MXPA96006585A - Human mucine (mu - Google Patents

Abstract

The present invention relates to the isolation and characterization of amino acid and nucleolide sequences of a new member of the mucingas family. In particular, it relates to the invention for preparing reagents intended for the diagnosis of patients and for vaccination, in order to treat certain diseases by stimulating the immunoresistance.

Description

MUCINA HUMANA (MUC8) - > The present invention relates to the isolation and characterization of amino acid and nucleotide sequences of a new member of the mucin gene family. The invention relates especially to the provision of reagents for the diagnosis of patients and for vaccination for the treatment of certain diseases by stimulating the immune defense. The epithelia of the respiratory, reproductive and gastrointestinal tracts of higher organisms are coated with ana protective secretion called mucus. This uucus gel is composed up to 95% by water and up to 5%, approximately, by mucins (Neutra and Forstner, 1987). Mucins are glycoproteins with two specific characteristics: first, at least 50% of their weight Molecular consists of oligosaccharides, which are linked by the glycosidic C to residues ñe > threonine and serine of the skeleton of 1-i protein, and secondly this strongly glycosylated region is composed of repetitive sesuenciales units. Mucins can be divided into two groups, on the one hand in the secretory mucins that, by means of intermolecular disulfide bridges, occur in the form of oligomers and, on the other hand, in the fixed mucins in roembrenas, which are "" anchored to the plasma membrane through a hydrophobic region (Gum, 1992; Metzgar et al, 1993; Strous and Dekker, 1992). Since 1987, DNA sequences of seven members of the family of musins have been isolated and characterized from human libraries. The sequence comparison did not provide any significant homology within the different mucins, nor in their composition of nucleotides n in their amino acid composition. However, a central region carrying the O-glyoans is common to all mucins and is composed of successive repetitive amino acid sequence and nucleotide sequences. 35 C lasses in posttranslational expulsion and modification of Mucins are associated with various diseases, such as carcinomas, cystic fibrosis, ulcerative colitis and Crohn's enteritis regionalis, and can make mucins the object of clinical investigations. Antibodies that recognize mucin epitopes (CA 19-9, CA 125 and M26) are used in diagnosis as markers (Magnani et al., 1984, Metzgar et al., 1984, Linsley et al., 1988). One purpose for many researchers is to obtain new information about mucin polypeptide and nucleotide sequences. Since the purification of mucin glycoproteins, of high molecular weight, often does not give results despite the progress of peptide chemistry, and the application of recombinant DNA technology in highly repetitive nucleotide sequences is also problematic. , there is a great demand for other mucins that would make possible advances in diagnosis and in the treatment of patients. Accordingly, the present invention had as its mission to find other mucins to jointly increase the value of the mucins in diagnosis and thereby provide an essential contribution to the diagnosis of tumors. The solution to this problem results from the provision of the embodiment described in the claims, especially the provision of a new mucin or mucin. The invention herein provides nucleotide sequences that encode a new human mucin. The nucleotide sequences are present as genomic DNA and cDNAs that are integrated into circular plasmid vectors. With these constructions, which contain mucin sequences, bacterial host organisms (E. coli) were transformed in a stable manner, which now allow amplifying the mucin sequences to unlimited amounts. By the expression of the coding sequence in a prokaryotic expression system the production and purification in large quantities of the recombinant polypeptide without impurities by other human glycoproteins is possible.

The bacterial host allows a non-problematic large-scale culture. For the isolation of the polypeptide from the cell lysate, a preparative affinity chromatography can be carried out by the monoclonal antibody (mA) Bl or maltose, since the polypeptide is synthesized as a fusion protein with the maltose binding protein (PUM). The expression of the polypeptide can be deliberately influenced by induction of the bacterial promoter with IPTG. This defined polypeptide allows the establishment of an ELISA system for screening in patient sera and can be used for the generation of other antibodies. By coupling the antibody with alkaline phosphatase or horseradish peroxidase, it is possible to develop an ELISA sandwich system which, through the binding of other antibodies defined to the solid phase, makes it possible to detect other antigenic determinants on the native human MUC8 glycoprotein. With the aid of the new invention, therefore, test kits can be developed for diagnosis that allow a routine clinical use. Since in the Southern blot analysis the localization of the MUC8 gene on the human chromosome ns 7 could be shown, the labeled DNA probes (radioactively or non-radioactively with biotin) are suitable as a genomic marker for the identification of neighboring genes or of the corresponding genes mutations (deletions, translocations and insertions). Since the MUC8 gene is very polymorphic, as could be demonstrated by hybridization with DNA samples from various patients, it is possible to use MUC8 DNA as a probe in fingerprint analysis for the identification of kinship relationships. Another aspect is the use of antisense RNA or antisense oligonucleotides that specifically block the translation of MUC8 mRNA. This could lead to a better prognosis of the tumor, since the mucin expression of tumor cells leads to an increased rate of metastasis in the animal model (Bresalier et al., 1991). Another possibility of application are vaccination trials. For 5 MUC1 peptides it could be shown that mucin epitopes of non-MHC-restricted cytotoxic T cells could be recognized and thus a tumor-specific immune response could be triggered (Hilkens et al., 1992; Barnd et al., 1989; Ioannides et al. al., 1993). 10 In immunohistochemical investigations of cryostat sections of various benign and malignant tissues, the model of expression of the antigen Bl in beings could be determined. "human." From these data can be deduced the possible site of employment of diagnostic systems for detection of mucin MUC8. In the framework of the invention described here, nucleotide sequences encoding human MUC8 mucin were isolated. A part of the repetitive nucleotide sequence was expressed in order to isolate and investigate larger quantities of the polypeptide with its biological and immunological properties. The DNA used for this was isolated by immuno- * "selection in an IZAP cDNA library, obtained from MRNA of the T84 cell line of colon carcinoma human. The clone was designated pBl and comprises 798 bp. The cDNA contains a 3 'untranslated portion of 282 bp, which consists of Alu repeats, contains a polyadenylation signal and ends with a poly (A) tail. The coding sequence of 517 nucleotides is composed of units repetitive of 53 bp and ends with an Opal termination codon. An EcoRI / HindII fragment of this cDNA comprising the repetitive coding region was used as a probe in Northern blot analysis. Three species of 4.8 kb, 1.9 kb and 1.35 kb mRNA hybridized in cell lines human tumors, of which the 4.8 kb band constituted the strongest signal. In Southern blot analyzes that were prepared with human genomic DNA probes digested with EcoRI and MspI it could be checked, with the EcoRI / HindII fragment as a probe, a longitudinal polymorphism of the restriction fragment, which stood out with maximum clarity in the samples digested with Mspl. To obtain additional sequence information, a human renal cDNA library in the IMAX1 phage and a genomic library in the cosmid vector pWE-15 was selected with the EcoRI / HindII fragment. From the IMAX1 library, a cDNA fragment could be isolated from a length of 854 bp. This clone pB1.2 contains a 5'-untranslated region of 576 bp which, after a potential initiation codon, follows a coding sequence of 278 bp. In the selection of the genomic DNA library, a positive clone pWE-Bl could be isolated, whose inserted DNA was cut with various restriction enzymes and then analyzed in Southern blotting. A Bslll / BamHI fragment of 5.9 kb in length hybridized with the cDNAs of the two Bl clones and was subcloned for further characterization in pBSK (+). The genomic DNA of the resulting pBl-Cos clone was sequenced thoroughly and the nucleotide sequence compared to that of the two cDNA clones. The comparison of homology resulted in the clone pBl-Cos containing an exon of 1,482 bp, "" - whose 5 'region is identical to parts of clone pB1.2, while the 3' region coincides with parts of clone pBl. The region The mean of the exon, which is not represented by DNA nucleotide sequences, consists of additional repetitive units, as revealed by partial digestion analysis with the restriction enzyme EarI. In total, the exon of clone pBl-Cos contains 19 EarI subfragments of a length of 53 bp. The genomic site of the MUC8 gene could be established in Southern blots with DNA from somatic cell hybrids on chromosome 7. MUC8 mucin-encoding DNA fragments can be inserted into eukaryotic expression vectors and used for the transformation (stable or transient) of mammalian cells, especially human cells.

* - Since the new generation of vectors usually presents stimulatory and promoter sequences and enables a selection in both prokaryotic and eukaryotic systems, a simple manipulation of the eukaryotic expression of MUC8 is guaranteed. With this, a polypeptide of co- and post-translational modification which should correspond to the native MUC8 mucin can be isolated and analyzed. This construct can be introduced stably into human B lymphocytes immortalized with Epstein-Barr virus, for to make possible a presentation of peptide fragments of MUC8 with MHC class I and II. By incubating the cells transfected with o-glycosylation inhibitors ': - Post-translational expression of epitopes associated with tumors can be further increased (Bu et al., 1993). The recognition, not restricted by MHC, of mucin epitopes by cytotoxic T cells, caused by the repetitive structure of mucin, could be shown repeatedly for MUC1 mucin. This idea is based on reintegrating transfectants that express large amounts of MUC8 to patients.

To elicit a specific immune response associated with tumors by recognition of antibodies or CTL. Another aspect is the detection of tumors or inflammation. in patients by detecting mucin antigens in tissues or sera. Immunological methods, such immunohistochemical staining of tissue sections, ELISA or Western blot with patient sera is possible with the hybridoma cell line Bl, which allows isolating antibodies in almost unlimited amounts from the culture supernatant. The hybridoma was obtained by Immunization of a Balb / c mouse with a high molecular weight protein fraction of a Mz-Sto-1 cell extract and subsequent fusion of spleen cells from the mouse immunized with immortalized myeloma cells (Herbsthofer, 1990). The Mz-Sto-1 cell line of stomach adenocarcinoma is established from a patient suffering from ring cell carcinoma (Dippold et al., 1987). In the analysis of "transference" Wéfetsrn could be identified, in sera of patients with pancreatic carcinoma exhibiting increased values of CA19-9, increased levels of MUC8 compared with sera of patients of carcinoma of 5 colon and healthy donors . This provides early indications for the establishment of MUC8 as a tumor marker in patient sera. By expressing the cDNA of pBl in the vector pMAL-c, which makes it possible, after induction with IPTG, to express 0 a fusion protein with the N-terminal end of a portion of the maltose binding protein, a polypeptide that is recognized by the Bl antibody is presented and, therefore, must contain the antigenic determinant. The computer analysis showed that the hexademer QDREEE (5-letter code) is the most probable epitope. This peptide is contained in each repeating unit, which is composed of amino acid sequences of 53 residues, encoded by 159 bp of nucleotides. Therefore, it can be used for specific blocking of an MUC8 antigen identification in immunological procedures. MUC8 polypeptides that can be isolated after prokaryotic or eukaryotic expression, can be applied directly as protective factors in various diseases (eg ulcer), or can be used for the generation of other MUC-8 specific antibodies. If the polypeptides are greater than 10 kD, they can be used directly as immunogens, otherwise a coupling to carrier proteins is necessary for an efficient immunization. After injection of the peptide into a vertebrate 0 (mouse, rat, rabbit, goat or sheep), MUC8-specific antibodies can be isolated from the animals in the form of polyclonal antiserum. For the production of monoclonal antibodies, proceed according to the method of Milstein and Kohler, which has already been used successfully for the production of mAk Bl. From 5 of the culture supernatants of hybridoma cell lines the antibodies can be purified following the corresponding protocols optimized for each subclass (see Harlow, E. and Lane, D. "Antibodies: A Laboratory Manual" (1988) Cold Spring Harbor Laborátory, Cold Spring Harbor, New York). The polypeptides as well as the antibodies can be modified by covalently or non-covalently conjugating substances that allow detection. These substances can constitute radionuclides, enzymes, cofactors, magnetic particles, fluorescence factors to chemiluminescence. These antibodies or polypeptides Modified 10 can be used in various immunological techniques for the detection of MUC8 mucin in biological materials, such as tissue, blood, urine, serum and tissue biopsies. < - • other body fluids. Tissue samples are usually investigated in the form of paraffin sections or cryostat on slides using a labeled MUC8 antibody or an unmodified antibody, which binding to a labeled second antibody should be detected. In ELISA systems and Western blot analysis, body fluid proteins can be easily investigated. There is A large number of competitive and non-competitive immunological assay systems, in which antigens or antibodies, are coupled to the solid phase (Harlow and Lane, 1988). For use in cancer therapy, the specific antibodies of MUC 8, as humanized as possible, can can be coupled with toxins or radionuclides and administered to patients whose tumor tissue shows an increased expression of MUC8, to damage or specifically mark the tumor tissue. Abbreviations: 30 bp base pairs BSA bovine serum albumin h kD kilodalton hour MBP maltose binding protein 35 RT room temperature The following examples explain the invention: - ^ Example 1 A) Selection in phage libraries An expression library was obtained of human cDNA in phage Uni-ZAP XR (Stratagene) from mRNA of line 5 of T84 cells of colon carcinoma. The phage were plated on soft agarose with a density of 5 x 10 * pfu / 150 mm plate and incubated for 3.5 h at 42aC. After this the nitrocellulose filters saturated with IPTG (isopropy1-β-D-thiogalactopyranoside) 0 were placed and incubated at 37 aC for another 3 hours. The filters were removed and after the separation of nonspecific junctions were incubated for 1 h at RT with supernatant of hybridomas Bl ** -; undiluted Anti-mouse antibody (DA-KO), diluted 1: 1000, coupled with alkaline phosphatase, was used as the second antibody. The transformation of the substrate was carried out with BCIP and NBT. After three resections, a positive plaque could be cloned. After in vivo cleavage by auxiliary phage R408 the plasmid pBluescript SK (-) was isolated with the Bl cDNA inserted. 0 A human kidney cDNA library was spiked in phage MAX1 (Clontech) with a density of 3 x 10 * pfu / 150 ml of plaque and, after 4 hours of incubation at 37aC, the filters were placed on top. nitrocellulose. After denaturation and neutralization, nitrocellulose filters are baked at 70 aC for 2 h for DNA fixation. The prehybridization as well as the hybridization were carried out in 5 x Denhardts, 0.5% SDS, NaCl IM, 5 mM EDTA (pH 8), NaH2PO «4 mM, Na2HPOt 37 mM, 10 mg / ml herring sperm DNA and 50% formamide, at 42aC. An EcoRI / HindII fragment from pBl cDNA 0 was radioactively labeled by the "random priming" method with "P a -dCTP and added to the hybridization solution After 16 h the filters were washed for 1 h at 65 aC in 1 h. x SSC + 0.1% SSD and exposed to an x-ray film.From four positive clones, after three 5 reselections a positive plaque was left which, by in vivo cleavage with the auxiliary phage R408, the plasmid pYEüra3 was obtained , whose inserted DNAs could be isolated after digestion with EcoRI The clone was named pBl.2 B) Selection in a genomic cosmid library A genomic library in the cosmid vector pWE-15 contains human DNA from peripheral blood lymphocytes and was made available by Prof. Dr. Elisabeth Weiß at the Institute of Human Genetics in Munich, NM544 bacteria were transformed with cosmid DNA by electroporation and 3 x 10 * colonies / 150 rare plaque were spread on nitrocellular filters After incubation for approximately 8 h at 37 aC, a replica filter was obtained by superimposing a second nitrocellulose membrane. The bacteria were denatured on the replica filter and after that it was neutralized, baked for 2 h for DNA fixation and hybridized with the EcoRI / -Hindll probe, as described for the Lambda-MAX1 library. After three resections, a clone whose insert of approximately 30 kb in length could be isolated was isolated. Isolate after digestion with Notl. The clone was named pWE-Bl. After double digestion with BamHI / BglII, a fragment of 5.9 kb in size could be isolated and ligated into a pBSK (+) vector linearized with Ba HI. This clone was designated pBl-Cos. C) DNA sequencing All sequencing was carried out with double-stranded plasmid DNA, according to the dideoxynucleotide method. Oligonucleotides of 18-20 units were used for the sequencing reactions, whose sequence was derived from already known nucleotide sequences. The Pharmacia T7 sequencing kit was used following exactly the manufacturer's recommendations, using "S a-dCTP." The reactions were separated on a 6% polyacrylamide gel and exposed to an X-ray film after drying of the gel. ) Transfer analysis of DNA and RNA The RNA was isolated following the method of guanidino isothiocyanate and subsequent ultracentrifugation in "" "gradients of cesium chloride (Sambrook, J. et al., 1989 Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). For the purification of poly (A) ^ RNA, the PolyATract System 5 III assay kit of the Promega brand name was used with biotinylated oligo (dT) primers. The samples thus obtained are separated on a 1.2% agarose gel in formaldehyde in 1 x MOPS buffer and traced on a nylon membrane by capillary transfer in 20 x SSC buffer. For prehybridization and hybridization, a solution of 1% BSA, 1 mM EDTA, Na, HPO, 250 mM and 7% SDS (pH 7) was used at 65 ° C. At the same temperature, the print is washed with a solution of 1 mM EDTA, 20 mM Na2HPO and 1% SDS (pH 7) and then exposed to a radiographic film. 5 DNA samples are obtained from peripheral blood lymphocytes, which were previously purified through a Ficoll gradient, by collecting the cells in guanidinium isothiocyanate buffer and subsequent precipitation in ethanol. After digestion with the corresponding restriction enzymes, the DNA samples were separated on a 0.8% agarose gel in TAE buffer. The samples are depurinated in the gel, denatured and then neutralized. The transfer to a nylon membrane and the hybridization are carried out as already described for RNA transfer. F) Expression of a MBP-B1 fusion protein The 798 bp insert of pBl cDNA was cloned into the Stul cleavage site of the pMAL-c vector (Biolabs) in the corresponding reading frames. TB1 bacteria (Biolabs) were transformed with the pMAL-Bl construct and the resulting transformants were incubated at 37 ° C to an OD600 of 0.5. For the recovery of the cell extract, 1 ml of culture was extracted and the remaining culture was induced with 0.3 mM IPTG (final conc.). After 2 h of incubation, 0.5 ml of the induced bacteria were removed and the two samples were brought into contact with SDS application buffer and separated on a 10% polyacrylamide gel (Sambrook et al., 1989) . G) In unotraiisfereilcía Once the proteins separated in SDS-PAGE were transferred to nitrocellulose by electroblotting (transfer buffer: 39 mM glycine, 38 mM Tris, 0.037% SDS and 20% methanol), the filter, after the TBS saturation + 10% FCS (for extracts of tumor cells) or TBS + 1% BSA (for bacterial lysates), is incubated with the respective antibodies. As the 2nd antibody, immunoglobulins conjugated with alkaline phosphatose were always used and the positioning of the substrate was carried out with BCIP and NBT tablets (Sigma). H) Immunohistochemical investigations From the freeze-dried human tissues 10 mm sections were prepared by a cryostat and incubated with undiluted hybridoma supernatant BlAnti-immunoglobulins coupled to mouse peroxidase were used as 2nd antibody. The transformation of the substrate was carried out in AEC buffer with HO.I) Isolation of clones of MUC8 (of cDNA and genomic origin) Since a biochemical purification of high molecular weight glycoproteins that would allow N-terminal sequencing of the protein, a clarification of the structure of the protein is practicable by sequencing and characterization of cDNA clones. The monoclonal antibody Bl was used to select from a colon cDNA library. A positive clone allowed to isolate, after selection, 2.4 x 10 * recombinant plaques. This clone, designated pBl, contains a cDNA insert of 798 bp in length. To obtain additional nucleotide sequences, it was selected in a second human cDNA library that had been obtained from renal mRNA, using a radiolabelled pBl cDNA fragment. From 6.3 x 105 plaques tested, after three resections, a positive clone designated pB1.2 could be isolated, whose inserted cDNA comprises * * - * * w- s ••, • - -13 * -854 bp. Since the two DNA libraries are distinguished by regions of repetitive sequences, but are not identical, they were selected in a genomic library with a pBl cDNA fragment. 3 x 105 colonies were tested, of which one clone contained genomic DNA that hybridized with the cDNA. The approximately 30 kb insert was cut with various restriction enzymes and hybridized successively with the two cDNA clones as a probe in a Southern blot analysis. The smaller isolable fragment of 5.9 kb in length was subcloned into pBluescript and the resulting clone was designated pBl-Cos. K) Analysis of the sequences of the mucin clones The nucleotide sequences of the three clones pBl, pB1.2 and pBl-Cos were determined, determining sequence-specific primers for the two strands of the DNA. The coding region of the pBl cDNA is composed of about 10 repetitive units of 53 bp each. Three of these repeats are found in the 3 'end in the pB1.2 cDNA. All repetitive units are characterized by an Earl cut site. The repetitive region of the Bl exon contained in the pBl-Cos clone is composed of 19 Earl fragments. Due to the repetitive units of the sequence so far it could not be determined directly by 225 pb sequencing, since both the generation of the sequence-specific primer and the assignment of sequences of deleted clones with Exol did not yield safe results. By sequences of the pBl-Cos exon, neither 321 bp is represented at the 5 'end of clone pB1.2 nor 229 bp at the 3' end of clone pBl. This indicates that there should still be at least two other exons containing these sequences. This assumption is confirmed since the exon of pBl-Cos is limited by the classical AG-GT sequences for intron / extron transitions. However, since the complete coding sequence of mucin MUC8 is contained in this exon, the exon can be used for the "" "cloning and expression of MUC8 over its entire length.The nucleotide sequence of the exon encodes 396 amino acids which, by ordinal analysis, it crushes a protein skeleton of PM 42178. The derived amino acid sequence contains approximately 6 repetitive units composed of 53 amino acids each Since the molecular weight determined by Western blot is approximately 170 kD, it must be specified oligomerization and / or intense co- and post-translational modifications, such as those also described for other mucins of secretion L) Analysis of genomic DNA transfer. Genomic DNA samples were isolated completely with EcoRI or MspI. peripheral lymphocytes from human donors, and hybridize on Southern blot with an EcoRI / PstI fragment of the pBl clone. The five samples showed a clear lesional polymorphism of the restriction fragments, which stood out especially in the digestion with Mspl. There, the bands oscillated in a length comprised between 1.5 and 2.9 kb. The size of the 0 fragments of EcoRI digestion was between 10 and 12 kb. With the same hybrid size also a DNA sample of somatic cell hybrids containing human chromosome 7. M) Transfer analysis of RNA and proteins. 5 Four micrograms of poly (A) + RNA from various tumor cell lines were hybridized in Northern blot with the EcoRI / HindII probe from 500 pB pBl cDNA. Three types of mRNA of 4.8 kb, 1.9 kb and 1.35 kb were recognized, of which the 4.8 kb band showed the strongest or strongest hybridization. If we are dealing here with precursors or degradation products of type 4.8, it can not be decided at the present time, since the length of the 5 'untranslated part or the starting point of the transcription is not known. In the Western blot, a protein with a size of 170 kD is recognized as antigen 5 Bl in reducing conditions. In non-reducing conditions, the band is approximately 5-1 / 4-kd. The part of O-glycosylation is unknown. N-glycosylation can be excluded due to the absence of the consensus sequence (Asn-X-Ser / Thr).

Description of the Figures Figure 1: Immunoblot of cell lysates, which were isolated from human tumor cell lines, with the anti-MUC8 of mouse mAk Bl. The proteins were separated by molecular weights electrophoretically in a 3-10% SDS-polyacrylamide gel and transferred to nitrocellulose. The antibody used was undiluted hybridoma supernatant, and, as the second antibody, mouse anti-immunoglobulins conjugated with alkaline phosphatase. Track 1: BED; Track 2: Capan-1; Track 3: Mz-Sto-1; Track 4: MKN-45; track 5: T84; Track 6: HT-29 MTX. Table 3: Collection of immunochemical data. Cryostat sections from various benign and malignant human tissues were prepared and incubated with undiluted Bl hybridoma supernatant as the antibody. Anti-mouse immunoglobulins conjugated with horseradish peroxidase were used as 2nd antibody. Figure 2: Nucleotide sequence of the cDNA of the pBl clone with the amino acid sequence derived in three letter code. The sequence was determined by the generation of sequence-specific primers by the T7 sequencer kit from Pharmacia, according to the Sanger method. Figure 3: Immunoblotting of lysates that had been isolated from bacterial cultures (TB1) after transformation with the pMAL-c vector or the pMal-Bl construct. The bacteria were harvested before and after the induction of the promoter with IPTG and the pellets were collected in loading buffer with SDS gel. After separation on -10% SDS-PAGE, the proteins were transferred to a nitrocellulose membrane and the following process was followed as described for Figure 1. Lane 1: pMAL-Bl induced with IPTG; lane 2: pMAL-c induced don IPÍG; Lane 3: pMAt-Bl not induced; track 5 4: pMAL-s not induced. Figure 4: Comparison of homology of the amino acid sequences of two MBP-B1 fusion proteins, both of which are specifically recognized by the mAk Bl and their 0 reading frames are displaced in one nucleotide. The identical amino acid sequences were subjected to an analysis for the calculation of antigenic determinants (Hopp and Woods, 1983) ** by which a QDREEE sequence could be determined as an antigenic determinant potential Figure 5: Northern blot analysis with various lines of human tumor cells The RNA samples purified with poly (A) + were separated by molecular weights on an agarose gel containing 0 formaldehyde and transferred to a nylon membrane, using an EcoRI / - Hindll fragment as radioactive probe of clone pBl (A) The normalization of the fingerprint was carried out with a β-actin probe (B) Track 1: HT-29, track 2: T84, track 3: BED, track 4: Mz-Sto-1 track 5: Capan-1, 5 Figure 6: Southern blot analysis with human genomic DNA: Genome DNA samples (15 mg) from five healthy or 0 different donors were completely digested with EcoRI (a) or MspI (b) -5) and separated by molecular weights in a 0.8% agarose gel. A 190 bp EcoRI / PstI fragment of the pBl clone was used as a radioactive probe. Figure 7: Nucleotide sequence and amino acid sequence 5 derived from the cDNA of clone pB1.2. The sequences were determined following the same procedure as described, * • for pBl. Figure 8: Complete nucleotide sequence of the pBl-Cos genomic clone compared to the two cDNA sequences of pBl 5 and pB1.2. The exon / intron transitions, the initiation and termination codon are highlighted by highlight printing.

The 225 bp of the non-sequenced exon are underlined. The "alignment" of the sequences was carried out by means of the "software" GeneWorks of IntelliGenetics. Figure 9: Schematic representation of the pBl-Cos exon and the two cDNA sequences. The identical regions are ** - arranged one below the other. The representation is not on a natural scale. 15 Figure 10: Partial digestion of the repetitive ression of the clone pBl-PCR with the restriction enzyme Earl. A: agarose gel; B: autoradiography of the transfer Southern with the EcoRI / HindII fragment of pBl as a radioactive probe; 2 mg of the insert DNA were respectively digested under the following conditions: lane 1: 2h 1 U / mg; lane 2: 5 min 0.5 U / mg; lane 3: 3 min 0.5 U / mg; track 4: 1 r min 0.5 U / mg; M: 123 bp standard length. ___ Figure 11: 25 Southern blot analysis with genomic DNA from human / rodent somatic cell hybrids. DNA samples of 10 mg each were completely digested with EcoRI and separated on an agarose 0.8% The EcoRI / -30 HindII fragment of pBl was used as a radioactive probe. The samples were applied so that DNA containing human chromosome 1 is found on lane 1, DNA containing human chromosome 2 on lane 2, and so on. Lane 25: IMR91 human cell line; lane 26: 35 3T6 mouse cell line; track 27: cell line of Hamster RJK88.

Figure 12: Western blot analysis with sera from patients with pancreatic and colon carcinoma and from a healthy donor. The Mz-Sto-1 cell extract serves as a positive control. 10 ml of all sera were applied. As the antibody, 2 mg / ml of mAk Bl were used as the 2a anti-mouse immunoglobulin antibody conjugated with alkaline phosphatase in a 1: 500 dilution.

Claims (1)

1. CLAIMS Ia.- cDNA encoding mucin MUC 8, characterized in that it essentially corresponds to the nucleotide sequence of pCos of Figure 8, from nucleotide acid 3,049 to 4,238. 2 * .- Recombinant DNA, characterized in that it contains the cDNA according to claim 1 ». 3* . - Mucina MUC 8, prepared using the cDNA according to claim 1 *. 4a.- Mucina MUC8, prepared with the use of the amino acid sequence that can be derived from the cDNA according to claim Ia. 5a.- A? NC according to claim Ia, characterized in that it essentially corresponds to the nucleotide sequence of pCos of figure 8, from nucleotide 3.049 to 3.325. 6 *. ~ CDNA according to claim Ia, characterized in that it essentially corresponds to the nucleotide sequence of the PCos of finite 8, from nucleotide 3.326 to 3.666. 7a.- cDNA according to claim Ia, characterized in that it essentially corresponds to the nucleotide sequence of pCos of figure 8, from nucleotide 3,667 to 4,238. 8 * .- DNA, characterized in that it corresponds essentially to the nucleotide sequence of pCos of Figure 8. 9a.- DNA, characterized in that it corresponds essentially to the nucleotide sequence of pB 1.2 of Figure 8. 10a.- DNA, characterized because it essentially corresponds to the nucleotide sequence of pB 1 of Figure 8. 11a.- Diagnostic procedure for the detection and determination of human antibodies against mucin MUC 8 as a tumora marker, characterized in that mucin peptides are used according to claim 3a or 4a or immunoreactive peptides derived therefrom. 12a.- Use of mucin according to one of claims 3 or 4a or immunoreactive peptides of mucin in a method for the detection of antibodies against mucin. 13a.- Use according to claim 11, wherein the detection is performed by ELISA. 14a.- Use of the mucin according to claim 3 or 4a or of immunoreactive peptides derived therefrom, to originate an antibody formation in a mammal. 15a.- Use according to claim 14, wherein the mammal is man. 16a.- Diagnostic procedure for the detection and determination of mucin MUC 8 as a tumor marker, characterized in that the pCos sequence of figure 8 or partial sequences derived from it are used as gene probes. 17a.- Use of DNA according to one of claims Ia and 2 * and 5a-10a for the recombinant production of mucin MUC 8 or of partial sequences thereof. 18a.- Mucina MUC8 or peptides derived from it that can be produced synthetically based on the amino acid sequence that can be derived from the cDNA according to claim la. 19. Monoclonal or polyclonal antibodies against mucin MUC 8 produced after immunization with MUC8 according to claims 34 or 4a. 20. Diagnosis for the detection of mucin MUC8) containing one or more antibodies against the MUC8 according to claim 19 S ..