Cell strain capable of stably expressing multi-protein tag as well as construction method and application of cell strain

Abstract

The invention discloses a cell strain capable of stably expressing a multi-protein tag as well as a construction method and application of the cell strain, and belongs to the technical field of biology. The cell strain is transfected by a lentiviral vector, a fusion expression vector containing P2A self-cleavage polypeptide and a plurality of protein tag coding sequences is integrated into a host cell, and a stable expression cell strain and a fusion protein expressing a plurality of protein tags including green fluorescent protein are obtained through antibiotic screening. The cell strain can be used for antibody specificity verification, protein interaction research and multi-label system standardization experiment platforms, and has the advantages of stable expression, simple operation, reliable result and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a cell line that stably expresses multiple protein tags, its construction method and application, and is particularly suitable for antibody verification, protein function research and the establishment of standardized experimental systems. Background Technology

[0002] In protein research, 2A peptides are a class of short peptides derived from viruses that can achieve multi-protein co-expression at the translational level. Among them, P2A has a high cleavage efficiency, typically exceeding 90%. Green fluorescent protein (GFP) is widely used as a reporter gene for cell tracking and expression detection. Protein tags such as His, Flag, and HA play important roles in protein purification, detection, and localization. Antibody specificity is crucial for the reliability of experimental data. Traditional antibody validation methods usually require the separate preparation of multiple protein tags, a cumbersome process lacking effective internal controls. Therefore, developing a cellular tool that can stably and efficiently express multiple protein tags simultaneously has significant application value. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a cell line stably expressing multiple protein tags, its construction method, and its applications. A fusion expression vector containing a P2A self-cleaving polypeptide and multiple protein tag coding sequences is integrated into the host cell genome via lentiviral vector transfection, and a stable expression cell line is obtained after antibiotic selection. This cell line simultaneously expresses green fluorescent protein (EGFP) and a fusion protein containing multiple tags, including Flag, HA, Myc, GST, His, V5, and SUMO.

[0004] The present invention adopts the following technical solution: In one aspect, the present invention provides a cell line that stably expresses multiple protein tags. The cell line expresses a fusion protein containing a P2A self-cleaving polypeptide and multiple protein tag coding sequences by transfecting the host cell with a fusion expression vector, which expresses a fusion protein including a green fluorescent protein tag.

[0005] Preferably, the multiple protein tags are two or more of Flag, HA, Myc, GST, His, V5, SUMO or EGFP.

[0006] Preferably, the backbone vector of the expression vector is a Plvx-puro lentiviral vector; the P2A self-cleaving polypeptide is derived from a virus; and the multiple protein tag coding sequences are sequentially linked to form a fusion protein coding region of multiple protein tags, the sequence of which is SEQ ID No:1.

[0007] Preferably, the host cell is HEK293 cell or a derivative thereof.

[0008] In another aspect of the present application, a method for constructing the cell strain is provided, comprising the following steps: (1) Constructing a fusion expression vector containing P2A and multiple protein tag coding sequences using a lentiviral vector; (2) Transfecting the expression vector into a host cell; (3) Obtaining a cell strain stably expressing multiple protein tags through antibiotic screening.

[0009] Preferably, in step 3, the antibiotic is puromycin, and the screening concentration is 0.5-4 μg / mL.

[0010] In another aspect of the present application, the cell strain is applied in antibody verification, Western Blot, immunoprecipitation or immunofluorescence experiments.

[0011] Preferably, the antibody verification is the specificity of the cell strain for simultaneously verifying multiple protein tag antibodies.

[0012] Compared with the prior art, the present application has the following beneficial effects: 1. One strain for multiple uses: a single cell strain can simultaneously verify multiple tag antibodies, greatly simplifying the experimental process.

[0013] 2. Built-in control: the double-band pattern produced by P2A cleavage provides internal quality control for antibody verification.

[0014] 3. Stable and reliable: stable cell strains avoid fluctuations in transient transfection efficiency, ensuring consistency of experimental results.

[0015] 4. Wide application: suitable for various protein research scenarios such as WB, Co-IP, IF, etc. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 FIG. 1 is a structural diagram of the pLVX-P2A-multiple protein tag fusion expression vector constructed in the present application; Figure 2 FIG. 2 is the result of gene expression verification, wherein the loading amount of each plasmid is 100 ng; Figure 3Figure 1 shows the results of Western Blot detection of the fusion expression of multiple protein tags (lane 1: protein Marker, lane 2: lysate of untransfected HEK293 cells, lane 3: lysate of HEK293 cells transfected with the fusion expression vector); wherein (a) is an Anti-EGFP detection antibody; (b) is an Anti-Flag detection antibody; (c) is an Anti-GST detection antibody; (d) is an Anti-HA detection antibody; (e) is an Anti-His detection antibody; (f) is an Anti-V5 detection antibody; (g) is an anti-Myc detection antibody; (h) is an anti-SUMO detection antibody. DETAILED DESCRIPTION

[0017] In order to make the present application more apparent and easy to understand, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0018] In order to verify whether the tag antibody can recognize the corresponding protein tag, a large number of proteins with different tags are usually required, which brings complexity to the experiment. The present application provides a cell strain stably expressing multiple protein tags, a construction method thereof and an application thereof. The cell strain is transfected by a lentiviral vector, a fusion expression vector containing a P2A self-cleavage polypeptide and multiple protein tag coding sequences is integrated into the host cell, and a stable expression cell strain is obtained through antibiotic screening, which simultaneously expresses a fusion protein of multiple tags including Flag, HA, Myc, GST, His, V5, SUMO and EGFP. The cell strain can be used for antibody specificity verification, protein interaction research and a multiple tag system standardized experimental platform, and has the advantages of stable expression, simple operation and reliable results.

[0019] The biochemical reagents and materials in the examples are all conventional commercially available reagents: HEK293 cells were purchased from the Chinese Academy of Sciences Typical Culture Collection Cell Bank; E. coli TOP10 was purchased from Shengong Bioengineering; the expression vector was constructed by Anshengda; RIPA cell lysis buffer was purchased from Aladdin Biochemical Technology; DMEM was purchased from Invitrogen; fetal bovine serum was purchased from Gicbo; puromycin was purchased from Aladdin Biochemical Technology; PEI was purchased from Aladdin Biochemical Technology; BCA kit was purchased from Thermo; BSA was purchased from Aladdin Biochemical Technology; endotoxin-free plasmid large extraction kit was purchased from Aladdin Biochemical Technology; Anti-EGFP detection antibody was purchased from Aladdin Biochemical Technology; Anti-Flag detection antibody was purchased from Aladdin Biochemical Technology; Anti-GST detection antibody was purchased from Aladdin Biochemical Technology; Anti-HA detection antibody was purchased from Aladdin Biochemical Technology; Anti-His detection antibody was purchased from Aladdin Biochemical Technology; Anti-V5 detection antibody was purchased from Aladdin Biochemical Technology; Anti-Myc detection antibody was purchased from Aladdin Biochemical Technology; anti-SUMO detection antibody was purchased from Aladdin Biochemical Technology; HPR rabbit secondary antibody and mouse secondary antibody were purchased from Aladdin Biochemical Technology; ECL chemiluminescence solution was purchased from Aladdin Biochemical Technology.

[0020] The culture medium of HEK293 cells is composed of 90% DMEM medium and 10% FBS, wherein % represents the percentage of the total medium volume. The culture medium of HEK293 cells after transfection of the expression vector is composed of 90% DMEM medium, 10% FBS and 2 μg / mL puromycin in final concentration, wherein % represents the percentage of the total medium volume. The technical means in the examples, including PCR, homologous recombination, vector construction and cell experiments, are also conventional means for biomedical field technicians.

[0021] Example 1: Transfection of HEK293 cells with expression vector plasmid 1.1 Construction of expression vector plasmid Vector ligation and transformation: the pLVX-puro backbone vector was linearized by double digestion with restriction enzymes BamHI and XbaI, and purified; the fusion protein coding region fragments of various protein tags obtained in the above step were mixed with the linearized vector at a molar ratio of 3:1, and Gibson Assembly reaction was performed; the reaction product was transformed into TOP10 E. coli competent cells, and coated on LB plates containing ampicillin and incubated at 37°C overnight; Colony PCR primary screening: single colonies were picked and subjected to PCR using specific primers spanning the internal elements to screen positive clones; Plasmid digestion identification: the plasmid of the positive clone was extracted and subjected to double digestion with BamHI and XbaI, and the correct size of the inserted fragment was confirmed by electrophoresis; Sequencing verification: the plasmid correctly identified by digestion was subjected to full-length DNA sequencing to confirm that all tags, linkers and P2A sequences were correct; the correctly constructed recombinant plasmid was named pLVX-Flag-HA-Myc-GST-P2A-EGFP-SUMO-V5-His-puro, and the structure was as shown in Figure 1 .

[0022] 1.2 Plasmid transformation and amplification Two sterile EP tubes were taken, and 50 μL of ice-melted competent E. coli trans5α was added to each, and 10 μL of 50 ng pLVX Flag-HA-Myc-GST-P2A-EGFP-SUMO-V5-His-puro expression vector was added to each, and mixed gently, and placed on ice for 40 min; heat shock at 42°C for 45 seconds, and quickly transferred to ice bath for 2 minutes; 200 μL of LB liquid antibiotic-free culture medium was added, and incubated at 37°C on a shaker for 1 hour for recovery; the recovered bacteria were centrifuged at 1000 rpm for 3 min, and the supernatant was discarded, and only about 50 μL was retained, and the E. coli was resuspended, and coated on LB plates containing 50 μg / mL Amp and LB plates without Amp, respectively, and incubated at 37°C overnight; single colonies were picked from the LB plate containing Amp, and placed in 200 mL of LB liquid medium containing 50 μg / mL Amp, and incubated at 37°C, 220 rpm for 16-24 h.

[0023] The overnight cultured bacteria were subjected to plasmid extraction using an endotoxin-free plasmid extraction kit, and pLVX Flag-HA-Myc-GST-P2A-EGFP-SUMO-V5-His-puro plasmid was obtained, and the concentration of the extracted plasmid was determined using a UV spectrophotometer.

[0024] 1.3 Cell transfection HEK293 cells were plated in 6-well plates, and when the density reached 40-50%, the cells in each well were replaced with 2 mL of complete culture medium; 100 μL of serum-free DMEM medium was gently mixed with 2 μg of pLVX Flag-HA-Myc-GST-P2A-EGFP-SUMO-V5-His-puro plasmid to prepare a plasmid diluent; 5 μL of 1 mg / mL PEI transfection reagent was immediately added to 100 μL of the plasmid diluent and gently mixed; incubate at room temperature for 10-25 min; add the incubation product to the 6-well plate at a volume of 100 μL per well; gently mix and incubate at 37°C in a 5% CO2 incubator; replace the medium after 24 h of incubation to obtain puro-Flag-HA-Myc-GST-P2A-EGFP-SUMO-V5-His Tag-HEK29 recombinant polyclonal (hereinafter referred to as pLVX puro-P2A EGFP Tag-HEK293); and take white light and fluorescence images of each group of cells using a microscope, count and calculate the transfection efficiency of each group, Transfection efficiency = number of green fluorescent cells / number of cells in the white light image x 100%.

[0025] Example 2: Screening of pLVX puro-P2A EGFP Tag-HEK293 recombinant polyclonal using puromycin 2.1 Determination of lethal concentration of puromycin on pLVX puro-P2A EGFP Tag-HEK293 recombinant polyclonal (1) pLVX puro-P2A EGFP Tag-HEK293 recombinant polyclonal cells in good growth condition in the culture medium were plated in a 96-well plate at a density of 3000 cells per well, and incubated in a 37°C, 5% CO2 incubator; (2) After the cells adhered, 0.0625, 0.125, 0.25, 0.5, 1, 2, and 4 μg / mL of puromycin were added to each well of the 96-well plate, respectively; (3) The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 48 h; (4) CCK8 solution was added to the 96-well plate at a volume of 10 μL per well, and the 96-well plate was incubated in a 37°C, 5% CO2 incubator for 4 h; (5) The absorbance of the test sample at 450 nm was measured using a microplate reader.

[0026] According to the results, 2 μg / mL of puromycin was selected to incubate with the pLVX puro-P2A EGFP Tag-HEK293 recombinant polyclonal cells for 48 h to screen them.

[0027] 2.2 Screening and enrichment of pLVX puro-P2A EGFP Tag-HEK293 recombinant polyclone After the untransfected HEK293 cells and the transfected pLVX puro-P2A EGFP Tag-HEK293 recombinant polyclone cells were fully grown, 0.25% trypsin was used for digestion, and the cells were transferred into 6 cm culture dishes. After the cells adhered, 2 μg / mL puromycin was added for screening for 48 h. Since the transfected pLVX puro-P2A EGFP Tag-HEK293 recombinant polyclone cells grew too slowly when cultured with 2 μg / mL puromycin, 1 μg / mL puromycin was used for continuous culture (the blank group was untransfected HEK293 cells). After 5 days, all the cells in the blank group died and floated, and part of the cells in the transfected group still survived. The surviving cells in the transfected group were subcultured into T25 bottles, and after fully growing, they were subcultured at a ratio of 1:3. One fifth of the cells were used for gene expression verification, and the results are shown in Figure 2 .

[0028] Example 3: Screening of pLVX puro-P2A EGFP Tag-HEK293 monoclonal cells and expansion of culture 3.1 Isolation of monoclonal cells by limiting dilution The cells were dispersed in a 96-well plate by gradient dilution, so that there was 1 cell per well. The numbers of the first column of wells in the 96-well plate were A1, B1, C1, D1, E1, F1, G1, and H1, and the numbers of the second column of wells were A2, B2, C2, D2, E2, F2, G2, and H2, and so on. The numbers of the twelfth column of wells were A12, B12, C12, D12, E12, F12, G12, and H12. The specific operation was as follows: 200 μL of cell suspension was added to well A1 of the sterilized 96-well plate, and 100 μL of medium was added to each of the other wells; 100 μL of cell suspension in A1 was mixed with the medium in B1 of the first column, avoiding the generation of bubbles during mixing, and the same 1:2 dilution was continued to H1, and the volume of each well in the first column was made up to 200 μL with medium, completing the dilution of the first column; the cell suspension in the first column was mixed, 100 μL of which was added to the second column, and the mixture was gently mixed with a pipette. The 1:2 dilution was repeated to the twelfth column, and the volume of each well was made up to 200 μL with medium, completing the dilution.

[0029] 3.2 Observation of green fluorescence of monoclonal cells by fluorescence microscope The diluted culture plates were incubated at 37°C for 8 days, and wells containing monoclonal colonies were labeled under a microscope. Once the monoclonal colonies had grown, they were sequentially transferred to 24-well plates, 6-well plates, and finally T25 culture flasks, resulting in two pLVX puro-P2A EGFP Tag-HEK293 monoclonal cell lines: pLVX puro-P2A EGFP Tag-HEK293 monoclonal cell line 1# and pLVX puro-P2A EGFP Tag-HEK293 monoclonal cell line 2#. Untransfected HEK293 cells and pLVX puro-P2A EGFP Tag-HEK293 monoclonal cells were photographed using a fluorescence microscope. Untransfected HEK293 cells did not fluoresce green, while pLVX puro-P2A EGFP Tag-HEK293 monoclonal cell line 1# and pLVX puro-P2A EGFP Tag-HEK293 monoclonal cell line 2# showed green fluorescence.

[0030] Example 4: Identification of pLVX puro-P2A EGFP Tag-HEK293 monoclonal strains 4.1 Detection of recombinant monoclonal protein expression by Western blotting 100 μL of pre-cooled RIPA (containing 1% protease inhibitor cocktail and PMSF at a final concentration of 1 mM, where % represents the percentage of total RIPA volume) was added to 2 × 10⁻⁶ cells / mL. 6In the cell pellets of untransfected HEK293 cells, pLVX puro-P2AEGFP Tag-HEK293 monoclonal cells 1#, and pLVX puro-P2A EGFP Tag-HEK293 monoclonal cells 2#, the pellets were vortexed to completely disperse them for total protein extraction. After centrifugation at 12000 rpm and 4°C for 20 min, the supernatant was collected and quantified using a BCA kit. Each protein sample was then mixed with 5×SDS-PAGE protein loading buffer at a volume ratio of 4:1 and boiled for 10 min. The target protein was separated by SDS-PAGE (4-20% gradient gel), transferred to a PVDF membrane, blocked with 5% bovine serum albumin (BSA) or skim milk for 1 hour, and incubated overnight at 4°C with primary antibody (Anti-EGFP detection antibody, Anti-Flag detection antibody, Anti-GST detection antibody, Anti-HA detection antibody, Anti-His detection antibody, Anti-V5 detection antibody, anti-Myc detection antibody, or anti-SUMO detection antibody). The membrane was washed three times with PBST (0.5 mL Tween 20 + 500 mL PBS), and incubated for 1 hour with either HPR rabbit or mouse secondary antibody (secondary antibody selection depends on the antibody host species). The membrane was washed three more times with PBST, and then incubated with ECL chemiluminescence buffer for 2 minutes. The protein bands were then exposed and photographed using a chemiluminescence imaging system. Figure 3 As shown.

[0031] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A cell line stably expressing multiple protein tags, characterized in that, The cell line expresses a fusion protein containing a P2A self-cleaving polypeptide and multiple protein tag coding sequences by transfecting it into host cells, thereby expressing a fusion protein with multiple protein tags, including a green fluorescent protein tag.

2. The cell line according to claim 1, characterized in that, The various protein tags are two or more of Flag, HA, Myc, GST, His, V5, SUMO or EGFP.

3. The cell line according to claim 1, characterized in that, The backbone vector of the expression vector is a Plvx-puro lentiviral vector; the P2A self-cleaving polypeptide is derived from a virus; the multiple protein tag coding sequences are sequentially linked to form a fusion protein coding region of multiple protein tags, the sequence of which is shown in SEQ ID No:

1.

4. The cell line according to claim 1, characterized in that, The host cell is HEK293 cell or a derivative thereof.

5. A method for constructing a cell line as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) Construct a fusion expression vector containing P2A and multiple protein tag coding sequences using lentiviral vectors; (2) Transfect the expression vector into the host cell; (3) Obtain cell lines that stably express multiple protein tags through antibiotic screening.

6. The construction method according to claim 5, characterized in that, In step 3, the antibiotic is puromycin; the screening concentration is 0.5-4 μg / mL.

7. The use of a cell line as described in any one of claims 1-4 in antibody validation, Western blotting, immunoprecipitation, or immunofluorescence experiments.

8. The application according to claim 7, characterized in that, The antibody validation refers to the use of cell lines to simultaneously validate the specificity of multiple protein-tagged antibodies.

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