Recombinant long-acting human growth hormone fusion protein as well as preparation method and application thereof

Abstract

The invention provides a novel nano antibody-based recombinant long-acting human growth hormone fusion protein, nucleic acid, a pharmaceutical composition, application and the like. Existing long-acting means of PEGylation, Fc fusion and the like often cause reduction of activity, the human growth hormone is innovatively fused with an anti-albumin nano antibody with a specific sequence, and protein with larger molecules is formed through combination of the nano antibody and albumin antigen, so that the half-life period of the human growth hormone is prolonged, the activity of the human growth hormone is prolonged, and the human growth hormone can be used for preparing the anti-albumin nano antibody. In addition, the activity of the fusion protein provided by the invention is higher than that of a human growth hormone standard substance. The fusion protein and the like have wide industrial application prospects in the field of biological medicines.

Description

A recombinant long-acting human growth hormone fusion protein, its preparation method and application Technical Field

[0001] This invention relates to the fields of molecular biology and biopharmaceutical engineering. Specifically, this invention relates to a recombinant long-acting human growth hormone fusion protein based on nanobodies, its preparation method, and its uses. Background Technology

[0002] Growth hormone deficiency (GHD) is a clinical syndrome characterized by metabolic disorders and is associated with many metabolic abnormalities, including abnormal body composition, decreased physical performance, altered lipid metabolism, reduced bone mass, increased insulin resistance, and decreased quality of life. Most metabolic abnormalities associated with GHD can be reversed with recombinant human growth hormone (rh-GH) substitutes, but poor adherence due to frequent dosing can weaken efficacy. Traditional treatment for GHD involves daily subcutaneous injections of rhGH; however, this treatment method is cumbersome, requiring daily injections, which is inconvenient for many patients, raising concerns about poor treatment adherence, which may lead to reduced efficacy. Long-acting rh-GH protein molecules or long-acting growth hormone preparations can not only reduce the number of injections and improve adherence but also help improve the efficacy of GH treatment. Therefore, there is still an urgent need to develop a new and effective fusion protein for the treatment of growth hormone deficiency.

[0003] Growth hormone (GH) is a polypeptide hormone composed of 191 amino acids, playing a central role in regulating metabolism, promoting bone growth, and tissue repair. Clinically, recombinant human growth hormone (rh-GH) is widely used to treat growth hormone deficiency, chronic kidney disease, and burns. However, natural GH has an extremely short half-life (approximately 20 minutes), requiring frequent injections to maintain efficacy, leading to poor patient compliance and high treatment costs. Therefore, developing long-acting GH has become an important research direction in the biopharmaceutical field, with the core focus on extending the drug's half-life through molecular modification (such as PEGylation and fusion protein design) or novel delivery systems.

[0004] The mechanism of action of growth hormone is generally believed to be through c-AMP, which, after binding to target cells, can alter the transport of amino acids and metabolites and induce the synthesis of certain specific proteins and nucleic acids.

[0005] I. Regulation of Bone Growth

[0006] IGF-1-mediated chondrogenesis: Growth hormone stimulates the liver to secrete insulin-like growth factor-1 (IGF-1), which directly acts on epiphyseal chondrocytes, activating the PI3K / Akt and MAPK signaling pathways within the chondrocytes, promoting cell division and differentiation, and driving longitudinal growth of long bones.

[0007] Bone metabolism regulation: IGF-1 works synergistically with growth hormone to enhance osteoblast activity, accelerate the deposition of minerals such as calcium and phosphorus in bone tissue, and improve bone density and mechanical strength.

[0008] II. Regulation of Protein Metabolism

[0009] Enhanced amino acid transport: Growth hormone activates the mTOR pathway, increases the expression of cell membrane amino acid transporters (such as SNAT2), and promotes the uptake of essential amino acids such as leucine and glutamine by muscle cells.

[0010] Ribosome function activation: Hormones upregulate the transcription rate of ribosomal RNA (rRNA), enhance protein synthesis efficiency, and at the same time inhibit the activity of protein-degrading enzymes (such as the ubiquitin-proteasome system) in muscle tissue.

[0011] III. Regulation of Fat and Glucose Metabolism

[0012] Fat mobilization: Growth hormone activates hormone-sensitive lipase (HSL) in adipose tissue, catalyzing the breakdown of triglycerides into free fatty acids, promoting β-oxidation for energy, while inhibiting the absorption of glucose by adipocytes and reducing lipid storage.

[0013] Insulin antagonistic effect: Growth hormone reduces glucose uptake by muscle and adipose tissue by inhibiting phosphorylation of insulin receptor substrate (IRS), resulting in elevated blood glucose levels and maintaining energy supply in a fasting state.

[0014] IV. Immune and Regenerative Functions

[0015] Immune cell activation: Growth hormone promotes the proliferation of thymic epithelial cells, upregulates the expression of CD28 co-stimulatory molecules on the surface of T cells, and enhances the activity of cytotoxic T lymphocytes (CTLs) and antibody production capacity.

[0016] Tissue repair: By activating the epidermal growth factor receptor (EGFR) and fibroblast growth factor (FGF) pathways, it stimulates the proliferation of adult stem cells in organs such as the skin and liver, accelerating wound repair and organ regeneration.

[0017] V. Endocrine Synergistic Effect

[0018] Growth hormone forms a cascade regulatory network with thyroid hormones and sex hormones:

[0019] Synergistic effect of thyroid hormones: T3 / T4 enhances the sensitivity of target tissues to growth hormone by upregulating the expression of growth hormone receptor (GHR); Synergistic effect of sex hormones: adolescent sex hormones (such as estrogen) promote the pulsatile secretion peak of pituitary growth hormone, jointly driving the peak of growth and development.

[0020] Currently, all commercially available long-acting growth hormones in China are PEGylated growth hormones. PEGylation increases the molecular weight of proteins, making them less likely to pass through the glomeruli, reducing the clearance rate, and preventing enzymatic degradation by proteases, thereby prolonging the half-life of the drug in the body. However, the PEGylation process of proteins will result in a loss of biological activity.

[0021] Specifically regarding human growth hormone, several studies have been conducted on its fusion Fc fragment. For example, CN102875683B (announced on June 11, 2014) discloses a long-acting recombinant human growth hormone Fc fusion protein, hGH-L-vFc, containing human growth hormone, a flexible peptide linker of approximately 2-20 amino acids, and a human IgG Fc variant. This human IgG Fc variant can be selected from the hinge region, CH2, and CH3 regions of human IgG4 containing S228P and L235A mutations. However, these existing studies may have resulted in reduced activity of the fusion protein. Therefore, further research is needed to develop human growth hormone derivatives with different long-acting mechanisms, high expression levels, and higher activity. Summary of the Invention

[0022] The technical problem this invention aims to solve is to provide a novel recombinant long-acting human growth hormone fusion protein based on nanobodies. Existing methods for achieving long-acting effects, such as PEGylation and fusion with Fc, often lead to a decrease in activity. This invention innovatively fuses human growth hormone with an anti-albumin nanobody. Through the binding of the nanobody to the albumin antigen, a larger protein molecule is formed, thereby increasing the half-life of human growth hormone and prolonging its activity, thus achieving a long-acting effect. Furthermore, the fusion protein of this invention exhibits higher activity than the human growth hormone standard.

[0023] This invention innovatively solves the technical problem of how to achieve efficient expression of long-acting growth hormone in *E. coli* and the preparation of samples. Specifically, it employs a technique of screening for fusion proteins that can improve expression levels, combined with optimized extraction and purification methods, namely, the selection of specific steps and parameters. This invention also provides nucleic acid molecules encoding fusion proteins, nucleic acid constructs, expression vectors, recombinant engineered strains, pharmaceutical compositions, preparation methods, and applications of the fusion proteins, etc.

[0024] One aspect of the present invention provides a long-acting recombinant human growth hormone fusion protein, characterized in that: the fusion protein comprises, from the N-terminus to the C-terminus, a lysin, an EK enzyme cleavage site, human growth hormone, a linker peptide, and an albumin nanobody fragment.

[0025] Furthermore, the solubilizing protein is selected from one of SUMO solubilizing protein, TrxA solubilizing protein, GST solubilizing protein, NusA solubilizing protein, and MBP solubilizing protein.

[0026] Further, the amino acid sequence of the SUMO lysosome is shown in SEQ ID NO.14, the amino acid sequence of the TrxA lysosome is shown in SEQ ID NO.1, the amino acid sequence of the GST lysosome is shown in SEQ ID NO.8, the amino acid sequence of the NusA lysosome is shown in SEQ ID NO.11, the amino acid sequence of the MBP lysosome is shown in SEQ ID NO.17, the amino acid sequence of the restriction site of the EK enzyme is shown in SEQ ID NO.2, the amino acid sequence of the human growth hormone is shown in SEQ ID NO.3, the amino acid sequence of the linker peptide is shown in SEQ ID NO.4, and the amino acid sequence of the albumin nanobody fragment is shown in SEQ ID NO.5.

[0027] Further, the amino acid sequence of the fusion protein is shown in any one of SEQ ID NO.15, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, and SEQ ID NO.18.

[0028] Another aspect of the present invention provides a recombinant nucleic acid molecule characterized in that it encodes the fusion protein described in the present invention.

[0029] Furthermore, the encoding nucleic acid sequence of the fusion protein is shown in any one of SEQ ID NO.16, SEQ ID NO.7, SEQ ID NO.10, SEQ ID NO.13, or SEQ ID NO.19.

[0030] Another aspect of the present invention provides a recombinant gene expression cassette, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention.

[0031] Another aspect of the present invention provides a recombinant vector, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention, or the recombinant gene expression cassette described in the present invention.

[0032] Another aspect of the present invention provides a recombinant host cell, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention, or the recombinant gene expression cassette described in the present invention, or the recombinant vector described in the present invention.

[0033] Another aspect of the present invention provides a pharmaceutical composition characterized in that it comprises the fusion protein of the present invention, and / or the recombinant nucleic acid molecule of the present invention, and / or the recombinant gene expression cassette of the present invention, and / or the recombinant vector of the present invention, and / or the recombinant host cell of the present invention.

[0034] Another aspect of the present invention provides a method for preparing recombinant long-acting growth hormone, characterized in that it includes fermentation and purification using the host cells described in the present invention to prepare recombinant long-acting growth hormone.

[0035] Another aspect of the present invention provides a method for purifying recombinant long-acting growth hormone, characterized by comprising the following steps:

[0036] (1) Extraction of the fusion protein of the present invention: the fermentation cells containing the fusion protein are broken to obtain the supernatant;

[0037] (2) Capture of the fusion protein described in this invention;

[0038] (3) Removal and purification of tag protein: The tag protein was removed by EK enzyme and further purified by chromatography.

[0039] Another aspect of the present invention provides the use of the fusion protein of the present invention, and / or the recombinant nucleic acid molecule of the present invention, and / or the recombinant gene expression cassette of the present invention, and / or the recombinant vector of the present invention, and / or the recombinant host cell of the present invention, and / or the pharmaceutical composition of the present invention in the preparation of a medicament for treating growth hormone deficiency.

[0040] Compared with existing technologies, the recombinant long-acting human growth hormone fusion protein of the present invention, its preparation method, and its applications have the following superior technical effects:

[0041] (1) This invention provides a new recombinant long-acting human growth hormone fusion protein based on nanobodies. Unlike existing long-acting methods such as PEGylation and fusion with Fc, this invention innovatively fuses human growth hormone with anti-albumin nanobodies. Through the binding of nanobodies with albumin antigens, a larger protein molecule is formed, thereby increasing the half-life of human growth hormone, prolonging its activity, and playing a long-acting role.

[0042] (2) This invention screened four solubilizing tags most conducive to the industrial production of human growth hormone, namely GST, NusA, SUMO, and MBP, and constructed fusion proteins with human growth hormone and anti-albumin nanobodies. The fusion protein expression level, the proportion of the target product, and the content of the target product were compared and screened. Finally, SUMO was selected as the solubilizing protein tag with the highest expression level.

[0043] (3) The purity of the recombinant long-acting growth hormone GH-ALB8 obtained by the present invention using a 3-step chromatography process is above 95%.

[0044] (4) In vitro bioactivity analysis of the present invention showed that the activity level of GH-ALB8 was higher than that of the GH standard. It can be seen that even though the recombinant human growth hormone fusion protein modified in the present invention uses albumin nanobody fragments with a larger molecular weight, its activity was not lost, but rather showed higher biological activity than that of the human growth hormone standard. Attached Figure Description

[0045] Figure 1 shows the pattern of the Escherichia coli plasmid pET28a-SUMO-GH-ALB8 expressing GH-ALB8 in this invention.

[0046] Figure 2. SDS-PAGE results of engineered bacteria expressing GH-ALB8 fusion protein with different tags after single-clone induction in shake flasks;

[0047] Figure 3. SDS-PAGE results of three high-expression strains capturing target proteins in one step;

[0048] Figure 4. Growth curve of BL21 (DE3) / pET28a-SUMO-GH-ALB8 strain in fermenter;

[0049] Figure 5. SDS-PAGE of BL21 (DE3) / pET28a-SUMO-GH-ALB8 fermenter cells at different induction times;

[0050] Figure 6. Results of one-step nickel chromatography capture chromatography and SDS-PAGE of SUMO-GH-ALB8 protein;

[0051] Figure 7. Results of SUMO-GH-ALB8 protein digestion and nickel column flow-through chromatography and SDS-PAGE of the samples;

[0052] Figure 8. SDS-PAGE results of purified GH-ALB8 protein;

[0053] Figure 9. Purity of GH-ALB8 sample determined by SEC-HPLC;

[0054] Figure 10. SDS-PAGE results of hydrophobic chromatography of GH-ALB8 protein;

[0055] Figure 11. ELISA results of GH-ALB8 combined with different albumins in vitro.

[0056] Figure 12. The ability of GH-ALB8 and GH to stimulate the proliferation of rat hepatocellular carcinoma cells H4IIE. Detailed Implementation

[0057] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to the described content. The fusion proteins and encoding nucleic acids and their elements provided by the present invention, as well as the preparation methods and applications, all use commercially available raw materials and reagents. Based on conventional knowledge of molecular biology and protein engineering in the art, those skilled in the art can implement the embodiments and methods of the present invention.

[0058] Example 1: Construction of GH-ALB8 expression vector with different fusion proteins

[0059] 1. Design and construct different modified long-acting fusion protein molecules

[0060] Five recombinant long-acting human growth hormone fusion proteins based on nanobodies with different solubilization tags were constructed.

[0061] The first type is a TrxA-GH-ALB8 fusion protein, which, from the N-terminus to the C-terminus, sequentially comprises TrxA lysolytic protein, an EK enzyme cleavage site, human growth hormone, a linker peptide, and an ALB8 albumin nanobody fragment. The amino acid sequence of the TrxA lysolytic protein is shown in SEQ ID NO.1, the amino acid sequence of the EK enzyme cleavage site is shown in SEQ ID NO.2, the amino acid sequence of the human growth hormone is shown in SEQ ID NO.3, the amino acid sequence of the linker peptide is shown in SEQ ID NO.4, the amino acid sequence of the ALB8 albumin nanobody fragment is shown in SEQ ID NO.5, the amino acid sequence of the TrxA-GH-ALB8 fusion protein is shown in SEQ ID NO.6, and the encoding nucleic acid sequence of the TrxA-GH-ALB8 fusion protein is shown in SEQ ID NO.7.

[0062] The second type is a GST-GH-ALB8 fusion protein, which, from the N-terminus to the C-terminus, sequentially comprises GST lysosome protein, an EK enzyme cleavage site, human growth hormone, a linker peptide, and an ALB8 albumin nanobody fragment. The amino acid sequence of the GST lysosome protein is shown in SEQ ID NO. 8, the amino acid sequence of the EK enzyme cleavage site is shown in SEQ ID NO. 2, the amino acid sequence of the human growth hormone is shown in SEQ ID NO. 3, the amino acid sequence of the linker peptide is shown in SEQ ID NO. 4, the amino acid sequence of the ALB8 albumin nanobody fragment is shown in SEQ ID NO. 5, the amino acid sequence of the GST-GH-ALB8 fusion protein is shown in SEQ ID NO. 9, and the encoding nucleic acid sequence of the GST-GH-ALB8 fusion protein is shown in SEQ ID NO. 10.

[0063] The third type is the NusA-GH-ALB8 fusion protein, which, from the N-terminus to the C-terminus, sequentially comprises NusA lysosomal protein, an EK enzyme cleavage site, human growth hormone, a linker peptide, and an ALB8 albumin nanobody fragment. The amino acid sequence of the NusA lysosomal protein is shown in SEQ ID NO.11, the amino acid sequence of the EK enzyme cleavage site is shown in SEQ ID NO.2, the amino acid sequence of the human growth hormone is shown in SEQ ID NO.3, the amino acid sequence of the linker peptide is shown in SEQ ID NO.4, the amino acid sequence of the ALB8 albumin nanobody fragment is shown in SEQ ID NO.5, the amino acid sequence of the NusA-GH-ALB8 fusion protein is shown in SEQ ID NO.12, and the encoding nucleic acid sequence of the NusA-GH-ALB8 fusion protein is shown in SEQ ID NO.13.

[0064] The fourth type is a SUMO-GH-ALB8 fusion protein, which, from the N-terminus to the C-terminus, sequentially comprises SUMO lysosomal protein, an EK enzyme cleavage site, human growth hormone, a linker peptide, and an ALB8 albumin nanobody fragment. The amino acid sequence of the SUMO lysosomal protein is shown in SEQ ID NO.14, the amino acid sequence of the EK enzyme cleavage site is shown in SEQ ID NO.2, the amino acid sequence of the human growth hormone is shown in SEQ ID NO.3, the amino acid sequence of the linker peptide is shown in SEQ ID NO.4, the amino acid sequence of the ALB8 albumin nanobody fragment is shown in SEQ ID NO.5, the amino acid sequence of the SUMO-GH-ALB8 fusion protein is shown in SEQ ID NO.15, and the encoding nucleic acid sequence of the SUMO-GH-ALB8 fusion protein is shown in SEQ ID NO.16.

[0065] The fifth type is an MBP-GH-ALB8 fusion protein, which, from the N-terminus to the C-terminus, sequentially comprises MBP lysosomal protein, an EK enzyme cleavage site, human growth hormone, a linker peptide, and an ALB8 albumin nanobody fragment. The amino acid sequence of the MBP lysosomal protein is shown in SEQ ID NO.17, the amino acid sequence of the EK enzyme cleavage site is shown in SEQ ID NO.2, the amino acid sequence of the human growth hormone is shown in SEQ ID NO.3, the amino acid sequence of the linker peptide is shown in SEQ ID NO.4, the amino acid sequence of the ALB8 albumin nanobody fragment is shown in SEQ ID NO.5, the amino acid sequence of the MBP-GH-ALB8 fusion protein is shown in SEQ ID NO.18, and the encoding nucleic acid sequence of the MBP-GH-ALB8 fusion protein is shown in SEQ ID NO.19.

[0066] Table 1. Elements and fusion protein sequences used in this invention.

[0067] surface

[0068] The corresponding gene sequence was synthesized by a gene synthesis company, and Nco I and Xho I restriction sites were introduced at both ends. These sites were then inserted into the pET28a plasmid vector via restriction enzyme ligation. For example, the map of the pET28a-SUMO-GH-ALB8 plasmid expressing the E. coli growth hormone and ALB8 albumin nanobody fragment fusion protein (GH-ALB8) is shown in Figure 1.

[0069] 2. Transform the recombinant plasmid constructed in step 1 into the host cell.

[0070] Take an appropriate amount of recombinant plasmid and gently mix it with E. coli C41(DE3) competent cells, then incubate on ice for 30 min, heat shock at 42℃ for 30 seconds, quickly place on ice for 2 min, add SOC medium and incubate at 37℃ and 220 rpm for 1 hour. Take an appropriate amount of the revived bacterial solution and spread it on LB agar plates containing 50 µg / ml Kan, and incubate at 37℃ overnight until obvious colonies grow.

[0071] 3. Amplification culture and induced expression

[0072] The components and proportions of the culture medium are as follows: LB medium (10 g / L yeast peptone, 5 g / L yeast extract, 10 g / L sodium chloride), TB medium (12 g / L yeast peptone, 24 g / L yeast extract, 2.3 g / L potassium dihydrogen phosphate, 12.5 g / L potassium dihydrogen phosphate, 4 ml / L glycerol).

[0073] Amplification culture: Single clones of the strain were picked from the transformation plates and inoculated into LB medium, and cultured at 37°C and 220 rpm with shaking until OD reached. 600 When the concentration reaches approximately 2.0, add sterile glycerol solution to mix and form a bacterial suspension with a final glycerol concentration of 20%. Dispense the suspension into sterile cryovials and store at -80°C.

[0074] Induction of expression: The above-mentioned glycerol bacteria were inoculated at a ratio of 0.1% into LB liquid medium and cultured at 37℃ and 220 rpm for 6-7 h with shaking until OD was reached. 600 When the OD reaches approximately 1.0, inoculate it into TB liquid medium at a ratio of 2-3% and incubate at 37°C and 220 rpm for approximately 2-3 hours until the OD reaches 1.0. 600 When the concentration reaches approximately 1.0, IPTG at a final concentration of 0.5 mM is added for induction, and the culture is continued overnight (approximately 16 h) with shaking at 25°C and 220 rpm. After induction, samples are taken to measure the OD of the culture. 600The culture was collected, and the bacterial cells were collected by centrifugation at 12000 rpm for 10 min. OD200 was induced. 600 The appropriate concentrations can be 0.5-2, IPTG concentrations can be 0.2 mM-1 mM, temperature can be 20℃-30℃, and induction time can be 5 h-16 h. The results are shown in Figure 2. Figure 2 shows the SDS-PAGE results of engineered bacteria expressing GH-ALB8 fusion protein in shake flasks after single-clone induction. The GST, NusA, and SUMO tags show clear target expression bands.

[0075] 0.5 g of bacterial cells containing GST, NusA, and SUMO lysin tags were captured. The captured proteins and enzyme digested proteins are shown in Figure 3, and the content of captured proteins is shown in Table 2. Among them, SUMO-GH-ALB8 had the highest expression level, and SUMO was the preferred lysin tag.

[0076] Table 2. One-step capture yield of 0.5 g cells of high-expression strains

[0077] Fusion Protein Name | Fusion Protein Expression Level (mg / g) | Percentage of Target Product (%) | Content of Target Product (mg / g) SUMO-GH-ALB | 85.52 | 47% | 2.594 | GST-GH-ALB | 82.88 | 65% | 1.872 | NusA-GH-ALB | 82.00 | 28% | 0.56 surface

[0078] Example 2: SUMO-GH-ALB8 cell fermentation in a fermenter

[0079] Place the sterile seed culture medium in a biosafety cabinet and irradiate with UV light for 30 min. Take the constructed glycerol bacterium BL21(DE3) / pET28a-SUMO-GH-ALB8. After the bacterial culture thaws, inoculate the bacterial culture into the seed culture medium at a rate of 0.10% in the biosafety cabinet, add 0.1% of 50 mg / ml kanamycin sulfate stock solution, and incubate on a constant temperature shaker at 25-37℃ and 220-250 r / min for 12-16 h. OD 600 Once the concentration reaches 4-8, the resulting liquid is harvested and ready for use.

[0080] For high-density fermentation (7 L), 3 L of prepared basal culture medium was added to the fermenter. The sterilization program was selected, and sterilization was performed at 121°C for 30 min. Before sterilization, the pH electrode was calibrated using standard buffer solutions of pH 4.01 and pH 6.86. The sterilization program was then selected, and sterilization was performed at 121°C for 90 min. After sterilization, the temperature was set to 37°C, and the aeration rate and rotation speed were set to 3 L / min and 200 rpm, respectively. The dissolved oxygen (DO) value was calibrated after the DO value stabilized. Ammonia was added to the alkali bottle inside the biosafety cabinet. The pH was set to 7.0, and automatic pH control was activated. Once pH 7.0 was reached, a sample was taken and measured with a pH meter. The pH electrode was calibrated based on the actual pH meter reading. After the value stabilized, a sample was taken and verified with a pH meter. Trace elements were added to the fermenter at a ratio of 20 ml / L. Kanamycin sulfate stock solution was added according to the experimental protocol. The prepared seed culture was then added to the fermenter at a ratio of 10% (300 ml). pH control: During fermentation, the acid-base pump is set to automatic control, maintaining the pH at approximately 7.0±0.1 until fermentation is complete. Temperature control: Temperature is automatically controlled. The temperature before induction is 37±1℃, and the temperature after induction is set according to the experimental protocol. Dissolved oxygen control: Dissolved oxygen is set at 30±10%. If dissolved oxygen is below 20%, the turbine speed is increased first, then pure oxygen is added to maintain dissolved oxygen at 30±10%. If dissolved oxygen is above 40%, pure oxygen is decreased first, then the turbine speed is decreased to maintain dissolved oxygen at 30±10%. Induction: Induction starting point OD. 600 The experimental protocol was followed, with a final IPTG concentration of 1 mM as the inducer. Induction was initiated until the stabilization period before the culture was transferred to the fermentation tank. Feeding was initiated when dissolved oxygen rapidly increased: 0-1 h, feed rate 22.4 ml / L / h; 1-2 h, feed rate 28.0 ml / L / h; 2-3 h, feed rate 33.6 ml / L / h; 3 h to induction, feed rate 39.2 ml / L / h; induction to culture, feed rate 22.4 ml / L / h. The feed rate was calculated based on the initial fermentation broth volume. OD was measured every 4 h. 600 Parameters were recorded every 4 hours, and the growth curve is shown in Figure 4. The SDS-PAGE spectra of bacterial expression levels at different time points are shown in Figure 5, and the bacterial expression levels are shown in Table 3. After fermentation, the target protein expression level was 1.49 g / L, and the bacterial cells were collected. The bacterial culture was centrifuged using a floor centrifuge. The fermentation broth was added to a centrifuge cup, balanced, and centrifuged. Centrifugation parameters: 8000 rpm (15189×g), 15 min, 4℃. After centrifugation, the supernatant was discarded, the precipitate was scraped into a sealed bag, and the net weight of the bacterial cells after centrifugation was recorded. The cells were stored at -20℃.

[0081] Table 3. Expression levels in fermenters over induction time

[0082] Induction duration and target protein expression level (g / L): 0 h 0.004 h 0.728 h 0.9112 h 1.49 surface

[0083] Example 3: Preparation of GH-ALB8 purified sample

[0084] a. Protein extraction: The bacterial cells collected in step 4 were resuspended in Lysis Buffer (20 mM phosphate buffer + 300 mM sodium chloride + 20 mM imidazole) at a ratio of 1:10 (w / v). The cells were then homogenized three times at 700-800 bar using a high-pressure homogenizer. The lysate was then centrifuged at 15000 g for 60 min at 4°C to collect the supernatant.

[0085] b. Protein Capture: The SUMO-GH-ALB8 fusion protein in the supernatant was captured using an AKTA pure150 protein purifier and a Nanomicro NW Rose Ni FF chromatography column. The standard procedure was as follows: the chromatography column was washed with 5 column volumes of purified water, then equilibrated with 5 column volumes of Lysis Buffer. The fragmented supernatant was then loaded at a certain flow rate to ensure that the fusion protein was fully incorporated into the chromatography column. After loading, the chromatography column was reequilibrated with 20 column volumes of Lysis Buffer. The SUMO-GH-ALB8 fusion protein was then linearly eluted with Elution Buffer (20 mM phosphate buffer + 300 mM sodium chloride + 300 mM imidazole). The results were obtained by standard SDS-PAGE assay, as shown in Figure 6, with a clear target band at approximately 50 kDa.

[0086] Removal and purification of tagged proteins:

[0087] Enzymatic digestion was performed using the Novizan EK enzyme to cleave the EK cleavage site between SUMO and GH-ALB8, yielding GH-ALB8, as shown in Figure 7. The digested sample was then purified again using an NW Rose Ni FF chromatography column. After loading, the flow-through fraction was GH-ALB8, while the SUMO portion of the cleaved fusion protein bound to the column. As shown in Figure 7, the flow-through fraction was 30 kDa recombinant GH-ALB8, while the eluted fraction consisted mainly of miscellaneous proteins, primarily the 20 kDa SUMO-tagged protein.

[0088] The flow-through fraction of recombinant GH-ALB8 was purified by nano-micro UniGel-30 DEAE chromatography as follows: the column was washed with 5 column volumes of purified water, and then equilibrated with 5 column volumes of DEAE equilibration buffer (20 mM Tris, pH 8.0). The flow-through fraction of recombinant GH-ALB8 was then loaded at a certain flow rate to ensure sufficient binding of impurities to the chromatographic material. The flow-through fraction after loading was GH-ALB8 with a purity higher than 95%, while other impurities remained bound to the column. The results are shown in Figure 8, where the flow-through fraction was 30 kDa recombinant GH-ALB8, and the eluted fraction contained impurities. The results, as shown in Figure 9, indicate that the SEC-HPLC purity of the obtained recombinant long-acting growth hormone GH-ALB8 was 97.09%.

[0089] The recombinant GH-ALB8 sample flow-through was purified by nano-micro Uni HR Butyl–30L chromatography as follows: The column was washed with 5 column volumes of purified water, then equilibrated with 5 column volumes of hydrophobic equilibration buffer (0.5 M (NH4)2SO4-10 mM Tris-HCl-mM EDTA pH 7.2) for 5 CV. Sample loading: 20 ml of the obtained sample solution was loaded at a flow rate of 120 cm / h. Post-equilibration: Post-equilibration was performed using equilibration buffer at a flow rate of 120 cm / h for 5 CV until the baseline stabilized. Elution: Linear elution with 0-100% B for 20 CV, followed by washing with 100% B (10 mM Tris-HCl-mM EDTA pH 7.2) for 5 CV. The eluted protein SDS-PAGE is shown in Figure 10, with a purity of approximately 80%, lower than that of the DEAE-mediated flow-through sample. Subsequent samples were processed using the DEAE flow-through method.

[0090] Example 4: Detection of GH-ALB8 binding albumin activity

[0091] To detect the binding ability of long-acting growth hormone (GH-ALB8) to serum albumin and accumulate data for further pharmacokinetic studies, the binding activity was detected using Elisi. 1. Coating: The 10X coating buffer was diluted 10-fold with ultrapure water to obtain the 1X coating buffer. Recombinant human serum albumin (HSA), monkey serum albumin, and bovine serum albumin (BSA) were diluted to 5 μg / ml using the 1X coating buffer. 100 μl of the solution was added to each well of the ELISA plate and incubated at 37°C for 1 hour. 2. Blocking: 950 ml of ultrapure water was mixed with 50 ml of 20X PBS and 1 ml of Tween 20 to prepare the PBST washing buffer. 5% BSA was prepared using PBST. The ELISA plate was washed three times with PBST, and 150 μl of 5% BSA was added to each well. The plate was incubated at 37°C for 1 hour. 3. Sample Addition: Concentrate the long-acting growth hormone sample GH-ALB8 5-fold to 0.75 mg / ml using a 3K ultrafiltration tube. Perform a series of serial dilutions using 5% BSA. Add 100 μl of serially diluted GH-ALB8 sample to each microplate coated with each protein. Incubate at 37°C and 300 rpm for 1 hour. 4. Primary Antibody: Wash the plate 3 times with PBST, adding 200 μl per well each time. Dilute rabbit anti-human growth hormone polyclonal antibody (primary antibody) 2000-fold with 5% BSA and add 100 μl per well; incubate at 37°C and 300 rpm for 1 hour. 5. Secondary Antibody: Wash the plate 3 times with PBST, adding 200 μl per well each time. Dilute HRP-labeled goat anti-rabbit IgG (secondary antibody) 2000-fold with 5% BSA and add 100 μl per well; incubate at 37°C and 300 rpm for 1 hour. 6. Color Development: Take the required volume of TMB solution and incubate at room temperature for at least 20 minutes. Wash the ELISA plate 6 times with PBST, adding 200 μl / well each time. Add 100 μl of TMB solution to each well and incubate at 25°C with a shaker for 5-15 minutes. Determine the termination time based on the color development intensity; add 50 μl of 5% sulfuric acid to each well to terminate the reaction. 7. Reading: Detect the absorbance using an ELISA reader at wavelengths of 450 nm / 630 nm, and calculate OD450-OD630. 8. Result Calculation: Perform a four-parameter curve fitting between sample concentration and OD value. The ELISA plate was coated with human serum albumin and monkey serum albumin, and detection was performed using rabbit anti-human growth hormone antibody. The results are shown in Figure 11. The long-acting growth hormone GH-ALB8 sample can bind to both human serum albumin and monkey serum albumin, and the color development OD decreases with gradient dilution of the sample, exhibiting an S-shaped curve. It does not bind to bovine serum albumin (BSA).

[0092] The parameters of the four-parameter fitting equation are shown in Table 4. The calculated EC50 of GH-ALB8 with human serum albumin is 246.5 ng / ml, and with monkey serum albumin is 323.0 ng / ml. This indicates that the binding capacity of long-acting growth hormone GH-ALB8 to both human and monkey serum albumin is on the same order of magnitude. The results demonstrate that the long-acting growth hormone GH-ALB8 of this invention can bind efficiently to albumin, even at low concentrations, to obtain larger protein molecules, thereby increasing the half-life of human growth hormone, prolonging its activity, and achieving a long-acting effect.

[0093] Table 4. Fitting equations for the binding of GH-ALB8 to monkey serum albumin and human albumin.

[0094] R2ABCDHSA0.9930.5041.300246.53.476MSA0.9900.2490.965323.03.538 surface

[0095] Example 5 In vitro bioactivity analysis

[0096] In vitro bioactivity can be determined by stimulating rat hepatocellular carcinoma cells H4IIE (highly expressing functional GHR, a classic GH activity detection model) with experimental samples (GH standard (national standard material, recombinant human growth hormone, batch number 140635-202407) and GH-ALB8) to assess their proliferation. H4IIE cells were resuscitated and cultured in complete culture medium in a CO2 incubator until the logarithmic growth phase (70%-80% confluence). Cells were digested with trypsin, collected by centrifugation, resuspended in complete culture medium, and the cell concentration was adjusted to 5 × 10⁶ cells / year. 4Cells / mL. Using complete culture medium as a diluent, GH standard and GH-ALB8 samples were diluted to gradient concentrations: 0.1, 0.2, 0.5, 0.8, 1.0, 2.0, 4.0, 5.0, 7.5, 10.0, 20.0, 50.0, and 100.0 ng / mL. 100 μL of cell suspension (approximately 5 × 10³ cells / well) was added to each well of a 96-well plate and incubated for 24 h to allow cell adhesion. The old culture medium was discarded. 100 μL of culture medium was added to the blank control group, and 100 μL of the corresponding concentration of standard / sample diluent was added to the remaining wells. Six replicates were made for each group (to minimize error). Incubation continued for 48 h (the optimal time window for GH-induced cell proliferation). 10 μL of CCK-8 reagent was added to each well, gently vortexed to mix, and incubated for 2 h. The absorbance (OD value) of each well was measured using a microplate reader at a wavelength of 450 nm, and the data was recorded. The net OD value was calculated as: OD value of each group - average OD value of the blank control group. The proliferation rate was calculated as: (Net OD value of sample group / Net OD value of negative control group) × 100%. The sample concentration (log) was used as the basis for calculation. 10 The values ​​were converted to x-axis and proliferation rate to y-axis, and the fitted S-shaped dose-response curve (four-parameter logistic model) is shown in Figure 12. The EC50 of GH standard was (11.14 nM) and that of GH-ALB8 was (9.28 nM), indicating that the activity level of GH-ALB8 was higher than that of GH standard. This experiment shows that even though the recombinant human growth hormone fusion protein modified in this invention uses albumin nanobody fragments with larger molecular weights, its activity was not only not lost, but it also showed higher biological activity than the human growth hormone standard.

[0097] The embodiments described above are merely examples for clearly illustrating the present disclosure and are not intended to limit the implementation of the present disclosure. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this disclosure.

Claims

1. A long-acting recombinant human growth hormone fusion protein, characterized in that: The fusion protein comprises, from N-terminus to C-terminus, a lysin, an EK enzyme cleavage site, human growth hormone, a linker peptide, and an albumin nanobody fragment.

2. The fusion protein according to claim 1, characterized in that, The lysosomal protein is selected from one of SUMO lysosomal protein, TrxA lysosomal protein, GST lysosomal protein, NusA lysosomal protein, and MBP lysosomal protein.

3. The fusion protein according to claim 1 or 2, characterized in that, The amino acid sequence of the SUMO lysosome is shown in SEQ ID NO.14, the amino acid sequence of the TrxA lysosome is shown in SEQ ID NO.1, the amino acid sequence of the GST lysosome is shown in SEQ ID NO.8, the amino acid sequence of the NusA lysosome is shown in SEQ ID NO.11, the amino acid sequence of the MBP lysosome is shown in SEQ ID NO.17, the amino acid sequence of the EK enzyme cleavage site is shown in SEQ ID NO.2, the amino acid sequence of the human growth hormone is shown in SEQ ID NO.3, the amino acid sequence of the linker peptide is shown in SEQ ID NO.4, and the amino acid sequence of the albumin nanobody fragment is shown in SEQ ID NO.

5.

4. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the fusion protein is shown in any one of SEQ ID NO.15, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, or SEQ ID NO.

18.

5. A recombinant nucleic acid molecule, characterized in that, The fusion protein is encoded according to any one of claims 1-4; preferably, the nucleic acid sequence encoding the fusion protein is shown in any one of SEQ ID NO.16, SEQ ID NO.7, SEQ ID NO.10, SEQ ID NO.13, and SEQ ID NO.

19.

6. A recombinant gene expression cassette, characterized in that, It includes the recombinant nucleic acid molecule as described in claim 5.

7. A recombinant vector, characterized in that, It comprises the recombinant nucleic acid molecule of claim 5 or the recombinant gene expression cassette of claim 6.

8. A recombinant host cell, characterized in that, It comprises the recombinant nucleic acid molecule of claim 5, the recombinant gene expression cassette of claim 6, or the recombinant vector of claim 7.

9. A pharmaceutical composition, characterized in that, It comprises the fusion protein of any one of claims 1-4, and / or the recombinant nucleic acid molecule of claim 5, and / or the recombinant gene expression cassette of claim 6, and / or the recombinant vector of claim 7, and / or the recombinant host cell of claim 8.

10. A method for preparing recombinant long-acting growth hormone, characterized in that, This includes using the host cell described in claim 8 for fermentation and purification to prepare recombinant long-acting growth hormone.

11. A method for purifying recombinant long-acting growth hormone, characterized in that, The method includes the following steps: (1) extraction of the fusion protein according to any one of claims 1-4: breaking the fermentation cells containing the fusion protein to obtain a supernatant; (2) capture of the fusion protein according to any one of claims 1-4; (3) removal and purification of the tag protein: removing the tag protein using EK enzyme and further purifying it by chromatography.

12. Use of the fusion protein of any one of claims 1-4, and / or the recombinant nucleic acid molecule of claim 5, and / or the recombinant gene expression cassette of claim 6, and / or the recombinant vector of claim 7, and / or the recombinant host cell of claim 8, and / or the pharmaceutical composition of claim 9 in the preparation of a medicament for treating growth hormone deficiency.

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