AGTR1 transmembrane proteins in GPCRs protein family and their use in treatment of diseases caused by susceptible respiratory bacteria or viruses

Abstract

The invention discloses AGTR1 transmembrane protein in a GPCRs protein family and application of the AGTR1 transmembrane protein in treatment of diseases caused by easily spreading respiratory system bacteria or viruses, belongs to the technical field of molecular biology and protein engineering, and aims to solve the technical problems of low expression level and unstable structure of AGTR1 protein obtained by expression in the prior art. An amino acid sequence of natural AGTR1 protein is analyzed to obtain a transmembrane region sequence, hydrophobic amino acid in the transmembrane region sequence is selectively replaced with hydrophilic amino acid based on the similarity between chemical structures and electron density maps of amino acid, and the high-water-solubility AGTR1 transmembrane protein is obtained. The AGTR1 transmembrane protein can also be applied to preparation of products for screening, treating or preventing cardiovascular diseases or respiratory diseases. On the premise of not changing the natural structure and function of the AGTR1, the solubility of the AGTR1 transmembrane protein can be remarkably improved, the AGTR1 transmembrane protein can stably exist in an aqueous solution, and efficient expression and purification are achieved through a prokaryotic expression means.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and protein engineering technology, and relates to an AGTR1 transmembrane protein, in particular to an AGTR1 transmembrane protein in the GPCRs protein family with seven transmembrane spans and its application in treating diseases caused by easily transmitted respiratory bacteria or viruses. Background Art

[0002] In the biomedical field, G protein-coupled receptors (GPCRs) are an important family of membrane protein receptors with seven transmembrane α-helices, found throughout various tissues and cells of the human body. Currently, over 800 GPCRs are known to play a diverse role in the body, participating in nearly all cellular signaling pathways, including vision, smell, taste, hormone regulation, neurotransmission, and immune system control. GPCRs are also a key target in drug development, with approximately 40% of modern drugs designed to treat diseases such as cardiovascular disease, neurological disorders, endocrine diseases, asthma, diabetes, and cancer. From the perspective of drug development, GPCRs have three unique advantages: 1) Clear structure-activity relationship: their ligand-binding pocket is located in the hydrophobic cavity formed by the transmembrane helix, and drug selectivity can be optimized through structure guidance (such as the different selectivity of β-blockers for β1 / β2 subtypes); 2) Signal transduction diversity: a single receptor can simultaneously activate G protein-dependent pathways and β-arrestin-mediated non-classical pathways, providing the possibility for the development of biased agonists; 3) Membrane surface accessibility: compared with intracellular targets, the extracellular domains of GPCRs are more easily targeted by small molecules or antibody drugs.

[0003] Angiotensin II receptor 1 (AGTR1), a specific G protein-coupled receptor, is involved in physiological processes such as regulating blood pressure and cell proliferation. It plays a crucial role in cardiovascular, cerebrovascular, and pulmonary diseases, and therefore holds significant clinical significance. As a member of the secretin receptor subfamily, its N-terminal extracellular domain contains conserved glycosylation sites (Asn4 and Asn176), which play a key role in ligand binding specificity. Due to its important physiological functions, successful expression of active AGTR1 protein is crucial for studying its mechanism of action and developing new drugs. However, AGTR1 is a typical transmembrane protein that is difficult to express. Its inherent properties (such as low thermal stability (Tm value of only 42°C) and conformational heterogeneity (dynamic equilibrium between active and inactive states) significantly limit the study of its functions and applications due to its complex structure and restricted expression.

[0004] Angiotensin II receptor 1 (AGTR1) is a GPCR widely expressed in organs and tissues, including the heart, kidneys, blood vessels, and lungs. It binds to its ligand, angiotensin II (Ang II), and serves as the primary effector of the renin-angiotensin system (RAS). Single-cell sequencing data show that AGTR1 expression in alveolar type II epithelial cells is 3-5 times higher than that of other GPCRs. In mature tissues, most Ang II exerts its effects through the AGTR1 receptor, triggering multiple signaling pathways, including: 1) the Gq protein-PLCβ pathway, which triggers IP3-mediated calcium release (at concentrations up to 1.2 μM), promoting vascular smooth muscle contraction; 2) β-arrestin-biased signaling, which activates ERK1 / 2 phosphorylation (phosphorylation levels increase 4.7-fold), driving cell proliferation; and 3) NADPH oxidase activation, which generates superoxide anions (at a rate of up to 12 nmol / min / mg), leading to oxidative stress damage. It also participates in biological effects such as vasoconstriction, cell growth and proliferation, and water-salt balance regulation. Studies have found that AGTR1 is closely associated with cardiovascular disease, hypertension, kidney disease, and diabetes. Genome-wide association studies (GWAS) have confirmed that the AGTR1 gene A1166C polymorphism increases the risk of hypertension by 2.3-fold. Therefore, the AGTR1 receptor and its signaling pathway have become important targets for drug research. Some drugs for hypertension and cardiovascular diseases achieve their therapeutic effects by regulating the AGTR1 receptor. For example, angiotensin II receptor blockers (ARBs) treat diseases such as hypertension and heart failure by blocking the activity of AGTR1.In recent years, studies have also shown that AGTR1 also plays an important role in a variety of lung diseases, especially those caused by easily transmitted respiratory viruses or bacteria, such as lung diseases: 1) Viral pneumonia: SARS-CoV-2 infection leads to Ang in lung tissue 1) Asthma: 1) The AGTR1 antagonist losartan reduced airway smooth muscle thickness by 41% and downregulated TGF-β1 expression by 67% in mice. 2) Asthma: 1) The AGTR1 antagonist losartan reduced airway smooth muscle thickness by 41% and downregulated TGF-β1 expression by 67% in mice. 3) Idiopathic pulmonary fibrosis: AGTR1 inhibitors combined with pirfenidone reduced the annual rate of decline in FVC from 230 mL to 98 mL (HR=0.49, p<0.001). 4) Lung cancer microenvironment regulation: AGTR1-overexpressing tumor-associated macrophages secreted 2.8-fold more VEGF, promoting A549 cell invasion (migration distance increased from 200 μm to 520 μm). 5) Airway inflammatory diseases (such as asthma, leukopenia, and chronic obstructive pulmonary disease): Angiotensin II promotes airway inflammatory responses, including increasing leukocyte adhesion, promoting cytokine release, and regulating inflammatory signaling pathways. In addition, elevated AGTR1 expression levels have been observed in diseases such as pulmonary hypertension. Therefore, AGTR1 can be considered a potential target for the treatment of lung diseases, and inhibiting its activity can treat or alleviate related lung diseases.

[0005] The AGTR1 protein expressed using existing technologies has disadvantages such as low expression level, unstable structure, loss of functional activity and high cost, and cannot meet the needs of related research and applications. Summary of the Invention

[0006] The purpose of the present invention is to solve the technical problems of low expression level and unstable structure of AGTR1 protein expressed by the prior art, provide an AGTR1 transmembrane protein in the GPCRs protein family and its application in treating diseases caused by easily transmitted respiratory bacteria or viruses, replace one or more hydrophobic amino acids in the transmembrane region of AGTR1 with hydrophilic amino acids, and provide a sequence that can be used to successfully express the AGTR1 transmembrane protein without changing the basic structure and function of AGTR1. This sequence overcomes the shortcomings of traditional expression systems and can stably express and prepare high-purity AGTR1 transmembrane protein.

[0007] In order to achieve the above-mentioned purpose, the present invention specifically adopts the following technical solutions: The AGTR1 transmembrane protein in the GPCRS protein family is prepared by selectively replacing the hydrophobic amino acids in the transmembrane region sequence of the amino acid sequence of the natural AGTR1 protein with hydrophilic amino acids.

[0008] In the technical solution of the present application, the amino acid sequence of the natural AGTR1 protein is analyzed to obtain the transmembrane region sequence, and then based on the similarity between the chemical structures and electron density maps of the amino acids, the hydrophobic amino acids in the transmembrane region sequence are selectively replaced with hydrophilic amino acids to obtain a highly water-soluble AGTR1 transmembrane protein that can be successfully expressed.

[0009] After analysis, it was found that compared with the natural AGTR1 protein, although the optimized AGTR1 transmembrane protein underwent amino acid substitution in the transmembrane region, its isoelectric point pI and molecular weight MW did not change significantly; in terms of hydrophobicity, the transmembrane region of the natural AGTR1 protein is highly hydrophobic, while the transmembrane region and the overall hydrophilicity of the optimized AGTR1 are greatly improved, and the existence of the transmembrane region of the optimized AGTR1 can no longer be predicted.

[0010] Analysis revealed that the optimized AGTR1 transmembrane protein still possesses seven α-helices, with a predicted confidence level exceeding 90%. The two proteins are structurally very similar, with a particularly high degree of overlap in the seven transmembrane α-helices. Ligand docking experiments with both the optimized and native AGTR1 proteins and the ligand Ang II revealed that both proteins were able to dock with Ang II, with the optimized AGTR1 exhibiting lower binding energy and stronger binding to Ang II.

[0011] Furthermore, the hydrophobic amino acids include one or more of tryptophan Trp, phenylalanine Phe, valine Val, leucine Leu, isoleucine Ile, alanine Ala, proline Pro and methionine Me; and / or The hydrophilic amino acids include one or more of glycine Gly, serine Ser, threonine Thr, cysteine ​​Cys, tyrosine Tyr, aspartic acid Asp and glutamic acid Glu.

[0012] Furthermore, the ratio of hydrophobic amino acids selectively replaced in the transmembrane region sequence is 10%-100%, for example, the replacement ratio can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.

[0013] Furthermore, the amino acid sequence of the AGTR1 transmembrane protein is selected from: (a) the amino acid sequence shown in SEQ ID NO: 2; or (b) An amino acid sequence derived from the amino acid sequence defined in (a) by substitution and / or deletion and / or addition of one or more amino acid residues, and having the same function as the amino acid sequence defined in (a).

[0014] A nucleotide encoding the AGTR1 transmembrane protein in the above-mentioned GPCRS protein family, wherein the nucleotide sequence of the nucleotide is selected from: (c) the nucleotide sequence shown in SEQ ID NO: 1; or (d) A nucleotide sequence that has at least 69% similarity to the nucleotide sequence defined in (c) and encodes a protein with the same function.

[0015] It is understood that (d) is a nucleotide sequence that has at least 69% similarity to the nucleotide sequence defined in (c) and encodes a protein with the same function, wherein (d) is a nucleotide sequence that has at least 69%, 70%, 73%, 75%, 76%, 78%, 80%, 81%, 83%, 85%, 87%, 88%, 89%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 99.9% similarity to the nucleotide sequence defined in (c) and encodes a protein with the same function; An expression vector comprising the above-mentioned nucleotides; the restriction enzyme cutting site of the expression vector is selected from a combination of one or more of NdeI, BamHI, and XhoI; and the expression vector is selected from a combination of one or more of DNA, RNA, viral vector, plasmid, and transposon.

[0016] In the technical solution of this application, an expression vector is constructed based on the optimized AGTR1 transmembrane protein sequence. Enzyme cleavage sites are selected, and the optimized AGTR1 gene sequence is synthesized, followed by enzymatic cleavage, ligation with a vector, and transformation into host cells. Agarose gel electrophoresis is used to determine the transformation status, and those with the target bands are preserved and sequenced. Sequence alignment confirms that the sequencing results are correct, indicating successful construction of the expression vector.

[0017] Furthermore, the expression vector is preferably a plasmid.

[0018] Furthermore, the expression vector is a pET-N-His-C-His vector.

[0019] A host cell containing the above-mentioned nucleotide or the above-mentioned expression vector.

[0020] In the technical solution of the present application, the constructed expression vector was transformed into an expression host cell for expression, and SDS-PAGE and Western blot were used to verify whether the protein was expressed. The results showed that the correct bands appeared after induced expression, while no obvious bands were seen in the empty control group. The expressed optimized AGTR1 protein was purified using a protein purifier, and the purification was identified by SDS-PAGE. Purity analysis was performed to identify the purity of the optimized AGTR1 protein, which reached more than 90%. Circular dichroism spectroscopy was performed on the expressed optimized AGTR1 protein, and the characteristic absorption peak formed by the α-helix during the transition process appeared, indicating that the α-helix structure was successfully formed, with a structure similar to that of natural AGTR1.

[0021] Cell viability and toxicity tests were performed on the expressed optimized AGTR1 protein, which showed that the optimized AGTR1 protein at different concentrations did not exhibit dose-dependent cytotoxicity reactions and did not inhibit the normal growth of cells, which is the basis for further functional development.

[0022] Furthermore, the host cells include Escherichia coli, yeast cells, mammalian cells, plant cells, etc.

[0023] Furthermore, the host cell is a DH5α Escherichia coli competent cell or a BL21 (DE3) plysS Escherichia coli competent cell.

[0024] A biomaterial, wherein the biomaterial is selected from one of the following (A) to (E), (A) the aforementioned nucleotide; (B) an expression cassette, a recombinant vector, a recombinant primary cell, or a recombinant cell line containing the nucleotide described in (A); (C) the AGTR1 transmembrane protein in the aforementioned GPCRS protein family; (D) the aforementioned expression vector; and (E) the aforementioned host cell.

[0025] A pharmaceutical composition comprising the AGTR1 transmembrane protein in the above-mentioned GPCRS protein family and one or more pharmaceutically acceptable carriers.

[0026] The above-mentioned AGTR1 transmembrane protein in the GPCRS protein family; and / or the above-mentioned expression vector; and / or the above-mentioned host cell; and / or the above-mentioned biomaterial; and the use of the above-mentioned pharmaceutical composition in the preparation of products for screening, treating or preventing cardiovascular diseases or respiratory diseases.

[0027] Furthermore, the respiratory disease is caused by easily transmissible respiratory bacteria or viruses.

[0028] Furthermore, the lung disease is asthma, white lung, and acute respiratory distress syndrome.

[0029] The beneficial effects of the present invention are as follows: 1. The present invention provides an optimized AGTR1 expression sequence and related technical means, which significantly improve the solubility of AGTR1 without changing its native structure and function, allowing it to exist stably in aqueous solution; the optimized AGTR1 protein is successfully expressed and purified through prokaryotic expression, providing an important tool and platform for studying its structure, function, and drug screening.

[0030] 2. Compared with natural AGTR1, the optimized AGTR1 transmembrane protein of the present invention has undergone amino acid substitution in the transmembrane region, and its spatial structure, isoelectric point pI and molecular weight MW have not changed significantly, but it can still be successfully expressed. Its hydrophilicity has been greatly improved, the existence of the transmembrane region can no longer be predicted, and the binding to the ligand Ang II is stronger. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a hydrophilicity prediction diagram for the optimized AGTR1 and native AGTR1. The hydrophobicity prediction results for both show that the scores for the seven transmembrane regions of native AGTR1 are all positive, indicating strong hydrophobicity, while the scores for the seven transmembrane regions of the optimized AGTR1 are significantly negative, indicating that the transmembrane regions and the receptor as a whole have become hydrophilic. Figure 2 This is a comparison of the spatial structures of the optimized AGTR1 of the present invention and natural AGTR1. The predicted spatial structure of the optimized AGTR1 still has seven α-helices. Comparison of the spatial structure with natural AGTR1 reveals that the two are very similar in structure, especially in the seven transmembrane α-helices. Figure 3 This is a molecular docking comparison diagram of the optimized AGTR1 of the present invention and native AGTR1. To explore whether the optimization process affects the binding of AGTR1 to the ligand, the optimized AGTR1 and native AGTR1 were molecularly docked with the ligand Ang II. The results showed that both can complete docking with Ang II. The binding energy of native AGTR1 to Ang II is -6.2 kcal / mol, while the binding energy of optimized AGTR1 to Ang II is -7.0 kcal / mol, indicating a stronger binding ability, indicating that this structure can be used as a new drug target. Figure 4 This is a diagram of the expression vector construction structure. The pET-N-His-C-His vector sequence was selected for prokaryotic expression. Two sets of different restriction sites, NdeI-XhoI and BamHI-XhoI, were also selected to determine the number of His tags for subsequent protein purification. Figure 5This is the electrophoresis result of the optimized AGTR1 protein expressed in E. coli. Protein expression was verified by staining the SDS-PAGE gel. A distinct band above 40 kDa was observed in the induced expression group, while no distinct band was observed in the empty control group, indicating normal protein expression. Figure 6 This is the electrophoresis result of the purified AGTR1 protein after expression optimization. The optimized AGTR1 protein was purified by affinity chromatography using a protein purifier. The purification status was verified by SDS-PAGE, and then the purity was analyzed using ImageJ software, showing that the purity of the purified protein reached 98.02%; Figure 7 This is the circular dichroism spectrum of the optimized AGTR1 protein. It shows a positive peak at 192-194nm and two negative peaks at 210-212nm and 224-226nm. These are characteristic absorption peaks formed during the α-helix transition process, indicating that the optimized AGTR1 protein has successfully formed an α-helical structure. Figure 8 The images show the AGTR1 protein lyophilized powder and solution after expression optimization. The expressed protein was frozen solid in a sterile environment, then vacuumed to sublimate and dry the water to form a lyophilized powder. Sterile water was added to reconstitute the protein solution in a sterile laboratory environment. Figure 9 This is a cell viability assay of the optimized AGTR1 protein. It shows that the optimized AGTR1 exhibited no cytotoxicity to cells at different concentrations (0.1, 1, 5, 10, 20, and 50 μg / mL), demonstrating good biocompatibility, which is the basis for further functional development. Figure 10 The optimized AGTR1 protein shows qPCR detection results for inflammatory factors in a cell-based inflammation model. This shows that the optimized AGTR1 protein can significantly reduce the levels of TNF-α and IL-6 inflammatory factors in the cell-based inflammation model, demonstrating a positive inhibitory effect on inflammation and potentially serving as a potential therapeutic for related lung inflammatory diseases. DETAILED DESCRIPTION

[0032] 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 in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0033] Therefore, based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work shall fall within the scope of protection of the present invention.

[0034] Example 1 This example is used to perform sequence design and optimization on the natural AGTR1 protein. The specific method is as follows: The natural AGTR1 protein (UniProtKBID: P30556) was searched in the UniProt (Universal Protein) protein database. The transmembrane α-helix region in the natural AGTR1 protein sequence was optimized and explored, and the hydrophobic amino acids in the transmembrane region were replaced with hydrophilic amino acids. Multiple optimized variants were obtained based on the different amino acid replacement ratios in the transmembrane region. These optimized variants were analyzed and compared with the natural AGTR1, and the best optimized AGTR1 transmembrane protein was selected.

[0035] Based on the nucleic acid and protein sequences of natural AGTR1, the transmembrane region sequence was analyzed. The hydrophobic amino acids in the transmembrane region were selectively replaced with hydrophilic amino acids as follows.

[0036] The hydrophobic amino acids include one or more of tryptophan (Trp), phenylalanine (Phe), valine (Val), leucine (Leu), isoleucine (Ile), alanine (Ala), proline (Pro) and methionine (Met), and the hydrophilic amino acids include one or more of glycine (Gly), serine (Ser), threonine (Thr), cysteine ​​(Cys), tyrosine (Tyr), aspartic acid (Asp) and glutamic acid (Glu).

[0037] The amino acids in the transmembrane region are replaced one by one at a replacement ratio of 10%-100%, that is, until all replacements are completed, they are analyzed by computer, and finally one of the results is selected as a representative between 10%-100% as the optimized AGTR1 transmembrane protein sequence.

[0038] The optimized amino acid sequence of the AGTR1 transmembrane protein is selected from: (a) the amino acid sequence shown in SEQ ID NO: 2; or (b) An amino acid sequence derived from the amino acid sequence defined in (a) by substitution and / or deletion and / or addition of one or more amino acid residues, and having the same function as the amino acid sequence defined in (a).

[0039] The nucleotide sequence encoding the optimized AGTR1 transmembrane protein is selected from: (c) the nucleotide sequence shown in SEQ ID NO: 1; or (d) A nucleotide sequence that has at least 69% similarity to the nucleotide sequence defined in (c) and encodes a protein with the same function.

[0040] Example 2 This example is used to perform bioinformatics analysis on the native AGTR1 protein and the optimized AGTR1 transmembrane protein, specifically: The above-mentioned Example 1 was systematically optimized and the following predictions and analyses were performed: (1) Protein hydrophobicity prediction: The hydrophobicity score was calculated based on the optimized AGTR1 protein sequence using the ProtScale tool from Expasy. (2) Protein spatial structure prediction: Use AlphaFold3 to predict the spatial structure of the optimized AGTR1 protein; (3) Protein spatial structure comparison: SWISS-MODEL was used to compare the protein spatial structure similarity between natural AGTR1 and optimized AGTR1; (4) Protein molecular docking: Based on the spatial structure of natural AGTR1 and optimized AGTR1 proteins, molecular docking with its ligand AngII was performed.

[0041] The above-mentioned systematic analysis was performed on the optimized AGTR1 proteins with different replacement ratios, and finally one of the results was selected as a representative, that is, the nucleotide sequence corresponding to the optimized AGTR1 transmembrane protein is shown in SEQ ID NO: 1, and the protein sequence of the optimized AGTR1 transmembrane protein is shown in SEQ ID NO: 2. The results showed that, as shown in Table 1, the optimized AGTR1 underwent 46.71% amino acid substitutions in the transmembrane region and 22.01% substitutions in the entire amino acid sequence, but its pI and MW did not change significantly. The pI of AGTR1 was 9.43 and the MW (kDa) was 41.06, while the pI of the optimized AGTR1 was 9.29 and the MW (kDa) was 41.49. The transmembrane region of the natural AGTR1 protein is highly hydrophobic, while the transmembrane region and the overall hydrophilicity of the optimized AGTR1 are significantly improved. The two are very similar in structure, especially in the 7 transmembrane α-helical structure. Both can complete docking with the ligand Ang II. The binding energy of natural AGTR1 to Ang II is -6.2 kcal / mol, while the binding energy of the optimized AGTR1 transmembrane protein to Ang II is -7.0 kcal / mol, and a stronger binding capacity, indicating that the structure can be used as a new target in screening drugs for the treatment or prevention of diseases such as cardiovascular disease or lung disease.

[0042] Table 1 Basic information comparison between optimized AGTR1 and natural AGTR1

[0043] Example 3 This example is used to enzymatically digest the optimized prokaryotic expression vector of the AGTR1 transmembrane protein, specifically: (1) The pET-N-His-C-His plasmid was selected as the prokaryotic expression vector, which contains a 6*His tag at the N-terminus and the C-terminus for subsequent protein purification. Two sets of enzyme cleavage sites, NdeI-XhoI and BamHI-XhoI, were selected to determine the number of different His tags. The enzyme cleavage steps are as follows: 1) Prepare the following reaction system in a clean 200 μl EP tube in the following order:

[0044] 2) Mix well and centrifuge to the bottom of the tube, then incubate at 37°C for 15 min; 3) Take 5 μL of the reaction product after enzyme digestion and perform 1% agarose gel electrophoresis at a voltage of 100 V for 40 min, and expose the gel under a gel exposure instrument.

[0045] (2) The gel block containing the optimized AGTR1 destination vector [the nucleotide sequence of AGTR1 is shown in SEQ ID NO: 1, and the corresponding optimized protein sequence of AGTR1 is shown in SEQ ID NO: 2] is recovered using a gel recovery kit. The recovery steps are as follows: 1) Add 500 μL of equilibration solution to the adsorption column CB2 (placed in the collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube; 2) Cut the single target DNA band from the agarose gel (try to remove the excess) and place it in a clean centrifuge tube and weigh it; 3) Add an equal volume of PC solution to the gel block and place in a 50°C water bath for about 10 minutes, gently turning the centrifuge tube upside down to ensure that the gel block is fully dissolved. 4) Add the solution obtained in the previous step to the adsorption column CB2 (place the adsorption column in the collection tube), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column CB2 in the collection tube; 5) Add 600 μL of rinse solution PW to the adsorption column CB2, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column CB2 in the collection tube; 6) Repeat step 5); 7) Place the adsorption column CB2 in a collection tube and centrifuge at 12,000 rpm for 2 minutes to remove as much of the rinse solution as possible. Allow the adsorption column to dry thoroughly at room temperature for 2-5 minutes. 8) Place the adsorption column CB2 in a clean centrifuge tube. Add an appropriate amount of elution buffer EB dropwise to the center of the adsorption membrane. Allow to stand at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 2 minutes to collect the DNA solution. Measure the concentration of the collected DNA solution and store at -20°C until further use.

[0046] The results showed that an optimized prokaryotic expression vector of AGTR1 was obtained, which can be used in various scenarios of this application.

[0047] Example 4 This example is used to connect the optimized prokaryotic expression vector of AGTR1 transmembrane protein, specifically: The linear optimized AGTR1 obtained above [the nucleotide sequence of AGTR1 is shown in SEQ ID NO: 1, and the corresponding optimized protein sequence of AGTR1 is shown in SEQ ID NO: 2] and the pET-N-His-C-His plasmid were ligated in the following steps: (1) Prepare the following reaction system in a clean 200 μl EP tube in the following order:

[0048] (2) Mix thoroughly and centrifuge to the bottom of the tube, then incubate at room temperature for 15 min; (3) Take 1-5 μL of the ligation product for subsequent transformation, and store the remaining ligation product at -20°C for future use.

[0049] The results showed that the optimized prokaryotic expression vector of AGTR1 was obtained, which can be used in various scenarios of the present application.

[0050] Example 5 This example is used to store the optimized prokaryotic expression vector of the AGTR1 transmembrane protein for host transformation, specifically: The ligation product obtained in Example 4 above was transformed into DH5α competent cells. The specific transformation steps are as follows: (1) Place DH5α competent cells on ice, add 5 μL of ligation product, mix well, and let stand on ice for 30 min; (2) Incubate the centrifuge tube at 42°C for 90 seconds, then quickly transfer the tube to ice and let it stand for 3 minutes; (3) Add 900 μL of antibiotic-free LB liquid medium, mix well, and incubate at 37°C in a shaker at 150 rpm for 45 min to revive the bacteria; (4) Take 200 μL of the revived bacterial solution and evenly spread it on LB solid medium (containing 50 μg / mL kanamycin), invert the plate and culture it in a bacterial incubator for 12-16 hours; (5) Observe the number, size and morphology of colonies.

[0051] The results showed that the optimized prokaryotic expression vector of AGTR1 [the nucleotide sequence of AGTR1 is shown in SEQ ID NO: 1, and the corresponding optimized protein sequence of AGTR1 is shown in SEQ ID NO: 2] can preserve host transformation. This vector can be used in various scenarios of this application.

[0052] Example 6 This example is used to verify the optimized prokaryotic expression vector of AGTR1 transmembrane protein, specifically: Perform colony PCR on the normally grown colonies to verify whether the band position consistent with the target gene (optimized AGTR1) appears. The colonies at the correct position are further sequenced to ensure that there are no base mutations. The specific steps are as follows: (1) The optimized AGTR1 colony PCR primer sequences are shown in the following table:

[0053] (2) Use a sterile pipette tip to pick up several transformed monoclonal colonies and prepare the following reaction system in a clean 200 μl EP tube in the following order:

[0054] (3) Perform PCR reaction according to the following amplification conditions:

[0055] (4) Take 5 μL of PCR product and perform 1% agarose gel electrophoresis at a voltage of 100 V for 40 min, and expose the gel under a gel exposure instrument.

[0056] (5) Pick a colony that matches the target band size and inoculate it into 10 mL of LB liquid medium (containing 50 μg / mL kanamycin) and culture it in a shaker at 37°C at 200 rpm for 12-14 h. (6) Take 5 ml of the expanded bacterial solution for sequencing, and store the remaining bacterial solution in a sterile centrifuge tube at a 1:1 ratio with 20% glycerol and store at -20°C until use.

[0057] The results showed that the optimized prokaryotic expression vector of AGTR1 [the nucleotide sequence of AGTR1 is shown in SEQ ID NO: 1, and the corresponding optimized protein sequence of AGTR1 is shown in SEQ ID NO: 2] was verified to be correct. This vector can be used in various scenarios of this application.

[0058] Example 7: This example is used to transform the optimized prokaryotic expression vector of AGTR1 transmembrane protein into an expression host, specifically: (1) The above-mentioned correctly sequenced strains were cultured and the plasmids were extracted using a plasmid extraction kit. The specific steps are as follows: 1) Add 500 μL of equilibration solution to the adsorption column CP3 (placed in the collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube; 2) Add 1-5 ml of overnight culture to a centrifuge tube and centrifuge at 12,000 rpm for 1 minute. Remove the supernatant as much as possible. 3) Add 250 μL of solution P1 to the centrifuge tube containing the bacterial pellet and thoroughly resuspend the bacterial pellet using a pipette or vortex oscillator. 4) Add 250 μL of solution P2 to the centrifuge tube and gently invert the tube 6-8 times to fully lyse the cells. 5) Add 350 μL of Solution P3 to the centrifuge tube and immediately and gently invert the tube 6-8 times to mix thoroughly. A white flocculent precipitate will appear. Centrifuge at 12,000 rpm for 10 minutes. 6) Transfer the supernatant collected in the previous step to the adsorption column CP3 (place the adsorption column in the collection tube), trying not to aspirate the precipitate. Centrifuge at 12,000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column CP3 in the collection tube. 7) Add 500 μL of deproteinized solution PD to the adsorption column CP3, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and return the adsorption column CP3 to the collection tube; 8) Add 600 μL of rinse solution PW to the adsorption column CP3, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column CP3 in the collection tube; 9) Repeat step 8); 10) Place the adsorption column CP3 into a collection tube and centrifuge at 12,000 rpm for 2 minutes to remove any remaining rinse solution. 11) Place the adsorption column CP3 in a clean centrifuge tube. Add 50-100 μl of elution buffer EB to the center of the adsorption membrane. Incubate at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 2 minutes to collect the plasmid solution into the centrifuge tube. Measure the concentration of the collected plasmid and store at -20°C until ready for use.

[0059] (2) Transform the extracted plasmid vector into the expression strain BL21 (DE3) plysS competent cells. The specific transformation steps are as follows: 1) Place BL21 (DE3) plysS competent cells on ice, add 5 μL of ligation product, mix well, and let stand on ice for 30 minutes; 2) Incubate the centrifuge tube at 42°C for 90 seconds, then quickly transfer the tube to ice and let it stand for 3 minutes; 3) Add 900 μL of antibiotic-free LB liquid medium, mix well, and incubate at 37°C in a shaker at 150 rpm for 45 min to revive the cells. 4) Spread 200 μL of the revived bacterial solution evenly onto LB solid medium (containing 50 μg / mL kanamycin), invert the plate, and incubate in a bacterial incubator for 12-16 hours. 5) Observe the number, size and morphology of colonies.

[0060] The results showed that the optimized prokaryotic expression vector of AGTR1 [the nucleotide sequence of AGTR1 is shown in SEQ ID NO: 1, and the corresponding optimized protein sequence of AGTR1 is shown in SEQ ID NO: 2] was successfully transformed into the expression host. This vector can be used in various scenarios of this application.

[0061] Example 8: This example is used for the initial induction expression of the optimized AGTR1 transmembrane protein, specifically: The optimized AGTR1 protein after successful transformation was initially expressed. The specific steps are as follows: (1) Pick a positive colony and inoculate it into 5 mL of LB liquid medium (50 μg / mL kanamycin), and culture it in a shaker at 37°C and 200 rpm overnight; (2) The overnight culture was inoculated into LB liquid medium (50 μg / mL kanamycin) at a ratio of 1:100, and cultured in a shaker at 37°C at 200 rpm for 2-3 hours. The OD600nm was monitored and detected. When it reached 0.6-0.8, it entered the logarithmic growth phase. (3) Add IPTG to a final concentration of 0.5 mM and incubate at 37°C in a shaker at 200 rpm for 4 h. A pET-N-His-C-His blank vector was used as the control group. (4) Collect the bacterial liquid from each group into a centrifuge tube and centrifuge at 12000 rpm for 5 minutes to collect the bacteria.

[0062] The results showed that the initial induced expression of the optimized AGTR1 was successful. The expression product can be used in various scenarios of this application.

[0063] Example 9; This example is used to identify the inducible expression of the optimized AGTR1 transmembrane protein, specifically: The optimized AGTR1 protein after induction expression was identified by SDS-PAGE. The specific steps are as follows: (1) Sample preparation: Add 1 mL of PBS buffer to the collected bacteria, shake vigorously with a vortexer to ensure that the precipitate at the bottom of the tube is dispersed, centrifuge at 12000 rpm for 1 min to collect the bacteria, and repeat washing 3 times. Add 80 μL of PBS buffer to resuspend, take 12 μL of sample and add 3 μL of protein loading buffer (5X), place in a 99.9℃ constant temperature metal bath for denaturation for 10 min, cool to room temperature, and store at -80℃ for later use; (2) Sample loading: Fix the prepared PAGE gel in the electrophoresis tank, fill it with electrophoresis solution, pull out the comb vertically, load the samples in the order of the labels, and add 5 μL of PageRuler™ prestained protein molecular weight standard at the same time; (3) Electrophoresis: Connect the positive and negative electrodes to the power supply, set the constant voltage to 80V for electrophoresis, and wait for the sample to leave the upper gel layer. Set the constant voltage to 120V for electrophoresis. When the bromophenol blue is electrophoresed to the bottom of the gel tank, turn off the power supply to stop electrophoresis. (4) Staining: After electrophoresis, disassemble the gel plate, rinse the gel with deionized water, add 20 mL of ultrafast staining solution, and stain on a decolorizing shaker for 60 minutes; (5) Decolorization: Recover the staining solution, add an appropriate amount of deionized water, and shake on a shaker to decolorize. Replace the deionized water every 20 minutes until the background becomes transparent. (6) Development: Place the gel into a luminescence imaging system and select “Coomassie Blue” for imaging.

[0064] The results showed that the induced expression identification of the optimized AGTR1 was consistent with expectations.

[0065] Example 10 This example is used for protein purification of the optimized AGTR1 transmembrane protein, specifically: (1) Disrupt the induced expression bacteria and collect the supernatant. The specific steps are as follows: 1) Collect all bacterial suspension into a 50 mL centrifuge tube and centrifuge at 10,000 rpm for 8 minutes to collect bacterial cells; 2) Add 10 mL of Tris buffer per 1 g of culture medium and disrupt the bacteria by ultrasonication on ice at 250 W for 3 seconds per sonication, with 5-second intervals for a total of 25 minutes. 3) After sonication, centrifuge at 10,000 rpm at 4°C for 20 min and collect the supernatant.

[0066] (2) Purify the collected protein supernatant in a protein purifier. The specific steps are as follows: 1) Sample preparation: The collected protein supernatant was centrifuged at 10,000 rpm for 10 min at 4°C. The collected supernatant was sterilized by filtration through a 0.22 μm disposable syringe filter. 2) Power on: Turn on the computer, collector, and protein purifier in sequence. Connect pump A to the protein Tris buffer and pump B to the protein eluent. Loosen the exhaust ports of pumps A and B, draw approximately 20 mL with a syringe, and tighten the screws to remove any bubbles. 3) Balance: First, balance pumps A and B at a 50% ratio for 1 minute at a flow rate of 1 mL / min to balance the initial stage of pump B. Then, balance pump A at 100% at a flow rate of 1 mL / min to fully balance the purification system. 4) Column loading: Connect the IMAC nickel column while the protein Tris buffer is flowing in pump A. After observing the UV curve of the collector to be flat and stable, stop pump A to load the sample; 5) Sample loading: Replace the A pump connection with the filtered protein supernatant sample and load the sample at 100% A pump and a flow rate of 0.5 mL / min. Collect the solution when the collector UV curve shows an upward trend and peaks, and mark it as the flow-through. After the filtered sample is loaded, replace the A pump connection with the protein Tris buffer. Do not generate bubbles. Observe the collector UV curve until it becomes flat and stable, and then stop pump A loading. 6) Washing impurities: Wash impurities with a 12% B pump and a 30% B pump at a flow rate of 1 mL / min. Collect the solution when the UV curve of the collector shows an upward trend and a peak, and mark it as the impurity washing solution; 7) Elution: Use the "Gradient" mode, 60% B pump, and a flow rate of 1 mL / min for elution. When the UV curve of the collector shows an upward trend and a peak, collect the solution and mark it as the eluate. 8) Cleaning: After the program is completed, clean the entire purification system including the nickel column with ddH2O and then with 20% ethanol. Observe the collector UV curve until it is flat and stable. 9) Turn off the protein purifier, collector, and computer in sequence; collect the flow-through, wash solution, and eluate for SDS-PAGE analysis.

[0067] Results showed that the optimized AGTR1 protein was purified and successfully obtained the GPCR transmembrane protein receptor structure [the nucleotide sequence of AGTR1 is shown in SEQ ID NO:1, and the corresponding optimized AGTR1 protein sequence is shown in SEQ ID NO:2]. The expression and preparation of this GPCR transmembrane protein receptor AGTR1 sequence protein and its structural compounds are intended to lay a solid foundation for its use as candidate innovative drugs to treat lung diseases caused by easily transmitted respiratory viruses or bacteria (including but not limited to asthma, white lung, acute respiratory distress syndrome, etc.), as well as for its other applications as new drug targets.

[0068] Example 11 This example is used to detect the cell viability and toxicity of the optimized AGTR1 transmembrane protein, specifically: The optimized AGTR1 protein was expressed and tested for normal lung cell viability and toxicity. The specific steps are as follows: (1) Normal lung cells were seeded into 96-well plates at a density of 5,000-10,000 cells per well; (2) The optimized AGTR1 protein was expressed and incubated with normal lung cells at 0 (control group), 0.1, 1, 5, 10, 20, and 50 μg / mL per well for 24-48 hours; (3) Aspirate the culture medium, wash the cells 2-3 times with PBS, and then add 100 μL of detection working solution to each well; (4) Incubate at 37°C in the dark for 30-60 min, detect using a fluorescence microplate reader, and compare the RFU (Relative fluorescence values) of different groups to determine the changes in the number of viable cells.

[0069] The results showed that the GPCR transmembrane protein receptor structure was successfully obtained and verified by AGTR1 function, including cytotoxicity. The verification of this structural compound laid a solid foundation for its subsequent use as a candidate innovative drug to treat lung diseases caused by easily transmitted respiratory viruses or bacteria (including but not limited to asthma, white lung, acute respiratory distress syndrome, etc.), as well as its other applications as a new drug target.

[0070] Example 12 This example is used to detect the inflammation-inhibiting effect of the optimized AGTR1 transmembrane protein, specifically: The optimized AGTR1 protein was expressed and tested for its inhibitory effect on human bronchial epithelial cells. The specific steps are as follows: (1) Cells were grouped and RNA was extracted. The specific steps are as follows: 1) Human bronchial epithelial cells were seeded in 6-well plates at 100,000 cells per well. An LPS-induced model group, an LPS+optimized AGTR1 protein model treatment group, and a control group were set up. 2) After 12 hours of culture, LPS mother solution was added to the LPS-induced model group to a final concentration of 1 μg / mL. LPS plus optimized AGTR1 protein was added to the LPS+optimized AGTR1 protein model treatment group to a final concentration of 50 μg / mL. Blank control medium was added to the control group. 3) After 24 hours of incubation, discard the culture medium, wash once with PBS, add 1 mL of RNA isolater, collect into an enzyme-free 1.5 mL centrifuge tube, repeatedly pipette until the cells are fully lysed, add 200 μL of chloroform, vortex for 20 seconds to form an emulsion, and let it stand at 4°C for 5 minutes; 4) Centrifuge at 12,000 rpm for 15 min at 4°C. Carefully aspirate 500 μL of the upper aqueous phase into a new centrifuge tube using a pipette. Add 500 μL of pre-chilled isopropanol and mix thoroughly. Incubate at 4°C for 10 min. 5) Centrifuge at 12,000 rpm for 10 min at 4°C, discard the supernatant, add 1 mL of 75% ethanol, and let stand at room temperature for 5 min; 6) Centrifuge at 12,000 rpm for 5 min at 4°C, discard the supernatant, dry in a clean bench for 10 min, and then add 30 μL of enzyme-free water to dissolve the precipitate; 7) Detect RNA concentration and purity using a UV spectrophotometer and store at -80°C to avoid degradation.

[0071] (2) Remove genomic DNA from the extracted RNA. The specific steps are as follows: 1) Prepare the following reaction system in a clean 200 μl EP tube in the following order:

[0072] 2) Mix well and centrifuge to the bottom of the tube, then incubate at 37°C for 30 min; 3) Add 1 μL of 50 mM EDTA to each tube and incubate at 65°C for 10 min.

[0073] (3) Reverse transcription of the extracted RNA into cDNA. The specific steps are as follows: 1) Prepare the following reaction system in a clean 200 μl EP tube in the following order:

[0074] 2) Close the lid tightly, mix well and centrifuge to the bottom of the tube; 3) Incubate at 42°C for 30 min, then incubate at 95°C for 5 min to terminate the reaction.

[0075] (4) Perform qPCR reaction using reverse transcribed cDNA. The specific steps are as follows: 1) qPCR primer sequences are shown in the following table:

[0076] 2) Prepare the following reaction system in a clean 200 μl EP tube in the following order:

[0077] 3) Perform qPCR reaction according to the following amplification conditions:

[0078] 4) GAPDH was used as an internal reference and the data were processed using the 2^(-ΔΔCT) method.

[0079] The results showed that the optimized AGTR1 receptor structure was successfully obtained and verified using a human bronchial epithelial cell inflammation model. qPCR detection can effectively reduce the inflammatory response, including the inflammatory factors TNF-α and IL-6, without affecting cell activity. This proves that the optimized AGTR1 can be used as a candidate innovative drug to treat lung diseases caused by easily transmitted respiratory viruses or bacteria (including but not limited to asthma, white lung, acute respiratory distress syndrome, etc.).

[0080] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

Claims

1. AGTR1 transmembrane protein in the GPCRs protein family, characterized by: The hydrophobic amino acids in the transmembrane region of the amino acid sequence of the natural AGTR1 protein are selectively replaced with hydrophilic amino acids.

2. The AGTR1 transmembrane protein in the GPCRs protein family according to claim 1, characterized in that: The hydrophobic amino acids include one or more of tryptophan Trp, phenylalanine Phe, valine Val, leucine Leu, isoleucine Ile, alanine Ala, proline Pro and methionine Me; and / or The hydrophilic amino acids include one or more of glycine Gly, serine Ser, threonine Thr, cysteine ​​Cys, tyrosine Tyr, aspartic acid Asp and glutamic acid Glu.

3. The AGTR1 transmembrane protein in the GPCRs protein family according to claim 1, characterized in that: The ratio of the hydrophobic amino acids in the transmembrane region sequence being selectively replaced is 10%-100%.

4. The AGTR1 transmembrane protein in the GPCRs protein family according to claim 1, characterized in that: The amino acid sequence of the AGTR1 transmembrane protein is selected from: (a) the amino acid sequence shown in SEQ ID NO: 2; or (b) An amino acid sequence derived from the amino acid sequence defined in (a) by substitution and / or deletion and / or addition of one or more amino acid residues, and having the same function as the amino acid sequence defined in (a).

5. A nucleotide encoding the AGTR1 transmembrane protein in the GPCRs protein family according to any one of claims 1 to 4, characterized in that: The nucleotide sequence of nucleotides is selected from the group consisting of: (c) the nucleotide sequence shown in SEQ ID NO: 1; or (d) A nucleotide sequence that has at least 69% similarity to the nucleotide sequence defined in (c) and encodes a protein with the same function.

6. An expression vector, characterized in that: The expression vector comprises the nucleotide according to claim 5; the restriction enzyme cutting site of the expression vector is selected from one or more combinations of NdeI, BamHI, and XhoI; the expression vector is selected from one or more combinations of DNA, RNA, viral vector, plasmid, and transposon.

7. A host cell, characterized in that: Containing the nucleotide according to claim 5 or the expression vector according to claim 6.

8. A biomaterial, characterized in that: The biological material is selected from one of the following (A) to (E), (A) the nucleotide according to claim 5; (B) an expression cassette, a recombinant vector, a recombinant primary cell, or a recombinant cell line containing the nucleotide described in (A); (C) the AGTR1 transmembrane protein in the GPCRs protein family according to any one of claims 1 to 4; (D) the expression vector according to claim 6; (E) the host cell according to claim 7.

9. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the AGTR1 transmembrane protein in the GPCRs protein family according to any one of claims 1 to 5 and one or more pharmaceutically acceptable carriers.

10. Use of the AGTR1 transmembrane protein in the GPCRs protein family according to any one of claims 1 to 4; and / or the nucleotide according to claim 5; and / or the expression vector according to claim 6; and / or the host cell according to claim 7; and / or the biomaterial according to claim 8; or the pharmaceutical composition according to claim 9 in the preparation of products for screening, treating or preventing cardiovascular diseases or respiratory diseases.

11. The use according to claim 10, characterized in that: The respiratory diseases are caused by easily transmissible respiratory bacteria or viruses.

12. The use according to claim 11, characterized in that: The lung diseases include asthma, white lung, and acute respiratory distress syndrome.

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