Genetically engineered anchorage-dependent cells and cba method detection kit

Abstract

One of the purposes of the present application is to provide a method and a plasmid for enhancing cell adhesion. The second purpose of the present application is to provide a cell for protein expression with enhanced adhesion. The third purpose of the present application is to provide a CBA method detection kit for autoimmune encephalitis based on the aforementioned cell. The fourth purpose of the present application is to provide a CBA method detection kit for autoimmune central nervous system demyelination disease based on the aforementioned cell.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the construction of a cell line with enhanced adherence and a CBA method kit based on this cell. Background Technology

[0002] Cell-based assays (CBA) are methods that involve transfecting eukaryotic cells with genes expressing specific antigens, followed by detection of specific antibodies in samples using transfected cells. In CBA methodology, cell selection is crucial for the reliability, sensitivity, and reproducibility of experimental results. 293T and CHO cells, with their high transfection efficiency, strong protein expression capacity, and low background characteristics, have become the preferred tools for eukaryotic protein expression, particularly suitable for studies requiring rapid gene manipulation and high-throughput screening. While CHO and 293T cells are key tools with outstanding advantages in high transfection efficiency and rapid proliferation, their weak adhesion is a major technical bottleneck. To address the shortcomings of CHO and 293T cells as CBA expression cell lines and improve adhesion, the inventors made the following invention. Summary of the Invention

[0003] One of the objectives of this invention is to provide a method and plasmid for enhancing cell adhesion.

[0004] The second objective of this invention is to provide a cell that enhances protein expression by improving its adhesion ability.

[0005] The third objective of this invention is to provide a CBA method detection kit for autoimmune encephalitis based on the aforementioned cells.

[0006] The fourth objective of this invention is to provide a CBA method detection kit for autoimmune central nervous system demyelinating diseases based on the aforementioned cells.

[0007] To achieve the above objectives, the present invention employs the following technical means.

[0008] 1. Insert the integrin β1 gene (NCBI: NM_002211.3) into the pCMV-Puro plasmid to obtain the pCMV-integrin-Puro plasmid (see sequence listing 1). The method and plasmid for obtaining cells with enhanced adhesion were obtained by transfecting cells with the pCMV-integrin-Puro plasmid and pre-coating the surface of cell culture material with poly-L-lysine and collagen I.

[0009] 2. Insert the target protein gene into the pCMV-integrin-Puro plasmid to obtain the pCMV-integrin-target gene-Puro plasmid. Transfect cells with this plasmid to achieve simultaneous and efficient dual expression of the target protein and integrin β1. While the target protein is expressed, integrin β1 is overexpressed, significantly improving cell adhesion and cell spread performance to meet the requirements for use in the CBA method.

[0010] 3. The gene sequences of NMDAR, AMPAR1, AMPAR2, LGI1, CASPR2, GABAB R1, GABAB R2, GAD65, IgLON5, mGluR5, GlyR, AQP4, MOG, GFAP, and MBP were inserted into the pCMV-integrin-Puro plasmid as target genes, resulting in 15 plasmids:

[0011] Plasmid 1: pCMV-NMDAR-integrin-Puro plasmid, see appendix Figure 1 ;

[0012] Plasmid 2: pCMV-AMPAR1-integrin-Puro plasmid, see appendix Figure 2 ;

[0013] Plasmid 3: pCMV-AMPAR2-integrin-Puro plasmid, see appendix Figure 3 ;

[0014] Plasmid 4: pCMV-LGI1-integrin-Puro plasmid, see appendix Figure 4 ;

[0015] Plasmid 5: pCMV-CASPR2-integrin-Puro plasmid, see appendix. Figure 5 ;

[0016] Plasmid 6: pCMV-GABAB R1-integrin-Puro plasmid, see appendix Figure 6 ;

[0017] Plasmid 7: pCMV-GABAB R2-integrin-Puro plasmid, see appendix Figure 7 ;

[0018] Plasmid 8: pCMV-GAD65-integrin-Puro plasmid, see appendix. Figure 8 ;

[0019] Plasmid 9: pCMV-IgLON5-integrin-Puro plasmid, see appendix Figure 9 ;

[0020] Plasmid 10: pCMV-mGluR5-integrin-Puro plasmid, see appendix. Figure 10 ;

[0021] Plasmid 11: pCMV-GlyR-integrin-Puro plasmid, see appendix Figure 11 ;

[0022] Plasmid 12: pCMV-AQP4-integrin-Puro plasmid, see appendix. Figure 12 ;

[0023] Plasmid 13: pCMV-MOG-integrin-Puro plasmid, see appendix. Figure 13 ;

[0024] Plasmid 14: pCMV-GFAP-integrin Puro plasmid, see appendix Figure 14 ;

[0025] Plasmid 15: pCMV-MBP-integrin-Puro plasmid, see appendix. Figure 15 .

[0026] Plasmids 6 and 7 were co-transformed into the same 293T cell line, and stable lines were selected to obtain cell line GABAB R1 / 2.

[0027] Plasmids 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 13, 14, and 15 were transfected into 293T cells to screen for stable cell lines, resulting in cell lines NMDAR, AMPA1, AMPA2, LGI1, CASPR2, GAD65, IgLON5, mGluR5, GlyR, AQP4, MOG, GFAP, and MBP, for a total of 14 stable 293T cell lines.

[0028] 4. Fourteen cell slides expressing the target protein were prepared by crawling 14 cell lines onto slides. The 14 cell slides were combined into different combinations as biological carriers in CBA detection. Combined with fluorescently labeled secondary antibodies, washing buffer, etc. required for immunofluorescence detection, a reagent can be prepared to realize the simultaneous detection of multiple autoantibody indicators in a single well.

[0029] 5. A CBA assay kit for detecting autoimmune encephalitis using cell lines NMDAR, AMPA1, AMPA2, LGI1, CASPR2, GABAB R1 / 2, GAD65, IgLON5, mGluR5, and GlyR to prepare cell slides that can simultaneously detect NMDAR, AMPA1, AMPA2, LGI1, CASPR2, GABAB R1 / 2, GAD65, IgLON5, mGluR5, and GlyR autoantibody markers.

[0030] 6. A CBA assay kit for autoimmune central nervous system demyelinating diseases, which uses cell lines AQP4, MOG, GFAP, and MBP to prepare cell slides and simultaneously detects autoantibody indicators of AQP4, MOG, GFAP, and MBP. Attached Figure Description

[0031] Appendix Figure 1 : pCMV-NMDAR-integrin-Puro plasmid diagram.

[0032] Appendix Figure 2 : pCMV-AMPAR1-integrin-Puro plasmid diagram.

[0033] Appendix Figure 3 : pCMV-AMPAR2-integrin-Puro plasmid diagram.

[0034] Appendix Figure 4 : pCMV-LGI1-integrin-Puro plasmid diagram.

[0035] Appendix Figure 5 : pCMV-CASPR2-integrin-Puro plasmid diagram.

[0036] Appendix Figure 6 : pCMV-GABAB R1-integrin-Puro plasmid diagram.

[0037] Appendix Figure 7 : pCMV-GABAB R2-integrin-Puro plasmid diagram.

[0038] Appendix Figure 8 Image of pCMV-GAD65-integrin-Puro plasmid.

[0039] Appendix Figure 9 Image of pCMV-IgLON5-integrin-Puro plasmid.

[0040] Appendix Figure 10 : pCMV-mGluR5-integrin-Puro plasmid diagram.

[0041] Appendix Figure 11 pCMV-GlyR-integrin-Puro plasmid diagram.

[0042] Appendix Figure 12 Image of pCMV-AQP4-integrin-Puro plasmid.

[0043] Appendix Figure 13 pCMV-MOG-integrin-Puro plasmid diagram.

[0044] Appendix Figure 14 : pCMV-GFAP-integrin Puro plasmid diagram.

[0045] Appendix Figure 15 : pCMV-MBP-integrin-Puro plasmid diagram.

[0046] Appendix Figure 16 : pCMV-integrin-Puro plasmid diagram. Detailed Implementation

[0047] Example 1: Verification of cell adhesion after transfection with pCMV-integrin-Puro plasmid.

[0048] Cell line 1: 293T cells;

[0049] Cell line 2: 293T cells were transfected with the pCMV-Puro empty plasmid;

[0050] Cell line 3: 293T cells were transfected into pCMV-integrin-Puro plasmid cells;

[0051] Cell line 4: CHO cells;

[0052] Cell line 5: CHO cells were transfected with the pCMV-Puro empty plasmid;

[0053] Cell line 6: CHO cells were transfected into pCMV-integrin-Puro plasmid cells.

[0054] Main materials: TC-treated 96-well cell culture plates, poly-L-lysine (Sigma-Aldrich, catalog number: P6282-5MG), collagen I (Sigma-Aldrich, catalog number: C3867-1VL), and CCK-8 assay reagent (Dojindo, catalog number: CK04).

[0055] The following reagents were used to pretreat the 96-well cell culture plates:

[0056] Condition 1: Do not process;

[0057] Condition 2: Poly-L-lysine (25 μg / ml);

[0058] Condition 3: Collagen I (5 μg / ml);

[0059] Condition 4: Poly-L-lysine (25 μg / ml) and collagen I (5 μg / ml) are mixed in equal proportions;

[0060] Condition 5: Poly-L-lysine (25 μg / ml) and collagen I (5 μg / ml) are mixed at a ratio of 1:2;

[0061] Using the above conditions, coat 100 μL / well at 2~8℃ overnight, discard the liquid, wash twice with sterile PBS, and air dry for 1 hour before use.

[0062] Procedure: Culture cell lines 1-6 to the logarithmic growth phase, digest with trypsin to count cells, and seed at a density of 5 × 10⁶ cells / year. 3 Cells / well were placed in a 37°C, 5% CO2 cell culture incubator after standing for 5 minutes. Cell adhesion was observed at 4, 8, and 24 hours. Before each observation, the cells were gently washed twice with PBS, and OD values ​​were measured after 2 hours in culture medium containing 10% CCK-8 reagent. After the measurement, the medium was changed and the cells were cultured again.

[0063] Evaluation method:

[0064] 1. Adhesion efficiency (%): Formula = [(OD_experimental - OD_blank) / (OD_control - OD_blank)] × 100%;

[0065] 2. Adhesion rate (ΔOD / h): Formula = (OD / h) t2 - OD t1 ) / (t2 - t1), where t is time (hours);

[0066] 3. Morphological scoring: observe the extension and flattening of pseudopodia, and score from 1 to 5 (5 being the best spread).

[0067] The experimental results are shown in the table below:

[0068]

[0069] Asterisk: indicates optimal performance under this condition.

[0070] The results showed that the cell adhesion performance of CHO cells and 293T cells was significantly increased after transfection with pCMV-integrin-Puro plasmid. The adhesion performance was further enhanced after pretreatment of the culture material under conditions 2-5, with condition 5 showing the best adhesion performance.

[0071] Example 2: Verification of dual expression of target protein and integrin.

[0072] Plasmid 1: pCMV-NMDAR-integrin-Puro plasmid, see appendix Figure 1 ;

[0073] Plasmid 2: pCMV-AMPAR1-integrin-Puro plasmid, see appendix Figure 2 ;

[0074] Plasmid 3: pCMV-AMPAR2-integrin-Puro plasmid, see appendix Figure 3 ;

[0075] Plasmid 4: pCMV-LGI1-integrin-Puro plasmid, see appendix Figure 4 ;

[0076] Plasmid 5: pCMV-CASPR2-integrin-Puro plasmid, see appendix. Figure 5 ;

[0077] Plasmid 6: pCMV-GABAB R1-integrin-Puro plasmid, see appendix Figure 6 ;

[0078] Plasmid 7: pCMV-GABAB R2-integrin-Puro plasmid, see appendix Figure 7 ;

[0079] Plasmid 8: pCMV-GAD65-integrin-Puro plasmid, see appendix. Figure 8 ;

[0080] Plasmid 9: pCMV-IgLON5-integrin-Puro plasmid, see appendix Figure 9 ;

[0081] Plasmid 10: pCMV-mGluR5-integrin-Puro plasmid, see appendix. Figure 10 ;

[0082] Plasmid 11: pCMV-GlyR-integrin-Puro plasmid, see appendix Figure 11 ;

[0083] Plasmid 12: pCMV-AQP4-integrin-Puro plasmid, see appendix. Figure 12 ;

[0084] Plasmid 13: pCMV-MOG-integrin-Puro plasmid, see appendix. Figure 13 ;

[0085] Plasmid 14: pCMV-GFAP-integrin Puro plasmid, see appendix Figure 14 ;

[0086] Plasmid 15: pCMV-MBP-integrin-Puro plasmid, see appendix. Figure 15 .

[0087] Expression cell line: 293T.

[0088] Plasmids 6 and 7 were co-transfected into the same 293T cell line and stable lines were selected to obtain cell line GABAB R1 / 2. Plasmids 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 13, 14, and 15 were transfected into 293T cells and stable lines were selected to obtain cell lines NMDAR, AMPAR1, AMPAR2, LGI1, CASPR2, GAD65, IgLON5, mGluR5, GlyR, AQP4, MOG, GFAP, and MBP, for a total of 14 stable 293T cell lines.

[0089] Verification using primary antibody against the target protein:

[0090] NMDAR (abcam part number: ab68144).

[0091] AMPAR1 (abcam part number: ab183797);

[0092] AMPAR2 (abcam part number: ab133477);

[0093] LGI1 (abcam part number: ab137045);

[0094] CASPR2 (abcam part number: ab137052);

[0095] GABAB R1 / 2 (abcam part numbers: ab238130, ab75838);

[0096] GAD65 (abcam part number: ab307489);

[0097] IgLON5 (Invitrogen part number: PA5-71122);

[0098] mGluR5 (abcam product number: ab76316);

[0099] GlyR (abcam product number: ab192508);

[0100] AQP4 (abcam part number: ab259318);

[0101] MOG (abcam part number: ab109746).

[0102] GFAP (abcam part number: ab68428);

[0103] MBP (abcam part number: ab65988).

[0104] The secondary antibody used for validation was Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, FITC (Invitrogen catalog number: A16105).

[0105] Materials for film climbing:

[0106] Slide 1: A slide coated overnight with a mixture of poly-L-lysine (25 μg / ml) and collagen I (5 μg / ml) in a 1:2 ratio;

[0107] Slide 2: Untreated slide.

[0108] Operating steps:

[0109] 1) Fourteen stable 293T cell lines were cultured to the logarithmic growth phase and counted by trypsin digestion;

[0110] 2) Place slides 1 and 2 at the bottom of a 6-well cell culture plate, moisten with culture medium for 5 minutes, then remove the medium. Inoculate at a density of 2 × 10⁻⁶. 5 2 ml of cells / ml was added to each well to inoculate 14 stable cell lines. After standing for 10 minutes, the cells were placed in a 37°C, 5% CO2 cell culture incubator and cultured overnight. The morphological score was then calculated based on the cell adhesion of the two types of slides.

[0111] 3) Remove the culture medium from the culture wells, take out the glass slides filled with cells, wash the glass slides twice with sterile PBS, treat with immunostaining permeabilization buffer (Saponin) for 10 minutes to obtain CBA cell slides, and cut the slides into 2*2mm cell slides.

[0112] 4) Fix the cell slide onto the glass slide using UV-curable adhesive.

[0113] 5) Dilute the anti-target protein primary antibody with PBS (0.01M pH 7.4) at a ratio of 1:2000 and add it to the cell slide containing the target protein. Use PBS as a blank control. React at room temperature for 30 minutes. After the reaction is complete, wash the cell slide three times with PBST (0.01M PBS pH 7.4 + 0.05% Tween-20).

[0114] 6) Dilute the secondary antibody with PBS (0.01M pH7.4) at a ratio of 1:2000 and add it to all cell slides. Incubate at room temperature for 30 minutes. After the reaction is complete, wash the cell slides three times with PBST (0.01M PBS pH7.4 + 0.05% Tween-20).

[0115] 7) Add mounting medium, cover with coverslip, and observe cell fluorescence morphology under a fluorescence microscope.

[0116] Evaluation method:

[0117] Morphological scoring: Observe the extension, flattening, and cell shedding of pseudopodia, and score from 1 to 5 (5 is the best spreading with no shedding).

[0118] Target protein expression: Observe the consistency of cell fluorescence morphology with the target protein and the percentage of positive signals, and score it as “-”, “+”, “++”, “+++” (“+++” means that the fluorescence morphology matches the target protein model and the positive percentage is over 90%).

[0119] The experimental results are shown in the table below:

[0120]

[0121] The results showed that all 14 cell lines expressed the target protein well. Morphological observation showed that the cell climbing ability on slide 1 was significantly improved by slide 2. Comparison between the cell climbing on slide 1 and the cell climbing after the reaction showed that the cell climbing stability on slide 1 was better and less detached, while there was obvious detachment when using slide 2.

[0122] Example 3: A CBA method detection kit for autoimmune encephalitis.

[0123] This kit consists of a slide substrate with cell slides attached and detection reagents. Cell slides are fixed to one or more reaction regions in the slide substrate using UV-curable adhesive. The cell slides include the following cell lines: NMDAR, AMPAR1, AMPAR2, LGI1, CASPR2, GABAB R1 / 2, GAD65, IgLON5, mGluR5, and GlyR. At least one of these 10 cell lines must be present on the slide. Based on the autoantibody marker for autoimmune encephalitis to be detected, the corresponding cell slides are selected and combined into a reaction region. The detection reagents contain at least a secondary antibody for detecting fluorescently labeled anti-human IgG antibodies. A complete kit also includes sample diluent, washing buffer, mounting medium, coverslips, and quality control materials.

[0124] As a preferred embodiment, this example uses the following combination to form a reagent kit for detection, with the following components:

[0125] 1) Cell slides containing cell lines NMDAR, AMPAR1, AMPAR2, LGI1, CASPR2, GABAB R1 / 2, GAD65, IgLON5, mGluR5, and GlyR were fixed in two reaction areas on a slide substrate.

[0126] 2) Fluorescent secondary antibody: FITC-labeled goat anti-human IgG;

[0127] 3) Sample dilution solution: PBST (0.01M PBS pH 7.4 + 0.05% Tween-20) containing 1% bovine serum albumin;

[0128] 4) Washing solution: PBST (0.01M PBS pH 7.4 + 0.05% Tween-20);

[0129] 5) Mounting tablets: Anti-quenching mounting tablets;

[0130] 6) Coverslip;

[0131] 7) Quality control materials: positive control and negative control.

[0132] Validation samples: 60 clinically positive serum samples covering 10 indicators, and 20 serum samples from normal physical examination subjects.

[0133] Operating steps:

[0134] 1) Sample dilution: Dilute the sample using sample diluent at a ratio of 1:10;

[0135] 2) Sample loading: Load the diluted sample, 30~50μL / reaction zone;

[0136] 3) Sample incubation: Incubate at room temperature for 30 minutes;

[0137] 4) Rinse: After rinsing with the cleaning solution, soak in the solution for 5 minutes;

[0138] 5) Secondary antibody loading: Add FITC-labeled goat anti-human IgG to each reaction area, 30~50μL / reaction area;

[0139] 6) Second antibody incubation: Incubate at room temperature for 30 minutes;

[0140] 7) Rinse: After rinsing with the cleaning solution, soak in the solution for 5 minutes;

[0141] 8) Mounting: Add mounting medium and cover with a coverslip;

[0142] 9) Reading the results: Use a fluorescence microscope to read the fluorescence results.

[0143] The test results are shown in the table below:

[0144]

[0145] Note: Results are expressed as (number of positive samples / number of samples).

[0146] The test results showed that the positive detection rate of LGI1 in clinically positive samples was 90%, and the detection rate of other clinically positive samples was 100%. The overall positive concordance rate was 98.3% (59 / 60), and the negative concordance rate was 100% (20 / 20). There was no cross-reaction between different indicators.

[0147] Example 4: A CBA method detection kit for autoimmune central nervous system demyelinating diseases.

[0148] This kit consists of a slide substrate with cell slides attached and detection reagents. The cell slides are fixed to the same reaction area in the slide substrate using UV-curable adhesive. The cell slides include the following cell lines: AQP4, MOG, GFAP, and MBP. The detection reagents contain at least a secondary antibody for detecting fluorescently labeled anti-human IgG antibodies. A complete kit also includes sample diluent, washing buffer, mounting medium, coverslips, and quality control materials.

[0149] As a preferred embodiment, this example uses the following combination to form a reagent kit for detection, with the following components:

[0150] 1) Cell slides containing four cell lines, namely AQP4, MOG, GFAP, and MBP, were fixed in a reaction area on a slide substrate.

[0151] 2) FITC-labeled goat anti-human IgG;

[0152] 3) Sample dilution solution: PBST (0.01M PBS pH 7.4 + 0.05% Tween-20) containing 1% bovine serum albumin;

[0153] 4) Washing solution: PBST (0.01M PBS pH 7.4 + 0.05% Tween-20);

[0154] 5) Mounting tablets: Anti-quenching mounting tablets;

[0155] 6) Coverslip;

[0156] 7) Quality control materials: positive control and negative control;

[0157] Validation samples: 19 clinically positive serum samples covering 4 indicators, and 20 serum samples from normal physical examination subjects.

[0158] Operating steps: Same as Example 3.

[0159] The test results are shown in the table below:

[0160]

[0161] Note: Results are expressed as (number of positive samples / number of samples).

[0162] The test results showed that the detection rate of this kit for clinical positive samples was 100%, the overall positive concordance rate was 100% (19 / 19), and the negative concordance rate was 100% (20 / 20). There was no cross-reaction between different indicators.

[0163] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any implementation by those skilled in the art using foreseeable alternatives is also within the scope of the present invention.

Claims

1. A modified plasmid that enhances cell adhesion, characterized in that, The modified plasmid is created by inserting the integrin β1 gene into the original plasmid.

2. The modified plasmid according to claim 1, characterized in that, The original plasmid is pCMV-Pure, and the gene sequence of the modified plasmid is sequence 1.

3. The modified plasmid according to either 1 or 2, wherein the target protein gene is further inserted into the plasmid, characterized in that, The target protein is one of NMDAR, AMPAR1, AMPAR2, LGI1, CASPR2, GABAB R1, GABAB R2, GAD65, IgLON5, mGluR5, GlyR, AQP4, MOG, GFAP, and MBP, and the plasmid structure is shown in Figures 1-15.

4. A cell with enhanced adhesion ability, characterized in that the plasmid of claims 1 to 3 is introduced into the cell, wherein the cell is one of 293T, CHO, or HeLa.

5. A method for enhancing cell adhesion, comprising cell construction and surface material treatment, characterized in that... Therefore, the cell construction refers to the construction of the cells as described in claim 4, and the surface material treatment is to pre-coat the material surface with one of poly-L-lysine, collagen I, or a mixture of both.

6. The method according to claim 5, characterized in that... The surface material treatment involves coating a mixture of poly-L-lysine and collagen I at a ratio of 1:

2.

7. A CBA method detection kit for autoimmune encephalitis, comprising a slide substrate and detection reagents, wherein the slide substrate has one or more reaction areas, and cell slides are fixed in the reaction areas. The cell slides are prepared from one of the following cell lines: NMDAR, AMPAR1, AMPAR2, LGI1, CASPR2, GABAB R1 / 2, GAD65, IgLON5, mGluR5, and GlyR, with at least one cell slide fixed in each reaction area of ​​the slide substrate. All cell slides were prepared using the method described in claim 5 or 6.

8. A CBA assay kit for detecting autoimmune central nervous system demyelinating diseases, comprising a slide substrate and detection reagents, wherein the slide substrate has one or more reaction areas, and cell slides are fixed within the reaction areas. The cell slides are prepared from one of the following cell lines: AQP4, MOG, GFAP, and MBP, with each cell line having one of these cell lines fixed within its reaction area. The kit is characterized in that... All cell slides were prepared using the method described in claim 5 or 6.

9. The CBA method detection kit according to any one of claims 7 or 8, wherein the detection reagent comprises the following components: fluorescent secondary antibody, sample diluent, washing buffer, mounting medium, quality control material, and coverslip, characterized in that, The fluorescent secondary antibody was FITC-labeled goat anti-human IgG.

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