Encoding gene of human stem cell factor, recombinant expression vector, engineering strain and preparation method thereof

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a human stem cell factor encoding gene, a recombinant expression vector, an engineered strain, and a preparation method thereof. The nucleotide sequence of the encoding gene is shown in SEQ ID NO:1. The recombinant expression vector includes an expression vector and the encoding gene shown in SEQ ID NO:1. The engineered strain is obtained by transforming the recombinant expression vector containing the encoding gene into a host strain and screening. This invention also provides a method for preparing human stem cell factors, comprising: (1) culturing the engineered strain in a culture medium until the OD reaches a suitable range; (2) obtaining a fermentation broth through induced culture; and (3) purifying and separating the fermentation broth to obtain recombinant human stem cell factors. This invention optimizes the protein expression system and purification process while ensuring the acquisition of high-purity human stem cell factors, making it more suitable for industrial-scale production and providing a reference for achieving large-scale and efficient preparation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a human stem cell factor encoding gene, recombinant expression vector, engineered strain, and preparation method thereof. Background Technology

[0002] Stem Cell Factor (SCF) is an important hematopoietic growth factor and a ligand for the tyrosine kinase receptor c-Kit. Its core function is achieved by binding to the c-Kit receptor on the surface of target cells, forming the SCF / c-Kit signaling axis, which activates multiple downstream signaling pathways (such as MAPK, PI3K / Akt, etc.), thereby regulating the biological processes of stem cells. In vivo, SCF exists as a non-covalently linked homodimer, with each monomer containing four cysteine ​​residues forming an intramolecular disulfide bond. SCF is a glycoprotein, with glycosylation modifications occurring at both the N-linked and O-linked sites of the polypeptide chain. SCF primarily acts on early hematopoietic stem cells and primitive hematopoietic progenitor cells, inducing their survival, proliferation, and differentiation. SCF has limited biological activity when used alone, but it exhibits strong synergistic effects with other cytokines such as G-CSF (granulocyte colony-stimulating factor), GM-CSF (granulocyte-macrophage colony-stimulating factor), EPO (erythropoietin), IL-3 (interleukin-3), IL-6 (interleukin-6), IL-11 (interleukin-11), and TPO (thrombopoietin). It shows promising applications in in vivo stem cell expansion therapy, in vitro cultured hematopoietic stem cells for gene therapy, and as adjuvant therapy after radiotherapy and chemotherapy for tumors.

[0003] Domestically and internationally, the production of recombinant SCF proteins primarily utilizes E. coli expression systems. The protein products are unmodified and often expressed as inclusion bodies. Subsequent purification processes require denaturation and renaturation to obtain the active protein. Overall recovery rates are lower than with soluble or secretory expression, costs are higher, and the final product may contain a mixture of different folding states, affecting product quality and batch-to-batch consistency.

[0004] To address the issue of inclusion body expression in E. coli expression systems, patent CN102559725A attempted to construct the pET-SUMO-rhSCF-α recombinant expression plasmid and express it in E. coli. The protein primarily exists in a soluble expression form. Subsequent processes, including Ni-NTA affinity chromatography, Sephadex G-25 chromatography for imidazole removal, SUMO protease cleavage of the SUMO tag, Ni-NTA affinity chromatography, and Sephadex G-25 chromatography for desalting, yielded a 98.3% pure rhSCF-α recombinant protein. While this method reduces the denaturation and renaturation steps in inclusion body purification, it still requires multiple rounds of affinity chromatography, desalting, and enzyme digestion. Furthermore, the use of coenzymes during production increases costs and hinders subsequent industrial-scale production.

[0005] To address the complex and costly purification of inclusion bodies, patent CN117624329A optimizes the protein purification method. It involves resuspending, centrifuging, and refolding stem cell cytokine inclusion bodies to obtain a crude protein extract. Chitosan, nano-ferric oxide, and stem cell cytokines are then mixed, followed by the addition of ethylene glycol dimethacrylate and sodium bisulfite for reaction. The stem cell cytokines are eluted with sodium dodecyl sulfate (SDS) solution to obtain a molecularly imprinted polymer. The crude protein extract and the molecularly imprinted polymer are then mixed and eluted with NaCl solution under an external magnetic field to obtain the stem cell cytokines. This method is relatively simple to operate and easy for subsequent industrial production; however, the protein purity obtained using this method is only 91.0%, which still needs improvement.

[0006] Therefore, optimizing protein expression systems and purification processes to ensure high product purity and better suit the needs of industrial-scale production is of great significance for achieving large-scale and efficient preparation. Summary of the Invention

[0007] This invention aims to provide a gene encoding human stem cell factor, a recombinant expression vector, an engineered strain, and a method for its preparation. The goal is to optimize the protein expression system and purification process while ensuring the acquisition of high-purity human stem cell factor, making it more suitable for industrial-scale production and providing a reference for achieving large-scale and efficient preparation.

[0008] In one aspect, a gene encoding a human stem cell factor is provided, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0009] SEQ ID NO:1 is: GAG GGT ATC TGT CGT AAC CGT GTC ACC AAC AAC GTT AAG GACGTT ACC AAA CTG GTC GCT AAC TTG CCT AAG GAT TAC ATG ATT ACC TTG AAG TAC GTTCCT GGT ATG GAT GTC TTG CCA TCT CAC TGT TGG ATCTCC GAA ATG GTC GTT CAA CTGTCC GAC TCT TTG ACC GAC TTA CTG GAT AAG TTC TCT AAC ATC TCC GAG GGT CTG TCTAAT TAC TCC ATTATC GAC AAG TTG GTT AAC ATC GTT GAC GAT TTG GTT GAG TGT GTCAAGGAG AAC TCT TCC AAG GAC CTG AAG AAA TCC TTC AAG TCT CCT GAG CCA AGA TTG TTCACC CCT GAG GAA TTC TTC CGT ATC TTC AAC CGT TCT ATT GAC GCT TTC AAA GAC TTCGTC GTT GCT TCT GAA ACC TCT GAT TGT GTT GTT GTC TCC TCT ACC TTG TCT CCT GAG AAGGAC TCTAGA GTC TCC GTT ACC AAA CCT TTC ATG TTG CCT CCA GTT GCT GCT.

[0010] In some embodiments, the encoding gene is a gene suitable for expression in Pichia pastoris strains.

[0011] In a second aspect, a recombinant expression vector is provided, comprising an expression vector and a coding gene as shown in SEQ ID NO:1.

[0012] In some embodiments, the expression vector is selected from at least one of pPIC9, pPIC9K, pPICZα, and pHIL-S1.

[0013] In some embodiments, the coding gene is constructed into an expression vector to form a recombinant expression vector using at least one method such as PCR, enzyme digestion and ligation, or homologous recombination. In some embodiments, the core elements of the recombinant expression vector consist of: a target protein gene, a promoter, a signal peptide, and an enhancement element.

[0014] In some implementations, the gene for human stem cell factor is introduced at the N-terminus of the sequence. Xho I. Restriction enzyme cleavage site and KR-EAEA- sequence, which are the recognition and cleavage sites for endopeptidase KEX2 and signal peptidase STE13, respectively; the C-terminus of the sequence is introduced... Not I. Restriction enzyme cleavage site.

[0015] In some embodiments, the method for preparing the coding gene includes: designing eight primers and obtaining a complete cDNA fragment by PCR splicing.

[0016] In some embodiments, the primers have nucleotide sequences as shown in SEQ ID NO:2~9.

[0017] In some embodiments, the DNA polymerase used for the PCR splicing is Phanta Max Super-Fidelity DNA Polymerase.

[0018] In some implementations, the PCR splicing process includes: Step a. The reaction system used for PCR splicing is as follows: total volume 50 μL, 25 μL 2×PhantaMax buffer, 1 μL dNTP mixture (10 mM each), 2 μL forward primer 01-UP3 (10 μM), 2 μL reverse primer 01-DW3 (10 μM), 1 μL PhantaMax high-fidelity DNA polymerase, and ddH2O to a final volume of 50 μL; The amplification program used was as follows: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 sec, annealing at 50℃ for 15 sec, extension at 72℃ for 11 sec, the denaturation, annealing and extension processes were cycled 35 times, followed by extension at 72℃ for 5 min, and stored at 4℃. The PCR product 01-S3 was recovered by 1% low-melting-point agarose gel electrophoresis; Step b. The reaction system is as follows: total volume 50 μL, 25 μL 2×PhantaMax buffer, 1 μL dNTP mixture (10 mM each), 1 μL PCR product 01-S3 obtained in step a, 2 μL forward primer 01-UP2 (10 μM), 2 μL reverse primer 01-DW2 (10 μM), 1 μL PhantaMax high-fidelity DNA polymerase, and ddH2O to a final volume of 50 μL. The amplification program was as follows: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 sec, annealing at 51℃ for 15 sec, extension at 72℃ for 21 sec, the denaturation, annealing and extension processes were cycled 35 times, followed by extension at 72℃ for 5 min. The PCR product 01-S2 was recovered by 1% low-melting-point agarose gel electrophoresis; Step c. The reaction system is as follows: total volume 50 μL, 25 μL 2×PhantaMax buffer, 1 μL dNTP mixture (10 mM each), 1 μL PCR product 01-S2 obtained in step b, 2 μL forward primer 01-UP1 (10 μM), 2 μL reverse primer 01-DW1 (10 μM), 1 μL PhantaMax high-fidelity DNA polymerase, and ddH2O to a final volume of 50 μL. The amplification program was as follows: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 sec, annealing at 50℃ for 15 sec, extension at 72℃ for 30 sec, the denaturation, annealing and extension processes were cycled 35 times, and then extension was performed at 72℃ for 5 min. The PCR product 01-S1 was recovered by 1% low-melting-point agarose gel electrophoresis; Step d. The reaction system is as follows: total volume 50 μL, 25 μL 2×PhantaMax buffer, 1 μL dNTP mixture (10 mM each), 1 μL PCR product 01-S1 obtained in step c, 2 μL forward primer 01-PIC (10 μM), 2 μL reverse primer 01-DW165PC (10 μM), 1 μL PhantaMax high-fidelity DNA polymerase, and ddH2O to a final volume of 50 μL. The amplification program was as follows: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 sec, annealing at 52℃ for 15 sec, extension at 72℃ for 37 sec, the denaturation, annealing and extension processes were cycled 35 times, and then extension was performed at 72℃ for 5 min. The PCR product was recovered by 1% low-melting-point agarose gel electrophoresis to obtain the complete rhSCF cDNA fragment.

[0019] Thirdly, an engineered strain is provided, the engineered strain containing the coding gene described in this invention.

[0020] In some embodiments, the engineered strain is obtained by transforming a recombinant expression vector containing the coding gene described in this invention into a host strain and then screening for it.

[0021] In some embodiments, the host strain is at least one of Pichia pastoris strain KM71, strain GS115, and strain X33.

[0022] In some embodiments, the recombinant expression vector is linearized with an endonuclease and then transformed into a host strain. Auxotrophic selection and / or resistance selection methods are then used to obtain the engineered strain (i.e., the Pichia pastoris expression strain). In this application, the transformation procedure was performed in accordance with the Invitrogen Pichia pastoris expression system operating manual.

[0023] In some embodiments, the recombinant expression vector is linearized and transformed into host strains KM71, GS115, or X33, plated on auxotrophic plates and / or resistance plates (bleomycin or genimycin), and screened to obtain Pichia pastoris expression strains.

[0024] In some embodiments, the auxotrophic plate is preferably Pichia pastoris regenerated glucose agar, comprising 2% (w / v) glucose, 1.34% (w / v) yeast nitrogen base, 0.00004% (w / v) biotin, 1.5% (w / v) agarose, and 18.2% (w / v) sorbitol. Preferably, the Pichia pastoris expression strain contains the pPIC9-act01-165aa plasmid.

[0025] Fourthly, a method for preparing human stem cell factors is provided, comprising: (1) The engineered strain was cultured in the culture medium until the OD reached a suitable range; (2) Fermentation broth was obtained through induction culture; (3) The fermentation broth was purified and separated to obtain recombinant human stem cell factor.

[0026] In some embodiments, the culture medium is selected from at least one of YPD medium, BMGY medium, or FBS fermentation medium.

[0027] In some embodiments, the YPD medium contains 1-5% (w / v) yeast extract, 1-5% (w / v) peptone, and 1-5% (w / v) glucose. In some embodiments, the YPD medium contains 1% (w / v) yeast extract, 2% (w / v) peptone, and 2% (w / v) glucose.

[0028] In some embodiments, the BMGY medium contains 1-5% (w / v) yeast extract, 1-5% (w / v) peptone, 0.1-0.5% (w / v) dipotassium hydrogen phosphate, 1-5% (w / v) potassium dihydrogen phosphate, 1-5% (w / v) yeast nitrogen base, 0.00001-0.00005% (w / v) biotin, and 1-5% (v / v) glycerol. In some embodiments, the BMGY medium contains 1% (w / v) yeast extract, 2% (w / v) peptone, 0.3% (w / v) dipotassium hydrogen phosphate, 1.18% (w / v) potassium dihydrogen phosphate, 1.34% (w / v) yeast nitrogen base, 0.00004% (w / v) biotin, and 1% (v / v) glycerol.

[0029] In some embodiments, the FBS fermentation medium contains 1-5% (w / v) yeast extract, 1-5% (w / v) peptone, 1-5% (v / v) glycerol, 0.1-0.5% (w / v) potassium hydroxide, 0.5-1% (w / v) potassium sulfate, 0.01-0.05% (w / v) calcium sulfate dihydrate, 0.1-0.5% (v / v) phosphoric acid, and 0.1-0.5 mL / L (v / v) polyether defoamer DF-103. In some embodiments, the FBS fermentation medium contains 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (v / v) glycerol, 0.16% (w / v) potassium hydroxide, 0.73% (w / v) potassium sulfate, 0.037% (w / v) calcium sulfate dihydrate, 0.22% (v / v) phosphoric acid, and 0.25 mL / L (v / v) polyether defoamer DF-103.

[0030] In some embodiments, in step (1), the YPD medium is used for primary culture at 200-350 rpm and 26-30°C for 14-16 hours; the BMGY medium is used for secondary culture at 26-30°C for 14-16 hours until the OD600nm value is 10-15; the FBS fermentation medium is used for fermentation culture at 26-30°C and pH 6.0-6.5, with dissolved oxygen controlled at 10%-40% (preferably any two values ​​from 10%, 15%, 20%, 25%, 30%, 35%, 40%, and above); and the culture is carried out until the OD600nm value is 100-200 (preferably any two values ​​from 100, 150, 200, and above).

[0031] In some embodiments, in step (2), after the culture in step (1) is completed, the temperature is lowered to 18~22℃ (preferably 18℃, 19℃, 20℃, 21℃, 22℃), and an inducer is added to induce fermentation for 35~50h (preferably 35h, 40h, 45h, 50h, or any two of the above values ​​within a range) to complete the fermentation.

[0032] In some implementations, the inducing agent in step (2) is methanol.

[0033] In some embodiments, in step (3), the purification separation is selected from at least one of cation exchange chromatography or molecular sieve chromatography.

[0034] In some embodiments, the cation exchange chromatography is performed using an SP-agarose gel FF ion exchange chromatography column. The specific process is as follows: After diluting the fermentation supernatant acetic acid (HAc) 1-5 times, adjust the pH to 3.5-4.5 and purify by column chromatography; cation exchange chromatography is performed using an SP-agarose gel FF ion exchange column; first, wash with 0.5-1M NaOH at no more than 200-300 cm / hr for 1-2 CV, then continue washing at no more than 100-150 cm / hr for at least 30 min; wash with water for injection for at least 1-3 CV; equilibrate with 50-100 mM NaAc-HAc at pH 3.5-4.5 for at least 1-3 CV, and the column effluent pH is 3.5-4.5 when usable; load the fermentation supernatant with a packing material flow rate not exceeding 15-25 OD / mL, and a flow rate not exceeding 200-300 cm / hr; at a flow rate not exceeding 200-300 cm / hr, use 50-100 mM NaAc-HAc and 20-100 mM (NH4)2SO4. Rinse at pH 3.5-4.5 for at least 1-3 CV; flow rate not exceeding 100-150 cm / hr; elute with 50-100 mM NaAc-HAc and 200-400 mM (NH4)2SO4 at pH 3.5-4.5 to collect the target protein.

[0035] In some embodiments, the diameter of the SP-agarose gel FF ion exchange chromatography column is 20-30 mm, preferably 20 mm, 25 mm, or 30 mm, and the column bed is 50-100 mL (preferably 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, or any two of the above values ​​forming a range). In some embodiments, the molecular sieve chromatography is performed using a Superdex 75 gel filtration chromatography column. The specific process is as follows: Molecular sieve chromatography is performed using a Superdex 75 gel filtration chromatography column; first, washing with 0.5-1M NaOH at a rate not exceeding 100-150 cm / hr for 1-2 CV, followed by continuous washing at a rate not exceeding 60-80 cm / hr for at least 30 min; washing with water for injection for at least 1-3 CV; equilibration with 50-100 mM PBNa (sodium phosphate) at pH 6.5-7.5 for at least 2.5-3 CV, and the column effluent pH is 7.0 ± 0.5 for use; elution and sample loading are performed using an SP-agarose gel FF ion exchange chromatography column, with the sample loading volume not exceeding 5% of the column bed volume and the flow rate not exceeding 120-150 cm / hr; protein is eluted using 50-100 mM PBNa at pH 6.5-7.5.

[0036] In some embodiments, the diameter of the Superdex 75 gel filtration chromatography column is 40-60 mm, preferably any one of any two values ​​from 40 mm, 50 mm, 60 mm, and above, and the column bed is 0.5-2 L (preferably any one of any two values ​​from 0.5 L, 1 L, 1.5 L, 2 L, and above).

[0037] In some embodiments, the human stem cell factor obtained in step (3) is sterilized by filtration using a 0.22 μm filter, aliquoted, and cryopreserved. Simultaneously, the purified sample is tested for purity, endotoxin content, and biological activity. Preferably, the purity is tested using SDS-PAGE or HPLC-SEC methods. Preferably, the endotoxin content is tested using Limulus Amebocyte Lysate (LAL) reagent. Preferably, the biological activity is tested using the UT-7 cell / MTT assay.

[0038] Compared with existing technologies, one of the above technical solutions has the following advantages or beneficial effects: This invention successfully obtained a Pichia pastoris strain that highly expresses recombinant stem cell factor. After induction, the recombinant stem cell factor can be secreted and expressed, with a protein expression level of not less than 260 mg / L (superior to the prior art CN1570119A, which can obtain recombinant human stem cell factor (rhSCF) with a purity of over 95% (yield of 0.13 g / L) after purification).

[0039] The purification steps of this invention are simpler. The target protein can be directly secreted and expressed in the culture medium. Then, only two purification steps, cation exchange chromatography and molecular sieve chromatography, are needed to obtain recombinant stem cell factors. The resulting fermentation broth has a protein yield of not less than 150 mg / L, a protein purity of ≥97%, an endotoxin test result of less than 0.01 EU / ug, and a specific activity comparable to WHO standards. Attached Figure Description

[0040] Figure 1 This is a simplified diagram of rhSCF cDNA PCR splicing in Example 1.

[0041] Figure 2 This is a diagram of plasmid enzyme digestion identification in Example 1.

[0042] Figure 3 This is a purification diagram from the SP-agarose gel FF ion exchange chromatography column in Example 3.

[0043] Figure 4 This is a purification diagram using the Superdex 75 gel filtration chromatography column in Example 3.

[0044] Figure 5 Electrophoresis diagram of purified human stem cell factor obtained in Example 3.

[0045] Figure 6 Electrophoresis diagram for detecting the purity of human stem cell factor stock solution.

[0046] Figure 7 The HPLC-SEC purity chromatogram of human stem cell factor stock solution.

[0047] Figure 8 A graph showing the activity of human stem cell factor stock solution.

[0048] Figure 9 This is a purified electrophoresis image of the human stem cell factor obtained in Comparative Example 1.

[0049] Figure 10 This is a purified electrophoresis image of the human stem cell factor obtained in Comparative Example 2.

[0050] Figure 11 This is a purified electrophoresis image of the human stem cell factor obtained in Comparative Example 3.

[0051] Unless otherwise stated, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. All patents and publications related to this invention are incorporated herein by reference in their entirety. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0053] All reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0054] Table 1 Main Product List

[0055] YPD medium: contains 1% (w / v) yeast extract, 2% (w / v) peptone, and 2% (w / v) glucose.

[0056] BMGY medium: contains 1% (w / v) yeast extract, 2% (w / v) peptone, 0.3% (w / v) dipotassium hydrogen phosphate, 1.18% (w / v) potassium dihydrogen phosphate, 1.34% (w / v) yeast nitrogen base, 0.00004% (w / v) biotin, and 1% (v / v) glycerol.

[0057] FBS fermentation medium: containing 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (v / v) glycerol, 0.16% (w / v) potassium hydroxide, 0.73% (w / v) potassium sulfate, 0.037% (w / v) calcium sulfate dihydrate, 0.22% (v / v) phosphoric acid, and 0.25 mL / L (v / v) polyether defoamer DF-103.

[0058] Example 1 Construction of expression strain 1. Selection of host strain: Pichia pastoris GS115.

[0059] 2. A recombinant expression vector was constructed using enzyme digestion and ligation methods, and then electroporated into the Pichia pastoris GS115 host strain. The specific steps are as follows: (1) The gene of human stem cell factor was modified according to the codon preference of Pichia pastoris, and Xho I (CTCGAG) restriction enzyme site and KR-EAEA- sequence (AAA AGA GAG GCT GAA GCT) were introduced at the N end of the sequence, which are the recognition and cleavage sites of endopeptidase KEX2 and signal peptidase STE13, respectively; Not I (GCGGCCGC) restriction enzyme site was introduced at the C end of the sequence.

[0060] (2) In order to synthesize the modified gene in step (1), eight primers were designed and complete cDNA fragments were obtained by PCR splicing, which have the nucleotide sequence shown in SEQ ID NO:1.

[0061] Primer 01-PIC, as shown in SEQ ID NO:2: 5`-GGGGT ATGCG GCCGC CTCTC GAGAA AAGAG AGGCT GAAGC TGAGG GTATC TGTCGTAACC GTGTC ACC-3`.

[0062] Primer 01-UP1, as shown in SEQ ID NO:3: 5`-TGTCG TAACC GTGTC ACCAA CAACG TTAAG GACGT TACCA AACTG GTCGC TAACTTGCCT AAGGA TTACA TGATT ACCTT GAAGT ACGTT-3`.

[0063] Primer 01-UP2, as shown in SEQ ID NO:4: 5`-ACATG ATTAC CTTGA AGTAC GTTCC TGGTA TGGAT GTCTT GCCAT CTCAC TGTTGGATCT CCGAA ATGGT CGTTC AACTG TCCGA CTCTT TGACC-3`.

[0064] Primer 01-UP3, as shown in SEQ ID NO:5: 5`-AACTG TCCGA CTCTT TGACC GACTT ACTGG ATAAG TTCTC TAACA TCTCC GAGGGTCTGT CTAAT TACTC CATTA TCGAC AAGTT GGTTA ACAT-3`.

[0065] Primer 01-DW1, as shown in SEQ ID NO:6: 5`-CTCTA GAGTC CTTCT CAGGA GACAA GGTAG AGGAG ACAAC ACAAT CAGAG GTTTCAGAAG CAACG ACGAA GTCTT TGAAA GCGTC AATAG AAC-3`.

[0066] Primer 01-DW2, as shown in SEQ ID NO:7: 5`-GTCTT TGAAA GCGTC AATAG AACGG TTGAA GATAC GGAAG AATTC CTCAG GGGTGAACAA TCTTG GCTCA GGAGA CTTGA AGGAT TTCTT CAG-3`.

[0067] Primer 01-DW3, as shown in SEQ ID NO:8: 5`-GGAGA CTTGA AGGAT TTCTT CAGGT CCTTG GAAGA GTTCT CCTTG ACACA CTCAACCAAA TCGTC AACGA TGTTA ACCAA CTTGT CGATA ATG-3`.

[0068] Primer 01-DW 165PC, as shown in SEQ ID NO:9: 5`-CAGAT TCGCG GCCGC CTAAG CAGCA ACTGG AGGCA ACATG AAAGG TTTGG TAACGGAGAC TCTAG AGTCC TTCTC AGGAG -3`.

[0069] PCR assembly was performed using a PCR instrument with Phanta Max Super-Fidelity DNA Polymerase as the DNA polymerase. Complete cDNA fragments were assembled via PCR. The assembly process is as follows: Figure 1 As shown, the specific operation steps are as follows: Step a. Primer 01-UP3 + Primer 01-DW3 The reaction system consisted of: a total volume of 50 μL, 25 μL of 2×PhantaMax buffer, 1 μL of dNTP mixture (10 mM each), 2 μL of forward primer 01-UP3 (10 μM), 2 μL of reverse primer 01-DW3 (10 μM), 1 μL of PhantaMax high-fidelity DNA polymerase, and ddH2O to a final volume of 50 μL.

[0070] The amplification program was as follows: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 sec, annealing at 50℃ for 15 sec, extension at 72℃ for 11 sec, and the denaturation, annealing and extension processes were cycled 35 times, followed by extension at 72℃ for 5 min.

[0071] The PCR products were recovered by 1% low-melting-point agarose gel electrophoresis, dissolved in 30 μL ddH2O, and stored at -18℃ or below, numbered 01-S3.

[0072] Step b. Primer 01-U2 + (Primer 01-UP3 + Primer 01-DW3) + Primer 01-DW2 The reaction system consisted of: a total volume of 50 μL, 25 μL of 2×PhantaMax buffer, 1 μL of dNTP mixture (10 mM each), 1 μL of PCR product 01-S3 obtained in step a, 2 μL of forward primer 01-UP2 (10 μM), 2 μL of reverse primer 01-DW2 (10 μM), 1 μL of PhantaMax high-fidelity DNA polymerase, and ddH2O to a final volume of 50 μL.

[0073] The amplification program was as follows: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 sec, annealing at 51℃ for 15 sec, extension at 72℃ for 21 sec, repeated 35 times for denaturation, annealing, and extension, followed by a final extension at 72℃ for 5 min, and stored at 4℃. The PCR products were recovered by 1% low-melting-point agarose gel electrophoresis, dissolved in 30 μL ddH2O, and labeled 01-S2.

[0074] Step c. Primer 01-U1 + (Primer 01-UP2 - Primer 01-DW2) + Primer 01-DW1 The reaction system consisted of: a total volume of 50 μL, 25 μL of 2×PhantaMax buffer, 1 μL of dNTP mixture (10 mM each), 1 μL of PCR product 01-S2 obtained in step b, 2 μL of forward primer 01-UP1 (10 μM), 2 μL of reverse primer 01-DW1 (10 μM), 1 μL of PhantaMax high-fidelity DNA polymerase, and ddH2O to a final volume of 50 μL.

[0075] The amplification program was as follows: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 sec, annealing at 50℃ for 15 sec, extension at 72℃ for 30 sec, repeated 35 times for denaturation, annealing, and extension, followed by a final extension at 72℃ for 5 min. The PCR products were recovered by 1% low-melting-point agarose gel electrophoresis, dissolved in 30 μL ddH2O, and labeled 01-S1.

[0076] Step d. Primer 01-PIC + (Primer 01-UP1-Primer 01-DW1) + Primer 01-DW165PC The reaction system consisted of: a total volume of 50 μL, 25 μL of 2×PhantaMax buffer, 1 μL of dNTP mixture (10 mM each), 1 μL of PCR product 01-S1 obtained in step c, 2 μL of forward primer 01-PIC (10 μM), 2 μL of reverse primer 01-DW165PC (10 μM), 1 μL of PhantaMax high-fidelity DNA polymerase, and ddH2O to a final volume of 50 μL.

[0077] The amplification program was as follows: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 sec, annealing at 52℃ for 15 sec, extension at 72℃ for 37 sec, repeated 35 times for denaturation, annealing, and extension, followed by a final extension at 72℃ for 5 min. The PCR products were recovered by 1% low-melting-point agarose gel electrophoresis, dissolved in 30 μL ddH2O, and labeled as rhSCF cDNA.

[0078] (3) Use Not I performed enzyme digestion and product recovery on the pGEM-T (Promega) vector and the amplified target gene rhSCF cDNA, and then transformed it into Escherichia coli DH5α strain using T4 ligase.

[0079] (4) After inoculating a single colony of the transformation plate into LB medium (10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride) and culturing at 37°C for 12-16 h, the plasmid was extracted and then... Nco I and Nde I double enzyme digestion identification yielded positive plasmid pGEM-T-act01-165aa. Sequencing of the ligated SEQ ID NO.1 sequence yielded the correctly sequenced pGEM-T-act01-165aa plasmid.

[0080] (5) Use Xho I and Not I performed enzyme digestion and product recovery on the correctly sequenced pGEM-T-act01-165aa plasmid and pPIC9 vector. The ligation was then carried out using T4 ligase. Xba I enzyme digestion confirmed the correct sequence of the pPIC9-act01-165aa plasmid.

[0081] (6) Using S al The pPIC9-act01-165aa plasmid was linearized by restriction enzyme digestion, and the recovered product was electroporated into the Pichia pastoris GS115 host strain. The product was then plated on RD plates (Pichia pastoris regeneration glucose agar: containing 2% (w / v) glucose, 1.34% (w / v) yeast nitrogen base, 0.00004% (w / v) biotin, 1.5% (w / v) agarose, and 18.2% (w / v) sorbitol), yielding the correctly sequenced pPIC9-act01-165aa Pichia pastoris expression strain. The plasmid digestion identification pattern is shown below. Figure 2 As shown.

[0082] like Figure 2 As shown, lane 3 is a DNA marker.

[0083] Lane 1 is the pPIC9 carrier via Xba I. Single enzyme digestion. Xba I is a single restriction site on pPIC9, which can linearize circular vectors and present them as a single, clear band.

[0084] Lane 2 is the recombinant vector pPIC9-act01-165aa Xba I. Single enzyme digestion. Because the target fragment contains... Xba At site I, enzyme digestion produces two DNA fragments of the theoretical size.

[0085] Lane 4 is the pGEMT carrier via Nde I + Nco Double digestion with enzyme I, both digestion sites are located in the multiple cloning site (MCS) region, with very few bases between the sites. After digestion, electrophoresis shows only a linear vector backbone band, which is clear and uniform.

[0086] Lane 5 pairs of recombinant vectors pGEMT-act01-165aa Nde I + Nco Double enzyme digestion. The target gene fragment and vector backbone were digested, and the two bands were clearly distinguishable, consistent with the theoretical size.

[0087] Lane 6 is a PCR product. The rhSCF cDNA fragment amplified by polymerase chain reaction (PCR) shows only a single bright band, indicating good amplification specificity and no non-specific bands.

[0088] Example 2 Fermentation of expression strain The pPIC9-act01-165aa Pichia pastoris expression strain was inoculated into 30 mL of YPD medium as primary culture medium and cultured at 200-350 rpm and 28°C for 14-16 hours. The next day, the primary culture was inoculated into two 300 mL bottles of BMGY medium as secondary culture medium and cultured at 28°C for 14-16 hours until the OD600 value reached 10-15. Then, it was transferred to FBS fermentation medium in a fermenter at 28°C, pH 6.0, and dissolved oxygen controlled at 10%-40%. After the OD600 value reached 100, the temperature was lowered to 22°C, and methanol was added for induction for about 40 hours. Fermentation was then completed, and the fermentation broth was obtained. The expression level of the target protein in the fermentation broth was detected by HPLC-RPC and was not less than 260 mg / L.

[0089] Example 3: Isolation, purification, and quality detection of the target protein The fermentation supernatant obtained from the fermentation broth in Example 2 was diluted 3 times with 5 mM HAc, and the pH of the diluted fermentation supernatant was adjusted to 4.0. The supernatant was then purified by column chromatography. The specific steps are as follows: 1. SP-agarose gel FF ion exchange chromatography column (SPFF), 25 mm in diameter, 60 mL column bed.

[0090] (1) First, use 0.5M NaOH to clean 1CV at a speed not exceeding 250cm / hr, and continue cleaning at a speed not exceeding 125cm / hr for at least 30 minutes.

[0091] (2) Rinse with water for at least 3 CV; use 50 mM NaAc-HAc buffer to adjust pH to 4.0 for at least 3 CV, and the column effluent pH is 4.0 ± 0.5 before use.

[0092] (3) For fermentation supernatant loading, the amount of packing material should not exceed 20 OD / ml, and the sample loading flow rate should not exceed 250 cm / hr.

[0093] (4) The flow rate should not exceed 250 cm / hr. Wash with 50 mM NaAc-HAc and 50 mM (NH4)2SO4 at pH 4.0 for at least 3 CV. Then, elute with 50 mM NaAc-HAc and 300 mM (NH4)2SO4 at pH 4.0 at a flow rate not exceeding 150 cm / hr to collect the target protein. The purification chromatogram is shown below. Figure 3 As shown, impurity proteins in the fermentation broth are removed by a low-salt buffer solution, while the target protein is eluted by a high-salt buffer solution.

[0094] 2. Superdex 75 gel filtration chromatography column, 50 mm in diameter, 1 L column bed.

[0095] (1) First, use 0.5M NaOH to clean 1CV at a speed not exceeding 150cm / hr, and continue cleaning at a speed not exceeding 80cm / hr for at least 30min.

[0096] (2) Rinse with water for at least 1 CV; use 50 mM PBNa pH 7.0 to equilibrate for at least 2.5 CV, and the pH of the column effluent is 7.0 ± 0.5 before use.

[0097] (3) Eluent sample loading on SP-agarose gel FF ion exchange chromatography column, the sample loading amount shall not exceed 5% of the column bed volume, and the flow rate shall not exceed 150 cm / hr.

[0098] (4) Elute the protein with 50 mM PBNa pH 7.0. The purification chromatogram is shown below. Figure 4 As shown.

[0099] (5) Filter sterilize with a 0.22μm filter, dispense, and freeze.

[0100] The purification yield was not less than 150 mg / L of fermentation broth. The protein purity, as determined by SDS-PAGE and HPLC-SEC, was ≥97%. The endotoxin content (LAL method) was less than 0.1 EU / μg. The activity, as determined by the UT-7 cell / MTT method, was comparable to the WHO standard, not less than 1.2 × 10⁻⁶. 6 U / mg. Relevant detection chromatograms are shown below. Figures 5-8 As shown.

[0101] Depend on Figures 5-8 The results show that by using SPFF chromatography column capture and S75 chromatography column purification, and optimizing the SPFF chromatography column capture buffer conditions, the purity can be improved while capturing, and the purity can be made not less than 97% through a two-step method.

[0102] Figure 5 and Figures 9-11 The electrophoresis images of the purified samples shown illustrate the different purification steps, with SPFF column capture and S75 column purification yielding the best purity results. Figure 5 As shown, the target protein and impurity proteins can be well separated through two-step purification.

[0103] Figure 6 The electrophoresis diagram shown indicates that the purity of the prepared rhSCF stock solution (reducing and non-reducing forms) is greater than 97%.

[0104] Figure 7 The HPLC-SEC purity chromatogram shown indicates that the liquid phase purity of the prepared rhSCF stock solution reached 99.2%.

[0105] Figure 8 The activity assay results shown indicate that the activity of the prepared rhSCF stock solution is comparable to that of the WHO standard, and is no less than 1.2 × 10⁻⁶. 6 U / mg.

[0106] Comparative Example 1 The fermentation supernatant was diluted 3-fold with 5 mM HAc, and the pH was adjusted to 4.0 with 2 M acetic acid. The supernatant was then captured using an SP-agarose gel FF ion exchange chromatography column (SPFF). Equilibration was performed using 50 mM NaAc-HAc at pH 4.0, followed by elution with 50 mM Na2HPO4 at pH 9.2. The SPFF eluted sample was purified by loading no more than 10% of the S200 column volume, and the equilibration buffer was 50 mM Na2HPO4 at pH 7.0. Peaks were collected aliquoted.

[0107] The results are as follows Figure 9 As shown, after capture by SPFF chromatography and purification by S200 chromatography, electrophoresis revealed that the protein had impurities that were difficult to remove.

[0108] Comparative Example 2 The fermentation supernatant was diluted 3-fold with 5 mM HAc, and the pH was adjusted to 4.0 with 2 M acetic acid for SPFF capture. Equilibration was performed using 50 mM NaAc-HAc at pH 4.0, followed by elution with 50 mM Na2HPO4 at pH 9.2. The resulting eluted sample was diluted 3-fold with 5 mM PBNa at pH 7.0, and the pH was adjusted to 7.0 with 2 M HCl. The column was purified using a strong anion exchange chromatography (QFF) column. The QFF column was equilibrated with 20 mM PBNa at pH 7.0, followed by gradient elution with 20 mM PBNa and 1 M NaCl at pH 7.0.

[0109] The results are as follows Figure 10 As shown, the SPFF chromatography column captured the protein and the QFF chromatography column was pure, with no obvious separation between impurities and proteins.

[0110] Comparative Example 3 The fermentation supernatant was diluted 3-fold with 5 mM HAc, and the pH was adjusted to 4.0 with 2 M acetic acid before being captured by SPFF chromatography. Equilibration was performed using 50 mM NaAc-HAc at pH 4.0, followed by elution with 50 mM Na2HPO4 at pH 9.2. The eluted sample from the SPFF column was diluted 3-fold with 5 mM PBNa at pH 7.0, and the pH was adjusted to 7.0 with 2 M HCl. The sample was then purified using a DEAE column. The DEAE column was equilibrated with 20 mM PBNa and 20 mM arginine at pH 7.0, followed by elution with a gradient of 20 mM PBNa, 20 mM arginine, and 500 mM NaCl at pH 7.0. Electrophoresis results showed no significant separation of impurities and proteins.

[0111] The results are as follows Figure 11 As shown, the SPFF chromatography column captured the protein and the DEAE chromatography column was pure, with no obvious separation between impurities and proteins.

[0112] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. A gene encoding a human stem cell factor, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

1.

2. A recombinant expression vector, characterized in that, It includes the expression vector and the encoding gene as described in claim 1.

3. The recombinant expression vector according to claim 1, characterized in that, The expression vector is selected from at least one of pPIC9, pPIC9K, pPICZα, and pHIL-S1; Alternatively, the coding gene may be constructed into an expression vector by at least one of PCR, enzyme digestion and ligation, or homologous recombination to form a recombinant expression vector.

4. An engineered bacterial strain, characterized in that, The engineered strain contains the encoding gene as described in claim 1.

5. The engineered strain according to claim 4, characterized in that, The engineered strain was obtained by transforming it into a host strain using a recombinant expression vector containing the encoding gene of claim 1, and then screening for it.

6. The engineered strain according to claim 4, characterized in that, The host strain is selected from at least one of Pichia pastoris strain KM71, strain GS115, and strain X33. Alternatively, the recombinant expression vector may be linearized with an endonuclease and transformed into a host strain, and engineered strains may be obtained by screening with auxotrophic plates and / or resistant plates.

7. A method for preparing human stem cell factors, characterized in that, include: (1) The engineered strain described in claim 4 is cultured in a culture medium until the OD reaches a suitable range; (2) Fermentation broth was obtained through induction culture; (3) The fermentation broth was purified and separated to obtain recombinant human stem cell factor.

8. The preparation method according to claim 7, characterized in that, The culture medium is selected from at least one of YPD medium, BMGY medium, or FBS fermentation medium; Alternatively, the YPD medium contains 1-5% (w / v) yeast extract, 1-5% (w / v) peptone and 1-5% (w / v) glucose; Alternatively, BMGY medium contains 1–5% (w / v) yeast extract, 1–5% (w / v) peptone, 0.1–0.5% (w / v) dipotassium hydrogen phosphate, 1–5% (w / v) potassium dihydrogen phosphate, 1–5% (w / v) yeast nitrogen base, 0.00001–0.00005% (w / v) biotin and 1–5% (v / v) glycerol; Alternatively, the FBS fermentation medium contains 1-5% (w / v) yeast extract, 1-5% (w / v) peptone, 1-5% (v / v) glycerol, 0.1-0.5% (w / v) potassium hydroxide, 0.5-1% (w / v) potassium sulfate, 0.01-0.05% (w / v) calcium sulfate dihydrate, 0.1-0.5% (v / v) phosphoric acid, and 0.1-0.5 mL / L (v / v) polyether defoamer DF-103.

9. The preparation method according to claim 8, characterized in that, In step (1), primary culture was performed in YPD medium at 200-350 rpm and 28±2℃ for 14-16 hours; secondary culture was performed in BMGY medium at 28±2℃ for 14-16 hours until the OD600nm value was 10-15; fermentation was performed in FBS fermentation medium at 28±2℃ and pH 6.0-6.5, with dissolved oxygen controlled at 10%-40%; culture was performed until the OD600nm value was 100-200. Alternatively, in step (2), after the culture in step (1) is completed, the temperature is lowered to 18~22℃, and an inducer is added to induce fermentation for 35~50h to complete the fermentation; Alternatively, in step (2), the inducing agent is methanol.

10. The preparation method according to claim 7, characterized in that, In step (3), the purification and separation is selected from at least one of cation exchange chromatography or molecular sieve chromatography; Alternatively, the cation exchange chromatography may be performed using an SP-agarose gel FF ion exchange chromatography column; Alternatively, the diameter of the SP-agarose gel FF ion exchange chromatography column is 20-30 mm, and the column bed is 50-100 mL; Alternatively, the molecular sieve chromatography may be performed using a Superdex 75 gel filtration chromatography column; Alternatively, the diameter of the Superdex 75 gel filtration chromatography column is 40~60mm, and the column bed is 0.5~2L.

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