WO2007112682A1 - Protéines humaines recombinées apparentées à l'hormone parathyroïde et leurs utilisations - Google Patents

Protéines humaines recombinées apparentées à l'hormone parathyroïde et leurs utilisations Download PDF

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WO2007112682A1
WO2007112682A1 PCT/CN2007/001039 CN2007001039W WO2007112682A1 WO 2007112682 A1 WO2007112682 A1 WO 2007112682A1 CN 2007001039 W CN2007001039 W CN 2007001039W WO 2007112682 A1 WO2007112682 A1 WO 2007112682A1
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parathyroid hormone
pthrpl
related protein
sequence
protein
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PCT/CN2007/001039
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WO2007112682A8 (fr
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Gang Guo
Jingyu Zhang
Dongchun Liang
Baoli Wang
Rui Zhang
Aijun Zuo
Bei Sun
Xinyu Liu
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Tianjin Medical University
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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the field of protein engineering.
  • the invention relates to recombinant human parathyroid hormone related proteins and uses thereof. Background technique
  • HMM hypercalcemia syndrome
  • PTH parathyroid hormone
  • PTH is secreted by the parathyroid glands into the blood and transported to target cells to exert their physiological effects, known as endocrine. This is not the case with PTHrP, which is mainly autocrine, paracrine and intracrine [6 ' 7 ' 11 ] .
  • PTHrP is considered to be the second hormone in the PTH family [7] .
  • PTHrP has a great similarity to the N-terminal sequence of the PTH amino acid sequence ⁇ PTHrP and PTH have 8 of the first 13 amino acids at the N-terminus (2nd, 3rd, 4th, 6th, 7th, 9th, 12th, 13th)
  • the exact same [ i the original researchers concluded that they have similar physiological functions.
  • Studies have confirmed that the PTHrPN-terminal fragment and PTH act on a common receptor, the PTH1 receptor (PTH1R) [7] , which has a similar effect in regulating calcium and phosphorus metabolism.
  • PTHrP has three mature protein forms after translation (PTHrPl-139, PTHrPl-141).
  • PTHrPl-173) [15] .
  • the PTHrP gene in rats and mice is simpler than humans, with only 5 exons.
  • the PTHrP mature peptides encoded by them are only PTHrPl-139 and PTHrPl-141 [6] .
  • the originally translated PTHrP precursor undergoes complex post-translational processing, including removal of the signal peptide (-36--1) and enzymatic cleavage of the endonuclease.
  • PTHrP is digested to produce a variety of different polypeptide fragments, forming a group of polypeptides with different biological activities.
  • Three bioactive secretory fragments have been confirmed: N-terminal fragment (1-36/37), intermediate fragment (38-94/95/101), C-terminal fragment (107-139/141) [12 ' 16 ' 17] .
  • Their biological functions are significantly different, and these polypeptide fragments are likely to have their own different receptors [12 ' 18] .
  • the N-terminal fragment is most similar to sputum in biological properties and exhibits similar physiological activities. They activate adenylate cyclase and/or phospholipase C by binding to a common receptor (PTH1R) to exert a variety of physiological functions [19-21 ] . Due to the different sites of protease action, the boundaries of the middle part are not well defined.
  • the intermediate part of the polypeptide fragments 88-91 and 89-106 also contain a nuclear localization sequence (NLS), which is similar in structure to viral and mammalian transcription factors. Under its action, PTHrP can enter the nucleus and play a regulatory role in gene transcription.
  • NLS nuclear localization sequence
  • the C-terminal fragment also contains NLS, which can enter the nucleus to exert transcriptional regulation and cause cell proliferation.
  • the biological effects that are not found in the other two fragments can be exerted by the corresponding receptors specifically distributed in bone and brain tissues [6 , 7] .
  • PTHrP is widely distributed in the body, and its target tissues and target organs are involved in almost all tissues and organs including bones, kidneys, blood vessels, smooth muscles, keratinocytes, placenta, islets, central nervous system, etc. [6 ' 7 ] .
  • PTHrP binds to its receptor (PTH1R), promotes chondrocyte proliferation, inhibits terminal differentiation, and inhibits chondrocyte apoptosis. This results in bone growth and endochondral osteogenesis.
  • PTHrP is very important for promoting fetal bone development and cannot be replaced by PTH.
  • PTHrP also promotes osteoblast proliferation, inhibits osteoclast activity, and stimulates bone formation [7 ' 9 ' 23 ] .
  • PTHrP and PTH are involved in the regulation of calcium and phosphorus metabolism, which indirectly affects bone tissue metabolism; it also reduces renal blood flow velocity and glomerular filtration rate, and also promotes the proliferation of renal tubular and glomerular cells in kidney tissue.
  • the repair process of injury plays a certain role [6 ' 23 _ 28] ; PTHrP is also expressed in smooth muscle cells (VSMC) and vascular endothelial cells, which has a strong relaxing effect on smooth muscle and can reduce the tension of hollow organs and blood vessels.
  • VSMC smooth muscle cells
  • vascular endothelial cells which has a strong relaxing effect on smooth muscle and can reduce the tension of hollow organs and blood vessels.
  • PTHrP In order to regulate digestion and absorption, lower blood pressure, maintain blood pressure balance [29 ' 34] ; In the pancreas, PTHrP can delay ⁇ cell apoptosis, promote ⁇ cell aggregation and insulin secretion [35 ⁇ 37] . In addition, PTHrP can exert its specific physiological effects in tissues such as breast, placenta, skin, small intestine, heart, pituitary, and central nervous system [7] . These physiological functions of PTHrP indicate that PTHrP will be clinically broad in the future. Application prospects can be used for the treatment of various diseases such as osteoporosis, renal failure, diabetes, and hypertension. In recent years, research on PTHrP has been increasing, and PTHrP has also been known. However, until now, many of its physiological characteristics and mechanisms of action have not been well understood. Due to the extremely low expression level of PTHrP in serum and histiocytes, it is difficult to meet the needs of scientific research by extracting samples by pure protein alone
  • Genetic engineering technology uses enzymatic methods to specifically cleave and recombine DNA molecules from different sources into a new hybrid DNA molecule, which is then transformed into a host cell, allowing the gene of interest to be expressed in vitro. After further purification, a large amount of the protein of interest can be obtained. For proteins with low expression levels in vivo, it is difficult to obtain a large number of high-purity samples by simple protein extraction and purification methods, and genetic engineering technology can obtain a large number of pure products in a short period of time, and thus has received increasing attention. At present, the use of genetic engineering at home and abroad to obtain PTHrP is limited to shorter fragments, and the synthesis of polypeptide fragments of such length as PTHrPl-141 has not been reported.
  • the Pichiapastoris expression system of Pichia pastoris has the advantages of convenient operation, economical and large-scale fermentation production of E. co., and correct folding and post-translational modification of recombinant proteins (eg glycosylation). And the secreted expression makes the product easy to purify. It can be grown in a permeate with methanol as the sole carbon source. This is because the peroxidase of the cell contains essential enzymes for the methanol metabolic pathway, such as: alcohol oxidation. Enzyme 1 (alcohol oxidasel, ⁇ 1) is the first enzyme in the methanol utilization pathway, and in methanol-cultured yeast, the enzyme accounts for more than 30% of the total cellular protein.
  • Pichia pastoris has a reduced degree of glycosylation of the recombinant protein, and the latter expresses a high degree of glycosylation of the recombinant protein, which may result in hypersensitivity.
  • clinical application of recombinant proteins from Pichia pastoris is safer.
  • Animal experiments are widely used to detect the biological activity of biological samples.
  • Animal experiments on different fragments of parathyroid hormone-related proteins can be used to determine the effects of parathyroid hormone-related proteins on osteogenesis in animals by testing biomechanics, bone metrology, and biochemical indicators that reflect bone remodeling. Confirm the biological activity of the different fragments. Summary of the invention
  • One aspect of the invention provides a parathyroid hormone related protein PTHrPl-86, the amino acid sequence comprising the SEQ
  • amino acid sequence is the sequence set forth in SEQ ID NO: 8.
  • nucleotide sequence encoding the parathyroid hormone-related protein PTHrP1-86 of the invention, comprising the sequence set forth in SEQ ID NO: 7 or comprising under stringent hybridization conditions and SEQ ID NO: 7. Shown Sequence hybridization sequence.
  • the nucleotide sequence is the sequence set forth in SEQ ID NO: 7.
  • hybridization under stringent conditions means that the nucleic acid can be subjected to the conditions described in T. Maniatis et al. (ed.), Molecular Cloning: A Laboratory Manual 2 nd ed., Cold Spring Harbor Laboratory (1989) or Hybridization was carried out under similar conditions.
  • nucleic acid-immobilized membrane was incubated with the probe at pH 50 for 6-20 hours in 6xSSC of nucleic acid (lxSSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0).
  • the membrane was washed in 2XSSC containing 0.5% SDS at 37 ° C while the SSC concentration was down-regulated to Ol x and the temperature was adjusted up to 50 ° C until the signal from the immobilized nucleic acid could be distinguished from the background, then Detection probe.
  • a vector and host cell comprising a nucleotide sequence encoding a parathyroid hormone related protein PTHrP1-86 are provided.
  • the host cell is a Pichia cell.
  • a human parathyroid hormone-related protein of the invention (PTHrPl-86 or PTHrPl-141) for the manufacture of a medicament for the treatment of osteoporosis.
  • a method of treating osteoporosis comprising administering to a patient a therapeutically effective amount of a human parathyroid hormone-related protein of the invention (PTHrPl-86 or PTHrPl-141) is provided.
  • a human parathyroid hormone-related protein of the invention PTHrPl-86 or PTHrPl-141
  • kits for treating osteoporosis comprising a therapeutically effective amount of a human parathyroid hormone-related protein of the invention (PTHrPl-86 or PTHrP1-141).
  • conservative substitution, deletion, addition means that the amino acid or nucleotide sequence modified as described above still has the same function as the original sequence. Based on the sequence, one of ordinary skill in the art can fully determine sequences having the same function obtained after conservative substitution, deletion, and addition.
  • terapéuticaally effective amount means a dose which a physician can determine to effectively exert a therapeutic effect, depending on the condition of the patient.
  • the present invention uses pGEM-T easy/PTHrPl-141, 1-86 as a template to obtain PTHrP1-141, 1-86 mature peptide cDNA by PCR, and clones the fragment into pGEM-T easy, and then uses pGEM-T
  • the Xhol and EcoRI sites in the easy multiple cloning site were ligated into the same endonuclease-treated pPIC9K linkage to achieve directional cloning.
  • the transformation methods of Pichia pastoris mainly include electroporation, protoplast method, PEG1000 method and lithium chloride method.
  • the electroporation method has the highest conversion efficiency, and can produce 10 3 - 10 4 transformants per ⁇ ⁇ plasmid DNA, but requires a special equipment electro-converter.
  • the protoplast method also obtains better conversion efficiency, but requires more expensive Zymolyase to partially hydrolyze the cell wall, and the experimental process is also cumbersome.
  • the PEG1000 conversion method is simple and economical. 10 2 - 10 3 transformants can be produced per ⁇ ⁇ plasmid DNA. In this study, a better conversion effect was obtained by this method, and an average of 100-200 transformants were obtained per 7 cm plate after the transformation. Advantages and effects
  • the recombinant human parathyroid hormone related protein 1-86, 1-141 is prepared by the method of the invention, and is simple, safe and reliable. After purification, the recombinant polypeptide PTHrPl-86, 1-141 can meet the requirements of biological activity determination and animal experiments, and can be used for the treatment of osteoporosis.
  • Rats, as experimental animals, are less expensive, easier to operate, and more reproducible.
  • the treatment and administration method are simple and convenient to operate, and the effect is obvious.
  • Figure 1 PCR amplification of PTHrPl-141 gene using genomic DNA as a template, wherein 1 is DNA Marker DL2000: 2000, 1000, 750, 500, 250, and lOObp 2 is a PCR amplification product of PTHrPl-141.
  • pGEM-T easy pGEM-T easy/ PTHrPl-141 recombinant plasmid was digested.
  • Marker DL2000 2000, 1000, 750, 500, 250, lOObp 2.
  • FIG. 1 Construction of pQE-30Xa/PTHrP 1-141 and pQE-3 OXa/PTHrP 1 -86 expression vectors.
  • FIG. 7 Restriction map of pQE-30Xa/PTHrPl-86 recombinant plasmid, wherein 1 is 1.
  • DNA Marker DL2000 2000, 1000, 750, 500, 250, 100 bp; 2.
  • Fig. 8 Results of SDS-PAGE electrophoresis of pQE-30Xa/PTHrPl-141 expression product in Ec M15[pREP4], wherein 1 is M15[pREP4] induced expression, fragmentation is precipitated; 2 is M15[pREP4] induced expression, fragmentation On 3; M15[pREP4] transformed with empty pQE-30Xa plasmid induced expression, fragmentation; 4 was induced by empty pQE-30Xa plasmid transformed with M15[pREP4], and the supernatant was broken; 5 was pQE-30Xa/ PTHrPl -141 Recombinant plasmid-transformed M15[pREP4]-induced expression (O.lmmol/L IPTG), fragmentation; 6 is pQE-30Xa/ PTHrPl-141 recombinant plasmid transformed M15[pREP4] induced expression (O.lmmol/L IPTG), the
  • Figure 9 Results of SDS-PAGE electrophoresis of pQE-30Xa/PTHrPl-86 expression product in E.co// M15[pREP4], where 1 is M15[pREP4] induced expression for 4 hours, and the supernatant is sterilized; M15[pREP4] transformed with pQE-30Xa plasmid induced expression for 4 hours, and the supernatant was pulverized; 3 was induced by p15E-30Xa/ PTHrPl-86 recombinant plasmid, and M15[pREP4] was induced to express for 4 hours (1.0 mmol/L IPTG).
  • the supernatant of the bacterium was 4; M15[pREP4] transformed with pQE-30Xa/ PTHrPl-86 recombinant plasmid was induced to express for 4 hours (2.0mmol/L IPTG), and the supernatant was broken; 5 was the molecular weight of the polypeptide: 16.95kD, 14.44kD , 10.60kD ; 6 is induced by M15[pREP4] for 4 hours, and the bacteria are precipitated; 7 is M15[pREP4] transformed with empty pQE-30Xa plasmid and induced to express for 4 hours, and the bacteria are precipitated; 8 is pQE-30Xa/ PTHrPl-86 The recombinant plasmid-transformed M15[pREP4] induced expression for 4 hours (1.0 mmol/L IPTG), and the pellet was precipitated; 9 was induced by pQE-30Xa/PTHrP1-86 recombinant
  • FIG. 10 Western Blotting of the recombinant protein PTHrP1-141, where 1 is the protein molecular weight marker (amino black staining): 66.2 kD, 45 kD, 35 kD, 25 kD, 18.4 kD, 14.4 kD; 2 is the color of the induced expression product result.
  • 1 is the protein molecular weight marker (amino black staining): 66.2 kD, 45 kD, 35 kD, 25 kD, 18.4 kD, 14.4 kD
  • 2 is the color of the induced expression product result.
  • FIG. 11 Western Blotting identification of the recombinant protein PTHrPl-86, wherein 1 is the molecular weight marker of the polypeptide (amino black staining): 16.95 kD, 14.44 kD, 10.60 kD ; 2 is the coloration result of the induced expression product.
  • Figure 14 PCR identification of plasmid pPIC9K-PTHrPl-86.
  • 1 PCR product with empty plasmid pPIC9K as template
  • 2, 3, 5, 6, 7 PCR product with recombinant plasmid pPIC9K-PTHrpl-86 as template
  • FIG. 15 Silver staining results of yeast medium supernatant after SDS-PAGE electrophoresis.
  • l pPIC9K transformant induced expression in 120-hour culture supernatant; 2: pPIC9K/PTHrP transformant induced expression on 12-hour medium Clear electrophoresis; 3: P PIC9K/PTHrP transformant induced expression 24 hours culture supernatant; 4: P PIC9K/PTHrP transformant induced expression 48 hours culture supernatant; 5: Protein molecular weight marker: 97.4KD, 66.2KD , 43.0KD, 31.0 D, 20.0KD, 14.4KD; 6: pPIC9K/PTHrP transformant induced expression 72 hours culture supernatant; 7: P PIC9K7PTHrP transformant induced expression 96 hours culture supernatant; 8: pPIC9K/ The PTHrP transformant induced expression of the culture supernatant for 108
  • FIG. 1 Effect of serum osteocalcin levels in rats.
  • Ovariectomized group PO.05, PTH1-34 high dose group, PTH1-141 high dose group and PTH1-141 low dose group P ⁇ 0.05.
  • FIG. 19 Effect on serum alkaline phosphatase in rats.
  • the high dose group of PTH1-34 and the high dose group of PTHrPl-141 were higher than the other groups.
  • Figure 20 Effect on serum calcium and phosphorus in rats. P ⁇ 0.05 in the sham operation group PO.05, the high-dose group of PST1-141 and the low-dose group of PTH1-141.
  • Figure 23 Effect of maximum deformability of the femur in rats.
  • Figure 26 Effect of rat femoral single-label volume on rats.
  • Figure 27 Effect of the double-labeled table volume on the rat femur.
  • Figure 28 Effect of the volume ratio of single and double markers on the femur of rats.
  • E. coli JM109 strain was purchased from QIAGEN company
  • E. coli M15 [pREP4] strain was purchased from QIAGEN company
  • rat osteosarcoma cell line UMR 106 was purchased from the United States ATCC (American Tissue Culture Collection)
  • the pGEM-T Easy carrier was purchased from Promega, and the pQE-30Xa was purchased from QIAGEN.
  • Tris Tri C ine, sodium dodecyl sulfate (SDS) were purchased from Huamei Bioengineering Co., Ltd. 3-isobutyl small methylxanthine (IBMX), agarose, glycine, ethidium bromide (EB), phenylmethylsulfonyl fluoride (PMSF), and cyclic adenosine monophosphate (cAMP) are products of Sigma, USA.
  • IBMX 3-isobutyl small methylxanthine
  • agarose glycine
  • EB ethidium bromide
  • PMSF phenylmethylsulfonyl fluoride
  • cAMP cyclic adenosine monophosphate
  • Ammonium persulfate (AP), tetramethylethylenediamine (TEMED), acrylamide and methylenebisacrylamide were purchased from USB Corporation of the United States.
  • HRP-labeled rabbit anti-goat IgG was purchased from Beijing Dingguo Bioengineering Co., Ltd.
  • Goat anti-human PTHrP (N-19) IgG was purchased from SANTA CRUZ.
  • hPTHl-34 was purchased from BACHEM. Other reagents are imported or domestically analyzed.
  • Yeast Extract Tryptone, Agar, and DMEM are all GIBCO products. Fetal bovine serum was purchased from Hyclone.
  • the DNA rapid purification and recovery kit was purchased from Beijing Dingguo Biotechnology Co., Ltd.
  • the ProbandTM M-NTA Affinity Chromatography Protein Purification Kit was purchased from Invitrogen.
  • the BCA protein concentration assay kit is a product of PIERCE.
  • the DAB chromogenic kit was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.
  • Human genomic DNA was extracted from whole blood according to the TKM method [38] .
  • the PTHrP1-141 gene was amplified by PCR using the following primers:
  • Upstream primer 5 --- GCG ⁇ 4I C i GCTGTGTCTGAACATCAGCTCCTC---3, ( SEQ ID NO: l ) , where is the start codon; the base indicated by the slash is the EcoRI restriction site.
  • the PCR reaction system was as follows: genomic DNA was ⁇ . ⁇ , lOxPCR buffer was 2.5 ⁇ 1, dNTP (2.5 mmol/L each) was 1.5 ⁇ 1, upstream primer (5 ⁇ 1/0 was ⁇ . ⁇ , downstream primer (5 mol/L) For 1.0 ⁇ l, r DNA polymerase (lU ⁇ L) was ⁇ . ⁇ , and dd3 ⁇ 40 was 17.0 ⁇ l.
  • the PCR reaction conditions were: pre-denaturation at 95 ° C for 5 min; denaturation at 95 ° C for 45 sec, annealing at 58 ° C for 45 sec, 72. C extends for 45 sec, 30 cycles; the last 72 ° C extends for 10 min.
  • the 2 ⁇ 1 PCR amplification product was subjected to 1.5% agarose gel electrophoresis, and the molecular weight of the amplified product was 423 bp (SEQ ID NO: 3) with reference to the nucleic acid molecular weight marker DL 2 000.
  • the PCR amplification product described above was recovered by a DNA rapid purification recovery kit (purchased from Beijing Dingguo Biotechnology Co., Ltd.) and then ligated to the pGEM-T easy vector. It was then transformed into competent bacteria E. co/JM109 prepared according to the Nishimmm method [39] .
  • plasmid was extracted by alkaline lysis [39 ' 4()] .
  • digestion two fragments of about 3000 bp and about 450 bp were observed, as shown in Fig. 2.
  • the recombinant plasmid was named P GEM-T/PTHrPl-141 o Example 2. Construction and expression of pQE-30Xa/PTHrPl-141 and pQE-30Xa/PTHrPl-86 recombinant plasmids (1) Amplification of PTHrPl-141 and PTHrPl- 86 fragment:
  • the PTHrPl-141 and PTHrPl-86 fragments were amplified by PCR as follows:
  • Downstream primer (for PTHrPl-141), wherein the base indicated by the slash is the I cleavage site:
  • the pQE-30Xa vector was digested with Stu I and Sal I, and ligated with the partially amplified product of the PCR amplification products PTHrPl-141 and PTHrPl-86 obtained in l. Plasmid pQE-30Xa ⁇ Both the PTHrPl-141 and PTHrPl-86 sequences have a restriction endonuclease restriction enzyme ⁇ ⁇ cleavage site. If the ligation product is pQE-30Xa/PTHrPl-141 recombinant, 3285 bp and 613 bp will be produced after digestion.
  • the recombinants pQE-30Xa/PTHrPl-141 and pQE-30Xa/PTHrPl-86 were transformed into E. coli M15 [pREP4].
  • IPTG final concentration 1 ⁇ ol/L
  • the expression product was induced by pQE-30Xa/PTHrPl-141 by SDS-PAGE [39 ' 4Q] .
  • the fusion protein expressed by the E. coli M15 [pREP4] host strain containing no pQE-30Xa plasmid and the host strain containing the blank pQE-30Xa plasmid included the carrier protein (2.2 kD) and PTHrPl-141 ( 16.2 kD). Therefore, deep-stained protein bands should be visible at approximately 18-19 kDa.
  • the target band is slightly larger than the predicted molecular weight. See Figure 8.
  • the expression product was induced by pDSE-30Xa/PTHrPl-86 by SDS-PAGE.
  • the fusion protein including the carrier protein (2.2 kD) and PTHrPl-86 (1O.OkD) was used as a control against the Eco/ 7 ' M15 [pREP4] host strain containing the pQE-30Xa plasmid and the host strain containing the blank pQE-30Xa plasmid. Therefore, a deep-stained protein band should be visible at about 12-13 kDa.
  • the results are shown in Figure 9.
  • the yield of the fusion protein in the supernatant of the bacterium increased with the increase of the final concentration of IPTG, and reached a peak at 1.0 mmol/L.
  • the protein in the gel was transferred to the PVDF membrane by semi-dry method, and the goat anti-human PTHrP (N-19) polyclonal antibody was used as the primary antibody, and the horseradish peroxidase-labeled rabbit anti-goat IgG antibody was used as the secondary antibody for the Western antibody.
  • Blot [43] identification The PVDF membrane used for Western Blotting assay has a 22kD deep-stained band after substrate color development, and its position is consistent with the specific band position of the recombinant plasmid-induced expression product, see Figure 10.
  • the PVDF membrane used for Western Blotting assay showed a 12kD deep-stained band after substrate color development, and its position was consistent with the specific band position of the recombinant plasmid-induced expression product, see Figure 11.
  • PTH and PTHrP play a physiological role through a common receptor [19 ' 2Q] , and bioactive PTH or PTHrP binds to a receptor and triggers a cascade of cascades triggered by intracellular second messenger cAMP, thus The intracellular cAMP concentration will increase significantly.
  • Whether the recombinant PTHrPl-86 and PTHrPl-141 were biologically active was determined by measuring changes in cAMP in the rat osteosarcoma cell line UMR106 (see Table 1 below).
  • the rat osteosarcoma cell line UMR 106 [47] was resuscitated and passaged to 24-well plates after 2-3 passages. TTC, 5 C0 2 , cultured in DMEM complete culture. When the cells reached 80% confluence (10 6 or more), discard the culture solution, rinse the cells twice with cold PBS, and add serum-free DMEM medium, 5% C0. 2 incubated at 37 ° C for 24 hr. The serum-free medium was discarded and rinsed twice with cold PBS solution. The above serum-free medium containing 1 mmol/L of IBMX was added for 60 minutes to inhibit the activity of phosphodiesterase.
  • the cAMP content was determined by HPLC.
  • the cAMP value of the sample to be tested is obtained by comparing the area above the peak with the area under the peak of the cAMP standard curve.
  • the cAMP standard was purchased from SIGMA and the standards were diluted to different concentrations to establish a standard curve. 3. Correct the cAMP content determination results by the total protein concentration of the cell lysate
  • the total protein concentration of the cell lysate was determined by the BCA method, and the measured intracellular cAMP ratio was used as the corrected cAMP content measurement result.
  • Example 3 Construction and expression of P PIC9k/PTHrPl-141 and pPIC9k/PTHrPl-86 recombinant plasmids (1). Construction of recombinant plasmid pPIC9K/PTHrPl-86 The PTHrP1-86 fragment obtained in Example 2 was ligated into pGEM-T easy (the same method as before), and the purified DNA fragment was recovered by digestion with EcoI and EcoRI, and the same expression plasmid pPIC9 was used. Figure 13) Make the connection. The recombinant plasmid pIC9/PTHrPl-86 was subjected to restriction enzyme digestion and sequencing (sequencing primer was 5'AOX1 supplied by Invitrogen) to determine the correct recombinant.
  • Pichia pastoris strain GS 115 (purchased from Invitrogen) was transformed by electroporation.
  • the additional product was 771 bp, which is the sum of the fragment 492 bp and the insert 279 bp between the corresponding upstream and downstream primers of pPIC9.
  • the recombinant plasmid pIC9/ PTHrPl-86 was integrated into the yeast chromosome.
  • BMMY medium 10 g/L yeast extract, 20 g/L peptone, 10 g/L glycerol, 13.4 g/L YNB, 0.4 mg/L biotin.
  • BMMY medium 10 g/L yeast extract, 20 g/L peptone, 10 g/L glycerol, 13.4 g/L YNB, 0.4 mg/L biotin.
  • 0.1 mol/L potassium phosphate buffer shaking culture was continued at 30 ° C, and 0.125 ml of methanol (final concentration of 0.5% V/V) was added every 24 hours to induce expression.
  • the culture was stopped, and the supernatant was centrifuged at 15 000 x g, and an appropriate amount was applied for SDS polyacrylamide gel electrophoresis.
  • a recombinant yeast strain containing the PTHrP mature peptide coding sequence showed a distinct protein staining band at 10 KD, whereas the control yeast strain (an empty plasmid-transformed yeast cell strain) did not have this band, see Figure 15.
  • Electrophoretic identification The supernatant was retained by centrifugation at 15 OOOxg, and a small amount was used as described above for SDS polyacrylamide gel electrophoresis.
  • Example 4 Construction of recombinant plasmid pPIC9K/PTHrPl-141
  • the pGEM-T easy/ PTHrPl-141 obtained above was transferred into Escherichia coli JM109, and the plasmid was extracted by alkaline lysis, and the DNA fragment was purified by XhoI and EcoRI digestion, and the same expression plasmid pPIC9 was used. connection.
  • the recombinant plasmid was identified by enzyme digestion. Simultaneous sequencing was performed, and the sequencing primers were 5' ⁇ 1 provided by Invitrogen.
  • Pichia pastoris GS115 transformed competent cells (simultaneously transformed with empty plasmid), coated on MD plates (1.34% YNB, 4xl0- 5% bio Inoculum, 2% glucose, 1.5% agar) Incubate for 30 days at 30 °C, inoculate each colony grown in YPD medium, and shake the culture, extract yeast genomic DNA as a template, and PCR (same example 2) Recombinant.
  • the recombinant plasmid pIC9/ PTHrPl-141 was integrated into the yeast chromosome.
  • Animal grouping The selected animals were grouped by weight and divided into 6 groups according to the random principle: sham operation group, negative control group, PTH1-34 high dose group, PTH1-34 low dose group, PTHrPl-86 high dose group, PTHrPl-86 Low dose group. Twenty animals in each group, except for the sham operation group, all the other groups underwent castration surgery. 2. Preparation of castrated animal models
  • Rats were anesthetized by intraperitoneal injection at a dose of 0.3 mL/100 g body weight using 10% chloral hydrate. After anesthesia, the rats were fixed in the prone position, and after the skin was disinfected, the muscle tissue was bluntly separated through the back incision, and then the peritoneal cavity was opened, and the ovary was confirmed to be ligated and excised. The tissue is returned to the body, and the gentamicin is irrigated and the wound is sutured, and the wound is naturally awakened. After 16 weeks of castration surgery, the bone density of the lumbar vertebrae of the rats was measured, and the animal model was confirmed to be successful.
  • the sham operation group was given vehicle (acidified saline), the negative control group, the vehicle, and the PTH1-34 high dose group was given PTH1-34 protein from Shanghai Saijin Biomedical Co., Ltd.
  • PTH1-34 low dose group to PTHl-34 (40nmol/kg body weight), PTHrPl-86 high dose group to recombinant PTHrPl-86 protein (40nmol/kg) Body weight), PTHrPl-86 low dose group was given recombinant PTHrPl-86 protein (5nmol/kg body weight), PTHrPl-141 high dose group was given recombinant PTHrPl-141 protein (40nmol/kg body weight), PTHrPl-141 low dose group was given recombinant PTHrPl -141 protein (5 nmol/kg body weight).
  • Tetracycline was injected intraperitoneally 10 days before the end of the administration and 3 days before the end of the administration. After the end of the administration, the animals were sacrificed by exsanguination of the femoral artery.
  • PTHrPl-86 and PTHrPl-141 can accelerate bone turnover, promote new bone formation, improve bone mechanical properties, have high flexural strength, enhanced resistance to deformation, increased toughness, reduced brittleness, and are less prone to fracture. It is proved that PTHrPl-86 can effectively improve the anti-fracture ability of bone and has certain curative effect on the treatment and prevention of osteoporosis. At the same time, it is proved that the method of the animal is effective, and the therapeutic effect of PTHrPl-86 on osteoporosis can be well determined. references
  • PTHrP Parathyroid hormone-related protein

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Abstract

L'invention concerne des protéines humaines recombinées apparentées à l'hormone parathyroïde (PTHrP1-86 et PTHrP1-141) et des séquences nucléotidiques codant ces protéines. L'invention concerne également l'application pharmaceutique de PTHrP1-86 et de PTHrP1-141 ainsi que des trousses thérapeutiques comprenant ces protéines. Les protéines recombinées et les trousses selon l'invention peuvent être utilisées pour le traitement de l'ostéoporose.
PCT/CN2007/001039 2006-03-30 2007-03-30 Protéines humaines recombinées apparentées à l'hormone parathyroïde et leurs utilisations WO2007112682A1 (fr)

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CN200610013398.8 2006-03-30
CN200610013404 2006-03-30
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CN200610013403 2006-03-30
CN200610013628 2006-03-30
CN200610013628.0 2006-03-30
CN200610013404.X 2006-03-30
CN200610013402 2006-03-30
CN200610013402.0 2006-03-30
CN200610013401 2006-03-30
CN200610013398 2006-03-30
CN200610013405.4 2006-03-30
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003059291A2 (fr) * 2002-01-10 2003-07-24 Osteotrophin Llc Traitement des affections osseuses avec des medicaments anabolisants du squelette

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003059291A2 (fr) * 2002-01-10 2003-07-24 Osteotrophin Llc Traitement des affections osseuses avec des medicaments anabolisants du squelette

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CELL BIOL. INT., vol. 28, no. 10, 2004, pages 661 - 673 *
DATABASE CNKI [online] LIU X.-Y.: "Expression and Bioactivity Analysis of PTH-related Protein 1-141 and 1-86" *
DATABASE GENBANK [online] 17 December 2003 (2003-12-17), OKOUMASSOUN L.E. ET AL.: "Parathyroid hormone-related protein (PTHrP) inhibits mitochondrial-dependent apoptosis through CK2", XP003018265, Database accession no. (NM_198966) *
DATABASE GENBANK [online] 8 February 2005 (2005-02-08), BARLING P.M. ET AL.: "Expression of PTHrP and the PTH/PTHrP receptor in growing red deer antler", XP003018266, Database accession no. (AY328402) *
J. CELL PHYSIOL., vol. 212, no. 3, 2007, pages 591 - 599 *
pages 13-25 - 13-35 *
PILBEAM C.C.: "Comparison of the Effects of Various Lengths of Synthetic Human Parathyroid Hormone-Related Peptide (hPTHrP) of Malignancy on Bone Resorption and Formation in Organ Culture", BONE, vol. 14, 1993, pages 717 - 720 *

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