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WO2015055026A1 - 重组灵芝免疫调节蛋白与人血清白蛋白融合蛋白及其制备方法与应用 - Google Patents

重组灵芝免疫调节蛋白与人血清白蛋白融合蛋白及其制备方法与应用 Download PDF

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WO2015055026A1
WO2015055026A1 PCT/CN2014/082031 CN2014082031W WO2015055026A1 WO 2015055026 A1 WO2015055026 A1 WO 2015055026A1 CN 2014082031 W CN2014082031 W CN 2014082031W WO 2015055026 A1 WO2015055026 A1 WO 2015055026A1
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rlz
protein
fusion
group
egg
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French (fr)
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张喜田
孙非
梁重阳
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Priority to EP14854197.2A priority Critical patent/EP3034515B1/en
Priority to AU2014336815A priority patent/AU2014336815B2/en
Priority to US14/914,258 priority patent/US20160289280A1/en
Priority to RU2016118398A priority patent/RU2665802C2/ru
Priority to CA2927632A priority patent/CA2927632A1/en
Priority to JP2016517518A priority patent/JP6475698B2/ja
Publication of WO2015055026A1 publication Critical patent/WO2015055026A1/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
    • 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
    • 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
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A61K36/074—Ganoderma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A61K38/385—Serum albumin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates to the construction, expression, purification and application of a fusion egg of a child group, and particularly relates to a recombinant Ganoderma lucidum immunoregulatory egg and human serum white egg yolk fusion for treating cell plaque and tumor embolism caused by phlegm White preparation and its application. Background technique
  • Antrodia camphorata immunomodulating egg ii LZ 8 The source is derived from the mycelium of Ganoderma lucidum, and its structural features are as follows: including an important domain required for the formation of a dimer at one end and a C-terminal FNI I: ⁇ The domain, the end domain of rLZ ⁇ S consists of one a-he.l .i . .:
  • A has important drugs that are transported to hepatocytes by diffusion and carrier transport, and then degraded by the granulocytic enzyme P4S0, the egg lysate or lysosome in the cytosol; and for larger peptides And proteins are degraded by uptake by hepatocytes by endo-mediated endocytosis.
  • P4S0 granulocytic enzyme
  • P4S0 the egg lysate or lysosome in the cytosol
  • P4S0 granulocytic enzyme
  • proteins are degraded by uptake by hepatocytes by endo-mediated endocytosis.
  • Ling The molecular weight of dimer of diacyloid is less than 26kEte, it may cause high clearance rate in vivo, and the pharmacokinetic parameters are difficult to meet the requirements of new drug burst. Therefore, it is extended by means of gene level fusion and other techniques. The time spent in the body lays a solid foundation for its application in clinical
  • Gene fusion technology is a fusion egg that connects different base functions to express a complex function, and can be extended by the principle of increasing the molecular weight of the polypeptide and egg tart or changing the affinity of the drug and the receptor by the fusion technique. The half-life of the drug.
  • the principle of constructing a fusion protein is to remove the terminating secret of a protein-encoding gene and then ligating the coding gene of another egg tick with a stop codon, that is, to achieve fusion of the coding genes of the two S, At the same time, two proteins are expressed.
  • the fusion egg has high stability, can regulate expression and is simple to prepare, and the product is uniform, and has little effect on the activity of the egg->peptide drug. It is a good way to study the long-acting polypeptide > egg drug.
  • the fusion gene of the current research S is human serum protein-based sleepy, human immunoglobulin (Ig (U, ig (; 4) gene, etc.)
  • Human blood albumin Itoro ⁇ Serufn Al tami tube is called human
  • the protein in plasma, its non-glycosylated single-chain polypeptide ft contains 585 gas, basal acid, molecular weight of 66k3 ⁇ 4 in plasma, its concentration is 42g / L, accounting for about 60% of total plasma eggs.
  • Human serum in body fluids Eggs 3 ⁇ 4 3 ⁇ 4 to transport fatty acids, biliary pigments, thioglycolic acids, steroid hormones, saponins and many therapeutic molecules; while maintaining normal blood osmotic pressure.
  • bloody white eggs can be used Treatment of body grams and burns, used to supplement blood loss caused by surgery, ft accident or major bleeding, can also be used as a blood pad compatibilizer.
  • Human immunoglobulin is the most abundant protein in human blood with a half-life of up to 2i days. It has been reported that the Fc fragment of IgG is fused with other proteins, which can significantly increase the biological activity and half-life of other proteins in vivo. Most of the experimenters choose the g subtype of g « J:g(;4 Pe segment as a fusion object, this method has been widely applied to some clinically important cytokines and soluble receptors, such as s NF a R, U 3 T! JV 4, 0-2, IFN ⁇ a, etc. Got a success.
  • the present invention adopts a gene recombination technology to construct a eukaryotic expression system by combining Ganoderma lucidum immunomodulatory protein with human blood stasis ⁇ egg, human rabbit globulin: After expression and purification, the protein of sputum was obtained, and the biological activity and pharmacological study of the target protein were carried out. The results showed that the fusion protein of Ganoderma lucidum immunomodulator (L2-8) and HSA fusion protein in pharmacological and biological activities and pathological application. There is a significant difference: different "invention content
  • the object of the present invention is to provide a recombinant Ganoderma lucidum immunoregulatory protein and human serum albumin fusion protein and a preparation method thereof, and to explore its application in the treatment of leukopenia caused by chemotherapeutic drugs and its application in antitumor drugs.
  • Fusion protein sequence the Ganoderma lucidum rabbit regulatory protein ⁇ 8) and the fusion protein i HHSA, the special ffi of which comprises 'Ganoderma lucidum immunoregulatory protein and HSA; the amino acid sequence thereof is: SDTM.
  • QMLGL (SEQ ID NO. i), wherein the cytosolic acid sequence of the peptide linked between the C-terminus of the Ganoderma lucidum immunomodulatory polypeptide and the human serum albumin polypeptide is " ⁇ 3 ⁇ 4SS;
  • the present invention provides the Ganoderma lucidum rabbit regulatory protein z 8) and the human IgGIFc segment! 1 8 ⁇ Fcl fusion protein, characterized by comprising Ganoderma lucidum immunoregulatory S and GlFc segments; the amino acid sequence thereof is:
  • the present invention provides a Ganoderma lucidum rabbit regulatory protein (LZ 8) and a human IgG4 Fc segment of rLZ 8 3 ⁇ 44 fusion egg, characterized by comprising Ganoderma lucidum immunoregulatory S and Ig (; 43 ⁇ 4 segment; the amino acid sequence thereof is:
  • KCKVSXK (; i.PSS ;:KT SM3 ⁇ 4;QPREPQVYn. PSQBEM'r QVSLTa ⁇
  • the conditions increase the expression level of the fusion protein; ⁇ gravity column affinity chromatography, molecular sieve, deuterated metal affinity affinity analysis (IMAC) method, hydrophobic g analysis (H1C), anion theory
  • the target product obtained the purity of different fusions of more than 99%, and provided the raw material medicine for the subsequent pharmaceutical experiment.
  • the leukopenia test was performed and the number of white blood cells was measured by an animal whole blood cell analyzer. The results showed that different fusion proteins and rLZ 8 were significantly different in the treatment of leukopenia. Among them, the fusion protein promoted the leukocyte growth cycle at the same dose.
  • the fusion egg rLZ 8 H promoted the growth of ⁇ cells more; indicating that HSA is more suitable as a fusion image of rLZ 8 , which can be obvious Strong ⁇ 8 in the treatment of cytopenia; inhibition of melanoma cell growth by fusion of g ⁇ l rLZ-8-HSA shows that the fusion protein rLZ 8 HSA is effectively inhibited by the same amount (as LZ 8 )
  • the growth of melanoma cells, before the fusion of r:L2 8 ⁇ treatment effect is significantly worse: different; at the same time in the fusion protein ⁇ 2 ⁇ 8 ⁇ ⁇ inhibition of liver cancer cell growth can be seen in the same During the treatment period, the cure rate of the fusion protein rLZ ⁇ 8TMHSA was significantly improved, which was unexpected by the inventors; the present invention also provides ft eggs.
  • the rLZ 8 HSA drug group has significantly stimulated platelet proliferation in mice at the initial stage of administration, and the difference is extremely significant, and returns to i in the middle of administration.
  • the experimental animal model of thrombocytopenia caused by injection of antiplatelet blood stasis has also achieved good results.
  • the beneficial effects of the present invention are as follows: the fusion protein provided by the present invention has a significantly longer half-life in vivo than rU 8; the fusion protein formed by the gene fusion technology will produce the biological activity of the target protein after fusion of the egg 6 , and The activity of the » protein-containing rL2-8-HSA of the invention was tested by ffi to show that its biological activity was significantly better than that of the previous rLZ 8; and the rLZ ⁇ 8-!iSA constructed by the present invention poked the Pichia pastoris project
  • the fermentation process of the strain is simple, the yield is high, and the expression product is simple and easy to purify, which provides a legal condition for industrial production; since the expression product of the Pichia pastoris expression system is easily degraded in the fermentation liquid, etc.
  • the invention greatly reduces the degradation of the target product during the fermentation expression process of Pichia pastoris by controlling the fermentation process conditions, and the yield of the nesting product: the invention uses the in vivo test to prove that the half-life in vivo is significantly longer than that before the fusion of rLZ 8
  • the lowest dose and onset time in the treatment of ⁇ -cell reduction study were excellent before rLZ 8 fusion; using melanoma and liver cancer as representative of anti-tumor research, respectively
  • the therapeutic effects of the two experimental methods are significantly better than the fusion Pre-rLZ 8, which was unexpected by the inventors; in the example of treatment of thrombocytopenia, it can be seen that the model group is compared with the rLZ 8 HSA drug group at the initial stage of administration. Plate hyperplasia, the difference is extremely significant, return to normal level in the middle of administration, which is the best effect that r
  • Lane 1 sample is protein Marker; Lane 2 sample is HSA standard; Swim 3 sample is; Lane 4 sample is induced 72h ri.. Z-8-fiSA on the ulcer; Lane 5 sample is induced 66h rLZ- 8-HSA supernatant; Lane 6 sample was induced 72h rLZ 8 Pel supernatant; Lane 7 sample was induced 66h Fci supernatant; Lane 8 sample was induced 72h rLZ 8 Fc4 supernatant; Lane 9 sample was induced 66h rLZ "8-Fc4 supernatant.
  • Lane ⁇ sample is egg t Marker; Swim 2 sample is rLZ 8; Lane 3 sample is rLZ HA; Beach ⁇ 4 sample is rLZ 8- Pel; Lane 5 sample is rLZ 8 Fc.'.i.
  • the constructs were constructed.
  • the target fragments rLZ8 HSA, rLZ8-Fcl and rLZ8 Pc4 sequences were synthesized according to yeast codon preference, and stored in puc57 plasmid.
  • Primers with ft-containing restriction sites St « j: and ⁇ were synthesized as follows:
  • LZ-8-HSA 5' CATAGGCCTTCTGATAGTGCTTIXiA 3'
  • ⁇ agarose electrophoresis identified the target fragment in 2184 bp (LZ 8 3 ⁇ 4), 1064 bp (LZ 8-Pcl), 774 bp (S, Z-8-Fc4), > pP C2a ⁇ vector and target j ⁇ according to GH BART Seamless Cloning arid Assembly Kit kit instructions, the carrier and l3 ⁇ 4 fragment molar ratio is ⁇ : 3, react with buffer, iOX enzyme mixture for 3 () minutes. The Wul ligation product was transformed into E. coli competent DH5 alpha, and after 1 hour of incubation, the LB plate of the sputum-resistant susceptor was picked and cultured overnight.
  • the sputum i2000 g was centrifuged to discard the supernatant, and the recombinant plasmid was extracted by the plasmid extraction kit.
  • the sequencing was carried out by double enzyme digestion and electrophoresis to identify whether the transformant was confirmed. Finally, the correctness of the sequence was confirmed by ⁇ ' ⁇ and A0X primers.
  • the recombinant plasmid was linearized with Sael enzyme, 37 ⁇ , about 1 hour.
  • Pichia pastoris was inoculated into YP culture in a SO'C ⁇ OOrpfn shaker overnight. Expanded culture to 500tnL, 0l «reach 1.: ⁇ : left and right, prepare yeast competent, ice bath After 3 () min, resuspend in the ice bath without water, 4 ⁇ :, centrifuge ⁇ ⁇ ' repeated 2 times. Change the ice bath! The sorbitol resuspends,
  • the specific method is as follows:
  • the fermentation broth is 10000 rpm/separated to obtain the supernatant, and then purified by a medium 3 ⁇ 4 fiber column with a diameter of 100 M (microfiltration) to remove small molecules of salts and steroids to obtain a yellow color containing the pigment and nucleic acid. Clarification solution.
  • rProtein A Gravi Trap gravity column affinity layer fold 3 ⁇ 4 buffer A phase 22M phosphate buffer, ⁇ 7. 7,0. 15M sodium chloride, prepare buffered sputum phase; 0. 1. ⁇ lemon Acidate buffer, ⁇ 4. 0, 0. 22 P m suction filtration, ultrasonic degassing; equilibrate column with phase A phosphate buffer, volume 10 ml. ⁇ ⁇ 1, rI, Z ⁇ 8TMFc4 fermentation broth 0ffl l.
  • Example treatment 90ml fermentation broth i() X phosphoric acid Salt buffer, 22 ⁇ ⁇ filter and sputum preservation
  • the inner volume was taken in a 1.5 ml centrifuge tube and the test was retained. After 5 uses, regeneration is required, 3 ⁇ 4 guanidine hydrochloride is added, in is placed, and the column is washed with phase A.
  • Step (1) Purify rLZ 8 ⁇ 3 ⁇ 43 ⁇ 4 packing with ⁇ 3 ⁇ 4 affinity chromatography (IC) method: i AC Seph rose 6Fast Flow 1 liter
  • IC affinity chromatography
  • Step (2) Purification is carried out by hydrophobic analysis, and the elution peak of the buffer solution obtained by the step (1) is subjected to hydrophobic 11 purification.
  • Phenyl SepharoseTM 6l t Flow (high sub) (GE) was used to fill the 5 cm diameter layer column, column 3 ⁇ 4 loom., the medium used 3 ⁇ column buffer before use.
  • C 0mm l I. Phosphate, (). 5M sodium gasification, pH 6.5 balance.
  • the eluate of the B-buffer solution containing the quinone protein was added to the deionized water, and the concentration of NaCl was diluted to adjust the value to 6.5 with phosphoric acid.
  • Polyester It was then eluted with 0-0.5 M NaCI over a 10 column volume gradient to collect the 3 ⁇ 4 main peak.
  • WWST-1 method was used to detect the effect of fusion egg on the spleen cell value of mice, reflecting the biological activity.
  • the invention ⁇ W ⁇ - ⁇ /c female small, weight 20-22g, neck The mice were sacrificed and the spleen was taken unconditionally.
  • the spleen was pre-added with 5 ml of 10% calf blood: Chuan! Tf smashed the spleen separately, and the gauze was filtered to remove the woven spleen.
  • Cellular cell solution Take H) 0 microliters of cell suspension to be added) Microliter of 2% glacial acetic acid solution "Microscope i".
  • 3 ⁇ 4 1. Different fusion 3 ⁇ 4 promotes cell value biological activity (X soil s
  • mice weighing about 18 22g were used as experimental mice, and 10 mice in each group were administered intravenously at a dose of 100 g / kg ( ⁇ - ⁇ ).
  • the design time period was 2 4, 6 8 , i0 hours after the blood test at different time periods, the results, the curve of the drug concentration and time (Garden 4) can be seen from the results of the actual results, with the drug into the mice With the prolongation of time, the concentration of the drug in the blood decreased to some extent.
  • Example 5 Fusion of egg rLZ 8 HSA in the treatment of cytoreductive disease
  • rLZ 8 is prepared with sterile physiological saline. Divided into yg/kg, 30 g/kg o ⁇ g/kg S group; rLZ iHI: SA 3 ⁇ 4 protein is prepared with sterile physiological saline, > 60 ⁇ g/kg, 30 ⁇ g/kg, l 5 ⁇ g / kg (in terms of LZ 8 ) dose group; gold S Saiqiang [child group human granulocyte colony stimulation a sub-injection (rM; CSF); j, production batch: 20130403; 7 ⁇ ⁇ g Z branch, Prepared with sterile physiological saline, 13, 5 yg / mL, 0.1 / single, cyclophosphamide (CP) for injection, production batch number 13020225 ; 20 mg / support with sput
  • normal control group )., Z"8 low dose group, ⁇ 8 medium dose group, )..7-8 high dose group, rLZ ⁇ 8 ⁇ HSA fusion protein low middle and high dose group, positive control group (Jin Lei Saiqiang).
  • each group of rats was given cyclophosphamide tail vein injection, 20ing / raL, 0. iniL / only, continuous: 3 ⁇ 4 ⁇ . Days, blood was taken from the tail vein of the rat, and the number of white blood cells was detected by the cell analyzer.
  • 3 ⁇ 4 egg, and positive drug golden 3 ⁇ 4 Saiqiang
  • the rats in the group and the CP group were given the same amount of normal saline, and blood was taken from the tail vein of the rats on the first day, on the 3rd day and the 7th day, and the number of white blood cells was detected.
  • the drug efficacy was analyzed by comparing the changes of the number of S3 ⁇ 4 cells before and after treatment.
  • the rLZHS i group had significantly increased the "rat cells, which were different and significant, and basically reached normal and gold on the 7th day of administration.
  • the number of S cells in the rLZ 8 HSA group increased on the first day of administration; on the 7th day after administration of H, ⁇ 3 ⁇ 4, the number of ⁇ 3 ⁇ 4 cells in the rLZ 8 HSA group was basically normal.
  • the focus is on the comparison of rL2- - group with rLZ 8 egg S l . It can be seen that under the same dosage conditions, rLZ 8 has obvious ⁇ cell proliferation on the first day after administration, numerically.
  • the rLZ-8 group compares ⁇ (). ⁇ , ⁇ (). 05.
  • mice melanoma cells B16-F 10 were selected from a medium containing 10% fetal bovine serum, and cultured in a 37" C, C te incubator.
  • the ventral flank K was slowly injected into the iU6-F10 cell suspension 200 ⁇ (cell content 1 ⁇ 10′) using an I ml syringe to establish a mouse xenograft model.
  • Grouping ' ⁇ treatment 24 hours after tumor cell inoculation, tail vein injection of rLZ-8 group (123 ⁇ g / kg, 246 g / kg, 492 g / 1 ⁇ 2), rLZ - 8- HSA group (with LZ - 8 S t ;: U. i 23 g/kg, 246 ⁇ g/kg, 492 ⁇ g/kg), dacarbazone group (2.5 rng/kg) or saline group.
  • rLZ-8, rLZ-8-HSA was injected once a day, and dacarbazine was injected continuously for 5 days in the tail vein. The injection was repeated 3 weeks later, and the treatment period was 28 days.
  • mice were observed for 3 ⁇ 4 state, ⁇ 13 ⁇ 4 7 days, and the body weight was taken every two or two weeks.
  • the tumor was removed at the end of the final experiment, and the tumor was weighed and recorded.
  • Growth inhibition rate (average of tumor weight in saline group - mean value of tumor weight in administration group) / Physiological value of epilepsy in saline group, and the inhibition rate of therapeutic drug on tumor growth in situ was calculated.
  • the experimental results show that after the tumors are weighed, the average number of tumor weights is calculated for each group. It can be seen from the table that the groups are administered for 28 days, and the rl.Z-8-USA dose group is smaller than other groups. In the rLZ-8 rLZ-8-USA group, as the drug concentration gradually increased, the subcutaneous tumor sputum gradually decreased.
  • 3 ⁇ 4 test method subcutaneous tumor inhibition test method: take well-grown cells, diluted with appropriate amount of lf-free saline to form a tumor cell suspension, the cell count is "0 7 per mouse right ⁇ 3 ⁇ 4 subcutaneous inoculation (). 2ml ( Except for the normal control group). After 2 hours of inoculation, treatment was given. ⁇ The normal control group and the negative control group were given 2 ⁇ 4 saline, 0.2ml ⁇ only 1 ⁇ d V-injection; the positive control group was given cyclophosphamide 20r «g ⁇ kg ', 0.2m I ⁇ only ' ⁇ ( ⁇ ⁇ , femoral injection. rLZ-8 treatment group
  • the tumor rate (%) (the average tumor weight of the control group - the average tumor weight of the experimental group) / the average tumor weight of the control group X 100% experimental results ; the results of the subcutaneous tumor of S180; as can be seen from Table 6, three doses rLZ 8 could inhibit the growth of Si8i>, and the tumor inhibition rate was 16.83 ⁇ 4-, 2 ⁇ .7% 4 ⁇ .53 ⁇ 4 owned rLZ-8 treatment group had a significant difference compared with the negative control group, ⁇ ⁇ 0.01). Compared with the negative control group, the tumor weight of the rLZ- 8- HSA treatment group was significantly different (P ⁇ 0.01) compared with the rLZ 8 treatment group (P ⁇ 0.0).
  • Test drug recombinant Ganoderma lucidum immunomodulatory protein (rLZ 8) was prepared by aseptic injection of water into a dose of 19.25 g ⁇ k ⁇ 9.625 u -kg ⁇ , 0.2m!./only; recombinant Ganoderma lucidum rabbit protein regulatory protein rLZU innocent injection
  • the dosage group of 19.25 ⁇ gji.kg ⁇ , 9, 625 u gj kg 1 (as 1?; ⁇ 8) was made with water K, >
  • ffi control drug resistance thrombopoietin ( ⁇ 0), Shen
  • Chemotherapeutic drugs cyclophosphamide (Cy), £: Su Hengyao Pharmaceutical Co., Ltd. production, production nickname] 2112121; 200mg / support. Sterile water for injection was formulated into WOrag on kg, 0, 2 ml/head.
  • Platelet dilution ⁇ urea L 3g, sodium sulphate; 0, 5g, A awake : 0. iml, add distilled water to lOOrai mixed, filtered for use.
  • mice were divided into 5 groups, each group was 0 mice, and the male and female were given an equal volume of normal saline in addition to the normal control group.
  • Each group of mice was intraperitoneally injected with guanidine, KR1 ⁇ 2g: :kg , () ⁇ 2ral * only d" 1 for 3 consecutive days.
  • the blood count is reduced to 300 ⁇ lOVl, the corresponding dose rLZ»S and fusion egg i.rLZ-8-HAS are given subcutaneously according to the upper sputum group.
  • the use of the rLZ 8 HSA of the present invention as an antitumor drug can be administered orally and parenterally.
  • the dose 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; non-gift administration 0-100 rag, several times per E1
  • the present invention C! medication tablets, pills capsules (including hard and soft capsules), these dosage forms include rLZ S and less - an inert diluent (such as lactose, mannitol, glucose, starch, Polyvinylpyrrolidone) may also be added to a pharmaceutically acceptable additive other than an inert diluent such as a moisturizer, a disintegrant, a stabilizer, and if necessary, the tablet or pill may be coated with a stomach-soluble or JS-soluble material. Or more than one layer of film.
  • Intestinal injections include rLZ 8 HSA and at least one inert water thinner (such as distilled water for injection, physiological saline).
  • RL2 8 HSA can also be made into lyophilized powder. It is dissolved in an inert water diluent for injection.

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Abstract

本发明属于生物制药领域，公开了利用基因工程技术制备的灵芝免疫调节蛋白与人血清白蛋白的融合蛋白rLZ-8-HSA、其制备方法和应用。所述融合蛋白与rLZ-8相比，体内半衰期延长、生物活性提高，可用作治疗白细胞减少症和抗肿瘤的药物。

Description

重组灵芝免疫调节蛋白与人血清甶蛋白融合蛋白及其制备
方法与应用 技术领域
[0001 ] 本发明涉及童组融合蛋 的构建、表达、纯化与应用，具体涉及用于治疗化疔导致 的 细胞较少症和肿瘤等症的重组灵芝免疫调节蛋 与人血清白蛋 ώ融合 S白的制备及 其应用。 背景技术
[0002] 臾芝免疫调节蛋 ii LZ 8)源说自于松杉灵芝的菌丝体,其结构特征如下：包括一个 端的形成二聚体所需的重要结构域和一个 C端的 FNI I： ί结构域， rLZ~S的 端结构域由一 个 a -he.l .i . .:|P '个 strand组成， LZ 8单体上的 N端 a helix和 β - strand与另一单体 上相阔的结构域通过空间交换形成了重耍的二聚体结合结构域，呈 *铃状。 已有文献报道 L2 8具有兔疫调节（专利申请号 201.1.1.022201.2, 5)和杀伤肿瘤细胞（专利 ZL20081.005020 &, K)的生物学活性。
[0003] 前，造成 类药物半衰期较短的原因主要有：①蛋白.质水解是绝大多数蛋 质的代谢方式之一，机体组织中蛋白水解醱的存在会使蛋白质药物活性降低；②肾赃是小 分子蛋白质分解代谢过程中最重要的器宫，相对分子质量小于 69000Da的绝大多数蛋白质 分子都能通过肾小球滤过作用而被排出体外；③肝賍在蛋白质药物代谢过埋中也 A有重要 作 药物通过扩散和载体转运等形式被转运至肝细胞中，然后再被徵粒体酶 P4S0,胞液中 的蛋 ¾酶或溶酶体所降解；而对于更大分子的肽和蛋白质则是通过受.体介导的内吞作用被 肝细胞摄取后被降解。由于灵:芝免疫调节蛋白二聚体分子量不足 26kEte，可能造成体内清除 率高，药代动力学参数难以满足其新药幵发的要求，因此,通过基因水平融合等技术手段延 长 ϋ···8在体内的祚用时间，为其在临床治疗中的应用奠定 ^实的基础。为了延长蛋白类药 物的半衰期,近几年各国学者主要从蛋 类药锪的白螢 融合、化学修饰、微囊化、构建突 变体、糖基化等方面迸行研究。随着研究的不断深入和进展，长效蛋白药物的新品种不断涌 现。
[0004] 基因融合技术是将不同的基函连接起来从而表迖具有复合功能的融合蛋 ，通过 基 Β融合技术增加多肽和蛋 ή药物分子量或改变药物与受体的亲和性等原理可以延长药 物的半衰期。构建融合蛋白的原则为：将一个蛋白的编码基因的终止密 ¾¾子去除,然后连接 上带有终止密码子的另 ·个蛋 ή的编码基因，即实现两种 S 的编码基因的融合，迸而同 时表达两种蛋白。融合蛋 基因稳定性高，可调控表达且制备简单，产物均一，对蛋 ^ >多肽 药物活性影响小等是 前研究长效多肽 >蛋 药物的一种良好的途径
[0005] 目前研究的较为广 S的融合基因有人血清 蛋白基困、人免疫球蛋白（Ig(U、 ig(;4)基因等。 人血淸白蛋白（Itoro^ Serufn Al tami 筒称 是人血浆中的蛋白质，其 非糖基化的单链多肽 ft含 585个氣,基酸，分子量为 66k¾ 在血浆中其浓度为 42g/L,约占 血浆总蛋 的 60%。 在体液中人血清 蛋¾ ¾以运输脂舫酸、胆色素、気基酸、类固醇激素、 金厲离子和许多治疗分子等；同时维持血液正常的镲透压。 在临床上人血淸白蛋 可用于 治疗体克与烧伤，用于补充因手术、 ft处事故或大出血所致的血液丢失,也可以作为血桨增 容剂,.
[0006] 人免筏球蛋白（IgG)是人体血液中最丰富的蛋白，其半衰期可达 2i天。 已有报遊 遝示将 IgG的 Fc片段与其他蛋白融合，可显著增加其他蛋白在体内的生物活性和半衰期， 其中大多数实验者选择〖g« J:g(;4这两种亚型的 Pe段作为融合对象，这种方法已经广泛应 于一些临床上很重要的细胞因子和 了溶性受体，如 s NF a R、 U 3 T!JV 4、 0-2, IFN~ a 等，并获得了成功。
[0007] 鉴于上述臂景技术,本发明采用了基因重组技术将灵芝免疫调节蛋白分别与人血 淸 β蛋 、人兔疫球蛋白： !^的! 段迸行 »合，构建真核表达体系，经过表达、纯化得到 Η的蛋白，并对目的蛋白迸行了相关生物学活性与药学研究，结果表明灵芝免疫调节蛋白 (L2-8)与 HSA融合蛋白在药学与生物学说活性以及病理应用方面有明显差:异《 发明内容
[0008] 本发明的目的是提供重组灵芝免疫调节蛋白与人血清白蛋白融合蛋白及其制备 方法，探讨其在治疗化疗药所致白細胞减少症等症及在抗肿瘤药物中的应用
[0009] 融合蛋白序列：本发明的灵芝兔疫调节蛋白 αΖ···8)与] 融合蛋白 i HHSA,其 特 ffi在于'包含灵芝免疫调节蛋白和 HSA；其氨基酸序列为： SDTM. FMAWiWKKLSFOTTP WGKG P NFI [)TV1'F<PKVLTDKA¥TY VAVS{J R L GVKPSYAVESDGSQ V FLEyNSGYGIADT TIQVFVVDPDT D pnAQW ;(X¾SSM膠 VTHS FLFSSAYSRGVFi冊) «ffiSEVM:RPKDWEE KM¥UAFAC|YLQQCPFEMW KI^N VTEFAKTCVADESAE (； DKSIJfIIJ'{;D ]. r] ATLJ¾i] (; ])(X¾i(QEPE:R iKI
P VDVMCTAFHD BB171IKYi^E!:A RHPYFMPE)JJ¾S(RYKAAR1jCCm
SSKLKECCEKPili SHC I A EVEND ) ADLPS ADPV fSKSWCK iMDW^
VSKNL} VGSKt;C iiPE PCAEDYi,SVVL^Q]^ViJ0; TPVSi)RyTK(;{;TfiS].V I PCFSAi^
AE1TTPHA!)!:CT[.SEKE Q:i gMi.VBLVKHKP ATKEQLlAV HDS¾AFVE CCKADni^
QMLGL (SEQ ID NO. i)，其中灵芝免疫调节蛋白多肽的 C端与人血清白蛋白多肽之间连接 肽的鼠基酸序列为《〕<¾SS；
本发明提供的灵芝兔疫调节蛋白 z 8)与人 IgGIFc段的！ 1 8· Fcl融合蛋白，特征 在于包含灵芝免疫调节 S 和 GlFc段；其氨基酸序列为：
PSl)TAIJ[?R WDV [(LSFDYTPNWGRG P NF]:DTVTFPKVi:rD AY1T VA¥SG 跳 GVKPS ViiSDG SQKVNFSJ^ SGYCnAi}T TiaVFVVDPi)T i)F]:i:AQW GCtGGS SEPKSCiiKT! rai^PAPELUXJPSVFU^^5 KFK0T!.lES^rPEVTCTVVDVSHfii)5EYKE^YV» (； V!iVH A TiiPREEQy SIT^
SDGSF'F-iTS LTVDK SR QQGNVFSCSV HEALiiNHYTQKSLSi.SreK{SEQ 〗:D NO.2),其中灵芝免疫调 节蛋 Θ多肽的 C端 人愈清 S蛋白多肽之间连接肽的氣基酸序列为 GGG (； SS；
本发明提供的灵芝兔疫调节蛋白（LZ 8)与人 IgG4Fc段的 rLZ 8 ¾4融合蛋 ^，特征 在于包含灵芝免疫调节 S 和 Ig(;4¾段；其氨基酸序列为：
PSi)TAIJ:F WDV L,SFD¥IPNSGRG P NF]:S)T¥TPPKVLTD AY1T VAVSG R S VKPSYAViiSDG SQKf l^i Y SG^G WDT TIQVFVVBPiyi' N f^i ! AQW XiGGS SYTQRFKD ASii/I^VTSA TAYMHi-SSL/rNE
[)SAVYYCSi:[YFi)MDF^: 0YWGQ{rrTV1^STASl, GFS¥S-PLAFCSRS1'SESTAAmCLViii)^
IJSGVHTFPAVLQXSGLYSLSSVVTVPSSSiiJlITYTCNVDHKPSHT VD
FPP PKI)1i;j:iSirrPEVTC¥VVDVSQii!)PEVQ[?NWYVD(;Vi l] AKTKPS?EEQ[¾^
KCKVSXK (； i.PSS ;:KT SM¾;QPREPQVYn. PSQBEM'r QVSLTa^
¥mSD{iSPFi;YSRl.;i^DRSSWQE(lN¥PSCSV HEAi,HNHYTQfiSLSLSii¾ (SEQ ID NO. ，其中灵芝兔疫 调节蛋 H多肽的 C端与人血清 β蛋白多肽之间连接肽的氨基酸序列为 GGGGSS；
工程菌株的构建与 S的蛋白表达及纯化；分别构建了 融合蛋白的核普酸序列的 载体；采用电转化技术得到以毕赤酵母作为融合蛋 胞外表达的宿主细胞；优化了毕赤酵 母发酵条件提高了融合蛋白的表达量；釆甩重力柱亲和层析、分子筛、阖定化金属 合亲和 愿析（IMAC)法、疏水g析（H1C)、阴离子说交涣歷析等方法纯化目的产物,得到不同融合 的纯度均达到 99%以上，为后续的药学实验提供了原料药。
[0010] 药学实验方面；将不同的融合蛋 与 Γ12···8作对照进行活性检奄实验,经过 法检测得出结论,结果说明融合蛋白 rLZ- 的生物学活性较融合前 rLZ 8生钩学活性 高，有显著差:异，具有统计学意义；而 rLZ- -8 Pel.与 r 8 Pc4的生物学活性没有达到融 合 f rLZ-8活性指标，具有显著差异；将不同的戳合蛋 β与 TLZ 8作对照进行体内半衰期 实验，经过 法检測得出不同的融合蛋白的体内半衰期均得到 . 著提高，约为 8 的 3倍；闻时将不同的融合蛋白与 r:L2 8作对照进行治疗白细胞减少症实验,经过动物全 血细胞分析仪检测白细胞数，结果表明不同的融合蛋白和 rLZ 8在治疗白细胞减少症疗效 明显差异；其中，在同一剂量下，融合蛋白 促进白细胞生长周期更短，在同一治 疗周期下，融合蛋 rLZ 8 H 促进 β细胞生长数量更多；说明 HSA更适合作为 rLZ 8的 融合 象，能够明显缯强〖 8在治疗 细胞减少症方面的应用；通过融合 g ^l rLZ-8-HSA 抑制黑色素瘤细胞生长实验表明，在同一剤量（以 LZ 8量计）下，融合蛋白 rLZ 8 HSA有 效抑制黑色素瘤細胞的生长， ¾融合前 r:L2 8铯疗效果比较具有明显差：异；同时在融合 蛋白 Γί2···8···Η 抑制肝癌细胞生长的实施俩中可以看到，在同一治疗周期内，融合蛋白 rLZ~8™HSA治愈率有明显的提高，是发明人始料未及的；本发明还提供 ft合蛋
对治疗血小扳减少症的实施例，从中可以看出，与模型组比较,在给药初期 rLZ 8 HSA药物 组已明显刺激小鼠血小板增生,差异极其显著,在给药中期时恢复到 i∑常水平，在治疗由注 射抗血小板血淸导致的血小板减少症实验动物模型也取得了很好的效果, >
[0011 ] 本发明有益效果如下：本发明中提供的融合蛋白，其体内半衰期的较 rU 8出现 显著延长；由于基因融合技术构建融合蛋白 会产生蛋 6融合后影响目的蛋白的生物活性， 而本发明构建的 »合蛋白 rL2-8-HSA的¾物活性经过试验 ffi明其生物活性明显优干 »合 前的 rLZ 8；且本发明构建的 rLZ~8-!iSA戳合蛋 毕赤酵母工程菌种发酵工艺简单，产量 高，表达产物单 ····，易纯化，为工业化生产提供了有科条件；由于采用毕赤酵母表达系统存 在表达产物在发酵液中易降解等! ¾題，本发明通过控制发酵工艺条件，大大减少了毕赤酵 母发酵表达过程中对目的产物的降解,提窩了产率：本发明采用体内试验证明体内半衰期 较:融合前 rLZ 8出现显著延长的同时在治疗 β细胞减少症研究中的最低给药剂量和起效 时间均优千融合前 rLZ 8；采用黑色素瘤与肝癌作为抗肿瘤研究的代表，分别开展了体内 和体 实验，在黑色素瘤与肝癌实施«中可以看到两种实验方式的治疗效果均明显优于融 合前 rLZ 8，这是发明人始料未及的；在治疗血小板减少症的实施例中可以看出，^模型组 比较，在给药初期 rLZ 8 HSA药物组已明 刺激小鼠的 .小板增生，差异极其显著，在给药 中期时恢复到正常水平，这也是 S3前发现的 rLZ 8融合蛋白所能达到的最佳效果。
[0012] 附图说明
[0013] 图 i 诱导表 ¾ 66h与 72h各融合蛋 β表达情况
图注 -泳道 1样品为蛋白 Marker；泳道 2样品为 HSA标准品；泳遒 3样品为 ；泳 道 4样品为诱导 72h ri..Z-8-fiSA上溃液；泳道 5样品为诱导 66h rLZ-8-HSA上清液；泳道 6 样品为诱导 72h rLZ 8 Pel上清液；泳道 7样品为诱导 66h Fci上清液；泳道 8样 品为诱导 72h rLZ 8 Fc4上清液；泳道说 9样品为诱导 66h rLZ"8-Fc4上清液。
C0014J 图 2不同融合蛋白 Western Bolt鉴定图谱
圏注：泳道 ί样品为蛋 t Marker；泳遨 2样品为 rLZ 8；泳道 3样品为 rLZ HA,；泳遒 4样品为 rLZ 8- Pel；泳道 5样品为 rLZ 8 Fc.'.i。
[0015] 图 3 rLZ 8 HSA融合蛋白分子筛纯化色谱图
图 4 不同融合蛋白^ rLZ 8蛋白体内半衰期对比 具体实施方式
[0016] 实施例 1 rLZ 8融合f白工程菌株构建与表达
构建部分；根据酵母密码子偏好性分别合成了目的片段 rLZ8 HSA、 rLZ8-Fcl、 rLZ8 Pc4序列，存于 puc57质粒中 设计 ft含酶切位点 St« j: 和 Κρηί 的引物 引物合成如 下：
(1) LZ-8-HSA :5' CATAGGCCTTCTGATAGTGCTTIXiA 3'
δ' CG<}(¾TACCGAATTCCTATTACA 3'
(2) LZ 8 1：5? GTTAGGCCTTCTGATACTGCTTTCA 3'
5' 'i'AG(K; CCTCA'rn'ACCA (； GG(; 3!
(3) LZ-8-Fc ：5' CC6AGGCCTTCTGATACTGCTT 3*
5' (½TGG'rAC CAC«rA(iCATGACt 3,
邋过 ΡΟί得到 S的片段， PCR条件为：幵始 9δ "C 30s,然后 9δ 'C 30s, 58 "C 30s, 72V 2! n，共 30个循环，最后 72V IQ in 16 ，待机。
[0017] ί 琼脂糖电泳鉴定目的片段分别在在 2184bp(LZ 8 ¾)、1064bp(LZ 8-Pcl) , 774bp(S,Z-8-Fc4) ,> pP C2a Λ载体和目的 j†段按照 GH BART Seamless Cloning arid Assembly Kit试剂盒说明书操诈，载体和 l¾的片段摩尔比是 ί :3，与 缓冲液， iOX酶混合 液反应 3()分钟。 取 Wul连接产物转化到大肠杆菌感受态 DH5 alpha中， 培养 1-小时 后，涂博来 «素抗性的 LB平板 挑取单脔落 摇床培养过夜。脔液 i2000g离心弃上清， 质粒小提试剂盒提取重組质粒，測序 用双酶切和电泳鉴定转化子是否 1£确，最后用 δ'ΛΟΧ 和： A0X引物证实序列的正确性.
[0018] 取重组质粒用 Sael酶线性化， 37Ό， i小时左右。 X33毕赤酵母接种到 YP 培养中 SO'C^OOrpfn摇床培养过夜。扩大培养到 500tnL，0l «达到 1.： ί：左右，制备酵母感受态,冰浴 3()min后至冰浴无瞎水重悬，4ΐ:， 离心 ·ίη'重复 2次。 换冰浴 ! 山梨醇重悬，
I SOOg离心 f¾in，分装每管 80ui酵母感受态，加入 的线性化质粒,， 冰浴 5分钟„转移 到电转杯中， L 5kV, oO μ F, 2&!3Α条件电转化。 摇床培养 2小时，涂博来霄素 Zeocm抗 性的 YPDS平板， 30 培养 3天¾
[0019] 筛选部分：无菌条件下在博来 «素 Zeoeii 抗性的 YPDS平板上分别挑选每种融合 蛋 齒 20个单克隆，祧菌置于 lOml YPD液体 it养基中 f 30Ό， 3()0rpffi摇瓶培养 Ϊ2小 时， 1500gf min离心弃 清，涣 BMGY培养基 : 30Γ , 300rpm摇瓶培养 18小时， 3細 g', Srain 离心弃上清，换 i ¾!Y ( i%甲醇）培养基，每 24小时补加 1%甲醇，诱导表达 72小时 loOOg, Sffiin离心， 2() 保存上清，经 SDS电泳及 Western Bol t.鉴 定量 ^窗性说明目的蛋 的 表达，以此筛选离表达工程脔株
[0020] 实施例 2 融合蛋 ¾纯化工艺说
由于融合蛋白有公共的 LZ~8结! ¾,所以以该结构的特点和性质，分别摸索了重力柱亲 和還析、分子筛、固定化金属¾合亲和愿析 Ci C)法,疏水愿析（HIC)、阴离子交换层析等 方法纯化，具体方法如下：
riH ci、： rLZ 8 Fc4纯化方法；
徵滤：将发酵液 10000转 /分离心得到上清，再用 径大小为 100M的中 ¾纤维柱纯化 (微滤)，除去小分子的盐类和瑭类，得到 含色素、核酸 蛋白的黃色澄清液。
[0021 ] rProtein A Gravi Trap 重力柱亲和层折：¾制缓冲液 A相 22M磷酸盐缓冲 液， ρίϋ 7. 7,0. 15M氯化钠，配制缓冲潑 Β相；0. 1.Μ柠檬酸盐缓冲液， ρΗ 4. 0, 0. 22 P m抽滤， 超声脱气；采用 A相磷酸盐缓冲液平衡色谱柱，体积 10ml。 分别将 Ϊ Ϊ处理的 .Ζ···8···! 1、 rI,Z~8™Fc4发酵液〗0ffl l.上样，反复结合两次 （样品处理：90ml发酵液 i()X磷酸盐 缓冲液 , 22 μ ηι过滤除鎩保存),采用 Α相辚酸盐缓冲液冲色谱柱，洗脱未结合, > B相柠檬 酸盐缓冲液洗脱样品，体积 iOmL放置 弃掉 的色谱柱内体积,用 1. 5ml离心管接 收样品，保留检测。 使用 5次之后，需要再生,加入 ¾ 盐酸胍，放置 in，再用 A 相冲 洗色谱柱。
[0022] 分子筛层析：用 Superdex 75填料填装柱（GE公司， XKJ6/ 0型， S 内径 ] 6麵， i 度 7()eiii)，填料高度 60αί)。用 100 y !」％丙醒进样捡测 測定柱效为 10000左右。蛋白按照流速 2fflg/ 浓度 L上样，然后用 pH7, 5， a¾TO ~^ιβ (50»«)缓冲液进行洗脱（如國 3)，收 集峰逬行取样，进行电泳和 检测
[0023] 实验结论：经过賴细纯化后，蛋 ¾ HPLC检测纯度为 99%以上， SI)S 电泳…-条 带。
[0024] r!,Z-8-HSA的纯化方法：
歩骤（1)：用阖定化金属¾合亲和层析（I C)法纯化 rLZ 8 ί¾¾填料: i AC Seph rose 6Fast Flow 1升购 |¾于 GE公司，装填 50/30柱子，填料高度 15cm,用纯化水置换保存液， I倍柱体积的 0. 1M硫酸铜溶液过柱，用 4倍体枳纯化水冲掉来被吸附的铜离子。然后使 用缓冲液 A : 20匪 o:!/L磷酸 ¾、(), 6mo l./L氣化钠、] ^平衡展析 tt 把含人血『¾蛋¾的发 酵上淸液加入磷酸盐、氯化钠、使之的达到 2(to ol/L磷酸盐、0. 6m /L氯化钠、 pH7. 3„ 然 后使用 MTA1* Purify层祈系统上样，流速 M)ral/!Bin»上样 ，用缓冲液 A洗涤,至吸收值达 到基点。 用缓冲液 B: 20誦 o l/L磷酸盐 , 0. 6mol /L氣化钠、0, 311咪睡、 ρίΙ7. 5来洗脱目标蛋 白，收¾缓冲溶液 Β的洗脱峰。
[0025] 歩骤（2)：用疏水愿析 One)进行纯化，对歩骤（1)收 ½的 ΰ缓冲溶液洗脱峰 迸行疏水 11析纯化。 用苯基疏水层析介质 Phenyl Sepharose™ 6l t Flow (high sub) (GE公司 )来装填直径 5cm的层桁柱，柱 ¾ loom., 介质在使用前需用 3侪柱体枳的缓沖液 C: 0mm l I.磷酸盐、().5M氣化钠、 pH6.5平衡。向步骤（1)得到含 Π标蛋白的 B缓冲溶液的 洗脱液屮加入去离 水，稀释 NaCl的浓度，用磷酸调 ^值至6.5。用 AKTA" urj i'y ^析系统上样，流逨 50ral/min，上枰结汆后，用 2侪柱体积的缓冲液 C:50mmol/L磷酸盐、 ().5M (化钠、 PI .5继续洗脱„收; ¾流 ^和用缓冲液 C的洗脱峰。与疏水^析柱结合的部 分,用 2倍柱体积的去离子水洗脱，流出液 ¾弃。 将疏水還析柱收 Φ液，加入终浓度为（).1 的四硼酸钠和氣化钙溶液，调节 ρίΐ至 9.0，处¾ (). δ-2 小时，然后 lOOOOrpm离心 20min，取 I：澝液，川 MTLUPOI仆:公司的 10K超滤膜说脱盐。
[0026] 歩骤（ ：阴离子交换层析精制柞品，装 Q SepharoseTHUi h I'reformaru^ ¾料 一根.1 径 2.6c.n,i¾' 15cm的^折柱子屮 , ¾料体积 80毫升„川 2倍杵体积的 -离 /-水洗涤， 然后用 5倍柱体枳的缓冲液 lK5()mMPli,pll7.0)平衡„ 上样后使用 2个柱休枳的缓冲液 I；洗
书
涤。 然后用 0-0.5M NaCI经 10倍柱体积梯度洗脱.收¾主峰。
[0027] 实验结论：经过精细纯化后，蛋白 IIPLC检测纯度为 99%以上， SDS-ΡΛ (； E电泳一条 带.，
[0028] 实施例 3 不同融合 3½促小鼠脾细胞增值比较
釆用 WST- 1 法检测融合蛋 对小鼠脾细胞^值作川的影响，反映出其生物学活性的 强弱，本发明釆 W ΒΛΙ-Β/c雌性小 ，体重 20-22g,拉颈处死小鼠，无 条件下取脾脏 , Έ 千预先加入 5ml含 10%小牛血 的隨 屮：川 !Tf分別将脾捣碎，并川纱布过滤 ¾ 织, :液以除去^织块.制备脾细胞细胞 液：取 H)0微升细胞悬液加入 )微升 2%冰醋酸 溶液 " 显微镜下 i「数。用含 2%小牛血淸的 l〕 :\调整细胞浓度为 5X lOVml：分别配制相问 摩尔浓度梯度的融合资白和 rLZ-8, ¾c 个梯度浓度，每个浓度设梵 个孔， 100微升 Z孔。 加细胞浓度为 5 X lOVral的细胞悬液 100微升 /孔。振荡混匀后放入 37t\ 5%C¾孵箱孵 fi 24h；孵育 Π加入 1，2()微升 Z孔,,放入: VTC、 5¾€()2孵箱继续孵宵: 、IW后，测 ( ;.w (ΒΙ0-ί?Λ0 酶标仪）。 ¾验结果见表 1。
[0029] ¾ 1. 不同融合 ¾ 促细胞 值生物活性（X土 s
\\蛋白
rLZ-8 rLZ-8- Fcl rLZr8-Fc4 rLZ-8-HSA 蛋白浓¾\^
5.0^1 (rtnol/L 0.739±0.23 0,543*0.26 0.452±0.43 0.936±0.3
ΙΟ.ϋχΙθ ηοΙ/L 1.197±0.31 0.937±0.29 0.840±0.27 1.680±0.38*
15.0χ10"'πιοΙ/ί 1.567士 0.27 1.114±0.31 0.929±0J1 2.373±0.35" 注：与 rl.Z-8 比较， *P <0. ()5。
[0030] 由表 1可以 出，随着 ¾β浓度的塯加，融合蛋白对脾细胞¾位作用也在增加， ί¾ 同一: ¾白浓度下，不向融合蛋 Α与 rLZ-8 作 比较可以看出， H.7- 8- HSA的促脾细胞 ¾ 值作用 ^干 ,Ζ- 8组，冇 ^著差 ; ¾有统计学意义„而融合蛋 A rI.Z»8-Fcl与 rLZ-8- 对 小 1¾脾细胞的 ifHii作川明显减弱；实验结果说明， l.Z-8 ¾ HSA融合后的活性位点没有受到
6
替换页（细则第 26条) 融合的影响，而 与 ig (； Pel和： igG Fc4的融合后，蛋 活性受到了影响，阻碼了 LZ 8 活性的发挥。
[0031] 实施例 4, 不同融合蛋白半衰期检測
釆用体重在 18 22g左右的 8ALB/C小鼠作为实验鼠，每组 10只小鼠，以融合蛋白 100g/ kg ( ΙΊ-Β量计）的剂量静脉注射给药，设计时间段为 2、4、6 8、i0小时后在不同时间段进行 采血检测,得出结果，做出药物浓度与时间的曲线阁（園 4)由实骏结果可以看出，随着药物 迸入小鼠体内时间的延长，血液中药物浓度出现不同程度的下降，其中，可以看到融合后蛋 Θ的半表期与 ¾合前 rLZ S的半衰期比较有极其 II著的增加（^d Ο(Μ)ί) ,大太延长了血液 中的药物浓度，提高 rLZ 8在小鼠体内的半衰期 ti
[0032] 实施例 5. 融合蛋 rLZ 8 HSA治疗 细胞减少症的研究
采用 Wistar大鼠作为实验动物，共说 i8只，体重】00g左右。 试剂配制方法如下； rLZ 8 用无菌生理盐水配制。 分为冊 y g/kg,30 g/kg o μ g/kg剂 S组； rLZ iHI:SA ¾合蛋白用 无菌生理盐水配制, > 分为 60 μ g/kg,30 μ g/kg、l 5 μ g/kg (以 LZ 8量计）剂量组；金 S赛强 【童组人粒细胞集落刺激 a子注射液（rM; CSF);j，生产批 ：20130403 ;7δ μ gZ支，用无菌 生理盐水配制成 13, 5 y g/mL，0. 1 /只，注射用环磷酰胺（CP) ,生产批号 13020225 ;20 mg/ 支 用无脔生理盐水配制 ;2(tog/¾l { ImL/只，即 20mg/kg。
[0033] 正常対照组， ).,Z"8低剂量組， ΠΖ~8中剂量组， )..7-8高剂量组， rLZ~8~HSA融合 蛋白的低中高剂量组，阳性对照组 (金磊赛强)。 除正常对照组 (给予等量生理盐水)舛，每组 大鼠均给予环磷酰胺尾静脉注射， 20ing/raL, 0. iniL/只，连续 :¾夭。 于第三天，大鼠尾静脉取 血,细胞分析仪检测白细胞数 造模成功后按上述分组分别给予相应剂量 rLZ 8、三种 |¾合 蛋 、阳性药（金 ¾赛强）治疗， i£常对照组和 CP组给予等量生理盐水，于治疗第 1天，第 :3 天和第 7天分别大鼠尾静脉取血，检測白细胞数。对比治疗前后 S¾细胞数变化，分析药物疗 效。
[0034] 表 2. rLZ 8对白细胞低下大鼠模型的影响（ iO)
Figure imgf000009_0001
注： ■¾ CP对照组比较， <0. 05, * * p <0. 0L>
[00353 由表 2可以看出，与 C 对照组比较，在给药第 1.天 rLZHS i组已明显升《大 鼠 细胞,幾异及其显著，在给药第 7天基本达到正常 与金 II赛强比较,在给药第 1天, rLZ 8 HSA组对大鼠的 S细胞数量增加;怍用明 H, ί¾给药第 7天时， rLZ 8 HSA组的大鼠 ί¾细胞数量基本达到正常，重点是 rL2- - 组和 rLZ 8蛋 S l相比较，可以看出，在相同 给药剂量条件下，而 rLZ 8 在给药后第一天，就有明显的 β细胞增值作用，数值上约为 rLZ 8蛋 ή增值数量的 2倍，在相同的给药时间上， L2~8-HSA低剂量组的白细胞增值作用 明. 高于 rLZ 8蛋白 Λ剂鱟组,都嚷显优于 rLZ»8蛋白组对大鼠 ί¾细胞增 ¾的促迸作用。 [0036] 实施例 6. 融合¾白 rLZ- 8- 1¾八对¾色素瘤的抑制作用
体外实验：采用 WST-1方法检测融合蛋 β rLZ-8-HSA抑制黑色素瘤细胞生长作用的影 响，制备 ^色索瘤细胞悬液；取 100微升细胞悬液加入 9()0微升 2%冰醋酸溶液于显微镜下 计数。 用含 2%小牛血清的 Ι)ΜΙ 调整细胞浓度为 2 Χ lOVtnl；分别配制相同摩尔浓度梯度 的融合蛋白 H Z- 8- HSA和 rLZ- 8，设 3个梯度浓度，每个浓度设置 9个孔， H)()微升 /孔。 加 细胞浓度为 2X lOVml的细胞悬液 100微升 /孔》 振荡混匀后放入 37"C、 5% C02孵箱孵 W ：孵育后加入 WST- 1， 20微升 /孔。放入 、 5%C0孵箱继续孵育 3小时后，测 ()Ι)ί;5 (B ! O-RAD酶标仪）。 实验结果见表 3„
[0037] 表 3. 融合蛋白 "LZ 8 HSA对 M色素瘤的抑制作用 （x ±s n=9)
^蛋白
rLZ-说8 . rLZr8-HSA
蛋白浓度^\_
5.(5x 1 (^raol/L 0.427±0.44 0. 11±0.31 *
JO.O lO'VoL/L 0.372±0.21 0.212±0.2 *
15.0x l(rl)mol/L 0.302±0.24 书 0.謙 0.1广
注： rLZ- 8组比较 <(). Οδ , <(). 05。
[0038] 体内实验： 先，建立小鼠肿瘤模型，小鼠黑色素瘤细胞 B16-F 10选用含 10%胎牛 血清的 培养基，置于 37"C、C te温箱中培养《在小鼠背腹侧皮 K用 I m l 注射器缓慢 注入 iU6-F10细胞悬液 200 μΐ (细胞含量为 1 X 10'个），建立小鼠移植瘤模型》
[0039] 分组' ϋ治疗方式：肿瘤细胞接种 24小时后，尾静脉注射 rLZ-8组（123 μ g/kg, 246 g/kg，492 g/½)、rLZ - 8- HSA组（以 LZ- 8 St;:U. i 23 g/kg, 246 μ g/kg, 492 μ g/kg)、 达卡巴嗉组（2. 5 rng/kg)或生理盐水组。 rLZ-8, rLZ-8-HSA每天注射一次，达卡巴嗪连续尾 静脉注射 5天，问隔 3周后再次注射，治疗周期为 28天。 实验期间观察小鼠的生 ¾状态，毎 1¾ 7天称 · 次体重，每隔两 2周进行小鼠 静脉采血，毎组最终实验结束时剥离瘤体，瘤体 称重并记录；按照公式肿瘤生长抑制率 = (生理盐水组瘤体重的平均值一给药组瘤体重的平 均值） /生理.盐水组痫休 ¾的平均值，计算治疗药物对原位肿瘤生长的抑制率。
[0040] 实验结果表明：瘤体称重后，每个组计算瘤体重的平均数，由表 可以看出各组给 药 28天， rl.Z-8-USA 剂量组瘤体重比其他组小， rLZ-8 rLZ-8-USA组内随着药物浓度的 逐渐 大，皮下瘤体 ΐ逐渐减小。 Γ^- 8- ilSA组与阴性对照组比较 ¾异特別显 ， rLZ-8组 内低、中、 之问的差异也很显著（ η= I () , P<Q, Οδ ) , rLZ-8-HSA组内低、中、 ¾之间的 ¾异也 很显著（η= 1 ()，尸 <0. ()5)。
[0041] 表 4. rLZ- 8对 B16- F10原位肿瘤: Ε量的影响
8
替换页（细则第 26条) ^药方式 持续给药 28天
组别 动物数 瘤体重量（g) 肿瘤生长抑制率 （％)
生理盐水组 10 1.2械 53
达卡 嗪组 10 0.52±0.12** 62.57±0.27 rLZ-8低剂量组 10 0.53±0.21**** 56.72±0.41
rLZ-8中剂量组 10 0.43±0.17**** 64.91±0.77
rLZ-8高剂 ¾组 10 0.25±0.04** 83.04x0.51*
rLZ-8-HSA低剂量组 10 0.35±0.11**** 71.77±0.31** rLZ-8-HSA中剂量组 10 0.27进 14**** 78.22±0.47*
rLZ-8-HSA高剂鼂组 10 0.13±0.05** 89.51±0.88M
说
注 对照组比较， * ρ<ο,
Figure imgf000011_0001
w实验进行了三次重复，整体结果趋势…致, 此表为其巾一组结. ； 1 达卡巴嚓组 \ ，") < (》5 -ρ < 0. or.实验进行了三次:? R复，整 体结果趋势一致，此表为其中一组结果。
书
[0042] 实施例 7融合蛋 rLZ-8-HSA对小鼠 艾氏腹水瘤的抑制作用
体外实验：采用 WST- 1方法检测融合 ¾白 rLZ- 8- HSA抑制小鼠 S180艾氏腹水痫细胞生 长作川的影响，制备 艾氏腹水瘤细胞悬液；取 100微升细胞悬液加入 900微升 2%冰醋 酸溶液千. 微铙下计数》用 2%小牛血淸的 DMHM调整细胞浓度为 2X l()5/ml；分别配制相 问靡尔浓度梯度的融合蛋 0 rLZ-8-HSA和 rl.Z- ,设 :3个梯度浓度，毎个浓度设 ¾ 9个孔， 100微升 /孔。加细胞浓度为 2X l( /m]的细胞恳液 100微升 /孔。掘荡混匀后放入 37V、 5% CO,孵筘孵宵 4h；孵育后加入 WST- i , 20微升 /孔。 放入 37Ό、 5¾C0 孵箱继续孵育 3 小时 测 0D4fl (H!O-KAI) 酶标仪）。 实验结果见表 5。
[0043] 表 5. 融合蛋 rLZ 8 HSA对 S180艾氏腹水瘤细胞的抑制作用 （ x±s n=9)
Figure imgf000011_0002
ΙΟ.Ο Ιθ τιοΙ/L 0.366±0. 1 0.233±0.18*
15.0χ1θ ηο1 ί 0.345士 0.27 0.127±0.15**
ΪΕ： ½" rI.Z-8组比较， <0.05, **/7 <0.05
体内¾验：实验材科：小鼠，体 l8-22g,由吉林大学实验动物中心提供。 小鼠艾氏腹 水细胞株 由本 ¾提供。环磷酰胺（CTX)，江苏恒瑞医药股份有限公司，批号：06101921； S18Q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 «组 (0.2.½g -kg 1) , LZ-8中剂量组 (0.5mg «kK ')、rI.Z- 8 ,5¾剂量组 (Img 'kg ')； rLZ- 8- IISA低剂 i (().25mg -kg ') , 8- HSA中剂細 1 (0.5mg -kg l) , rLZ-8-llSA 剂S:组 (Img -kR 1) ' rLZ-8-i]SA各刖^ U均以 LZ- 8量计。 每组 10只小鼠。
[0044] ¾验方法： 皮下抑瘤实验方法：取生长良好的 细胞，以适量无 l f生理盐 水稀释成瘤细胞悬液，细胞计数为」07 每鼠右腋¾皮下接种 ().2ml (正常对照组除外）。 接种 2 h后，给予治疗。 ^常对照组和阴性对照组给予生 ¾盐水 0.2ml♦只— 1 · d V股腔注 射；阳件对照组给予环磷酰胺 20r«g · kg '，0.2m I ·只 ' ·（Γι，股腔注射。 rLZ- 8治疗组分别
9
替换页（细则第 26条) 剂量尾静脉注射， 0.2ml *只 · d"! ti 连续！0天,， 分别于给药前、给药 iOd后从小 鼠眼眶静脉丛取血，送检于 大 · 院检验科溯白细胞数,,并在停药次 B ,颈椎脱臼处死全部 小鼠，解剖取出瘤块,称瘤重。 按下式计算捭瘤率：
神瘤率（％) =(对照组平均瘤重一实验组平均瘤重）/对照组平均瘤重 X 100% 实验结果 ;S180皮下裨瘤实验结果；由表 6可以看出， 3个剂量的 rLZ 8均能抑制 Si8i> 的生长，抑瘤率分别为 16.8¾-,2δ.7% 4δ.5¾„ rLZ-8治^组瘤歉与阴性对照组比较,均 具有显著差异 ίΡ < 0.01), rLZ- 8- HSA抬疗组瘤重与阴性对照组比较,均具有 . 著差异 (P < 0.01) 的闻时与 rLZ 8治疗组比较均具有显著差异 (P < 0.0].)。
[00*5] 表 6. i'LZ S对小鼠移植性肿瘤 SifM x±s, η~1ϋ ) 说
Figure imgf000012_0002
与阴性对照组比较， * ·Ρ < 0.05, < 0.01 rl,Z-S组比较， P< {).05, p
< {).01
[0046] 实施例 8融合蛋白 rLZ 8™HSA治疗环磷酰胺引起的小鼠血小板减:
验
试验用药：重组灵芝免疫调节蛋白（rLZ 8)无菌注射用水配制成 19.25 g^k \ 9.625 u -kg^剂量组 ,0.2m!./只；重组灵芝兔疫调节蛋白融合蛋白 rLZU 无脔注射用 水 K制成 19.25 μ gji.kg■、9, 625 u gj kg 1 (以 1?；~8量计）剂量组, >
[0047] ffi性对照药：血小板生成素 ( ΊΉΡ0 ),沈 |¾ .；；；-：生制药股扮 :有限公司生产， ^O ugjLkg d.O.2ml./只。
[0048] 化疗药物：环磷酰胺（Cy)， £:苏恒瑶制药有限公司生产，生产枇号】2112121； 200mg/支。 无菌注射用水配制成 WOrag上 kg ，0, 2ml/只。
[0049] 血小板稀释液 ί尿素；L 3g、枸橼黢钠；0, 5g、甲醒 :0. iml,加蒸馏水至 lOOrai混 合，过滤备用。
[0050] 实验方法：实验动物分成 5组，每组】 0只小鼠，雌雄各半 除正常对照组 给予 等体积生理盐水）夕卜 ,每组小鼠均腹腔注射环辚銑胺， KR½g::kg , ()· 2ral *只 d"1 ,连 续 3天。 血小扳数降到 300Χ lOVl以下时，按上逑分组分别皮下给予相应剂量 rLZ»S和 融合蛋 i .rLZ-8-HAS(l .25 μ
Figure imgf000012_0001
μ gjLkg"1 0.2m〖《只 1 · d"1)及闭性药 (THPO 770ugJLkg^0.2mi · R 1 · cf1)治疗，正常对照组和模型组（CP)组给予等体积生理盐水， 于治疗第 3d和第 7i、14d分别小鼠尾静脉取血，高倍镜下血小板计数，实验结果见表 7。
[00513 实验结果；与模型组比较，各药物剂量组每只小鼠给药第 3天血小板数已明显升 ί ，在给药第 7天基本达到 i£常， fi.有显著性差异 ( <0.05) ； rLZ ·8···!¾Λ各組血小扳数 增 长明 L给药第 3天基本达到正常，与模型组比较 有显著差异 <ΐ).05) 具有统计学意 义。 L)
Figure imgf000013_0001
注；与模型组比较存在凝著性差异， ¾?<0. 05, Οί ,
[0053] 实施例 9 :rL2-8-HSA抗肿瘤组合物制剂
L通过上述药理实验证明， rLZ 8说 HSA的抗肿瘤作用维持机体1¾细胞水平的效果是显 著的，而 无毒副作用，因此， '以认为 rLZ 8 HSA适于药物使用而 li是安全的。
[0054] 2.本发明的 rLZ 8 HSA作为抗肿瘤药物的应用可以通过口服和非肠遨给药。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 10-1000 mg，每日数 次；非賜遒给药】0-100 rag,每 E1数次
[0055] 3. 本发明 C!服药包片剂、丸剂胶囊（包括硬胶囊和软胶囊），这些剂型包括 rLZ S 和 ^少―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潸剂、崩解剂、稳定剂 如果需要，片剂 或丸剂可用胃溶或 JS溶材料涂敷上一层或一层以上的膜》 非肠道用注射剂包括 rLZ 8 HSA 和至少一种惰性水稀释剂（如注射用蒸溜水、生理盐水），也可以将 rL2 8 HSA制成冻干粉， 使用前将其溶解于惰性水稀释剂供注射用。
[0056] (： I；)制剂 I
取 .rL2 8 H&\ .1000 mg,溶于 i(M) ml无 if生理盐水中，混合均匀后，分装成 rLZ 10 rag/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 2010年版注射液项下要求
[0057] (2)制剂锊 2
取 rL2 8 HSA 100 g，药兩淀粉 0, 5 kg按公知的跤囊制备技术和设备制成胶囊， rLZ-8-HSA 10 mg/粒。 其他项目应符合中华人民共和国药典 2010年版胶囊项下要求。
[0058] ( 3 )制剂铜 3
取 rLZ ·8 H¾ 100 8>微晶纤维素 560 无水乳糖 380 g，硬脂酸镁 2( ) 按公知的制 片技术和设备制成片剂, rLZ 8 HSA 10 rag/片，其他项目应符合中华人！ ¾共和国药典 2030 年版片剂项下要求。
[0059] ( 4 )制剂例 4
取 rL2 8 HSA适量，中华人民共和国药典 2010年口服液项下要求，按公知的制片技术

Claims

•ku ¾i ¾^ ： i
'J ¾
1. 重组灵芝免疫调节蛋 与人 III淸 蛋白融合蛋白（rLZ 8 SA),其特征在于灵芝免 疫调节蛋白的 C末端 f人血清白蛋 之间由连接肽连接,其氨基酸序列如 Si¾ ID NO. 1.所 示"
2.如权利耍求 1-所述的融合蛋 Θ 8 - HSA在制备治疗化疗药所致 β缁胞减少症药物 中的应用。
3. 如权利要求 i所述的融合蛋 在 S备治疗抗顯色素瘤、肝癌药物中的应 f-\L'
4. 如权利要求 1所述的融合蛋白 rLZ- 8- HSA在制备治疗倉小板减少症药物中的应用 δ. 如权利要求 2、3、4所述药物,其特征在于该药物制剂核心成分是由权利要求 .所述 的 »合蛋白和任选的药学可接受的辅剂组成。
6. 如权利要求 δ所述的药物制剂给药途径为□服和非肠道给药，其中， 服包括口服 液,片剂，丸剂和胶囊；非肠道给药包括外用药和注射剂。
PCT/CN2014/082031 2013-10-15 2014-07-11 重组灵芝免疫调节蛋白与人血清白蛋白融合蛋白及其制备方法与应用 Ceased WO2015055026A1 (zh)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190352345A1 (en) * 2017-04-08 2019-11-21 Xitian Zhang Mutants of recombinant immunoregulatory protein of Ganoderma lucidum and applications thereof
CN120060422A (zh) * 2024-07-11 2025-05-30 山东福德健康产业有限公司 一种具有抗肿瘤功效的灵芝低聚肽及其制备方法

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI608846B (zh) * 2014-03-13 2017-12-21 益生生技開發股份有限公司 用於改善化學治療所造成之不良副作用的組合療法
CN111484547B (zh) * 2020-04-28 2022-09-20 上海市农业科学院 一种抗肿瘤蛋白ael1、融合蛋白和抗肿瘤制剂及应用
CN112516288B (zh) * 2020-12-22 2023-04-18 西藏阿那达生物医药科技有限责任公司 N-糖基化修饰之灵芝免疫调节蛋白的应用
EP4386008A4 (en) * 2021-08-09 2025-03-12 Changchun Intellicrown Pharmaceutical Co., Ltd. New mutant of recombinant ganoderma lucidum immunoregulatory protein and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040063635A1 (en) * 2002-07-01 2004-04-01 Zailin Yu Recombinant human albumin fusion proteins with long-lasting biological effects
CN101463089A (zh) * 2009-01-08 2009-06-24 中国人民解放军军事医学科学院生物工程研究所 人血清白蛋白-干扰素融合蛋白及其编码基因与应用
WO2013067355A1 (en) * 2011-11-04 2013-05-10 Novartis Ag Low density lipoprotein-related protein 6 (lrp6) - half life extender constructs

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2004117638A (ru) * 2004-06-09 2005-11-20 Институт цитологии и генетики Сибирского отделени Российской академии наук (СО РАН) (RU) Состав, обладающий противоопухолевой, гепатопротекторной и иммуностимулирующей активностью
US8629096B2 (en) * 2005-09-23 2014-01-14 Yeastern Biotech Co., Ltd. Compositions comprising fungal immunomodulatory protein and use thereof
CN101475632B (zh) * 2008-01-03 2012-01-04 张喜田 具有抗肿瘤作用的重组灵芝免疫调节蛋白及其药物制剂
CN102274487B (zh) * 2008-01-03 2016-09-28 张喜田 重组灵芝免疫蛋白在制备治疗白细胞减少症药物中的应用
CN101612385B (zh) * 2009-05-25 2012-06-20 张喜田 rLZ-8在治疗血小板减少症中的应用及其制剂
KR20120079492A (ko) * 2010-12-06 2012-07-13 시티옌 쨩 재조합 영지버섯 면역조절 단백질 ｒＬＺ?８ 및 그의 용도
CN102199201B (zh) * 2011-03-22 2013-05-01 上海交通大学 灵芝属内重组的真菌免疫调节蛋白基因、该基因编码的蛋白及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040063635A1 (en) * 2002-07-01 2004-04-01 Zailin Yu Recombinant human albumin fusion proteins with long-lasting biological effects
US20090053173A1 (en) * 2002-07-01 2009-02-26 Yan Fu Long acting human interferon analogs
CN101463089A (zh) * 2009-01-08 2009-06-24 中国人民解放军军事医学科学院生物工程研究所 人血清白蛋白-干扰素融合蛋白及其编码基因与应用
WO2013067355A1 (en) * 2011-11-04 2013-05-10 Novartis Ag Low density lipoprotein-related protein 6 (lrp6) - half life extender constructs

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LEI, J.Y. ET AL.: "Expression, purification and characterization of recombinant human interleukin-2-serum albumin (rhIL-2-HSA) fusion protein in Pichia pastoris.", PROTEIN EXPRESSION AND PURIFICATION, vol. 84, no. 1, 17 May 2012 (2012-05-17), pages 154 - 160, XP028493249 *
PHARMACOPOEIA OF THE PEOPLE'S REPUBLIC OF CHINA, 2010
YU , DAN.: "Preliminary Study on Recombinant protein Ganoderma Lucidum Immunoregulatory (rLZ-8) anti-mouse ascites hepatoma cell line S1800 part of search.", CHINESE MASTER'S THESES FULL-TEXT DATABASE (ELECTRONIC JOURNALS), MEDICINE AND HEALTH SCIENCES., 15 September 2009 (2009-09-15), pages E072 - 131, XP008181821 *
ZHOU, HUI., MEDICINE AND HEALTH SCIENCES, 15 August 2013 (2013-08-15), XP008181822 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190352345A1 (en) * 2017-04-08 2019-11-21 Xitian Zhang Mutants of recombinant immunoregulatory protein of Ganoderma lucidum and applications thereof
US12077566B2 (en) * 2017-04-08 2024-09-03 Xitian Zhang Mutants of recombinant immunoregulatory protein of Ganoderma lucidum and applications thereof
CN120060422A (zh) * 2024-07-11 2025-05-30 山东福德健康产业有限公司 一种具有抗肿瘤功效的灵芝低聚肽及其制备方法

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