KV1.3 expression vector, cell-free expression system, recombination channel and application thereof

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a KV1.3 expression vector, a cell-free expression system, a recombination channel and application thereof. The recombinant expression vector is obtained by inserting a DNA (deoxyribonucleic acid) sequence as shown in SEQ ID NO.1 between an Nde I site and an Xho I site of a pIVEX-2. 4d vector, and the recombinant expression vector is named as pIVEX-K. The cell-free expression system is obtained by transferring the expression vector into a cell-free system. The recombination channel is obtained by recombining a cell-free expression system product and lipidosome. The recombinant channel can be applied to high-throughput screening of compounds influencing the potassium ion channel function, the influence of the compounds on the potassium channel function is directly displayed by monitoring the change of a fluorescence signal before and after a to-be-detected object is added, and the recombinant channel has the characteristics of simplicity in operation, short test period and high sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a K V 1.3 Expression vectors, cell-free expression systems, recombinant channels and their applications. Background Technology

[0002] Studies have shown that voltage-gated potassium channels (K) V 1.3 Encoded by the KCNA3 gene, it is mainly distributed in the brain, lungs, pancreas, and immune organs and cells, such as the thymus, spleen, lymph nodes, B lymphocytes, T lymphocytes, macrophages, and microglia. V 1.3 This pathway is associated with various physiological diseases, such as asthma, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, and psoriasis, as well as other autoimmune and chronic diseases. Furthermore, K is present in the pathogenesis of many of these diseases. V 1.3 Ion channel expression is upregulated. K V 1.3 Abnormal expression of the K channel is associated with the occurrence of various cancers, including breast cancer, prostate cancer, kidney cancer, and head and neck cancer. It is an important indicator of cancer occurrence and development and is considered an important potential target for the treatment of various cancers. V 1.3 Potassium channels are tetrameric structures composed of four identical subunits. Potassium ions can selectively pass through the central pore region. However, due to the highly complex structure of channel proteins, potassium ions cannot pass through the central pore region. V 1.3 It is difficult to express and is often unstable in eukaryotic cell expression systems, which to some extent limits the study of this protein.

[0003] Cell-free protein synthesis (CFPS) is an open system for synthesizing proteins in vitro. Using exogenous mRNA or DNA as a template, it achieves and controls gene transcription and protein translation by supplementing the enzyme system of cell extracts with substrates and energy substances such as amino acids, NTPs, tRNA, and DNA. CFPS was initially applied to the analysis of the genetic code of E. coli. CFPS has the following advantages in the synthesis of membrane proteins: (1) Manipulation: It is an open system without the limitation of cell membranes and can directly add exogenous substances, which facilitates the post-translational modification of proteins; (2) Short reaction cycle: It does not need to maintain the ability of DNA to inherit and does not have a complex transcription and translation process. It can rapidly transcribe and translate the target gene into the target protein in vitro; (3) Small reaction volume: It can perform high-throughput synthesis screening and is expected to solve the problem of the difficulty in rapidly and massively producing ion channels.

[0004] Therefore, there is a need to design a synthetic expression vector that utilizes a cell-free protein synthesis system based on *E. coli*, and to achieve K synthesis by adding surfactant peptides that mimic the bilayer structure to the reaction system. V 1.3 Rapid and large-scale in vitro synthesis of the channel; via recombinant K V 1.3-Liposome vesicle channels were used, and their recombinant channel ion selectivity was evaluated using a fluorescent probe method. A voltage-gated potassium ion channel, K0, was also established. V 1.3 High-throughput screening method for compounds. Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant expression vector, a cell-free expression system, a recombinant channel, and their applications, to achieve K V 1.3 Rapid and large-scale in vitro synthesis of the channel; via recombinant K V 1.3-Liposome vesicle channels were used, and their recombinant channel ion selectivity was evaluated using fluorescent probes. A voltage-gated potassium ion channel, K0, was also established. V 1.3 High-throughput screening method for compounds.

[0006] The present invention is achieved through the following technical solution.

[0007] In a first aspect, the present invention provides an expression vector pUC-K, wherein the expression vector pUC-K is a vector in which the DNA sequence shown in SEQ ID NO.1 is inserted between the Nde I and Xho I sites of the pIVEX-2.4d vector.

[0008] Preferably, the preparation method of the expression vector pUC-K is as follows: the pIVEX-2.4d vector and the sequence shown in SEQ ID NO.1 are double-digested with restriction endonucleases Nde I and Xho I (NEB) at 37°C for 3 h, the SEQ ID NO.1 fragment and the pIVEX-2.4d vector backbone are recovered, and the SEQ ID NO.1 fragment and the pIVEX-2.4d vector backbone are ligated using T4 DNA ligase.

[0009] Secondly, this invention provides a potassium ion channel K V 1.3 Cell-free expression system, comprising an Escherichia coli cell-free system transfected with the expression vector pUC-K described in the first aspect, wherein the Escherichia coli cell-free system contains the surfactant peptide A6K.

[0010] Preferably, the cell-free expression system further includes Escherichia coli cell extract and a mixture of several amino acids.

[0011] Thirdly, the present invention provides a recombination channel, wherein the recombination channel is generated by the cell-free expression system product K described in the first aspect. V1.3 Obtained by recombination with liposomes.

[0012] Preferably, the recombination method comprises: preparing liposome vesicles; mixing the prepared liposome vesicles with a detergent, and adding K... V 1.3 The protein was added to a liposome-detergent solution, incubated, and then the detergent was removed to obtain the recombinant channel.

[0013] Preferably, the preparation of liposome vesicles includes the following steps: washing soybean phosphatidylcholine powder with acetone, stirring at room temperature, and discarding the supernatant; removing the remaining acetone until the powder is dry; resuspending the dry liposome powder in R buffer, rotating and incubating, and sonicating; diluting the liposomes with R buffer and then mixing and incubating; adding dried Bio-Beads equilibrated with R buffer in batches and mixing and incubating.

[0014] Fourthly, the present invention provides a method for screening toxins that affect the function of potassium ion channels, comprising the following steps:

[0015] Step 1: Taking the recombinant channels described in the second aspect as the object, to indirectly reflect the K+ within the vesicles + ACMA, a fluorescent dye with varying concentrations, was added to the recombinant channel as an indicator molecule. A high-throughput drug screening method was established using a positive regulator, and the activity of the recombinant potassium ion channel was determined.

[0016] Step 2: Using the aforementioned high-throughput drug screening method, a positive agonist was used to open the recombinant potassium ion channel as a positive control group; and a recombinant potassium ion channel without the addition of an agonist was used as a negative control group.

[0017] Step 3: Add the toxin to the control group system and monitor the changes in fluorescence signal before and after the addition of the toxin;

[0018] Compared with the positive control group, if the fluorescence signal decreases and gradually recovers to the level of the negative control group as the toxin concentration increases, it indicates that the test toxin can inhibit the closure of potassium ion channels and is a specific inhibitor of potassium ion channels. Conversely, if the fluorescence signal does not change as the concentration of the test toxin increases, it indicates that the test toxin is not a specific toxin of potassium ion channels.

[0019] Furthermore, the positive agonist is monocyte chemoattractant protein 1 (MCP-1).

[0020] Preferably, the fluorescence excitation wavelength is 470~495 nm and the emission wavelength is 515~574 nm.

[0021] Preferably, only step 1 is performed to screen for positive agonists that affect potassium ion channel function.

[0022] The nucleotide sequence of SEQ ID No. 1 is as follows:

[0023]

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. Compared with eukaryotic cell expression lines, the potassium ion channel expression vector of the present invention has the advantages of short reaction time and high expression level in cell-free expression systems, and is suitable for large-scale expression of potassium ion channels.

[0026] 2. The recombination channel in this invention has the advantages of high specificity, good stability, easy preservation and good reproducibility.

[0027] 3. Compared with voltage clamp electrophysiology, this invention is simple to operate, has a short experimental cycle, and high sensitivity. It can directly demonstrate the effect of the analyte on potassium ion channel function by simply observing changes in fluorescence signals. It is suitable for high-throughput screening and can also be applied to the field of biosensors and protein interaction research. Attached Figure Description

[0028] Figure 1 This refers to the recombinant vector K described in this invention. V Image of 1.3-pIVEX-2.4d enzyme digestion results

[0029] Figure 2 This refers to the cell-free system for synthesizing K as described in this invention. V 1.3 Western Blot identification results

[0030] Figure 3 This refers to the cell-free system for synthesizing K as described in this invention. V Figure 1.3 shows the purification results of Rho1D4.

[0031] Figure 4 This refers to the cell-free system for synthesizing K as described in this invention. V Circular dichroism chromatogram 1.3

[0032] Horizontal axis: wavelength, in nm; Vertical axis: average residue ellipticity * 10 -3 The unit is deg·cm 2 ·dmol -1

[0033] Figure 5 The image shown is a low-pressure transmission electron microscope image of the preparation of liposome vesicles according to the present invention.

[0034] Figure 6 This refers to the recombination channel K described in this invention. V 1.3 Low-pressure transmission electron microscopy image of liposome vesicles

[0035] Figure 7This indicates that the positive drug MCP-1 affects the recombinant channel K described in this invention. V 1,3-Liposome vesicles

[0036] Excitation effect diagram

[0037] x-axis: reaction time, in seconds; y-axis: relative fluorescence intensity change.

[0038] Figure 8 This indicates that the positive drug, Marguerite scorpion venom, affects the recombinant channel K described in this invention. V 1.3-Inhibition of liposome vesicles (Diagram)

[0039] x-axis: reaction time, in seconds; y-axis: relative fluorescence intensity change. Detailed Implementation

[0040] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0041] The compound described in this invention is Figure 1 The compounds shown are not particularly limited, and those skilled in the art can prepare them based on common knowledge in the field, or obtain the compounds described in this invention through commercial sales.

[0042] The present invention will be described in detail below through embodiments.

[0043] In the following examples, unless otherwise specified, all reagents and materials used are commercially available.

[0044] In the following examples, the effect of the compounds on potassium ion channels was detected using a FLIPR-TERA instrument (Molecular Devices). FLIPR is a homogeneous, dynamic, cellular fluorescence detection method that uses ACMA as a fluorescence-sensitive surrogate for hydrogen ions (H⁺) to indirectly reflect potassium ion (K⁺) ion influx by detecting hydrogen ion influx. + The reaction of potassium channels leads to their opening and closing.

[0045] Example 1

[0046] Recombinant expression vector K V Construction of 1.3-pIVEX-2.4d

[0047] Original human source K was obtained from the NCBI database. V 1.3 (KCNA3) protein sequence, in K V1.3 A rho1D4 purification tag (TETSQVAPA) was added to the C-terminus of the protein sequence. To achieve the purpose of this invention, prokaryotic codon optimization was performed, and Nde I and Xho I restriction sites were introduced at the 5' and 3' ends, respectively, to obtain the artificially synthesized DNA sequence SEQ ID NO.1.

[0048] The pIVEX-2.4d vector and the sequence shown in SEQ ID NO.1 were double-digested with restriction endonucleases Nde I and Xho I (NEB) at 37°C for 3 h. The fragment of SEQ ID NO.1 and the pIVEX-2.4d vector backbone were recovered, and the fragment of SEQ ID NO.1 and the pIVEX-2.4d vector backbone were ligated using T4 DNA ligase (16°C, 4 h). The ligation product was transformed into E. coli DH5α competent cells using the CaCl2 method. Single clones were picked, and positive clones were identified by restriction enzyme digestion and sequencing to obtain K. V 1.3-pIVEX-2.4d. Figure 1 K is shown. V Image showing the digestion results of 1.3-pIVEX-2.4d enzyme.

[0049] Example 2

[0050] Using the recombinant expression vector from Case 1 as a template, potassium ion channel K was synthesized using the E. coli CFPS system. V 1.3

[0051] 1. Using the recombinant expression vector from Example 1 as a template, the amphiphilic surfactant peptide A6K was added to a cell-free E. coli reaction system to mimic the natural membrane structure. The total reaction volume was 50 µL. The reaction system consisted of: 25 μL E. coli cell extract, 7 μL reaction buffer, 7 μL of a mixture of 20 amino acids, 0.5 μg of recombinant expression vector, 2 mA6K, reacted at 900 rpm and 30°C for 12 h, centrifuged at 13000×g for 10 min, and the supernatant was collected to obtain the recombinant protein, which was then identified by Western blotting. The protein size was approximately 75 kDa. Figure 2 Synthesizing K for cell-free systems V Figure 1.3 shows the Western Blot identification results.

[0052] 2. Collect the supernatant from the above reaction product and purify recombinant protein K using rho1D4 immunomagnetic beads. V1.3. The cell-free reaction product supernatant and precipitate were mixed with magnetic beads and incubated overnight at 4°C. The supernatant was then separated using a magnetic microtube scaffold. The mixture was washed five times with binding buffer (0.01 M PBS, 0.02 w / v FC-14) and eluted five times with elution buffer (0.01 M PBS, 0.02 w / v FC-14, 400 µM rho1D4 eluting peptide). All eluted fractions were analyzed for purity using 15% SDS-PAGE. The results showed that the purified K... V 1.3 The purity can reach over 90%, and the yield can reach 1 mg / mL. Figure 3 For K V The purification results are shown in Figure 1.3.

[0053] 3. The purified K V 1.3 Ion channel protein solutions were diluted to a concentration of 0.5 mg / mL, and their secondary structure was characterized using circular dichroism spectroscopy (CD). Recordings were performed on a circular dichroism spectrometer at 25 °C, with a wavelength range of 190–260 nm, a bandwidth of 1 nm at each measurement point, and a duration of 0.5 seconds. A 111-QS quartz sample cell with an optical diameter of 1 mm was used for the synthesis of K... V Secondary structure characterization in section 1.3. Background subtraction was performed on the CD spectral data, and Origin software was used for fitting. The results showed that K purified using detergent FC-14... V 1.3 It exhibits typical α-helical structure characteristics, with characteristic minimums at 208 nm and 222 nm, and shows an inverted double peak. Figure 4 For K V CD spectrum of 1.3.

[0054] Example 3

[0055] The recombinant channel was obtained by recombining the cell-free synthesized potassium ion channel from Case 2 with liposome vesicles.

[0056] 1. Preparation of liposome vesicles. 10 g of soybean phosphatidylcholine powder was washed with 100 mL of acetone and stirred at room temperature for 2 hours. The supernatant was discarded, and the washing was repeated three times. The remaining acetone was evaporated under vacuum until the powder was dry, and stored at -20 °C. The dried liposome powder was resuspended in R buffer (20 mM succinic acid, 80 mM NaCl, 0.6 mM KOH, pH 8.0) and incubated at 30 °C for 30 min. The liposomes were treated with a probe sonicator at 30% power for 10 cycles (3 s on, 7 s off). The liposomes were diluted to a concentration of 16 mg / mL with R buffer containing 5 mM MgCl2 and 20% (wt / vol) octyl-β-D-glucopyranoside, and incubated at 30 °C for 1 h. Take 1 mL of liposome solution and add dried Bio-Beads equilibrated with R buffer in batches (add 0.4 g Bio-Beads first, mix and incubate at 30°C for 30 min; add 0.6 g Bio-Beads second, mix and incubate at 30°C for 90 min). Store the prepared liposomes at 4 °C for later use. Observe the morphology of the liposomes using low-voltage transmission electron microscopy. Figure 5 Low-pressure transmission electron microscopy image for preparing liposome vesicles.

[0057] 2. K V 1.3-Recombination of liposome vesicles

[0058] The prepared liposome vesicles were mixed with detergent (DDM) at a weight ratio of 1:2 and incubated at room temperature for 30 min. The purified K... V 1.3 The protein was added to the above liposome-detergent solution, and recombinant buffer (150 mM KCl, 1 mM EDTA, 20 mM HEPES-KOH, pH 7.4) was added and incubated at room temperature for 1 h. Bio-beads were added in four portions and incubated at room temperature for 30 min, 4 °C for 1 h, overnight, and 2 h, respectively. The weight ratio of Bio-beads to detergent was 10:1. The supernatant was collected to obtain the recombinant channel K. V 1.3-Liposome vesicles were desalted and concentrated using a 30 kDa ultrafiltration tube and stored at 4 °C. The morphology of the liposome vesicles was observed by low-voltage transmission electron microscopy. The results showed that the formed vesicles were regular and uniform in morphology, and the size was calculated to be approximately 130 nm using ImageJ software. Figure 6 To prepare recombinant channel K V 1.3-Low-pressure transmission electron microscopy image of liposome vesicles.

[0059] Example 4

[0060] Using the restructuring channel K in Implementation Case 3 V 1.3-Liposome vesicles for compound screening

[0061] 1. Establishment of a high-throughput detection method based on the hydrogen ion concentration-dependent dye ACMA

[0062] Prepare 3 384-well plates, each equipped with K V 1.3-A 384-well liposome plate was used as the detection plate for fluorescence signal reading. A 384-well V-shaped plate containing ACMA and drug solution was designated as Plate 1, and a 384-well plate containing CCCP was designated as Plate 2. A FLIPR automated sample loading system was used to add different concentrations of positive control drug and 1.5 µM ACMA to a solution containing 10 µL of K. V 1.3-A liposome detection plate was used, and the fluorescence baseline was recorded. Excitation wavelength was 470–495 nm, emission wavelength was 515–574 nm, exposure time was 0.05 s, and data was collected once per second for 10 s. 3.5 µM CCCP was added to the detection plate using a FLIPR automated sample loading system, and fluorescence signals were read at 5 s / cycle for a total of 50 cycles. Real-time sample loading and recording were used to detect changes in fluorescence signals over time caused by different concentrations of positive drugs. For toxin screening, K+ was pre-treated... V 1.3-Liposomes were incubated with a certain concentration of toxin for 20 min; all assays were performed at room temperature, with 6 replicates for each concentration, and the assays were repeated three times.

[0063] according to Figure 7 Even 10 nM MCP-1 can induce changes in fluorescence signal in a dose-dependent manner. The changes in fluorescence signal gradually increase with increasing MCP-1 concentration, and the largest change occurs when the MCP-1 concentration reaches 300 nM. ECG was fitted using GraphPad. 50 The value is 22.57 nM.

[0064] according to Figure 8 Open the recombination channel K at 300 nM MCP-1 V 1.3-In the presence of liposome vesicles, different concentrations of mackerel scorpion venom inhibited the recombinant channel K. V 1.3-Liposome vesicles induced changes in fluorescence signal, and a clear dose-response relationship was observed. The half-maximal inhibitory effect (IC50) of mackerel scorpion vesicle venom on the recombinant channel was [not specified in the original text]. 50 The value is 12.09 pM. These results indicate that the recombination channel K used in this invention... V 1,3-Liposome vesicles can well reflect the effect of compounds on K. V 1.3 The function of the channel, applicable to the analysis of compound K V 1.3 In vitro evaluation of the channel.

[0065] 2. Stability and reliability evaluation of ACMA-based high-throughput detection methods

[0066] To evaluate the stability and reliability of the detection method, the coefficient of variation (CV) and Z' factor of the detection method were calculated.

[0067] CV is a statistical metric used to measure the dispersion of data. It is expressed as the ratio of the standard deviation to the mean, usually as a percentage.

[0068]

[0069] Where σ is the standard deviation of the data, representing the dispersion of the data. μ is the mean of the data, representing the central tendency of the data. The smaller the CV value, the lower the dispersion of the data and the higher the stability of the data. For agonist and inhibitor detection, each group has 6 parallel experiments and 3 replicates. We use the inter-group CV value of the three replicates to measure the stability of the detection method, where σ and μ represent the standard deviation and mean of the inter-group experiments, respectively. CV < 10% indicates high stability and good data repeatability of the detection method.

[0070] The Z' factor, as an evaluation metric, is a widely used evaluation method in high-throughput screening. The Z' factor is calculated using the following formula:

[0071]

[0072] Where σ p The standard deviation of the positive control is σ. n The standard deviation of the negative control is μ. p The mean value of the positive control is μ. n This represents the average value of the negative control. For agonist and inhibitor detection, each group had 6 parallel experiments, repeated three times, for a total of 18 replicates. Data from each well after fluorescence signal stabilization were collected, and the Z' factor was calculated to measure the reliability of the detection method. Detections with Z' factor values ​​between 0.5 and 1 are considered reliable.

[0073] The results showed that for agonist detection, the calculated CV value was 4.18% and the Z' factor was 0.767. For toxin screening, the calculated CV value was 3.57% and the Z' factor was 0.722.

[0074] The above results demonstrate that the screening method established in this invention has excellent stability and reliability, and is suitable for K V 1.3-channel high-throughput drug screening.

Claims

1. An expression vector pUC-K, characterized in that, The expression vector pUC-K is a vector in which the DNA sequence shown in SEQ ID NO.1 is inserted between the Nde I and Xho I sites of the pIVEX-2.4d vector.

2. The expression vector pUC-K according to claim 1, characterized in that, The expression vector pUC-K was prepared as follows: the pIVEX-2.4d vector and the sequence shown in SEQ ID NO.1 were double-digested with restriction endonucleases Nde I and Xho I (NEB) at 37°C for 3 h. The SEQ ID NO.1 fragment and the pIVEX-2.4d vector backbone were recovered, and the SEQ ID NO.1 fragment and the pIVEX-2.4d vector backbone were ligated using T4 DNA ligase.

3. A potassium ion channel K V 1.3 Cell-free expression system, characterized in that, The invention includes a cell-free E. coli system in which the expression vector pUC-K of claim 1 or 2 is transferred, and the cell-free E. coli system contains the surfactant peptide A6K.

4. The potassium ion channel K according to claim 3 V 1.3 Cell-free expression system, characterized in that, The cell-free expression system also includes E. coli cell extract and a mixture of several amino acids.

5. A potassium ion channel K V 1.3 Recombination channel, characterized in that, The recombination pathway is via the cell-free expression system product K described in the first aspect. V 1.3 Obtained by recombination with liposomes.

6. The potassium ion channel K according to claim 5 V 1.3 Recombination channel, characterized in that, The recombination method is as follows: preparing liposome vesicles; mixing the prepared liposome vesicles with a detergent; and adding K... V 1.3 The protein was added to a liposome-detergent solution, incubated, and then the detergent was removed to obtain the recombinant channel.

7. The potassium ion channel K according to claim 5 V 1.3 Recombination channel, characterized in that, The preparation of liposome vesicles includes the following steps: washing soybean phosphatidylcholine powder with acetone, stirring at room temperature, and discarding the supernatant; removing the remaining acetone until the powder is dry; resuspending the dry liposome powder in R buffer, rotating and incubating, and sonicating; diluting the liposomes with R buffer and incubating for 1 h; adding dried Bio-Beads equilibrated with R buffer in batches and incubating.

8. A method for screening toxins that affect potassium ion channel function, characterized in that, Includes the following steps: Step 1: Using the recombinant channel described in any one of claims 4 to 6 as the object, to indirectly reflect the K+ within the vesicle + ACMA, a fluorescent dye with varying concentrations, was added to the recombinant channel as an indicator molecule. A high-throughput drug screening method was established using a positive regulator, and the activity of the recombinant potassium ion channel was determined. Step 2: Using the aforementioned high-throughput drug screening method, a positive agonist was used to open the recombinant potassium ion channel as a positive control group; and a recombinant potassium ion channel without the addition of an agonist was used as a negative control group. Step 3: Add the toxin to the control group system and monitor the changes in fluorescence signal before and after the addition of the toxin; Compared with the positive control group, if the fluorescence signal decreases and gradually recovers to the level of the negative control group as the toxin concentration increases, it indicates that the test toxin can inhibit the closure of potassium ion channels and is a specific inhibitor of potassium ion channels. Conversely, if the fluorescence signal does not change as the concentration of the test toxin increases, it indicates that the test toxin is not a specific toxin of potassium ion channels.

9. The method for screening toxins affecting potassium ion channel function according to claim 8, characterized in that, The positive agonist is monocyte chemoattractant protein 1 (MCP-1).

10. The method for screening toxins affecting potassium ion channel function according to claim 7, characterized in that, Fluorescence excitation wavelength is 470~495 nm, and emission wavelength is 515~574 nm.

Images