CN116445417B - Positive modulators of olfactory receptors, recombinant cells or recombinant cell lines containing same and methods for detecting odorant compounds - Google Patents
Positive modulators of olfactory receptors, recombinant cells or recombinant cell lines containing same and methods for detecting odorant compounds Download PDFInfo
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Abstract
The application discloses a positive regulator of olfactory receptor, recombinant cells or recombinant cell lines containing the positive regulator and a method for detecting odor compounds. RTP1S protein can be used as positive regulator of olfactory receptor, and RTP1S protein is derived from at least one of wild boar, cattle and Ind ear bats; recombinant cells or recombinant cell lines include: carrying a nucleotide sequence encoding an RTP1S protein and a nucleotide sequence encoding an olfactory receptor, or expressing an RTP1S protein and an olfactory receptor; the method comprises the steps of contacting a sample to be tested with the recombinant cells and the recombinant cell line, and determining whether the sample to be tested contains the odor compound or not based on the response result of the recombinant cells or the recombinant cell line after the contact, wherein an olfactory receptor in the recombinant cells or the recombinant cell line is an olfactory receptor capable of recognizing the odor compound. The functional expression of olfactory receptors in recombinant cells or recombinant cell lines containing positive modulators of olfactory receptors is better.
Description
Technical Field
The application belongs to the technical field of biochemical detection, in particular to a forward regulator of an olfactory receptor, a recombinant cell or a recombinant cell line containing the forward regulator and a method for detecting an odor compound, and more particularly, the application relates to a recombinant cell or a recombinant cell line, an application of RTP1S protein in serving as the forward regulator of the olfactory receptor, a method for promoting functional expression of the olfactory receptor in a heterogeneous cell and a method for detecting the odor compound.
Background
The mammal has excellent olfactory perception capability, can sensitively and rapidly identify and distinguish the odor in food, individuals or environment, and is important for foraging, defense, migration and other behaviors. Olfactory receptors (Odorant receptors (ORs)) play an important role in the connection between odorant molecules and the sense of smell. However, olfaction is one of the most complex and unintelligible sensory systems, and a great deal of research is required to elucidate its working mechanism. The experimental method based on living or isolated olfactory tissues can not detect the response condition of the olfactory receptor to the odor molecules with high flux, for example, the functional expression problem of the olfactory receptor in a heterologous cell line can be solved, the research work of the arc removal and olfactory recognition mechanism of the olfactory receptor can be facilitated, and the development of basic science and industrial application can be promoted. Olfactory receptor proteins belong to the GPCR superfamily, have seven transmembrane structures, are synthesized in the endoplasmic reticulum, and are finally transported to the cell membrane of olfactory neurons to act.
In the heterologous expression process, most olfactory receptors encounter problems of blocking the endoplasmic reticulum, and functional expression cannot be achieved, thus limiting the use thereof. Thus, there is a need to provide an accessory protein or method that can enhance the functional expression of olfactory receptors in heterologous cell lines.
Disclosure of Invention
The present application aims to solve at least one of the technical problems existing in the prior art to at least some extent. Therefore, the application provides a recombinant cell or a recombinant cell line containing a positive regulator of an olfactory receptor, wherein the recombinant cell or the recombinant cell line thereof has high functional expression of the olfactory receptor and can increase the activation effect of the olfactory receptor and a corresponding ligand compound thereof.
The present application has been completed based on the following findings by the inventors:
it has now been found that various accessory proteins such as RTP1, RTP2, REEP1, M3R, ric8A, ric B, hsc t, etc. are reported to promote functional expression of olfactory receptors in heterologous cell lines. Among them, RTP1 has the most remarkable effect of enhancing functional expression of olfactory receptor, and particularly, the short subtype of RTP1 (RTP 1S) has better effect than the full-length subtype (RTP 1L).
Based on this, in one aspect of the application, the application proposes a recombinant cell or recombinant cell line. According to an embodiment of the application, the recombinant cell or recombinant cell line comprises: carrying a nucleotide sequence encoding an RTP1S protein and a nucleotide sequence encoding an olfactory receptor; or express RTP1S protein and olfactory receptor; wherein said at least one ofThe RTP1S protein is derived from wild boarSus scrofaCattleBos taurusHeyingzhu ear batsMyotis lucifugusAt least one of them. The recombinant cell or the recombinant cell line has better effect of functional expression of the olfactory receptor, and can increase the activation effect of the olfactory receptor and the corresponding ligand compound.
According to an embodiment of the present application, the recombinant cell or recombinant cell line may further comprise at least one of the following technical features:
according to an embodiment of the application, derived from wild boarsSus scrofaThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 1; or from a mouse ear batMyotis lucifugusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 2; or of bovine originBos taurusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 3.
According to an embodiment of the application, the olfactory receptor comprises at least one of MOR41-1, MOR106-1, MOR103-12 and MOR179-7, and optionally MOR213-1.
According to an embodiment of the application, the recombinant cell or recombinant cell line is obtained by introducing into a host cell an expression vector carrying the following sequences: a nucleotide sequence encoding the RTP1S protein and a nucleotide sequence encoding the olfactory receptor.
According to an embodiment of the application, the host cell is a eukaryotic cell or a prokaryotic cell.
According to an embodiment of the present application, the RTP1S protein has an amino acid sequence as shown in SEQ ID NO:1 and/or SEQ ID NO:2, and the olfactory receptor comprises at least one of MOR41-1, MOR106-1, MOR103-12 and MOR 179-7; or the RTP1S protein has an amino acid sequence as shown in SEQ ID NO. 3, and the olfactory receptor comprises at least one of MOR41-1, MOR179-7, MOR106-1, MOR103-12 and MOR213-1.
In another aspect of the application, the application proposes the use of an RTP1S protein as a positive modulator of the olfactory receptor; the RTP1S protein is derived from wild boarsSus scrofaCattleBos taurusHeyingzhu ear batsMyotis lucifugusAt least one of them. The RTP1S protein from the source can be used as a positive regulator of an olfactory receptor, and can improve the functional expression of the olfactory receptor in a heterologous cell.
According to an embodiment of the application, derived from wild boarsSus scrofaThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 1; or from a mouse ear batMyotis lucifugusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 2; or of bovine originBos taurusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 3.
According to an embodiment of the application, the olfactory receptor comprises at least one of MOR41-1, MOR106-1, MOR103-12 and MOR179-7, and optionally MOR213-1.
According to an embodiment of the present application, the RTP1S protein has an amino acid sequence as shown in SEQ ID NO:1 and/or SEQ ID NO:2, and the olfactory receptor comprises at least one of MOR41-1, MOR106-1, MOR103-12 and MOR 179-7; or the RTP1S protein has an amino acid sequence as shown in SEQ ID NO. 3, and the olfactory receptor comprises at least one of MOR41-1, MOR179-7, MOR106-1, MOR103-12 and MOR213-1.
In yet another aspect of the application, the application provides a method of promoting functional expression of an olfactory receptor in a heterologous cell. According to an embodiment of the application, the method comprises: introducing a nucleotide sequence encoding an RTP1S protein and a nucleotide sequence encoding an olfactory receptor into the heterologous cell; wherein the RTP1S protein is derived from wild boarSus scrofaCattleBos taurusHeyingzhu ear batsMyotis lucifugusAt least one of them. Thus, the functional expression of the olfactory receptor in the heterologous cell can be promoted, and the activating effect of the ligand corresponding to the olfactory receptor on the olfactory receptor can be improved.
According to an embodiment of the application, derived from wild boarsSus scrofaThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 1; or from a mouse ear batMyotis lucifugusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 2; or of bovine originBos taurusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 3.
According to an embodiment of the application, the olfactory receptor comprises at least one of MOR41-1, MOR106-1, MOR103-12 and MOR179-7, and optionally MOR213-1.
According to an embodiment of the application, the introduction of the nucleotide sequence encoding the RTP1S protein and the nucleotide sequence encoding the olfactory receptor into the heterologous cell is achieved by the following steps: constructing an expression vector carrying a nucleotide sequence encoding the RTP1S protein and a nucleotide sequence encoding an olfactory receptor; introducing the expression vector into the heterologous cell.
According to an embodiment of the present application, the RTP1S protein has an amino acid sequence as shown in SEQ ID NO:1 and/or SEQ ID NO:2, and the olfactory receptor comprises at least one of MOR41-1, MOR106-1, MOR103-12 and MOR 179-7; or the RTP1S protein has an amino acid sequence as shown in SEQ ID NO. 3, and the olfactory receptor comprises at least one of MOR41-1, MOR179-7, MOR106-1, MOR103-12 and MOR213-1.
In yet another aspect of the application, the application provides a method of detecting an odor compound. According to an embodiment of the application, the method comprises: contacting a sample to be tested with the recombinant cell or recombinant cell line; determining whether the sample to be tested contains the odor compound based on the response result of the recombinant cells or recombinant cell lines after the contacting; wherein the olfactory receptor in the recombinant cell or recombinant cell line is an olfactory receptor that recognizes the odorant compound. As can be seen from the foregoing, the olfactory receptor in the recombinant cell or recombinant cell line has a better functional expression effect. Therefore, the detection of the odor compound by the method comprising the recombinant cell or the recombinant cell line can further improve the sensitivity of detecting the odor compound.
According to an embodiment of the application, the olfactory receptor comprises at least one of MOR41-1, MOR106-1, MOR103-12 and MOR179-7, and optionally MOR213-1.
According to an embodiment of the application, the presence response value of the recombinant cell or recombinant cell line is an indication that the test sample contains the odorant compound; alternatively, the absence of a response value of the recombinant cell or recombinant cell line is an indication that the test sample does not contain the odorant compound.
According to an embodiment of the present application, the RTP1S protein has an amino acid sequence as shown in SEQ ID NO:1 and/or SEQ ID NO:2, and the olfactory receptor comprises at least one of MOR41-1, MOR106-1, MOR103-12 and MOR 179-7; or the RTP1S protein has an amino acid sequence as shown in SEQ ID NO. 3, and the olfactory receptor comprises at least one of MOR41-1, MOR179-7, MOR106-1, MOR103-12 and MOR213-1.
According to an embodiment of the application, the response value is obtained by detecting a change in the activity of the olfactory receptor.
According to an embodiment of the application, the change in activity is determined by at least one of the following detection methods: luciferase assay, secretory alkaline phosphatase assay, fluorescent protein assay, fluorescent probe assay, ca 2+ Concentration detection, amperometric detection, isotopic labeling, antibody detection and pH detection.
Additional aspects and advantages of the 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 the application.
Drawings
The foregoing and/or additional aspects and advantages of the application will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a graph showing the results of activation of 14 olfactory receptor/ligand pairs of example 1 of the present application;
FIG. 2 shows the results of a multi-species RTP1S screening of 5 uOR/ligand pairs of example 2 of the present application.
Detailed Description
Embodiments of the present application are described in detail below. The following examples are illustrative only and are not to be construed as limiting the application.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present application, unless otherwise indicated, the meaning of "a plurality" is two or more.
In order that the application may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present application, but not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
The present application discloses a recombinant cell or recombinant cell line, the use of RTP1S protein as positive modulator of olfactory receptor, a method for promoting functional expression of olfactory receptor in heterologous cells and a method for detecting an odorant compound, which are described in detail below, respectively.
Recombinant cells or recombinant cell lines
In one aspect of the application, the application provides a recombinant cell or recombinant cell line. According to an embodiment of the application, the recombinant cell or recombinant cell line comprises: carrying a nucleotide sequence encoding an RTP1S protein and a nucleotide sequence encoding an olfactory receptor; or express RTP1S protein and olfactory receptor; wherein the RTP1S protein is derived from wild boarSus scrofaCattleBos taurusHeyingzhu ear batsMyotis lucifugusAt least one of them. The inventors found that RTP1S protein was used as an auxiliary protein,RTP1S protein derived from wild boar compared with murine RTP1S proteinSus scrofaCattleBos taurusHeyingzhu ear batsMyotis lucifugusThe RTP1S protein has better activating effect on olfactory receptor and corresponding ligand compound. Thus, the recombinant cells or recombinant cell lines of the present application have a better effect of functionally expressing the olfactory receptor, and can increase the activating effect of the olfactory receptor and the ligand compound corresponding thereto.
According to an embodiment of the application, the RTP1S protein is derived from a wild boarSus scrofaCattleBos taurusHeyingzhu ear batsMyotis lucifugusOne of them.
According to an embodiment of the application, derived from wild boarsSus scrofaThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 1; or from a mouse ear batMyotis lucifugusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 2; or from cattle (or so-called common cattle)Bos taurusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 3.
RTP1S（Sus scrofa) Amino acid sequence:
MCKSVTTEEWKKVFYEKMEEAKPADSWDLIIDPNLKHNVLAPGWKQYLELHASGRFHCSWCWHTWQSPHVVILFHMHLDRAQRAGSVRMRVFKQLCYECGTSRLDESSMLEENIESLVDNLITSLREQCYGERGGQYRIHVASRQDNRRHRGEFCEACQEGIVHWKPSEKLLEEEATTYTFSRAPSPTKQQAEGGSGCNFCSIPWCLFWATVLLLIIYLQFSFRSSV（SEQ ID NO:1）。
RTP1S（Myotis lucifugus) Amino acid sequence:
MCKSVTTGEWKKAFYEKMEVAKPADSWDLIIDPNLKHNVLAPGWKQYLELHASGRFHCSWCWHTWQSPHVVILFHMYLDRAQRAGSVRMRVFKQLCYECGTAHLDESSMLEENIESLVDNLITSLREQCYGERGGHYRIHVASRQDNRRHRSEFCEACQEGIVHWKPSQKLLEEEATIYTFKPAPSPSKPQAEMGSGSNFCSIPWCLFWATVLLLIIYLQFSFRSSV（SEQ ID NO:2）。
RTP1S（Bos taurus) Amino acid sequence:
MCKSVTTDEWKKIFYEKMEEAKPADSWDLIIDPNLKHNVLAPGWKQYLELHASGRFHCSWCWHTWQSPHLVILFHMYLDRAQRAGSVRMRVFKQLCYECGTARLDESSMLEENIECLVDNLITSLREQCYGERGGQYRIHVASRQDTRRHRGEFCEACQEGIVHWKPSEKLLEEEATTYTFSRAPSPTKPQAEEGSGCNFFSIPWCLFWATVLLLIIYLQFSFRSSV（SEQ ID NO:3）。
according to an embodiment of the application, the olfactory receptor comprises at least one of MOR41-1, MOR106-1, MOR103-12 and MOR179-7, and optionally MOR213-1.
According to an embodiment of the present application, the RTP1S protein has an amino acid sequence as shown in SEQ ID NO:1 and/or SEQ ID NO:2, and the olfactory receptor comprises at least one of MOR41-1, MOR106-1, MOR103-12 and MOR 179-7; or the RTP1S protein has an amino acid sequence as shown in SEQ ID NO. 3, and the olfactory receptor comprises at least one of MOR41-1, MOR179-7, MOR106-1, MOR103-12 and MOR213-1.
According to an embodiment of the application, the recombinant cell or recombinant cell line is obtained by introducing into a host cell an expression vector carrying the following sequences: a nucleotide sequence encoding the RTP1S protein and a nucleotide sequence encoding the olfactory receptor.
In this context, the "expression vector" may be one expression vector, two expression vectors, or a plurality of expression vectors. In the case of an expression vector, the nucleotide sequence encoding the RTP1S protein and the nucleotide sequence carrying the coding olfactory receptor are located on the same expression vector; or on two expression vectors, wherein the nucleotide sequence for encoding the RTP1S protein is exemplarily located on a first expression vector, the nucleotide sequence for encoding the olfactory receptor is located on a second expression vector, and the types of the first expression vector and the second expression vector can be the same or different; or on three expression vectors, wherein three nucleotide sequences for encoding three proteins are respectively positioned on one expression vector, and the three expression vectors can be the same or different.
It is noted that, based on the amino acid sequence of the known protein, the nucleotide sequence of the protein can be obtained by methods conventional in the art, for example, available from the ORDB database (Olfactory Receptor DataBase: https:// senselab. Med. Yale. Edu/ORDB /).
In some alternative embodiments of the application, the recombinant cell or recombinant cell line further comprises: carrying a nucleotide sequence encoding a Golf protein; or expresses a Golf protein.
In some alternative embodiments of the present application, the expression vector may further comprise a nucleotide sequence encoding the Golf protein. It may be located in the same expression vector as the nucleotide sequence encoding the RTP1S protein and the nucleotide sequence encoding the olfactory receptor, or may be on a different expression vector, particularly without limitation.
In some embodiments of the present application, the expression vector may be a prokaryotic expression vector or a eukaryotic expression vector, and the specific type is not limited, as long as expression of proteins such as RTP1S protein and olfactory receptor can be achieved.
Illustratively, the expression vector may be a viral expression vector, a lentiviral expression vector, or a plasmid expression vector.
In some alternative embodiments of the application, the host cell may be eukaryotic or prokaryotic.
In some alternative embodiments of the application, the host cell may be a heterologous cell to the olfactory receptor, or may be a homologous cell to the olfactory receptor, such as a cell selected from the group of cells isolated from the olfactory substrate.
Those skilled in the art will recognize that most olfactory receptor proteins are specifically expressed within olfactory neuronal cells, but olfactory neuronal cells are inconvenient for long-term culture in vitro. Thus, herein, "heterologous cell" refers to a cell other than an olfactory neuronal cell. In some alternative embodiments of the application, the host cell includes, but is not limited to, a HEK293 cell, a CHO cell, a Xenopus oocyte, a Hela cell, a COS cell, or a yeast cell.
In another aspect of the application, the application proposes the use of an RTP1S protein as a positive modulator of the olfactory receptor; the RTP1S protein is derived from wild boarsSus scrofaCattleBos taurusHeyingzhu ear batsMyotis lucifugusAt least one of them. The RTP1S protein from the source can be used as a positive regulator of an olfactory receptor, and can improve the functional expression of the olfactory receptor in a heterologous cell.
As used herein, a "positive modulator" refers to a substance that increases the functional expression of an olfactory receptor.
As used herein, "functional expression" refers to the ability of an olfactory receptor to be expressed on a cell as well as to be activated by its ligand, i.e., to be stably anchored to the cell membrane after translational modification of an olfactory receptor protein, and to be normally activated by its ligand and to transmit a signal (i.e., to complete the conversion of a chemical signal and an electrical signal).
According to an embodiment of the application, derived from wild boarsSus scrofaThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 1; or from a mouse ear batMyotis lucifugusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 2; or of bovine originBos taurusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 3.
According to an embodiment of the application, the olfactory receptor comprises at least one of MOR41-1, MOR106-1, MOR103-12 and MOR179-7, and optionally MOR213-1.
According to an embodiment of the present application, the RTP1S protein has an amino acid sequence as shown in SEQ ID NO:1 and/or SEQ ID NO:2, and the olfactory receptor comprises at least one of MOR41-1, MOR106-1, MOR103-12 and MOR 179-7; or the RTP1S protein has an amino acid sequence as shown in SEQ ID NO. 3, and the olfactory receptor comprises at least one of MOR41-1, MOR179-7, MOR106-1, MOR103-12 and MOR213-1.
In yet another aspect of the application, the application provides a method of promoting functional expression of an olfactory receptor in a heterologous cell. According to an embodiment of the application, the method comprises: introducing a nucleotide sequence encoding an RTP1S protein and a nucleotide sequence encoding an olfactory receptor into the heterologous cell; wherein the RTP1S protein is derived from wild boarSus scrofaCattleBos taurusHeyingzhu ear batsMyotis lucifugusAt least one of them. Thus, the functional expression of the olfactory receptor in the heterologous cell can be promoted, and the activating effect of the ligand corresponding to the olfactory receptor on the olfactory receptor can be improved.
According to an embodiment of the application, derived from wild boarsSus scrofaThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 1; or from a mouse ear batMyotis lucifugusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 2; or of bovine originBos taurusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 3.
According to an embodiment of the application, the olfactory receptor comprises at least one of MOR41-1, MOR106-1, MOR103-12 and MOR179-7, and optionally MOR213-1.
According to an embodiment of the application, the introduction of the nucleotide sequence encoding the RTP1S protein and the nucleotide sequence encoding the olfactory receptor into the heterologous cell is achieved by the following steps: constructing an expression vector carrying a nucleotide sequence encoding the RTP1S protein and a nucleotide sequence encoding an olfactory receptor; introducing the expression vector into the heterologous cell.
Herein, "constructing an expression vector carrying a nucleotide sequence encoding the RTP1S protein and carrying a nucleotide sequence encoding an olfactory receptor" may be one expression vector or two expression vectors. In the case of an expression vector, the nucleotide sequence encoding the RTP1S protein and the nucleotide sequence carrying the coding olfactory receptor are located on the same expression vector. In the case of two expression vectors, the nucleotide sequence encoding the RTP1S protein is located in a first expression vector, the nucleotide sequence encoding the olfactory receptor is located in a second expression vector, and the types selected by the first expression vector and the second expression vector can be the same or different.
It is noted that, based on the amino acid sequence of the known protein, the nucleotide sequence of the protein can be obtained by methods conventional in the art, for example, available from the ORDB database (Olfactory Receptor DataBase: https:// senselab. Med. Yale. Edu/ORDB /).
In some alternative embodiments of the present application, the expression vector may further comprise a nucleotide sequence encoding the Golf protein. It may be located in the same expression vector as the nucleotide sequence encoding the RTP1S protein and the nucleotide sequence encoding the olfactory receptor, or may be on a different expression vector, particularly without limitation.
In some embodiments of the present application, the expression vector may be a prokaryotic expression vector or a eukaryotic expression vector, and the specific type is not limited, as long as expression of proteins such as RTP1S protein and olfactory receptor can be achieved.
Illustratively, the expression vector may be a viral expression vector, a lentiviral expression vector, or a plasmid expression vector.
According to an embodiment of the present application, the RTP1S protein has an amino acid sequence as shown in SEQ ID NO:1 and/or SEQ ID NO:2, and the olfactory receptor comprises at least one of MOR41-1, MOR106-1, MOR103-12 and MOR 179-7; or the RTP1S protein has an amino acid sequence as shown in SEQ ID NO. 3, and the olfactory receptor comprises at least one of MOR41-1, MOR179-7, MOR106-1, MOR103-12 and MOR213-1.
In yet another aspect of the application, the application provides a method of detecting an odor compound. According to an embodiment of the application, the method comprises: contacting a sample to be tested with the recombinant cell or recombinant cell line; determining whether the sample to be tested contains the odor compound based on the response result of the recombinant cells or recombinant cell lines after the contacting; wherein the olfactory receptor in the recombinant cell or recombinant cell line is an olfactory receptor that recognizes the odorant compound. As can be seen from the foregoing, the olfactory receptor in the recombinant cell or recombinant cell line has a better functional expression effect. Therefore, the detection of the odor compound by the method comprising the recombinant cell or the recombinant cell line can further improve the sensitivity of detecting the odor compound.
As known to those skilled in the art, olfactory receptors are a class of membrane proteins that are capable of binding to odorant compounds (ligands) and causing intracellular signaling. Thus, herein, "olfactory receptor is an olfactory receptor capable of recognizing the odor compound" means an olfactory receptor capable of binding to the odor compound to be detected (specifically, refer to table 1), in other words, an olfactory receptor capable of being activated according to the odor compound to be detected can be selected.
According to an embodiment of the application, the olfactory receptor comprises at least one of MOR41-1, MOR106-1, MOR103-12 and MOR179-7, and optionally MOR213-1.
According to an embodiment of the application, the presence response value of the recombinant cell or recombinant cell line is an indication that the test sample contains the odorant compound; alternatively, the absence of a response value of the recombinant cell or recombinant cell line is an indication that the test sample does not contain the odorant compound.
According to an embodiment of the present application, the RTP1S protein has an amino acid sequence as shown in SEQ ID NO:1 and/or SEQ ID NO:2, and the olfactory receptor comprises at least one of MOR41-1, MOR106-1, MOR103-12 and MOR 179-7; or the RTP1S protein has an amino acid sequence as shown in SEQ ID NO. 3, and the olfactory receptor comprises at least one of MOR41-1, MOR179-7, MOR106-1, MOR103-12 and MOR213-1.
According to an embodiment of the application, the response value is obtained by detecting a change in the activity of the olfactory receptor.
According to an embodiment of the application, the change in activity is determined by at least one of the following detection methods: luciferase assay, secretory alkaline phosphatase assay, fluorescent protein assay, fluorescent probe assay, ca 2+ Concentration detection, amperometric detection, isotopic labeling, antibody detection and pH detection.
According to an embodiment of the application, the recognition is by cAMP increase.
In some alternative embodiments of the application, the change in cAMP concentration in the cells is achieved by employing GloSensor TM cAMP detection kit or by Dual-Glo ™ double luciferase assay.
Illustratively, the change in cAMP concentration in the cell is accomplished by employing a GloSensor TM cAMP detection kit. The GloSensor-20F cAMP gene construct can pre-express a luciferase variant, and the increase of cAMP concentration can cause conformational change of the luciferase variant,transition of luciferase from inactive to active state, gloSensor TM The cAMP detection kit provides a substrate of a luciferase variant, and the method can measure the concentration change of cAMP in real time, so that the response value of an olfactory receptor can be rapidly and sensitively detected.
The scheme of the present application will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present application and should not be construed as limiting the scope of the application. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The DNA sequences of the olfactory receptors in the present application are obtained from the ORDB database (Olfactory Receptor DataBase: https:// senselab med.
Example 1: screening uOR/ligand
The activity of olfactory receptors (abbreviated as OR) was detected using the Dual-Glo ™ Dual luciferase system. Co-transfecting OR and mouse RTP1S (mRTP 1S), mouse Golf and CRE-luciferase, pLR-SV40 into HEK293T cells, setting a plurality of test groups according to the difference of OR, setting a control group according to each group, wherein the control group and the test group are different in that the mouse RTP1S is not added, and setting a blank control group, wherein the blank control group is not added with any compound (namely a subsequent compound-free stimulation group). After 24 hours, the OR was stimulated with ligand and the activity dependent mRTP1S was screened as OR uOR, see in particular fig. 1.
The results are shown in fig. 1, with the horizontal axis representing the different olfactory receptor/ligand pairs used and the vertical axis representing the RRR luminescence intensity fold (ratio of the luminescence intensity of the compound stimulated group to the luminescence intensity of the non-compound stimulated group). FIG. 1 a) shows the activation of 14 olfactory receptor/ligand pairs in the absence of mRTP1S (w/o mRTP 1S) and in the presence of mRTP1S (w/mRTP 1S), wherein the activation of 7 olfactory receptor/ligand pairs is expressed as an accessory protein dependency, i.e.7 accessory protein dependent olfactory receptors (uOR) are initially screened out. FIG. 1 b) is a duplicate of 7 uOR, with t-test statistical analysis, the activation effect of these 7 olfactory receptors (MOR 41-1, MOR203-1, MOR106-1, MOR103-12, MOR179-7, MOR213-1, MOR 272-1) was significantly different from the corresponding ligand in the absence of mRTP1S (w/o mRTP 1S) and in the presence of mRTP1S (w/mRTP 1S). Wherein the uOR/ligand pairs used for the screening are shown in table 1.
Table 1: uOR and ligand use concentration meter for RTP1s function screening
Example 2: screening of multi-species RTP1S
The Dual-Glo ™ Dual luciferase system was used to detect OR activity. For 7 uOR selected in example 1, 5 of them were selected (MOR 41-1 (Genbank Gene ID:258749,protein ID:NP_666965.2), MOR106-1 (Genbank Gene ID:56858,protein ID:NP_064684.2), MOR103-12 (Genbank Gene ID:258228,protein ID:NP_001011525.1), MOR179-7 (Genbank Gene ID:258362,protein ID:NP_666477.1), MOR213-1 (Genbank Gene ID:258255,protein ID:NP_997032.3)) were co-transfected into HEK293T cells with RTP1S, mouse Golf, CRE-luciferase, pLR-SV40, respectively, a plurality of test groups were set according to uOR, each group was set with a control group which differed from the test group by not adding RTPS in its corresponding test group, and a blank control group was set without adding any compound (i.e., the following no compound-stimulated group). After 24 hours, OR was stimulated with ligand (concentrations of ligand used see table 1), and other species RTP1S were selected that promoted OR activity and were functionally comparable OR even better than mouse OR human RTP1S, see in particular fig. 2.
As shown in FIG. 2, the horizontal axis represents RTP1S (wild boar RTP1S #)Sus scrofa) The amino acid sequence of RTP1S protein of Bos taurus is shown as SEQ ID NO. 1, the amino acid sequence of RTP1S protein of Myotosis lucifugus of Yingkou bata is shown as SEQ ID NO. 2, the amino acid sequence of RTP1S protein of Bos taurus of cattle is shown as SEQ ID NO. 3, the otherThe amino acid sequence of the species RTP1S protein is shown as SEQ ID NO. 4-SEQ ID NO. 17, and the vertical axis is the RRR luminous intensity multiple (the ratio of the luminous intensity of the compound stimulated group to the luminous intensity of the compound stimulated group). Wherein, the wild boar RTP 1S%Sus scrofa) The positive regulation effect of 4/5 uOR/ligand pairs is superior to that of mRTP1S (RTP 1S of murine origin), namely MOR 41-1/coumarin, MOR 106-1/phenylacetaldehyde, MOR 103-12/benzaldehyde and MOR179-7/2,3, 5-trimethyl pyrazine; RTP1S of common cattleBos taurus) The positive regulation effect of 5/5 uOR/ligand pairs is superior to that of mRTP1S, and MOR 41-1/coumarin, MOR 106-1/phenylacetaldehyde, MOR 103-12/benzaldehyde, MOR213-1/2,3, 5-trimethyl pyrazine and MOR179-7/2,3, 5-trimethyl pyrazine are respectively adopted; RTP1S of Yingkou ear batsMyotis lucifugus) The positive regulation effect of 4/5 uOR/ligand pairs is superior to that of mRTP1S, namely MOR 41-1/coumarin, MOR 106-1/phenylacetaldehyde, MOR 103-12/benzaldehyde and MOR179-7/2,3, 5-trimethyl pyrazine.
RTP1S (mouse)mouse) Amino acid sequence:
MCKSVTTGEWKKVFYEKMEEVKPADSWDFIIDPNLKHNVLAPGWKQYLELHASGRFHCSWCWHTWQSPHVVILFHMYLDKAQRAGSVRMRVFKQLCYECGTARLDESSMLEENIESLVDNLITSLREQCYGERGGHYRIHVASRQDNRRHRGEFCEACQEGIVHWKPSEKLLEEEATTYTFSRAPSPTKPQAETGSGCNFCSIPWCLFWATVLMLIIYLQFSFRTSV（SEQ ID NO:4）；
RTP1S (human-human) Amino acid sequence:
MCKSVTTDEWKKVFYEKMEEAKPADSWDLIIDPNLKHNVLSPGWKQYLELHASGRFHCSWCWHTWQSPYVVILFHMFLDRAQRAGSVRMRVFKQLCYECGTARLDESSMLEENIEGLVDNLITSLREQCYGERGGQYRIHVASRQDNRRHRGEFCEACQEGIVHWKPSEKLLEEEATTYTFSRAPSPTKSQDQTGSGWNFCSIPWCLFWATVLLLIIYLQFSFRSSV（SEQ ID NO:5）；
RTP1S (bag badger)Sarcophilus harrisii) Amino acid sequence:
MCKSLTTAEWKKIFYEKMEEAKPADSWDLIMDPNLQHNVLAPGWKQYLEQHASGRFHCSWCWHSWQSSHVVILFHMFLDPTQRMGCVRMRVFRQLCYECGSARQDESSMLEENIEGLVDNLITSLREQCYGERGGQYRIHVAGRQDSRRHRGESCEACLAGINHWKPSEKMLDEEATTYTFSRPASPAKAAEGGGQGCNFCSVPWCLFWATVLLLIIYLQFSFRSSA（SEQ ID NO:6）；
RTP1S (big fox bats)Pteropus vampyrus) Amino acid sequence:
MADKLLRFHCSWCWHTWQSPHVVILFHMYLDRAQRAGSVRMRVFKQLCYECGTARLDESSMLEENIESLVDNLITSLREQCYGERGGHYRIHVASRQDNRRHRGEFCEACNEGIVHWKPSEKLLEEEATTYTFTRAPSPTKPQAEAGSGCNFCSIPWCLFWATVLLLIIYLQFSFRSSV（SEQ ID NO:7）；
RTP1S (macaque)Macaca mulatta) Amino acid sequence:
MCKSVTTDEWKKVFYEKMEEAKPADSWDLIIDPNLKHNVLSPGWKQYVELHASGRFHCSWCWHTWQSPHLVILFHMFLDRAQRAGSVRMRVFKQLCYECGSARLDESSMLEENIEGLVDNLITSLREQCYGERGGQYRIHVASRQDNRRHRGEFCEACQEGIVHWKPSEKLLEEEATTYTFSRAPSPTKPQDETGSGWNFCSIPWCLFWATVLLLIIYLQLSFRSSV（SEQ ID NO:8）；
RTP1S (pilot whale)Globicephala melas) Amino acid sequence:
MCKSVTTGEWKKIFYEKMEAAKPADSWDLIIDPNLKHNVLAPGWKQYLELHASGRFHCSWCWHTWQSPHVVILFHMYLDRAQQAGSVRMRVFKQLCYKCGTARLDESSMLEENIESLVDNLITSLREQCYEERNGQYRIHMASRQDNRRHRGEFCEACQEGIVHWKPSEKLLEEEATTYTFSRAPSPTKPQAKAGSGCNFCSIPWCLFWATVLLLIIYLQFSFHSSV（SEQ ID NO:9）；
RTP1S (Pacific short kistrodon)Lagenorhynchus obliquidens) Amino acid sequence:
MCKSVTTGEWKKIFYEKMEAAKPADSWGLIIDPNLKHNVLAPGWKQYLELHASGRFHCSWCWHTWQSPHVVILFHMYLDRAQQAGSVRMRVFKQLCYKCGTARLDESSMLEENIESLVDNLITSLREQCYEERNGQYRIHMASRQDNRRHRGEFCEACQEGIVHWKPSEKLLEEEATTYTFSRAPSPTKPQAEAGSGCNFCSIPWCLFWATVLLLIIYLQFSFHSSV（SEQ ID NO:10）；
RTP1S (bottle nose dolphin)Tursiops truncatus) Amino acid sequence:
MCKSVTTDEWKKIFYEKMEAAKPADSWDLIIDPNLKHNVLAPGWKQYLELHASGRFHCSWCWHTWQSPHVVILFHMYLDRTQRARSVRVRVFKQLCYECGTARLDESSMLEEDIESLVGNLISSLREQRYGESGGQYRIHVASRQDNRRHRGEFCEACQEGIVHWKPSEKLLEEEAATYTFSRAPSPTKPQAEAGSGCNFCSIPWCLFWATVLLLIIYLQFSFHSSV（SEQ ID NO:11）；
RTP1S (white whale)Delphinapterus leucas) Amino acid sequence:
MCKSVTAGEWKKIFYEKMGAAKPADSWDLIIDPNLKHNVLAPGWKQYLELRASGRFHCSWCWHTWQSPHVVILFHMYLDHAQRAGSVHMRVFKQLCYKCGTARLDESSMLEENIESLVDNLITSLREQCYGESGGQYRIHMASRQDNRRHRREFCEACQEGIVHWKPSEKLLEEEATTYTFSRAPSPTKPQAKAGSGCNFCSIPWCLFWATVLLLIIYLQFSFHSSV（SEQ ID NO:12）；
RTP1S (Momordica grosvenori)Microcebus murinus) Amino acid sequence:
MCKSVTTGEWKKIFYEKMEEAKPADSWDLIIDPNLKHNVLAPGWKQYLELHASGRFHCSWCWHTWQSPHVVILFHMHLDRTVRAGSVRMRVFKQLCYECGTARLDESSMLEENIESLVDNLITSLREQCYGEDPGQYRIHVANRQDTRRHRREFCEACQEGIVHWKPSQKLLEEEANTYIFTPAPSPTKPQTETGSGCNFCSIPWCLFWATVLLLIIYLQFSFRTSV（SEQ ID NO:13）；
RTP1S (African elephant-Loxodonta africana) Amino acid sequence:
MCKSVTTGEWKKVFYEKMEEAKPADSWDLIIDPNLKHNVLAPGWKQYLELHASGRFHCSWCWHTWQSPHVVILFHMHLDRAQRMGSVRMRVFKQLCYECGTSHLDESSMLEENIESLVDNLITSLREQCYGEPGGQYRIHVASRQDTRRHRGEFCEACQEGIVHWKPSEKLLEEEATTYTFTRAPSPTKPQAEGGSGCNFCSIPWCLFWATVLLLLIYLQFSFRSSF（SEQ ID NO:14）；
RTP1S (wild yak)Bos mutus) Amino acid sequence:
MCKSVTTDEWKKIFYEKMEEAKPADSWDLIIDPNLKHNVLAPGWKQYLELHASGRFHCSWCWHTWQSPHLVILFHMYLDRAQRAGSVRMRVFKQLCYEGGTARLDESSMLEENIECLVDNLITSLREQCYGERGGQYRIHVASRQDTRRHRGEFCEACQEGIVHWKPSEKLLEEEATTYTFSRAPSPTKPQAEEGSGCNFFSIPWCLFWATVLLLIIYLQFSFRSSV（SEQ ID NO:15）；
RTP1S (American lion)Puma concolor) Amino acid sequence:
MCKSVTTGEWKKVFYEKMEEAKPADSWDLIIDPNLKHNVLAPGWKQYLELHASGRFHCSWCWHTWQSPHVVILFHMYLDREQRAGSVRMRVFKQLCYECGTARLDESSMLEENIESLVDNLITSLREQCYGEPGGQYRIHVASRQDTRRHRGEFCEACQEGIVHWKPSEKLLEEEATTYTFSRAPSPTKPQAEAGSGCNFCSIPWCLFWATVLLLIIYLQFSFRSSV（SEQ ID NO:16）；
RTP1S (Tiger whale)Orcinus orca) Amino acid sequence:
MCKSVTTDEWKKIFYEKMEAAKPADSWDLIIDPNLKHNVLAPGWKQYLELHASGRFHCSWCWHTWQSPHVVILFHMYLDRAQQAGSVRMRVFKQLCYKCGTARLDESSMLEENIESLVDHLITSLREQCYEERNGLYRIHMASRQDNRRHRGEFCEACQEGIVHWKPSEKLLEEEATTYTFSRAPSPTKPQAEAGSGCNFCSIPWCLFWATVLLLIIYLQFSFHSSV（SEQ ID NO:17）。
in the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present application have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the application, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the application.
Claims (8)
1. A recombinant cell or recombinant cell line comprising:
carrying a nucleotide sequence encoding an RTP1S protein and a nucleotide sequence encoding an olfactory receptor; or alternatively
Expression of RTP1S protein and olfactory receptor;
wherein the recombinant cell or recombinant cell line is obtained by introducing into a host cell an expression vector carrying the following sequences:
a nucleotide sequence encoding the RTP1S protein and a nucleotide sequence encoding the olfactory receptor;
the RTP1S protein is derived from wild boarsSus scrofaOr a mouse ear batMyotis lucifugus；
Derived from wild boarsSus scrofaThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 1;
derived from Yingzhu ear batsMyotis lucifugusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 2;
the olfactory receptor is MOR41-1, MOR106-1, MOR103-12 or MOR179-7.
2. A recombinant cell or recombinant cell line comprising:
carrying a nucleotide sequence encoding an RTP1S protein and a nucleotide sequence encoding an olfactory receptor; or alternatively
Expression of RTP1S protein and olfactory receptor;
wherein the recombinant cell or recombinant cell line is obtained by introducing into a host cell an expression vector carrying the following sequences:
a nucleotide sequence encoding the RTP1S protein and a nucleotide sequence encoding the olfactory receptor;
the RTP1S protein is derived from cattleBos taurus；
Derived from cattleBos taurusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 3;
the olfactory receptor is MOR41-1, MOR179-7, MOR106-1, MOR103-12 or MOR213-1.
3. Use of RTP1S protein as a positive modulator of olfactory receptors;
wherein the RTP1S protein is derived from wild boarSus scrofaOr mouse ear batsMyotis lucifugus；
Derived from wild boarsSus scrofaThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 1;
derived from Yingzhu ear batsMyotis lucifugusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 2;
the olfactory receptor is MOR41-1, MOR106-1, MOR103-12 or MOR179-7.
4. Use of RTP1S protein as a positive modulator of olfactory receptors;
wherein the RTP1S protein is derived from cattleBos taurus；
Derived from cattleBos taurusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 3;
the olfactory receptor is MOR41-1, MOR179-7, MOR106-1, MOR103-12 or MOR213-1.
5. A method of promoting functional expression of an olfactory receptor in a heterologous cell, comprising:
introducing a nucleotide sequence encoding an RTP1S protein and a nucleotide sequence encoding an olfactory receptor into the heterologous cell;
wherein the RTP1S protein is derived from wild boarSus scrofaOr mouse ear batsMyotis lucifugus；
Derived from wild boarsSus scrofaThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 1;
derived from Yingzhu ear batsMyotis lucifugusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 2;
the olfactory receptor is MOR41-1, MOR106-1, MOR103-12 or MOR179-7.
6. A method of promoting functional expression of an olfactory receptor in a heterologous cell, comprising:
introducing a nucleotide sequence encoding an RTP1S protein and a nucleotide sequence encoding an olfactory receptor into the heterologous cell;
wherein the RTP1S protein is derived from cattleBos taurus；
Derived from cattleBos taurusThe RTP1S protein has an amino acid sequence shown as SEQ ID NO. 3;
the olfactory receptor is MOR41-1, MOR179-7, MOR106-1, MOR103-12 or MOR213-1.
7. The method according to claim 5 or 6, wherein said introducing a nucleotide sequence encoding an RTP1S protein and a nucleotide sequence encoding an olfactory receptor into said heterologous cell is accomplished by:
constructing an expression vector carrying a nucleotide sequence encoding the RTP1S protein and a nucleotide sequence encoding an olfactory receptor;
introducing the expression vector into the heterologous cell.
8. A method of detecting an odor compound, comprising:
contacting a sample to be tested with the recombinant cell or recombinant cell line of any one of claims 1-2;
determining whether the sample to be tested contains the odor compound based on the response result of the recombinant cells or recombinant cell lines after the contacting;
wherein the olfactory receptor in the recombinant cell or recombinant cell line is an olfactory receptor that recognizes the odorant compound;
the presence response value of the recombinant cell or recombinant cell line is an indication that the test sample contains the odorant compound; alternatively, the absence of a response value of the recombinant cell or recombinant cell line is an indication that the test sample does not contain the odorant compound;
the response value is obtained by detecting a change in the activity of the olfactory receptor;
the change in activity is determined by at least one of the following detection methods:
luciferase assay, secretory alkaline phosphatase assay, fluorescent protein assay, fluorescent probe assay, ca 2+ Concentration detection, amperometric detection, isotopic labeling and antibody detection.
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