CN115838425B - Antibody targeting angiotensin II type 1 receptor extracellular second loop and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and provides an antibody targeting an 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 variable region gene is 336bp; the nucleotide sequence of the heavy chain is shown in SEQ ID NO:1 is shown in the specification; the heavy chain amino acid sequence is shown in SEQ ID NO:2 is shown in the figure; the nucleotide sequence of the light chain is shown in SEQ ID NO:3 is shown in the figure; the amino acid sequence of the light chain is shown in SEQ ID NO: 4. After cDNA is reverse transcribed by extracting RNA from hybridoma cells, antibody genes are amplified by PCR to determine the heavy chain and light chain amino acid sequences and CDR regions of the antibody. The amino acid sequence of the AT1-AA is originally obtained by the method, thereby providing necessary basic work for preparing the monoclonal AT1-AA with low cost for mass production by adopting a recombinant protein expression method in the later period.

Description

An antibody targeting the second extracellular loop of angiotensin II type 1 receptor and its application

Technical Field

The invention belongs to the technical field of genetic engineering, and specifically relates to an antibody targeting the second extracellular loop of angiotensin II type 1 receptor and an application thereof.

Background technique

At present, cardiovascular disease is still the leading cause of death in the world. The renin-angiotensin-aldosterone system (RAAS) plays an important role in maintaining the stability of the cardiovascular system, normal development, and blood pressure regulation. The main effector of RAAS is angiotensin II (AngII), which exerts various physiological and pathophysiological effects by activating angiotensin II type 1 receptor (AT1R) signal transduction.

AT1R is a typical GPCR seven-transmembrane α-helical structure with an extracellular N-terminus, three extracellular loops (ECL1-3), three intracellular loops (ICL1-3), an amphipathic helix VIII (H8) and an intracellular C-terminus, among which ECL2 has the strongest immunogenicity. Overactivation of AT1R can lead to vascular system dysfunction, such as increased vascular tension, inflammation, fibrosis and thrombosis. Under pathophysiological conditions, Ang II chronically activates AT1R, leading to receptor overactivation, which subsequently triggers a variety of diseases such as hypertension, heart failure, vascular remodeling, diabetic nephropathy and atherosclerosis. However, in some cardiovascular diseases, it is found that AT1R is overactivated, but the level of Ang II is not high, which suggests that other factors may be involved.

At the end of the 20th century, researchers discovered an autoantibody against AT1R (angiotensin II type 1 receptor autoantibody, AT1-AA) in the serum of patients with various cardiovascular diseases such as preeclampsia, coronary heart disease, hypertension, and peripheral arterial disease. This autoantibody can exert a receptor agonist-like effect similar to Ang II. More and more studies have confirmed that although both can activate AT1R, the sites of Ang II and AT1-AA binding to AT1R and the activation patterns of the receptor are different. Ang II binds to the 5th and 6th transmembrane regions of AT1R, causing transient activation of AT1R; while AT1-AA can specifically recognize the second extracellular loop of AT1R (AT1R-ECL2), resulting in continuous activation of AT1R and downstream signals, causing pathological effects such as endothelial damage, vascular smooth muscle cell transformation, and myocardial hypertrophy. Therefore, it is currently believed that AT1-AA is an important cause of AT1R overactivation in various cardiovascular diseases. However, the molecular regulatory mechanism of AT1-AA in cardiovascular diseases has not been fully elucidated, which brings difficulties to targeted intervention.

Obtaining sufficient and highly pure AT1-AA is the primary prerequisite for studying its pathological significance and specific molecular mechanisms. At present, there are two main ways to obtain AT1-AA: one is to purify AT1-AA from the serum of clinical patients. However, the source of clinical patient serum is limited, the titer is low, and it is difficult to collect. If the purification is not timely, the titer of AT1-AA may decrease or be lost, so it is difficult to provide sufficient guarantee for the mechanism research of AT1-AA; the second is to obtain AT1-AA through active immunization, that is, to replicate the immune response, but active immunization takes a long time, and the antibody is polyclonal, which makes it difficult to carry out fine molecular regulation mechanism research. Therefore, obtaining AT1-AA with high efficacy and specificity and relatively low cost is an important bottleneck problem that restricts the development of this field. To this end, we used the human AT1R-ECL2 peptide to prepare hybridoma cells that can produce monoclonal antibodies AT1-AA. Although hybridoma cells can produce highly pure monoclonal AT1-AA, the high purification cost limits its application.

Summary of the invention

In order to realize large-scale mass production of AT1-AA with highly uniform physical and chemical properties, the present invention provides an antibody targeting the second extracellular loop of angiotensin II type 1 receptor and its application. RNA of hybridoma cells is extracted, cDNA is reverse transcribed, and antibody genes are amplified by PCR to determine the heavy chain and light chain amino acid sequences and CDR regions of the antibody. The amino acid sequence of AT1-AA is originally obtained by the above method, thereby providing the necessary basic work for the later preparation of low-cost monoclonal AT1-AA by mass production using a recombinant protein expression method.

The present invention is achieved by the following technical scheme: an antibody targeting the second extracellular loop of angiotensin II type 1 receptor, wherein the variable region gene of the antibody is 336 bp; the nucleotide sequence of its heavy chain is shown in SEQ ID NO: 1; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 2; the nucleotide sequence of the light chain is shown in SEQ ID NO: 3; and the amino acid sequence of the light chain is shown in SEQ ID NO: 4.

The method for preparing the antibody targeting the second extracellular loop of angiotensin II type 1 receptor comprises extracting RNA of hybridoma cells, reversely transcribing cDNA, amplifying antibody genes by PCR, determining the amino acid sequences of the heavy chain and light chain of the antibody and the CDR region; and obtaining the amino acid sequence of the antibody AT1-AA targeting the second extracellular loop of angiotensin II type 1 receptor.

The specific method is:

(1) Obtaining hybridoma cells: Balb/C mice were actively immunized with AT1R-ECL2, the second extracellular loop of the angiotensin II type 1 receptor; mouse spleen lymphocytes were fused with myeloma cells to produce monoclonal hybridomas that secrete AT1-AA and cultured, and then the cells in the logarithmic phase were injected into the mouse peritoneum to obtain ascites. After 1×10 ⁷ injection, highly purified AT1-AA hybridomas were isolated from the mouse ascites. The method described in the reference (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 Angiotensin II Type 1 Receptor. J Immunol Res. 2016;2016:1858252.) was used.

(2) Extraction and purification of AT1-AA from hybridoma cells: AT1-AA was obtained from hybridoma cells using affinity chromatography, and extracted and purified using Mab Trap ^TM Protein G HP 5 mL high-efficiency purification columns (GE Heathcare Life Sciences) to obtain high-purity AT1-AA; specifically, the hybridoma cell suspension was injected into the peritoneal cavity of mice, and after 14-18 days of abdominal distension, ascites was extracted for purification. First, the purification column was rinsed with binding buffer, and the ascites and binding buffer were mixed in equal volumes and then passed through the purification column. The column was then washed with binding buffer, and finally the IgG bound to the column was eluted with elution buffer, and the purification column was revived by rinsing with binding buffer;

The obtained AT1-AA was used to detect the beating frequency of neonatal rat cardiomyocytes using isolated and cultured primary neonatal rat cardiomyocytes to verify the biological activity of the purified AT1-AA; specifically, the neonatal rats born 0-4 days were quickly decapitated, the heart was taken out to wash the blood and remove the connective tissue, the heart was cut into small pieces, a mixed enzyme solution was added to repeatedly digest the tissue, a single cell group suspension was obtained by filtration and then inoculated in a culture dish, and the suspension mainly containing cardiomyocytes was collected after 2 hours of adhesion, and resuspended in a new culture medium after centrifugation, and the medium was changed after 36 hours of adhesion;

The culture medium containing fetal bovine serum was removed from the neonatal rat cardiomyocytes cultured normally in 6-well plates and replaced with ordinary DMEM high-glucose culture medium. After 24 hours, the basal beating frequency of the cardiomyocytes was counted first, and then AT1-AA and negative IgG with a concentration of 1×10 ⁶ were added respectively. After incubation at 37°C for 5 minutes, each group was counted separately. Each counting time was 15 seconds, and finally the beating frequency of neonatal rat cardiomyocytes per minute was obtained.

(3) Amplification of antibody genes: RNA extraction and cDNA synthesis were performed on hybridoma cells with verified biological activity, and antibody genes were amplified by PCR. After the hybridoma cells were revived, the RNA in the cells was extracted using Qiagen's RNeasyMiniKit, and a 10µl amplification reaction system was prepared using Roche's first-strand synthesis kit. After cDNA was synthesized, the cDNA formed by reverse transcription of 2-20ug total RNA was used as a template, and the heavy and light chain genes were amplified by PCR using 30 total systems and 27 reaction systems. The amplified products were verified by 1.5% agarose electrophoresis and were all 330bp bands.

The present invention obtains the amino acid sequence of AT1-AA, thereby providing necessary basic work for the subsequent mass production of low-cost monoclonal AT1-AA using a recombinant protein expression method. The AT1-AA prepared by the recombinant protein expression method is not only low-cost but also uniform in quality, and can provide sufficient raw materials for subsequent pathological experiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph for identifying the purity and biological activity of AT1-AA produced by hybridoma cells; in the graph: A is the SDS-PAGE result; B is the result of detecting the beating frequency of neonatal rat myocardial cells using isolated and cultured primary neonatal rat myocardial cells;

FIG2 is a PCR verification result of the amplified antibody gene; in the figure: lanes 1-3 are the PCR results of different heavy chain primers, and lane 4 corresponds to the light chain PCR result;

Figure 3 shows the heavy chain CDR and FR regions and the light chain CDR and FR regions; A in the figure shows the heavy chain CDR and FR regions; B shows the light chain CDR and FR regions;

FIG4 shows VH and VL templates; in the figure: A is a VH template, and B is a VL template;

Figure 5 is the VH homology modeling evaluation result; in the figure: A is the ERRAT result of VH; B is the VERIFY3D result of VH; C is the Ramachandran plot evaluation result in PROCHECK of VH;

FIG6 is a VL homology modeling evaluation result; in the figure: A is the ERRAT result of VL; B is the VERIFY3D result of VL; C is the Ramachandran plot evaluation result in PROCHECK of VL;

Figure 7 is the 3D structure of the variable region;

FIG8 is a diagram showing the molecular docking of the AT1-AA variable region and AT1R using Zdock using the AT1R structure in AlphaFold.

Detailed ways

In order to make the purpose, 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. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection 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 disclosure and materials cited therein are hereby incorporated by reference.

Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

1. Identify the purity and biological activity of AT1-AA produced by hybridoma cells. AT1-AA was obtained from the ascites produced by hybridoma cells by affinity chromatography, and extracted and purified using Mab Trap ^TM Protein G HP 5 mL high-efficiency purification column (GE Heathcare Life Sciences) to obtain high-purity AT1-AA. The specific method is:

A. Purification of AT1-AA from mouse ascites produced by hybridoma cells

(1) The synthesized human AT1 receptor extracellular second loop antigen epitope peptide was used to immunize Balb/c mice to prepare high-titer polyclonal antibody serum, and cell fusion was performed to prepare hybridoma cell lines.

(2) Select clean grade AT1-AA negative Balb/c mice aged 10-12 weeks, regardless of gender. Room temperature 18℃-22℃, humidity 40%-70%, light and dark alternate every 12 hours. Feed with free food and water. Adaptive feeding for 3-5 days.

(3) The mice were randomly divided into two groups for pre-immunization treatment: one group of mice was intraperitoneally injected with 0.4 mL-0.5 mL paraffin wax, and the other group of mice was intraperitoneally injected with Freund's incomplete adjuvant (0.02 mL/g). Both paraffin wax and Freund's incomplete adjuvant were used for pre-immunization treatment of mice. The mice were fed normally for one week.

(4) One week later, treat the hybridoma cells that are growing in the logarithmic growth phase. Remove the cells from the culture dish by blowing them off and transfer them to a 50 mL centrifuge tube. Centrifuge at 800 RPM for 5 minutes and discard the culture medium to make a cell suspension with a final concentration of 2 × 10 ⁶ -1 × 10 ⁷ cells/mL.

(5) Inject the treated hybridoma cell suspension into the peritoneal cavity of pre-immunized mice, 0.5 mL per mouse, and continue to feed normally. After 14-18 days, when the mice develop obvious ascites, the ascites is extracted.

(6) Rinse the purification column with binding buffer, mix equal volumes of ascites and binding buffer and pass them through the purification column, then wash with binding buffer and elute the IgG on the column with elution buffer. Collect the eluate in an Eppendorf tube pre-filled with neutral buffer to detect the concentration and set aside.

Buffer formulation:

10× Binding Buffer A (10×Na ₂ HPO 4 • 12H ₂ O solution): 38.5 g Na 2 HPO ₄ • 12H ₂ O was fully dissolved in 490 mL double distilled water, the volume was fixed to 500 ml, mixed and filtered through a 0.22 μm filter membrane, and stored at 4°C.

10× Binding Buffer B (10×NaH ₂ PO4•2H ₂ O solution): Dissolve 15.6 g of NaH ₂ PO4•2H ₂ O powder in 450 mL of double distilled water, make up to 500 mL, mix well, filter through a 0.22 μm filter membrane, and store at 4°C.

Binding buffer (20mmol/L _Na2HPO4 , pH= _7.0 ): Measure 27mL of 10× _Na2HPO4 • _12H2O solution and 23mL of 10× _Na2HPO4 • _12H2O solution, add 440mL of double distilled water, fully dissolve, adjust the pH to 7.0, and then make up to 500ml with double distilled water. After mixing, filter with a 0.22μm filter and store at 4℃.

Elution buffer (100mmol/L Glycine-HCl, pH=2.7): Dissolve 3.75g glycine in 450mL double distilled water, adjust the pH to 2.7, make up to 500mL, mix well, filter with a 0.22μm filter, and store at 4℃.

Neutral buffer (1 mol/L Tris, pH=8.0): Dissolve 60.57 g Tris in 450 mL double distilled water, adjust the pH to 8.0, make up to 500 mL, mix well, filter with a 0.45 μm filter, and store at 4°C.

B. Extraction of neonatal rat cardiomyocytes and drug treatment:

(1) Prepare a 50 mL sterile centrifuge tube and add 25 ml of 10% FBS/PS/DMED high-glucose medium to the centrifuge tube; take out the 0-4 day old suckling mouse and soak it in 75% alcohol for disinfection. Use large forceps in the right hand to take out the suckling mouse, use the thumb and index finger of the left hand to fix the body of the suckling mouse, leaving the head, and use tissue scissors in the right hand to quickly cut off the head along the neck, dripping 3-4 drops of blood (try to remove blood cells). Use ophthalmic scissors to cut the sternum from the left edge of the xiphoid process, expose the heart, and use curved forceps to clamp it out and place it in pre-cooled PBS for washing.

(2) The removed heart is passed through 2-3 plates containing pre-cooled PBS to fully wash away the blood. Use clean ophthalmic scissors to remove the connective tissue from the washed heart. Then place the heart in a small glass bottle, add an appropriate amount of PBS, and use ophthalmic scissors to cut the heart tissue into ¹ mm3 tissue blocks. Let it stand for 5 minutes, discard the supernatant, and repeat 3-4 times until the PBS is clear. The purpose is to remove the blood that was not pumped out before.

(3) Discard the PBS in the glass bottle (be careful not to suck away the tissue pieces), put it into a small rotor, add 2-3 mL of mixed enzyme solution to the tissue, and use a temperature-controlled magnetic stirrer to repeatedly digest the tissue at a constant temperature and speed of 37°C. The speed is controlled at 60 RPM per minute. Digest for 5 minutes and discard the first digestion solution; digest the tissue multiple times in a short time. The total number of digestions should not exceed 10 times. The digestion time for each time should not exceed 7 minutes. Each digestion is also collected in a centrifuge tube containing culture medium to terminate the digestion reaction.

(4) After all digestion is completed, filter the mixture with a 200-mesh sieve to obtain a single-cell suspension. Centrifuge at 800 RPM for 5 minutes, discard the supernatant, resuspend the cells in new 10% FBS/PS/DMED high-glucose medium, inoculate and culture in a ¹⁰ cm2 culture dish, and place in a 37°C, 5% CO2 incubator for differential attachment for 2 hours (to remove a large number of fibroblasts).

(5) After 2 hours, the fibroblasts have attached to the wall, but the cardiomyocytes have not yet attached to the wall. Collect the suspension containing mainly cardiomyocytes, centrifuge and resuspend in new culture medium, add 0.1 mmol/L 5-bromodeoxyuridine (5-BrdU), count the cells and inoculate them at an appropriate concentration in a 6-well plate or 96-well plate. Change the medium for the first time after 36 hours. Do not shake the plate during this period to prevent the cardiomyocytes from not firmly attaching to the wall.

(6) After 72 hours, the culture medium containing fetal bovine serum was removed from the neonatal rat cardiomyocytes in the 6-well plate and replaced with ordinary DMEM high-glucose culture medium for continued culture. After 24 hours, the basal beating frequency of the cardiomyocytes was counted, and then AT1-AA and negative IgG were added at a concentration of 1×10 ⁶ , respectively. After incubation at 37°C for 5 minutes, each group was counted separately, and each counting time was 15 seconds. Finally, the beating frequency of neonatal rat cardiomyocytes per minute was obtained.

The SDS-PAGE results are shown in Figure 1A. The results show that both have obvious 55 kDa heavy chain and 25 kDa light chain bands, indicating high purity. The primary neonatal rat cardiomyocytes are shown in Figure 1B, upper figure. The primary neonatal rat cardiomyocytes isolated and cultured were used to detect the beating frequency of neonatal rat cardiomyocytes. The test results are shown in Figure 1B, lower figure. The results show that AT1-AA can significantly stimulate the beating frequency of neonatal rat cardiomyocytes to accelerate.

(2) Verify the amplified antibody gene. After RNA extraction and cDNA synthesis of the hybridoma cells with verified activity, the amplified antibody gene was verified by PCR. The results are shown in Figure 2. The target antibody gene is 300 bp. Lanes 1-3 are the PCR results of different heavy chain primers, and lane 4 corresponds to the light chain PCR results.

RNA extraction (QIAGEN, RNeasy Mini Kit) method:

(1) Resuscitate the hybridoma cells, transfer them to a 50 mL centrifuge tube, add 20 mL of DMEM, and centrifuge at 300 g for 10 min.

(2) Discard the supernatant, add 30 mL of PBS to a 50 mL centrifuge tube, centrifuge at 300 g for 10 min, discard the supernatant, add 700 uL of RLT to the cell pellet, and pipette thoroughly to completely lyse the cells.

(3) Take 350uL of lymphocyte sample from the above lysis solution, pipette it thoroughly, homogenize it, and oscillate it to lyse it thoroughly; add 350uL of 70﹪ ethanol and pipette it back and forth to mix it evenly.

(4) Add 700uL of the liquid to the center of the column, centrifuge at room temperature for 15s, and discard the waste liquid; add 700uL of Buffer RW1 to the column, centrifuge at room temperature for 15s, and discard the waste liquid and collection tube.

(5) Place the column in a new collection tube, add 500uL Buffer RPE (add 4 times the volume of ethanol to Buffer RPE for the first use), centrifuge at room temperature for 15s, and discard the waste liquid. Add another 500uL Buffer RPE, centrifuge at room temperature for 2min. Empty for 1min, and discard the collection tube. Elute with 50uL DEPC water.

cDNA synthesis (20uL system): Roche's first-strand synthesis kit (Transcriptor First Strand cDNA Synthesis Kit.) was used. The amplification system preparation and reaction are shown in Table 1.

Table 1: Amplification system preparation and reaction

PCR amplification of antibody genes: Using cDNA formed by reverse transcription of 2-20ug total RNA as a template, configure the PCR reaction system according to Table 2 to amplify antibody light chain and heavy chain genes (30 total systems, 27 reaction system PCR):

Table 2: Amplification system preparation and reaction

The PCR products were subjected to 1.5% agarose electrophoresis, and both VH and VL should be bands of about 330 bp. After recovery and quantification, the next round of PCR can be carried out or frozen at -20°C for future use.

(3) The heavy chain base sequence and amino acid sequence of AT1-AA. The nucleotide sequence of the heavy chain is shown in SEQ ID NO: 1; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 2; the nucleotide sequence of the light chain is shown in SEQ ID NO: 3; the amino acid sequence of the light chain is shown in SEQ ID NO: 4; the heavy chain CDR region and FR region and the light chain CDR region and FR region are shown in Figure 3.

To analyze the binding mode of AT1-AA and AT1R, homology modeling was first performed based on the measured AT1-AA variable region sequence, and VH and VL were modeled online using Swiss-model. The modeling results are shown in Figure 4. After sequence alignment, the PDB number of the VH modeling template is 6j5d, the sequence consistency is 80.36% (greater than 30% is reasonable modeling), and the GMQE (global model quality evaluation) is 0.87 (the credibility range is between 0-1, and the larger the value, the better the quality). The PDB number of the VL modeling template is 5myx, the sequence consistency is 90.18%, and the GMQE is 0.89.

The VH and VL modeling results were then evaluated by SAVES v6.0, and the results are shown in Figures 5 and 6. In the ERRAT results, the higher the Overall quality factor value, the better, and the score>85 is better. The crystal can reach 95, and the score of VH is 94.2308, and the score of VL is 100. In the VERIFY3D results, more than 80% of the residues have a 3D/1D value greater than 0.2, and the model quality is qualified, and both VH and VL are 100%. In the PROCHECK results, the Ramachandran plot evaluation shows that more than 90% of the amino acid residues in the core area are reasonable. VH has 91 amino acid residues located in the core area (95.8%), 4 residues in the allowed area (4.2%), and no residues in the prohibited area and the roughly allowed area; VL has 87 amino acid residues located in the core area (94.6%), 4 residues in the allowed area (4.3%), no residues in the roughly allowed area, and 1 residue in the prohibited area (1.1%). In summary, the VH and VL modeling was reasonable, and then the final structure of the homology modeling of the AT1-AA variable region was obtained by the superposition command (align) of pymol.

Using the AT1R structure in AlphaFold, Zdock was used to dock the AT1-AA variable region with the AT1R molecule. The results showed that S32 of VL formed two hydrogen bonds with T190 of AT1R, S56 of VH formed a hydrogen bond with T94 of AT1R, and G98 of VH formed a hydrogen bond with D9 of AT1R. G10, I11, K12, R13, P95, N98, F171, N176, T178, H183, and E185 of AT1R had a hydrophobic effect, D31, D33, Y37, F99, and R101 of VL had a hydrophobic effect, and T30, Y31, W32, N34, Q49, F51, S54, and Y58 of VL had a hydrophobic effect.

By performing homology modeling and molecular docking experiments on the sequencing results of the variable region of AT1-AA, it was demonstrated that AT1-AA can form hydrogen bonds and hydrophobic interactions with AT1R and that the sequence is effective, providing a theoretical basis for the subsequent mass production of AT1-AA based on this sequence.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims (1)

1. An antibody targeting the extracellular second loop of an angiotensin II type 1 receptor, characterized in that: the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO: 1. shown; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2. shown; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 3. shown; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4. as shown.