WO2009094850A1 - Protéine immunomodulatrice recombinante de reishi (ganoderma lucidium) (rlz-8) et ses utilisations - Google Patents

Protéine immunomodulatrice recombinante de reishi (ganoderma lucidium) (rlz-8) et ses utilisations Download PDF

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WO2009094850A1
WO2009094850A1 PCT/CN2008/002142 CN2008002142W WO2009094850A1 WO 2009094850 A1 WO2009094850 A1 WO 2009094850A1 CN 2008002142 W CN2008002142 W CN 2008002142W WO 2009094850 A1 WO2009094850 A1 WO 2009094850A1
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rlz
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Xitian Zhang
Fei Sun
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Priority to CA2711207A priority patent/CA2711207C/en
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Priority to US12/829,653 priority patent/US20110009597A1/en
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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
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    • 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
    • C07K14/375—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Basidiomycetes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61P31/04—Antibacterial agents
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    • A61P31/12—Antivirals
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    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a fungal immunoregulatory protein, in particular to a specific spatial structure of recombinant Ganoderma lucidum immunoregulatory protein for adaptation to tumors, leukopenia and allergic diseases. Therapeutic use of the disease.
  • BACKGROUND OF THE INVENTION The prior literature indicates that certain proteins contained in the fungus Ganoderma lucidum have a wide range of immunomodulatory activities, which can promote lymphocyte proliferation, promote the production of cytokines by human peripheral blood lymphocytes, agglutinate red blood cell activity, and exert influence on adhesion molecules. It inhibits allergic reactions and immune anti-tumor effects.
  • an immunomodulatory protein (Imnmnoregulatory Protein from Ganoderma Lucidium, LZ-8) was isolated from the mycelium extract of Ganoderma lucidum for the first time by Kino et al., Japan, named LZ- 8, and determined its gene sequence, amino acid sequence and immunophysiological activity. Protein sequencing indicated that LZ-8 consists of 110 amino acid residues and is acetylated at the amino terminus. The molecular weight is 12. 4 kD and the isoelectric point is 4. 4.
  • the inventors have for the first time disclosed the crystal spatial structure of the genetically recombinant Ganoderma lucidum immunoregulatory protein, the main features of which are: an important domain required for the formation of a dimer at the N-terminus and a FNIII domain at the C-terminus, N-terminal structure
  • the domain consists of an a-hel ix (sequence is 2-SDTA- LIFRLAWDVK- 15, 14 amino acids) and P-strand (sequence is 16-KLSFD-20, composed of 5 amino acids), which is on a-hel ix
  • the residue of Ser is blocked by acetylation.
  • the N-terminus on the Lz_8 monomer a-hel ix and ⁇ -strand form an important dimer-binding domain by spatial exchange with the same domain on the other monomer, which is dumbbell-shaped.
  • the C-terminal FNIII domain belongs to the immunoglobulin sandwich structure and contains two structures, ⁇ -sheet I and ⁇ -sheet II, which are formed by ⁇ -strands A-B-E and 0-strands G-F-C-D, respectively.
  • the results suggest that the essential role of the recombinant Ganoderma lucidum immune protein may be the formation of homodimers through the N-terminal ⁇ -helix A (research shows that its N-terminal 13 amino acid residues are necessary for Fip to form functional homodimers). ), thereby binding to lymphocytes, through a series of information transmission, and promoting the secretion of various cytokines by lymphocytes to achieve immunoregulatory functions.
  • Human Granulo- cyte Macrophage Colony Stimulating Fa is the only one used to treat leukocyte or neutropenia and bone marrow caused by various causes.
  • all products have been noted for its clinical adverse reactions to fever, bone pain, myalgia, rash, itching, abdominal pain, diarrhea and so on.
  • a small number of patients may have a first dose response when they first take the drug, which is characterized by facial flushing, sweating and decreased blood pressure, and decreased oxygen saturation.
  • Ganoderma lucidum immune protein The main function of Ganoderma lucidum immune protein is that it can promote the proliferation of peripheral lymphocytes and spleen cells, and induce macrophages of animals and humans to secrete various cytokines (interleukins, tumor necrosis factors and interferons, etc.), and thus Eliminate the damage of pathogens, maintain the health of the body, and complete the immune regulation function, but the detailed adjustment mechanism is not completely clear.
  • the protein can also significantly inhibit systemic allergic reactions, inhibit tumor growth, and counteract the rejection after organ transplantation.
  • An object of the present invention is to find a crystal structure of a recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) and its novel use in the field of medical treatment.
  • the invention relates to the application of recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) in the field of medical treatment such as anti-tumor, elevated white blood cells and inhibition of immune rejection.
  • rLZ-8 recombinant Ganoderma lucidum immunoregulatory protein
  • the recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) of the present invention is obtained by recombinant expression in Pichia pastoris by genetic engineering technology, and the nucleotide sequence of the protein is based on the published gene sequence of the natural Lingzhi immunomodulatory protein ( M58032) Redesigned and synthetic.
  • the crystal structure of the recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) is obtained for the first time by X-ray diffraction.
  • the crystal culture conditions of the protein were as follows: 1. 75M ammonium sulfate, 0.1 M Tris pH 6. 0, 6. 4% PEG400 o
  • a single crystal having a space group of P32 was obtained by a hanging drop gas phase diffusion method.
  • a set of crystal data of 1.8 angstrom resolution was collected in the image plate diffraction data collection system of X-ray diffractometer, and then analyzed and calculated by using structural analysis software such as CCP4, and then a set of phase exchange method was used to solve the set. Crystal structure.
  • each asymmetric unit Four molecules can be found in each asymmetric unit, wherein the two molecules form a dimer with a hydrophobic interaction between the N-terminal alpha-helix and a hydrogen bond between the antiparallel beta-strand.
  • the C-terminus of a single molecule has a FNIII-type immunoglobulin fold that is formed by two beta-sheets. At the surface and at the junction of the beta-sheet, there is a speculative sugar-binding site.
  • rLZ-8 prepared by using IMDM cell culture solution are added to human promyelocytic leukemia cell line NB4 and human chronic myeloid leukemia cell K562, and rLZ can be clearly observed.
  • the purified rLZ-8 was administered to mice bearing S180 and H22 for 10 days. The mice of the ascites tumor were weighed daily, and were sacrificed by cervical dislocation.
  • the fluorescent dye FITC was also used to label rLZ-8, and human acute myeloid leukemia cell line HL-60 cells, rat cardiomyocytes, rabbit chondrocytes were collected with FITC-rLZ-8, and the cells were collected several times. Washing, under the microscope, showed high brightness of green fluorescence in HL-60 cells, which was significantly different from normal control group, while rat cardiomyocytes and rabbit chondrocytes were not stained, indicating that HL-60 cells can be Recognized by rLZ-8, but cannot mark the other two Seed cells.
  • This effect may be related to the presence of more highly folded oligosaccharide chains on the surface of tumor cells, since there are two sugar chain binding sites in the C-terminal FNIII immunoglobulin domain of rLZ-8, whereas cells of normal tissues The surface does not have this feature, such as rat cardiomyocytes and rabbit chondrocytes.
  • rLZ-8 has a strong killing effect on HL-60 cells in this experiment, but has no obvious effect on the other two cells. It is speculated that the selectivity of this rLZ-8 killer cell may be recognized by it. Cell surface receptors are involved.
  • the present invention also uses PI single staining and ANNEXIN V& FITC double staining to detect apoptosis of tumor cells such as K562 and NB4 directly killed by rLZ-8, and the result further clarifies rLZ.
  • the mechanism of -8 killing of tumor cells may be to induce apoptosis of tumor cells.
  • the present invention discloses that rLZ-8 has an effect of preventing and treating leukopenia.
  • rLZ-8 prepared by physiological saline was administered to a rat model of low leukocyte induced by cyclophosphamide, and stimulated with Jin Leisai (colony cell stimulation).
  • Factor was a positive drug, continuous administration for 7 days, blood was taken from the tail vein on the 3rd and 7th day, the number of white blood cells in the blood was measured, and the changes in white blood cell count before and after treatment were compared to analyze the drug efficacy.
  • the rLZ-8 drug group had significantly increased rat leukocytes on the third day of administration, and the difference was significant, and returned to normal level on the 7th day of administration.
  • each group of mice began intraperitoneal injection of the same volume of normal saline, positive drug (Jin Lei Saiqiang), different doses on the same day of radiation. rLZ-8.
  • the irradiation conditions were 7. 50 Gy, 180 mV, 15 mA, and the dose rate was 450 len/min.
  • the number of white blood cells in the tail veins of each group of animals was measured before the administration, and on the 5th, 7th, and 9th day after irradiation.
  • the body was weighed and deprived 7 days after the irradiation, and the spleen was weighed to calculate the spleen coefficient.
  • CFU-S spleen surface hematopoietic foci
  • the present invention proves that rLZ-8 does not produce hemolysis, has no agglutination effect on human blood four types of red blood cells, and does not produce abnormal changes in rat bone marrow images.
  • the rLZ-8 of the present invention can be used as an immunosuppressive agent.
  • rLZ-8 was found to inhibit systemic allergic reactions in mice, and mice sensitized with bovine serum albumin BSA did not have positive reactions such as convulsions, decreased activity or death.
  • the present invention includes preparation of a pharmaceutical preparation containing rLZ-8 as an active ingredient, which is mainly administered by a parenteral route, such as a common injection, a freeze-dried injection, a liposome injection, and an injection for targeted administration.
  • a parenteral route such as a common injection, a freeze-dried injection, a liposome injection, and an injection for targeted administration.
  • Etc. can also be formulated into a gastrointestinal route, such as tablets, capsules, sprays, gels, gel absorbents, oral drugs, suspensions, granules, patches, pills, powders , injections, infusion solutions, suppositories, diluted preparations, controlled release preparations, and the like.
  • the pharmaceutical preparation of the present invention wherein the content of the recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) is
  • other pharmaceutically acceptable carriers may be, starch, dextrin, sucrose, lactose, cellulose, magnesium stearate, Tween-80, carbomer, methylcellulose, carboxymethyl Cellulose sodium, sodium alginate, glycerin, gelatin, polyethylene glycol, thimerosal, propylparaben, methylparaben, ethylparaben, chlorobutanol, sodium benzoate, borax, neger, Sorbitol, propylene glycol isopropanol, lauryl table ketone, isopropanol/propylene glycol (1:1), isopropanol/(1:2) and isopropanol/propylene glycol (2:1), triethanolamine, hydroxide Sodium petrolatum, stearic acid, liquid paraffin, lanolin, stearyl alcohol, cetyl alcohol, glyceryl monostearate, sodium lauryl sulfate, Span 60,
  • FIG. 1 Schematic diagram of crystal structure of rLZ-8Fig. 2 Results of in vitro killing of NB4 tumor cells by rLZ-8Fig. 3 Results of in vitro killing of K562 tumor cells by rLZ-8Fig.
  • the rLZ-8 gene (SEQ1) was redesigned for artificial whole gene synthesis, which is identical to the amino acid sequence encoded by the native Ganoderma lucidum immunoregulatory protein, and the difference is that it is more suitable for utilization. Codon for the Pichia pastoris expression system.
  • This sequence (SEQ1) was ligated to the yeast ⁇ -factor leader peptide coding sequence to form a fusion gene, which was cloned into the pMD18-T vector.
  • the correctly sequenced vector was linearized and transferred into the yeast genome, and methanol was screened on MM and MD plates using a highly efficient Mut+ strain.
  • Natural Ganoderma immunomodulatory protein gene sequence (M58032) coding region sequence: AGCATCATGTCCGACACTGCCTTGATCTTCAGGCTCGCCTGGGACGT GAAGAAGCTCTCGTTCGACTACACCCCGAACTGGGGCCGCGGCAAC CCCAACAACTTCATCGACACTGTCACCTTCCCGAAAGTCTTGACCGA CAAGGCGTACACGTACCGCGTCGCCGTCTCCGGACGGAACCTCGGC GTGAAACCCTCGTACGCGGTCGAGAGCGACGGCTCGCAGAAGGTCA ACTTCCTCGAGTACAACTCCGGGTATGGCATAGCGGACACGAACACG ATCCAGGTGTTCGTTGTCGACCCCGACACCAACAACGACTTCATCAT CGCCC AGTGG AACTAGGAGGAGGC AG.
  • Recombinant Ganoderma Lucidum Immune redesigned regulatory proteins is the nucleotide sequence (SEO 1) is: ATGTCTGATACTGCTTTGATCTTCAGATTGGCTTGGGATGTTAAGAAG TTGTCTTTTGATTACACTCCAAACTGGGGTAGAGGTAACCCAAACAA CTTCATTGATACTGTTACTTTTCCTAAGGTTTTGACTGATAAGGCTTAC ACTTACAGAGTTGCTGTTTCTGGTAGAAACTTGGGTGTTAAGCCATCT TACGCTGTTGAATCTGATGGTTCTCAAAAGGTTAACTTCTTGGAATAC AACTCTGGTTACGGTATTGCTGATACTAACACTATTCAAGTTTTCGTTG TTGATCCAGATACTAACAACGATTTCATTATCGCTCAATGGAACTAGT AA;
  • the amino acid sequence of the recombinant Ganoderma lucidum immunoregulatory protein irLZ-8 is:
  • the temperature, rotation speed, pH value, liquid volume and methanol addition amount of scale fermentation expression were detected, and the optimization method for the process conditions of yeast engineering bacteria expressing rLZ-8 at 80L fermenter scale was established. According to the physical and chemical properties of LZ-8, the formulation of the fermentation medium was designed. The yield of the target protein was about SOOmg.I/
  • the fermentation broth separator is centrifuged ⁇ the supernatant is separated by a tube separator ⁇ ultrafiltration ⁇ cation exchange purification column - AKTA protein purification workstation to prepare the target protein ⁇ strong anion exchange chromatography ⁇ hydrophobication purification column ⁇ gel filtration chromatography.
  • the purity of the isolated and purified product was analyzed by reverse phase liquid chromatography, and the purity of rLZ-8 was >99%.
  • the molecular weight of rLZ-8 recombinantly expressed by laser flight mass spectrometry was 12722 Da.
  • Single crystals with space group P32 were obtained by hanging drop gas diffusion method.
  • the crystal culture conditions were: 1.75 M ammonium sulfate, O.lM Tris pH 6.0, 6.4% PEG400. Collected in MarResearch 345 dtd image plate diffraction data collection system. A set of crystal diffraction data with a resolution of 1.8 angstroms.
  • LZ-8 includes an N-terminal important domain required for the formation of a dimer and a C-terminal FNIII domain.
  • the N-terminal domain of rLZ-8 consists of an a-helix (sequence is 2-SDTAL)
  • IFRLAWDVK-15 composed of 14 amino acids
  • ⁇ -strand sequence is 16-KLSFD-20, composed of 5 amino acids
  • the N-terminus on the Lz-8 monomer a-helix and ⁇ -strand form an important dimer-binding domain by spatial exchange with the same domain on the other monomer, which is 3 ⁇ 4 bell-like.
  • the C-terminal FNIII domain belongs to the immunoglobulin sandwich structure and contains two structures, ⁇ -sheet l and ⁇ -sheet II. As shown in Fig. 1, the two are formed by ⁇ -strands ABE and ⁇ -strands GFCD, respectively.
  • the -strand sequence is:
  • Example 2 Killing effect of rLZ-8 on human promyelocytic leukemia cell line NB4
  • 8 concentrations were prepared using IMDM medium, respectively ⁇ 1 , l ⁇ g'mr 1 , 3.125 ⁇ ⁇ 1" 1 , 6.25 ⁇ ⁇ ⁇ 1 , 12.5 g'ml , 25 ⁇ ⁇ ⁇ ⁇ , 50 ⁇ ⁇ ⁇ 1" 1 , 100 g'mr'.
  • Table 1 and Figure 2 show that the rLZ-8 drug group is at OD 57 .
  • the light absorption value of nm was significantly different from that of the normal control group of NB4, indicating that rLZ-8 has a strong killing effect on NB4 tumor cells in vitro.
  • the concentration of SDM was determined by using IMDM culture solution, which were respectively S. S ml' 1 , 6.25 ⁇ 1 .
  • test wells were added with 0.1 ml of K562 tumor cells and 0.1 ml of rLZ-8, and the concentration of rLZ-8 was from low to high; the negative control group plus K562 cells and the culture medium were each 0.1 ml; the positive drug arsenic trioxide; each group Make 6 duplicate holes.
  • Positive drug control group 10 0.22 ⁇ 0.03* 78.2
  • K562 cells and NB4 cells were separately added to a 24-well plate, 1 ml/well, and different concentrations of rLZ-8, 1 ml/well were added, and 3 replicate wells were set in each group.
  • the cells were collected in a 37 ° C, 5% C0 2 incubator for 24 h.
  • the cells in each concentration group were collected, washed twice with PBS and adjusted to a cell concentration of lxl 0 6 /ml, fixed in 70% ice ethanol, and placed at 20 ° C overnight.
  • Annexin V-FITC kit flow cytometry to detect apoptosis
  • ⁇ 4 cells and HL-60 cells were separately added to a 24-well plate, 1 ml/well, and different concentrations of rLZ-8, 1 ml/well were added, and 3 replicate wells were set in each group.
  • the cells were collected in a 37 ° C, 5 % C0 2 incubator for 24 hours.
  • the cells in each concentration group were collected, washed twice with pre-cooled PBS at 4 ° C, and resuspended in 250 ⁇ l binding buffer to adjust the concentration to lx. L0 6 /ml.
  • apoptosis rate of NB4-rLZ-8 group and HL-60-rLZ-8 group was significantly higher than that of normal control group, and HL-60-rLZ-8 group, with rLZ As the concentration of -8 increases, the rate of apoptosis also increases.
  • mice weighing 18-22 g, half male and half, were provided by the Experimental Animal Center of Jilin University.
  • Mouse Ehrlich ascites cell line S180 was provided by the present laboratory.
  • S180 ascites tumor and solid tumor experimental group were divided into normal control group, negative control group, positive control group, rLZ-8 low dose treatment group (OJSmgAg' 1 ) rLZ-8 medium dose treatment group (O ⁇ mg ⁇ g' rLZ -8 high dose treatment group (lmg'k ⁇ 10 per group).
  • S180 subcutaneous tumor inhibition test method Take well-grown S180 cells, and dilute into tumor cell suspension with appropriate amount of sterile physiological saline. The cell count is lxlo nl ' 1 , and 0.2 ml of subcutaneous inoculation into the right axilla. (except for the normal control group). After 24 hours of inoculation, treatment was given. Normal control group and negative control group were given normal saline 0.2 ⁇ 1 ⁇ only -1 1 , intraperitoneal injection; positive control group was given cyclophosphamide 20 mg*kg,
  • 0.2ml-A-'-d" 1 intraperitoneal injection.
  • the rLZ-8 treatment group was given the corresponding dose of tail vein injection, 0.2ml - only 1 ⁇ 1 for 10 consecutive days.
  • 10d after administration Blood was taken from the orbital venous plexus of the mice, and the number of white blood cells was measured in the laboratory of the First affiliated Hospital of Jilin University. On the next day after the drug was stopped, all the mice were sacrificed by cervical dislocation, and the tumor was dissected and the tumor weight was calculated. :
  • Tumor inhibition rate (%) 2 (negative tumor weight in the negative control group, average tumor weight in the experimental group) / negative control group, average tumor weight, ⁇ 100%
  • SI 80 ascites tumor inhibition test method Take well-grown S180 cells, and dilute into tumor cell suspension with appropriate amount of sterile physiological saline. The cell count is lxloSnl- 1 , and each mouse is inoculated with 0.2ml (except normal control group). After 24 hours of inoculation, treatment was given. Normal control group and negative control group were given normal saline 0.2ml_only 1 , ⁇ 1 , intraperitoneal injection; positive control group was given cyclophosphamide 20mg, kg 0.2ml - only ⁇ cf 1 , intraperitoneal injection.
  • the rLZ-8 treatment group was given the corresponding dose of tail vein injection, 0.2 ml, only ⁇ cT' a for 10 consecutive days. Weighed daily, observe the degree of change in body weight of the mice, and make a weight gain curve.
  • Subcutaneous tumor inhibition test results of S180 It can be seen from Table 5 that three doses of rLZ-8 can inhibit the growth of S180, and the tumor inhibition rates are 16.8%, 25.7% and 45.5 %, respectively.
  • the tumor weight of the rLZ-8 treatment group was significantly different from that of the negative control group (> ⁇ 0.01).
  • CTX group 20 9.56 ⁇ 0.31* 4.41 ⁇ 0.25* rLZ-8 low dose group 0.25 9.48 ⁇ 0.30* 10.44 ⁇ 0.18**
  • mice weighing 18-22 g, half male and half, were provided by the Experimental Animal Center of Jilin University.
  • Mouse Hepatocellular carcinoma cell line H22 provided by the laboratory.
  • the H22 hepatoma cells were divided into normal control group, negative control group, positive control group, rLZ-8 low dose treatment group (0.25 mg * kg-'), rLZ-8 medium dose treatment group (0.5 mg ⁇ kg). — ' ), rLZ-8 high dose treatment group (lmg ⁇ kg - '). 10 in each group. -
  • H22 inhibition experiment subcutaneous Method: H22 cells grow well, sterile saline diluted to an appropriate amount of tumor cell suspension, cell counts of 107 per mouse ⁇ 0. 2ml were inoculated subcutaneously in the right armpit
  • the rLZ-8 treatment group was given the corresponding dose of tail vein injection, 0.2 ml - only for 10 consecutive days.
  • Blood was taken from the orbital venous plexus of the mice before administration and 10 days after administration, and the number of white blood cells was measured in the laboratory of the First affiliated Hospital of Jilin University.
  • the average tumor weight of the control group ⁇ ⁇ %
  • H22 Ascites anti-tumor test method Take well-grown H22 cells, and dilute into tumor cell suspension with appropriate amount of sterile physiological saline. The cell count is 10 7 per mouse peritoneal cavity 0.2 ml (except normal control group). After 24 hours of inoculation, treatment was given. Normal control group and negative control group were given normal saline 0.2ml-only 1 1 intraperitoneal injection; positive control group was given cyclophosphamide SOmg'kg' 1 , O ⁇ ml- -'-d- 1 , intraperitoneal injection. The rLZ-8 treatment group was given the corresponding dose of tail vein injection, 0.2 ⁇ 1 ⁇ c ⁇ cT continuous
  • CTX group 20 0.45 ⁇ 0.02 * 62.5
  • the cadaver ⁇ 0.01 was seen from Table 8.
  • the number of white blood cells in each group was at the same level, and there was no difference compared with the negative control group (P>0.05).
  • the number of white blood cells in the negative control group was higher than that in the normal control group.
  • the number of white blood cells between the low dose group and the middle dose group in the rLZ-8 group was no difference in the number of white blood cells between the low dose group and the middle dose group in the rLZ-8 group (P>0.05).
  • the high-dose group and the normal control group P>0.05
  • the number of white blood cells in the positive control group was significantly lower than that in the normal control group and the negative control group ( ⁇ ⁇ 0.01).
  • CTX group 20 9.67 ⁇ 0.43* 4.49 ⁇ 0.21 * rLZ-8 low dose group 0.25 9.65 ⁇ 0.33* 10.53 ⁇ 0.24**
  • H22 ascites inhibition test results The experimental results showed that the survival time of the rLZ-8 group was longer than that of the negative control group, and the negative control group had decreased appetite, but the weight gain increased rapidly and the activity decreased. As can be seen from Figure 7, the average weight gain trend of the rLZ-8 group was larger than that of the normal group, but smaller than that of the negative control group. This indicates that rLZ-8 can inhibit the growth of H22 tumor cells in the peritoneal cavity of mice to some extent.
  • Example 7 rLZ-8 fluorescent labeling and its effect on normal tissue cells and HL-60 cells
  • Fluorescent pigment Fluorescent pigment (Fluorescein-5-Lsothiocyanalte, FITC), Gil Biochemical (Shanghai); Dimethyl sulfoxide; Carbonate buffer (pH 8 ⁇ 9.5) (Na 2 C0 3 4.3g, NaHC0 3 8.6g plus ddH 2 0 to 500 ml); phosphate buffer (PBS); Desalting Hiprep 26/10 desalting column; AKTA purifier; Hitachi spectrophotometer.
  • rLZ-8 (7.5 mg-ml" 1 ) 20 ml was dialyzed against carbonate buffer (pH 8.3) overnight, 3.75 mg of FITC was weighed, and 3.75 ml of dimethyl sulfoxide (DMSO) was added to prepare FITC-DMSO.
  • DMSO dimethyl sulfoxide
  • This room is prepared.
  • the rats were sacrificed by neck removal, the heart was taken, placed on a cryostat, and the temperature was lowered to -20 ⁇ for sectioning.
  • the myocardial tissue sections were incubated with FITC-rLZ-8 solution (100 ng 'mr 1 ) prepared in PBS at 37 ° C for 1 h. After observation under a fluorescence microscope, a blank control group was set up.
  • Fluorescence microscope 80i (Nikon), IMDM cell culture medium (Hyclone), fetal bovine serum.
  • HL-60 was inoculated into a 24-well culture plate at 2x1 ⁇ ml' 1 with 0.5 ml per well.
  • the cells were taken out at 1.5 h and 6 h, respectively, and added to a 1.5 ml EP tube, centrifuged at 100 ° min- 1 , the supernatant was discarded, washed 3 times with PBS, washed and resuspended.
  • HL-60 cells incubated with FITC-rLZ-8 for 1 h and 6 h showed strong green fluorescence, and cells in 6 h group showed agglutination. There was no green fluorescence in the blank control group, which was significantly different from the former.
  • Recombinant Ganoderma lucidum immunomodulatory protein (rLZ-8) was prepared as a sterile saline solution to prepare 5 ⁇ g'kg 2.5 ⁇ g-kg 1 , 1.25 u g'kg and O SS g'kg dose groups, 0.2 ml/mouse.
  • Jin Lei Saiqiang (recombinant human granulocyte colony-stimulating factor injection (rhG-CSF)), production batch number:
  • Cyclophosphamide (CTX) for injection production batch number 07218821; 200 mg / support.
  • CX Cyclophosphamide
  • mice were divided into 7 groups, 10 in each group, half male and half female. Except for the normal control group (administered with equal volume of normal saline), each group of mice was given intraperitoneal injection of cyclophosphamide, U ⁇ mg g' 1 , 0.2 ml / for 3 consecutive days. On day 3, blood was taken from the tail vein of the mouse and white blood cell counts were performed under the microscope.
  • White blood cell counting method The blood was diluted 20 times with 3% dilute acetic acid, the red blood cells were all dissolved, dropped into a white blood cell counting cell, and counted under a microscope to obtain the number of white blood cells per cubic millimeter of blood.
  • rLZ-8 (5 yg'kg ) 9.92 ⁇ 0.2: 1.3 ⁇ 0.32 5.0 ⁇ 0.12* 20.3 ⁇ 0.11* 14.3 ⁇ 0.21* rLZ-8 (2.5 ygk '10.25 ⁇ 0.3 1.5 ⁇ 0.54 6.6 ⁇ 0.77* 22.8 ⁇ 0.15* 15.2 ⁇ 0.11*
  • Example 9 Treatment experiment of rat leukopenia induced by cyclophosphamide
  • Recombinant Ganoderma lucidum immunomodulatory protein (rLZ-8) is formulated into sterile saline to prepare SO g ⁇ g ⁇
  • Jin Lei Saiqiang (recombinant human granulocyte colony-stimulating factor injection (rhG-CSF)), production batch number: 20071104; 15 ( ⁇ g / branch. Prepared with sterile saline to make Q g'kg' 1 , 0.2ml / only .
  • Cyclophosphamide (CTX) for injection production batch number 07118821; 200 mg / support.
  • AQm ⁇ kg' 1 , 0.2 ml/only was prepared with sterile physiological saline.
  • the experimental animals were divided into 10 groups of 10 animals each, half male and half female.
  • each group of rats were given intraperitoneal injection of cyclophosphamide, AQmg-kg '1, 0.2ml / only: 3 consecutive days.
  • blood was taken from the tail vein of the rat and white blood cell counts were performed under the microscope.
  • the corresponding doses of rLZ-8 and positive drug Jin Lei Saiqiang
  • CTX normal control group and cyclophosphamide
  • White blood cell counting method The blood was diluted 20 times with 3% dilute acetic acid, the red blood cells were all dissolved, dropped into a white blood cell counting cell, and counted under a microscope to obtain the number of white blood cells per cubic millimeter of blood. 3. Experimental results
  • Example 10 Prevention of leukopenia caused by xenon radiation in mice
  • Recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) is formulated into sterile saline to prepare 5 y g, kg human
  • Jin Lei Saiqiang (recombinant human granulocyte colony stimulating factor injection (rhG-CSF)), production batch number: 20060403; 150 ⁇ / support. Prepared with sterile saline to prepare S ⁇ g'kg- 1 , 0.2 ml/head.
  • Cyclophosphamide (CTX) for injection production batch number 07218821; 200 mg / support. Prepared with sterile physiological saline to make 12.5 mg.kg ⁇ 1 , 0.2 ml/head.
  • the experimental animals were divided into 7 groups of 10 animals each, half male and half female.
  • each group was given a positive drug (Jin Lei Saiqiang), different doses of rLZ-8, once a day for 5 consecutive days, and irradiated on the 5th day.
  • the irradiation conditions were 7.50 Gy, 180 mV, 15 mA, and the dose rate was 450 rpm.
  • the number of white blood cells was counted in the tail vein before administration, on the 5th day after administration, on the 5th day after the irradiation, and on the 7th day.
  • the counting method is the same as above.
  • each drug concentration group was administered on the 5th day of administration.
  • the cells were significantly elevated, and there was a significant difference (PO.05).
  • the number of cells was the lowest on the 5th day of irradiation and began to recover on the 7th day.
  • Example 11 Treatment experiment of leukopenia caused by xenon radiation in mice
  • Recombinant Ganoderma lucidum immunomodulatory protein (rLZ-8) was prepared as a sterile saline solution to prepare 5 ⁇ g-kg 1 2.5 ⁇ g.kg, 1.25 ⁇ g-kg 1. 0.625 g-kg 1 ⁇ , 0.2 ml/only.
  • Jin Lei Saiqiang (recombinant human granulocyte colony-stimulating factor injection (rhG-CSF)), production batch number:
  • Cyclophosphamide for injection (CTX), production batch number 07121821; 200 mg / support. Formulated with sterile physiological water to make 12.5 mg'kg, 0.2 ml/head.
  • mice The experimental animals were divided into 7 groups of 10 animals each, half male and half female. Except the normal control group, each group of mice began intraperitoneal injection of an equal volume of normal saline, a positive drug (Jin Lei Saiqiang), and different doses of rLZ-8 on the same day of radiation.
  • the irradiation conditions were 7.50 Gy, 180 mV, 15 mA, and the dose rate was 450 ⁇ /min.
  • the number of white blood cells in the tail veins of each group of animals was measured before the administration, and on the 5th, 7th, and 9th day after irradiation.
  • the body was weighed and deprived 7 days after the irradiation, and the spleen was weighed to calculate the spleen coefficient. Then, it was fixed in Bouin's solution, and the number of spleen surface hematopoietic foci (CFU-S) was counted 6 hours later.
  • the number of white blood cells was the lowest on the 7th day after the irradiation, and the recovery began on the 9th day, and the drug-administered group and the model group were significantly different ⁇ 0.05).
  • each drug group can promote spleen hyperplasia, and the spleen coefficient and spleen nodule production unit (CFU-S) are significantly higher than the model group (P ⁇ 0.05).
  • Table 12 Results of the therapeutic effect of rLZ-8 on the mouse leukopenia model induced by ⁇ -ray irradiation.
  • rLZ-8 5 ⁇ g-kg 1 11 ⁇ 2 ⁇ 0.21 0.84 ⁇ 0.32* 0.31 ⁇ 0.32* 0.90 ⁇ 0.14* rLZ-8 2.5 ⁇ g-kg" 12.9 ⁇ 0.39 0.69 ⁇ 0.54 0.56 ⁇ 0.54* 0.54 ⁇ 0.13* rLZ -8 1.25 ⁇ g-kg' 9.6 ⁇ 0.31 0.82 ⁇ 0.45* 0.65 ⁇ 0.45* 0 ⁇ 70 ⁇ 0.12* rLZ-8 0.62 ⁇ g-kg 10.4 ⁇ 0.91 0.73 ⁇ 0.43* 0.26 ⁇ 0.43* 0.64 ⁇ 0.01
  • rLZ-8 was formulated into 700 ⁇ g ⁇ ml" 1 with physiological saline, and aluminum hydroxide suspension (adjuvant) was formulated into ISmg ' mr 1 , bovine serum albumin (BSA), and ovalbumin (OA) with PBS.
  • mice of 15 weeks old, 18-22 g, 40 were placed in a heat-free environment.
  • the subjects were divided into normal control group, rLZ-8 drug group 1, rLZ-8 drug group 2, and positive control group.
  • Sensitization after administration 2 times 1 mg of BSA was mixed with 0.2 ml of a suspension of 1 Smg ⁇ ml - 1 aluminum hydroxide, and sensitized mice were intraperitoneally injected.
  • the normal control group was given an equal amount of physiological saline instead of rLZ-8.
  • rLZ-8 drug group 2 on the 17th day after sensitization, a mixture of BSA-rLZ-8 (rLZ-8 70 ⁇ g ⁇ ml" 1 and BSA 5 mg ⁇ ml" 1 , prepared with PBS) was intravenously administered 0.1 ml. lOg 1 body weight.
  • the positive control group was injected with 1 mg of OA instead of BSA tail vein on the 17th day after sensitization.
  • Observation criteria The systemic allergic reaction was observed after 30 minutes of intravenous administration of BSA in mice. Positive reaction criteria: mice with convulsions, decreased activity or death; negative reaction, normal mouse activity.
  • Table 14 shows that rLZ-8 inhibits systemic allergic reactions in mice induced by bovine serum albumin (BSA); simultaneous injection of rLZ-8 with shock-dose BSA does not prevent systemic allergic reactions.
  • BSA bovine serum albumin
  • rLZ-8 was formulated into 1 mg'mr 1 with 5% glucose solution; blood cell suspension was prepared: 4 ml of human blood, centrifuged at 1000 r/min for 10 min, and the supernatant was removed. Add the red blood cell pellet to the 5% glucose solution about 10 times, shake well, centrifuge at 1000r/min for 20min, remove the supernatant, and repeat the washing 2-3 times until the supernatant is not red. The resulting red blood cells were mixed with a 5% glucose solution to form a 2% suspension for experimental use.
  • No. 1 to No. 5 was the rLZ-8 drug group
  • No. 6 tube was the negative control group (5% glucose solution)
  • No. 7 tube was the positive control tube (distilled water).
  • a total of 4 sets of parallel control tubes were set up. Add 2% red blood cell suspension, 5% glucose or distilled water in turn, mix well, and immediately incubate in an incubator at 37 °C ⁇ 0.5 °C. Start observation once every 15 minutes, and observe it once every lh after lh for 3 hours. After the end, the solution in each tube was placed in a dry centrifuge tube and centrifuged at 1500 r/min for 25 min. The supernatant was taken, and the OD value of each tube was read at 545 nm of the spectrophotometer with distilled water as a blank to calculate the hemolysis rate.
  • the hemolysis rate of the 1-5 rLZ-8 drug group was less than 5%, indicating that no hemolysis reaction occurred.
  • Rats in the rLZ-8 drug group were given different doses of rLZ-8 in the tail vein, once a day; the control group was given the same amount of normal saline. On the seventh day of dosing, the right thigh bone marrow smear was taken.
  • Figure 11 shows that there was no abnormality in the rat bone marrow smear compared with the normal control group.
  • the subjects were divided into rLZ-8 concentration group, positive drug control group and normal control group. 96 holes
  • the positive control drug PHA is for four people.
  • rLZ-8 has no agglutination effect on human four red blood cells
  • the recombinant Ganoderma lucidum immunomodulatory protein rLZ-8 of the present invention can be administered orally and parenterally as an antitumor drug.
  • the dosage to be taken is determined by factors such as symptoms, age, and weight. For adults, oral administration, 10-1000 mg per dose per day, several times a day; parenteral administration 10-100 mg, several times per week.
  • the oral tablet, pill capsule (including hard capsules and soft capsules) of the present invention comprise recombinant Ganoderma lucidum immunomodulatory protein rLZ-8 and at least one inert diluent (for example, lactose, nectar) Sugar alcohols, glucose, starch, polyvinylpyrrolidone) It is also possible to add pharmaceutically acceptable additives other than inert diluents such as lubricants, disintegrants, stabilizers. If desired, the tablets or pills may be coated with one or more layers of the stomach or enteric material.
  • the parenteral injection comprises recombinant Ganoderma lucidum immunoregulatory protein rLZ-8 and at least one inert water diluent (such as distilled water for injection, physiological saline), and the recombinant Ganoderma lucidum immunoregulatory protein rLZ-8 can also be used as a lyophilized powder. It is previously dissolved in an inert water diluent for injection.
  • inert water diluent such as distilled water for injection, physiological saline
  • Recombinant Ganoderma lucidum immunoregulatory protein rLZ-8 lOOOOmg dissolved in 100ml sterile saline, mixed evenly, and then dispensed into rLZ-8 10mg/ml / concentration of the injection in the vial, sealed, sterilized product.
  • Other items shall comply with the requirements of the 2005 edition of the Pharmacopoeia of the People's Republic of China.
  • Other items It should meet the requirements of the 2005 edition of the Pharmacopoeia of the People's Republic of China.
  • the results of the present invention indicate that the recombinant Ganoderma lucidum immunomodulatory protein has obvious treatment and prevention of leukopenia caused by various reasons, and is superior to the existing clinical application drugs in clinical application dosage, therapeutic effect and clinical discomfort.
  • Animal experiments have shown that the side effects of Ganoderma lucidum immunoregulatory protein in the gene group are much lower than those of recombinant colony cell stimulating factor.
  • the inventors expressed the Ganoderma lucidum immunoregulatory protein using the Pichia pastoris eukaryotic expression system; in the international similar study, we achieved the first fermentation production of 100 liters of rLZ-8, and established a complete production and purification process plan; The measurement of the spatial structure of rLZ-8 was completed at one time, which laid a solid foundation for further study of the anti-tumor mechanism of rLZ-8.
  • LZ-8 can increase the number of white blood cells in the experimental animal lymphocyte hypoxia model, and the pharmacodynamic effect is significantly stronger than the colony stimulating factor; rLZ-8 can induce leukemia cells NB4, K562 and HL-60 cells Apoptosis, which is the first time we have elucidated another possible pathway for fungal immunoregulators to kill tumor cells.
  • Therapeutic experiments in blastoma mice further indicate that rLZ-8 can effectively inhibit the growth of transplanted tumors in vivo; Experiments show that rLZ-8 has no obvious toxicity to rat blood cells; no pathological damage to the aorta and main vessel wall; no effect of bone marrow smear on hematopoietic function.
  • the present invention discloses LZ-8 for the first time. Crystal space structure; It is disclosed that LZ-8 can directly kill tumor cells without destroying normal cells; and discloses a new use of LZ-8 for preventing and treating leukopenia caused by various causes.

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Description

重组灵芝免疫调节蛋白 （rLZ-8 ) 及其用途 技术领域 本发明涉及一种真菌免疫调节蛋白， 特别是一种特定空间结构的重组 灵芝免疫调节蛋白对肿瘤、 白细胞减少症及过敏性疾病等适应症的治疗用 途。 背景技术 现有文献表明真菌灵芝含有的某类蛋白具有广泛的免疫调节活性， 它 可以促进淋巴细胞增殖， 促进人外周血淋巴细胞产生细胞因子的作用， 凝 集红细胞活性， 对粘附分子施加影响， 抑制过敏反应和免疫抗肿瘤作用。
1989年， 由日本 Kino等人第一次从赤灵芝菌丝体提取物中分离得到了一 种免疫调节蛋白， 即灵芝免疫调节蛋白 （Imnmnoregulatory Protein from Ganoderma Lucidium, LZ-8)， 命名为 LZ- 8， 并测定了其基因序列、 氨基酸 顺序和免疫生理活性。 蛋白质测序表明 LZ- 8 由 110个氨基酸残基组成，氨基 端乙酰化。 分子量为 12. 4 kD， 等电点为 4. 4。
发明人首次公开了基因重组灵芝免疫调节蛋白的晶体空间结构， 其结构 的主要特征为： 包括一个 N-端的形成二聚体所需的重要结构域和一个 C端的 FNIII结构域， N-端结构域由一个 a - hel ix (序列为 2- SDTA- LIFRLAWDVK- 15， 14个氨基酸组成） 和 P -strand (序列为 16- KLSFD- 20， 5个氨基酸组成）， 其中在 a -hel ix上的 Ser的残基被乙酰化封闭。 Lz_8单体上的 N端 a -hel ix 和 β -strand与另一单体上相同的结构域通过空间交换形成了重要的二聚体 结合结构域， 呈哑铃状。 C-端的 FNIII结构域属于免疫球蛋白三文治结构， 包含 β - sheet I和 β -sheet II两个结构， 二者分别由 β -strands A- B- E 和 0 - strands G- F- C-D形成。 研究结果表明基因重组灵芝免疫蛋白的本质 作用可能是通过 N端 α螺旋 A形成同源二聚体 （研究表明， 其 N端 13个氨基 酸残基对 Fip形成有功能的同源二聚体十分必要）， 借此结合到淋巴细胞， 通 过一系列信息传递， 并促进淋巴细胞分泌各种细胞因子， 实现免疫调节功能。
现临床所应用具有升高白细胞作用的药物中， 重组集落细胞刺激因子 ( Human Granulo- cyte Macrophage Colony Stimulating Fa) 是唯一 一种用于治疗各种原因所导致的白细胞或粒细胞减少症及骨髓抑治疗法所引 起的白细胞减少症的基因工程重组蛋白制剂。 尽管多年的临床应用证明具有 明显的疗效， 但所有产品均注明了它的临床不良反应为发热、 骨痛、 肌痛、 皮疹、 瘙痒、 腹痛、 腹泻等。 少数患者初次用药可出现首次剂量反应， 表现 为面部潮红、 出汗及血压下降、 血氧饱和度降低。 严重但罕见的不良反应有. - 变态反应、 支气管痉挛、 心力衰竭、 室上性心动过速、 毛细血管渗漏综合征、 脑血管疾病、 精神错乱、 惊厥、 血压下降、 颅内压升高、 肺水肿等。 以往的研究报道认为， 真菌免疫调节蛋白抗肿瘤的作用均是通过免疫途 径实现的， 发明人的研究发现灵芝免疫调节蛋白对人急性髓性粒细胞白血 病细胞 HL- 60、 人早幼粒白血病细胞 NB4、 人慢性髓性白血病细胞 K562等 肿瘤细胞具有直接杀伤作用， 而不是免疫途径杀伤肿瘤细胞。
灵芝免疫蛋白的主要功能还在于它可以促进末梢淋巴细胞和脾脏细 胞的增生， 诱导动物和人体的巨噬细胞分泌多种细胞因子（白细胞介素， 肿瘤坏死因子和干扰素等）， 进而防御及消除病原体的侵害， 维护机体的 健康， 完成免疫调节功能， 但详尽的调节机制目前还不完全明确。 本发明 认为， 该种蛋白还可以明显抑制全身性过敏反应， 抑制肿瘤生长， 对抗器 官移植后的排异现象。
发明内容 本发明的目的是发现重组灵芝免疫调节蛋白 （rLZ- 8 ) 的晶体结构及其在 医学治疗领域的新用途。
本发明的内容为重组灵芝免疫调节蛋白 （rLZ-8 ) 在抗肿瘤、 升高白细胞 及抑制免疫排斥反应等医学治疗领域中的应用。
本发明的重组灵芝免疫调节蛋白 （rLZ- 8 ) 是通过基因工程技术在毕赤酵 母中重组表达而获得的， 该蛋白的核苷酸序列是根据已公开的天然灵芝免疫调 节蛋白的基因序列 （M58032) 重新设计并人工合成的。
本发明通过 X射线衍射的方法首次获得了重组灵芝免疫调节蛋白 （rLZ- 8) 的晶体结构。 该蛋白的晶体培养条件为： 1. 75M ammonium sulfate, 0. 1M Tris pH6. 0, 6. 4% PEG400o 利用悬滴气相扩散法可以获得了空间群为 P32的单晶。 在 X射线衍射仪的像板衍射数据收集系统中收集了一套 1. 8埃分辨率的晶体 数据， 再通过利用 CCP4等结构解析软件进行分析计算， 再以相位交换的方法 来解得一套晶体结构。 在每一个非对称单位中可发现 4个分子， 其中两个分 子间各以 N端 alpha-helix之间的疏水作用和反向平行的 beta-strand之间 的氢键形成二聚体。 单个分子的 C端有一个 FNIII型类免疫球蛋白折叠， 该 结构域由两个 beta- sheet形成。 在 beta-sheet的表面及交界处各有一个推 测为糖连结位点。
本发明在体外抗肿瘤实验中， 用 IMDM细胞培养液配制的不同浓度的 rLZ-8加入到体外培养的人早幼粒白血病细胞 NB4和人慢性髓性白血病细 胞 K562中， 可明显的观察到 rLZ-8对两种肿瘤细胞的杀伤作用， 用 MTT法 检 】， 实验数据表明药效与剂量呈显著的依赖关系。 在体内抑瘤实验中， 将 纯化后 rLZ- 8对荷 S180、 H22小鼠给药， 连续给药 10天， 对腹水瘤小鼠 每日称体重， 脱颈处死， 对皮下接种肿瘤小鼠剖瘤称重， 计算肿瘤抑制率， 并测量各组给药前后的血液中白细胞数。 结果表明， rLZ- 8对肿瘤呈剂量 依赖性抑制， 对环磷酰胺组对比不会显著降低小鼠白细胞数量。 体外杀伤 肿瘤细胞、 体内抑瘤实验都显示， 本发明中的 rLZ- 8具有显著的抗肿瘤作 用 o
° 本发明在实验中还用荧光染料 FITC标记 rLZ- 8， 用 FITC-rLZ-8孵育 人急性髓性粒细胞白血病细胞 HL-60细胞、 大鼠心肌细胞， 兔软骨细胞， 收集细胞并多次洗涤， 置于显微镜下观察， 在 HL- 60细胞样本发出亮度很 高的绿色荧光， 与其正常对照组呈极显著差异， 而大鼠心肌细胞和兔软骨 细胞没有被染色， 说明 HL-60细胞可以被 rLZ-8识别， 但不能标记另外两 种细胞。 这种作用可能与肿瘤细胞表面有较多高度折叠卷曲的寡糖链有关， 因为 rLZ-8的 C端 FNIII型类免疫球蛋白结构域中存在两个糖链结合位点， 而正常组织的细胞表面则不具备这个特点， 如大鼠心肌细胞和兔软骨细胞。 同时， 我们在这个实验中还发现 rLZ- 8对 HL- 60细胞有较强的杀伤作用， 而对另外两种细胞则无明显作用效果， 推测这种 rLZ-8杀伤细胞的选择性 可能与其识别细胞表面受体有关。
为进一步明确 rLZ- 8的抗肿瘤机制， 本发明还应用 PI单染和 ANNEXIN V& FITC双染两种方法对经 rLZ- 8直接杀伤 K562和 NB4等肿瘤细胞进行 细胞凋亡检测， 结果进一步阐明 rLZ-8对肿瘤细胞的杀伤作用机制可能是 诱导肿瘤细胞发生细胞凋亡。 - 本发明公开了 rLZ-8具有预防和治疗白细胞减少症的作用。 在环磷酰 胺所致大、 小鼠白细胞减少症的治疗实验中， 将生理盐水配制的 rLZ- 8对经 环磷酰胺作用的大鼠白细胞低下模型进行给药， 用金磊赛强 （集落细胞刺激 因子） 为阳性药物， 连续给药 7天， 于第 3天和第 7天尾静脉取血， 检测血 液中白细胞数量， 对比治疗前后白细胞数变化， 分析药物疗效。 与环磷酰胺 对照组比较， 在给药第 3天 rLZ-8药物组已明显升高大鼠白细胞， 差异及其 显著， 在给药第 7天时恢复到正常水平。
在 Y辐射所致小鼠白细胞减少症的治疗实验中， 除正常对照组外， 每组 小鼠于辐射的同一天开始腹腔注射等体积的生理盐水、 阳性药物 （金磊赛强） 、 不同剂量的 rLZ- 8。 照射条件为 7. 50 Gy， 180 mV, 15 mA ， 剂量率为 450 伦 /分。 各组动物分别于给药前、 照射第 5、 7、 9天尾静脉采血计白细胞数。 照射后 7天称体重脱臼处死， 取脾脏称重， 计算脾脏系数。 然后固定于 Bouin 氏液， 6h后计脾脏表面造血灶 （CFU-S) 数。 在 Y辐射所致小鼠白细胞减少 症的预防实验中， 在进行辐射前 5天开始给药， 其后的实验方法与治疗实验 一致。 二者的实验数据均表明 rLZ-8可以预防并治疗 γ辐射所导致的白细胞 减少症。
本发明证明其 rLZ-8不产生溶血作用、 对人血四种血型红细胞没有凝集 作用和不产大鼠骨髓像的异常变化。
本发明所述的 rLZ-8可以作为一种免疫抑制剂。 在实验中发现 rLZ- 8可 以抑制小鼠系统性过敏反应，使经牛血清白蛋白 BSA致敏的小鼠没有出现抽搐、 活动减少或死亡等阳性反应。
本发明的内容包括制备一种 rLZ-8为活性成分的药物制剂， 该药物制剂 主要以非胃肠道途径给药， 如普通注射剂、 冷冻干燥注射剂、 脂质体注射剂、 靶向给药的注射剂等， 也可制成经胃肠道途径给药的制剂， 如片剂、 胶囊剂、 喷雾剂、 凝胶剂、 凝胶吸收剂、 口服药、 混悬剂、 冲剂、 贴剂、 丸剂、 散剂、 注射剂、 输液剂、 栓剂、 稀释制剂、 控释制剂等。
本发明的药物制剂其中重组灵芝免疫调节蛋白 （rLZ-8) 的含量为
1-99%, 其他为药物可接受的载体可以是， 淀粉、 糊精、 蔗糖、 乳糖、 纤维 素类、 硬脂酸镁、 吐温- 80、 卡波姆、 甲基纤维素、 羧甲基纤维素钠、 海藻 酸钠、 甘油、 明胶、 聚乙二醇、 硫柳汞、 尼泊金丙酯、 尼泊金甲酯、 尼泊 金乙酯、 三氯叔丁醇、 苯甲酸钠、 硼砂、 新洁尔灭、 山梨醇、 丙二醇异丙 醇、 月桂氮桌酮、 异丙醇 /丙二醇 （1 : 1 )、 异丙醇 / ( 1： 2) 和异丙醇 /丙二 醇 （2: 1 )、 三乙醇胺、 氢氧化钠凡士林、 硬脂酸、 液体石蜡、 羊毛脂、 十 八醇、 十六醇、 单硬脂酸酸甘油酯、 月桂醇硫酸钠、 司盘 60、 平平加 0、 甘 露醇、 山梨醇等。 本领域的药物制剂可以通过本领域通常的方法制备。 说明书附图说明： 图 1 rLZ-8的晶体结构示意图 图 2 rLZ-8对 NB4肿瘤细胞体外杀伤实验结果 图 3 rLZ-8对 K562肿瘤细胞体外杀伤实验结果 图 4 rLZ-8诱导 K562和 NB4细胞凋亡的 PI单染检测结果 图 5 rLZ-8诱导 K562和 NB4细胞凋亡的 Annexin V/PI双染检测结果 图 6 接种 S180艾氏腹水瘤细胞小鼠的体重变化实验结果 图 7 接种 H22移植性肿瘤细胞小鼠的体重变化实验结果 图 8 FITC-rLZ-8对大鼠心肌组织的标记实验结果 （暗、 明场） 图 9 FITC-rLZ-8对兔软骨细胞的标记实验结果 （暗、 明场） 图 10 FITC-rLZ-8对 HL-60细胞的标记实验结果 （暗、 明场） 图 11 大鼠骨髓涂片结果 具体实施方式: 实施例 1: 重组灵芝免疫调节蛋白的获得
1. rLZ>8基因人工合成、 工程菌构建和筛选
根据毕赤酵母遗传密码偏爱性， 在天然灵芝免疫调节蛋白基因序列
(M58032)编码区的基础上， 重新设计编码了 rLZ-8基因 （SEQ1 )进行人工全 基因合成， 该序列与天然灵芝免疫调节蛋白基因编码的氨基酸序列相同， 所不 同的地方在于利用了更适合毕赤酵母表达系统的密码子。
用该序列 （SEQ1 )与酵母 α-因子前导肽编码序列相连成为融合基因， 克隆 入 pMD18-T载体中。将测序正确的载体线性化， 转入酵母基因组中， 在 MM和 MD平板上筛选甲醇利用高效型 Mut+菌株。
天然灵芝免疫调节蛋白基因序列 （M58032 ) 序列编码区为： AGCATCATGTCCGACACTGCCTTGATCTTCAGGCTCGCCTGGGACGT GAAGAAGCTCTCGTTCGACTACACCCCGAACTGGGGCCGCGGCAAC CCCAACAACTTCATCGACACTGTCACCTTCCCGAAAGTCTTGACCGA CAAGGCGTACACGTACCGCGTCGCCGTCTCCGGACGGAACCTCGGC GTGAAACCCTCGTACGCGGTCGAGAGCGACGGCTCGCAGAAGGTCA ACTTCCTCGAGTACAACTCCGGGTATGGCATAGCGGACACGAACACG ATCCAGGTGTTCGTTGTCGACCCCGACACCAACAACGACTTCATCAT CGCCC AGTGG AACTAGGAGGAGGC AG。
经重新设计的重组灵芝免疫调节蛋白 （rLZ-8) 的核苷酸序列 (SEO 1 )是： ATGTCTGATACTGCTTTGATCTTCAGATTGGCTTGGGATGTTAAGAAG TTGTCTTTTGATTACACTCCAAACTGGGGTAGAGGTAACCCAAACAA CTTCATTGATACTGTTACTTTTCCTAAGGTTTTGACTGATAAGGCTTAC ACTTACAGAGTTGCTGTTTCTGGTAGAAACTTGGGTGTTAAGCCATCT TACGCTGTTGAATCTGATGGTTCTCAAAAGGTTAACTTCTTGGAATAC AACTCTGGTTACGGTATTGCTGATACTAACACTATTCAAGTTTTCGTTG TTGATCCAGATACTAACAACGATTTCATTATCGCTCAATGGAACTAGT AA;
所述重组灵芝免疫调节蛋白 irLZ-8)的氨基酸序列是：
MSDTALIFRLAWDVKKLSFDYTPNWGRGNPNNFIDTVTFPKVLTDKAY
VVDPDTNNDFIIAQWN;
2. rL 8工程菌的表达
对规模发酵表达的温度、 转速、 pH值、 装液体积、 甲醇添加量等条 进行检测， 确立了酵母工程菌在 80L发酵罐规模下表达 rLZ-8的工艺条件 优化方法。 根据! "LZ-8的理化特性， 设计了发酵培养基的配方。 目的蛋白 产量约为 SOOmg.I/
3. rLZ-8纯化工艺
发酵液分离机离心→将上清用管式分离机分离→超滤→阳离子交换纯 化柱—AKTA蛋白质纯化工作站制备目的蛋白→强阴离子交换作用层析→疏 水作用纯化柱→凝胶过滤层析。
4. rLZ-8纯度鉴定及分子量测定
利用反相液相色谱对分离纯化的产物进行纯度分析， rLZ-8纯度为 >99%。 激光飞行质谱鉴定重组表达的 rLZ-8分子量为 12722Da。
rLZ~8高级空间结构的确定
" 利用悬滴气相扩散法获得了空间群为 P32的单晶。 晶体培养条件为： 1.75 M ammonium sulfate, O.lM Tris pH 6.0, 6.4% PEG400。 在 MarResearch 345 dtd像板衍射数据收集系统收集了一套 1.8埃分辨率的晶体衍射数据。 利用 CCP4等软件进行结构解析， 通过相位交换， 解得一套晶体结构， 以下为晶 体 rLZ-8的结构简要描述:
LZ-8的结构包括一个 N-端的形成二聚体所需的重要结构域和一个 C端 的 FNIII结构域。 rLZ-8的 N-端结构域由一个 a-helix (序列为 2-SDTAL
IFRLAWDVK-15, 14个氨基酸组成） 和 β-strand (序列为 16-KLSFD-20, 5个 氨基酸组成）， 其中在 α-helix上的 Ser的残基被乙酰化封闭。 Lz-8单体上的 N端 a-helix和 β-strand与另一单体上相同的结构域通过空间交换形成了重要 的二聚体结合结构域， 呈 ¾铃状。 C-端的 FNIII结构域属于免疫球蛋白三文 治结构， 包含 β-sheet l和 β-sheet II两个结构， 如图 1所示二者分别由 β-strands A-B-E和 β-strands G-F-C-D形成， 其中的 β-strand序列为：
A. 21-TPNWGRG-27; B. 34-IDTVTFP-39; C. 48-YTYRVAV-54;D. 57-RNLGVKP -63; E. 72-SQKVN-76; F. 91-TIQVFVVDPD-100; G. 102-NNDFIIAQW-110o 实施例 2: rLZ-8对人早幼粒白血病细胞 NB4的杀伤作用
1.试剂
rLZ-8 除菌后用 IMDM 培养液配制成 8 个浓度， 分别为 Ο^δμ^ιηΓ1 , l^g'mr1 , 3.125μ ·πι1"1 , 6.25μ ·ηιΓ1 , 12.5 g'ml ， 25μ ·ηιΓι , 50μ ·ηι1"1 , 100 g'mr'。
Figure imgf000007_0001
3. 实验结果
表 1和图 2显示， rLZ-8药物组在 OD57。nm的光吸收值与 NB4正常对 照组比较， 有明显差异， 说明 rLZ-8在体外对 NB4肿瘤细胞有较强杀伤作 用。
表 1 rLZ~8对 NB4细胞体外杀伤作用 （《±s, n=6) 分组 剂量 ( μ§ ml"1 ) OD570nm 生长抑制率 （％)
正常对照 1.16士 0.020
~ 20
阳性药对照组 10 0.33士 0.01 * 72
rLZ-8组 0.78 0.71±0.03* 39
1.56 0.65±0.05* 44
3.125 0.53±0.04* 54
6.25 0.45±0.02 * 61
12.5 0.30±0.04* 74
25 0.22±0.01* 81 25
50 0.11±0.01 * 91
100 0.08±0.01* 93
*与 NB4正常对照组比较， P<0.01 实施例 3: rLZ-8对人慢性髓性白血病细胞 K562的杀伤作用
1. 试剂
rLZ-8除菌后用 IMDM培养液配制成 6个浓度， 分别为 S. S ml'1 , 6.25μΒ·ιηΓ1,
Figure imgf000007_0002
2. 实验方法
96 孔培养板中， 试验孔加 K562肿瘤细胞 0.1 ml和 rLZ-8 0.1 ml, rLZ-8浓度由低到 高； 阴性对照组加 K562细胞和培养液各 O.lml; 阳性药物三氧化二砷； 每组作 6个复孔。 置 37°C、 5% C02培养箱中 48h， 在细胞培养终止前 4h加入 ΜΤΤ15μ1 (Smg-ml"1), 细胞 培养终止后加入 ΙΟΟμΙ O.lmol-L"1盐酸异丙醇,在酶联免疫检测仪上 570nm测 OD值。 . 实验结果 表 2 和图 3所示， rLZ-8药物组在 OD57()nm的光吸收值与 k562正常对照组比较， 有明显差异， 说明 rLZ-8在体外对 K562细胞同样具有杀伤作用。 rLZ-8对 K562肿瘤细胞体外杀伤作用 （《±s, n=6)
分组 剂量 （ g'mr1 ) OD570nm 生长抑制率 （％) 正常对照 1.01±0.01
阳性药对照组 10 0.22±0.03* 78.2
rLZ-8组 3.125 0.66±0.03* 34.7
6.25 0.58±0.03* 42.6
12.5 0.52±0.05* 48.5
25 0.31士 0.02* 69.3
50 0.25±0.04* 75.2
100 0.19±0.03* 81.2
*与 K562正常对照组比较， <0.01 实施例 4: rLZ-8对血液肿瘤细胞凋亡作用的影响
1. PI单染流式细胞仪检测
1仪器和细胞株
荧光显微镜， 型号 Leica ASLMD 、 K562、 ΝΒ4。
)
Figure imgf000008_0001
1.4 实验方法
将 K562细胞与 NB4细胞分别加入 24孔板， 1ml/孔， 并加入不同浓度 rLZ-8, 1ml/孔， 每组设 3个复孔。 置 37°C、 5% C02培养箱 24h， 将每个浓度 组细胞收集， PBS洗涤 2次并调整细胞浓度为 l x l 06/ml， 70 %冰乙醇固定， 置 — 20°C过夜。 固定后细胞用 PBS洗涤 2次， 加入 PI (终浓度为 Si^g'mr1)室温避 光孵育 10min， lOOOrmin—1离心 5min， 弃上清液用 400μ1ΡΒ8重悬沉淀。 lh之 内上机检测。
1.5实验结果
由表 3和图 4可见， 与 K562及 NB4正常对照组比较， rLZ-8药物组细 胞调亡率均有所提高，可以得出使细胞凋亡是 rLZ-8杀死肿瘤细胞的途径之一 表 3 rLZ-8诱导 K562及 NB4细胞凋亡的实验结果 （％) K562 8.89 16.43 17.91 21.57
NB4 4.06 19.23 24.51 38.60
2. Annexin V-FITC试剂盒流式细胞仪检测细胞凋亡
2.1仪器和细胞株
FACS Calibur型流式细胞仪美国 Becton-Dickinson公司。 NB4、 HL-60。
Figure imgf000009_0001
2.4实验方法
将 ΝΒ4细胞和 HL-60细胞分别加入 24孔板， 1ml/孔， 并加入不同 浓度 rLZ-8， 1ml/孔， 每组设 3个复孔。 置 37 °C、 5 % C02培养箱中 24小 时， 将每个浓度组细胞收集， 用 4°C预冷的 PBS洗细胞 2 次， 用 250μ1结 合缓冲液重新悬浮细胞， 调节其浓度为 l x l06/ml。 取 ΙΟΟμΙ的细胞悬液于 5ml流式管中， 加入 5μ1 的 Annexin V/FITC和 ΙΟμΙ的 SC^g'mr1 PI溶液， 混匀后于室温避光孵育 15min， 在反应管中加 40(^1PBS， 流式细胞仪分析。
2.5实验结果
由图 5及表 4可以看出， NB4-rLZ-8组和 HL-60-rLZ-8组凋亡率明显 高于其正常对照组， 并且， HL-60-rLZ-8组， 随着 rLZ-8浓度的增加， 凋亡 率亦增加。
表 4 rLZ-8诱导 NB4及 HL-60细胞凋亡的实验结果 （％) 调亡率 正常对照組 阳性药组 ^1^8,.,、 Z"8 ,- n Z"8 rl.
(0.^g.ml ) (0.5μ^ιη1 ) (2.5 g.ml ',
NB4 6.1 23.4 21.0 22.3 34.0
HL-60 0.4 39.7 30.5 407 47.7 实施例 5: rLZ-8对小鼠 S180艾氏腹水瘤的抑制实验
1. 实验材料
小鼠， 体重 18— 22g， 雌雄各半， 由吉林大学实验动物中心提供。 小鼠 艾氏腹水细胞株 S180由本室提供。 环磷酰胺 （CTX) ，江苏恒瑞医药股份有 限公司， 批号： 06101921。 S180腹水瘤及实体瘤实验组均分为正常对照组、 阴性对照组、 阳性对照组、 rLZ-8低剂量治疗组 （OJSmgAg'1 ) rLZ-8中 剂量治疗组 （O^mg^g' rLZ-8高剂量治疗组 （lmg'k^ 每组 10只。
2. 实验方法
S180皮下抑瘤实验方法： 取生长良好的 S180细胞， 以适量无菌生理盐 水稀释成瘤细胞悬液， 细胞计数为 lxlo nl '1 , 每鼠右腋窝皮下接种 0.2ml (正常对照组除外）。 接种 24h后， 给予治疗。 正常对照组和阴性对照组给予 生理盐水 0.2ιη1·只—1 1 , 腹腔注射； 阳性对照组给予环磷酰胺 20mg*kg ，
0.2ml- A-'-d"1 , 腹腔注射。 rLZ-8治疗组分别给予相应剂量尾静脉注射， 0.2ml- 只―1^1。 连续 10天。 分别于给药前、 给药 10d后从小鼠眼眶静脉丛取血， 送 检于吉大一院检验科测白细胞数。 并在停药次日， 颈椎脱臼处死全部小鼠， 解剖取出瘤块， 称瘤重。 按下式计算抑瘤率：
抑瘤率 （％) 二 （阴性对照组平均瘤重一实验组平均瘤重） /阴性对照组 平均瘤重 χ 100%
SI 80腹水抑瘤实验方法： 取生长良好的 S180细胞， 以适量无菌生理盐 水稀释成瘤细胞悬液， 细胞计数为 lxloSnl—1 , 每鼠腹腔接种 0.2ml (正常对 照组除外）。 接种 24h后， 给予治疗。 正常对照组和阴性对照组给予生理 盐水 0.2ml_只―1，^1， 腹腔注射； 阳性对照组给予环磷酰胺 20mg，kg 0.2ml- 只 ^cf1 , 腹腔注射。 rLZ-8治疗组分别给予相应剂量尾静脉注射， 0.2ml，只 ·cT' a 连续 10天。 每日称重， 观察小鼠体重变化程度， 作体重增长曲线。
3. 实验结果
S180皮下抑瘤实验结果： 由表 5可以看出， 3个剂量的 rLZ-8均能抑制 S180的生长， 抑瘤率分别为 16.8%、 25.7%和 45.5 %。 rLZ-8治疗组瘤重 与阴性对照组比较， 均具有显著差异 （ > < 0.01 )。
由表 6可以看出， 给药前， 各组小鼠白细胞数均在同一水平上， 与阴性 对照组比较无差异 （Ρ〉0.05 )。 治疗 10天后， 阴性对照组白细胞数较正常对 照组高， rLZ-8低剂量组和中剂量组白细胞数与阴性对照组比较无差异
(P>0.05 ), 高剂量组与正常对照组比较无差异 （P>0.05 )， 阳性对照组白 细胞数明显降低， 与正常对照组及阴性对照组比较均有显著差异 （尸 < 0.01 )。
表 5. rLZ-8对小鼠移植性肿瘤 S180的抑制作用 («±s， n=10)
分组 剂量 (mg-kg"1 ) 瘤重 (g) 抑瘤率 （％) 阴性对照组 ― 1.01士0.03 ―
CTX组 20 0.35±0.02* 65.3
rLZ-8低剂量组 0.25 0.84±0.03* 16.8
rLZ-8中剂量组 0.5 0.75±0.02* 25.7
rLZ-8高剂量组 1 0.55±0.03* 45.5
* 与阴性对照组比较， 尸< 0.01
表 6. rLZ-8对小鼠移植性肿瘤 S180白细胞影响 （《±s， n=10)
白细胞数
分组 剂量 (mg-kg"1 )
给药前 给药 10d
正常对照组 ― 9.49±0.27 9.54±0.33
阴性对照组 ― 9.54±0.25 10.44±0.34
CTX组 20 9.56±0.31* 4.41±0.25* rLZ-8低剂量组 0.25 9.48±0.30* 10.44±0.18**
rLZ-8中剂量组 0.5 9.43±0.44* 10.34±0.31**
rLZ-8高剂量组 1 9.49±0.36* 9.55±0.36**
*与阴性对照组比较， 尸<0.01； **与阴性对照组比较， 尸 >0.05
S180腹水抑瘤实验结果： 实验结果表明， 各组小鼠腹水出现时间基本一 致， 阴性对照组小鼠体重增长迅速， 存活时间减少。 由图 6可见， rLZ- 8组 小鼠平均体重增长趋势比正常组大， 但比阴性对照组比较小。 说明 rLZ- 8能 够在一定程度上抑制小鼠腹腔 S180肿瘤细胞的增长。 实施例 6: rLZ-8对小鼠肝癌细胞 H22的抑制实验
实验材料
小鼠， 体重 18— 22g， 雌雄各半， 由吉林大学实验动物中心提供。 小鼠 肝癌细胞株 H22， 由本室提供。 环磷酰胺（CTX)， 江苏恒瑞医药股份有限公司， 批号： 06101921。
2. 实验方法
H22肝癌细胞实验组均分为正常对照组、 阴性对照组、 阳性对照组、 rLZ- 8低剂量治疗组 (0. 25mg * kg—' )、 rLZ- 8中剂量治疗组 （0. 5mg · kg— ' )、 rLZ-8高剂量治疗组 （lmg · kg— ' )。 每组 10只。 -
H22皮下抑瘤实验方法： 取生长良好的 H22细胞， 以适量无菌生理盐 水稀释成瘤细胞悬液， 细胞计数为 107 · 每鼠右腋窝皮下接种 0. 2ml
(正常对照组除外）。 接种 24h后， 给予治疗。 正常对照组和阴性对照组给 予生理盐水 0.2ηι1·只 d—1 , 腹腔注射； 阳性对照组给予环磷酰胺 20mg，kg ，
0.2ιη1·只'1 ^1， 腹腔注射。 rLZ-8治疗组分别给予相应剂量尾静脉注射， 0.2ml- 只 连续 10天。 分别于给药前、 给药 10d后从小鼠眼眶静脉丛取血， 送 检于吉大一院检验科测白细胞数。 并在第停药次日， 颈椎脱臼处死全部小鼠， 解剖取出瘤块， 称瘤重。 按下式计算抑瘤率- 抑瘤率 （％) = (对照组平均瘤重一实验组平均瘤重） I
对照组平均瘤重 χ ΐοο %
H22腹水抑瘤实验方法： 取生长良好的 H22细胞， 以适量无菌生理盐水 稀释成瘤细胞悬液， 细胞计数为 107 每鼠腹腔接种 0.2ml (正常对照组 除外）。 接种 24h后， 给予治疗。 正常对照组和阴性对照组给予生理盐水 0.2ml- 只—1 1， 腹腔注射； 阳性对照组给予环磷酰胺 SOmg'kg'1, O^ml- -'-d-1, 腹 腔注射。 rLZ-8治疗组分别给予相应剂量尾静脉注射， 0.2πι1·只 ·cT 连续
10天。 每日称重， 观察小鼠体重变化程度， 作体重增长曲线。
3. 实验结果
' H22皮下抑瘤实验结果： 由表 7可以看出， 3个剂量的 rLZ-8均能抑制 S180的生长， 抑瘤率分别为 16.7%、 30.0%、 42.5 %。 rLZ-8治疗组瘤重与阴 性对照组比较， 均有极显著差异 （P < 0.01 )。
表 7. rLZ-8对小鼠移植性肿瘤 H22的抑制作用 （¾±s， n=10)
分组 剂量 （mg'kg-1 ) 瘤重 (g) 抑瘤率 （％) 阴性对照组 1.20±0.02 ―
CTX组 20 0.45±0.02* 62.5
rLZ-8低剂量组 0.25 1.00±0.03* 16.7
rLZ-8中剂量组 0.5 0.84±0.02* 30.0
rLZ-8高剂量组 . 1 0.69±0.03* 42.5
与阴性对照组比较， 尸 < 0.01 由表 8看出， 给药前， 各组小鼠白细胞数均在同一水平上， 与阴性对 照组比较无差异 （尸>0.05 )。 治疗 10天后， 阴性对照组白细胞数较正常对照 组高， rLZ-8低剂量组和中剂量组白细胞数与阴性对照组比较无差异 (P>0.05 )， 高剂量组与正常对照组比较无差异（P〉0.05 )， 阳性对照组白细胞数明显降低, 与正常对照组及阴性对照组比较均有显著差异 （Ρ < 0.01 )。
表 8. rLZ-8对小鼠移植性肿瘤 Η22白细胞影响 （《±s， n=10)
白细胞数
Figure imgf000012_0001
齐 ij量 (mg-kg-1 )
给药前 给药 10d
正常对照组 ― 9.65±0.28 9.69±0.26
阴性对照组 ― 9.76±0.31 10.49±0.33
CTX组 20 9.67±0.43* 4.49±0.21 * rLZ-8低剂量组 0.25 9.65±0.33* 10.53±0.24**
rLZ-8中剂量组 0.5 9.65±0.38* 10.43士 0.27**
rLZ-8高剂量组 1 9.63±0.41 * 9.86±0.27**
*与阴性对照组比较， **与阴性对照组比较， 尸 >0.05
H22腹水抑瘤实验结果： 实验结果显示 rLZ-8组小鼠生存时间比阴性 对照组延长， 阴性对照组小鼠食欲减退， 但体重增长迅速， 活动减少。 由 图 7可见， rLZ-8组小鼠平均体重增长趋势比正常组大， 但比阴性对照组 比较小。 说明 rLZ-8能够在一定程度上抑制小鼠腹腔 H22肿瘤细胞的增长。 实施例 7: rLZ-8荧光标记及其对正常组织细胞和 HL-60细胞的影响
1. rL 8的 FITC荧光素标记
( 1 ) 试剂与仪器
荧光色素 （Fluorescein-5-Lsothiocyanalte， FITC), 吉尔生化 （上海） ； 二甲基亚砜； 碳酸盐缓冲液 （pH 8〜9.5 ) (Na2C03 4.3g, NaHC03 8.6g加 ddH20至 500ml) ； 磷酸盐缓冲液 （PBS) ； Desalting Hiprep 26/10脱盐柱； AKTA purifier ； 日立分光光度计等。
(2) 实验方法
将纯化的 rLZ-8 (7.5mg- ml"1 ) 20ml对碳酸盐缓冲液 （pH8.3 ) 透析过 夜， 称取 3.75mg FITC， 加入二甲亚砜 (DMSO)3.75ml配制成 FITC-DMSO溶 液。 在 50ml小烧杯中先放入 rLZ-8按 FITC-DMSO溶液逐滴加入 rLZ-8溶液 中， 用 PBS加至 30ml， 磁力搅拌器室温下避光搅拌 4h， 用 Desalting Hiprep 26/10脱盐柱于 AKTA purifier系统上除去游离荧光素， 75ml PBS洗脱，
280nm 495nm检测， 峰收集。
(3) 实验结果
将制备的 FITC-rLZ-8 ( 10倍稀释） 于 220nm〜520nm扫描， A495=0.445， A280=0.67, 计算标记效率 （F/P) 为 3.80。
2. 对大鼠心肌组织的标记作用
(1 ) 试剂与仪器
Leica CM1850 冷冻切片机； wistar大鼠； 荧光显微镜 80i (Nikon);
等渗 PBS缓冲液 （pH7.2); 胎牛血清 （FBS, Gibco); FITC-rLZ-8
本室制备。
(2) 实验方法
将大鼠脱颈处死， 取心脏， 置于冷冻切片机， 待温度降至 -20Ό进行切片, 将心肌组织切片与 PBS配制的 FITC-rLZ-8溶液 （ lOOng'mr1 ) 37°C孵育 lh 后， 荧光显微镜下观察， 并设空白对照组。
(3) 实验结果
荧光显微镜下观察心肌组织无可见荧光， 与空白对照组无差异， 见图 8。
Figure imgf000013_0001
7min), 以等渗 PBS洗 3次， 向 EP管中加入 0.1ml PBS， 重悬细胞。 取悬液 于荧光显微镜观察。
(3) 实验结果
荧光显微镜下观察， 兔软骨细胞形态完好， 无绿色荧光， 与对照组相比无 显著差异。 各实验组照片如图 9所示。
4. 对 H1-60细胞的标记作用
( 1 )试剂与仪器
荧光显微镜 80i (Nikon), IMDM细胞培养液 （Hyclone), 胎牛血清 .
(FBS, Gibco), FITC-rLZ-8和等渗 PBS缓冲液 （pH7.2) 本室制备。
(2) 实验方法
将 HL-60以 2x1 {^ml'1接种于 24孔培养板中， 每孔 0.5ml， 以 IMDM (2%FBS) 培养液配制 FITC-rLZ-8为 lOOng'ml'1，每孔 0.5ml孵育 （37°C )， 设空白对照组、 lh实验组和 6h实验组。 分别于 lh和 6h取出细胞加入至 1.5ml EP管中， lOOOrmin—1离心， 弃上清， 以 PBS清洗 3次， 洗净重悬。
(3) 实验结果
如图 10， 荧光显微镜下观察， 经 FITC-rLZ-8孵育 lh和 6h的 HL-60细 胞绿色荧光强， 其中 6h组细胞出现凝集， 空白对照组无绿色荧光， 与前者具 显著差异。
实施例^ 环磷酰胺所致小鼠白细胞减少症的治疗实验
1. 药物配制
重组灵芝免疫调节蛋白（rLZ-8)无菌生理盐水配制成 5 μ g'kg 2.5 μ g-kg 1, 1.25 u g'kg 、 O SS g'kg 剂量组， 0.2ml/只。
金磊赛强 （重组人粒细胞集落刺激因子注射液 （rhG-CSF) )， 生产批号：
20060403； 150 g/支。 用无菌生理盐水配制成 3.2 g，kg 0.2ml/只。
注射用环磷酰胺 (CTX), 生产批号 07121821 ; 200mg/支。 用无菌生理盐 水配制成 Jmg'kg—1 , 0.2ml/只。
3%乙酸溶液配制： 即取冰乙酸 3ml， 加蒸馏水至 100ml混合， 过滤。
2. 实验方法 实验动物分成 7组， 每组 10只， 雌雄各半。 除正常对照组 （给予等体积 生理盐水） 夕卜， 每组小鼠均给予环磷酰胺腹腔注射， U^mg g'1, 0.2ml/只， 连续 3天。 于第 3天， 小鼠尾静脉取血， 显微镜下白细胞计数。 造模成功后 按上述分组分别给予相应剂量 rLZ-8及阳性药 （金磊赛强） 治疗， 正常对照 组和环磷酰胺 （CTX) 组给予等体积生理盐水， 于治疗第 3d和第 7d、 14d分 别小鼠尾静脉取血， 对比治疗前后白细胞数变化， 分析药物疗效。
白细胞计数方法： 用 3%稀乙酸将血液稀释 20倍， 使红细胞全部溶解， 滴入白细胞计数池中， 在显微镜下计数， 求得每立方毫米血液中的白细胞数。
3.实验结果
由表 9可以看出， 与 CTX模型组比较， 在给药第 3天 rLZ-8药物组白 细胞已明显升高， 在给药第 7天基本达到正常， 且有显著性差异 (P<0.05)。 表 9. rLZ-8对小鼠白细胞减少症模型治疗作用的结果 （^土 S, n=10)
组别 造模前 造模后 给药第 3天给药第 7天给药第 14^ 正常对照组 10.5±0.2( 11.0±0.25 10.8±0.51 10.2±0.11 11.2±0.31 模型组 （CTX组） 10.7±0.5< 1.5±0.71 4.6±0.34 8.3 ±0.34 12.0±0.32
金磊赛强组 10.8±0.2： 1.5±0.35 5.5±0.12* 22.7±0.12* 13.0±0.19* (3.2 g - kg-')
rLZ-8 (5 yg'kg ) 9.92 ±0.2: 1.3±0.32 5.0±0.12* 20.3±0.11* 14.3±0.21* rLZ-8 (2.5 ygk '10.25 ±0.3 1.5 ±0.54 6.6±0.77* 22.8±0.15* 15.2±0.11*
Z-8 ( 1.25 g-kg-110.4 ±0.3: 1.7±0.45 8.1±0.17* 19.7±0.17* 13.9±0.14*
,Z-8 (0.62yg'kg-110.4土 0.9: 1.4 + 0.45 6.4±0.41* 27.2±0.10* 13.9±0.17*
*与模型组比较， <0.05
实施例 9: 环磷酰胺所致大鼠白细胞减少症的治疗实验
1. 药物配制
重组灵芝免疫调节蛋白 （rLZ-8) 无菌生理盐水配制成 SO g^g^
lO g'kg^ 5μ^ 2.5μβ^β- \.25μ^ 0.625 μ g-kg1, OJl g-kg1 剂量组， 0.2ml/只。
金磊赛强 （重组人粒细胞集落刺激因子注射液 （rhG-CSF)), 生产批 号： 20071104; 15(^g/支。 用无菌生理盐水配制成 Q g'kg'1, 0.2ml/只。
注射用环磷酰胺 (CTX)， 生产批号 07121821; 200mg/支。 用无菌生理 盐水配制成 AQm^kg'1， 0.2ml/只。
3%乙酸溶液配制： 即取冰乙酸 3ml， 加蒸馏水至 lOOmr混合， 过滤。
2. 实验方法
实验动物分成 10组， 每组 10只， 雌雄各半。 除正常对照组 （给予等 体积生理盐水） 夕卜， 每组大鼠均给予环磷酰胺腹腔注射， AQmg-kg'1, 0.2ml/只: 连续 3天。 于第 3天， 大鼠尾静脉取血， 显微镜下白细胞计数。 造模成功 后按上述分组分别给予相应剂量 rLZ-8及阳性药 （金磊赛强） 治疗， 正常 对照组和环磷酰胺 （CTX) 组给予等体积生理盐水， 于治疗第 3d和第 7d、 14d分别小鼠尾静脉取血， 对比治疗前后白细胞数变化， 分析药物疗效。
白细胞计数方法： 用 3%稀乙酸将血液稀释 20倍， 使红细胞全部溶解， 滴入白细胞计数池中， 在显微镜下计数， 求得每立方毫米血液中的白细胞数。 3. 实验结果
由表 10可以看出， 与 CTX模型组比较， 在给药第 3天 rLZ-8药物组 白细胞已明显升高， 在给药第 7天基本达到正常， 且有显著性差异 (P <0.05)。 表 10. rLZ~8对大鼠白细胞减少症模型治疗作用的结果 （*±S， n=10)
组别 造模前 造模后 rLZ-8 第 3 rLZ-8 第 7 rLZ-8第 14
天 天 天
正常对照组 Π .8±0.21 11.2±0·41 10.5 ±0.29 11.0±0.85 12.0±0.11 阴性对照组 11·56±0·89 0.8 ±0.71 0.6±0.32 11·2±0·12 11.5 ±0·12 金磊赛强 11·7±0.14 0.6±0.23 1,4±0.11* 13.1 ±0.21* 10.3 ±0.14 rLZ-8 20 μ 11.2±0.11 0.7±0.34 1.6±0.33* 15.8±0.13* 9.8±0·19*
g-kg 1
rLZ-8 10 μ 11.1 ±0.34 0·6±0·56 1.6±0.71 * 15.8±0.33* 10.9±0.21*
g-kg 1
rLZ-8 5 u 11.4 ±0.22 0.8 ±0.79 2.6土 0.64* 17.6±0.23* 11.2 ±0.26
g-kg 1
rLZ-8 2.5 μ 11.0±0.98 0.8 ±0.12 1.7±0.18* 12.8±0.11* 10.6±0.29*
g-kg 1
rLZ-8 1.25 μ 12.0±0.24 0,7±0.11 2.8 ±0.12* 11.0±0.47* 10.3 ±0.45*
g-kg'1
rLZ-8 0.62 μ 11.7 ±0.45 0·8±0.74 5.2±0.14* 12.8±0.74* 12.1 ±0.24*
g-kg 1
rLZ-8 0.31 μ 14.2±0.11 0.9±0.12 1.9±0.17* 13.5 ±0.13* 11.0±0.31
g-kg 1
*与模型组比较， / 0.05
实施例 10: Υ辐射所致小鼠白细胞减少症的预防实验
1. 药物配制
重组灵芝免疫调节蛋白 （rLZ-8) 无菌生理盐水配制成 5 y g，kg人
2.5 μ g-kg 1 1.25 g-kg 1 > 0.625 μ g'kg-1剂量组， 0.2ml/只。
金磊赛强（重组人粒细胞集落剌激因子注射液 (rhG-CSF))，生产批号：20060403; 150μΕ/支。 用无菌生理盐水配制成 S^ g'kg—1 , 0.2ml/只。
注射用环磷酰胺 (CTX)， 生产批号 07121821 ; 200mg/支。 用无菌生理盐水 配制成 12.5mg.kg·1， 0.2ml/只。
2. 实验方法
实验动物分成 7组， 每组 10只， 雌雄各半。 除正常对照组 （给予等体 积的生理盐水） 夕卜、 每组分别给予阳性药物 （金磊赛强）、 不同剂量的 rLZ-8， 每日 1次，连续 5天，于第 5天进行辐射。照射条件为 7.50 Gy， 180 mV， 15 mA ， 剂量率为 450伦 /分。 各组于给药前、 给药第 5天、 辐射后第 5天、 第 7天 分别尾静脉采血计白细胞数。计数方法同上。后称体重脱 Q处死， 取脾脏称重， 计算脾脏系数。 然后固定于 Bouin氏液， 6h后计脾脏表面造血灶（CFU-S)数。 3. 实验结果
由表 11可以看出， 与 CTX模型组比较， 各药物浓度组在给药第 5天白 细胞已明显升高， 且有显著性差异 (PO.05), 照射第 5天细胞数最低， 第 7 天开始恢复。 给药组与模型组有显著性差异 <0.05)o
表 11. rLZ-8对 Y射线辐射所致小鼠白细胞减少症模型预防作用的结果
(*±S， n=10)
组别 给药前 给药第 5天辐射后第 5？辐射后第 7？ 正常对照组 11.5 ±0.20 11.0±0.25 10.8±0.21 9.8±0.11
模型组 11.5±0.56 14·4±0.71 0·30±0.33 0.26±0.33 金磊赛强组 11.4 ±0.22 21.8±0·35* 0.26 ±0.20 0.22±0.12
3.2 g · kg1
rLZ-8 5 μ g-kg" 10.3±0·21 19.5±0.32* 0.32±0·18 0.53±0·35*
•-LZ-8 2.5 μ g-kg 11.7±0.39 12.8±0·54* 0.32±0.15 0.45 ±0.20*
rLZ-81.25 μ g-kg 10.4 ±0.31 12.5 ±0.45* 0.25±0.11 0.46±0.11*
rLZ-80.62 μ g-kg 11·2±0·91 10.7±0·45* 0.16±0.31 0.40±0.12*
*与模型组比较， / 0.05
实施例 11: Υ辐射所致小鼠白细胞减少症的治疗实验
1.药物配制
重组灵芝免疫调节蛋白（rLZ-8)无菌生理盐水配制成 5 μ g-kg1 2.5 μ g.kg 、 1.25 μ g-kg1. 0.625 g-kg1 Μ^Ά, 0.2ml/只。
金磊赛强 （重组人粒细胞集落刺激因子注射液 （rhG-CSF))， 生产批号：
20060403； 15(^g/支。 用无菌生理盐水配制成 S^ g'kg-1, 0.2ml/只。
注射用环磷酰胺 (CTX)， 生产批号 07121821; 200mg/支。 用无菌生理水 配制成 12.5mg'kg ， 0.2ml/只。
. 实验方法：
实验动物分成 7组， 每组 10只， 雌雄各半。 除正常对照组外， 每组小鼠 于辐射的同一天开始腹腔注射等体积的生理盐水、 阳性药物 （金磊赛强）、 不 同剂量的 rLZ-8。 照射条件为 7.50 Gy， 180 mV， 15mA ， 剂量率为 450伦 /分。 各组动物分别于给药前、 照射第 5、 7、 9天尾静脉采血计白细胞数。 照射后 7天称体重脱臼处死， 取脾脏称重， 计算脾脏系数。 然后固定于 Bouin氏液， 6h后计脾脏表面造血灶 （CFU-S) 数。
. 实验结果：
由表 12可以看出， 照射后第 7天白细胞数最低， 第 9天开始恢复， 且 给药组与模型组有显著性差异 <0.05)o
由表 13可以看出， 各药物组均能促进脾脏增生， 脾脏系数、 脾结节生成 单位 (CFU-S)明显高于模型组 (尸<0.05)。 表 12 rLZ-8对 γ射线辐射所致小鼠白细胞减少症模型治疗作用的结果.
(*±S， n=10)
组别 照射前 照射第 5天 照射第 7天 照射第 9天 正常对照组 10.5 ±0.20 11.0±0.25 10.0 ±0.25 12·8±0.51
模型组 11.0±0.56 0.7±0.71 0.2±0.71 0.61 ±0.01 金磊赛强组 11.9 ±0.22 0.68±0.35 0.58±0·35* 0.80±0.11* 3.2 μ g · kg 1
rLZ-8 5 μ g-kg 1 11·2±0.21 0.84±0.32* 0.31 ±0.32* 0.90±0.14* rLZ-8 2.5 μ g-kg" 12.9±0.39 0.69±0.54 0.56 ±0.54* 0.54±0.13* rLZ-8 1.25 μ g-kg' 9.6±0.31 0.82±0.45* 0.65 ±0.45* 0·70±0.12* rLZ-8 0.62 μ g-kg 10.4±0.91 0.73 ±0.43* 0.26 ±0.43* 0.64 ±0.01
*与模型组比较， ^<0.05 表 13. rLZ-8对 γ射线辐射所致小鼠白细胞减少症模型脾脏结节数的影响
G±S， n=10) 组别 脾结节数 脾脏系数 增长率
模型组 4.4±0.10 0.17±0.11
金磊赛强组 6.0±0.11* 0.20±0.10* 26%
3.2 μ g · kg"
rLZ-8 5 μ g-kg" 9.7±0.16* 0.22 ±0.09 54%
rLZ-8 2.5 μ g-kg 14.3 ±0.03* 0.52±0.12* 69%
rLZ-8 1.25 μ g-kg' 6.3 ±0.21* 0.24 ±0.13 30%
rLZ-8 0.62 μ g-kg' 8.9±0.41* 0.50±0.10* 50%
*与模型组比较， / 0.01 实施例 12: rLZ~8的系统性过敏反应实验
1.试剂
rLZ-8用生理盐水配制成 700 μ g · ml"1 , 氢氧化铝悬液 (佐剂)用 PBS配 成 ISmg ' mr1 , 牛血清白蛋白 (BSA)， 卵白蛋白 (OA)。
2. 实验方法
15周龄雄性小鼠 18— 22g， 40只， 放置于无热源环境。 将实验对象分 为正常对照组， rLZ-8药物组 1， rLZ-8药物组 2， 阳性对照组。 rLZ-8药物 组 1， 给予 rLZ-8 0.1ml/10g体重， 腹腔注射， 2次 /周， 共给药 6次。 在给 药 2次后致敏： 将 lmg BSA与 lSmg ^ ml—1氢氧化铝悬液 0.2ml混合， 腹腔 注射致敏小鼠。 致敏后第 17天以 lmg BSA加入 0.2mlPBS， 尾静脉注射小 鼠， 观察过敏反应。 正常对照组给予等量生理盐水代替 rLZ-8。 rLZ-8药物 组 2， 在致敏后第 17天给予 BSA- rLZ-8混合液 (rLZ-8 70 μ g · ml"1与 BSA 5mg · ml"1, 用 PBS配制)静脉注射 0.1ml · lOg 1体重。 阳性对照组， 致敏 后第 17天用 lmg OA代替 BSA尾静脉注射小鼠。 观察标准： 小鼠给予静 脉注射 BSA 30min后观察全身过敏反应。 阳性反应标准： 小鼠抽搐、 活动 减少或死亡； 阴性反应， 小鼠活动正常。
3. 实验结果
表 14所示： rLZ-8能够抑制牛血清白蛋白 （BSA) 诱导的小鼠的系统 性过敏反应； rLZ-8与休克剂量的 BSA同时注射则不能阻止系统性过敏反 应的发生。
表 14 rLZ- 8对 BSA诱导小鼠系统性过敏反应影响的实验结果
分组 致敏注射 休克注射 灵芝蛋白 ϋ 治疗 过敏只数 /总 死亡只数 /总 只数 只数 正常对 BSA腹腔注 BSA静脉注射
― 10/10 1/10 照组 射 (i.P.) (i.v.)
阳性对 BSA腹腔注
OA静脉注射（i.v.) ― 0/10 0/10 照组 射 (i.P.)
rLZ-8药 BSA腹腔注 BSA静脉注射
1 射 (i.P.) (i.v.) + 5/10 0/10 物组
rLZ-8药 BSA腹腔注 BSA- rLZ-8静脉
物组 2 射 (i.P.) 注射 （i.v.) + 9/10 0/10 实施例 13: rLZ~8的溶血实验
1. 试剂
rLZ-8用 5 %葡萄糖溶液配制成 lmg'mr1 ; 血细胞悬液配制： 人血 4ml， 1000r/min离心 10min， 去上清。 将红细胞沉淀加入 5%葡萄糖溶液约 10倍 量， 摇匀， 1000r/min离心 20min， 去上清， 重复洗涤 2— 3次， 至上清液不 显红色为止。将所得红细胞用 5%葡萄糖溶液配成 2%的混悬液， 供实验用。
2. 实验方法
取洁净的试管 28只， 进行编号。 1一 5号为 rLZ-8药物组， 6号管为阴 性对照组 （5 %葡萄糖溶液）， 7号管为阳性对照管 （蒸馏水）。 共设 4组平 行对照管。 依次加入 2%红细胞悬液、 5 %葡萄糖或蒸馏水， 混匀后， 立即 置 37°C±0.5°C的恒温箱中温育。 开始每隔 15min观察 1 次， lh后每隔 lh 观察 1 次， 共 3 小时。 结束后将各管中的溶液置入干燥离心管中离心， 1500r/min， 25min。 取上清， 在分光光度计 545nm处， 以蒸馏水为空白读 取各管 OD值， 计算溶血率。
3. 实验结果
由表 15可以看出， 1一 5组 rLZ-8药物组溶血率均小于 5 %， 可以说明 未出现溶血反应。
rLZ~8对人红细胞致溶作用的实验结果 （《±s， n=4)
试管编号 1 2 3 4 5 6 7 溶血率 0.70±0. 0.74±0. 0.63±0. 0.65士 0. 0.59±0. 0.52±0. 100±0.0
( % ) 03 04 04 04 07 08 0 实施例 14: rLZ>8对大鼠骨髓像的影响
1. 实验材料
Wister大鼠 9只， 100g左右。 rLZ-8用无菌生理盐水配制。分为 όθπ^·]^·1、 30 mg-kg"1、 15 mg-kg 1剂量组。
2. 实验方法
正常对照组 3只， 蛋白低剂量组 2只， 蛋白中剂量组 2只， 蛋白高剂 量组 2只。 rLZ-8药物组大鼠， 分别给予不同剂量的 rLZ-8尾静脉注射， 1 次 /日； 对照组给予等量生理盐水。 给药第七天， 取右侧大腿骨髓涂片。
3. 实验结果
' 图 11 表明， 大鼠骨髓像涂片与正常对照组比较未见异常。
Figure imgf000019_0001
2. 实验方法
将实验对象分为 rLZ-8各浓度组、 阳性药对照组， 正常对照组。 96孔
Figure imgf000019_0002
3.试验结果
由表 16所示， 阳性对照药 PHA对人四种
凝集作用； rLZ-8 对人四种红细胞无凝集作用，
Figure imgf000019_0003
浓度时对绵羊红细胞表现出凝集活性。 表 16 rLZ~8对人四种型别红细胞凝集作用实验结果 正 rLZ-8药物组 g'mr1 )
分组 常
12.5 6.25 3.13 1.56 0.78 0.39 0.20 0.10 0.05 0.03
A
B
0
AB
绵羊红细
胞 实施例 16: 重组灵芝免疫调节蛋白抗肿瘤组合物制剂
1. 通过上述药理实验证明， 重组灵芝免疫调节蛋白 rLZ-8的抗肿瘤作用和 提高白细胞水平的效果是显著的， 而且无毒副作用， 因此。 可以认为重组 灵芝免疫调节蛋白 Ζ-8适于药物使用而且是安全的。
2. 本发明的重组灵芝免疫调节蛋白 rLZ-8作为抗肿瘤药物的应用可以通过 口服和非肠道给药。 服用的剂量由症状、 年龄、 体重等因素决定。 对成年 人来说， 口服， 每人每剂 10-1000mg， 每日数次； 非肠道给药 10-100mg， 每曰数次。
3. 本发明口服药包片剂、 丸剂胶囊 （包括硬胶囊和软胶囊） ， 这些剂型包 括重组灵芝免疫调节蛋白 rLZ-8 和至少一种惰性稀释剂 （例如乳糖、 甘露 糖醇、 葡萄糖、 淀粉、 聚乙烯吡咯烷酮） 也可以加入惰性稀释剂以外的药 物学上可以接受的添加物如润滑剂、 崩解剂、 稳定剂。 如果需要， 片剂或 丸剂可用胃溶或肠溶材料涂敷上一层或一层以上的膜。 非肠道用注射剂包 括重组灵芝免疫调节蛋白 rLZ-8 和至少一种惰性水稀释剂 （如注射用蒸馏 水、 生理盐水） ， 也可以将重组灵芝免疫调节蛋白 rLZ-8 制成冻干粉， 使 用前将其溶解于惰性水稀释剂供注射用。
( 1 )制剂例 1
取重组灵芝免疫调节蛋白 rLZ-8 lOOOmg, 溶于 100ml无菌生理盐水中， 混合均匀后， 分装成 rLZ-8 10mg/ml/支浓度的注射液于药瓶中， 密封， 灭菌 制成产品。其他项目应符合中华人民共和国药典 2005年版注射液项下要求。
(2)制剂例 2
取重组灵芝免疫调节蛋白 rLZ-8 100g，,药用淀粉 0.5kg按公知的胶囊制 备技术和设备制成胶囊， rlz8 10mg/粒.其他项目应符合中华人民共和国药典 2005年版胶囊项下要求。
(3)制剂例 3
取重组灵芝免疫调节蛋白 rLZ-8 100g, 微晶纤维素 560g， 无水乳糖 380g， 硬脂酸镁 200g， 按公知的制片技术和设备制成片剂， rLZ-8 10mg/片. 其他项目应符合中华人民共和国药典 2005年版片剂项下要求。
(4)制剂例 4
取重组灵芝免疫调节蛋白 rLZ-8 适量， 中华人民共和国药典 2005年口 服液项下要求， 按公知的制片技术和设备制口服液。 工业应用性
本发明结果表明， 基因重组灵芝免疫调节蛋白具有明显的治疗及预防 由多种原因导致的白细胞减少症， 在临床应用剂量、 治疗效果及临床不适 症等方面均优于现有的临床应用药物。 动物实验表明， 基因童组灵芝免疫 调节蛋白的副作用远低于重组集落细胞刺激因子。
发明人利用毕赤酵母真核表达系统表达了灵芝免疫调节蛋白； 在国际 同类研究中， 我们第一次实现了 100升 rLZ-8的发酵生产， 并且建立了完 整的生产及纯化工艺方案； 第一次完成了 rLZ-8空间结构的测定工作， 为 深入研究 rLZ-8抗肿瘤机制打下了坚实的基础。
发明人创新性发现! "LZ-8可以提升实验动物淋巴细胞低下模型的白细 胞数量， 且药效学作用明显强于集落刺激因子； rLZ-8可以诱导白血病细 胞 NB4、 K562和 HL-60发生细胞凋亡， 这也是我们第一次阐明了真菌免 疫调节蛋白杀伤肿瘤细胞的另一可能途径。苛瘤小鼠治疗实验进一步表明， rLZ-8可以有效的抑制移植性肿瘤在体内的生长； 安全性实验表明， rLZ-8 对大鼠血细胞无明显毒性；对主动脉、主静脉血管壁未见病理性破坏作用; 骨髓涂片结果表明对造血机能也无病变作用。 本发明首次公开了 LZ-8的 晶体空间结构； 公开了 LZ-8可以直接杀伤肿瘤细胞而对正常细胞没有破 坏作用； 公开了 LZ-8对预防及治疗多种原因导致的白细胞减少症的新用 途。

Claims

权利 要求
1. 一种由核苷酸序列 （SEQ1 ) 所编码的具有特定空间结构的重组灵芝免 疫调节蛋白 （rLZ-8)， 该蛋白在制备用于抗肿瘤、 升高白细胞和抑制免 疫排斥反应等药物中的用途。
2. 根据权利要求 1所述的用途， 所述重组灵芝免疫调节蛋白 （rLZ-8) 的 核苷酸序列 (SEQ 1 )是： ATGTCTGATACTGCTTTGATCTTCAGAT
TGGCTTGGGATGTTAAGAAGTTGTCTTTTGATTACACTCCAAACTG GGGTAGAGGTAACCCAAACAACTTCATTGATACTGTTACTTTTCCT AAGGTTTTGACTGATAAGGCTTAC ACTTAC AGAGTTGCTGTTTCTG GTAGAAACTTGGGTGTTAAGCCATCTTACGCTGTTGAATCTGATGG TTCTCAAAAGGTTAACTTCTTGGAATACAACTCTGGTTACGGTATT GCTGATACTAACACTATTCAAGTTTTCGTTGTTGATCCAGATACTAA CAACGATTTCATTATCGCTCAATGGAACTAGTAA;
所述重组灵芝免疫调节蛋白 (rLZ-8)的蛋白序列是：
MSDTALIFRLAWDVKKLSFDYTPNWGRGNPNNFIDTVTFPKVLTDK
VF WDPDTNNDFIIAQWN;
所述重组灵芝免疫调节蛋白 (rLZ-8)的高级结构是： rLZ-8的蛋白结 构包括一个 N-端的形成二聚体所需的重要结构域和一个 C端的 FNIII 结构域， rLZ-8的 N-端结构域由一个 α-helix (序列为 2-SDTALIFRLA WDVK- 15, 14个氨基酸组成） 和 β-strand (序列为 16-KLSFD-20, 5个 氨基酸组成）， 其中在 α-helix上的 Ser的残基被乙酰化封闭， Lz-8单体 上的 N端 a-helix和 β-strand与另一单体上相同的结构域通过空间交换 形成了重要的二聚体结合结构域， 呈哑铃状， C-端的 FNIII结构域属于 免疫球蛋白三明治结构， 包含 β-sheet l 和 β- sheet II两个结构， 二者 分别由 β-strands A-B-E和 β-strands G-F -C-D形成， 其中的 β-strand序 列为： A. 21-TPNW GRG-27; B. 34-IDTVTFP-39; C. 48-YTYRVAV-54; D. 57-RNLGVK P-63; E. 72 -SQKVN-76; F. 91-TIQVFV VD PD-100; G.
102-NNDFIIAQW-110。
3. 根据权利要求 1所述的用途， 所述的肿瘤是： 血癌， 肺癌， 胰腺癌， 肝 癌， 肠癌， 淋巴癌， 前列腺癌， 子宫癌， 骨癌， 乳腺癌等肿瘤。
4. 根据权利要求 1所述的用途，升高白细胞作用相关适应症可以是癌症化 疗、 放疗等原因导致中性粒细胞减少症。
5. 根据权利要求 4 所述的用途,升高白细胞作用相关适应症可以是骨髓移 植后的中性粒细胞减少症或骨髓发育不良综合征引起的中性粒细胞减 少症或骨髓增生异常综合征伴中性粒细胞减少症。
6. 根据权利要求 4所述的用途， 升高白细胞作用相关适应症可以是再生障 碍性贫血引起的中性粒细胞减少症或先天性、特发性中性粒细胞减少症 或周期性中性粒细胞减少症。
7. 根据权利要求 4所述的用途,升高白细胞作用相关适应症可以是化学中 毒、 受辐射所导致的白细胞减少症。
8. 根据权利要求 4 所述的用途,升高白细胞作用相关适应症可以是由传染 病引起的白细胞减少症， 包括伤害、 病毒感染、 支原体肺炎、 传染性肺 炎、 粟粒性肺结核等。
9. 根据权利要求 4所述迫用途，升高白细胞作用相关适应症可以是由常规 用药引起的白细胞减少症， 所述常规用药包括使用青霉素类、 头孢菌素 类、 氨基糖苷类、 大环内酯类、 氯霉素类、 林可酰胺类、 多肽类等抗生 素药物及治疗甲亢的药物、 治疗糖尿病的药物。
10.根据权利要求 1所述的用途，其中预防白细胞减少作用适用于在癌症化 疗、放疗或骨髓移植前使用， 并且预防由这些治疗手段导致的白细胞减 少症。
11.根据权利要求 1所述的用途， 其中抑制免疫排斥反应， 其特征为抑制由 器官移植引起的免疫排斥反应， 可以有效的进行抗原覆盖， 抑制整个抗 原递呈过程。
12.根据权利要求 10 所述的用途，抑制免疫排斥反应的适应症可以是肝移 植、 肾移植后引起的免疫排斥反应症或肝移植、 肾移植后引起的免疫排 斥反应对传统免疫抑制剂的耐药症。
13.—种包含权利要求 1 所述的重组灵芝免疫调节蛋白 (rLZ-8)和任选的药 学可接受的辅剂的药物组合物。
14.根据权利要求 13所述的药物组合物， 其可以通过口服和非肠道给药。：
15.根据权利要求 13所述的药物组合物， 其中口服药包括口服药、 片剂丸 剂和胶囊。
16.根据权利要求 13所述的药物组合物， 其中非肠道给药包括外用药和注 射剂。
17.根据权利要求 13所述的药物组合物， 其中注射剂型包括冻干粉针剂和 水针剂。
PCT/CN2008/002142 2008-01-03 2008-12-30 Protéine immunomodulatrice recombinante de reishi (ganoderma lucidium) (rlz-8) et ses utilisations Ceased WO2009094850A1 (fr)

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