Expression vector for RSPO1 protein and use thereof

Abstract

The present invention belongs to the field of biology, and relates to a vector for expressing an RSPO1 protein and the use thereof. A plasmid containing a strong cell expression promoter is constructed, a 293 cell line with efficient and stable expression is obtained by means of screening, and a downstream purification process is optimized, thereby achieving mass production of an RSPO1 recombinant protein. The obtained 293 cell line with stable expression can be cultured in suspension in a serum-free medium for growth and cytokine expression and production, thus eliminating the influence of components in serum on the product quality. Moreover, the production volume can be scaled up to 2 L, 10 L or even 300 L, eradicating the limitations of the small volume of a plate culture system. In addition, the operations of plasmid extraction and transfection are omitted from production, which can reduce the batch-to-batch variation in cytokine expression, laying a foundation for large-scale expression.

Description

Expression vector of RSP01 protein and application thereof

[0001] The present application claims priority to the following:

[0002] Application No.: CN202411017335.4; Application Date: July 29, 2024. TECHNICAL FIELD

[0003] The present application belongs to the field of biology, and specifically relates to an expression vector of RSP01 protein and application thereof. BACKGROUND

[0004] RSPO1 (R-spondin 1) is a member of the R-spondin family, which includes four members (Rspo1-4) and is a microenvironment factor for maintaining adult stem cells in multiple organs. RSPO1 is a secreted activin protein with two cysteine-rich furin-like domains (FU-like CR) and a thrombospondin type 1 domain (TSR).

[0005] Rspo1 protein appears as a pluripotent signaling ligand, and the most famous molecular function is its ability to stimulate the Wnt / β-catenin signaling pathway, which plays a role in multiple aspects by enhancing Wnt signaling, and is a commonly used growth factor for culturing gastric epithelial organoids, small intestinal and colonic organoids, liver organoids, prostate organoids, pancreatic organoids, and breast organoids.

[0006] Rspo1 as a cell-protecting and proliferating agent for beta cells has been used in several animal models to treat chemotherapy or radiation-induced intestinal mucositis and inflammatory bowel disease. Other studies have shown that it can be used for joint diseases such as arthritis and cancer; it can also act as a tumor suppressor gene in lymphocytic leukemia. Recent studies have also found that Rspo1 can also promote osteoblast differentiation, reduce the symptoms of bone damage in arthritis in mouse models, and help bone repair and remodeling. Rspo1 is also expected to be applied to the treatment of various myelomas. Therefore, Rspo1 can be a therapeutic potential target for treating various diseases.

[0007] As a key growth factor in organoid culture, the high activity, batch stability, and non-pollution of Rspo1 are the keys to the success of organoid experiments.

[0008] SUMMARY

[0009] The present application aims to construct a plasmid containing a strong cell expression promoter, obtain a high-efficiency and stable 293 cell strain through screening, and optimize the downstream purification process, so as to realize mass production of Rspo1 recombinant protein.

[0010] The nucleic acid molecule R-spondin of the first aspect of the application is codon-optimized according to the 293 host based on the R-spondin reference sequence SEQ ID NO: 1 provided by NCBI, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 2.

[0011] The second aspect of the application discloses a method for constructing a recombinant plasmid R-spondin-pCDNA3.1, wherein the nucleic acid molecule R-spondin with the nucleotide sequence shown in SEQ ID NO: 2 is synthesized and inserted into the vector OPM-V02 to obtain the plasmid R-spondin-V02, and then the plasmid R-spondin-V02 is subjected to HindIII / EcoRI double enzyme digestion together with pCDNA3.1, and then the corresponding target fragment is recovered by gel running, and the recombinant plasmid R-spondin-pCDNA3.1 is obtained after ligation.

[0012] The third aspect of the application discloses a cell strain expressing Rspo1 protein, and the construction method of the cell strain is as follows: the recombinant plasmid R-spondin-pCDNA3.1 is linearized by PvuI and then transfected into 293 cells, and the cell strain expressing Rspo1 protein is obtained after transfection.

[0013] Preferably, the obtained positive cell population is inoculated into a multi-well plate, and when the cell confluence is 80-90%, the cell population is subcultured multiple times, and the supernatant is taken for protein quantitative detection to screen a cell strain with high expression of Rspo1 protein.

[0014] Preferably, the obtained cell strain Expi293-Rspondin4-P7 OPM-RD2104 is registered in the China General Microbiological Culture Collection Center with the registration number CGMCC No. 45941.

[0015] The fourth aspect of the application discloses a culture method of the above-mentioned cell strain, and the specific method is as follows:

[0016] S1: the cells are resuscitated in a culture medium and inoculated after being subcultured for more than three times;

[0017] S2: the cells after subculture are diluted and then transfected with the recombinant plasmid R-spondin-pCDNA3.1;

[0018] S3: the cells are centrifuged and the medium is replaced every 3-5 days after transfection, and then the culture is completed.

[0019] S4: the cells cryopreserved in S3 are recovered, and after subculturing to a viability of greater than 90%, monoclone plating is performed, and the expression amount (ForteBio) of the cell supernatant is detected;

[0020] S5: the cell strain with high expression amount is screened and suspended domestication culture is performed to obtain a cell strain expressing Rspo1 protein.

[0021] Preferably, the culture conditions in S1 and S2 are 120 rpm, 37℃, 8% CO2, 80% humidity, and shaking culture.

[0022] Preferably, the R-spondin-pCDNA3.1 plasmid in S2 is filtered through a 0.22 μm membrane and then added to OPM-293 CD05, and mixed with PEI diluted with OPM-293 CD05 for incubation.

[0023] Preferably, in S4, the sugar concentration during culture is controlled to be 2-8 g / L, and when the cell viability is 60%, the sample is centrifuged, and the supernatant is used for expression amount detection by ForteBio.

[0024] The fifth aspect of the present application discloses the application of the above-mentioned nucleic acid molecule R-spondin, the recombinant plasmid R-spondin-pCDNA3.1 or the above-mentioned cell strain in the production of a recombinant protein. The recombinant protein is specifically Rspo1 protein.

[0025] The 293 stable expression cell line obtained by the technical scheme of the present application can be cultured in a serum-free medium to grow and express and produce cytokines, and the volume can be scaled up to 2L, 10L or even 300L, breaking the small limitation of the well plate culture system and reducing the batch difference of factor expression, thereby providing a basis for large-scale expression.

[0026] The 293 stable expression cell line obtained by the technical scheme of the present application has the following advantages compared with the corresponding transient expression system: 1. No need to prepare a large amount of plasmid, reducing labor and consumable costs. 2. The expression amount is obviously improved compared with transient expression. 3. The purification method and freeze-drying process are confirmed, which can provide a basis for commercial production and product transportation.

[0027] The cell strain Expi293-Rspondin4-P7 OPM-RD2104 belongs to human embryonic kidney cell (HEK293), and was received by the China General Microbiological Culture Collection Center (CGMCC) on June 11, 2024, and was registered, with the registration number of the preservation center being CGMCC No. 45941. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a graph of cell viability of Expi293 cells under different concentrations of G418;

[0029] Figure 2 is a graph of cell viability of 293 cells under different concentrations of G418;

[0030] Figure 3 is a graph of viable cell density of 293 cells under different concentrations of G418;

[0031] Figure 4 is a graph of protein concentration detection of cell supernatant during plasmid transfection;

[0032] Figure 5 is a graph of protein concentration detection of cell supernatant during monoclonal plating;

[0033] Figure 6 is a graph of WB detection of Rspondin expression of different cell strains;

[0034] Figure 7 is a graph of detection of Rspondin expression of cell strains screened by the application;

[0035] Figure 8 is a chromatogram of Example 3;

[0036] Figure 9 is a graph of protein electrophoresis detection of the product of Example 3, 1, Marker; 2, standard protein (SST); 3, eluate of nickel metal chelate chromatography. DETAILED DESCRIPTION

[0037] The application will be further described in the following examples, but the application is not limited to the scope of the examples. In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0038] Example 1 Design of Protein

[0039] The sequence of R-spondin 1 is 243 aa in full length, with a signal peptide added at the N-terminus to promote secretory expression. A 6-his tag is added at the C-terminus for easy purification. An enterokinase cleavage site is inserted between the his tag and the protein for easy removal of the tag when needed. The specific sequence is as follows:

[0040] After codon optimization of the amino acid sequence, the sequence is ligated to the vector pcDNA3.1.

[0041] Example 2 Construction of 293 Cell Strains Stably Expressing R-spondin Protein

[0042] 1. Determine the optimal screening concentration of G418:

[0043] Experimental apparatus:

[0044] 1) Instrumentation

[0045] 2) Reagents and supplies

[0046] Experimental procedure:

[0047] Select 0 / 100 / 200 / 400 / 800 ug / mL G418 concentration to press Expi293 cells, centrifugal change liquid every 3-5 days, regularly sample count, find 7-10 days can make Expi293 cell all death G418 concentration as working concentration.

[0048] The experimental results are shown in Figure 1, 200 and 400 ug / mL of G418 has good killing effect on Expi293, can make Expi293 cell all death in 7-11 days, therefore selected 200 and 400 ug / mL of G418 concentration as working concentration.

[0049] 2, recombinant pcDNA3.1 construction:

[0050] According to the R-spondin reference amino acid sequence provided by NCBI, the coding gene R-spondin is synthesized after codon optimization, which is inserted into the vector OPM-V02 to obtain the plasmid R-spondin-V02. Then the plasmid R-spondin-V02 and pCDNA3.1 are digested by HindIII / EcoRI, and the target fragment is recovered and connected to construct the recombinant plasmid R-spondin-pCDNA3.1. After confirming the correctness of the plasmid by sequencing, the E. coli is transformed for plasmid extraction, and finally the recombinant plasmid R-spondin-pCDNA3.1 is linearized by PvuI for 293 cell transfection.

[0051] 1) Experimental apparatus:

[0052] 2) Experimental materials:

[0053] 3) Primer information

[0054] 3, recombinant pcDNA3.1 transfection of 293 cells and screening:

[0055] 1) Experimental apparatus:

[0056] 2) Experimental materials:

[0057] 3) Reagents:

[0058] The cell viability and cell density were detected after the cells were pressurized with G418 concentrations of 200 and 400 ug / mL, respectively, and the results are shown in Figures 2-3.

[0059] 4. Plasmid transfection experiment steps:

[0060] 1) Expi293 cells were resuscitated in OPM-293 CD05, and 30 mL was cultured in a 125 mL shaker flask, 120 rpm, 37°C, 8% CO2, 80% humidity shaker, and cultured for more than three passages before inoculation.

[0061] 2) The cell density was diluted to 1.5 x 10 6 cell / ml, with a viability of more than 95%, and cultured in a 120 rpm, 37°C, 8% CO2, 80% humidity shaker.

[0062] 3) The cell transfection density should be 3-5 x 10 6 cell / ml, and if the cell density is too high, dilute it to the appropriate density with OPM-293 CD05. Take 0.01 mg of plasmid R-spondin-pCDNA3.1 filtered through a 0.22 μm membrane and add it to 0.5 mL of OPM-293 CD05, and take another 0.03 mg of PEI to 0.5 mL of OPM-293 CD05, mix the diluted PEI with the plasmid R-spondin-pCDNA3.1, and let it stand at room temperature for 20 minutes.

[0063] 4) Pour the DNA-PEI mixture into the Expi 293 cells, mix well, and culture in a 120 rpm, 37°C, 8% CO2, 80% humidity shaker.

[0064] 5) Add 400 ug / mL or 200 ug / mL G418 for selection the day after transfection, and at the same time, take samples at regular intervals to record cell density and cell viability 1-26 days after pressurization, centrifuge and change the liquid every 3-5 days, and then transfer to a 120 rpm, 37°C, 8% CO2, 80% humidity shaker for culture. After the viability density recovers, expand the culture, and perform Fed-Batch and cryopreservation, respectively.

[0065] 5. Pool Fed-Batch sample preparation:

[0066] Inoculate cells at 1E6 / mL, total volume 10 mL, feed 5% OPM-293ProFeed when cell density is 4-6E6 / mL, control sugar 2-8g / L throughout the culture process, centrifugal sampling when cell viability is about 60%, and supernatant expression is detected using ForteBio. The identification results are shown in Figure 4, and the results show that the expression of Rspondin: 400ug / mL G418 pressure (G1: Rspondin-D10-400ug / mL G418) > 200ug / mL G418 pressure (F1: Rspondin-D10-200ug / mL G418) > transient sample (E1: Rspondin transient D6 shake flask) > blank control (H1).

[0067] 6、Monoclonal plating:

[0068] According to the test results, select cells with 200ug / mL and 400ug / mL pressure after transfection for recovery, and subculture to a viability of greater than 93%, then use limited dilution method to plate at 0.5 cells / well, and after plating, observe under a microscope on D1 / 4 / 7 / 10, exclude multi-clonal wells, and only leave single-clonal wells for labeling. On D20, select single-clonal cells for expansion and transfer into 24-well plates, 6-well plates, and finally take the cell supernatant in the 6-well plates for ForteBio detection.

[0069] According to the ForteBio results, select clones R4 / 10 / 13 / 25 / 28 / 46 / 41 with high expression for transfer into shake tubes for suspension acclimation, and then perform Fed-Batch and ForteBio detection of expression.

[0070] Clone Fed-Batch sample preparation:

[0071] Inoculate cells at 1E6 / mL, total volume 10 mL, feed 5% OPM-293ProFeed when cell density is 4-6E6 / mL, control sugar 2-8g / L throughout the culture process, centrifugal sampling when cell viability is about 60%, and supernatant expression is detected using ForteBio. The results are shown in Figure 5, and according to the results in Figure 5, select the top four clones for WB detection verification,

[0072] 7、WB detection of Rspondin expression

[0073] 1) Experimental instruments:

[0074] 2) Experimental materials:

[0075] 3) Experimental method

[0076] Protein sample preparation:

[0077] Take 40 μL of cell supernatant and add 10 μL of 5xSample Buffer, mix, heat at 95°C for 10 min,

[0078] SDS-PAGE:

[0079] After installing the precast gel plate to the electrophoresis device, add MOPS Running Buffer, take 10 μL of protein Marker and an appropriate amount of protein sample, and load them in order. Perform SDS-PAGE at a voltage of 130 V for about 60-70 min, until the protein Marker runs to the bottom of the separation gel, and prepare for membrane transfer (the membrane transfer solution needs to be pre-cooled in the 4°C refrigerator in advance).

[0080] Membrane transfer:

[0081] Transfer the protein to the PVDF membrane at a constant current of 400 mA for 70 min.

[0082] Blocking:

[0083] After the membrane transfer is completed, immerse the PVDF membrane in the rapid blocking solution and incubate at room temperature for 15-25 min.

[0084] Antibody incubation:

[0085] Dilute the primary antibody (rabbit monoclonal antibody [H169-1-5] Anti-His IgG Fc) and the rapid blocking solution at a concentration of 1:25000, cut the membrane according to the size of the target protein based on the position of the protein Marker, and place it in the diluted primary antibody, and incubate at 37°C for 1.5 h. Wash with TBST for 3 times, each for 10 min. Dilute the secondary antibody (goat anti-rabbit IgG H&L-HRP) and the rapid blocking solution at a concentration of 1:20000, and place the PVDF membrane in the secondary antibody dilution solution, and incubate at 37°C for 1.5 h. Wash with TBST for 3 times, each for 10 min.

[0086] Development

[0087] Mix the Enhancer Buffer Solution and the Peroxide Buffer of the ECL kit at a ratio of 1:1. Immerse the PVDF membrane in the mixed solution and expose it to the full-automatic luminescence imaging system.

[0088] The WB detection results of the supernatant of the Rspondin molecule FB are shown in FIG. 6; in combination with the Fortie and WB expression detection results, the optimal cell strains of Rspondin are R4, R10, and R28.

[0089] 8. Detection of target gene

[0090] Combined with the cell growth and expression results, R4 was finally selected as the main clone for the target gene detection.

[0091] 1) Materials:

[0092] 2) Reagents:

[0093] 3) Instruments:

[0094] 4) Experimental steps:

[0095] 4.1 DNA extraction

[0096] DNA extraction was performed according to the DNA extraction kit (DP304-02) kit.

[0097] 4.2 Primer

[0098] 4.3 Formal experiment

[0099] 4.3.1 Target gene detection

[0100] 4.3.2 PCR reaction system

[0101] 4.3.3 PCR program

[0102] 4.3.4 Electrophoresis recovery

[0103] After running the PCR product on the gel, the gel was cut and recovered at 1093bp for sequencing.

[0104] 4.3.5 Sequencing

[0105] Sequencing primer:

[0106] 4.3.6 Result comparison

[0107] The comparison results are shown in Figure 7; the results show that they are consistent with the reference sequence.

[0108] Example 3 Purification of expression supernatant using nickel filler

[0109] 1. Chromatography column information

[0110] 2. Equipment information

[0111] 3. Process flow

[0112] 4. Experimental results

[0113] The results of the chromatogram are shown in Figure 8; the SDS-PAGE detection is shown in Figure 9. The target protein can be captured using this process and the filler, and obvious target protein bands can be seen in the sample detected by SDS-PAGE.

[0114] The above description is merely preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can make equivalent replacements or changes to the technical solutions and the inventive concept of the present application within the technical scope disclosed by the present application, which should be encompassed in the protection scope of the present application.

[0115]

Claims

1. A nucleic acid molecule R-spondin, characterized in that, The nucleotide sequence codon-optimized as shown in SEQ ID NO: 2 expresses an amino acid sequence as shown in SEQ ID NO:

1.

2. A recombinant plasmid R-spondin-pCDNA3.1, characterized in that, The nucleic acid molecule R-spondin of claim 1 is synthesized and inserted into a vector OPM-V02 to obtain a synthetic plasmid R-spondin-V02, and the synthetic plasmid R-spondin-V02 is digested by HindIII / EcoRI, and then the target fragment is recovered by gel electrophoresis and connected to construct a recombinant plasmid R-spondin-pCDNA3.

1.

3. A cell line expressing Rspol protein, characterized in that, The recombinant plasmid R-spondin-pCDNA3.1 of claim 2 is linearized by PvuI and then transfected into 293 cells to obtain a cell strain expressing Rspo1 protein.

4. The cell line expressing Rspol protein according to claim 3, characterized in that, The obtained positive cell population is inoculated into a multi-well plate, and when the cell confluence is 80-90%, the cell population is subcultured multiple times, and the supernatant is subjected to protein quantification detection to screen a cell strain highly expressing Rspo1 protein.

5. The cell line expressing Rspol protein according to claim 4, characterized in that, Its registration number in the China General Microbiological Culture Collection Center is CGMCC No. 45941.

6. The construction method of the cell strain expressing Rspo1 protein of claim 4, characterized in that, S1: the cells are resuscitated in a culture medium and inoculated after being subcultured more than three times; S2: the cells after subculture are diluted and then transfected with the recombinant plasmid R-spondin-pCDNA3.1 of claim 2; S3: the cells are centrifuged and the medium is replaced every 3-5 days after transfection, and then the culture is completed by Fed-Batch and cryopreservation. S4: the cells cryopreserved in S3 are resuscitated, subcultured to a viability of more than 90%, and then monoclonal plated, and the expression amount of the cell supernatant is detected; S5: the cell strain with high expression amount is screened and subjected to suspension domestication culture to obtain a cell strain expressing Rspo1 protein.

7. The method of claim 6, wherein the cell line expressing Rspo1 protein is constructed by, The culture conditions in S1 and S2 are 120 rpm, 37℃, 8% CO2, 80% humidity, and shaking culture.

8. The method of claim 6, wherein the cell line expressing Rspo1 protein is constructed by, The recombinant plasmid R-spondin-pCDNA3.1 in S2 is filtered through a 0.22μm membrane and then added to OPM-293 CD05 and mixed with PEI diluted with OPM-293 CD05 for incubation.

9. The method of claim 6, wherein the cell line expressing Rspo1 protein is constructed by, In S4, the sugar concentration is controlled at 2-8g / L during the culture process, and the sample is collected by centrifugation when the cell viability is 60%, and the expression amount of the supernatant is detected by ForteBio.

10. The nucleic acid molecule R-spondin of claim 1, the recombinant plasmid R-spondin-pCDNA3.1 of claim 2, or the cell strain of any one of claims 3-5 for use in producing a recombinant protein.

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