Monoclonal cell strain for over-expression of Nav1.5 and construction method thereof

Abstract

The invention provides a monoclonal cell strain for over-expression of Nav1.5 and a construction method of the monoclonal cell strain, and belongs to the technical field of biology. The monoclonal cell strain capable of overexpressing Nav1.5 is prepared by optimizing a codon construction vector of the SCN5A gene, and the cell strain capable of overexpressing Nav1.5 has wide application prospects in the fields of cardiac electrophysiology, pharmacology, hereditary arrhythmia disease research, new drug development and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a monoclonal cell line overexpressing Nav1.5 and a construction method thereof. Background Art

[0002] Nav1.5 is a voltage-gated sodium channel specifically expressed in cardiac tissues, distributed in atrial and ventricular myocytes and Purkinje fibers, and is an essential participant in the initiation of cardiac action potentials and cardiac electrical conduction. The human cardiac sodium channel (Nav1.5) is a member of the voltage-gated sodium channel family, which has 9 members (Nav1.1 to 1.9) and is encoded by 9 genes (SCN1-5A, SCN8-11A).

[0003] Nav1.5 plays a key role in the initiation and propagation of cardiac electrical activity. It is also the target of many antiarrhythmic drugs and is essential for maintaining normal heart rate and cardiac function. Many antiarrhythmic drugs work by acting on Nav1.5 channels. Using cell lines expressing Nav1.5, it is possible to evaluate the effects of drugs on Nav1.5 channel function, as well as the cardiac safety and potential toxicity of the drugs. By expressing Nav1.5 in cell lines, researchers can study the initiation and conduction characteristics of cardiac action potentials and how these processes are affected by different physiological and pathological conditions.

[0004] Cell lines expressing Nav1.5 have a wide range of applications in cardiac electrophysiology, pharmacology, genetic arrhythmia research, and new drug development. Through these cell lines, researchers can gain a deeper understanding of the role of Nav1.5 in cardiac function and disease, and provide new strategies and targets for the treatment of related diseases. Summary of the invention

[0005] The object of the present invention is to provide a monoclonal cell line overexpressing Nav1.5 and a construction method thereof.

[0006] The present invention provides a gene fragment, the nucleotide sequence of which is shown as SEQ ID No.1.

[0007] The present invention also provides a recombinant vector, which comprises the above gene fragment.

[0008] Furthermore, the recombinant vector comprises the gene fragment according to claim 1, and the vector in the recombinant vector is preferably a pCDNA3 plasmid vector.

[0009] The present invention also provides a cell line for over-expressing Nav1.5, which is obtained by transfecting cells with the above-mentioned recombinant vector.

[0010] Furthermore, the cells are HEK293 cells.

[0011] The present invention also provides a method for constructing the above cell line, the method comprising the following steps:

[0012] (1) Take the gene fragment with the nucleotide sequence shown in SEQ ID No. 1 and the pCDNA3 plasmid vector to construct a recombinant vector;

[0013] (2) Transfecting cells with the recombinant vector obtained in step (1), culturing, and screening to obtain a cell line that overexpresses Nav1.5.

[0014] Furthermore, in step (1), the pCDNA3 plasmid vector is a pCDNA3.1(+) vector.

[0015] Furthermore, in step (2), the cells are HEK293 cells.

[0016] Furthermore, in step (2), the screening is performed using 1100-1300 μg / mL (G-418Sulfate) screening medium culture screening.

[0017] Further, the screening was performed using 1200 μg / mL (G-418Sulfate) screening medium culture screening.

[0018] The present invention has achieved the following beneficial effects:

[0019] The method optimizes the codons of the SCN5A gene, redesigns the gene with a nucleotide sequence as shown in SEQ ID No. 1, enhances mRNA stability, increases the translation efficiency of the target gene SCN5A, and improves the protein expression level.

[0020] This method describes in detail how to construct a monoclonal cell line that overexpresses Nav1.5, and provides three cell identification methods: PCR, IFC, and Nav1.5 characteristic current detection. Nav1.5-HEK293 cells are identified and detected from three perspectives: DNA, protein expression, and characteristic current. This method optimizes the indirect immunofluorescence experimental steps, does not require membrane permeabilization, can eliminate the interference of intracellular proteins, and can more accurately and quickly detect the correct expression of membrane proteins. It can be used for the detection of cell membrane proteins. Indirect immunofluorescence shows the high expression of Nav1.5 protein on the HEK293 cell membrane.

[0021] In summary, the present invention provides a monoclonal cell line overexpressing Nav1.5 and a construction method thereof. The present invention constructs a vector by optimizing the codons of the SCN5A gene to prepare a monoclonal cell line capable of overexpressing Nav1.5. The cell line overexpressing Nav1.5 has broad application prospects in the fields of cardiac electrophysiology, pharmacology, genetic arrhythmia disease research and new drug development.

[0022] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0023] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the PCR identification result.

[0025] Figure 2 The results of indirect immunofluorescence identification.

[0026] Figure 3 Results of the identification of the characteristic current of Nav1.5: (A) is the continuous step voltage stimulation program and the characteristic current of Nav1.5; (B) is the current-voltage relationship curve (IV curve). DETAILED DESCRIPTION

[0027] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0028] The "room temperature" referred to in the present invention is 10-30°C, and the time of "overnight" is 7-17 hours.

[0029] The information of the cell lines, strains and plasmids used in the present invention are as follows:

[0030] Cell line: HEK293

[0031] Plasmid: pCDNA3 vector

[0032] Gene information: SCN5A sodium voltage-gated channel alpha subunit 5 [Homosapiens (human)], also known as Nav1.5; Gene ID: 6331.

[0033] The main reagent information is shown in Table 1:

[0034] Table 1 Main reagent information

[0035]

[0036] The main solutions and their configurations are shown in Table 2:

[0037] Table 2 Main components of intracellular and extracellular fluid recorded by Na current (1L)

[0038]

[0039] The main instruments are shown in Table 3:

[0040] Table 3 Main instruments

[0041]

[0042] Example 1: Study on monoclonal cell lines overexpressing Nav1.5

[0043] 1. Construct vector and prepare plasmid

[0044] After optimizing the codons, the SCN5ACDs region (Genebank: NM_000335, 6135 bp) was cloned into the BamHI / EcoRI site of the pCDNA3.1(+) vector. The vector contains a CMV promoter and an SV40 promoter, which drive the expression of the inserted NaV1.5 and neomycin resistance genes, respectively. TM 3.1(+) is a non-fusion vector. The insert should contain the Kozak translation initiation sequence and the ATG initiation codon, i.e. GCC ACCATG, to correctly initiate translation. Transform into E. coli, select and culture on LB plates containing 50-100 μg / ml ampicillin, and sequence to confirm whether the gene is cloned in the correct position.

[0045] The gene sequence SCN5A-0920-7 inserted into the pCDNA3.1(+) vector is as follows (SEQ ID No. 1):

[0046]

[0047] The SCN5A gene synthesis information is shown in Table 4:

[0048] Table 4 Synthesis information

[0049]

[0050] 2. Cell transfection

[0051] (1) HBS formula: 20 mM HEPES (cell culture grade), 150 mM NaCl, pH 7.4.

[0052] Dissolve 876 mg of NaCl in 90 ml of double distilled water. Add 2 ml of 1M HEPES to adjust the pH to 7.4. Make up to 100 ml and filter through a 0.2 μm pore size filter.

[0053] (2) Cell plating: 0.5-1.0×10 6 293 cells (specifically 1×10 6 HEK293) in a 60 mm small dish. After 16 to 24 hours, the cell density should reach 50-60% confluence during transfection. Change the medium 2 hours before transfection and replace the old medium with 3 mL of complete medium.

[0054] (3) Preparation of transfection complex:

[0055] ① Take a 2mL EP tube and dilute 5μg of DNA (RNA) in HBS buffer to 0.1μg / μL (final volume 50μL) in a high pressure reaction tube.

[0056] ② Take a 2mL EP tube and mix 30μL DOTAP with HBS buffer to a final volume of 100μL.

[0057] ③ Transfer the nucleic acid solution from step ① (50 μL) to the reaction tube containing DOTAP in DPBS buffer from step ② (100 μL). Mix the transfection solution carefully by gently pipetting several times. Do not rotate or centrifuge.

[0058] ④ Incubate the transfection solution at 15-25°C for 10-15 minutes. The appearance of turbidity in the reaction mixture does not affect the transfection efficiency.

[0059] ⑤ Gently mix the DOTAP / nucleic acid mixture with 5 mL of culture medium and incubate at room temperature for 3 to 5 minutes.

[0060] (4) Transfection: Discard the culture medium, wash the cells with 2 mL of DPBS, and then add the mixed liposome and plasmid complex dropwise. Incubate in an incubator. After 6 hours, remove the culture medium, slowly add 4 mL of complete medium containing 20% ​​serum, and continue culturing.

[0061] (5) After 24 h of culture, add 4 mL of 1200 μg / mL (G-418Sulfate) 10% serum complete medium, continue to culture. Set up a non-transfected control group, culture for 7 days until the control group begins to die, change the medium every 3-5 days depending on the degree of cell death, and the cells in the control group will die completely after 10-15 days.

[0062] (6) Add 5 mL of complete culture medium to the transfected test group and maintain 1200 μg / mL (G-418Sulfate) resistance screening of resistance-positive cells; if the cells are overcrowded, continue to passage. After passaging, collect cells for subsequent verification.

[0063] 3. Construction of monoclonal stable transfectant

[0064] The mixed cloned stable cells after transfection and screening are diluted and cultured, and the cell clones grown from single cells are picked and expanded to obtain cell lines with single traits and stable expression.

[0065] The specific steps are as follows:

[0066] (1) After digestion, the cells were inoculated in a 96-well plate at a cell density of 1 cell / well, 100uL per well (limiting dilution or flow cytometry) and a density of 50 cells / plate. The culture medium contained 800μg / mL of The culture was continued in complete medium supplemented with 10% serum (G-418 Sulfate).

[0067] (2) On the 4th to 5th day after inoculation, add 800 μg / mL of (G-418Sulfate) 10% serum complete medium 100uL / well, continue to culture; 10-15 days after inoculation, mark the wells containing only single cell colonies and change the medium. 15-20 days after inoculation, digest the surviving cell colonies to 24-well plates, and choose a medium containing 400μg / mL of (G-418Sulfate) complete medium with 15-20% serum, continue culturing. Mark the wells with single cells and use (G-418Sulfate) antibiotics were used for selection.

[0068] (3) After the cells have grown to full size, the culture is expanded and the cells are collected for PCR, IHC identification, and Nav1.5 current detection. Monoclonal cells with normal identification results are selected and cryopreserved.

[0069] 4. Identification of monoclonal cell lines

[0070] 4.1 PCR identification

[0071] (1) Primer information

[0072] Three pairs of qPCR primers (shown in Table 5) were designed for the codon-optimized sequence SCN5A-O920-7 in the CDs region of the SCN5A gene and used for backup after verification. DNA was extracted from cells in the transfection group and the control group, and PCR was used to verify whether the transfection was successful using a high-fidelity PCR enzyme.

[0073] Table 5 PCR primers

[0074]

[0075] (2) PCR amplification system

[0076] Table 6 PCR amplification system

[0077]

[0078] (3) PCR amplification procedure

[0079] Table 7 PCR amplification program

[0080]

[0081]

[0082] PCR amplification was performed according to the above PCR amplification system and PCR amplification procedure. After the reaction, 5 μL of PCR product was taken and detected by 1% agarose gel electrophoresis, and a 5000 bp DNA marker was added as a control.

[0083] 4.2 Indirect immunofluorescence

[0084] (1) Laying plate: Nunc TM The glass-bottomed culture dish was coated with a diluted Poly-L-lysine solution (1:50 dilution) overnight, washed 3 times with PBS and completely air-dried. Then, HEK-293 and NaV1.5-293 cells were digested and plated on Nunc TM Glass-bottom culture dish, set aside after it is full of cells.

[0085] (2) Fixation: Add 500 μL to 1 mL of fixative (4% paraformaldehyde) to each well to cover the cells, incubate at room temperature for 15 min, and discard the fixative.

[0086] (3) Blocking: Add 500 μL to 1 mL of QuickBlock to each well TM Cover the cells with immunostaining blocking solution and block for 10 to 30 minutes at room temperature without washing.

[0087] (5) Incubation with primary antibody: dilute Anti-NaV1.5 antibody 1:500 and add it to the culture dish at once, incubate at 4°C overnight, and wash once with 1 mL of PBS.

[0088] Primary antibody: 2uL antibody + 1mL QuickBlock TM Immunostaining primary antibody dilution

[0089] (6) Incubate with secondary antibody: 488-conjugated Goat Anti-Rabbit IgG (H+L) (AS053) was diluted with QuickBlock at a ratio of 1:500 TM Dilute the primary antibody diluent for immunostaining in the dark, cover the cells, and incubate at room temperature in the dark for 1 h.

[0090] Secondary antibody: 2uL antibody + 1mL QuickBlock TM Immunostaining secondary antibody dilution

[0091] (7) DAPI staining: DAPI (1 μg / mL) was used for nuclear staining for 1 minute. The cells were washed once with PBS. The cells were observed and photographed using an upright fluorescence microscope.

[0092] 4.3Nav1.5 characteristic current detection

[0093] 4.3.1 Electrophysiological recording

[0094] (1) The patch clamp recording system used an amplifier, a digital-to-analog converter, and pClamp10.6 acquisition and analysis software (Molecular devices, USA);

[0095] (2) Use a borosilicate hard glass electrode. Use a drawing instrument (P-97, Sutter) to draw the glass electrode. After filling the electrode with liquid, the electrode resistance is between 2-5MΩ;

[0096] (3) During the experiment, the pre-treated cell slides were placed in a bath, and independent single cells were selected to make the field of view clear;

[0097] (4) Fix the microelectrode filled with electrode liquid on the amplifier probe, use the microelectrode manipulator to put the electrode into the liquid and slowly approach the cell;

[0098] (5) After the electrode contacts the cell, negative pressure is applied to form a GΩ seal between the electrode tip and the cell membrane surface. After compensating the electrode capacitance, negative pressure is applied to break the cell membrane and compensate for the series resistance and cell membrane capacitance.

[0099] (6) Electrophysiological recording was performed using 6 kHz filtering;

[0100] (7) Before administering the drug preparation, the current value should reach a stable state for at least 1 minute. If there is no change after 1 minute of perfusion administration or after perfusion until the current reaches a new stable state, measure the current value again. The drug is administered from low to high concentrations of the drug solution. Each concentration change must be performed under the premise that the current is stable at the previous concentration.

[0101] 4.3.2 Voltage stimulation protocol

[0102] Nav1.5-peak current recording: the cell membrane voltage was clamped at -100 mV, the depolarization voltage was from -100 mV to +50 mV, the step voltage was 5 mV, maintained for 200 ms, and each sweep was 5 s.

[0103] 4.3.3 Drawing

[0104] Clampfit and Graphpad were used for data analysis, drawing and image processing. The voltage-gated sodium channel current was induced by a continuous step voltage stimulation program, and the IV curve of the sodium channel was drawn with the maximum value (i.e., peak value) of the sodium current at each voltage as the ordinate and each stimulation voltage as the abscissa.

[0105] 5. Experimental results

[0106] Indirect immunofluorescence was performed without membrane permeabilization during the experiment. The cells showed strong green fluorescence signals, indicating that the target protein was located on the cell membrane, excluding the interference of intracellular proteins, indicating that Nav1.5 was overexpressed on the cell membrane. Finally, the results of PCR identification ( Figure 1 ), indirect immunofluorescence results ( Figure 2 ) and Nav1.5 characteristic current results ( Figure 3 ), confirming that the monoclonal cell line constructed by optimizing the codons of the SCN5A gene in the present invention can overexpress Nav1.5.

[0107] In summary, the present invention provides a monoclonal cell line overexpressing Nav1.5 and a construction method thereof. The present invention constructs a vector by optimizing the codons of the SCN5A gene to prepare a monoclonal cell line capable of overexpressing Nav1.5. The cell line overexpressing Nav1.5 has broad application prospects in the fields of cardiac electrophysiology, pharmacology, genetic arrhythmia disease research and new drug development.

Claims

1. A gene fragment, characterized in that: Its nucleotide sequence is shown in SEQ ID No.

1.

2. A recombinant vector, characterized in that: It comprises the gene fragment described in claim 1.

3. The recombinant vector according to claim 2, characterized in that The recombinant vector comprises the gene fragment according to claim 1, and the vector in the recombinant vector is preferably a pCDNA3 plasmid vector.

4. A cell line overexpressing Nav1.5, characterized in that: The invention is obtained by transfecting cells with the recombinant vector described in claim 2 or 3.

5. The cell line according to claim 4, characterized in that The cells are HEK293 cells.

6. A method for constructing the cell line according to claim 4 or 5, characterized in that: The method comprises the following steps: (1) Take the gene fragment with the nucleotide sequence shown in SEQ ID No. 1 and the pCDNA3 plasmid vector to construct a recombinant vector; (2) Transfecting cells with the recombinant vector obtained in step (1), culturing, and screening to obtain a cell line that overexpresses Nav1.

5.

7. The construction method according to claim 6, characterized in that: In step (1), the pCDNA3 plasmid vector is a pCDNA3.1(+) vector.

8. The construction method according to claim 6, characterized in that: In step (2), the cells are HEK293 cells.

9. The construction method according to claim 6, characterized in that: In step (2), the screening is performed using 1100-1300 μg / mL (G-418Sulfate) screening medium culture screening.

10. The construction method according to claim 9, characterized in that: The screening was performed with 1200 μg / mL (G-418Sulfate) screening medium culture screening.

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