FR2636231A1 - N-glycosylated mature interleukin-1 ss-producing recombinant eukaryotic cells, process and vectors required for producing them and process for producing N-glycosylated mature interleukin-1 ss - Google Patents

Abstract

The present invention relates to mature interleukin-1 ss-producing eukaryotic cells which contain, with the means required for its expression, a DNA sequence which encodes mature interleukin-1 ss preceded by a DNA sequence which encodes the signal peptide of one of the natural precursors of human growth hormone. Application: production of mature interleukin-1 ss.

Description

Recombinant eukaryotic cells producing N-glycosylated mature interleukin-1 B, process and vectors necessary for their production and method for obtaining mature N-glycosylated interleukin-1 X.

 The present invention relates to recombinant eukaryotic cells producing mature N-glycosylated interleukin-1.

It also relates to a method and the vectors for obtaining these cells. Finally, it also aims to obtain inter-leukine-1 mature N-glycosylated by culturing said cells.

 The eukaryotic cells according to the invention contain, with the means necessary for its expression, a DNA sequence coding for the mature form of interleukin-1, preceded by a DNA sequence coding for the signal peptide of the one of the natural precursors of human growth hormone.

 These recombinant eukaryotic cells are obtained by the method consisting in transfecting eukaryotic cells using a vector carrying, with the means necessary for its expression, a DNA sequence coding for the mature form of interleukin-1. S preceded by a DNA sequence encoding the signal peptide of one of the natural precursors of human growth hormone, and to select the transfected cells, producing intermediate interleukin-1 p, by growth in medium selective.

 Human interleukin-1, hereinafter referred to as human IL-1, is a polypeptide mediator produced primarily by activated monocytes, which plays an important role in the immune defense system.

 From liposaccharide-stimulated macrophage messenger RNA, two complementary DNA clones have recently been isolated representing two distinct molecular forms of human IL-1. These two forms, designated IL-1 x and IL-1 9 are described in detail by MARCH et al. in NATURE, vo. 315, 1985, p.

641-647.

 The mode of secretion of IL-1, B is still unknown.

This protein is synthesized by monocytes or other cells in the form of an intracellular precursor with a molecular weight of about 31,000 daltons, which is cleaved by proteolytic enzymes into a secreted active form having a molecular weight of about 17500, also referred to as mature form (MARCH et al in NATURE, vol 315, 1985, pp. 641-647).

 The intracellular precursor of IL-1 p does not contain a conventional N-terminal signal peptide or internal hydrophobic region.

 AURON et al. in J. MOL. CELL. IMMUNOL. 1985, 2: 169, however, have described a slightly hydrophobic sequence of 17 amino acids in the N-terminal part of this protein, however, the possible role of this sequence in the excretion process is still hypothetical.

 Eukaryotic cells transfected with complementary DNA encoding the IL-1 9 precursor have been shown to be relatively ineffective for the secretion of mature IL-1 in culture media.

 So far, it has not been possible to obtain large amounts of mature IL-1 p from transfected eukaryotic cells.

 We have now found a method which makes it possible to obtain mature IL-1 p in significant amounts from eukaryotic cells. In addition, this method makes it possible to obtain mature IL-1β in the glycosylated form, which can be used as an active ingredient of medicaments for the treatment of diseases, such as cancers and certain infectious or parasitic diseases. Indeed, it is known that IL-1 o (or p have multiple biological properties, and that they play a mediating role in the mechanisms of immune defenses (C. A. DINARELLO Bull Inst.

Pasteur 1987, 85, 267-285).

 According to a first aspect, the invention thus relates to recombinant eukaryotic cells producing mature interleukin-I, which contain, with the means necessary for its expression, a DNA sequence coding for the mature form of interleukin-1. preceded by a DNA sequence encoding the signal peptide of one of the natural precursors of human growth hormone.

 It also relates to a process for obtaining said cells consisting of 1) transfecting eukaryotic cells using a vector carrying, with the means necessary for its expression, a DNA sequence coding for the mature form of the interleukin-1 ss preceded by a DNA sequence coding for the signal peptide of one of the precursors of human growth hormone, and then 2) to select the transfected cells producing intermediate interleukin-1 by selective culture.

 Finally, it relates to the process for obtaining a mature interleukin-1 by culturing recombinant eukaryotic cells, in the presence of a selection system, to collect the culture medium and to separate the interleukin-1. 1 p contained in the culture medium of the other constituents thereof, in which the cultured cells are derived from eukaryotic cells 1) transfected with a vector carrying, with the means necessary for its expression, a DNA sequence coding for the mature form of p-interleukin preceded by a DNA sequence coding for the signal peptide of one of the natural precursors of human growth hormone, and then 2) selected by selective culture.

It should be specified that said method can be applied, not only and in a particularly suitable manner, to the production of interleukin-1p of human origin, but also to the production of interleukin-1 p of other origin. animal, insofar as such molecules have a particular intoret justifying an industrial application, for example as a diagnostic agent or active principle of a drug especially for veterinary use (CR MALISZEWSKI et al., Molecular
Immunology, vol. 25, no. 5, p 429-437, 1988).

 The present invention also relates to the recombinant vectors for carrying out said method as well as to the mature IL-1B X producing cell lines.

 The eukaryotic cells useful for the implementation of the invention are cells of animal origin capable of glycosylation. Among these cells, the cells commonly referred to by the name CHO by reference to their origin (Chinese hamster ovary cells) are particularly suitable.

 Techniques related to the use of these cells, whether their propagation or their transfection by the vectors or the selection of cells actually transfected, are known to those skilled in the art. Some will be more precisely described during the presentation of the examples.

 The vectors necessary for the implementation of the invention can take various forms. It may be all or part of a viral genome and in particular the genome of a retrovirus or a plasmid or a cosmid. Advantageously, a plasmid is used.

 The recombinant vectors according to the invention, hereinafter referred to as vectors, carry, with the means necessary for its expression, a DNA sequence coding for the mature form of interleukin-1 B, preceded by a DNA sequence. encoding the signal peptide of one of the natural precursors of human growth hormone.

 The DNA sequence encoding the signal peptide of one of the natural precursors of human growth hormone, hereinafter referred to as "a DNA sequence coding for the hGH signal peptide or DNA sequence coding for ps-hGH "may have one of the sequences that allows the degeneracy of the genetic code: a lead amino acid - except the methion: ne - can be coded by 2, 3, 4 or even for some, 6 different codons. A preferred nucleotide sequence coding for ps-hGH is the sequence below, where the corresponding amino acid was noted for each of 26 codons.

ATG GCT ACA GGC TCC CGG ACG TCC CTG CTC CTG GCT TTT GGC CTG
Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Leu Ala Phe Gly Leu
CTC TGC CTG CCC TGG CTT CAA GAG GGC AGT GCC 3
Leu Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala
The DNA sequence coding for mature IL-1β used in the process according to the invention is a sequence obtained according to standard techniques, from a complementary DNA or by way of synthesis.

 The synthesis of the DNA sequence coding for the mature form of human IL-1β will be described in detail below; the synthetic sequence obtained is shown in FIG. 1, in which the corresponding amino acid was recorded for each of the 153 codons.

 It is known that to enable, after a transfection, the detection, within a population of eukaryotic cells, the cells that have actually integrated a particular vector, it is necessary to use a selection system. which allows said cells to live in lethal conditions for untransformed cells. This selection system comprises a gene coding for a protein called selection marker allowing selection under the above conditions.

The DNA sequence coding for the selection marker may belong to either an autonomous expression unit carried by the same vector or a different vector, or to the same expression unit as that of the ILw
The means necessary for the expression of these sequences are chosen from those generically used for the construction of vectors intended for the transfection of eukaryotic cells.

Advantageously, they are derived from the genome of the SV40 virus from which, in particular, DNA sequences can be prepared including, in particular, the early promoter, introns and / or the early or late polyadenylation signals (Proud,
W. (1978) Nature, 273, 113-120).

 Examples of suitable selection markers are thymidine kinase, guanine phosphoribosyltransferase, neomycin phosphotransferase and dihydrofolate reductase; for this purpose, reference can be made to the article by KAUFMAN et al. in J.

Mol. Biol. t1982) 159, 601-621, cited in this specification for reference.

 A particularly suitable gene for the purposes of the invention is a DNA sequence coding for dihydrofolate reductase (enzyme hereinafter denoted by the abbreviation DHFR); described by Subraman, et al (1981 Mol Cell 1, 854-864), such a sequence, carried by an expression vector, allows, after transfection of cells incapable of synthesizing in a functional form of DHFR ( DHFR cells), growth on a medium deficient in hypoxanthine, glycine and thymidine, the only cells that actually incorporated the vector.

 The interest given to the use of a vector carrying, with the means necessary for its expression, a DNA sequence coding for DHFR is accentuated by the fact that it can be at the origin, that the eukaryotic cell transfected either capable (DHFR cell) or not (DHFR- cell) of synthesizing in a functional form the DHFR, of an amplification process leading to an increased obtaining of a protein of interest encoded by a DNA sequence carried , with the means necessary for its expression, by said vector. The mechanism of this amplification remains to be clarified. DHFR is known to be inhibited by the compound known as methotrexate (N - ((((2,4-diamino-2-pteridinyl) methyl) methylamino) -4-benzoyl) L-glutamic acid) and the presence of Methotrexate in the selective culture medium causes the death of most cells and only cells that have become capable of synthesizing large quantities of DHFR are still alive. And it has been found (Kaufman, R.J.

et al. (1982) J. Mol. Biol., 159, 601-621), in cells having incorporated a vector carrying, with the means necessary for their expression, a DNA sequence coding for DHFR and a DNA sequence coding for another protein, which this amplified production of DHFR was accompanied by an amplified production of said protein.

 The construction of the vectors according to the invention makes use of techniques now well known to those skilled in the art.

 It comprises in particular the isolation of DNA fragments using restriction enzymes from pre-existing vectors, the chemical synthesis of oligonucleotides, the assembly, possibly after modification of their ends, of these various fragments. using an enzyme such as bacteriophage T4 DNA ligase, cloning - after bacterial transformation in Escherichia coli - vectors and their purification according to techniques also well known to those skilled in the art.

These techniques are described in particular in the book entitled Moiecular Cloning: a Laboratory Manual by Maniatis, T. et al. published in 1982 by the Cold Spring Harbor Laboratory
New York (USA).

 The set of restriction enzymes required for the construction of the vectors presented below is sold in particular by New England Biolabs (USA).

 The bacteriophage T4 DNA ligase is available from the company PHARMACIA (Sweden).

 In another aspect, the invention relates to selected cell lines, from eukaryotic cells having incorporated a vector according to the invention, by culturing said cells in a selective medium.

 The present invention will now be described in more detail with reference to human mature IL-1 w without limiting the scope thereof to obtain this particular IL-1.

I - Synthesis of the DNA Sequence Encoding the Mature Form of Human IL-1 ss (FIG.
Oligonucleotides were synthesized with a
"Biosearch 4600" synthesizer using diisopropylphosphoramidite nucleosides, and purified by electrophoresis on 12% polyacrylamide gel - 8M urea.

 The synthetic gene of IL-1 9 was obtained by assembling 19 oligonucleotides containing from 35 to 58 nucleotides, indicated by arrows, in the appended FIG. 1, the length of each fragment being specified in parentheses. This synthetic sequence differs from the natural sequence by a few nucleotides; the bases of the natural sequence which have been modified above the synthetic sequence have been noted. On the other hand, the restriction sites introduced in this sequence were noted.

 The fragments (1 g), with the exception of fragments 1 and 11, were phosphorylated either in complementary pairs or in a group of 3 oligonucleotides (fragments 4, 5 and 16) in a standard libation mixture containing the following ingredients: 50 mM Tris-HCl, pH 7.4; 10 mM MgCl 2; 10. mM dithiothreitol; 1 mM spermidine; ATP I mM; 1 mg / ml of bovine serum albumin and 10 units of T4-polynucleotide kinase (-1 μl) in a total volume of 1 μl.

 After 30 minutes at 37 ° C., the mixtures were placed in a 650 ° C water bath and held for 10 minutes at this temperature.

 The solutions were pooled, Fragments 1 and 11 were added and the resulting mixture was heated for 10 minutes at 650C.

 The mixture was then allowed to cool to 22 ° C, then bacteriophage T4 DNA ligase (1> 1 - 5 units) was added and the mixture so treated was maintained for 10 minutes at 220 ° C.

 The synthetic gene (shown in Figure 1) was purified by non-denaturing polyacrylamide gel electrophoresis (5%) and the desired DNA fragment was electroeluted for 16 hours in 20 mM Tris-acetate pH 7 buffer. , 9; 500 mM EDTA and 0.1 X sodium dodecyl sulfate (SDS).

The DNA fragment was then purified by calibration chromatography of DE-52 (Pharmacia) eluting with a buffer composed of 1.5M NaCl; 50 mM Tris-HCl pH 7.6; 5 mM EDTA and precipitated with ethanol. This DNA fragment was introduced into the M13mp18 vector (see MESSING et al.
METHODS in Enzymology 101 (part C) Recombinant DNA, Academic
Press, New York 1983, p. 20) previously digested with enzymes
EcoRI and HindIII and cloned into E. coli strain JM 103.

 The clones obtained were analyzed by digestion with restriction enzymes. Among the clones exhibiting the expected restriction map, clone 18IL9 was found to effectively contain the synthetic gene after sequencing each of its DNA strands by the method of SANGER et al (Proc Nat Sci.

USA 74 (1977) 5463-5467).

II - Coupling of the DNA Sequence Encoding the Mature Form of Human IL-1 B with the DNA Sequence Encoding the ps-hGH
The DNA sequence coding for the hGH signal peptide and the DNA sequence coding for the first amino acids of mature IL-1 X as well as the appropriate control sequences were inserted at the unique BglII site of the clone 18IL9, such that the DNA sequence coding for the mature IL-1p is directly preceded by the DNA sequence coding for the signal peptide of
I'hGH.

 At the 3 'end of the DNA sequence encoding mature IL-1, a SacI site adjacent to the HindIII site was inserted to facilitate the introduction of a eukaryotic polyadenylation signal.

 The resulting clone was digested with HindIII and BamHI restriction enzymes and the HindIII-BamHI fragment containing the IL-1 X coding sequence preceded by the coding sequence for ps-hGH was used to make plasmid pSV1003.

III - Construction of the plasmid pSV1003
The plasmid pSV1003, represented in FIG. 2, results from the assembly of the 8 DNA fragments by implementing the methods indicated previously: a) a 278 base pair PbuII-BglI fragment (bp) derived from
SV40 virus genome (Fiers, W. (1978), Nature 273, 113-120) and
containing the 5 'portion of the early promoter of this virus.

b) A 329 bp BglI-PstI fragment from the plasmid pL1 (Okayama,
H. and Berg, P. (1983) Mol. Cell. Biol. 3, 280-289), and
containing the 3 'part of the SV40 early promoter followed
late introns of this same virus.

The BamHI site initially present in this fragment was
removed by filling the site in the presence of the fragment of
Klenow of DNA polymerase I (Pharmacia).

c) A PstI-HindIII fragment of 12 bp derived from the plasmid pUC18 (Yanisch
Perron, C., Vieira, J. and Messing, J. (1985) Gene 33, 103-119).

d) The HindIII-BamHI fragment containing the coding sequence for
mature IL-1 p, preceded by the coding sequence for ps-hGH,
described in II above.

e) A 237bp BamHI-BclI fragment originating from the genome of the SV40 virus and
containing the late signal of polyadenylation of this virus.

f) A BamHI-EcoRV fragment of 190bp resulting from plasmid pBR322
(Bolivar, F. (1977) Gene 2, 95-113).

g) A PvuII-PvuI Fragment of 2556bp Derived from the Plasmid pSV2-dhfr
(Subramani, S., Mulligan, R. and Berg, P. (1981) Cell Mol.

Biol. 1, 854-864) and comprising an expression unit for the
DHFR. This plasmid was deposited at ATCC under No. 37146. This
transcription unit contains, between the PvuII sites and
EcoRI, the prime promoter of SV40 virus, the coding sequence
for mouse DHFR, the intron of SV40 virus antigen
and the early signal of polyadenylation of this same virus. This
unit of expression is followed by the 626bp EcoRI-PvuI fragment
from plasmid pBR322.

The HindIII, BamHI and EcoRI sites initially present in this
fragment g, as well as the entire region of DNA included
between the BamHI and EcoRI sites, were removed by filling,
as previously described for fragment b.

h) a PvuI-PvuII fragment of 1438bp derived from plasmid pUC9 (Vieira,
J. and Messing, J. (1982) Gene 19, 259-268). This fragment
does not contain sequences close to the replication origin
bacterial that are harmful to gene expression
Eukaryotic (Peterson, DO, Beifuss, KK and Morley, KL

 (1987) Mol. Cell. Biol. 7, 1563-1567).

The plasmid psV1003 therefore contains: an expression unit for the fusion protein (ps - hGH)
IL-1 B, which unit has the early promoter of the virus
SV40, followed by late introns of this same virus, and downstream of
these, the coding sequence for the fusion protein (ps
hGH) - IL-1p, followed by the late polyadenylation signal of
SV40 virus.

an expression unit for mouse DHFR, which unit
comprises the SV40 early promoter, the coding sequence
for the mouse DHFR and en.aval of the latter the intron of
the SV40 virus antigen, followed by the early signal of
polyadenylation of this same virus.

séquence d'ADN issue du génome du virus SV40 séquence d'ADN codant pour la DHFR séquence d'ADN codant pour l'interleukine-1 p précédée de la séquence codant pour ps-hGH séquence d'ADN issue du plasmide pBR322 séquence d'ADN issue du plasmide pUC9 séquence d'ADN issue du plasmide pUC18. The following legend has been adopted for Figure 2

DNA sequence derived from SV40 virus genome DNA sequence coding for DHFR DNA sequence coding for interleukin-1 p preceded by coding sequence for ps-hGH DNA sequence derived from plasmid pBR322 sequence DNA from plasmid pUC9 DNA sequence from plasmid pUC18.

IV - Transfection of CHO Cells and Expression of IL-1 in Them

 The transient expression of the plasmid pSV1003 was carried out in CHO DHFR cells selected by Urlaub et al. (Proc Natl Acad Sci USA, 77, 4216-4220, 1980) using the DEAE-dextran method described by LUPKER et al. (Gene 1983, 24: 281-287). This expression was carried out in 10cm culture dishes; the cells were inoculated the day before the transfection at a density of 106 cells per dish.

 The culture media were collected 4 days after transfection and clarified by centrifugation. Supernatants were collected.

The monolayers of the cells remaining on the culture dishes were washed twice with the phosphate buffer or buffer
PBS (R. DULBECCO and M. VOGT, J. Exp.Med 99 (1954) 167), scraped resuspended in 5 ml of this buffer, and then subjected to ultrasonic treatment. Cell lysates were thus obtained.

 The amount of IL-1β contained in the supernatants and the lysates was evaluated by a biological assay by measuring the proliferation of the D10.G4.1 lymphocyte line in the presence of Concanavalin A according to the method of KAYE et al. (Lymphokine Res.

3 (1984) 175-182).

 As shown by the results in Table 1 below, plasmid pSV1003 allows synthesis of IL-1 in biologically active form. Almost all the IL-1ss produced is exported to the culture medium.

Table I

<tb><SEP> Biological Activity <SEP>
<tb><SEP> Plasmid <SEP> (Units <SEP> x <SEP> 1b2 / box) <SEP> X <SEP> of <SEP> IL-1
<tb> recombinant <SEP> secreted <SEP>
<tb><SEP> supernatant <SEP> Lysate
<tb> pSV1003 <SEP> 986 <SEP> 21 <SEP> 98
<Tb>
It will be specified that a unit corresponds to 10 μg When the reference IL-1 obtained from the National Institute for
Biological Standards and Control (London).

 CHO DHFR cells were transformed with plasmid pSV003 to establish stable cell lines secreting p-IL-I.

 The stable transformation of CHO DHFR- cells, the selection of the transformed clones and the amplification of the genes were carried out according to the techniques described by FERRARA et al.

(FEBS Lett 226, (1987) 47-52).

 50 DHFR colonies (clones 1003.1 to 1003.50) were isolated and cultured. The presence of IL-1 p in the culture medium was determined by immunoenzymatic assay (ELISA marketed by Cistron). This assay indicated that clones transformed with plasmid pSV1303 very efficiently secreted IL-1) 3.

 The clones that secreted the highest amounts of IL-1a were subjected to an amplification procedure, gradually increasing the concentrations of methotrexate in the selective medium.

 Several clones were highly productive of IL-1 p at the concentration of 100 nM methotrexate.

 The best expression was obtained for clone 1003,26. When 4 x 10 5 cells of this clone were seeded in a 6 cm Petri dish in a selective medium containing 100 nM methotrexate and cultured for 4 days, it produced 215 ng / ml mature IL-1.

V - Characterization of IL-1 B produced by clone 1003,26
The cells of clone 1003,26 as well as CHO cells
DHFR selected as controls were seeded in boxes of
Petri dish of 35 mm, at the rate of 4 x 105 cells per box.

24 hours later, the cells were washed twice with phosphate buffer (PBS), and preincubated in medium
MEM lacking methionine (market marketed by society
GIBCO) containing 0.2% dialyzed fetal calf serum (GIBCO), 300 g / ml L-glutamine and 150 μg / ml L-proline.

 After two hours, the preincubation medium was removed and replaced with 0.8 ml of the same medium containing 100 μCi methionine (35S) (Amersham 37TBq / mmol).

 After incubation for 16 hours at 37C, the culture media were collected and clarified by centrifugation at 1000 rpm for 10 minutes at room temperature.

200 μl of each labeled supernatant were incubated for 2 hours at 40 ° C with shaking with 10 μl of pre-immune rabbit serum in the presence of 200 μl of
Staphylococcus aureus (sold under the trademark Pansorbin by Calbiochem) set to limit non-specific interactions.

 The resulting mixture was centrifuged for 5 minutes at 5000 rpm; To the resulting supernatants, 5 μl of human anti-human IL-1 rabbit serum (commercially available from CISTRON) and 200 μl of fixed Staphylococcus aureus cells were added.

 This last mixture was incubated for 16 hours at 4 ° C. with stirring.

 The immunoprecipitates were then treated according to the procedure described by KESSLER (Meth Enzymol 73 (1981) 442-459) and analyzed on 20% polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS) according to the LAENMLI method ( Nature 1970, 277, 680-685) using the device "Phast System" of Pharmacia; the gels were dried and autoradiographed.

As a control, recombinant IL-1 was used.
125 produced by E. coli and labeled with iodine 125 I.

Under these conditions, a predominant form having a molecular weight of 22,000 and two minor forms of 21,000 and 17,500 were immunoprecipitated from the supernatants of cells of Lineage 1003,26, as is apparent from Figure 3 which is the photograph of the autoradiogram on which a represents IL-1 p produced by E. coli and labeled with iodine 125 I; b and d represent IL-1 s immunoprecipitated from culture supernatants of clone 1003.26 cells (b in the absence of tunicamycin and in the presence of tunicamycin); c corresponds to CHO-DHFR cells
It was verified that the difference in molecular weight between IL-1 produced by CHO cells and that produced by E. coli was due to N-glycolysation of the protein produced by CHO cells.

This check was carried out using tunicamycin which is known to be an inhibitory agent of
N-glycosylation.

Tunicamycin (SIGMA) was added to the culture medium of cells of the 1003,26 line during preincubation and labeling, at a rate of 10 g / ml, and
Immunoprecipitation was performed as indicated above.

The disappearance of the two bands corresponding to the molecular weights removed and their replacement by a single band corresponding to a molecular weight of 17,500 show that IL-1 secreted by the cells of the 1003,26 line is in the form
N-glycosylated.

Claims (18)

1. Eukaryotic cells producing mature N-glycosylated interleukin-1, characterized in that they contain, with the means necessary for its expression, a DNA sequence coding for mature interleukin-1 ss preceded by a sequence of DNA encoding the signal peptide of one of the natural precursors of human growth hormone. 2. Eukaryotic cells according to claim 1, characterized in that the DNA sequence coding for interleukin-1 is the DNA sequence coding for interleukin-1 of human origin. 3. Eukaryotic cells according to one of claims 1 or 2, characterized in that they are CHO cells. 4. Process for producing mature interleukin-producing eukaryotic cells according to any one of claims 1 to 3, characterized in that it consists in transfecting eukaryotic cells with the aid of a vector carrying, with the means necessary for its expression, a DNA sequence coding for interleukin-1ss preceded by a DNA sequence coding for the signal peptide of one of the natural precursors of human growth hormone, and then to select transfected cells producing interleukin-Ifi grown by culture in selective medium. 5. Method according to claim 4, characterized in that the eukaryotic cells are CHO cells. 6. Method according to one of claims 4 or 5, characterized in that the vector further carries a coding sequence for a selection marker. 7. Process according to any one of claims 4 to 6, wherein the selection marker is dihydrofolate reductase. 8. Method according to claim 7, characterized in that the vector carries a single expression unit for dihydrofolate reductase and interleukin-1 9. 9. Process according to any one of claims 4 to 8, characterized in that the vector carries two distinct expression units, one for dihydrofolate reductase and the other for InterIeukin-I. 10. Method according to any one of claims 4 to 9, characterized in that the DNA sequence coding for the interleukin-1 9 mature is that coding for interleukin-I, B of human origin 11. Method according to any one of claims 4 to 10, characterized in that the vector has the characteristics of the plasmid pSV1003. 12. Expression vector characterized in that it carries, with the means necessary for its expression, a DNA sequence coding for mature interleukin-19, preceded by a DNA sequence coding for the signal peptide. of one of the precursors of human growth hormone. 13. Vector according to claim 12, characterized in that it further carries a coding sequence for a selection marker. 14. Vector according to one of claims 12 or 13, characterized in that the selection marker is dihydrofolate reductase. 15. The vector of claim 14, characterized in that it carries a single unit of expression for dihydrofotate reductase and interleukin-1ss. 16. The vector of claim 14, characterized in that it carries two distinct expression units, one for dihydrofolate reductase and the other for interleukin-1 9. 17. Vector according to any one of claims 12 to 16, characterized in that it has the characteristics of the plasmid pSV1003. 18. Method for obtaining mature interleukin-I X N-glycosylated culture of eukaryotic cells producing interleukin-1 ss, to collect the culture medium and to separate other interleukin-1 components | contained in said medium, characterized in that the cells cultured are derived from eukaryotic cells transfected with a vector carrying both, with the means necessary for their expression, a DNA sequence coding for L '. Mature interleukin-1 preceded by a DNA sequence coding for the signal peptide of one of the natural precursors of human growth hormone, then selected by selective culture.