Rat taar8a mutants and uses thereof

Abstract

The application discloses a rat TAAR8a mutant and application thereof. Compared with a wild type rTAAR8a, the rTAAR8a mutant has a mutation at the 108th position and / or the 191st position. Compared with the wild type rTAAR8a, the rTAAR8a mutant of the application can enhance the activity of the rTAAR8a mutant in binding NH3 through the mutation of the above-mentioned positions.

Description

Technical Field

[0001] This application relates to the field of chemical detection technology, specifically to a rat TAAR8a mutant and its application, and more specifically to an rTAAR8a mutant, biomaterials, products and their uses, and methods for detecting ammonia. Background Technology

[0002] In the fields of environmental monitoring, animal husbandry, medical diagnosis, and biosensing, there are numerous volatile amines with varying physicochemical properties, release characteristics, and physiological / environmental impacts. Ammonia (NH3) is a highly representative gaseous substance, widely generated in animal metabolism, agricultural production, industrial emissions, and human physiological processes. Its trace detection and accurate identification have significant application value for environmental quality control, animal health monitoring, and early disease diagnosis. Excessive ammonia concentrations can cause environmental pollution and animal respiratory diseases. Furthermore, abnormal ammonia levels in human exhaled breath can serve as potential diagnostic markers for kidney dysfunction and gastrointestinal diseases. Therefore, achieving highly sensitive and specific identification of ammonia has significant practical implications and promising application prospects.

[0003] The recognition mechanism of volatile amines in mammals (such as rats) mainly involves the binding of amine molecules to specific olfactory receptors on olfactory sensory neurons in the olfactory epithelium of the nasal cavity. This activates the receptor-mediated G protein-coupled signaling pathway, converting the chemical signal into an electrical signal and transmitting it to the central nervous system, thus completing the odor perception process. It has been reported that rats possess 16 functional trace amine-associated receptors (TAARs), belonging to the G protein-coupled receptor (GPCR) superfamily. Except for TAAR1, the remaining TAARs are mainly distributed in the olfactory epithelium and are specifically responsible for sensing odor molecules such as volatile amines, playing a crucial role in innate olfactory recognition, environmental warning, and physiological metabolic regulation. The rat trace amine receptor TAAR8a, as an important member of the TAAR family, selectively recognizes ammonia (NH3) and can serve as a specific molecular target for ammonia recognition. It plays an important role in the development of ammonia trace detection sensors, the design of ammonia-responsive probes, and the development of auxiliary diagnostic reagents for related diseases. However, wild-type TAAR8a has a weak response to ammonia (NH3) and low sensitivity, and is easily interfered with by other volatile amines in the environment, which is not conducive to achieving efficient and accurate identification and detection of ammonia and limits its practical application in related fields. Summary of the Invention

[0004] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides an olfactory receptor rTAAR8a mutant and its uses.

[0005] This application is based on the inventor's following discoveries: Currently, researchers have confirmed that some members of the rat TAAR family have the ability to recognize volatile amines. Among them, TAAR8a has been found to specifically bind to ammonia. However, related research has only reached the level of basic binding verification of receptor and ligand, and the structure of TAAR8a has not been specifically modified to improve its response performance to ammonia.

[0006] This application is the first to use computer simulation combined with biological screening to precisely anchor the ligand binding pocket of rat TAAR8a (rTAAR8a) for ammonia (NH3) recognition, identify the key amino acid sites in the binding pocket involved in ammonia recognition, and perform site-specific modification of these key amino acids to enhance the response intensity of rTAAR8a to ammonia. This provides a new technical basis for the targeted modification of trace amine receptors.

[0007] Therefore, in a first aspect of this application, an rTAAR8a mutant is proposed. According to an embodiment of this application, compared with wild-type rTAAR8a, the rTAAR8a mutant has mutations at positions 108 and / or 191. Compared with wild-type rTAAR8a, the rTAAR8a mutant of this application, through mutations at the aforementioned sites, can improve its response intensity and signal output level to ammonia, thereby enhancing the activity of the rTAAR8a mutant in binding NH3.

[0008] In a second aspect of this application, a fusion protein is proposed. According to an embodiment of this application, the fusion protein comprises the rTAAR8a mutant described in the first aspect and a protein fragment, wherein the protein fragment is linked to the rTAAR8a mutant.

[0009] In a third aspect of this application, a nucleic acid molecule is provided. According to embodiments of this application, the nucleic acid molecule encodes the rTAAR8a mutant described in the first aspect or the fusion protein described in the second aspect. The nucleic acid molecule according to embodiments of this application encodes the aforementioned rTAAR8a mutant.

[0010] In a fourth aspect, this application provides an expression vector. According to an embodiment of this application, the expression vector carries the nucleic acid molecule described in the third aspect. The expression vector according to an embodiment of this application encodes the aforementioned rTAAR8a mutant.

[0011] In a fifth aspect, this application provides a recombinant cell or recombinant cell line. According to embodiments of this application, the recombinant cell or recombinant cell line carries the nucleic acid molecule described in the third aspect, or the expression vector described in the fourth aspect; or expresses the rTAAR8a mutant described in the first aspect or the fusion protein described in the second aspect. Using this recombinant cell or recombinant cell line under suitable conditions, the aforementioned rTAAR8a mutant can be effectively expressed intracellularly, resulting in recombinant cells or recombinant cell lines expressing the rTAAR8a mutant.

[0012] In a sixth aspect of this application, a product is proposed. According to embodiments of this application, the product comprises: the rTAAR8a mutant described in the first aspect, the fusion protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, or the recombinant cell or recombinant cell line described in the fifth aspect. As is known prior, the rTAAR8a mutant, through mutation at the aforementioned sites, can enhance its response to ammonia, particularly enhancing the activity of rTAAR8a in binding to ammonia. Therefore, products containing the rTAAR8a mutant, etc., can identify ammonia.

[0013] In a seventh aspect of this application, the use of an rTAAR8a mutant or the product described in the sixth aspect in the identification of ammonia is proposed, wherein the rTAAR8a mutant is as defined in the first aspect. As is previously known, the rTAAR8a mutant, through mutation at the aforementioned site, can enhance its response to ammonia. When the rTAAR8a mutant is stimulated with ammonia-containing substances, it can be activated. Therefore, the rTAAR8a mutant can be used to effectively identify ammonia, laying the foundation for the identification and detection of ammonia concentration.

[0014] In an eighth aspect of this application, the use of ammonia in activating an rTAAR8a mutant, as defined in the first aspect, is disclosed. As is previously known, the rTAAR8a mutant, through mutation at the aforementioned site, can enhance its response to ammonia. Stimulation of the rTAAR8a mutant with ammonia activates it. Therefore, ammonia can be effectively identified using the rTAAR8a mutant, laying the foundation for ammonia identification and concentration detection.

[0015] In a ninth aspect of this application, a method for detecting ammonia is proposed. According to an embodiment of this application, the method includes: contacting a sample to be tested with an rTAAR8a mutant, determining the response value of the rTAAR8a mutant after contact; and determining whether the sample to be tested contains ammonia based on the response value; wherein the rTAAR8a mutant is defined as the rTAAR8a mutant described in the first aspect. The method of this application involves contacting the sample to be tested with an rTAAR8a mutant. If the sample to be tested contains ammonia, the rTAAR8a mutant can be activated, and a response value after activation of the rTAAR8a mutant is obtained. Based on the response value, it can be determined whether the sample to be tested contains ammonia, laying the foundation for the identification of ammonia and the detection of ammonia concentration.

[0016] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0017] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a schematic diagram of the predicted binding mode of the rTAAR8a mutant with ammonia in Example 1 of this application; Figure 2 This is the dose-dependent curve of rTAAR8a and its mutants rTAAR8a S108D and rTAAR8a I191N in response to ammonia in Example 2 of this application. Detailed Implementation

[0018] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0019] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0020] This application details Definitions and General Terms The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0021] To facilitate understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0023] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0024] In this document, "conserved amino acid" refers to an amino acid sequence whose binding properties are not significantly affected or altered after substitution. Substitutions can be introduced into the olfactory receptor rTAAR8a of this application using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Exemplarily, the conserved amino acids of D or N are shown in the table below:

[0025] In this article, "protein tag" generally refers to a polypeptide or protein fused together with the target protein (rTAAR8a mutant) for expression, detection, elicitation, or purification of the target protein. This includes, but is not limited to, His tags (also known as His-Tag, sequence HHHHHH), Flag tags (also known as Flag-Tag, sequence DYKDDDDK), GST tags (also known as GST-Tag, glutathione thiotransferase tag), MBP tags (also known as MBP-Tag, maltose-binding protein tag), SUMO tags, and C-Myc tags.

[0026] In this article, "signal peptide" usually refers to a short peptide sequence located at the N-terminus of the target protein (rTAAR8a mutant). It acts as a "navigation system" or "address tag" for the protein, guiding the target protein to a specific destination. After that, it is usually cleaved by a signal peptidase.

[0027] In this document, the term "expression vector" generally refers to a nucleic acid fragment capable of self-replication within a suitable host, containing a target nucleic acid molecule, which transfers the inserted target nucleic acid molecule to host cells and / or between host cells. The expression vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The expression vector also includes expression vectors having multiple of the above-described functions. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the expression vector, the expression vector can produce the desired expression product.

[0028] In this article, "operably ligated" refers to ligating a foreign gene into an expression vector, enabling the control elements within the expression vector, such as transcriptional and translational control sequences, to perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used expression vectors include plasmids and bacteriophages. According to some specific embodiments of this application, after the expression vector is introduced into suitable recipient cells, the aforementioned rTAAR8a mutant can be effectively obtained in large quantities in vitro under the mediation of a regulatory system.

[0029] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of this application and can be used for the expression and / or secretion of target proteins (e.g., rTAAR8a mutants).

[0030] In this document, the term "suitable conditions" refers to conditions suitable for the expression of the rTAAR8a mutant described in this application. It will be readily understood by those skilled in the art that suitable conditions for the expression of the rTAAR8a mutant include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy recipient cell state, suitable recipient cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the rTAAR8a mutant according to the specific environment of their laboratory.

[0031] In this article, “original cells” refers to cells selected from a group of cells isolated from animal tissues (e.g., olfactory substrates) that express the rTAAR8a mutant.

[0032] In this document, the term "contact" should be interpreted broadly, encompassing both direct and indirect contact, without limitation. For example, contact can be achieved by mixing the test sample (which is itself a liquid or prepared as a liquid using a solvent) with the rTAAR8a mutant (i.e., direct contact between the test sample and the rTAAR8a mutant); alternatively, the test sample and the rTAAR8a mutant can be placed in the same space, allowing odor molecules (e.g., ammonia) released from the test sample to contact the rTAAR8a mutant (i.e., indirect contact between the test sample and the rTAAR8a mutant).

[0033] In this paper, the term "fluorescent protein" refers to a protein that is capable of spontaneous fluorescence, emitting fluorescence when excited by light of a specific wavelength without the addition of an external substrate or cofactor, including but not limited to green fluorescent protein.

[0034] This application discloses an rTAAR8a mutant, biomaterials, products and their uses, and a method for detecting ammonia, which will be described in detail below.

[0035] rTAAR8a mutant In a first aspect, this application proposes an rTAAR8a mutant. According to embodiments of this application, compared to wild-type rTAAR8a, the rTAAR8a mutant has mutations at positions 108 and / or 191. Compared to wild-type rTAAR8a, the rTAAR8a mutant of this application, through mutations at the aforementioned sites, can improve its response intensity and signal output level to ammonia (CAS: 7664-41-7), enhancing the activity of the rTAAR8a mutant in binding NH3, and can be used for the detection of ammonia.

[0036] According to embodiments of this application, the above-mentioned rTAAR8a mutant may further include at least one of the following technical features: In an optional embodiment of this application, the amino acid at position 108 is mutated to D or a conserved amino acid thereof.

[0037] In an optional embodiment of this application, the amino acid at position 191 is mutated to N or a conserved amino acid thereof.

[0038] In an optional embodiment of this application, the amino acid sequence of the wild-type rTAAR8a is shown in SEQ ID NO:1.

[0039] In an optional embodiment of this application, the mutation at position 108 is S108D or a conserved amino acid thereof. This enhances the binding affinity of rTAAR8a to NH3.

[0040] In an optional embodiment of this application, the mutation at position 191 is I191N or a conserved amino acid thereof. This enhances the binding affinity of rTAAR8a to NH3.

[0041] In an optional embodiment of this application, the mutation is S108D.

[0042] According to the embodiments of this application, the inventors discovered that the 108th amino acid is located in the core region of the binding pocket, and the S108D mutation optimizes the binding effect by changing the amino acid characteristics: (1) Change in charge characteristics: the aspartic acid (D) side chain contains a carboxyl group (-COOH), which can dissociate into a negatively charged -COO. - NH3 is easily protonated in physiological environments to form positively charged NH4. + The two can form a strong electrostatic attraction, which significantly enhances the binding affinity of the receptor to NH3 (the electrostatic interaction strength is increased several times compared to the weak hydrogen bonds of the wild type). (2) Enhanced hydrogen bonding: The carboxyl group of the aspartic acid side chain can bind to NH3 / NH3 + Stable double hydrogen bonds are formed, which further strengthens the binding effect, reduces the dissociation of NH3, prolongs the binding duration, and makes the receptor activation signal more persistent and stronger. (3) Spatial adaptation optimization: The aspartic acid side chain is slightly larger than that of serine, which can finely adjust the spatial conformation of the binding pocket, making the pocket more closely fit the molecular size of NH3, reducing the steric hindrance in the binding process, and improving the matching efficiency between NH3 and the binding pocket.

[0043] In an optional embodiment of this application, the mutation is I191N.

[0044] According to the embodiments of this application, the inventors found that the introduction of the I191N mutation mainly improves the binding performance by improving the polarity and spatial environment of the binding pocket: (1) Reversal of polarity: Isoleucine (I) is a nonpolar amino acid. After being replaced by polar asparagine (N), the local polarity of the binding pocket is significantly enhanced, and the polar interaction with polar NH3 is significantly improved, which solves the problem of NH3 binding difficulty caused by the excessive hydrophobicity of the wild-type pocket. (2) Increase in hydrogen bond binding sites: The side chain of asparagine contains an amide group (-CONH3), and both its amino and carbonyl groups can bind with NH3 / NH4. + (3) Reduced steric hindrance: Asparagine side chain is a polar amide group, which is smaller in volume than the nonpolar aliphatic chain of isoleucine. This can widen the local space of the binding pocket, reduce the steric hindrance when NH3 enters the pocket, and at the same time avoid the repulsive effect of nonpolar side chain on NH3, thus improving the binding efficiency.

[0045] In an optional embodiment of this application, the mutations are I191N and S108D.

[0046] According to embodiments of this application, the inventors discovered that the two mutants, I191N and S108D, enhance electrostatic interactions, hydrogen bonding, and polar interactions with NH3 by specifically altering the charge characteristics, polarity, and spatial conformation of key amino acids in the binding pocket. This reduces binding steric hindrance, increases binding affinity and stability, making NH3 easier to bind and less prone to dissociation, thereby activating a stronger receptor-mediated signaling pathway and ultimately exhibiting a stronger NH3 binding response than wild-type rTAAR8a.

[0047] In an optional embodiment of this application, the mutation site of the rTAAR8a mutant is S108D, compared with the wild-type rTAAR8a whose amino acid sequence is shown in SEQ ID NO:1, and the amino acid sequence of the rTAAR8a mutant is shown in SEQ ID NO:2.

[0048] In another optional embodiment of this application, the mutation site of the rTAAR8a mutant is I191N, compared with the wild-type rTAAR8a with an amino acid sequence as shown in SEQ ID NO:1, and the amino acid sequence of the rTAAR8a mutant is shown in SEQ ID NO:3.

[0049] In an optional embodiment of this application, the amino acid sequence of the rTAAR8a mutant is shown in SEQ ID NO:2 or SEQ ID NO:3.

[0050] The amino acid sequence of rTAAR8a (UniProt ID: Q923X9): MTSNFSQAPLQLCYENVNASCIKTPYSPGLRVLLYMVFGFGAVLAVCGNLLVVISVLHFKQLHSPANFLIASLASADFLVGISVMPFSMVRSIESCWYFGDTFCSLHSCCDAAFCYSSLFHLCFISVDRYIAVTDPLVYPTKFTVSVSGICISISWILPLVYSSAVFYTGISAT GIENLVSALNCVGGCQIVVNQDWVLIDFLLFLIPTLVMIILYSKIFLVAKQQAVKIETSISGSKGESSLESHKARVAKRERKAAKTLGVTVVAFMVSWLPYTIDTLIDAFMGFITPAYVYEICCWSAYYNSAMNPLIYAFFYPWFRKAIKLILSGEILKSHSSTMSLFSE (SEQ ID NO:1); The amino acid sequence of rTAAR8a S108D: MTSNFSQAPLQLCYENVNASCIKTPYSPGLRVLLYMVFGFGAVLAVCGNLLVVISVLHFKQLHSPANFLIASLASADFLVGISVMPFSMVRSIESCWYFGDTFCSLHDCCDAAFCYSSLFHLCFISVDRYIAVTDPLVYPTKFTVSVSGICISISWILPLVYSSAVFYTGISAT GIENLVSALNCVGGCQIVVNQDWVLIDFLLFLIPTLVMIILYSKIFLVAKQQAVKIETSISGSKGESSLESHKARVAKRERKAAKTLGVTVVAFMVSWLPYTIDTLIDAFMGFITPAYVYEICCWSAYYNSAMNPLIYAFFYPWFRKAIKLILSGEILKSHSSTMSLFSE (SEQ ID NO:2); The amino acid sequence of rTAAR8a I191N: MTSNFSQAPLQLCYENVNASCIKTPYSPGLRVLLYMVFGFGAVLAVCGNLLVVISVLHFKQLHSPANFLIASLASADFLVGISVMPFSMVRSIESCWYFGDTFCSLHSCCDAAFCYSSLFHLCFISVDRYIAVTDPLVYPTKFTVSVSGICISISWILPLVYSSAVFYTGISAT GIENLVSALNCVGGCQNVVNQDWVLIDFLLFLIPTLVMIILYSKIFLVAKQQAVKIETSISGSKGESSLESHKARVAKRERKAAKTLGVTVVAFMVSWLPYTIDTLIDAFMGFITPAYVYEICCWSAYYNSAMNPLIYAFFYPWFRKAIKLILSGEILKSHSSTMSLFSE (SEQ ID NO:3).

[0051] It should be noted that the rTAAR8a mutant in this application refers to the active part. Adding conjugates (such as protein tags) to the N-terminus or C-terminus of the rTAAR8a mutant in order to obtain the rTAAR8a mutant in this application is also within the scope of protection of this application.

[0052] biomaterials The biological materials in this application include fusion proteins, nucleic acid molecules, expression vectors, recombinant cells, or recombinant cell lines.

[0053] In a second aspect of this application, a fusion protein is proposed. According to an embodiment of this application, the fusion protein comprises the rTAAR8a mutant described in the first aspect and a protein fragment, wherein the protein fragment is linked to the rTAAR8a mutant.

[0054] In one alternative embodiment of this application, the protein fragment is attached to the N-terminus or C-terminus of the rTAAR8a mutant.

[0055] In one alternative embodiment of this application, the protein fragment is selected from signal peptides or tag proteins.

[0056] In a third aspect of this application, a nucleic acid molecule is provided. According to embodiments of this application, the nucleic acid molecule encodes the rTAAR8a mutant described in the first aspect or the fusion protein described in the second aspect. The nucleic acid molecule according to embodiments of this application encodes the aforementioned rTAAR8a mutant.

[0057] In one alternative embodiment of this application, the nucleic acid molecule is DNA.

[0058] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is also disclosed. Furthermore, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0059] In a fourth aspect, this application provides an expression vector. According to an embodiment of this application, the expression vector carries the nucleic acid molecule described in the third aspect. The expression vector according to an embodiment of this application encodes the aforementioned rTAAR8a mutant.

[0060] Furthermore, when linking the nucleic acid molecule described in the second aspect to the expression vector, the nucleic acid molecule can be directly or indirectly connected to control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the expression vector itself, or they can be exogenous, i.e., not derived from the expression vector itself. Naturally, the connection between the nucleic acid molecule and the control elements simply needs to be operably established.

[0061] According to embodiments of this application, the expression vector may further include at least one of the following technical features: In one optional embodiment of this application, the expression vector includes a prokaryotic expression vector or a eukaryotic expression vector.

[0062] In one alternative embodiment of this application, the expression vector includes at least one selected from plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

[0063] In an optional embodiment of this application, a recombinant cell or recombinant cell line is provided. According to embodiments of this application, the recombinant cell or recombinant cell line carries the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect; or expresses the rTAAR8a mutant described in the first aspect or the fusion protein described in the second aspect. Using this recombinant cell or recombinant cell line under suitable conditions, the aforementioned rTAAR8a mutant can be effectively expressed intracellularly, resulting in recombinant cells or recombinant cell lines expressing the rTAAR8a mutant.

[0064] In one alternative embodiment of this application, the recombinant cells or recombinant cell lines are obtained by introducing an expression vector into a host cell.

[0065] In one optional embodiment of this application, the host cell is a eukaryotic cell or a prokaryotic cell.

[0066] In some optional embodiments of this application, the eukaryotic cells include, but are not limited to, cells selected from cell groups isolated from the olfactory substrate, HEK293 cells, CHO cells, Xenopus oocytes, HeLa cells, COS cells, and yeast cells.

[0067] In one alternative embodiment of this application, the prokaryotic cells are selected from bacteria.

[0068] Furthermore, those skilled in the art will understand that the features and advantages described above for the rTAAR8a mutant also apply to this biological material (fusion protein, nucleic acid molecule, expression vector, recombinant cell or recombinant cell line), and will not be repeated here.

[0069] product In a sixth aspect of this application, a product is proposed. According to embodiments of this application, the product comprises: the rTAAR8a mutant described in the first aspect, the fusion protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, or the recombinant cell or recombinant cell line described in the fifth aspect. As is known prior, the rTAAR8a mutant, through mutation at the aforementioned sites, can enhance its response to ammonia, particularly enhancing the activity of rTAAR8a in binding to ammonia. Therefore, a product containing the rTAAR8a mutant can identify ammonia and is used for the detection or identification of ammonia.

[0070] In one optional embodiment of this application, the product is selected from products used for detecting ammonia, and the specific type is not limited. Any product used for detecting ammonia is within the scope of protection of this application. Exemplarily, the product includes, but is not limited to, reagents, kits, ammonia trace detection sensors, and ammonia-responsive probes.

[0071] In one optional embodiment of this application, the reagent or kit may be a reagent or kit for ammonia detection, or a reagent or kit for ammonia-based auxiliary diagnosis of diseases. The specific type is not limited and is within the scope of protection of this application.

[0072] In an optional embodiment of this application, the kit may further include other reagents, including but not limited to buffer solutions.

[0073] use In a seventh aspect of this application, the use of an rTAAR8a mutant or the product described in the sixth aspect in the identification of ammonia is proposed, wherein the rTAAR8a mutant is as defined in the first aspect. As is previously known, the rTAAR8a mutant, through mutation at the aforementioned site, can enhance its response to ammonia. When the rTAAR8a mutant is stimulated with the aforementioned ammonia substance, it can be activated. Therefore, the rTAAR8a mutant can be used to effectively identify ammonia, laying the foundation for the identification of ammonia and the detection of ammonia concentration.

[0074] It should be noted that if the rTAAR8a mutant is activated after being stimulated with ammonia, that is, "the rTAAR8a mutant can recognize ammonia"; if the rTAAR8a mutant is not activated after being stimulated with ammonia, that is, "the rTAAR8a mutant cannot recognize ammonia".

[0075] According to embodiments of this application, the above-described uses may further include at least one of the following technical features: In an optional embodiment of this application, the identification is manifested through changes in the activity of the rTAAR8a mutant.

[0076] In one optional embodiment of this application, the activity of the rTAAR8a mutant is obtained based on changes in the concentration or value of at least one of the following: rTAAR8a mutant protein conformation, G protein binding or dissociation, cAMP, IP3, calcium ions, current, and pH.

[0077] In some alternative embodiments of this application, the recognition is manifested by an increase in cAMP downstream of the rTAAR8a mutant. The inventors discovered through experiments that when ammonia gas stimulates cells expressing the rTAAR8a mutant, the intracellular cAMP concentration increases after the rTAAR8a mutant is activated. By detecting changes in cAMP concentration, it can be determined whether the rTAAR8a mutant can recognize ammonia gas.

[0078] In an optional embodiment of this application, the change in cAMP concentration of the rTAAR8a mutant is detected by at least one of luciferase and secretory alkaline phosphatase.

[0079] In an optional embodiment of this application, the change in cAMP concentration is obtained by detecting changes in the signal of a fluorescent protein or a fluorescent probe.

[0080] In an optional embodiment of this application, the change in current can be detected by a field-effect transistor sensor.

[0081] In some alternative embodiments of this application, the recognition is manifested by an increase in intracellular calcium ions in cells expressing the rTAAR8a mutant. The inventors discovered through experiments that when ammonia gas stimulates cells expressing the rTAAR8a mutant, the intracellular calcium ion level increases after the rTAAR8a mutant is activated. By detecting changes in calcium ion concentration, it can be determined whether the rTAAR8a mutant can recognize ammonia gas.

[0082] In an optional embodiment of this application, the change in calcium ion concentration of the rTAAR8a mutant is detected by calcium ion flux detection.

[0083] In some optional embodiments of this application, the activity of the rTAAR8a mutant can also be obtained by surface plasmon resonance (SPR).

[0084] In an eighth aspect of this application, the use of ammonia in activating an rTAAR8a mutant, as defined in the first aspect, is proposed. As is previously known, the rTAAR8a mutant, through mutation at the aforementioned site, can enhance its response to ammonia. Stimulation of the rTAAR8a mutant with ammonia activates it. Therefore, ammonia can be effectively identified using the rTAAR8a mutant, laying the foundation for ammonia identification and concentration detection.

[0085] According to embodiments of this application, the above-described uses may further include at least one of the following technical features: In an optional embodiment of this application, the activation is manifested through changes in the activity of the rTAAR8a mutant.

[0086] In one optional embodiment of this application, the activity of the rTAAR8a mutant is obtained based on changes in the concentration or value of at least one of the following: rTAAR8a mutant protein conformation, G protein binding or dissociation, cAMP, IP3, calcium ions, current, and pH.

[0087] In some alternative embodiments of this application, the activation is manifested through an increase in cAMP downstream of the rTAAR8a mutant. The inventors discovered through experiments that when ammonia gas stimulates cells expressing the rTAAR8a mutant, the intracellular cAMP concentration increases after the rTAAR8a mutant is activated. By detecting changes in cAMP concentration, it can be determined whether the rTAAR8a mutant can recognize ammonia gas.

[0088] In an optional embodiment of this application, the change in cAMP concentration of the rTAAR8a mutant is detected by at least one of luciferase and secretory alkaline phosphatase.

[0089] In an optional embodiment of this application, the change in cAMP concentration is obtained by detecting changes in the signal of a fluorescent protein or a fluorescent probe.

[0090] In an optional embodiment of this application, the change in current can be detected by a field-effect transistor sensor.

[0091] In some alternative embodiments of this application, the activation is manifested by an increase in intracellular calcium ions in cells expressing the rTAAR8a mutant. The inventors discovered through experiments that when ammonia gas stimulates cells expressing the rTAAR8a mutant, the rTAAR8a mutant is activated, and the intracellular calcium ion level increases. By detecting changes in calcium ion concentration, it can be determined whether the rTAAR8a mutant can recognize ammonia gas.

[0092] In an optional embodiment of this application, the change in calcium ion concentration of the rTAAR8a mutant is detected by calcium ion flux detection.

[0093] In some optional embodiments of this application, the activity of the rTAAR8a mutant can also be obtained by surface plasmon resonance (SPR).

[0094] Methods for detecting ammonia In a ninth aspect of this application, a method for detecting ammonia is proposed. According to an embodiment of this application, the method includes: contacting a sample to be tested with an rTAAR8a mutant, determining the response value of the rTAAR8a mutant after contact; and determining whether the sample to be tested contains ammonia based on the response value; wherein the rTAAR8a mutant is defined as the rTAAR8a mutant described in the first aspect. According to the method of this application embodiment, contacting the sample to be tested with an RTAAR8a mutant, if the sample to be tested contains ammonia, the rTAAR8a mutant can be activated, and a response value after activation of the rTAAR8a mutant is obtained. Based on the response value, it can be determined whether the sample to be tested contains ammonia, laying the foundation for the identification of ammonia and the detection of ammonia concentration.

[0095] According to embodiments of this application, the above method may further include at least one of the following technical features: In one embodiment of this application, the sample to be tested may be an aromatic product, including but not limited to perfumes, fragrances, or cosmetics.

[0096] In one optional embodiment of this application, the sample to be tested is diluted before contact, with a dilution factor of 10 to 10,000 times, such as 10 to 1,000 times, 10 to 500 times, 10 to 400 times, 10 to 300 times, 10 to 200 times, 10 to 100 times, 10 to 90 times, 20 to 80 times, 30 to 70 times, etc.

[0097] In one optional embodiment of this application, the presence of the rTAAR8a mutant is an indication that the test sample contains the ammonia gas; or, the absence of the rTAAR8a mutant is an indication that the test sample does not contain the ammonia gas.

[0098] It should be noted that "indication of absence of ammonia" means that ammonia is completely absent from the sample being tested; or that a small amount of ammonia is present in the sample being tested, but cannot be detected. The activation concentration of the rTAAR8a mutant to different ammonia concentrations can be obtained by stimulating the rTAAR8a mutant with different concentrations of ammonia.

[0099] In one embodiment of this application, the mutation site of the rTAAR8a mutant is S108D, and the presence response value of the rTAAR8a mutant is an indication that the sample to be tested contains ammonia or contains ammonia of not less than 3 μM; or, the mutation site of the rTAAR8a mutant is I191N, and the absence response value of the rTAAR8a mutant is an indication that the sample to be tested does not contain ammonia or contains ammonia of less than 1 μM.

[0100] In an optional embodiment of this application, the method further includes: determining the ammonia content in the sample to be tested based on a standard curve, wherein the standard curve is a curve corresponding to a predetermined amount of ammonia and the response value of the rTAAR8a mutant.

[0101] In an alternative embodiment of this application, the rTAAR8a mutant is provided by cells expressing the rTAAR8a mutant.

[0102] In one alternative embodiment of this application, the cells include primitive cells or transgenic cells.

[0103] In some alternative embodiments of this application, the transgenic cells may be derived from eukaryotic cells and / or prokaryotic cells.

[0104] In some optional embodiments of this application, the eukaryotic cells include, but are not limited to, cells selected from cell groups isolated from the olfactory substrate, HEK293 cells, CHO cells, Xenopus oocytes, HeLa cells, COS cells, and yeast cells.

[0105] In one alternative embodiment of this application, the prokaryotic cells are selected from bacteria.

[0106] In an optional embodiment of this application, the response value is obtained by detecting changes in the activity of the rTAAR8a mutant in the cells.

[0107] In one optional embodiment of this application, the activity of the rTAAR8a mutant is obtained based on changes in the concentration or value of at least one of the following: rTAAR8a mutant protein conformation, G protein binding or dissociation, cAMP, IP3, calcium ions, current, and pH.

[0108] In an optional embodiment of this application, the change in cAMP concentration of the rTAAR8a mutant is obtained by detecting changes in the signal of a fluorescent protein or a fluorescent probe.

[0109] In an optional embodiment of this application, the change in cAMP concentration of the rTAAR8a mutant is detected by at least one of luciferase and secretory alkaline phosphatase.

[0110] For example, the detection method of luciferase is as follows: when cells containing rTAAR8a mutant are stimulated with ammonia or a sample containing ammonia, if the rTAAR8a mutant is activated, the intracellular cAMP concentration increases. cAMP binds to the promoter region of CRE-luciferase and promotes the transcription and translation of luciferase. Therefore, by detecting the activity of luciferase, the response of rTAAR8a mutant can be characterized, and it can be determined whether rTAAR8a mutant can recognize ammonia.

[0111] In an optional embodiment of this application, the change in cAMP concentration is obtained by detecting changes in the signal of a fluorescent protein or a fluorescent probe.

[0112] In an optional embodiment of this application, the change in current can be detected by a field-effect transistor sensor.

[0113] In some optional embodiments of this application, the response value is reflected by detecting an increase in intracellular calcium ions in cells expressing the rTAAR8a mutant. The inventors discovered through experiments that when ammonia gas stimulates cells expressing the rTAAR8a mutant, the intracellular calcium ion level increases after the rTAAR8a mutant is activated. By detecting changes in calcium ion concentration, it can be determined whether the rTAAR8a mutant can recognize ammonia gas.

[0114] In an optional embodiment of this application, the change in calcium ion concentration of the rTAAR8a mutant is detected by calcium ion flux detection. This allows for immediate detection of ammonia by the rTAAR8a mutant, offering advantages such as simple operation and short detection time.

[0115] In some optional embodiments of this application, the activity of the rTAAR8a mutant can also be obtained by surface plasmon resonance (SPR).

[0116] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0117] Example 1: The olfactory receptor rTAAR8a can selectively recognize the odorant NH3, but its response fold change is slightly low. To improve its activity, this embodiment uses a combination of computer simulation and biological screening to construct an enhanced rTAAR8a. Specifically: A three-dimensional structural prediction model of rTAAR8a was established, and molecular docking between the rTAAR8a model and NH3 was performed using AutoDock Vina. The results are as follows: Figure 1 As shown, this illustrates the amino acids within NH35Å in molecular docking.

[0118] Based on the docking results, mutations were performed on two amino acid sites to screen for rTAAR8a mutants with increased response intensity. The activities of the two mutants were measured using the Dual-Glo™ Luciferase Assay System (Promega). Gene constructs of the human olfactory receptor, Golf, CRE-Luciferase, and pRL-SV40 were transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen). After 24 hours, the cells were stimulated with a specific concentration of NH3 and incubated for 3-4 hours. If the olfactory receptor is activated, the intracellular cAMP concentration increases. cAMP binds to the promoter region of CRE-luciferase, promoting the transcription and translation of luciferase. Therefore, luciferase activity characterizes the response of the olfactory receptor.

[0119] Different concentrations of NH3 were diluted in culture medium to stimulate rTAAR8a and its mutants, and their responses were measured. The response results are as follows: Figure 2 As shown, there are two mutants, rTAAR8a S108D (amino acid sequence as shown in SEQ ID NO:2) and rTAAR8a I191N (amino acid sequence as shown in SEQ ID NO:3), which have significantly increased response folds, and the minimum response odor concentration of rTAAR8a S108D is significantly reduced. Figure 2 The dose-dependent curves of rTAAR8a WT (i.e., wild-type rTAAR8a, amino acid sequence as shown in SEQ ID NO:1), rTAAR8a S108D, and rTAAR8a I191N in response to NH3 are shown. The horizontal axis represents the logarithm of NH3 concentration (M), and the vertical axis represents the fold change in response relative to the blank control (no NH3). Among them, rTAAR8a WT showed the strongest fold change in response to NH3, reaching 5-fold. The fold changes in response of the rTAAR8a S108D mutant and the rTAAR8a I191N mutant increased to 8-fold and 11-fold, respectively.

[0120] The above results indicate that by combining computer simulation with biological screening, the binding pocket of the olfactory receptor rTAAR8a can be anchored, and the amino acids in the binding pocket can be modified to enhance the response intensity of the olfactory receptor rTAAR8a to the odor molecule ammonia and accelerate the response time. This provides a basis for the modification of olfactory receptors.

[0121] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0122] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. An rTAAR8a mutant, characterized in that, Compared to wild-type rTAAR8a, the rTAAR8a mutant has mutations at positions 108 and / or 191.

2. The rTAAR8a mutant according to claim 1, characterized in that, The rTAAR8a mutant further satisfies one or more of the following conditions: 1) The amino acid at position 108 is mutated to D or its conserved amino acid; 2) The amino acid at position 191 is mutated to N or its conserved amino acid; 3) The amino acid sequence of the wild-type rTAAR8a is shown in SEQ ID NO:

1.

3. The rTAAR8a mutant according to claim 1, characterized in that, The rTAAR8a mutant further satisfies one or more of the following conditions: 1) The mutation at position 108 is S108D or its conserved amino acid; 2) The mutation at position 191 is I191N or its conserved amino acid; 3) The mutation is S108D; 4) The mutation is I191N; 5) The mutations are I191N and S108D.

4. A biomaterial, characterized in that, The biomaterial includes any one of the following (i) to (iv): (i) A fusion protein comprising the rTAAR8a mutant and a protein fragment as described in any one of claims 1 to 3, wherein the protein fragment is linked to the rTAAR8a mutant; Optionally, the protein fragment is selected from signal peptides or tag proteins; (ii) a nucleic acid molecule that encodes the rTAAR8a mutant according to any one of claims 1 to 3 or the fusion protein according to (i); (iii) A vector carrying the nucleic acid molecule described in (ii); (iv) Recombinant cells or recombinant cell lines comprising the nucleic acid molecule described in (ii) or the vector described in (iii), or expressing the rTAAR8a mutant of any one of claims 1 to 3 or the fusion protein described in (i); Optionally, the recombinant cells or recombinant cell lines are obtained by introducing an expression vector into host cells; Optionally, the host cell is a eukaryotic cell or a prokaryotic cell.

5. A product characterized in that, include: The rTAAR8a mutant as described in any one of claims 1 to 3, or the biological material as described in claim 4.

6. Use of the rTAAR8a mutant or the product of claim 5 in the identification of ammonia, wherein the rTAAR8a mutant is defined as the rTAAR8a mutant of any one of claims 1 to 3.

7. Use of ammonia in activating the rTAAR8a mutant, as defined in any one of claims 1 to 3.

8. A method for detecting ammonia, characterized in that, include: The sample to be tested was brought into contact with the rTAAR8a mutant, and the response value of the rTAAR8a mutant after contact was determined. Based on the response value, determine whether the sample to be tested contains the ammonia gas; The rTAAR8a mutant is defined as the rTAAR8a mutant as described in any one of claims 1 to 3.

9. The method according to claim 8, characterized in that, The method further satisfies one or more of the following conditions: 1) Based on the standard curve, determine the ammonia content in the sample to be tested, wherein the standard curve is the corresponding curve of a predetermined amount of ammonia and the response value of the rTAAR8a mutant; 2) The rTAAR8a mutant is provided by cells expressing the rTAAR8a mutant; Optionally, the cells may include primitive cells or transgenic cells; 3) The response value was obtained by detecting changes in the activity of the rTAAR8a mutant in the cells.

10. The method according to claim 9, characterized in that, The activity of the rTAAR8a mutant is obtained based on changes in the concentration or value of at least one of the following: rTAAR8a mutant protein conformation, G protein binding or dissociation, cAMP, IP3, calcium ions, electric current, and pH. Optionally, the change in cAMP concentration of the rTAAR8a mutant is obtained by detecting changes in the signal of a fluorescent protein or a fluorescent probe, or the change in cAMP concentration of the rTAAR8a mutant is obtained by detecting at least one of luciferase and secretory alkaline phosphatase. Optionally, the change in calcium ion concentration of the rTAAR8a mutant is detected by calcium ion flux detection.

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