CN118909077A - Preparation method of tumor suppressor M - Google Patents

Abstract

The invention belongs to the field of biotechnology, and provides a preparation method of Oncoinhibin M, which comprises the steps of synthesizing a nucleic acid construct corresponding to Oncoinhibin M recombinant protein, constructing the nucleic acid construct on a eukaryotic expression vector to obtain an Oncoinhibin M recombinant expression vector, and transfecting HEK293 cells with the Oncoinhibin M recombinant expression vector through a transient expression system; culturing transfected HEK293 cells, centrifuging, collecting supernatant, and filtering and purifying to obtain Oncoinhibin M. The invention adopts transient transfection technique, which has short time and can obtain a large amount of Oncoinhibin M within 2 weeks; the purity of the product obtained by the affinity chromatography is up to more than 95%, and the method provides support for research of tumor formation, immunoregulation, diagnosis, targeted treatment of diseases such as tumor and the like.

Description

A preparation method of oncostatin M

Technical Field

The invention belongs to the field of biotechnology and relates to a method for preparing oncostatin M.

Background Art

Oncostatin M (OSM) is a pleiotropic cytokine that belongs to the interleukin 6 (IL-6) family. Oncostatin M binds to its receptor gp130 and recruits OSMRβ or LIFRβ, thereby activating multiple signaling pathways, including activation of Janus kinase (JAK) signal transducer, transcription activator (STAT) signaling pathway, mitogen-activated protein kinase (MAPK), JNK and PI3K/AKT signaling pathways. These signaling pathways play an important regulatory role in cell proliferation, growth and differentiation, and thus participate in a variety of inflammatory responses, such as wound healing, liver regeneration and bone remodeling.

Oncostatin M has been identified as an important cause of many inflammatory diseases, including arthritis, inflammatory bowel disease, lung and skin diseases, cardiovascular diseases, and COVID-19. In addition, Oncostatin M is also considered to be an important cause of many cancers, such as breast cancer, cervical cancer, ovarian cancer, testicular cancer, colon cancer, gastrointestinal cancer, brain cancer, lung cancer, skin cancer, etc. However, there is currently no approved treatment for Oncostatin M, and its related mechanisms and clinical applications also need to be further studied.

Currently, there are no cases of obtaining oncostatin M through recombinant expression on the market, so there is a need for a method to quickly obtain a large amount of oncostatin M to provide more research samples for scientific research or clinical research.

Summary of the invention

In view of the deficiencies in the prior art, the present invention provides a method for preparing oncostatin M, and detects and evaluates the activity of the oncostatin M prepared thereby. The method of the present invention rapidly obtains a large amount of highly active oncostatin M through the HEK293 eukaryotic expression system, providing good support for the use of related proteins in scientific and clinical research.

The technical solution provided by the present invention is as follows:

A method for preparing oncostatin M comprises the steps of synthesizing a nucleic acid construct corresponding to an oncostatin M recombinant protein, constructing the nucleic acid construct onto a eukaryotic expression vector, and obtaining an oncostatin M recombinant expression vector; transfecting the oncostatin M recombinant expression vector into HEK293 cells through a transient expression system; culturing the transfected HEK293 cells, collecting the supernatant after centrifugation, and filtering and purifying to obtain oncostatin M; the sequence of the nucleic acid construct corresponding to the oncostatin M recombinant protein is shown in SEQ ID NO:5.

Furthermore, the amino acid sequence of the oncostatin M recombinant protein is shown in SEQ ID NO:1.

Furthermore, the synthesis method of the nucleic acid construct corresponding to the oncostatin M recombinant protein includes: introducing an artificial signal peptide amino acid sequence for secretory expression at the N-terminus of the oncostatin M recombinant protein to obtain an oncostatin M recombinant protein introduced with artificial signal peptide amino acids, wherein the artificial signal peptide amino acid sequence is shown in SEQ ID NO:2; then introducing a tag for protein purification after the N-terminal signal peptide of the oncostatin M recombinant protein introduced with artificial signal peptide amino acids to obtain an oncostatin M recombinant protein introduced with artificial signal peptide amino acids and a protein purification tag, wherein the amino acid sequence of the tag is shown in SEQ ID NO:3; then codon optimization is performed on the oncostatin M recombinant protein introduced with artificial signal peptide amino acids and a protein purification tag to obtain a nucleic acid construct corresponding to the oncostatin M recombinant protein.

Furthermore, the sequence of the oncostatin M recombinant protein into which the artificial signal peptide amino acids and protein purification tags are introduced is shown in SEQ ID NO:4.

Furthermore, the promoter used to construct the eukaryotic expression vector is the CMV promoter, and the promoter and the nucleic acid construct corresponding to the oncostatin M recombinant protein are connected after enzyme digestion.

Furthermore, the steps of constructing the oncostatin M recombinant expression vector include: using restriction endonuclease NheⅠ at the N-terminus of the nucleic acid construct corresponding to the oncostatin M recombinant protein, adding TAG and restriction endonuclease HindⅢ at the C-terminus, and constructing it into a eukaryotic expression vector.

Furthermore, the method for identifying oncostatin M comprises: culturing TF-1 cells, and using the cells to identify the in vitro activity of the purified oncostatin M.

Furthermore, the step of transfecting HEK293 cells by the transient expression system includes: resuscitating the HEK293 cells for at least two generations; ensuring that the cell density is above 2.0×10 ⁶ cells/mL and the cell viability is greater than 95% on the day of transfection; and adding the oncostatin M recombinant expression vector and PEI transfection reagent dropwise into the HEK293 cells for transfection.

Furthermore, oncostatin M was purified using a His tag affinity chromatography column.

Furthermore, the culture conditions of the transfected HEK293 cells were: temperature of 37° C., 5% volume percent CO ₂ , and rotation speed of 110-120 rpm.

Beneficial Effects

The present invention uses transient transfection technology, which is simple and easy to master, and can obtain a large amount of recombinant OSM protein within 2 weeks, with an expression level as high as 120 mg/L.

The present invention fuses the his tag to perform affinity chromatography, and the purity of the recombinant protein obtained by one-step purification reaches more than 95%, which is simple to operate, high in purification, convenient for subsequent operations, and can improve the protein recovery rate.

The present invention adopts the HEK293 eukaryotic expression system to make the recombinantly expressed oncostatin M protein have complete post-translational modifications (mainly including glycosylation, phosphorylation, ubiquitination and acetylation, etc.), and also make the recombinantly expressed oncostatin M have correct protein folding and form correct disulfide bonds. With these modifications and folding, the product can be closer to the natural structure and thus have higher activity; the present invention obtains highly active oncostatin M through transient transfection of eukaryotic cells HEK293, thereby providing a new strategy for the treatment of diseases such as subsequent experimental research, the development of OSM signal transduction inhibitors, and clinical drug development.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram of the construction of the expression vector pcDNA3.1-His-OSM, Human;

Figure 2 is a SDS-PAGE detection diagram of the dialyzed recombinant expressed OSM under reducing and non-reducing conditions in an embodiment of the present invention. Lane 1: 2 μg dialyzed OSM, non-reducing conditions; Lane 2: 2 μg dialyzed OSM, reducing conditions; M: protein marker;

FIG. 3 is a graph showing the activity detection results of the recombinantly expressed OSM protein in TF-1 cells after dialysis in Example 4 of the present invention.

DETAILED DESCRIPTION

The detailed operation method of the present invention is further illustrated below in conjunction with specific examples. However, these examples are only used to illustrate the present invention in detail and do not limit the scope of the present invention.

The present invention provides a method for preparing oncostatin M, which mainly comprises the following steps:

(1) Construction of a vector for recombinant expression of oncostatin M;

(2) transfecting HEK293 cells with the recombinant expression vector constructed in step (1) via a transient expression system;

(3) Cultivate cells at 37°C and 5% _CO2 in a shaking incubator, take samples and count them at intermediate intervals, and monitor cell status;

(4) When the cell viability is lower than 60%, the culture supernatant is collected by centrifugation, filtered, and then OSM is purified from the supernatant using an affinity chromatography column (Ni);

(5) Cultivating TF-1 cells and using them to identify the in vitro activity of purified oncostatin M (OSM).

Example 1

The method for constructing an oncostatin M (OSM) recombinant expression vector comprises the following steps:

(1) Based on the protein sequence of oncostatin M (uniport Accession #: P13725), the expression range was determined by analyzing with the assistance of bioinformatics software. The amino acid sequence of the oncostatin M recombinant protein is shown in SEQ ID NO: 1;

(2) introducing an artificial signal peptide amino acid for secretory expression at the N-terminus of SEQ ID NO:1, wherein the amino acid sequence of the artificial signal peptide is as shown in SEQ ID NO:2, thereby obtaining an oncostatin M recombinant protein into which the artificial signal peptide amino acid is introduced, so that the desired protein is secreted into the culture medium, facilitating subsequent purification;

(3) introducing a tag (6×His tag) for protein purification after the N-terminal signal peptide of the oncostatin M recombinant protein into which the artificial signal peptide amino acids are introduced, wherein the amino acid sequence of the tag for protein purification is shown in SEQ ID NO:3, thereby obtaining the oncostatin M recombinant protein into which the artificial signal peptide amino acids and the protein purification tag are introduced; the sequence of the oncostatin M recombinant protein into which the artificial signal peptide amino acids and the protein purification tag are introduced is shown in SEQ ID NO:4;

(4) codon optimization was performed on the sequence of SEQ ID NO:4 to obtain a nucleic acid construct corresponding to the oncostatin M recombinant protein, wherein the sequence of the nucleic acid construct corresponding to the oncostatin M recombinant protein is shown in SEQ ID NO:5;

(5) The sequence shown in SEQ ID NO:5 was synthesized and constructed into the eukaryotic expression vector pcDNA3.1(+) by using restriction endonuclease NheⅠ(GCTAGC) at the N-terminus and TAG and restriction endonuclease HindⅢ(AAGCTT) at the C-terminus, and then 500ug of plasmid was extracted for transfection level. The promoter used was CMV promoter, which was connected to the nucleic acid construct after enzyme digestion.

Example 2

Transfection of HEK293 cells with the transient expression system includes the following steps:

(1) Resuscitate human embryonic kidney 293 (HEK293) cells for at least two generations;

(2) The cell density was adjusted to 1.0×10 ⁶ cells/mL one day before transfection, and the cell density was ensured to be 2.0×10 ⁶ cells/mL on the day of transfection, and the cell viability was greater than 95%; the recombinant expression vector constructed in Example 1 was mixed with PEI transfection reagent at a mass ratio of 1:3, and then added dropwise to the HEK293 cells prepared above;

(3) The transfected HEK293 cells were cultured at 37°C, 5% CO ₂ , and 115 rpm. Samples were taken and counted during the culture to monitor the cell status.

Example 3

Purification of Oncostatin M (OSM) from the supernatant using affinity chromatography column (Ni)

The His tag affinity chromatography column is used to separate and purify oncostatin M, comprising the following steps:

(1) Collect the supernatant of the cultured cells by centrifugation at 8000 rpm for 30 min, filter the supernatant with a 0.45 μm filter membrane, and then load it onto a pre-equilibrated Ni column (equilibration solution includes: 50 mM Tris, 150 mM NaCl, pH 8.0);

(2) Use imidazole for gradient competitive elution (using equilibrium solutions containing 0, 20, 250, and 500 mM imidazole, respectively) to elute the target protein, and collect the proteins according to the peaks emitted by the purification instrument;

(3) Collect the product and analyze its purity by running SDS-PAGE;

(4) According to the SDS-PAGE results, the target protein was collected and dialyzed (dialysis fluid included: 50 mM Tris, 150 mM NaCl, pH 8.0) in a refrigerator at 4°C for 16 h;

(5) The dialyzed samples were subjected to SDS-PAGE under reducing and non-reducing conditions to identify the final protein purity.

The identification results are shown in FIG2 , indicating that high-purity oncostatin M was obtained through the HEK293 eukaryotic expression system.

Example 4

Cultivating TF-1 cells and using the cells to identify the in vitro activity of purified oncostatin M (OSM) comprises the following steps:

(1) Resuscitate TF-1 cells and culture them in a 37°C, 5% _CO2 incubator. Protein activity testing can be performed only after they have been passaged at least twice and can double normally.

(2) Take out the cells in the logarithmic growth phase and count them, then calculate the required cell volume according to 1*10 ⁶ cells/well and 100uL/well;

(3) Take out the required volume of cells, centrifuge them, wash them with 1640, add the required volume of viability culture medium to resuspend the cells, inoculate them into a 96-well plate using a 12-channel pipette, and place them in a 37°C, 5% _CO2 incubator for later use;

(4) Dilute the protein in a gradient manner and then add it to a 96-well plate prepared in advance;

(5) Place in a 37°C, 5% _CO2 incubator for 48 hours and then add colorimetric reagent;

(6) Then use GraphPad to select the four-parameter fitting curve and calculate the EC50.

Results of oncostatin M protein activity test in TF-1 cells:

The identification results are shown in Figure 3 and the table above, indicating that oncostatin M expressed by HEK293 eukaryotic cells has a promoting effect on the proliferation of TF-1 cells, wherein the EC50 of oncostatin M expressed by eukaryotic cells is 0.6956 ng/mL, and the EC50 of oncostatin M expressed by prokaryotic cells is 1.163 ng/ml, and the EC50 of oncostatin M expressed by eukaryotic cells is lower than that of oncostatin M expressed by prokaryotic cells, indicating that oncostatin M expressed by HEK293 eukaryotic cells has better activity.

Claims (10)

1. A method for preparing oncostatin M, characterized in that a nucleic acid construct corresponding to an oncostatin M recombinant protein is synthesized and constructed into a eukaryotic expression vector to obtain an oncostatin M recombinant expression vector; the oncostatin M recombinant expression vector is transfected into HEK293 cells through a transient expression system; the transfected HEK293 cells are cultured, the supernatant is collected after centrifugation, and oncostatin M is obtained after filtration and purification; the sequence of the nucleic acid construct corresponding to the oncostatin M recombinant protein is shown in SEQ ID NO:5. 2. The method for preparing oncostatin M according to claim 1, characterized in that the amino acid sequence of the oncostatin M recombinant protein is as shown in SEQ ID NO: 1. 3. The method for preparing oncostatin M according to claim 1, characterized in that the method for synthesizing the nucleic acid construct corresponding to the oncostatin M recombinant protein comprises: introducing an artificial signal peptide amino acid sequence for secretory expression at the N-terminus of the oncostatin M recombinant protein to obtain an oncostatin M recombinant protein introduced with artificial signal peptide amino acids, wherein the artificial signal peptide amino acid sequence is as shown in SEQ ID NO: 2; then introducing a tag for protein purification after the N-terminal signal peptide of the oncostatin M recombinant protein introduced with artificial signal peptide amino acids to obtain an oncostatin M recombinant protein introduced with artificial signal peptide amino acids and a protein purification tag, wherein the amino acid sequence of the tag is as shown in SEQ ID NO: 3; then codon optimization is performed on the oncostatin M recombinant protein introduced with artificial signal peptide amino acids and a protein purification tag to obtain the nucleic acid construct corresponding to the oncostatin M recombinant protein. 4. The method for preparing oncostatin M according to claim 3, characterized in that the sequence of the oncostatin M recombinant protein into which the artificial signal peptide amino acids and protein purification tags are introduced is as shown in SEQ ID NO: 4. 5. The method for preparing oncostatin M according to claim 1, characterized in that the promoter used to construct the eukaryotic expression vector is a CMV promoter, and the promoter and the nucleic acid construct corresponding to the oncostatin M recombinant protein are connected after enzyme digestion. 6. The method for preparing oncostatin M according to claim 1 is characterized in that the step of constructing the oncostatin M recombinant expression vector comprises: using restriction endonuclease NheⅠ at the N-terminus of the nucleic acid construct corresponding to the oncostatin M recombinant protein, adding TAG and restriction endonuclease HindⅢ at the C-terminus, and constructing it into a eukaryotic expression vector. 7. The method for preparing oncostatin M according to claim 1, characterized in that the method for identifying oncostatin M comprises: culturing TF-1 cells, and using the cells to identify the in vitro activity of the purified oncostatin M. 8. The method for preparing oncostatin M according to claim 1, characterized in that the step of transfecting HEK293 cells by means of a transient expression system comprises: resuscitating the HEK293 cells for at least two generations; ensuring that the cell density is above 2.0×10 ⁶ cells/mL and the cell viability is greater than 95% on the day of transfection; and adding the oncostatin M recombinant expression vector and PEI transfection reagent dropwise into the HEK293 cells for transfection. 9 . The method for preparing oncostatin M according to claim 1 , characterized in that oncostatin M is purified using a His tag affinity chromatography column. 10 . The method for preparing oncostatin M according to claim 1 , wherein the culture conditions of the HEK293 cells after transfection are: a temperature of 37° C., 5% volume percent CO ₂ , and a rotation speed of 110-120 rpm.