Recombinant human growth hormone-collagen fusion protein and preparation method and application thereof

Abstract

This invention discloses a recombinant human growth hormone-collagen fusion protein, its preparation method, and its applications, relating to the field of fusion protein technology. The fusion protein comprises a human growth hormone sequence and a human-like collagen sequence. The amino acid sequence of the fusion protein is shown in SEQ ID NO.7. The first homologous region of the human growth hormone is located at the N-terminus of the fusion protein, and the second homologous region of the human-like collagen is located at the C-terminus. In this invention, recombinant human growth hormone and human collagen with tissue repair function are fused through genetic engineering, achieving efficient expression in the periplasmic space in *E. coli*. High-purity products are obtained by GH affinity chromatography and weak cation exchange chromatography. The protein has a normal structure and good in vitro activity, and can be used to prepare long-acting growth hormone preparations or wound repair materials, showing significant clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of fusion protein technology, and in particular to recombinant human growth hormone-collagen fusion protein, its preparation method, and its application. Background Technology

[0002] Growth hormone (GH) is an important protein hormone in the human body, which promotes growth, cell proliferation, and tissue repair. Recombinant human growth hormone (rhGH) has been used to treat growth hormone deficiency and promote wound healing, but its short half-life requires frequent dosing, leading to poor patient compliance. Existing long-acting technologies (such as PEGylation) may introduce immunogenic risks. Collagen is an important component of tissue repair, especially type III collagen, which plays a crucial role in wound healing and reducing scar formation.

[0003] Collagen is an important component of human tissue, playing a vital role in promoting platelet aggregation, repairing epidermal cells, and accelerating wound healing. Recombinant human collagen has lower immunogenicity compared to animal-derived collagen. Most existing wound repair materials are physical mixtures of type I collagen and growth factors; however, these materials are non-fusion proteins, and because they primarily use animal-derived type I collagen, they are costly and possess a certain degree of immunogenicity. Type III collagen is also an important collagen in the dermis, playing a crucial role in the proliferative and tissue remodeling phases of wound healing. Furthermore, studies have found that type III collagen may reduce scar formation.

[0004] Therefore, developing a fusion protein that is structurally stable, has a long half-life, low immunogenicity, and combines rhGH activity with collagen wound repair function is of great clinical significance and application value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a recombinant human growth hormone-collagen fusion protein with long half-life, low immunogenicity, and both growth hormone activity and collagen repair function, as well as its preparation method and application.

[0006] The technical solution adopted to solve the above technical problems is: the recombinant human growth hormone-collagen fusion protein contains a human growth hormone sequence and a human-like collagen sequence. The amino acid sequence of the fusion protein is shown in SEQ ID NO.7. The first homologous region of the human growth hormone is located at the N-terminus of the fusion protein, and the second homologous region of the human-like collagen is located at the C-terminus of the fusion protein.

[0007] Furthermore, the fusion protein is linked by a linker peptide to form a single polypeptide chain, and the linker peptide is GGGSGGGSGGGS.

[0008] An isolated nucleic acid molecule encoding a recombinant human growth hormone-collagen fusion protein, the nucleotide sequence of which is shown in SEQ ID NO.8.

[0009] A recombinant expression vector for secretory expression comprises a phosphate promoter and an STII signal peptide sequence, wherein the signal peptide is located before the N-terminus of a human growth hormone sequence, and is used to guide the secretion of the recombinant fusion protein into the periplasmic space for secretory expression using the phosphate promoter.

[0010] Furthermore, the bacteria are Escherichia coli, specifically any one of GI724, BL21, DH5α, JM109, HB101, Rosetta, and Origami; the yeast cells are Pichia pastoris cells; and the animal cells are Chinese hamster ovary cells.

[0011] The application of the signal peptide in the expression of recombinant human growth hormone-collagen fusion protein, wherein the nucleotide sequence of the signal peptide is shown in SEQ ID NO.9.

[0012] A method for preparing recombinant human growth hormone-collagen fusion protein includes the following steps:

[0013] S801, culture engineered bacteria containing an expression vector encoding a fusion protein to express the fusion protein;

[0014] S802, collect and break the bacterial cells to obtain a crude extract containing the fusion protein;

[0015] S803, the crude extract was subjected to growth hormone affinity chromatography and ion exchange chromatography in sequence to purify and obtain the fusion protein.

[0016] Furthermore, the method for obtaining the crude extract containing the fusion protein in S802 is as follows:

[0017] S80201, the fermentation broth of Escherichia coli expressing recombinant human growth hormone-collagen fusion protein was centrifuged at 8000 rpm and 4℃ for 40 min, the supernatant was discarded, and the bacterial precipitate was collected.

[0018] S80202 involves placing the bacterial cell precipitate in an ultra-low temperature freezer at -80℃ for repeated freeze-thaw cycles to break it up, repeating the cycle three times to fully rupture the bacterial cell walls and release the target protein.

[0019] S80203, according to the ratio of cell mass to resuspension volume (mL) = 1:9, add resuspension to the ruptured cells. The resuspension composition is: 50 mM Tris, 0.2 M arginine, 0.3 M NaCl, pH 7.4. Before adding the resuspension, place the resuspension system in a 4℃ cold storage for 1 h to fully resuspend the target protein and maintain its stability.

[0020] S80204: Centrifuge the resuspended solution at 8000 rpm and 4℃ for 40 min to remove cell debris and insoluble impurities. Collect the supernatant and store it at 4℃ for later purification.

[0021] Furthermore, the method for purifying the fusion protein in S803 is as follows:

[0022] S80301, GH affinity chromatography: A GH affinity chromatography column was used. The equilibration buffer was 50 mM Tris, 0.2 M arginine, and 0.3 M NaCl at pH 7.4. The elution buffer was 50 mM glycine at pH 3.0. A linear gradient elution was performed from 100% equilibration buffer to 100% elution buffer, with an elution volume of 5 column volumes. The elution peak of the target protein was collected.

[0023] S80302, Weak Cation Exchange Chromatography: A weak cation exchange chromatography column was used. The equilibration buffer was 20 mM Tris and 10 mM PB at pH 6.0, and the elution buffer was 20 mM Tris and 10 mM PB at pH 9.0. A pH gradient elution was performed from 100% equilibration buffer to 60% elution buffer, with an elution volume of 20 column volumes. The elution peak of the target protein was collected to obtain the high-purity fusion protein.

[0024] The recombinant human growth hormone-collagen fusion protein, isolated nucleic acid molecules, recombinant expression vectors, and host cells are used in drug preparation to prolong the half-life of growth hormone for the treatment of various growth and development disorders and to promote wound healing.

[0025] The beneficial effects of the present invention are as follows: (1) The present invention uses recombinant human growth hormone and human collagen with tissue repair function to fuse through genetic engineering, achieve high efficiency expression in the periplasmic space in Escherichia coli, and obtain high purity products by GH affinity chromatography and weak cation exchange chromatography. The protein has a normal structure and good in vitro activity, and can be used to prepare long-acting growth hormone preparations or wound repair materials, which has important clinical application prospects.

[0026] (2) The fusion protein of the present invention has the advantages of stable structure, long half-life and low immunogenicity. It also has the wound repair function of human growth hormone and collagen. It can prolong the half-life of growth hormone, be used to treat various growth and development disorders, and promote wound healing. When combined with appropriate excipients, it can be further prepared into injections, dressings, gels and other preparations, which are suitable for the research and development of long-acting growth hormone drugs and wound repair drugs. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the structure of the recombinant human growth hormone-collagen fusion protein of the present invention.

[0028] Figure 2 This is a PCR result image of the fusion protein gene fragment.

[0029] Figure 3 This is a diagram showing the results of double enzyme digestion of the vector and fragment.

[0030] Figure 4 This is a graph showing the PCR identification results of a single-clone colony expressing the fusion protein.

[0031] Figure 5 This is a diagram showing the results of double enzyme digestion identification of the pACYC-GH-Col plasmid.

[0032] Figure 6 It is a growth hormone affinity chromatography pattern.

[0033] Figure 7 This is a graph showing the electrophoresis results of growth hormone.

[0034] Figure 8 It is a weak cation chromatography pattern.

[0035] Figure 9 This is a graph showing the results of weak cation electrophoresis.

[0036] Figure 10 This is a graph showing the purity results of growth hormone affinity elution samples.

[0037] Figure 11 This is a graph showing the purity results of samples eluted by weak cation chromatography.

[0038] Figure 12 This is a graph showing the results of liquid chromatography-mass spectrometry (LC-MS) analysis for determining the complete molecular weight of rhGH-Col protein.

[0039] Figure 13 This is a result of the peptide map's amino acid coverage.

[0040] Figure 14 This is a diagram showing the results of peptide mapping modification.

[0041] Figure 15 This is an infographic for identifying disulfide bonds.

[0042] Figure 16 This is a graph showing the results of in vitro biological activity.

[0043] Figure 17 This is a bar chart showing the weight changes of patients who took GH-Col stock solution once every 6 days.

[0044] Figure 18 This is a bar chart showing the weight changes when GH-Col stock solution is administered once every 3 days.

[0045] Figure 19 This is a blood drug concentration-time curve for the GH stock solution group.

[0046] Figure 20 This is a blood drug concentration-time curve for the GH-Col group.

[0047] Figure 21 It is a photograph of the wound. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0049] like Figure 1 As shown, the recombinant human growth hormone-collagen fusion protein of this embodiment comprises a human growth hormone sequence and a human-like collagen sequence. The amino acid sequence of the fusion protein is shown in SEQ ID NO.7. The first homologous region of human growth hormone is located at the N-terminus of the fusion protein, and the second homologous region of human-like collagen is located at the C-terminus of the fusion protein. The fusion protein is linked by a linker peptide to form a single polypeptide chain, and the linker peptide is GGGSGGGSGGGS.

[0050] The amino acid sequence of human growth hormone is shown in SEQ ID NO.1:

[0051] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

[0052] The nucleotide sequence encoding human growth hormone is shown in SEQ ID NO.2:

[0053] TCCCAACTATACCACTAAGTCGACTATTCGATAACGCTATGCTTCGGGCCCATCGTCTTCATCAGCTAGCCTTTGACACCTACCAGGAGTTTGAAGAGGCCTATATCCCCAAGGAACAGAAGTATTCATTCCTGCAGAACCCCCAGACCTCCCTCTGTTTCTCAGAATCGATTCCGACACCCTCCAATCGCGAGGAAACACAACAGAAATCCAACCTAGAGCTCCTCCGCATAAGCTTGCTGCTCATCCAGTCGTGGCTCGAGCCCGTGCAGTTCCTGAGGAGTGTCTTCGCCAACAGCCTGGTCTACGGCGCCTCTGATTCGAACGTGTACGACCTGCTGAAGGACCTAGAGGAAGGGATCCAAACGCTGATGGGGAGGCTGGAAGATGGCAGCCCGCGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGATGACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCTTC

[0054] The amino acid sequence of the collagen is shown in SEQ ID NO.3:

[0055] LVPRGSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGLHM

[0056] The nucleotide sequence encoding the collagen is shown in SEQ ID NO.4:

[0057] CTGGTTCCGCGTGGTAGTCCGGGTCTGCCGGGTCCTCGTGGTGAACAGGGCCCTACCGGTCCGACCGGTCCTGCAGGTCCTCGTGGACTGCAGGGCCTGCAGGGTCTGCAGGGTGAACGCGGTGAACAGGGTCCGACCGGCCCTGCAGGTCCAAGAGGTCTGCAGGGCGAACGCGGCGAACAGGGTCCTACCGGTCTGGCAGGTAAAGCCGGTGAAGCCGGTGCCAAAGGCGAAACCGGTCCGGCTGGCCCTCAGGGTCCTAGAGGTGAACAGGGACCGCAGGGCCTGCCGGGTAAAGATGGCGAAGCCGGCGCACAGGGCCCGGCAGGTCCTATGGGTCCTGCAGGA

[0058] The primer sequence F is as shown in SEQ ID NO.5:

[0059] CCAATGCATTCCCAACTATACCACTAAGTCGAC

[0060] The primer sequence R is as shown in SEQ ID NO.6:

[0061] GGAATTCCATATGTAGTCCTGCAGGACCCATAGG

[0062] The amino acid sequence of recombinant human growth hormone - collagen fusion protein is as shown in SEQ ID NO.7:

[0063] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGSGGGSGGGSLVPRGSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGLHM

[0064] An isolated nucleic acid molecule encoding a recombinant human growth hormone-collagen fusion protein, the nucleotide sequence of which is shown in SEQ ID NO.8.

[0065] The nucleotide sequence encoding the recombinant human growth hormone-collagen fusion protein is shown in SEQ ID NO. 8:

[0066] TCCCAACTATACCACTAAGTCGACTATTCGATAACGCTATGCTTCGGGCCCATCGTCTTCATCAGCTAGCCTTTGACACCTACCAGGAGTTTGAAGAGGCCTATATCCCCAAGGAACAGAAGTATTCATTCCTGCAGAACCCCCAGACCTCCCTCTGTTTCTCAGAATCGATTCCGACACCCTCCAATCGCGAGGAAACACAACAGAAATCCAACCTAGAGCTCCTCCGCATAAGCTTGCTGCTCATCCAGTCGTGGCTCGAGCCCGTGCAGTTCCTGAGGAGTGTCTTCGCCAACAGCCTGGTCTACGGCGCCTCTGATTCGAACGTGTACGACCTGCTGAAGGACCTAGAGGAAGGGATCCAAACGCTGATGGGGAGGCTGGAAGATGGCAGCCCGCGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGATGACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCTTCGGTGGTGGTAGTGGTGGTGGTTCAGGCGGCGGTAGCCTGGTTCCGCGTGGTAGTCCGGGTCTGCCGGGTCCTCGTGGTGAACAGGGCCCTACCGGTCCGACCGGTCCTGCAGGTCCTCGTGGACTGCAGGGCCTGCAGGGTCTGCAGGGTGAACGCGGTGAACAGGGTCCGACCGGCCCTGCAGGTCCAAGAGGTCTGCAGGGCGAACGCGGCGAACAGGGTCCTACCGGTCTGGCAGGTAAAGCCGGTGAAGCCGGTGCCAAAGGCGAAACCGGTCCGGCTGGCCCTCAGGGTCCTAGAGGTGAACAGGGACCGCAGGGCCTGCCGGGTAAAGATGGCGAAGCCGGCGCACAGGGCCCGGCAGGTCCTATGGGTCCTGCAGGA

[0067] A recombinant expression vector comprising a separated nucleic acid molecule.

[0068] A host cell comprising a recombinant expression vector, wherein the host cell is a bacterium, a yeast cell, or an animal cell. The bacterium is *Escherichia coli*, specifically any one of GI724, BL21, DH5α, JM109, HB101, Rosetta, or Origami; the yeast cell is *Pichia pastoris* cells; and the animal cell is a Chinese hamster ovary cell.

[0069] The method for preparing recombinant human growth hormone-collagen fusion protein in this embodiment includes the following steps:

[0070] S801, culture engineered bacteria containing an expression vector encoding a fusion protein to express the fusion protein;

[0071] S802, collect and break the bacterial cells to obtain a crude extract containing the fusion protein;

[0072] The method for obtaining the crude extract containing the fusion protein in S802 is as follows:

[0073] S80201, the fermentation broth of Escherichia coli expressing recombinant human growth hormone-collagen fusion protein was centrifuged at 8000 rpm and 4℃ for 40 min, the supernatant was discarded, and the bacterial precipitate was collected.

[0074] S80202 involves placing the bacterial cell precipitate in an ultra-low temperature freezer at -80℃ for repeated freeze-thaw cycles to break it up, repeating the cycle three times to fully rupture the bacterial cell walls and release the target protein.

[0075] S80203, according to the ratio of cell mass to resuspension volume (mL) = 1:9, add resuspension to the ruptured cells. The resuspension composition is: 50 mM Tris, 0.2 M arginine, 0.3 M NaCl, pH 7.4. Before adding the resuspension, place the resuspension system in a 4℃ cold storage for 1 h to fully resuspend the target protein and maintain its stability.

[0076] S80204: Centrifuge the resuspended solution at 8000 rpm and 4℃ for 40 min to remove cell debris and insoluble impurities. Collect the supernatant and store it at 4℃ for later purification.

[0077] S803, the crude extract was subjected to growth hormone affinity chromatography and ion exchange chromatography in sequence to purify and obtain the fusion protein.

[0078] S80301, GH affinity chromatography: A GH affinity chromatography column was used. The equilibration buffer was 50 mM Tris, 0.2 M arginine, and 0.3 M NaCl, pH 7.4. The elution buffer was 50 mM glycine, pH 3.0. A linear gradient elution was performed from 100% equilibration buffer to 100% elution buffer, with an elution volume of 5 column volumes. The elution peak of the target protein was collected, such as... Figure 6 and Figure 7 As shown, Figure 7 In the diagram, M stands for Premixed Protein Marker (Low), 1 is the sample before loading, 2 is the flow-through sample, and 3 is the elution sample.

[0079] S80302, Weak Cation Exchange Chromatography: A weak cation exchange chromatography column was used. The equilibration buffer was 20 mM Tris, 10 mM PB, pH 6.0, and the elution buffer was 20 mM Tris, 10 mM PB, pH 9.0. Elution was performed using a pH gradient from 100% equilibration buffer to 60% elution buffer, with an elution volume of 20 column volumes. The elution peak of the target protein was collected, yielding a high-purity fusion protein. Figure 8 and Figure 9 As shown, Figure 9 In the diagram, M stands for Premixed Protein Marker (Low), 1 is the sample before loading, 2 is the flow-through sample, and 3-9 are the eluted samples. The sample with the highest purity, number 9, was selected to elute sample 7 for subsequent experiments.

[0080] The recombinant human growth hormone-collagen fusion protein of this embodiment is expressed in Escherichia coli.

[0081] 1. Obtaining the vector and the GH-Col gene fragment

[0082] Plasmids were extracted from preserved PACYC strains using an endotoxin-free plasmid extraction kit according to the manufacturer's instructions. The vector was digested with NsiⅠ and NdeⅠ restriction endonucleases at 37℃ for 30 min. The digestion products were separated by 1% agarose gel electrophoresis at 120V for 40 min. Figure 3 The band shown is approximately 2959 bp in size. This large fragment was gel-cleaved and recovered into the vector PACYC for subsequent T4 ligation. An STII signal peptide coding sequence was introduced at the N-terminus of the GH-Col gene to ensure the fusion protein is secreted into the periplasmic space.

[0083] The nucleotide sequence encoding the STⅡ signal peptide is shown in SEQ ID NO.9:

[0084] ATGAAAAAGAATATCGCATTTCTTCTTGCATCTATGTTCGTTTTTTCTATTGCTACAAATGCCTATGCA

[0085] Using the extracted vector as a template, PCR primers were designed based on the GH-Col gene sequence, and two restriction nuclease sites, NsiⅠ and NdeⅠ, were added. The GH-Col gene fragment was amplified by PCR. The PCR reaction conditions were: 95℃ for 2 min; 95℃ for 20 s, 65.8℃ for 20 s, 72℃ for 1 min, for a total of 34 cycles; extension at 72℃ for 5 min. The amplified products were separated by 1% agarose gel electrophoresis at 120V for 40 min, as shown below. Figure 2 The strip shown is approximately 981bp in size. This small fragment of GH-Col was cleaved and recycled. Figure 2 In the diagram, M represents DNA marker DL8000, and 1-4 are PCR products of fusion protein gene fragments, digested with NsiⅠ and NdeⅠ restriction nucleases at 37℃ for 30 min. The digested products were then separated by 1% agarose gel electrophoresis at 120V for 40 min. Figure 3 The strip shown is approximately 962bp in size. This small fragment of GH-Col is cut and recycled for subsequent T4 bonding. Figure 3 In the diagram, M stands for DNA marker DL8000, 1-4 are double digestion products of the expression vector, and 5-8 are double digestion products of the fusion protein gene fragment.

[0086] 2. Construction of recombinant plasmid PACYC-GH-Col

[0087] Mix 1 μL T4 DNA Ligase, 2 μL 5×T4 DNA Ligase Buffer, PCR GH-Col gene fragment and PACYC vector digestion product (molar ratio 3:1), add sterile water to make up to 10 μL, react at 25℃ for 1 h, cool the resulting ligation product on ice for a few seconds, transform GI724 chemocompetent cells, plate the transformation product on LB agar plates containing 20 μg / mL tetracycline, and incubate overnight in an inverted 37 ℃ incubator.

[0088] 3. Identification of recombinant plasmid PACYC-GH-Col

[0089] Five single colonies of suitable size and clear outline were selected and inoculated into 5 mL of liquid LB medium containing 20 μg / mL tetracycline. The colonies were incubated at 37 ℃ with a shaking incubator until the OD600 reached 0.6-0.8. 0.5 μL of the bacterial culture was used as a template for bacterial PCR using the primers described above. Figure 4The band shown is approximately 981bp in size. Figure 4 In the diagram, M represents the DNA marker DL8000, and 1–5 are PCR results from five randomly selected single-clone colonies. The extracted plasmid from colony number 1 was randomly selected and subjected to double enzyme digestion and sequencing. (Example:) Figure 5 The GH-Col band of approximately 962 bp and the carrier band of approximately 2959 bp shown are both consistent with the theoretical values. Figure 5 In the diagram, M represents DNA marker DL8000, and 1 represents the double enzyme digestion product of the target plasmid. 3 mL of the remaining bacterial culture from the identified positive clones was used to extract the plasmid, which was then sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing. The sequencing results, shown in SEQ ID NO.8, indicate that the recombinant plasmid PACYC-GH-Col was constructed correctly.

[0090] 4. Expression of recombinant plasmid PACYC-GH-Col

[0091] The correctly identified bacterial strains were inoculated into liquid LB medium containing 20 μg / mL tetracycline and cultured in a constant temperature incubator at 37 ℃ with shaking until the OD600 reached 0.6–0.8. 20 mL of the bacterial culture was then inoculated into 1 L of liquid LB medium containing 20 μg / mL tetracycline (containing 10 μL of antifoaming agent) for further expansion and cultured at 37 ℃ and 220 rpm for 20 h with shaking.

[0092] Identification of the recombinant human growth hormone-collagen fusion protein in this embodiment

[0093] 1. Purity testing

[0094] Size exclusion high-performance liquid chromatography (HPLC) conditions: BioCore SEC-150 column (7.8 mm × 300 mm, 5 μm); sample loading volume not less than 20 μg, flow rate 1.0 mL / min, detection time 15 min, detection wavelength 280 nm, column temperature room temperature, sample chamber temperature 10℃. Mobile phase was 0.1 M phosphate-0.1 M NaCl buffer. Elution method was isocratic elution. Figure 10 and Figure 11 The SEC-HPLC results show that the purity of the main protein component after purification by chromatography 1 was 82.843%, but multiple secondary chromatographic peaks were still visible, indicating the presence of certain impurities or polymers. After further purification by chromatography 2, the purity of the target protein significantly increased to 99.327%, with a sharp main peak and good symmetry (tailing factor ≈ 1.24), and the impurity peaks essentially disappeared. These results demonstrate that chromatography 2 can effectively remove relevant impurities and significantly improve the chromatographic purity of the target protein.

[0095] 2. Determination of complete molecular weight

[0096] The molecular weight of intact proteins was determined by LC-MS. LC conditions: column temperature 80℃; sample storage temperature 10℃; flow rate 0.4 mL / min; sample loading volume 5 μL. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution, eluted according to the program in Table 1. Mass spectrometry conditions: positive ion, MS mode; capillary voltage: 2500V; Cone voltage: 40V; desolvent gas temperature: 350℃; source temperature: 120℃; desolvent gas flow rate: 800 L / h; scan range (m / z): 400–4000. Figure 12 The liquid chromatography-mass spectrometry (LC-MS) analysis results for the intact molecular weight of the rhGH-Col protein show that the molecular weight of the fusion protein is 34065.9293 Da, which is basically consistent with its theoretical molecular weight of 34065.589 Da, with a mass deviation of 10 ppm. This result indicates that the target protein structure in the sample is intact, without significant degradation or unexpected modification.

[0097] Table 1 Gradient elution procedure for mass spectrometry molecular weight detection

[0098] Time (min) Flow rate (mL / min) A（%） B（%） 0.00 0.4 95 5 1.00 0.4 95 5 1.01 0.2 95 5 3.50 0.2 5 95 3.70 0.4 5 95 4.00 0.4 95 5 4.50 0.4 5 95 5.00 0.4 95 5 7.00 0.4 95 5

[0099] 3. Peptide mapping detection

[0100] Take 100 μL of sample, add 300 μL of guanidine hydrochloride and 2 μL of DTT (1M), incubate at 37℃ for 1 h, let it cool to room temperature, add 4 μL of LIM (1M), incubate at room temperature in the dark for 0.5 h, then place the sample in an ultrafiltration concentration tube (3K), centrifuge at 12000 rpm, 4℃ for 8 min, replace with 50 mM NH4HCO3 buffer (pH 8.0) at least 4 times, adjust the final sample concentration to 0.3 mg / mL, add trypsin at a ratio of 15:1, digest at 37℃ for 4 h, terminate the reaction with formic acid at a stop concentration of 1%, and inject the sample. HPLC conditions: column temperature 65℃; sample storage temperature 10℃; flow rate 0.3 mL / min; sample loading volume 5 μL. Mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile solution, and gradient elution is performed according to the program in Table 2. Mass spectrometry conditions: MSE mode for data acquisition; capillary voltage: 2500V; Cone voltage: 40V; desolvent gas temperature: 350℃; source temperature: 120℃; desolvent gas flow rate: 600L / h; scan range (m / z): 100-2000; acquisition time: 0-42min; collision energy: 35-45V.

[0101] The amino acid coverage results of the peptide map are as follows: Figure 13 As shown, the amino acid sequence coverage of this fusion protein is 100%.

[0102] The modification results of the peptide map are as follows Figure 14As shown, multiple peptides, including LFDNAMIR, SNELLR, and FDTNSHNDDALLK, were identified, and Met was detected. 6 Oxidation and Asn 4 Asn 7 Post-translational modifications such as deamidation were observed. The mass spectrometry observations of all identified peptides deviated from the theoretical values ​​by less than 11 ppm, demonstrating the accuracy and reliability of the data, the thoroughness of peptide identification, and the crucial role of modification analysis in providing key evidence for protein structure confirmation and quality control.

[0103] Table 2 Gradient elution procedure for mass spectrometry peptide mapping detection

[0104] Time (min) Flow rate (mL / min) A（%） B（%） 0.00 0.3 100 0 2.00 0.3 100 0 32.00 0.3 60 40 33.00 0.3 5 95 39.00 0.3 5 95 40.00 0.3 100 0 42.00 0.3 100 0

[0105] 4. Disulfide bond detection

[0106] Add 300 μL of guanidine hydrochloride and 4 μL of IAM to 100 μL of sample. Incubate at 37 °C for 1 h, then add another 4 μL of IAM and incubate at room temperature in the dark for 0.5 h. Transfer the sample to an ultrafiltration concentration tube (3K), centrifuge at 12000 rpm and 4 °C for 8 min, and replace with 50 mM Tris-HCl buffer (pH 7.35) at least 4 times. Adjust the sample concentration to 0.15 mg / mL with 50 mM Tris-HCl buffer (pH 7.35). Add trypsin at a ratio of 7.5:1 and digest at 37 °C for 4 h. Terminate the reaction with formic acid at a stop concentration of 1%. Inject the sample. HPLC conditions: column temperature 65 °C; sample storage temperature 10 °C; flow rate 0.3 mL / min; loading volume 5 μL. Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is 0.1% formic acid acetonitrile solution. Perform gradient elution according to the program in Table 3. Mass spectrometry conditions: MSE mode data acquisition; capillary voltage: 2500V; Cone voltage: 40V; desolventizing gas temperature: 350℃; source temperature: 120℃; desolventizing gas flow rate: 600L / h; scan range (m / z): 100~2000; acquisition time: 0~42min; collision energy: 35~45V. Disulfide bond analysis results are as follows... Figure 15 As shown, the sequence coverage of rhGH-Col protein under non-reductive digestion conditions was 19%, and two pairs of disulfide-linked peptides were identified, namely YSFLQNPQTSLCFSEISIPTPSNR and NYGLLYCFR, and IVQCR and SVEGSCFGGSGGGSGGGSGLVPR, which are consistent with the theoretical sites.

[0107] Table 3 Gradient elution procedure for disulfide bond detection by mass spectrometry

[0108] Time (min) Flow rate (mL / min) A（%） B（%） 0.00 0.3 100 0 2.00 0.3 100 0 32.00 0.3 60 40 33.00 0.3 5 95 39.00 0.3 5 95 40.00 0.3 100 0 42.00 0.3 100 0

[0109] 5. Preliminary analysis of biological activity

[0110] The reporter gene assay was used. The national standard for the biological activity of recombinant human growth hormone, pre-diluted to 0.003 IU / ml (1 μg / ml), was serially diluted 3-fold in 96-well cell culture plates, resulting in 8 dilutions, with 2 wells for each dilution. The test sample was diluted to 20 μg / mL and serially diluted 3-fold in 96-well cell culture plates, resulting in 8 dilutions, with 2 wells for each dilution. Cells in good growth condition were prepared into a cell suspension containing 3.5 × 10⁵ to 3.8 × 10⁵ cells per mL using complete culture medium. The prepared standard and test sample solutions were transferred to 70 μL per well of a 96-well cell culture plate. The cell suspension was then seeded into the same 96-well cell culture plate, 70 μL per well, and incubated at 37°C in a 5% CO₂ incubator for 18–24 h. Add luciferase substrate, 80 μL / well, and mix thoroughly at room temperature in the dark for 2 min; measure the luminescence intensity using a chemiluminescence microplate reader. The reporter gene assay results are as follows: Figure 16 As shown, the blue curve represents the activity curve of the standard, the orange curve represents the blank buffer curve, the red curve represents the activity curve of the test sample after a 50-fold dilution, the yellow curve represents the activity curve of the test sample after a 500-fold dilution, and the green curve represents the activity curve of the test sample after a 1000-fold dilution. The rhGH-Col fusion protein exhibits dose-dependent activity similar to that of natural rhGH, with a calculated specific activity of 2.66 IU / mg.

[0111] The recombinant human growth hormone-collagen fusion protein, isolated nucleic acid molecules, recombinant expression vectors, and host cells are used in drug preparation to prolong the half-life of growth hormone for the treatment of various growth and development disorders and to promote wound healing.

[0112] In vivo activity verification of the rhGH-Col fusion protein in this embodiment

[0113] 1. Growth-promoting activity

[0114] To evaluate the growth-promoting activity of the rhGH-Col fusion protein in vivo, a hypophysectomized rat model was established using transauricular aspiration of the pituitary gland for multiple-dose experiments. Experimental groups included equimolar amounts of GH stock solution and GH-Col stock solution. GH stock solution was administered once daily, while GH-Col stock solution was administered subcutaneously via either a 6-day or 3-day dosing regimen. Figure 17 and Figure 18 As shown, the GH-Col group exhibited a significant growth-promoting effect after administration, with a weight gain trend comparable to that of the GH stock solution group. Furthermore, it maintained a good growth-promoting effect even with a long dosing interval, indicating that GH-Col has good biological activity.

[0115] 2. Pharmacokinetics and Long-Term Effect Evaluation

[0116] To compare the metabolic behavior of GH-Col and GH stock solution in vivo, further pharmacokinetic studies were conducted. Healthy adult rats were administered the same molar amount of growth hormone in GH stock solution and GH-Col stock solution, respectively. Blood samples were collected at multiple time points after administration to determine serum drug concentrations. Figure 19 and Figure 20 As shown, the blood drug concentration in the GH stock solution group reached its peak at 4–6 hours and then rapidly decreased after 9 hours; while the blood drug concentration in the GH-Col group remained at a high level at 4–9 hours and only gradually decreased after 12 hours, exhibiting obvious sustained-release characteristics, suggesting that GH-Col has a longer half-life and more prolonged pharmacological effects in rats.

[0117] 3. Promotes wound healing

[0118] To evaluate the role of GH-Col in tissue repair, a rat model of deep second-degree burns was established, with a back wound diameter of approximately 1.6 cm. Treatment began 24 hours post-surgery. The experiment included a control group, an Fc-GH group, a GH group, a GH-Col group (once daily or once every three days), and a burn ointment control group. Treatment lasted for 20 consecutive days, and wound area and body weight changes were recorded on days 0, 6, 12, and 20. As shown in Tables 4 and 5, the GH-Col group was significantly superior to the control group and the GH undiluted group in promoting wound healing. The wound healing rate reached 99.2% on day 20 in the once-daily GH-Col group and 96% in the once-every-three-day GH-Col group, both significantly higher than the GH undiluted group (48.2%) and the control group (89%). Regarding body weight changes, the GH-Col group showed stable weight gain during treatment, comparable to the GH undiluted group, indicating that it promoted growth without causing significant metabolic disturbances. Furthermore, wound photographs were recorded as follows: Figure 21 As shown, the wound healing process in the GH-Col group was faster and the healing quality was higher, demonstrating the significant advantage of GH-Col in promoting tissue repair.

[0119] Table 4 Results of wound healing rates in each group

[0120]

[0121] Table 5. Results of animal weight monitoring in each group

[0122]

[0123] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention.

Claims

1. A recombinant human growth hormone-collagen fusion protein, characterized in that: The fusion protein contains a human growth hormone sequence and a human-like collagen sequence. The amino acid sequence of the fusion protein is shown in SEQ ID NO.

7. The first homologous region of the human growth hormone is located at the N-terminus of the fusion protein, and the second homologous region of the human-like collagen is located at the C-terminus of the fusion protein.

2. The recombinant human growth hormone-collagen fusion protein according to claim 1, characterized in that: The fusion protein is formed into a single polypeptide chain by linking peptides, the linking peptide being GGGSGGGSGGGS.

3. An isolated nucleic acid molecule encoding the recombinant human growth hormone-collagen fusion protein as described in any one of claims 1-2, wherein the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

8.

4. A recombinant expression vector for secretory expression, comprising a phosphate promoter and an STII signal peptide sequence, wherein the signal peptide is located before the N-terminus of a human growth hormone sequence, for guiding the secretion of a recombinant fusion protein into the periplasmic space, and for secretory expression using the phosphate promoter.

5. A host cell comprising the recombinant expression vector of claim 4, wherein the host cell is a bacterium, yeast cell, or animal cell.

6. The host cell according to claim 5, characterized in that: The bacteria are Escherichia coli, specifically any one of GI724, BL21, DH5α, JM109, HB101, Rosetta, and Origami. The yeast cells are Pichia pastoris cells, and the animal cells are Chinese hamster ovary cells.

7. A method for preparing the recombinant human growth hormone-collagen fusion protein according to any one of claims 1-2, characterized in that, Includes the following steps: S801, culture engineered bacteria containing an expression vector encoding a fusion protein to express the fusion protein; S802, collect and break the bacterial cells to obtain a crude extract containing the fusion protein; S803, the crude extract was subjected to growth hormone affinity chromatography and ion exchange chromatography in sequence to purify and obtain the fusion protein.

8. The method for preparing recombinant human growth hormone-collagen fusion protein according to claim 7, characterized in that, The method for obtaining the crude extract containing the fusion protein in S802 is as follows: S80201, the fermentation broth of Escherichia coli expressing recombinant human growth hormone-collagen fusion protein was centrifuged at 8000 rpm and 4℃ for 40 min, the supernatant was discarded, and the bacterial precipitate was collected; S80202 involves placing the bacterial cell precipitate in an ultra-low temperature freezer at -80℃ for repeated freeze-thaw cycles to break it up, repeating the cycle three times to fully rupture the bacterial cell walls and release the target protein. S80203, according to the ratio of cell mass to resuspension volume (mL) = 1:9, add resuspension to the ruptured cells. The resuspension composition is: 50 mM Tris, 0.2 M arginine, 0.3 M NaCl, pH 7.

4. Before adding the resuspension, place the resuspension system in a 4℃ cold storage for 1 h to fully resuspend the target protein and maintain its stability. S80204: Centrifuge the resuspended solution at 8000 rpm and 4℃ for 40 min to remove cell debris and insoluble impurities. Collect the supernatant and store it at 4℃ for later purification.

9. The method for preparing recombinant human growth hormone-collagen fusion protein according to claim 7, characterized in that, The method for purifying the fusion protein from S803 is as follows: S80301, GH affinity chromatography: A GH affinity chromatography column was used. The equilibration buffer was 50 mM Tris, 0.2 M arginine, and 0.3 M NaCl at pH 7.

4. The elution buffer was 50 mM glycine at pH 3.

0. A linear gradient elution was performed from 100% equilibration buffer to 100% elution buffer, with an elution volume of 5 column volumes. The elution peak of the target protein was collected. S80302, weak cation exchange chromatography: A weak cation exchange chromatography column was used. The equilibration buffer was 20 mM Tris, 10 mM PB, pH 6.0, and the elution buffer was 20 mM Tris, 10 mM PB, pH 9.

0. A pH gradient elution was performed from 100% equilibration buffer to 60% elution buffer, with an elution volume of 20 column volumes. The elution peak of the target protein was collected to obtain the high-purity fusion protein.

10. The use of the recombinant human growth hormone-collagen fusion protein according to any one of claims 1-2, the isolated nucleic acid molecule according to claim 3, the recombinant expression vector according to claim 4, and the host cell according to claims 5-6 in the preparation of the drug, to prolong the half-life of growth hormone, for the treatment of various growth and development disorders, and to promote wound healing.

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