Recombinant cell as well as preparation method and application thereof

Abstract

The invention provides a recombinant cell and a preparation method and application thereof, and relates to the technical field of biology, and a recombinant expression vector comprises an MRC1 gene sequence; the sequence of the MRC1 gene is as shown in SEQ ID NO. 1. In the technical scheme provided by the invention, the MRC1 gene sequence is constructed into the recombinant expression vector, and then the recombinant expression vector is transfected into the host cell, so that the MRC1 protein can be stably expressed and synthesized in the host cell body; cHO-K1 cells are adopted as host cells, so that exogenous genes can be efficiently expressed, and complicated post-translation modification such as glycosylation and phosphorylation can be performed, so that the stability and activity of protein are improved; by transfecting the recombinant expression vector into the CHO-K1 cell, an exogenous gene can be stably integrated into the genome of the CHO-K1 cell to form a stable expression cell strain, so that the consistency and reliability of long-term expression are ensured, and the MRC1 protein can be produced and synthesized in batches.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant cell and a preparation method and application thereof. Background Art

[0002] MRC1 protein refers to Mannose Receptor C type 1, also known as CD206. It is a transmembrane protein with a relative molecular mass of approximately 166 kDa and plays an important role in the immune system. MRC1 is mainly distributed on the surface of macrophages and immature dendritic cells. As a pattern recognition receptor (PRR), it can recognize specific sugar structures on the surface of pathogens, such as mannose residues or sulfated carbohydrates.

[0003] Currently, MRC1 protein is obtained by selecting appropriate host cells for expression and purification. However, the expression level of MRC1 protein in conventional host cells is relatively low, and the purity and activity of the synthesized protein are both low. Summary of the invention

[0004] The main purpose of the present invention is to provide a recombinant cell and a preparation method and application thereof, aiming to provide a recombinant cell that stably expresses MRC1 protein.

[0005] To achieve the above object, the present invention provides a method for preparing a recombinant cell, comprising: S10. synthesizing the MRC1 gene sequence, cloning the MRC1 gene sequence into a plasmid backbone to obtain a recombinant expression vector, wherein the MRC1 gene sequence is as shown in SEQ ID NO.1; S20. transfecting the recombinant expression vector into a host cell to obtain a transfected cell, wherein the host cell comprises a CHO-K1 cell; S30. Subculture the transfected cells to obtain recombinant cells.

[0006] In one embodiment, step S20 includes: The domesticated host cell monoclones are sorted and amplified, and the recombinant expression vector is transfected into the amplified domesticated host cell to obtain transfected cells.

[0007] In one embodiment, in step S30, the method of transfecting cells is electroporation.

[0008] In one embodiment, in step S30, culture medium A is used for culture during subculture.

[0009] The present invention also provides a recombinant cell, which includes the recombinant cell prepared according to the above-mentioned method for preparing the recombinant cell.

[0010] The present invention also provides a use of a recombinant cell in synthesizing MRC1 protein, wherein the recombinant cell includes the recombinant cell as described above.

[0011] In the technical scheme of the present invention, by constructing the MRC1 gene sequence into a recombinant expression vector and then transfecting it into a host cell, it can be stably expressed in the host cell to synthesize the MRC1 protein; by using CHO-K1 cells as host cells, the exogenous gene can be efficiently expressed, and complex post-translational modifications such as glycosylation and phosphorylation can be performed, thereby improving the stability and activity of the protein; by transfecting the recombinant expression vector into CHO-K1 cells, the exogenous gene can be stably integrated into the genome of the CHO-K1 cells to form a stable expression cell line, thereby ensuring the consistency and reliability of long-term expression, and facilitating batch amplification of cells expressing the MRC1 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 This is a schematic diagram of the structure of the recombinant expression vector pWX039-Pr-Z-108_MRC1 in Example 1 of the present invention; Figure 2 is a cell growth curve diagram during the recovery period of the cell group in Example 2 of the present invention; Figure 3 This is a diagram of the monoclonality of cloned cells in Example 3 of the present invention; Figure 4 This is a diagram showing the results of FACS staining of recombinant cells after the cell population is recovered in Example 2 of the present invention; Figure 5 This is a diagram showing the results of FACS staining of cloned cells in Example 3 of the present invention; Figure 6 This is a diagram showing the FACS staining detection results of the top 6 clone cells PDL0 and PDL30 in terms of MRC1 protein expression level in Example 3 of the present invention.

[0014] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings in conjunction with the embodiments. DETAILED DESCRIPTION

[0015] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0016] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to normal conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, and "A and / or B" is taken as an example, including schemes A, B, or A and B that meet the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work, all belong to the scope of protection of the present invention.

[0017] MRC1 protein refers to Mannose Receptor C type 1, also known as CD206. It is a transmembrane protein with a relative molecular mass of approximately 166 kDa and plays an important role in the immune system. MRC1 is mainly distributed on the surface of macrophages and immature dendritic cells. As a pattern recognition receptor (PRR), it can recognize specific sugar structures on the surface of pathogens, such as mannose residues or sulfated carbohydrates.

[0018] Currently, MRC1 protein is obtained by selecting appropriate host cells for expression and purification. However, the expression level of MRC1 protein in conventional host cells is relatively low, and the purity and activity of the synthesized protein are both low.

[0019] For this reason, please see Figures 1 to 6 The present invention provides a method for preparing a recombinant cell, comprising: S10. synthesizing the MRC1 gene sequence, cloning the MRC1 gene sequence into a plasmid backbone to obtain a recombinant expression vector, wherein the MRC1 gene sequence is as shown in SEQ ID NO.1; S20. transfecting the recombinant expression vector into a host cell to obtain a transfected cell, wherein the host cell comprises a CHO-K1 cell; S30. Subculture the transfected cells to obtain recombinant cells.

[0020] In the technical scheme of the present invention, by constructing the MRC1 gene sequence into a recombinant expression vector and then transfecting it into a host cell, it can be stably expressed in the host cell to synthesize the MRC1 protein; by using CHO-K1 cells as host cells, the exogenous gene can be efficiently expressed, and complex post-translational modifications such as glycosylation and phosphorylation can be performed; by transfecting the recombinant expression vector into CHO-K1 cells, the exogenous gene can be stably integrated into the genome of the CHO-K1 cells to form a stable expression cell strain, thereby ensuring the consistency and reliability of long-term expression and establishing a cell strain that highly expresses the MRC1 protein.

[0021] The sequence of SEQ ID NO.1 is as follows:

[0022] In one embodiment, step S20 includes: The domesticated host cell single clones are sorted and amplified, and the recombinant expression vector is transfected into the amplified domesticated host cell to obtain transfected cells.

[0023] In one embodiment, in step S30, the method of transfecting cells is electroporation.

[0024] In the technical solution of the present invention, by adopting electroporation transfection, larger DNA molecules or substances that are difficult to pass through the cell membrane can be more effectively introduced into cells, and the transfection speed is fast and the operation is simple. It can maintain a good cell survival rate while maintaining a high transfection efficiency, and is suitable for large-scale production of genetically modified cells.

[0025] In one embodiment, in step S30, culture medium A is used for subculture, and the culture medium A includes CD CHO AGT culture medium, L-glutamine and HT supplement solution.

[0026] In the technical solution of the present invention, by using culture medium A for recovery, sufficient nutrients such as amino acids, vitamins, lipids, etc. can be supplemented for cells to support the cells to quickly resume normal metabolic activities. At the same time, the cryoprotectant is diluted to reduce the concentration of the cryoprotectant to avoid high concentrations of cryoprotectant toxicity to the cells; by using culture medium A, the cells can be maintained within an appropriate pH range to ensure the normal physiological function of the cells.

[0027] The present invention also provides a recombinant cell, which includes the recombinant cell prepared according to the above-mentioned method for preparing the recombinant cell.

[0028] Since the recombinant cell adopts all the technical solutions of all the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments, which will not be described one by one here.

[0029] The present invention also provides a use of a recombinant cell in synthesizing MRC1 protein, wherein the recombinant cell includes the recombinant cell as described above.

[0030] In the technical scheme of the present invention, by adopting recombinant cells to express MRC1 protein, MRC1 protein can be stably synthesized, and effective expression of MRC1 protein is achieved, ensuring that the final product has good biological activity and stability, thereby improving the quality of the synthesized protein; using the recombinant cells provided by the present invention, functional research and drug screening of MRC1 protein as well as its application as a diagnostic marker can be more conveniently carried out, thereby expanding its use in basic research and clinical medicine.

[0031] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0032] Experimental Materials Unless otherwise specified, the reagents and materials used in the examples of the present invention are commercially available.

[0033] CHO-K1 cells: purchased from ATCC, catalog number ATCC CCL-61; Medium A: 24.30 g / kg CD CHO AGT medium, 0.5846 g / kg L-glutamine, 10 g / kg 100× HT supplement; CD CHO AGT medium: supplier Gibco, product number 12490-001; L-Glutamine: Supplier: Sigma, catalog number G8540; 100×HT supplement solution: supplier Gibco, product number 11067-030; Medium AZ8: Medium A + 800 μg / mL bleomycin; Medium AZ4: Medium A + 400 μg / mL bleomycin; Medium AZ2: Medium A + 200 μg / mL bleomycin; Zeocin: Supplier: Gibco, Product No. R250-05; DMSO: supplier Sigma, product number D2650; DPBS: supplier Corning, product number 21-031-CV; 154 Reagent: Supplier: WuXi Biologics, catalog number DPT-WBP154-051.

[0034] Example 1 This embodiment provides a recombinant expression vector, and the preparation method thereof is as follows: The nucleotide sequence was synthesized and shown in SEQ ID NO. 1, and cloned into the plasmid backbone pWX039 to obtain the expression vector pWX039-Pr-Z-108_MRC1. After verification by Sanger sequencing analysis, no mutations, insertions or deletions were observed in the sequence.

[0035] Figure 1 is a map of the recombinant expression vector.

[0036] Example 2 This embodiment provides a recombinant cell, and the preparation method thereof is as follows: Resuscitate a CHO-K1 WCB host cell line using medium A. CHO-K1 host cells were passaged every 3 or 4 days, and the cell density on day 3 was approximately 1×10 5 cells / mL, and the cell density after 4 days was about 0.4×10 5 cells / mL.

[0037] CHO-K1 host cells were transfected by electroporation. During transfection, 1.0×10 7 The CHO-K1 host cells were centrifuged at 290×g for 5 min, and the supernatant was removed and resuspended in 300 μL DPBS. The recombinant expression vector and 154 Reagent in Example 1 were added to 250 μL DPBS and gently mixed. Carefully mix the plasmid reagent mixture and host cells, transfer them to an electroporation cup, and perform electroporation transfection using a Bio-Rad electroporator. After the transfection is completed, the cells in the electroporation cup are aspirated into a preheated shake tube containing 10 mL of culture medium A. The shake tube was cultured in a KUHNER shaker at a temperature of 36.5°C, a humidity of 75%, a carbon dioxide concentration of 6%, and a speed of 225 rpm. The transfected cells were cultured in a shaker for 24 hours and then pressurized. The pressurized culture medium used was culture medium AZ8. After 24 hours of pressurization, the cells were counted and passaged for 3 days, with 3×10 5 cells / mL, and the subculture medium was medium AZ4.

[0038] The cell growth curve during the recovery period of the cell population is as follows Figure 2 shown.

[0039] During the process of cell population expansion and subculture, the cells are frozen. The freezing medium is: 90% medium A + 10% DMSO (v / v). The cryopreservation tube containing the cell suspension is placed in a program cooling box, placed in a -80℃ refrigerator overnight, and then placed in a liquid nitrogen tank for storage.

[0040] Example 3 The cell population of the recombinant cells in Example 2 is cloned by using the limiting dilution cloning (LDC) method, which includes the following steps: Centrifuge the host cell suspension that was subcultured on the same day (300 g, 10 min) to obtain the host cell culture supernatant. The cloning medium is 69% medium A + 20% CHO-N079 host cell culture supernatant + 1% ClonaCell™-CHOACF Supplement + 10% cell suspension. Filter the prepared cloning medium with a 0.1 μm filter and place it on a Kuhner shaker for preheating. The shaking parameters are: temperature 36.5°C, humidity 75%, and carbon dioxide 6%.

[0041] A cell population of recombinant cells was revived in advance. After 2 weeks of pressurized screening culture, the doubling time was restored. When the viability was >95%, the cell population was plated through a round of LDC: the cells were counted by Vi-CELL XR (cell type: CHO-K1) and then the cells were diluted to 40 cells / mL with preheated culture medium A, and then diluted to 4 cells / mL with cloning culture medium; using a liquid workstation, the diluted cells were plated into 96-well plates at 100 μL per well, and 6 plates were plated for each cell population; after plating, the 96-well plates were placed in a carbon dioxide incubator for static culture, and the culture conditions were: temperature 36.5℃, carbon dioxide 6%.

[0042] On day 0 (the day of plating), day 1, day 2, day 5, and day 9 after plating, cells in the wells were photographed using a cell imaging system (Cell Metric CLD System, Solentim, UK) to monitor cell growth (see the monoclonality images of the final clones for details). Figure 3 ).

[0043] On day 7, 90 μL of fresh culture medium AZ4 with selection pressure was added to each well of the 96-well plate. Thirty clones were expanded into centrifuge tubes and then expanded into 24-well plates using culture medium AZ2 to obtain clonal cells.

[0044] Performance Testing The recombinant cells after the cell population recovery in Example 2 were subjected to FACS staining to verify the expression level of MRC1. The detection results are as follows: Figure 4 shown.

[0045] The cloned cells in Example 3 were subjected to FACS staining, and the top 6 clones with the highest expression levels of MRC1 protein were selected according to the different expression levels of the cloned MRC1 protein. The detection results are shown in FIG. Figure 5 shown.

[0046] Resuscitate one tube of RCB cells from each of the top 6 clones with the highest MRC1 protein expression level into a shake tube, and then continue to subculture for 4 weeks using the culture medium AZ2. After the stable subculture, resuscitate one tube of PDL0 (RCB) and one tube of PDL30 (subcultured for 4 weeks) for each clone. After 2 weeks of subculture, perform FACS staining to compare the expression levels of MRC1 head to head. The test results are as follows: Figure 6 shown.

[0047] like Figure 6 As shown, 108_MRC1-03, 108_MRC1-17, 108_MRC1-21, and 108_MRC1-31 all showed stable MRC1 expression.

[0048] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A method for preparing a recombinant cell, characterized in that: include: S10. synthesizing the MRC1 gene sequence, cloning the MRC1 gene sequence into a plasmid backbone to obtain a recombinant expression vector, wherein the MRC1 gene sequence is as shown in SEQ ID NO.1; S20. transfecting the recombinant expression vector into a host cell to obtain a transfected cell, wherein the host cell comprises a CHO-K1 cell; S30. Subculture the transfected cells to obtain recombinant cells.

2. The method for preparing a recombinant cell according to claim 1, characterized in that: Step S20 includes: The domesticated host cell monoclones are sorted and amplified, and the recombinant expression vector is transfected into the amplified domesticated host cell to obtain transfected cells.

3. The method for preparing a recombinant cell according to claim 1, characterized in that: In step S30, the method of transfecting cells is electroporation.

4. The method for preparing a recombinant cell according to claim 1, characterized in that: In step S30, culture medium A is used for culture during subculture, and the culture medium A includes CD CHO AGT culture medium, L-glutamine and HT supplement solution.

5. A recombinant cell, characterized in that The recombinant cell includes a recombinant cell prepared by the method for preparing a recombinant cell according to any one of claims 1 to 4.

6. Use of a recombinant cell in synthesizing MRC1 protein, characterized in that: The recombinant cell comprises the recombinant cell according to claim 5.

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