Angiotensin II-1 receptor polypeptide or fusion and application thereof

Abstract

The embodiment of the invention discloses an angiotensin II-1 type receptor polypeptide or a fusion compound and application of the angiotensin II-1 type receptor polypeptide or the fusion compound. The invention provides an angiotensin type 1 receptor polypeptide or fusion, which is coated in a solid phase material (porous plate, magnetic beads, agarose particles, latex particles and the like) and is used for detecting an antibody aiming at an angiotensin II-1 receptor in the body of a patient suffering from diseases such as eclampsia and the like. The compound can also be used for blocking the combination of autoantibodies in the bodies of pregnant women and angiotensin II-1 type receptors, and treating various cardiovascular diseases such as dilated cardiomyopathy, just-gauge disease, heart failure and the like.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of biotechnology, and specifically to an angiotensin II-1 receptor polypeptide or fusion and its application, and more specifically to an angiotensin II-1 receptor (ATII-1R) polypeptide and its modified product, and its application in disease diagnosis and treatment. Background Art

[0002] Preeclampsia is a serious disease that occurs during pregnancy. A large number of experiments have confirmed that endothelial damage and dysfunction are the initiating factors of this series of lesions. Overactivation of angiotensin II-1 receptor (ATII-1R) is a typical pathological change of preeclampsia. The presence of an autoantibody (ATII-1R-AA) against ATII-1R in patients with preeclampsia may be the main cause of ATII-1R overactivation. This autoantibody can specifically recognize the extracellular second loop functional epitope peptide of ATII-1R, showing a receptor agonist-like effect, and is associated with a variety of pathological changes in preeclampsia, suggesting that the detection of this autoantibody in patients has a pathological diagnostic significance that cannot be ignored.

[0003] In 1999, Wallukat et al. first discovered ATII-1R autoantibodies in preeclampsia. This antibody specifically recognizes the extracellular region of ATII-1R and exerts a receptor agonist-like effect. Studies have confirmed that this autoantibody can act on vascular smooth muscle cells, causing increased expression of cell tissue factor, calcium influx, NADPH oxidase activation and cell proliferation, which may induce a variety of cardiovascular diseases. It has also been found that this autoantibody in the mother can cause increased permeability of the uterine endothelium, affect the development of the fetus, and even leave it with metabolic abnormalities in adulthood.

[0004] Detecting ATII-1R autoantibodies in pregnant women or patients can not only help doctors make pathological diagnoses of diseases such as preeclampsia, but also warn of possible negative impacts on fetal development.

[0005] At the same time, using drugs to block this autoantibody can reduce its damage to the blood vessels and uterine endothelial cells of pregnant women, inhibiting or treating the occurrence and development of the disease. Summary of the invention

[0006] To this end, an embodiment of the present invention provides an angiotensin II type 1 receptor polypeptide or fusion and applications thereof.

[0007] The first object of the present invention is to provide the use of such polypeptides and their human protein conjugates in disease diagnosis.

[0008] The second object of the present invention is to provide the use of such polypeptides or fatty acid modifications thereof or human albumin fusions in the treatment of diseases.

[0009] Specifically, a 96-well plate or other solid phase material is coated with a polypeptide-human albumin conjugate or polypeptide-human albumin fusion protein that can bind to the autoantibody ATII-1R-AA, and immunological methodologies such as indirect ELISA, chemiluminescence, double antigen sandwich method, gold-labeled test strips / fluorescent test strips are used to quantitatively or semi-quantitatively detect the concentration of autoantibody ATII-1R-AA in the patient's body.

[0010] According to a first aspect of an embodiment of the present invention, the present invention provides an angiotensin type 1 receptor polypeptide or fusion, comprising:

[0011] 1)IHRNVFFIENTNITVCAFHYESQNST

[0012] 2) IHRNVFFIENTNITVCAFHYESR (16 / 18 carbon palm fatty acid) NST;

[0013] 3) IHRNVFFIENTNITV;

[0014] 4) IHRNVFFIENTNITVCAFHYESR (16 / 18 carbon palm fatty acid);

[0015] 5)IHRNVFFIENTNITVCAFHYESQNST-people white.

[0016] Among them, the amino acid sequence of IHRNVFFIENTNITVCAFHYESQNST is shown in SEQ ID No. 1;

[0017] The amino acid sequence of IHRNVFFIENTNITVCAFHYKSRNST is shown in SEQ ID No. 2;

[0018] The amino acid sequence of IHRNVFFIENTNITV is shown in SEQ ID No.3.

[0019] According to a second aspect of an embodiment of the present invention, the present invention provides an antibody, wherein the angiotensin type 1 receptor polypeptide or fusion as described above is coated on a solid phase material.

[0020] Furthermore, the solid phase material is a porous plate, magnetic beads, agarose particles, or latex particles.

[0021] According to a third aspect of an embodiment of the present invention, the present invention provides use of the above-mentioned antibody in the detection of eclampsia.

[0022] According to a fourth aspect of an embodiment of the present invention, the present invention provides the use of angiotensin type 1 receptor polypeptide or fusion as described above, wherein the polypeptide or fusion blocks the binding of autoantibodies in pregnant women to angiotensin II type 1 receptor, and is used to treat cardiovascular diseases.

[0023] Furthermore, the cardiovascular diseases include dilated cardiomyopathy, Chagas disease and heart failure.

[0024] The embodiments of the present invention have the following advantages:

[0025] The present invention provides a class of polypeptides and their use in the diagnosis and treatment of diseases such as eclampsia. The present invention provides an angiotensin II-1 receptor polypeptide or fusion, which is coated on a solid phase material (multi-well plate, magnetic beads, agarose particles, latex particles, etc.) for detecting antibodies against angiotensin II-1 receptor in patients with diseases such as eclampsia; it can also be used to block the binding of autoantibodies in pregnant women with angiotensin II-1 receptors, and treat various cardiovascular diseases such as dilated cardiomyopathy, Chagas disease, and heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0027] Figure 1 The ATII-1R extracellular region protein sequence alignment of human, monkey, rat and mouse provided in the embodiments of the present invention;

[0028] Figure 2 This is the recombinant ATII-1R extracellular region-human albumin fusion protein SDS-PAGE gel provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0029] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Abbreviations and definitions

[0031] "Polypeptide" refers to any form of a polypeptide that exhibits a desired biological activity (e.g., binding to an antibody or inhibiting the binding of an antibody to its antigen). Therefore, the polypeptide is used in the broadest sense and explicitly includes, but is not limited to, polypeptides, coupled polypeptides, modified polypeptides, and polypeptide fusion proteins.

[0032] As used herein, "detect" or "diagnosis" includes discovery, concomitant diagnosis, and pathology associated with a disease.

[0033] As used herein, "inhibit" or "treat" or "treatment" includes delaying the development of symptoms associated with a disease and / or alleviating the severity of these symptoms that will or are expected to develop in the disease. The term also includes alleviating existing symptoms, preventing additional symptoms, and alleviating or preventing the underlying causes of these symptoms.

[0034] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0035] Example 1. Peptide synthesis, modification and coupling

[0036] In this case, the human ATII-1R extracellular region was used as the target protein, and its sequence was compared with the homologous proteins of rat and monkey. Figure 1 As shown, it comes from the angiotensin II type-1 receptor, which is highly conserved and almost consistent among different species. The following is the comparison result between human (Homo), monkey (Macaca), rat (Rattus), mouse (Mouse) and the sequence.

[0037] A polypeptide sequence was selected from it, the sequence is as follows:

[0038] 1)IHRNVFFIENTNITVCAFHYESQNST;

[0039] 2) IHRNVFFIENTNITV;

[0040] 3)IHRNVFFIENTNITVCAFHYESRNST.

[0041] The two polypeptides are prepared by chemical synthesis, and the purity is required to be greater than 98%. The two polypeptides are respectively coupled with human albumin to prepare antigens for ATII-1R-AA autoantibody detection.

[0042] The side chain fatty acid of polypeptide sequence 3 is modified by chemical synthesis method, and the purity is required to be greater than 99%, so as to be used as ATII-1R-AA autoantibody blocker.

[0043] Example 2: Synthesis, expression and purification of recombinant polypeptide-human leukemia fusion gene

[0044] 1. The sequences of the fully synthesized human ATII-1R extracellular region fragment and human albumin fusion protein are as follows:

[0045] IHRNVFFIENTNITVCAFHYESQNST-GGGGSGGGS Human White.

[0046] 2. E. coli soluble expression of the above fusion protein

[0047] The fusion protein was purified by HPLC.

[0048] 3. Purify to obtain high purity protein such as Figure 2 shown.

[0049] Example 3: Detection of ATII-1R Antibody by Polypeptide Coating ELISA

[0050] 1. Experimental Materials

[0051] 1. IHRNVFFIENTNITVCAFHYESQNST-GGGGSGGGS human white;

[0052] 2. Serum from immunized mice;

[0053] 3. Patient serum.

[0054] 2. Experimental Results

[0055] IHRNVFFIENTNITVCAFHYESQNST-GGGGSGGGS Human white coated, mouse tail blood or patient serum was diluted with 1% BSA-PBS sample diluent, incubated for 2 hours, washed, and then the corresponding HRP enzyme-labeled secondary antibody was added for color development. The reaction results of different samples with coupled HSA at different dilution multiples are shown in Tables 1 and 2.

[0056] Table 1

[0057]

[0058] Table 2

[0059]

[0060] The results showed that the polypeptide conjugate could detect ATII-1R-AA autoantibodies in the patient's serum.

[0061] Example 4: Modified polypeptide inhibits the killing effect of autoantibodies on vascular endothelial cells

[0062] 1. Experimental Materials

[0063] 1. Rat embryonic cardiomyocytes H9C2;

[0064] 2. ATII-1R autoantibodies immunoaffinity purified from patient sera;

[0065] 3. Peptide modifications.

[0066] 2. Experimental Results

[0067] As shown in Table 3, ATII-1R autoantibodies can induce the death of rat embryonic cardiomyocytes, while peptide modifications can inhibit the killing effect of ATII-1R autoantibodies on cardiomyocytes.

[0068] Table 3

[0069] blank ATII-1R-AA Peptide Modifications ATII-1R-AA+ peptide modification Cell death rate 10.00％ 35％ 12％ 10％

[0070] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

[0071] Sequence Listing

[0072] <110> Beijing Tiancheng Biotechnology Co., Ltd.

[0073] <120> Angiotensin II type 1 receptor polypeptide or fusion and its application

[0074] <130> GG241401370A

[0075] <160> 3

[0076] <170> PatentIn version 3.5

[0077] <210> 1

[0078] <211> 26

[0079] <212> PRT

[0080] <213> Artificial sequence

[0081] <400> 1

[0082] Ile His Arg Asn Val Phe Phe Ile Glu Asn Thr Asn Ile Thr Val Cys 1 5 10 15 Ala Phe His Tyr Glu Ser Gln Asn Ser Thr 20 25

[0084] <210>2

[0085] <211>26

[0086] <212>PRT

[0087] <213>Artificial Sequence

[0088] <400>2

[0089] Ile His Arg Asn Val Phe Phe Ile Glu Asn Thr Asn Ile Thr Val Cys 1 5 10 15 Ala Phe His Tyr Lys Ser Arg Asn Ser Thr 20 25

[0091] <210>3

[0092] <211>15

[0093] <212>PRT

[0094] <213>Artificial Sequence

[0095] <400>3

[0096] Ile His Arg Asn Val Phe Phe Ile Glu Asn Thr Asn Ile Thr Val 1 5 10 15。

Claims

1. An angiotensin II type 1 receptor polypeptide or fusion, characterized in that: include: 1)IHRNVFFIENTNITVCAFHYESQNST 2) IHRNVFFIENTNITVCAFHYESR (16 / 18 carbon palm fatty acid) NST; 3) IHRNVFFIENTNITV; 4) IHRNVFFIENTNITVCAFHYESR (16 / 18 carbon palm fatty acid); 5)IHRNVFFIENTNITVCAFHYESQNST-people white.

2. An antibody, characterized in that The angiotensin II type 1 receptor polypeptide or fusion product as claimed in claim 1 is coated on a solid phase material.

3. The antibody according to claim 2, characterized in that The solid phase material is a porous plate, magnetic beads, agarose particles, and latex particles.

4. Use of the antibody according to claim 2 in the detection of eclampsia.

5. The use of the angiotensin II type 1 receptor polypeptide or fusion according to claim 1, characterized in that: The angiotensin type 1 receptor polypeptide or fusion blocks the binding of autoantibodies in pregnant women with angiotensin type 1 receptors, and is used for treating cardiovascular diseases.

6. The use according to claim 5, characterized in that: The cardiovascular diseases include dilated cardiomyopathy, Chagas disease and heart failure.

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