A collagen short peptide mutant fusion protein and application thereof in preparation of lactoferricin

Abstract

The application discloses a collagen short peptide mutant fusion protein and application thereof in preparation of lactoferrin peptides, and the amino acid sequence of the collagen short peptide mutant fusion protein is shown as SEQ ID NO:3. The application can better guide the expression of bovine lactoferrin peptides by modifying the collagen short peptide as a carrier protein, increase the expression amount, and improve the expression amount by 10-15 times. Moreover, the collagen short peptide and the connected acidolysis peptide can be degraded into amino acids by acid in the subsequent acidolysis process, and the lactoferrin peptides can be released. The lactoferrin peptides can be used in the fields of medicine, food and feed and the like.

Description

(I) Technical Field

[0001] This invention relates to a collagen short peptide mutant fusion protein and its application in the preparation of lactoferrin peptide. (II) Background Technology

[0002] Lactoferrin (LF) was first isolated from bovine milk by Groves in 1960. Because lactoferrin binds to iron to form a red complex, it was initially called "red protein." Lactoferrin is a glycoprotein found in multiple glands of mammals, such as milk, tears, saliva, and other exocrine fluids, as well as in neutrophils and plasma. Studies have shown that it is most abundant in colostrum.

[0003] Bovine lactoferrin is broken down in the digestive tract into smaller polypeptides composed of 25 amino acid residues arranged in sequence. These polypeptides are amino acid residues 17-41 of bovine lactoferrin. These active peptides have an isoelectric point (PI) > 12, exhibiting strong cationicity. The cysteine ​​residues at positions 19 and 36 form intramolecular disulfide bonds, thus giving natural lactoferrin peptides an α-helix structure. These properties endow bovine lactoferrin with antibacterial, bactericidal, and cell-protective effects.

[0004] Currently reported, the secretion yield of lactoferrin peptide is low. Isui and Jose et al. successfully cloned the cDNA gene of bovine lactoferrin peptide (LfcinB), formed a fusion protein with thioredoxin, constructed the pET32-bLf recombinant expression vector, and expressed it in *E. coli*. After nickel column purification, the yield was 15.3 mg / L. Zigang Tian et al. designed and synthesized an LfcinB-derived peptide multimer sequence, inserted it into the expression vector pET32a, and induced expression with IPTG, achieving a maximum expression level of 10 mg / L. Liu Jingying et al. fused bovine lactoferrin peptide with Cry60Ba crystal protein in *Bacillus thuringiensis* for expression as inclusion bodies. Hydrolysis products were obtained by processing the inclusion bodies, but they were unable to isolate and purify the lactoferrin peptide, and whether expression in inclusion body form affects lactoferrin activity is unknown. Yi Junbo et al. cloned the LfcinB gene fragment into a secretory expression vector. In pPIC9K, a recombinant plasmid was obtained, linearized and electroporated into Pichia pastoris. After methanol induction, LfcinB was expressed in yeast, but it needed to be concentrated 10-fold to show a clear band on SDS-PAGE gel. Liu Chun et al. fused lactoferrin peptide and collagen in Pichia pastoris for expression, but the large molecular weight of collagen in this fusion method resulted in lactoferrin peptide accounting for only 6.5% of the total molecular weight of the fusion protein. At the same time, the lack of a means to prepare lactoferrin peptide from the fusion protein meant that the fusion protein could only be used as a whole, which reduced the value of lactoferrin peptide.

[0005] Collagen (COL) is a biological macromolecule widely found in animal connective tissues. It possesses a highly repeating triple helix structure, exhibits good acid degradation properties, and shows high expression levels in Pichia pastoris. Werten et al. expressed recombinant gelatin (a product of partial hydrolysis of collagen) in Pichia pastoris, achieving single-copy expression levels of 3-6 g / L. In summary, using collagen as a carrier protein to enhance lactoferrin peptide expression is feasible. However, collagen itself has a large molecular weight, resulting in a significant proportion in the fusion protein and relatively low lactoferrin peptide yield. Furthermore, the numerous repeating structures increase the difficulty of subsequent acid hydrolysis for lactoferrin peptide preparation. Therefore, the rational selection of collagen peptides is crucial for fusion protein expression, requiring the following three criteria: 1. Low molecular weight while retaining high collagen expression capacity; 2. Good hydrophilicity; 3. Ability to neutralize the strong cationicity of lactoferrin peptides.

[0006] This invention preferably targets a short peptide composed of 178 amino acids (amino acids 608-785) in collagen. Following the characteristic repeating structure rules of collagen and the amino acid preference of Pichia pastoris, hydrophobic amino acids are mutated to hydrophilic amino acids, while simultaneously lowering the isoelectric point as much as possible. Specifically, isoleucine at position 614 is mutated to aspartic acid, leucine at position 633 to glycine, isoleucine at position 636 to proline, phenylalanine at position 658 to glycine, valine at position 706 to proline, and phenylalanine at position 721 to glutamic acid to improve the hydrophilicity of the collagen short peptide. After mutation, the hydrophilicity is -1.09 and the isoelectric point is 4.83. The fusion protein has a hydrophilicity of -1.05 and an isoelectric point of 9.78, significantly reducing the strong cationicity of the lactoferrin peptide during expression. This invention uses Pichia pastoris GS115 as a host to construct a recombinant Pichia pastoris strain that secretes and expresses a preferred collagen short peptide-acid hydrolysate-bovine lactoferrin peptide-6HIS fusion protein, and provides a method for preparing lactoferrin peptide from the fusion protein. (III) Summary of the Invention

[0007] The purpose of this invention is to provide a collagen short peptide mutant fusion protein and its application in the preparation of lactoferrin peptide, which solves the problems of low yield and difficult purification of bovine lactoferrin peptide in the prior art.

[0008] The technical solution adopted in this invention is:

[0009] This invention provides a collagen short peptide mutant fusion protein (HlfcinB), which uses a collagen short peptide mutant as a carrier protein, with an acid-hydrolyzed peptide linked to its C-terminus, a lactoferrin peptide linked to the C-terminus of the acid-hydrolyzed peptide, and a 6HIS linked to the C-terminus of the lactoferrin peptide, thus constructing a novel fusion protein for lactoferrin peptide expression. The amino acid sequence of the collagen short peptide mutant is shown in SEQ ID NO:1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO:2. The acid-hydrolyzed peptide is aspartic acid-proline-glutamic acid-tryptophan, wherein the peptide bond between aspartic acid and proline can be cleaved under acidic conditions, and glutamic acid and tryptophan play a protective role. The nucleotide sequence encoding the acid-hydrolyzed peptide is GACCCTGAGTGG, and its amino acid sequence is DPEW.

[0010] The lactoferrin peptide (LfcinB) sequence is a small active peptide with antibacterial function composed of 25 amino acid residues of full-length bovine lactoferrin (NCBI sequence number: NM_180998.2, derived from Bos taurus) after being broken down in the digestive tract. The nucleotide sequence encoding bovine lactoferrin peptide (LfcinB) is shown in SEQ ID NO:5, and its amino acid sequence is shown in SEQ ID NO:6.

[0011] The 6HIS is a histidine tag with the structure HIS-HIS-HIS-HIS-HIS-HIS, which can be used for nickel column purification of fusion proteins. Its nucleotide sequence is CACCATCATCACCACCAT, and its amino acid sequence is HHHHHH.

[0012] Preferably, the amino acid sequence of the collagen short peptide mutant fusion protein is shown in SEQ ID NO:3.

[0013] The present invention also provides a gene encoding the collagen short peptide mutant fusion protein, the nucleotide sequence of which is shown in SEQ ID NO:4.

[0014] The present invention also provides a recombinant plasmid containing the gene encoding the collagen short peptide mutant fusion protein, and a recombinant genetically engineered bacterium constructed with the recombinant plasmid; the recombinant plasmid is based on the pPIC9K plasmid as the vector, and the EAEA residue at the C-terminus of the α signal peptide of the pPIC9K plasmid is mutated to a methionine residue to improve the stability of the expressed protein.

[0015] The recombinant plasmid was constructed as follows: (1) The nucleotide sequence of the collagen short peptide mutant fusion protein was digested with EcoRI and NotRI and then ligated into the pPIC3.5K plasmid to construct pPIC3.5K-HlfcinB;

[0016] (2) Using pPIC9K plasmid as a template, the pPIC9K vector fragment with the α signal peptide EAEA residue mutated to methionine residue was amplified using the following primers to obtain the pPIC9K vector fragment.

[0017] pPIC9K-F:GGGGTATCTCTCGAGAAAAGAATG

[0018] pPIC9K-R:CATTCTTTTCTCGAGAGATACCCCT

[0019] The HlfcinB fragment was amplified using the following primers with pPIC3.5K-HlfcinB as a template;

[0020] HlfcinB-F:AAGGGGTATCTCTCGAGAAAAGAATGGGTCCAACTGGTCCTGACGGTCC

[0021] HlfcinB-R:CTAGGGAATTCTACGTATTAATGGTGGTGATGATGGTGGAAAGCTCTTCTAACACAAGTAATAG

[0022] (3) The pPIC9K vector fragment and HLfcinB fragment described in step (2) were seamlessly ligated using a one-step cloning kit. The resulting product was transformed into Escherichia coli DH5α competent cells and cultured on plates containing ampicillin and kanamycin. Positive clones were picked and plasmids were extracted to obtain a recombinant plasmid containing the gene encoding the collagen short peptide mutant fusion protein.

[0023] This invention also relates to a recombinant genetically engineered bacterium containing the gene encoding the collagen short peptide mutant fusion protein. The engineered bacterium uses Pichia pastoris strain GS115 as the host bacterium. The engineered bacterium is obtained by linearizing the recombinant plasmid containing the gene encoding the fusion protein with the restriction endonuclease SacⅠ, then transforming it into competent cells of Pichia pastoris GS115 by electroporation, screening for positive transformants, and obtaining the recombinant genetically engineered bacterium.

[0024] This invention provides an application of the collagen short peptide mutant fusion protein in the preparation of lactoferrin peptides. The method of application is as follows: the fermentation broth obtained by fermentation culture of recombinant genetically engineered bacteria containing the encoding gene of the collagen short peptide mutant fusion protein is centrifuged, the supernatant is collected to extract pure enzyme, and the supernatant is freeze-dried to obtain lyophilized fusion protein powder; the lyophilized fusion protein powder is dissolved in 100-200mM HCl aqueous solution, reacted in a metal bath at 80℃ for 8-12h, and the pH is adjusted to 7.0 using 1M NaOH to obtain lactoferrin peptide solution.

[0025] The fermentation broth was prepared according to the following method:

[0026] The recombinant genetically engineered bacteria were streaked onto YPD solid plates containing 100 μg / mL G418 resistance, and incubated at 30°C for 3 days. Single colonies were selected and inoculated into MD medium (30 mL / 250 mL, 50 mL / 500 mL), and cultured at 30°C and 200 rpm for 16-18 h to serve as the seed culture for fermentation.

[0027] The seed culture was inoculated at a volume concentration of 10% into a 5L fermenter containing 3L of BSM medium. The pH was adjusted to 5.0, the temperature was set at 30℃, the initial agitation speed was set to 500 rpm, and the dissolved oxygen was controlled to be above 20%. After the glycerol in the medium was consumed, the dissolved oxygen rose rapidly (DO > 60%), and then the glycerol solution was fed in continuously until the cell volume weight (WCW) reached 150 g / L. After the feeding was completed, the cell was starved for 30 min, and then methanol solution was fed in continuously for induction, with the specific growth rate controlled at 0.015 h⁻¹. -1 The fermentation broth was obtained by adjusting the rotation speed and aeration rate to control dissolved oxygen to be greater than 20%. The glycerol solution was a 50% glycerol aqueous solution with 12 mL / L of trace element (PTM1) added. The methanol solution was anhydrous methanol with 12 mL / L of trace element (PTM1) added.

[0028] BSM medium: 85% H3PO4 26.7mL / L, KOH 4.13g / L, K2SO4 18.2g / L, CaSO4 0.93g / L, MgSO4·7H2O 14.9g / L, glycerol 40.0g / L, trace element (PTM1) 4.35mL / L, solvent is water, pH 5.0-5.5; prepared as follows: 85% H3PO4 26.7mL, KOH 4.13g, K2SO4 18.2g, CaSO4 0.93g, MgSO4·7H2O 14.9g, glycerol 40.0g. Before inoculation, adjust the pH to 5.0-5.5 with ammonia water and add PTM1 4.35mL / L.

[0029] Trace element PTM1: H3BO3 0.02g / L, CuSO4·5H2O 6.0g / L, MnSO4·H2O 3.0g / L, Na2MoO4·2H2O 0.2g / L, CoCl2 0.5g / L, NaI 0.08g / L, ZnCl2 20.0g / L, FeSO4·7H2O 65.0g / L, Biotin 0.2g / L, 5.0mL / L H2SO4, solvent ddH2O, filtered for sterilization, stored at 4℃ protected from light.

[0030] Preferably, the fermentation is divided into four stages, namely, a batch fermentation stage, a fed-batch stage, a starvation stage, and a methanol-induced stage:

[0031] 1) Batch fermentation stage

[0032] The seed culture was inoculated into a 5L fermenter containing 3L of fermentation medium at a volume concentration of 10%. The pH was adjusted to 5.0 with ammonia, the temperature was set at 30℃, the initial rotation speed was set at 500 rpm, and the aeration rate was controlled to ensure dissolved oxygen (DO) above 20% until the glycerol in the medium was depleted.

[0033] 2) Feeding stage

[0034] When the glycerol in the culture medium was depleted, the dissolved oxygen rose rapidly (DO > 60%). Then, the glycerol solution was fed in at a rate of 6.2 mL / h / L for 5 hours. The flow rate was then adjusted to 12 mL / h / L until the WCW of the yeast cells reached 150 g / L.

[0035] 3) Starvation stage

[0036] When the carbon source (glycerol) in the fermenter is exhausted, the fermenting cells will no longer consume a lot of oxygen due to the lack of nutrient source. At this time, the dissolved oxygen in the fermenter will rise rapidly. Wait for 30 minutes until the metabolic pathway of glycerol as a substrate in the cells is completely finished before methanol induction.

[0037] 4) Methanol induction stage

[0038] After the starvation phase, methanol solution was fed in to induce acclimatization. Initially, this was the methanol adaptation phase, with the feed rate controlled at 1.1 mL / h / L. After adaptation (the dissolved oxygen level changed sequentially after methanol feeding began: high dissolved oxygen (low methanol utilization and gradual accumulation), gradually decreasing to a low level (gradual adaptation and methanol utilization), and then rising to a relatively high level with fluctuations (accumulated methanol was consumed, and new methanol was consumed immediately upon replenishment), indicating that the Pichia pastoris had completed methanol adaptation), the methanol feed rate was appropriately increased to 3.6 mL / h / L. After 24 hours of feeding, the feed rate was further increased, controlled at 6.2-10.5 mL / h / L based on dissolved oxygen feedback, maintaining a specific growth rate of 0.015 h⁻¹. -1 Adjust the rotation speed and aeration rate to control dissolved oxygen to be greater than 20% until fermentation is complete.

[0039] The lyophilized fusion protein powder was prepared according to the following steps:

[0040] 1) Fermentation broth pretreatment

[0041] The fermentation broth was centrifuged at 12,000 rpm for 20 min, and then the supernatant was filtered through 0.45 μm and 0.22 μm filter membranes, and the filtrate was collected.

[0042] (2) Nickel affinity chromatography

[0043] Because the HLfcinB gene expressed in this experiment contains a histidine tag, it can be purified using nickel affinity chromatography. Buffer A was 20 mM Tris-HCl (pH 8.0); Buffer B was 20 mM Tris-HCl (pH 8.0) buffer containing 500 mM imidazole; the packing material was Ni Beastrose FF (20 mL column volume). The purification steps are as follows:

[0044] ① Column packing and equilibration: Pour Ni Beastrose FF packing material into a vertically placed XK16 chromatography column and allow it to settle overnight. After settling, wash the column with ddH2O for 5 column volumes, then equilibrate the column with Buffer A containing 1% Buffer B for 3-4 column volumes at a flow rate of 5 mL / min.

[0045] ② Sample loading: Replace the filtrate from step (1) with Buffer A equilibration buffer containing 1% Buffer B and then load the sample. Set the flow rate to 2 mL / min and the loading volume to 100 mL. Collect the flow-through liquid.

[0046] ③ Reequilibration: Equilibrate the chromatography column with Buffer A containing 1% Buffer B, and elute after the conductivity and UV absorbance have stabilized;

[0047] ④ Elution: Gradient elution was performed using a mixture of Buffer A and Buffer B containing 8%, 20%, 30%, 50%, and 100% Buffer B. Elution peaks were monitored under UV280, and elution peaks with absorbance were collected. Two column volumes were eluted for each concentration, and the elution rate was 2 mL / min.

[0048] ⑤ Washing: Rinse with 1M NaOH aqueous solution for 5 column volumes, then rinse with degassed ddH2O for 3-4 column volumes;

[0049] ⑥ Storage: Wash with 20% ethanol for 5 column volumes, shut down the instrument according to standard operating procedures, and remove the chromatography column;

[0050] (3) Fusion protein freeze-dried powder

[0051] The eluent from step (2) containing 30% Buffer B and Buffer A (i.e., 150mM imidazole 20mM Tris-HCl (pH 8.0)) was freeze-dried at -60℃ for 48h to obtain the fusion protein lyophilized powder.

[0052] Compared with the prior art, the beneficial effects of the present invention are as follows: Since the expression level of bovine lactoferrin peptide in the prior art is low (10-30 mg / L), the present invention, by modifying collagen short peptides as carrier proteins, can better guide the expression of bovine lactoferrin peptides and increase its expression level to as high as 183.7 mg / L, which is 10-15 times higher. Moreover, through the rational design of collagen short peptides and linked acid-hydrolyzable peptides, they can be degraded into amino acids by acid in the subsequent acid hydrolysis process, releasing lactoferrin peptides. These lactoferrin peptides can be used in the fields of medicine, food, and feed. (iv) Description of the attached drawings

[0053] Figure 1 The image shows the recombinant plasmid pPIC9K-HLfcinB, which is the collagen short peptide mutant-acid hydrolysate-bovine lactoferrin peptide-6HIS fusion protein of Example 1.

[0054] Figure 2 This is an agarose gel image of positive transformants of Escherichia coli DH5α transformed by the recombinant plasmid of Example 1; lane M represents the marker, and lanes 1-10 represent transformant colonies.

[0055] Figure 3 This is an agarose gel image of a positive transformant of Pichia pastoris GS115 transformed by the recombinant plasmid in Example 2; lane M represents the marker, and lanes 1-8 represent transformant colonies.

[0056] Figure 4 This is an SDS-PAGE analysis diagram of the collagen short peptide mutant-acid hydrolysate-bovine lactoferrin peptide-6HIS fusion protein expression in Example 3. Lane M represents the marker, and lanes 1-8 represent the uninduced control, fermentation broth at 12h, 18h, 24h, 36h, 48h, 60h, and 72h, respectively.

[0057] Figure 5 The image shows the SDS-PAGE of the collagen short peptide mutant-acid hydrolyzed peptide-bovine lactoferrin peptide-6HIS fusion protein from Example 4 after purification using a nickel column. Lane M represents the marker, and lanes 1-9 represent the electrophoresis images of each component in the purification steps.

[0058] Figure 6 This is a small molecule SDS-PAGE verification image of lactoferrin peptide obtained after acid hydrolysis of collagen short peptide mutant-acid hydrolyzed peptide-bovine lactoferrin peptide-6HIS fusion protein in Example 4. Lane M represents the marker, and lanes 1-9 represent the electrophoresis images of lactoferrin peptide after acid hydrolysis.

[0059] Figure 7 The inhibition curves of GS115 fermentation broth and lactoferrin peptide on Escherichia coli K88 are shown. (V) Detailed Implementation

[0060] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0061] The Pichia pastoris GS115 strain and expression vector pPIC9K used in this invention were both purchased from Invitrogen, USA.

[0062] YPD complete medium: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, solvent is water.

[0063] YPD solid medium is made by adding 20 g / L agar to YPD complete medium.

[0064] MD medium (selective medium): YNB 13.4 g / L, glucose 20 g / L, biotin 4 × 10⁻⁶ -4 g / L, agarose 20 g / L, solvent is water. Preparation method per 100 mL: Add 2 g (20 g / L) of agarose to 80 mL of water, sterilize at 121℃ for 20 minutes. After the temperature drops to 60℃, add 10 mL (13.4 g / L) of 10×YNB, 10 mL (20 g / L) of 10×glucose, and 0.2 mL (4×10 g / L) of 500×biotin to a clean bench. -4 g / L).

[0065] BMG medium (yeast growth medium): K2HPO4 3g / L, KH2PO4 11.8g / L, YNB (amino-free yeast nitrogen source) 3.4g / L, ammonium sulfate 10g / L, glycerol 10mL / L, 4×10 -4 g / L biotin, in water as the solvent. Prepare as follows: 3g K₂HPO₄, 11.8g KH₂PO₄, 3.4g YNB (amino-free yeast nitrogen source), 10g ammonium sulfate, 10mL glycerol, completely dissolved, and diluted to 1L with deionized water. Autoclave at 115℃ for 30min. After cooling, add 2mL of 0.2% 500× biotin to a clean bench.

[0066] BMM medium (yeast induction medium): K2HPO4 3g / L, KH2PO4 11.8g / L, YNB (amino-free yeast nitrogen source) 3.4g / L, ammonium sulfate 10g / L, methanol 10mL / L, 4×10 -4g / L biotin, in water as the solvent. Prepare as follows: Completely dissolve 3g K₂HPO₄, 11.8g KH₂PO₄, 3.4g YNB (amino-free yeast nitrogen source), and 10g ammonium sulfate, and bring the volume to 1L with deionized water. Autoclave at 115℃ for 30min. After cooling, add 2mL of 500× biotin and 10mL of methanol to a clean bench.

[0067] BSM medium (g / L): 85% H3PO4 26.7mL / L, KOH 4.13g / L, K2SO4 18.2g / L, CaSO4 0.93g / L, MgSO4·7H2O 14.9g / L, glycerol 40.0g / L, trace elements (PTM1) 4.35mL / L, solvent is water, pH 5.0-5.5; prepared as follows: 85% H3PO4 26.7mL, KOH 4.13g, K2SO4 18.2g, CaSO4 0.93g, MgSO4·7H2O 14.9g, glycerol 40.0g. Before inoculation, adjust the pH to 5.0-5.5 with ammonia water and add PTM1 4.35mL / L.

[0068] Trace elements (PTM1): H3BO3 0.02g / L, CuSO4·5H2O 6.0g / L, MnSO4·H2O 3.0g / L, Na2MoO4·2H2O 0.2g / L, CoCl2 0.5g / L, NaI 0.08g / L, ZnCl2 20.0g / L, FeSO4·7H2O 65.0g / L, Biotin 0.2g / L, 5.0mL / L H2SO4, ddH2O to a final volume of 1L, filter sterilize, store at 4℃ protected from light.

[0069] Example 1

[0070] 1. Collagen short peptide mutant

[0071] Using collagen amino acid sequences from NCBI and modeling with the SWISS-MODEL database, a short collagen peptide amino acid sequence with stable structure and good hydrophilicity was selected from the amino acid sequence of human type III collagen αⅠ chain (NCBI sequence number: NM_000090.4, from Homosapiens) based on the collagen model. This sequence is located at amino acids 608-785 of the telopeptide-free type III collagen αⅠ chain sequence, totaling 177 amino acids. Following the characteristic repetitive structure rules of collagen, hydrophobic amino acids were mutated to hydrophilic amino acids to increase the hydrophilicity of the collagen short peptide while minimizing the isoelectric point. Through preliminary screening of mutation sites, the specific mutation points were determined as follows: isoleucine at position 614 was mutated to aspartic acid, leucine at position 633 to glycine, isoleucine at position 636 to proline, phenylalanine at position 658 to glycine, valine at position 706 to proline, and phenylalanine at position 721 to glutamic acid. The mutated collagen short peptide mutant contains 177 amino acids, and its amino acid sequence is shown in SEQ ID NO:1; the coding gene is 531 bp in length, and its nucleotide sequence is shown in SEQ ID NO:2, with a hydrophilicity of -1.09 and an isoelectric point of 4.83.

[0072] SEQ ID NO:1:

[0073] GPTGPDGPPGPAGQPGDKGEGGAPGGPGPAGPRGSPGERGETGPPGPAGGPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQ.

[0074] SEQ ID NO:2:

[0075] .

[0076] 2. Synthesis and expression of collagen short peptide mutant fusion proteins

[0077] (1) Synthesis of collagen short peptide mutant fusion protein

[0078] Using a collagen short peptide mutant as the carrier protein, an acid-hydrolyzed peptide was linked to the C-terminus, and a lactoferrin peptide (nucleotide sequence shown in SEQ ID NO:5, amino acid sequence shown in SEQ ID NO:6) was linked to the C-terminus of the acid-hydrolyzed peptide. A 6HIS (nucleotide sequence CACCATCATCACCACCAT, amino acid sequence HHHHHH) was then linked to the C-terminus of the lactoferrin peptide to construct a novel fusion protein for lactoferrin peptide expression. This fusion protein, known as collagen short peptide mutant-acid-hydrolyzed peptide-bovine lactoferrin peptide-6HIS fusion protein, or simply collagen short peptide mutant fusion protein HlfcinB, was synthesized by Nanjing Genscript Biotech Co., Ltd. It is 630 bp in length, consisting of 210 amino acids, with the nucleotide sequence shown in SEQ ID NO:4 and the amino acid sequence shown in SEQ ID NO:3.

[0079] SEQ ID NO:3:

[0080] GPTGPDGPPGPAGQPGDKGEGGAPGGPGPAGPRGSPGERGETGPPGPAGGPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQDPEWFKCRRWQWRMKKLGAPSITCVRRAFHHHHHH。

[0081] SEQ ID NO:4

[0082] GGTCCAACTGGTCCTGACGGTCCACCTGGACCAGCCGGTCAACCAGGAGATAAAGGTGAAGGTGGTGCTCCAGGTGGTCCTGGTCCTGCTGGTCCTAGAGGTTCTCCAGGTGAAAGAGGTGAGACTGGTCCACCTGGACCTGCTGGTGGTCCAGGTGCTCCTGGTCAAAACGGTGAACCAGGTGGTAAAGGAGAAAGAGGTGCTCCAGGAGAAAAAGGAGAGGGTGGTCCACCTGGTGTTGCCGGTCCACCTGGTGGTTCTGGTCCAGCCGGTCCACCTGGACCTCAAGGTCCAAAGGGTGAAAGAGGTTCTCCAGGTGGTCCTGGTGCTGCTGGTGAACCTGGTGCTAGAGGTTTGCCTGGTCCACCTGGATCTAACGGTAACCCTGGTCCACCTGGACCTTCTGGTTCTCCAGGTAAAGACGGTCCACCTGGTCCAGCTGGTAACACTGGTGCTCCAGGTTCTCCTGGTGTTTCTGGTCCAAAGGGAGATGCCGGTCAACCTGGAGAGAAAGGTTCTCCAGGTGCTCAAGACCCTGAGTGGTTTAAGTGTAGAAGATGGCAATGGAGAATGAAGAAGTTGGGTGCTCCATCTATTACTTGTGTTAGAAGAGCTTTCCACCATCATCACCACCATTAA。

[0083] SEQ ID NO:5:

[0084] TTTAAGTGTAGAAGATGGCAATGGAGAATGAAGAAGTTGGGTGCTCCATCTATTACTTGTGTTAGAAGAGCTTTC.

[0085] SEQ ID NO:6:

[0086] FKCRRWQWRMKKLGAPSITCVRRAF.

[0087] (2) Expression of collagen short peptide mutant fusion protein

[0088] Nanjing GenScript Biotechnology Co., Ltd. will produce the collagen short peptide mutant fusion protein from step (1).

[0089] The HlfcinB encoding gene (nucleotide sequence shown in SEQ ID NO:4) was digested with EcoRI and NotI and ligated to the multiple cloning site of the pPIC3.5K plasmid to construct the plasmid pPIC3.5K-HLfcinB. This plasmid was then transformed into *E. coli* DH5α competent cells and stored at -80°C. After culturing on plates containing ampicillin and kanamycin, positive clones were picked, and plasmids were extracted. Two cloning vectors, pPIC9K plasmid and pPIC3.5K-HlfcinB, were obtained from the corresponding bacterial plasmids.

[0090] Constructing the pPIC9K-HlfcinB fusion expression cloning vector using seamless cloning technology:

[0091] Using the pPIC9K plasmid as a template, the pPIC9K vector fragment with the α signal peptide EAEA residue mutated to a methionine residue was amplified using primers pPIC9K-F and pPIC9K-R.

[0092] pPIC9K-F:GGGGTATCTCTCGAGAAAAGAATG;

[0093] pPIC9K-R:CATTCTTTTCTCGAGAGATACCCCT.

[0094] Using pPIC3.5K-HlfcinB as a template, the HlfcinB fragment was amplified using primers HlfcinB-F and HlfcinB-R.

[0095] HlfcinB-F:AAGGGGTATCTCTCGAGAAAAGAATGGGTCCAACTGGTCCTGAC GGTCC;

[0096] HlfcinB-R:CTAGGGAATTCTACGTATTAATGGTGGTGATGATGGTGGAAAGCTCTTCTAACACAAGTAATAG.

[0097] The PCR system is as follows: primer F 1 μL, primer R 1 μL, DNA 1 μL, high-fidelity enzyme 25 μL, water to make up to 50 μL.

[0098] The PCR products were digested with DPNⅠ enzyme to remove interference from the original template. The digestion system was as follows: 50 μL PCR stock solution, 1 μL DPNⅠ, and 5 μL 10×BUFFER. The digested PCR products were then purified using a purification kit (purchased from Shanghai Sangon Biotech Co., Ltd.). The specific procedures were performed according to the kit's instructions.

[0099] The purified PCR products (pPIC9K vector fragment and HLfcinB fragment) were recombinantly ligated using a one-step cloning kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.) to obtain the ligation product. The specific recombinant ligation procedure was performed according to the kit instructions. The reaction mixture consisted of: 1 μL recombinase, 2 μL buffer, 2 μL pPIC9K fragment, 3 μL HLfcinB fragment, and 2 μL sterile water.

[0100] The ligation product was transformed into E. coli DH5α (commercial DH5α competent cells were purchased from Beijing Jinsha Biotechnology Co., Ltd.). The specific procedures were as follows: 100 μL of competent cells thawed on ice was taken, the target DNA (ligation product) was added, and the mixture was gently mixed and incubated on ice for 5 min; the cells were heat-shocked in a 42℃ water bath for 45 s, and then quickly returned to ice and incubated for 2 min; 700 μL of antibiotic-free sterile LB medium was added to a centrifuge tube, mixed, and then incubated at 37℃, 200 rpm for at least 20 min; 100 μL of the incubated cells were aspirated and added to an LB solid medium plate containing a final concentration of 0.1 mg / ml ampicillin and 0.05 mg / ml kanamycin, spread evenly, and the plate was dried; the plate was inverted and incubated overnight at 37℃.

[0101] Single colonies on transformation plates were selected for agarose gel electrophoresis verification of positive transformants, such as... Figure 2 As shown, lanes 3-10 contained 8 transformants, all of which were positive transformants. The validation sequence length was 496 bp of the universal sequence plus 630 bp of the fusion protein gene sequence, totaling 1126 bp. The plasmid pPIC9K-HLfcinB was extracted from the positive transformants (see figure). Figure 1 Sequencing was performed and the correct strain was stored in a -80°C freezer.

[0102] The validation primers used were the universal primers for the 9K plasmid, 5AOX and 3AOX, with the sequences as follows:

[0103] 3AOX:GCAAATGGCATTCTGACATCC;

[0104] 5AOX GACTGGTTCCAATTGACAAGCTT.

[0105] Example 2: Construction of recombinant Pichia pastoris engineered strain GS115-HLfcinB;

[0106] 1. Linearization of expression vector pPIC9K-HLfcinB

[0107] The plasmid pPIC9K-HLfcinB constructed in Example 1 was digested with restriction endonuclease SacⅠ overnight at 37°C. The complete digestion was then detected by 1% agarose gel electrophoresis. After complete digestion, the digestion solution was purified using a PCR product purification kit, and the linearized plasmid was recovered.

[0108] The enzyme digestion reaction system is as follows:

[0109] Plasmid pPIC9K-HLfcinB 1 μg, 10×L buffer 2 μL, SacⅠ 1 μL, sterile water added to 20 μL.

[0110] 2. Preparation of Pichia pastoris GS115 competent cells

[0111] (1) Pick a single colony of Pichia pastoris GS115 from a YPD plate and inoculate it into a test tube containing 3 mL of YPD liquid medium. Incubate overnight at 30°C and 220 rpm with shaking. (2) Take 500 μL of the overnight culture and inoculate it into a 500 mL Erlenmeyer flask containing 50 mL of fresh YPD liquid medium. Incubate overnight at 30°C and 220 rpm with shaking until the OD600 reaches 1.3-1.5. (3) Transfer the above culture into a sterile centrifuge tube and centrifuge at 4°C and 5000 rpm for 5 min. Remove the supernatant and place on ice. (4) Resuspend the above cells in 20 mL of LiAc-DTT solution (100 mM LiAc, 10 mM DTT, 0.6 M sorbitol, 10 mM Tris-HCl, pH 7.5). Incubate at 30°C with shaking for 30 min. Centrifuge at 4°C and 5000 rpm for 5 min. Remove the supernatant.

[0112] Repeat step (4) three times or add 1 mL of 1 M sorbitol pre-cooled on ice to resuspend the bacterial cells, then transfer to a 1.5 mL EP tube, centrifuge at 3000 rpm for 5 min, remove the supernatant, and repeat this step three times.

[0113] The collected bacterial cells were resuspended in 1M sorbitol pre-chilled on ice to a final volume of approximately 0.5 mL; aliquoted into 80 μL tubes and stored at -80 °C for later use.

[0114] 3. Electroconversion of Pichia pastoris

[0115] Remove the Pichia pastoris GS115 competent cells prepared in step 2 from the -80℃ freezer and place them on ice. Mix 1 μg of the linearized plasmid prepared in step 1 with 80 μL of the competent cells prepared in step 2, transfer the mixture to a pre-chilled 0.2 cm electroporation cuvette, gently tap it to ensure it is at the bottom of the cuvette, and place it on ice for 5-10 min. Following the operating instructions of the Bio-Rad electroporator, set the mode to Pic mode, wipe the outer wall of the electroporator dry and place it in the electroporation position, set the electroporation voltage to 1.5 kV, capacitance to 25 μF, resistance to 200 Ω, and electroporation time to 5 msec. Immediately after electroporation, add 1 mL of pre-chilled 1 M sorbitol to the electroporation cuvette, gently pipette to mix, and quickly transfer to a 1.5 mL container. In EP tubes, incubate statically at 30℃ and 220rpm for 1-2 hours; spread 100-200μL of bacterial cells onto MD plates containing 0.1mg / ml ampicillin and 0.05mg / ml kanamycin, and incubate upside down in a 30℃ incubator for 2-4 days until single colonies appear.

[0116] 4. PCR identification of recombinant transformants

[0117] (1) Cell disruption of yeast transformants:

[0118] Select a single colony from the transformation plate and place it in a PCR tube containing 0.1M NaOH aqueous solution. Mix by pipetting until visible turbidity is achieved. Place the PCR tube in a microwave oven and heat for 5 minutes. Then quickly place it in liquid nitrogen and freeze for 5 minutes. Repeat this process twice. Finally, heat the tube in a microwave oven for 5 minutes. Centrifuge and place the precipitate at the bottom of the PCR tube.

[0119] (2) Identification by colony PCR:

[0120] PCR verification was performed using α-F, 3AOX primers as amplification primers. The enzyme used was T5 Super PCR Mix (Colony) (purchased from Beijing Qingke Biotechnology Co., Ltd.). The PCR conditions were: 98℃ pre-denaturation for 5 min, one thermal cycle; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 15 s, 30 thermal cycles; final extension at 72℃ for 5 min. The PCR products were detected by 1% agarose gel electrophoresis. A band of approximately 835 bp was the target gene, and the corresponding bacterial species were positive transformants. (The gel electrophoresis results are shown in the image.) Figure 3As shown, lanes 1-8 were all positive transformants. Single colonies of the corresponding positive transformants from the MD transformation plate were inoculated into 250 mL Erlenmeyer flasks containing 50 mL of YPD medium and cultured overnight at 30°C and 220 rpm to obtain recombinant yeast GS115-pPIC9K-HLfcinB. The culture was preserved at -80°C using the glycerol method.

[0121] The primer sequences for verification are as follows:

[0122] α-F: TACTATTGCCAGCATTGCTGCT

[0123] 3AOX: GCAAATGGCATTCTGACATCC

[0124] 5. Inducible expression of recombinant yeast GS115-pPIC9K-HLfcinB

[0125] (1) Seed culture

[0126] Recombinant yeast GS115-pPIC9K-HLfcinB, stored at -80℃, was streaked onto YPD solid plates containing 100 μg / mL G418 resistance and incubated statically at 30℃ for 3 days. Single colonies were selected and inoculated into MD medium (30 mL / 250 mL, 50 mL / 500 mL) and cultured at 30℃ and 200 rpm for 16-18 h as the seed culture for fermentation.

[0127] (2) Fermentation culture

[0128] The fermentation process in a bioreactor mainly consists of four stages: batch fermentation, fed-batch fermentation, starvation, and methanol induction. The specific steps are as follows:

[0129] 1) Batch fermentation stage

[0130] The cultured seed culture was inoculated into a 5L fermenter containing 3L of fermentation medium at a volume concentration of 10%. The pH was adjusted to 5.0 with ammonia, the temperature was set at 30℃, the initial rotation speed was set at 500 rpm, and the aeration rate was controlled to ensure dissolved oxygen (DO) above 20% until the glycerol in the medium was depleted.

[0131] 2) Feeding stage

[0132] Once the glycerol in the culture medium was depleted, dissolved oxygen rose rapidly (DO > 60%). Then, a glycerol solution (50% glycerol aqueous solution with 12 mL / L PTM1 added) was added to the feed at a rate of 6.2 mL / h / L for 5 hours. The flow rate was then adjusted to 12 mL / h / L until the wet cell weight (WCW) of the yeast reached 150 g / L.

[0133] 3) Starvation stage

[0134] When the carbon source (glycerol) in the fermenter is exhausted, the fermenting cells will no longer consume large amounts of oxygen due to the lack of nutrient source. At this time, the dissolved oxygen in the fermenter will rise rapidly. Wait 30 minutes until the metabolic pathway of glycerol as a substrate in the cells is completely completed before methanol induction.

[0135] 4) Methanol induction stage

[0136] After the starvation phase, induction was initiated by feeding a methanol solution (12 mL / L of PTM1 in anhydrous methanol). Initially, this was a methanol acclimatization phase, with the feed rate controlled at 1.1 mL / h / L. After acclimatization, the methanol feed rate was appropriately increased to 3.6 mL / h / L. After 24 hours of feeding, the feed rate was further increased, controlled between 6.2 and 10.5 mL / h / L based on dissolved oxygen feedback, maintaining a specific growth rate of 0.015 h⁻¹. -1 Adjust the rotation speed and aeration rate to control dissolved oxygen to be greater than 20% until fermentation is complete.

[0137] Every 12 hours, 1 mL of bacterial culture was collected, centrifuged at 12,000 rpm for 2 minutes at room temperature, and the supernatant was collected for SDS-PAGE electrophoresis. After induction, samples were taken every 12 hours to determine the content of the fusion protein HLfcinB and the wet weight (WCW) of the bacterial cells.

[0138] The sample to be tested should be stored in a -80℃ refrigerator for later use.

[0139] Example 3: Detection of recombinant yeast fusion protein

[0140] The supernatant collected in Example 2 was analyzed by SDS-PAGE, and the results are as follows: Figure 4 As shown, lane M represents the marker, and lanes 1-8 represent the uninduced control, fermentation broth at 12h, 18h, 24h, 36h, 48h, 60h, and 72h, respectively. The band around 25kDa represents the fusion protein of collagen short peptide mutant-acid hydrolysate-bovine lactoferrin peptide-6HIS. Due to the special repeating structure of collagen, the fusion protein is larger than expected on SDS-PAGE, and the protein expression level increases over time. However, the fusion mode of collagen short peptide also caused collagen degradation, specifically resulting in multiple bands.

[0141] The expression level of the fusion protein was quantitatively determined using a BCA kit (purchased from Shanghai Sangon Biotech Co., Ltd.), following the instructions in the BCA kit manual. The total expression level of the recombinant Pichia pastoris GS115-pPIC9K-HLfcinB fusion protein in a 72-hour fermentation broth was found to be 2.8 g / L.

[0142] Example 4: Isolation and purification of fusion protein

[0143] 1. The fermentation broth from Example 2 was separated and purified to obtain the fusion protein. The 6HIS tag carried by the fusion protein itself can be used to separate and purify an AKTA-packed nickel column. The operation procedure is as follows:

[0144] (1) Fermentation broth pretreatment

[0145] After induction with methanol for 3 days, the fermentation broth was centrifuged at 12,000 rpm for 20 min. The supernatant was then filtered through 0.45 μm and 0.22 μm filter membranes to remove solid impurities, and the filtrate was collected.

[0146] (2) Nickel affinity chromatography

[0147] Because the HLfcinB gene expressed in this experiment contains a histidine tag, it can be purified using nickel affinity chromatography. Buffer A was 20 mM Tris-HCl (pH 8.0); Buffer B was 20 mM Tris-HCl (pH 8.0) buffer containing 500 mM imidazole; the packing material was Ni Beastrose FF (20 mL column volume). The purification steps are as follows:

[0148] ① Column packing and equilibration: Pour Ni Beastrose FF packing material into a vertically placed XK16 chromatography column and allow it to settle overnight. After settling, wash the column with ddH2O for 5 column volumes, then equilibrate the column with Buffer A containing 1% Buffer B for 3-4 column volumes at a flow rate of 5 mL / min.

[0149] ② Sample loading: Replace the filtrate from step (1) with Buffer A equilibration buffer containing 1% Buffer B and then load the sample. Set the flow rate to 2 mL / min and the loading volume to 100 mL. Collect the flow-through liquid.

[0150] ③ Reequilibration: Equilibrate the chromatography column with Buffer A containing 1% Buffer B, and elute after the conductivity and UV absorbance have stabilized;

[0151] ④ Elution: Gradient elution was performed using a mixture of Buffer A and Buffer B containing 8%, 20%, 30%, 50%, and 100% Buffer B. Elution peaks were monitored under UV280, and elution peaks with absorbance were collected. Two column volumes were eluted for each concentration, and the elution rate was 2 ml / min.

[0152] ⑤ Washing: Rinse with 1M NaOH aqueous solution for 5 column volumes, then rinse with degassed ddH2O for 3-4 column volumes;

[0153] ⑥ Preservation: Wash with 20% ethanol for 5 column volumes, shut down the instrument according to standard operating procedures, and remove the chromatography column.

[0154] (3) Fusion protein freeze-dried powder

[0155] The eluent from step (2) containing 30% Buffer B and Buffer A (i.e., 150mM imidazole 20mM Tris-HCl (pH 8.0)) was freeze-dried at -60℃ for 48h to obtain 0.5g of fusion protein lyophilized powder.

[0156] Dissolve 0.05g of the lyophilized fusion protein powder in 1mL of water and perform SDS-PAGE analysis. The results are as follows: Figure 5 As shown, lanes 1-2 are unpurified samples, specifically the supernatants from fermentation for 24h and 72h in Example 2. Lanes 3, 6, 7, and 9 are aqueous solutions of the lyophilized fusion protein powder obtained in step (3) above. Lane 4 is the flow-through liquid from step ②. Lanes 5 and 8 are the concentrated flow-through liquid from step ② (concentration method: ultrafiltration).

[0157] It can be seen that compared with the unpurified sample, the purified sample contained a significant reduction in endogenous proteins of Pichia pastoris, such as AOX, but it could not separate collagen peptides from their de novo degradation.

[0158] 2. Preparation of lactoferrin peptides from fusion proteins by acid hydrolysis

[0159] (1) Dissolve 0.1 g of the fusion protein lyophilized powder in 1 ml of 100 mM HCl, react in a metal bath at 80 °C for 8 h, and then adjust the pH to 7 with an equimolar amount of NaOH to obtain the fusion protein acid hydrolysate. The protein content was determined to be 183.7 mg / L by the BCA method. Small molecule SDS-PAGE detection and mass spectrometry identification results are as follows: Figure 6 As shown.

[0160] (2) Dissolve 0.1 g of the fusion protein lyophilized powder in 1 ml of 50 mM HCl, react in a metal bath at 60 °C for 8 h, then adjust the pH to 7 with an equimolar amount of NaOH. Detect by small molecule SDS-PAGE and mass spectrometry. The results are as follows: Figure 6 As shown.

[0161] Figure 6 Lanes 1-4 were hydrolyzed at 80℃ with 100mM HCl for 8 hours; lane 5 was the control supernatant from step 1; lane M was the marker; and lanes 6-10 were hydrolyzed at 60℃ with 50mM HCl for 8 hours. It can be seen that the fusion protein can be processed to obtain pure lactoferrin peptides.

[0162] 3. Performance testing of lactoferrin peptides

[0163] The antibacterial properties of the fusion protein hydrolysate from step 2(1) and the fermentation broth from Example 2 were tested. The antibacterial properties were tested using the dynamic growth curve method, with Escherichia coli K88 as the indicator bacterium.

[0164] The results are as follows Figure 7 Compared with the GS115 fermentation broth, lactoferrin peptides showed a certain inhibitory effect on Escherichia coli K88 in the initial stage, but the inhibition weakened over time. The results show that the lactoferrin peptides prepared by the method provided in this invention have significant antibacterial activity.

Claims

1. A collagen short peptide mutant fusion protein, characterized in that, The amino acid sequence of the collagen short peptide mutant fusion protein is shown in SEQ ID NO:

3.

2. The encoding gene of the collagen short peptide mutant fusion protein of claim 1.

3. A recombinant plasmid containing the gene encoding the collagen short peptide mutant fusion protein as described in claim 1.

4. A recombinant genetically engineered bacterium constructed from the recombinant plasmid as described in claim 3.

5. The use of the collagen short peptide mutant fusion protein of claim 1 in the preparation of lactoferrin peptide.

6. The application as described in claim 5, characterized in that, The application method is as follows: the fermentation broth obtained by fermenting recombinant genetically engineered bacteria containing the collagen short peptide mutant fusion protein encoding gene is centrifuged, the supernatant is collected to extract pure enzyme, and the fusion protein is freeze-dried to obtain lyophilized powder; the lyophilized powder is dissolved in 50-200mM HCl aqueous solution, reacted in a metal bath at 60-80℃ for 8-12h, and the pH is adjusted to 7.0 with 1M NaOH to obtain lactoferrin peptide solution.

7. The application as described in claim 6, characterized in that, The fermentation broth was prepared according to the following method: The recombinant genetically engineered bacteria were streaked onto YPD solid plates containing 100 μg / mL G418 resistance, and incubated at 30°C for 3 days. Single colonies were selected and inoculated into MD medium, and cultured at 30°C and 200 rpm for 16-18 hours to obtain the seed culture for fermentation. The seed culture was inoculated at a volume concentration of 10% into a 5L fermenter containing 3L of BSM medium. The pH was adjusted to 5.0, the temperature was set at 30℃, the initial agitation speed was set to 500 rpm, and the dissolved oxygen was controlled to be above 20%. After the glycerol in the medium was consumed and the dissolved oxygen rose rapidly, a glycerol solution was added sequentially, and the culture was continued until the cell concentration reached 150 g / L. After the addition was completed, the culture was starved for 30 min, and then a methanol solution was added sequentially for induction, with the specific growth rate controlled at 0.015 h⁻¹. -1 The fermentation broth was obtained by adjusting the rotation speed and aeration rate to control dissolved oxygen to be greater than 20%; the glycerol solution was prepared by adding 12 mL / L of trace elements to a 50% glycerol aqueous solution. The methanol solution refers to anhydrous methanol with 12 mL / L of trace elements added. BSM medium: 85% H3PO4 26.7mL / L, KOH 4.13g / L, K2SO4 18.2g / L, CaSO4 0.93g / L, MgSO4·7H2O 14.9g / L, glycerol 40.0g / L, trace elements 4.35mL / L, solvent: water, pH 5.0-5.5; Trace elements: H3BO3 0.02g / L, CuSO4·5H2O 6.0g / L, MnSO4·H2O 3.0g / L, Na2MoO4·2H2O 0.2g / L, CoCl2 0.5g / L, NaI 0.08g / L, ZnCl2 20.0g / L, FeSO4·7H2O 65.0g / L, biotin 0.2g / L, 5.0mL / L H2SO4, solvent: ddH2O.

8. The application as described in claim 6, characterized in that, The fermentation is divided into four stages: batch fermentation, fed-batch fermentation, starvation, and methanol induction. 1) Batch fermentation stage The seed culture was inoculated into a 5L fermenter containing 3L of fermentation medium at a volume concentration of 10%. The pH was adjusted to 5.0 with ammonia, the temperature was set at 30℃, the initial rotation speed was set at 500 rpm, and the aeration rate was controlled to be above 20% dissolved oxygen until the glycerol in the medium was depleted. 2) Feeding stage When the glycerol in the culture medium is depleted, the dissolved oxygen rises rapidly. Then, the glycerol solution is fed in at a rate of 6.2 mL / h / L for 5 hours. Then the flow rate is adjusted to 12 mL / h / L until the cell concentration reaches 150 g / L. 3) Starvation stage After the feeding process is completed, starvation induction is performed for 30 minutes. 4) Methanol induction stage After the starvation phase, methanol solution was fed in to induce growth. Initially, this was a methanol acclimatization phase, with the feed rate controlled at 1.1 mL / h / L. After acclimatization, this was adjusted to 3.6 mL / h / L. After 24 hours of feeding, the feed rate was maintained between 6.2 and 10.5 mL / h / L based on dissolved oxygen feedback, with the specific growth rate controlled at 0.015 h⁻¹. -1 Adjust the rotation speed and aeration rate to control dissolved oxygen to be greater than 20% until fermentation is complete.

9. The application as described in claim 6, characterized in that... The lyophilized fusion protein powder was prepared according to the following steps: 1) Fermentation broth pretreatment The fermentation broth was centrifuged at 12,000 rpm for 20 min, and then the supernatant was filtered through 0.45 μm and 0.22 μm filter membranes, and the filtrate was collected. (2) Nickel affinity chromatography Buffer A was 20 mM Tris-HCl, pH 8.0; Buffer B was 20 mM Tris-HCl containing 500 mM imidazole, pH 8.0; the packing material was Ni Beastrose FF, the column volume was 20 mL, and the purification experimental steps were as follows: ① Column packing and equilibration: Pour Ni Beastrose FF packing material into a vertically placed XK16 chromatography column and allow it to settle overnight. After settling, wash the column with ddH2O for 5 column volumes, and then equilibrate the column with Buffer A containing 1% Buffer B for 3-4 column volumes at a flow rate of 5 mL / min. ② Sample loading: After replacing the filtrate from step (1) with Buffer A equilibration buffer containing 1% Buffer B, load the sample at a flow rate of 2 mL / min and a loading volume of 100 mL. ③ Reequilibration: Equilibrate the chromatography column with Buffer A containing 1% Buffer B, and elute after the conductivity and UV absorbance have stabilized; ④ Elution: Gradient elution was performed using Buffer A and Buffer B containing 8%, 20%, 30%, 50%, and 100% Buffer B, respectively. Elution peaks were monitored under UV280, and elution peaks with absorbance were collected. Two column volumes were eluted for each concentration, and the elution rate was 2 mL / min. ⑤ Washing: Rinse with 1M NaOH aqueous solution for 5 column volumes, then rinse with degassed ddH2O for 3-4 column volumes; ⑥ Storage: Wash with 20% ethanol for 5 column volumes, shut down the instrument according to standard operating procedures, and remove the chromatography column; (3) Fusion protein freeze-dried powder The eluent from step (2) containing 30% Buffer B and Buffer A was freeze-dried at -60℃ for 48 hours to obtain the fusion protein freeze-dried powder.

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