Strain for transforming shil-27 pichia pastoris high-efficiency expression gene and application thereof

Abstract

The present application provides shIL-27 Pichia high-efficiency expression gene and the corresponding expression production method, the Pichia high expression gene includes Kex2 signal cleavage site coding sequence, the coding sequence of IL-27B, linker sequence and the coding sequence of IL-27A.The present application is transformed by using the constructed shIL-27 Pichia expression vector, and the extracellular secretion expression of Pichia X-33 can obtain 400mg / L expression amount.The obtained shIL-27 has good purity and activity, and can be used in anti-tumor treatment, immunoregulation, inflammation inhibition, weight loss and diabetes treatment and the like.

Description

[0001] This patent application is a divisional application of Chinese Patent 202211386037.3 entitled “High-efficiency expression of shIL-27 Pichia pastoris gene and expression production method”, the full text of which is incorporated herein by reference. Field of Invention: This invention relates to the field of genetic engineering, and in particular to the establishment of a highly efficient expression gene for single-stranded human interleukin-27 (shIL-27) in Pichia pastoris and the corresponding expression and production method. Background technology: In 1996, Devergne et al. discovered a novel cellular gene, EBI3, regulated by Epstein-Barr virus (EBV), and proposed that EBI3 binds non-covalently to p35 or related molecules to form a secreted heterodimer. In 2002, Pflanz discovered a new family member of long-chain quadrature helical bundle cytokines using database calculations. Its molar mass was determined to be 28 by SDS-PAGE, and it was named p28. p28 binds to EBI3 to form a soluble heterodimer, interleukin-27 (IL-27). In 1998, Sprecher et al. successfully cloned a new type I cytokine receptor, named WSX-1 because its C-terminal domain contains a highly conserved Trp-Ser-X-Trp-Ser (WSXWS) sequence. It shares structural homology with members of the IL-6 / IL-12 cytokine signaling receptor family, including IL-12 receptor β1, IL-12 receptor β2, leukemia inhibitory factor receptor, oncogene receptor, gp130, and granulocyte colony-stimulating factor receptor, revealing the important role of WSX-1 in immune response regulation. Subsequently, Chen et al. identified the T-cell cytokine receptor (TCCR) (same as WSX) and confirmed that CD4+ T cells, CD8+ T cells, B cells, natural killer cells (NK cells), and macrophages in mouse spleen all express TCCR. IL-27 can bind to the receptor WSX-1 alone but is insufficient for signal transduction. However, when WSX-1 and gp130 form a receptor complex and bind to the ligand IL-27, downstream signal transduction can occur, inducing cellular effects.

[0002] IL-27 is primarily secreted by antigen-presenting cells and participates in both adaptive and innate immune responses. IL-27 promotes the rapid clonal expansion of naive CD4+ T cells, IFN-γ production, and Th1 differentiation, playing a crucial regulatory role in adaptive immune responses. WSX-1 and gp130 are co-expressed in monocytes, dendritic cells (DCs), T lymphocytes, B lymphocytes, NK cells, and mast cells. Previous studies suggested that IL-27 exerts its anti-inflammatory effect by promoting IL-10 production from Treg cells or by inhibiting Th17 cell development through the STAT1 pathway, thus improving Th17-related inflammatory diseases. Recent studies have shown that neutralizing endogenous IL-27 promotes the production of inflammatory cytokines by monocytes. IL-27 negatively regulates the differentiation of Ly6C+ monocytes into Tip-DCs by inhibiting IFN-γ secretion from CD4+ T cells, thereby improving the hepatic inflammatory response during Trypanosoma africanum infection. IL-27 significantly inhibits mast cell activation, reduces the expression levels of inflammatory cytokines, and improves mast cell-mediated inflammatory responses. Furthermore, the mechanism of action of the immunomodulatory drug resiquimod in alleviating allergic bronchial asthma is closely related to IL-27. Resiquimod stimulates antigen-presenting cells in the lungs of mice to secrete IL-27, activates antigen-presenting cells to secrete IFN-γ, thereby inhibiting Th2 cell polarization and upregulating PDL-1 on their surface to enhance immune tolerance, indicating that IL-27 has an immunoprotective role in Th2 cell-mediated allergic asthma. In mice infected with lymphocytic choroid plexus meningitis virus, IL-27 produced by effector B cells promotes the survival of virus-specific CD4+ T cells and maintains the function of follicular helper T cells. IL-27R signaling on follicular helper T cells drives the production of IFN-γ and IL-21, generating antiviral antibodies to promote the control of viral infection. The target cells of IL-27 are not limited to immune cells. Recent studies have shown that IL-27 exhibits significant anti-tumor activity in tumors such as melanoma, chronic B-lymphoblastic leukemia, endometrial cancer, prostate cancer, and lung cancer. Experiments have confirmed that IL-27 can directly target subcutaneous adipocytes in mice, promoting adipocyte thermogenesis and accelerating energy consumption through the p38-MAPK-PGC-1 signaling pathway, significantly improving obesity symptoms and increasing insulin sensitivity in mice. Therefore, IL-27 exerts anti-inflammatory, antiviral, and anti-tumor effects through immunomodulation, and can also promote adipocyte thermogenesis to aid in weight loss, improve obesity, and alleviate type 2 diabetes.

[0003] Nearly 20 years have passed since the discovery of IL-27. During these 20 years, researchers have conducted in-depth studies on the functions of IL-27, discovering that it has anti-inflammatory, antiviral and anti-tumor effects, and can also promote thermogenesis in adipocytes to help with weight loss, improve obesity and type 2 diabetes. However, the problem of its efficient preparation has not yet been well solved. At present, its preparation is mainly carried out using mammalian cells, which results in high yield and low cost, which greatly limits its practical development and application. Summary of the Invention

[0004] Regarding the above issues: This application provides a shIL-27 Pichia pastoris high-expression gene, which includes a Kex2 signal cleavage site coding sequence, an IL-27B coding sequence, a linker sequence, and an IL-27A coding sequence.

[0005] Furthermore, the nucleotide sequence of the gene is SEQ ID NO.1.

[0006] On the other hand, this application provides a Pichia pastoris strain that highly expresses shIL-27, wherein the aforementioned gene has been introduced. Preferably, the Pichia pastoris strain is Pichia pastoris X-33.

[0007] On the other hand, this application provides a method for preparing the above-mentioned strain, including: 1) The DNA sequence encoding shIL-27 was artificially synthesized; 2) Constructing the expression cassette for shIL-27; 3) Constructing expression carriers; 4) Expression and purification of shIL-27 protein; 5) Identify shIL-27 and test its biological activity.

[0008] Furthermore, the expression cassette includes a Kex2 signal cleavage site coding sequence, an IL-27B coding sequence, a linker sequence, and an IL-27A coding sequence; the nucleotide sequence of the expression cassette is SEQ ID NO.1.

[0009] Furthermore, the expression vector is a eukaryotic expression vector.

[0010] Furthermore, the expression vector is pPICZα.

[0011] On the other hand, this application provides the application of the above-mentioned strain in the preparation of shIL-27.

[0012] Furthermore, the above-mentioned strain was cultured using BMMY fermentation in the application, with an initial pH of 4.6 and a fermentation time of 72 hours.

[0013] Furthermore, shIL-27 is used for anti-tumor therapy, immune modulation, anti-inflammatory purposes, weight loss, or diabetes treatment.

[0014] The Pichia pastoris used in this application is not limited to X-33; any Pichia pastoris strain that can be used for genetic engineering may be used.

[0015] The vector is not limited to pPICZα; other vectors, especially commercially available or self-made vectors suitable for eukaryotes / yeasts, can also be used to construct the strains of this application. The method of transforming the expression vector is not limited to electroconversion; other transformation methods can be routinely evaluated for feasibility and used by those skilled in the art. Attached Figure Description

[0016] Figure 1 Agarose gel electrophoresis pattern of enzyme digestion identification of pPICZα-IL-27 recombinant plasmid. M1: DL 15000 DNA Marker; M2: DL 2000 DNA Marker; 1: Xho I, Xba I double digestion results; 2: Hind III single digestion results.

[0017] Figure 2 Agarose gel electrophoresis patterns of yeast genomic DNA extracted from pPICZα-IL-27 plasmid for PCR screening of recombinants. M: DL 2000 DNA Marker; 1-16: PCR products of different yeast transformants; 17: Negative control.

[0018] Figure 3 SDS-PAGE results of fermentation broth supernatant taken at different time points. M: Protein Marker; 1: Culture supernatant before methanol induction; 2, 3, 4, 5, 6: Fermentation broth supernatant after methanol induction for 24h, 48h, 72h, 96h, and 120h.

[0019] Figure 4 Results of SDS-PAGE electrophoresis screening for the optimal induction pH of the target protein shIL-27 fusion protein, where M is the protein marker; 1: supernatant before methanol induction; 2-7: fermentation broth supernatant induced under pH conditions of 2.2, 2.8, 3.4, 4.0, 4.6, and 5.2, respectively.

[0020] Figure 5 SDS-PAGE results of ion exchange chromatography samples, M is the protein marker; 1-6 are fractional elution peaks of the samples.

[0021] Figure 6Western blotting results of ion exchange chromatography samples with elution peaks of 300 mmol / L NaCl. Samples 1-3 have elution peaks of 300 mmol / L NaCl.

[0022] Figure 7 Results of assay of the inhibitory activity of recombinant shIL-27 fusion protein on PC-3 tumor cell proliferation. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the embodiments are used only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0024] Example 1. Synthetic DNA sequence encoding shIL-27 fusion protein SEQ ID NO: 1 According to literature reports, DNA sequences encoding the shIL-27 protein were artificially synthesized. IL-27B is a human-derived mature IL-27B with the signal peptide removed, containing 209 amino acid residues; IL-27A is a human-derived mature IL-27A with the signal peptide removed, containing 215 amino acid residues. The IL-27B and IL-27A sequences are linked by a linker consisting of the "GGGSGGGSGGGS" sequence. The amino acid sequences were artificially synthesized based on yeast-preferred codons. The inventors designed six DNA sequences using various yeast codon preference algorithms. After further screening, the optimal sequence for stable and efficient expression of shIL-27 was finally determined as SEQ ID NO. 1. During sequence synthesis, an XhoI site and a Kex2 cleavage site Glu-Lys-Arg sequence were introduced upstream of the gene, and a stop codon and an XbaI site were introduced downstream of the gene. The encoded protein sequence is shown in SEQ ID NO. 2.

[0025] Example 2. Construction of expression vector The synthesized gene fragment and vector pPICZα were digested with XhoⅠ and XbaⅠ respectively, and incubated overnight at 37℃. The digestion products were excised using agarose gel electrophoresis to remove the target fragment, which was then recovered using an agarose gel DNA recovery kit. The recovered target gene and vector were mixed at a 3:1 molar ratio and ligated overnight using T4 DNA Ligase ligase in a PCR instrument at 16℃ to construct the pPICZα-shIL-27 recombinant plasmid. The ligation product was transformed into competent E. coli cells. The transformed competent cells were evenly spread on low-salt LB agar plates containing Zeocin (25 μg / mL) and incubated upside down at 37℃ for 12-16 h after complete absorption. Using a sterile pipette tip, 4-8 healthy single colonies were picked from the transformation plate and inoculated into 5 mL of LB agar containing Zeocin (25 μg / mL), and incubated overnight at 37℃ with vigorous shaking (225 rpm). The restriction enzyme sites XhoⅠ and XbaⅠ are located at opposite ends of the inserted gene fragment. The recombinant vector contains two HindⅢ restriction sites, one located within the inserted target fragment and the other on the vector itself. The pPICZα-IL-27 recombinant plasmid extracted using the kit was identified by double digestion with XhoⅠ and XbaⅠ and single digestion with HindⅢ. Plasmid DNA was extracted using a rapid plasmid miniprep kit, and the recombinant plasmid pPICZα-shIL-27 was identified by double digestion with XhoⅠ and XbaⅠ and single digestion with HindⅢ. After digestion at 37℃ for 4 h, the correct clone was identified based on the restriction enzyme profile. After double digestion with XhoⅠ and XbaⅠ, two bands appeared at 3500 bp and 1300 bp. After single digestion with HindⅢ, two bands appeared at 3600 bp and 1200 bp (e.g., ...). Figure 1 (As shown). Select clones with correct restriction enzyme digestion patterns and send them to a sequencing company for sequencing verification.

[0026] Example 3. Expression and purification of shIL-27 fusion protein 1. Electroporation of Pichia pastoris X-33 and screening for positive transformants. Take 20 μg of the recombinant plasmid pPICZα-shIL-27 with correct sequencing results, digest the plasmid with restriction endonuclease Pme I, and electroporate it into Pichia pastoris X-33 competent cells prepared by the D-sorbitol method. Spread 50-100 μL of bacterial culture evenly on YPD plates containing Zeocin (100 μg / mL), and incubate upside down at 30℃ for 2-3 days, observing the growth of transformants. Select Zeocin-resistant clones and inoculate them into 5 mL of YPD medium, shaking at 230 rpm for 30 h. Collect the bacterial cells by centrifugation, and extract the yeast genomic DNA using the "boil-freeze-boil" method. Perform PCR using primers P1: 5'-TCGCTGTTGACTGTTCTTGGACTTTG-3' (SEQ ID NO.3) and primer P2: 5'-TGTGGAATCTAGCAGCACCTTGTCTC-3' (SEQ ID NO.4), and perform 0.8% agarose gel electrophoresis on the amplified products. Observe whether a gene fragment of approximately 414 bp can be obtained, and analyze whether the gene is integrated into the yeast genome (e.g., Figure 2 (As shown).

[0027] 2. Induction of recombinant Pichia pastoris X-33 expression and screening of strains expressing high levels of shIL-27 fusion protein (1) Select positive recombinant bacteria and inoculate them into 10 mL of BMGY medium. Incubate at 30 °C with shaking for 24 h until OD reaches 0.5. 600 Cells were collected when the pH reached 2.0-6.0. (2) Resuspend the cell pellet in an equal volume (10 mL) of BMMY, and culture at 30 °C with shaking to induce expression. During the induction process, methanol was added every 24 h until the final concentration reached 0.5%, and sterile distilled water was added at the same time to keep the total volume of the fermentation broth constant; (3) After continuous induction culture for 5 days, 1 mL of fermentation broth was taken every 24 hours, the cells were centrifuged, and the supernatant and precipitate were separated. The supernatant was used for SDS-PAGE protein analysis to screen strains that highly express the target protein and to determine the optimal fermentation time. The results showed that the target protein was expressed on the first day of induction, and the expression level increased with the extension of induction time, reaching its peak on the third day (e.g., Figure 3 (As shown).

[0028] 3. Determination and analysis of the optimal pH value for Pichia pastoris X-33-induced expression of shIL-27 fusion protein (1) Select Pichia pastoris engineered strain with high expression of shIL-27 fusion protein and culture it in 10mLYPD medium at 30℃ and 225rpm for 24h; (2) The amplified Pichia pastoris engineered strain was inoculated into 10 mL of BMGY at pH 6.0 and 28 °C. Incubate at 220 rpm with shaking for approximately 24 hours to allow its OD to adjust. 600 The pH ranges from 2.0 to 6.0. This method ensures consistent starting conditions for yeast amplification, laying the foundation for determining the optimal induction pH later. (3) Centrifuge at room temperature (4000 rpm) for 5 min, discard the supernatant, add 9 mL of the aforementioned BMMY without buffer, add 1 mol / L Na2HPO4 and 0.5 mol / L citric acid according to the table below to prepare BMMY with different pH values, culture at 30℃ and 225 rpm with shaking, add methanol every 24 h during the induction process until the final concentration is 0.5%, and add sterile distilled water at the same time to keep the total volume of fermentation broth constant;

[0029] (4) SDS-PAGE analysis was performed on the supernatant of each pH sample on day 3 to determine the optimal induction pH. The results showed that the optimal induction pH was pH 4.6 (e.g., ...). Figure 4 (As shown).

[0030] After preliminary optimization of fermentation expression conditions, the results of SDS-PAGE were calculated using ImageJ software, and the results showed that an expression level of 400 mg / L could be achieved.

[0031] 4. Purification of shIL-27 fusion protein 1) Concentration of fermentation broth (1) After fermentation, centrifuge the bacterial solution at 4°C and 4000 rpm to collect the supernatant.

[0032] (2) The supernatant of the fermentation broth was concentrated by ultrafiltration using a 10 kDa ultrafiltration membrane by about 10 times, and the buffer was replaced with 50 mM phosphate buffer (pH 6.4) for subsequent gel chromatography purification.

[0033] 2) Ion exchange chromatography Equilibrate the SP Sepharose FF column with 3-5 column volumes of 50 mmol / L sodium phosphate (pH 6.4) buffer, then load the sample and flush to baseline with 3-5 column volumes of 50 mmol / L phosphate (pH 6.4) buffer. Perform gradient elution with 0.1, 0.2, 0.3, 0.4, 0.5, and 1 mol / L NaCl-50 mmol / L sodium phosphate (pH 6.4) eluent.

[0034] The protein peaks were collected separately, and SDS-PAGE was used to determine the peak position of the target protein. The results showed that 0.3 mol / L NaCl could effectively elute the shIL-27 fusion protein, obtaining the target protein with a purity greater than 95% (e.g., ...). Figure 5(As shown). The fraction containing the target protein was concentrated using an ultrafiltration membrane (molecular weight cutoff of 10 kDa).

[0035] 3) Gel filtration chromatography Wash the Superdex 75 prepgrade gel column with 3-5 column volumes of 50 mmol / L sodium phosphate (pH 7.0) buffer until baseline stable. Further purify the eluent containing the target protein fraction using a Superdex 75 prepgrade gel column, and then quantify it for bioactivity assay.

[0036] Example 4. Identification of shIL-27 fusion protein The target protein was separated by 10% SDS-PAGE, followed by wet transfer. Before use, the PVDF membrane was activated in methanol for 30 seconds. In the transfer buffer, the membrane was placed in the transfer tank in the following order from bottom to top: sponge, filter paper, gel, PVDF membrane, filter paper, sponge, avoiding air bubbles. Transfer was performed at a constant current of 200 mA on ice for 30 minutes. After transfer, the PVDF membrane was removed and rinsed in TBST to prevent drying and affecting the experimental results. Blocking was performed at room temperature with 1% BSA on a shaker for 1 hour. The blocking solution was removed, and the anti-hIL-27 specific antibody was diluted with 1% BSA according to the antibody instructions. The membrane was incubated overnight at 4°C with shaking. After primary antibody recovery, the membrane was washed three times with TBST for 10 minutes each time. The secondary antibody was diluted with TBST according to the instructions. The membrane was incubated at room temperature for 1 hour. The membrane was washed three times with TBST for 10 minutes each time. The TBST wash was discarded, and ECL developing solution was added and incubated briefly. The membrane was then developed under imaging conditions, and the results were analyzed. The result showed a band at the expected molecular weight position, indicating that the recombinant protein can bind to the hIL-27 specific antibody (e.g., Figure 6 (As shown).

[0037] Example 5. Detection of the bioactivity of shIL-27 fusion protein 1) Detection of the inhibitory activity of shIL-27 fusion protein on tumor cell proliferation (1) PC-3 cell culture: DMEM / F12 containing 10% (V / V) FBS was used as the culture medium and cultured at 37℃ and 5% carbon dioxide. (2) Cell seeding: After passage for 24-36 hours, the cells were digested and centrifuged, resuspended in DMEM / F12 medium containing 10% (V / V) FBS, and diluted to a concentration of 3.0 × 10⁻⁶ cells per 100 μL. 3 The concentration of cells was seeded into 96-well plates, with 100 μl of the above-diluted cell suspension added to each well, and cultured at 37°C and 5% CO2 for 24 hours. (3) Add shIL-27 fusion protein: Dilute the recombinant shIL-27 fusion protein to different concentrations (25 μg / mL, 50 μg / mL, 100 μg / mL) with culture medium and replace the original culture medium. Add culture medium without reference and test samples to the control group. Incubate at 37°C and 5% carbon dioxide for 96 hours. (4) Cell viability detection: Discard the culture medium, wash twice with PBS, and then use a cell viability assay kit to detect cell proliferation. Add 100 μL of maintenance medium containing 20 μL of cell viability assay reagent to each well, incubate at 37°C and 5% CO2 for 1-2 hours, and detect cell proliferation at 490 nm using a microplate reader, recording and calculating (e.g., ...). Figure 7 (As shown).

Claims

1. A Pichia pastoris strain that highly expresses shIL-27, characterized in that, The Pichia pastoris strain was transformed with the shIL-27 Pichia pastoris high-efficiency expression gene, which contains the coding sequence for the Kex2 signal cleavage site, the coding sequence for IL-27B, the linker sequence, and the coding sequence for IL-27A; the nucleotide sequence of the gene is SEQ ID NO.1; SEQ ID NO.1 stably and efficiently expresses shIL-27 to an expression level of 400 mg / L; The method for preparing the Pichia pastoris strain that highly expresses shIL-27 includes: 1) The artificially synthesized shIL-27 Pichia pastoris gene with the sequence SEQ ID NO.1 is expressed at a high efficiency. 2) Constructing an expression carrier: The synthesized gene fragment and vector pPICZα were digested with XhoⅠ and XbaⅠ respectively, and incubated overnight at 37℃. The digestion products were excised by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit. The recovered digested target gene and vector were mixed at a molar ratio of 3:1 and ligated overnight at 16℃ using T4 DNA Ligase ligase in a PCR instrument to construct the pPICZα-shIL-27 recombinant plasmid. 3) Expression and purification of shIL-27 protein; 4) Identify shIL-27 and test its biological activity; Pichia pastoris specifically refers to Pichia pastoris X-33.

2. The application of the strain according to claim 1 in the preparation of shIL-27.

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