CN115838748A

CN115838748A - High-efficiency expression gene of shIL-27 pichia pastoris and expression production method

Info

Publication number: CN115838748A
Application number: CN202211386037.3A
Authority: CN
Inventors: 马杰; 王伟; 焦平; 孙丹丹; 赵赫; 孟祥锋
Original assignee: Jilin University
Current assignee: Saipu Biotechnology Changchun Co ltd
Priority date: 2022-11-07
Filing date: 2022-11-07
Publication date: 2023-03-24
Anticipated expiration: 2042-11-07
Also published as: CN116790644A; CN115838748B

Abstract

The invention provides a high-efficiency expression gene of shIL-27 pichia pastoris and a corresponding expression production method, wherein the high-expression gene of pichia pastoris comprises a Kex2 signal cutting site coding sequence, an IL-27B coding sequence, a linker sequence and an IL-27A coding sequence. The constructed shIL-27 pichia pastoris expression vector is used for transforming pichia pastoris X-33 to carry out extracellular secretion expression, so that the expression quantity of 400mg/L can be obtained. The obtained shIL-27 has good purity and activity, and can be used for antitumor therapy, immunoregulation, inflammation inhibition, weight reduction, diabetes treatment and the like.

Description

High-efficiency expression gene of shIL-27 pichia pastoris and expression production method

The invention belongs to the field of the following:

the invention relates to the field of genetic engineering, in particular to a high-efficiency expression gene of single-chain human interleukin-27 (shIL-27) pichia pastoris and establishment of a corresponding expression production method.

Background art:

in 1996, devergne et al discovered a new EB virus regulated cellular gene, EBI3, and suggested that EBI3 bound to p35 or related molecules in a non-covalent fashion to form a secreted heterodimer. In 2002, pfalaz found a new family member of long-chain four-helix bundle cytokines by database calculation, and designated p28 by a molar mass of 28 as determined by SDS-PAGE. p28 binds to EBI3 to form a soluble heterodimer, interleukin-27 (IL-27). In 1998, sprecher et al succeeded in cloning a novel type I cytokine receptor, which was designated WSX-1 because the c-terminal domain contained a highly conserved Trp-Ser-X-Trp-Ser (WSXWS) sequence. It has structural homology with IL-12 receptor beta 1, IL-12 receptor beta 2, leukemia inhibitory factor receptor, tumor suppressor receptor, gp130 and granulocyte colony stimulating factor receptor, which are IL-6/IL-12 cytokine signaling receptor family members, revealing that WSX-1 plays an important role in immune response regulation. Subsequently, chen et al identified T-cell cytokine receptor (TCCR) (as WSX), confirming that CD4+ T cells, CD8+ T cells, B cells, natural killer cells (NK cells), and macrophages all express TCCR in the spleen of mice. IL-27 binds to the receptor WSX-1 alone but is not sufficient to conduct a signal. If WSX-1 and gp130 constitute a receptor complex, binding to the ligand IL-27, signals can be transmitted downstream, causing a cellular effect.

IL-27 is secreted primarily by antigen presenting cells and is involved in both adaptive and innate immune responses. IL-27 can promote the rapid clonal expansion of the naive CD4+ T cells, the generation of IFN-gamma and the differentiation of Th1, and has important regulation effect on adaptive immune response. Co-expression of WSX-1 and gp130 was found in monocytes, DC cells, T lymphocytes, B lymphocytes, NK cells, mast cells. Previous studies suggest that IL-27 exerts an anti-inflammatory effect by promoting IL-10 production by Treg cells, or that Th 17-associated inflammatory diseases are ameliorated by inhibiting the development of Th17 cells via the STAT1 pathway. Recent studies have shown that neutralization of endogenous IL-27 promotes the production of inflammatory cytokines by monocytes. IL-27 negatively regulates the differentiation of Ly6C + monocytes to Tip-DCs by inhibiting CD4+ T cells from secreting INF-gamma, and improves the inflammatory response of the liver in the process of African trypanosome infection. IL-27 significantly inhibits mast cell activation, reduces the expression level of inflammatory cytokines, and improves mast cell-mediated inflammatory responses. In addition, the mechanism of action of the immunomodulatory drug Resiquimod (Resiquimod) in alleviating allergic bronchial asthma is closely related to IL-27. The resiquimod stimulates antigen presenting cells in the lung of a mouse to secrete IL-27, activates the antigen presenting cells to secrete IFN-gamma so as to inhibit polarization of Th2 cells, and up-regulates PDL-1 on the surface of the antigen presenting cells to enhance immune tolerance, so that the IL-27 has an immune protection effect in Th2 cell-mediated allergic asthma. In mice infected with lymphocytic choriomeningitis virus, the production of IL-27 by effector B cells promotes the survival of virus-specific CD4+ T cells, maintaining follicular helper T cell function. IL-27R signaling on follicular helper T cells drives the production of IFN-. Gamma.and IL-21, and the production of anti-viral antibodies facilitates control of viral infection. The target cells for IL-27 action are not just immune cells. In recent years, research shows that IL-27 shows remarkable antitumor activity in tumors such as melanoma, chronic B lymphocyte leukemia, endometrial cancer, prostate cancer, lung cancer and the like. Experiments prove that IL-27 can directly target subcutaneous fat cells of mice, promote the heat generation of the fat cells through a p38-MAPK-PGC-1 signal pathway, accelerate energy consumption, obviously improve the obesity symptoms of the mice and improve insulin sensitivity. Therefore, IL-27 exerts anti-inflammatory, antiviral and antitumor effects through immune regulation, and also can promote adipocyte thermogenesis to reduce weight, improve obesity and type 2 diabetes.

The discovery of IL-27 has been for nearly 20 years so far, researchers have deeply studied the function of IL-27 in 20 years, find that it has anti-inflammatory, antiviral and anti-tumor effects, can promote adipocyte to produce heat, in order to lose weight, improve functions such as obesity and type 2 diabetes, etc., but its high-efficient preparation problem has not been solved well yet, its preparation is mainly carried on with mammalian cell at present, the output is low with high costs, has greatly limited its actual development and application.

Disclosure of Invention

Aiming at the problems:

the application provides a shIL-27 pichia pastoris high expression gene, which comprises a Kex2 signal cleavage site coding sequence, an IL-27B coding sequence, a linker sequence and an IL-27A coding sequence.

Further, the nucleotide sequence of the gene is SEQ ID NO.1.

On the other hand, the application provides a pichia pastoris strain with high shIL-27 expression, wherein the gene is transferred into the pichia pastoris strain. Preferably, the Pichia pastoris strain is Pichia pastoris X-33.

In another aspect, the present application provides a method for preparing the above strain, comprising:

1) Artificially synthesizing a DNA sequence for coding shIL-27;

2) Constructing an expression cassette of the shIL-27;

3) Constructing an expression vector;

4) Expressing and purifying the shIL-27 protein;

5) The shIL-27 was identified and tested for biological activity.

Further, the expression cassette comprises 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.

Further, the expression vector is a eukaryotic expression vector.

Further, the expression vector is pPICZ alpha.

In another aspect, the application provides an application of the strain in preparing the shIL-27.

Further, BMMY is used for fermentation culture of the strain, the initial pH of the fermentation culture is 4.6, and the fermentation culture time is 72 hours.

Further, the shIL-27 is used for antitumor therapy, immunomodulation, inflammation suppression, weight reduction or diabetes treatment.

The Pichia pastoris in the present application is not limited to X-33, and various Pichia pastoris strains useful for genetic engineering can be used.

The vector is not limited to pPICZ alpha, other vectors, in particular commercially available or self-made vectors suitable for eukaryotic/yeast applications, can also be used for the construction of the strains of the present application; the method of transforming the expression vector is not limited to the electric transformation, and other transformation methods can be routinely evaluated and used by those skilled in the art.

Drawings

FIG. 1: agarose gel electrophoresis pattern of enzyme digestion identification of pPICZ alpha-IL-27 recombinant plasmid. M1: DL15000DNA Marker; m2: DL 2000DNA Marker;1: xho I and Xba I double enzyme digestion results; 2: hind III single cleavage results.

FIG. 2: and (3) extracting pPICZ alpha-IL-27 plasmid to transform yeast genome DNA to carry out PCR screening of agarose gel electrophoresis maps of recombinants. M: DL 2000DNA Marker;1-16: PCR products of different yeast transformant genomes; 17: and (5) negative control.

FIG. 3: SDS-PAGE results of the supernatant of the fermentation broth were sampled at different time points. M: protein Marker;1: supernatant of the culture solution before methanol induction; 2. 3, 4, 5, 6: and (3) inducing and expressing fermentation liquid supernatants for 24h, 48h, 72h, 96h and 120h by using methanol.

FIG. 4: performing SDS-PAGE electrophoresis to screen the optimal induced pH value result of the target protein shIL-27 fusion protein, wherein M is a protein Marker;1: methanol induced pre-supernatant; 2-7: respectively are fermentation liquid supernatants of induced expression under the conditions of pH value of 2.2,2.8,3.4,4.0,4.6 and 5.2.

FIG. 5: detecting the result of SDS-PAGE of the ion exchange chromatography sample, wherein M is a protein Marker;1-6 are samples of fractional elution peaks.

FIG. 6: western Blot detection results of 300mmol/L NaCl elution peak ion exchange chromatography samples, and 1-3 are 300mmol/L NaCl elution peak samples.

FIG. 7 is a schematic view of: the result of the determination of the proliferation activity of the recombinant shIL-27 fusion protein on the tumor cell PC-3.

Detailed Description

The present invention is further specifically illustrated below with reference to specific examples, which are intended to be purely exemplary of the invention and are not intended to limit its scope.

Example 1. Artificially synthesized DNA sequence encoding the shIL-27 fusion protein SEQ ID NO:1

Artificially synthesizing a DNA sequence for encoding the shIL-27 protein according to a literature report, wherein the IL-27B is a mature human IL-27B with a signal peptide removed and comprises 209 amino acid residues; IL-27A is a mature human IL-27A with the signal peptide removed and comprises 215 amino acid residues. The IL-27B and IL-27A sequences are connected by a linker consisting of a sequence of 'GGGSGGGSGGGS', and the amino acid sequences are artificially synthesized according to codons preferred by yeast. The inventor designs 6 DNA sequences in sequence by various algorithms of yeast codon preference, and finally determines that the optimal sequence capable of stably and efficiently expressing the shIL-27 is SEQ ID NO.1 through further screening. When the sequence is synthesized, xhoI site and Kex2 cleavage site Glu-Lys-Arg sequence are introduced upstream of the gene, and stop codon and XbaI site are introduced downstream of the gene. The sequence of the encoded protein is shown in SEQ ID NO. 2.

Example 2 construction of expression vectors

The synthesized gene fragment and the vector pPICZ alpha are subjected to double enzyme digestion by Xho I and Xba I respectively, and are kept at the constant temperature of 37 ℃ overnight. And (4) cutting the enzyme digestion product by agarose gel electrophoresis to obtain a target fragment, and then recovering by using an agarose gel DNA recovery kit. And (3) mixing the recovered target gene subjected to enzyme digestion with a vector according to the weight ratio of 3:1, and connecting overnight at constant temperature by using a T4 DNA Ligase 16 ℃ PCR instrument to construct the pPICZ alpha-shIL-27 recombinant plasmid. And (3) transforming the connecting product into escherichia coli competent cells, uniformly coating the transformed competent cells on a low-salt LB plate culture medium containing Zeocin (25 mu g/mL), and performing inverted culture at 37 ℃ for 12-16h after the bacterial liquid is completely absorbed. 4-8 well-grown single colonies were picked from the transformation plate using a sterile pipette tip, inoculated into 5mL of LB medium containing Zeocin (25. Mu.g/mL), and cultured overnight at 37 ℃ with vigorous shaking (225 rpm). The restriction sites Xho I and Xba I are located at the two ends of the inserted gene fragment. The recombinant vector has two HindIII sites, one of which is located in the inserted target fragment and the other is located on the vector. The pPICZ alpha-IL-27 recombinant plasmid extracted by the kit is subjected to Xho I and Xba I double enzyme digestion and Hind III single enzyme digestion identification. Extracting plasmid DNA by using a rapid plasmid miniprep kit, identifying the recombinant plasmid pPICZ alpha-shIL-27 by respectively carrying out double digestion on Xho I, xba I and Hind III single enzyme, digesting for 4h at 37 ℃, identifying correct clone according to an endonuclease spectrum, and generating two bands at 3500bp and 1300bp positions after carrying out double digestion on Xho I and Xba I. Two bands appear at 3600bp and 1200bp after single enzyme digestion of Hind III (shown in FIG. 1). Selecting the clone with the correct restriction enzyme map, and sending the clone to a sequencing company for sequencing verification.

Example 3 expression and purification of shIL-27 fusion proteins

1. Electrotransformation of Pichia pastoris X-33 and screening of positive transformants

And (3) taking 20 mu g of recombinant plasmid pPICZ alpha-shIL-27 with correct sequencing result, digesting the plasmid by using restriction enzyme Pme I, and electrically transforming the Pichia pastoris X-33 competent cells prepared by a D-sorbitol method. 50-100 mu L of the bacterial liquid is uniformly spread on YPD plates containing Zeocin (100 mu g/mL), inverted cultured in an incubator at 30 ℃ for 2-3 days, and the growth of transformants is observed. Zeocin-resistant clones were selected and inoculated into 5mL YPD medium and shake-cultured at 230rpm for 30h. The cells were collected by centrifugation, and the genomic DNA of yeast was extracted by the "boiling-freeze-boiling" method using a primer P1:5 'TCGCTGTTGACTGTTCTTGGACTTTG-3' (SEQ ID NO. 3) and primer P2:5' (SEQ ID NO. 4), PCR was performed, and the amplified product was subjected to 0.8% agarose gel electrophoresis. Whether a gene fragment of about 414bp could be obtained or not was observed, and whether the gene was integrated into the yeast genome or not was analyzed (see FIG. 2).

2. Induction expression of recombinant pichia pastoris X-33 and screening of high-expression shIL-27 fusion protein strain

(1) Selecting positive recombinant bacteria, inoculating into 10mL BMGY medium, performing shake culture at 30 deg.C for 24h to OD ₆₀₀ Collecting cells when the number of cells reaches 2.0-6.0;

(2) Equal volume (10 mL) of BMMY resuspended cell pellet, cultured with shaking at 30 ℃ and expression induced. During the induction process, methanol is supplemented every 24h until the final concentration is 0.5%, and meanwhile, sterilized distilled water is supplemented, so that the total volume of the fermentation liquor is kept unchanged;

(3) Continuously performing induction culture for 5 days, taking 1mL of fermentation liquor every 24h, centrifuging thalli, separating supernatant and precipitate, performing SDS-PAGE protein analysis on the supernatant, screening a strain with high expression of a target protein, and determining the optimal fermentation time length, wherein the result shows that the expression of the target protein is increased on the induction 1 day, and the expression reaches a peak value on the 3 rd day (as shown in figure 3).

3. Determination and analysis of optimal pH value of pichia pastoris X-33 inducible expression shIL-27 fusion protein

(1) Selecting Pichia pastoris engineering bacteria with high shIL-27 fusion protein expression quantity, and carrying out shake culture in a 10mLYPD culture medium at 30 ℃ and 225rpm for 24h;

(2) Inoculating the amplified Pichia pastoris engineering bacteria in 10mL BMGY, performing shake culture at pH 6.0, 28 ℃ and 220rpm for about 24h to make the Pichia pastoris engineering bacteria OD ₆₀₀ Up to 2.0-6.0. The method ensures that the initial conditions of yeast amplification are the same, and lays a foundation for determining the optimal pH value for induction in the future;

(3) Centrifuging at room temperature (4000 rpm) for 5min, discarding the supernatant, adding 9mL of the unbuffered BMMY, and adding 1mol/L Na in the amount shown in the following table ₂ HPO ₄ Preparing BMMY with different pH values by 0.5mol/L citric acid, performing shake culture at 30 ℃ and 225rpm, supplementing methanol every 24h in the induction process until the final concentration is 0.5%, and supplementing sterilized distilled water to keep the total volume of the fermentation liquor unchanged;

pH value	1mol/L Na ₂ HPO ₄ (μL)	0.5mol/L citric acid (. Mu.L)
			2.2	20	980
2.8	160	840
			3.4	290	710
4.0	390	610
			4.6	470	530
5.2	540	460

(4) The supernatants from each pH sample at 3d were analyzed by SDS-PAGE to determine the pH for optimal induction, which was pH4.6 (FIG. 4).

Through the preliminary optimization of fermentation expression conditions, the result of SDS-PAGE is calculated by Image J software, and the result shows that the expression quantity of 400mg/L can be realized.

Purification of shIL-27 fusion proteins

1) Concentration of fermentation broth

(1) After the fermentation is finished, the bacterial liquid is centrifuged at 4000rpm and 4 ℃ to obtain the supernatant.

(2) The supernatant of the fermentation broth was concentrated by about 10-fold ultrafiltration using a 10kDa ultrafiltration membrane, and subjected to subsequent purification by gel chromatography by replacing the buffer with 50mM phosphate buffer (pH 6.4).

2) Ion exchange chromatography

The SP Sepharose F.F. column was equilibrated with 3-5 bed volumes of 50mmol/L sodium phosphate (pH 6.4) buffer and then loaded, flushed to baseline with 3-5 bed volumes of 50mmol/L phosphate (pH 6.4) buffer, and gradient eluted using 0.1, 0.2, 0.3, 0.4, 0.5, 1mol/LNaCl-50mmol/L sodium phosphate (pH 6.4) eluent in that order.

The protein peaks are collected in parts, and SDS-PAGE determines the peak position of the target protein, so that 0.3mol/L NaCl can better elute the shIL-27 fusion protein, and the target protein with the purity of more than 95% is obtained (as shown in FIG. 5). The fractions containing the target protein were concentrated using an ultrafiltration membrane (cut-off molecular weight 10 kDa).

3) Gel filtration chromatography

The Superdex 75prep grade gel column was washed with 3-5 bed volumes of 50mmol/L sodium phosphate (pH 7.0) buffer until the baseline stabilized. And (3) further purifying the eluent containing the target protein component by using a Superdex 75prep grade gel column, and quantitatively detecting the bioactivity.

Example 4 identification of shIL-27 fusion proteins

The target protein was separated by 10% SDS-PAGE, followed by wet membrane transfer. Activating the PVDF membrane in methanol for 30 seconds before use, clamping and placing the PVDF membrane, the filter paper, the glue, the filter paper and the sponge in a membrane transferring solution in sequence from bottom to top into a membrane transferring groove to avoid generating bubbles, and transferring the PVDF membrane for 30min in a 200mA constant-current ice bath. And (4) taking out the PVDF membrane after the membrane conversion is finished, and rinsing the PVDF membrane in TBST to avoid the influence of the drying of the PVDF on the experimental result. 1% BSA shake-blocking for 1h at room temperature, removing blocking solution, diluting the specific antibody against hIL-27 with 1% BSA according to the antibody specification, incubating overnight with shaking at 4 ℃. After recovery of the primary antibody, the column was washed 3 times with TBST for 10 min/time. The secondary antibodies were diluted proportionally with TBST according to secondary antibody instructions. Incubate at room temperature for 1h. TBST washing 3 times, 10 min/time. And (4) absorbing TBST washing liquor, adding ECL developing solution for incubation for a moment, developing under an imaging instrument, and analyzing the result. The result showed that a band appeared at the expected molecular weight position, indicating that the recombinant protein could bind to the hIL-27 specific antibody (as shown in FIG. 6).

Example 5 detection of the biological Activity of the shIL-27 fusion protein

1) Detection of shIL-27 fusion protein for inhibiting tumor cell proliferation activity

(1) PC-3 cell culture: the culture medium is DMEM/F12 containing 10% (V/V) FBS, and is cultured at 37 ℃ under 5% carbon dioxide;

(2) Cell inoculation: after 24-36 hours of passage, the cells were digested, centrifuged, resuspended in DMEM/F12 medium containing 10% (V/V) FBS, diluted to 3.0X 10 per 100. Mu.L ³ Inoculating the cell with the concentration of each cell in a 96-well plate, adding 100 mu l of the diluted cell suspension into each well, and culturing for 24 hours at 37 ℃ under the condition of 5% carbon dioxide;

(3) Addition of the shIL-27 fusion protein: diluting the recombinant shIL-27 fusion protein into different concentrations (25 mug/mL, 50 mug/mL and 100 mug/mL) by using a culture medium, replacing the original culture medium, adding a culture medium without a reference substance and a test substance into a control group, and culturing for 96 hours at 37 ℃ under the condition of 5% carbon dioxide;

(4) And (3) detecting the activity of the cells: the culture medium was discarded, washed twice with PBS, and then the proliferation of the cells was detected using a cell viability kit. mu.L of maintenance medium containing 20. Mu.L of cell viability assay reagent was added to each well, incubated at 37 ℃ for 1-2 hours under 5% carbon dioxide, and cell proliferation was measured using a microplate reader at 490nm wavelength and recorded and counted (as shown in FIG. 7).

Claims

1. The high-efficiency expression gene of the shIL-27 pichia pastoris is characterized by comprising a Kex2 signal cleavage site coding sequence, an IL-27B coding sequence, a linker sequence and an IL-27A coding sequence.

2. The gene according to claim 1, wherein the nucleotide sequence of the gene is SEQ ID No.1.

3. A Pichia pastoris strain highly expressing shIL-27, characterized in that the gene according to claim 1 or 2 is transferred into the Pichia pastoris strain.

4. The method for preparing the strain according to claim 3, comprising:

1) Artificially synthesizing a DNA sequence for coding shIL-27;

2) Constructing an expression cassette of the shIL-27;

3) Constructing an expression vector;

4) Expressing and purifying the shIL-27 protein;

5) The shIL-27 was identified and tested for biological activity.

5. The method of claim 4, wherein the expression cassette comprises a Kex2 signal cleavage site coding sequence, a coding sequence for IL-27B, a linker sequence, and a coding sequence for IL-27A.

6. The production method according to claim 4 or 5, wherein the expression vector is a eukaryotic expression vector.

7. The production method according to claim 6, wherein the expression vector is pPICZ α.

8. The use of the strain according to claim 3 for the preparation of shIL-27.

9. The use of claim 8, wherein said use comprises fermentatively culturing said strain in BMMY at an initial pH of 4.6 for a fermentation time of 72 hours.

10. The use of claim 8 or 9, wherein the shIL-27 is used for anti-tumor therapy, immunomodulation, inhibition of inflammation, weight loss, or diabetes therapy.