+

WO1993005151A1 - Antigene cenp-c humain clone - Google Patents

Antigene cenp-c humain clone Download PDF

Info

Publication number
WO1993005151A1
WO1993005151A1 PCT/US1992/007649 US9207649W WO9305151A1 WO 1993005151 A1 WO1993005151 A1 WO 1993005151A1 US 9207649 W US9207649 W US 9207649W WO 9305151 A1 WO9305151 A1 WO 9305151A1
Authority
WO
WIPO (PCT)
Prior art keywords
cenp
polypeptide
ser
antibodies
lys
Prior art date
Application number
PCT/US1992/007649
Other languages
English (en)
Inventor
William C. Earnshaw
Hisato Saitoh
John E. Tomkiel
Original Assignee
The Johns Hopkins University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Johns Hopkins University filed Critical The Johns Hopkins University
Publication of WO1993005151A1 publication Critical patent/WO1993005151A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens

Definitions

  • the present invention relates to the use of recombinant DNA technology for the transformation of a host with a eukaryotic genetic sequence for the expression of a centromere polypeptide and methods of using recombinantly produced centromere polypeptides to detect anticentromere antibodies.
  • Scleroderma is a serious autoimmune disease of the connective tissue which has a five year survival rate of about 60%.
  • the disease is rare, occurring at a rate of 1/100,000 per year in the United States (Rodnan, et al., supra).
  • Patients with scleroderma often have autoantibodies to nuclear proteins or structures, including centromeres (anti-centromere antibodies, or ACA), topoisomerase I and nucleoli.
  • ACA were discovered in 1980, when it was found that certain patients with the calcinosis/Raynauds phenomenon/esophegeal dysmotility/ sclerodactyly/telangiectasiae (CREST) variant of scleroderma produce autoantibodies that recognize the centromere region of chromosomes.
  • CREST calcinosis/Raynauds phenomenon/esophegeal dysmotility/ sclerodactyly/telangiectasiae
  • ACA are closely associated with the CREST syndrome, the only clinical finding common to all ACA-positive individuals is Raynaud's phenomenon. The role of these antibodies in the etiology or pathogenesis of the disease is unknown.
  • ACA recognize three chromosomal antigens: CEN P-A ( 17 kDa), CENP-B (80 kDa) and CENP-C (140 kDa).
  • CEN P-A 17 kDa
  • CENP-B 80 kDa
  • CENP-C 140 kDa
  • the autoimmune response to these antigens has been analyzed, indicating multiple epitopes on CENP-B and CENP-C
  • Auto-antisera may be affinity purified by immunoblotting and eluting the absorbed antibodies.
  • CENP-B has been cloned, and purified antigenic fragments of CENP-B have been prepared.
  • Cloned CENP-B prote a for the serological test is readily produced in E. coli, and the use of cloned autoantigen CENP-B for the detection of ACA has been described in U.S. Application Ser. No. 07/174,854, filed March 29, 1988. The test appears to detect ACA in sera of approximately 95% of individuals with the autoantibodies. Monoclonal antibodies specific for CENP-B have also been reported.
  • Yet another object of the present invention is to provide a biologically functional plasmid or viral DNA vector which includes a DNA sequence encoding human CENP-C polypeptide.
  • Another object of the present invention is to provide a host cell transformed or transfected with a DNA sequence which encodes human CENP-C polypeptide such that the host cell can express this polypeptide.
  • It is still another object of the present invention is to provide a method of detecting autoantibodies to human CENP-C polypeptide.
  • a DNA sequence is provided which encodes a portion of the sequence of human CENP-C, that portion of CENP-C being immunologically bound by autoantibodies of CREST patients.
  • a biologically functional plasmid or viral DNA vector is also provided which includes a DNA sequence encoding for human CENP-C polypeptide.
  • a method of producing human CENP-C polypeptide comprises obtaining a host cell transformed with an expression vector containing an intron-free DNA sequence of human CENP-C polypeptide, growing the host cell to express the CENP-C sequence, and recovering a preparation of the human CENP-C polypeptide. Also contemplated by the present invention are the transformed or transfected host cells themselves.
  • a method of detecting autoantibodies to human CENP-C polypeptide comprises contacting a sample with the polypeptide expressed by the DNA sequence of the invention, incubating the sample with the polypeptide for a period of time and under conditions sufficient for binding of autoantibodies to the polypeptide, and detecting the presence of the autoantibodies which are bound to the polypeptide or eluted therefrom.
  • the present invention provides polypeptides possessing part or all of the primary structural conformation for at least one epitope for binding autoantibodies to human CENP-C. These polypeptides are highly useful for the immunological detection of autoantibodies reactive with them, since such antibodies are indicative of autoimmune rheumatic diseases.
  • Antibodies reactive with CENP-C but not with CENP-B were obtained from an auto-antiserum that contained such antibodies by pre-adsorbing anti-CENP-B antibodies with a CENP-B fusion protein. Using this pre-adsorbed serum, a commercially available human cDNA expression library was screened and, a reactive clone was found. Auto-antisera which were affinity purified by purification from phage plaques of this clone were found to bind CENP-C in immunoblots of human chromosomes.
  • DNA from the reactive clone was sub-cloned, and a region encoding epitopes reactive with auto-antisera was identified.
  • the DNA sequence of the various clones encoding CENP-C peptide was determined. Approximately 95% of the human CENP-C polypeptide has been cloned and sequenced, and the CENP-C sequence is novel.
  • the cDNA sequence encoding CENP-C is shown as SEQ ID NO. 1, and the amino acid sequence of CENP-C is shown as SEQ ID NO. 2.
  • a major advantage of the present invention is that it provides the art with a ready source of human CENP-C polypeptide. It is difficult to separate naturally occurring human CENP-C polypeptide from other eukaryotic non-CENP-C polypeptides when isolating CENP-C from natural sources. The absence of non-CENP-C eukaryotic polypeptides is important for the development of test systems which will only detect autoantibodies specifically reactive with human CENP-C polypeptide. By producing human CEN P-C polypeptide in recombinant host cells, it is possible to obtain much larger quantities of the polypeptide. Not only is it possible to use the polypeptide of the invention to more accurately classify patients with such autoimmune rheumatic diseases as CREST syndrome, but it is now possible to also provide commercially useful quantities of human CENP-C polypeptide for use in diagnostic systems.
  • Novel polyclonal and monoclonal antibodies provided by this invention bind an epitope on CENP-C and, in so doing, can prevent the binding of autoantibodies to the corresponding autoimmune epitope.
  • these monoclonal antibodies are useful in sensitive immunoassays, such as competitive immunoasssays to measure the presence of autoantibodies to this epitope.
  • Still another advantage of these monoclonal antibodies is that they can be used to affinity purify CENP-C polypeptide from natural sources or from the surrounding polypeptide milieu, such as when the CENP-C polypeptide is cloned in eukaryotes.
  • FIG. 1 Map of CENP-C clones, showing immunoreactivity and Kyle-Doolittle hydropathy plot.
  • FIG. 1 Bacterial expression of CENP-C polypeptides shown by SDS gel and immunoblot.
  • double-stranded DNA molecule refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its normal, double-stranded helix.
  • deoxyribonucleotides adenine, guanine, thymine, or cytosine
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed stand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • heterologous region or domain of a DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
  • the gene when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • Another example of a heterologous region is a construct where the coding sequence itself is not found in nature (e.g., an intron-free coding sequence (cDNA) where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally occurring mutational events do not give se to a heterologous region of DNA as defined herein.
  • a DNA “coding sequence” is a DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • a coding sequence is "under the control" of the promoter sequence in a cell when RNA polymerase which binds the promoter sequence transcribes the coding sequence into mRNA which is then in turn translated into the protein encoded by the coding sequence.
  • a cell has been "transformed” by exogenous DNA when such exogenous DNA has been introduced inside the cell wall.
  • Exogenous DNA may or may not be integrated (covalently linked) to chromosomal DNA making up the genome of the cell.
  • the exogenous DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the exogenous DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish ceil lines or clones comprised of a population of daughter cells containing the exogenous DNA.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
  • a “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment (a heterologous segment) may be attached so as to bring about the replication of the attached segment.
  • a heterologous segment a DNA segment that is translated into a polypeptide when a host cell transformed by the expression vector is grown under conditions conducive to expression.
  • probe refers to a structure comprised of a poiynucleotide which forms a hybrid structure with a target sequence, due to complementarity of at least one sequence in the probe with a sequence in the target region.
  • the poiynucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs.
  • target region refers to a region of the nucleic acid which is to be amplified and/or detected.
  • target sequence refers to a sequence with which a probe or primer will form a stable hybrid under desired conditions.
  • Coupled refers to attachment by covalent bonds or by strong non-covalent interactions (e.g., hydrophobic interactions, hydrogen bonds, etc.). Covalent bonds may be, for example, ester, ether, phosphoester, amide, peptide, imide, carbon-sulfur bonds, carbon-phosphorus bonds, and the like.
  • support refers to any solid or semisolid surface to which a desired binding partner may be anchored. Suitable supports include glass, plastic, metal, polymer gels, and the like, and may take the form of beads, wells, dipsticks, membranes, and the like.
  • label refers to any atom or moiety which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to poiynucleotide or polypeptide.
  • a "biological sample” refers to a sample of tissue or fluid isolated from a individual, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood ceils, tumors, organs, and also samples of in vivo cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components).
  • Human tissue is an aggregate of human cells which may constitute a solid mass. This term also encompasses a suspension of human cells, such as blood cells, or a human cell line.
  • Two amino acid sequences correspond to each other if any differences in the amino acid sequences of the two proteins involve only conservative amino acid substitutions. Conservative amino acid substitutions occur when an amino acid has substantially the same charge as the amino acid for which it is substituted and the substitution has no significant effect on the local conformation of the protein.
  • Two DNA sequences correspond to each other if the sequences or their complementary sequences encode amino acid sequences which correspond to each other.
  • Two epitopes correspond to each other if they can be specifically bound by the same antibody.
  • Two antibodies correspond to each other if both are capable of binding to the same epitope, and binding of one antibody to its epitope prevents binding by the other antibody (i.e., one antibody "blocks" the binding of the other).
  • CENP-C polypeptides are polypeptides whoss amino acid sequence corresponds to a portion of the amino acid sequence of the CENP-C protein.
  • CENP-C polypeptides may additionally contain an amino-acid sequence which does not correspond to any portion of the sequence of CENP-C.
  • fusion proteins expressed by recombinant organisms which contain a portion of a protein contributed by the expression vector fused to a portion of the CENP-C sequence are contemplated within the term CENP-C polypeptides.
  • a preparation containing CENP-C polypeptides is substantially free of other nuclear antigens when the preparation is not significantly immunologically reactive with any antinuclear antibodies other than those reactive with CENP-C. Antinuclear antibodies react with one or more antigenic components of the human cell nucleus, including D NA.
  • An antibody is "cross-reactive” if it binds to an epitope on each of two different proteins via the antibody's antigen-combining site. Two proteins possess cross-reactive epitopes if a single antibody will bind to each of the two proteins.
  • An antigen is "immunologically bound” by an antibody if the antigen-combining site specifically binds to an epitope on the antigen.
  • Unique sequences either amino acid sequences or nucleic acid sequences which encode them, are sequences which are identical to a sequence of a CENP-C polypeptide, but which are not found elsewhere in the human genome.
  • An epitope is "unique" to CENP-C polypeptides if it is found on CENP-C polypeptides but not found on any other nuclear antigens.
  • CENP-C may be purified from natural sources as described below, great care is required to avoid contamination of such CEN P-C with other nuclear antigens. Therefore, it is preferable to obtain CENP-C by recombinant methods from a DNA sequence encoding such a protein or polypeptide, which DNA sequence can be synthesized chemically or isolated by one of several approaches.
  • the DNA sequence to be synthesized can be designed with the appropriate codons for the CENP-C amino acid sequence. In general, one will select preferred codons for the intended host, if the sequence will be used for expression.
  • the complete sequence may be assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge ( 1981) Nature 292:756; Nambair, et al. (1984) Science 223:1299; Jay, et al. (1984) J. Biol. Chem., 259:6311.
  • CENP-C polypeptides of this invention may be prepared by chemical synthesis.
  • the Merrifield technique Journal of American Chemical Society, vol. 85, pp. 2149-2154, 1968, can be used.
  • RNA libraries are obtained.
  • the library can consist of a genomic DNA library from a human source. Human genomic libraries are known in the art. More preferred are DNA libraries constructed of cDNA, prepared from poly-A-plus RNA (mRNA) by reverse transcription. The mRNA is isolated from a cell line or tissue believed to express the protein.
  • mRNA poly-A-plus RNA
  • Suitable sources of mRNA for cDNA library construction include cultured cells, such as HeLa cells, placental umbilical cord endothelial cells, colon cells, and lymphoid cells.
  • the genomic DNA or cDNA is cloned into a vector suitable for construction of a library.
  • the construction of an appropriate library is within the skill of the art. See, e.g., B. Perbal, supra.
  • suitable DNA libraries are available from suppliers of biological research materials, such as Clonetech and Stratagene, as well as public depositories such as the American Type Culture Collection.
  • selection may be accomplished by expressing the library sequences and detecting the expressed peptides immunologically. Clones which express peptides that bind to the antibodies of this invention are selected. These selection procedures are well known to those of ordinary skill in the art (see, e.g., Sambrook, et al.), and they are exemplified in the Examples below. Antibody preparations which are specific for CENP-C are required for such selection. Methods for obtaining such antibody preparations are provided below.
  • oligonucleotides corresponding to a portion of the nucleotide sequence of SEQ ID NO. 1 may be used to probe the library to identify the segment carrying a sequence encoding CENP-C.
  • the isolation methods may rely in part on nucleic acid hybridization using appropriate single stranded or double stranded nucleotide or oligonucleotide probes.
  • Such probes can be constructed synthetically, based on the DNA or amino acid sequences disclosed herein, or isolated from genomic or cDNA clones also described herein.
  • the probes may be synthesized chemically, based upon the nucleic acid sequence of CENP-C provided by this invention, shown in SEQ ID NO.1.
  • DNA probes may also be prepared by random labelling of cDNA clones identified as above. While the exact length of any probe employed is not critical, typical probe sequences are no greater than 1000 nucleotides in length, more typically they are not greater than 500 nucleotides, even more typically they are no greater than 250 nucleotides; they may be no greater than 100 nucleotides, and also may be no greater than 75 nucleotides in length. Generally it is recognized in the art that probes from about 14 to about 20 base pairs are usually effective.
  • oligonucleotide probes are usually labeled with a marker, such as a radionucleotide or biotin, using standard procedures.
  • the labeled set of probes is then used in the screening step, which consists of allowing the single-stranded probe to hybridize to isolated ssDNA from the library, according to standard techniques.
  • Either stringent or permissive hybridization conditions could be appropriate, depending upon several factors including, but not limited to, the length of the probe, whether the probe and library are from the same species, and whether the species are evolutionarily close or distant. It is within the skill of the art to optimize hybridization conditions so that homologous sequences are isolated and detectable above background hybridizations.
  • hybridization conditions be of sufficient stringency so that selective hybridization occurs; i.e., hybridization is due to a minimum degree of nucleic acid homology (e.g., at least about 75%). as opposed to nonspecific binding or hybridization due to a lower degree of homology. See generally, “Nucleic Acid Hybridization,” (1985) B.D. Hames and S.J. Higgins, eds.
  • the sequence can be cloned into any suitable vector or replicon and thereby maintained in a composition which is substantially free of vectors that do not contain the coding sequence (e.g., free of other clones from the library).
  • suitable vector or replicon e.g., any suitable vector or replicon and thereby maintained in a composition which is substantially free of vectors that do not contain the coding sequence (e.g., free of other clones from the library).
  • Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice (see, e.g., Sambrook, et al., incorporated herein by reference).
  • the DNA sequences and DNA molecules of the present invention may be expressed using a wide variety of host/vector combinations.
  • the coding sequence for the polypeptide is placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence is transcribed into RNA in the host cell transformed by a vector containing this expression construct.
  • the coding sequence may or may not contain a signal peptide or leader sequence.
  • Hosts may be prokaryotie, such as Escherichia, Bacillus, and other bacteria, or eukaryotic, including fungi, yeasts, and mammalian cells in culture; vectors may be plasmids, cosmids, bacteriophage or other viruses and the like, and host cells are chosen which may replicate and/or express the particular vector.
  • Preferred systems use prokaryotic host cells with plasmid or bacteriophage vectors.
  • not all host/expression vector combinations function with equal efficiency in expressing the DNA sequences of this invention or in producing the polypeptides of this invention.
  • a particular selection of a host/expression vector combination may be made by those skilled in the art.
  • the selection should be based on a balancing of a number of factors. These include compatibility of the host and vector, toxicity of the proteins encoded by the DNA sequence to the host, ease of recovery of the desired protein, expression of characteristics of the DNA sequences and the expression control sequences operatively linked to them, biosafety, costs and the folding, form or any other necessary post-expression modifications of the desired protein.
  • the host cell will not express proteases which degrade the recombinant polypeptide of this invention.
  • Fusion proteins provide an alternative to direct expression.
  • a DNA sequence encoding the N-terminal portion of an endogenous protein, or other stable protein is fused to the 5' end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences.
  • the protein is produced by growing host cells transformed by an expression vector containing the coding sequence for a polypeptide cross-reactive with CENP-C under conditions whereby the polypeptide is expressed.
  • the selection of the appropriate growth conditions is within the skill of the art.
  • the polypeptide is then isolated from the host cells and purified. Purification of the protein can be accomplished according to techniques which are well-known in the protein purification art. For example, various types of chromatography may be used.
  • CENP-C polypeptides can also be purified using immunoaffinity techniques. Antibodies are provided herein which are specific for epitopes found on CENP-C but not found on other nuclear antigens, and the desired polypeptides can be positively selected from a mixture of many proteins by binding to these antibodies. In a preferred method, antibodies specific for CENP-C polypeptides are attached to an insoluble support. The use of the antibodies of the present invention to purify the proteins allows good separation from the proteins found in the nucleus, particularly from CENP-B. In particular, some antibodies to CENP-C produced to fusion proteins made in E. coli do not cross-react with CENP-B.
  • such antibodies may be produced by immunization of a mammal with CENP-C polypeptide, preferably a fusion protein produced in a prokaryotic system.
  • CENP-C is positively selected by the antibodies, which do not bind other nuclear antigens.
  • a preparation of CENP-C which is substantially free of other nuclear antigens may be prepared using antibodies specific for CENP-C and immunoaffinity techniques that are well known in the art.
  • other techniques of purification are known in the art and can be used to purify the proteins of this invention.
  • CENP-C polypeptides uses the pT7-7 vectors, a phage T7-based system, for expression of human proteins in E. coli. These vectors utilize the promoter and 5' coding region of the T7 protein 10, which are fused to a cloning polylinker, containing sites for a number of commonly used restriction enzymes (Tabor, et al. (1985), Proc. Natl. Acad. Sci. (USA). 82:1074-1078). Expression of the cloned inserts is induced by addition of IPTG to cultures of E. coli carrying recombinant plasmids.
  • Expression vector pT7-7-CENP-C-1 based on pT7-7 which has a sequence encoding a CENP-C fusion protein, was deposited with the American Type Culture Collection 12301 Parklawn Drive. Rockville, Maryland, on September 12, 1991, under Accession No. 75100 .
  • Cloned protein may be identified by immunoblotting.
  • fusion protein expression by bacteria transformed with an expression vector encoding a CENP-C polypeptide is induced for 4 hours, following which the bacteria are harvested by centrifugation, washed once with buffer, and lysed by boiling in SDS-PAGE sample buffer.
  • This cloning system typically gives high levels of expression of the cloned proteins, which are readily detected by Coomassie blue staining. Staining of such cloned protein is shown in Figure 2 (panels A and C). SDS gels of bacterial lysates are electroblotted to nitrocellulose, which is then incubated in an appropriate dilution of test serum, and then bound antibody is detected with 125 I protein A (see Figure 2, panels B and D).
  • CENP-C fusion proteins expressed in the pT7-7 vectors have the unusual property of being highly insoluble and remaining tightly associated with bacterial debris that pellets at low speed. This has been demonstrated for the fusion protiens M8 (also called CENP-C-1), EA, AA and AE. Preparations where the CENP-C M8 fusion protein makes up approximately 10-20% of the total protein present have been obtained.
  • the CENP-C protein in such a preparation is of sufficient purity that it may be used for screening of patient sera for anti-CENP-C antibodies.
  • anrigen preparations of this sort which are suitable for immunoblotting are also suitable as substrates in solid phase binding assays.
  • Immunoblotting of a series of subclones that span the cloned portion of CENP-C shows that an antigenic determinant for autoantibodies is found in a 120 amino acid region in the carboxy-terminal portion of the CENP-C molecule between residues 677 and 797 of SEQ ID NO. 2 (see the immunoblots in Figure 2 and summary in Figure 1).
  • Results of a screen of patient sera show that cloned CENP-C is suitable for detection of ACA.
  • the protein from clone M8 was used to screen 6 ACA-positive sera and 4 controls.
  • SDS gels of bacterial lysates are electroblotted to nitrocellulose, which is labeled and sliced vertically into 20-30 thin strips per gel. Each strip is then incubated in an appropriate dilution of test serum, and then bound antibocy is detected with 125 I protein A. All 6 ACA-positive sera recognized the CENP-C fusion protein, while none of the control sera did (see Figure 3).
  • CENP-C polypeptide in general, can provide compositions substantially free of other human proteins.
  • the ability to obtain high levels of purity is one benefit of using recombinant expression systems, which also can produce CENP-C polypeptides in substantial quantities vis-a-vis in vivo sources.
  • recombinant expression systems which also can produce CENP-C polypeptides in substantial quantities vis-a-vis in vivo sources.
  • CENP-C polypeptide compositions free of other nuclear antigens can readily be produced. Those of ordinary skill in the art can select among the above techniques to prepare the substantially pure peptides of this invention.
  • CENP-C polypeptides Mammals immunized with CENP-C polypeptide will produce polyclonal antibodies contemplated by this invention.
  • Preferred CENP-C polypeptides are those whose sequence corresponds to SEQ ID NO.2 from residue 677 to residue 797.
  • a particulary preferred pep tide immunogen contains the sequence of SEQ ID NO. 2 from residue 331 to 980. Immunization of mammals such as rabbits, mice, goats, etc.. to produce antibodies is well known in the art.
  • a preferred method of making antibodies involves transfecting an E. coli host cell with expression vector pT7-7-CENP-C-1, deposited under ATCC Accession No. 75100 , and growing a population of the transformed cells then inducing expression of CENP-C fusion protein with IPTG.
  • the fusion protein is injected into mammals (preferrably rabbits), to produce antibodies according to well known procedures. Antibodies can be detected by their ability to bind to the nucleus of human cells.
  • Polyclonal antibody preparations can be partially purified using immunoaffinity techniques, employing a polypeptide of the present invention. Immunoaffinity purification of polyclonal antibodies may also employ proteins purified from cell nuclei, or polypeptide fragments of such proteins. Such purification methods are well-known in the art. Immunoaffinity techniques can be used to purify monospecific polyclonal antibodies specifically reactive with CENP-C but not with other nuclear antigens, such as CENP-B. Similar binding properties are employed as in the tests described for screening monoclonal antibodies b elow. That is to say, that antibodies which immunoreact with CENP-C are positively selected, while those that immunoreact with other nuclear antigens are removed from the desired antibody preparation.
  • CENP-C polypeptides immobilized on insoluble supports can be used for positive selection.
  • CENP-B polypeptides produced according to U.S. Ser. No. 07/174,854, incorporated herein by reference, can be used for negative selection if desired.
  • the present invention also relates to monoclonal antibodies which recognize an epitope on human CENP-C which corresponds to the epitope recognized by autoantibodies to human CENP-C. These monoclonal antibodies are highly useful for immunological detection of CENP-C and in connection with the diagnosis of autoimmune disease.
  • Monoclonal antibodies can also be raised which are specific for CENP-C polypeptides provided by this invention according to techniques that are well known in the art.
  • a rat or mouse will be immunized with the polypeptide of the present invention (or a protein cross-reactive with a peptide according to SEQ ID NO.2) and the rodent will later be sacrificed and spleen cells recovered for fusion with myeloma cells.
  • Hybrid cells can be selected according to techniques known in the art, for example, selections involving complimentation of two phenotypes, one from each parental cell.
  • Antibody production of each hybrid cell can be screened individually. Antibodies which bind to epitopes on proteins cross-reactive with CENP-C protein but not on other nuclear antigens are selected.
  • Antibodies can be tested for immunoreactivity with CENP-C polypeptide using a substantially pure preparation of CENP-C polypeptide isolated from a recombinant host cell transformed with an expression vector containing DNA encoding the CENP-C polypeptide, optionally conjugated to an immunogenic moiety such as bovine serum albumin.
  • the desired specific antibodies should be positive in this test.
  • the antibodies should also be tested for immunoreactivity with other nuclear antigens, preferably including CENP-B; desired antibodies having absolute specificity for CENP-C should be negative in such tests.
  • the cDNA sequence of the invention encodes essentially the entire human CENP-C molecule it is now a matter of routine to prepare, subclone and express smaller polypeptide fragments of cDNA from this or a corresponding cDNA sequence which would encode as few as one epitope for autoantibodies to human CENP-C.
  • the presence of such an epitope on a cloned polypeptide can then be determined using, for example, sera from a patient with auto-antibodies to CENP-C as described herein.
  • the human CENP-C polypeptide of the invention is particularly suited for use in immunoassays in which it can be utilized in liquid phase or bound to a solid phase carrier.
  • the human CENP- C polypeptide used in these assays can be detectably labeled in various ways.
  • immunoassays which can utilize human CENP-C polypeptide of the invention are competitive and non-competitive immunoassays in either a direct or indirect format.
  • immunoassays are the radioimmunoassay (RIA), the sand vich (immunometric) assay and the Western blot assay.
  • Detection of autoantibodies which bind to the human CENP-C polypeptide of the invention can be done utilizing immunoassays which run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Regardless of the type of immunoassay which is used, the concentration of human CENP-C polypeptide utilized can be readily determined by one of ordinary skill in the art.
  • the human CENP-C polypeptide of the invention can be bound to many different carriers and used to detect the presence of antibody specifically reactive with the polypeptide.
  • carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses and magnetite.
  • the nature of the carrier can be either soluble or insoluble for purposes of the invention.
  • Those skilled in the art will know of other suitable carriers for binding human CENP-C poiypeptide. or will be able to ascertain such, using routine experimentation.
  • labels There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioiso- topes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds.
  • the autoantibody which binds to the human CENP-C polypeptide of the invention may be present in various biological fluids and tissues. Any sample containing a detectable amount of autoantibodies to human CENP-C polypeptide can be used. Normally, a sample is a liquid such as urine, saliva, cerebrospinal fluid, blood, serum and the like, or a solid or semi-solid such as tissue, feces and the like. Most preferably, the sample contains serum from a patient.
  • immunoassays based on one or more CENP-C polypeptides according to this invention optionally with immunoassays using CENP-B polypeptides, it is possible to detect ACA in human sera and to subclassify patients whose sera contain ACA, depending on whether their sera contain antibodies specific for CENP-B, antibodies specific for CENP-C and/or antibodies cross-reactive with both proteins.
  • Example 1 Isolation of CENP-C cDNA Clones
  • a lambda-gtl l expression library was screened using serum from a patient with anticentromere antibodies (ACAs).
  • ACAs anticentromere antibodies
  • One cDNA clone was isolated using the lambda-gt11 expression method of Young and Davis (Proceedings of the National Academy of Sciences, USA. 80:1194, 1983) from a cDNA library constructed from human placental cell mRNA (Clonetech).
  • Approximately 4 x 10 6 recombinant phage were plated on lawns of Escherichia coli RY1090, production of fusion protein induced by overlaying nitrocellulose filters (BA83, Schleicher & Schuell, Inc., Keene. NH) impregnated with isopropylthiogalactoside (IPTG, Sigma Chemical Co., St. Louis, MO), and immunopositive colonies detected by processing the filter as though for immunoblotting (see below).
  • Serum GS from a pateint with Raynaud's phenomenon and scleroderma was chosen as the most suitable of the 38 ACA-positive antisera available.
  • This serum contains IgG specific for the 80kD centromere autoantigen CENP-B and the 140kD centromere autoantigen CENP-C; it is negative for other common autoantibodies, including Ro, La, Sm, DNA (ss and ds), RNP, Scl- 70 (topoisomerase I), actin, and intermediate filaments.
  • Serum GS binds detectibly to CENP-C in immunoblots of isolated chromosomal proteins at dilutions of up to 1:10,000. Filters were screened with serum GS which had been depleted of anti-CENP-B antibodies by preabsorption with bacterially produced CENP-B fusion protein, prepared as described in U.S. Application Ser. No. 07/174.854, incorporated herein by reference. By this method, three cones were detected and eventually plaque purified. After plaque purification, EcoRI digestion of purified DNA of one of these phage, designated lambda CENP-C-1 (also called M8 in some experiments), liberated a cDNA fragment of 2084 base pairs (bps). EcoRI digestion of purified DNA from a second phage, designated PM, liberated a cDNA fragment of 1619 bps.
  • EcoRI digestion of purified DNA from a second phage designated PM, liberated a cDNA fragment of 1619 bps.
  • Phage clones containing longer cDNA inserts were obtained by screening cDNA libraries constructed from human colon mRNA (Clonetech), human fetal brain mRNA (Stratagene), or HeLa cell mRNA (Stratagene) by DNA plaque hybridization. In all cases the plaque lifts were processed by standard procedures (Maniatis, et al., in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1981).
  • Probes were labeled with alpha- 32 P- dATP or alpha- 32 P-dCTP using random priming with DNA polymerase I, Klenow fragment (New England Biolabs, Beverly, MA) (Feinberg, et al., Annals of Biochemistry, 132:6, 1983).
  • DNA polymerase I Klenow fragment
  • Klenow fragment New England Biolabs, Beverly, MA
  • additional libraries from human colon, fetal brain or HeLa cells yielded two overlapping clones: CENP-C-2 (1359bp) and CENP-C-3 (1095 bp).
  • CENP-C is encoded by a single human chromosomal locus that is transcribed into a single 3.5k base mRNA of which about 90% has been cloned herein.
  • a clone map is shown in Figure 1.
  • the nucleotide sequence is shown in SEQ ID No. 1.
  • the predicted peptide sequence is shown in SEQ ID NO. 2.
  • the predicted partial polypeptide sequence of 980 amino acides has an estimated pi of 9.5 and a calculated molecular weight of 111kD.
  • Figure 1 also shows a Kyle-Doolittle hydropathy plot for the CENP-C protein. No sequences with significant homologies to the CENP-C sequence were detected in the GENEBANK or EMBO sequence banks.
  • the nucleotide sequence of CENP-C is provided among the three deposited plasmids as indicated in the map of Figure 1, showing the relationship of these fragments to the open reading frame.
  • the sequence of the deposits should be considered controlling.
  • cDNA from CENP-C appropriate regions of cDNA were cloned into one of the PT7-7 series of bacterial expression plasmids (S. Tabor, Harvard Medical School). This series of plasmids was specifically engineered to contain T7 RNA promoter and the translation start site for the T7 protein 10. The plasmids were modified by the addition of a polylinker of unique restriction enzyme sites following the translation start site.
  • any exogenous cDNA fragment can conveniently be cloned such that its encoded polypeptide is in frame with the protein 10 translation start site in one of the plasmids, as long as compatible restriction sites are available on the fragment.
  • each cDNA was excised with Eco RI from the original lamda clone, or from Bluescript (Stratagene) subclones and the fragment isolated using Geneciean (Bio101. Inc.) after electrophoresis on agarose gels.
  • the gel fragments were added to the appropriate PT7-7 plasmid that had been linearized with Eco RI and treated with alkaline phosphatase.
  • the mixtures were then diluted into DNA ligase buffer (New England Biolabs), brought to room temperature, and DNA ligase added for 2 h to overnight.
  • DNA from the ligation reactions were transformed by standard methods into Escherichia coli strains BL21 (DE3) which is a T7 lysogen (Hanahan, Journal of Molecular Biology, 166:557, 1983). Transformants were selected by ampicillin resistance. Restriction mapping of isolated plasmid DNA identified those plasmids into which the cDNA had been cloned in the proper orientation.
  • CENP-C-1 Subclones of CENP-C-1 were generated by digestion of CENP-C-1 and pT-7-7 DNA with the appropriate restriction enzymes.
  • the appropriate CENP-C-1 derived fragment and the linearized pT7-7 plasmid were isolated and ligated as described above.
  • the following CENP-C-1 fragments were subcloned into pT7-7 for expression in E. coli: EA (EcoRI-Alu I, bp 992-1398), ET (EcoRI-RaqI, bp 992-1670), AA (Alul-Alul. bp 1409-2026), EH (EcoRI-Haelll, bp 992-2393). and AE (AluI-EcoRI. bp 2027-3094).
  • T7/CENP-C fusion proteins were induced from overnight cultures of BL21 (DE3) transformants harboring the desired plasmids that were grown at 37 °C in LB (Maniatis, et al., ibid) supplemented with 20 ug/ml ampicillin.
  • the overnight culture was diluted 1:100 into the same medium.
  • the bacteria were incubated at 37° C until the O.D. 600 of the culture was 0.5.
  • IPTG was added at a final concentration of 0.5 mM, and the incubation was continued at 37 ° for three hours.
  • Bacteria were lysed by boiling in SDS gel sample buffer and proteins resolved by PAGE (Laemili; Nature, 227, 680, 1970).
  • Lane 1 in all panels shows control lysates from bacteria that contained the pT7-7 plasmid without insert.
  • Panel A bacterial lysates expressing M8-protein (lane 2), EH-protein (lane 3), and PM-protein (lane 4) were subjected to 7.5% SDS-PAGE and stained with Coomassie blue.
  • panel C bacterial lysates expressing EA-protein (lane 2), ET-protein (lane 3), AA-protein (lane 4) and- AE-protein (lane 5) were subjected to 15% SDS-PAGE and stained with Coomassie-blue.
  • Panels B and D show immunoblots of gels identical to those in to panels A and C, respectively, probed with ACA-positive serum. Dots of right side of each lane mark the positions of the major induced peptide. The lines at the left side of each panel indicate the molecular markers: 200, 116, 95, 68, 60, 43, 40.29 and 16.9 kDa.
  • Antibodies were affinity purified from antigens immobilized on nitrocellulose blots by exposure to 3M NH4-K-thiocyanate (Earnshaw, et al., Chromosoma, 91: 313, 1985).
  • antibodies were affinity-purified from autoimmune serum using protein encoded by the CENP-C-1 clone in lambda gtll (Synder, et al., 1988, Methods Enzymol, 154:107-128, Earnshaw. Ratr e and Stetton. 1989. Chromosoma. 98:1-12). Phage were plated on a lawn of E. coli Y 1090 at a density of 1 x 10 4 cells per 9 cm petri dish at 42 ° C. When plagues were visible (at approximately four hours), a 9 cm diameter nitrocellulose filter (which had been presoaked in 10 mM IPTG) was placed on the bacterial lawn for 4 h at 37°C.
  • This filter was then removed from the plate, washed in PTX buffer (Earnshaw, et al., 1984), blocked by incubation with 4% BSA in PTX for 20 min., and incubated with GS autoimmune serum for 16 hr. at room temperature. The filter was then washed in PTX buffer for 5 min., and bound antibodies were eluted by treatment with 3.5 ml of 0.2 M glycine/HCL, pH 2.5, for 2 min. at 37°C. The solution of freshly eluted, affinity-purified antibodies was neutralized by addition of 1.75 ml 1 M potassium phosphate, pH 9.1.
  • Indirect immunofluorescence was performed using colcemid- arrested HeLa ee ls spread by centrifugation on coverslips (Earnshaw, et al., Chromosoma, 1989). Briefly, bound antibody was detected with biotinylated second antibody (anti-human, anti-rabbit, or anti-mouse; Vector Laboratories. Inc., Burlingame, CA) and streptavidin linked to Texas Red (Bethesda Research Laboratories, Bethesda, Md). Slides were examined with an Olympus BH-2 microscope and images were obtained on Kodak Tri-X film, ASA-1000.
  • T7-CENP-C fusion protein was isolated from BL21 (DE3) lysogens harboring pT7-7-CENP-C plasmids by a simple differential sedimentation procedure. Briefly, frozen induced lysogens were disrupted with lysozyme, EDTA, sonication, and detergent, releasing the fusion protein. The fusion protein was separated from soluble and membrane proteins by sedimentation at 10,000 g (10 min) in the presence of 1% NP-40 in TEN buffer (10 mM Tris, pH 7.4, 1 mM Na EDTA, 50 mM NaCl). The fusion protein was electrophoresed on a 7.5% polyacrylamide gel and excised for use in producing polyclonal antibodies.
  • Injections were given at multiple sites: subcutaneous, intramuscular, and intraperitoneal. Animals were bled from the ear on days 0, 38, 92, 135, and 169 by venipuncture. All four rabbits responded by producing antibodies that recognize CENP-C in immunoblots of isolated chromosomes. These polyclonal anticentromere antisera were designed raCENP-C-1A, raCENP-C-1B, raCENP-C-1EA, and raCENP-C-1AE, respectively.
  • Immunoblotting assays were carried out using sera fro m 6 ACA-positive patients and four normal controls. Patients were evaluated at the time of the study when blood was obtained. Immunoblotting was performed as described in Example 2 using CENP-C-1 fusion protein. SDS gels of bacterial lysates were electroblotted to nitrocellulose, which was labeled and sliced vertically into 20-30 thin strips per gel. Each strip was then incubated in an appropriate dilution of test serum, and then bound antibody was detected with 125 I protein A.
  • Figure 3 shows immunoblotting of the CENP-C-1 (M8) fusion protein with 10 patient sera (all used at a dilution of 1:500). Plus (-) indicates the ACA-positive sera. Minus (-) indicates the ACA-nega- tive sera. The location of the CENP-C-1 fusion protein is indicated by the arrow at the right.
  • AAG AAA AGG ATC TGT CTT GAT AAC GAT GAA AGA AAG ACT AAC TTA ATG 2543 Lys Lys Arg Il e Cys Leu Asp Asn Asp Glu Arg Lys Thr Asn Leu Met
  • GTA AAT CTA GGT ATA CCT CTT GGA GAT CCT TTG CAG CCA ACG AGG GTA 2591 Val Asn Leu Gly Il e Pro Leu Gly Asp Pro Leu Gln Pro Thr Arg Val

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Rheumatology (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Rehabilitation Therapy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • Diabetes (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention se rapporte à des polypeptides possédant une partie ou la totalité de la conformation structurelle principale permettant à un épitope de lier des autoanticorps au CENP-C humain. Ces polypeptides sont très utiles pour la détection immunologique d'autoanticorps qui réagissent en leur présence, ces anticorps indiquant des maladies rhumatismales auto-immunes.
PCT/US1992/007649 1991-09-12 1992-09-11 Antigene cenp-c humain clone WO1993005151A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US75900191A 1991-09-12 1991-09-12
US759,001 1991-09-12

Publications (1)

Publication Number Publication Date
WO1993005151A1 true WO1993005151A1 (fr) 1993-03-18

Family

ID=25054001

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/007649 WO1993005151A1 (fr) 1991-09-12 1992-09-11 Antigene cenp-c humain clone

Country Status (1)

Country Link
WO (1) WO1993005151A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994025604A3 (fr) * 1993-04-24 1996-05-02 R S R Limited Fragments de steroide hydroxylase 21 et dosages les utilisant
WO1999024605A3 (fr) * 1997-11-12 1999-07-29 Deutsches Krebsforsch Adn a activite de promoteur pour gene a cycle cellulaire

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CELL vol. 70, no. 1, 10 July 1992, CELL PRESS, CAMBRIDGE, NA.; pages 115 - 125 H. SAITOH ET AL. 'CENP-C, an autoantigen in scleroderma, is a component of the human inner kinetochore plate' *
CHROMOSOMA vol. 98, 1989, SPRINGER VERLAG, BERLIN, BRD; pages 1 - 12 W.C. EARNSHAW ET AL. 'Visualization of centromere proteins CENP-B and CENP-C on a stable dicentric chromosome in cytological spreads' cited in the application *
J. CELL BIOL. vol. 104, no. 4, April 1987, ROCKEFELLER UNIV. PRESS, NY, US; pages 817 - 829 W.C. EARNSHAW ET AL. 'Molecular cloning of cDNA for CENP-B the major human centromere autoantigen' cited in the application *
PROC. NATL. ACAD SCI. vol. 84, no. 14, July 1987, WASHINGTON, DC, US; pages 4979 - 4983 W.C. EARNSHAW ET AL. 'Analysis of anticentromere autoantibodies using cloned autoantigen CENP-B' cited in the application *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994025604A3 (fr) * 1993-04-24 1996-05-02 R S R Limited Fragments de steroide hydroxylase 21 et dosages les utilisant
WO1999024605A3 (fr) * 1997-11-12 1999-07-29 Deutsches Krebsforsch Adn a activite de promoteur pour gene a cycle cellulaire

Similar Documents

Publication Publication Date Title
JPH07143888A (ja) 型特異的乳頭腫ウイルスｄｎａ配列およびペプチド
EP0698093B1 (fr) GENE DE LA MALADIE DE VON HIPPEL-LINDAU (VHL) ET ADNc CORRESPONDANT, ET PROCEDES DE DETECTION DE PORTEURS DU GENE DE LA MALADIE DE VHL
US4918162A (en) Assays and antibodies for N-MYC proteins
JPH06500688A (ja) 神経線維腫遺伝子
WO1996038467A1 (fr) Proteines liees a l'x fragile, leurs compositions et procedes de production et d'utilisation
AU730811B2 (en) AFC1 and RCE1:isoprenylated CAAX processing enzymes
US5759803A (en) Recombinant retinoblastoma-associated protein 1 (E2F-1) polypeptides and cDNA
JPH10210991A (ja) トキソプラスマ症のための診断上の遺伝子
CA2250452A1 (fr) Sequence d'introns partielle du gene de la maladie de von hippel-lindau (vhl) et son utilisation dans le diagnostic de maladies
EP0750637B1 (fr) Adn codant des proteines conferant une resistance au cai et leurs utilisations
WO1993005151A1 (fr) Antigene cenp-c humain clone
AU596670B2 (en) Assays and antibodies for n-myc proteins
EP0809698B1 (fr) NUCLEOTIDES ET SEQUENCES AMINOACIDES DEDUITES DU GENE TUMORAL Int6
EP0406305A1 (fr) Procedes et compositions integrant des epitopes antigeniques, autoimmuns
JP2002503952A (ja) 多嚢胞性腎疾患遺伝子
US7189810B2 (en) Polypeptides, use thereof and process for producing the same
US7029911B1 (en) AFC1 and RCE1: isoprenylated CAAX processing enzymes
WO1998020025A1 (fr) Methodes diagnostiques permettant de rechercher la sclerodermie chez un patient
US6433137B1 (en) TUP1 sequences from Candida albicans and methods for screening agents for inhibiting virulence in Candida albicans using TUP1
EP1130030A1 (fr) Facteur associé à la différentiation érytroide
US6344554B1 (en) Polynucleotide sequences from Candida albicans encoding polypeptides associated with filamentous growth
AU2004212565A1 (en) Polycystic kidney disease gene
JP2003523758A (ja) 新規転写因子ｃａｒｐ−２
JPH1180196A (ja) ＬｃｋＳＨ３ドメイン結合蛋白質及びそれをコードするＤＮＡ
WO1986006494A1 (fr) Moyens pour le diagnostic in vitro de cellules malignesorigi naires du tube digestif

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载