FR2852325A1 - New nucleic acid that directs expression of protein in silk glands of silkworm, useful for preparation of pharmaceutical proteins and for modifying textile properties of silk - Google Patents

Abstract

Nucleic acid (I) that directs expression of a protein of interest (II) specifically in the cells of the posterior silk-producing glands (A) of Bombyx mori, is new. Nucleic acid (I) that directs expression of a protein of interest (II) specifically in cells of the posterior silk-producing glands (A) of Bombyx mori. (I) comprises, in the 5' to 3' direction: a regulatory region (RR) comprising signals for regulating expression of a polynucleotide of interest (III) in the specified cells and, under control of RR, a region that encodes a modified signal peptide (mSP). (I) is at least 90% identical with the 1150-2026 nucleotide (nt) region of a 2721 nt sequence (1; reproduced), in which the codon containing nt 1486-1488 encodes Ala, Ile or Leu. Independent claims are also included for: (1) sequence (Ia) complementary to (I); (2) expression cassette (EC) containing (III), under control of (I); (3) recombinant integration vector (RIV) comprising (I), (Ia) or EC; (4) recombinant cloning vector (RCV) containing the sequence of RIV; (5) recombinant host cell containing (I), (Ia), EC, RIV or RCV, integrated into its genome; (6) method for preparing recombinant B. mori; (7) method for preparing a silk thread by culturing the animals of (6); and (8) method for preparing (II) by recovering it from the silk thread of method (8).

Description

<Desc / Clms Page number 1>

Field of the invention:
The invention relates to the field of synthesis and secretion of proteins of industrial interest, such as fibrous proteins for textile use, in particular new bristles or proteins for biomedical use. It relates to the targeted expression of polynucleotides and / or polypeptides of interest specifically in certain tissues of an animal and more specifically in the seric gland of Bombyx mori. According to the invention, the seric gland of the silkworm is used as a "bioreactor" capable of producing proteins in large quantities and of exporting them to the silk cocoon from where they can be purified.

State of the art:
There are methods of producing fibrous proteins for textile use such as the method of producing spider silk thread proteins by the mammary glands of transgenic goats (Nexia Biotechnologies Inc. 1000 St-Charles Avenue, Bloc B, Vaudreuil-Dorion , QC, J7V 8P5, Canada).

However, these methods do not allow the fibrous protein to be organized in the form of a wire. With regard to the production of soluble proteins for biomedical purposes, the known methods use bacterial cultures, cultures of cell lines or vertebrate organisms such as cows, goats or even transgenic rabbits. This type of production of recombinant proteins is very expensive. On the other hand, when the recombinant proteins are synthesized from vertebrate systems, their industrial use, in particular their food or medical use, obligatorily implies to check beforehand that these proteins or the compositions which contain them, are free of transmissible pathogenic contaminants to the man.

<Desc / Clms Page number 2>

 Given the important economic role of the production of recombinant proteins, there is a need in the state of the art for new means of producing recombinant proteins, in large quantity, at low cost, and from '' an organism sufficiently distant from the human species to avoid contamination problems, especially when the protein produced has a biomedical interest. There is also a need for new means of producing recombinant proteins which make it possible to organize the protein in the form of a wire when said recombinant protein is fibrous.

 For production to be significant, the protein must be synthesized specifically from a tissue, and exported to the outside of the animal, preferably an invertebrate.

However, to the knowledge of the applicant, no invertebrate organism is likely to meet the previously formulated requirements. The transgenesis of Bombyx mori for example is known (Tamura et al. Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon derived vector. Nature Biotechnology Vol 18,2000), but the use of this method has not yet allowed to lead to the production of recombinant proteins in large quantities.

 The applicant therefore set out to develop an expression system which in particular made it possible to eliminate the safety and high cost problems of the previously known systems.

Summary of the invention:
Such a recombinant protein expression system is now provided according to the invention.

 The invention relates to a nucleic acid directing the expression of a protein of interest specifically in the cells of the posterior seric glands of Bombyx mori, characterized in that said nucleic acid comprises, from the 5 ′ end to the 3 end ', (i) a regulatory region

<Desc / Clms Page number 3>

 comprising signals for regulating the expression of a polynucleotide of interest specifically in said sericogenic cells and (ii) a region coding for a modified signal peptide, placed under the control of said regulatory region, said nucleic acid having at least 90% identity in nucleotides with the polynucleotide ranging from nucleotide 1150 to nucleotide 2026 of the sequence SEQ ID N 1 and in which the trinucleotide ranging from nucleotide 1486 to nucleotide 1488 of the sequence SEQ ID N 1 codes for an amino acid chosen from the group including alanine, isoleucine and leucine.

 The invention also relates to an expression cassette comprising a polynucleotide of interest placed under the control of a nucleic acid as defined above, the polynucleotide of interest being a sense polynucleotide, coding for a polypeptide.

 The invention also relates to a recombinant integration vector comprising a nucleic acid or an expression cassette as defined above, as well as to a host cell, modified by the integration of this nucleic acid or of this expression cassette in its genome.

 Another subject of the invention is the use of a nucleic acid, an expression cassette, a recombinant vector or a recombinant host cell as defined above for obtaining an animal. transgenic.

 The invention also relates to methods for obtaining a transgenic animal modified by integration into its genome of a nucleic acid, an expression cassette, a recombinant vector or a recombinant host cell such as as defined above.

Detailed description of the invention:
It has been shown according to the invention that a nucleic acid which comprises both (i) a regulatory region comprising at least one copy of the target nucleotide motif of the factor

<Desc / Clms Page number 4>

 transcription of the SGF1 forkhead type, a suitable transcription promoter site and (ii) a region coding for a modified signal peptide, said region being placed under the control of said regulatory region, is capable of directing the synthesis of a protein of interest specifically in the posterior sericogenic cells of the Bombyx mori lepidoptera, when the nucleotide sequence encoding said protein of interest is fused in phase with the nucleotide sequence encoding said modified signal peptide.

 According to the invention, the nucleotide sequence encoding the protein of interest is fused in phase with the nucleotide sequence encoding said modified signal peptide when the nucleotide located at the 3 ′ end of the last codon of the sequence encoding the modified signal peptide precedes the nucleotide located at the 5 'end of the first codon of the sequence coding for the protein of interest.

 Surprisingly, the applicant has shown that the replacement of the codon for the cysteine of the signal peptide included in the preproprotein fibrohexamerine of the fiber of the silk thread by a hydrophobic amino acid such as alanine, isoleucine or else leucine, allowed a protein fused with said modified signal peptide to be exported outside the seric glands of Bombyx mori, like the proteins constituting the silk thread.

 The subject of the invention is a nucleic acid directing the expression of a protein of interest specifically in the cells of the posterior sericogenic glands of Bombyx mori, characterized in that said nucleic acid comprises, from the 5 ′ end towards the 3 'end, (i) a regulatory region comprising signals regulating the expression of a polynucleotide of interest specifically in said sericogenic cells and (ii) a region coding for a modified signal peptide, placed under the control of said region regulatory, said nucleic acid having at least 90% nucleotide identity with the polynucleotide ranging from nucleotide 1150 to the nucleotide

<Desc / Clms Page number 5>

 2026 of the sequence SEQ ID N 1 and in which the trinucleotide ranging from nucleotide 1486 to nucleotide 1488 of the sequence SEQ ID N 1 codes for an amino acid chosen from the group comprising alanine, isoleucine and leucine.

 Preferably, any nucleic acid and any polypeptide according to the invention is in an isolated or purified form.

 The term "isolated" in the sense of the present invention designates a biological material which has been removed from its original environment (the environment in which it is naturally located). For example, a polynucleotide found naturally in an organism is not isolated. The same polynucleotide separated from the adjacent nucleic acids in which it is naturally inserted into the genome of the organism is isolated. Such a polynucleotide may be included in a vector and / or such a polynucleotide may be included in a composition and nevertheless remain in an isolated state since the vector or the composition does not constitute its natural environment. The same reasoning also applies to a polypeptide extracted from its natural environment.

 The term "purified" does not require that the material be present in a form of absolute purity, exclusive of the presence of other compounds. Rather, it is a relative definition.

 A polynucleotide or a polypeptide is in the purified state after purification of the starting material or of the natural material of at least one order of magnitude, preferably two or three and preferably four or five orders of magnitude.

 For the purposes of the present description, the expression "nucleotide sequence" can be used to denote either a polynucleotide or a nucleic acid. The term "nucleotide sequence" encompasses the genetic material itself and is therefore not limited to information regarding its sequence.

<Desc / Clms Page number 6>

 The terms "nucleic acid", "polynucleotide", "oligonucleotide or even nucleotide sequence include RNA, DNA, cDNA sequences or even RNA / DNA hybrid sequences of more than one nucleotide, either in the form single strand or in the form of duplex.

 The term "nucleotide" designates natural nucleotides (A, T, G, C and U).

 For the purposes of the present invention, a first polynucleotide is considered to be "complementary" to a second polynucleotide when each base of the first nucleotide is paired with the base complementary to the second polynucleotide whose orientation is reversed. The bases, complementary are A and T (or A and U), and C and G.

 According to the invention, a first nucleic acid having at least 90% identity with a second reference nucleic acid will have at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.3% 98.6%, 99%, 99.6% of amino acid identity with said second reference nucleic acid.

 The percentage of identity between two nucleic acid sequences, within the meaning of the present invention, is determined by comparing the two optimally aligned sequences, through a comparison window.

 The part of the nucleotide sequence in the comparison window can thus include additions or deletions (for example gaps) with respect to the reference sequence (which does not include these additions or these deletions) so as to obtain an optimal alignment between the two sequences.

 The percentage of identity is calculated by determining the number of positions at which an identical nucleic base is observed for the two sequences compared, then by dividing the number of positions at which there is identity between the two nucleic bases by the total number of positions in the window

<Desc / Clms Page number 7>

 comparison, then multiplying the result by one hundred in order to obtain the percentage of nucleotide identity of the two sequences between them.

 The optimal alignment of the sequences for the comparison can be carried out by computer using known algorithms.

 Most preferably, the percentage of sequence identity is determined using the CLUSTAL W software (version 1.82), the parameters being fixed as follows: (1) CPU MODE = ClustalW mp; (2) ALIGNMENT = full; (3) OUTPUT FORMAT = aln w / numbers (4) OUTPUT ORDER = aligned; (5) COLOR ALIGNMENT = no; (6) KTUP (word size) = default; (7) WINDOW LENGTH = default; (8) SCORE TYPE = percent; (9) TOPDIAG = default; (10) PAIRGAP = default; (11) PHYLOGENETIC TREE / TREE TYPE = none; (12) MATRIX = default; (13) GAP OPEN = default; (14) END GAPS = default; (15) GAP EXTENSION = default; (16) GAP DISTANCES = default; (17) TREE TYPE = cladogram and (18) TREE GRAP DISTANCES = hide.

 A nucleic acid having at least 90% nucleotide identity with a nucleic acid according to the invention includes variants of a nucleic acid according to the invention.

 By variant of a nucleic acid according to the invention, is meant a nucleic acid which differs from the reference nucleic acid by one or more substitutions, additions or deletions of a nucleotide, with respect to the reference nucleic acid. A variant of a nucleic acid according to the invention can be of natural origin, such as an allelic variant which exists naturally. Such a variant nucleic acid can also be an unnatural nucleic acid obtained, for example by mutagenesis techniques.

 In general, the differences between the reference nucleic acid and the variant nucleic acid are reduced so that the reference nucleic acid and the variant nucleic acid

<Desc / Clms Page number 8>

 have very similar nucleotide sequences and in many identical regions.

 The variants of the regulatory nucleic acid according to the invention all comprise 100% of nucleotides identity with the region going from nucleotide 1379 to nucleotide 1390 of the sequence SEQ ID N 1. Furthermore, the trinucleotide of variants of l the regulatory nucleic acid according to the invention corresponding to the trinucleotide ranging from nucleotide 1486 to nucleotide 1488 of the sequence SEQ ID N 1 always codes for an amino acid chosen from the group comprising alanine, isoleucine, and leucine.

Thus, in the regulatory nucleic acid according to the invention, the trinucleotide going from nucleotide 1486 to nucleotide 1488 of the sequence SEQ ID N 1 is chosen from the following trinucleotides: (i) the following trinucleotides coding for the amino acid alanine: GCU, GCT, GCC, GCA and GCG; (ii) the following trinucleotides encoding the amino acid isoleucine: AUU, ATT, AUC, ATC, AUA and ATA; (iii) the following trinucleotides coding for the amino acid leucine:
CUU, CTT, CUC, CTC, CUA, CTA, CUG, and CTG.

 A variant of a regulatory nucleic acid according to the invention retains the ability to direct the expression of a polynucleotide of interest specifically in the cells of the posterior seric glands of a lepidoptera. To verify the functional character of a variant of a nucleic acid according to the invention, including a nucleic acid comprising 90% of nucleotides identity with the sequence SEQ ID N 1, a person skilled in the art can # opens the specific expression tests for a reporter or marker gene described in the examples.

 By polynucleotide of interest within the meaning of the invention is meant a polynucleotide capable of directing the synthesis of a fusion polypeptide between a modified signal peptide and a polypeptide of interest.

<Desc / Clms Page number 9>

 A nucleic acid according to the invention comprises a sequence with a promoter function, said promoter constituting a regulatory signal making it possible to direct the expression of a polynucleotide of interest, specifically in the cells of the posterior sericigenic gland of a lepidoptera.

 For the purposes of the present description, a nucleic acid with a promoter function, also designated promoter or promoter sequence, consists of a nucleic acid which has the recognition patterns of RNA polymerase, and more specifically of a TATA box and of a CAAT box, the structure of which is well known to those skilled in the art.

It has been shown according to the invention that the nucleic acid of sequence SEQ ID N 1 comprises the regulatory signals necessary for the expression of a polynucleotide of interest, specifically in the cells of the posterior sericogenic glands of Bombyx mori. Such specificity had not been observed with the regulatory sequences described by Tamura (Tamura et al. Germline transformation of the silkworm Bombyx mari L. using a piggyBac transposon derived vector.

Nature Biotechnology Vol 18,2000) whose expression was not limited to the cells of the posterior seric glands of Bombyx mori. It has also been shown according to the invention that the nucleic acid comprising, from the 5 ′ to the 3 ′ end, the polynucleotide ranging from nucleotide 1150, to nucleotide 2026 of the sequence SEQ ID N 1, is capable of direct the expression of a polynucleotide of interest specifically in the cells of the posterior sericogenic glands of Bombyx mori.

 The sequence SEQ ID N 1 comprises, from the 5 ′ to the 3 ′ end, the promoter of the fibrohexamerine gene between nucleotide 1 and nucleotide 1452. This promoter comprises a localized TATA box of the nucleotide in position 1420 up to 'to the nucleotide at position 1423 of the sequence SEQ ID N 1. This promoter sequence also contains a CAAT box located from the nucleotide at position 1362 to the nucleotide at position 1365 of the sequence SEQ ID N 1. This

<Desc / Clms Page number 10>

 promoter sequence finally contains a TATTTATTTAA sequence located from the nucleotide at position 1379 to the nucleotide at position 1390 of the sequence SEQ ID N 1. The TATA box constitutes the promoter element proper, which is generally located at a distance of approximately 30 bases of the transcription initiation site. The CAAT box is a cis-acting element which is commonly found in the promoter and activator region (enhancer).

 The sequence SEQ ID N 1 comprises exon 1 of fibrohexamerine between the nucleotide at position 1452 and the nucleotide at position 1525, which codes for the modified signal peptide.

The sequence SEQ ID N 1 comprises the intron 1 of fibrohexamerine between the nucleotide at position 1526 and the nucleotide at position 2026. The sequence SEQ ID N 1 comprises a polynucleotide between the nucleotide at position 2027 and the nucleotide at position 2040. This last polynucleotide is not essential to the invention and only facilitated the construction of the sequence SEQ ID N 1 which further comprises a polynucleotide coding for a reporter protein Ds-Red between the nucleotide at position 2041 and the nucleotide at position 2721 .

 A nucleic acid comprising (i) the regulatory region extending from the nucleotide in position 1 to the nucleotide in position 1452 of the sequence SEQ ID N 1 or any biologically active fragment of this nucleic acid containing the boxes TATA, CAAT and at least one sequence CTATTTATTTAA as defined above and (ii) a region coding for a modified signal peptide placed under the control of such a nucleic acid constitutes another object of the present invention.

 It has been shown according to the invention that exon 1 of the fibrohexamerine gene as defined above must carry a modification of the trinucleotide in position 1486 to 1488 of the sequence SEQ ID N 1, so that the addressing of the polypeptide encoded by the polynucleotide of interest is correct, i.e. so that

<Desc / Clms Page number 11>

 the polypeptide encoded by the polynucleotide of interest is secreted with the silk thread. The trinucleotide at position 1486 to 1488 of the sequence SEQ ID N 1 coding for cysteine must be modified into a trinucleotide coding for an amino acid chosen from the group comprising alanine, isoleucine and leucine. Thanks to the production of DNA constructs comprising all or more restricted parts of the sequence SEQ ID N 1, it has been shown according to the invention that the nucleic acid of the invention makes it possible to specifically direct the expression of '' a polynucleotide of interest in the cells of the posterior seric glands of Bombyx mori. The presence of the reporter gene Ds-Red also made it possible to show that the modification of exon 1 of the fibrohexamerin gene makes it possible to obtain correct secretion of the polypeptide encoded by the polynucleotide of interest.

 Expression cassettes comprising a polynucleotide of interest coding for a reporter gene, the gene for the protein Ds-Red, which was placed under the control of a regulatory nucleic acid as defined above, were produced. both tissue specific regulatory signals, modified fibrohexamerin exon 1, all fibrohexamerin intron 1, and a promoter-serving nucleic acid.

 These expression cassettes were used to transform Bombyx mori cells, and more precisely gametic precursors, at the origin of a transgenic animal, after which the expression of the reporter gene was followed in the various tissues of the 'animal. The results show that the regulatory signals necessary for tissue specific transcription activity and for strong expression in the cells of the posterior sericogenic gland of Bombyx mori are included in particular in the region going from nucleotide 1150 to nucleotide 2026 of the sequence SEQ ID N 1.

 More specifically, it has been shown according to the invention that this region contains a motif: CTATTTATTTAA (sequence SEQ ID N 2) localized from the nucleotide in position 1379 to the nucleotide in position 1390 of the sequence SEQ ID N 1. This

<Desc / Clms Page number 12>

motif constitutes a binding site to transcription factors of the SGF1 forkhead type (Julien, E., Bordeaux, MC, Garel, A. & Couble, P. (2002). Fork head alternative binding drives stage specific expression in the silk gland of Bombyx mori. Insect Biochem Molecul. Biol. 32, 377-387.)
Without wishing to be bound by any theory, the inventors believe that the CTATTTATTTAA motif constitutes an important structural characteristic of the nucleic acid of the invention having a function of sequence of activating transcription (enhancer) and regulator of specific expression of tissue.

 According to the invention, the insertion of new CTATTTATTTAA motifs upstream of the motif naturally contained in the fibrohexamerine promoter makes it possible to increase the expression rate of the polypeptide encoded by the polynucleotide of interest, without altering the specificity.

 The introduction of DNA carried by the piggyBac integration vector into the Bombyx mori genome, obtained after microinjection in the eggs of the animal, also made it possible to surprisingly show that the modification of exon 1 of the fibrohexamerin gene makes it possible to obtain an addressing of the protein of interest compatible with a secretion of this in the silk thread. In other words, when a polynucleotide of interest is fused in phase at the 3 ′ end of exon 1 of the fibrohexamerin gene, the modification of exon 1 of this gene, which codes for the signal peptide modified allows to keep the secretion of the protein of interest identical to that of fibrohexamerine.

Regulatory nucleic acids according to the invention
The subject of the invention is a nucleic acid directing the expression of a protein of interest specifically in the cells of the posterior sericogenic glands of Bombyx mori, characterized in that said nucleic acid comprises,

<Desc / Clms Page number 13>

 the 5 'end towards the 3' end, (i) a regulatory region comprising signals regulating the expression of a polynucleotide of interest specifically in said sericogenic cells and (ii) a region coding for a modified signal peptide , placed under the control of said regulatory region, said nucleic acid having at least 90% nucleotide identity with the polynucleotide ranging from nucleotide 1150 to nucleotide 2026 of the sequence SEQ ID N 1 and in which the trinucleotide ranging from nucleotide 1486 to nucleotide 1488 of the sequence SEQ ID N 1 codes for an amino acid chosen from the group comprising alanine, isoleucine, and leucine.

 According to a first embodiment, specific to the above nucleic acid, said nucleic acid is characterized in that it comprises, from the 5 ′ end towards the 3 ′ end, (i) a regulatory region comprising signals for regulating the expression of a polynucleotide of interest specifically in said sericogenic cells and (ii) a region coding for a modified signal peptide, placed under the control of said regulatory region, said nucleic acid comprising, from the 5 end 'at the 3' end, the polynucleotide ranging from nucleotide 1150 to nucleotide 2026 of the sequence SEQ ID N 1 and in which the trinucleotide ranging from nucleotide 1486 to nucleotide 1488 of the sequence SEQ ID N 1 codes for an amino acid chosen from the group comprising alanine, isoleucine, and leucine.

 According to a second particular embodiment of the above nucleic acid, said nucleic acid is characterized in that it comprises, from the 5 ′ end towards the 3 ′ end, (i) a regulatory region comprising signals for regulating the expression of a polynucleotide of interest specifically in said sericogenic cells and (ii) a region coding for a modified signal peptide, placed under the control of said regulatory region, said nucleic acid comprising, from the 5 'end at the 3 'end, the polynucleotide ranging from nucleotide 1 to nucleotide 2026 of the sequence SEQ ID N 1 and in which the trinucleotide ranging from nucleotide 1486 to nucleotide 1488 of the sequence SEQ ID N 1

<Desc / Clms Page number 14>

 code for an amino acid selected from the group consisting of alanine, isoleucine, and leucine.

 According to a third particular embodiment, the above nucleic acid is modified by the introduction of at least one and at most four copies of a polynucleotide of sequence SEQ ID N 2, between nucleotides 1 and 1379 of the sequence SEQ ID N 1. Such a modified nucleic acid corresponds for example to the nucleic acid of sequence SEQ ID N 3.

 The present invention also relates to a nucleic acid whose sequence is complementary to the sequence of any of the nucleic acids as defined above.

 As already specified, one of the objectives pursued by the present invention is to obtain expression at a high level of a polypeptide in the cells of the posterior sericogenic glands of Bombyx mori encoded by a polynucleotide of interest .

 The subject of the invention is therefore also a nucleic acid comprising a polynucleotide of interest encoding a polypeptide of interest, fused with the regulatory nucleic acid directing the expression of the polypeptide of interest specifically in the posterior sericogenic cells of Bombyx mori, as defined in the present description. Such a nucleic acid comprising a polynucleotide encoding a polypeptide of interest is also designated an expression cassette for the purposes of the present description.

 The invention therefore also relates to an expression cassette comprising a polynucleotide of interest placed under the control of a regulatory nucleic acid as defined above.

Expression cassettes according to the invention:
According to a first embodiment, an expression cassette according to the invention is characterized in that the

<Desc / Clms Page number 15>

 polynucleotide of interest which it comprises codes for a polypeptide.

 Preferably, the polynucleotide of interest encodes a polypeptide chosen from proteins such as spider spideroid (genus Nephila), Galleria fibroin or more generally any other polypeptide which makes it possible to obtain a silk thread whose resistance properties ductility or elasticity, for example are changed.

 The polynucleotide of interest can also code for a soluble polypeptide of biomedical interest such as, for example, hormones, antigens, enzymes, growth factors, or even receptors.

Recombinant vectors according to the invention:
The invention relates to recombinant integration vectors comprising a regulatory nucleic acid or an expression cassette as defined above.

 The invention therefore consists of recombinant vectors into which a regulatory nucleic acid according to the invention has been inserted, comprising regulatory signals allowing the expression of a polynucleotide of interest, specifically in the cells of the posterior sericogenic glands of Bombyx. mori, when this polynucleotide of interest is placed under its control.

 Part of the invention thus forms recombinant integration vectors comprising: a) a regulatory nucleic acid as defined in the present description; and b) A polynucleotide of interest placed under the control of the regulatory nucleic acid defined in a).

 The polynucleotide of interest can consist of a polynucleotide coding for a detectable polypeptide or marker polypeptide such as for example a polypeptide coding for the protein DsRed.

<Desc / Clms Page number 16>

 A recombinant integration vector according to the invention meeting the above definition is the vector Pig fhx (sp *) DsRed- (3xP3-GFP) deposited on February 20, 2003 at the National Collection of Cultures of Microorganisms (CNCM) of the Institut Pasteur under the registration number CNCM 1-2975.

 The invention also relates to recombinant cloning vectors comprising the nucleotide sequence of a recombinant integration vector as described above.

 To obtain any of the regulatory sequences according to the invention, it is possible to use the techniques and primers described in the examples. Those skilled in the art can also use the vector Pig fhx (sp *) DsRed- (3xP3-GFP) deposited on February 20, 2003 at the National Collection of Cultures of Microorganisms (CNCM) of the Institut Pasteur under the registration number CNCM 1-2975.

General characteristics of the recombinant vectors according to the invention:
By vector within the meaning of the present invention, is meant a circular or linear DNA or RNA molecule which is either in single strand or double strand form. A vector according to the invention can be a cloning vector or an integration vector.

 According to a preferred embodiment, a recombinant vector according to the invention is an integrative vector allowing the insertion of copies of the DNA sequence inserted in this vector into the genome of the animal of the species Bombyx mori.

 Advantageously, the recombinant vector, or in other cases the expression cassette contained in this vector can also contain 3 'untranslated sequences called terminators. Among the terminators which can be used in the constructions of the invention, mention may be made of the polyadenylation sequence of SV40.

<Desc / Clms Page number 17>

The preferred integration vector according to the invention is derived from the piggyBac transposon originating from Trichoplusia ni, existing in the natural state in this species (Cary, LC et al. Transposon mutagenesis of baculoviruses: analysis of Trichoplusia nor transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology 172, 156-69 (1989))
The piggyBac transposon includes DNA fragments that can move from one location in the genome to another through transposition. It includes feet, left and right, composed of two short reverse repeated sequences framing the genes coding for its mobility functions.

 The integration vector described according to the invention is a modified piggyBac transposon in which the nucleic acid of interest replaces part of the genes coding for the mobility functions of the transposon which have been deleted.

 The integration vector is used in combination with an assistant vector allowing the expression of the deleted mobility genes in the integration vector.

 The integration vector associated with the assistant vector allows the integration of the nucleic acid or the expression casette according to the invention into the genome of the host cell by means of transposition.

 The integration vector according to the invention can also be derived from the Hobo element of Drosophila or from the Minos transposon of Drosophila.

 According to another embodiment, a recombinant cloning vector is used in order to amplify the nucleotide sequence of an integration vector as described above. According to this other embodiment, the above recombinant integration vector is linearized prior to its insertion into a recipient vector at a site or a cloning polysite of said recipient vector, before ligation. The cloning vectors used are of bacterial or viral origin. For example,

<Desc / Clms Page number 18>

 plasmids of the PUC, pBR or pSK type, known to those skilled in the art, which will not be described in detail.

Recombinant host cells according to the invention
To allow the expression of a polynucleotide of interest placed under the control of a regulatory nucleic acid according to the invention, the regulatory nucleic acids, the expression cassettes or also the recombinant vectors defined in the present description must be introduced. in a host cell. The introduction of the polynucleotides according to the invention into a host cell can be carried out in vitro, according to techniques well known to those skilled in the art. By host cell comprising a nucleic acid is meant a cell having integrated into its genome one or more copies of a nucleic acid.

 The invention further relates to a host cell comprising a regulatory nucleic acid, an expression cassette or also a recombinant vector as defined above.

 Such a host cell is preferably a cell from an animal belonging to the genus Bombyx, more particularly to the species Bombyx mori, whatever the strain considered. Such a host cell can also be a cell from an animal belonging to another species of lepidoptera such as Philosamia cynthia or more generally, to the genus Bombyx, Antheraea, Galleria, Philosamia, Spodoptera or Drosophila.

 Most preferably, a host cell according to the invention consists of a cell of the posterior sericogenic glands of Bombyx mori.

Transgenic animals according to the invention:
Another subject of the invention is the use of a nucleic acid, an expression cassette, a vector or a recombinant host cell as described above for

<Desc / Clms Page number 19>

 obtaining a transgenic silk-producing animal belonging to the genus Bombyx.

 Preferably, said transgenic animal belongs to the Bombyx mori species and secretes a protein of interest into the silk thread.

 The invention also relates to a transgenic multicellular organism, comprising a host cell or a plurality of host cells, an expression cassette or also a recombinant vector as defined above.

 The subject of the invention is also a transgenic animal, comprising, in a form integrated into its genome, a nucleic acid or an expression cassette as defined in the present description.

 In general, the invention also relates to the use of a regulatory nucleic acid, an expression cassette, a recombinant vector or also a host cell as defined above for the obtaining a transgenic animal.

 The transgenic animals according to the invention preferably belong to the species and to the genus which have already been listed above concerning the origin of the animal host cells of the invention.

 Another subject of the invention is a process for obtaining a transgenic animal belonging to the Bombyx mori species, characterized in that it comprises the following steps: a) Injection of a nucleic acid, of a cassette expression or vector as defined above in Bombyx mori eggs from one to five hours after egg laying, before blastoderm formation; b) Selection of transgenic animals having integrated into their genome a nucleic acid or an expression cassette as defined in the present description.

The subject of the invention is also a process for obtaining a transgenic animal belonging to the Bombyx mori species, characterized in that it further comprises the following steps:

<Desc / Clms Page number 20>

 c) Crossing between them of two transgenic animals as obtained in step b; d) Selection of animals homozygous for the transgene.

 The invention also relates to a process for obtaining a silk thread, characterized in that it comprises the following steps: a) breeding a transgenic animal as defined in the present description; b) Recover the silk produced by the transgenic animal.

 The subject of the invention is also a process for obtaining a protein of interest characterized in that it comprises the following steps: a) breeding a transgenic animal as defined in the present description; b) Recover the silk produced by the transgenic animal; c) Recover the protein of interest within the silk thread.

The invention is further illustrated, without being limited, by the following figures and examples, in which:
FIG. 1 illustrates a protocol for constructing a vector according to the invention.

FIG. 2 illustrates a protocol substituting the codon cysteine for an isoleucine codon in the signal peptide of the gene coding for fibrohexamerine
FIG. 3 illustrates a protocol for modifying the fibrohexamerin promoter by the addition of three copies of a polynucleotide of sequence SEQ ID N 2.

 Figure 4 is a schematic representation of the sequence SEQ ID N 3.

 FIG. 5 illustrates the tissue-specific accumulation of the Ds-Red protein placed under the control of a nucleic acid according to the invention.

 FIG. 6 illustrates the detection of the protein Ds-Red, placed under the control of a nucleic acid according to the invention, in the sericogenic glands of silkworms.

<Desc / Clms Page number 21>

 FIG. 7 illustrates the accumulation, in the silk thread, of the Ds-Red protein placed under the control of a nucleic acid according to the invention.

 FIG. 8 illustrates the detection of the protein Ds-Red, placed under the control of a nucleic acid according to the invention, in the cocoons of silkworms.

EXAMPLES: General materials and methods used in the examples.

A) Material:
The animals used belong to the Bombyx mori species, to the Nistari breed from the National Sericultural Unit (25, quai Jean-Jacques Rousseau 69350 La Mulatière, France) It is a multivoltine breed, developing without diapause embryonic. The bacteria used for cloning are the E.coli DH5alpha-TI Max strains. Efficiency available from INVITROGEN (reference 440097).

B) Methods # Animal breeding conditions:
The silkworms are reared under controlled conditions in a type A1 pet store at temperatures varying according to the age of the larvae (between 22 C and 25 C), and at a humidity of 75%. They are fed on an artificial medium prepared from ground mulberry leaves.

# Extraction of nucleic acids:
The DNA of the plasmid constructs was extracted from the bacteria by the Qiaprep Spin Miniprep kit from the company Qiagen (reference 27106). The genomic DNA of the silkworms was extracted by phenol-chloroform-isoamyl alcohol.

<Desc / Clms Page number 22>

# Production of transgenic animals:
The injection of the integration vector into the eggs of the animal is carried out 2 to 5 hours after laying, before the formation of the blastoderm. A solution comprising the integration vector and the "assistant" vector in equal amounts is prepared in a 0.5 mM phosphate buffer (pH 7.0) containing 5 mM KCI. One to 5 nL of this solution is injected using an Eppendorf microinjector (Transjector 5246). The hole formed by the injection is then covered with glue (Loctite Superglue 3 from Henkel France SA) and the eggs are kept at 25 C to continue their development.

 # Selection of foreign animals: The selection of animals made transgenic with the vector Pigfhx (sp *) DsRed- (3xP3-GFP) is carried out thanks to the early expression of the 3xP3-GFP gene in the eyes of the animal.

# Test for detecting the presence of the protein Ds-Red in the transgenic animal:
The Ds-Red protein is visualized in situ (in the cells of the seric gland) and in the cocoon, under a Leica fluorescence magnifier (MZFL2) equipped with an excitation light emission system (558 nm) and a 583 nm emission light filter and a digital camera. Analysis of the fluorescence of the cocoons is carried out dry. The dissected seric glands are observed under a magnifying glass in PBS. The detection of the Ds-Red protein was carried out at different stages of development and, in particular, at the 5th larval stage at the time of the massive synthesis of silk proteins.

 On protein extracts from seric glands or silk cocoons, detection is carried out by Western

<Desc / Clms Page number 23>

 Blot using a double antibody: an anti-Ds-Red rabbit antibody (Clonetech Company, reference 8370-1) and an anti-Goat Rabbit antibody coupled to peroxidase (Biorad Company, reference 170-6515).

# Western blot DS-red protein detection test with samples of seric glands, cocoon and silkworm blaze:
The Western Blot DS-red protein detection test includes several steps: - Extraction of protein samples:
We take a third of the cocoon or the pair of seric glands (secretor separated from the reservoir) which we transfer into a microtube. The cocoon is then fragmented into small pieces or the gland is ground in 1 ml of LiSCN 10M and the protein extracts are incubated for at least 1 hour at room temperature, shaking periodically using a vortex. Finally, the protein samples are diluted in 4/5 of 10 mM tris buffer - 2% SDS - 5% mercaptoethanol.

 The outer envelope of the cocoon or blaze is transferred to a microtube to which 150 L of H20 are added to rinse the blaze. The soluble DsRed protein is recovered by passing the sample to ultrasound for 10 minutes.

 The samples are prepared before deposition by denaturing the protein extracts in the Laemmli 5X buffer, 5 min. at 100 C.

- Electrophoresis:
The protein extracts are deposited on precast gels (In Vitrogen Novex TrisGlycine, 10 wells, 1.0 mm). The proteins are migrated at 120 Volts, 40 mA for 2 h 30 min with a migration buffer (In Vitrogen Novex SDS Running buffer (10X)).

<Desc / Clms Page number 24>

- Protein transfer:
The proteins are transferred to nitrocellulose membranes 9 cm by 6 cm (Hybond ECL Amersham Pharmacia) The size of the whatman paper used is 10 cm by 7.5 cm. Protein transfer is carried out at 120 Volts, 350 mA for 1 h 15 min with transfer buffer (InVitrogen Novex Tris Glycine Transfer Buffer (25X)).

- Staining of proteins with Rouge Ponceau:
The nitrocellulose membrane is incubated with Ponceau red (Sigma) for 1 minute to reveal the proteins which have been transferred. If necessary, the strips of the size marker are marked with a pen.

- Blocking of the membrane:
The membrane is incubated in PLT (PBS-Tween 0.2% - 2% milk) overnight at 4 C.

- Incubation with the first antibody:
The primary anti-DsRed antibody (Living Color DsPeptide Clontech) is diluted in the PLT to 1 / 500th, and incubated for 2 hours at room temperature. The incubation is followed by rinsing twice for 5 minutes in 0.02% PBS-tween.

- Incubation with the second antibody:
The secondary antibody (Goat Anti-Rabbit IgG (H + L), Horseradish Peroxidase Conjugate Biorad) is diluted in the PLT to 1 / 3000th, and incubated for 1 hour at room temperature.

The incubation is followed by rinsing 4 times 10 minutes in PBS-tween 0.02%.

- Revelation of membranes:
Use solution A (5 mL of 100 mM Tris, pH 8.5, 22 L of coumaric acid and 50 l of Luminol to add in the dark) and solution B (5 mL of 100 mM Tris, pH 8. 5 , 3 L of H202).

A mixture of solutions A and B at

<Desc / Clms Page number 25>

 in the dark, then the membrane to be revealed is incubated for 1 minute in the mixture of solution A and B. The membrane is exposed on an autoradiographic film for approximately 3 minutes and the film is exposed after exposure.

Example 1: Construction of the integration vector comprising an expression cassette comprising a polynucleotide coding for the Ds-Red polypeptide, placed under the control of a regulatory nucleic acid according to the invention and, in particular, construction of the cassette d expression comprising part of the sequence SEQ ID N 1.

 The first stage of construction is shown in Figure 1A. Two plasmids are required for this first step.

 It is the plasmid pDsRed1-N1 marketed by the company Clonetech (reference 6921-1), comprising, from the 5 ′ end, towards the 3 ′ end: - The CMV IE promoter, - The Ds-Red gene, - The SV40 polyadenylation signal, - The kanamycin resistance gene, - The origin of replication of the plasmid pUC.

 The Ds-Red protein gene is delimited at its ends by the nucleotide sequence of the restriction enzymes Age # and Not I.

The second plasmid used, called pfhx (sp *) GFP, from the 5 'end to the 3' end: - The modified fibrohexamerine promoter - The gene coding for the green fluorescent protein (GFP) - The signal for polyadenylation SV40 - The kanamycin resistance gene - The origin of replication of the plasmid pUC
As shown in FIG. 1A, the plasmid pfhx (sp *) GFP, also includes a nucleotide sequence

<Desc / Clms Page number 26>

 delimited by the nucleotide sequence of two restriction enzymes, Age # and Not I, placed downstream of the promoter sequence of the fibrohexamerine gene.

 The Age I-Not I fragment, originating from the plasmid pDSred1-N1 and comprising the sequence coding for the protein Ds-Red is cloned into the plasmid pfhx (sp *) GFP, in place of the sequence Age I-Not I.

 The plasmid resulting from this cloning, called pfhx (sp *) DsRed is represented in FIG. 1B, and comprises from the 5 ′ end, towards the 3 ′ end: - The promoter of the modified fibrohexamerine, - The Ds gene- Red, - The SV40 polyadenylation signal, - The kanamycin resistance gene, - The origin of replication of the plasmid pUC.

 As shown in FIG. 1B, the modified fibrohexamerin promoter and the Ds-Red gene are included in a fragment delimited by the sequences Bgl II and Nae 1. This fragment is cloned in the vector pPigA3cyp6a2- (3xP3- GFP) 2 derived from the piggyBac transposon, the structure of which is shown in FIG. 1B, in place of the Bgl II-Stu # fragment included in the latter, according to a method identical to that described above. The vector pPigA3cyp6a2- (3xP3-GFP) 2 comprises a left foot (L), and a right foot (R) shown in FIG. 1 B.

 The vector obtained at the end of this second cloning, represented in FIG. 1C, named Pigfhx (sp *) DsRed- (3xP3-GFP) meets the definition of a vector according to the invention.

 Example 2: Method of substitution of the codon cysteine in an isoleucine codon in the gene coding the signal peptide of fibrohexamerine. The method used is shown in Figure 2 and includes several steps. First of all, a fragment of the fibrohexamerin promoter is isolated by digestion with the restriction enzymes Hind III and Pst I. The second step, designated PCR1 in FIG. 2A, consists in carrying out a

<Desc / Clms Page number 27>

 polymerase chain reaction using the primers of sequence SEQ ID N 4 and SEQ ID N 5 shown in FIG. 2A. The third step, designated PCR2 in FIG. 2A, consists in carrying out a polymerase chain reaction using the primers of sequence SEQ ID N 6 and SEQ ID N 7.

The primers of sequence SEQ ID N 5 and SEQ ID N 6 comprise an isoleucine codon, intended to replace the cysteine codon of the natural signal peptide of fibrohexamerine. The amplified nucleic acids are then ligated, and the nucleic acid shown in FIG. 2B is obtained, delimited by the sequences of the restriction enzymes Sac11 and Pstl. This nucleic acid comprises an isoleucine codon, shown in bold in FIG. 1B, replacing the natural codon cysteine. This fragment is then cloned in place of the corresponding natural sequence in the plasmid pfhx (sp *) GFP.

EXAMPLE 3 Protocol for Modifying the Fibrohexamerine Promoter by Adding Three Copies of a Polynucleotide of Sequence ID N 2, Permitting the Fixation of the Transcription Factor SGF-1 / Forkhead Between Nucleotides 1 and 1379 sequence SEQ ID N 1. This protocol is illustrated in Figure 3.

 In a first step, a polymerase chain reaction of the fibrohexamerin promoter sequence is carried out. This amplification is carried out using primers A, B, C and D, represented in FIG. 3, of sequence SEQ ID N 8 for primer B, and of sequence SEQ ID N 9 for primer C.

 In a second step, the nucleic acids obtained are subjected to a ligation, in order to obtain the nucleic acid whose coding strand is represented in FIG. 3. The nucleic acid obtained comprises part of the sequence of the fibrohexamerine promoter modified by the addition of three copies of a polynucleotide of sequence SEQ ID N 2. This nucleic acid is then cloned into the plasmid pfhx (sp *) GFP, in place of the

<Desc / Clms Page number 28>

 natural sequence of the fibrohexamerin promoter, delimited by the Hind III and Pst I restriction sites.

 The diagram of the sequence SEQ ID N 3 is shown in FIG. 4. The sequence SEQ ID N 3 comprises, from the 5 ′ end to the 3 ′ end: # The promoter (1) of the fibrohexamerine gene.

 # Three sequences (2) for fixing the element SGF1 / Forkhead (3), in addition to the natural sequence.

 # Exon 1 (4) of fibrohexamerine, whose codon cysteine is modified.

 # Intron 1 (5) of fibrohexamerine.

 # A polynucleotide (6) which facilitated the construction of the sequence SEQ ID N 1 # A polynucleotide (7) coding for the reporter protein Ds-Red.

Example 4: Accumulation of the Ds-Red Protein in the Sericigenic Glands of Silkworms Transgenic with the Vector pPigFbx (sp *) DsRed- (3xP3-GFP) Derived from the PiggyBac Transposon Comprising the Sequence ID N 1

Photographs a, b, c, d, e and f presented in FIG. 5 illustrate the tissue-specific expression of the Ds-Red protein in a silkworm rendered transgenic with the vector derived from the piggyBac transposon comprising the sequence SEQ ID N 3 Photographs a, b and c were taken in daylight at three magnifications. The test for detecting the presence of the Ds-Red protein was carried out according to the protocol described above. The photographs d, e and f are shots corresponding to the photographs a, b and c but taken in the fluorescence spectrum of the protein Ds-Red.

 These photographs show that the fluorescence of the protein Ds-Red, and therefore its expression is limited to the posterior sericogenic cells (PSG, 2) of Bombyx morio

<Desc / Clms Page number 29>

No fluorescence corresponding to the presence of the Ds-Red protein is observed in the median sericogenic cells (MSG, 1). It can be deduced therefrom that the nucleic acids according to the invention make it possible to direct the expression of a polypeptide of interest specifically in the posterior sericogenic glands of a lepidoptera.

 This result is confirmed by the Western Blot represented in FIG. 6. This Western Blot produced under the conditions described above, makes it possible to detect the presence of the protein Ds-Red in the sericogenic glands of silkworms. An SDS-PAGE gel (14%; Tris-glycine; 0.1% SDS) was transferred to a nitrocellulose filter and stained with Coomassie blue. The nitrocellulose filter is presented in FIG. 6A. This same filter was then incubated with a primary polyclonal antibody against the Ds-Red protein, then with a secondary anti-rabbit goat antibody conjugated to horseradish peroxidase. The result is shown in Figure 6B. Bands detected by chemiluminescence are observed, corresponding to the presence of the protein Ds-Red.

 Four protein deposits from the seric glands of a silkworm are visible on the filter presented in FIGS. 6A and 6B. The proteins were extracted from the seric glands according to the protocol described above. The four deposits are as follows: - Track 1: proteins from the median seric glands of a non-transgenic silkworm.

- Lane 2: proteins from the median seric glands of a transgenic silkworm, heterozygous for the transgene contained in the sequence SEQ ID N 1.

- Lane 3: proteins from the posterior seric glands of a non-transgenic silkworm.

- Lane 4: proteins from the posterior seric glands of a transgenic silkworm, heterozygous for the transgene contained in the sequence SEQ ID N 1.

 The filter shown in FIG. 6A presents bands of comparable intensity, from one track to another. This leads to the conclusion that

<Desc / Clms Page number 30>

 equivalent amounts of protein were deposited on the SDS-PAGE gel. The filter presented also includes a size marker visible on the left side of the filter. The same filter, subjected to antibody treatment, as described above is shown in Figure 6B. It is observed that the protein Ds-Red is present in large quantities in the posterior seric glands. The protein Ds-Red is also present in the median seric glands, but to a lesser extent. The inventors believe that this presence is due to the migration of the Ds-Red protein secreted by the posterior sericogenic cells into the median sericogenic glands, and not to a synthesis of the Ds-Red protein by the median sericogenic glands.

Example 5 Accumulation of the Ds-Red Protein in Silk in Silk Worms Made Transgenic with the Vector Pigfhx (sp *) DsRed- (3xP3-GFP) Derived from the PiggyBac Transposon Comprising the Sequence ID N 1.

 The test for detecting the presence of the protein Ds-Red was carried out according to the protocol described above. As observed in FIG. 7, the silk threads exhibit a fluorescence corresponding to the presence of the protein Ds-Red.

It can be deduced therefrom that the nucleic acids according to the invention make it possible to direct the secretion of a polypeptide of interest in the silk of a lepidoptera.

 This result is confirmed by the Western Blot shown in FIG. 8. This Western Blot, carried out under the conditions described above, makes it possible to detect the presence of the protein Ds-Red, in the cocoons of silkworms. An SDSPAGE gel (14%; Tris-glycine; 0.1% SDS) was transferred to a nitrocellulose filter and stained with Ponceau red. The nitrocellulose filter is presented in FIG. 8A. This same filter was then incubated with a primary polyclonal antibody against the Ds-Red protein, then with a secondary anti-rabbit goat antibody conjugated to horseradish peroxidase. The result is presented on

<Desc / Clms Page number 31>

 Figure 6B. Bands detected by chemiluminescence are observed, corresponding to the presence of the protein Ds-Red.

 Nine protein deposits from silkworm cocoons are visible on the filter presented in FIGS. 8A and 8B. The proteins were extracted according to the protocol described above. These are: - Tracks C1 and C2: proteins from a non-transgenic silkworm cocoon.

 - Tracks T1 and T2: from a transgenic silkworm cocoon, heterozygous for the transgene described in the sequence SEQ ID N 1.

 The first six deposits, starting from the size marker, present on the left of the filter, correspond to proteins from the cocoon of a silkworm stripped of the outer envelope called blaze. The last three deposits correspond to proteins from rinsing the blaze of the cocoon of a silkworm with water.

 The filter shown in FIG. 8A makes it possible to observe that the quantities of proteins initially deposited on the SDSPAGE gel are equivalent. The same filter, subjected to the processing described above is represented in FIG. 8B. It is observed that the protein Ds-Red is present in large quantity in the fraction resulting from the rinsing of the blaze of the transgenic silkworm cocoon. It can be deduced therefrom that the nucleic acids according to the invention make it possible to direct the secretion of a polypeptide of interest in the silk of a lepidoptera.

Claims (22)

SEQ ID N 1 codes for an amino acid chosen from the group comprising alanine, isoleucine and leucine. 3 ', (i) a regulatory region comprising signals regulating the expression of a polynucleotide of interest specifically in said sericogenic cells and (ii) a region coding for a modified signal peptide, placed under the control of said regulatory region , said nucleic acid having at least 90% nucleotide identity with the polynucleotide ranging from nucleotide 1150 to nucleotide 2026 of the sequence SEQ ID N 1 and in which the trinucleotide ranging from nucleotide 1486 to nucleotide 1488 of the sequence  CLAIMS 1. Nucleic acid directing the expression of a protein of interest specifically in the cells of the posterior sericogenic glands of Bombyx mori, characterized in that said nucleic acid comprises, from the 5 ′ end towards the end 2. Nucleic acid according to claim 1, characterized in that said nucleic acid comprises, from the 5 ′ end towards the 3 ′ end, (i) a regulatory region comprising signals for regulating the expression of a polynucleotide of interest specifically in said sericogenic cells and (ii) a region coding for a modified signal peptide, placed under the control of said regulatory region, said nucleic acid comprising, from the 5 ′ to the 3 ′ end, the polynucleotide ranging from nucleotide 1150 to nucleotide 2026 of the sequence SEQ ID N 1 and in which the trinucleotide ranging from nucleotide 1486 to nucleotide 1488 of the sequence SEQ ID N 1 codes for an amino acid chosen from the group comprising alanine, isoleucine and leucine. 3. Nucleic acid according to claim 1, characterized in that said nucleic acid comprises, from the 5 ′ end towards the 3 ′ end, (i) a regulatory region comprising <Desc / Clms Page number 33>  signals for regulating the expression of a polynucleotide of interest specifically in said sericogenic cells and (ii) a region coding for a modified signal peptide, placed under the control of said regulatory region, said nucleic acid comprising, from the 5 end 'at the 3' end, the polynucleotide ranging from nucleotide 1 to nucleotide 2026 of the sequence SEQ ID N 1 and in which the trinucleotide ranging from nucleotide 1486 to nucleotide 1488 of the sequence SEQ ID N 1 codes for an amino acid chosen from the group comprising alanine, isoleucine and leucine. 4. Nucleic acid according to one of claims 1 to 3, characterized in that it is modified by the introduction of at least one and at most four copies of a polynucleotide of sequence SEQ ID N 2, between nucleotides 1 and 1379 of the sequence SEQ ID N 1. 5. Nucleic acid of sequence complementary to a nucleic acid according to one of claims 1 to 4. 6. Expression cassette comprising a polynucleotide of interest placed under the control of a nucleic acid according to one of claims 1 to 5. 7. Expression cassette according to claim 6, characterized in that the polynucleotide of interest codes for a polypeptide. 8. Expression cassette according to claim 7, characterized in that the polynucleotide of interest codes for a polypeptide chosen from the following proteins: - spider spidroin; and - fibroin from animals belonging to the genus Galleria. <Desc / Clms Page number 34> 9. Recombinant integration vector comprising a nucleic acid according to one of claims 1 to 5 or an expression cassette according to one of claims 6 to 8. 10. Recombinant cloning vector comprising the nucleotide sequence of a recombinant integration vector according to claim 9. 11. Recombinant integration vector according to claim 9, characterized in that it is the vector Pigfhx (sp *) DsRed- (3xP3-GFP) deposited on February 20, 2003 in the Collection Nationale de Cultures de Microorganismes (CNCM) of the Institut Pasteur under the registration number CNCM 1- 2975. 12. Recombinant host cell comprising a nucleic acid according to one of claims 1 to 5 or an expression cassette according to one of claims 6 to 8 or a vector according to one of claims 9 to 11. 13. Recombinant host cell according to claim 12, characterized in that it is a cell of a silk-producing animal belonging to a genus chosen from Bombyx, Antheraea, Galleria, Philasomia, or Spodoptera Drosophila. 14. Recombinant host cell according to claim 12, characterized in that it is a Bombyx morio cell. 15. Recombinant host cell according to claim 12, characterized in that it is a cell of the posterior sericogenic glands of Bombyx mori. 16. Use of a nucleic acid according to one of claims 1 to 5, of an expression cassette according to one <Desc / Clms Page number 35>  of claims 6 to 8, of a vector according to one of claims 9 to 11 or of a recombinant host cell according to one of claims 12 to 15 for obtaining a transgenic animal belonging to the genus Bombyx. 17. Use according to claim 16 characterized in that the transgenic animal belonging to the Bombyx mori species secretes a protein of interest in the silk thread. 18. Transgenic animal comprising, in a form integrated into its genome, a nucleic acid according to one of claims 1 to 5, an expression cassette according to one of claims 6 to 8 or a vector according to one of claims 9 to 11. 19. Method for obtaining a transgenic animal belonging to the Bombyx mori species, characterized in that it comprises the following steps: a) Injection of a nucleic acid according to one of claims 1 to 5, of a expression cassette according to one of claims 6 to 8 or of a vector according to one of claims 9 to 11 in Bombyx mori eggs from one to five hours after egg laying, before the formation of the blastoderm; b) Selection of transgenic animals having integrated into their genome a nucleic acid according to one of claims 1 to 5 or an expression cassette according to one of claims 6 to 8. 20. A method of obtaining an animal according to claim 19 characterized in that it further comprises the following steps: c) Crossing between them of two transgenic animals as obtained in step b); d) Selection of animals homozygous for the transgene. <Desc / Clms Page number 36> 21. Method for obtaining a silk thread, characterized in that it comprises the following steps: a) Breeding a transgenic animal according to claim 18 b) Recovering the silk produced by the transgenic animal. 22. A method of obtaining a protein of interest characterized in that it comprises the following steps: a) breeding a transgenic animal according to claim 18 b) recovering the silk produced by the transgenic animal c) recovering the protein of interest within the silk thread.