WO2005040375A1 - A fungal immuromodulatory protein produced by microorganisme and uses thereof - Google Patents

A fungal immuromodulatory protein produced by microorganisme and uses thereof Download PDF

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Publication number
WO2005040375A1
WO2005040375A1 PCT/CN2004/001044 CN2004001044W WO2005040375A1 WO 2005040375 A1 WO2005040375 A1 WO 2005040375A1 CN 2004001044 W CN2004001044 W CN 2004001044W WO 2005040375 A1 WO2005040375 A1 WO 2005040375A1
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protein
host cell
immunomodulatory protein
ganoderma
yeast
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PCT/CN2004/001044
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English (en)
French (fr)
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WO2005040375A9 (fr
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Jiunn-Liang Ko
Yu-Lu Huang
Tzu-Chih Chen
Hsu-Wei Hung
Ho-Lung Jiang
Ching-Lung Hu
Cheng-Chun Kuan
Hsuan-Ju Thou
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Yeastern Biotech Co., Ltd.
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Priority to CNB2004800268679A priority Critical patent/CN100462437C/zh
Priority to JP2006526506A priority patent/JP2007535299A/ja
Priority to KR1020067005474A priority patent/KR100888850B1/ko
Priority to EP04762178A priority patent/EP1666592A4/en
Priority to US10/572,563 priority patent/US8163519B2/en
Publication of WO2005040375A1 publication Critical patent/WO2005040375A1/zh
Publication of WO2005040375A9 publication Critical patent/WO2005040375A9/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/064—Saccharomycetales, e.g. baker's yeast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/04—Antipruritics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a modified nucleic acid molecule, which translates a fungal immunomodulatory protein (Fungal Immunomodulatory Protein) and can be expressed better in fungi.
  • the present invention also relates to an expression vector comprising the nucleic acid molecule, a host cell transformed with the expression vector, a method for expressing a protein of the present invention in a transformed host cell, use of the host cell containing the protein of the present invention, and purification of a fungal immunomodulatory protein Methods.
  • the protein of the present invention has a wide range of immunomodulatory activities.
  • the invention therefore further relates to the use of the protein of the invention in a cosmetic or pharmaceutical composition and to a food or feed additive composition containing the protein of the invention.
  • the present invention relates to a method for orally ingesting a fungal immunomodulatory protein or a protein fused with a fungal immunomodulatory protein to modulate immune activity. Background technique
  • glycophilic proteins There are some glycophilic proteins (Lectin) in the fruit bodies or mycelia of plants or mushrooms. They have immunomodulatory and patoprotective activities in medical reports, and may have the function of scavenging free radicals (Lin JM et al., Aw J Chin Med. 1993; 21 (1): 59-69).
  • Glycoprotein ⁇ -galactoside ( ⁇ -galactoside) isolated from mistotoe plants is a specific glycoprotein, which promotes both in vitro and in vivo Hyperplasia of cytokine (Gabius HJ et al., Anticancer Res. 1992 May-Jun; 12 (3): 669-75).
  • Ganoderma is a Chinese herbal medicine with great economic value.
  • ganoderma lucidum red
  • tree tongue ganoderma i. G. applanatum (brown)
  • ganoderma lucidum G. tsugae
  • red ganoderma G. sinense
  • black G. oregonense
  • Some edible mushrooms such as Ganoderma lucidum, straw mushroom, and enoki mushroom, have similar amino acid sequences and immunoregulatory functions in purified proteins.
  • Such proteins are named fungal immunomodulatory proteins (Fungal Immunomodulatory Protein) or FTP (Ko JL Eur. J. Biochem. 1995; 228: 224-249).
  • Figure 3 shows a comparison of the amino acid sequences of three fungal immunomodulatory proteins. Among them, ganoderma is the most representative. Ganoderma lucidum is a member of healthy foods, which is of great help in maintaining human health. '
  • Ganoderma has antiallergic properties (Chen H. Y et al., J. Med. My col. 1992; 33: 505-512) and protects liver function (Lin JM et al., Am J Chin Med. 1993; 21 (1): 59-69), anti-tumor and enhance immune function, but mostly limited to crude extracts (Horner WE et al., Allergy 1993; 48: 110-116) or small molecule compounds (Kawagi shi H., et al., Phytochemistry 1993; 32: 239-241).
  • Baker's yeast is the safest food microorganism and the main model cell for modern studies of genetics, physiological metabolism, and molecular biology. Because it is safer than other microorganisms, in addition to traditional applications in food processing (such as bread and wine), in recent years, the bread yeast genetic engineering system has been successfully applied to the production of medicinal proteins, such as hirudin, Hemoglobin, Urokinase, human serum albumin (HAS), insulin-like growth factor-1 (IGF-1), granulocyte macrophage colony ⁇ Granulocyte macrophage colony stimulating factor (GM-CSF), hepatitis B vaccine, etc. (Romanos MA et al., Yeast 1992; 8: 423-488).
  • the appropriate host system can be selected according to the characteristics of the heterologous protein (food and pharmaceutical grade proteins are mainly Saccharomyces cerevisiae).
  • Ganoderma immunomodulatory protein can be obtained from Ganoderma lucidum by many complicated extraction methods at this stage (US Patent No. 5334704).
  • US Patent No. 5334704 Because ganoderma only grows in the natural environment and grows on old trees in high mountains, the production of ganoderma cannot provide a large number of high-quality ganoderma immunomodulatory proteins for research.
  • Prior to the present invention a large number of inventions for the preparation of Ganoderma immunomodulatory protein have not been made.
  • glycoproteins isolated from Ganoderma lucidum have immunomodulatory activity.
  • the invention pointed out that the preparation of the glycoprotein can directly cultivate Ganoderma ganoderma (raycelia), and then extract it from the hypha to obtain an aqueous solution, and the glycoprotein can be purified from the extract. Because of the high purity requirements of glycoproteins, it is difficult to apply the patented technology to industrial applications. Summary of the invention
  • the present invention provides a nucleic acid molecule comprising the nucleic acid sequence shown in FIG. 1 (1).
  • the invention also provides an expression vector and a host cell transformed with the vector. .
  • the present invention further provides a method for preparing a host cell containing a Ganoderma immunomodulatory protein.
  • the present invention further provides ganoderma immunomodulatory protein prepared according to this method.
  • the invention further provides the use of a host cell containing a ganoderma immunomodulatory protein.
  • the invention further provides a method for purifying Ganoderma immunomodulatory protein from a host cell transformed by the expression vector.
  • the present invention further provides a composition containing the Ganoderma immunomodulatory protein of the present invention.
  • the composition can be applied to cosmetics, pharmaceuticals, food and serving additives.
  • the present invention also provides a method for orally administering Ganoderma lucidum immunomodulatory protein or a protein fused with a fungal immunomodulatory protein to an individual to regulate immune activity.
  • yeast can more faithfully express heterologous proteins from ganoderma, including protein biological activity and various protein modification effects (such as saccharification and formation of disulfide chains).
  • the DNA sequence (S E Q I D N O: 1) of the codons of the ganoderma immunomodulatory protein yeast (Sacc 3 ⁇ 4r ⁇ . Ces cere 'sise) is as follows:
  • the nucleic acid molecule provided by the present invention can be connected to a section of extracellular secreted information and sequences, and can allow the ganoderma immunomodulatory protein produced by the host cell to be directly secreted into the extracellular culture medium, thereby reducing the cost during purification and isolation.
  • the nucleic acid molecule provided by the present invention is interchanged with nucleic acid molecules of other genes to form a new nucleic acid sequence.
  • This new nucleic acid sequence can be expressed in a performance system.
  • a new recombinant protein not only has the original immunoregulatory ability of Ganoderma immunomodulatory protein, but also has the characteristics of expressing another gene (Wei sbart RH, Cancer Lett. 2003; 195 ( 2): 211- 9.).
  • ganoderma immunomodulatory protein still retains biological activity in the digestive system, using ganoderma immunomodulatory protein and another protein as a fusion protein, the ganoderma immunomodulatory protein can be used as a de livery system.
  • Another protein Bringing to the target cell produces the biological activity of the protein.
  • the fusion protein can be used orally to enable all the proteins constituting the fusion protein to perform their functions.
  • the present invention provides a vector comprising the nucleic acid molecule of the present invention.
  • This vector can be transformed into a host cell. Depending on the application and condition, the vector can be circular or integrated.
  • the present invention provides a host cell transformed with the expression vector. It can be a bacterial, fungal, or yeast cell. This host cell may be in the form of a whole or a broken cell.
  • This host cell can be Saccharorayces cerevisiae, Pichia pastoris, Hansenula polymorpha, Candida uti lis, Candida boidinii), Candida mal tosa, Kluyverorayces lactis, Yarrowia lipolytica, Western yeast (Schwanniomyces occidentalis), Schizosaccaromyces pombe ), To lopsis, (Arxula adeninivorans), or Aspergillus (Aspergillus nidulans. (A. nidulans), Aspergillus niger (A. niger), Aspergillus awamori (A. a araori) , A. oryzae), Tricoderma (T. reesei). Plant cell-can also be used as a host cell.
  • the invention provides a method for preparing a host cell containing ganoderma immunomodulatory protein.
  • This method includes (a) constructing an expression vector containing the modified Ganoderma immunomodulatory protein gene sequence, (b) using the expression vector to transform into a host cell, and (c) using an appropriate culture method so that the host cell can express Produce protein.
  • the protein sequence referred to in the present invention is shown in FIG. 3.
  • the host cell referred to in the present invention may be Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Candida uti lis, Candida boidin Yeast (Candida boidinii), Candida maltosa, Kluyveromyces lactis, Yarrowia lipolytica, Schwanniomyces occidentalis, Western yeast (Schizosaccaromyce s pombe), Torulopsis, (Arxula adeninivorans), or Aspergillus (A. nidulans), A. niger, A. awaraori ), A. oryzae), Tricoderma (T. reesei).
  • Plant cells can also be used as a host cell.
  • the most suitable host cell is Saccharomyces cerevisiae).
  • the expression vector referred to in (b) is pYBlOlFIP- yeast or pYBlOlFIP- lz (Fig. 4 (II) and Fig. 4 (III)).
  • the nucleic acid sequence of the specific codon expressed by the fungus that expresses the protein is shown in Figure 1 (1), which is the DNA nucleic acid sequence of the preferred codon of baker's yeast i Saccharomyces cerevisiae.
  • the nucleic acid sequence of a fungal preference codon is determined by the rate at which tRNA translates during translation (Sorensen MA, Pedersen S. J Mol Biol. 1991; 222 (2): 265-80).
  • the invention provides the use of a host cell containing ganoderma immunomodulatory protein.
  • yeasts containing ganoderma immunomodulatory protein intact cells can be directly administered orally and can exert biological activity in the organism without any purification steps, thereby reducing production costs.
  • the present invention provides a method for purifying Ganoderma immunomodulatory protein from transformed host cells.
  • This method includes (a) dissolving the fermented cells in a solvent; (b) breaking the cells; (c) adjusting the pH to between 4 and 5; (d) removing cell fragments; (e) centrifuging the supernatant Remove large impurities; (f) add the supernatant to a column that has been equilibrated to pH (4- 5); (g) precipitate Ganoderma immunomodulatory protein.
  • step (a) 5-50 mM phosphate buffer (pH 7.0) is added to the fermented cells at a ratio of 1: 5-1: 20; (C) Adjust the pH to between 4-5; (d) Centrifuge at 3,000xg for 15 minutes to remove cell fragments; (e) Centrifuge at 12,000xg for 1 minute to remove large impurities; (f) Remove The supernatant was added to a CM Sepharose column (Amersham Biosciences) at an equilibrium pH of 4-5; (g) Ganoderma immunomodulatory protein was precipitated with 20-50 mM acetic acid at pH 4-5.
  • CM Sepharose column Amersham Biosciences
  • the present invention also provides ganoderma immunomodulatory protein prepared by this method.
  • the present invention relates to the service route of purified Ganoderma immunomodulatory protein or host cells containing Ganoderma immunomodulatory protein. It can be administered by intravenous injection, intraperitoneal injection, intramuscular injection, oral, transmucosal absorption, skin contact absorption or immersion in liquid or other applicable delivery routes. Among them, oral administration
  • the application method is a priority method. When applied to freshwater or marine animals, such as fish, it can also be immersed in a liquid containing Ganoderma immunomodulatory protein and absorbed into the fish through the gills.
  • the purified ganoderma immunoregulatory protein or host cell containing ganoderma lucidum regulatory protein provided by the present invention can be widely used in milks, fish, crustaceans, livestock products, and poultry.
  • the mammals and poultry here are pigs and chickens.
  • the fish referred to are grouper, salmon, trout.
  • the nail shells are shrimp, lobster, grass shrimp, monk shrimp, white shrimp.
  • the present invention provides an oral application of the Ganoderma immunomodulating protein composition to achieve the function of immunoregulation.
  • the Ganoderma immunomodulatory protein can be prepared from Ganoderma or by the method disclosed in the present invention.
  • Relevant research based on Ganoderma immunomodulatory protein points out that the use in medicine can reduce inflammation, reduce and avoid allergic reactions, regulate immunity, prevent diabetes, improve asthma, antiviral and bacterial infections, and resist organ rejection. Applied to food or feed, it can prolong life, improve immunomodulatory activity, feed conversion rate, and reduce the immune response caused by urgency (Black PH, Brain Behav Immun. 2003 Oct; 17 (5): 350-64.) .
  • the ganoderma immunomodulatory protein provided by the present invention can be used for direct oral administration to regulate immune disorders.
  • Ganoderma immunomodulatory protein can be obtained from E. coli (£ coli or fungi such as baker's yeast i Sacchar ces cerevisiae).
  • the present invention provides an immunological test, such as Western blot or Enzyme-Linked Immunosorbent Assay (ELISA) to check Chinese medicine (such as Ganoderma), food, beverages, The content of cosmetics, feed additives, medicines or other fungal immunomodulatory protein-containing compositions, Test basis for the content of epidemic-regulated protein.
  • ELISA Enzyme-Linked Immunosorbent Assay
  • composition containing the protein of the present invention will include other dosage-formed additives known in the prior art. These additives include sweeteners, thickeners, flavoring agents and the like suitable for oral use.
  • the proper dosage of the composition of the present invention depends on the type, severity and stage of the disease, and also varies from patient to patient. Determining the proper dosage generally requires weighing the benefits of the treatment of the invention against the dangers or adverse physical and mental side effects of the treatment of the invention. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 (1) is a preferred codon, FIP-yeast, of a modified baker's yeast ⁇ Saccharomyces cerevisiae) using the gene to synthesize Ganoderma immunomodulatory protein according to the present invention
  • Figure 1 (II) shows the original codon & (gene sequence, FIP-lz) of Ganoderma lucidium.
  • Fig. 2 is a gene synthesis of Ganoderma lucidum immunomodulatory protein gene using the present invention, wherein Fig. 2 (I) is a yeast carei s se) The forward and reverse primers required for the preferred codons.
  • Figure 2 (II) shows the forward and reverse primers required for the original codons of Ganoderma lucidium.
  • Figure 3 is a comparison of the amino acid sequences of Ganoderma lucidium, Ganodejrma tsugae and Flamnulina velutips. Bold text indicates that the three amino acids are the same.
  • Figure 4 shows the construction of three plasmids (I) pYBlOl (II) pYB101-FIP-yeast (III) ⁇ ) ⁇ -FIP-lz.
  • Figure 5 shows the contents of Ganoderma immunomodulatory protein expressed by yeast (DY150 or BY4741).
  • Figure 5 (I) shows the transformation with pYBlOl-FIP-yeast expression vector;
  • Figure 5 (II) shows the transformation with pYBlOl-FIP-lz expression vector.
  • M represents protein molecular weight standards: 175, '83, 62, 47. 5, 32. 5, 25, 16. 5 and 6. 5 Kd.
  • S stands for Ganoderma immunomodulatory protein standard prepared from transformed E. coli.
  • Lanes 1-2 show two transformants of yeast DY150 transformed by pYB101-FIP-yeast expression vector
  • lanes 3-4 show two transformations of BY4741 yeast transformed by pYB101-FIP-yeast expression vector Strains
  • lanes 5-6 show two transformed strains of BY4741 yeast transformed by the pYB101-FIP-lz expression vector
  • lanes 7-8 show two transformed DY150 yeast transformed by pYBlOl-FIP-lz expression vector Transformation strain.
  • Figure 6 shows the nucleic acid sequence and restriction enzyme positions of Ganoderma immunomodulatory protein extracellularly expressed, where (I) is a forward primer and (II) is a reverse primer.
  • Figure 7 shows the nucleic acid sequence and restriction enzyme positions of the ct-factor leader sequence contained in pYBlOls and pYBlOls-FIP vectors. Restriction enzyme positions are indicated in bold type. The a-factor leader sequence and the Ganoderma immunomodulatory protein gene are indicated by arrows.
  • Fig. 8 is a construction diagram of pYBlOls plasmid (I) and pYB101s-FIP plasmid (II).
  • Figure 9 shows the Ganoderma immunomodulatory protein expressed extracellularly by pYBlOls-FIP transformed BY4741.
  • M represents a protein molecular weight standard: 175, 83, 62, 47. 5, 32. 5, 25, 16. 5 and 6.5 Kd.
  • S represents a standard of Ganoderma immunomodulatory protein prepared from transformed E. coli Product.
  • Lanes 1-3 show the expression levels of pYBlOls-FIP-transformed BY4741 on the first, second and third days after galactose induction.
  • Figure 10 shows the amount of IFN-r produced by human peripheral blood cells under different preparations of Ganoderma lucidum immunoregulatory protein, cultured with human peripheral blood cells (PBL) with different concentrations of protein.
  • blank indicates the PBS control group.
  • Batchl represents the supernatant after the first 50 liter fermentation process was broken.
  • Batch2 represents the supernatant after the second 50-liter fermentation process.
  • Yeast represents the generally commercially available yeast powder and the supernatant after sterilization.
  • PHA stands for phytohemagglutinin.
  • Figure 11 (1) is the purification analysis of Ganoderma lucidum immunomodulatory protein in AKTA TM explorer 10S system.
  • F3, F4 and F5 are the third, fourth and fifth tubes to collect samples.
  • FIG. 11 (II) SDS-PAGE electrophoresis picture of Ganoderma immunomodulatory protein purified by AKTA TM explorer 10S system.
  • M represents a protein molecular weight standard: 175, 83, 62, 47. 5, 32. 5, 25, 16. 5 and 6. 5 Kd.
  • STD represents a Ganoderma immunomodulatory protein standard prepared from transformed E. coli. F3, F4, and F5 collect samples for the third, fourth, and fifth tubes.
  • Figure 12 shows the results of the detection of Ganoderma immunomodulatory protein in a 50L fermenter by yeast containing Ganoderma immunomodulatory protein.
  • M represents a protein molecular weight standard;
  • S represents a Ganoderma immunomodulatory protein purification standard produced by E. coli.
  • Figure 13 shows the results of NBT analysis of grouper head kidney cells purified by different concentrations of Ganoderma immunomodulatory protein. Cont represents the PBS control group.
  • Figure 14 shows intraperitoneal injection of Ganoderma lucidum immunomodulatory protein into the abdominal cavity of a grouper, and then the grouper was infected with the red color virus to observe its mortality.
  • PBS means intraperitoneal injection of 0.1 ml of PBS before injection of virus, 0.25 ⁇ g / 0.1ml of PBS means intraperitoneal injection of 0.25 ⁇ g / 0.1ml of purified Ganoderma immunoregulatory egg before injection of virus, white, 1 ⁇ g / 0.
  • Lml means intraperitoneal injection of 1 ⁇ g / 0.1 ml of purified Ganoderma immunomodulatory protein before virus injection
  • 5 ⁇ g / 0.1 ml means intraperitoneal injection of 5 ⁇ g / 0.1 ml of purified Ganoderma immunomodulatory protein before virus injection .
  • FIG. 15 shows that the grouper juveniles are infected with Vibrio spp. After being fed with yeast containing Ganoderma immunomodulatory protein, and the survival rate of grouper juveniles is observed.
  • PBS represents the phosphate buffer control group. 20 mg, 4 mg, and 0.8 mg indicate that the grouper was fed the yeast containing Ganoderma immunomodulatory protein at the above-mentioned dose before the Vibrio inoculation. Control indicated that the grouper was fed 20 mg of yeast—commercially available.
  • FIG. 16 shows the sensitized Balb / c mice treated with Z3 ⁇ 4rp.
  • mice The spleen cells of the mice were taken out, and then stimulated with Ganoderma immunomodulatory protein or Der extract to observe the production of IFN-r in spleen cells.
  • FlP-gts represents Ganoderma immunomodulatory protein.
  • i3 ⁇ 4r stands for dust mite allergen (der p 2).
  • Control represents the PBS control group.
  • Figure 1 (I) shows baker's yeast ( ⁇ feccAarowces The DNA sequence of your favorite codon! J FIP- yeast;
  • Figure 1 (II) is the DNA sequence FIP- lz of the original codon of G. lucidum (Gmode band_ / i / cii i). According to the known method Biol. Chem.
  • the known ganoderma immunomodulatory protein gene sequence is used to design nucleic acid sequences with different codon preferences, and according to these two different codes (J, order the required DNA forward primer and reverse primer (Mission biotech, Taiwan), and set the restriction enzyme cleavage site DNA sequence of the expression vector on both sides of the gene (k / I and excitation) primers, as shown in Figure 2 (I) and Figure 2 ( ⁇ ), followed by DNA polymerization, polymerase chain reaction, and finally sequenced (Miss ion biotech Co., Ltd., Taiwan) confirmed that the gene synthesis of ganoderma immunomodulatory protein with two different codons was completed.
  • a galactose promoter, an auxotrophic screening gene Ura3, and a 2 ⁇ ori yeast proliferation origin were added to a pYB plasmid (Yeas tern Biotech Co., Ltd, Taiwan), that is, Complete ⁇ (1). And can be transformed into general baker's yeast (such as: BY4741 or DY150).
  • the expression vector ⁇ and the above-synthesized gene fragments of different preference codons each function with an appropriate DNA restriction enzyme ⁇ Bam Hi and Pml I, New England Biolabs, USA).
  • Each of the DNA solutions subjected to the restriction enzymes was purified through a purification DNA column (PCR Cleaning Up Purification Kit, VIogene, Taiwan) to obtain ganoderma immunomodulatory protein gene DNA and expression vector DNA synthesized by two different codons. Then the ratio of moles of moles of DNA concentration to the ratio of 3: 1 (synthetic gene: vector) was ligated with Ganoderma immunomodulatory protein DNA and yeast expression vector DNA by T 4 DNA ligase (New England Biolabs, Beverly, USA). (L igation). Named as pYBlOl- FIP- yeast (the yeast codon, (II)) and pYBlOl- FIP-LZ (the original codon for Ganoderma, (111)).
  • ⁇ -FIP- yeast or pYBlOl-FIP-LZ was transformed into E. coli DH5 ⁇ , respectively.
  • DNA extraction reagents Mini-M Plasmid DNA Extraction Kit, Viogene, Taiwan
  • pYB101-FIP-yeast and ⁇ 1 ⁇ -FIP-LZ can be obtained from E. coli by transformation.
  • Ganoderma immunomodulatory protein expression vector DNA (pYB101-FIP-yeast and pYBlCH-FIP-lz) of two different codons obtained in Example 1 were transformed into 1 ⁇ g of baker's yeast (BY4741 or DY150) and yeast. ⁇ ⁇ ⁇ ⁇ ] work together (Yeast Transformation Kit, Yeastern Biotech Co., Ltd., Taiwan).
  • Example 3 Quantitative Transformation of Baker's Yeast Expressing Ganoderma Immunomodulatory Protein by Western Blot 1. Yeast The collected samples were diluted with the PBS (Sigma, USA) cells into 0. 5 0D M. nm , dissolved in 2x SDS sample buffer (Sigma, USA), heated at 100 ° C for three minutes, after cooling, take 10 ⁇ 1 of each diluted sample and perform SDS-PAGE electrophoresis at 100 volts. Electrophoresis is completed after electrophoresis to the appropriate location (Tfe tore (1970) 227, 680-685).
  • M represents a protein molecular weight standard
  • S represents a Ganoderma immunomodulating protein purification standard produced by E. coli
  • groups 1-4 are codons for improving the Ganoderma immunomodulating protein.
  • Groups 2-5 are Ganoderma immunomodulatory proteins expressed by codons of the original Ganoderma immunomodulatory protein.
  • Fig. 5 (1) the expression of Ganoderma lucidum immunomodulatory protein is clearly seen in groups 1-3, while the group of 5--8 cannot detect the raw protein of Ganoderma lucidum. This result shows that the codons of the modified Ganoderma immunomodulatory protein can be highly expressed in yeast.
  • the FIP nucleic acid sequence of the yeast preferred codon was selected as a template for exogenous production genes, and BY4741 was selected as a host cell for exocrine production.
  • the FIP of the yeast preferred codon was selected as the template DNA (see Figure 1 (I) for the sequence), and the pYBl Ols expression vector (Yeastern Biotech Co., Ltd. Taiwan) forward primer and reverse primer ( Figure 6 (I and II)) (Mission Biotech Co., Ltd. Taiwan) 0 FIP of yeast preference codons as template DNA ( P YB101-FIP -yeast), and the above-mentioned forward and reverse primers were used to synthesize a DNA fragment of the Ganoderma immunomodulatory protein gene by PCR.
  • the pYBlOls-FIP expression vector was constructed according to the steps of Example 1, as shown in FIG. 8. DNA sequencing was used to determine the entire gene sequence (Mission Biotech Co., Ltd. Tai
  • the pYBlOls-FIP expression vector was transformed into DH5 ⁇ according to the procedure of Example 1. Extract a large amount of DNA from the pYBlOls- FIP expression vector.
  • the pYBlOls-FIP Meda vector DNA extracted above was transformed into Baker's Yeast BY4741 according to the procedure of the second embodiment.
  • the above-mentioned correct transformant containing pYBlOls-FIP expression vector was selected from YNBD agar (containing 0.024% histidine, 0.0072% leucine, 0.0012% methionine).
  • Example 2 According to the procedure of Example 2, the above-mentioned correct transformant containing the pYBlOls-FIP expression vector was inoculated into 50 ml of a culture solution. Galactose induction was used to produce Ganoderma lucidum immunoregulatory protein, and samples were collected daily.
  • Example 6 Testing the transformed baker's yeast expressing Ganoderma immunomodulatory protein yield using SDS-PAGE method: According to the procedure of Example 2, the collected sample was subjected to SDS-PAGE. This SDS-PAGE was then stained with Coomassie brilliant blue G-250 (sigma) for 30 minutes, and then decolored to the protein band with a destain solution (20% methanol, 10% acetic acid) ( protein band) is clearly visible (see Figure 9).
  • Example seven 50 liter fermentation process.
  • a Baker's Yeast strain (BY4741 / pYB101-FIP- yeast) determined to have the yield of Ganoderma lucidum disease-free regulatory protein was selected by Western blotting method and inoculated to 50ml YNBD Relevant drugs required for the defective strain were added to the culture medium (Example 2: Step 2), and cultured at 30 ° C. and a rotation speed of 250 ⁇ 111 for 24 hours.
  • the cultured 500ml bacterial solution was centrifuged and washed with YNB culture solution three times to remove glucose, and then 50 liters of YNBG culture solution (0.17% Bacto amino acid-free yeast nitrogen group, 0.5% Ammonium sulfate, 2% galacto Sugar (Sigma, USA)) and related drugs required for the defective strain (Example 2: Step 2), the concentration of bacteria was adjusted to 0.1 0D 6 commandeelle nm at a culture temperature of 25-30 ° C, 400- 500 rpm, pH 4. 5-5. 5 (acid and alkali are adjusted by 2M NaOH and HC1 (Sigma, USA)), pressure in the tank 0.2-2. 4kg / cm 2 , filtered through 0.2 ⁇ m Fermentation was performed for 3 days under the conditions of air ventilation of 40-50 L / min.
  • the sampled bacterial solution was used in the third embodiment to determine the yield of Ganoderma lucidum immunomodulatory protein by Western blotting method. The results are shown in Figure 12. It is known from FIG. 12 that 50 liters of fermented product contains Ganoderma immunomodulatory protein.
  • Example 8 Activity test of Ganoderma immunomodulatory protein expressed by baker's yeast
  • Glass bead grinding method The yeast samples collected after cultivation are centrifuged, and the yeast pellets that have been collected are washed with PBS, and centrifuged three times. Collect the yeast pellets and add 0.4 mm glass beads with the same volume as the pellet. Shake the mixture for 20 seconds at 4 ° C and ice bath for 1 minute. Repeat this step five times. After five minutes of centrifugation at 15,000 rpm at 4 ° C, the supernatant was used as the total cell extract. Centrifuge the specimen and leave the supernatant. Filter the supernatant with a 0.2um membrane (Sartorius) and leave to set aside.
  • Human peripheral blood mononuclear cells were isolated by adding heparin to prevent heparinized peripheral blood from adults and adding Ficoll-paque culture medium (Amersham Biosciences) to centrifugation.
  • 1 X 10 6 cells / ml cells were cultured in RPMI1640 medium (GIBC0, 10% FBS, 100 g / ml streptomycin, 100 units / ml penicillin, and 200 mM sodium L-glutamate). Grand Island, NY) in a 24-well tissue culture plate (Nunc, Roskilde, Denmark). Incubate at 37 ° C for 0, 24 or 48 hours.
  • the control group and normal yeast group did not stimulate the production of IFN-Y.
  • the amount of IFN-Y produced by purified Ganoderma immunomodulatory protein at different doses were compared with ganoderma immunomodulatory protein produced by other methods, and a higher IFN-Y content was obtained after 48 hours of culture. It is also shown that the 50 liter fermentation sample will stimulate the content, which means that the FIP produced by the present invention is biologically active, not because of yeast.
  • Example 9 Purification of Ganoderma lucidum immunoregulatory protein in 50L fermentation process 1. After fermentation, the yeast must be reconstituted into 10% bacteria solution with 20 mM phosphate buffer solution (Sigma) pH 6.0. The pH value is adjusted by 1M acetic acid. The cells were then broken with a sterilizer (Basic Z model, Constant System Ltd. UK) at a pressure of 30 Kpsi, and adjusted to pH 4-5.
  • a sterilizer Basic Z model, Constant System Ltd. UK
  • FIG. 1 The analysis chart is shown in Figure 1 1 (I).
  • F3, F4 and F5 represent the 3rd, 4th and 5th collection tube samples, and the 3rd and 4th collection tube samples have significantly higher mAU values.
  • Samples from the 3rd, 4th, and 5th collection tubes were subjected to SDS-PAGE.
  • the results are shown in Figure 11 (II), where M is the protein molecular weight standard and STD is the Ganoderma immunomodulatory protein purification standard produced by E. coli.
  • Figure 11 (II) shows that the third and fourth collection tube samples have the same molecular weight as the standard, and their purity is quite high. And the subsequent use of Western blot method is considered FIP.
  • the fish species is Epinephelus malabaricus, which was purchased from a private farm in the Tingding District, Kaohsiung.
  • the average body length was 7. 25 cm and the average weight was 5.9 g. It is kept in an environment with a salinity of 33 ppt and a water temperature of 25 ° C.
  • the Ganoderma immunomodulatory protein solution purified from Example IX was injected intraperitoneally into grouper fry at a concentration of 5, 1, 0.25 ⁇ g / tail / 0.1 ml, and a PBS solution was injected as a control group.
  • the environment was kept in the original word for 14 days. Observe and record deaths every day. No death occurred in all groups after two weeks, showing that the concentration of Ganoderma immunomodulatory protein used in the experiment did not cause a toxic response to grouper.
  • Example 11 Zfer-treated Balb / c mice were sensitized, and spleen cells of the mice were removed, and then stimulated again with Ganoderma immunomodulatory protein or Der extract to observe the production of IFN-r in spleen cells. .
  • mice fed with Ganoderma immunomodulin group fed once every two days (2 ⁇ g) until Balb / c mice were killed on day 15 and spleen cells were removed.
  • mice intraperitoneal injection of 10 on the first day, 10 g of intraperitoneal injection every 7 days, and 10 ⁇ g of spray every 7 days, killed Balb / c mice on the 15th day, and removed the spleen cell.
  • spleen cells Treatment of spleen cells: The isolated spleen cells were cultured at 1 X 10 ⁇ cell s / ml in RPMI 1640 medium containing 5% FBS, and then treated with 10 ⁇ g / ral Zfer ° or 2 ⁇ g / ml FIP. deal with. After 48 hours of culture, the culture fluid was collected and analyzed for IFN-r content.
  • the spleen cells will increase after stimulated with Ganoderma immunomodulatory protein IFN-r aio (0.7 pg / ml). This shows that oral ganoderma immunomodulatory proteins can immunomodulate, especially directly inhibiting allergic reactions.

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Description

利用微生物制备的真菌免疫调节蛋白及其用途 技术领域
本发明涉及一段改良后的核酸分子， 该核酸分子转译真菌免疫调节蛋白（Fungal Immunomodulatory Protein) , 可以在真菌有较佳的表达。 本发明还涉及包含该核酸分子的 表达载体、 此表达载体转形的宿主细胞、 表达本发明的蛋白质于此转形宿主细胞的方法、 包含本发明蛋白质的宿主细胞的用途及纯化真菌免疫调节蛋白的方法。 本发明的蛋白质具 有广泛的免疫调节功能（immunomodulatory activity)。 因此本发明进一步涉及本发明蛋白 质在化妆品或医药组合物的用途及含本发明蛋白质的食品或饲料添加物组合物。 最后， 本 发明涉及个体口服真菌免疫调节蛋白或与真菌免疫调节蛋白融合的蛋白以调节免疫活性的 方法。 背景技术
在植物或蕈类植物的子实体或菌丝体有些亲醣蛋白质 (Lectin)，在医学报导上有免疫 调节（Immunomodulatory)及保护肝脏 ( patoprotective)的活性， 可能具有清除自由基的 功能（Lin J. M. et al. , Aw J Chin Med. 1993 ; 21 (1)： 59-69)。 由桑寄生（mistletoe)植 物分离出的亲醣蛋白 β _半乳糖苷 （β -galactoside ) 为专一性的亲醣蛋白， 不论在活体 外 n vi tro)及活体中 ( n vivo)皆会促进细胞激素（cytokine)的增生（Gabius H. J. et al.， Anticancer Res. 1992 May- Jun ; 12 (3)： 669-75)。 在以 Balb/C小鼠作为疾病模式测试中， 亲醣蛋白质及重组亲醣蛋白质都可抑制肿瘤的形成。 （Couraud P. O. et al. , J. Biol. Chem. 1989 ; 264 : 1310—1316)。
灵芝（Ganoderma)是很有经济价值的中药，灵芝的种类很多例如： 灵芝 lucidum) (红色）, 树舌灵芝 i G. applanatum) (棕色), 松衫灵芝 (G. tsugae) (红色 紫炅芝 ( G. sinense) (黑色) ( G. oregonense) (暗核色）。
一些食用菇类， 例如灵芝、草菇及金针菇中所纯化的蛋白质中皆具有类似的氨基酸序 列及免疫调节功能， 这类蛋白质被命名为真菌类免疫调节蛋白（Fungal Immunomodulatory Protein)简称 FTP (Ko J. L. , Eur. J. Biochem. 1995 ; 228 : 224-249)。 图 3为三种真菌 类免疫调节蛋白氨基酸序列的比较。其中尤以灵芝最具代表性。灵芝为健康食品中的一员， 对维持人体健康有莫大助益。 '
过去研究发现灵芝具有抗过敏性 （Chen H. Y et al. , J. Med. My col. 1992 ; 33： 505-512) 、 保护肝脏功能 (Lin J. M. et al. , Am J Chin Med. 1993 ; 21 (1)： 59-69)、 抗 肿瘤及增强免疫功能， 但大多局限于粗萃取物 (Horner W. E. et al. , Allergy 1993 ; 48： 110-116)或小分子化合物方面研究 (Kawagi shi H.， et al. , Phytochemistry 1993 ; 32： 239-241)。 日本明治制药则从灵芝纯化出免疫调节蛋白（LZ- 8)， 发现具有抑制全身过敏反 应、 治疗肝癌及预防糖尿病等功能 （Kino K. et al. , J. Biol Chem. 1989 ; Jan 5 ; 264 (1)： 472- 8)。柯（1995)亦从另一可食用的菇类- 金针菇中获得免疫调节蛋白（FlP-fve) ;发现该 蛋白质与 LZ - 8及免疫球蛋白的重链区的蛋白结构有相当程度的相似性，在老鼠足趾肿胀实 验中发现有抑制全身性过敏反应， 因此得知这类蛋白质具有免疫调节活性， 其调节机制为 促进 IFN- γ， IL-2 及 TNF- α 基因的转录作用。 （Ko J. L. , Eur. J. Biochem. 1995 ; 228 : 224-249)。 '
面包酵母菌是最安全的食品微生物，也是近代研究遗传、生理代谢及分子生物学的主 要模式细胞。 由于其安全性高于其它微生物， 除了传统运用于食品加工 (如面包、酒类)外， 近年来面包酵母菌遗传工程系统已被成功应用于药用蛋白质的生产，如水蛭素（Hirudin)、 血红蛋白 （Hemoglobin) 、 尿激酶 （Urokinase ) 、 人类血清蛋白 （human serum albumin, HAS ) 、 似胰岛素生长因子- 1 (insul in-like growth factor- 1， IGF- 1)、 粒细胞巨噬细 胞集落朿 Ij激因子 (granulocyte macrophage colony stimulating factor, GM-CSF)、 B型 肝炎疫苗等（Romanos M. A. et al. , Yeast 1992 ; 8 : 423-488)。 可依异源蛋白质的特性， 选择适当的宿主系统 (食品及药品级蛋白质以面包酵母 Saccharomyces cerevisiae为主）。
在现阶段可以利用许多复杂的萃取方法从灵芝中获得灵芝免疫调节蛋白（美国专利第 5334704号）。 然而由于灵芝只长在自然环 i竟中， 且多生长于高山的老树上， 因此灵芝的产 量无法稳定提供大量高品质的灵芝免疫调节蛋白供研究使用。 在本发明之前亦未曾有大量 制备灵芝免疫调节蛋白的发明。
先前的研究 (美国专利第 5334704号）禾 !j用萃取纯化的方法生产灵芝免疫调节蛋白的 产量并不好。若直接利用大肠杆菌生产， 则有内毒素（endotoxin)的问题， 必须花费高额经 费来纯化， 从而造成成本的增加。 利用哺乳类细胞生产所需成本甚高， 亦有病毒或朊毒体 (prion ) 污染的危险。
在先前的研究中（美国专利第 5334704号）公开了从灵芝 Ganode皿)分离的醣蛋白 具有免疫调节活性。 该发明指出， 此醣蛋白的制备可以直接培养灵芝 Ganoderma) 躯 (raycel ia) , 再从菌丝萃取并得到一水溶液， 自此萃取物纯化出该醣蛋白。 因为醣蛋白纯度 要求很高， 该专利的技术要用在工业的应用上是很困难。 发明内容
发明摘述
本发明提供一种核酸分子， 包含图 1 (1)所示的核酸序列。 本发明还提供表达载体及该载体转形的宿主细胞。 .
本发明另提供制备含灵芝免疫调节蛋白的宿主细胞的方法。
本发明另提供依此方法所制备的灵芝免疫调节蛋白。
本发明另提供含灵芝免疫调节蛋白的宿主细胞的用途。
本发明另提供由该表达载体所转形的宿主细胞纯化灵芝免疫调节蛋白的方法。
本发明另提供含本发明灵芝免疫调节蛋白的组合物。该组合物能应用于化妆品、 医药 品、 食品及伺料添加物。
本发明另提供给个体口服灵芝免疫调节蛋白或与真菌免疫调节蛋白融合的蛋白以调 节免疫活性的方法。
发明详述
由于面包酵母菌与灵芝同属真菌类， 故酵母菌能较忠实地表达来自灵芝的异源蛋白 质， 包括蛋白质的生物活性及各种蛋白质修饰作用 （如醣化作用， 及双硫链的形成） 。
本发明中灵芝免疫调节蛋白酵母菌（Sacc ¾r^. ces cere 'sise)喜好密码子的 DNA核 酸序列（S E Q I D N O : 1 )如下:
ATGTCTGATAC TGCTTTGATT TTCAGATTGG CTTGGGATGT
TAAGAAGTTG TCTTTCGATT ACACTCCAAA CTGGGGTAGA
GGTAACCCAA ACAACTTCAT TGATACTGTT ACTTTCCCAA
AGGTTTTGAC TGATAAGGCT TACACTTACA GAGTTGCTGT
TTCTGGTAGA AACTTGGGTG TTAAGCCATC TTACGCTGTT
GAATCTGATG GTTCTCAAAA GGTTAACTTC TTGGAATACA
ACTCTGGTTA CGGTATTGCT GATACTAACA CTATTCAAGT
TTTCGTTGTT GATCCAGATA CTAACAACGA TTTCATTATTG
CTCAATGGA ACTGA
本发明提供的核酸分子能衔接一段胞外分泌的讯息、序列，可以让宿主细胞生产的灵芝 免疫调节蛋白直接分泌到细胞外的培养液中， 从而使得纯化分离时可以降低成本。
本发明提供的核酸分子与其它基因的核酸分子连換形成一新的核酸序列。此新的核酸 序列可以表现在一表现系统中。 而由此新的核酸序列产生新的重组蛋白（fusion protein) 不只具有灵芝免疫调节蛋白原有的免疫调节能力， 也同时具备表现另一个基因的特性 (Wei sbart RH, Cancer Lett. 2003 ; 195 (2) : 211- 9. )。 因为灵芝免疫调节蛋白在消化系统 中仍保有生物活性， 利用灵芝免疫调节蛋白与另一个蛋白质作为融合蛋白（fus ion protein) , 灵芝免疫调节蛋白可以作为一种携带系统（de l ivery system)， 将另一个蛋白质 带到标的细胞产生该蛋白的生物活性。 此外口服该融合蛋白， 也能使组成融合蛋白质的蛋 白质皆发挥其功能。
本发明提供一载体， 该载体包含本发明的核酸分子。 此载体可以转形至宿主细胞。 视 应用于不同状况及用途， 该载体可以是环状 (circular)或嵌入（integrated)的质粒。
本发明提供被上述表达载体转形的宿主细胞。 It匕宿主细胞可以是细菌、真菌细胞或酵 母菌细胞。 此宿主细胞可以是完整的或打破的细胞形式。
此宿主细胞可以是面包酵母菌（Saccharorayces cerevisiae )、巴氏毕赤酵母（ Pichia pastoris ) 、 汉逊酵母 (Hansenula polymorpha) 、 产朊假丝酵母 ( Candida uti lis ) 、 博伊丁假丝酵母 （Candida boidinii ) 、 麦牙糖念珠菌（Candida mal tosa) , 乳酸克鲁维 酵母 (Kluyverorayces lactis) , 解月旨酵母 (Yarrowia lipolytica) , 西方酵母 (Schwanniomyces occidentalis) , 粟酒裂殖酉孝母 (Schizosaccaromyces pombe) , 球拟酵母 属 (To lopsis)， (Arxula adeninivorans) , 或曲毒属 (Aspergillus ) (构巢曲霉. ( A. nidulans) , 黑曲霉（A. niger) , 泡盛曲霉（A. a araori) , 米曲菌（ A. oryzae) ) ,木霉 (Tricoderma) (里氏木霉 (T. reesei) )。 植物细胞-也可当作是一种宿主细胞。
本发明提供一个制备含灵芝免疫调节蛋白宿主细胞的方法。 此方法包括 (a)构筑一个 含改良后灵芝免疫调节蛋白基因序列的表达载体，（b)利用此表达载体转形到一个宿主细胞 内， （c)使用适当的培养方法， 使宿主细胞可以表达产生蛋白质。 本发明所指蛋白质序列在 图 3。 本发明所指宿主细胞可为面包酵母菌 (Saccharomyces cerevisiae)，巴氏毕赤酵母 (Pichia pastoris) ,汉逊酵母 (Hansenula polymorpha) , 产阮假丝酵母 (Candida uti lis) , 博伊丁假丝酵母（Candida boidinii) , 麦牙糖念珠菌 (Candida maltosa) , 乳酸克鲁维酵母 (Kluyveromyces lactis) , 角军月旨酵母 (Yarrowia lipolytica)， 西方酵母 (Schwanniomyces occidentalis) , 粟酒裂殖酉孝母 (Schizosaccaromyce s pombe) , 球拟酵母属 (Torulopsis)， (Arxula adeninivorans) , 或曲霉属 (Aspergillus ) (构巢曲霉. ( A. nidulans) , 黑曲霉 (A. niger) , 泡盛曲霉 (A. awaraori) , 米曲菌 ( A. oryzae) ) ,木霉（Tricoderma) (里氏木 霉 (T. reesei) ) . 植物细胞也可当作是一种宿主细胞。 在步骤 b与 c中， 最适的宿主细胞 为面包酵母 Saccharomyces cerevisiae) 。 在（b)所指的表达载体为 pYBlOlFIP- yeast 或 pYBlOlFIP- lz (图 4 (II)及图 4 ( III ) )。 所指具体的表达蛋白质真菌喜好密码子的核 酸序列说明见图 1 (1)， 为面包酵母 i Saccharomyces cerevisiae) 喜好的密码子的 DNA 核酸序列。 真菌喜好密码子的核酸序列是取决于其在转译（translation)时 tRNA转译的速 率（Sorensen MA， Pedersen S. J Mol Biol. 1991 ; 222 (2) : 265—80)。 本发明提供含灵芝免疫调节蛋白宿主细胞的应用。例如包含有灵芝免疫调节蛋白完整 细胞的酵母菌， 可以直接以口服方式施用并可以在生物体中发挥生物活性， 不用任何纯化 的步骤， 从而减少生产上的成本。
本发明提供一种从转形的宿主细胞中纯化出灵芝免疫调节蛋白的方法。此方法包含 (a) 将发酵后的细胞溶于一溶剂中； （b)打破细胞； （c)调整 pH值到 4- 5之间； （d) 去除细胞破 片；（e)上清液离心去除大的杂质； （f)将上清液加入已经平衡好 pH值（4- 5 )的管柱中； （g) 析出灵芝免疫调节蛋白。 其中纯化步骤中的（a)用 5-50 mM 磷酸盐缓冲液（phosphate buffer) pH 7. 0, 以 1 : 5-1 : 20比例加入发酵后的细 '胞； （b)以破菌机进行破菌； （c) 调整 pH值到 4-5之间; (d) 3, 000xg离心 15分钟， 去除细胞破片； (e) 12， OOOxg离心 1分钟， 去除大的杂质； （f)将上清液加入已平衡 pH值为 4-5 的 CM Sepharose管柱中（Amersham Biosciences) ； (g)以 20- 50mM pH4- 5的醋酸析出灵芝免疫调节蛋白。
本发明也提供由此方法制备的灵芝免疫调节蛋白。
本发明提俱有关纯化的灵芝免疫调节蛋白或含灵芝免疫调节蛋白宿主细胞的服用途 径。 服用方式可以是用静脉注射、 腹腔注射、 肌肉注 ¾"、 口服、 经黏膜层吸收、 皮肤接触 吸收或以浸泡于液体中的形式或其它可应用的传递 (del ivery)路径。 而其中以口服应用方 式为优先考虑方法。 应用在淡水或海水动物， 如鱼类时， 也可以浸泡在含有灵芝免疫调节 蛋白的液体中， 通过鳃吸附到鱼体内。
本发明提供的纯化的灵芝免疫调节蛋白或含灵芝兔疫调节蛋白宿主细胞可以广泛使 用于喃乳类、 鱼类、 甲壳类及畜产、 家禽类。 这里所 的哺乳类及家禽类为猪、 鸡。 所指 鱼类是石斑鱼、 鲑鱼、 鳟鱼。 所指甲壳类是虾、 龙虾、 草虾、 斑节虾、 白虾。
本发明提供口服包含本发明灵芝免疫调节蛋白组合物的应用而达到免疫调节的功能。 此灵芝免疫调节蛋白可以是从灵芝制备出来或由本发明所公开的方法而来。 基于灵芝免疫 调节蛋白的相关研究指出， 在医药上使用可以减少发炎、 降低及避免过敏反应、 调节免疫 力、 预防糖尿病、 改善气喘、 抗病毒及细菌的感染及抗器官排斥。 应用于食品或饲料的添 加上， 可以延长生命、 增进免疫调节活性、 饲料转换率以及降低紧迫所引起的免疫反应 (Black PH, Brain Behav Immun. 2003 Oct ; 17 (5) : 350—64. )。
本发明提供的灵芝免疫调节蛋白可以以直接口服的使用方式来调节免疫失调的疾病。 灵芝免疫调节蛋白可以是从大肠杆菌 （£ coli 或真菌例如面包酵母菌 i Sacchar呵 ces cerevisiae)制备获得 。
本发明提供一种免疫学试验， 例如西方墨点法 (Western blot)或酶联免疫吸附反应 (Enzyme-Linked Immunosorbent Assay (ELISA) )用来检领 !j中药（如灵芝）、 食品、 饮料、 化 妆品、 饲料添加物、 药物或其它含真菌免疫调节蛋白组合物的含量， 作为各产品中真菌免 疫调节蛋白含量的检验基准。 (Molecular Cloning: A Labora tory Manual, 2nd Ed.， pp : 18. 60-18. 75. )。
包含本发明蛋白质的组合物会包含其它先前技术所知的 有剂型的添加物质，这些添 加物质包括适合口服使用的甜味剂（sweetener)、 填充物 (thickener)及调味物质 (flavoring agent)等。
本发明组合物的适当剂量依照疾病的类型、 严重程度及吋期（stage)而定， 也因病人 的差异而不同。 确定适当的剂量一般须衡量本发明治疗所带来的好处与本发明治疗所产生 的危险或对身体身心有害副作用的平衡。 附图说明
图 1 (1)为本发明利用基因合成灵芝免疫调节蛋 白的改良成面包酵母菌 { Saccharomyces cerevisiae) 所喜好的密码子， FIP- yeast；
图 1 (II)为灵芝 Ganoderma lucidium)原有的密码子 & (基因序列， FIP- lz。
图 2 为本发明利用基因合成灵芝免疫调节蛋白基因， 其中图 2 (I)为酵母菌
Figure imgf000008_0001
carei s se)喜好的密码子所需的正向及反向引物， 图 2 (II) 为灵芝 (Ganoderma lucidium)原有的密码子所需的正向及反向引物。
图 3为灵芝 ( Ganoderma lucidium)， 松衫灵芝 ( Ganodejrma tsugae)禾口 Flamnulina velutips的氨基酸序列比较。 粗体字表示三者相同的氨基酸。
图 4为（I) pYBlOl (II) pYB101-FIP-yeast (III) ρΥΒΙΟΙ - FIP- lz三种质粒构筑图。 图 5为酵母菌 (DY150或 BY4741)所表达灵芝免疫调节蛋白的含量。 其中图 5 (I)表示 以 pYBlOl- FIP-yeast表达载体进行转形； 图 5 (II) 表示以 pYBlOl-FIP-lz表达载体进行 转形。 图中， M表示蛋白质分子量标准品: 175, '83， 62， 47. 5, 32. 5, 25, 16. 5及 6. 5 Kd。 S 表示由转形的大肠杆菌所制备的灵芝免疫调节蛋白标准品。 第 1-2 道表示 pYB101-FIP-yeast 表达载体所转形 DY150 酵母菌的两个转形株， 第 3-4 道表示 pYB101-FIP-yeast 表达载体所转形 BY4741 酵母菌的两个转形株， 第 5-6 道表示 pYB101-FIP-lz表达载体所转形 BY4741酵母菌的两个转形株，第 7-8道表示 pYBlOl- FIP-lz 表达载体所转形 DY150酵母菌的两个转形株。
图 6为胞外表达灵芝免疫调节蛋白的核酸序列及限制酶位置， 其中 （I)为正向引物， (II)为反向引物。
图 7为 pYBlOls及 pYBlOls- FIP载体所包含的 ct -因子领导序列的核酸序列及限制酶 位置。 限制酶位置以粗体字标示。 a -因子领导序列及灵芝免疫调节蛋白基因以箭头表示。
图 8为 pYBlOls质粒 (I)及 pYB101s-FIP质粒 (II)构筑图。 图 9为 pYBlOls - FIP转形 BY4741所胞外表达出来的灵芝免疫调节蛋白。 其中， M表 示蛋白质分子量标准品： 175, 83, 62， 47. 5, 32. 5, 25, 16. 5及 6. 5 Kd. S表示由转形 的大肠杆菌所制备的灵芝免疫调节蛋白标准品。 第 1-3道表示半乳醣诱导后第一、 第二及 第三天 pYBlOls- FIP转形 BY4741的表达量。
图 10为不同灵芝免疫调节蛋白制备下，用不同浓度的蛋白质与人类周边血细胞 (PBL) 一起培养下， 人类周边血细胞产生 IFN- r的量。 blank表示 PBS对照组。 Batchl表示第一 次 50升发酵产程破菌后的上清液。 Batch2表示第二次 50升发酵产程破菌后的上清液。 Yeast 表示一般市售酵母粉， 经破菌后的上清液。 PHA 表示植物血凝素 (phytohemagglutinin) 。 图 11 (1)为灵芝免疫调节蛋白在 AKTA™ explorer 10S system的纯化分析图， F3、 F4 及 F5为第三、 第四及第五管收集样品。
图 11 (II) 通过 AKTA™ explorer 10S system纯化出的灵芝免疫调节蛋白的 SDS- PAGE 电泳图。 M表示蛋白质分子量标准品: 175, 83， 62, 47. 5, 32. 5, 25, 16. 5及 6. 5 Kd. STD 表示由转形的大肠杆菌所制备的灵芝免疫调节蛋白标准品。 F3、 F4及 F5为第三、 第四及 第五管收集样品。
图 12为含灵芝免疫调节蛋白的酵母菌进行 50L发酵槽发酵， 以西方墨点法检测灵芝 免疫调节蛋白的产量结果。 M表示蛋白质分子量标准品.; S表示由大肠杆菌生产的灵芝免疫 调节蛋白质纯化标准品。
图 13为不同浓度纯化的灵芝免疫调节蛋白对石斑鱼头肾细胞的 NBT分析结果。 Cont 表示 PBS对照组。
图 14为腹腔注射不同浓度灵芝免疫调节蛋白于石斑鱼腹腔内， 再将石斑鱼感染红彩 病毒观察其死亡率。 PBS表示注射病毒前腹腔注射 0. 1 ml 的 PBS， 0. 25 μ g/0. lml表示注 射病毒前腹腔注射 0. 25 μ g/0. lml的纯化灵芝免疫调节蛋.白， 1 μ g/0. lml表示注射病毒前 腹腔注射 1 μ g/0. lml 的纯化灵芝免疫调节蛋白， 5 μ g/0. lml 表示注射病毒前腹腔注射 5 μ g/0. lml的纯化灵芝免疫调节蛋白。 '
图 15为喂食石斑幼鱼含灵芝免疫调节蛋白的酵母菌后， 将石斑幼鱼感染弧菌， 观察 石斑幼鱼的存活率。 PBS表示磷酸盐缓冲剂对照组。 20mg、 4mg、 0. 8mg表示弧菌接种前给 石斑鱼喂食上述剂量含灵芝免疫调节蛋白的酵母菌。 Control表示给石斑鱼喂食 20mg—般 市售酵母菌。 图 16为将 Z¾rp处理过已致敏过的 Balb/c小鼠， 取出该小鼠脾脏细胞， 再利用灵芝 免疫调节蛋白或 Der 萃取物来再次刺激， 观察脾脏细胞其 IFN-r的产量。 FlP-gts表示 灵芝免疫调节蛋白。 i¾r 表示尘螨过敏原（der p 2)。 Control表示 PBS对照组。 具体实施方式
下面结合具体实施例对本发明作进一步阐述， 但不限制本发明。
实施例一： pYBlOl-FIP- yeast 和 ρΥΒΙΟΙ- FIP- LZ表达载体的制备
1. FIP- yeast和 FIP- lz的 DNA和引物核酸序列的制备：
参照已知方法（Z^oc. Natl. Acad. Sci. USA. 1991 May 15 ; 88 (10)： 4084 - 4088) , 以基因合成的方式来合成不同密码子喜好的灵芝免疫调节蛋白 DNA 核酸序列， 如图 1 (I) 为面包酵母（■feccAarowces
Figure imgf000010_0001
喜好的密码子的 DNA序歹! J FIP- yeast ;图 1 (II) 为灵芝（ Gmode匪 _/i/cii i )原始密码子的 DNA序列 FIP- lz。依据已知方法 Biol. Chem. 266 (4) , 2486-2493 (1991) ) ， 利用已知的灵芝免疫调节蛋白基因序列， 分别设计出不同 密码子喜好的核酸序列， 并依照这两种不同密码子喜好的核酸序歹 (J , 订购所需的 DNA正向 引物及反向引物 （Mission biotech, Taiwan) ， 并在基因两侧设 i十要接入表达载体的限制 酶切割位 DNA序列 （ k/I and 勵 ) 的引物， 见图 2 (I) 和 图 2 (Π) ， 而后进行 DNA 聚合酉每链反应 (polymerase chain reaction) ，最后经定序 (Mi ss ion biotech Co. , Ltd. , Taiwan)确定， 便完成两种不同喜好密码子的灵芝免疫调节蛋白的基因合成。
2. ρΥΒΙΟΙ, ρΥΒΙΟΙ- FIP- yeast 和 pYB101_FIP_LZ质粒的构筑：
如图 4所示， 将半乳糖启动子 (galactose promoter)、营养缺陷筛选基因 Ura3及 2 μ ori酵母菌的增殖起点（implication origin)加入 pYB质粒中（Yeas tern Biotech Co. , Ltd, Taiwan)即完成 ρΥΒΙΟΙ (1)。 并可以被转形应用于一般面包酵母（伊 U如： BY4741或 DY150)。
表达载体 ρΥΒΙΟΙ 及上述合成好的不同喜好密码子的基因片段各以适当 DNA 限制酶 {Bam Hi 和 Pml I, New England Biolabs, USA)—起作用。 各将经限制酶作用过后的 DNA 溶液， 经过纯化 DNA管柱（PCR Cleaning Up Purification Kit, VIogene, Taiwan)纯化出 两种不同喜好密码子合成的灵芝免疫调节蛋白基因 DNA及表达载体 DNA。 再将 DNA浓度调 成分子摩尔数比为 3: 1 (合成基因：载体），经 T4DNA连接酶 (New England Biolabs, Beverly, USA)进行灵芝免疫调节蛋白 DNA及酵母菌表达载体 DNA 的连接（l igation)。 分别命名为 pYBlOl- FIP- yeast (酵母菌喜好的密码子 ， （II) )及 pYBlOl- FIP - LZ (灵芝原始密码子， (111) )。
3. pYBlOl-FIP- yeast 或 pYBlOl- FIP-LZ转形至大肠杆菌中：
参照已知方法 Molecular Cloning A Laboratory Manual, 2nd Ed. , , pp : l. 82—1. 84) 分别将 ρΥΒΙΟΙ-FIP- yeast 或 pYBlOl- FIP- LZ转形至大肠杆菌 DH5 α 中。使用市售 DNA萃 取试剂（Mini-M Plasmid DNA Extraction Kit, Viogene, Taiwan) , 可由转形至大肠杆菌 中得到大量 pYB101-FIP-yeast及 ρΥΒ1(Π- FIP- LZ。
实施例二： 小量生产 FIP- yeast及 FIP-LZ
1. 分别将 pYBlOl-FIP- yeast 和 pYBlOl- FIP- LZ 表达载体 DNA 转形至面包酵母 (BY4741或 DY150)中:
将实施例一所获得的两种不同喜好密码子的灵芝免疫调节蛋白表达载体 DNA (pYB101-FIP-yeast与 pYBlCH- FIP- lz)各 1 μ g及面包酵母（BY4741或 DY150)与酵母菌 转形试齐 ί]一起作用 (Yeast Transformation Kit, Yeastern Biotech Co. , Ltd. , Taiwan)。
2. 利用营养需求筛选正确的转形面包酵母菌株- 根据 BY4741 及 DY150 营养缺陷上的特性（BY4741： MATa his3deltal Ieu2delta0 raetl5delta0 ura3delta0。 DY150: MATa ura3-52 leu2-3 112 trpl~l his3 - 11 15 ade2- 1 canl-100)， 上述各个转形酵母菌平均涂抹在 YNBD培养基上培养 (0. 17% Bacto不含氨基酸 的酵母氮基 (Difco, England)、 0. 5%硫酸铵、 2%葡萄糖， 2%琼脂， （Sigma, USA) ) ΰ 因此 分别在 YNBD培养基上事先还需要加入 0. 0024%组氨酸、 0. 0072%亮氨酸、 0. 0012% 蛋 氨酸（BY4741)或 0. 0072%亮氨酸、 0. 0048%色氨酸、 0. 0024%组氨酸、 0. 0024% 腺嘌呤 (DY150) (Sigma, USA) , 置于 30 °C培养箱中培养 2-3天。 因为尿嘧啶 （uracil ) 并未添加 在培养基中， 因此未转形带有上述表达载体的酵母菌株， 无法自行产生尿嘧啶 （uracil ) 而会死亡。 本试验利用营养需求筛选正确带有表达载体的转形面包酵母菌株。
3. 带有灵芝免疫调节蛋白转形面包酵母的小量摇瓶测试- 将上述利用营养需求筛选出正确的带有灵芝免疫调节蛋白转形面包酵母菌株，各取两 株菌， 点菌至 50ral的 YNBD培养液及缺陷株所需的相关药品 （实施例二： 步骤 2) 中， 培 养温度为 30°C， 250 rpm的转速进行培养 3天， 而后离心菌液， 用 YNB培养液清洗三次去 除葡萄糖， 再以 50ml的 YNBG培养液 （0. 17% Bacto不含氨基酸的酵母氮基、 0. 5%硫酸 铵、 2%半乳糖 (Sigraa, USA) ) 及缺陷株所需的相关药品 （实施例二： 步骤 2)， 将菌的浓度 调成 0. 1 0D600nm, 以培养温度为 30Ό、 250 rpm 的转速进行培养， 再利用分光光度计 (Spectrophotometer) (Ultrospec 2100 pro, Amersham pharmacia biotech, USA) , 每日记 录 0D6。。„m并收集 lml酵母样品经过 10, OOOx rpm离心（Microfuge 18 Centrifug, Bechraan Coulter, USA) , 水洗三次后将酵母菌菌体保存在- 20 °C中。
实施例三： 利用西方墨渍法 (Western Blot)定量转形的面包酵母表达灵芝免疫调节蛋 白质- 1.将上述收集后的酵母菌样品以 PBS (Sigma, USA)将菌体稀释成 0. 5 0DM。nm，溶于 2x SDS 样品缓冲液 （sample buffer ) (S igma, USA)后， 100 °C下加热三分钟， 冷却后每个稀释样 品取 10 μ 1并以 100伏特进行 SDS- PAGE电泳， 待电泳至适当位置后即完成电泳（Tfe tore (1970) 227, 680-685)。
2. 西方墨渍法方式 （参考 Molecular Cloning A Labora tory Manual, 2nd Ed. ,， pp : 18. 60-18. 75)：将胶体上蛋白质转渍到 PVDF膜 (Hybond-P, Amersham Biosci ence, USA)上， 通入 50mA电流直到蛋白质皆转渍到转渍试 έ氏上， 再将转渍试纸取出， 加入阻断缓 冲液 (blocking buffer)以 37°C作用一小时， 而^在 4 °C作用隔夜， 将阻断缓冲液倒掉， 加 入 1( 稀释兔子抗灵芝免疫调节蛋白血清（Taiwan Advance Bio- Pharm Inc. , Taiwan)， 以 . 37 Ό作用一小时后去上清液并经清洗缓冲液 (washing buffer)洗过，再加入 1CT4稀释含 HRP 标示的老鼠抗兔子血清的抗血清 (Sigma, USA) , 以 37 Ό作用一小时后去上清液并经清洗缓 冲液洗过， 加入呈色液 TMB (sigma, USA) , 待出现颜色即可用水终止呈色反应， 完成西方 转渍法测试。 结果见图 5。
由图 5 (I)及图 5 (Π)得知， M表示蛋白质分子量标准品， S表示由大肠杆菌生产的灵 芝免疫调节蛋白质纯化标准品， 1-4 群为改良灵芝免疫调节蛋白质的密码子所表达的灵芝 免疫调节蛋白质。 2-5群为原灵芝免疫调节蛋白质的密码子所表达的灵芝免疫调节蛋白质。 如图 5 (1)所示， 1-3群清楚可见灵芝免疫调节蛋白表达， 而 5- 8群无法侦测生灵芝免疫调 节蛋白。 此结果显示， 改良灵芝免疫调节蛋白质的密码子能在酵母菌中高度表达出来。
实施例四：构筑表达载体 pYBlOl s- FIP
1.构筑 FIP
根据实施例三的结果选择酵母菌喜好密码子的 FIP 核酸序列作为外泌生产基因的模 板（template)， 并选择 BY4741作为外泌生产的宿主细胞。
2.制备 FIP引物
根据实施例三的结果及步骤选择酵母菌喜好密码子的 FIP作为模板 DNA (序列见图 1 (I) )，依照此模板 DNA设计并购买两端接入 pYBl Ols表达载体 (Yeastern Biotech Co.， Ltd. Taiwan) 的正向引物及反向引物（图 6 (I及 II) ) (Mis sion Biotech Co.， Ltd. Taiwan) 0 以酵母菌喜好密码子的 FIP做为模板 DNA (PYB101-FIP- yeast) , 及上述正向引物及反向引 物利用 PCR反应合成灵芝免疫调节蛋白基因的 DNA片段。
3.构筑 pYBlOl s及 pYB101s-FIP
将半乳糖启动子（galactose promoter)、 α -因子领导序列 （图 7 )、 营养缺陷筛选基 因 Ura3及 2 μ ori 酵母菌的增殖起点（repl ication origin)加入 pYB质粒中（Yeastern
Biotech Co. , Ltd, Taiwan)即完成 pYBlOl s (图 8 (I) )。 并可以被转形应用于一般面包酵 母（例如： BY4741)。
根据实施例一的步骤构筑 pYBlOls-FIP表达载体，如图 8所示。利用 DNA定序确定整 个基因序列（Mission Biotech Co. , Ltd. Tai
4. pYBlOls- FIP转形至大肠杆菌中
根据实施例一的步骤将 pYBlOls- FIP表达载体转形至 DH5 α 中。抽取大量 pYBlOls- FIP 表达载体的 DNA。
实施例五：制备小量灵芝免疫调节蛋白
1.将 pYBlOls- FIP转形至酵母菌 BY4741中
根据实施例二的步骤将上述抽出的 pYBlOls-FIP 麦达载体 DNA 转形至烘焙酵母 (Baker' s Yeast ) BY4741中。
2. 利用营养需求筛选正确的转形面包酵母菌株：
根据实施例二的步骤将上述含 pYBlOls- FIP表达载体的正确转形株从 YNBD琼脂 (含 0. 0024%组氨酸， 0. 0072%亮氨酸， 0. 0012%蛋氨酸）中挑 ttS。
3. 带有灵芝免疫调节蛋白转形面包酵母的小量摇瓶测试：
根据实施例二的步骤将上述含 pYBlOls-FIP表达载体的正确转形株接种至 50ml的培 养液中。 利用半乳醣 (galactose)诱导（induction)生产出夕卜泌的灵芝免疫调节蛋白， 并且 每日收集样品。
实施例六： 利用 SDS- PAGE法测试转形的面包酵母表达灵芝免疫调节蛋白质产量： 根据实施例二的步骤将上述收集的样品进行 SDS-PAGE。 而后再将此 SDS-PAGE利用考 麻萨亮蓝（Coomassie brilliant blue) G-250 (sigma)进行染色 30分钟，用去染液（destain solution, 20% 甲醇， 10% 乙酸）退色至蛋白质带（protein band)清晰可见（见图 9 )。
实施例七： 50升发酵产程 .
1. 接种 50ml锥形瓶培养- 上述实施例二中，挑选经西方墨点法确定具有灵芝免疾调节蛋白产量的转形面包酵母 —株 (BY4741/pYB101- FIP- yeast)接种至 50ml的 YNBD培养基中，加入务缺陷株所需的相关 药品 （实施例二： 步骤 2)， 以 30°C、 250卬111转速培养24 /』、时。
2. 接种 500ml锥形瓶培养：
将上述培养后的 50ml菌液接种至同样的 450ral的培养液中， 以 30°C、 250rpm转速培 养 24小时。
3. 接种至 50升发酵槽 (Chuan Tai Factory, Taiwan) 行发酵：
将上述培养后的 500ml菌液以利用离心方式， 并用 YNB培养液清洗三次去除葡萄糖， 再以 50升的 YNBG培养液 （0. 17% Bacto不含胺基酸之酵母氮基， 0. 5%硫酸铵， 2%半乳 糖 (Sigma, USA) ) 及缺陷株所需的相关药品 （实施例二：步骤 2)，将菌的浓度调成 0. 1 0D6„„nm 以培养温度为 25- 30°C、 400-500 rpm转速、 pH 4. 5-5. 5 (由 2M NaOH及 HC1 (Sigma, USA) 来调节酸碱）、 槽内压力 0. 2-0. 4kg/cm2, 经 0. 2 μ m过滤空气通气量为 40- 50L/min的条件 进行发酵 3天。
4. 灵芝免疫调节蛋白 50升发酵槽产程的产量测定：
取样的菌液利用上述实施例三， 以西方墨点、法的方法测定灵芝免疫调节蛋白的产量。 结果见图 1 2。 由图 1 2得知， 50升发酵后的产物含有灵芝免疫调节蛋白。
实施例八： 转形面包酵母表达灵芝免疫调节蛋白质的活性测试
1. 全部细胞萃取物的制备：
玻璃珠（glass bead)磨菌方法： 培养后釆集的酵母菌样品， 经离心， 用 PBS清洗已收 获酵母菌团块，离心三次。收集酵母菌丸粒，加入与 pellet等体积的 0. 4mm玻璃珠。在 4°C 下震动混合物 20秒且冰浴 1分钟。重复此步骤五次。在 4°C 15000rpm离心五分钟后， 用上 清液当作全部细胞萃取物。 离心检体， 留置上清液。 用 0. 2um膜 (Sartorius) 过滤上清液 且留置备用。
2. 人类周边血单核球细胞制备：
利用成人已加肝素防止凝固（heparinized)的周边血液加入 Ficoll- paque 培养液 (Amersham Biosciences)离心分离出人类周边血单核球细胞。 1 X 106 cells/ml的细胞培 养在含有 10 % FBS、 lOO g/ml 链霉素、 100 units/ml 青霉素及 200 mM L-谷氨酸钠 ( L-glutamate ) 的 RPMI1640 培养基 (GIBC0, Grand Island, NY)的 24-孔组织培养盘 (Nunc, Roskilde, Denmark)中。 以 37°C培养 0、 24或 48小时。
3. 细胞激素（cytokine)的测定：
上述 1 X 10'; cells/ml的细胞培养在 24-孔组织培养盘（Nunc, Roskilde, Denmark) , 加入上述萃取出不同浓度的灵芝免疫调节蛋白共同培养。 IFN- γ活性是用商品试剂组 (R&D Systems, Minneapolis, MN) 来 检 测 。 结 果 见 图 1 0 ， 由 图 1 0 得 知 PHA (phytohaemagglutinin, Sigma)在此 IFN- γ ELISA测试中作为标准品使用 (Jhe Journal of Immunology, 1998, 161： 2114-2119)。 第 1批及第 2批代表第一次 50升发酵样品及第 二次 50升发酵样品， 使用不同剂量下产生的 IFN- γ 量。 对照组及一般酵母组不会刺激生 成 IFN- Y量。纯化后灵芝免疫调节蛋白不同剂量也会产生的 IFN- Y量。其中 4 y g浓度的 纯化后灵芝免疫调节蛋白与其它方式产生的灵芝免疫调节蛋白比较，培养 48小时后得到较 高的 IFN- Y 含量。 另外也显示 50升发酵样品会朿激 含量， 代表本发明所产生的 FIP具有生物活性， 而并非因为酵母菌的关系。
实施例九： 50L发酵产程灵芝免疫调节蛋白的纯化 1. 发酵后酵母菌须以 20 mM磷酸盐缓冲液（Sigma) pH 6. 0 回溶成 10%菌液， pH值 由 1M醋酸调整。 再利用破菌机（Basic Z model, Constant System Ltd. UK)以 30Kpsi的 压力打破细胞， 再调整至 pH 4-5。
2. 以 3， 000 xg离心 15 min， 取上清液， 再以 12， 000 xg离心 1 min。
3. 取 10 mL 的上清液注入 30 niL的（1琼脂凝胶 (AmershamBiosciences) (已先用 20-50 mM醋酸平衡， 使周围的酸度维持 PH 4- 5)的管柱中。
4.以 20-50 mM pH4-5醋酸， 流洗两倍管柱体积、， 收取流出液， 即得到灵芝酵母蛋白。
5.分析图见图 1 1 (I)， F3、 F4及 F5表示第 3、 第 4及第 5个收集管样品， 第 3及 第 4个收集管样品明显有较高的 mAU值， 因此收集第 3、 4及第 5个收集管样品， 并且进行 SDS-PAGE. 结果见图 1 1 (II) ， M表示蛋白质分子量标准品， STD表示由大肠杆菌生产的 灵芝免疫调节蛋白质纯化标准品， 由图 1 1 (II) 得知第 3、 4收集管样品与标准品有相同 的分子量， 并且其纯度相当高。 并且后续利用西方墨点法却认为 FIP。
实施例十：灵芝免疫调节蛋白的应用
1.伺料添加物
A.灵芝蛋白对石斑鱼的毒性试验：
鱼种为花鬼斑 (： Epinephelus malabaricus) ， 购自高雄茄定地区的私人养殖场。 平 均体长为 7. 25cm、 平均体重为 5. 9g。 蓄养在海水盐度 33 ppt、 水温 25°C的环境中。
取由实施例九纯化的灵芝免疫调节蛋白溶液， 分别以 5、 1、 0. 25 μ g/尾 /0. lml的浓 度腹腔注射入石斑鱼苗， 另外注射 PBS溶液作为对照组。 以原词养环境流水饲养 14天。每 天观察并记录死亡情形。 两周后所有组别皆没有死亡发生， 显示实验所选用的灵芝免疫调 节蛋白浓度对石斑鱼并不会导致毒性反应。
B.灵芝免疫调节蛋白对体外石斑鱼头肾细胞 特异免疫的影响：
由市场购入约 600g石斑鱼，取头肾、分离吞 P筮细胞并以 AL- 10培养液 (L15 (Gibco) ： AIM5 (Gibco) =1： 1混合）悬浮， 种入 0. 1 ml的 lX107/ml细胞在 96孔培养盘（Corning)， 静置吸附 2 小时。 鱼的头肾细胞是鱼类非专一†生免疫的主要细胞 （Engelsma MY, Fish Shellfish Immunol. 2003 Nov ; 15 (5) : 397-410) a 力口入 2、 1、 0. 5、 0. 2、 0. 02 μ g/0. lml 由实施例九纯化的灵芝免疫调节蛋白溶液，于室温作用 2小时。进行 NBT分析套组 (Sigma)： 根据 NBT分析套组可得知添加灵芝免疫调节蛋白后， 石斑鱼头肾细胞的吞噬作用反应。
结果见图 1 3。 由图 1 3得知， 添加 2、 1 禾口 0. 5 μ §/0. lml 由实施例九纯化的灵芝 免疫调节蛋白， 可增加头肾细胞的吞噬作用。 由 it匕推知灵芝免疫调节蛋白可增强石斑鱼的 非特异性免疫反应。
C. 石斑鱼腹腔注射灵芝免疫调节蛋白两周后进行虹彩病毒（Iridovirus)攻击试验的 预备试验：
对前述毒性试验的鱼进行腹腔注身寸 PBS或灵芝免疫调节蛋白两周后， 以腹腔注射方式 注射 0. lml的 10, 000XTCID5。病毒剂量， 每日观察鱼死亡率。 一般， 经腹腔注射虹彩病毒的 石斑鱼两周内死亡。 由图 1 4得知， 以最高剂量 5 g FIP/0. 1显示两周后有 70%死亡率， 因此， 两周内达到 30%保护率。
、 D.对哈氏弧菌 Vibrio harvey) 的保护率
a. 测试鱼种为石斑鱼
Figure imgf000016_0001
， 平均体重为 52. 9克。 喂食石斑 鱼 20mg、 ½g及 0. 8 mg /60g三种不同浓度的含灵芝免疫调节蛋白的酵母菌与对照一般酵 母菌组 20 mg/60g石斑鱼及 PBS对照组， 共五组， 进行喂食， 每组 20尾石斑鱼， 每两天喂 食一次， 连续喂食七天后， 进行攻毒试验。
b. 以 10XLD5。(3X106CFU/ml)的哈氏弧菌 Vibrio ?sr ej^进行攻毒试验， 以求其保 护力。 攻毒前先行采集血液， 每组 10尾， 进行测试鱼的非特异性免疫反应包括 Lysozyme 活性、 吞噬能力（Phagocytosis)及 NBT 阳性细胞数量三项， 其它 10尾进行攻毒细菌试验。 每日观察记录鱼的活力、 食欲及死亡率。
c 结果见图 1 5， 经由弧菌感染后的鱼， 五天后， 喂食一般酵母菌的鱼的存活率只 有 10-20%, 喂食含灵芝免疫调节蛋白的酵母菌的鱼的则其存活率有 60%- 80%， 并且死亡时 间会延后。 此结果指出口服含灵芝免疫调节蛋白酵母菌增强鱼类免疫力及延长鱼类生命。
实施例十一： 将 Zfer 处理过已致敏过的 Balb/c小鼠， 取出该小鼠脾脏细胞， 再利 用灵芝免疫调节蛋白或 Der 萃取物来再次刺激， 观察脾脏细胞的 IFN-r的产量。
致敏实验的步骤：
a.喂食灵芝免疫调节蛋白组的小鼠：两天喂食一次 (2 μ g)直到第 15天杀死 Balb/c小 鼠， 取出脾脏细胞（spleen cell)。
b. /fer 处理的小鼠： 第一天进行腹腔注射 10 ，隔七天再进行腹腔注射 lO g,再 隔 7天进行喷雾一次 10 μ g，第 15天杀死 Balb/c小鼠， 取出脾脏细胞。
c.脾脏细胞处理： 上述分离出的脾脏细胞， 培养 1 X 10β cell s/ml在 RPMI 1640含 5% FBS的培养基中， 然后再以 lO y g/ral Zfer °或 2 μ g/ml FIP处理。 培养 48小时后收集 培养液并分析 IFN-r的含量。
结果见图 1 6， 其中 Der 7表示尘螨（dust mite)过敏原 Der p II； FIP-gts表示灵 芝免疫调节蛋白。结果显示长期喂食灵芝免疫调节蛋白给 Balb/c小鼠，其脾脏细胞再经灵 芝免疫调节蛋白剌激并不会增加产生 IFN - r (81. 9 pg/ral)。 而未喂食灵芝免疫调节蛋白的 对照组中 Balb/c 小鼠的脾脏细胞再经灵芝免疫调节蛋白刺激会增加产生 IFN- r (463. 8 pg/ml)。 经过/ ¾ ^致敏过的 Balb/c小鼠， 其脾脏细胞再经灵芝免疫调节蛋白刺激会增加 产生 IFN-r aiOO. 7 pg/ml)。 这显示口服灵芝免疫调节蛋白可以免疫调节， 特别是直接抑 制过敏反应。
本发明所描述及实施例中显示足够的细节及结果证明其实施性及应用性。

Claims

权 利 要 求
1.一种核酸分子， 包含以下核酸序列， 其能转译出灵芝免疫调节蛋白-
ATGTCTGATAC TGCTTTGATT TTCAGATTGG CTTGGGATGT
TAAGAAGTTG TCTTTCGATT ACACTCCAAA CTGGGGTAGA
GGTAACCCAA ACAACTTCAT TGATACTGTT ACTTTCCCAA
AGGTTTTGAC TGATAAGGCT TACACTTACA GAGTTGCTGT
TTCTGGTAGA AACTTGGGTG TTAAGCCATC TTACGCTGTT
GAATCTGATG GTTCTCAAAA GGTTAACTTC TTGGAATACA
ACTCTGGTTA CGGTATTGCT GATACTAACA CTATTCAAGT
TTTCGTTGTT GATCCAGATA CTAACAACGA TTTCATTATTG
CTCAATGGA ACTGA
2.根据权利要求 1所述的核酸分子，其特征在于在同- -个携带系统中连接另
的核酸分子。
3.一种表达载体， 其特征在于包括权利要求 1所述的核酸分子。
4.一种表达载体， 其特征在于包括权利要求 2所述的经连接核酸分子。
5.—种宿主细胞， 其特征在于由权利要求 2所述的表达载体所转形。
6. 根据权利要求 5所述的宿主细胞， 其特征在于为细菌、 真菌或酵母菌。
7. 根据权利要求 5 所述的宿主细胞， 其特征在于为选自包括面包酵母菌 {Saccharowyces cerevisiae) , 巴氏毕赤酉孝母 0¾'c/^'a pas tor is) , ¾.¾I#¾ (Hansen ul a polyworpha) , 产朊假丝酵母（ί¾/? '6¾ "i J s) , 博伊丁假丝酵母（ί¾/7ώ'ο¾ , 麦 牙糖念珠菌（ 7 'ο¾ wal tosa) , 乳酸克鲁维酵母（/i //(^r^ c^ 1 act is) , 解脂酵母 ( Yarrowia lipolytics)， 西方酵母 {Schwann iomyces occi ental is)， 粟酒裂殖酵母 Schizosaccaromyces pombe) , 球拟酵母属 ( /"om/opsis) , (Arxula adeninivorans) ,曲霉 Aspergillus (构巢曲霉. ( A. nidulans), 黑曲霉 niger) , 泡盛曲霉 awamori) , 米曲菌（ A oryzae) ,木 i ricoderma (里氏木霉（7. reese ) )所组成的群组。
8. 根据权利要求 6所述的宿主细胞，其特征在于酵母菌是面包酵母菌（ Saccharowyces cerevisiae) 0
9. 根据权利要求 5所述的宿主细胞，其特征在于其存在形式为完整细胞或破菌细胞。
10. 根据权利要求 9所述的宿主细胞， 其特征在于完整细胞可分泌灵芝免疫调节蛋 白。 '
11. 根据权利要求 5所述的宿主细胞， 其特征在于在选自哺乳类、鱼类、 甲壳类及家 禽类动物群组中施用。
12. 根据权利要求 11所述的宿主细胞， 其特征在于其给予方式为静脉注射、 腹腔注 射、 口 ]¾、 黏膜吸附、 皮肤吸附及液体浸泡。
13. 根据权利要求 12所述的宿主细胞， 其特征在于其给予方式为口服。
14. 根据权利要求 11所述的宿主细胞， 其特征在于哺乳类指猪， 家禽类指鸡。
15. 根据权利要求 11所述的宿主细胞， 其特征在于鱼类指石斑鱼、 鲑鱼或鳟鱼。
16. 根据权利要求 11所述的宿主细胞， 其特征在于甲壳类指虾、 龙虾、 草虾、 斑节 虾、 白虾。
17.一种制备含灵芝免疫调节蛋白宿主细胞的方法，其方法程序为 (a)构筑一个表达载 体， 该载体插入具有改良后真菌免疫调节蛋白核酸序列； （b)用此载体转形宿主细胞； （c) 在表达的适当环境下， 培养宿主细胞。
18. 根据权利要求 17所述的方法， 其特征在于改良后真菌免疫调节蛋白的核酸序列 为权利要求 1所示的核酸分子序列。
19. 根据权利要求 17 所述的方法， 其特征在于步骤 (a)所指的真菌为面包酵母菌 ( Saccharomyces cerevisiae) 。
20. 根据权利要求 17 所述的方法， 其特征在于步骤（b)所指的载体为 pYB101-FIP-yeast o
21.一种真菌免疫调节蛋白， 其特征在于根据权利要求 17的方法制备，及自宿主细胞 分离。
22.一种由转形灵芝免疫调节蛋白的核酸序列的宿主细胞中纯化灵芝免疫调节蛋白的 方法， 包含：
(a)将发酵后的转形细胞溶于一溶剂中；
(b)打破细胞；
(c)调整 pH值到 5-6间；
(d)分出细胞萃取物中的破片；
(e)离心上清液；
(f)将上清液加入已经 pH值 5-6平衡好的管柱中；.
(g)离析出灵芝免疫调节蛋白。
23.根据权利要求 22所述的方法，其特征在于灵芝免疫调节蛋白是由.20-50πιΜ醋酸 pH 4 - 5所离析出。
24.一种用口服方式来调节免疫活性的组合物， 其特征在于包含真菌免疫调节蛋白。
25.根据权利要求 24所述的组合物，其特征在于真菌免疫调节蛋白是由天然灵芝或根 据权禾 U要求 22所述的方法所制备。
26. 根据权利要求 24所述的组合物， 其特征在于应用于化妆用途以减低发炎及过敏 反应。
27. 根据权利要求 24所述的组合物， 其特征在于应用于药品用来减低发炎与过敏反 应、 调节免疫活性、 预防糖尿病、 改善气喘、 增加抗菌能力、 增加抗病毒能力及降低器官 转移的排斥反应。
28. 根据权利要求 24所述的组合物， 其特征在于应用于食物或饲料添加， 用以延长 寿命、 增加免疫反应、 '饲料转换率及减低紧迫症。
29.一种调节免疫活性的方法， 其特征在于包括口服给予真菌免疫调节蛋白或真菌免 疫调节蛋白与另一种蛋白质融合至个体。
30. 根据权利要求 2 9所述的方法， 其特征在于蛋白质由大肠杆菌 co/i 或面 包酵母 ( Saccharomyces cerevisiae)所制备。 ·
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