CAMK2D K461R plasmid and application thereof

Abstract

The invention provides a CAMK2D K461R plasmid and application thereof, the CAMK2D K461R plasmid has a nucleotide sequence shown in a sequence table SEQ ID NO.1, 3-hydroxyl butyrylation modification of mutated CAMK2D obtained by adopting the CAMK2D K461R plasmid is reduced, the activity of the CAMK2D is reduced, atrial fibrillation is induced, and on the basis, a cell model related to the atrial fibrillation can be successfully constructed.

Description

Technical Field

[0001] The invention relates to the field of molecular biology, in particular to a CAMK2D K461R plasmid and an application thereof. Background Art

[0002] Atrial fibrillation (AF) is the most commonly diagnosed and treated arrhythmia in clinical practice, and is also the most commonly hospitalized arrhythmia. Approximately 33% of patients hospitalized for arrhythmia suffer from AF; therefore, AF is an increasingly serious clinical problem. AF is also one of the major complications of cardiac surgery. The incidence of AF is related to age and gender. The incidence of AF increases by 0.1% for every additional year of age under 40 years old, and increases by 1.5% for every additional year of age for women over 80 years old and 2% for every additional year of age for men. Therefore, the middle-aged and elderly population is a high-risk population for the development of AF. Studies in my country have shown that compared with the 51-60 year old population, the incidence of AF in the 71-80 year old population increases by 5 times, and the incidence of AF in the 80 year old population increases by about 6 times. The prevalence of AF increases in patients with various etiologies, including heart failure, hypertension, and coronary heart disease.

[0003] Coronary artery bypass grafting (CABG) is the best means and main surgical treatment for multi-vessel coronary artery vascular disease. Although cardiac surgical techniques and perioperative management are becoming increasingly mature, postoperative atrial fibrillation (POAF) after CABG is still one of the main complications of CABG at home and abroad. It is reported that the incidence of postoperative atrial fibrillation after CABG is 24.6%. New postoperative atrial fibrillation after CABG not only increases the incidence of long-term postoperative atrial fibrillation by 3 times, but also increases ischemic stroke, heart failure and mortality. Therefore, studying the mechanism of atrial fibrillation complicated by coronary heart disease and treating it in a targeted manner is of great significance to improving the health level of the Chinese people.

[0004] Post-translational modifications of proteins constitute important epigenetic mechanisms regulating different biological events. In the past decade, different short-chain and long-chain acylations on histone lysine (lysine, K) have been identified. Lysine 3-hydroxybutyrylation (Kbhb) refers to the addition of 3-hydroxyisobutyryl groups to histone / non-histone lysine residues. There are few studies on the role of Kbhb in human diseases. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a CAMK2D K461R plasmid.

[0006] Another technical problem to be solved by the present invention is to provide a new use of the CAMK2D K461R plasmid.

[0007] The technical solution adopted by the present invention is:

[0008] A CAMK2D K461R plasmid is pcDNA3.1-CAMK2D K461R, having a nucleotide sequence shown in SEQ ID NO.1 in the sequence table.

[0009] Use of the CAMK2D K461R plasmid in increasing CAMK2D enzyme activity.

[0010] The use of the above-mentioned CAMK2D K461R plasmid is that the CAMK2D K461R plasmid is used as a vector to be introduced into cells for expression, and a mutated CAMK2D (K461R) is obtained by transfection. The mutated CAMK2D is a normal CAMK2D in which the lysine (K) at the amino acid position 461 is mutated to arginine (R). The mutated CAMK2D has a reduced level of 3-hydroxybutyrylation modification after protein translation.

[0011] The normal CAMK2D has an amino acid sequence shown in SEQ ID NO.3 in the sequence listing.

[0012] The mutated CAMK2D has an amino acid sequence shown in SEQ ID NO.4 in the sequence listing.

[0013] Use of the CAMK2D K461R plasmid in preparing a reagent for establishing an atrial fibrillation-related cell model or an atrial fibrillation animal model.

[0014] Preferably, in the new use of the CAMK2D K461R plasmid, the atrial fibrillation-related cell model is a cell model that simulates the down-regulation of 3-hydroxybutyrylation modification.

[0015] Preferably, the new use of the CAMK2D K461R plasmid is that the model established by the reagent is used to study the therapeutic effect of atrial fibrillation drugs.

[0016] Use of the CAMK2D K461R plasmid in preparing a drug for treating atrial fibrillation.

[0017] The beneficial effects of the present invention are:

[0018] The 3-hydroxybutyrylation modification of the mutated CAMK2D obtained by using the above-mentioned CAMK2D K461R plasmid was reduced, which increased the activity of CAMK2D and induced atrial fibrillation. Based on this, an atrial fibrillation-related cell model can be successfully constructed. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the proteins identified by 3-hydroxybutyrylation modification and their differential acylation modification sites;

[0020] Figure 2 CAMK2D protein 3-hydroxybutyrylation modification level protein expression results diagram;

[0021] Figure 3 is the pcDNA3.1 vector map;

[0022] Figure 4 It is a comparison chart of the enzyme activity detection results between CAMK2D (normal) and CAMK2D (K461R) groups. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0024] The present invention conducted a Kbhb modification omics study on atrial tissue specimens from patients with atrial fibrillation and those maintaining sinus rhythm after coronary heart disease surgery. Through omics data and patient phenotype analysis, the 3-hydroxybutyrylation modification of CAMK2D was reduced in the atrial fibrillation group, thereby increasing the activity of the CAMK2D enzyme and inducing atrial fibrillation.

[0025] Example 1

[0026] 3-Hydroxybutyrylation Modification Omics Research

[0027] 1. Protein Extraction

[0028] The samples were taken out from -80℃, and appropriate amount of tissue samples were weighed into a mortar pre-cooled with liquid nitrogen, and then fully ground into powder with liquid nitrogen. Four times the volume of lysis buffer (1% Triton X-100, 1% protease inhibitor, 3μMTSA, 50mM NAM) was added to each group of samples, and ultrasonic lysis was performed. Centrifuge at 4℃, 12000g for 10min to remove cell debris, transfer the supernatant to a new centrifuge tube, and use the BCA kit to determine the protein concentration.

[0029] 2. Enzymatic digestion with pancreatin

[0030] Add dithiothreitol to the protein solution to make the final concentration 5mM, and reduce it at 56℃ for 30min. Then add iodoacetamide to make the final concentration 11mM, and incubate at room temperature in the dark for 15min. Finally, dilute the urea concentration of the sample to less than 2M. Add pancreatin at a mass ratio of 1:50 (pancreatin: protein) and enzymolysis at 37℃ overnight. Then add pancreatin at a mass ratio of 1:100 (pancreatin: protein) and continue enzymolysis for 4h.

[0031] 3. TMT labeling

[0032] The peptides digested by trypsin were desalted with Strata X C18 (Phenomenex) and then freeze-dried in vacuum. The peptides were dissolved with 0.5 MTEAB and labeled according to the TMT kit instructions. The simple operation was as follows: the labeling reagent was thawed and dissolved in acetonitrile, mixed with the peptides and incubated at room temperature for 2 h. The labeled peptides were mixed and desalted and freeze-dried in vacuum.

[0033] 4. Modification Enrichment

[0034] Dissolve the peptide in IP buffer solution (100mMNaCl, 1mM EDTA, 50mMTris-HCl, 0.5% NP-40, pH 8.0), transfer the supernatant to the pre-washed 3-hydroxybutyrylated resin, place it on a rotary shaker at 4°C, shake gently and incubate overnight. After the incubation, wash the resin 4 times with IP buffer solution and twice with deionized water. Finally, use 0.1% trifluoroacetic acid eluent to elute the peptide bound to the resin, elute three times in total, collect the eluent and vacuum freeze-dry. After drying, desalt according to the C18ZipTips instructions, and vacuum freeze-dry for liquid chromatography-mass spectrometry analysis.

[0035] 5. Liquid chromatography-mass spectrometry analysis

[0036] The peptides were dissolved in liquid chromatography mobile phase A (0.1% (v / v) formic acid in water) and separated using the EASY-nLC 1000 ultra-high performance liquid phase system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. Liquid phase gradient settings: 0-40 min, 10%-25% B; 40-52 min, 25%-38% B; 52-56 min, 38%-80% B; 56-60 min, 80% B, and the flow rate was maintained at 700 nL / min.

[0037] The peptides were separated by ultra-high performance liquid chromatography (UPLC) and injected into the NS1 ion source for ionization and then into the OrbitrapFusion TMThe peptide precursor ions and their secondary fragments were analyzed by mass spectrometry. The ion source voltage was set to 2.0 kV, and the peptide parent ions and their secondary fragments were detected and analyzed using a high-resolution Orbitrap. The primary mass spectrometry scan range was set to 350-1550 m / z, and the scan resolution was set to 60,000; the secondary mass spectrometry scan range had a fixed starting point of 100 m / z, and the Orbitrap scan resolution was set to 30,000. The data acquisition mode used a data-dependent scan (DDA) program, that is, after the primary scan, the top 20 peptide precursor ions with the highest signal intensity were selected to enter the HCD collision cell in turn and use 35% fragmentation energy for fragmentation, and the secondary mass spectrometry analysis was also performed in turn. In order to improve the effective utilization of the mass spectrometer, the automatic gain control (AGC) was set to 5E4, the signal threshold was set to 5000 ions / s, the maximum injection time was set to 100 ms, and the dynamic exclusion time of the tandem mass spectrometry scan was set to 15 seconds to avoid repeated scanning of the parent ion.

[0038] 6. Database Search

[0039] The secondary mass spectrometry data were searched using Maxquant (v1.5.2.8). Search parameter settings: the database was SwissProt Human (20317 sequences), a reverse library was added to calculate the false positive rate (FDR) caused by random matching, and a common contamination library was added to the database to eliminate the impact of contaminating proteins in the identification results; the enzyme cleavage method was set to Trypsin / P; the number of missed cleavage sites was set to 4; the minimum length of the peptide was set to 7 amino acid residues; the maximum number of peptide modifications was set to 5; the primary parent ion mass error tolerance of the First search and Main search was set to 20ppm and 5ppm, respectively, and the mass error tolerance of the secondary fragment ion was 0.02Da. Cysteine ​​alkylation was set as a fixed modification, and the variable modifications were oxidation of methionine, acetylation of the protein N-terminus, and 3-hydroxybutyrylation of lysine. The quantitative method was set to TMT-10plex, and the FDR of protein identification and PSM identification was set to 1%.

[0040] A total of 4534 peptides were identified through spectrum analysis, and 3172 3-hydroxybutyrylated modified peptides were identified on 1292 proteins. Finally, 29 differentially modified sites of 27 proteins were identified to be upregulated, and 3 differentially modified sites of 3 proteins were identified to be downregulated (CAMK2D was included in them), see Figure 1 .

[0041] Example 2

[0042] CAMK2D protein 3-hydroxybutyrylation modification level protein expression

[0043] Tissue protein extraction: Cut the tissue into small pieces. Add RIPA lysis buffer at a ratio of 150-250ul lysis buffer per 20mg tissue. Homogenize with a glass homogenizer until fully lysed. Centrifuge the lysed sample at 10000-14000g for 3-5 minutes, take the supernatant, quantify the protein, and denature it.

[0044] Take a 1.5ml centrifuge tube, add 25μL magnetic beads, add 500μL buffer (made of crosslinker, lysis buffer, and deionized water) and wash repeatedly twice, each time with an interval of 1min, and retain the magnetic beads. Add CAMK2D antibody (diluted with crosslinker, lysis buffer, and deionized water), mix at room temperature for 15min, elute once with 100μL buffer, elute twice with 300μL buffer, and retain the magnetic beads. Add 2.5μL crosslinker, 43.5μL deionized water, 4μL. 0.25mM disuccinimidyl suberate (DSS), mix at room temperature for 30min, repeat elution 3 times with 100μL buffer, and then elute twice with 200μL lysis buffer. Add the sample with the same concentration of total protein, mix at room temperature for 3 hours, elute twice with 500 μL lysis buffer, elute once with deionized water, and then elute twice with 100 μL elution buffer, retain the liquid, add 10 μL neutralization solution, 27.5 μL electrophoresis loading buffer, and 7 μL 1M DTT.

[0045] Prepare separation gel and seal it with isopropanol, and let it stand at room temperature for about 20 minutes. After the separation gel solidifies, wash away the isopropanol remaining on the top of the separation gel with deionized water, then prepare the concentrated gel and insert the comb to form the loading lane. Let it stand at room temperature for about 20 minutes; after the concentrated gel solidifies, prepare the protein sample and 95℃ water bath; mix 50-100μg protein sample with loading buffer, and heat denature at 95℃ for 5 minutes; pull out the comb on the concentrated gel, assemble the SDS-PAGE electrophoresis tank, and add SDS-PAGE Running buffer to the designated position according to the instructions on the instrument; load the sample, add the denatured sample to the lane of the concentrated gel; connect the power cord, and pay attention to the correct connection of the positive and negative poles. 110V electrophoresis for 90 minutes; after the electrophoresis is completed, turn off the power, recycle the SDS-PAGE Running buffer, and disassemble the electrophoresis tank. Cut the gel according to the size of the protein marker and the size of the experimental target protein; cut the filter paper and PVDF membrane of the same size according to the size of the cut gel. Soak the PVDF membrane in methanol for 3-5 seconds, and soak the filter paper in transfer buffer; assemble the transfer sandwich: the order from positive to negative is: positive electrode, filter paper, PVDF membrane, gel, filter paper, negative electrode. Use a glass rod to remove bubbles; place the transfer sandwich on the electroporator, with the gel on the negative electrode side, and connect the power supply. 100V, electrotransfer for 65 minutes; after the transfer is completed, cut off the power supply and remove the PVDF membrane; wash the membrane with 25ml TBS for 5 minutes at room temperature, shake on a horizontal shaker. Place the membrane in 25 ml blocking buffer (TBST) at 4°C overnight; wash three times with 15 ml TBS / T; add primary antibody (3-hydroxybutyrylated, 1:1000) at 4°C overnight; wash three times with 15 ml TBS / T; add horseradish peroxidase (HRP)-labeled secondary antibody (1:2000), incubate at room temperature for 1 hour with slow shaking; wash three times with 15 ml TBS / T; wash once with 15 ml TBS; perform protein detection by colorimetric method; finally, expose and image the gel using a gel imager.

[0046] Results Figure 2 .

[0047] Example 3

[0048] CAMK2D K461R plasmid (pcDNA3.1-CAMK2DK461R) vector construction

[0049] 1. Vector and target gene information

[0050] The map of pcDNA3.1 vector is as follows Figure 3 shown.

[0051] 2. Vector digestion

[0052] Add each reagent in the order shown in Table 1, gently pipette and mix, and place in a 37°C water bath for 1-2 hours; after the enzyme digestion is completed, perform agarose gel electrophoresis to recover the target fragment;

[0053] The vector enzyme digestion system is as follows:

[0054] Table 1

[0055]

[0056] 3. Acquisition of target fragments

[0057] 3.1 Primer design:

[0058] The target sequence is directly synthesized into the vector

[0059] 3.2 PCR amplification of target fragment

[0060] Prepare the system as shown in Table 2 below, mix gently, and place in a PCR instrument for reaction;

[0061] Fragment PCR amplification system:

[0062] Table 2

[0063]

[0064] PCR program, see Table 3 below:

[0065] Table 3

[0066]

[0067] 4. Connect the target fragment to the vector

[0068] HB infusion TM One-step cloning and ligation system:

[0069] Prepare the reaction system shown in Table 4 in an ice-water bath. If the liquid accidentally sticks to the tube wall, it can be centrifuged briefly to sink to the bottom of the tube. After the ligation reaction solution reacts at 50°C for 30 minutes, place it on ice for 5 minutes and transform immediately.

[0070] Table 4

[0071]

[0072] 5. Conversion

[0073] 1) After taking out the DH5α competent cells from the -80℃ refrigerator, they should be immediately placed on ice to thaw. The competent cell packaging process should be gentle to reduce mechanical damage to them;

[0074] 2) After the competent medium is thawed, aliquot it into 50 μL volumes per tube (20 μL is sufficient for plasmid transformation). After aliquoting, add the ligation product in an amount not exceeding 1 / 10 of the competent medium volume (currently add 5 μL of ligation product) and place it on ice for 20-30 minutes.

[0075] 3) Heat shock at 42℃ for 90s (this time must be very strict), and immediately put it on ice for 2-3min after heat shock;

[0076] In a clean bench, add 500 μL LB medium (note that it must be LB medium without antibiotics) and gently invert it upside down 3-5 times;

[0077] 5) Incubate at 37°C, 230 rpm with shaking for 45-60 min;

[0078] 6) Apply the bacterial solution to the solid plate of the corresponding resistance, spread it evenly, and then incubate the plate upside down at 37°C incubator for 12-16 hours;

[0079] 6. PCR Identification of Bacterial Fluid

[0080] 6.1 Bacterial liquid PCR identification system, see Table 5 below:

[0081] Table 5

[0082]

[0083] Note: When configuring the mix, the proportions in the mix should be amplified in equal proportions. Number of clones to be verified; Select verification primers based on the vector used

[0084] 6.2 Bacterial liquid PCR identification procedure, see Table 6 below:

[0085] Table 6

[0086]

[0087]

[0088] 7. Sequencing

[0089] Two clones were selected from the positive clones screened out for sequencing, and the sequencing results were compared and analyzed. If the sequencing results were consistent with the target sequence, the target plasmid was successfully constructed, and the obtained CAMK2D (K461R) plasmid had the nucleotide sequence shown in SEQ ID NO.1 in the sequence table.

[0090] 8. Plasmid extraction

[0091] Carry out bacterial liquid amplification and plasmid extraction and purification.

[0092] 2. Enzyme activity detection

[0093] 1. Resuscitate mouse atrial myocytes (HL1) and culture them. Subculture 5*10^5 HL1 cells in advance to a 6-well plate for transfection (assuming that the cells have been cultured to meet the needs of subsequent transfection experiments). After the operation, place at 37°C, 5% CO 2 in an incubator;

[0094] 2. On the second day, when the cells adhered to the wall and grew to 50% density, CAMK2D (normal) plasmid (having the nucleotide sequence shown in SEQ ID NO.2 in the sequence table) and CAMK2D (K461R) plasmid were transfected into HL1 cells respectively. The components of the transfected 6-well plate are shown in Table 7 below:

[0095] Table 7

[0096]

[0097] 3. Collect cells 48 hours after transfection, wash twice with PBS, add lysis buffer at 1*10^7 / ml, and incubate on ice for 10 minutes;

[0098] 4. After lysis, centrifuge at 12000rpm, 4℃ for 5min, take the supernatant and place it on ice;

[0099] 5. After measuring the protein concentration by BCA method (Biyuntian P0012S), the CAMK2D enzyme activity was measured. The results were as follows: Figure 4 As shown. Figure 4 It can be seen that the CAMK2D enzyme activity in the CAMK2D (K461R) plasmid transfection group was significantly higher than that in the CAMK2D (normal) plasmid group.

[0100] The above-described embodiments are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineering and technical personnel in the field shall fall within the protection scope of the claims of the present invention.

Claims

1. A CAMK2D K461R plasmid, characterized in that: It is pcDNA3.1-CAMK2D K461R, having the nucleotide sequence shown in SEQ ID NO.1 in the sequence listing.

2. Use of the CAMK2D K461R plasmid according to claim 1 in increasing CAMK2D enzyme activity.

3. The use of the CAMK2D K461R plasmid according to claim 2, characterized in that: The CAMK2D K461R plasmid is used as a vector to be introduced into cells for expression, and a mutated CAMK2D is obtained by transfection. The lysine at the amino acid position 461 of the normal CAMK2D is mutated to arginine in the mutated CAMK2D. The 3-hydroxybutyrylation modification level of the mutated CAMK2D protein after translation is reduced.

4. The use of the CAMK2D K461R plasmid according to claim 3, characterized in that: Normal CAMK2D has the amino acid sequence shown in SEQ ID NO.3 in the sequence listing; the mutated CAMK2D has the amino acid sequence shown in SEQ ID NO.4 in the sequence listing.

5. Use of the CAMK2D K461R plasmid according to claim 1 in preparing a reagent for establishing an atrial fibrillation-related cell model or an atrial fibrillation animal model.

6. The use of the CAMK2D K461R plasmid according to claim 5, characterized in that: The atrial fibrillation-related cell model is a cell model that simulates the downregulation of 3-hydroxybutyrylation modification.

7. Use of the CAMK2D K461R plasmid according to claim 5 or 6, characterized in that: The model is used to study the therapeutic effects of atrial fibrillation drugs.

8. Use of the CAMK2D K461R plasmid according to claim 1 in the preparation of a medicament for treating atrial fibrillation.

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