Long-acting parathyroid hormone mutant, fusion protein containing same and application of long-acting parathyroid hormone mutant

Abstract

The invention provides a long-acting parathyroid hormone mutant, a fusion protein containing the same and application of the long-acting parathyroid hormone mutant, the amino acid sequence of the parathyroid hormone PTH34 mutant is shown as SEQ ID NO.1, and the parathyroid hormone PTH34 mutant is obtained through mutation of any one of the following mutations: L7A, N16S, M18H, L7A / N16S, L7A / M18H, N16S / M18H and L7A / N16S / M18H. The fusion protein formed after the mutant and HSA are connected through a connecting peptide is good in stability, long in half-life period and high in activity, and can be used for preparing the long-acting parathyroid hormone mutant. The traditional Chinese medicine composition can be well applied to preparation of medicines for treating osteoporosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological products, and in particular relates to a long-acting parathyroid hormone mutant and a fusion protein containing the same and an application thereof. Background Art

[0002] Osteoporosis is a metabolic bone disease characterized by decreased bone density and destruction of bone microarchitecture, which will lead to an increased risk of fractures. Parathyroid hormone (PTH) is a peptide hormone secreted by the parathyroid glands. It is a basic single-chain polypeptide hormone secreted by parathyroid chief cells. The main function of PTH is to regulate the metabolism of calcium and phosphorus in vertebrates. It acts on bones, kidneys and intestines to increase blood calcium levels and decrease blood phosphorus levels. Its main function is to regulate calcium and phosphorus metabolism. The secretion of PTH is mainly regulated by plasma calcium ion concentration. When the plasma calcium ion concentration increases, the secretion of PTH will be inhibited; when the plasma calcium ion concentration decreases, the secretion of PTH is stimulated. PTH can stimulate bone formation at physiological doses, but promote bone resorption at high doses. PTH mainly acts by binding to its receptor PTH1R, which is a member of the class B G protein-coupled receptor (GPCR) family. （1） .

[0003] Natural PTH is composed of 84 amino acids (PTH84), but studies have found that its biological activity mainly depends on the 1st to 34th amino acid residues (PTH34) at its amino terminus. PTH34 is an active fragment of PTH and has the same biological activity as full-length PTH (1-84). In the treatment of osteoporosis, Teriparatide is a synthetic PTH34 fragment that can promote bone formation and reduce the risk of fractures. It has been used in many countries around the world to treat postmenopausal osteoporotic women and men with high fracture risks. It is currently one of the main drugs used in clinical treatment of osteoporosis.

[0004] However, the half-life of PTH34 in the circulating blood is about 10 minutes. The half-life of teriparatide in the human body after subcutaneous injection is 1 hour. PTH84 can also be used as a drug, and its half-life in the human body after subcutaneous injection is 2.5 hours. （2） The short half-life requires teriparatide to be injected once a day, which seriously reduces patient compliance and limits its clinical application. Therefore, it is necessary to increase the half-life of PTH and reduce the injection frequency.

[0005] However, studies have found that intermittent PTH exposure, produced by daily pulse injections of PTH34, is associated with increased cancellous bone volume, density, and strength. Intermittent PTH exposure has complex effects on cortical bone, including increases in cortical bone porosity and cortical bone thickness, resulting in neutral to positive effects on cortical bone strength. However, continuous PTH exposure, associated with hyperparathyroidism, generally has a neutral effect on cancellous bone and a net decomposition (resorption) effect on cortical bone, which is not conducive to increases in bone density. （3） .

[0006] Kostenuik et al. reported an IgG Fc-fused PTH34 that extended the half-life of PTH34 by 33-fold in rats, making it possible to experiment with once- or twice-weekly injections. Once- or twice-weekly subcutaneous injections of PTH-Fc significantly increased bone mass, density, and strength in osteoporotic ovariectomized mice and rats, much more significantly than what could be achieved with daily high-concentration PTH(1-34). PTH-Fc also significantly improved cortical bone volume and density under conditions in which daily PTH(1-34) was ineffective. These results challenge the view that daily, brief exposures are required for the anabolic effects of PTH on both cortical and cancellous bone. PTH molecules with extended half-lives may represent a powerful and previously undefined anabolic regimen for both cortical and cancellous bone. （4） .

[0007] However, current studies on PTH34 have shown that the activity of PTH34 is low, and the half-life of Fc-fused PTH34 is still short, and the expression and preparation are difficult. In Kostenuik's study, Escherichia coli was used for expression and obtained by renaturation, which had low yield and high cost.

[0008] The documents mentioned in the above background technology are specifically referenced as follows: (1), Pioszak&Xu, PNAS, 2008, 105(13): 5034-5039; Zhao LH, et al., Science, 2019, 364: 148-153. (2), Tay D, et al., Br J ClinPharmacol. 2018 Feb;84(2):252-267. (3), Kostenuik, et al., Journal of Bone and Mineral Research, 2007, 22(10): 1534-1547. (4), Kostenuik, et al., Journal of Bone and Mineral Research, 2007, 22(10): 1534-1547. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides a long-acting parathyroid hormone mutant and a fusion protein containing the same and its application.

[0010] The purpose of the present invention is achieved through the following technical solutions: Long-acting parathyroid hormone mutant, the amino acid sequence of the parathyroid hormone PTH34 mutant is SEQ ID NO.1 obtained by any one of the following mutations: The leucine at position 7 is mutated to alanine, and the amino acid sequence of the mutant L7A is shown in SEQ ID NO.2; Or the 16th asparagine of the parathyroid hormone of the amino acid sequence as shown in SEQ ID NO.1 is mutated to serine, and the amino acid sequence of the mutant N16S is shown in SEQ ID NO.3; Or the 18th methionine of the parathyroid hormone with an amino acid sequence as shown in SEQ ID NO.1 is mutated to histidine, and the amino acid sequence of the mutant M18H is shown in SEQ ID NO.4; Or the mutant is a combination of mutating the leucine at position 7 of the parathyroid hormone shown in the amino acid sequence of SEQ ID NO.1 to alanine and mutating the asparagine at position 16 to serine, and the amino acid sequence of the combined mutant L7A / N16S is shown in SEQ ID NO.5; Or a combination of mutating the leucine at position 7 of the parathyroid hormone shown in the amino acid sequence of SEQ ID NO.1 to alanine and mutating the methionine at position 18 to histidine, wherein the amino acid sequence of the combined mutant L7A / M18H is shown in SEQ ID NO.6; Or a combination of mutating the asparagine at position 16 of the parathyroid hormone shown in the amino acid sequence of SEQ ID NO.1 to serine and mutating the methionine at position 18 to histidine, wherein the amino acid sequence of the combined mutant N16S / M18H is shown in SEQ ID NO.7; Or a combination of mutating the leucine at position 7 to alanine, mutating the asparagine at position 16 to serine, and mutating the methionine at position 18 to histidine. The amino acid sequence of the combined mutant L7A / N16S / M18H is shown in SEQ ID NO.8.

[0011] A fusion protein is formed by fusing a protein with any of the long-acting parathyroid hormone mutants described above via a connecting peptide, wherein the amino acid sequence of the connecting peptide is shown in SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11.

[0012] Preferably, the fusion protein is formed by fusing the protein with the long-acting parathyroid hormone mutant with the sequence of SEQ ID NO.8 via a connecting peptide, and the amino acid sequence of the fusion protein is: SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14.

[0013] Preferably, a buffer for storing the fusion protein is a phosphate buffer with a pH of 7.4, and the buffer comprises mannitol, Tween80 and Arg.

[0014] Preferably, a buffer for storing a fusion protein comprises 5 mg / mL mannitol, 0.02% Tween80, and 100 mM Arg.

[0015] Preferably, a buffer for storing a fusion protein comprises 5 mg / mL mannitol, 0.02% Tween80, 0.01% phenol, and 100 mM Arg.

[0016] Preferably, a method for preparing the fusion protein comprises the following steps: S1. Designing gene sequence: connecting the gene of the long-acting parathyroid hormone mutant formed after the long-acting parathyroid hormone is mutated with the protein HSA gene through a connecting peptide to form a fusion protein gene; S2, constructing a vector, inserting the fusion protein gene sequence in S1 into an expression vector after double enzyme digestion to construct a recombinant vector, wherein the expression vector includes pPICZαA, pPICZαB, pPICZαC, pGAPZαA, pGAPZαB, and pGAPZαC; S3, preparing and obtaining a high-expression cell strain; transferring the recombinant vector obtained in S2 into a Pichia pastoris strain, and obtaining a high-expression cell strain after screening; the Pichia pastoris strains include Pichia pastoris strain X33 and Pichia pastoris strain GS115.

[0017] S4. Protein expression and purification: The colonies of the fusion protein were selected and cultured in the culture medium. The obtained supernatant was purified by Ni-NTA and Q-HP elution in turn to finally obtain a high-purity fusion protein.

[0018] Preferably, the fusion protein is used in the preparation of a drug for treating osteoporosis.

[0019] Preferably, a drug for treating osteoporosis comprises the fusion protein as described above.

[0020] The beneficial effects of the present invention are embodied in that the PTH34 mutant provided by the present invention forms a long-acting fusion protein with high activity, long half-life and stability by fusing with HSA, which can be effectively used in the preparation of drugs for treating osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0022] Figure 1 : Alignment diagram of the first 34 amino acid sequences of PTH from different sources in Example 1.

[0023] Figure 2 : The activities of S1 site and L7 site mutants were demonstrated.

[0024] Figure 3 : The activities of N16 and M18 site mutants were demonstrated.

[0025] Figure 4 : The activity of each combination mutant is shown in comparison with the wild type (WT).

[0026] Figure 5 : Purified fusion proteins are shown.

[0027] Figure 6 : Demonstrated the effect of the connecting peptide on the in vitro activity of the fusion protein.

[0028] Figure 7 : Shows the effect of the fusion protein on serum calcium concentration in OVX mice.

[0029] Figure 8 : Shown the effect of fusion protein on bone density in OVX mice.

[0030] Fig. 9 : Shows the changes in the concentration of the fusion protein in rats over time. DETAILED DESCRIPTION

[0031] Example 1: Saturation mutation improves the activity of PTH34

[0032] 1) Site and sequence Sequence comparison of the first 34 amino acids from humans, cattle, and horses revealed that there were variations in the 1st, 7th, 16th, and 18th amino acids. Figure 1 As shown, it is suggested that these positions may determine the specific functions or activity differences of different sequences in different species. Single-point saturation mutations were performed on these positions, and the four amino acids were mutated using the degenerate codon NNK.

[0033] The amino acid sequence of human PTH34 is SEQ ID NO.1: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF DNA sequence of human PTH34: AGTGTGTCTGAGATACAGCTGATGCATAACCTTGGAAAGCATTTGAACTCCATGGAAAGAGTCGAATGGTTACGAAAAAAACTGCAAGACGTGCATAACTTT The primers are as follows: 1S-F: (the underline is the XhoI restriction site, and the italic is the mutation site) AGATA CTCGAG AAAAGAGAGGCTGAAGCT NNK GTGTCTGAGATACAGCTG Univ-R: The underline is the KpnI restriction site ATAAGT GGTACC CCAAAGTTATGCACGTCTTGC 7L-F: (underlined is the XhoI restriction site, italics are mutation sites) AGATA CTCGAG AAAAGAGAGGCTGAAGCTAGTGTGTCTGAGATACAG NNK ATGCATAACCTTGG 16N-F1: Italics indicate mutation sites GCATTTG NNK TCCATGGAAAGAGTCGAATGG 16N-R1: Italics indicate mutation sites CCATGGA MNN CAAATGCTTTCCAAGGTT 18M-F1: Italics indicate mutation sites GAACTCC NNK GAAAGAGTCGAATGGTTACG 18M-R1: Italics indicate mutation sites CTCTTTC MNN GGAGTTCAAATGCTTT 16-18-F2: The underline indicates the expression XhoI restriction site; AGATA CTCGAG AAAAGAGAGGCTGAAGCTAGTGTGTCTGAGATACAGCTG 2) The DNA sequence of human PTH34 was fully synthesized by Qingke Biotechnology (Beijing) and constructed into the pUC-18 vector (pUC-18-PTH34): 3) PCR was performed on the vector using 1S-F / Univ-R primers to construct a mutant library; after gel extraction and recovery, double restriction digestion was performed using XhoI / KpnI; after gel extraction, the vector was connected with the Pichia pastoris expression vector pPICZαA vector that was digested with the same double restriction enzymes to obtain the pPICZαA-PTH34-1S saturated mutant library; 4) The establishment of the 7L mutant library was similar to that of 1S. PCR was performed on the pUC-18-PTH34 expression vector using 7L-F / Univ-R, and the remaining steps were the same as above to construct the pPICZαA-PTH34-7L saturation mutant library; 5) The saturation mutant library of 16N and 18M was prepared using a two-step method. First, primers 16N-F1 / 16N-R1 were used to perform PCR on pUC-18-PTH34. Then, the primers 16-18F2 / Univ-R were used to perform a second round of PCR. After double restriction digestion, the PCR product was ligated with the Pichia pastoris expression vector pPICZαA that was digested with the same double restriction enzymes to obtain the pPICZαA-PTH34-16N saturation mutant library. 6) The saturation mutant library of 18M is similar to that of 16N. First, 18M-F1 / 18M-R1 was used to perform PCR on pUC-18-PTH34. After restriction digestion and ligation, the pPICZαA-PTH34-18M saturation mutant library was constructed. Example 2: Transformation of Pichia pastoris and secretory expression of target protein

[0034] 1) Preparation of competent cells of Pichia pastoris: In a 50 mL Erlenmeyer flask, culture 5 mL of Pichia pastoris strain X33 with YPD medium at 30°C overnight. In a 2 L Erlenmeyer flask, inoculate 500 mL of fresh medium with 0.1-0.5 mL of overnight culture. Culture again overnight until OD600 reaches 1.3-1.5.

[0035] Centrifuge the cells at 1500 × g for 5 minutes at 4°C. Resuspend the pellet in 500 mL of ice-cold (0°C) sterile water. Centrifuge the cells again and resuspend the pellet in 250 mL of ice-cold (0°C) sterile water. Centrifuge the cells again and resuspend the pellet in 20 mL of ice-cold (0°C) 1 M sorbitol. Centrifuge the cells again and resuspend the pellet in 1 mL of ice-cold 1 M sorbitol to a final volume of approximately 1.5 mL. Keep the cells on ice and use the same day.

[0036] 2) After mixing the plasmids of each mutant library obtained above with the competent cells, electrotransform them using a Bio-Rad electroporator, and then immediately spread them on a YPDS plate containing 100 μg / mL Zeocin; 3) On the third and fifth days after coating, select at least 100 colonies and transfer them to plates with higher concentrations of Zeocin, with the highest concentration being 800 μg / mL; 4) After 7 days, the colonies on the plate are the constructed mutant library.

[0037] 5) Inoculate each colony into a 96-deep-well plate, first add BMGY medium and culture overnight at 30°C; then remove the medium, replace it with BMMY medium, and add fresh methanol every 24 hours to the final concentration of 0.5%, and continue to culture at 30°C with shaking.

[0038] 6) After 5 days of expression, centrifuge the plate and remove the supernatant for the following activity test. Example 3: Protein expression and activity identification

[0039] 1) Activity detection method: The proliferation experiment was performed using the MC3T3-E1 cell line, and the activity of PTH34 and its mutants was evaluated by detecting the effects of different concentrations of PTH34 and its mutants on cell proliferation.

[0040] 2) Cell culture: MC3T3-E1 cells were cultured in cell culture medium containing 10% FBS until 80%-90% confluence. The cells were digested with trypsin-EDTA solution, centrifuged and resuspended in fresh culture medium.

[0041] 3) Cell seeding: Adjust the cell suspension to an appropriate cell density (1×10 4 cells / mL), added to a 96-well plate, 100 μL per well; placed the 96-well plate in a 37°C CO 2 Incubate in an incubator for 24 hours.

[0042] 4) Add drugs: According to the results of the previous preliminary experiments, the mutants were diluted 100 times with serum-free medium, and then 100 μL of the protein supernatant containing the mutants was added to each well of the cells; the 96-well plate was returned to the incubator and cultured for 24 h; 5) Cell proliferation detection: After 24 hours of culture, add 10 μL of CCK-8 solution to each well according to the instructions of the CCK-8 kit; continue to culture for 2 hours to allow CCK-8 to fully react.

[0043] 6) Data collection: Use a microplate reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0044] 7) Results: Saturation mutations were performed on the four sites S1, L7, N16, and M18, and activity was tested. The results showed that for the S1 site, mutation to other amino acids would not further increase its activity, so it was kept as S amino acid. For the L7 site, when it was mutated to A (L7A), its activity increased by 1.68 times, while when it was mutated to F (L7F), the activity decreased significantly. The results are as follows: Figure 2 As shown, * indicates that the mutant is compared with the wild type, p < 0.05; *** indicates that the mutant is compared with the wild type, p < 0.001. For the N16 site, when it is mutated to S (N16S), its activity is increased by nearly 2 times, while when it is mutated to W (N16W), its activity is significantly reduced. For the M18 site, when it is mutated to H (M18H), its activity is increased by 2.16 times, while when it is mutated to V (M18V), its activity does not change much. The results are shown in Figure 3 As shown, * indicates that the mutant is compared with the wild type, p < 0.05; *** indicates that the mutant is compared with the wild type, p < 0.001.

[0045] The amino acid sequence after mutation is as follows: SEQ ID NO.2: SVSEIQAMHNLGKHLNSMERVEWLRKKLQDVHNF SEQID NO.3:SVSEIQLMHNLGKHLSSMERVEWLRKKLQDVHNFSEQ ID NO.4:SVSEIQAMHNLGKHLNSHERVEWLRKKLQDVHNF Example 4: Combination mutation

[0046] 1) Combinatorial mutations are performed on the above key sites with improved activity to further improve their activity; 2) PTH3 L7A / N16S, L7A / M18H, N16S / M18H, and L7A / N16S / M18H mutants were constructed using the above primers and methods, and were respectively constructed into the vector pPICZαA.

[0047] The amino acid sequence of PTH34 L7A / N16S is SEQ ID NO.5: SVSEIQ A MHNLGKHL S SMERVEWLRKKLQDVHNF The amino acid sequence of PTH34 L7A / M18H is SEQ ID NO.6: SVSEIQ A MHNLGKHLNS H ERVEWLRKKLQDVHNF The amino acid sequence of PTH34 N16S / M18H is SEQ ID NO.7: SVSEIQLMHNLGKHL S S H ERVEWLRKKLQDVHNF The amino acid sequence of PTH34 L7A / N16S / M18H is SEQ ID NO.8: SVSEIQ A MHNLGKHL S S H ERVEWLRKKLQDVHNF 3) The vector was transformed into Pichia pastoris X33 strain using the above method and cultured at 30°C for 4-5 days.

[0048] 4) Take the protein supernatant and perform activity detection as above.

[0049] 5) Results: The combined mutations of each key site can further improve its activity. The activity of the L7A / N16S mutant is 2.65 times higher than that of the wild type, the activity of the L7A / M18H mutant is 2.98 times higher than that of the wild type, the activity of the N16S / M18H mutant is 2.39 times higher than that of the wild type, and the activity of the L7A / N16S / M18H mutant is nearly 4 times higher than that of the wild type. Figure 4 As shown, *** indicates p < 0.001 compared with the wild type. Example 5: Fusion expression of PTH34 L7A / N16S / M18H mutant and HSA

[0050] 1) Gene sequence design: The PTH34 L7A / N16S / M18H (PTH34-ASH) gene was connected to the HSA gene, and a connecting peptide was inserted in the middle. The connecting peptide was in three different forms: flexible linker (GS), rigid linker (PG) and mixed linker (GSPG); and a control without linker was set up.

[0051] Amino acid sequence of GS SEQ ID NO.9: GGGGSGGGGSGGGGS Amino acid sequence of PG SEQ ID NO.10: PPTPGPSPTPGPSPG Amino acid sequence of GSPG SEQ ID NO.11: GGGSPGPGPTPGGGS The amino acid sequence of PTH34-ASH-HSA (without linker) is SEQ ID NO.15: The underlined part is PTH34-ASH, followed by the amino acid sequence of HSA SVSEIQAMHNLGKHLSSHERVEWLRKKLQDVHNF DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRH PYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAE VENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCC KHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL The amino acid sequence of PTH34-ASH-GS-HSA is SEQ ID NO.12: wherein the underline is PTH34-ASH, the italics are linker, and the following is the amino acid sequence of HSA.

[0052] SVSEIQAMHNLGKHLSSHERVEWLRKKLQDVHNF GGGGSGGGGSGGGGS DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRH PYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAE VENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCC KHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL The amino acid sequence of PTH34-ASH-PG-HSA is SEQ ID NO.13: wherein the underline is PTH34-ASH, the italics are linker, and the following is the amino acid sequence of HSA.

[0053] SVSEIQAMHNLGKHLSSHERVEWLRKKLQDVHNF PPTPGPSPTPGPSPGDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL Amino acid sequence of PTH34-ASH-GSPG-HSA SEQ ID NO.14: The underlined part is PTH34-ASH, the italic part is the linker, and the following is the amino acid sequence of HSA SVSEIQAMHNLGKHLSSHERVEWLRKKLQDVHNF GGGSPGPGPTPGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRH PYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAE VENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCC KHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 2) Vector construction: Using the method described above, the gene sequence of the fusion protein was double-digested with XhoI / KpnI and inserted into the pPICZαA expression vector to construct pPICZαA-PTH34-ASH-GS-HSA, pPICZαA-PTH34-ASH-PG-HSA, and pPICZαA-PTH34-ASH-GSPG-HSA expression vectors, and a control pPICZαA-PTH34-ASH-HSA without linker was set up: the vector contains a C-His-tag for easy purification and detection.

[0054] 3) Obtaining high-yield cell lines: Prepare competent cells of Pichia pastoris X33 using the above method, and transfer the above expression vector into Pichia pastoris by electroporation; and obtain a high-expression cell line after Zeocin screening; 4) Protein expression and purification: Select one colony for each fusion protein and inoculate it into a shake flask containing 100 mL of culture medium for cultivation; first add BMGY culture medium and culture overnight at 30°C; then remove the culture medium, replace with BMMY culture medium, and add fresh methanol every 24 hours to a final concentration of 0.5%, and continue shaking culture at 30°C; after 5 days of expression, centrifuge to obtain the supernatant. Load the supernatant onto the Ni-NTA column, balance the column and load the sample with equilibration buffer (25mM Tris / HCl, 150mM NaCl, pH8.0), use wash buffer (25mM Tris / HCl, 150mM NaCl, 30mM imidazole, pH 8.0) for thorough washing; then use elution buffer (25mM Tris / HCl, 150mM NaCl, 500mM imidazole, pH 8.0) for direct elution. The eluted buffer containing the target protein was then exchanged into 25 mM Tris / HCl, pH 8.0 using a G-25 desalting column.

[0055] The above protein solution was loaded onto Q-HP and linearly eluted with 25 mM Tris / HCl, 500 mM NaCl, pH 8.0; the eluted buffer containing the target protein was then exchanged into PBS, pH 7.4 using a G-25 desalting column. After purification, a high-purity fusion protein sample was obtained, such as Figure 5 As shown, 1 represents PTH34-ASH-GS-HSA, 2 represents PTH34-ASH-PG-HSA, 3 represents PTH34-ASH-GSPG-HSA, and 4 represents PTH34-ASH-HSA. Example 6. In vitro activity detection of fusion protein

[0056] 1) As described above, the activity of PTH34 and each fusion protein was detected using a cell-based method; 2) Take 25 μg of each expressed protein and add it to the 96-well plate containing cells. After culturing for 24 hours, add 10 μL CCK-8 solution; continue culturing for 2 hours, and then use a microplate reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0057] 3) Results: Effect of connecting peptide on in vitro activity of fusion protein Figure 6 As shown, * indicates that the mutant is compared with the wild type, p < 0.05. In the in vitro activity test, it was found that after PTH34-ASH was fused with HSA, its in vitro activity was significantly reduced if the linker was not added; after the linker was added, regardless of whether it was a flexible, rigid or mixed linker, the fusion protein did not affect the binding of PTH34-ASH to the receptor and its function, indicating that the linker plays an important role in it. Example 7: Stability formula screening

[0058] In order to improve protein stability during purification and storage, the drug formulation was further optimized based on the main phosphate buffer (PBS, pH 7.4). Protein drugs were stabilized by adding different proportions of mannitol (1-20 mg / mL), phenol (0.01%-0.05%), Tween 80 (0.01%-0.04%), and Arg (50-200 mM).

[0059] The 1 mg / mL PTH34-ASH-GSPG-HSA protein was exchanged into the buffer solution containing the above-mentioned components, filtered and sterilized, and an accelerated experiment was performed at 37°C. After incubation for 10 days, the protein was removed and the residual protein activity was detected using the above-mentioned cell activity method, with 0 day as 100%.

[0060] The results are shown in Table 1. When only PBS was used, the activity decreased significantly after incubation at 37°C for 10 days; after adding mannitol, the rate of activity decrease was significantly slowed down; after adding phenol, Tween80 or Arg, the activity was further improved. There was little difference between formula F and formula G. Finally, formula F (PBS, 5 mg / mL mannitol, 0.02% Tween80, 100mM Arg, pH 7.4) was selected as the storage buffer for the protein drug of the present invention.

[0061] Table 1: Effect of different buffer formulations on activity formula composition Residual activity (0 days is 100%) A PBS, pH 7.4 56.5 ± 6.5% B PBS, 5 mg / mL mannitol, pH 7.4 78.9 ± 4.6% C PBS, 10 mg / mL mannitol, pH 7.4 83.1 ± 6.2% D PBS, 5 mg / mL mannitol, 0.01% phenol, pH 7.4 86.4 ± 5.8% E PBS, 5 mg / mL mannitol, 0.02% Tween80, pH 7.4 87.7 ± 6.3% F PBS, 5 mg / mL mannitol, 0.02% Tween80, 100 mM Arg, pH 7.4 97.8 ± 7.1% G PBS, 5 mg / mL mannitol, 0.02% Tween80, 0.01% phenol, 100 mM Arg, pH 7.4 98.2 ± 7.4% Example 8. In vivo activity detection of fusion protein

[0062] 1) Ovariectomy model. 8-week-old female C57BL / 6 mice were housed in an individually ventilated cage system at 24±2℃, 12-hour light / 12-hour dark cycle. Mice had free access to food and water. Surgical procedure for ovariectomized (OVX) mice: After the mice were anesthetized, a small incision was made on the back skin on both sides of the mice, through which the bilateral ovaries were removed, and after the skin was sutured, the mice were returned to the cage.

[0063] 2) Effect on serum calcium ion concentration. Another 9 OVX mice were divided into 3 groups. One group was injected with PBS, one group was injected with 20 nmol / kg PTH34, and the other group was injected with 20 nmol / kg PTH34-ASH-GSPG-HSA. The serum was collected at 2h and 10h after injection and the serum calcium ion concentration was detected using a kit.

[0064] The results showed that: the calcium level in serum could not be significantly increased after PBS injection; the calcium level could be significantly increased 2 hours after PTH34 injection, but it decreased at 10 hours; and the calcium level was still significantly increased at 10 hours after PTH34-ASH-GSPG-HSA injection. Figure 7 As shown, * indicates that the treatment group is compared with the PBS group, p<0.05; ** indicates that the treatment group is compared with the PBS group, p<0.01; *** indicates that the treatment group is compared with the PBS group, p<0.001. This shows that PTH34-ASH-GSPG-HSA has a long-term effect and can increase the calcium ion level in serum for a long time in the body.

[0065] 3) Therapeutic effect on osteoporosis. The mice were randomly divided into 4 groups (4 mice in each group): control group (Sham), ovariectomy group (OVX), PTH34-ASH-GSPG-HSA group and PTH34 group. Ovariectomy was performed in the OVX group, PTH34-ASH-GSPG-HSA group and PTH34 group, while only adipose tissue was removed from the control group (Sham) mice, and bilateral ovaries were intact.

[0066] The PTH34-ASH-GSPG-HSA group received a subcutaneous injection once a week at a dose of 20 nmol / kg; the PTH34 group received an injection every other day at a dose of 20 nmol / kg; the control and OVX groups received the same volume of PBS buffer. All mice continued to be housed for 8 weeks. After the experiment, the femur of one hind limb was removed and quickly fixed with 4% paraformaldehyde. Bone mineral density (BMD) was analyzed using a micro-computed tomography (Micro-CT) device (SkyScan 1174; Bruker, Kontich, Belgium).

[0067] The results showed that compared with the Sham group, the BMD of OVX mice decreased significantly; after the injection of PTH34-ASH-GSPG-HSA and PTH34, the BMD increased significantly compared with OVX and almost returned to the level of the Sham group. Figure 8 As shown, *** indicates that the OVX group was compared with the Sham group, p < 0.001. It is proved that PTH34-ASH-GSPG-HSA has the same function as PTH34 in treating osteoporosis, but PTH34-ASH-GSPG-HSA can be injected once a week, which greatly reduces the frequency of injection. Example 9: In vivo half-life detection

[0068] In order to determine the pharmacokinetic characteristics of the fusion protein, the PTH34-ASH-GSPG-HSA fusion protein was subcutaneously injected into female SD rats at a dose of 5 nmol / rat. 100 μL of blood was drawn from the caudal vein at different time points and transferred to a 5% paraformaldehyde-containing EDTA-Na 2 Eppendorf tubes were used for anticoagulation. After centrifugation, plasma was stored at -80°C for further analysis. The concentration of fusion protein in plasma was determined by ELISA. Mouse anti-HSA monoclonal antibody (ab10241, Abcam) was spread on the bottom of a 96-well plate; 100 μL of rat serum was added to the 96-well plate and incubated for 2 hours; Rabbit anti-PTH monoclonal antibody (ab16632, Abcam) was added and incubated for 1 hour; Goat Anti-Rabbit IgG (HRPConjugated) (ab6721, Abcam) was added and incubated for 1 hour; finally, TMB solution was added for 25 minutes and 1M HCl was used to terminate the reaction, and the detection was performed at 450 nm on an ELISA reader. The standard curve was drawn using the standard of PTH34-ASH-GSPG-HSA.

[0069] The results are as follows Fig. 9 It showed that the half-life of PTH34-ASH-GSPG-HSA in rats reached 13.2 h, while the half-life of PTH34 in rats was only 0.26 h (Rmezanipour N, et al., Endocrinology, 2022, 163(2): 1-9); the half-life of PTH34-ASH-GSPG-HSA was 50.7 times higher than that of PHT34.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Long-acting parathyroid hormone mutant, Features: The amino acid sequence of the parathyroid hormone PTH34 mutant is obtained by mutation of any one of the following: L7A, N16S, M18H, L7A / N16S, L7A / M18H, N16S / M18H, L7A / N16S / M18H; wherein, The sequence of the mutant L7A is SEQ ID NO.2; The sequence of the mutant N16S is SEQ ID NO.3; The sequence of the mutant M18H is SEQ ID NO.4; The sequence of the mutant L7A / N16S is SEQ ID NO.5; The sequence of the mutant L7A / M18H is SEQ ID NO.6; The sequence of the mutant N16S / M18H is SEQ ID NO.7; The sequence of the mutant L7A / N16S / M18H is SEQ ID NO.

8.

2. A fusion protein, Features: The protein is fused with the long-acting parathyroid hormone mutant according to claim 1 via a connecting peptide, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.

11.

3. A fusion protein according to claim 2, Features: The protein is fused with the long-acting parathyroid hormone mutant with the sequence of SEQ ID NO.8 through a connecting peptide, and the amino acid sequence of the fusion protein is: SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.

14.

4. A buffer for storing the fusion protein according to claim 2, Features: The buffer is a phosphate buffer with a pH of 7.4, and the buffer comprises mannitol, Tween 80 and Arg.

5. A buffer for storing fusion protein as claimed in claim 4, Features: The buffer contained 5 mg / mL mannitol, 0.02% Tween80, and 100 mM Arg.

6. A phosphate buffer for storing fusion protein as claimed in claim 4, Features: The buffer contained 5 mg / mL mannitol, 0.02% Tween80, 0.01% phenol, and 100 mM Arg.

7. A method for preparing the fusion protein according to claim 2, Features: The steps include: S1. Designing gene sequence: connecting the gene of the long-acting parathyroid hormone mutant formed after the long-acting parathyroid hormone is mutated with the protein HSA gene through a connecting peptide to form a fusion protein gene; S2, constructing a vector, inserting the fusion protein gene sequence in S1 into an expression vector after double enzyme digestion to construct a recombinant vector, wherein the expression vector includes pPICZαA, pPICZαB, pPICZαC, pGAPZαA, pGAPZαB, and pGAPZαC; S3. Prepare and obtain high-expressing cell lines; The recombinant vector obtained in S2 was transferred into the Pichia pastoris strain, and a high-expressing cell strain was obtained after screening; S4. Protein expression and purification: The colonies of the fusion protein were selected and cultured in the culture medium. The obtained supernatant was purified by Ni-NTA and Q-HP elution in turn to finally obtain a high-purity fusion protein.

8. Use of the fusion protein according to claim 2 in the preparation of a drug for treating osteoporosis.

9. A drug for treating osteoporosis, Features: Comprising the fusion protein as described in claim 2.

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