FR2751346A1 - PROCESS FOR THE PREPARATION OF NON-HUMAN TRANSGENIC MAMMALIAN ORGANS FOR THEIR TRANSPLANTATION IN MAN, AND NUCLEOTIDE SEQUENCES FOR THE IMPLEMENTATION OF SAID METHOD - Google Patents

Abstract

The use of nucleotide sequences coding for antibodies to any molecule involved in a graft versus host reaction (hereinafter anti-molecule antibodies), for preparing transgenic cells or organs of non-human mammals, wherein said molecules at least partially form immune complexes with said anti-molecule antibodies, such that the activity of said molecules in graft versus host reactions is at least partially inhibited, said transgenic cells or organs of non-human mammals being intended for transplantation into a patient, is disclosed.

Description

PROCESS FOR PREPARING NON-MAMMALIAN ORGANS
TRANSGENIC HUMANS FOR THEIR TRANSPLANTATION
IN MAN, AND NUCLEOTIDE SEQUENCES FOR
IMPLEMENTATION OF THIS PROCESS
The subject of the present invention is the use of nucleotide sequences for the implementation of a process for the preparation of transgenic non-human mammalian organs capable of being grafted in humans by substantially reducing the risks of rejection of grafts.

 Transplantation of animal organs in humans, or xenotransplantation, is a technique currently considered as a possible alternative, making it possible to overcome the crucial lack of donor in allograft. Spectacular advances in this area have been made in recent years, and pork as a donor animal is needed by the scientific and medical community. The reasons for this choice, as opposed to the choice of primates as organ donors, are many: the risk of transmission of pathogenic viruses to humans is much lower from pork (Allan, 1996), husbandry and time Primate breeding, unlike pork, makes their use difficult, and a significant ethical barrier exists for the use of monkeys for human medicine purposes. The major obstacle to the use of pig grafts in humans is above all immunological: the man possesses natural antibodies (called XAN- natural xeno-antibodies) directed against molecules expressed by porcine cells. When a pig organ is grafted in humans (and in higher primates that also have these antibodies), these antibodies react with the vascular endothelium resulting in hyperacute rejection of the organ. This is the XAN- and complement-induced loss of vascular endothelium integrity resulting in edema, cell infiltration, and organ destruction (Bach et al., 1995). The porcine antigens recognized by XAN were analyzed: it is mainly a carbohydrate structure consisting of galactose connected in configuration a1,3 on N-acetyl lactosamine (epitope cc-galactosyl) present on glycolipids and membrane glycoproteins (Sandrin et al., 1993).

 The work done by D. White's team (Cambridge) partially solved this problem of hyperacute rejection: by blocking the activation of human complement on the surface of porcine endothelial cells (Carrington et al, 1995) they managed to perform pig heart transplants in the macaque, whose survival seems similar to the survival of an allograft (organ transplant of the same species) carried out under the same conditions, insofar as the XAN deposit is not too massive.

Technically, this has been achieved by insertion, by germinal transgenesis, of a regulatory molecule of human complement, DAF (for decay accelerating factor, or CD55), in the genome of pork. This species-specific molecule prevents human C3 convertase from triggering the complement activation chain reaction on the surface of porcine cells. This molecule seems however "outdated" when the amount of complement fixed is too important. In this case, a rejection occurs anyway.

 The presence of α-galactosyl epitopes, the major xenoantigen on porcine cells, is due to the action of a Golgi enzyme, UDP-Gal: Galal-4GlcNacal, 3-galactosyltransferase (also referred to as al, 3 GT enzyme).

The al, 3GT enzyme participates in the galactose binding, in a1,3 configuration, on the galactose of N-acetyl lactosamine membrane glycoproteins and glycolipids, which creates a xenoantigenic epitope called a-galactosyl consisting of the Galal complex, 3Gal -N-acetyllactosamine. Inactivation of the gene coding for this enzyme in pigs could block the synthesis of α-galactosyl epitopes and induce non-recognition of pork cells by human XAN. This inactivation, used in conjunction with the expression of human DAF in pigs, could represent a decisive advantage for the success of the xenograft of pig organs in humans. However, the inactivation of a gene by homologous recombination ("gene knock out") is a technique whose use is currently restricted to the mouse, because its implementation requires the availability of embryonic stem cells, called ES cells.

These cells, despite intense research, could never be obtained from another species than the mouse. The inactivation of al, 3GT in pigs can not be achieved by this technique.

Apart from the xenoantigenic molecules and the molecules involved in the biosynthesis of the latter, there are other molecules whose inhibition could be useful in xenotransplantation, in particular molecules whose biological activity contributes to rejection of transplant, without these The molecules are either xenoantigens or participate in the synthesis of xenoantigens. These include all the inflammatory molecules produced by the endothelium of the graft, in particular during a xenograft cytokines (IL-1, IL-6), chemotactic factors (IL-8, IP-10 , RANTES,
MCP / JE, pl5E inhibitors, GFM-CSF, G-CSF), adhesion molecules (ELAM-1, VCAM-1, ICAM-1, c-sis, GPlba, vWF, ligand LAM-1), transcription factors (c-fos, NFkB, Gem), vascular tone regulators (iNO synthase, PGH synthase, endothelin), factors involved in coagulation (PAF acetyltransferase, tissue factor, PAI-1), immunocompetence factors (MHC I, CMII II). They are also the receptors, expressed by the endothelium of the graft, to molecules originating from the recipient and having a biological activity contributing to the graft rejection: C5a receptor, TNFα receptor, IFN receptor, cell receptors NK.

 The object of the present invention is to provide transplantable transgenic non-human mammalian organs in patients requiring organ transplantation, with a reduction in the risk of graft rejection phenomenon, or even complete cancellation of this risk.

 The present invention also aims to provide nucleotide sequences that can be used for the genetic transformation of animals to obtain the aforementioned organs.

 Another object of the invention is to provide transgenic non-human mammals from which the above-mentioned organs can be taken.

 The subject of the present invention is the use of nucleotide sequences encoding antibodies directed against any molecule involved in a graft rejection phenomenon (hereinafter referred to as antimolecule antibodies), and especially against any molecule having an activity such that said molecule represents a xenoantigenic epitope, or participates in the biosynthesis of xenoantigenic epitopes in non-human mammalian cells (and more particularly on the surface of these cells), these epitopes being capable of being recognized by human natural xeno-antibodies when said cells, or organs consisting of these cells, of non-human mammals, are grafted in humans, for the preparation of transgenic cells, or transgenic organs, of non-human mammals, in which said molecules form, in whole or in part, immune complexes with said anti-molecule antibodies, of such so that the activity of said molecules in the graft rejection phenomena is totally or partially inhibited, in particular that the aforementioned xenoantibodies no longer recognize, in whole or in part, the aforesaid epitopes, or in which the biosynthesis of said xenoantigenic epitopes is partially or totally inhibited, said cells or said transgenic organs of non-human mammals being intended to be grafted in a patient.

The nucleotide sequences used in the context of the present invention are advantageously those coding for antibodies directed against
any xenoantigenic, carbohydrate or non-carbohydrate epitope recognized by human xenoantibodies, and more particularly the galactosylated carbohydrate epitopes located on the surface of the membranes of non-human mammalian cells, in particular the β-galactosyl epitope present on the surface of pork and consisting of the above-mentioned Galol complex, 3Gal-N-acetyllactosamine,
any molecule participating in the biosynthesis of xenoantigenic epitopes in humans, of carbohydrate or non-carbohydrate nature, and more particularly the galactosyltransferases present in non-human mammalian cells, in particular the x1,3GT enzyme present in pork cells ,
any molecule of inflammatory nature synthesized in the endothelium of non-human mammals, and whose biological activity notably leads to the modification of the anticoagulant properties of the endothelium in vivo in xenogeneic medium, such as cytokines and chemoattractors (IL-1 , IL-6, IL-8, IP10, RANTES, MCP / JE, pl5E inhibitors, GM-CSF, G-CSF), adhesion molecules (ELAM-1, VCAM-1, ICAM-1, c-sis , GPlba, vWF, ligand LAM-1) transcription factors or modifying transcription (c-fos, NFkB, Gem), vascular tone regulators (iNO synthase, PGH synthase, endothelin), factors involved in coagulation ( PAF acetyltransferase, tissue factor, PAI-1), immunocompetence factors (MHC I, CMII II),
membrane receptors with molecules present in the recipient, the action of these molecules being directed towards a graft rejection, of which for example C5aR, or TNFaR, or NK cell receptors.

 The invention more particularly relates to the use of molecules involved in a graft rejection phenomenon, as described above, for the implementation of a method of obtaining, in particular according to the methods described below. of nucleotide sequences coding for antibodies directed against the aforesaid molecules, these nucleotide sequences themselves being capable of being used for the preparation of transgenic cells, or of transgenic organs as defined above according to the invention.

 Advantageously, the nucleotide sequences used in the context of the invention are obtained by selection of the aforesaid anti-molecule antibodies, namely antibodies directed against the molecules involved in a graft rejection phenomenon, this selection being carried out using said molecules. used as target molecules recognized by said antibodies, and sequencing the nucleotide sequences encoding said selected antibodies.

 Said anti-molecule antibodies are themselves advantageously obtained from any hybridoma capable of being formed, by conventional methods, from spleen cells of an animal, in particular of mouse or rat, immunized against one of said molecules involved in a graft rejection phenomenon, on the one hand, and cells of a suitable myeloma, on the other hand, said hybridoma being selected by its ability to produce monoclonal antibodies recognizing said molecule initially used for the immunization of animals.

 The hybridoma DNA thus selected is then cloned, according to techniques well known to those skilled in the art, into cellular hosts capable of producing said monoclonal antibodies, the nucleotide sequences coding for said antibodies thus selected being then sequenced.

The said anti-molecule antibodies can also be obtained by screening an antibody library (such as the library described in the article of
Nissim et al., 1994) with said molecules, said antibody library being constructed from human or murine immunoglobulin DNA sequences, or from another species, in a cellular host (in particular in phages) capable of expressing the antibodies encoded by said immunoglobulin DNA sequences.

 Advantageously, the abovementioned antibodies, from which are deduced the nucleotide sequences used in the context of the present invention are single-stranded antibodies (also called ScFv antibodies).

 These ScFv antibodies are advantageously obtained from an antibody library as described above, in which the DNA sequences coding for these antibodies are treated, in particular according to the method described in the article by Nissim et al. , 1994, so as to obtain single-stranded antibodies, in particular by joining all or part of a DNA sequence coding for an immunoglobulin heavy chain with all or part of a DNA sequence coding for a light chain immunoglobulin.

 These ScFv antibodies can also be obtained by cloning complementary DNA (cDNA) derived from hybridomas, as described above, coding for an immunoglobulin directed against one of the said molecules involved in a graft rejection phenomenon, followed by a treatment, as described above, so as to obtain single-stranded antibodies by joining all or part of the fraction of said cDNA coding for the immunoglobulin heavy chain with all or part of the fraction of said cDNA encoding the light chain of this immunoglobulin.

 According to a preferred embodiment of the invention, the nucleotide sequences used for the preparation of transgenic cells or organs mentioned above are those encoding antibodies directed against any molecule involved in the biosynthesis of xenoantigenic epitopes in non-human mammalian cells. .

 Advantageously, the nucleotide sequences used in the context of the present invention are those coding for antibodies directed against at least one isoform (and advantageously against all isoforms) of galactosyltransferases present in non-human mammals, and more particularly against at least one of o: 1,3GT isoforms present in pigs, for the preparation of transgenic non-human mammalian organs in which the biosynthesis of α-galactosyl epitopes in the cells of said organs is partially or totally inhibited .

 As such, the invention more particularly relates to antibodies, where appropriate single-stranded, directed against at least one of the isoforms of the cI 3, 3GT said antibodies being as obtained by implementation of a methods described above carried out using one or more isoforms of 1'xx1,3GT, as molecules involved in a graft rejection phenomenon.

The invention more particularly relates to the antibodies as described above, directed against at least one of the isoforms of the oci, 3GT present in pigs, and in particular against at least one of the isoforms represented by the sequences in amino acids SEQ ID NO 2 (corresponding to isoform 1),
SEQ ID NO 4 (corresponding to isoform 2), SEQ ID NO 6 (corresponding to
Isoform 3) and SEQ ID NO 8 (corresponding to isoform 4), these isoforms 1 to 4 being respectively coded by the nucleotide sequences represented by
SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7, or encoded by any nucleotide sequence derived therefrom, in particular by degeneracy of the genetic code.

 The invention more particularly relates to the antibodies as described above, directed against at least one of the isoforms 3 and 4 of oL1,3GT present in the pig and represented by the amino acid sequences SEQ ID NO6 and SEQ ID NO 8.

 Advantageously, the above-mentioned anti-1,3-GT antibodies of the invention are single-stranded antibodies whose amino acid sequences are such that they contain the CDR1, CDR2 and CDR3 portions of the heavy chain of said bound anti-ce1,3GT antibodies. by a linker sequence of about 6 to about 36 amino acids, all or part of the VX3 light chain of the above-mentioned anti-oc1,3GT antibodies.

A particularly preferred linker is that consisting of three successive repetitions of the following acid sequence
Gly - Gly- Gly- Gly- Ser
Particular antibodies according to the invention are those represented by the amino acid sequences SEQ ID NO 10 (antibody designated ScFv1), SEQ ID
NO 12 (antibody designated ScFv2), SEQ ID NO 14 (antibody designated ScFv3),
SEQ ID NO 16 (designated antibody ScFv4) and SEQ ID NO 18 (designated antibody)
ScFv5), or by any amino acid sequence derived therefrom, in particular by addition, deletion or substitution of one or more amino acids, or by any fragment of the aforementioned sequences or their derived sequences, said derived sequences and said fragments being likely to recognize at least one isoform of the oci, 3GT.

The subject of the invention is also the nucleotide sequences coding for an anti-cc1,3GT antibody as described above, and comprising in particular the nucleotide sequences represented by SEQ ID NO 9 (coding for ScFv1), SEQ ID No. 11 (coding for ScFv2), SEQ ID NO 13 (coding for
ScFv3), SEQ ID NO 15 (coding for ScFv4), SEQ ID NO 17 (coding for
ScFv5), or any nucleotide sequence derived therefrom, in particular sequences derived by degeneracy of the genetic code, and nevertheless being capable of encoding the ScFv1, ScFv2, ScFv3, ScFv4 or ScFv5 antibodies, or the sequences derived by addition, deletion or substitution of one or more nucleotides, or any fragment of the aforementioned nucleotide sequences or of their derived sequences, said derived sequences and said fragments being capable of coding for an antibody capable of recognizing at least one of the isoforms of the α1,3GT .

The nucleotide sequences SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15 and SEQ ID NO 17 mentioned above are advantageously obtained by sequencing the cDNAs encoding the antibodies represented by
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID.
NO 18, respectively, these cDNAs from cell clones selected for their ability to produce said antibodies, the selection of said cellular clones being carried out using isoform 2 of α1,3 GT (i.e., polypeptide represented by SEQ ID No. 4) used as a target molecule recognized by said antibodies, said cellular clones being themselves obtained by transformation of cells, in particular bacteria or phages, with nucleotide sequences from a cDNA library coding for antibodies, such as the aforementioned library described by Nissim et al., 1994, or from hybridomas obtained by the technique indicated above by immunization of an animal with said isoform No. 2 of 1'1,3GT.

 The subject of the invention is also any vector, in particular a plasmid, containing at least one of the nucleotide sequences described above according to the invention, in particular at least one of the sequences SEQ ID No. 9, SEQ ID No. 11, SEQ ID NO 13, SEQ ID NO 15 or SEQ ID NO 17, and the elements necessary for the expression of the anti-molecule antibodies, and more particularly the anti-α1, 3GT antibodies mentioned above.

 The invention also relates to any cellular host (such as a eukaryotic cell) containing at least one of the vectors as described above, according to the invention.

 The invention also relates to any non-human transgenic mammal, or any non-human transgenic mammalian cell, comprising in their genome at least one nucleotide sequence described above, coding for anti-molecule antibodies as described above, and more particularly for the aforementioned anti-α1,3GT antibodies.

The invention more particularly relates to any non-human transgenic mammal, or any transgenic non-human mammalian cell, as described above, comprising in their genome at least one of the nucleotide sequences represented by SEQ ID NO 9, SEQ ID NO 11
SEQ ID No. 13, SEQ ID No. 15 and SEQ ID No. 17, or at least one derived sequence or fragment as defined above, of said nucleotide sequences.

 Advantageously, the non-human transgenic mammals according to the invention are as obtained by introducing, in particular by microinjection into a fertilized oocyte, at least one nucleotide sequence as defined above, coding for an anti-molecule antibody. as defined above, and more particularly at least one of the nucleotide sequences represented by SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ NO 15 and SEQ ID NO 17, or their derived sequence or fragment such as defined above, and insertion of the embryo into the matrix of a surrogate mother and development of the term embryo.

The subject of the invention is also any non-human transgenic mammal defined above, as produced by crossing transgenic animals expressing at least one nucleotide sequence as defined above, coding for an anti-molecule antibody, and more particularly at least one of the nucleotide sequences represented by SEQ ID NO 9, SEQ ID NO 11,
SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17, or their derived sequence or fragment as defined above.

By way of illustration, the non-human transgenic mammals according to the invention can be obtained according to the method described in the article of
DePamphilis ML, et al., 1988.

 Among the non-human transgenic mammals obtainable within the scope of the present invention, mention may be made of the mouse, the rat, the pig or the rabbit.

 The invention more particularly relates to transgenic pigs containing in their genome at least one of the nucleotide sequences represented by SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15 and SEQ ID NO 17, or their derived sequence or fragment as defined above.

 The invention also relates to cells cultured from non-human transgenic animals as described above.

The subject of the invention is also the non-human mammalian organs, and more particularly the porcine organs, in particular the kidneys, the liver, the pancreas or the heart, comprising in the genome cells constituting them at least one nucleotide sequence such that defined above, coding for an anti-molecule antibody, and more particularly at least one of the nucleotide sequences represented by SEQ ID NO 9, SEQ ID No. 11,
SEQ ID No. 13, SEQ ID No. 15 and SEQ ID No. 17, or their derived sequence or fragment as defined above, said organs being as obtained by sampling from transgenic non-human mammals according to the invention.

 The invention also relates to any polypeptide comprising the amino acid sequence as represented by SEQ ID NO 6 (corresponding to isoform 3 of the oci, 3GT) or SEQ ID NO 8 (corresponding to isoform 4 of the invention). α-1,3-GT), or any polypeptide containing any fragment of at least about 6 amino acids, of one of said amino acid sequences, said polypeptide containing said fragment being capable of generating antibodies recognizing one of said amino acid sequences; at least one of the four isoforms of the oci, 3GT, or any sequence derived therefrom, in particular by adding, deleting or modifying one or more amino acids, provided that the derived sequence is capable of generating antibodies recognizing the at least one of the four aforesaid isoforms.

 The subject of the invention is also the nucleotide sequences comprising the sequences represented by SEQ ID No. 5 and SEQ ID No. 7, or any derived sequences, in particular the sequences derived by degeneracy of the genetic code, and nevertheless being capable of encoding the polypeptides represented by the amino acid sequences represented by SEQ ID NO 6 and SEQ ID NO 8 respectively, or the sequences derived by addition, deletion or substitution of one or more nucleotides, or any fragment of the aforementioned nucleotide sequences or their derived sequences, said derived sequences and said fragments being capable of encoding an antibody capable of recognizing at least one of the isoforms of ocl, 3GT.

 The invention also relates to any vector, in particular a plasmid, comprising at least one nucleotide sequence coding for the sequences represented by SEQ ID No. 5 or SEQ ID No. 6, or by a derived or fragmentary sequence thereof, as defined above. , as well as the elements necessary for the expression of the isoforms of the oci, 3GT encoded by said nucleotide sequences.

 The invention also relates to any cell host transformed by a vector as described above, as well as any process for the preparation of the abovementioned isoforms 3 and 4 of the oci, 3GT by culturing said cell host in an appropriate culture medium. and separating said isoforms from the other constituents of the culture medium.

 The subject of the invention is more particularly any nucleotide sequences advantageously obtained by sequencing the cDNAs encoding the antibodies directed against the abovementioned isoforms 3 and 4 of the oci, 3GT, these cDNAs originating from cell clones selected for their capacity to produce said antibodies. , the selection of said cellular clones being carried out using the isoform 3 and / or 4 of the IC1,3 GT (ie polypeptides represented by SEQ ID NO 6 and SEQ ID NO 8, respectively) used as that target molecules recognized by said antibodies, said cell clones, being themselves obtained by transformation of cells, in particular bacteria or phages, with nucleotide sequences from a cDNA library encoding antibodies, such as the library mentioned above by Nissim et al., 1994, or from hybridomas obtained according to the technique indicated above by immunization of an animal with it isoform 3 or said isoform 4 of the ocl, 3GT.

 The subject of the invention is also any method, in particular a surgical method, for treating patients requiring a transplant of cells or organs, by implantation of said transgenic cells according to the invention in appropriate tissues of said patient, or by removal of the organ defective patient and replacement of this defective organ by a non-human transgenic mammalian organ according to the invention.

 The invention will be further illustrated with the aid of the following detailed description of the obtaining of the different isoforms of porcine 1,3 GT, as well as the transformation of animal cells using nucleotide sequences coding for anti-oe1,3GT antibodies according to the invention, and procedures for obtaining transgenic animals according to the invention.

I - Obtaining the different isoforms of the a1.3 GT
1) Experimental procedures
a) Cells and tissues
The porcine organs were obtained from pigs of about 200 to 300 kg. Enzyme isolation and culture of porcine aortic endothelial cells (PAECs) were performed according to the method described in Warren, 1990. PAECs were stimulated with 100 U / ml TNF (human) (Genzyme,
Cambridge, USA) with or without delO jug / ml of cycloheximide.

(b) Oligonucleotides
The following oligonucleotides were used
oligonucleotide 1 5 '-AGGAAGAGTGGTTCTGTC-3' hybridizing
oligonucleotide 6 5 '-GAGTCACTTGTCATCGTCGTCCTTGTAATCGATGTTATTTCTAACCA
AATT-3 'hybridizing to nucleotides 1105 to 1123 of the sequence SEQ ID NO 1, and added in phase to an oligonucleotide encoding a FLAG peptide (Kodak, New Haven, USA) a stop codon and an XhoI site.

c) cloning and construction
A 2 Kb ocl, 3GT cDNA clone (pCDNA-ccGT) was isolated from a cDNA library constructed in the pCDNA-1 vector (Invitrogen, Leek, Country).
Bas) by transfer hybridization using an amplified PCR probe with primers 1 and 2 (derived from the sequence published in the article by Sandrin et al., 1994. This clone corresponds to that of isoform # 2.

 Isoforms 1, 3 and 4 of porcine α1, 3T were isolated by PCR amplification of a region comprising exons 4-7 with primers 1 and 2, using the PAEC retro-transcript RNA. The amplified fragments are treated with Klenow polymerase to obtain blunt ends and cloned into the SmaI site of plasmid pUC18. Three clones containing fragments of 300, 237 and 201 bp were obtained and used as a template to introduce, by secondary PCR amplification, an EcoRI site at the 5 'end (with oligo 3) and a SpeI site in Exon 7 (with oligo 4).

The introduction of this SpeI site does not modify the amino acid sequence. The PCR products were ligated into an EcoRI-SpeI digested pKS plasmid. The remaining 3 'portion of the coding sequence was amplified by PCR from the clone pCDNA-ocGT with primers 5 and 6, treated to obtain blunt ends and cloned into the EcoRV site of a pKS plasmid. From this recombinant plasmid, a SpeI fragment carrying the coding region of nucleotide 262 to 1125, in phase with a FLAG sequence (contained in oligonucleotide 6), was isolated and cloned into the SpeI sites of the pKS plasmids containing the regions. 5 'variables. The resulting constructs were sequenced, and the EcoRI fragments containing the entire coding sequences were subcloned into the pCDAAS-neo encaryotic expression vector driving expression of the transgene under control of the CMV promoter. Isoform 2 of porcine αGT was isolated in the same way, using the clone pcDNA-oe
GT as initial matrix.

d) transfection of cells
HeLa cells cultured in RPMI-10% FCS were treated with trypsin, resuspended at 5.106 cells / ml in ice-cold medium and mixed with 20 μg / ml of transgene-bearing plasmid DNA and a resistance gene. neomycin. The cells were treated by electroporation at 250
V, 350, uF, left on ice for 10 minutes and seeded at 2000 cells / cm 2. After 24 h, 500 μg / ml G418 was added and the cells were cultured for two weeks. The clones were isolated and cultured in the presence of G418.

e) immunofluorescence
HeLa cell clones expressing one of the four isoforms of the <x1.3GT were seeded on 4-chambered microscope slides (Nunc, Rookilde, Denmark), for analysis of x-galactosyl epitope expression. on cell membranes. Two days after seeding, the cells were washed twice with PBS, fixed for 10 min at room temperature with 3% paraformaldehyde, further washed and incubated for one hour at room temperature with B4-FITC isolectin.
Banderaea simplicifolia (Sigma, St Louis, USA), diluted to 1,100 in
PBS. The coverslips were washed 4 times in PBS before mounting. For subcellular localization, cells were fixed with 3% paraformaldehyde and permeabilized by three sequential 5 min incubations with 50%, 100% and 50% ice-cold acetone. After three washes with 0.5% PBS / Tween-20, the cells were incubated with anti-FLAG M2 antibody (1: 200;
New Haven, USA), then incubated for one hour with a FITC-labeled anti-mouse donkey secondary antibody (1: 500, Jackson, Baltimore,
USA). The preparations were mounted in a FA fluid (Difco, Grayson,
USA) and visualized with a Nikon epifluorescence microscope.

f) Northern blot analysis
Total cellular RNA was isolated from cultured cells and organs described by Zipfel et al., 1989. Ten μg of total RNA was fractionated on an agarose / formaldehyde gel, transferred to a Hybond membrane. N (Amersham, Little Chalfont, UK) by capillary action in 20X SSC for 16h and immobilized by UV crosslinking. The filters were pre-hybridized for 6 h in a solution containing 50% formamide, 5 X SSPE (0.18
M NaCl, 10 mM sodium phosphate, pH7.7, 1 mM EDTA), 5 X Denhart's, 0.5% SDS, and 100 μg / ml denatured salmon sperm DNA. Membrane-bound RNA was hybridized with P32-labeled cDNA probes corresponding to the coding region of Au, 3 GT isoform 1, porcine p-actin or 28S RNA. Hybridization was performed overnight at 420C in a pre-hybridization solution containing 106 cpm / ml of a probe. The filters were washed twice in 2X SSPE, 0.5% SDS at room temperature for 15 min, and three times in 0.5 X SSPE, 0.5% SDS at 65 ° C for 15 min. Hybridization was visualized and the signals quantized with a phosphorimage device (Molecular Dynamics, Sunnyvale, USA).

g) RNase protection test
The RNase protection tests were carried out according to the method described by Melton et al., 1984. Two RNA probes, complementary to exons 5 and 6, and exons. 4 and 7 were prepared in the following manner: two EcoRI-SpeI fragments, corresponding to nucleotides 7 to 266 of clone αG Tiso 1, and nucleotides - 7 to 174 of clone aGT iso4 (FIG. cloned into the EcoRI-SpeI sites of the pKS plasmids. Using RNA polymerase
T7, antisense RNA probes were synthesized and 32P dUTP was incorporated. The template plasmid DNA was digested with DNAase I, and 500,000 cpm of each probe was hybridized for 16 h to 10 μg of total porcine organ RNA or cells in 30 μl of buffer. hybridization containing 80% formamide at 50 ° C. The control samples contain 10 μg of yeast RNA The hybridization mixtures were diluted to 400 μl before the addition of 5 μg / ml of RNase A and 10 U / ml RNase T1 After 1 h at 30 ° C, 50% g Proteinase K and 0.5% SDS were added. The mixture was then phenol / chloroform extracted and ethanol precipitated. Digestion products were analyzed on 6% polyacrylamide gels. The gels were exposed for 16 hours against Kodak AR films at -80 ° C.

2) Results
a) Cloning of four isoforms of the oci, 3GT
As previously described, a porcine cDNA expression library constructed using LLC-PK1 RNA (Guerif et al, 1995) was screened using a probe corresponding to the 5 'end of the coding sequence. A cDNA clone, pcDNA-aGT, with the coding region corresponding to a porcine oc1,3GT already described (Sandrin et al., 1994), was isolated. This α1,3 GT corresponds to the isoform 2 represented by SEQ ID NO 4 and encoded by the sequence SEQ ID NO 3. This pcDNA-αGT clone contains a 3 'untranslated region 770 bp long, a coding region of 1080 bp. containing the porcine homologues of what has been defined in the case of the mouse as exons. 4, and 6 to 9 (Joziasse et al., 1992) and a 5 'untranslated region of 150 bp. Using primers adjacent to the exons. 4 and 7, amplification
PCR of the PAEC DNA was performed. Amplification products of about 200 bp at 300 bp were found, suggesting the existence of additional transcripts. Three different products were cloned, one being identical to the sequence described by Strahan et al., 1995 (and referred to herein as isoform 1, Fig. 1, i.e. the isoform represented by SEQ ID NO 2 encoded by the sequence SEQ ID NO 1). In comparison with the aforementioned isoform 2 (from clone pcDNA-ccGT), it contains an additional 36 bp segment after nucleotide 80. Another product, isoform 3, ie the isoform represented by SEQ ID NO6 encoded by the sequence SEQ ID
NOS, does not comprise a 63 bp segment of nucleotide 117 to nucleotide 179, and the last product, isoform 4, namely the isoform represented by SEQ
ID NO 8, and encoded by the sequence SEQ ID NO7, does not include the 36 bp segment from nucleotide 81 to nucleotide 116, and the 63 bp segment from nucleotide 117 to nucleotide 179 (Fig. 1). The translation of the mRNAs corresponding to each of the identified transcripts predicts the synthesis of four forms of the cc1,3GT polypeptide, 372, 360, 351 and 339 amino acids that differ only in the length of their respective "stem" regions (stems).

The situation described here is comparable to that described in the case of the mouse (Joziasse et al., 1992), since four different mRNAs were found, containing exons 5 and 6, or containing only exon 5 or exon. 6, or containing neither exon 5 nor exon 6. The porcine segments of 36 bp and 63 bp correspond to exons 5 and 6 murine, except that, in the mouse, the exon 6 is 66 bp long instead of 63 bp in pigs (Fig. 1).

b) oc1,3GT activity of isoforms
The presence or absence of exons 5 and 6 determines four length variations in the stem region of the oci, 3GT. This region, located within the Golgi apparatus, binds the transmembrane anchor signal to the catalytically active luminal domain. In order to verify whether the four isoforms defined by the alternative splicing of exons 5 and 6 are catalytically active, cells
Human HeLa were transformed with recombinant plasmids expressing each of the four isoforms of porcine ocl, 3GT under the control of the CMV promoter. The product of this enzyme on cell surfaces was analyzed by immunofluorescence, using IB-4 lectin reacting specifically with Gala residues. The Gala epitope could be detected on the surface of HeLa cells translated with the four isoforms of the enzyme, as shown by lectin binding to IB-4 (Fig. 2), indicating that these isoenzymes all have galactosyltransferase. Expression levels of the oci, 3GT mRNA in porcine HeLa-α1, 3GT clones, were controlled by Northern blot, and were slightly higher in the clones containing isoforms 1 and 2, and slightly lower for the clones. clone containing isoforms 2 and 3, compared to the level found in the PAECs.

c) Subcellular location of isoforms oci, 3GT
The Golgi location of the oci, 3GT is due to the presence in exon 4 of an anchor signal domain. Variations in length in the stem region, ie the presence or absence of exons 5 and 6 may also influence the retention in Golgi (Dahdal et al., 1993) or expose a sensitive cleavage site proteases on certain isoforms (Weinstein et al., 1987
Lammers and Jamieson, 1989, Shaper et al., 1986; Yadav and Brew, 1991).

In order to verify if these variations of the region stem from the oci, 3GT could modify the localization in the Golgi, the cellular localization of the different isoforms was analyzed. The four CMV-based expression vectors described above express the al, 3 GT isoforms with a FLAG tag fused at the C-terminus which allows the protein to be detected by indirect immunofluorescence in HeLa cells transfected with an anti-FLAG antibody. As observed by fluorescence microscopy, the four isoforms are similarly localized and form a compact juxtanuclear structure corresponding to that of the Golgi apparatus (Roth et al.
Berger, 1982). Surprisingly, while 100% of the cells express the enzyme, as evidenced by uniform staining obtained with IB4 lectin, the enzyme is not detectable on all of these cells using the antibody. anti-FLAG. This may be due to an irregular expression of the enzyme associated with a limited sensitivity of the antibody, or the reorganization of the Golgi apparatus during the cell cycle.

d) Expression of isoforms of the oci, 3GT
The Galal epitope, 3Gal, resulting from the activity of the oci, 3GT, is not expressed homogeneously in pig tissues (Oriol et al., 1993). In order to study these variations at the enzymatic level, the level of 1,3GT transcripts was studied by Northern blot, and the distribution of isoforms was studied by RNase protection test.

 Northern blot analysis shows expression in all tissues studied, but with a striking difference between tissues. The kidneys contain about 50% of what is found in the spleen, the thymus 40% and the heart and ovaries, 20 to 25%. The lungs and liver contain less than 10% of what has been found in the spleen. Stimulation of endothelial cells with inflammatory cytokines induces an increase in the regulation or synthesis of many genes, including adhesion molecules, namely molecules related to inflammation and coagulation. An increase in oci regulation, 3GT has never been described in endothelial cells. Using PAECs stimulated for 6 hours with 100 U / ml of human TNFα, an increase of twice the overall level of mRNA encoding ocl, 3GT was observed. Incubation of PAECs in the presence of cycloheximide also increases the level of mx1,3GT-encoding mRNA likely by messenger stabilization. Surprisingly, stimulation with TNFo + cycloheximide does not lead to an increase in the level of the mRNA encoding the oci, 3GT, but rather to a slight decrease.

This effect may be related to the discovery that cycloheximide facilitates apoptosis due to TNFoc leading to a decrease in the regulation of certain antigens (Malorni et al., 1993).

 The expression of the isoforms of the oci, 3GT was analyzed by protection test with RNase in pig tissues. Two radiolabelled antisense probes were used to analyze the four identified RNA species.

 The mRNA transcript of hybrid isoform 1 with a 273 bp fragment on probe 1 corresponding to a sequence delimited by nucleotides 7 to 266 on the transcript (the nucleotide numbers correspond to the sequence shown in FIG. 1 isoform 1). Hybridization of probe 1 with the mRNA encoding isoform 2 leads to a fragment of 87 bp delimited by nucleotides - 7 to 80, and a larger fragment of 150 bp delimited by nucleotides 117 to 266. L hybridization of probe 1 with the mRNA encoding isoform 3 leads to a fragment of 123 bp delimited by nucleotides - 7 to 116 and a smaller fragment of 87 bp delimited by nucleotides 180 to 266. Hybridization of probe 1 with the mRNA of isoform 4 of OC1,3 GT leads to two fragments of 87 bp delimited by nucleotides - 7 to 80 and by nucleotides 180 to 266. The second probe, probe 4, is a 174 bp mRNA fragment corresponding to isoform 4. It is fully protected by the mRNA encoding isoform 4, and leads to two 87 bp fragments with isoforms 1, 2 and 3.

 The probe fragments protected by the mRNAs encoding the isoforms 1, 2 and 3 are those of 273, 150 and 123 bp, respectively, the probe 4 protected by the mRNA coding for the isoform 4 leads to a 174 bp band. . The distribution of transcripts differs from one tissue to another: in the kidney, isoform 3 alone is expressed, whereas in the ovaries only isoform 4 is expressed. In the heart, lungs and spleen, isoforms 1 and 2 are found, while isoforms 2 and 4 are found in the liver. Endothelial cells express isoforms 1, 2 and 4. In the thymus, all 4 isoforms can be observed.

II-Transformation of Animal Cells Using Nucleotide Sequences Coding for α-α-1,3-GT Antibodies According to the Invention
1) Materials and methods
a) cDNA libraries, cells and plasmids
The cDNA library is derived from LLC-PK1 porcine epithelial cells.

It is constructed in the pCDNA-1 vector. The bacterial strains used are E. coli-TG1 sup E, E. coli-XL-1, SURE, M15, MC 1061 / P3, and DH5cc.

The prokaryotic expression plasmid pQE-30 is from Qiagen. The eukaryotic expression plasmid pCDAAS-9 contains a neomycin resistance gene and drives the transcription of cDNA that it carries under the control of the cytomegalovirus promoter. The human immunoglobulin ScFv library is constructed in plasmid pHEN-1 and was used as described in the article by Nissim et al., 1994. The preparation of recombinant phages is also described.
b) Recombinant expression system and purification
The production and purification system of the recombinant 3GT oci comes from Qiagen. Transcription was induced by culturing E.coli-M1S transformed with the enzyme cDNA with 2mM IPTG for 5 hours. The recombinant protein in the cell lysate was then purified on an ion exchange column under denaturing conditions. The purified protein was dialysed in PBS and stored at -20 C until used.

c) ELISA
Microtitration plates (Nunc) were ice-cold overnight at 4 ° C with 5 t / ml purified al, 3GT protein in carbonate buffer pH 9.5, then saturated for 2 h at 37 ° C in 2%. skim milk, then incubated for 2h, 37 ° C with a recombinant phage suspension expressing a ScFv fragment of antibodies from the ScFv library After washing, the plates were incubated 1 h, 37 ° C with a labeled anti-M13 phage antibody with peroxidase (Pharmacia), diluted 1/500. The revelation was carried out by reaction of the peroxidase with diaminobenzidine in the presence of hydrogen peroxide.

d) Transfection and porcine cells
LLC-PK1 pork cells were transfected with pCDAAS plasmids carrying the cDNA of anti-α1,3 GT positive clones. Transfection was performed by electroporation of a mixture of 500,000 cells and 20 μg of plasmid DNA at 250V and 350μF. The clones were selected by 15 day culture in the presence of 1500 u / ml neomycin (G418), isolated using cloning rings and grown separately in the presence of G418 until assayed.

e) Immunofluorescence and cytofluorometry:
The cells were labeled with fluorescein-labeled Bandeiraea simplicifolia isolate B4 (IB-4-FITC, Sigma), lectin specific for α-galactose structures: the cells were washed at 40C in PBS-2% BSA, incubated at 4 ° C for 30 min with 20 μg / ml IB-4-FITC, washed 3 times and analyzed by cytofluorometry using a FACS-can (Becton Dickinson).

f) Sequence:
Sequence reactions were performed with D-taq (Amersham), using as primers two oligonucleotides located in the 5 'portion of the VX3 segment. Oligo 1, allowing the reading of the 5 'part of the 5' cDNA
TTCTGAGGCTGTCTCCTT-3 '. Oligo 2, allowing the reading of the 3 'part of the 5' cDNA -ATCGTCTGAGCTGACTCA-3 '.

g) Molecular targeting of anti-al, 3-GT ScFv in Golgi
In order to express ScFv antibodies in the same cell compartment as α1,3 GT, i.e. in Golgi, signal and Golgi retention sequences were added to ScFv clones,
N-terminal and C-terminal, respectively. The selected signal and retention sequences were adapted from the data reported by
Richardson et al., 1995. They were introduced by PCR amplification of the starting ScFv clones, using primers containing the cDNA of these sequences, and restriction sites for cloning. The sequence of these primers is as follows
- primer encoding the signal sequence
5 '-TTTGAATTCATGGAAAGGCACTGGATCTTTCTCTTCCAGGT
GCAGCTGGTGCAG-3 '.

- primer encoding the Golgi retention sequence:
5 '-TTTCTCGAGTTATTACAGCTCGTCCTTTTCGCTACCTAGGA
CGGTGCAGCTT-3 '.

2) RESULTS
a) Cloning of the oci, pork 3GT
A 313bp fragment corresponding to the 5 'end of the porcine 1.3GT cDNA was amplified by RT-PCR from porcine aortic endothelial cell RNA. This fragment served as a probe for cloning the entire cDNA into a porcine epithelial cell bank expressed in E.

Coli MC1061 P3, in the plasmid pCDNA-1. The isolated clone measures approximately 2 Kb, has a 5 'non-coding part of 147 bp followed by an open reading phase of 1104 bp and a 3' non-coding part of approximately 750 bp. It corresponds to the isoform of the enzyme whose exon 5 is absent (namely the isoform 2 represented by SEQ ID NO 4). The isolated porcine cDNA is functional because it confers al, 3GT activity on Cos cells, naturally devoid of this activity, after transfection. This activity can be demonstrated by acquiring reactivity with Bande ira e simplicifolia isolate B4, specifically labeling α-galactosyl residues.

 b) Protein Expression of the oci, recombinant 3GT.

 The coding part of the oci, 3GT was subcloned by PCR into the prokaryotic expression vector pQE30 (Quiagen), in 5 'fusion with a tail of six histidines serving for the purification of the recombinant protein on a template. charged (Ni-NTA). The clone thus obtained was completely sequenced to ensure the absence of mutations compared to the starting clone, which could have been introduced during the PCR amplification. Plasmid pQE30-α-1,3GT was introduced into E. coli M15 and the recombinant protein produced in vitro by induction of transcription at IPTG, and purified.

c) Production of anti-al, 3GT ScFv antibodies
The human immunoglobulin (Ig) library consists of 50 sequences of Ig heavy chain variable portions in the germinal configuration, associated with a peptide containing from 4 to 12 random amino acids, encoding the CDR3 portion of the heavy chain, and inserted into the phagemid pHEN1-VR3, in fusion with the plasmid portion encoding the g3p protein of the phage envelope. The phages that can be derived from these plasmids thus express a fragment (ScFv) of functional Ig merged with an envelope protein, and behave, in terms of their antigen recognition properties, such as Ig which they express the sequence. Phages derived from this Ig library were subjected to 4 successive sorts by "panning" on the purified recombinant protein, as described in Nissim et al., 1994.
d) ELISA test of the positivity of anti-al, 3GT ScFv antibodies.

Ninety-six individual colonies of E. coli-TG1 supE bacteria each containing a pHEN1-ScFv clone were randomly isolated after the 4 rounds of enrichment against al, 3GT protein. These colonies were grown overnight in 200 μl of LB medium in microtiter plate. The supernatant, containing phage exposing on their envelope 1 to 3 ScFv antibody molecule molecules, was tested by ELISA to measure the relative reactivity of the different clones against the oci, 3GT. Of the 96 clones tested, 15 showed a reactivity greater than three times the control level (Figure 2). The six most positive clones were reamplified and purified phage were prepared from 500 ml of bacteria supernatant
TG-1. Purified phage, and concentrated by precipitation and resuspension in PBS have a titer greater than 1010 cfu / ml. This concentrated phage preparation was re-tested in ELISA. The results, shown in FIG. 2, reproduce the order of reactivity observed in FIG. 2.

e) cDNA Sequence of ScFv Anti-alX3GT Antibody Fragments
The selected anti-al, 3GT ScFv clones were sequenced to ensure the non-introduction of errors during PCR subcloning and to determine the genomic-random peptide heavy chain associations (from 4 to 12 amino acids). VR3 light chain that produce an antibody reactive against the oci, 3GT. Of the six sequences analyzed, two are identical (n "2 and 6), and two differ by only two amino acids in the CDR3 part (n" 1 and 5). Sequence # 3 has an amber codon (codon recognized as a translational stop in a eukaryotic system, but considered a glutamine in the E. coli-TG1 supE strain used for cloning) at the CD3-peptide linker junction linking the chains. heavy on the VX3 light chain.

A stop codon at this position is a predictable consequence of the random association of cDNA fragments that occurs during the manufacture of the ScFv library. The replacement of the nucleotide T at position 313 by a C makes it possible to obtain a codon corresponding to a glutamine at position 105 (see FIG.
SEQ ID NO 13). The heavy chains (designated VH sequences) selected as well as the sequence of the CDR3 regions are described in Table 1; the ScFv1 clone sequence corresponds to the sequence represented by SEQ ID NO 9, the sequence of the ScFv2 clone and that of the ScFv6 clone correspond to the sequence represented by SEQ ID NO 11, the sequence of the ScFv3 clone corresponds to the sequence represented by SEQ ID NO 13, the sequence of the clone
ScFv4 corresponds to the sequence represented by SEQ ID NO 15 and the sequence of the ScFv5 clone corresponds to the sequence represented by SEQ ID NO 17.

Table 1
Clone <R

(g) Evaluation of the efficacy of intracellular immunization
The cDNAs encoding the native anti-α1, δT1-6 antibodies, or having signal and Golgi location sequences, were subcloned into the eukaryotic expression vector pCDAAS, under the control of the CMV promoter. To test the consequence of the expression of the anti-al, 3GT ScFv on the decrease of the expression of the α-galactosyl epitope on the pork cell membrane, LLC-PK1 cells were transfected with the different pCDAAS plasmids. -ScFv, and clones were isolated The presence of the α-galactosyl xenoantigenic epitope was evaluated by immunofluorescence with IB4-FITC lectin (o-patterned galactose specific), in comparison with the same non-transfected cells The results show that the ScFv # 2 antibody (namely that represented by SEQ ID NO 12) encoding the VH DP-5 sequence, associated an artificial CD3 part composed of amino acids PEIDQ, linked to the sequence VX3, san Addition of signal sequences and Golgi retention, when expressed in a porcine cell, leads to up to 95% decrease in the expression of galactose connected in oc configuration. Indeed, the average fluorescence of the cells
LLC-PK1 is 465 units when labeling is performed with 20 μg / ml IB-4
FITC, and it drops to 49 units for the LLC-PK1-ScFv6 clone.

 In conclusion, in the context of xenotransplantation, the possibility of inhibiting the expression of oci, 3GT in a pork cell by using this technique leads to very clear applications: the availability of transgenic pigs expressing no or few oc-galactosyl epitopes, consequence of the inhibition of the oci, 3GT, and expressing a human complement inhibitor, as it already exists, makes it possible to carry out xenografts by avoiding the reaction of the graft cells with antibodies and with the complement of the human recipient.

The results which are presented above, show that cells of porcine origin expressing a ScFv anti-oe1,3GT antibody can be obtained, and that this expression results in a significant decrease in the level of expressed xenoepitopes.
III - Anti-ol1,3GT Monoclonal Antibodies
The coding portion of the tag, 3GT, isoform # 2 was subcloned by PCR into the prokaryotic expression vector pQE30 (Quiagen), in 5 'fusion with a tail of six histidines serving for the purification of the recombinant protein on a charged matrix (Ni-NTA) The clone thus obtained was completely sequenced to ensure the absence of mutations compared to the starting clone, which could have been introduced during the PCR amplification. Plasmid pQE30-oc1, 3GT was introduced into E. coli M15 and the recombinant protein produced in vitro by induction of transcription with IPTG.After purification, α1,3GT was dialysed in PBS and injected into a BALB mouse. Each spleen was collected and fused with the SP2-O mouse hybridoma, at a rate of 3 injections of 40 μg every 15 days.The selection of hybridomas secreting an immunoglobulin recognizing α1,3GT was performed by ELISA test: plates of m Microtitration was ice-cold with 10 microgram / ml recombinant ce1,3GT protein, 16h at 4 ° C at pH 9.5, then saturated 2h, 37 ° C with PBS-BSA1%, 0.5% Tween 20. Hybridoma supernatants were incubated 1 h, 37 C and immunoabsorbed antibodies were revealed with peroxidase-labeled anti-Ig. Hybridomas providing an optical density at least three times higher than the background noise were retained.

IV - Cloning of ScFv antibodies (single chain Fv) from hybridomas
Cloning fragments of immunoglobulins into fragments
ScFv is described in Clackson et al. (1991)). Briefly, RNA is prepared from 5x108 hybridoma cells, and the messenger RNA is selected on oligo (dT) -cellulose. A first strand of cDNA is synthesized as follows: 10 μg of mRNA is incubated with 20 pmoles of VHlFOR or VKlFOR oligos (Clackson et al., 1991), 250 M of each dNTP, 10 m M DTT, 100 m M Tris.HCl (pH8.3), 10 mM MgCl2, and 140 mM KCl, 10 min. at 70 ° C, then 1h at 42 ° C in the presence of 50 units of Reverse Transcriptase.
PCR of the VH and VL fragments was performed by mixing 2 μl RT reaction with 25 pmoles of the VHlFOR or VKlFOR primers, and VHlBACK or VKlBACK (Clackson et al., 1991), 250 M of each dNTP, 67 mM Tris.HCl. (pH 8.8), 17 mM (NH4) 2SO4, 10 mM MgCl2, and 2 units Taq polymerase, for 30 rounds of amplification. The fragments obtained are then purified and 1 .mu.g of each are mixed with 300 ng of linker (gly4Ser) 3, and amplified by 7 cycles of PCR intended to bind the fragments. The linked VH and VL fragments are then amplified in 20 cycles with the VHlBACK oligos and
VK4FOR. The fragments obtained are then re-amplified with the oligos VHlBACK-BamH1 and VK4FOR-BamH1, digested with BamH1 and cloned into the eukaryotic expression vector PCDAAS, under the control of the CMV promoter.

V -Voies of intracellular action of anti-al, 3GT antibodies
The molecular mechanism by which an antibody expressed in a cell interferes with the function of a molecule carrying the antigenic site against which that antibody is expressed is unclear. However, several explanations have been proposed. In the case of inhibition of HIV reverse transcriptase (Maciejewski et al., 1995), the anti-RT antibody is not neutralizing in an in vitro test. The authors propose that the antigen / antibody complex formed in the cell may sterically block the movement of the enzyme along the RNA molecule, or may modify its secondary structure. In the case of IL-2 receptor a-chain inhibition (Richardson et al., 1995), a degradation process of the antigen / antibody complex has been proposed. This process appears non-lysosomal because methionine methyl ester (a lysosomal inhibitor) does not cause accumulation of the complex. Degradation does not occur in the Golgian compartment either because the brefeidin A (which destroys the Golgi) has no effect. Degradation occurs therefore early, probably at the level of the endoplasmic reticulum. In several cases where the intracellular localization of the target molecule conditions its function, it is possible for the intracellular antibody to retain this molecule in a compartment which is foreign to it.

Richardson et al. have indeed shown that the expression of an anti-RT antibody, retained in the endoplasmic reticulum by incorporation of a retention sequence KDEL, retains the RT in this compartment.

 In the case of inhibition of the oci, 3GT by ScFv antibodies, it can be assumed that: 1) either the antibodies modulate the activity of the enzyme (for example by modifying its structure), 2) or they retain in an intracellular compartment other than trans-Golgi (compartment in which the galactose branching at a1,3 on galactose of N-acelyllactosamine is carried out), or 3) they cause the degradation of the antigen / antibody complex by a mechanism elucidated. It could be, for example, ubiquitination followed by degradation by the proteasome complex.

VI - Construction of transgenic animals with proposed ScFvs
Obtaining transgenic animals for an anti-al, 3GT ScFv has research and clinical interests. For research, transgenesis in the rat is particularly suitable because its organs can be grafted in the monkey, where they undergo a rejection similar to xenograft rejection in the porcine-primate model For the therapeutic use of grafts, the pig is the animal of choice.

a) Transgenesis in the rat
Unicellular stage embryos are obtained from a super-ovulation protocol applied to CD-aged female rats aged 28 to 38 days (Armstrong and Opavoky, 1988). This protocol makes it possible to regularly obtain an average of 40 to 50 embryos per female. The construct carrying the cDNA encoding the anti-α1, 3GT antibody (two examples of constructs that can be used are shown in Figures 4 and 5) is injected at the rate of a few thousand copies per embryo. The treated embryos are then reimplanted into the matrix. Rats from these embryos are then tested to select animals that have actually incorporated transgenic DNA into their genome. These tests are performed at the level of DNA and RNA, which can be detected by hybridization with a DNA probe corresponding to the transgene, and at the protein level. The detection of the ScFv protein can be carried out by reaction with a human anti-Fab antibody, or by an anti-epitope antibody, if the ScFv cDNA is in fusion with a cDNA encoding a peptide carrying this epitope.

b) Transgenesis in pigs
Pronuclear-stage fertilized embryos are obtained by laparotomy from oviducts of mature sows aged 7 to 10 months, 50 hours after the induction of superovulation and 20 to 26 hours after insemination. Pronucleus, into which the DNA is injected, is visualized by centrifugation of the oocyte at 15,000 g for 3 minutes. The results after reimplantation of oocytes treated in this way show that 10% of them develop and give an animal, and that 15% of them have incorporated the transgenic DNA, ie 1.5% of the oocytes treated. Of these animals positive for DNA integration, 67% actually express the protein corresponding to the transgene (White et al., 1994, Cozzi and White, 1995).

Legends of the figures
- Figure 1:
Analysis of transcript sequences of the isoforms of the porcine 3GT, and alignment on a murine primary transcript map. The isoforms 1, 3 and 4 cDNAs were cloned from PCR-amplified, retrotranscripts mRNAs from PAEC and the cDNA corresponding to isoform 2 was cloned from an LLC-pK1 library as described. above. The nucleotide sequence was determined using Sequenase (USB), and compared by control with sequences corresponding to Isoforms 1 and 2 described by
Strahan et al., 1995 and Sandrin et al., 1994. Numbers in bold on the left side of the figure corresponding to isoforms 1 to 4. The first bases of exons 5, 6 and 7 are numbered on the sequence corresponding to 1 isoform 1.

Figures in italics indicate the number of missing bases in the corresponding spliced exon. The primary mouse transcription map with its 9 exons, as described by Joziasse et al., 1992, is compared with the sequences of isoforms 1 to 4.

 The cytoplasmic tail (black box) and the transmembrane region (boxed with vertical lines) are coded by exon 4. The regions of origin (gray box) include exons 5 and 6. Exons 5 and 6 are spliced alternately in the mouse and the pig. The catalytic domain is coded by exons 7, 8 and 9.

- Figure 2:
Reactivity measured by ELISA of unpurified preparations of phages corresponding to 15 clones recognizing the al, 3GT protein: Ninety-six individual colonies of TG1 bacteria containing a pHEN-1-ScFv clone, preselected by an enrichment procedure described in Nissim et al. ., 1994, were cultured and their supernatant (containing phages expressing the antibody) tested in ELISA as described in Materials and Methods above.

The positive supernatants (numbered 1 to 15) are shown in abscissa in the figure. The negative control (-) consists of a blank and the positive control (+) by the measurement of the optical density obtained by the reaction of an M13 phage (Pharmacia) on an anti-phage M13 mouse IgG (Pharmacia) deposited on the plate. The optical density (OD) values are indicated on the ordinate.

- Figure 3
Reactivity measured by ELISA purified phage corresponding to 6 clones recognizing the a1,3GT protein: The six most positive clones in the above ELISA (Fig.2) were amplified for the preparation of purified phage.

The purified and concentrated phages were re-tested in ELISA, similar to that described in Figure 2. These phages were tested at a dilution of 1 to 1/8. The dilutions made are represented in abscissa, and the measured optical density (OD) is indicated on the ordinate. the curve corresponding to the positive control passes through points represented by crosses, that corresponding to the phage containing ScFv1 (M13-ScFv1) passes through points represented by a black dot in the center of white squares, that corresponding to the phage containing ScFv2 (M13- ScFv2) passes through points represented by black diamonds, that corresponding to the phage containing ScFv3 (M13-ScFv3) passes through points represented by points represented by a white dot in the center of black squares, that corresponding to the phage containing ScFv4 (M13
ScFv4) passes through points represented by white diamonds, that corresponding to the phage containing ScFv5 (M13-ScFv5) passes through points represented by black squares, that corresponding to the phage containing ScFv6 (M13-ScFv6) passes through points represented by white squares, white being represented by a black circle.

- Figure 4
This construction example consists of the use of the promoter of the
MHC-1 of H-2Kb mice, allowing the insertion of the transgene (0.7Kb ScFv cDNA) between the promoter part proper (H-2kb promoter, 4Kb) and a series of introns / exons (5.5 Kb) belonging to the H-2Kb gene, this series being followed by SV40 pA of 0.3 Kb. These introns contain regulatory sequences which make it possible to obtain a good activity of the promoter, and therefore a good expression of the transgene. The H-2Kb promoter allows constitutive and ubiquitous expression of the cDNA whose expression it controls (Kimura et al., 1986, Byrne et al., 1995).

- Figure 5
This construction example consists of the use of the minigene DAF (Cozzi et al., 1996), whose efficiency has already been demonstrated in transgenesis in pigs. In a similar way to the H-2Kb construct, this minigene places the cDNA
ScFv (0.7 Kb) under the control of the DAF promoter, itself controlled by intronic sequences (the group consisting of the DAF promoter, the exons and the introns representing 4.6 Kb), the ScFv cDNA being followed by pA
SV40 of 0.3 Kb.

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(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: PatentIn Release # 1.0, Version 1.25 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 1128 base pairs
(B) TYPE: nucleic acid
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) MOLECULE TYPE: mRNA cDNA
(ix) ADDITIONAL CHARACTERISTIC:
(A) NAME / KEY: CDS
(B) LOCATION: 1..1128
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: AAT TCG AAA ATA ATG AAT GTC AAA GGA AGA GTG GTT CTG TCA ATG CTG 48
Asn Ser Lys Met Asn Val Lys Gly Arg Valley Val Val Leu Ser Met Leu
1 5 10 15
CTT GTC ACT TCA GTA ATG GTT GTG TT TGG GAA TAC ATC AAC AGC CCA 96
Leu Val Ser Thr Val Met Val Val Phe Trp Glu Tire Ile Asn Ser Pro
GAAT GGT TCT TTG TTC TGG ATA TAC CAG TCA AAA AAC CCA GAA GTT GGC 144
Glu Gly Ser Leu Phe Trp Tire Island Gln Ser Lys Asn Glu Val Gly
35 40 45
AGC AGT GCT CAG AGG GGC TGG TGG TTT CCG AGC TGG TTT AAC AAT GGG 192
Ser Ser Ala Gln Arg Gly Trp Trp Ser Phe Pro Trp Phe Asn Asn Gly
50 55 60
ACT CAC AGT TAC CAC GAA GAA GAA GAC GCT ATA GGC AAC GAA AAG GAA 240
Thr His Ser Tyr Glu Glu Glu Glu Asp Ala Gly Asn Glu Glu Glu
65 70 75 80 CAA AGA ARA GAA GAC AAC AGA GGA GAG CTT CCA CTA GTG GAC TGG TT 288 Gln Arg Glu Asp Lys Asn Arg Gly Glu Pro Leu Leu Val Asp Trp Phe
85 90 95 AAT CCT GAG AAA CGC CCA GAG GTC GTG ACC ATA ACC AGM TGG AAG GCT 336
Asn Pro Glu Arg Lys Arg Glu Val Thr Val Thr Thr Arg Trp Lys Ala
100 105 110
CCA GTG GTA TGG GAA GGC ACT TAC AAC AGM GCC GTC TTA GAT AAT TAT 384
Pro Val Val Trp Glu Gly Thr Tyr Asn Arg Ala Val Leu Asp Asn Tyr
115 120 125
TAT GCC AAA CAG ARA ATT ACC GTG GGC TTG ACG GTT CTT GCT GTC GGA 432
Tyr Ala Lys Gln Lys Thr Island Gly Val Leu Thr Val Leu Ala Val Gly
130 135 140
AGA TAC ATT GAG TAC CAT TTG GAG GAG TTC TTA ATA TCT GCA AAT ACA 480
Arg Tyr Isle Glu His Tyr Leu Glu Glu Phe Leu Isle Ser Ala Asn Thr 145 150 155 160
TAC TTC ATG GTT GGC CAC ARA GTC ATC TTT ATC ATC ATG GTG GAT GAT 528
Tyr Phe Met Val Gly His Lys Val Ile Phe Tyr Ile Met Val Asp Asp Asp
165 170 175
ATC TCC AGG ATG CCT TTG ATA GAG CTG GGT CCT CTG CGC TCC TTT ARA 576
Ile Ser Arg Met Pro Leu Ile Glu Leu Gly Pro Leu Arg Ser Phe Lily
180 185 190
GTG TTT GAG ATC AAG TCC GAG AAG AGG TGG CAA GAC ATC AGC ATG ATG 624
Val Phe Glu Island Lys Ser Glu Lys Arg Trp Gln Asp Ser Island Met Met
195 200 205
CGC ATG AAG ACC ATC GGG GAG CAC ATC CTG GCC CAC ATC CAG CAC GAG 672
Arg Met Lys Thr Ile Gly Glu His Ile Leu Ala His Gln His Glu Island
210 215 220
GTG GAC TTC CTC TTC TGC ATG GAC GTG GAT CAG GTC TTC CAA AAC AAC 720
Val Asp Phe Leu Phe Cys Met Asp Asp Val Gln Val Phe Gln Asn Asn 225 230 235 240
TTT GGG GTG ACC GAG CT GGC CAG TCG GTG GCT CTA CAG CAG GCC TGG 768
Phe Gly Val Glu Thr Leu Gly Gln Ser Val Ala Gln Leu Gln Ala Trp
245 250 255
TGG TAC AAG GCA CAT CCT GAC GAG TTC ACC TAC AGG AGG CGG AAG GAG 816
Trp Tyr Lys Ala His Asp Asp Glu Phe Thr Tyr Glu Arg Arg Lys Glu
260,265,270
TCC GCA GCC TAC ATT CCG TT GGC CAG GGG TAT GAT TAT TAC CAC GCA 864
Ser Ala Ala Tire Pro Island Phe Gly Gln Gly Asp Phe Tire Tyr His Ala
275 280 285
GCC ATT TT GGG GGA ACA CCC ACT CAG GTT CTA AAC ATC ACT CAG GAG 912
Ala Island Phe Gly Gly Thr Pro Thr Gln Val Leu Asn Thr Thr Gln Glu
290,295,300
TGC TTC AAG GGA ATC CTC CAG GAC AAG GAA AAT GAC ATA GAA GCC GAG 960
Cys Phe Lys Gly Island Leu Gln Asp Lys Glu Asp Aspn Glu Ala Glu 305 310 315 320
TGG CAT GAT GAA AGC CAT CTA AAC AAG TAT TTC TTC CTC AAC ARA CCC 1008
Trp His Asp Glu Ser His Leu Asn Lys Tyr Phe Leu Leu Asn Lys Pro
325 330 335
ACT AAA ATC TTA TCC CCA GAA TAC TGC TGG CAT TAT CAT ATA GGC ATG 1056
Thr Lys Island Leu Ser Pro Glu Tire Cys Trp Asp Tire His Island Gly Met
340,345,350
TCT GTG GAT ATT AGG ATT GTC AAG ATA GCT TGG CAG ARA AAA GAG TAT 1104
Ser Val Asp Ile Arg Ile Val Lys Ile Ala Trp Gln Lilies Lilies Glu Tyr
355 360 365 AAT TTG GTT AGA AAT ATC ATC TG 1128
Asn Leu Val Arg Asn Asn Island
370,375
(2) INFORMATION FOR SEQ ID NO: 2:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 375 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
Asn Ser Lys Met Asn Val Lys Gly Arg Valley Val Val Leu Ser Met Leu
1 5 10 15
Leu Val Ser Thr Val Met Val Val Phe Trp Glu Tire Ile Asn Ser Pro
20 25 30
Glu Gly Ser Leu Phe Trp Tire Island Gln Ser Lys Asn Glu Val Gly
35 40 45
Ser Ser Ala Gln Arg Gly Trp Trp Ser Phe Pro Trp Phe Asn Asn Gly
50 55 60
Thr His Ser Tyr Glu Glu Glu Glu Asp Ala Gly Asn Glu Glu Glu
65 70 75 80 Gln Arg Lys Glu Asp Asn Arg Gly Glu Leu Pro Leu Val Asp Trp Phe
85 90 95
Asn Pro Glu Arg Lys Arg Glu Val Thr Val Thr Thr Arg Trp Lys Ala
100 105 110
Pro Val Val Trp Glu Gly Thr Tyr Asn Arg Ala Val Leu Asp Asn Tyr
115 120 125
Tyr Ala Lys Gln Lys Thr Island Gly Val Leu Thr Val Leu Ala Val Gly
130 135 140
Arg Tyr Isle Glu His Tyr Leu Glu Glu Phe Leu Isle Ser Ala Asn Thr 145 150 155 160
Tyr Phe Met Val Gly His Lys Val Ile Phe Tyr Ile Met Val Asp Asp Asp
165 170 175
Ile Ser Arg Met Pro Leu Ile Glu Leu Gly Pro Leu Arg Ser Phe Lily
180 185 190
Val Phe Glu Island Lys Ser Glu Lys Arg Trp Gln Asp Ser Island Met Met
195 200 205
Arg Met Lys Thr Ile Gly Glu His Ile Leu Ala His Gln His Glu Island
210 215 220
Val Asp Phe Leu Phe Cys Met Asp Asp Val Gln Val Phe Gln Asn Asn 225 230 235 240
Phe Gly Val Glu Thr Leu Gly Gln Ser Val Ala Gln Leu Gln Ala Trp
245 250 255
Trp Tyr Lys Ala His Asp Asp Glu Phe Thr Tyr Glu Arg Arg Lys Glu
260,265,270
Ser Ala Ala Tire Pro Island Phe Gly Gln Gly Asp Phe Tire Tyr His Ala
275 280 285
Ala Island Phe Gly Gly Thr Pro Thr Gln Val Leu Asn Thr Thr Gln Glu
290,295,300
C (2) INFORMATION FOR SEQ ID NO: 3:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 1092 base pairs
(B) TYPE: nucleic acid
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) MOLECULE TYPE: mRNA cDNA
(ix) ADDITIONAL CHARACTERISTIC:
(A) NAME / KEY: CDS
(B) LOCATION: 1..1092
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: AAT TCG AAA ATA ATG AAT GTC AAA GGA AGA GTG GTT CTG TCA ATG CTG 48
Asn Ser Lys Met Asn Val Lys Gly Arg Valley Val Val Leu Ser Met Leu
1 5 10 15
CTT GTC TCA ACT GTA ATG GTT GTG TT TGG GAA TAC ATC AAC AGA AAC 96
Leu Val Ser Thr Val Met Val Val Phe Trp Glu Tire Ile Asn Arg Asn
20 25 30
CCA GAA GTT GGC AGC AGT GCT AG AGG GGC TGG TGG AG CCG AGC TGG 144
Pro Glu Val Gly Ser Ser Ala Gln Arg Gly Trp Trp Ser Phe Pro Trp
35 40 45
TTT AAC AAT GGG ACT CAC AGT TAC CAC GAA GAA GAA GAC GCT ATA GGC 192
Phe Asn Asn Gly Thr His Ser Tyr His Glu Glu Glu Asp Ala Gly Island
50 55 60
ARC GAA GAA GAA CAA AGA AAA GAC GAA GAC AGA GGA GAG CTT CCA CTA 240
Asn Glu Lys Glu Gln Arg Lys Glu Asp Asn Arg Gly Glu Leu Pro Leu
65 70 75 80
GTG GAC TGG TT AAT CCT GAG ARA CGC CCA GAG GTC GTG ACC ATA ACC 288
Val Asp Trp Phe Asn Pro Glu Lys Arg Glu Pro Val Val Thr Thr Thr
85 90 95
AGA TGG AAG GCT CCA GTG GTA TGG GAA GGC ACT TAC AAC AGA GCC GTC 336
Arg Trp Lys Ala Pro Val Val Trp Glu Gly Thr Tyr Asn Arg Ala Val
100 105 110
TTA GAT AAT TAT TAT GCC AAA CAG AAA ATT ACC GTG GGC TTG ACG GTT 384
Leu Asp Asn Tire Tyr Ala Lys Gln Lys Thr Island Val Gly Leu Thr Val
115 120 125
CTT GCT GTC GGA AGA CAT GAG CAT TAC TTG GAG GAG TTC TTA ATA 432
Leu Ala Val Gly Arg Tire Glu Island His Tyr Leu Glu Glu Phe Leu Ile
130 135 140
TCT GCA AAT ACA TTC TAC ATG GTT GGC CAC AAA GTC ATC TTT TAC ATC 480
Ser Ala Asn Thr Tyr Phe Met Val Gly His Lys Val Ile Phe Tyr Ile 145 150 155 160
ATG GTG GAT GAT ATC ATG CAG ATG CCT TTG ATAG GAG CTG GGT CCT CTG 528
Met Asp Val Asp Asp Ser Arg Arg Pro Leu Leu Glu Leu Gly Pro Leu
165 170 175
CGC TCC TT AAA GTG TTT GAG ATC AAG TCC GAG AAG AGG TGG CAA GAC 576
Arg Ser Phe Lys Val Phe Glu Ile Lys Ser Glu Lys Arg Trp Gln Asp
180 185 190
ATC AGC ATG ATG CGC ATG AAG ACC ATC GGG ATC GAC ATC CTG GCC CAC 624
Ser Is Met Met Arg Met Lys Thr Ile Gly Glu His Ile Leu Ala His
195 200 205
ATC CAG CAC GAG GTG GAC TTC CTC TTC TGC ATG GAC GTG GAT CAG GTC 672
Gln Island His Glu Val Asp Asp Phe Leu Phe Cys Asp Asp Val Gln Val
210 215 220
VAT included CAA AAC AAC TT GGG GTG GAG ACC CTG GGC CAG TCG GTG GCT CAG 720
Phe Gln Asn Asn Phe Gly Glu Val Thr Leu Gly Gln Val Ala Gln 225 230 235 240
CTA CAG GCC TGG TGG TAC AAG GCA CAT CCT GAC GAG TTC ACC TAC GAG 768
Leu Gln Ala Trp Trp Tyr Lys Ala His Asp Asp Glu Phe Thr Tyr Glu
245 250 255
AGG CGG AAG GAG TCC GCA GCC TAC ATT CCG TTT GGC CAG GGG GAT TTT 816
Arg Arg Lys Glu Ser Ala Ala Tire Pro Island Phe Gly Gln Gly Asp Phe
260,265,270
TAT TAC TAC CAC GCA GCC AT ATT GGG GGA ACA CCC ACT CAG GTT CTA AAC 864
Tyr Tire His Ala Ala Island Phe Gly Gly Thr Pro Thr Gln Val Leu Asn
275 280 285
ATC ACT CAG GAG TGC TTC AAG GGA ATC CTC CAG GAC AAG GAA AAT GAC 912
Thr Gln Island Glu Cys Phe Lys Gly Island Leu Gln Asp Lily Glu Asn Asp
290,295,300
ATA GAA GCC GAG TGG CAT GAT AGC GAA CAT CTA AAC AAG TAT TTC CTT 960
Ile Glu Ala Glu Trp His Asp Glu Ser His Leu Asn Lys Tyr Phe Leu 305 310 315 320
CTC AAC AAA CAC ACT AAA ATC TTA TCC CCA GAA TAC TGC TGG GAT TAT 1008
Leu Asn Lys Pro Thr Lys Island Leu Ser Pro Glu Tyr Cys Trp Asp Tyr
325 330 335
CAT ATA GGC ATG TCT GTG GAT ATT AGG ATT GTC AAG ATA GCT TGG CAG 1056
His Island Gly Met Ser Val Asp Ile Arg Ile Val Lys Ile Ala Trp Gln
340,345,350
AAA AAA GAG TAT AAT TTG AGA AGT AAT AAC ATC TG 1092
Lys Glou Tyr Lys Asn Leu Val Arg Asn Asn Ile
355 360 (2) INFORMATION FOR SEQ ID NO: 4:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 363 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
Asn Ser Lys Met Asn Val Lys Gly Arg Valley Val Val Leu Ser Met Leu
1 5 10 15
Leu Val Ser Thr Val Met Val Val Phe Trp Glu Tire Ile Asn Arg Asn
20 25 30
Pro Glu Val Gly Ser Ser Ala Gln Arg Gly Trp Trp Ser Phe Pro Trp
35 40 45
Phe Asn Asn Gly Thr His Ser Tyr His Glu Glu Glu Asp Ala Gly Island
50 55 60
Asn Glu Lys Glu Gln Arg Lys Glu Asp Asn Arg Gly Glu Leu Pro Leu
65 70 75 80
Val Asp Trp Phe Asn Pro Glu Lys Arg Glu Pro Val Val Thr Thr Thr
85 90 95
Arg Trp Lys Ala Pro Val Val Trp Glu Gly Thr Tyr Asn Arg Ala Val
100 105 110
Leu Asp Asn Tire Tyr Ala Lys Gln Lys Thr Island Val Gly Leu Thr Val
115 120 125
Leu Ala Val Gly Arg Tire Glu Island His Tyr Leu Glu Glu Phe Leu Ile
130 135 140
Ser Ala Asn Thr Tyr Phe Met Val Gly His Lys Val Ile Phe Tyr Ile 145 150 155 160
Met Asp Val Asp Asp Ser Arg Arg Pro Leu Leu Glu Leu Gly Pro Leu
165 170 175
Arg Ser Phe Lys Val Phe Glu Ile Lys Ser Glu Lys Arg Trp Gln Asp
180 185 190
Ser Is Met Met Arg Met Lys Thr Ile Gly Glu His Ile Leu Ala His
195 200 205
Gln Island His Glu Val Asp Asp Phe Leu Phe Cys Asp Asp Val Gln Val
210 215 220
Phe Gln Asn Asn Phe Gly Glu Val Thr Leu Gly Gln Val Ala Gln 225 230 235 240
Leu Gln Ala Trp Trp Tyr Lys Ala His Asp Asp Glu Phe Thr Tyr Glu
245 250 255
Arg Arg Lys Glu Ser Ala Ala Tire Pro Island Phe Gly Gln Gly Asp Phe
260,265,270
Tyr Tire His Ala Ala Island Phe Gly Gly Thr Pro Thr Gln Val Leu Asn
275 280 285
Thr Gln Island Glu Cys Phe Lys Gly Island Leu Gln Asp Lily Glu Asn Asp
290,295,300
Ile Glu Ala Glu Trp His Asp Glu Ser His Leu Asn Lys Tyr Phe Leu 305 310 315 320
Leu Asn Lys Pro Thr Lys Island Leu Ser Pro Glu Tyr Cys Trp Asp Tyr
325 330 335
His Island Gly Met Ser Val Asp Ile Arg Ile Val Lys Ile Ala Trp Gln
340,345,350
Lys Glou Tyr Lys Asn Leu Val Arg Asn Asn Ile
355,360
(2) INFORMATION FOR SEQ ID NO: 5:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 1065 base pairs
(B) TYPE: nucleic acid
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) MOLECULE TYPE: mRNA cDNA
(ix) ADDITIONAL CHARACTERISTIC:
(A) NAME / KEY: CDS
(B) LOCATION: 1..1065
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: AAT TCG AAA ATA ATG AAT GTC AAA GGA AGA GTG GTT CTG TCA ATG CTG 48
Asn Ser Lys Met Asn Val Lys Gly Arg Valley Val Val Leu Ser Met Leu
1 5 10 15
CTT GTC ACT TCA GTA ATG GTT GTG TT TGG GAA TAC ATC AAC AGC CCA 96
Leu Val Ser Thr Val Met Val Val Phe Trp Glu Tire Ile Asn Ser Pro
GAAT GGT TCT TTG TTC TGG ATA TAC CAG TCA AGA ACT CAC AGT TAC CAC 144
Glu Gly Ser Leu Phe Trp Tyr Island Gln Ser Lys Thr His Ser Tyr His
35 40 45 GAA GAA GAA GAC GCT ATA GGC AAC GAA GAA GAA CAA AGA AAA GAA GAC 192
Glu Glu Glu Asp Ala Gly Asn Glu Glu Glu Arg Glu Asp Glu Asp Glu
50 55 60
ARC AGA GGA GAG CTT CCA CTA GTG GAC TGG TTT AAT CCT GAG AAA CGC 240
Asn Arg Gly Glu Pro Leu Leu Val Asp Trp Ashe Phe Pro Glu Lys Arg
65 70 75 80
CCA GAG GTC GTG ACC ATA ACC AG TGG AAG GCT CCA GTG GTA TGG GAA 288
Pro Glu Val Thr Val Thr Island Thr Trp Lys Ala Pro Val Val Trp Glu
85 90 95
GGC ACT TAC AAC AGA GCC GTC TTA GAT TAT AAT TAT GCC ARA CAG ARA 336
Gly Thr Tyr Asn Arg Ala Val Leu Asp Asn Tire Tyr Ala Lys Gln Lys
100 105 110
ATT ACC GTG GGC TTG ACG GTT CTT GCT GTC GGA AGA TAC ATT GAG CAT 384
Thr Island Thr Val Gly Leu Thr Al Leu Val Val Gly Arg Tyr Glu His Island
115 120 125
TAC TTG GAG GAG TTC TTA ATA TCT GCA AAT ACA TAC TTC ATG GTT GGC 432
Tyr Leu Glu Glu Phe Leu Ser Ala Asn Thr Island Tyr Phe Met Val Gly
130 135 140
CAC AAA GTC ATC ATC TAC ATC ATG GTG ATC ATC ATG ATG CCT 480
His Lys Val Isle Phe Tyr Island Met Asp Val Asp Asp Ser Arg Met Pro 145 150 155 160
TTG ATA GAG CTG GGT CCT CTG CGC TCC TT ARA GTG TT GAG ATC AAG 528
Leu Ile Glu Leu Gly Pro Leu Arg Ser Phe Lily Val Phe Glu Ile Lys
165 170 175
TCC GAG AAG AGG TGG CAA GAC ATC AGC ATG ATG CGC ATG AAG ACC ATC 576
Ser Glu Lys Arg Trp Gln Asp Ser Island Met Met Arg Met Lys Thr Ile
180 185 190
GGG GAG CAC ATC CTG GCC CAC ATC CAG CAC GAG GTG GAC TTC CTC TTC 624
Gly Glu His Island Leu Ala His Gln Island His Glu Val Asp Phe Phe Phe
195 200 205
TGC ATG GAC GTG GAT CAG GTC TTC CAA AAC AAC TT GGG GTG GAG ACC 672
Cys Met Asp Asp Val Gln Gln Val Phe Gln Asn Phe Gly Val Glu Thr
210 215 220
CTG GGC CAG TCG GTG GCT CAG CTA CAG GCC TGG TGG TAC AAG GCA CAT 720
Leu Gly Gln Ser Val Ala Gln Gln Ala Trp Trp Tyr Lys Ala His 225 230 235 240
CCT GAC GAG TTC ACC TAC GAG AGG CGG AAG GAG TCC GCA GCC TAC ATT 768
Pro Asp Glu Phe Thr Tyr Glu Arg Arg Lys Glu Ser Ala Ala Tyr Ile
245 250 255
CCG TT GGC CAG GGG GAT TT TAT TAC CAC GCA GCC ATT TT GGG GGA 816
Pro Phe Gly Gln Gly Asp Phe Tire Tyr His Ala Ala Phe Gly Gly
260,265,270
ACA CCC ACT CAG GTT CTA AAC ATC ACT CAG GAG TGC TTC AAG GGA ATC 864
Thr Pro Thr Gln Val Leu Asn Thr Thrn Gln Cys Phe Lys Gly Ile
275 280 285
CTC CAG GAC AAG GAA AAT GAC ATA GAA GCC GAG TGG CAT GAT GAA AGC 912
Leu Gln Asp Lys Glu Asp Aspn Glu Glu Ala Glu Trp His Asp Glu Ser
290,295,300
CAT CTA ARC AAG TAT TTC TTC CTC AAC AAA CCC ACT AAA ATC TTA CBT 960
His Leu Asn Lys Tyr Phe Leu Leu Asn Lys Pro Thr Lys Island Leu Ser 305 310 315 320
CCA GAA TAC TGC TGG GAT TAT CAT ATG GGC ATG TCT GTG GAT ATT AGG 1008
Pro Glu Tyr Cys Trp Asp Tire His Island Gly Met Ser Val Asp Island Arg
325 330 335
ATT GTC AAG ATA GCT TGG AGC AAA AAA AGG TAT AAT AGG GTT AGA AAT 1056
Ile Val Lys Ala Island Trp Gln Lys Glu Tyr Lys Asn Leu Val Arg Asn
340 345 350 AAC ATC TG 1065
Asn Island
355
(2) INFORMATION FOR SEQ ID NO: 6:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 354 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:
Asn Ser Lys Met Asn Val Lys Gly Arg Valley Val Val Leu Ser Met Leu
1 5 10 15
Leu Val Ser Thr Val Met Val Val Phe Trp Glu Tire Ile Asn Ser Pro
20 25 30
Glu Gly Ser Leu Phe Trp Tyr Island Gln Ser Lys Thr His Ser Tyr His
35 40 45
Glu Glu Glu Asp Ala Gly Asn Glu Glu Glu Arg Glu Asp Glu Asp Glu
50 55 60
Asn Arg Gly Glu Pro Leu Leu Val Asp Trp Ashe Phe Pro Glu Lys Arg
65 70 75 80
Pro Glu Val Thr Val Thr Island Thr Trp Lys Ala Pro Val Val Trp Glu
85 90 95
Gly Thr Tyr Asn Arg Ala Val Leu Asp Asn Tire Tyr Ala Lys Gln Lys
100 105 110
Thr Island Thr Val Gly Leu Thr Al Leu Val Val Gly Arg Tyr Glu His Island
115 120 125
Tyr Leu Glu Glu Phe Leu Ser Ala Asn Thr Island Tyr Phe Met Val Gly
130 135 140
His Lys Val Isle Phe Tyr Island Met Asp Val Asp Asp Ser Arg Met Pro 145 150 155 160
Leu Ile Glu Leu Gly Pro Leu Arg Ser Phe Lily Val Phe Glu Ile Lys
165 170 175
Ser Glu Lys Arg Trp Gln Asp Ser Island Met Met Arg Met Lys Thr Island
180 185 190
Gly Glu His Island Leu Ala His Gln Island His Glu Val Asp Phe Phe Phe
195 200 205
Cys Met Asp Asp Val Gln Gln Val Phe Gln Asn Phe Gly Val Glu Thr
210 215 220
Leu Gly Gln Ser Val Ala Gln Gln Ala Trp Trp Tyr Lys Ala His 225 230 235 240
Pro Asp Glu Phe Thr Tyr Glu Arg Arg Lys Glu Ser Ala Ala Tyr Ile
245 250 255
Pro Phe Gly Gln Gly Asp Phe Tire Tyr His Ala Ala Phe Gly Gly
260,265,270
Thr Pro Thr Gln Val Leu Asn Thr Thrn Gln Cys Phe Lys Gly Ile
275 280 285
Leu Gln Asp Lys Glu Asp Aspn Glu Glu Ala Glu Trp His Asp Glu Ser
290,295,300
His Leu Asn Lys Tyr Phe Leu Leu Asn Lys Pro Thr Lys Island Leu Ser 305 310 315 320
Pro Glu Tyr Cys Trp Asp Tire His Island Gly Met Ser Val Asp Island Arg
325 330 335
Ile Val Lys Ala Island Trp Gln Lys Glu Tyr Lys Asn Leu Val Arg Asn
340,345,350
Asn Island
(2) INFORMATION FOR SEQ ID NO: 7:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 1029 base pairs
(B) TYPE: nucleic acid
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) MOLECULE TYPE: mRNA cDNA
(ix) ADDITIONAL CHARACTERISTIC:
(A) NAME / KEY: CDS
(B) LOCATION: 1..1029
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: AAT TCG AAA ATA ATG AAT GTC AAA GGA AGA GTG GTT CTG TCA ATG CTG 48
Asn Ser Lys Met Asn Val Lys Gly Arg Valley Val Val Leu Ser Met Leu
1 5 10 15
CTT GTC ACT TCA GTA ATG GTT GTG TT TGG GAA TAC ATC AAC AGG ACT 96
Leu Val Ser Thr Val Met Val Val Phe Trp Glu Tire Ile Asn Arg Thr
20 25 30
CAC AGT TAC CAC GAA GAA GAC GAC GTA ATA GGC GAA GAA AAG GAA CAA 144
His Ser Tyr His Glu Asp Glu Glu Ala Gly Asn Glu Glu Gln Gln
35 40 45
AGA AAA GAA GAC AAC AGA GGA GAG CTT CCA CTA GTG GAC TGG TTT AAT 192
Arg Lys Glu Asp Asn Arg Gly Glu Leu Pro Leu Val Asp Trp Phe Asn
50 55 60
CCT GAG ARA CGC CCA GAG GTC GTG ACC ATA ACC AGM TGG AAG GCT CCA 240
Pro Glu Lys Arg Glu Pro Val Val Thr Thr Thr Arg Trp Lys Ala Pro
65 70 75 80
GTG GTA TGG GAA GGC ACT TAC AAC AGM GCC GTC TTA GAT AAT TAT TAT 288
Val Val Trp Glu Gly Thr Tyr Asn Arg Ala Val Leu Asp Asn Tire Tyr
85 90 95
GCC AAA CAG AAA ACC ACC GTG GGC TTG ACG GTT CTT GCT GTC GGA AGA 336
Ala Lys Gln Lys Thr Island Val Gly Leu Thr Al Leu Val Val Gly Arg
100 105 110
TAC ATT GAG TAC CAT TTG GAG GAG TTC TTA ATA TCT GCA AAT ACA TAC 384
Tyr Isle Glu His Tyr Glu Glu Phe Leu Leu Ser Ser Ala Asn Thr Tyr
115 120 125
ATG GTT GGC CAC AAA GTC ATC ATC TAC ATC ATG GTG ATC GAT ATC 432
Phe Met Val Gly His Lys Val Ile Phe Tyr Ile Met Val Asp Asp Asp Ile
130 135 140
TCC AGG ATG CCT TTG ATA GAG CTG GGT CCT CTG CGC TCC TT AAA GTG 480
Ser Arg Met Pro Leu Ile Glu Leu Gly Pro Leu Arg Ser Phe Lys Val 145 150 155 160
TT GAG ATC AAG TCC GAG AAG AGG TGG CAA GAC ATC AGC ATG ATG CGC 528
Phe Glu Isle Lys Ser Glu Lys Arg Trp Gln Asp Ser Island Met Met Arg
165 170 175
ATG AAG ACC ATC GGG GAG CAC ATC CTG GCC CAC ATC CAG CAC GAG GTG 576
Met Lys Thr Ile Gly Glu His Ile Leu Ala His Gln Island His Glu Val
180 185 190
GAC TTC CTC TTC TGC ATG GAC GTG GAT CAG GTC TTC CAA AAC AAC TT 624
Asp Phe Leu Phe Cys Met Asp Asp Val Gln Gln Phe Gln Asn Asn Phe
195 200 205
GGG GTG GAG ACC CTG GGC CAG TCG GTG CAG GAG CTA CAG GCC TGG TGG 672
Gly Val Glu Thr Leu Gly Gln Val Al Gln Leu Gln Ala Trp Trp
210 215 220
TAC AAG GCA CAT CCT GAC GAG TTC ACC TAC AGG AGG CGG AAG GAG TCC 720
Tyr Lys Ala His Pro Glu Asp Asp Phe Thr Tyr Glu Arg Arg Lys Glu Ser 225 230 235 240
GCA GCC TAC ATT CCG TTT GGC CAG GGG GAT TTT TAT TAC CAC GCA GCC 768
Ala Ala Tire Pro Island Phe Gly Gln Gly Asp Phe Tire Tyr His Ala Ala
245 250 255
ATT TTT GGG GGA ACA CCC ACT CAG GTT CTA AAC ATC ACT CAG GAG TGC 816
Ile Phe Gly Gly Thr Thr Thr Gln Val Leu Asn Thr Thr Gln Glu Cys
260,265,270
AAG GGA ATC CTC CAG GAC AAG GAA AAT GAC ATA GAA GCC GAG TGG 864
Phe Lys Gly Island Leu Gln Asp Lily Glu Asn Asp Glu Island Ala Glu Trp
275 280 285
CAT GAT GAA AGC CTA CAT AAC AAG TAT TTC TTC CTC AAC AAA CCC ACT 912
His Asp Glu Ser His Leu Asn Lys Tyr Phe Leu Leu Asn Lys Pro Thr
290 295 300 AAA ATC TTA TCC CCA GAAT TAC TGC TGG CAT TAT CAT ATG GGC ATG TCT 960
Lilies Ile Leu Ser Pro Glu Tyr Cys Trp Asp Tire His Island Gly Met Ser 305 310 315 320
GTG GAT ATT AGG ATT GTC AAG ATA GCT TGG CAG ARA AAA GAG TAT AAT 1008
Val Asp Arg Island Val Lys Island Ala Island Trp Gln Lily Lys Glu Tyr Asn
325 330 335
TTG GTT AGA AAT ATC ATC TG 1029
Leu Val Arg Asn Asn Island
340
(2) INFORMATION FOR SEQ ID NO: 8:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 342 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:
Asn Ser Lys Met Asn Val Lys Gly Arg Valley Val Val Leu Ser Met Leu
1 5 10 15
Leu Val Ser Thr Val Met Val Val Phe Trp Glu Tire Ile Asn Arg Thr
20 25 30
His Ser Tyr His Glu Asp Glu Glu Ala Gly Asn Glu Glu Gln Gln
35 40 45
Arg Lys Glu Asp Asn Arg Gly Glu Leu Pro Leu Val Asp Trp Phe Asn
50 55 60
Pro Glu Lys Arg Glu Pro Val Val Thr Thr Thr Arg Trp Lys Ala Pro
65 70 75 80
Val Val Trp Glu Gly Thr Tyr Asn Arg Ala Val Leu Asp Asn Tire Tyr
85 90 95
Ala Lys Gln Lys Thr Island Val Gly Leu Thr Al Leu Val Val Gly Arg
100 105 110
Tyr Isle Glu His Tyr Glu Glu Phe Leu Leu Ser Ser Ala Asn Thr Tyr
115 120 125
Phe Met Val Gly His Lys Val Ile Phe Tyr Ile Met Val Asp Asp Asp Ile
130 135 140
Ser Arg Met Pro Leu Ile Glu Leu Gly Pro Leu Arg Ser Phe Lys Val 145 150 155 160
Phe Glu Isle Lys Ser Glu Lys Arg Trp Gln Asp Ser Island Met Met Arg
165 170 175
Met Lys Thr Ile Gly Glu His Ile Leu Ala His Gln Island His Glu Val
180 185 190
Asp Phe Leu Phe Cys Met Asp Asp Val Gln Gln Phe Gln Asn Asn Phe
195 200 205
Gly Val Glu Thr Leu Gly Gln Val Al Gln Leu Gln Ala Trp Trp
210 215 220
Tyr Lys Ala His Pro Glu Asp Asp Phe Thr Tyr Glu Arg Arg Lys Glu Ser 225 230 235 240
Ala Ala Tire Pro Island Phe Gly Gln Gly Asp Phe Tire Tyr His Ala Ala
245 250 255
Ile Phe Gly Gly Thr Thr Thr Gln Val Leu Asn Thr Thr Gln Glu Cys
260,265,270
Phe Lys Gly Island Leu Gln Asp Lily Glu Asn Asp Glu Island Ala Glu Trp
275 280 285
His Asp Glu Ser His Leu Asn Lys Tyr Phe Leu Leu Asn Lys Pro Thr
290,295,300
Lilies Ile Leu Ser Pro Glu Tyr Cys Trp Asp Tire His Island Gly Met Ser 305 310 315 320
Val Asp Arg Island Val Lys Island Ala Island Trp Gln Lily Lys Glu Tyr Asn
325 330 335
Leu Val Arg Asn Asn Island
340
(2) INFORMATION FOR SEQ ID NO: 9:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 708 base pairs
(B) TYPE: nucleic acid
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) MOLECULE TYPE: mRNA cDNA
(ix) ADDITIONAL CHARACTERISTIC:
(A) NAME / KEY: CDS
(B) LOCATION: 1.708
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9:
CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG CCT GGG GCC 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
TCA GTG AAG GTT TCC TGC AAG GCT TCT GGA TAC ACC TTC ACT AGC TAT 96
Ser Val Lys Ser Ser Cys Lys Ser Ala Ser Gly Tyr Ser Thr Threat
20 25 30
GCT ATG CAT TGG GTG CGC CAG GCC CCC GGA CAA AGG CTT GAG TGG ATG 144
Ala Met His Trp Arg Val Gln Ala Pro Arg Gly Gln Arg Leu Glu Trp Met
35 40 45
GGA TGG ATC AAC GCT GGC AAT GGT AAC ACA ARA TAT TCA AGC AAG TTC 192
Gly Trp Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe
50 55 60
CAG GGC AGA GTC ACC ATT ACC AGG GAC ACA TCC GCG AGC ACA GCC TAC 240 Gln Gly Arg Val Thr Thr Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
ATG GAG CTG AGC AGC CTG AGA TCT GAA GAC ACG GCC GTG TAT TAC TGT 288
Met Glu Leu Ser Ser Leu Arg Ser Asp Glu Asp Ala Val Tyr Tyr Cys
85 90 95
GCA AGA AGT GGT ATG TT TGG GGC CAA GGT ACC CTG GTC ACC GTG TCG 336
Ala Arg Ser Gly Met Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
AGA GGT GGA GGC GGT TCA GGC GGA GGT GGC TCT GGC GGT GGC GGA TCG 384
Arg Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
TCT GAG CTG ACT CAG GAC CCT GCT GTG TCT GTG GCC TTG GGA CAG ACA 432
Ser Glu Leu Thr Asp Asp Gln Ala Val Ser Ala Val Leu Gly Gln Thr
130 135 140
GTC AGG ATC ACA TGC CAA GGA GAC AGC CTC AGA AGC TAT TAT GCA AGC 480
Val Arg Thr Island Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser 145 150 155 160
TGG TAC CAG AGC AGC AGC GGA CAG AGC GTC CTM GTC AGC ATC TAT GGT 528
Trp Gln Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Tyr Gly
165 170 175 AAA AAC AAC CGG CCC TCA GGG ATC CCA GAC CGA TTC TCT GGC TCC AGC 576
Lys Asn Asn Arg Pro Ser Gly Pro Asp Arg Arg Ser Ser Gly Ser Ser
180 185 190
TCA GGA AAC ACA GCT TCC TTG ACC ATC ACT GGG GCT CAG GCG GAA GAT 624
Ser Gly Asn Thr Ala Ser Leu Thr Thr Gly Ala Gln Ala Glu Asp
195 200 205
GAG GCT GAC TAT TAC TGT AAC TCC CGG GAC AGC AGT GGT AAC CAT GTG 672
Glu Ala Asp Tyr Tire Cys Asn Ser Arg Asp Ser Ser Gly Asn His Val
210 215 220
GTA TTC GGC GGA GGG ACC AAG CTG ACC GTC CTA GGT 708
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 225 230 235
(2) INFORMATION FOR SEQ ID NO: 10:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 236 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ser Ser Cys Lys Ser Ala Ser Gly Tyr Ser Thr Threat
20 25 30
Ala Met His Trp Arg Val Gln Ala Pro Arg Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Trp Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Thr Thr Arg Asp Asp Ser Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Asp Glu Asp Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Met Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Arg Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Ser Glu Leu Thr Asp Asp Gln Ala Val Ser Ala Val Leu Gly Gln Thr
130 135 140
Val Arg Thr Island Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser 145 150 155 160
Trp Gln Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Tyr Gly
165 170 175
Lys Asn Asn Arg Pro Ser Gly Pro Asp Arg Arg Ser Ser Gly Ser Ser
180 185 190
Ser Gly Asn Thr Ala Ser Leu Thr Island Thr Gly Ala Gln Ala Glu Asp
195 200 205
Glu Ala Asp Tyr Tire Cys Asn Ser Arg Asp Ser Ser Gly Asn His Val
210 215 220
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 225 230 235
(2) INFORMATION FOR SEQ ID NO: 11:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 711 base pairs
(B) TYPE: nucleic acid
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) MOLECULE TYPE: mRNA cDNA
(ix) ADDITIONAL CHARACTERISTIC:
(A) NAME / KEY: CDS
(B) LOCATION: 1..711
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11:
CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG CCT GGG GCC 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
TCA GTG AAG GTC TCC TGC AAG GTT TCC GGA TAC ACC TAC ACT GAA TTA 96
Ser Val Lys Val Ser Cys Lys Ser Val Gly Tyr Thr Leu Thr Glu Leu
20 25 30
TCC ATG CAC TGG GTG CGA CAG GCT CCT GGA AAA GGG CTT GAG TGG ATG 144
Ser Met His Trp Arg Val Gln Ala Gly Lys Pro Gly Leu Glu Trp Met
35 40 45
GGA GGT TT GAT CCT GAA GAT GGT GAA ACA ATC TAC GAC CAG AAG TTC 192
Gly Gly Phe Asp Asp Glu Asp Gly Glu Thr Tire Tyr Ala Gln Lys Phe
50 55 60
CAG GGC AGA GTC ACC ATG ACC GAG GAC ACA TCT ACA GAC ACA GCC TAC 240 Gln Gly Arg Val Thr Met Thr Asp Thru Thr Thr Asp Thr Asp Ala Tyr
65 70 75 80
ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG TAT TAC TGT 288
Met Glu Leu Ser Ser Leu Arg Ser Asp Glu Asp Ala Val Tyr Tyr Cys
85 90 95
GCA AGC CCT GAG ATT GAT CAG TGG GGC CAA GGT ACC CTG GTC ACC GTG 336
Ala Arg Glu Asp Asp Gln Trp Gly Gln Gl Gly Thr Leu Val Thr Val
100 105 110
TCG AGA GGT GGA GGC GGT TCA GGC GGA GGT GGC TCT GGC GGT GGC GGA 384
Ser Arg Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
TCG TCT GAG CTG ACT CAG GAC CCT GCT GTG TCT GTG GCC TTG GGA CAG 432
Ser Ser Glu Leu Thr Asp Asp Gln Ala Val Val Ser Val Ala Leu Gly Gln
130 135 140
ACA GTC AGG ATC ACA TGC CAA GGA GAC AGC CTC AGA AGC TAT TAT GCA 480
Thr Val Arg Thr Island Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 145 150 155 160
AGC TGG TAC CAG CAG AGC AGC GGA AGC GCC CTC GTA CTM AGC ATC TAT 528
Ser Trp Tyr Gln Gln Lys Gly Gln Ala Pro Val Leu Val Tyr
165 170 175
GGT AAA AAC AAC CGG CCC TCA GGG ATC CCA GAC CGA TTC TCT GGC TCC 576
Gly Lys Asn Asn Arg Pro Ser Gly Pro Island Arg Arg Phe Ser Gly Ser
180 185 190
AGC TCA GGA AAC
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
l 5 10 15
Ser Val Lys Val Ser Cys Lys Ser Val Gly Tyr Thr Leu Thr Glu Leu
20 25 30
Ser Met His Trp Arg Val Gln Ala Gly Lys Pro Gly Leu Glu Trp Met
35 40 45
Gly Gly Phe Asp Asp Glu Asp Gly Glu Thr Tire Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Thr Thr Thr Thr Thr Asp Asp Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Asp Glu Asp Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Asp Asp Gln Trp Gly Gln Gl Gly Thr Leu Val Thr Val
100 105 110
Ser Arg Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Ser Glu Leu Thr Asp Asp Gln Ala Val Val Ser Val Ala Leu Gly Gln
130 135 140
Thr Val Arg Thr Island Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 145 150 155 160
Ser Trp Tyr Gln Gln Lys Gly Gln Ala Pro Val Leu Val Tyr
165 170 175
Gly Lys Asn Asn Arg Pro Ser Gly Pro Island Arg Arg Phe Ser Gly Ser
180 185 190
Ser Ser Gly Asn Thr Ala Ser Leu Thr Thr Gly Ala Gln Ala Glu
195 200 205
Asp Glu Ala Asp Tire Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His
210 215 220
Val Val Phe Gly Gly Gly Thr Lys Leu Th Val Leu Gly 225 230 235
(2) INFORMATION FOR SEQ ID NO: 13:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 717 base pairs
(B) TYPE: nucleic acid
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) MOLECULE TYPE: mRNA cDNA
(ix) ADDITIONAL CHARACTERISTIC:
(A) NAME / KEY: CDS
(B) LOCATION: 1..717
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13:
CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG CCT GGG GCC 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
TCA GTG AAG GTT TCC TGC AAG GCT TCT GGA TAC ACC TTC ACT AGC TAT 96
Ser Val Lys Ser Ser Cys Lys Ser Ala Ser Gly Tyr Ser Thr Threat
20 25 30
GCT ATG AAT TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT GAG TGG ATG 144
Ala Met Asn Trp Arg Val Gln Ala Gly Gln Pro Gly Leu Glu Trp Met
35 40 45
GGA TGG ATC AAC ACC AAC ACT GGG AAC CCA ACG TAT GCC CAG GGC TTC 192
Gly Trp Asn Thr Asn Thr Gly Asn Pro Thr Ala Gln Gly Phe
50 55 60
ACA GGA CGG TT GTC TTC TTC TTG GAC ACC TCT GTC AGC ACG GCA TAT 240
Thr Gly Arg Phe Val Ser Phe Ser Asp Thr Ser Ser Val Ser Thr Ala Tyr
65 70 75 80
CTG CAG ATC TGC AGC CTA AAG GCT GAG GAC ACG GCC GTG TAT TAC TGT 288
Leu Gln Island Cys Ser Leu Lys Ala Asp Glu Thr Ala Val Tyr Tyr Cys
85 90 95
GCA AGA TCT AAT GAT CCT GCT GAT CAG TGG GGC CAA GGT ACC CTG GTC 336
Ala Arg Ser Asp Aspn Ala Asp Gln Gln Trp Glly Gly Thr Leu Val
100 105 110
ACC GTG TCG AGA GGT GGA GGC GGT TCA GGC GGA GGT GGC TCT GGC GGT 384
Thr Val Ser Arg Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
115 120 125
GGC GGA TCG TCT GAG CTG ACT CAG GAC CCT GCT GTG TCT GTG GCC TTG 432
Gly Gly Ser Glu Gln Leu Thr Gln Pro Asp Ala Val Val Ser Val Ala Leu
130 135 140
GGA CAG ACA GTC AGG ATC ACA TGC CAA GGA GAC AGC CTC AGA AGC TAT 480
Gly Gln Thr Val Arg Thr Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr 145 150 155 160
TAT GCA AGC TGG TAC CAG CAG AGC GGA GAC AGC GCC CCT GTA CTT GTC 528
Tyr Ala Ser Trp Gln Gln Gln Lys Gly Gln Ala Pro Val Leu Val
165 170 175
ATC TAT GGT ARA AAC AAC CGG CCC TCA GGG ATC CCA GAC CGA TTC TCT 576
Tyr Gly Lys Asn Asn Arg Pro Ser Gly Pro Asp Arg Phe Ser
180 185 190
GGC TCC AGC TCA GGA AAC ACA GCT TCC TTG ACC ATC ACT GGG GCT CAG 624
Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Thr Gly Ala Gln
195 200 205 GCG GAA GAT GAG GCT GAC TAT TAC TGT AAC TCC CGG GAC AGC AGT GGT 672
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Ser Gly
210 215 220 AAC CAT GTG GTA TTC GGC GGA GGG ACC AAG CTG ACC GTC CTA GGT 717
Asn His Val Val Phe Gly Gly Gly Thr Leu Leu Leu Leu Leuk 225 230 235 (2) INFORMATION FOR SEQ ID NO: 14:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 239 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14: Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ser Ser Cys Lys Ser Ala Ser Gly Tyr Ser Thr Threat
20 25 30
Ala Met Asn Trp Arg Val Gln Ala Gly Gln Pro Gly Leu Glu Trp Met
35 40 45
Gly Trp Asn Thr Asn Thr Gly Asn Pro Thr Ala Gln Gly Phe
50 55 60
Thr Gly Arg Phe Val Ser Phe Ser Asp Thr Ser Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Island Cys Ser Leu Lys Ala Asp Glu Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Asp Aspn Ala Asp Gln Gln Trp Glly Gly Thr Leu Val
100 105 110
Thr Val Ser Arg Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Glu Gln Leu Thr Gln Pro Asp Ala Val Val Ser Val Ala Leu
130 135 140
Gly Gln Thr Val Arg Thr Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr 145 150 155 160
Tyr Ala Ser Trp Gln Gln Gln Lys Gly Gln Ala Pro Val Leu Val
165 170 175
Tyr Gly Lys Asn Asn Arg Pro Ser Gly Pro Asp Arg Phe Ser
180 185 190
Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Thr Gly Ala Gln
195 200 205
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Ser Gly
210 215 220
Asn His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 225 230 235
(2) INFORMATION FOR SEQ ID NO: 15:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 762 base pairs
(B) TYPE: nucleic acid
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) MOLECULE TYPE: mRNA cDNA
(ix) ADDITIONAL CHARACTERISTIC:
(A) NAME / KEY: CDS
(B) LOCATION: 1.762
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15:
CGA TTC ATG GAA AGG CAC TGG ATC TT CTC TTC CAG GTG CAG CTG GTG 48
Arg Phe Met Glu Arg His Trp Ile Phe Leu Phe Gln Val Gln Leu Val
1 5 10 15
CAG TCT GGG GGA GGC TTG GTA CAG CCT GGG GGG TCC CTG AGC CTC TCC 96 Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
20 25 30 TGT GCA GCC TCT GGA TTC ACC INCL. AGT AGC TAT AGC ATG AAC TGG GTC 144
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser Ser Ser Asn Trp Val
35 40 45
CGC AGC GCT CCA GGG GGG AGG CTG GAG TGG GTC TCA TAC ATT AGT AGT 192
Arg Gln Ala Pro Gly Lys Gly Glu Leu Trp Val Ser Ser Ser Ser Ser
50 55 60
AGT AGT AGT ACC ATA TAC GAC TAC GAC TCT GTG AGG GAC CGA TTC 240
Ser Ser Ser Thr Island Tire Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
65 70 75 80
ATC TCC AGA GAC AAT GCC AAG AAC TCA CTG TAT CTG CAA ATG AAC AGC 288
Ser Ser Arg Asn Asp Ala Lys Asn Ser Leu Leu Leu Gln Asn Ser
85 90 95
CTG AGA GAC GAG GAC ACG GCC GTG TAT TAC TGT ACA AGA GCT TGG AGG 336
Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Ala Trp Arg
100 105 110 ACG GAT TGG GGC CAA GGT ACC CTG GTC ACC GTG TCG AGA GGT GGA GGC 384
Thr Asp Trp Gly Gln Gly Thr Val Thr Val Ser Arg Gly Gly Gly
115 120 125
GGT TCA GGC GGA GGT GGC TCT GGC GGT GGC GGA TCG TCT GAG CTG ACT 432
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Leu Thr
130 135 140
CAG GAC CCT GCT GTG TCT GTG GCC TTG GGA CAG ACA GTC AGG ATC ACA 480 Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln Thr Val Arg Thr Island 145 150 155 160
TGC CAA GGA GAC AGC CTC AGA AGC TAT TAT GCA AGC TGG TAC CAG CAG 528
Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tire Tyr Ala Ser Trp Tyr Gln Gln
165 170 175 AAG CCA GGA CAG GCC CCT GTA CTT GTC ATC TAT GGT ARA AAC AAC CGG 576
Lys Pro Gly Gln Ala Pro Val Leu Val Tyr Island Gly Lys Asn Asn Arg
180 185 190
CCC TCA GGG ATC CCA GAC CGA TTC TCT GGC TCC AGC TCA GGA AAC ACA 624
Pro Ser Gly Pro Asp Arg Arg Ser Ser Gly Ser Ser Ser Gly Asn Thr
195 200 205
GCT TCC TTG ATC ACT ACC GGG GCT CAG GCG GAT GAAT GAG GCT GAC TAT 672
Ala Ser Leu Thr Island Thr Gly Ala Gln Ala Asp Glu Glu Ala Asp Tyr
210 215 220
TAC TGT AAC TCC CGG GAC AGC AGT GGT AAC CAT GTG GTA TTC GGC GGA 720
Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His Val Val Phe Gly Gly 225 230 235 240
GGG ACC AAG CTG ACC GTC CTA GGT AGC GAA AAG GAC GAG CTG 762
Gly Thr Lys Leu Thr Val Leu Gly Ser Glu Lys Asp Glu Leu
245 250 (2) INFORMATION FOR SEQ ID NO: 16:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 254 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16:
Arg Phe Met Glu Arg His Trp Ile Phe Leu Phe Gln Val Gln Leu Val
1 5 10 15 Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
20 25 30
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser Ser Ser Asn Trp Val
35 40 45
Arg Gln Ala Pro Gly Lys Gly Glu Leu Trp Val Ser Ser Ser Ser Ser
50 55 60
Ser Ser Ser Thr Island Tire Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
65 70 75 80
Ser Ser Arg Asn Asp Ala Lys Asn Ser Leu Leu Leu Gln Asn Ser
85 90 95
Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Ala Trp Arg
100 105 110
Thr Asp Trp Gly Gln Gly Thr Val Thr Val Ser Arg Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Leu Thr
130 135 140 Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln Thr Arg Val Thr Island 145 150 155 160
Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tire Tyr Ala Ser Trp Tyr Gln Gln
165 170 175
Lys Pro Gly Gln Ala Pro Val Leu Val Tyr Island Gly Lys Asn Asn Arg
180 185 190
Pro Ser Gly Pro Asp Arg Arg Ser Ser Gly Ser Ser Ser Gly Asn Thr
195 200 205
Ala Ser Leu Thr Island Thr Gly Ala Gln Ala Asp Glu Glu Ala Asp Tyr
210 215 220
Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His Val Val Phe Gly Gly 225 230 235 240
Gly Thr Lys Leu Thr Val Leu Gly Ser Glu Lys Asp Glu Leu
245,250
(2) INFORMATION FOR SEQ ID NO: 17:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 687 base pairs
(B) TYPE: nucleic acid
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) MOLECULE TYPE: mRNA cDNA
(ix) ADDITIONAL CHARACTERISTIC:
(A) NAME / KEY: CDS
(B) LOCATION: 1..687
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17:
CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCT TCT GGA TAC ACC TTC 48
Pro Gly Ala Ser Val Lys Ser Val Cys Lys Ser Ala Ser Gly Tyr Thr Phe
1 5 10 15
ACT AGC TAT GCT ATG CAT TGG GTG CGC CAG GCC CCC GGA CAA AGG CTT 96
Thr Ser Tyr Ala Met His Trp Arg Val Gln Ala Pro Gly Gln Arg Leu
20 25 30
GAG TGG ATG GGA TGG ATC AAC GCT GGC AAT GGT AAC ACA AAA TAT TCA 144
Glu Trp Met Gly Trp Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser
35 40 45
CAG AAG TTC CAG GGC AGA GTC ACC ACC AGG GAC ACA TCC GCG AGC 192 Gln Lys Phe Gln Gly Arg Val Thr Thr Thr Arg Asp Asp Ser Ser Ala Ser
50 55 60
ACA GCC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAA GAC ACG GCC GTG 240
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
65 70 75 80
TAT TAC TGT GCA AGA AGT GGG GTG TAT TGG GGC CAA GGT ACC CTG GTC 288
Tyr Tyr Cys Ala Arg Ser Gly Val Tyr Trp Gly Gln Gly Thr Leu Val
85 90 95
ACC GTG TCG AGA GGT GGA GGC GGT TCA GGC GGA GGT GGC TCT GGC GGT 336
Thr Val Ser Arg Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
100 105 110
GGC GGA TCG TCT GAG CTG ACT CAG GAC CCT GCT GTG TCT GTG GCC TTG 384
Gly Gly Ser Glu Gln Leu Thr Gln Pro Asp Ala Val Val Ser Val Ala Leu
115 120 125
GGA CAG ACA GTC AGG ATC ACA TGC CAA GGA GAC AGC CTC AGA AGC TAT 432
Gly Gln Thr Val Arg Thr Island Cys Gln Gly Asp Ser Leu Arg Ser Tyr
130 135 140
TAT GCA AGC TGG TAC CAG CAG AGC AGC GGA AGC GCC CCT GTA CTT GTC 480
Tyr Ala Ser Trp Tyr Gln Gln Lys Gly Gln Pro Ala Val Leu Val 145 150 155 160
ATC TAT GGT AAA AAC AAC CGG CCC TCA GGG ATC CCA GAC CGA TTC TCT 528
Tyr Gly Lys Asn Asn Arg Pro Ser Gly Pro Asp Arg Phe Ser
165 170 175
GGC TCC AGC TCA GGA AAC ACA GCT TCC TTG ACC ATC ACT GGG GCT CAG 576
Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Thr Gly Ala Gln
180 185 190 GCG GAA GAT GAG GCT GAC TAT TAC TGT ARC TCC CGG AGC GAC AGT GGT 624
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Ser Gly
195 200 205 AAC CAT GTG GTA TTC GGC GGA GGG ACC AAG CTG ACC GTC CTA GGT AGC 672
Asn His Val Val Phe Gly Gly Gly Thr Lys Leu Th Val Leu Gly Ser
210 215 220 GAA AAG GAC GAG CTG 687
Glu Lys Asp Glu Leu 225
(2) INFORMATION FOR SEQ ID NO: 18:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 229 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18:
Pro Gly Ala Ser Val Lys Ser Val Cys Lys Ser Ala Ser Gly Tyr Thr Phe
1 5 10 15
Thr Ser Tyr Ala Met His Trp Arg Val Gln Ala Pro Gly Gln Arg Leu
20 25 30
Glu Trp Met Gly Trp Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser
35 40 45 Gln Lys Phe Gln Gly Arg Val Thr Thr Thr Arg Asp Asp Ser Ser Ala Ser
50 55 60
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
65 70 75 80
Tyr Tyr Cys Ala Arg Ser Gly Val Tyr Trp Gly Gln Gly Thr Leu Val
85 90 95
Thr Val Ser Arg Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
100 105 110
Gly Gly Ser Glu Gln Leu Thr Gln Pro Asp Ala Val Val Ser Val Ala Leu
115 120 125
Gly Gln Thr Val Arg Thr Island Cys Gln Gly Asp Ser Leu Arg Ser Tyr
130 135 140
Tyr Ala Ser Trp Tyr Gln Gln Lys Gly Gln Pro Ala Val Leu Val 145 150 155 160
Tyr Island Gly Lys Asn Asn Arg Pro Ser Gly Island Pro Asp Arg Phe Ser
165 170 175
Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Thr Gly Ala Gln
180 185 190
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Ser Gly
195 200 205
Asn His Val Val Phe Gly Gly Gly Thr Lys Leu Th Val Leu Gly Ser
210 215 220
Glu Lys Asp Glu Leu 225

Claims (15)

 1. Use of Nucleotide Sequences Coding for Antibodies Directed Against Any Molecule Involved in a Graft Rejection Phenomenon (Also Referred to Hereinafter as Anti-Molecule Antibodies), and in particular against Any Molecule Having an Activity Such That the Molecule Represents a Xenoantigenic Epitope or participates in the biosynthesis of xenoantigenic epitopes in non-human mammalian cells (and more particularly on the surface of these cells), these epitopes being capable of being recognized by human natural xeno-antibodies when said cells, or organs consisting of these cells, of non-human mammals, are grafted in humans, for the preparation of transgenic cells, or transgenic organs, of non-human mammals, in which said molecules form, in whole or in part, immune complexes with said anti-human antibodies; molecules, so that the activity of these mo lecules in the graft rejection phenomena is totally or partially inhibited, in particular that the aforementioned xeno-antibodies do not recognize, in whole or in part, the aforesaid epitopes, or in which the biosynthesis of said xenoantigenic epitopes is partially or totally inhibited, said cells or said transgenic organs of non-human mammals being intended to be grafted in a patient.  2. Use according to claim 1 of nucleotide sequences coding for antibodies directed against  any xenoantigenic, carbohydrate or non-carbohydrate epitope recognized by human xenoantibodies, and more particularly the galactosylated carbohydrate epitopes located on the surface of the membranes of non-human mammalian cells, in particular the β-galactosyl epitope present on the surface of pig and consisting of the above-mentioned Gaia 1,3 Gal-N-acetyllacto-samine complex,  any molecule participating in the biosynthesis of xenoantigenic epitopes in humans, of carbohydrate or non-carbohydrate nature, and more particularly the galactosyltransferases present in non-human mammalian cells, in particular the oe1,3GT enzyme present in pork cells ,  any molecule of inflammatory nature synthesized in the endothelium of non-human mammals, and whose biological activity notably leads to the modification of the anticoagulant properties of the endothelium in vivo in xenogeneic medium, such as cytokines and chemoattractors (IL-I) , IL-6, IL-5, DP-10, RANTES, MCP / JE, p15E inhibitors, GM-CSF, G-CSF), adhesion molecules (ELAM-I, VCAM-1, ICAM-1, -sis, Galba, vWF, LAM-I ligand) transcription factors or modifying transcription (c-fos, NFkB, Gem), regulators of vascular tone (iNO synthase, PGH synthase, endothelin), the factors involved in coagulation (PAF acetyltransferase, tissue factor, PAF1), immunocompetence factors (CMII I, CMII II),  membrane receptors with molecules present in the recipient, the action of these molecules being directed towards a graft rejection, of which for example the C5aR, or TNFotR or NK cell receptors.  3. Use according to claim 1 or claim 2, of nucleotide sequences coding for antibodies directed against at least one isoform (and advantageously against all isoforms) of galactosyl transferases present in non-human mammals, and more particularly of the α-1,3GT enzyme present in pigs, for the preparation of transgenic non-human mammalian organs in which the biosynthesis of α-galactosyl epitopes in the cells of said organs is partially or totally inhibited.  4. Antibodies, where appropriate single-strand, directed against at least one isoform of α-1,3GT, and more particularly directed against at least one of the isoforms of α-1,3GT present in the pig and represented by the amino acid sequences SEQ ID NO 2 (corresponding to isoform 1), SEQ ID NO 4 (corresponding to isoform 2), SEQ ID NO 6 (corresponding to isoform 3) and SEQ ID NO 8 (corresponding to isoform 4), these isoforms 1 to 4 being respectively encoded by the nucleotide sequences represented by SEQ ID NO 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7, or encoded by any nucleotide sequence derived therefrom, in particular by degeneracy of the genetic code.  5. Antibody according to claim 4, directed against at least one of the a-1,3GT isoforms 3 and 4 present in the pig and represented by the amino acid sequences SEQ ID NO 6 and SEQ ID NO 8.  6. Antibodies according to claim 4 or claim 5, characterized in that they are represented by the amino acid sequences SEQ ID NO 10 (antibody designated ScFv1), SEQ ID NO 12 (antibody designated ScFv2), SEQ ID NO 14 (ScFv3 designated antibody), SEQ ID NO 16 (designated antibody ScFv4) and SEQ  ID No. 18 (ScFvS), or by any amino acid sequence derived therefrom, in particular by addition, deletion or substitution of one or more amino acids, or by any fragment of the aforementioned sequences or their derived sequences, said sequences and said fragments being capable of recognizing at least one isoform of o-1,3GT.  7. Nucleotide sequences encoding an antibody according to one of claims 4 to 6, and comprising in particular the nucleotide sequences represented by SEQ ID NO 9 (coding for ScFv1), SEQ ID NO 11 (coding for ScFv2), SEQ ID NO 13 (coding for ScFv3), SEQ ID NO 15 (coding for ScFv4), SEQ ID NO 17 (coding for ScFvS), or any nucleotide sequence derived from the latter, in particular the sequences derived by degeneracy of the code genetics, and yet being able to code for ScFv1 antibodies, ScFv2, ScFv3, ScFv4 or ScFvS, or the sequences derived by addition, deletion or substitution of one or more nucleotides, or any fragment of the aforementioned nucleotide sequences or their derived sequences, said derived sequences and said fragments being capable of coding for a antibody capable of recognizing at least one of the isoforms of 1'1,3GT.  8. Vector containing at least one of the nucleotide sequences according to claim 7 and the elements necessary for the expression of the antibodies according to one of claims 4 to 6.  Cellular host containing at least one of the vectors according to claim 8.  10. Nonhuman transgenic mammal, or mammalian cells, comprising in its genome at least one nucleotide sequence coding for antibodies directed against any molecule involved in a graft rejection phenomenon (hereinafter referred to as anti-molecule antibodies), and in particular against any molecule having an activity such that said molecule represents a xenoantigenic epitope, or participates in the biosynthesis of xenoantigenic epitopes in non-human mammalian cells (and more particularly at the surface of these cells), these epitopes being capable of be recognized by human natural xeno-antibodies when said cells, or organs consisting of these cells, of non-human mammals, are grafted in humans, and more particularly at least one nucleotide sequence according to claim 7.  11. Transgenic non-human mammal according to claim 10, as obtained by introduction, in particular by microinjection into a fertilized oocyte, of at least one nucleotide sequence coding for antibodies directed against any molecule involved in a graft rejection phenomenon. , and more particularly of one of the nucleotide sequences according to claim 7, and insertion of the embryo into the matrix of a surrogate mother and development of the term embryo.  12. Cells grown from non-human transgenic animals according to claim 10 or claim 11.  13. Non-human mammalian organs, comprising in the genome cells constituting at least one nucleotide sequence coding for antibodies directed against any molecule involved in a graft rejection phenomenon (hereinafter referred to as anti-molecule antibodies), and in particular against any molecule having an activity such that said molecule represents a xenoantigenic epitope, or participates in the biosynthesis of xenoantigenic epitopes in non-human mammalian cells (and more particularly at the surface of these cells), these epitopes being capable of be recognized by human natural xeno-antibodies when said cells, or organs consisting of these cells, of non-human mammals, are grafted in humans, and more particularly at least one of the nucleotide sequences according to claim 7, as obtained by sampling on non-human transgenic mammals according to the endication 10 or claim 11.  14. A polypeptide comprising the amino acid sequence as represented by SEQ ID No. 6 (corresponding to isoform 3 of α1,3-GT) or SEQ ID NO 8 (corresponding to isoform 4 of the o 1, 3-GT), or any polypeptide containing any fragment of at least about 6 amino acids, of one of said amino acid sequences, said polypeptide containing said fragment being capable of generating antibodies recognizing one at least four isoforms of o-1,3GT, or any sequence derived therefrom, in particular by addition, deletion or modification of one or more amino acids, provided that the derived sequence is capable of generating antibodies recognizing at least one of the four aforesaid isoforms.  15. Nucleotide sequences comprising the sequences represented by SEQ ID No. 5 and SEQ ID No. 7, or any derivative sequences, in particular by the sequences derived by degeneracy of the genetic code, and nevertheless being capable of encoding the polypeptides represented by the amino acid sequences represented by SEQ ID No. 6 and SEQ ID NO 8 respectively, or sequences derived by addition, deletion or substitution of one or more nucleotides, or any fragment of the aforementioned nucleotide sequences or their derived sequences, said derived sequences and said fragments being capable of encoding then an antibody capable of recognize at least one isoform of cel, 3 GT.