CN115838425A - Antibody targeting second extracellular loop of angiotensin II type 1 receptor and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of genetic engineering, and provides an antibody targeting an extracellular second ring of an angiotensin II type 1 receptor and application thereof in order to realize large-scale mass production of AT1-AA with highly uniform physicochemical properties. The variable region gene of the antibody is 336bp; the nucleotide sequence of the heavy chain is shown as SEQ ID NO:1 is shown in the specification; the heavy chain amino acid sequence is shown as SEQ ID NO:2 is shown in the specification; the nucleotide sequence of the light chain is shown as SEQ ID NO:3 is shown in the specification; the amino acid sequence of the light chain is shown as SEQ ID NO:4, respectively. The antibody heavy chain, light chain amino acid sequence and CDR region are determined by extracting RNA of hybridoma cell, performing reverse transcription on cDNA, and performing PCR amplification on antibody gene. The amino acid sequence of the AT1-AA is obtained originally by the method, thereby providing necessary basic work for preparing the monoclonal AT1-AA with low cost in batch production by adopting a recombinant protein expression method in the later period.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an antibody targeting an extracellular second ring of an angiotensin II type 1 receptor and application thereof.
Background
At present, cardiovascular diseases are the leading cause of death in the world, and the renin-angiotensin-aldosterone system (RAAS) plays an important role in maintaining the stability and normal development of the cardiovascular system, regulating blood pressure, and the like. The primary effector of RAAS is Angiotensin II (Ang II), which exerts various physiological and pathophysiological effects by activating Angiotensin II type 1 receptor (at1r) signaling.
AT1R is a typical GPCR seven-transmembrane α -helix structure with one extracellular N-terminus, three extracellular loops (ECL 1-3), three intracellular loops (ICL 1-3), one amphipathic helix VIII (H8) and one intracellular C-terminus, with ECL2 being the most immunogenic. Excessive activation of AT1R can lead to vascular system dysfunction such as increased vascular tone, inflammation, fibrosis, and thrombosis, among others. Under pathophysiological conditions, ang II chronically activates AT1R, leading to excessive receptor activation, which in turn can lead to a variety of diseases such as hypertension, heart failure, vascular remodeling, diabetic nephropathy and atherosclerosis. However, in some cardiovascular diseases, AT1R is over-activated and Ang II levels are not high, suggesting that other factors may be involved.
AT the end of the 20 th century, researchers found that autoantibodies against AT1R (angiotensin II type 1 receptor autoantibody, at1-AA) were present in the serum of patients with various cardiovascular diseases such as preeclampsia, coronary heart disease, hypertension, and peripheral arterial disease. The autoantibodies can exert Ang II-like receptor agonist-like effects. More and more studies have demonstrated that Ang II binds AT1R differently from AT1-AA in a site and in a pattern that activates the receptor, although AT1R is activated. Ang II binds to the 5 th, 6 th transmembrane region of AT1R, causing AT1R transient activation; and AT1-AA can specifically recognize the extracellular second ring (AT 1R-ECL 2) of AT1R, so that AT1R and downstream signals are continuously activated, and pathological effects such as endothelial injury, vascular smooth muscle cell transformation, cardiac hypertrophy and the like are triggered. AT1-AA is therefore currently believed to be a significant cause of AT1R overactivation in a variety of cardiovascular diseases. However, the molecular regulation mechanism of AT1-AA involved in cardiovascular diseases is not completely elucidated, and brings difficulty to targeted intervention.
Obtaining AT1-AA in sufficient quantity and high purity is the first prerequisite for studying the pathological significance and specific molecular mechanism. There are two main ways to obtain AT1-AA AT present: firstly, AT1-AA is purified from the serum of clinical patients. However, the serum source of clinical patients is limited, the titer is low, the collection is difficult, and if the purification is not timely, the titer of AT1-AA can be reduced or lost, so that sufficient guarantee is difficult to provide for the mechanism research of AT1-AA; secondly, AT1-AA is obtained through active immunization, namely, the immune reaction is replicated, but the active immunization takes longer time, and the antibody is polyclonal, so that the fine molecular regulation mechanism research is difficult to develop. Thus, obtaining AT1-AA with high potency and specificity and relatively low cost is a significant bottleneck problem limiting the development of the art. For this purpose, we prepared hybridoma cells producing monoclonal antibody AT1-AA using human AT1R-ECL2 peptide fragment. Although hybridoma cells can produce high purity monoclonal AT1-AA, the purification cost is high, which limits the application.
Disclosure of Invention
The invention provides an antibody targeting the extracellular second loop of an angiotensin II type 1 receptor and application thereof, in order to realize large-scale mass production of AT1-AA with highly uniform physicochemical properties. The antibody heavy chain, light chain amino acid sequence and CDR region are determined by extracting RNA of hybridoma cell, performing reverse transcription on cDNA, and performing PCR amplification on antibody gene. The amino acid sequence of the AT1-AA is obtained originally by the method, thereby providing necessary basic work for preparing the monoclonal AT1-AA with low cost in batch production by adopting a recombinant protein expression method in the later period.
The invention is realized by the following technical scheme: an antibody targeting the extracellular second loop of angiotensin II type 1 receptor, said antibody variable region gene being 336bp; the nucleotide sequence of the heavy chain is shown as SEQ ID NO:1 is shown in the specification; the heavy chain amino acid sequence is shown as SEQ ID NO:2 is shown in the specification; the nucleotide sequence of the light chain is shown as SEQ ID NO:3 is shown in the figure; the amino acid sequence of the light chain is shown as SEQ ID NO:4, respectively.
The method for preparing the antibody of the second ring outside the target angiotensin II type 1 receptor cell comprises the steps of extracting RNA of hybridoma cells, carrying out reverse transcription on cDNA, carrying out PCR amplification on antibody genes, and determining heavy chain amino acid sequences, light chain amino acid sequences and CDR regions of the antibody; obtaining the amino acid sequence of an antibody AT1-AA targeting the extracellular second loop of the angiotensin II type 1 receptor.
The specific method comprises the following steps:
(1) Obtaining of hybridoma cells: actively immunizing Balb/C mice with an extracellular second loop AT1R-ECL2 of an angiotensin II type 1 receptor; fusing mouse spleen lymphocyte and myeloma cell to generate monoclonal hybridoma secreting AT1-AA and culturing, and injecting the cell in logarithmic phase into mouse abdominal cavity to take ascites. By 1X 10 7 Highly purified AT1-AA hybridomas were isolated from the mouse ascites after injection. The methods described in references (Wei M, ZHao C, zhang S, wang L, liu H, ma X. Preparation and Biological Activity of the Monoclonal Antibody against the Second Extracellular Loop of the antibiotic Type II1 Receptor 2016. J Immunol Res. 1858252).
(2) Extracting and purifying AT1-AA from hybridoma cells: obtaining AT1-AA from hybridoma cells by affinity chromatography using Mab Trap TM Extracting and purifying Protein G HP 5mL high-efficiency purification column (GE healthcare Life Sciences) to obtain high-purity AT1-AA; the method specifically comprises the following steps: injecting the hybridoma cell suspension into abdominal cavity of mouse, and extracting ascites for purification after the abdominal cavity of mouse has been expanded for 14-18 days. Rinsing the purification column with a binding buffer solution, mixing the ascites with the binding buffer solution in equal volume, passing through the purification column, continuing to rinse impurities with the binding buffer solution, eluting IgG bound to the column with an elution buffer solution, and rinsing with the binding buffer solution to revive the purification column;
detecting the beating frequency of the myocardial cells of the suckling mice by using the primary suckling mouse myocardial cells which are separated and cultured to obtain AT1-AA, and verifying the biological activity of the purified AT1-AA; the method comprises the following specific steps: breaking the head of a suckling mouse born for 0-4 days quickly, taking out the heart to wash blood and remove connective tissues, shearing the heart into small pieces, adding mixed enzyme liquid to digest the tissues repeatedly, filtering to obtain a single cell group suspension, inoculating the single cell group suspension into a culture dish, collecting the suspension mainly containing the myocardial cells after adhering to the wall for 2 hours, centrifuging, then re-suspending by using a new culture medium, and replacing the liquid after adhering to the wall for 36 hours;
removing culture medium containing fetal calf serum from myocardial cells of suckling mice cultured in 6-well plate, replacing with common DMEM high-sugar culture medium, counting myocardial cell basic beating frequency after 24 hr, and adding 1 × 10 concentration 6 After incubating the AT1-AA and the negative IgG for 5 minutes AT 37 ℃, counting each group respectively, wherein the counting time is 15 seconds each time, and finally obtaining the myocardial cell beating frequency of the suckling mouse per minute;
(3) Amplification of antibody genes: performing RNA extraction, cDNA synthesis and PCR amplification of antibody genes on the hybridoma cells with verified biological activity; after the hybridoma cells are recovered, RNA in the cells is extracted by using an RNeasy Minikit of Qiagen, a 10 mu l amplification reaction system is prepared by using a first strand synthesis kit of Roche company, after cDNA is synthesized, cDNA formed by reverse transcription of 2-20ug of total RNA is used as a template, heavy chain and light chain genes are amplified by PCR through 30 total systems and 27 reaction systems, and amplification products are verified to be 330bp bands through 1.5% agarose electrophoresis.
The invention obtains the amino acid sequence of the AT1-AA, thereby providing necessary basic work for preparing the monoclonal AT1-AA with low cost in batch production by adopting a recombinant protein expression method in the later period, and the AT1-AA prepared by the recombinant protein expression method has low cost and uniform quality and can provide sufficient raw materials for later-period pathological experiments.
Drawings
FIG. 1 is a graph showing the purity and bioactivity of AT1-AA produced by hybridoma cells; in the figure: a is the result of SDS-PAGE; b is a detection result graph of the beating frequency of the myocardial cells of the suckling mice by using the primary suckling mouse myocardial cells which are subjected to isolated culture;
FIG. 2 shows the results of PCR verification of the amplified antibody gene; in the figure: lane 1-3 show PCR results for different heavy chain primers, and Lane 4 shows PCR results for the corresponding light chain;
FIG. 3 is a heavy chain CDR region and FR region and a light chain CDR region and FR region; in the figure, A is a heavy chain CDR region and a FR region; b is a light chain CDR region and a FR region;
FIG. 4 shows VH and VL templates; in the figure: a is a VH template, B is a VL template;
FIG. 5 shows the results of VH homology modeling evaluation; in the figure: a is ERRAT result of VH; b is VERIFY3D result for VH; the evaluation result of the Laplace graph in PROCHECK with C as VH;
FIG. 6 shows the results of VL homology modeling evaluation; in the figure: a is ERRAT result of VL; b is VERIFY3D result of VL; results of evaluation of a Laplace graph in PROCHECK with C as VL;
FIG. 7 is a variable region 3D structure;
FIG. 8 is a diagram of the molecular docking of the AT1-AA variable region with AT1R using the structure of AT1R in AlphaFold using ZDock.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.
Those skilled in the art will recognize that equivalents to the specific embodiments described, as may be learned by routine experimentation, are encompassed by the present application.
The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
1. Identification of hybridoma cellsThe AT1-AA purity and the biological activity are generated. Obtaining AT1-AA from ascites fluid produced by hybridoma cells by affinity chromatography using Mab Trap TM And (3) extracting and purifying the Protein G HP 5mL high-efficiency purification column (GE healthcare Life Sciences) to obtain high-purity AT1-AA. The specific method comprises the following steps:
A. purification of AT1-AA in ascites of mice produced by hybridoma cells
(1) Immunizing Balb/c mice with the synthesized extracellular second loop epitope peptide segment of the human AT1 receptor to prepare high-titer multi-antiserum, and performing cell fusion to prepare a hybridoma cell strain.
(2) Selecting clean-grade Balb/c mice with negative AT1-AA and age of 10-12 weeks without limit. The room temperature is 18-22 ℃, the humidity is 40-70%, and the illumination and the darkness alternate every 12 hours. Free diet and drinking water feeding. Adaptive feeding for 3-5 days.
(3) Mice were randomly divided into two groups for pre-immunization treatment: one group of mice is injected with 0.4-0.5 mL of paraffin in the abdominal cavity, the other group of mice is injected with Freund's incomplete adjuvant (0.02 mL/g) in the abdominal cavity, and the paraffin and the Freund's incomplete adjuvant are used for pre-immunizing the mice. And continuing normal feeding for one week.
(4) One week later, hybridoma cells growing to logarithmic growth phase in culture were treated. Blowing off the cells in the culture dish, transferring into a 50mL centrifuge tube, centrifuging at 800RPM for 5 minutes, discarding the culture medium to obtain a cell suspension with a final concentration of 2 × 10 6 -1×10 7 Individual cells/mL.
(5) The treated hybridoma cell suspension was injected into the abdominal cavity of mice that had been pre-immunized, 0.5mL per mouse, and normal feeding was continued. After obvious ascites appears in the mice at 14-18 days, the ascites is extracted.
(6) Rinsing the purification column with a binding buffer solution, mixing the ascites with the binding buffer solution in equal volume, passing through the purification column, rinsing impurities with the binding buffer solution, eluting IgG on the column with the elution buffer solution, and receiving the eluted liquid in an Ep tube which is pre-placed in a neutral buffer solution for detection of concentration for later use.
The buffer solution formula comprises:
binding buffer solution A (10 × Na) 2 HPO4•12H 2 O solution): 38.5g Na2HPO 4 •12H 2 Dissolving O in 490mL double distilled water, diluting to a constant volume of 500mL, mixing, filtering with 0.22 μm filter membrane, and storing at 4 deg.C.
Binding buffer B solution (10 × NaH) 2 PO4•2H 2 O solution): 15.6g of NaH 2 PO4•2H 2 Fully dissolving the O powder in 450mL of double distilled water, fixing the volume to 500mL, uniformly mixing, filtering by a 0.22 mu m filter membrane, and storing at 4 ℃.
Binding buffer (20 mmol/L Na) 2 HPO 4 pH = 7.0): 27mL of 10 XNa was measured 2 HPO4•12H 2 O solution and 23mL of 10 XNa 2HPO 4 •12H 2 Adding 440mL of double distilled water into the O solution, fully dissolving, adjusting the pH value to 7.0, then using the double distilled water to fix the volume to 500mL, uniformly mixing, filtering by a 0.22 mu m filter, and storing at 4 ℃.
Elution buffer (100 mmol/L Glycine-HCl, pH = 2.7): 3.75g of glycine is fully dissolved in 450mL of double distilled water, the pH value is adjusted to 2.7, the volume is adjusted to 500mL, the glycine and the water are uniformly mixed, filtered by a 0.22 mu m filter and stored at 4 ℃.
Neutral buffer (1 mol/L Tris, pH = 8.0): and fully dissolving 60.57g of Tris in 450mL of double distilled water, adjusting the pH to 8.0, metering the volume to 500mL, uniformly mixing, filtering by using a 0.45-micron filter, and storing at 4 ℃.
B. Extracting myocardial cells of suckling mice and adding medicine:
(1) Preparing a 50mL sterile centrifuge tube, and adding 25 mL of 10% FBS/PS/DMED high-sugar medium into the centrifuge tube; soaking suckling mouse 0-4 days after birth in 75% alcohol for disinfection. The right hand holds the large forceps to take out the suckling mouse, the left thumb and the index finger are used for fixing the body of the suckling mouse, the head is left, the right hand rapidly cuts the head along the neck by using tissue scissors, and 3-4 drops of blood are dripped (blood cells are removed as much as possible). The sternum is cut from the left edge of the xiphoid process by changing the ophthalmic scissors, and the exposed heart is clipped by a pair of forceps and is washed in precooled PBS.
(2) Continuously passing the heart taken out through 2-3 plates containing precooled PBS to fully wash the blood; removing connective tissue from cleaned heart with clean ophthalmic scissors(ii) a Then placing into a glass bottle, adding a proper amount of PBS, and shearing heart tissue into 1mm by using an ophthalmic scissors 3 The tissue mass was allowed to stand for 5 minutes, the supernatant was discarded and repeated 3-4 times until the PBS was clear in order to remove blood that had not been pumped out previously.
(3) Discarding PBS in the glass bottle (paying attention to not sucking away tissue blocks), putting into a small rotor, adding 2-3 mL of mixed enzyme solution into the tissue, repeatedly digesting the tissue by a temperature-controlled magnetic stirrer rotating at a constant temperature and a constant speed of 37 ℃, controlling the rotating speed at 60 RPM per minute, digesting for 5 minutes in an oscillating way, and discarding the first digestive juice; the tissue is digested for a plurality of times in a short period of time, the total number of times of digestion is preferably not more than 10 times, each time of digestion is not more than 7 minutes, and each time of digestion is also collected in a centrifuge tube filled with culture medium to terminate the digestion reaction.
(4) After complete digestion, the mixture was filtered through a 200 mesh screen to obtain a single cell suspension. Centrifuging at 800RPM for 5 min, discarding supernatant, resuspending cells in new 10% FBS/PS/DMED high sugar medium, inoculating and culturing at 10cm 2 The culture dish was placed in a 5% CO2 incubator at 37 ℃ for differential adherence for 2 hours (removing a large amount of fibroblasts).
(5) After 2 hours, fibroblasts had adhered to the wall and cardiomyocytes had not adhered to the wall, a suspension containing predominantly cardiomyocytes was collected, resuspended in fresh medium after centrifugation and 0.1 mmol/L5-bromodeoxyuridine (5-BrdU) was added, and the cells were seeded at an appropriate concentration after counting and cultured in 6-well plates or 96-well plates. The liquid is changed for the first time after 36 hours, and the orifice plate is not shaken during the liquid changing period, so that the myocardial cells are prevented from adhering to the wall firmly.
(6) The culture medium containing fetal calf serum is removed after the suckling mouse myocardial cells planted in the 6-well plate are cultured for 72 hours, and the culture is continued by replacing the culture medium with the common DMEM high-sugar medium. After 24 hours, the frequency of the basic beating of the myocardial cells is counted, and then the frequency is added to the cells at a concentration of 1 × 10 6 After incubating the AT1-AA and the negative IgG for 5 minutes AT 37 ℃, counting each group respectively, wherein the counting time is 15 seconds, and finally obtaining the beating frequency of myocardial cells of the suckling mice per minute.
The SDS-PAGE results are shown in FIG. 1, A, and the results show that both have significant bands of heavy and light chains of 55 kDa and 25 kDa, indicating higher purity. The primary suckling mouse myocardial cell is shown in the upper graph B in the graph 1, the primary suckling mouse myocardial cell which is subjected to isolated culture is used for detecting the beating frequency of the suckling mouse myocardial cell, the detection result is shown in the lower graph B in the graph 1, and the result shows that AT1-AA can obviously stimulate the beating frequency of the suckling mouse myocardial cell to be accelerated.
(2) And (5) verifying the amplified antibody gene. After RNA extraction and cDNA synthesis of the hybridoma cells with verified activity, the amplified antibody gene is verified by PCR. The results are shown in FIG. 2, where the target antibody gene is 300bp, the PCR results of the different heavy chain primers in lanes 1-3, and the PCR result of the light chain in lane 4.
RNA extraction (QIAGEN, RNeasy Mini Kit) method:
(1) The hybridoma cells were recovered, transferred to a 50mL centrifuge tube, added to 20mLDMEM, and centrifuged at 300g for 10min.
(2) Discard the supernatant, add 30mLPBS to a 50mL centrifuge tube, centrifuge for 10min at 300g, discard the supernatant, add 700uL RLT to the cell pellet, and blow-blow to completely lyse the cells.
(3) Sucking 350uL lymphocyte sample from the upper lysis solution, fully blowing, homogenizing, and shaking to suspend for complete lysis; and adding 350uL 70% of ethanol, and blowing back and forth to mix evenly.
(4) Adding 700uL of the liquid into the center of the column, centrifuging at room temperature for 15s, and discarding the waste liquid; 700uL Buffer RW1 was added to the column, centrifuged at room temperature for 15s, and the waste and collection tubes were discarded.
(5) The column was placed in a fresh collection tube, 500uLBuffer RPE (first use required 4 volumes of ethanol to Buffer RPE) was added and centrifuged at room temperature for 15s, and the waste was discarded. Then 500uL Buffer RPE was added and centrifuged at room temperature for 2min. And (5) separating for 1min, and discarding the collecting pipe. 50uL DEPC water.
cDNA synthesis (20 uL system): a First Strand synthesis kit (Transcriptor First Strand cDNAsynthesis kit) from Roche was used. The amplification system formulation and reaction are shown in Table 1.
Table 1: amplification system preparation and reaction
PCR amplification of antibody genes: using cDNA reverse transcribed from 2-20ug total RNA as template, PCR reaction system was configured to amplify antibody light and heavy chain genes (30 total systems, 27 reaction systems PCR) as shown in Table 2:
table 2: amplification system preparation and reaction
The PCR product is subjected to 1.5% agarose electrophoresis, the VH and VL are both bands of about 330bp, and the PCR product is subjected to the next round of PCR or frozen at-20 ℃ for standby after recovery and quantification.
(3) The base sequence and amino acid sequence of the heavy chain of AT1-AA. The nucleotide sequence of the heavy chain is shown as SEQ ID NO:1 is shown in the specification; the heavy chain amino acid sequence is shown as SEQ ID NO:2 is shown in the specification; the nucleotide sequence of the light chain is shown as SEQ ID NO:3 is shown in the specification; the amino acid sequence of the light chain is shown as SEQ ID NO:4 is shown in the specification; the heavy chain CDR and FR regions and the light chain CDR and FR regions are shown in FIG. 3.
For analyzing the binding mode of AT1-AA and AT1R, firstly, performing homologous modeling according to the measured AT1-AA variable region sequence, and modeling VH and VL on line by using a Swiss-model, wherein the modeling result is shown in FIG. 4, through sequence comparison, the PDB number of the modeling template of VH is 6j5d, the sequence consistency reaches 80.36 percent (more than 30 percent is reasonable for modeling), and the GMQE (global model quality evaluation) is 0.87 (the confidence level is between 0 and 1, the larger the value is the better the quality). The PDB number of the modeled template for VL was 5myx, with a sequence identity of 90.18% and a GMQE of 0.89.
VH and VL modeling results were then evaluated by SAVES v6.0, and the results are shown in fig. 5, 6. The higher the Overall quality factor value in the ERRAT results the better, the better the score >85, the crystal can reach 95, the VH score is 94.2308, and the VL score is 100. When VERIFY3D results exceed 80% of residues with 3D/1D values greater than 0.2, the model is of acceptable quality, with 100% for both VH and VL. It is reasonable to evaluate the laplace map in PROCHECK results that more than 90% of the amino acid residues fall within the core region, 91 amino acid residues in VH are all located in the core region (95.8%), 4 residues fall within the permissive region (4.2%), and no residues fall within the forbidden and substantially permissive regions; VL 87 amino acid residues were all located in the core region (94.6%), 4 residues falling in the permissive region (4.3%), no residues falling in the approximate permissive region, and 1 residue falling in the forbidden region (1.1%). In conclusion, VH and VL are reasonably modeled, and then the homologous modeling final structure of the AT1-AA variable region is obtained through pymol's superposition command (align).
The AT1-AA variable region was docked to the AT1R molecule using the AT1R structure in AlphaFold using ZDock. As a result, it was found that S32 of VL forms two hydrogen bonds with T190 of AT1R, S56 of VH forms a hydrogen bond with T94 of AT1R, and G98 of VH and D9 of AT1R form a hydrogen bond. G10, I11, K12, R13, P95, N98, F171, N176, T178, H183, E185 of AT1R have a hydrophobic effect, D31, D33, Y37, F99, R101 of VL have a hydrophobic effect, and T30, Y31, W32, N34, Q49, F51, S54, Y58 of VL have a hydrophobic effect.
A homology modeling and molecular docking experiment are carried out on a sequencing result of the AT1-AA variable region, so that the AT1-AA can form hydrogen bonds and hydrophobic interaction with the AT1R and sequence effectiveness are proved, and a theoretical basis is provided for the follow-up batch production of the AT1-AA according to the sequence.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (4)
1. An antibody that targets the extracellular second loop of an angiotensin II type 1 receptor, characterized by: the variable region gene of the antibody is 336bp; the nucleotide sequence of the heavy chain is shown as SEQ ID NO:1 is shown in the specification; the heavy chain amino acid sequence is shown as SEQ ID NO:2 is shown in the specification; the nucleotide sequence of the light chain is shown as SEQ ID NO:3 is shown in the specification; the amino acid sequence of the light chain is shown as SEQ ID NO:4, respectively.
2. A process for preparing an antibody targeting the extracellular second loop of angiotensin II type 1 receptor according to claim 1, characterized in that: extracting RNA of hybridoma cells, performing reverse transcription on cDNA, performing PCR amplification on antibody genes, and determining amino acid sequences and CDR regions of heavy chains and light chains of the antibodies; obtaining the amino acid sequence of an antibody AT1-AA targeting the extracellular second loop of the angiotensin II type 1 receptor.
3. The production method according to claim 2, characterized in that: the specific method comprises the following steps:
(1) Obtaining of hybridoma: actively immunizing Balb/C mouse with angiotensin II type 1 receptor extracellular second loop AT1R-ECL2, fusing mouse spleen lymphocyte and myeloma cell to generate AT1-AA secreting monoclonal hybridoma, culturing, and then 1 × 10 7 Introducing the hybridoma cells in the logarithmic phase into the abdominal cavity of a mouse to take ascites; isolating highly purified AT1-AA hybridomas from the ascites of mice;
(2) Extracting and purifying AT1-AA from hybridoma cells: obtaining AT1-AA from hybridoma cells by affinity chromatography using Mab Trap TM Extracting and purifying the Protein G HP 5mL high-efficiency purification column to obtain high-purity AT1-AA; the method specifically comprises the following steps: introducing the hybridoma cell suspension into the abdominal cavity of the mouse, and taking ascites for purification after the abdomen of the mouse expands for 14-18 days; rinsing the purification column with a binding buffer solution, mixing the ascites with the binding buffer solution in equal volume, passing through the purification column, continuing to rinse impurities with the binding buffer solution, eluting IgG bound to the column with an elution buffer solution, and rinsing with the binding buffer solution to revive the purification column;
detecting the beating frequency of the myocardial cells of the suckling mice by using the primary suckling mouse myocardial cells obtained by the separation culture to verify the biological activity of the purified AT1-AA; the method specifically comprises the following steps: taking myocardial tissue of a suckling mouse born for 0-4 days, adding mixed enzyme liquid to repeatedly digest the tissue, filtering to obtain a single cell group suspension, inoculating the single cell group suspension into a culture dish, attaching the single cell group suspension to the wall for 2 hours, collecting the suspension mainly containing the myocardial cells, suspending the suspension by using a new culture medium after centrifugation, and changing the liquid after attaching the wall for 36 hours;
removing culture medium containing fetal calf serum from myocardial cells of suckling mice cultured in 6-well plate, replacing with common DMEM high-sugar culture medium, counting myocardial cell basic beating frequency after 24 hr, and adding 1 × 10 concentration 6 After incubating the AT1-AA and the negatiable IgG for 5 minutes AT 37 ℃, counting each group respectively, wherein the counting time is 15 seconds each time, and finally obtaining the myocardial cell beating frequency of the suckling mouse per minute;
(3) Amplification of antibody genes: performing RNA extraction, cDNA synthesis and PCR amplification of antibody genes on the hybridoma cells with verified biological activity; after the hybridoma cells were recovered, RNA in the cells was extracted using RNeasyMiniKit from Qiagen, 10 μ l of an amplification reaction system was prepared using a first strand synthesis kit from Roche corporation, cDNA was synthesized, and then heavy and light chain genes were amplified by PCR using 30 total systems and 27 reaction systems using cDNA reverse transcribed from 2 to 20ug of total RNA as a template, and the amplification products were verified by 1.5% agarose electrophoresis.
4. The production method according to claim 3, characterized in that: the formula of the binding buffer solution is as follows: 10 Xbinding buffer solution A namely 10 XNa 2 HPO4•12H 2 Solution O: 38.5gNa2HPO 4 •12H 2 Fully dissolving O in 490mL double distilled water, fixing the volume to 500mL, uniformly mixing, filtering with a 0.22 mu m filter membrane, and storing at 4 ℃;
10 × binding buffer solution B namely 10 × NaH 2 PO4•2H 2 Solution O: 15.6g of NaH 2 PO4•2H 2 Fully dissolving O powder in 450mL double distilled water, fixing the volume to 500mL, uniformly mixing, filtering with a 0.22-micron filter membrane, and storing at 4 ℃;
binding buffer 20mmol/L Na 2 HPO 4 pH = 7.0: 27mL of 10 XNa was measured 2 HPO4•12H 2 O solution and 23mL of 10 XNa 2HPO 4 •12H 2 Adding 440mL of double distilled water into the O solution, fully dissolving, adjusting the pH value to 7.0, then using the double distilled water to fix the volume to 500mL, uniformly mixing, filtering by a 0.22 mu m filter, and storing at 4 ℃;
the elution buffer is 100mmol/L Glycine-HCl, and the formula of the pH =2.7 is as follows: fully dissolving 3.75g of glycine in 450mL of double distilled water, adjusting the pH to 2.7, metering the volume to 500mL, uniformly mixing, filtering by using a 0.22-micron filter, and storing at 4 ℃;
the formula of the mixed enzyme solution is as follows: preparing a collagenase solution: weighing 10mg of type II collagenase, fully dissolving the collagenase in a super clean bench by using 16ml PBS buffer solution, uniformly mixing, and filtering the mixture by using a 0.22 mu m filter for later use; preparing a mixed enzyme solution: a commercially available 0.25% EDTA-free pancreatin and collagenase solution were mixed to prepare a mixed enzyme solution at a volume ratio of 1.
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