Method for expressing B16 cells of human CD22

Abstract

The invention relates to the fields of plasmid construction, plasmid transfection, flow detection and confocal microscopy imaging, and discloses a method for expressing a B16 cell of human CD22, and the method comprises the following steps: S1, plasmid construction, S2, cell transfection, and S3, monoclonal culture. A CD22 expression plasmid is designed on the basis of a PiggyBac plasmid vector system, the plasmid system is transfected into a B16 cell by using a transfection reagent, monoclonal sorting is performed on the fluorescent expression positive cell by using a flow sorter, continuous expanding culture is performed from a monoclonal cell strain, and finally, the expression of the CD22 is detected by using an antibody.

Description

Technical Field

[0001] The present invention relates to the fields of plasmid construction, plasmid transfection, flow cytometry and confocal microscope imaging, and in particular to a method for expressing human CD22 in B16 cells. Background Art

[0002] CD22 CAR-M is a cell therapy with the ability to phagocytose tumors. It enables T cells to recognize and attack tumor cells with CD22 antigens in the body by marking CD22 on them. This method has many advantages, such as being able to overcome the limitations of traditional immunotherapy and reducing damage to normal tissue cells. Currently, in the study of the ability of CD22 CAR-M to phagocytose tumors, suspended cells expressing CD22 are used as the target. CAR-M can specifically recognize and bind to tumor markers through its extracellular scFv, thereby achieving the effect of phagocytosis and killing tumors.

[0003] However, most of the tumor cells expressing CD22 are suspension cells. Even breast cancer and lung cancer cells reported to express CD22 have an expression level of only 0.5% of that of suspension cells. However, in order to better study the special phagocytic killing ability of CD22 CAR-M on solid tumors, a solid tumor cell that can stably express CD22 is needed as a target in the study of CAR-M function. Summary of the invention

[0004] The present invention designs a CD22 expression plasmid based on the PiggyBac plasmid vector system, and uses a transfection reagent to transfect the plasmid system into B16 cells. The cells expressing positive fluorescence are sorted by a flow cytometry instrument for monoclonal sorting, and then the monoclonal cell line is continuously expanded, and finally an antibody is used to detect the expression of CD22.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for expressing human CD22 B16 cells, the method comprising the following steps:

[0006] S1. Plasmid construction

[0007] (I) Experimental equipment:

[0008] Agarose, Goldview nucleic acid dye, related primers, Novozyme high-fidelity PCR enzyme, Novozyme DNA purification and recovery kit, restriction endonuclease, PCR instrument, Novozyme homologous recombinase, DH5α competent cells, Tiangen endotoxin-free plasmid extraction kit;

[0009] (II) Experimental steps:

[0010] ①Use primer 1 and primer 2 in Table 1, follow the reaction procedure in Table 2, use the plasmid in the plasmid library of the research group as a template, PCR to obtain the CD22-P2A-mCherry fragment, and use the PCR product recovery kit to recover the fragment;

[0011] Table 1 CD22-P2A-mCherry primers

[0012]

[0013] Table 2 PCR reaction procedure for CD22-P2A-mCherry fragment

[0014]

[0015] ② Use the restriction endonucleases in Table 3 and the reaction procedures in Table 4 to digest the pB vector plasmid in the group, and use a PCR product recovery kit to recover the fragments;

[0016] Table 3 Double restriction digestion of pB vector

[0017]

[0018] Table 4 Restriction endonuclease reaction procedure

[0019]

[0020] ③ Using homologous recombinase, mix the nucleic acid fragments obtained above according to the ratio in Table 6;

[0021] ④ Use DH5α to transform the homologous recombination product;

[0022] ⑤Pick a single colony for expansion and send it for sequencing;

[0023] ⑥Use the endotoxin-free plasmid extraction kit to extract the plasmid for subsequent transfection;

[0024] S2. Cell transfection

[0025] (I) Experimental equipment:

[0026] 2000 reagents, Opti-Men medium, 1640 antibiotic-free medium, 1640 complete medium, pB-CD22-P2A-mCherry plasmid, B16 cells, 24-well plates;

[0027] (II) Experimental plan:

[0028] ① One night in advance, 24-well plate, 1×10 5 B16 cells were cultured in 800 μL 1640 complete medium;

[0029] ② Before the experiment, replace the culture medium in each well with culture medium without double antibodies;

[0030] ③ Mix the lipo2000 reagent thoroughly by inverting, dilute 1.6 μL of Lipofectamine reagent with 50 μL of Opti-MEM medium, and let stand for 5 minutes;

[0031] ④ Dilute 0.8 μg of plasmid with 50 μL Opti-MEM medium and let stand for 5 minutes;

[0032] ⑤ Add the diluted Lipo2000 reagent dropwise to the diluted DNA, i.e., a 1:1 ratio, then pipette to mix well and let stand at room temperature for 15 minutes;

[0033] ⑥ Thoroughly mix the DNAlipo2000 mixture again and add 50 μL of DNA lipid complex to each well of cells;

[0034] ⑦After 6 hours, replace the medium with complete culture medium for cells, and observe the cell transfection efficiency under fluorescence microscope after 24 hours;

[0035] S3. Monoclonal culture

[0036] (I) Experimental equipment:

[0037] BD flow cytometry sorter, 96-well plate, 24-well plate, 2% double-antibody 1640 complete medium;

[0038] (II) Experimental plan:

[0039] ① Add 100 μL of 2% double antibody-containing 1640 complete medium to the 96-well plate;

[0040] ② Use BD flow cytometer to sort transfected positive cells into 96-well plates, with 1 cell per well;

[0041] ③Observe the cell growth. When the cells fill the 96-well plate and maintain a positive signal, expand the cells to a 24-well plate.

[0042] Preferably, in step S1, the reaction is carried out in a PCR instrument at 50° C. for 15 min.

[0043] Preferably, in step S2, the final amount of reagent used in each well is 1.6 μL.

[0044] Preferably, in step S2, the final amount of plasmid per well is 0.3 μg.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] 1. The present invention designs a CD22 expression plasmid based on the PiggyBac plasmid vector system, and uses a transfection reagent to transfect the plasmid system into B16 cells;

[0047] 2. Use a flow cytometer to isolate the fluorescent-positive cells for monoclonal sorting, then continue to expand the monoclonal cell line, and finally use antibodies to detect the expression of CD22. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is the gel imaging result diagram of the present invention;

[0049] Figure 2 Transformation diagram of homologous recombination products into DH5α;

[0050] Figure 3 It is the area division diagram of the two-dimensional parabolic equation under the rectangular coordinate system;

[0051] Figure 4 This is a diagram showing the transfection efficiency of B16 cells after transfection with the pB-CD22-P2A-mCherry plasmid of the present invention and detection of transfection efficiency using a confocal microscope;

[0052] Figure 5 This is a diagram of the preliminary detection of CD22 expression after transfection using CD22-FITC antibody in the present invention. DETAILED DESCRIPTION

[0053] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] A method for expressing human CD22 B16 cells, the method comprising the following steps:

[0055] S1. Plasmid construction

[0056] (I) Experimental equipment:

[0057] Agarose, Goldview nucleic acid dye, related primers, Novozyme high-fidelity PCR enzyme, Novozyme DNA purification and recovery kit, restriction endonuclease, PCR instrument, Novozyme homologous recombinase, DH5α competent cells, Tiangen endotoxin-free plasmid extraction kit;

[0058] (II) Experimental steps:

[0059] ①Use primers 1 and 2 in Table 1, follow the reaction procedure in Table 2, and use the plasmid in the plasmid library of the research group as a template to obtain the CD22-P2A-mCherry fragment by PCR. The gel imaging results are shown in Figure 1 , use the PCR product recovery kit to recover the fragments;

[0060] Table 1 CD22-P2A-mCherry primers

[0061]

[0062] Table 2 PCR reaction procedure for CD22-P2A-mCherry fragment

[0063]

[0064] ②Use the restriction endonucleases in Table 3 and follow the reaction procedure in Table 4 to digest the pB vector plasmid in the group. The gel imaging results are shown in Figure 1 , use the PCR product recovery kit to recover the fragments;

[0065] Table 3 Double restriction digestion of pB vector

[0066]

[0067] Table 4 Restriction endonuclease reaction procedure

[0068]

[0069] ③ Using homologous recombinase, mix the nucleic acid fragments obtained above according to the ratio in Table 6;

[0070] ④ Use DH5α to transform the homologous recombination product. The results are shown in Figure 2 ;

[0071] ⑤Pick a single colony for expansion and send it for sequencing;

[0072] ⑥Use the endotoxin-free plasmid extraction kit to extract the plasmid for subsequent transfection;

[0073] S2. Cell transfection

[0074] (I) Experimental equipment:

[0075] 2000 reagents, Opti-Men medium, 1640 antibiotic-free medium, 1640 complete medium, pB-CD22-P2A-mCherry plasmid, B16 cells, 24-well plates;

[0076] (II) Experimental plan:

[0077] ① One night in advance, 24-well plate, 1×105 B16 cells were cultured in 800 μL 1640 complete medium;

[0078] ② Before the experiment, replace the culture medium in each well with culture medium without double antibodies;

[0079] ③ Mix the lipo2000 reagent thoroughly by inverting, dilute 1.6 μL of Lipofectamine reagent with 50 μL of Opti-MEM medium, and let stand for 5 minutes;

[0080] ④ Dilute 0.8 μg of plasmid with 50 μL Opti-MEM medium and let stand for 5 minutes;

[0081] ⑤ Add the diluted Lipo2000 reagent dropwise to the diluted DNA, i.e., a 1:1 ratio, then pipette to mix well and let stand at room temperature for 15 minutes;

[0082] ⑥ Thoroughly mix the DNAlipo2000 mixture again and add 50 μL of DNA lipid complex to each well of cells;

[0083] ⑦After 6 hours, the cells were given complete culture medium and the cell transfection efficiency was observed under a fluorescence microscope after 24 hours. The results are shown in Figure 3 , CD22: This label refers to the CD22 expression sequence, including the extracellular sequence, transmembrane sequence and intracellular sequence, P2A: P2A is the linker element of CD22 and mCherry fluorescent protein, mCherry: used for preliminary detection of transfection efficiency and flow cytometry sorting; pB-CD22-P2A-mCherry plasmid transfected B16, and confocal microscopy was used to detect transfection efficiency. Figure 4 The CD22-FITC antibody was used to preliminarily detect the expression of CD22 after transfection. Figure 5 ;

[0084] S3. Monoclonal culture

[0085] (I) Experimental equipment:

[0086] BD flow cytometry sorter, 96-well plate, 24-well plate, 2% double-antibody 1640 complete medium;

[0087] (II) Experimental plan:

[0088] ① Add 100 μL of 2% double antibody-containing 1640 complete medium to the 96-well plate;

[0089] ② Use BD flow cytometer to sort transfected positive cells into 96-well plates, with 1 cell per well;

[0090] ③Observe the cell growth. When the cells fill the 96-well plate and maintain a positive signal, expand the cells to a 24-well plate.

[0091] In this embodiment: in step S1, the reaction is carried out in a PCR instrument at 50° C. for 15 minutes.

[0092] In this embodiment: in step S2, the final reagent used in each well is 1.6 μL.

[0093] In this embodiment: in step S2, the final amount of plasmid per well is 0.3 μg.

[0094] In summary, the present invention designs a CD22 expression plasmid based on the PiggyBac plasmid vector system, and transfects the plasmid system into B16 cells using a transfection reagent. The cells expressing positive fluorescence are sorted by a flow cytometer for monoclonal sorting, and then the monoclonal cell line is continuously expanded, and finally an antibody is used to detect the expression of CD22.

[0095] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0096] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for expressing human CD22 B16 cells, characterized in that: The method comprises the following steps: S1. Plasmid construction (I) Experimental equipment: Agarose, Goldview nucleic acid dye, related primers, Novozyme high-fidelity PCR enzyme, Novozyme DNA purification and recovery kit, restriction endonuclease, PCR instrument, Novozyme homologous recombinase, DH5α competent cells, Tiangen endotoxin-free plasmid extraction kit; (II) Experimental steps: ①Use primer 1 and primer 2 in Table 1, follow the reaction procedure in Table 2, use the plasmid in the plasmid library of the research group as a template, PCR to obtain the CD22-P2A-mCherry fragment, and use the PCR product recovery kit to recover the fragment; Table 1 CD22-P2A-mCherry primers Table 2 PCR reaction procedure for CD22-P2A-mCherry fragment ② Use the restriction endonucleases in Table 3 and the reaction procedures in Table 4 to digest the pB vector plasmid in the group, and use a PCR product recovery kit to recover the fragments; Table 3 Double restriction digestion of pB vector Table 4 Restriction endonuclease reaction procedure ③ Using homologous recombinase, mix the nucleic acid fragments obtained above according to the ratio in Table 6; ④ Use DH5α to transform the homologous recombination product; ⑤Pick a single colony for expansion and send it for sequencing; ⑥Use the endotoxin-free plasmid extraction kit to extract the plasmid for subsequent transfection; S2. Cell transfection (I) Experimental equipment: 2000 reagents, Opti-Men medium, 1640 antibiotic-free medium, 1640 complete medium, pB-CD22-P2A-mCherry plasmid, B16 cells, 24-well plates; (II) Experimental plan: ① One night in advance, 24-well plate, 1×10 5 B16 cells were cultured in 800 μL 1640 complete medium; ② Before the experiment, replace the culture medium in each well with culture medium without double antibodies; ③ Mix the lipo2000 reagent thoroughly by inverting, dilute 1.6 μL of Lipofectamine reagent with 50 μL of Opti-MEM medium, and let stand for 5 minutes; ④ Dilute 0.8 μg of plasmid with 50 μL Opti-MEM medium and let stand for 5 minutes; ⑤ Add the diluted Lipo2000 reagent dropwise to the diluted DNA, i.e., a 1:1 ratio, then pipette to mix well and let stand at room temperature for 15 minutes; ⑥ Thoroughly mix the DNAlipo2000 mixture again and add 50 μL of DNA lipid complex to each well of cells; ⑦After 6 hours, replace the medium with complete culture medium for cells, and observe the cell transfection efficiency under fluorescence microscope after 24 hours; S3. Monoclonal culture (I) Experimental equipment: BD flow cytometry sorter, 96-well plate, 24-well plate, 2% double-antibody 1640 complete medium; (II) Experimental plan: ① Add 100 μL of 2% double antibody-containing 1640 complete medium to the 96-well plate; ② Use BD flow cytometer to sort transfected positive cells into 96-well plates, with 1 cell per well; ③Observe the cell growth. When the cells fill the 96-well plate and maintain a positive signal, expand the cells to a 24-well plate.

2. The method for expressing human CD22 in B16 cells according to claim 1, characterized in that: In step S1, the reaction was carried out in a PCR instrument at 50°C for 15 minutes.

3. The method for expressing human CD22 in B16 cells according to claim 1, characterized in that: In step S2, the final amount of reagent used in each well was 1.6 μL.

4. The method for expressing human CD22 in B16 cells according to claim 1, characterized in that: In step S2, the final amount of plasmid per well was 0.3 μg.

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