 M,

  pH 8.8, containing 0.03% SDS at 30 ° C for 18 hours.

  After digestion, the total reaction medium is centrifuged and the supernatant is immediately injected onto a High Pressure Liquid Chromatography (HPLC) column (Vydac, 2.1 x 250 mm). The peptide fragments are separated on a linear gradient of acetonitrile (0-55%) containing 0.1%

  trifluoroacetic acid for more than 60 minutes.

  A flow of 200 pl / min. is applied and the absorbance is measured at 214 and 280 nm. Each peak is collected manually

  and stored at -20 ° C for later analysis.

  6. Analysis of amino acid sequences The intact protein immobilized on the PVDF membrane or the peptides resulting from the enzymatic digestion are separated

  by CHLP and are then subjected to automatic sequencing.

  Automatic Edman degradation is carried out on a gas phase microsequencer 477A (Applied Biosystems),

  connected to a phenylthiohydantoin analyzer (model 120A).

  7. Analysis of the complementary DNAs obtained by amplification in a mixture of oligonucleotides

(MOPAC analysis).

  All DNA and RNA manipulations were performed using standard techniques described by Sambrook et al., (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, NY, 2nd Ed. ). Two pairs of primers were synthesized: P1 (sense strand) having the following sequence:

  P1: 5'-AA (A, G) ATGAC (A, C, G, T) (C, T) T (A, C, G, T) TT (T, C) GC (A, C, G, T)

  TT-3 and P2 which is the anti-sense of Pi: These two sequences include internal sequences complementary to all possible codons for the sequence

  amino acid Lys-Met-Thr-Leu-Phe-Ala-Phe.

  Two other primers P3 (sense strand) and P4 (anti-strand

  meaning) have on the other hand been synthesized. P3 has the following sequence:

  P3; 5'-GT (A, C, G, T) TA (T, C) TA (T, C) AA (T, C) GC (A, C, G, T) GT (A, C, G , T)

TT-3 '

  P3 and P4 comprise sequences corresponding to all the possibilities of codons for the peptide sequence Val-Tyr-Tyr-Asn-Ala- Val-Phe. For the preparation of template RNA, a silkworm larva in the 5th stage (day 5) was immunized with 10 μl of a

  solution of the lipopolysaccharide complex (2 mg / ml).

  After 6 hours, the fatty substance is recovered and the total RNA is extracted as described by Auffray and Rougeon (1980,

Eur. J. Biochem. 107: 303-314).

  Single-stranded complementary DNA is synthesized from total RNA using antisense primers (P2

  or P4) and MuLV reverse transcriptase (BRL).

  The complementary single-stranded DNA is amplified with 1 mM of the oligonucleotides assembled in pairs (P1 and P4, or P2 and P3) in a solution containing 10 mM of Tris-HCl, pH 8, 50 mM KCl, 1.5 mM MgC12, 200 MM dNTP and 2.5 polymerase units from Thermus aquaticus (Lee et al. 1988, Science 239: 1288-1291, and

  Lee and Caskey 1992, Methods in Enzymology 216: 69-72).

  The polymerase chain reaction (PCR) is carried out by denaturing at a temperature of 94 ° C. for 30 seconds, ringing at 52 ° C. for 1 minute, then carrying out the extension at 72 ° C. for 2 minutes, for 30 seconds.

  cycles using the Tempcycler II, Model 110P (Coy Corp.).

* The aliquots of the amplified mixtures are analyzed on 2% agarose gels. The bands dyed using ethidium bromide are cut from the gel and the isolated DNA is cloned into the plasmid pCR-Script SK (+) using the PCR cloning kit marketed by Stratagene. 8. Screening of complementary DNAs A library of complementary DNAs obtained from the Bombyx mori fatty substance, which was injected 10 hours before heat-killed E. cloacae bacteria, is prepared in

  using the lambda- complementary DNA synthesis kit

gt-10 marketed by Amersham.

  A PCR product with a length of 160 base pairs obtained by MOPAC analysis (combinations of P1 and P4) was labeled with phosphorus 32 by random labeling (Mega Prime DNA Labeling System, Amersham) and used to screen the

complementary DNA bank.

  About 75,000 colonies were spread at a density of about 2,500 colonies per 90 mm diameter dish and

  screened using Nylon filters (Amersham Hybond-

  NOT). Hybridization is carried out using a solution comprising 6 x SSC, 2 x Denhardt solution, 0.1% SDS, 50%

  formamide and DNA from salmon sperm sonicated at 100 mg / ml.

  It is carried out at 42 ° C. for 14 hours with a probe having a radioactivity of 106 cpm / Ml. (1 x SSC = 0.15M NaCl / 0.015M sodium citrate, pH 7; Denhardt solution 0.02%, polyvinylpyrrolidone 0.02%, Ficoll 0.02%, serum

bovine albumin).

  The filters are briefly washed in a solution containing 2 x SSC, 0.1% SDS at room temperature and are then washed twice at 50 ° C. in a solution containing 2 x SSC, 0.1% SDS for 1 hour and autoradiographed at -70 C in

  using intensifying screens.

  The colonies are hybridized with the probe then

  re-screened and selected by sequencing.

  9. Subcloning and sequencing strategies One of the lambda gt 10 positive clones containing 2.3 kb a

  has been subcloned into pBluescript vectors.

  Two Pst I digestion fragments, 0.8 kb and 0.3 kb respectively, were again subcloned into

  pBluescript vectors. The 1.2 kb fragment from the sub-

  initial cloning was religated (Figure 4).

  The sequencing of the two strands was carried out with a universal primer M13, a primer T3 and synthetic primers using the sequencing kit T7 marketed by Pharmacia or the sequencing kit Taq Track

marketed by Promega.

  10. Analysis by the Northern Transfer Technique The total RNA was extracted from the fatty substance and from the epidermal cells by direct precipitation in a solution containing 3M LiCl and 6M urea (Auffray

  and Rougeon, 1980 previously cited).

  The RNA is separated by denaturing electrophoresis on a 1% formaldehyde agarose gel with MOPS buffer and then is

  transferred to nylon membranes (Hybond-N, Amersham).

  The filter is firstly hybridized overnight at 42 ° C. with complementary DNA digested with EcoRI / Pst 1 and is randomly labeled with phosphate 32 (fragment corresponding to the open reading sequences of the fragment of 1.2 kb encoding the majority of the P-50 protein sequence) in a solution containing: 50% formamide, 5 x SSC, 2x Denhardt solution, 100 µg / ml sperm DNA

denatured salmon and 0.1% SDS.

  The filter is then briefly washed with a solution containing 4 x SSC and 0.1% SDS at room temperature, twice at 50 ° C for 20 minutes and finally with a solution containing 2 x SSC and 0.1% SDS for 20 minutes at C. After autoradiography the filter is dehybridized according to the manufacturer's instructions and rehybridized with the Cecropin B24 probe from Bombyx mori (Taniai et al. 1992, Biochem. Biophys. Acta, 1132: 203-206; Brey et al. 1993 , Proc. Natl. Acad. Sci. USA 90, 6275-6279) and the α-tubulin probe as

  internal witness (Kalfayan and Winsink, 1981, Cell 24: 97-106).

  11. In vitro transcriDtion, in vitro translation and fluorography The synthesis of synthetic messenger RNA was carried out using a linearized pBluescript containing the P-50 insert and an RNA polymerase dependent DNA coded by bacteriophage T7 polymerase and using the kit of

  RNA transcription marketed by Stratagene.

  The rabbit reticulocyte lysate system (Promega) was used for the translation of synthetic messenger RNA

  using 35S sulfur labeled methionine.

  The in vitro translation products were separated on an SDS-PAGE gel. After electrophoresis the gel is treated with

  En3Hance (DuPont) and fluorographed at -70'C.

  12. Computer analysis of sequence homologies The FASTA, TFASTA and SwissProt software were used to determine the sequence homologies between P-50 and other known proteins (Argos, 1990, Methods in

Enzymol. 182: 751-776).

  DNA strider 1.1 software was used to

hydrophobicity analysis.

B. RESULTS

1. Link test

  A binding test with a gram-negative bacterium (E.

  cloacae), or gram-positive (B. licheniformis), followed by washing with NaCl (incubation time 2.5 minutes) and acid extraction was carried out in order to selectively purify the proteins of the hemolymph having an affinity

  important for bacterial surfaces.

  The bacteria tests were then analyzed on

  SDS-PAGE gel. The SDS-PAGE gel of gram bacteria extracts

  negative shows a predominant band corresponding to a

50 kD protein (Figure 1A).

  By comparing the binding of proteins from immune hemolymph (Figure 2, well 2) and non-immune hemolymph (Figure 2, well 3), a significant increase in the

  amount of 50 kD protein (P-50) in the immune hemolymph.

  When the immune hemolymph is incubated with the bacteria tested during different incubation times (1, 2, 5, 5, 15 and minutes), the amount of P-50 increases over time up to an incubation time of 15 minutes and thus becomes the most abundant protein linked to the bacterial surface

  (Figure 2, columns 1, 2, 4, 5 and 6).

  In this in vitro binding test (FIG. 2), the bacteria are washed twice with a 0.5 M NaCl solution, instead of performing successive washes, due to the

large number of samples.

  After the 10 minute incubation period, the binding test supernatant is reincubated with a new batch of bacteria to determine if P-50 remains in the supernatant. Only insignificant amounts of P-50 can be detected during the second 10-minute incubation. Thus, P-50 is largely eliminated from the immune hemolymph during the first 10-minute incubation. An insoluble P-1,3-glucan (curdlan) was used in other in vitro binding tests, at final concentrations of 3 mg / ml of immune hemolymph. After a 10-minute incubation, the pellet is washed twice with 0.5 M NaCl solution and the acid extract is analyzed on SDS-PAGE. P-50 is detected, with proteins

minor contaminants.

  2. Purification and sequencing of amino acids The proteins eluted during the binding test are separated on Tricine-SDS-PAGE and the protein band corresponding to a molecular weight of 50 kD is subjected to digestion with a proteinase in situ in order to perform an internal amino acid sequence analysis, as the intact 50 kD protein is not sensitive to Edman degradation

  due to its blocked a-amino groups.

  A series of strips corresponding to P-50 (approximately 15 μg) is cut from the gel, dehydrated and digested in

  situ using endopeptidase Lys-C.

  The resulting peptide fragments are separated by reverse phase CHLP (FIG. 1B) and their primary structure is determined by automatic sequencing of the amino acids. The sequences which have been determined are as follows: - peak corresponding to the black arrow: 2 bands - major band: DVVYYNAVFSINK - minor band: MTLFAFXGNLNXK - peak corresponding to the white arrow:

LDSTSVGTLSAEVLDPVNGRXVYEEPDLK

  3. MOPAC analysis and isolation of the complementary DNA coding for PEn using the data obtained concerning the internal amino acid sequence of P-50, degenerate primers were synthesized (P 1 and P3 for the sense strand,

  P 2 and P 4 for the anti-sense strand) (Figure 3A).

  PCR amplification is carried out as described in the Materials and Methods chapter using complementary single-stranded DNA from fatty bodies of immune silkworms (combination of P 1 and P 4 or P3 and P2). Only a combination of primers (P 1 and P 4) gives rise to a product of 160

  base pairs (bp) as shown in Figure 3B.

  The 160 base pair product is cloned and sequenced.

  The amino acid sequence deduced from the 160 base pair sequence contains the three peptide fragments of P-50, as analyzed by Edman degradation. The complementary DNA library constructed from an immunized silkworm fatty substance is screened using the product of 160 base pairs randomly labeled with phosphorus 32, as probe. positive clones are isolated after screening of approximately 75,000 independent clones. Among thirteen positive clones chosen at random, all have the same insert of identical size (2.3 kb) with the exception of one clone which

has a 1.8 kb insert.

  The nucleotide sequence of one of these clones (lambda

  BP5OO1) containing one of the 2.3 kb inserts has been analyzed above

  below. 4. Nucleotide sequence and deduced amino acid sequence

  The EcoR I fragment of the lambdaBP5001 clone was sub-

  cloned into the plasmid pBluescript.

  Subcloning has been carried out as described in the Materials and Methods chapter. The restriction card

  used for subcloning is that of FIG. 4.

  The nucleotide sequence and the deduced amino acid sequence are those listed SEQ ID N ′ 1 and SEQ ID N'2. The lambdaPB5001 sequence contains a non-coding region at its 5 'end and an open reading phase of 1401 nucleotides corresponding to 467 amino acids. An untranslated region of 1100 nucleotides is also present between the stop codon and the polyadenylation site. This sequence contains two possible polyadenylation signals (AATAAA), which could correspond to the existence of messenger RNAs from

different sizes.

  Six ATTTA sequences were also found in the region near the 3 'end of the open reading phase, suggesting some instability of the messenger RNAs of the

protein P-50.

  The calculated molecular weight of the protein deduced from

  the complementary DNA sequence is 52.225D.

  Analysis of the hydrophobicity of the sequence shows that the first eighteen amino acids correspond to a typical signal peptide profile (Figure 5). The C-terminal part of the P-50 protein is also very hydrophobic, which suggests the existence of a membrane anchor. The deduced protein also contains a potential N-glycosylation site on its amino acid in position

182.

  5. Comparison of the amino acid sequence of the P-50 protein and other proteins The complementary DNA of the P-50 protein has significant sequence homology with certain domains of four different proteins presenting a point of view immune: 3-1, 3 bacterial glucanase, protein that binds the human lipopolysaccharide complex (LBP), protein

  increasing human permeability (BPI) and human CD14.

  a) B-1,3 glucanase The most important sequence homology is obtained with the DNA complementary to P-1,3 glucanase A1 from Bacillus circulans WL-12 (Yahata et al., 1990, Gene 86, 113 -117). 34% identity and 56% similarity across 124 amino acids

acids (Figure 6).

  This region of 124 amino acids contains the supposedly highly conserved active site (19 amino acids) of both P-1,3-1,4 glucanases and / -1,3-1,4 glucanases (Schimming et al. , 1992, Eur. J. Biochem., 204, 13-19; Hofemeister et al.,

  1986, Gene 49: 177-187) as shown in Figure 6.

  In this region, P-50 shows a structural identity of 53% and a similarity of 63% with 3-1.3 glucanase from Bacillus circulans and an identity of 53% and a similarity of 90% with P-1 , 3- 1,4 Fibrobacter glucanase

  succinogenes (Teather and Erfle, 1990, J. Bacteriol. 172: 3837-

  3841) including histidine and aspartic acid residues

  strictly retained from the assumed active site.

  b) LBP / BPI A significant sequence homology is also observed with the LPS binding domain of human LBP and BPI (Schumann et al., 1990, Science 249: 1429; Hoess et al., 1993, EMBO J. Vol. 12 N 9 3351-3356) as shown in Figure 7. In this domain of 19 amino acids, we observe an identity of 21%, and a similarity of 47% with LBP and a

  21% identity and 53% similarity to BPI.

  If the consensus binding domain of LBP or BPI is compared with P-50, an identity of 37% is obtained and

68% similarity. (figure 7).

  c) CD14 Another human protein involved in the complex

  LPS / LBP binding or in the LPS binding is CD14.

  This protein shows an identity of 21% and a similarity of 46%. 6. In Vitro Translation In order to confirm the identity of the cloned gene product, lambdaBP5001 is linearized by Bgl II digestion and the

  transcription is performed in vitro.

  The resulting messenger RNAs are translated in vitro using the rabbit reticulocyte lysate in the presence of S-methionine.

  The reaction mixtures are then separated by SDS-

  PAGE and fluorographed. The translated protein has an estimated molecular weight of around 52 kD as shown in Figure 8. This is in agreement with the calculated molecular weight

  from the deduced amino acid sequence.

  7. Induction of messaqer RNA encoding P-50 in response to bacterial infection using axenic Bombyx mori larvae The amount of P-50 protein in the hemolymph increases significantly after contact with bacteria

as shown in figure 1.

  The induction of messenger RNAs encoding P-50 following prothicular abrasion was examined with

axenic Bombyx mori larvae.

  LambdaBP5001 was digested with Pst 1 / EcoR i and the resulting 1.2 kb fragment, containing the major part of the region coding for the protein P-50, was purified, randomly labeled with 32p and used as a probe for a Northern blot. P-50 messenger RNA is constitutively expressed at low levels in the fat and in the epidermal cells of non-abraded larvae

procuticular (Figure 9A).

  Six hours after the abrasion of the skin in the presence of bacteria, the messenger RNAs are strongly induced in the fatty substance and, to a lesser degree, in the epidermal cells. Even sterile abrasion in the strict absence of bacteria is capable of inducing messenger RNA in

  two tissues six hours after abrasion (Figure 9A).

  In order to have a positive control, the transferred DNA (blot) is dehybridized and rehybridized with the gene expressed constitutively of α-tubulin and of another protein.

  involved in induced immunity, cecropin B2.

  The results of FIG. 9B show that P-50 is an acute phase protein which plays an important role in the

immune response from Bombyx mori.

  All the results of this example show that the P50 protein binds LPS and plays an important role in the

immune response.

EXAMPLE 2:

  1) - OBTAINING TRANSGENIOUS TOBACCO PLANTS AND

  EVIDENCE OF RESISTANCE TO INFECTION

FUNGIOUE

  The complementary DNA of the P-50 protein obtained in Example 1 is cloned into the EcoRI sites of the plasmids pMON530 and pBI 121 (Jaynes et al., 1993, Plant Science, 89, -53). The inserted complementary DNA is placed under the control of the proteinase II promoter from the potato

  (Jaynes et al. 1993, Science, 89, 43-53).

  Leaf discs about 1 cm in diameter are cut from the leaves of six-week-old tobacco plants (Nicotiana tabacum var. Xanthii and var. Samsung) which have been aseptically grown in boxes of

Magenta.

  Leaf discs are obtained by punching young leaves using a sterilized punch. The explants are precultured for 1 to 2 days on an MS104 medium (MSO, 7 g / l of phytoagar, 1 mg / ml of benzyladenine and 0.1 mg / ml of naphthalene acetic acid) to stimulate growth. The MSO medium comprises 4.3 g / l of Murashige salt

and Skoog and 30 g / l of sucrose.

  The explants are inoculated by immersing them for 2 to seconds in an overnight culture of Agrobacterium tumefaciens. The explants' water is then absorbed and the explants are placed in culture on boxes of nurse culture. Each canister culture dish is prepared using MS104 medium to which 1.5 ml of tobacco culture suspension has been added. These boxes are

  covered with a piece of Whatman 3mm filter paper.

  The explants are then left to incubate for 2 days and then transferred to an MS selection medium (MS104, 500 mg / ml of carbenicillin or cefatoxime, 300 mg / ml of kanamycin). When the stems appear, the explants are transferred to an MS rooting medium (MSO with 0.6% agar, 500 mg / ml of carbenicillin and 100 mg / ml of kanamycin). The feet emerging from the explants and having well-defined stems are cut and transplanted into a new rooting medium. The seedlings which develop roots for around 2 weeks in the rooting medium are then transferred to sterile soil in Jiffy pots and placed in boxes

of sterilized Magenta.

  After 7 to 10 days, the lids of the Magenta boxes are reopened to acclimatize the plants. The plants are then transferred to pots for two weeks, then to larger pots and grown in a greenhouse under natural light. They are watered daily with a 20:20:20 solution (N / K / P). The average temperature is around 32 C. Cultures nourishing tobacco cells (Nicotiana tabacum var. Xanthii) are used during the co-culture of the explants with the Agrobacterium bacteria in order to improve the transformation efficiency. Culture suspensions are maintained in Erlenmeyer flasks containing 50 ml of culture suspension (4.3 g / l of MS salt, 30 g / l of sucrose, 5 ml / l of vitamin B5, 1 mg / l of 2,4-D). The cultures are stored at room temperature with constant stirring of 150 revolutions / minute. The cell suspensions are maintained by making subcultures of 10

  ml of culture, filtered and washed, in 50 ml of medium.

  The expression of the gene coding for P-50 is demonstrated by measuring the RNA level by a transfer analysis.

Northern blot.

  The expression of P-50 in tobacco plants is analyzed using the method described by Sanchez-Serrano

et al. (1987, EMBO J. 6, 303-306).

  Dialysis tongs are placed on leaves of tobacco plants 60 to 80 cm high. The RNAs are isolated from the leaves of the plants on which the dialysis tongs were placed, 6, 12 and 24 hours after installation. The Poly RNAs (A) are isolated using the FastTrackTM oligo dT cellulose kit (sold by Invitrogen). 10 μg per sample are loaded onto the gel. The DNA insert

  complementary to the P-50 of 1200 bp is used as a probe.

  Total proteins are extracted from tobacco leaves with a solution containing 80 mM tris-HCl (pH 6.8), 2% SDS, 10% glycerol and are separated on 13% denaturing polyacrylamide gels, at a rate of 20 Mg. total protein per sample. The proteins are then transferred to an Immobilon-P membrane sold by Millipore. The membrane is incubated with an antiserum directed against P-50 in

  using conventional Western analysis techniques.

  The detection of immunoreactive proteins is carried out in

  using the Amersham ECL luminescence technique.

  2) - RESISTANCE OF PLANTS OBTAINED AGAINST A

FUNGAL INFECTION

  Phytophtera syringae, which is very virulent at

  Tobacco is used in inoculation tests.

  Zoospores suspended in water at different cell densities are used as an inoculum. Young tobacco plants resistant to kanamycin are obtained from seeds of the R1 generation, either controls or derived from transgenic plants for P50, and transplanted directly into pots containing artificial soil (Pro-Mix) and then cultivated in a greenhouse. Control plants are transgenic for the transfer vector not containing

insert P-50.

  The plants used in the fungal infection test are grown in small pots until they reach a height of about 10 cm, then the border of 4 to 5 leaves is cut. . All the roots on the same side of the plant, halfway between the base and the wall of the pot, are cut. The pots are placed in saucers and 5 ml of zoospores suspended in water at the rate of 107 cells per ml are poured into the ground. The

  control plants receive only water.

  Plants are randomly arranged and coded to obtain single-blind conditions. The plants are incubated at 30 ° C in growth chambers and the soil is kept moist by soaking in saucers. The percentage of notched leaves that wither away is noted for each plant, each day, and the average percentage of leaves that have perished depending on each treatment is calculated. When all the notched leaves

  have perished, the plant is considered to have perished

  even. New leaves that appear during the test are not counted even if they appear to wither away considerably. Results are analyzed using the Wilcoxon two sample test by comparing the values

  means between controls and transgenic plants.

  Another test is to put the inoculation test plants growing in pots until they reach a height of 15 cm and the edges of 8 to 10 leaves are notched as described above. Each of the plants receives 20 mm of an inoculum containing 106 cells / ml or ml of water applied via a knife wound made at the base. The scalloped leaves on each plant are estimated each day for their degree of decline. The percentage of leaves and plants having perished is calculated as in the case of the method

root inoculation.

  The process described in this example makes it possible to obtain

  plants resistant to fungal infections.

EXAMPLE 3:

NEUTRALIZATION OF ENDOTOXINS

  The complementary DNA of the P-50 protein is expressed in a eukaryotic or bacterial system according to the techniques

  described by Sambrook et al. (1989, previously cited). The P-

  is purified according to the techniques described by Das, (1990, Methods in Enzymology 182, 93-112) and Bradley (1990, Methods in Enzymology 182, 112-132). The purified P-50 obtained in this way is used in biological activity tests.

  ENDOTOXIN NEUTRALIZATION TEST

  pg of endotoxin of Escherichia coli 0113 are

  added to serial dilutions of the P-50 protein.

  The activity of the endotoxin is quantified by the turbidimetric test described by Novisky et al., (1985, J. Clin.

  Micro, 20, 211-216) for the anti-LPS factor of Limulus.

  Other samples of LPS are tested such as those of Klebsiella pneumoniae, Serratia marcesens and Serratia minnesota. The neutralization of these different species is carried out with a five-fold excess of P-50 by

report to the LPS.

SERUM NEUTRALIZATION TEST

  In order to test the potential efficacy of P-50 in vivo, the protein was tested for its ability to inactivate

  endotoxin in the presence of a total rat serum.

  A test is carried out in 96-well plates as

  described by Novitsky et al. (previously cited).

  0.05 ml of serum or 0.5 ml of serum with 25 mg / ml of P-50 is added in order to each well, then 0.5 ml

  endotoxin from Escherichia coli 0113.

  The plates are covered with a Parafilm film to avoid evaporation, shaken on a mechanical shaking platform for 15 minutes and incubated at 37 ° C

for 1 hour.

  The plastic film covering the plates is removed and then 0.1 ml of LAL is added to each well which is tested as

described above.

  The LR50 is defined as being half of the maximum growth of the optical density compared to the

witness.

EXAMPLE 4:

  Protection of animals with the P-50 protein Male Sprague-Dawley rats (230-450g) are lightly anesthetized with Halothane. They are then injected with a test solution through the vein

dorsal of the penis.

  The control rats receive a solution containing a phosphatesaline buffer (0.15 NaCl) buffered to pH 7.4 and containing 0.02 M of sodium phosphate. A second group of rats receives LPS (E. coli 0111: B4, Sigma Chemical Company)

in buffer.

  A third group of rats is tested with a suspension of protein P50 and LPS mixed in equal amounts by weight in buffer and incubated for 1

hour at 37 C before injection.

  A fourth group of rats receives albumin mixed with LPS in equal amounts by weight and

incubated in a similar manner.

  The last group of rats is treated by injection with

  only P-50 incubated in buffer.

  The volume injected (1.7 - 2.9 ml) depends on the weight of the rat which is adjusted so as to provide a dose of 15 mg of LPS per

body kilo.

  The survival of the rats is followed for 24 hours.

  The method described above makes it possible to demonstrate the therapeutic activity of the P50 protein, on a

representative animal model.

  Conclusion The results of these four examples highlight the remarkable properties of the P50 protein and its

  pharmaceutical and agronomic therapeutic applications.

LIST OF SEQUENCES

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  (ii) TITLE OF THE INVENTION: Proteins binding the lipopolysaccharide complex (LPS), and their uses (iii) NUMBER OF SEQUENCES: 5 (iv) COMPUTER-READABLE FORM: (A) TYPE OF MEDIUM: Floppy disk (B) COMPUTER : IBM PC compatible

  (C) OPERATING SYSTEM: PC-DOS / MS-DOS

  (D) SOFTWARE: PatentIn Release $ 1.0, Version X1.25 (EPO)

  (2) INFORMATION FOR SEQ ID NO: 1:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGIN: (A) ORGANIZATION: Bombyx mori

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:

  Arg Ile Ser Val Gln Asp Val Pro Lys Met Thr Leu Phe Ala Phe Gln

1 5 10 15

Gly Asn Leu

  (2) INFORMATION FOR SEQ ID NO: 2:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGIN: (A) ORGANIZATION: Bombyx mori

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:

  Phe His Thr Tyr Ser Val Gln? Rp Thr Pro Asp Phe Ile Ala Leu Ser

1 5 10 15

Val Asp Gly

  (2) INFORMATION FOR SEQ ID NO: 3:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 455 amino acids (B) TYPE: amino acid (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGIN: (A) ORGANIZATION: Bombyx mori

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:

  Lys Ile Ser Tyr Ala Gln Met Pro Asp Vai Lys Ile Gln Ala Phe Arg

1 5 10 15

  Pro Lys Gly Leu Arg Ile Ser Val Gln Asp Val Pro Lys Met Thr Leu

25 30

  Phe Ala Phe Gln Glv Asn Leu Asn His Lys Leu Asp Ser Thr Ser Val

40 45

  Gly Thr Leu Ser Ala Glu Val Leu AsD Pro Val Asn Gly Arg Trp Val

55 50

  Tyr Glu Glu Pro Asp Leu Lys Leu ys Val Lys Asp Val Val Tyr Tyr

70 75 80

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  AID Od o ah sTH UID UID nesz elyV us eli STH nah- USV AID ds

SLI OLI 991

  T1V J.141 1a9 IA 2L [1 na- us S usV sb. UlD zAl 2aS leA a4d o0d IA1 091 991 091 9t1 nID oad sTH 2A1 al1 od alI zAl UTD niD al UID d.zú 1eA uSV nlD 0t1 úE1 OE0 dsv naf% 6aS dsv a14d UID nID niV aD al 21

SZI 0Z1 9S1

  sAI AID AID bl. TeA 1 D2aV V 14 sAz o141i O s a sAD nlD Old sXIq UID

011 901 001

  2aS dsv 1qL jaS 21v USV o -d as. nId nea n h 14d 1 12f lea 1qL atd UID

96 06 98

  UlD US at1L SAT nlO aAa 2li sAz SAT USV ali.aS a64d leA 21V USV Pro Phe Asp Asp His Phe Tyr Ile Thr Leu Gly Val Ala Ala Gly Gly

385 390 395 400

  Ile Thr Glu Phe Arg Asp Gly Ser Ile Thr Ser Gly Gly Val Thr Lys

405 410 415

  Pro Trp Arg Asp Ser Ala Arg Lys Ala Ser Val His Phe Trp Arg His

420 425 430

  Met Ser Asp Trp Phe Pro Arg Trp Ser Gln Pro Ser Leu Ile Val Asp

435 440 445

Phe Val Lys Val Ile Ala Leu

450 455

  (2) INFORMATION FOR SEQ ID NO: 4:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2271 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: double (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA for miRNA (iii ) HYPOTHETIC: NO (vi) ORIGIN: (A) ORGANISM: Bombyx mori (ix) ADDITIONAL CHARACTERISTICS:

(A) NAME / KEY: CDS

(B) LOCATION: 16..1415

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:

  GAGGATCCGG GTACC ATG GGT GGA CGC GTG.TG TGT CTG ATT TTA TTT ATA 51

  Met Gly Gly Arg Val Leu Cys Leu Ile Leu Phe Ile

1 5 10

  AAA ATA TCG TAC GCT CAA ATG CCC GAT GTG.AkA ATA CAA GCT TTT CGC 99 Lys Ile Ser Tyr Ala Gln Met Pro Asp Val Lys Ile Gln Ala Phe Arg

20 25

  CCG AAA GGA CTT CGA ATA TCC GTT CAA GAT GTC CCC AAA ATG ACA CTG 147

  Pro Lys Gly Leu Arg Ile Ser Val Gln Asp Val Pro Lys Met Thr Leu

35 40

  TTC GCG TTC CAA GGC AAC TTG AAC CAT AAA CTG GAC AGC ACC AGC GTC 195

  Phe Ala Phe Gln Gly Asn Leu Asn His Lys Leu Asp Ser Thr Ser Val

50 55 60

  GGG ACA CTG AGT GCA GAG GTA CTG GAT CCA GTC AAC GGC CGA TGG GTG 243

  Gly Thr Leu Ser Ala Glu Val Leu Aso Pro Val Asn Gly Arg Trp Val

70 75

00ú 55Z 06Z 58Z

  AID 52V PIV sXD Pi a I s .; l :. LeA AID ias eP '2AI us We ias 516 LDD DDD DDD ID- DDD VI D -D'D VED DDD DDl DDD LVI IVV DIEV iD 0OL AID 7Aî sAX sA- nea, ae ca f hi nath na aTI nID oid iAI nae dil

  L98 VDD LVI DV DV 'I DI 9E D'D D LS D D L VD L V VVD DDD IV DID DDL

59Z O9Z 5

  dsV AID UID ozc n2l sAq TI z aTI nID TiA 141 AID 7AI q4L aad 618 DVD DDD VVD DD '' lt VV 3D- D DLV VD DID VDV VDD DVI DD IIIL eIV a4d AID aiI 2aS -L s az e AiTD îas LS A aII o0d O2d na aTI I LL VDD ILi VDD VI DI VDV DI '* D' DD9 DD DV DLD DVL VDD VDD VID LIV dsV eIV AID aS rT uTD si. C- sAD eT nID h IV las jas aS q sAD úZL LVD IDD IDD DDI & D, DVD Vf DIV DDI VDD DVD VDD VDI DDL IDV LDL

OZZ 51 Z 0 1 Z N / A

  AID.iS a4d n7 us na 141 LiTD îaS îAi alI îaS dsV dsV na 'aqd 9L9 99DDD IDE DII DIT iVV ITD fD- DD3 LD DI VI VV LDI DVD DVD DLD LiL

0 961 061

  AID oîd qaK sTH UI UiD n 9- sL uIV uSV 91I STH ne- usv AID dsV LZ9 DDD DDD DIV iD YvD VrD LID VVf DDD DVV lv iVD VID DVV VDD IVD

981 081 SLI

  PIV qLI îaS T / 2li4 ne- us us, 52V UiD 1Aî -'S TeA 8a4d Od îAI 6L9 VDD DDV LDL VID VDV ILI IVL 'Il ILDD DVD DVIL DDI DID DIL DDD DVI

OLI 991,091

  nid old STH JAi alI o: d a-: A: 1 ulD nest aiI UID dîL TeA usv nhi 1ú5 EVVD DDD D-D IlV 11V DDD 'VL IVI DYD DVD DIV VVD DDL LID DVV VVD

551 05 5 L 1

  dsV nea :: ras ds a4a uiD niD niD aqd aII jqL UlD AID pl sAD vIV ú8bt DVD DID DDI IYD LLi WVD DVD DYD DLI LV VD VD 'D VDD DDD IDL 3DD 0 t, 1 5D 0 E1 SZI sAq AID AID bi îa 61f ql- s7 14, L o2d SA 'sAD nID O d sA'I UlD 58 WVVV DDD DDD VDD DILD VDV 9D' Dv VDV VDD DVV IDL VD VDD VVV YD OZI Sll 011 zeS ds U4L niS us UsV oCa dsd nI D aia a IPA q & J aqd UID L88 IDI IVLD VDV DDV YDD LVY lDD _'D VVD VeD DVD VDV VID DDV DII SYD

501 001 96

  UID USV 2qL sAX nld Az aTl sA-r sA 'usv ai: isS aud IPA eIV usV 6úú VVD DVV vDv VV DYD DVL VIY VVY DVV DVV DIV VDL DL DID DDD DVV

06 98 08

  JA & .A TVA TîA dse sA 'A. sA' ne- sA 'nae dsv o0d nID nlD.AI 16Z V'i Livi DID DLD DYD DV'f Di VVV & D Vv ILID V D DDD DVD VVD Dvil 9E

  AAT GCA GAA CTC TAT TCC GGA CCT AAT GAC TAC AGC AAT ACG GTT TTG 963

  Asn Ala Glu Leu Tyr Ser Gly Pro Asn Asp Tyr Ser Asn Thr Val Leu

305 310 315

  TAC GGA GGA CCG ATC ATG GAT CTG GAG TGC CGC GAG AAC TTT CTC TCC 1011

  Tyr Gly Gly Pro Ile Met Asp Leu Glu Cys Arg Glu Asn Phe Leu Ser

320 325 330

  ACA AAA AGA CGC AGA GAC GGC ACG TCG TGG GGC GAC AGT TTT CAT ACA 1059

  Thr Lys Arg Arg Arg Asp Gly Thr Ser Trp Gly Asp Ser Phe His Thr

335 340 345

  TAT TCC GTT CAA TGG ACA CCT GAT TTC ATA GCC CTG TCT GTG GAC GGC 1107

  Tyr Ser Val Gin Trp Thr Pro Asp Phe Ile Ala Leu Ser Val Asp Gly

350 355 360

  GAG GAG TGG GCG CGA GTG GAG GCG CCG CGG GAC GCC CTG CCG GCT GTC 1155

  Glu Glu Trp Ala Arg Val Glu Ala Pro Arg Asp Ala Leu Pro Ala Val

365 370 375 380

  TGC GCG CAC GCC CCG CGG CAC CTG CTG CAG GCC GGC TCG CAG ATG GCG 1203

  Cys Ala His Ala Pro Arg His Leu Leu Gln Ala Gly Ser Gln Met Ala

385 390 395

  CCA TTC GAT GAC CAC TTC TAC ATA ACG CTA GGC GTG GCG GCA GGA GGC 1251

  Pro Phe Asp Asp His Phe Tyr Ile Thr Leu Gly Val Ala Ala Gly Gly

400 405 410

  ATA ACG GAG TTC CGC GAC GGG TCT ATA ACC TCC GGG GGA GTC ACC AAG 1299

  Ile Thr Glu Phe Arg Asp Gly Ser Ile Thr Ser Gly Gly Val Thr Lys

415 420 425

  CCC TGG AGA GAC AGC GCT CGG AAG GCA TCC GTA CAT TTC TGG CGG CAC 1347

  Pro Trp Arg Asp Ser Ala Arg Lys Ala Ser Val His Phe Trp Arg His

430 435 440

  ATG TCC GAT TGG TTC CCA CGG TGG AGT CAG CCA TCT TTA ATC GTG GAC 1395

  Met Ser Asp Trp Phe Pro Arg Trp Ser Gln Pro Ser Leu Ile Val Asp

445 450 455 460

  TTT GTC AAA GTT ATC GCC TTA TGATCTAG7C ATTTAGATTT TGTATAATTA 1446

Phe Val Lys Val Ile Ala Leu

  AGTTATTAGT ATAGTTTCTG CAGGCAAAAT TAATTGCATT CTGAGTTTTT ATTGTCAAAC 1506

  TACAATTTAA AATTTAATTC CTAAAAGATC TCTCTTCT-TT CTAAATCTGT AATTTAGCTA 1566

  ATTTTATTTG TATCATTTTC TTTTTTTAAA TTTTGATAGA GCGGCGAATG ACTAGAAGAT 1626

  GTGATCTGCG.AAATAAAAC TAAGTGACAT GC-CATLGC.AAG GCATACACAC AACAGAGTTT 1686

  TGCATTATTA TACCTTTCT TGAGCGAGAT GAGGACTACT GACGTGACCT GGAGTGATAT 1746

  TTTGGAGCAA GCCTAAGACA GGGTCAAGTG GAGACAGCTC TAAGGCCCCT ATGTACCGGC 1806

  GGGGTGTCTT AGGGCTGCAA TAACAACATT ACC-TTCTA ACCAAACAGC GATATTATCT 1866

  GTAATATTAT AGTTAGAAAG CCTGCATCTT ATACTCTTAT TGACACACGT ACCTGGATGA 1926

  TTTTAACGAT GAAGAAATAA CATCGTGTAA AAAATTTAGC TGGAAAAGAT TTTGAGGAAT 1986

  CAAGAAATAC CCTATGAACC AGCACAGGTA CGTGTCACCA CTCTGGCTAA TTCTGCCACG 2046

  TTGCGGTTGG TTTGAAGTTT GAGACAACCT TTGCACGATA CAATTGAGAC TAAGACCTCA 2106

  TGGCTCGGAG TGAGTGGAGT CATTCAAGTT GCGACTGGCT CCAGTAACCA CTTATAACCA 2166

  GGTAGATCGT GAGCCTATCA GCTCATTTAG AGCAACAAAA AACAAAATAA TGTACTCTTG 2226

  AAAAAATCAT TTTAATAAAA GACCAGAGTG AAAAAAAAAA AAAAA 2271

  (2) INFORMATION FOR SEQ ID NO: 5:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 467 amino acids (B) TYPE: amino acid (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: protein

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:

  Met Gly Giy Arg Val Leu Cys Leu Ile Leu Phe Ile Lys Ile Ser Tyr

1 5 10 15

  Ala Gln Met Pro Asp Val Lys Ile Gln Ala Phe Arg Pro Lys Gly Leu

25 30

  Arg Ile Ser Val Gln Asp Val Pro Lys Met Thr Leu Phe Ala Phe Gln

40 45

  Gly Asn Leu Asn His Lys Leu Asp Ser Thr Ser Val Gly Thr Leu Ser

55 60

  Ala Glu Val Leu Asp Pro Val Asn Gly Arg Trp Val Tyr Glu Glu Pro

70 75 80

  Asp Leu Lys Leu Lys Val Lys Asp Val Val Tyr Tyr Asn Ala Val Phe

90 95

  Ser Ile Asn Lys Lys Ile Tyr Glu Lys Thr Asn Gln Gln Phe Thr Val

105 110

  Thr Glu Leu Glu Asp Pro Asn Ala Ser Thr Asp Ser Gin Lys Pro Glu

120 125

  Cys Lys Pro Thr Lys Thr Arg Val Arg Gly Gly Lys Ala Cys Ala Gly

135 140

  Gln Thr Ile Phe Glu Glu Gin Phe Asp Ser Leu Asp Glu Asn Val Trp

150 155 160

  dsV 6z dili od SÀ7 ji _7. ff I7D jaS.WL alI u as AID dsv 6z Su'0 aqd nhid. alI a11 D TD P- T ilA TD narIa zI all zIL aUd sTH

00O 56ú 06ú 58E

  dsv dsV aqd Old PTV; S: ui as LITD Pl Ul nal ne1 sTH 51v ozd

08E SLE OLE

  PIV STH PTV sAD IPA PT o: _c na PIV dsv bV o: d PIV nIo IPA úIV

S9ú 09úE SE

  vIV dL nid nid AID dsV e :, aS na-ï ela aII ad dsv oid j LI d: j, OSE 't 0 úOb UI [ITA jaS 2A J9, J STH aqd: as ds AID d: aS qi, AID dsv 5: V

Súú 0úú SZú

  DrV bzr sXq zjq jas nsa ayc usv nhi klv SAD nT naq dsV qaW al 0Zú SlE OúE 01O oid AIE AIS i rI nel ITA: ry usv 9as 2AL dsV usV old A I jaS AiL

00E 56Z 065

  na% niD eLV usv AIO 52 jv s7D el alI s I A IPA XAI laS PIW

58Z 08Z SLZ

  X, us' q; aN jaS aIu IAl sL? sÀ na, aqd o.d nIu nar nat alI nIE

OLZ 59Z 09Z

  o0d: X j na 'd2, dsv AID ulD Cod na, ski IV b: V aTI nID iPA rqL SSZ 05Z Sb5Z ID aI ..ZqjI a14d VlV aqd A1i ai- aas Z: e senior 5V LIT jaS IPA alI ObZ SúcZ OúZ SZZ old od na1 alI dsv pT peT L z: as pT uTD s% aTI SAD PIV nie p V

OZZ 51Z 01Z

  aS aS jas l4J SD IS 1aS ae4j nae us nael aIq AID iaS aXúL alI u aS

SOZ 00 561

  dsv dsV nal a 'd ITD oad aX; - STH UlD ulD nae sÀl i1v us alI STH

061 581 081

  nfl usV AiE ds wlv pv. oS iea A T., L nao usV usV 6zV ule zAL jas

SLI OLI 91

  IPA aMd 0ol X h od si nie d STh eal oid alI iL UlD nli alI uIE 6E 99b naeevi alI TeA sA'I TA aqd ds lzA eaI nae aS O d uID eas daI 5bv oa and 9bb Ott 9út djI dsV jeS qap; sTH 5r dl- eia sTH TPA: aS PIV sÀZ L: V PIV eaS Ot

Claims (20)

  1. Protein characterized in that it comprises in whole or in part the following sequence SEQ ID N'1: Arg Ile Ser Val Gln Asp Val Pro Lys Met Thr Leu Phe Ala Phe Gln Gly Asn Leu. 2. Protein characterized in that it comprises in whole or in part the following sequence SEQ ID N'2: Phe His Thr Tyr Ser Val Gln Trp Thr Pro Asp Phe Ile Ala Leu Ser Val Asp Gly.   3. Protein according to one of claims 1 or 2   characterized in that it has a molecular weight of around 50 kD.   4. Protein according to one of claims 1 to 3,   characterized in that it comprises the sequence SEQ ID N 3, or part of the following sequence SEQ ID N 3 Lys Ile Ser Tyr Ala Gln Met Pro Asp Val Lys Ile Gln Ala Phe Arg Pro Lys Gly Leu Arg Ile Ser Val Gln Asp Val Pro Lys Met Thr Leu Phe Ala Phe Gln Gly Asn Leu Asn His Lys Leu Asp Ser Thr Ser Val Gly Thr Leu Ser Ala Glu Val Leu Asp Pro Val Asn Gly Arg Trp Val Tyr Glu Glu Pro Asp Leu Lys Leu Lys Val Lys Asp Val Val Tyr Tyr Asn Ala Val Phe Ser Ile Asn Lys Lys Ile Tyr Glu Lys Thr Asn Gln Gln Phe Thr Val Thr Glu Leu Glu Asp Pro Asn Ala Ser Thr Asp Ser Gln Lys Pro Glu Cys Lys Pro Thr Lys Thr Arg Val Arg Gly Gly Lys Ala Cys Ala Gly Gln Thr Ile Phe Glu Glu Gln Phe Asp Ser Leu Asp Glu Asn Val Trp Gln Ile Glu Gln Tyr Ile Pro Ile Tyr His Pro Glu Tyr Pro Phe Val Ser Tyr Gln Arg Asn Asn Leu Thr Val Ser Thr Ala Asp Gly Asn Leu His Ile Asn Ala Lys Leu Gln Gln His Met Pro Gly Phe Leu Asp Asp Ser Ile Tyr Ser Gly Thr Leu Asn Leu Phe Ser Gly Cys Thr Ser Ser Ala Glu Ala Cys Ile Lys Gln Ala Ser Gly Ala Asp Ile Leu Pro Pro Ile Val Ser Gly Arg Ile Thr Ser Ile Gly Phe Ala Phe Thr Tyr Gly Thr Val Glu Ile Arg Ala Lys Leu Pro Gln Gly Asp Trp Leu Tyr Pro Glu Ile Leu Glu Pro Phe Leu Lys Lys Tyr Gly Ser Met Asn Tyr Ala Ser Gly Val Val Lys Ile Ala Cys Ala Arg Gly Asn Ala Glu Leu Tyr Ser Gly Pro Asn Asp Tyr Ser Asn Thr Val Leu Tyr Gly Gly Pro Ile Met Asp Leu Glu Cys Arg Glu Asn Phe Leu Ser Thr Lys Arg Arg Arg Asp Gly Thr Ser Trp Gly Asp Ser Phe His Thr Tyr Ser Val Gln Trp Thr Pro Asp Phe Ile Ala Leu Ser Val Asp Gly Glu Glu Trp Ala Arg Val Glu Ala Pro Arg Asp Ala Leu Pro Ala Val Cys Ala His Ala Pro Arg His Leu Leu Gln Ala Gly Ser Gln Met Ala Pro Phe Asp Asp His Phe Tyr Ile Thr Leu Gly Val Ala Ala Gly Gly Ile Thr Glu Phe Arg Asp Gly Ser Ile Thr Ser Gly Gly Val Thr Lys Pro Trp Arg Asp Ser Ala Arg Lys Ala Ser Val His Phe Trp Arg His Met Ser Asp Trp Phe Pro Arg Trp Ser Gln Pro Ser Leu Ile Val Asp Phe Val Lys Val Ile Ala Leu   5. Proteins according to one of claims 1 to 3,   having the sequence ID N 5 or part of the following sequence SEQ ID N'5: Met Gly Gly Arg Val Leu Cys Leu Ile Leu Phe Ile Lys Ile Ser Tyr Ala Gln Met Pro Asp Val Lys Ile Gln Ala Phe Arg Pro Lys Gly Leu Arg Ile Ser Val Gln Asp Val Pro Lys Met Thr Leu Phe Ala Phe Gln Gly Asn Leu Asn His Lys Leu Asp Ser Thr Ser Val Gly Thr Leu Ser Ala Glu Val Leu Asp Pro Val Asn Gly Arg Trp Val Tyr Glu Glu Pro Asp Leu Lys Leu Lys Val Lys Asp Val Val Tyr Tyr Asn Ala Val Phe Ser Ile Asn Lys Lys Ile Tyr Glu Lys Thr Asn Gln Gln Phe Thr Val Thr Glu Leu Glu Asp Pro Asn Ala Ser Thr Asp Ser Gln Lys Pro Glu Cys Lys Pro Thr Lys Thr Arg Val Arg Gly Gly Lys Ala Cys Ala Gly Gln Thr Ile Phe Glu Glu Gln Phe Asp Ser Leu Asp Glu Asn Val Trp Gln Ile Glu Gln Tyr Ile Pro Ile Tyr His Pro Glu Tyr Pro Phe Val Ser Tyr Gln Arg Asn Asn Leu Thr Val Ser Thr Ala Asp Gly Asn Leu His Ile Asn Ala Lys Leu Gin Gln His Met Pro Gly Phe Leu Asp Asp Ser Ile Tyr Ser Gly Thr Leu Asn Leu Phe Ser Gly Cys Thr Ser Ser Ala Glu Ala Cys Ile L ys Gln Ala Ser Gly Ala Asp Ile Leu Pro Pro Ile Val Ser Gly Arg Ile Thr Ser Ile Gly Phe Ala Phe Thr Tyr Gly Thr Val Glu Ile Arg Ala Lys Leu Pro Gln Gly Asp Trp Leu Tyr Pro Glu Ile Leu Leu Glu Pro Phe Leu Lys Lys Tyr Gly Ser Met Asn Tyr Ala Ser Gly Val Val Lys Ile Ala Cys Ala Arg Gly Asn Ala Glu Leu Tyr Ser Gly Pro Asn Asp Tyr Ser Asn Thr Val Leu Tyr Gly Gly Pro Ile Met Asp Leu Glu Cys Arg Glu Asn Phe Leu Ser Thr Lys Arg Arg Arg Asp Gly Thr Ser Trp Gly Asp Ser Phe His Thr Tyr Ser Val Gln Trp Thr Pro Asp Phe Ile Ala Leu Ser Val Asp Gly Glu Glu Trp Ala Arg Val Glu Ala Pro Arg Asp Ala Leu Pro Ala Val Cys Ala His Ala Pro Arg His Leu Leu Gln Ala Gly Ser Gln Met Ala Pro Phe Asp Asp His Phe Tyr Ile Thr Leu Gly Val Ala Ala Gly Gly Ile Thr Glu Phe Arg Asp Gly Ser Ile Thr Ser Gly Gly Val Thr Lys Pro Trp Arg Asp Ser Ala Arg Lys Ala Ser Val His Phe Trp Arg His Met Ser Asp Trp Phe Pro Arg Trp Ser Gln Pro Ser Leu Ile Val Asp Phe Val Lys Val Ile Ala Leu 6. Protein according to claim 5, characterized in   that it has a glycosylation in position 182.   7. Fragment of a protein according to one of claims 1 to 6.   8. Oligonucleotide coding at least for the protein or   one of its fragments according to one of claims i & 7.   9. DNA according to claim 8 comprising the following sequence SEQ ID N'4:   GAGGATCCGG GTACC ATG GGT GGA CGC GTG TTG TGT CTG ATT TTA TTT   ATA AAA ATA TCG TAC GCT CAA ATG CCC GAT GTG AAA ATA CAA GCT   TTT CGC CCG AAA GGA CTT CGA ATA TCC GTT CAA GAT GTC CCC AAA   ATG ACA CTG TTC GCG TTC CAA GGC AAC TTG AAC CAT AAA CTG GAC   AGC ACC AGC GTC GGG ACA CTG AGT GCA GAG GTA CTG GAT CCA GTC   AAC GGC CGA TGG GTG TAC GAA GAG CCC GAT CTT AAA CTA AAA GTC   AAG GAC GTC GTC TAT TAT AAC GCG GTC TTC TCA ATC AAC AAG AAA   ATA TAC GAG AAA ACA AAC CAA CAG TTC ACC GTA ACA GAG CTA GAA   GAT CCT AAT GCA AGC ACA GAT TCT CAG AAA CCA GAA TGT AAG CCA   ACA AAG ACG AGA GTG CGA GGC GGC AAA GCG TGT GCC GGA CAA ACA   ATA TTC GAG GAG CAA TTT GAT TCC CTG GAC GAA AAC GTT TGG CAA   ATC GAG CAG TAT ATA CCG ATT TAT CAC CCC GAA TAC CCC TTC GTG   TCC TAC CAG CGT AAT AAT TTA ACA GTA TCT ACC GCA GAT GGA AAC   CTA CAT ATT AAC GCC AAA CTT CAA CAA CAT ATG CCC GGC TTT CTG   GAC GAC TCT ATA TAT TCT GGC ACA CTT AAT TTG TTC AGT GGG TGT   ACT TCG TCA GCA GAG GCA TGC ATC AAA CAG GCT TCC GGT GCT GAT   ATT CTA CCA CCA ATC GTC AGC GGC AGA ATC ACA TCA ATA GGA TTT   GCA TTT ACG TAC GGA ACA GTC GAA ATC AGA GCG AAA TTA CCG CAA   GGG GAC TGG CTG TAT CCG GAA ATT CTA TTG GAG CCG TTC TTA AAG   AAG TAT GGA AGT ATG AAT TAT GCG TCC GGC GTA GTG AAG ATA GCG   TGT GCG CGC GGT AAT GCA GAA CTC TAT TCC GGA CCT AAT GAC TAC   AGC AAT ACG GTT TTG TAC GGA GGA CCG ATC ATG GAT CTG GAG TGC   CGC GAG AAC TTT CTC TCC ACA AAA AGA CGC AGA GAC GGC ACG TCG   TGG GGC GAC AGT TTT CAT ACA TAT TCC GTT CAA TGG ACA CCT GAT   TTC ATA GCC CTG TCT GTG GAC GGC GAG GAG TGG GCG CGA GTG GAG   GCG CCG CGG GAC GCC CTG CCG GCT GTC TGC GCG CAC GCC CCG CGG   CAC CTG CTG CAG GCC GGC TCG CAG ATG GCG CCA TTC GAT GAC CAC   TTC TAC ATA ACG CTA GGC GTG GCG GCA GGA GGC ATA ACG GAG TTC   CGC GAC GGG TCT ATA ACC TCC GGG GGA GTC ACC AAG CCC TGG AGA   GAC AGC GCT CGG AAG GCA TCC GTA CAT TTC TGG CGG CAC ATG TCC   GAT TGG TTC CCA CGG TGG AGT CAG CCA TCT TTA ATC GTG GAC TTT   GTC AAA GTT ATC GCC TTA TGATCTAGTC ATTTAGATTT TGTATAATTA   AGTTATTAGT ATAGTTTCTG CAGGCAAAAT TAATTGCATT CTGAGTTTTT   ATTGTCAAAC TACAATTTAA AATTTAATTC CTAAAAGATC TCTCTTCTTT   CTAAATCTGT AATTTAGCTA ATTTTATTTG TATCATTTTC TTTTTTTAAA   TTTTGATAGA GCGGCGAATG ACTAGAAGAT GTGATCTGCG AAAATAAAAC   TAAGTGACAT GCATGCAAAG GCATACACAC AACAGAGTTT TGCATTATTA   TTACCTTTCT TGAGCGAGAT GAGGACTACT GACGTGACCT GGAGTGATAT   TTTGGAGCAA GCCTAAGACA GGGTCAAGTG GAGACAGCTC TAAGGCCCCT   ATGTACCGGC GGGGTGTCTT AGGGCTGCAA TAACAACATT ACCTTTCTTA   ACCAAACAGC GATATTATCT GTAATATTAT AGTTAGAAAG CCTGCATCTT   ATACTCTTAT TGACACACGT ACCTGGATGA TTTTAACGAT GAAGAAATAA   CATCGTGTAA AAAATTTAGC TGGAAAAGAT TTTGAGGAAT CAAGAAATAC   CCTATGAACC AGCACAGGTA CGTGTCACCA CTCTGGCTAA TTCTGCCACG   TTGCGGTTGG TTTGAAGTTT GAGACAACCT TTGCACGATA CAATTGAGAC   TAAGACCTCA TGGCTCGGAG TGAGTGGAGT CATTCAAGTT GCGACTGGCT   CCAGTAACCA CTTATAACCA GGTAGATCGT GAGCCTATCA GCTCATTTAG   AGCAACAAAA AACAAAATAA TGTACTCTTG AAAAAATCAT TTTAATAAAA GACCAGAGTG AAAAAAAAAAAA AAAAA   10. Eukaryotic or prokaryotic cell expressing a protein or one of its fragments according to one of the claims 1 to 7.   11. Eukaryotic or prokaryotic cell carrying at least   part of an oligonucleotide according to one of claims 8 and 9.   12. Plant or bacterial cell according to one of the claims 10 and 11.   13. Plant cell according to claim 12, characterized in that it comes from a plant belonging to the families of the vine, tobacco, tomato or potato.   14. Method for obtaining a protein or one of its   fragments according to one of claims 1 to 7, characterized in   what an oligonucleotide according to one of claims 8 and 9   is expressed in a suitable prokaryotic or eukaryotic cell.   15. Medicine containing a protein or one of its proteins   fragments according to one of claims 1 to 7 or obtained according to claim 14.   16. Use of a protein or one of its proteins   fragments according to one of claims 1 to 7 for the   manufacture of a medicament for the treatment of ailments   linked to the lipopolysaccharide complex.   17. Use according to claim 16 for treating septic shock.   18. Method for removing the lipopoly complex   saccharide of preparations containing it, said process consisting in binding the lipopolysaccharide complex to a protein or to one of its fragments according to one of claims 1 to 7.   19. The method of claim 18, characterized in that the protein binding the lipopolysaccharide complex is fixed on a support.   20. Use of fragments of one of the oligonucleotides   according to claim 8 for gene amplification.