CN115838425B

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

Info

Publication number: CN115838425B
Application number: CN202211054756.5A
Authority: CN
Inventors: 刘慧荣; 张苏丽; 张茜; 武烨; 白丽娜; 王鹏丽
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2022-08-31
Filing date: 2022-08-31
Publication date: 2024-06-18
Anticipated expiration: 2042-08-31
Also published as: CN115838425A

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

Antibody targeting angiotensin II type 1 receptor extracellular second loop and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to an antibody targeting an extracellular second loop of an angiotensin II1 type receptor and application thereof.

Background

At present, cardiovascular diseases are still the most leading cause of death worldwide, and the renin-angiotensin-aldosterone system (renin-angiotenin-aldosterone system, RAAS) has important roles in maintaining stable and normal development of the cardiovascular system, blood pressure regulation, and the like. The main effector of RAAS is angiotensin II (Ang II), which exerts various physiological and pathophysiological effects by activating angiotensin II type 1 receptor (at1 r) signaling.

AT1R is a typical GPCR seven transmembrane α -helical 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, thrombosis, and the like. Under pathophysiological conditions, ang II chronically activates AT1R, resulting in excessive activation of the receptor, which may subsequently trigger various diseases such as hypertension, heart failure, vascular remodeling, diabetic nephropathy and atherosclerosis. However, in part of cardiovascular disease, AT1R is over-activated, while Ang II levels are not high, suggesting that other factors may be involved.

AT the end of the 20 th century, researchers have subsequently found the presence of an autoantibody (angiotensin II type 1 receptor autoantibody,AT1-AA) to AT1R in serum from patients with various cardiovascular diseases such as preeclampsia, coronary heart disease, hypertension, peripheral arterial disease, etc. The autoantibody may exert an Ang II-like receptor agonist-like effect. More and more studies confirm that although both activate AT1R, ang II has a difference in both the site of binding to AT1R and the pattern of activation of the receptor from AT 1-AA. Ang II binds to the 5 th, 6 th transmembrane region of AT1R, causing transient activation of AT 1R; the AT1-AA can specifically recognize the extracellular second loop (AT 1R-ECL 2) of the AT1R, so that the 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 caused. Thus, AT1-AA is currently considered to be a significant cause of over-activation of AT1R in a variety of cardiovascular diseases. However, the molecular regulatory mechanisms of AT1-AA involved in cardiovascular diseases have not been fully elucidated, which presents difficulties for targeted intervention.

Obtaining a sufficient amount of high purity of AT1-AA is a primary premise for studying its pathological significance and specific molecular mechanism. There are two main ways to obtain AT 1-AA: firstly, AT1-AA is purified from serum of clinical patients. However, the serum of clinical patients has limited sources, low titer and difficult collection, and if the purification is not timely, the titer of AT1-AA can be reduced or lost, so that sufficient guarantee is difficult to be provided for the mechanism research of AT 1-AA; secondly, AT1-AA is obtained through active immunization, namely replication immune reaction, but the active immunization takes a long time, and the antibody is polyclonal, so that the research on a fine molecular regulation mechanism is difficult to develop. Thus, obtaining an AT1-AA with high potency and specificity and relatively low cost is an important bottleneck limiting the development in the art. For this purpose, we have prepared hybridoma cells producing the monoclonal antibody AT1-AA using the human AT1R-ECL2 peptide fragment. Although hybridoma cells can produce monoclonal AT1-AA in high purity, purification costs are high, limiting its use.

Disclosure of Invention

The invention provides an antibody targeting an extracellular second loop of an angiotensin II type 1 receptor and application thereof, in order to realize AT1-AA with highly uniform mass production physical properties. 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.

The invention is realized by the following technical scheme: an antibody targeting the extracellular second loop of an angiotensin II type 1 receptor, said antibody variable region gene being 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.

The method for preparing the antibody targeting the extracellular second loop of the angiotensin II type 1 receptor comprises the steps of extracting RNA of hybridoma cells, carrying out reverse transcription on cDNA, amplifying antibody genes by PCR, and determining the heavy chain and light chain amino acid sequences and CDR regions of the antibody; the amino acid sequence of the antibody AT1-AA targeting the extracellular second loop of the angiotensin II type 1 receptor was obtained.

The specific method comprises the following steps:

(1) Obtaining hybridoma cells: actively immunizing a Balb/C mouse with an angiotensin II type 1 receptor extracellular second loop AT1R-ECL 2; spleen lymphocytes from mice are fused with myeloma cells to produce a monoclonal hybridoma that secretes AT1-AA and cultured, and then cells in the logarithmic phase are injected into the abdominal cavity of the mice to take ascites. Highly purified AT1-AA hybridomas were isolated from ascites of mice following injection with 1X 10 ⁷. The method described in 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.）.

(2) Extraction and purification of AT1-AA from hybridoma cells: obtaining AT1-AA from the hybridoma cells by utilizing affinity chromatography, and extracting and purifying by using a mAb Trap ^TM Protein G HP 5 mL high-efficiency purification column (GE HEATHCARE LIFE SCIENCES) to obtain high-purity AT1-AA; the method comprises the following steps: injecting the hybridoma cell suspension into the abdominal cavity of the mouse, and extracting ascites for purification after 14-18 days of abdominal bulge of the mouse. Washing the purification column with a binding buffer solution, mixing ascites with the binding buffer solution in equal volume, passing through the purification column, continuing washing impurities with the binding buffer solution, eluting IgG bound to the column with an eluting buffer solution, and washing with the binding buffer solution to rejuvenate the purification column;

Detecting the beating frequency of the obtained AT1-AA by utilizing the isolated and cultured primary mammary rat myocardial cells, and verifying the biological activity of the purified AT 1-AA; the method comprises the following steps: taking out the heart, washing blood and removing connective tissue from the milk mouse after birth for 0-4 days, cutting the heart into small pieces, adding mixed enzyme solution, repeatedly digesting the tissue, filtering to obtain single cell group suspension, inoculating in a culture dish, attaching for 2 hours, collecting suspension mainly containing myocardial cells, centrifuging, re-suspending with new culture medium, attaching for 36 hours, and changing the liquid;

removing the culture medium containing fetal bovine serum from the normal milk mouse myocardial cells cultured in a 6-well plate, replacing the culture medium with a common DMEM high-sugar culture medium, counting the basic beating frequency of the myocardial cells after 24 hours, then respectively adding AT1-AA and NEGATIVE IGG with the concentration of 1X 10 ⁶, incubating for 5 minutes AT 37 ℃, counting each component for 15 seconds, and finally obtaining the beating frequency of the milk mouse myocardial cells per minute;

(3) Amplification of antibody genes: performing RNA extraction and cDNA synthesis on the hybridoma cells with verified biological activity, and amplifying antibody genes by PCR; after resuscitating hybridoma cells, extracting RNA in the cells by using RNEASYMINIKIT of Qiagen, preparing a 10 mu l amplification reaction system by using a first strand synthesis kit of Roche company, synthesizing cDNA, and amplifying heavy chain genes and light chain genes by using 2-20ug of total RNA reverse transcription as templates through PCR of 30 total systems and 27 reaction systems, wherein amplified 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 by adopting a recombinant protein expression method in the later period, and the AT1-AA prepared by adopting the recombinant protein expression method has low cost and uniform quality and can provide sufficient raw materials for the later period pathological experiments.

Drawings

FIG. 1 is a graph of the purity and bioactivity of the identification of hybridoma cells producing AT 1-AA; in the figure: a is SDS-PAGE result; b is a diagram of the detection result of the beating frequency of the primary milk mouse cardiac myocytes which are separated and cultured;

FIG. 2 shows the results of PCR for verifying amplified antibody genes; in the figure: lanes 1-3 are different heavy chain primer PCR results, lane 4 corresponds to the light chain PCR result;

FIG. 3 is a heavy chain CDR region and an FR region and a light chain CDR region and an FR region; in the figure, A is the heavy chain CDR region and FR region; b is a light chain CDR region and an FR region;

FIG. 4 is a VH and VL template; 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 the ERRAT results of VH; b is the VERIFY3D result of VH; c is the evaluation result of the Lagrangian diagram in PROCHECK of VH;

FIG. 6 is a VL homology modeling evaluation result; in the figure: a is ERRAT results for VL; b is the VERIFY3D result of VL; c is the evaluation result of the Lagrangian diagram in PROCHECK of VL;

FIG. 7 is a variable region 3D structure;

FIG. 8 is a diagram of the docking of the AT1-AA variable region to an AT1R molecule using Zdock using the AT1R structure in AlphaFold.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments; all other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the 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, the disclosure of which is incorporated herein by reference as is commonly understood by reference.

Those skilled in the art will recognize that equivalents of the specific embodiments described, as well as those known by routine experimentation, are intended to be encompassed within the present application.

The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments used in the following examples are laboratory conventional instruments unless otherwise specified; the experimental materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

1. The purity and bioactivity of AT1-AA produced by the hybridoma cells are identified. AT1-AA is obtained from ascites produced by hybridoma cells by an affinity chromatography method, and is extracted and purified by using a 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 comprises the following steps:

A. purification of AT1-AA in ascites of mice produced by hybridoma cells

(1) The synthesized extracellular second cyclic antigen epitope peptide of the human AT1 receptor is immunized with Balb/c mice to prepare high-titer polyclonal serum, and cell fusion is carried out to prepare hybridoma cell strains.

(2) The Balb/c mice with the cleaning grade and the detection of being negative to the AT1-AA and the age of 10-12 weeks are selected, and the sex is unlimited. The room temperature is 18-22 ℃, the humidity is 40-70%, and the light and the darkness are alternated every 12 hours. Free diet and drinking water feeding. The feed is suitable for 3-5 days.

(3) Mice were randomly divided into two groups and preimmunized: one group of mice is injected with paraffin wax of 0.4-0.5 mL 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 wax and Freund's incomplete adjuvant are used for pre-immunizing the mice. Normal feeding was continued for one week.

(4) After one week, the hybridoma cells growing to the logarithmic growth phase, which are being cultured, are treated. Cells in the dish were blown off, transferred to a 50mL centrifuge tube, centrifuged at 800RPM for 5 minutes, and the medium was discarded to give a final cell suspension at a concentration of 2X 10 ⁶-1×10⁷ cells/mL.

(5) The treated hybridoma cell suspension is injected into the abdominal cavity of mice which are subjected to preimmunization treatment, and each mouse is injected with 0.5mL to continue normal feeding. After 14-18 days, the mice showed obvious ascites, and the ascites was extracted.

(6) Washing the purification column with a binding buffer solution, mixing ascites with the binding buffer solution in equal volume, passing through the purification column, washing impurities with the binding buffer solution, eluting IgG on the column with an eluting buffer solution, and receiving the eluted solution into an Ep tube which is placed in a neutral buffer solution in advance for concentration detection for later use.

The formula of the buffer solution comprises the following steps:

10 Xbinding buffer A (10 XNa ₂HPO4•12H₂ O solution): 38.5g Na2HPO ₄•12H₂ O is fully dissolved in 490mL double distilled water, the volume is fixed to 500 mL, the mixture is filtered by a 0.22 mu m filter membrane after being evenly mixed, and the mixture is preserved at 4 ℃.

10 Xbinding buffer solution B (10 XNaH ₂PO4•2H₂ O solution): 15.6g of NaH ₂PO4•2H₂ O powder is fully dissolved in 450mL of double distilled water, the volume is fixed to 500mL, and the mixture is uniformly mixed, filtered by a 0.22 mu m filter membrane and stored at 4 ℃.

Binding buffer (20 mmol/L Na ₂HPO₄, ph=7.0): 27mL of 10 XNa ₂HPO4•12H₂ O solution and 23mL of 10 XNa 2HPO ₄•12H₂ O solution are measured, 440mL of double distilled water is added for full dissolution, the pH is adjusted to 7.0, the double distilled water is used for constant volume to 500mL, and after uniform mixing, a 0.22 μm filter is used for filtration and the mixture is preserved at 4 ℃.

Elution buffer (100 mmol/L Glycine-HCl, ph=2.7): 3.75g glycine was fully dissolved in 450mL double distilled water, pH was adjusted to 2.7, volume was adjusted to 500mL, and after mixing well, 0.22 μm filter was filtered and stored at 4 ℃.

Neutral buffer (1 mol/L Tris, ph=8.0): 60.57g of Tris is fully dissolved in 450mL of double distilled water, the pH is regulated to 8.0, the volume is fixed to 500mL, and after uniform mixing, a 0.45 mu m filter is used for filtration and the mixture is preserved at 4 ℃.

B. extracting and dosing myocardial cells of a milk mouse:

(1) Preparing a sterile centrifuge tube of 50 mL, and adding 10% FBS/PS/DMED high sugar culture medium of 25ml into the centrifuge tube; taking the suckling mice born for 0-4 days, soaking and sterilizing in 75% alcohol. The right hand holding forceps is used for taking out the suckling mouse, the body of the suckling mouse is fixed by the thumb and the index finger of the left hand, the head is left, the tissue of the right hand is sheared along the neck to quickly break, and 3-4 drops of blood are dropped (blood cells are removed as much as possible). The replacement ophthalmic scissors cut the sternum from the left edge of the xiphoid process, and after exposing the heart, the heart is clamped out by using bent forceps and placed in precooled PBS for cleaning.

(2) The extracted hearts were serially passed through 2-3 plates containing pre-chilled PBS to thoroughly wash the blood; the cleaned ophthalmology scissors remove connective tissue from the cleaned heart; then put into a small glass bottle, add an appropriate amount of PBS, cut the heart tissue into 1mm ³ pieces of tissue with ophthalmic scissors, stand for 5 minutes, discard the supernatant, repeat 3-4 times until PBS is clear, in order to remove the blood that was not pumped out before.

(3) Discarding PBS (taking care of not sucking away tissue blocks) in a glass bottle, putting a small rotor, adding 2-3 mL mixed enzyme solution into the tissue, repeatedly digesting the tissue by rotating at constant temperature and constant speed of 37 ℃ by using a temperature-controlled magnetic stirrer, controlling the rotating speed to be 60 RPM per minute, carrying out shaking digestion for 5 minutes, and discarding the first digested solution; the tissue is digested a short number of times, preferably not more than 10 times, each time for not more than 7 minutes, and each time the digestion is collected in a centrifuge tube containing the medium to terminate the digestion reaction.

(4) After complete digestion, the mixture was filtered through a 200 mesh screen to give a single cell suspension. Centrifugation at 800RPM for 5 min, supernatant was discarded, cells resuspended in fresh 10% FBS/PS/DMED high sugar medium, inoculated in 10cm ² dishes, and placed in a 5% CO2 incubator at 37℃for differential adherence for 2 hours (removal of large amounts of fibroblasts).

(5) After 2 hours, the fibroblasts had attached, but the cardiomyocytes had not attached, the suspension containing mainly the cardiomyocytes was collected, resuspended in fresh medium after centrifugation, and 0.1 mmol/L of 5-bromodeoxyuridine (5-BrdU) was added, and after cell counting, inoculated in 6-or 96-well plates at the appropriate concentration. After 36 hours, the liquid is changed for the first time, the pore plate is not required to be rocked during the period, and the myocardial cells are prevented from being infirm attached to the wall.

(6) After 72 hours, the culture medium containing fetal calf serum is removed from the milk mouse myocardial cells planted in the 6-hole plate, and the culture medium is replaced by a common DMEM high-sugar culture medium for continuous culture. After 24 hours, firstly counting the basic beating frequency of the myocardial cells, then respectively adding AT1-AA with the concentration of 1X 10 ⁶ and NEGATIVE IGG, incubating for 5 minutes AT 37 ℃, respectively counting each group, wherein the counting time is 15 seconds each time, and finally obtaining the beating frequency of the myocardial cells of the milk mice per minute.

SDS-PAGE results are shown in FIG. 1A, and the results show that the two have obvious 55 kDa heavy chain and 25 kDa light chain bands, which indicate that the purity is higher. The primary milk mouse myocardial cells are shown in the upper graph in the figure 1B, the isolated and cultured primary milk mouse myocardial cells are used for detecting the beating frequency of the milk mouse myocardial cells, the detection result is shown in the lower graph in the figure 1B, and the result shows that AT1-AA can obviously stimulate the beating frequency of the milk mouse myocardial cells to be accelerated.

(2) Verifying the amplified antibody genes. After RNA extraction and cDNA synthesis of the hybridoma cells with verified activity, the amplified antibody genes are verified by PCR. As a result, the target antibody gene was 300bp, and lanes 1 to 3 were the results of PCR with different heavy chain primers, while lane 4 corresponds to the result of PCR with light chain.

RNA extraction (QIAGEN, RNeasy Mini Kit) method:

(1) The hybridoma cells were resuscitated, transferred to a 50mL centrifuge tube, added to 20mL dmem and centrifuged at 300g for 10min.

(2) The supernatant was discarded, 30mL of LPBS was added to a 50mL centrifuge tube, and after centrifugation for 10min, the supernatant was discarded, 700uL of RLT was added to the cell pellet, and the cells were completely lysed by blowing.

(3) Sucking 350uL of lymphocyte sample from the lysate, fully blowing, homogenizing, and shaking to thoroughly lyse; adding 350uL 70% ethanol, and blowing back and forth to mix uniformly.

(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 liquid and collection tube were discarded.

(5) The column was placed in a new collection tube, 500uLBuffer RPE (4 volumes of ethanol were added to Buffer RPE for the first time) was added and centrifuged at room temperature for 15s, and the waste solution was discarded. Then 500uL Buffer RPE was added and the mixture was centrifuged at room temperature for 2min. Air-free for 1min, discard collection tube. 50uL DEPC water was eluted.

CDNA Synthesis (20 uL System): a first strand synthesis kit (Transcriptor FIRST STRAND CDNASYNTHESIS kit) from Roche was used. The formulation and reaction of the amplification system are shown in Table 1.

Table 1: preparation and reaction of amplification system

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

Table 2: preparation and reaction of amplification system

The PCR product is subjected to agarose electrophoresis with the concentration of 1.5%, VH and VL are bands of about 330bp, and the PCR product is recovered and quantified for the next round or frozen at the temperature of minus 20 ℃ for later use.

(3) Heavy chain base sequence and amino acid sequence of AT 1-AA. 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 is shown in the figure; the heavy chain CDR region and the FR region are shown in FIG. 3, as well as the light chain CDR region and the FR region.

In order to analyze the binding mode of AT1-AA and AT1R, firstly, homologous modeling is carried out according to the sequence of the AT1-AA variable region, the modeling result is shown in figure 4, the modeling result is shown in the following, the PDB number of the modeling template of the VH is 6j5d, the sequence consistency is 80.36% (more than 30% is reasonable in modeling), GMQE (global model quality assessment) is 0.87 (the reliability range is between 0 and 1, and the larger the value is the better the quality). The modeling template for VL had a PDB number of 5myx, a sequence identity of 90.18% and GMQE of 0.89.

VH and VL modeling results were then evaluated by SAVES v 6.0.0 and are shown in fig. 5 and 6. The higher Overall quality factor value in ERRAT results, the better the score >85, the crystal can reach 95, the vh score is 94.2308, and the vl score is 100. If more than 80% of residues in the VERIFY3D result have a 3D/1D value of greater than 0.2, the model quality is acceptable, and both VH and VL are 100%. The evaluation of the Lagrangian diagram in the PROCHECK result is reasonable that more than 90% of amino acid residues fall in the core region, 91 amino acid residues of VH are all in the core region (95.8%), 4 residues fall in the allowed region (4.2%), and no residues fall in the forbidden region and the approximate allowed region; VL has 87 amino acid residues in the core region (94.6%), 4 residues in the allowed region (4.3%), no residues in the approximate allowed region, and 1 residue in the forbidden region (1.1%). Taken together, VH and VL modeling was rational, after which the final structure of homology modeling of the AT1-AA variable region was obtained by a superposition command (align) of pymol.

The AT1-AA variable region was docked to the AT1R molecule using Zdock using the AT1R structure in AlphaFold. 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 forms a hydrogen bond with D9 of AT 1R. G10, I11, K12, R13, P95, N98, F171, N176, T178, H183, E185 of AT1R have hydrophobic effects, D31, D33, Y37, F99, R101 of VL have hydrophobic effects, T30, Y31, W32, N34, Q49, F51, S54, Y58 of VL have hydrophobic effects.

Through carrying out homologous modeling and molecular docking experiments on the sequencing result of the AT1-AA variable region, the AT1-AA can form hydrogen bond and hydrophobic interaction with AT1R, and the sequence effectiveness is proved, so that a theoretical basis is provided for subsequent batch production of AT1-AA according to the sequence.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

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.