CN116790644A

CN116790644A - High-efficiency expression gene of shIL-27 pichia pastoris and expression production method

Info

Publication number: CN116790644A
Application number: CN202311017272.8A
Authority: CN
Inventors: 马杰; 王伟; 焦平; 孙丹丹; 赵赫; 孟祥锋
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2022-11-07
Filing date: 2022-11-07
Publication date: 2023-09-22
Also published as: CN115838748A; CN115838748B

Abstract

The application provides a high-efficiency expression gene of Pichia pastoris and a corresponding expression production method, wherein the high-efficiency 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. According to the application, the constructed shIL-27 Pichia pastoris expression vector is used for converting 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 anti-tumor treatment, immunoregulation, inflammation inhibition, weight loss, diabetes treatment and other aspects.

Description

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

The present patent application is a divisional application of Chinese patent 202211386037.3 entitled "high-efficiency Pichia pastoris expression Gene and expression production method", which is incorporated herein by reference in its entirety.

The application field:

the application 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.

The background technology is as follows:

in 1996, devergne et al discovered a new cellular gene EBI3 regulated by EB virus and proposed that EBI3 bind to p35 or related molecules in a non-covalent form to form a secreted heterodimer. In 2002, pflanz found a new family member of long-chain four-helix bundle cytokines by database calculations, whose molar mass was 28 as determined by SDS-PAGE, and designated p 28. 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, designated WSX-1, because its c-terminal domain contains a highly conserved Trp-Ser-X-Trp-Ser (WSXWS) sequence. It has structural homology with IL-6/IL-12 cytokine signaling receptor family members IL-12 receptor beta 1, IL-12 receptor beta 2, leukemia inhibitory factor receptor, oncoinhibin receptor, gp130 and granulocyte colony stimulating factor receptor, revealing that WSX-1 has important roles in immune response regulation. Subsequently, chen et al identified T cell cytokine receptors (T-cell cytokine receptor, TCCR) (with WSX), confirming that cd4+ T cells, cd8+ T cells, B cells, natural killer cells (Natural killer cell, NK cells) and macrophages all expressed TCCR in the spleen of mice. IL-27 can bind to receptor WSX-1 alone but is insufficient to signal. If WSX-1 and gp130 constitute a receptor complex, binding to the ligand IL-27 can transmit a signal downstream, causing a cellular effect.

IL-27 is secreted primarily by antigen presenting cells and is involved in adaptive and innate immune responses. IL-27 can promote the rapid cloning and amplification of naive CD4+ T cells, and the production of IFN-gamma and Th1 differentiation have important regulation effect on adaptive immune response. WSX-1 and gp130 co-expression were 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 inhibition of Th17 cell development by STAT1 pathway ameliorates Th 17-related inflammatory diseases. Recent studies have shown that neutralization of endogenous IL-27 promotes the production of inflammatory cytokines by monocytes. IL-27 negatively regulates Ly6C+ monocyte differentiation into Tip-DCs by inhibiting secretion of INF-gamma by CD4+ T cells, thereby improving liver inflammatory response during infection of African trypanosomes. 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) to reduce allergic bronchial asthma is closely related to IL-27. The resiquimod stimulates the antigen presenting cells in the lung of the mice 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, which proves that the IL-27 has an immune protection effect in Th2 cell-mediated allergic asthma. IL-27 produced by effector B cells promotes survival of virus-specific CD4+ T cells upon infection of mice with lymphocytic choriomeningitis virus, maintaining follicular helper T cell function. IL-27R signaling on follicular helper T cells drives IFN-gamma and IL-21 production, which produces antiviral antibodies that facilitate control of viral infection. The target cells for IL-27 action are not only immune cells. In recent years, research shows that IL-27 has obvious anti-tumor activity in melanoma, chronic B lymphocyte leukemia, endometrial cancer, prostate cancer, lung cancer and other tumors. Experiments prove that the 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 path, accelerate energy consumption, obviously improve obesity symptoms of the mice and improve insulin sensitivity. Therefore, IL-27 exerts anti-inflammatory, antiviral and antitumor effects through immunomodulation, and can also promote heat production of adipocytes to reduce weight, improve obesity and type 2 diabetes.

IL-27 has been found for nearly 20 years, researchers have conducted intensive studies on the functions of IL-27 for 20 years, and have found that the IL-27 has anti-inflammatory, antiviral and antitumor effects, and can promote the heat production of fat cells so as to reduce weight, improve obesity, type 2 diabetes and other functions, but the problem of efficient preparation of the IL-27 is not solved well, and the preparation of the IL-27 is mainly conducted by using mammalian cells at present, so that the production is low in cost and the practical development and application of the IL-27 are greatly limited.

Disclosure of Invention

Aiming at the problems as follows:

the application provides a high-expression gene of shIL-27 Pichia pastoris, which comprises a Kex2 signal cutting 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.

In another aspect, the application provides a Pichia pastoris strain with high expression of shIL-27, wherein the gene is transferred. Preferably, the Pichia pastoris strain is Pichia pastoris X-33.

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

1) Artificially synthesizing a DNA sequence for encoding the 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) And identifying the shIL-27 and detecting the biological activity of the shIL-27.

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 ppiczα.

In another aspect, the application provides the use of the strain described above in the preparation of shIL-27.

Further, the above strain was cultivated by fermentation using BMMY in the above application, the initial pH of the fermentation cultivation was 4.6, and the fermentation cultivation time was 72 hours.

Further, the shIL-27 is used for anti-tumor treatment, immunomodulation, inflammation inhibition, weight loss or diabetes treatment.

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

The vector is not limited to ppiczα, other vectors, particularly commercially available or homemade vectors suitable for eukaryotic/yeast use, may also be used in the construction of the strains of the application; the method of transforming the expression vector is not limited to electrotransformation, and other transformation methods may be routinely evaluated for feasibility 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: DL 15000DNA Marker; m2: DL 2000DNA markers; 1: double digestion results of Xho I and Xba I; 2: hindIII single cleavage results.

Fig. 2: extracting pPICZ alpha-IL-27 plasmid transformed saccharomycete genome DNA, and carrying out PCR to screen agarose gel electrophoresis pattern of recombinants. M: DL 2000DNA markers; 1-16: different yeast transformant genome PCR products; 17: negative control.

Fig. 3: SDS-PAGE results of the broth supernatants were sampled at different time points. M: protein markers; 1: supernatant of the culture solution before methanol induction; 2. 3, 4, 5, 6: methanol is used for inducing expression for 24h, 48h, 72h, 96h and 120h of fermentation broth supernatant.

Fig. 4: screening the optimal induced pH value result of the target protein shIL-27 fusion protein by SDS-PAGE electrophoresis, wherein M is a protein Marker;1: supernatant before methanol induction; 2-7: respectively, are the supernatants of the fermentation broth which are induced to express under the condition of pH value 2.2,2.8,3.4,4.0,4.6,5.2.

Fig. 5: SDS-PAGE detection results of the ion exchange chromatography sample, wherein M is a protein Marker;1-6 are fraction eluted peak samples.

Fig. 6: the detection result of Western Blot of the ion exchange chromatography sample with 300mmol/L NaCl elution peak is that with 1-3 being 300mmol/L NaCl elution peak.

Fig. 7: the recombinant shIL-27 fusion protein inhibits the proliferation activity determination result of tumor cells PC-3.

Detailed Description

The application is further specifically illustrated below in connection with specific embodiments, it being understood that the examples are cited merely to illustrate the application and are not intended to limit the scope thereof.

Example 1. Synthetic DNA sequence encoding a sil-27 fusion protein SEQ ID NO:1

Artificially synthesizing a DNA sequence encoding an shIL-27 protein according to a literature report, wherein IL-27B is human mature IL-27B with a signal peptide removed, and comprises 209 amino acid residues; IL-27A is a mature human IL-27A comprising 215 amino acid residues with the signal peptide removed. The IL-27B and IL-27A sequences are linked by a linker composed of a GGGSGGGSGGGS sequence, and the amino acid sequences are artificially synthesized according to codons favored by yeasts. The inventor designs 6 DNA sequences successively through a plurality of algorithms with preferred yeast codons, and finally determines that the optimal sequence capable of stably and efficiently expressing the shIL-27 is SEQ ID NO.1 through further screening. During the synthesis of the sequence, an XhoI site and a Kex2 cleavage site Glu-Lys-Arg sequence are introduced upstream of the gene, and a stop codon and an XbaI site are introduced downstream of the gene. The coded protein sequence is shown as SEQ ID NO. 2.

EXAMPLE 2 construction of expression vectors

The synthesized gene fragment and vector pPICZα were digested with XhoI and XbaI, respectively, and incubated overnight at 37 ℃. The target fragment was excised from the digested product by agarose gel electrophoresis, and then recovered by using an agarose gel DNA recovery kit. The recovered target gene after enzyme digestion and a vector are subjected to enzyme digestion according to the following steps of 3:1, and connecting overnight at constant temperature by using a PCR instrument of a T4 DNA Ligase at 16 ℃ to construct the pPICZ alpha-shIL-27 recombinant plasmid. The ligation product was transformed into E.coli competent cells, and the transformed competent cells were spread on a low-salt LB plate medium containing Zeocin (25. Mu.g/mL) and after complete absorption of the bacterial liquid, were cultured upside down at 37℃for 12-16h. 4-8 single colonies of good growth status were picked from the transformation plate with a sterile gun head and inoculated into 5mL of LB medium containing Zeocin (25. Mu.g/mL) and cultured overnight with vigorous shaking (225 rpm) at 37 ℃. The cleavage sites XhoI and XbaI are located at the two ends of the inserted gene fragment, respectively. The recombinant vector has two HindIII sites, one of which is located in the inserted fragment of interest and the other on the vector. And (3) carrying out XhoI and XbaI double digestion and HindIII single digestion identification on the pPICZ alpha-IL-27 recombinant plasmid extracted by the kit. Plasmid DNA is extracted by using a rapid plasmid small extraction kit, recombinant plasmid pPICZ alpha-shIL-27 is identified by double enzyme digestion of XhoI and XbaI and single enzyme double digestion of HindIII respectively, digestion is carried out for 4 hours at 37 ℃, correct clone is identified according to an endonuclease spectrum, and after double enzyme digestion of XhoI and XbaI, two bands appear at 3500bp and 1300 bp. Two bands appeared at 3600bp and 1200bp after single HindIII cleavage (as shown in FIG. 1). And selecting clones with correct enzyme digestion patterns, and sending the clones to a sequencing company for sequencing verification.

EXAMPLE 3 expression and purification of the shIL-27 fusion protein

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

And taking 20 mug of recombinant plasmid pPICZ alpha-shIL-27 with correct sequencing result, digesting the plasmid by using restriction enzyme Pme I, and electrically transforming Pichia pastoris X-33 competent cells prepared by a D-sorbitol method. And (3) uniformly coating 50-100 mu L of bacterial liquid on a YPD plate containing Zeocin (100 mu g/mL), culturing in a 30 ℃ incubator for 2-3 days in an inverted mode, and observing the growth of the transformant. 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 genomic DNA of yeast was extracted by "boil-freeze-boil" method, using primer P1:5'-TCGCTGTTGACTGTTCTTGGACTTTG-3' (SEQ ID NO. 3) and primer P2:5'-TGTGGAATCTAGCAGCACCTTGTCTC-3' (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 was obtained or not was examined, and whether the gene was integrated into the yeast genome or not was analyzed (see FIG. 2).

2. Inducible 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 culture medium, shake culturing at 30deg.C for 24 hr, and reaching OD ₆₀₀ Collecting cells when the cell number reaches 2.0-6.0;

(2) Equal volumes (10 mL) of BMMY resuspended cell pellet, cultured with shaking at 30℃and induced to express. In the induction process, methanol is supplemented every 24 hours 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) And (3) continuously carrying out induction culture for 5 days, taking 1mL of fermentation liquor every 24 hours, centrifuging thalli, separating supernatant and sediment, using the supernatant for SDS-PAGE protein analysis, screening strains with high expression of target proteins, determining the optimal fermentation time length, and indicating that the expression of target proteins is induced on the 1 st day, and the expression quantity is increased along with the extension of the induction time, wherein the expression quantity reaches a peak value on the 3 rd day (shown in figure 3).

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

(1) Selecting Pichia pastoris engineering bacteria with high expression quantity of the shIL-27 fusion protein, and culturing for 24 hours in a 10mLYPD culture medium at 30 ℃ and 225rpm in an oscillating way;

(2) Inoculating amplified Pichia pastoris engineering bacteria into 10mL BMGY, and shake culturing at pH 6.0 and 28deg.C and 220rpm for about 24 hr to make 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 the subsequent determination of the optimal pH value for inductionA foundation;

(3) Centrifuging at room temperature (4000 rpm) for 5min, discarding supernatant, adding 9mL of unbuffered BMMY, and adding 1mol/L Na according to the amount shown in the following table ₂ HPO ₄ And 0.5mol/L citric acid to prepare BMMY with different pH values, and carrying out shaking culture at 30 ℃ and 225rpm, wherein methanol is supplemented to a final concentration of 0.5% every 24h in the induction process, and meanwhile, sterilized distilled water is supplemented, so that the total volume of fermentation liquor is kept unchanged;

pH value of	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) SDS-PAGE analysis was performed on the supernatants of each pH sample at 3d to determine the pH of the best induction, which showed the best induction to be pH4.6 (as shown in FIG. 4).

Through preliminary optimization of fermentation expression conditions, SDS-PAGE results are calculated through Image J software, and the results show that the expression quantity of 400mg/L can be realized.

Purification of the shIL-27 fusion protein

1) Concentrating the fermentation liquor

(1) After the fermentation, the bacterial liquid was centrifuged at 4000rpm at 4℃to obtain a supernatant.

(2) The supernatant of the fermentation broth was concentrated by ultrafiltration using an ultrafiltration membrane of 10kDa by about 10 times, and then subjected to subsequent gel chromatography purification by replacing the supernatant 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, then loaded, flushed to baseline with 3-5 bed volumes of 50mmol/L phosphate (pH 6.4) buffer, and eluted with 0.1, 0.2, 0.3, 0.4, 0.5, 1mol/LNaCl-50mmol/L sodium phosphate (pH 6.4) sequentially.

The peak positions of the target proteins are determined by SDS-PAGE after the protein peaks are collected, and the result shows that 0.3mol/L NaCl can well elute the shIL-27 fusion protein to obtain the target proteins with the purity of more than 95 percent (shown in figure 5). The fractions containing the protein of interest were concentrated using ultrafiltration membranes (molecular weight cut-off 10 kDa).

3) Gel filtration chromatography

The Superdex 75prep grade gel column was washed with 3-5 bed volumes of 50mmol/L sodium phosphate buffer (pH 7.0) to baseline stability. The eluent containing the target protein component is subjected to further purification treatment by using a Superdex 75prep grade gel column, and is quantitatively used for biological activity detection.

EXAMPLE 4 identification of the shIL-27 fusion protein

The target protein was separated by 10% SDS-PAGE and then wet-transferred. Before using, the PVDF film is activated in methanol for 30 seconds, and is clamped in a film transferring liquid in the order of sponge, filter paper, glue, PVDF film, filter paper and sponge from bottom to top, and then is put into a film transferring groove, so that bubbles are avoided, and a 200mA constant flow ice bath film transferring is performed for 30 minutes. And taking out the PVDF membrane after membrane transfer, and rinsing in TBST to prevent PVDF drying from affecting the experimental result. Blocking was performed for 1h at room temperature with a 1% BSA shaker, blocking solution was removed, and anti-hIL-27 specific antibody was diluted with 1% BSA according to the antibody specification ratio, and incubated overnight at 4 ℃. After recovery of the primary antibody, TBST was washed 3 times for 10 min/time. The secondary antibodies were diluted proportionally using TBST according to the secondary antibody instructions. Incubate for 1h at room temperature. TBST was washed 3 times for 10 min/time. The TBST wash was removed, incubated for a short time with ECL developer, developed under an imaging instrument, and the results analyzed. As a result, bands were present at the expected molecular weight positions, indicating that the recombinant protein could bind to hIL-27 specific antibodies (as shown in FIG. 6).

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

1) Detection of tumor cell proliferation inhibition activity by shIL-27 fusion protein

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

(2) Cell inoculation: after 24-36 hours of passage, cells were centrifuged by digestion, resuspended in DMEM/F12 medium containing 10% (V/V) FBS, and diluted to 3.0X10 per 100. Mu.L ³ The concentration of individual cells is inoculated in a 96-well plate, 100 mu l of the diluted cell suspension is added into each well, and the culture is carried out for 24 hours under the conditions of 37 ℃ and 5% carbon dioxide;

(3) Addition of a shIL-27 fusion protein: diluting 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) Detecting cell activity: the 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 activity detection reagent was added to each well, incubated at 37℃under 5% carbon dioxide for 1-2 hours, and cell proliferation was detected at 490nm by using an enzyme-labeled instrument, and recorded and calculated (as shown in FIG. 7).

Claims

1. The high-efficiency expression gene of the Pichia pastoris is characterized by comprising a Kex2 signal cutting site coding sequence, an IL-27B coding sequence, a linker sequence and an IL-27A coding sequence; the nucleotide sequence of the gene is SEQ ID NO.1; the SEQ ID NO.1 stably and efficiently expresses the shIL-27 to reach the expression quantity of 400 mg/L.

2. A pichia pastoris strain highly expressing sil-27, wherein the pichia pastoris strain has been transformed with the gene according to claim 1.

3. The application of the strain according to claim 2 in preparing the shIL-27, wherein the high-expression shIL-27 Pichia pastoris strain is transformed with a shIL-27 Pichia pastoris efficient expression gene with a nucleotide sequence shown as SEQ ID NO.1, and the shIL-27 Pichia pastoris efficient expression gene comprises a Kex2 signal cleavage site coding sequence, an IL-27B coding sequence, a linker sequence and an IL-27A coding sequence;

the preparation method of the Pichia pastoris strain with high expression of the shIL-27 comprises the following steps:

1) The high-efficiency expression gene of the Pichia pastoris with the artificial synthetic sequence of SEQ ID NO.1;

2) Constructing a pPICZ alpha expression vector;

3) Expressing and purifying the shIL-27 protein;

4) Identifying the shIL-27 and detecting the biological activity thereof;

in the application, the BMMY culture medium is used for fermenting and culturing the Pichia pastoris strain with high expression of the shIL-27, the initial pH of the fermenting and culturing is 4.6, and the fermenting and culturing time is 72 hours.

4. The use according to claim 3, wherein the hil-27 is for anti-tumor therapy, immunomodulation, inhibition of inflammation, weight loss or diabetes therapy.