WO2003097833A2

WO2003097833A2 - Method for the functional artificial evolution of polynucleotide sequences, recombinant polynucleotides obtained and peptides, polypeptides and proteins coded by same

Info

Publication number: WO2003097833A2
Application number: PCT/FR2003/001405
Authority: WO
Inventors: Michèle Legrain
Original assignee: Entomed
Priority date: 2002-05-06
Filing date: 2003-05-06
Publication date: 2003-11-27
Also published as: AU2003260538A8; WO2003097833A3; AU2003260538A1; FR2839317A1

Abstract

The invention relates to a method of preparing recombinant double-stranded polynucleotides or peptides, polypeptides or proteins coded by same. The inventive method is characterised in that it comprises: (a) the assembly of overlapping single-stranded nucleotide sequences which cover the sequence of all or part of the polynucleotides coding for a source population of peptides, polypeptides or proteins, in order to obtain recombinant double-stranded polynucleotides, said recombinant double-stranded polynucleotides optionally comprising at at least one of the ends thereof a sequence for regulating the expression of a gene or a 3’ or 5’ part of said regulatory sequence; and (b) the introduction of complementary nucleotide sequences of a nucleotide sequence of a cloning and/or expression vector at the 3’ and/or 5’ ends of each of the recombinant double-stranded polynucleotides obtained in step (a). The invention also relates to recombinant double-stranded polynucleotides obtained, to peptides, polypeptides or proteins obtained which have one or more improved properties and to the uses of the inventive method.

Description

METHOD OF ARTIFICIAL FUNCTIONAL EVOLUTION OF SEQUENCES

POLYNUCLEOTIDES, RECOMBINANT POLYNUCLEOTIDES OBTAINED AND

PEPTIDES, POLYPEPTIDES AND PROTEINS ENCODED THEREBY

The subject of the present invention is a process for the preparation of recombinant double-stranded polynucleotides or the peptides, polypeptides or proteins for which they code. The invention also relates to the recombinant double-stranded polynucleotides obtained and the peptides, polypeptides or proteins obtained exhibiting one or more improved properties as well as to the uses of the method.

Various techniques have been described in the prior art for artificially accelerating the functional evolution of genes. Among the most widespread, we can cite cassette replacement mutagenesis, Erroreprone PCR and DNA shuffling.

Cassette replacement mutagenesis is a technique based on the replacement of a plasmid DNA fragment containing a portion of a wild-type sequence of interest with a DNA fragment (cassette) containing at least one mutation, desired or random. The mutant cassette is composed of 2 synthetic complementary oligonucleotides of predefined sequence (Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites, JA Wells, M. Vasser and DB Powers, Gene 34: 315-323 (1985)) or 2 degenerate complementary oligonucleotides or 2 doped complementary oligonucleotides (Recombinant DNA, Watson, Gilman, Witkowski and Zoller, Freeman 204-205 (1992)), so as to generate a library of mutant plasmids containing several different sequences. Error-prone PCR is a technique using the low degree of fidelity of DNA polymerases to introduce in small proportion mutations points along the amplified sequences (A method for random mutagenesis of a defined DNA fragment using a modified polymerase chain reaction, DW Leung, E. Chen and DV Goeddel, Technique 1: 11-15 (1989)).

DNA shuffling is a technique based on homologous in vi tro recombination of double-stranded polynucleotide sequences by random fragmentation using DNAse I and reassembly by PCR (DNA shuffling by random fragmentation and reassembly: In vi tro recombination for molecular evolution, WPC Stemmer, Proceedings of the national Academy of Sci enc es, USA 91: 10747-10751 (1994); DNA Shuffling of a family of genes from diverse species accelerantes directed evolution, A. Cremeri, S. Raillard, S. Bermudez and WPC Stemmer, Nature 391: 288-291 (1998); Protein evolution by molecular breeding, J. Minshull and WPC Stemmer, Current Opinion in Chemical Biology, 3: 284-290 (1999); WO 95/22625; EP 0 911 396; WO 00/09679).

Cassette replacement mutagenesis and errorprone PCR are techniques that quickly become limiting when one seeks to rapidly generate libraries of peptides or recombinant proteins with improved properties. These limitations are notably linked to the drawbacks caused by the repetition of the basic process which is required to ensure functional evolution (eg accumulation of neutral mutations). DNA shuffling is an artificial evolution technique which forces the user to isolate as well as to fragment beforehand the polynucleotides to be assembled. Furthermore, the fragmentation of the polynucleotides being random (DNAse I), a significant proportion of non-functional recombinant polynucleotides is inevitably generated. Indeed, the functional properties of a peptide, polypeptide or protein having a defined three-dimensional structure, are based on the spatial distribution of the chemical groups carrying the activity. Thus, the fragmentation of polynucleotides within sequences involved in elementary structural motifs is likely to alter the overall folding of the peptide, polypeptide or protein chain and consequently alter its functionality. Finally, the random fragmentation of small genes generates only a few fragments capable of reappearing during the assembly process. All of these drawbacks thus contribute to slowing down and making the artificial evolution of genes coding for peptides, polypeptides or functional proteins more complex.

The present invention aims precisely to overcome the above-mentioned drawbacks by proposing a simple and rapid method for preparing recombinant polynucleotides or peptides, polypeptides or proteins for which they code, said peptides, polypeptides or proteins advantageously having one or more improved properties. The expression peptides, polypeptides and proteins according to the present invention respectively means a chain of 2 to 20 amino acid residues, chain of 20 to 50 amino acid residues and chain of more than 50 amino acid residues.

The main object of the present invention therefore is a process for preparing double-stranded polynucleotides recombinant or peptides, polypeptides or proteins for which they code, characterized in that it comprises: a) the assembly of overlapping single-stranded nucleotide sequences which cover the sequence of all or part of polynucleotides encoding a starting population of peptides, polypeptides or proteins, to obtain recombinant double-stranded polynucleotides, said recombinant double-stranded polynucleotides optionally comprising, at at least one of their ends, a sequence for regulating the expression of a gene or a 3 'or 5' part of this regulatory sequence, b) the introduction at the 3 ′ and / or 5 ′ ends of each of the recombinant double-stranded polynucleotides obtained in step (a) of nucleotide sequences complementary to a nucleotide sequence of a cloning vector and / or expression.

At least one of the ends of said cloning and / or linearized expression vector may correspond to the 3 ′ or 5 ′ part of sequence (s) for regulating the expression of a gene including the 3 ′ or 5 part 'complementary corresponds to one of the ends of the recombined double-stranded polynucleotides obtained in step (b) of the process of the invention. The term “sequence (s) for regulating the expression of a gene according to the present invention is intended to mean sequences which regulate the transcription, translation and / or maturation of peptides, polypeptides or proteins such as a promoter, a sequence coding for a signal or transit peptide (pre sequences, pro sequence), a sequence coding for an endoprotease, or a terminator. All of these regulatory sequences are known to those skilled in the art and the latter is able to choose them according to the host organisms. in which the peptides, polypeptides or proteins encoded by the recombinant double-stranded polynucleotides obtained by the process of the invention are expressed.

In step (a) of the method of the invention, the recombinant double-stranded polynucleotides comprise at at least one of their ends all or part of a sequence for regulating the expression of a gene, which is provided to said double-stranded polynucleotides recombined by additional single-stranded nucleotide sequences present: i) either at one of the ends of the overlapping single-stranded nucleotide sequences constituting the ends of the recombined double-stranded polynucleotides, before assembly (a), ii) either, during assembly (a), in the form of one or more single-stranded nucleic acids, each consisting of a first and a second nucleic region, of which: - a first group consists of first regions complementary to the sequences overlapping single-stranded nucleotides constituting at least one of the ends of the recombined double-stranded polynucleotides, before assembly (a), and the second he regions of this first group correspond to all or part of a sequence for regulating the expression of a gene, - the second group, if present, consists of first regions and second regions, partly complementary second regions of the first group and for a second part complementary to each other, so as to form said sequence for regulating the expression of a gene or a part thereof, iii) either by the combination of (i ) and (ii). Each of the single-stranded nucleotide sequences used in step (a) of the process of the invention consists or comprises: an internal nucleotide segment coding for an amino acid sequence of one of the variable regions of one of the peptides, polypeptides or proteins of the starting population, said variable nucleotide segment being flanked in 5 ′ by a nucleotide segment coding for the amino acid sequence of all or part of any of the substantially conserved regions of the peptides, polypeptides or proteins of the starting population, which is immediately adjacent to the N terminus of a variable region, said nucleotide segment itself being optionally flanked 5 ′ by an additional single-stranded nucleotide sequence corresponding to all or part of a regulatory sequence for gene expression,

- in 3 ′ by a nucleotide segment coding for the amino acid sequence of all or part of any of the substantially conserved regions of the peptides, polypeptides or proteins of the starting population, which is immediately adjacent to the C-terminus d 'a variable region, said nucleotide segment itself being optionally flanked 3' of an additional single-stranded nucleotide sequence corresponding to all or part of a sequence for regulating the expression of a gene.

The peptides, polypeptides or proteins of the starting population can be natural peptides, polypeptides or proteins, more particularly, variants from different families, orders, genera or species or allelic variants.

The peptides, polypeptides or proteins of the starting population can be mutated peptides, polypeptides or proteins.

The peptides, polypeptides or proteins of the starting population are chosen from peptides, polypeptides or proteins of animal, plant, bacterial or viral origin. According to the present invention, the peptides, polypeptides or proteins of the starting population are preferably natural variants of peptides, polypeptides or insect proteins and / or peptides, polypeptides or mutated proteins derived from natural variants of peptides, polypeptides or insect proteins.

The peptides, polypeptides or proteins of the starting population can in particular have a three-dimensional structure comprising at least one helix 0. and / or at least one β strand, optionally linked by at least one disulfide bridge, or fragments thereof. . Among the peptides, polypeptides or proteins corresponding to such a three-dimensional conformation, there may be mentioned in particular:

- peptides, polypeptides or proteins having a three-dimensional structure of the type comprising at least one α helix and optionally at least one disulfide bridge such as for example those of the family of cecropins (2 α helices), melittins (2 α helices), magainins (1 α helix), brévinines (1 α helix and 1 disulfide bridge), esculentines (1 α helix and 1 disulfide bridge), ranatuerins (1 α helix and 1 disulfide bridge), dermaseptins (1 α helix) , transport proteins lipids (4 α helices linked by 4 disulfide bridges) or myoemerythine (4 α helices);

- peptides, polypeptides or proteins having a three-dimensional structure of the type comprising at least one β strand and optionally at least one disulfide bridge such as, for example, those of the family of classical or α defensins of mammals (3 anti-parallel β strands connected by 3 disulfide bridges), mammalian β defensins (3 antiparallel β strands connected by 3 disulfide bridges), protectins (2 anti-parallel β strands connected by 2 disulfide bridges), thanatin (2 anti-parallel β strands connected by 1 disulfide bridge), androctonine (2 anti-parallel β strands linked by 2 disulfide bridges), plant knottin-like-peptides (3 anti-parallel β strands linked by 3 disulfide bridges), superoxide dismutase (8 anti-parallel β strands), the retinol-binding plasma protein (8 anti-parallel β strands), influenza virus neuraminidase (6 identical identical patterns of 4 antiparallel β strands), Immu noglobulins (constant regions: 7 anti-parallel β strands and 1 disulfide bridge; variable regions: 9 anti-parallel β strands and 1 disulfide bridge), toxins isolated from molluscs (for example conotoxin ω: 3 anti-parallel β strands and 3 disulfide bridges) or toxins isolated from spiders ( for example agatoxin μ: 3 anti-parallel β strands and 3 disulfide bridges);

- Peptides, polypeptides or proteins having a three-dimensional structure of the type comprising at least one α helix and at least one β strand, connected by at least one disulfide bridge. This three-dimensional structure called CSαβ is notably represented in the peptides or proteins of the insect defensin family (1 α helix and 2 β strands anti-parallels connected by 3 disulfide bridges), drosomycin (1 α helix and 3 anti-parallel β strands connected 4 disulfide bridges), plant defensins (1 α helix and 3 anti-parallel β strands connected by 4 disulfide bridges ), plant heveins (1 α helix and 3 antiparallel β strands connected by 4 disulfide bridges), plant γ-thionines (2 α helix and 2 anti-parallel β strands connected by 2 to 4 disulfide bridges) or toxins isolated from scorpions (for example charibdtoxine: 1 α helix and 3 anti-parallel β strands linked by 3 disulfide bridges).

According to the present invention, the peptides, polypeptides or proteins of the starting population preferably have a three-dimensional structure of the type comprising an α helix and two antiparallel β strands linked by three disulfide bridges, or the fragments thereof. This three-dimensional structure called CSαβ is characteristic of peptides and polypeptides isolated from insects, called insect defensins. These insect defensins have antibacterial properties, in particular against Gram-positive bacteria, which are useful in the treatment and / or prevention of infections both in humans and animals and in plants. The defensins of insects have been the subject of numerous publications, patents and patent applications among which we can cite: Cystêines-rich antimicrobial peptides in invertebrates, JL. Dimarcq, P. Bulet, C. Hetru, J. Hoffmann, Biopolymers (Peptide Science), Vol. 47, 465-477 (1998); Antimicrobial peptides in insects: structure and function, P. Bulet, C. Hetru, JL Dimarcq, D. Hoffmann, Developmental and Comparative Immunology 23 (1999) 329-344; EP 0349451; EP 0546213, FR 2695392 and WO 99/53053. The peptides, polypeptides or proteins of the starting population have regions of variable amino acid sequences and regions of amino acid sequences substantially conserved within said population.

The term “substantially conserved amino acid sequence regions” according to the present invention means 2 or more peptide, polypeptide or protein subsequences having at least 60%, preferably 80% and in particular 90 to 95% of the acid residues. amines that are identical when these subsequences are aligned for maximum match.

The Applicant has defined, prior to step (a) of assembly, the alignment of the peptide, polypeptide or protein sequences of the starting population to identify said variable regions and said substantially conserved regions.

The invention also relates to a single-stranded nucleotide sequence for use in step (a) of the process of the invention, characterized in that it consists or comprises:

an internal nucleotide segment coding for an amino acid sequence of one of the variable regions of one of the peptides, polypeptides or proteins of the starting population, said variable nucleotide segment being flanked in 5 ′ by a nucleotide segment coding for the acid sequence amines of all or part of any of the substantially conserved regions of the peptides, polypeptides or proteins of the starting population, which is immediately adjacent to the N-terminus of a variable region, said segment nucleotide itself being optionally flanked 5 ′ by an additional single-stranded nucleotide sequence corresponding to all or part of a gene expression regulation sequence, - in 3 ′ by a nucleotide segment coding for the sequence in amino acids of all or part of any of the substantially conserved regions of the peptides, polypeptides or proteins of the starting population, which is immediately adjacent to the C-terminus of a variable region, said nucleotide segment itself optionally flanked 3 ′ by an additional single-stranded nucleotide sequence corresponding to all or part of a sequence for regulating the expression of a gene.

The number of overlapping single-stranded nucleotide sequences of step (a) is a function of the number of peptides, polypeptides or proteins of the starting population and of the number of variable regions within said population.

The size of the overlapping single-stranded nucleotide sequences of step (a) is essentially a function of the size of the variable regions of said peptides, polypeptides or proteins.

Steps (a) and (b) are carried out by polymerization preferably by a polymerization chain reaction (PCR).

The polymerization of step (a) comprises several cycles of denaturation, hybridization, extension in the presence of a polymerase, under conditions such that each single-stranded nucleotide sequence and single-stranded nucleic acid if present, serves as a template for elongation another single-stranded nucleotide sequence or single-stranded nucleic acid if present.

The polymerization step (b) is carried out with primers comprising nucleotide sequences complementary to a nucleotide sequence of a cloning and / or expression vector.

The method of the invention is also characterized in that it comprises, after step (b), the insertion of each of the recombined double-stranded polynucleotides into the cloning and / or expression vector comprising 3 ′ ends and / or 5 ′ complementary to those introduced into said recombinant double strand polynucleotides obtained in step

(b), then screening or selecting said recombinant double strand polynucleotides or peptides, polypeptides or proteins encoded by them for one or more desired properties.

According to another form of the invention, the method comprises, after step (b), the co-transformation of a host organism with the cloning and / or expression vector comprising 3 ′ and / or 5 ′ ends complementary to those introduced into said recombined double strand polynucleotides obtained in step (b) in the presence of said recombined double strand polynucleotides, the expression in the host organism and optionally the secretion of the peptides, polypeptides or proteins encoded by them, then screening or selecting said peptides, polypeptides or proteins for one or more desired properties.

The desired property or properties correspond, according to the present invention, to one or more improved properties compared to that (s) of the peptides, polypeptides or proteins of the starting population. The property or properties are chosen from the following group: anti-bacterial properties, antifungal properties, anti-inflammatory properties, anti-tumor properties, anti-cancer properties, wound healing and ability to bind a receptor, in particular an ion channel.

The invention also relates to a recombinant double-stranded polynucleotide, characterized in that it is obtained by the method of the invention.

The invention also relates to a cloning and / or expression vector characterized in that it comprises a recombinant double-stranded polynucleotide obtained by the method of the invention for transforming a host organism and expressing in the latter the peptide, polypeptide or the protein encoded by the recombinant polynucleotide. The vector can be a plasmid, a cosmid, a bacteriophage or a virus, in particular a baculovirus. The expression vector is advantageously a vector with autonomous replication comprising elements allowing its maintenance and its replication in the host organism as an origin of replication. In addition, the vector may include elements allowing its selection in the host organism such as for example an antibiotic resistance gene or a selection gene which ensure complementation with the respective gene deleted at the genome of the host organism. . The vector can also be a shuttle vector comprising elements allowing its maintenance and its replication in each of the host organisms and elements allowing its selection in each of the host organisms. Such cloning and / or expression vectors are well known to those skilled in the art and widely described in the literature. The invention also relates to a host organism characterized in that it is transformed by a cloning and / or expression vector of the invention. The term “host organism” according to the present invention means any mono or multicellular organism, lower or higher, in which a recombinant double-stranded polynucleotide obtained by the process of the invention is introduced for the production of a peptide, polypeptide or of a protein obtained by the process of the invention. The host organism can be a microorganism such as a yeast (for example a yeast of the genus Saccharomyces, Kluyveromyces, Hansenula, Pichia or Schizosaccharomyces), a bacterium (for example Escherichia coli) or a fungus (for example Aspergil l us ni dulans ). The host organism can also be an animal cell, in particular an arthropod cell (for example a Spodoptera frugip erda or Tri chopl usi a ni cell) or a Mammalian cell. The host organism can also be a plant cell or a plant.

The invention therefore also relates to a peptide, polypeptide or protein characterized in that it (it) is obtained by the process of the invention.

The invention also relates to a library, characterized in that it consists of recombinant double-stranded polynucleotides or of cloning and / or expression vectors or of host organisms or of peptides, polypeptides or proteins.

The invention also relates to the use of the method of the invention, for the construction of a cloning and / or expression vector. The implementation of the process of the invention thus makes it possible to easily and quickly prepare recombinant double-stranded polynucleotides or peptides, polypeptides or proteins for which they code, which advantageously have one or more properties improved compared to that (s) of peptides, polypeptides or proteins of the starting population. The use of single-stranded nucleotide sequences as defined above, in step (a) of the method of the invention makes it possible to preserve the integrity of the elementary structural patterns of the peptides, polypeptides or proteins encoded by the recombinant double-stranded polynucleotides obtained. by the method of the invention. The overall folding of the peptide, polypeptide or protein chains and consequently the functionality of the peptides, polypeptides or proteins are not altered.

The use of primers comprising nucleotide sequences complementary to a nucleotide sequence of a cloning and / or expression vector in step (b) of the method of the invention, and optionally of one or more acids single-stranded nucleic acids as defined above, in step (a) of the process of the invention allow, for their part, to build a library of recombinant double-stranded polynucleotides capable of being inserted directly into a cloning vector and / or expression, by homologous recombination inside a host organism and to express and possibly secrete directly the peptides, polypeptides or proteins for which the recombined double-stranded polynucleotides code. The amplification step in a bacterium, in particular E. coli being henceforth unnecessary, the user not only saves time but also avoids the possible loss of sequences which would be toxic in the bacterium. Other advantages and characteristics of the process of the invention will appear from the examples which follow concerning the preparation of a bank of defensins of recombinant insects having improved antibacterial properties and in which reference will be made to the accompanying drawings in which:

FIG. 1 represents the alignment of the peptide sequences of 40 insect defensins;

-Figure 2 represents the modeling of the three-dimensional structure of the dipteran defensin

Anopheles gambiae;

FIG. 3 represents the list of overlapping single-stranded nucleotide sequences used in step (a) of the method; FIG. 4 represents the construction of a bank of defensins of recombinant insects;

FIG. 5 represents the yeast expression vector S. cerevisiae pEM105;

FIG. 6 represents the in vitro activities (IC90, CMI and CMB; μg / ml) of hybrid insect defensins against Gram positive bacteria);

FIG. 7 represents the efficiency in vivo

(percentage of survival and mean health scores in relation to post-infection days) of the defensins of recombinant insects ETD-P1255, ETD-P1263 and ETD-P1268 in the mouse model of methicillin-sensitive Staphylococcus aureus peritonitis with administration by intraperitoneal route;

-Figure 8 represents the in vivo efficacy (percentage of survival and average health scores compared to the post-infection days) of defensins ETD-P1255, ETD-P1263 and ETD-P1268 recombinant insects in the mouse model of methicillin-sensitive Staphylococcus aureus peritonitis with intravenous administration; FIG. 9 represents the efficiency in vivo

(percentage of survival and mean health scores in relation to post-infection days) of the recombinant insect defensin ETD-P1263 in the mouse model of methicillin-resistant Staphylococcus aureus peritonitis with intraperitoneal administration.

Example 1: Preparation of a bank of defensins of recombinant insects having improved antibacterial properties.

A) Construction of the bank of defensins of recombinant insects.

) Choice of overlapping single-stranded nucleotide sequences and single-stranded nucleic acids used in step (a) of the process.

Figure 1 in the appendix represents the alignment of the peptide sequences of 40 insect defensins (31 described in the literature and 9 newly isolated by the applicant according to a conventional process known to those skilled in the art, notified (*)) from 6 different insect orders. Analysis of the alignment revealed the presence of 3 variable regions (in bold, Figure 1) separated by substantially conserved regions. 32 different sequences have been identified for variable region No. 1, 28 for variable region No. 2 and 33 for variable region No. 3. As shown in Figure 2 in the appendix, the variable regions No. 1, No. 2 and No. 3 correspond respectively to the N-terminal loop, to the loop located between the helix α and the first strand β and to the C-terminal loop located between the 2 β strands of insect defensins.

In order to prepare a library of mutants associating these different regions, the applicant has designated and synthesized 94 overlapping single-stranded nucleotide sequences which cover the sequence of all or part of polynucleotides encoding a starting population of insect defensins (acid residues amines in parentheses in FIG. 1 not being covered by the overlapping single-stranded nucleotide sequences), in order to obtain, at the end of step (a), recombinant double-stranded polynucleotides which comprise at their 5 'ends (coding strand) the end of the pro sequence of factor MF l of S. cerevisiae and at their 3 'ends (coding strand) the beginning of the transcription terminator sequence of the PGK1 gene. Each of these single-stranded nucleotide sequences consists of or includes:

- an internal nucleotide segment coding for an amino acid sequence of one of the variable regions of one of the insect defensins of the starting population, said variable nucleotide segment being flanked - in 5 'by a nucleotide segment coding for the acid sequence amines of the substantially conserved region of the defensin of the dipteran Anopheles gambiae from the starting population, which is immediately adjacent to the N terminus of the variable region, - in 3 'by a nucleotide segment coding for the amino acid sequence of the substantially preserved region of the Diptera defensin Anopheles gambiae from the population of start, which is immediately adjacent to the C-terminus of the variable region, said nucleotide segment itself being optionally flanked 3 ′ by an additional single-stranded nucleotide sequence corresponding to the start of the transcription terminator sequence of the PGK1 gene .

The overlapping single-stranded nucleotide sequences which comprise an additional single-stranded nucleotide sequence corresponding to the start of the transcription terminator sequence of the PGK1 gene are the overlapping single-stranded nucleotide sequences constituting the 3 'ends (coding strand) of recombinant double-stranded polynucleotides assembly (a), that is to say those which constitute or comprise internal nucleotide segments coding for the amino acid sequence of one of the variable regions No. 3 of the insect defensins of the starting population.

The list of overlapping single-stranded nucleotide sequences is shown in Figure 3. The overlapping single-stranded nucleotide sequences are described in the sequence list under the sequence identifiers SEQ ID NO: 1 to 94.

3 series of single-stranded nucleic acids have also been synthesized: EM363 (SEQ ID NO: 95), EM351 (SEQ ID NO: 96) and EM357 (SEQ ID NO: 97).

The single-stranded nucleic acid EM363 consists of a first region which is complementary to the sequence of the nucleotide segment coding for the amino acid sequence of the substantially conserved region of the defensin of the dipteran Anopheles gambiae of the starting population, which is immediately adjacent to the N terminus of variable region # 1 encoded by the internal nucleotide segment, single-stranded nucleotide sequences overlapping constituting the 3 'end (coding strand) of the recombined double stranded polynucleotides, before assembly

(at) . The single-stranded nucleic acid EM363 also consists of a second region which corresponds to a first part of the end of the pro sequence of the factor MFα1 of

S. cerevisiae.

EM351 single-stranded nucleic acid consists of a first region which is complementary to the second region of EM363 nucleic acid and a second region which corresponds to the rest of the end of the pro sequence of the factor

MFαl of S. cerevisiae.

The single-stranded nucleic acid EM357 consists of a first region which is complementary to the sequence corresponding to the start of the transcription terminator of the PGK1 gene of the overlapping single-stranded nucleotide sequences constituting the 3 'ends (coding strand) of the recombinant double-stranded polynucleotides , before assembly

(at) . The single-stranded nucleic acid EM357 also consists of a second region which corresponds to the rest of the start of the transcription terminator of the PGK1 gene.

The single-stranded nucleic acids EM351, EM357 and EM363 are used to reconstitute sequences allowing the insertion of the single-stranded nucleidic sequences assembled in step (a) of the method, into the yeast expression and secretion vector S. cerevisiae pEM105. EM357 single stranded nucleic acid is also used as a primer in step (b) of the process.

2) Assembly step (a) of the process. The assembly step (a) is carried out by PCR as shown in FIG. 4 in the appendix. EM351, EM357 and EM363 single stranded nucleic acids are used at a concentration of 10 picomoles in a final reaction volume of 60 μl. Mixtures of overlapping single-stranded nucleotide sequences are used at a rate of 10 picomoles for each of the following series of single-stranded nucleotide sequences: 0.31 picomoles per single-stranded nucleotide sequence consisting of or having an internal nucleotide segment encoding an amino acid sequence of one of the variable regions No. 1 of one of the insect defensins of the starting population; 0.36 picomoles per single-stranded nucleotide sequence consisting of or having an internal nucleotide segment encoding an amino acid sequence of one of the variable regions no. 2 of one of the insect defensins of the starting population and 0.30 picomoles per single-stranded nucleotide sequence consisting of or having an internal nucleotide segment encoding an amino acid sequence of one of the variable regions No. 3 of one of the insect defensins of the starting population.

The assembly PCR reaction begins with denaturation at a temperature of 94 ° C for 3 minutes. The following steps are repeated 5 times: i) denaturing at a temperature of 9 ° C for

45 seconds, ii) hybridization at a temperature of 50 ° C for 45 seconds, iii) extension in the presence of a polymerase at a temperature of 72 ° C for 45 seconds. The PCR reaction ends with an extension step in the presence of a polymerase at a temperature of 72 ° C for 10 minutes. At the end of step (a), recombinant double-stranded polynucleotides are obtained which comprise at their 5 ′ ends (coding strand) the end of the pro sequence of the factor MFα1 of S. cerevisiae and at their 3 'ends (coding strand) the beginning of the transcription terminator sequence of the PGK1 gene.

3) Step (b) of the process. The recombinant double-stranded polynucleotides from step (a) of assembly are then amplified in the presence of 2 of the primers EM349 (SEQ ID NO: 98) and EM357 (SEQ ID NO: 97) (Figure 4). These two primers comprise a sequence respectively complementary to each of the ends of the recombinant double-stranded polynucleotides originating from step (a) and a sequence of 30 nucleotides complementary to the ends of the yeast expression vector S. cerevisiae pEM105 linearized.

Step (b) of the process of the invention begins with denaturation at a temperature of 94 ° C for 3 minutes. The following steps are repeated 25 times: i) denaturation at a temperature of 9 ° C for 45 seconds, ii) hybridization at a temperature of 60 ° C for 45 seconds, iii) extension in the presence of a polymerase at a temperature of 72 ° C for 45 seconds. The amplification reaction ends with an extension step in the presence of a polymerase at a temperature of 72 ° C for 10 minutes.

Step (b) of the method leads to the generation of recombinant double-stranded polynucleotides having extensions capable of hybridizing with the ends of the yeast expression vector S. cerevisiae pEM105 linearized.

4) Construction of the expression library by co-transformation of a strain of yeast S. cerevisiae. The yeast expression vector S. cerevisiae pEM105

(Figure 5) carrier of the strong constitutive promoter TDH3

(glyceraldehyde 3 phosphate dehydrogenase) fused to the sequences coding for the pre secretion signals of BGL2 (Beta-1,3 glucanase) and pro of the factor MFα1 of S. cerevisiae and the transcription terminator for the PGK1 (phosphoglycerate kinase) gene was constructed. It also carries sequences allowing its replication and its selection in yeast (2μ ori and URA3) and in Ξ. coli (ColEl ori and Amp) as well as the iC .--. X2 gene coding for the endoprotease Yscf which cleaves the pro sequence during the secretion process (insufficient expression of genomic KEX2). This vector carries 2 unique restriction sites Nhel and Sali allowing its linearization. The Nhel site is located in the sequence coding for the MFα1 pro (silent mutation introduced at the level of the sequence coding for residues 51 to 52 of the MFα1 pro) and the Sal i site is upstream of the P GK1 terminator. A yeast strain YEM1 (MATα, ura3-Δ5, pral, prbl, prcl, cpsl) was co-transformed by the plasmid pEM105 digested with Sali and Nhel and the pool of recombinant double-stranded polynucleotides having extensions obtained after step (b) of the process (lμg of pEM105 digested with Nhel and Sal i + 115ng of PCR fragment by transformation). The transformation was carried out by the lithium acetate method known to those skilled in the art. The transformants were selected on selective YΝBG medium supplemented with 0.5% casamino acids. In order to check the quality of the library, 100 transformants were analyzed for the production of peptide after growth in a Kapeli selective medium (14.2 g KH2P04 SDS 2370017; 4g MgS04 SDS 460012; 1.6mL H3P04; ImL of a solution d '' trace elements: 16g MnS04, H20, 0.4g CuS04, 5H20, 15g ZnSO4,7H20, 2.8g CoC12, 6H20, 2.48g Νa2Mo04, 7.5g H3B03, 0.2g Citric acid, lg KC1, 2.5g NiS04, 6H20, 25g

Trisidium citrate 2H20, qs 200mL; 400mL glucose at 500g / L; lmL FeCl3 / CaCl2; 500mL of Casaaminoacids at 200g / L; lOmL of a solution of vitamins: 0.2g Thiamine HC1, 0.5g Pyridoxine HC1, 0.8g Nicotinic acid, 0.005g D-Biotin, lg Ca-D-Pantothenate, 8g Meso Inositol, qs 200mL; qs 5L). The culture supernatants harvested after 48 hours of growth were analyzed by electrophoresis on acrylamide gel (Tris-tricine gel) and staining of the proteins with silver nitrate. Peptide production was detected in 65% of these clones. 45 clones producing defensins of recombinant insects were analyzed by nucleotide sequencing. 45 defensins of different recombinant insects could be characterized: 22 different sequences among the 32 possibilities were identified for variable region no. 1, 24 different sequences among the 28 possibilities were identified for variable region no. 2 and 20 sequences different among the 33 possibilities have been identified for variable region n ° 3.

B) Screening or selection of defensins of recombinant insects having activity against Gram-positive bacteria Staphylococcus aureus.

1) Culture and isolation.

The bank of defensins of recombinant insects obtained according to the protocol previously described in part A, is spread on Petri dishes (YNBG casamino acids agar). The number of clones per dish is chosen so as to form isolated colonies. These colonies are pricked and cultured automatically in 384-well microplates, in 60 μl of medium (YNBG, casamino acids, 13% glycerol). The plates are incubated at 30 ° C for 72 hours, then two copies are stored at -80 ° C, and another copy is kept at 4 ° C before being used after the process.

2) Culture and production. From a 384-well plate, 4 96-well DeepWell plates containing 1 ml of Kapeli culture medium are inoculated. The Deepwell plates are covered with a gas permeable membrane and are incubated at 30 ° C with shaking for 96 hours. After 72 hours, an addition of 100 μl of glucose at 500 g / 1 per well is made.

3) Purification.

1 ml of sterile ultrapure water is added to all the wells of the 96-well plates. These plates are then centrifuged for 30 min at 4000 rpm and at 20 ° C. The supernatant is purified on Oasis HLB plates (WATERS): 1.3 ml of supernatant are deposited on each of the 96 microcolumns, subjected to a vacuum drawing to pass the liquid. Wash with 800 μl per column of water supplemented with 0.05% Trifluoroacetate (TFA). The elution is carried out with 500 μl of 60% acetonitrile supplemented with 0.05% TFA. The eluate is collected in a 1 well DeepWell 96-well plate.

4) Evaporation of the solvent.

The elution solvent is then evaporated under vacuum (30 ° C, 9 mbar, 11 h). The plates are then covered with a self-adhesive silicone film and stored at 4 ° C.

5) Analysis by High Performance Liquid Chromatography (HPLC) on reverse phase column: Mass control and quantification for activity tests. The equivalent of 40 to 200 μl of purified supernatant according to the previous step, is analyzed by HPLC (Alliance type,

Waters Associates) coupled with mass spectrometry (type

ZQ 2000, Waters Associates), driven by Masslynx software

(Waters Associates). The samples are injected automatically and sequentially (injector 2790, Waters

Associates) on an analytical column of Uptisphere type

250 x 4.6 mm, filled with silica with a C18 graft (reverse phase) with a particle size of 5 μ and a porosity of 300 A °

(Interchim). Elution is carried out by a gradient of acetonitrile in 0.05% TFA from 15 to 60% in 30 min. with a constant flow rate of 0.8 ml / min.

The UV spectrum (variation in absorbance at 225 nm; diode array detector type 996, Waters Associates) and the mass spectrum of each sample are analyzed individually. The mass corresponding to each peak of the UV spectrum is calculated by deconvolution software (Transform software, Waters Associates). When the measured mass corresponds to the theoretical mass (range from 3500 to 5500 for insect defensins), the peak of interest is integrated by the Masslynx software and then quantified.

The quantification is made with respect to a calibration curve (area of the peak as a function of the quantity of sample) established by injection of known quantities of defensin to Anopheles gambiae under the same analysis conditions. The quantification of the recombinant defensins (in μg) is calculated by individual integration of the peak of the chromatogram corresponding to each recombined defensin.

6) Preparation of the Staphylococcus aureus inoculum.

A preculture in 4 ml of LB medium is obtained by seeding a colony of Staphylococcus aureus (strain clinical 21, sensitive to: amoxicillin, pristinamycin, erythromycin, pefloxacin, suifamethoxazole, trimethoprime, kanamycin, fusidic acid, fosfomycin, gentamycin, amikacin, rifampicin, oxacillin, teicoplanin, chloricphenin, methamycinine, chloramphenicol _; ; Donated by the Institute of Molecular and Cellular Biology, Strasbourg), and incubated at 37 ° C with shaking for 16h. 200 μl of this preculture are distributed in 8 tubes of 4 ml of LB medium, then incubated for 4 h at 37 ° C. with shaking to obtain the primary solution. An inoculum is produced by diluting this primary solution (approximately 1/100), so as to obtain a concentration of S t aphyl ococcus aureus of 1.1 × 10 ⁶ bacteria / ml. The concentrations are calculated by measuring the OD at 600 nm of the primary solution according to the formula: Concentration of Staphylococcus aureus = 5.25.10 ⁸ x OD - 3.107 (for OD ranging from 0.05 to 0.3 approximately) and controlled via a cascade dilution (10 ^"1 , 10 ^{" 2} , 10 ^"3 , ...) of the suspension at 2.10 ⁶ / ml and counting of the Colony-forming Units (CFU) after spreading 100 μL of the 10 ^{" 6} dilutions, 10 ^"5 and 10 ^{" 4} on LB agar boxes. After incubation at 30 ° C for 16 to 18 hours, the colonies are counted (CFU / ml).

7) Screening or selection of samples. The dried samples are taken up in 200 μl of water and tested at 3 different dilutions: 40 μl, 25 μl and 16 μl of these samples are distributed in the test plates before adding 180 μl of inoculum per well. The microplates are incubated at 30 ° C for 16 to 18 hours, then the optical density (OD) of the wells is measured at 595 nm. The results are interpreted by calculating the percentage of growth for each well according to the formula:% Sprout = (DO well - DO TS) / (DO TP - DO TS) * 100 where DO TP = DO growth indicator (inoculum alone), DO of TS = OD of sterility control (culture medium without bacteria) and DO well = DO of the well for which the percentage of growth is to be calculated (inoculum + defensin of recombinant insect). Defensins of recombinant insects are considered active when the growth percentage is less than or equal to 10. The recombinant double-stranded polynucleotides ETD-P1255, ETD-P1263, ETD-P1268, ETD-P1354, ETD-P1364, ETD-P1377 , ETD-P1447 and ETD-P1449 are described in the sequence list under the sequence identifiers SEQ ID NO: 99 to 106. The defensins of recombinant insects ETD-P1255, ETD-P1263, ETD-P1268, ETD-P1354, ETD-P1364, ETD-P1377, ETD-P1447 and ETD-P1449 are described in the sequence list under the sequence identifiers SEQ ID NO: 107 to 114.

C) Antibacterial activities in vi tro.

The anti-bacterial activities of each of the defensins of hybrid insects (ETD-P) were evaluated by determination of the IC90 (growth inhibition> 90%), the minimum inhibitory concentration (MIC) and the minimum concentration. Bacteria (CMB). The activities of each of the hybrid defensins are compared with those of the defensin of the Anopheles gambiae diptera as well as with those of conventionally used antibiotics (vancomycin and linezolid) tested under the same conditions.

1) Isolation.

The hybrid defensin clones of interest, identified according to the protocol previously described in part B), are taken from one of the stocks of 384-well microplates stored at −80 ° C. (part ie B), paragraph 1)). A inoculation is carried out on a YNBG Casamino acid agar agar and placed in an incubator at 30 ° C. for 48 to

72 hours. After observing the state of growth of the yeasts, a colony is isolated, spread on a YNBG Casamino acids agar dish and incubated for 24 to 48 hours at 30 ° C.

Expression is controlled by PCR.

2) Production.

A preculture is prepared by suspending a colony in 4 ml of Kapeli medium and then the preculture is incubated for 18 to 24 hours at 30 ° C. with shaking.

50mL of Kapeli medium are distributed in 500mL vials. The OD of the precultures is measured at a dilution to l / 100th (990 μL of water + 10 μL of homogenized preculture) to determine the OD of the undiluted preculture. The preculture is then diluted so as to obtain a solution of OD = 0.05 according to the formula: d = 0.05 / OD preculture undiluted. A volume of the preculture, defined according to the formula (preculture) = 50 / d, is added to the 50 ml of Kapeli medium to obtain a solution of OD = 0.05. The flasks are incubated at 30 ° C for 48 hours with shaking (250 rpm / min).

3) Purification.

The culture obtained is centrifuged in 50 ml conical centrifuge tubes for 15 min at 4000 rpm. The supernatant is transferred to another 50mL tube and is purified with the Sep-pack, on Oasis HLB lg of phase according to the following steps: Equilibration of the phase with 15mL of pure methanol, washing with 15mL of water / TFA 0.05% mixture, loading of the supernatant to be purified, washing with 15mL of water / TFA 0.05% mixture and elution with lOmL of water / Acetonitrile 60% / TFA 0.05% mixture. The acetonitrile / H2O mixture is evaporated under vacuum (30 ° C., 9 mbar, 24 hours) and then the samples are taken up in 400μL of water and placed on an agitator.

4) Quantification and mass control for activity tests. Quantification and mass control is carried out according to the protocol previously described in part B), paragraph 5).

5) Determination of IC90.

The IC90 is determined by a liquid test in 96-well microplates. The test is carried out on sensitive Gram-positive bacterial strains Staphylococcus aureus

(clinical strain 21; Gift from the Institute of Biology

Molecular and Cellular, Strasbourg) and resistant (clinical strain 4; Gift of Dr. Gilles Prévost, Institute of Bacteriology, Strasbourg) so as to obtain an inoculum according to the protocol described in part B) paragraph 6). 100 μl of samples to be tested are distributed in duplicates so as to obtain a concentration range of 25 to 0.05 μg / ml in the wells. The samples are added to 100 μl of bacterial suspension at 2.10 ^s / ml in each well so as to obtain a final concentration of 10 ⁶ / ml. The microtiter plates are incubated at 30 ° C for 16 to

18 hours. The colonies are counted by counting CFUs

(Colony-forming Units) and the result is reported in CFU / ml. The optical density of the wells is measured at 600 nm and the results are interpreted by calculating the percentage of growth in each well according to the formula described in part B) paragraph 7). Growth inhibitions 90 (IC 90) are the wells where the growth percentage is less than or equal to 10%. The results are shown in Figure 6 in the appendix.

6) Determination of CMI and CMB.

The MICs (Minimum Inhibitory Concentrations) and the CMBs (Minimum Bactericidal Concentrations) were determined on the following Gram positive bacterial strains:

Staphylococcus aureus susceptible (clinical strain 21;

Donation from the Institute of Molecular and Cellular Biology,

Strasbourg);

Resistant Staphylococcus aureus (clinical strain 4;

Gift of Dr. Gilles Prévost, Institute of Bacteriology,

Strasbourg);

Staphylococcus epidermidis sensitive (clinical strain

34; Gift of the Institute of Molecular Biology and

Cellular, Strasbourg).

The bacterial strains are from cultures on Mueller-Hinton blood agar medium, freshly transplanted. A preculture of 3 ml in Mueller-Hinton medium of each of the strains is incubated at 35 ° C for 6 hours with shaking.

An inoculum of the desired strain is prepared from the 5-6 hour preculture. The inoculum is brought to a concentration of 2.10 ⁶ CFU / ml by dilution in Mueller-Hinton medium. a) Determination of MICs.

The MIC is determined by a liquid test in 96-well microplates (Costar 3599).

The 60 central wells (wells marked B to G and 2 to 11) of the 96-well microplate are used. Column 1 of the microplate serves as a sterility control and column 12 as a growth control. Lines A2 to Ail and H2 to H11 serve either as a sterility control or as a growth control.

100 μl of Mueller-Hinton medium are distributed in columns 3 to 12 and 200 μl in column 1. When the lines A2 to Ail and H2 to H11 are used as growth indicators, 100 μl of Mueller-Hinton medium are deposited in the well. When lines A2 to Garlic and H2 to H11 are used as sterility controls, 200 μl of Mueller-Hinton medium are deposited.

The samples to be tested (in powder form) are dissolved in the appropriate solvent, so as to have a minimum volume of 100 μl to 640 μg / ml. 400 μl of Mueller-Hinton medium are added to the solutions prepared to obtain solutions of 500 μl to 128 μg / ml in fine. 200 μl of each of the samples to be tested (in solution at 128 μg / ml) are then distributed in the wells (wells B2 and C2 of column 2 for the first sample to be tested, wells D2 and E2 for the second sample to be tested, etc ...). A 2-by-2 cascade dilution is carried out using a multichannel pipette. The cascade dilution is carried out by taking 100 μl of sample in solution from the wells of column 2, by placing them in column 3 which contains 100 μl of Mueller-Hinton medium and mixing several times, and so on until in column 11. The excess 100 μl in column 11 is discarded. 100 μl of bacterial inoculum are distributed in wells B2 to Gll, as well as in the growth control column. By adding the inoculum, a dilution is carried out with _ in each well to finally obtain a concentration range from 64 to 0.125 μg / ml. Cascade dilutions to the 10 ^th of 10 ^"1 to 10 ^{" ε} of the inoculum at 2.10 ^s CFU / ml are made, and 100 μl of the dilutions 10 ^"s , 10 ^{" 5} and 10 ^"4 are spread out on boxes of Muller-Hinton agar. The microplate is incubated at 35-37 ° C for 16 to 18 hours and then the optical density of the microplate is read at 600-620 nm. The inoculum count boxes (Muller-Hinton agar) are incubated at 35-37 ° C for 16 to 18 hours then the count of CFU / ml of the inoculum is carried out. The results are interpreted by calculating the percentage of growth in each well according to the formula:

((DO well - mean negative control) / (mean DO positive control - mean DO negative control)) * 100 The MIC is defined as the lowest concentration for which 0 to 10% growth is obtained. b) Determination of CMBs.

The CMBs are determined from the test microplate. 100 μl of each well of the microplate which does not present a disorder (no bacterial growth visible to the naked eye) as well as of the well at the highest concentration having a visible disorder (this for each line) are deposited on medium. adequate agar. The dishes are incubated at 35-37 ° C overnight before a CFU / ml count.

The CMB is defined as the lowest concentration of drug, giving a bacterial count less than 0.1% of the starting inoculum (ie a reduction of 3 logarithmic values). The results of the CMI and CMB are set out in Figure 6 in the appendix.

D) Effectiveness in vivo.

1) Protocol - Model of Staphylococcus aureus peritonitis.

The effectiveness of the various defensins of recombinant insects (ETD-P), of the defensin of Anopheles gambiae

(DefA) and conventional antibiotics (Imipenem, IMP and

Vancomycin, Van) was evaluated in vivo in mice in a model of Staphylococcus aureus peritonitis. The tested samples were produced and purified according to the protocol previously described. Staphylococcus strains methicillin-sensitive aureus (MSSA) (Clinical strain 21;

Donation from the Institute of Molecular and Cellular Biology,

Strasbourg) or methicillin-resistant (MRSA) (Clinical strain 4; Gift of Dr. Gilles Prévost, Institute of Bacteriology, Strasbourg) are cultured overnight at 37 ° C in Muller-Hinton medium. The bacteria are washed and taken up in a 0.9% NaCl solution. After quantification by measuring OD at 600 nm, the bacteria are diluted to obtain a bacterial density of 2.109 CFU / ml. The bacterial suspension is then diluted to half with a 0.9% NaCl solution containing 10% gastric mucin.

Groups of 6 19-20 g OF1 male mice are infected intraperitoneally with 500 μl of the S suspension. aureus containing 5.108 CFU in 0.9% NaCl, 5% mucin. The treatments with the samples are carried out one hour after infection with a single injection by intraperitoneal or intravenous route in the case of defensins (ETD- and DefA) and Vancomycin (Vanco), or by sub- cutaneous in the case of Imipenem (IMP). Control experiments (placebo) are systematically carried out in which the sample volumes are replaced by 0.9% NaCl. The therapeutic efficacy of the treatments is evaluated by several criteria: the percentage of survival on day 7 after infection, the change in body weight and health score. This last parameter, relating to the state of health of the mice, takes account of the condition of the coat, of the mobility, of the state of hydration, of the presence or not of diarrhea. It is defined as follows by a value from 0 to 5: 0 = dead, 1 = dying, 2 = very sick, 3 = sick, 4 = slightly sick and 5 = healthy.

2) Comparison of the efficacy of the recombinant defensins ETD-P1255, ETD-P1263 and ETD-P1268 with that of the defensin of Anopheles gambiae (DefA) and of the Imipenem (IMP) in the methicillin-sensitive Staphylococcus aureus peritonitis (MSSA) model.

-Administration intraperitoneally.

Figure 7 in the appendix represents the percentage of survival and the average health scores compared to the post-infection days. In this experiment, DefA and the 3 recombinant defensins administered in a single dose of 3 mg / kg intraperitoneally, show good therapeutic efficacy in a model where all the control mice treated with placebo (NaCl 0.9%) died 2 to 5 days after infection. Only 1 in 6 mice died in the groups treated with DefA, ETD-1263 and ETD-1268. No mortality was observed in the group treated with ETD-P1255. Analysis of the health scores and of the weight evolution, shows that ETD-P1255 and ETD-P1263 are more active than DefA: the mice treated with ETD-P1263 and ETD-P1255 recover more quickly than the mice treated with the other recombinant defensins, with an average health score of 4 to 5 from the first day after infection. -Administration by intravenous route.

Figure 8 in the appendix represents the percentage of survival and the average health scores in relation to the post-infection days. In this experiment, DefA and the 3 recombinant defensins administered in a single dose of 3 mg / kg intravenously, have a lower therapeutic efficacy compared to their administration by intraperitoneal route. While 50% of the mice treated with placebo (0.9% NaCl) died 3 days after infection, only treatment with 1ΕTD-P1263 was completely effective with weight gain from the 2nd day after infection and a maximum health score (5/5) on day 5 after infection. ETD-P1255 shows good survival efficiency with 84% survival compared to 67% for the groups treated with DefA and the other recombinant defensins. Analysis of weight curves and health scores, however, shows that ETD-1255 is less effective than DefA. 3) Comparison of the efficacy of the recombinant defensin ETD-1263 with that of the defensin of Anophèl es gambiae (DefA) and Vancomycin (Van) in the methicillin-resistant Staphylococcus aureus peritonitis model (MRSA) -Administration intraperitoneally. Figure 9 in the appendix represents the percentage of survival and the average health scores in relation to the post-infection days. In a model where 100% of the mice treated with placebo (0.9% NaCl) died on day 3 after infection, ETD-1263 shows very good therapeutic efficacy at a dose of 3 mg / kg administered intravenously -péritonéale. With the exception of a dead mouse, all the treated mice quickly regained weight and returned to good health from the 2nd day after infection. The efficacy of ETD-P1263 at a dose of 3 mg / kg is comparable to that of DefA at a dose of 10 mg / kg and Vancomycin at a dose of 10 or 30 mg / kg, the evolution of weight and health scores being very similar for these four treatment groups.

Claims

1) Method for preparing recombinant double-stranded polynucleotides or peptides, polypeptides or proteins for which they code, characterized in that it comprises: a) the assembly of overlapping single-stranded nucleotide sequences which cover the sequence of all or part of polynucleotides encoding a starting population of peptides, polypeptides or proteins, in order to obtain recombinant double-stranded polynucleotides, said recombinant double-stranded polynucleotides optionally comprising at one of their ends a sequence for regulating the expression of a gene or a 3 ′ or 5 ′ part of this regulatory sequence, b) the introduction at the 3 ′ and / or 5 ′ ends of each of the recombinant double-stranded polynucleotides obtained in step (a) of nucleotide sequences complementary to a sequence nucleotide of a cloning and / or expression vector, then optionally c) the insertion of each of the polynucleotides es double strands recombined in the cloning and / or expression vector comprising 3 ′ and / or 5 ′ ends complementary to those introduced into said recombinant double strand polynucleotides obtained in step (b), optionally followed by d) la co-transformation of a host organism with the cloning and / or expression vector comprising 3 ′ and / or 5 ′ ends complementary to those introduced into said recombined double-stranded polynucleotides obtained in step (b) in the presence of said recombinant double strand polynucleotides, and expression in the host organism and possibly secretion of peptides, polypeptides or proteins encoded by them.

2) Method according to claim 1, characterized in that in step (a) of the method of the invention, the recombinant double-stranded polynucleotides comprise at at least one of their ends all or part of a sequence of regulation of the expression of a gene, which is supplied to said recombined double-stranded polynucleotides by additional single-stranded nucleotide sequences present: i) either at one of the ends of the overlapping single-stranded nucleotide sequences constituting the ends of the recombined double-stranded polynucleotides , before assembly (a), ii) either, during assembly (a), in the form of one or more single-stranded nucleic acids, each consisting of a first and a second nucleic region, of which :

a first group consists of first regions complementary to the overlapping single-stranded nucleotide sequences constituting at least one of the ends of the recombined double-stranded polynucleotides, before assembly (a), and the second regions of this first group correspond to all or part of a gene expression regulation sequence,

the second group, if present, consists of first regions and second regions, partly complementary to the second regions of the first group and secondly complementary to each other, so as to form said sequence for regulating the expression of a gene or a part thereof, iii) or by the combination of (i) and (ii).

3) Method according to claim 1 or 2, characterized in that steps (a) and (b) are carried out by polymerization preferably by a polymerization chain reaction (PCR).

4) Method according to claim 3, characterized in that the polymerization of step (a) comprises several cycles of denaturation, hybridization, extension in the presence of a polymerase, under conditions such that each single-stranded nucleotide sequence and nucleic acid single strand if present, serves as a template for the extension of another single strand nucleotide sequence or single strand nucleic acid if present.

5) Method according to one of claims 3 or 4, characterized in that in step (b) of polymerization is carried out with primers comprising nucleotide sequences complementary to a nucleotide sequence of a vector of cloning and / or expression.

6) Method according to any one of the preceding claims, characterized in that the peptides, polypeptides or proteins of the starting population have regions of variable amino acid sequences and regions of amino acid sequences substantially conserved within of said population.

7) Method according to any one of the preceding claims, characterized in that each of the single-stranded nucleotide sequences consists or comprises: an internal nucleotide segment coding for an amino acid sequence of one of the variable regions of one of the peptides, polypeptides or proteins of the starting population, said variable nucleotide segment being flanked

- in 5 ′ by a nucleotide segment encoding the amino acid sequence of all or part of any of the substantially conserved regions of the peptides, polypeptides or proteins of the starting population, which is immediately adjacent to the N-terminus d 'a variable region, said nucleotide segment itself being optionally flanked 5' of an additional single-stranded nucleotide sequence corresponding to all or part of a gene expression regulation sequence, 3 'by a nucleotide segment encoding the amino acid sequence of all or part of any of the substantially conserved regions of the peptides, polypeptides or proteins of the starting population, which is immediately adjacent to the C terminus of a variable region, said nucleotide segment itself being optionally flanked 3 'of an additional single-stranded nucleotide sequence corresponding to to ut or part of a gene expression regulation sequence.

8) Method according to any one of the preceding claims, characterized in that it comprises after step

(c), screening or selecting said recombinant double strand polynucleotides for one or more desired properties. 9) Method according to any one of the preceding claims, characterized in that it comprises after step

(d), screening or selecting said peptides, polypeptides or proteins for one or more desired properties.

10) Method according to one of claims 8 or 9, characterized in that the desired property or properties correspond to one or more improved properties compared to that (s) of the starting population.

11) Method according to any one of the preceding claims, characterized in that it comprises before step

(a) aligning the peptide, polypeptide or protein sequences of the starting population to identify the variable regions and the substantially conserved regions.

12) Method according to any one of the preceding claims, characterized in that the number of overlapping single-stranded nucleotide sequences of step (a) is a function of the number of peptides, polypeptides or proteins of the starting population and of the number of variable regions within said population.

13) Method according to any one of the preceding claims, characterized in that the size of the overlapping single-stranded nucleotide sequences of step (a) is essentially a function of the size of the variable regions of said peptides, polypeptides or proteins. 14) Method according to any one of the preceding claims, characterized in that the peptides, polypeptides or proteins of the starting population are natural.

15) Method according to any one of the preceding claims, characterized in that the peptides, polypeptides or proteins of the starting population are variants originating from different families, orders, genera or species or allelic variants.

16) Method according to any one of the preceding claims, characterized in that the peptides, polypeptides or proteins of the starting population are peptides, polypeptides or proteins mutated.

17) Method according to any one of the preceding claims, characterized in that the peptides, polypeptides or proteins of the starting population are of animal, plant, bacterial or viral origin.

18) Method according to any one of the preceding claims, characterized in that the peptides, polypeptides or proteins of the starting population are natural variants of peptides, polypeptides or insect proteins and / or mutated peptides, polypeptides or proteins derived from natural variants of insect peptides, polypeptides or proteins.

19) Method according to any one of the preceding claims, characterized in that the peptides, polypeptides or proteins of the starting population have a three-dimensional structure comprising at least one α-helix and / or at at least one β strand, optionally connected by at least one disulfide bridge, or fragments thereof.

20) Method according to any one of the preceding claims, characterized in that the peptides, polypeptides or proteins of the starting population have a three-dimensional structure comprising an α helix and two antiparallel β strands linked by three disulfide bridges, or fragments of them.

21) Method according to any one of claims from 8 to 20, characterized in that the property or properties are chosen from the group comprising: antibacterial properties, anti-fungal properties, anti-inflammatory properties, anti- tumor, anti-cancer properties, wound healing and ability to bind a receptor, in particular an ion channel.

22) A single-stranded nucleotide sequence for use in step a) of a method according to any one of claims 1 to 21, characterized in that it consists or comprises: an internal nucleotide segment encoding a sequence in amino acids of one of the variable regions of one of the peptides, polypeptides or proteins of the starting population, said variable nucleotide segment being flanked in 5 'by a nucleotide segment coding for the amino acid sequence of all or part of one any of the substantially conserved regions of the peptides, polypeptides, or proteins of the population of start, which is immediately adjacent to the N terminus of a variable region, said nucleotide segment itself being optionally flanked 5 ′ by an additional single-stranded nucleotide sequence corresponding to all or part of a regulatory sequence of the expression of a gene, in 3 ′ by a nucleotide segment coding for the amino acid sequence of all or part of any of the substantially conserved regions of the peptides, polypeptides or proteins of the starting population, which is immediately adjacent at the C terminus of a variable region, said nucleotide segment itself being optionally flanked 3 ′ by an additional single-stranded nucleotide sequence corresponding to all or part of a sequence for regulating the expression of a uncomfortable.

23) A recombinant double-strand polynucleotide characterized in that it is obtained by the method according to any one of claims from 1 to 21.

24) A cloning and / or expression vector characterized in that it comprises a recombinant double-stranded polynucleotide according to claim 23.

25) A host organism characterized in that it is transformed by a cloning and / or expression vector according to claim 24.

26) A peptide, polypeptide or protein characterized in that it (it) is obtained by the process according to any one of claims from 1 to 21. 27) A library characterized in that it consists of recombinant double-stranded polynucleotides according to claim 23 or of cloning and / or expression vectors according to claim 24 or of host organisms according to claim 25 or of peptides, polypeptides or proteins according to claim 26.

28) Use of the method according to any one of claims 1 to 21, for the construction of a cloning and / or expression vector.