EP0871659A1 - Proteines de fusion comprenant des proteines regulatrices du cycle cellulaire - Google Patents

Proteines de fusion comprenant des proteines regulatrices du cycle cellulaire

Info

Publication number
EP0871659A1
EP0871659A1 EP97901350A EP97901350A EP0871659A1 EP 0871659 A1 EP0871659 A1 EP 0871659A1 EP 97901350 A EP97901350 A EP 97901350A EP 97901350 A EP97901350 A EP 97901350A EP 0871659 A1 EP0871659 A1 EP 0871659A1
Authority
EP
European Patent Office
Prior art keywords
fusion proteins
seq
purification
cyclin
human
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP97901350A
Other languages
German (de)
English (en)
Other versions
EP0871659A4 (fr
Inventor
Steven Kovacevic
Keith A. Otto
Ramachandra N. Rao
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eli Lilly and Co
Original Assignee
Eli Lilly and Co
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 Eli Lilly and Co filed Critical Eli Lilly and Co
Publication of EP0871659A1 publication Critical patent/EP0871659A1/fr
Publication of EP0871659A4 publication Critical patent/EP0871659A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the present invention provides novel fusion proteins comprising cyclins and CDKs.
  • a preferred embodiement of the invention provides fusion proteins comprising human cyclin Dl and human CDK4.
  • the fusion proteins of the invention optionally contain modifications, which facilitate their purification. Addition of histidine residues to selected constructs allows purification via immobilized metal affinity chromatography. Antigenic determinants allowing monoclonal antibody-based affinity chromatography purification are provided in selected embodiments of the invention. Proetease cleavage sites are incorporated in selected constructs to allow cleavage of the regions incorporated in the cyclin-CDK fusion proteins for purification. Additional modifications which fctcilitate purification include strepavadin binding domains and antigenic determinants for antibody affinity chromatography.
  • Figure 1 is a restriction site and function map of plasmid PK415.
  • Figure 2 is a restriction site and function map of plasmid PK485.
  • Figure 3 is a restriction site and function map of plasmid pK480.
  • the fusion proteins of the present invention comprise cyclins and CDKs linked via various peptide spacers and optionally contain amino acid sequences, which are incorporated to facilitate purification.
  • the DNA sequence (SEQ ID NO:l) encoding a preferred embodiment of the present invention is provided below. 1 GACGAAAGGG CCTCGTGATA CGCCTATTTT TATAGGTTAA TGTCATGATA 51 ATAATGGTTT CTTAGACGTC AGGTGGCACT TTTCGGGGAA ATGTGCGCGG
  • SEQ ID NO:2 The polypeptide encoded by SEQ ID N0:1 is presented below as SEQ ID NO:2.
  • the DNA sequence of SEQ ID NO:l is the preferred coding sequence for the polypeptide of SEQ ID NO:2. Numerous other DNA sequences will also encode the polypeptide of SEQ ID NO:2 due to the degeneracy of the genetic code. All DNA sequences encoding the polypeptide of SEQ ID NO:2 are contemplated by the present invention and thus are within the scope of the present invention.
  • the DNA sequence of SEQ ID N0:1 is a component of the plasmid K415.
  • a restriction site and function map of plasmid K415 is provided in Figure 1.
  • E. coli host cells transformed with K415 were deposited in the NRRL, Northern Regional Research Laboratory, 1815 North University Street, Peoria, Illinois 61604 on or before August 9, 1995 and will be available pursuant to Budapest Treaty requirements upon issuance of a patent in a Budapest signatory country.
  • the NRRL accession number for E. coli/K415 is B-21490.
  • Residues 18 through 27 comprise the epitope recognized by the monoclonal antibody designated myc.
  • Residues 31 though 327 correspond to human cyclin Dl .
  • Residues 331 through 345 are an illustrative "linker” or polypedtide connector.
  • linker or polypedtide connector.
  • linker or polypedtide connector.
  • linker or polypedtide connector.
  • linker polypeptide connector
  • shinge are used interchangeably in describing the present invention and all three terms refer to the sequences of amino acids which are used to connect the cyclin and CDK components of the fusion proteins of the present invention.
  • Residues 346 through 648 correspond to human CDK4.
  • Residues 651 through 660 correspond to strepavadin and were engineered into the molecule to allow facile purification.
  • polypeptide of SEQ ID NO:2 has numerous components which allow great flexibility in purification, but are not required for the ultimate benefit provided by the present invention-a biologically active fusion protein comprising cyclin and CDK components .
  • a most preferred aspect of this embodiment of the present invention is the cyclin Dl-linker-CDK4 component of the molecule. This most preferred aspect is provided below as SEQ ID NO:3.
  • Biologically active fusion protein comprising a member of the cyclin family and the CDK family are further illustrated by the DNA sequence of SEQ ID NO: and the corresponding polypeptide sequence, SEQ ID NO:5.
  • SEQ ID NO:4 is provided immediately below.
  • polypeptide encoded by the sequence of SEQ ID NO:4 is provided below as SEQ ID NO:5.
  • the DNA sequence of SEQ ID NO:4 is the preferred coding sequence for the polypeptide of SEQ ID NO: 5. Numerous other DNA sequences will also encode the polypeptide of SEQ ID NO:4 due to the degeneracy of the genetic code. All DNA sequences encoding the polypeptide of SEQ ID NO:5 are contemplated by the present invention and thus are within the scope of the present invention.
  • the DNA sequence of SEQ ID NO:4 is a component of the plasmid K485.
  • a restriction site and function map of plasmid K485 is provided in Figure 2.
  • E. coli host cells transformed with K485 were deposited in the NRRL, Northern Regional Research Laboratory, 1815 North University Street, Peoria, Illinois 61604 on or before August 9, 1995 and will be available pursuant to Budapest Treaty requirements upon issuance of a patent in a Budapest signatory country.
  • the NRRL accession number for E. coli/K485 is B-21492.
  • the DNA sequence of Sequence ID 4 and the polypeptide encoded thereby comprise human cyclin Dl and human CDK4 which are joined by a polypeptide linker.
  • the distinct functional subcomponents of the polypeptide of SEQ ID NO:5 are described by reference to the amino acid residue numbers provided in SEQ ID NO:5.
  • Amino acid residues 2 through 8 are Histidine residues which were incorporated to allow immobilized metal affinity chromatography purification.
  • Residues 14 through 23 contain the antigenic determinant recognized by the myc monoclonal antibody and thereby allow myc monoclonal antibody based affinity purification.
  • Residues 24 through 28 contain a thrombin cleavage site and were engineered into the polypeptide of SEQ ID NO:5 to allow cleavage of the molecule on the amino side of the human cyclin Dl component.
  • Residues 43 through 329 correspond to human cyclin Dl.
  • Residues 333 through 347 are the polypeptide linker used to join the human cyclin Dl and human CDK4 components of the molecule.
  • Residues 348 through 650 correspond to human CDK4.
  • Residues 653 through 662 were engineered into the molecule to provide a sequence which binds to paramagnetic streptavadin beads and thus allows facile purification of the molecule.
  • the present invention also provides the DNA sequence of SEQ ID NO:6, which is presented below.
  • SEQ ID NO:6 The polypeptide encoded by SEQ ID NO:6 is presented below as SEQ ID NO:7.
  • the DNA sequence of SEQ ID NO:6 is the preferred coding sequence for the polypeptide of SEQ ID NO:7. Numerous other DNA sequences will also encode the polypeptide of SEQ ID NO:6 due to the degeneracy of the genetic code. All DNA sequences encoding the polypeptide of SEQ ID NO:7 are contemplated by the present invention and thus are within the scope of the present invention.
  • the DNA sequence of SEQ ID NO:6 is a component of the plasmid K480.
  • a restriction site and function map of plasmid K480 is provided in Figure 3.
  • E. coli host cells transformed with K480 were deposited in the NRRL, Northern Regional Research Laboratory, 1815 North University Street, Peoria, Illinois 61604 on or before August 9, 1995 and will be available pursuant to Budapest Treaty requirements upon issuance of a patent in a Budapest signatory country.
  • the NRRL accession number for E. coJi/K480 is B-21491.
  • the DNA sequence of SEQ ID NO:6 and the polypeptide encoded thereby comprise human cyclin Dl and human CDK4 which are joined by a polypeptide linker.
  • the distinct functional subcomponents of the polypeptide of SEQ ID NO:7 are described by reference to the amino acid residue numbers provided in
  • Amino acid residues 17 through 22 are Histidine residues which were incorporated to allow immobilized metal affinity chromatography purification.
  • Residues 28 through 37 contain the antigenic determinant recognized by the myc monoclonal antibody and thereby allow myc monoclonal antibody based affinity purification.
  • Residues 38 through 43 contain a thrombin cleavage site and were engineered into the polypeptide of Sequence ID 7 to allow cleavage of the molecule on the amino side of the human cyclin Dl. component.
  • Residues 47 through 343 correspond to human cycl:.n Dl.
  • Residues 347 through 390 are the polypeptide linker used to join the human cyclin Dl and human CDK4 components of the molecule. Residues 391 through 693 correspond to human CDK4. Residues 696 through 705 were engineered into the molecule to provide a sequence which binds to paramagnetic streptavadin beads and thus allows facile purification of the molecule.
  • the molecule of SEQ ID NO:7 shares several features with the molecules of SEQ ID Nos:2 and 5.
  • the polypeptide linker which joins the human cyclin Dl and the human CDK4 portions of the molecule of SEQ ID NO:7 is substantially different from the polypeptide linkers of the molecules of SEQ ID Nos: 2 and 5.
  • the structural dissimilarity of the linkers combined with the biological activity of the fusion proteins of the invention underscores the flexibility in linker selection. Accordingly, the fusion proteins of the present invention are not limited to cyclin-CDK fusion proteins containing the linkers which are specifically exemplified.
  • the fusion protein of SEQ ID NO:7 has the additional features discussed above for allowing great flexibility in choice of purification schemes.
  • the preferred aspect of this embodiment of the present invention is the segment of the molecule comprising the biologically active human cyclin Dl-linker-human CDK4 sequence. This preferred sequence is set forth below as SEQ ID NO:8.
  • proteins of the present invention can be synthesized by a number of different methods. All of the amino acid compounds of the invention can be made by chemical methods well known in the art, including solid phase peptide synthesis, or recombinant methods. Both methods are described in U.S. Patent 4,617,149, herein incorporated by reference.
  • polypeptides may be synthesized by solid-phase methodology utilizing an Applied Biosystems 430A peptide synthesizer (commercially available from Applied Biosystems, Foster City California) and synthesis cycles supplied by Applied Biosystems.
  • Applied Biosystems 430A peptide synthesizer commercially available from Applied Biosystems, Foster City California
  • Protected amino acids such as t- butoxycarbonyl-protected amino acids, and other reagents are commercially available from many chemical supply houses.
  • Sequential t-butoxycarbonyl chemistry using double couple protocols are applied to the starting p-methyl benzhydryl amine resins for the production of C-terminal carboxamides .
  • C-terminal acids the corresponding pyridine-2-aldoxime methiodide resm is used.
  • Asparagine, glutamine, and arginine are coupled preformed hydroxy benzotriazoie esters.
  • the following side chain protection may be used: Arg, Tosyl Asp, cyclohexyl Glu, cyclohexyl Ser, Benzyl
  • the peptide/resin is washed with ether, and the peptide extracted with glacial acetic acid and then lyophilized. Purification is accomplished by size-exclusion chromatography on a Sephadex G-10 (Pharmacia) column in 10% acetic acid.
  • the proteins of the present invention may also be produced by recombinant methods. Recombinant methods are preferred if a high yield is desired.
  • a general method for the construction of any desired DNA sequence is provided in J. Brown, et al.. Methods in Enzvmolocrv. 68:109 (1979) . See also, J. Sambrook, et al.. supra.
  • prokaryotes are used for cloning of DNA sequences in constructing the vectors of this invention.
  • Prokaryotes may also be employed in the production of the protein of interest.
  • the Escherichia coli K12 strain 294 (ATCC No. 31446) is particularly useful for the prokaryotic expression of foreign proteins.
  • a commercially available E. coli strain which is preferred for prokaryotic expression of the fusion proteins of the invention is designated DH10B.
  • DH10B is available from Gibco BRL, P.O. Box 68, Grand Island, N.Y. 14072-0068.
  • Other strains of E. coli which may be used (and their relevant genotypes) include the following.
  • DH5a F ⁇ ( ⁇ 80dlacZDM15) , D(lacZYA-argF)U169 supE44, hsdR17(r ⁇ -/ m ⁇ + ) , recAl, endAl, gyrA96, thi-1, relAl HB101 supE44, hsdS20(rB ⁇ IT ⁇ -) ' recA13, ara-14, proA2 lacYl, galK2, rpsL20, xyl-5, mtl-1, mcrB, mrr JM109 recAl, el4 ⁇ (mcrA), supE44, endAl, hsdRl7(r ⁇ ⁇ , m ⁇ + ), gyrA96, relAl, thi-1, ⁇ (lac-proAB) ,
  • strains are all commercially available from suppliers such as: Bethesda Research Laboratories, Gaithersburg, Maryland 20877 and Stratagene Cloning Systems, La Jolla, California 92037; or are readily available to the public from sources such as the American Type Culture
  • bacterial strains can be used interchangeably.
  • the genotypes listed are illustrative of many of the desired characteristics for choosing a bacterial host and are not meant to limit the invention in any way.
  • the genotype designations are in accordance with standard nomenclature. See, for example, J. Sambrook, et al. , supra.
  • a preferred strain of E. coli employed in the cloning and expression of the genes of this invention is RV308, which is available from the ATCC under accession number ATCC 31608, and is described in United States Patent 4,551,433, issued November 5, 1985. The three E.
  • coli host cells transformed with the vectors described in Figures 1,2 and 3 and discussed in preceding sections will be publicly available upon issuance of a patent in a "Budapest Treaty" country and thus are the preferred means for prokaryotic expression of the fusion proteins which are described herein as illustrative of the fusion proteins of the invention.
  • the fusion proteins produced by the E. coli "deposits" of the invention require solubilization, folding and phosphorylation for complete biological activity. While they are still preferred when substantial amounts of fusion protein are desired, the facile nature of numerous eukaryotic expression systems results in a preference for these systems when modest amounts of the biologically active fusion proteins are desired.
  • the strains of E. coli discussed supra In addition to the strains of E. coli discussed supra.
  • bacilli such as Bacillus subtilis.
  • enterobacteriaceae such as Salmonella tvphimuriu or Serratia marcescans, and various Pseudomonas species may also be used.
  • other bacteria especially Streptomvces, spp., may be employed in the prokaryotic cloning and expression of the proteins of this invention.
  • Promoters suitable for use with prokaryotic hosts include the b-lactamase [vector pGX2907 (ATCC 39344) contains the replicon and b-lactamase gene] and lactose promoter systems [Chang £L_al. , Nature (London) . 275:615 (1978) ; and Goeddel e _ai., Nature (London) .
  • alkaline phosphatase the tryptophan (trp) promoter system
  • vector pATHl ATCC 37695
  • hybrid promoters such as the tac promoter (isolatable from plasmid pDR540 ATCC-37282)
  • other functional bacterial promoters whose nucleotide sequences are generally known, enable one of skill in the art to ligate them to DNA encoding the proteins of the instant invention using linkers or adapters to supply any required restriction sites. Promoters for use in bacterial systems will also contain a Shine-Dalgarno sequence operably linked to the DNA encoding the desired polypeptides. These examples are illustrative rather than limiting.
  • the proteins of this invention may be synthesized either by direct expression or as a fusion protein comprising the protein of interest as a translational fusion with another protein or peptide which may be removable by enzymatic or chemical cleavage. It is often observed in the production of certain peptides in recombinant systems that expression as a fusion protein prolongs the lifespan, increases the yield of the desired peptide, or provides a convenient means of purifying the protein of interest.
  • a variety of peptidases e.g. trypsin which cleave a polypeptide at specific sites or digest the peptides from the amino or carboxy termini (e.g. diaminopeptidase) of the peptide chain are known.
  • particular chemicals e.g.
  • cyanogen bromide will cleave a polypeptide chain at specific sites.
  • the skilled artisan will appreciate the modifications necessary to the amino acid sequence (and synthetic or semi-synthetic coding sequence if recombinant means are employed) to incorporate site-specific internal cleavage sites. See e.g. , P. Carter, "Site Specific Proteolysis of Fusion Proteins", Chapter 13 in PROTEIN
  • the proteins of the present invention may also be produced in eukaryotic systems.
  • the present invention is not limited to use in a particular eukaryotic host cell.
  • a variety of eukaryotic host cells are available from depositories such as the American Type Culture Collection (ATCC) and are suitable for use with the vectors of the present invention.
  • ATCC American Type Culture Collection
  • the choice of a particular host cell depends to some extent on the particular expression vector used to drive expression of the cyclin-CDK fusion protein-encoding nucleic acids of the present invention.
  • Exemplary host cells suitable for use in the present invention are listed in Table I
  • H4IIEC3 Rat Hepatoma ATCC CCL 1600 C127I Mouse Fibroblast ATCC CCL 1616
  • a preferred eukaryotic cell line of use in expressing the fusion proteins of this invention is the widely available cell line AV12-664 (hereinafter "AV12").
  • AV12 This cell line is available from the American Type Culture Collection under the accession number ATCC CRL 9595.
  • the AV12 cell line was constructed by injecting a Syrian hamster in the scruff of the neck with human adenovirus 12 and isolating cells from the resulting tumor.
  • a wide variety of vectors some of which are discussed below, exists for the transformation of such mammalian host cells, but the specific vectors described herein are in no way intended to limit the scope of the present invention.
  • the sequences encoding the illustrative fusion proteins of the invention are easily removed from the deposited E.
  • the pSV2-type vectors comprise segments of the simian virus 40 (SV40) genome that constitute a defined eukaryotic transcription unit-promoter, intervening sequence, and polyadenylation site.
  • SV40 simian virus 40
  • the plasmid pSV2-type vectors transform mammalian and other eukaryotic host cells by integrating into the host cell chromosomal DNA.
  • Plasmid pSV2-type vectors such as plasmid pSV2-gpt, pSV2-neo, pSV2-dhfr, pSV2-hyg, and pSV2-b-globin, in which the SV40 promoter drives transcription of an inserted gene.
  • These vectors are suitable for use with the coding sequences of the present invention and are widely available from sources such as the ATCC or the Northern Regional Research Laboratory (NRRL), 1815 N. University Street, Peoria, Illinois, 61604.
  • the plasmid pSV2-dhfr (ATCC 37146) comprises a murine dihydrofolate reductase (dhfr) gene under the control of the SV40 early promoter. Under the appropriate conditions, the dhfr gene is known to be amplified, or copied, in the host chromosome. This amplification can result in the amplification of closely-associated DNA sequences and can, therefore, be used to increase production of a protein of interest. See, e.g.. R. T. Schimke, Cell,
  • Plasmids constructed for expression of the proteins of the present invention in mammalian and other eukaryotic host cells can utilize a wide variety of promoters.
  • the present invention is in no way limited to the use of the particular promoters exemplified herein.
  • Promoters such as the SV40 late promoter, promoters from eukaryotic genes, such as, for example, the estrogen-inducible chicken ovalbumin gene, the interferon genes, the gluco-corticoid-inducible tyrosine aminotransferase gene, and the thymidine kinase gene, and the major early and late adenovirus genes can be readily isolated and modified to express the genes of the present invention.
  • Eukaryotic promoters can also be used in tandem to drive expression of a coding sequence of this invention. Furthermore, a large number of retroviruses are known that infect a wide range of eukaryotic host cells. The long terminal repeats in the retroviral DNA frequently encode functional promoters and, therefore, may be used to drive expression of the nucleic acids of the present invention.
  • Plasmid pRSVcat (ATCC 37152) comprises portions of a long terminal repeat of the Rous Sarcoma virus, a virus known to infect chickens and other host cells. This long terminal repeat contains a promoter which is suitable for use in the vectors of this invention. H. Gorman, et al. ,
  • the plasmid pMSVi (NRRL B-15929) comprises -34-
  • the mouse metallothionein promoter has also been well characterized for use in eukaryotic host cells and is suitable for use in the expression of the nucleic acids of the present invention.
  • the mouse metallothionein promoter is present in the plasmid pdBPV-MMTneo (ATCC 37224) which can serve as the starting material of other plasmids of the present invention.
  • An especially useful expression vector system employs one of a series of vectors containing the BK enhancer, an enhancer derived from the BK virus, a human papovavirus.
  • the most preferred such vector systems are those which employ not only the BK enhancer but also the adenovirus-2-early region IA (ElA) gene product.
  • the ElA gene product (actually, the ElA gene produces twc products, which are collectively referred to herein as "the ElA gene product”) is an immediate-early gene product of adenovirus, a large DNA virus.
  • a preferred eukaryotic expression vectcr employed in the present invention is the phd series of vectors which comprise a BK enhancer in tandem with the adenovirus late promoter to drive expression of useful products in eukaryotic host cells.
  • the construction and method of using the phd plasmid, as well as related plasmids, are described in U.S. Patents 5,242,688, issued September 7, 1993, and 4,992,373, issued February 12, 1991, all of which are herein incorporated by reference.
  • Escherichia coli K12 GM48 cells harboring the plasmid phd are available as part of the permanent stock collection of the Northern Regional Research Laboratory under accession number NRRL B-18525.
  • the plasmid may be isolated from this culture using standard techniques.
  • the plasmid phd contains a unique Bell site which may be utilized for the insertion of the gene encoding the protein of interest.
  • linkers or adapters may be employed in cloning the gene of interest into this Bell site.
  • the phd series of plasmids functions most efficiently when introduced into a host cell which produces the ElA gene product, cell lines such as AV12- 664, 293 cells, and others, described supra.
  • Transformation of the mammalian cells can be performed by any of the known processes including, but not limited to, the protoplast fusion method, the calcium phosphate co-precipitation method, electroporation and the like. See, e.g. , J. Sambrook, et al. , supra. at 3:16.30-
  • viruses are also appropriate vectors.
  • the adenovirus, the adeno- associated virus, the vaccinia virus, the herpes virus, the baculovirus, and the rous sarcoma virus are useful.
  • Such a method is described in U.S. Patent 4,775,624, herein incorporated by reference.
  • Several alternate methods of expression are described in J. Sambrook, et al. , supra. at 16.3-17.44.
  • eukaryotic microbes such as yeast cultures may also be used.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used eukaryotic microorganism, although a number of other strains are commonly available.
  • the plasmid YRp7 (ATCC-40053) , for example, is commonly used. See, e.g. , L. Stinchcomb, ⁇ t fii. Nature (London) . 282:39 (1979); J. Kingsman e_L_al• Gene. 7:141 (1979); S. Tschemper et al. , Gene, 10:157 (1980).
  • This plasmid already contains the trp gene which provides a selectable marker for a mutant strain of yeast lacking the ability to grow in tryptophan.
  • Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [found on plasmid pAP12BD (ATCC 53231) and described in U.S. Patent No. 4,935,350, issued June 19, 1990, herein incorporated by reference] or other glycolytic enzymes such as enolase [found on plasmid pACl (ATCC 39532)], glyceraldehyde-3-phosphate dehydrogenase [derived from plasmid pHcGAPCl (ATCC 57090, 57091)], hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase, as well as the alcohol dehydrogenase and pyruvate de
  • yeast promoters which are inducible promoters, having the additional advantage of their transcription being controllable by varying growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein [contained on plasmid vector pCL28XhoLHBPV (ATCC 39475) and described in United States Patent No. 4,840,896, herein incorporated by reference], glyceraldehyde 3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose [e.g. GAL1 found on plasmid pRYl21 (ATCC 37658)] utilization.
  • alcohol dehydrogenase 2 isocytochrome C
  • acid phosphatase degradative enzymes associated with nitrogen metabolism
  • metallothionein obtained on plasmid vector pCL28XhoLHBPV (ATCC 39475) and described in United States Patent No
  • yeast enhancers such as the UAS Gal from Saccharomyces cerevisiae (found in conjuction with the CYC1 promoter on plasmid YEpsec--hIlbeta ATCC 67024) , also are advantageously used with yeast promoters.
  • substitutions may be introduced into the protein in a variety of ways, such as during the chemical synthesis or by chemical modification of an amino acid side chain after the protein has been prepared.
  • Alterations of the protein having a sequence which corresponds to the sequences of SEQ ID NO:2, 3, 5, 7 or 8 may also be induced by alterations of the nucleic acid compounds which encodes these proteins.
  • These mutations of the nucleic acid compound may be generated by either random mutagenesis techniques, such as those techniques employing chemical mutagens, or by site-specific mutagenesis employing oligonucleotides.
  • Those nucleic acid compounds which confer substantially the same function in substantially the same manner as the exemplified nucleic acid compounds are also encompassed within the present invention.
  • nucleic acid compounds which comprise isolated nucleic acid sequences which encode SEQ ID NO: 2, 3, 5, 7, and 8.
  • amino acid compounds of the invention can be encoded by a multitude of different nucleic acid sequences because most of the amino acids are encoded by more than one nucleic acid triplet due to the degeneracy of the amino acid code. Because these alternative nucleic acid sequences would encode the same amino acid sequences, the present invention further comprises these alternate nucleic acid sequences.
  • the genes encoding the DNA molecules of the present invention may be produced using synthetic methodology. This synthesis of nucleic acids is well known in the cirt. See, e.g.. E.L. Brown, R. Belagaje, M.J.
  • the DNA segments corresponding to the fusion proteins are generated using conventional DNA synthesizing apparatus such as the Applied Biosystems Model 380A or 380B DNA synthesizers (commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404) which employ phosphoramidite chemistry.
  • Applied Biosystems Model 380A or 380B DNA synthesizers commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404
  • the more traditional phosphotriester chemistry may be employed to synthesize the nucleic acids of this invention. See, e.g. , M.J. Gait, ed.,
  • the DNA sequences of the present invention may be designed to possess restriction endonuclease cleavage sites at either end of the transcript to facilitate isolation from and integration into expression and amplification plasmids.
  • restriction sites are chosen so as to properly orient the coding sequence with control sequences to achieve proper in-frame reading and expression of the molecule.
  • a variety of other such cleavage sites may be incorporated depending on the particular plasmid constructs employed and may be generated by techniques well known in the art.
  • the human cyclin and human CDK coding regions of the desired DNA sequences can be generated using the polymerase chain reaction as described in U.S. Patent No. 4,889,818, which is herein incorporated by reference.
  • the preferred expression systems for use in the present invention are the various Baculovirus systems.
  • the pFastBacl expression system which is commercially available from the Life Technologies group of Gibco BRL Products as
  • Baculovirus expression systems are well known in the art and numerous scientific articles and "methods" books are available on the subject. The present inventors have found the Life Technologies technical literature to provide excellent guidance for producing products of interest via -40 -
  • Baculovirus expression The preferred techniques for Baculovirus expression of the sequences of the present invention are those provided in the product literature. Minor variations such as linker construction and the like are considered in light of the advanced state of this art as too trivial to warrant discussion. In the event skilled artisans elect to depart from the commercially available Baculovirus systems, the present inventors recommend Baculovirus Expression Vectors-A Laboratory Manual, O'Reilly, David R., Miller, Lois K., and Luckow, Verne A., W. H. Freeman and Company, New York, New York as a source of additional information on any protocol required for successful expression of polypeptides in Baculovirus systems.
  • Skilled artisans wishing to practice the recombinant DNA aspects of the present invention are directed to the NIH guidelines for information on research involving recombinant DNA molecules.
  • a copy of the current guidelines can be obtained from Office of Recombinant DNA Activities, National Institutes of Health, Building 31, Room 4B11, Bethesda, MD 20892. Compliance with all such current regulations regarding vector selection, expression of human and animal genes and containment requirements is required by law.
  • the examples are intended to further illustrate the present invention and are not to be interpreted as limiting on the scope thereof.
  • a sample of NRRL B-21490 is obtained from the NRRL.
  • the sample is cultured according to well known procedures using standard media containing Ampicillin for selection of the desired transformed phenotype.
  • Plasmid isolation is accomplished in accordance with standard methodology. See e.g. Sambrook and Maniatis, supra.
  • the desired fragment is excised from plasmid pK415 (See Figure 1) by sequential digestion with the restriction endonucleases, Ascl and Sse 83871.
  • the Ascl digestion is performed using New England Biolabs reagents and protocols.
  • the restriction endonuclease Sse 83871 is available from Takara Biomedicals via PanVera Corp., 565 Science Drive, Madison, WI 53711 (1 800 791-1400) .
  • the vendors instructions on digestion procedures are recommended.
  • pFastBacI is digested with BssHII (New England Biolabs) and PstI (New England Biolabs) in accordance with vendors instructions and the large fragment is isolated.
  • a restriction site and function map of pFastBacI is provided at page 5 of the GibcoBRL/Life Technologies Catalog Number 10359-016 (Instruction Manual-BAC-TO-BACTM Baculovisur Expression System) .
  • the catalog is herein incorporated by reference.
  • the fusion protein encoding sequence is then ligated into the pFastBacl vector using standard ligation reagents and conditions.
  • Preferred ligation rea ⁇ rents and conditions are set forth at pages 7 and 8, Section 3.3, of GibcoBRL/Life Technogies Catalog Number 10359-016.
  • Page 5 of GibcoBRL/Life Technogies Catalog Number 10359-016 provides DNA sequence information and restriction endonuclease cleavage sites for the multiple cloning site of pFastBacl and is therefore useful in the event skilled artisans elect to fragment the sequence from p415 or excise it by other than the restriction endonucleases suggested above and utilize linkers to facilitate the subsequent ligation into pFastBacl.
  • Transposition of the pFastBacl vector comprising the fusion protein of plasmid pK415 into DHlOBaclO (competent cells are provided as part of the expression kit accompanying pFastBacl in Catalog Number 10359-16) is conducted in accordance with the teachings of page 8 of GibcoBRL/Life Technogies Catalog Number 10359-016.
  • Isolation of Recombinant Bacmid DNA is accomplished in accordance with the teachings of pages 8 and 9 of GibcoBRL/Life Technogies Catalog Number 10359-016.
  • Transfection of Sf9 cells with recombinant Bacmid DNA, harvesting and storage of the recombinant Baculovirus, and Infection of Insect Cells with recombinant Baculovirus particles is accomplished with the teachings at pages 9 and 10 of GibcoBRL/Life Technogies Catalog Number 10359-016.
  • Baculovirus expression systems were constructed in substantial accordance with the teachings of Example 1.
  • Plasmid pK480 from E. coli/pK485 was used in place of plasmid pk415 as the source of the DNA sequence encoding the fusion protein of interest.
  • Baculovirus expression systems were constructed in substantial accordance with the teachings of Example 1. Plasmid pK485 from E. coli/pK480, NRRL number B21491, was used in place of plasmid pK415 as the source of the DNA sequence encoding the fusion protein of interest. With the exception of the substitution of plasmid pK480 for plasmid pK415 all steps of this Example 3 were carried out in conformance with the teachings of Example 1.
  • Affinity chromatography resins for fusion protein purification are readily constructed from commercially available reagents using techniques well known in the art.
  • CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals) is the preferred matrix for linkage of appropriate monoclonal or polyclonal antibodies to allow antibody-based affinity purification of the fusion proteins.
  • Pharmacia Fine Chemicals publishes "Affinity Chromatography-Principles and Methods". This manual sets forth all steps in preparing the affinity resin and performing the antibody-based affinity purification steps. The manual is available from Pharmacia Fine Chemicals, Box 175, S-751 04 Uppsala 1, Sweden.
  • Resuspension Buffer 50mM HEPES pH 7.5, 0.32M Sucrose, 0.1 mM PMSF, l.OmM DTT, ImM EDTA and 80mM ⁇ -glycerophosphate.
  • 500 ⁇ L of the SF9 extract was added to 200 ⁇ L of Streptavidin Paramagnetic Beads (Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711-5399) and the mixture was incubated at room temperature for 45 minutes .
  • the paramagnetic beads were pelleted at room temperature using a MagneSphere Technology Magnetic separation stand (Promega) .
  • the beads were washed three times with 1 mL of 1XPBS/25 mg/ml BSA (or 0.1% Tween 20) at room temperature.
  • Elution Buffer A is 25mM HEPES pH 7.5, 0.1 mM PMSF, ImM d- Biotin O.lmM DTT, 20mM ⁇ -glycerophosphate, ImMNaF, lOmM Sodium Orthovanadate and 10% glycerol.
  • the purified fusion protein was stored at -70' C until ready for use.
  • the extract agarose mixture was incubated at 4'C for 4 hours. The mixture was gently agitated during the incubation. The agarose was then pelleted by centrifugation at 2000xg for two minutes and then washed three times with
  • Elution Buffer B is 50mM HEPES pH 7.5, 300mM NaCl, 250mM Imidizole, 0.1 mM PMSF,, lOmM Sodium Orthovanadate, ImM NaF and 20mM ⁇ -glycerophosphate.
  • the eluted fusion protein was dialyzed in 3.0L of Dialysis Buffer overnight at 4'C.
  • Dialysis Buffer is 25mM HEPES ph 7.5, 10% glycerol, 0.01% Triton-X, 0.ImM PMSF,20mM ⁇ -glycerophosphate, ImM NaF and lOmM Sodium Orthovanadate.
  • the dialyzed fusion protein was stored at -70 * C.
  • the lysates were loaded onto a Poros Q column at 5ml/L of infected insect cells.
  • the Poros Q column was washed with 10-column volumes of Equilibration buffer.
  • the column was eluted with 0-1M NaCl gradient collecting 2ml/fraction.
  • the column fractions were assayed for activity and peak fractions were pooled.
  • the resulting pool was diluted to give a final NaCl concentration of lOOmM.
  • the dilute pool fractions were loaded onto a Hydroxapatite column equilibrated with 25mM Tris pH 8.0, 0.1 mM PMSF, ImM EDTA, and 20 ⁇ g/ml leupeptin.
  • the Hydroxapatite column was washed with 10-column volumes of Equilibration buffer and eluted cyclin Dl and cdk4 with 0- 400mM potassium phosphate, pH 7.5. Column fractions were assayed for activity and the peak fractions pooled. The eluted protein was stored at -70C.
  • IP Lysis Buffer 50mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, ImM DTT, 2.5mM EGTA, 0.1% Tween 20, 10% Glycerol, O.lmM PMSF, 500UM ATP, lOmM ⁇ -glycerophosphate, ImM NaF, and O.lmM orthovanadate
  • the cells were sonicated three times on ice for 10 seconds each time, and the lysates were clarified for 5 minutes at 10,000 rpm and °C.
  • kinase redactions with various amounts of partially purified cyclin Dl and cdk4 from insect cells contained: 50mM HEPES pH 7.5, lOmM MgCl2, 0.2 ⁇ Ci [gamma- 32 P]ATP (Amersham, 6,000 Ci/mmol) , 0.12 ⁇ g pRb (full-length protein from Immuno Pharmaceutics), O.lmM sodium orthovanadate, lOmM ⁇ -glycerophate and ImM NaF in a total of 100 ⁇ L.
  • Kinase reactions with immunoprecipitated fusion protein on Protein-G-Agarose (Boehringer Mannheim) from the REF cell line were resuspended in 50 ⁇ l of Kinase Reaction Buffer (50mM HEPES pH 7.5, lOmM MgCl2, lO.O ⁇ Ci
  • Kinase Reaction Buffer 50mM HEPES pH 7.5, lOmM MgCl2, lO.O ⁇ Ci
  • nitrocellulose membranes were blocked with 5% dry milk in 1 x PBS for 30 to 60 minutes. Membranes were washed 3x, 10 minutes for each wash, in lx PBS/0.1% Tween 20. The membrane was incubated with primary antibody (cyclin Dl or cdk4) at a 1:2000 dilution in IX PBS/0.1% Tween 20/1% Milk for 1 hour at room temperature then washed 3X for 10 minutes each in IX PBS/0.1% Tween 20.
  • primary antibody cyclin Dl or cdk4
  • the membrane was then incubated with a secondary antibody (horse radish peroxidase conjugated goat anti-mouse or rabbit antibody from Amersham) at a 1:1000 dilution in IX PBS/0.1% Tween 20/1% Milk for 25 minutes at room temperature.
  • the membrane was washed 6X in PBS/0.1% Tween 20, 2X in IX PBS, and developed with Amersham ECL detection reagents. The results indicated that the fusion protein had substantially the same amount of activity as the individual subunits.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne de nouvelles protéines de fusion comprenant des cyclines et des CDK. Une réalisation préférée de l'invention porte sur des protéines de fusion comprenant de la cycline D1 humaine et de la CDK4 humaine. Les protéines de l'invention présentent facultativement des modifications qui en facilitent la purification. L'addition de restes histidine à des produits de recombinaison sélectionnés permet une purification par chromatographie d'affinité à métaux immobilisés. Certaines réalisations de l'invention concernent des déterminants antigéniques permettant la purification par chromatographie d'affinité aux anticorps monoclonaux. Des sites de clivage de protéase sont incorporés dans les produits de recombinaison sélectionnés pour permettre le clivage des régions incorporées dans les protéines de fusion cycline-CDK destinées à la purification. Des modifications supplémentaires facilitant la purification concernent des domaines de liaison de la strépavadine et des déterminants antigéniques destinés à la chromatographie d'affinité aux anticorps.
EP97901350A 1996-01-05 1997-01-03 Proteines de fusion comprenant des proteines regulatrices du cycle cellulaire Withdrawn EP0871659A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US962996P 1996-01-05 1996-01-05
US9629P 1996-01-05
PCT/US1997/000140 WO1997025345A1 (fr) 1996-01-05 1997-01-03 Proteines de fusion comprenant des proteines regulatrices du cycle cellulaire

Publications (2)

Publication Number Publication Date
EP0871659A1 true EP0871659A1 (fr) 1998-10-21
EP0871659A4 EP0871659A4 (fr) 2000-04-26

Family

ID=21738815

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97901350A Withdrawn EP0871659A4 (fr) 1996-01-05 1997-01-03 Proteines de fusion comprenant des proteines regulatrices du cycle cellulaire

Country Status (4)

Country Link
EP (1) EP0871659A4 (fr)
AU (1) AU1526897A (fr)
CA (1) CA2241849A1 (fr)
WO (1) WO1997025345A1 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5986055A (en) 1997-11-13 1999-11-16 Curagen Corporation CDK2 interactions
US6432668B1 (en) 1997-12-30 2002-08-13 Chiron Corporation Polynucleotides encoding human cyclin-dependent kinase (hPFTAIRE)
KR20000019889A (ko) * 1998-09-16 2000-04-15 성재갑 곤충세포를 이용한 cdk4/p16 복합체의 제조방법
WO2000036118A2 (fr) 1998-12-16 2000-06-22 Chiron Corporation KINASE DEPENDANT DE LA CYCLINE DE TYPE HUMAIN (hPNQALRE)
US6716626B1 (en) 1999-11-18 2004-04-06 Chiron Corporation Human FGF-21 nucleic acids
JP2003516731A (ja) 1999-11-18 2003-05-20 カイロン コーポレイション ヒトｆｇｆ−２１遺伝子および遺伝子発現産物
US20020082205A1 (en) 2000-03-08 2002-06-27 Nobuyuki Itoh Human FGF-23 gene and gene expression products
DE10113776B4 (de) * 2001-03-21 2012-08-09 "Iba Gmbh" Isoliertes streptavidinbindendes, kompetitiv eluierbares Peptid, dieses umfassendes Fusionspeptid, dafür codierende Nukleinsäure, Expressionsvektor, Verfahren zur Herstellung eines rekombinanten Fusionsproteins und Verfahren zum Nachweis und/oder zur Gewinnung des Fusionsproteins
US20150299764A1 (en) * 2012-09-05 2015-10-22 Universite Libre De Bruxelles Immobilised cyclin-dependent kinase 4 fusion proteins and uses thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5196320A (en) * 1989-09-20 1993-03-23 Abbott Biotech, Inc. Method of producing engineered binding proteins

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
EMBL Database, Heidelberg, FRG Swissprot accession number P11802 01 October 1989 HANKS, S.K.: "Cell division protein kinase 4 (EC 2.7.1.-) (cyclin-dependent kinase 4) (PSK-J3)" XP002131314 *
EMBL Database, Heidelberg, FRG Swissprot accession number P24385 01 March 1992 MOTOKURA, T. ET AL.: "G1/S-specific cyclin D1 (PRAD1 oncogene) (BCL-1 oncogene)" XP002131313 *
RAO, R.N. ET AL.: "Conditional transformation of rat embryo fibroblast cells by a cyclin D1-cdk4 fusion gene" ONCOGENE, vol. 18, no. 46, November 1999 (1999-11), pages 6343-6356, XP000870164 *
See also references of WO9725345A1 *
TAM, S.W. ET AL.: "Differential expression and regulation of Cyclin D1 protein in normal and tumor human cells: association with Cdk4 is required for Cyclin D1 function in G1 progression" ONCOGENE, vol. 9, no. 9, September 1994 (1994-09), pages 2663-2674, XP000864419 *

Also Published As

Publication number Publication date
CA2241849A1 (fr) 1997-07-17
EP0871659A4 (fr) 2000-04-26
WO1997025345A1 (fr) 1997-07-17
AU1526897A (en) 1997-08-01

Similar Documents

Publication Publication Date Title
CN108949721B (zh) 表达磷脂酶d的重组菌株及应用
CN112375748B (zh) 基于水疱性口炎病毒载体的新型冠状病毒嵌合重组疫苗及其制备方法与应用
CN1930299B (zh) 红球菌属的腈水合酶
WO1995016044A2 (fr) Fusions de peptides et de proteines a des molecules de thioredoxine, des molecules apparentees a la thioredoxine, et a des molecules apparentees a la thioredoxine modifiees
CN107541525A (zh) 一种基于CRISPR/Cas9技术介导山羊Tβ4基因定点敲入的方法
CN102146371B (zh) 高抗草甘膦突变基因及其改良方法和应用
CN112941038B (zh) 基于水疱性口炎病毒载体的重组新型冠状病毒及其制备方法与应用
KR20130098089A (ko) 인터루킨-6와 결합할 수 있는 신규한 폴리펩타이드
EP0871659A1 (fr) Proteines de fusion comprenant des proteines regulatrices du cycle cellulaire
CN112251464B (zh) 一种基因点突变的诱导方法
WO1995034664A1 (fr) Procede de detection d'interactions de ligands
US20030145345A1 (en) LexA DNA binding domain optimized for arabidopsis species
CN111748546B (zh) 一种产生基因点突变的融合蛋白及基因点突变的诱导方法
CN107058367A (zh) 高产普鲁兰酶的重组枯草芽孢杆菌的构建方法
CN110066801B (zh) 一种枯草芽孢杆菌中唾液酸诱导表达元件及构建方法
KR20150007077A (ko) 면역글로불린 g에 대한 리피바디 및 그 용도
CN114317584B (zh) 新型转座子突变株文库的构建系统、新型转座子突变文库和应用
KR102064475B1 (ko) 뮤코닉산 생산용 미생물 및 그 제조방법
DK2516702T3 (en) PROTEIN DISPLAY
KR20210053932A (ko) 사람 백혈구 항원에 의해 제공된 무작위화된 펩타이드 라이브러리
CN110857320A (zh) 能够结合诺如病毒的抗体、使用其的复合材料、检测装置和方法
AU758623B2 (en) Kinase wee1 fusion protein compositions, nucleotide sequences, expression systems, and methods of use
CN110857321B (zh) 能够结合诺如病毒的抗体、使用其的复合材料、检测装置和方法
CN106676135A (zh) Alb‑uPA‑teton慢病毒载体及其制备方法和应用
CN112625138A (zh) 新冠状病毒检测用多位点重组抗原及其制备方法

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19980703

AK Designated contracting states

Kind code of ref document: A1

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

A4 Supplementary search report drawn up and despatched

Effective date: 20000309

AK Designated contracting states

Kind code of ref document: A4

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

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20000518