Recombinant cell, construction method and application of recombinant cell in characterization of activity of nicotiana tabacum alkaloid activated nicotinic acetylcholine receptor
By constructing recombinant cells overexpressing nicotine acetylcholine receptors α4 and β2 subunits, and using calcium ion fluorescence probes to detect fluorescence intensity, the problem of cumbersome activity of tobacco alkaloids in the prior art is solved, and a fast and convenient detection effect is achieved.
Patent Information
- Application Number
- CN202510412856.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-02
- Publication Date
- 2025-07-11
AI Technical Summary
In the prior art, detection of the vitality of tobacco alkaloid activation of nicotine-type acetylcholine receptors is complicated and difficult to evaluate quickly and conveniently.
Recombinant cells were constructed, and the α4 and β2 subunits of nicotinic acetylcholine receptors were overexpressed. The fluorescence intensity was detected by calcium ion fluorescence probes to characterize the activity of tobacco alkaloids activated nicotinic acetylcholine receptors.
It provides a fast and convenient method that can characterize the ability of tobacco alkaloids, tobacco extracts and tobacco flue gas to activate nicotine-type acetylcholine receptors at the cellular level. The detection results are accurate and sensitive, and are suitable for evaluating the binding ability and nervous system excitation of cigarettes and nicotine-type acetylcholine receptors.
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Figure CN120290486A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of biological evaluation of tobacco alkaloids and receptor activity, and particularly relates to a recombinant cell, a construction method thereof, and an application thereof in characterizing the activity of tobacco alkaloids in activating nicotinic acetylcholine receptors. Background Art
[0002] Nicotinic acetylcholine receptors (nAChRs) are cysteine-loop ligand-gated ion channels, which are pentamers composed of five identical or different subunits surrounding a central ion channel. When smoking, nicotine enters the lungs with the smoke, and then is absorbed into the blood circulation through the capillaries in the alveoli. The nicotine ingested into the body can quickly pass through the blood-brain barrier, bind to the nicotinic acetylcholine receptors on the surface of central neurons, act on the mesolimbic dopamine system, and then produce a rewarding effect. Repeated nicotine exposure prompts smokers to develop nicotine dependence. The components of the smoke are complex, and more than 7,000 substances have been discovered. In in vitro cytological experiments, it has been found that in addition to nicotine, other alkaloids in the smoke, such as nornicotine, anabasine, anatabine, neonicotine, cotinine, nornicotine, and isonicotine, can all activate or inhibit nAChRs, and then produce neurobiological effects. Therefore, the excitatory effect of smoking on the nerves does not only come from nicotine. The ability of various tobacco alkaloids to activate nAChRs can reflect the activity of the excitatory nervous system to produce a rewarding effect.
[0003] Currently, the detection of the activation ability of nAChRs mainly relies on calcium ion detection methods, and the operation is cumbersome. Summary of the Invention
[0004] Based on this, it is necessary to provide a recombinant cell, a construction method thereof, and an application thereof in characterizing the activity of tobacco alkaloids in activating nicotinic acetylcholine receptors, so as to quickly and conveniently detect the activity of tobacco alkaloids in activating nicotinic acetylcholine receptors.
[0005] In some embodiments, a recombinant cell is provided, and the recombinant cell overexpresses the α4 subunit of nicotinic acetylcholine receptor and the β2 subunit of nicotinic acetylcholine receptor.
[0006] In some embodiments of the provided recombinant cell, the amino acid sequence of the α4 subunit of nicotinic acetylcholine receptor is as shown in SEQ ID NO:1, and / or, the amino acid sequence of the β2 subunit of nicotinic acetylcholine receptor is as shown in SEQ ID NO:2;
[0007] Optionally, the recombinant cell contains the nucleotide sequence encoding the α4 subunit of nicotinic acetylcholine receptor and the nucleotide sequence encoding the β2 subunit of nicotinic acetylcholine receptor;
[0008] Optionally, the nucleotide sequence encoding the nicotinic acetylcholine receptor α4 subunit is as shown in SEQ ID NO:3, and / or the nucleotide sequence encoding the nicotinic acetylcholine receptor β2 subunit is as shown in SEQ ID NO:4.
[0009] In some embodiments, in the provided recombinant cells, the host cell of the recombinant cells is a mammalian cell;
[0010] Optionally, the mammalian cell includes one or more of 293 cells, PC12 cells, and N2A cells.
[0011] In some embodiments, the recombinant cells contain a recombinant expression vector, and the recombinant expression vector carries at least one of the nucleotide sequence encoding the nicotinic acetylcholine receptor α4 subunit and the nucleotide sequence encoding the nicotinic acetylcholine receptor β2 subunit;
[0012] Optionally, the expression vector used for the recombinant expression vector includes one or both of the pCMV-3×Flag-Puro plasmid and the pCDNA3.1-Hyg plasmid.
[0013] In some embodiments, in the provided recombinant cells, the expression vector used for the recombinant expression vector is provided with a selection marker, and the selection marker includes an antibiotic resistance gene.
[0014] Optionally, the antibiotic resistance gene includes one or both of the G418 resistance gene and the hygromycin resistance gene.
[0015] In some embodiments, a method for constructing recombinant cells is provided, including the following steps: transferring the nucleotide of the nicotinic acetylcholine receptor α4 subunit and the nucleotide of the nicotinic acetylcholine receptor β2 subunit into a host cell to obtain the recombinant cells, and the recombinant cells overexpress the nicotinic acetylcholine receptor α4 subunit and the nicotinic acetylcholine receptor β2 subunit.
[0016] In some embodiments, in the method for constructing the provided recombinant cells, the following steps are included: transferring a recombinant expression vector carrying at least one of the nucleotide of the nicotinic acetylcholine receptor α4 subunit and the nucleotide of the nicotinic acetylcholine receptor β2 subunit into the host cell to obtain the recombinant cells.
[0017] In some embodiments, provided is the use of the recombinant cells or the recombinant cells constructed by the construction method in characterizing the activity of activating nicotinic acetylcholine receptors in at least one of tobacco alkaloids, tobacco extracts, and tobacco smoke.
[0018] In some embodiments, a method for characterizing the activity of activating nicotinic acetylcholine receptors by at least one of tobacco alkaloids, tobacco extracts, and tobacco smoke is provided. The recombinant cells or the recombinant cells constructed by the described construction method are used to characterize the activity of activating nicotinic acetylcholine receptors by at least one of tobacco alkaloids, tobacco extracts, and tobacco smoke;
[0019] Optionally, the method includes the following steps:
[0020] In the presence of a buffer, the starved recombinant cells are contacted with a calcium ion fluorescent probe to prepare cells containing the calcium ion fluorescent probe; and,
[0021] At least one of the tobacco alkaloids, tobacco extracts, and tobacco smoke to be tested is contacted with the cells containing the calcium ion fluorescent probe, and the fluorescence intensity is measured.
[0022] In some embodiments, the method for characterizing the activity of activating nicotinic acetylcholine receptors by at least one of tobacco alkaloids, tobacco extracts, and tobacco smoke further includes the following steps: establishing a cumulative dose curve of at least one of the tobacco alkaloids, tobacco extracts, and tobacco smoke to be tested, and calculating the half-maximal effective concentration.
[0023] The aforementioned provided recombinant cells can express α4β2-type nicotinic acetylcholine receptors and can be used to characterize the ability of tobacco alkaloids, tobacco extracts, tobacco smoke, etc. to activate nicotinic acetylcholine receptors. The provided method for characterizing the activity of activating nicotinic acetylcholine receptors by at least one of tobacco alkaloids, tobacco extracts, and tobacco smoke uses α4β2-nAChRs as the target receptor and a calcium ion fluorescent probe as the final detection index, and can characterize the ability of tobacco alkaloids to activate nicotinic acetylcholine receptors at the cell level, with the advantages of convenient, rapid, and efficient detection. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the technical solutions in the embodiments and implementations of the present application and to more fully understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments or implementations. Obviously, the following described drawings are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative efforts.
[0025] Figure 1 It is the plasmid map of pCMV-CHRNA4 (human)-3×flag-Puro in Example 1;
[0026] Figure 2 It is the plasmid map of pCDNA3.1-CHRNB2-Hyg in Example 1;
[0027] Figure 3 To show the activation effect of CSE at different concentrations on α4β2-nAChRs in Example 2;
[0028] Figure 4 To show the cumulative dose-response curves of nicotine and nornicotine on the receptor binding and activation of α4β2-nAChRs in Example 3, where the abscissa is the Log value of the added dose and the ordinate is the percentage of α4β2-nAChRs receptor activation. Detailed implementation manners
[0029] To facilitate the understanding of the present application, the present application will be described more comprehensively below with reference to the relevant drawings. Preferred embodiments of the present application are shown in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present application more thorough and comprehensive.
[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this application belongs. The terms used in the description of the present application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.
[0031] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:
[0032] In the present application, the terms "a plurality of", "multiple", "multiple times", "plurality" etc., unless otherwise specified, refer to a quantity greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.
[0033] The "combinations thereof", "any combinations thereof", "any combination manners thereof" etc. used in the present application include all suitable combination manners of any two or more of the listed items.
[0034] In the present application, the "suitable combination manners", "suitable manners", "any suitable manners" etc. described as "suitable" are subject to being able to implement the technical solution of the present application, solve the technical problems of the present application, and achieve the expected technical effects of the present application.
[0035] In the present application, "preferred", "better", "more preferable", "preferably" are only used to describe the embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of the present application.
[0036] In this application, terms such as "further", "furthermore", "especially", etc. are used for descriptive purposes, indicating differences in content, but should not be construed as limiting the scope of protection of this application.
[0037] In this application, "optionally", "optional", "option" mean that it can be either present or absent, that is, it refers to any one of two alternative options of "present" or "absent". If "optional" appears multiple times in a technical solution, without special instructions, and without contradictions or mutual constraints, each "optional" is independent of each other.
[0038] In this application, in relation to "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.
[0039] In this application, for the technical features described in an open-ended manner, it includes both a closed technical solution composed of the listed features and an open technical solution containing the listed features.
[0040] In this application, regarding numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical values are considered continuous within the above numerical intervals and include the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this article should be understood to include any and all sub-ranges subsumed therein.
[0041] For the temperature parameter in this application, unless otherwise specifically limited, it allows both constant temperature treatment and fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.
[0042] In this application, %(w / w) and wt% both represent weight percentages, %(v / v) refers to volume percentages, and %(w / v) refers to mass-volume percentages.
[0043] "Room temperature" in this application generally refers to 5°C to 30°C, preferably 25 ± 5°C.
[0044] Tobacco alkaloids are structurally similar to acetylcholine and are agonists of nAChRs, which can bind to and activate nAChRs.
[0045] In some embodiments, a recombinant cell is provided, which overexpresses the α4 subunit of nicotinic acetylcholine receptor and the β2 subunit of nicotinic acetylcholine receptor.
[0046] In some embodiments, in the provided recombinant cell, the amino acid sequence of the α4 subunit of nicotinic acetylcholine receptor is as shown in SEQ ID NO:1, and / or, the amino acid sequence of the β2 subunit of nicotinic acetylcholine receptor is as shown in SEQ ID NO:2.
[0047] In some embodiments, the provided recombinant cell contains a nucleotide sequence encoding the α4 subunit of nicotinic acetylcholine receptor and a nucleotide sequence encoding the β2 subunit of nicotinic acetylcholine receptor.
[0048] In some embodiments, the nucleotide sequence encoding the α4 subunit of nicotinic acetylcholine receptor is as shown in SEQ ID NO:3, and / or, the nucleotide sequence encoding the β2 subunit of nicotinic acetylcholine receptor is as shown in SEQ ID NO:4.
[0049] In some embodiments, in the provided recombinant cell, the host cell of the recombinant cell is a mammalian cell.
[0050] In some embodiments, the mammalian cell includes one or more of 293 cells, PC12 cells, and N2A cells.
[0051] In some embodiments, the recombinant cell contains a recombinant expression vector, which carries at least one of the nucleotide sequence encoding the α4 subunit of nicotinic acetylcholine receptor and the nucleotide sequence encoding the β2 subunit of nicotinic acetylcholine receptor.
[0052] In some embodiments, the expression vector used for the recombinant expression vector includes one or both of pCMV-3×Flag-Puro plasmid and pCDNA3.1-Hyg plasmid.
[0053] In some embodiments, in the provided recombinant cell, the expression vector used for the recombinant expression vector is provided with a selection marker, and the selection marker includes an antibiotic resistance gene.
[0054] In some embodiments, the antibiotic resistance gene includes one or both of G418 resistance gene and hygromycin resistance gene.
[0055] In some embodiments, a method for constructing a recombinant cell is provided, comprising the following steps: transferring nucleotides encoding the α4 subunit of the nicotinic acetylcholine receptor and nucleotides of the β2 subunit of the nicotinic acetylcholine receptor into a host cell to obtain a recombinant cell, wherein the recombinant cell overexpresses the α4 subunit of the nicotinic acetylcholine receptor and the β2 subunit of the nicotinic acetylcholine receptor.
[0056] In some embodiments, in the method for constructing a recombinant cell provided, the following steps are included: transferring a recombinant expression vector carrying at least one of nucleotides encoding the α4 subunit of the nicotinic acetylcholine receptor and nucleotides of the β2 subunit of the nicotinic acetylcholine receptor into a host cell to obtain a recombinant cell.
[0057] In some embodiments, provided is the use of a recombinant cell or a recombinant cell constructed by the construction method in characterizing the activity of activating nicotinic acetylcholine receptors in at least one of tobacco alkaloids, tobacco extracts, and tobacco smoke.
[0058] In some embodiments, a method for characterizing the activity of activating nicotinic acetylcholine receptors in at least one of tobacco alkaloids, tobacco extracts, and tobacco smoke is provided, and a recombinant cell or a recombinant cell constructed by the construction method is used to characterize the activity of activating nicotinic acetylcholine receptors in at least one of tobacco alkaloids, tobacco extracts, and tobacco smoke.
[0059] In the traditional detection process, the patch clamp technique is often used to detect the calcium ion flow after the activation of certain receptors in nerve cells. This detection method requires professional equipment to insert an electrode into a single cell, and then add various stimulating substances to detect the weak current between the two electrodes. This detection method is complex, requires professional equipment and professional personnel with rich experience to operate, and the result can only reflect the activation effect of a single nerve cell. In this method, after a calcium ion probe penetrates the cell membrane, a fluorescence detection method is used. This method is simple to operate, can reflect the average calcium ion change of about 5000 cells in a well, and further reflects the binding and activation ability of the stimulant to the α4β2-nAChRs receptor, has better universality, accurate detection results, and high sensitivity.
[0060] In some embodiments, the method for characterizing the activity of activating nicotinic acetylcholine receptors in at least one of tobacco alkaloids, tobacco extracts, and tobacco smoke provided includes the following steps:
[0061] In the presence of a buffer solution, contacting the starved cultured recombinant cell with a calcium ion fluorescence probe to prepare a cell containing the calcium ion fluorescence probe; and,
[0062] Contacting at least one of the tobacco alkaloids, tobacco extracts, and tobacco smoke to be tested with the cell containing the calcium ion fluorescence probe, and measuring the fluorescence intensity.
[0063] In some embodiments, the provided method for characterizing the activity of activating nicotinic acetylcholine receptors in at least one of tobacco alkaloids, tobacco extracts, and tobacco smoke further includes the following steps: establishing a cumulative dose curve of at least one of the tobacco alkaloids, tobacco extracts, and tobacco smoke to be tested, and calculating the half-maximal effective concentration.
[0064] In some embodiments, a method for characterizing the activity of activating nicotinic acetylcholine receptors by tobacco alkaloids at the cellular level is established to explore the agonist ability of at least one of various tobacco alkaloids, tobacco extracts, and tobacco smoke on nAChRs at the cytological level, which is of great significance for the effects, mechanisms, and material basis of the excitatory effect of mainstream smoke on the nervous system.
[0065] In some embodiments, the calcium ion probe is selected as Fluo-8, and the final concentration is 1 μM to 10 μM.
[0066] The provided method for characterizing the activity of activating nicotinic acetylcholine receptors in at least one of tobacco alkaloids, tobacco extracts, and tobacco smoke can be characterized at the cellular level. Using the established α4β2-nAChRs stable cell line to characterize the activity of activating nicotinic acetylcholine receptors in at least one of tobacco alkaloids, tobacco extracts, and tobacco smoke, without repeatedly transiently transfecting plasmids, making the screening more stable and reliable. Using α4β2-nAChRs as the target receptor, this subtype of receptor can better reflect the ability of tobacco alkaloids to excite the nervous system. Using the calcium ion fluorescence probe as the final detection index, the sensitivity is high. The provided method is not limited to tobacco alkaloids and can be extended to tobacco extracts, condensate, or smoke cell exposure systems for evaluating the binding ability of cigarettes and nicotinic acetylcholine receptors and the potential nervous system excitatory ability.
[0067] The present application is further described by the following specific examples, but does not limit the present application.
[0068] The 293 cells used in the examples were derived from the American Type Culture Collection (ATCC), ATCC NO.CRL-1573.
[0069] Example 1 Construction of Recombinant Cells
[0070] (1) Construction of overexpression vectors for human α4 and β2 nicotinic acetylcholine receptor subunits
[0071] a. Obtain mRNA of human cells and synthesize cDNA;
[0072] b. According to the comparison of the sequences of the human nicotinic acetylcholine receptor α4 subunit and the human nicotinic acetylcholine receptor β2 subunit on the website of the National Center for Biotechnology Information, the following primers are designed:
[0073] CHRNA4: 5'-CTTTCTAGAGAATTCATGGAGCTAGGGGGC-3' and 5'-GTCACCGGTCTCGAGGATCATGCCAGCCAG-3',
[0074] CHRNB2: 5'-CTCGGATCCGCCACCATGGCCCGGCGCTGC-3' and 5'-CCCTCTAGACTCGAGCTTGGAGCTGGGGGC-3'.
[0075] Using cDNA as a template, the CDS sequences of CHRNA4 (α4 subunit) and CHRNB2 (β2 subunit) were amplified respectively under the action of high-fidelity enzyme. Among them, the amino acid sequence of the nicotinic acetylcholine receptor α4 subunit is shown in SEQ ID NO:1, and the amino acid sequence of the nicotinic acetylcholine receptor β2 subunit is shown in SEQ ID NO:2. The nucleotide sequence encoding the nicotinic acetylcholine receptor α4 subunit is shown in SEQ ID NO:3, and the nucleotide sequence encoding the nicotinic acetylcholine receptor β2 subunit is shown in SEQ ID NO:4.
[0076] CHRNA4 (α4 subunit) vector ligation: The expression vector was linearized by double digestion. The expression vector was the pCMV-3×Flag-Puro plasmid vector. The amplified CDS sequence of CHRNA4 (removing the stop codon) and the recovered product of the linearized expression vector were recombined by homologous recombination to obtain the homologous recombination product.
[0077] CHRNB2 (β2 subunit) vector ligation: The expression vector was linearized by double digestion. The expression vector was the pCDNA3.1-Hyg plasmid vector, and the plasmid map is as Figure 2 shown. The amplified CDS sequence of CHRNB2 (removing the stop codon) and the recovered product of the linearized expression vector were recombined by homologous recombination to obtain the homologous recombination product.
[0078] The expression vectors of CHRNA4 and CHRNB2 were eukaryotic screening pressures G418 and puromycin respectively.
[0079] c. Transform the above homologous recombination products of CHRNA4 (α4 subunit) and CHRNB2 (β2 subunit) into Escherichia coli for amplification of the target plasmid.
[0080] d. Pick monoclonal Escherichia coli after amplification, and perform PCR identification and sequencing identification after expanded culture. The plasmids with correct sequencing were named pCMV-CHRNA4-3×Flag-Puro (α4 subunit) and pCDNA3.1-CHRNB2-Hyg (β2 subunit) overexpression vectors respectively. The plasmid map of the obtained pCMV-CHRNA4 (human)-3×flag-Puro is as Figure 1 shown, and the plasmid map of the obtained pCDNA3.1-CHRNB2-Hyg is as Figure 2 shown.
[0081] (2)Screening of α4β2-nAChRs stable transfected cell line
[0082] a. Seed 293 cells in a 12-well plate, and co-transfect the above-obtained pCMV-CHRNA4 (human)-3×flag-Puro and pCDNA3.1-CHRNB2-Hyg plasmids into 293 cells.
[0083] b. Add G418 and puromycin screening pressures to screen cells 24 hours after transfection. Change the medium every 2 days and screen continuously for two weeks. The G418 screening pressure is 1000 ng / ml, and the puromycin screening pressure is 120 ng / ml.
[0084] c. Remove the screening pressure and amplify the remaining positive cells. These 293 cells are the positive cells stably transfected with α4-nAChRs and β2-nAChRs plasmids. These cells will stably express the human nicotinic acetylcholine receptor α4 subunit and β2 subunit, and assemble into α4β2-type nicotinic acetylcholine receptor. Name the obtained cells α4β2-nAChRs-293 cells.
[0085] The amino acid sequence of SEQ ID NO:1 is shown as follows:
[0086] MELGGPGAPRLLPPLLLLLGTGLLRASSHVETRAHAEERLLKKLFSGYNKWSRPVANISDVVLVRFGLSIAQLIDVDEKNQMMTTNVWVKQEWHDYKLRWDPADYENVTSIRIPSELIWRPDIVLYNNADGDFAVTHLTKAHLFHDGRVQWTPPAIYKSSCSIDVTFFPFDQQNCTMKFGSWTYDKAKIDLVNMHSRVDQLDFWESGEWVIVDAVGTYNTRKYECCAEIYPDITYAFVIRRLPLFYTINLIIPCLLISCLTVLVFYLPSECGEKITLCISVLLSLTVFLLLITEIIPSTSLVIPLIGEYLLFTMIFVTLSIVITVFVLNVHHRSPRTHTMPTWVRRVFLDIVPRLLLMKRPSVVKDNCRRLIESMHKMASAPRFWPEPEGEPPATSGTQSLHPPSPSFCVPLDVPAEPGPSCKSPSDQLPPQQPLEAEKASPHPSPGPCRPPHGTQAPGLAKARSLSVQHMSSPGEAVEGGVRCRSRSIQYCVPRDDAAPEADGQAAGALASRNTHSAELPPPDQPSPCKCTCKKEPSSVSPSATVKTRSTKAPPPHLPLSPALTRAVEGVQYIADHLKAEDTDFSVKEDWKYVAMVIDRIFLWMFIIVCLLGTVGLFLPPWLAGMI。
[0087] The amino acid sequence of SEQ ID NO:2 is shown below:
[0088] MARRCGPVALLLGFGLLRLCSGVWGTDTEERLVEHLLDPSRYNKLIRPATNGSELVTVQLMVSLAQLISVHEREQIMTTNVWLTQEWEDYRLTWKPEEFDNMKKVRLPSKHIWLPDVVLYNNADGMYEVSFYSNAVVSYDGSIFWLPPAIYKSACKIEVKHFPFDQQNCTMKFRSWTYDRTEIDLVLKSEVASLDDFTPSGEWDIVALPGRRNENPDDSTYVDITYDFIIRRKPLFYTINLIIPCVLITSLAILVFYLPSDCGEKMTLCISVLLALTVFLLLISKIVPPTSLDVPLVGKYLMFTMVLVTFSIVTSVCVLNVHHRSPTTHTMAPWVKVVFLEKLPALLFMQQPRHHCARQRLRLRRRQREREGAGALFFREAPGADSCTCFVNRASVQGLAGAFGAEPAPVAGPGRSGEPCGCGLREAVDGVRFIADHMRSEDDDQSVSEDWKYVAMVIDRLFLWIFVFVCVFGTIGMFLQPLFQNYTTTTFLHSDHSAPSSK。
[0089] The nucleotide sequence of SEQ ID NO:3 is shown below:
[0090]
[0091] The nucleotide sequence of SEQ ID NO: 4 is shown below:
[0092]
[0093] Example 2 Activation of α4β2 nicotinic acetylcholine receptor by cigarette smoke extract (CSE)
[0094] a. α4β2-nAChRs-293 cells were seeded in 24-well plates at a density of 20,000 cells per well. The cell culture plates were specifically for fluorescence detection with a black transparent bottom. After culturing for 12 hours, the medium was changed to DMEM 1640 + 1% FBS and the cells were starved for 12 hours.
[0095] b. The cell culture medium was replaced with HBSS buffer solution, and the calcium ion fluorescence probe Fluo-8 with a final concentration of 10 μM was added. After incubation at 37 °C for 1 h, the cells were washed twice with HBSS to remove the excess probe that did not enter the cells.
[0096] c. Cigarette smoke extracts (CSE) with final concentrations of 1 wt%, 2.5 wt% and 5 wt% were added. After the action, the average fluorescence intensity was measured on a microplate reader under the conditions of an excitation wavelength of 488 nm and an emission wavelength of 520 nm. For easy comparison, the fluorescence intensity after adding acetylcholine was used as the maximum binding capacity (100%). The action time of the test substance was 5 min - 30 min. 10 μM acetylcholine was used as the maximum stimulation concentration.
[0097] The detection results are as Figure 3 shown. From the Figure 3 results, it can be seen that CSE can dose-dependently induce an increase in the cell fluorescence reading, indicating that CSE can significantly activate the α4β2-nAChRs receptor in α4β2-nAChRs-293 cells, and when the CSE concentration reaches 5%, its activation effect can reach 70% (using 10 μM acetylcholine as 100% agonist).
[0098] Example 3 Activation of α4β2 nicotinic acetylcholine receptor by tobacco alkaloids
[0099] Tobacco alkaloids are one of the main substances in the mainstream cigarette smoke, mainly including nicotine, nornicotine, myosmine, anatabine, etc. Tobacco alkaloids are considered to be the main substances that enter the body and activate the reward circuit of the nervous system - the dopamine circuit, and they mainly play this role by binding to nicotinic acetylcholine receptors. Currently, it is generally recognized that nicotine is the main smoke component that activates the dopamine circuit, and there is less research on other tobacco alkaloids.
[0100] Nicotine, nornicotine, anabasine, myosmine, anatabine, cotinine, anhalamine, N-methylanatabine were selected as examples for detecting the binding capacity of nicotinic acetylcholine receptors.
[0101] The α4β2-nAChRs stable transfected cells were seeded into a 96-well black clear bottom fluorescence detection plate at a density of 5000 cells / well. After 24 hours of culture, the cells were starved for 12 hours in low serum medium RPMI 1640 + 1% FBS. Then the cell medium was replaced with HBSS buffer solution, and the calcium fluorescent probe Fluo-8 was added at a final concentration of 5 μM. After incubation at 37 °C for 1 h, the cells were washed twice with HBSS to remove the excess probe that did not enter the cells. Nicotine, nornicotine, anatabine, anabasine, neonicotine, cotinine, anhalamine, and N-methylneonicotine were added gradually from low to high at concentrations of 10 -9 、10 -8 、10 -7 、10 -6 、10 -5 、10 -4 、10 -3 M (with 10 μM acetylcholine as the positive control). After acting for 5 min, the average fluorescence intensity was measured on a microplate reader under the conditions of an excitation wavelength of 488 nm and an emission wavelength of 520 nm. Thus, the cumulative dose curve of this tobacco alkaloid was established, and the EC50 was calculated. For convenient comparison, the fluorescence intensity of adding 10 μM acetylcholine was taken as the maximum binding capacity (100%).
[0102] The cumulative curves of nicotine and nornicotine are shown in Figure 4 , and based on this curve, the EC50 values for nicotine and nornicotine binding to and activating α4β2-nAChRs were calculated to be 0.75 ± 0.29 μM and 7.88 ± 1.48 μM, respectively. This difference in EC50 can reflect to a certain extent the strength of the dependence produced by this substance.
[0103] Table 1 shows the results of the activation effects of different tobacco alkaloids on α4β2-nAChRs.
[0104] Table 1. EC50 of tobacco alkaloids for activating α4β2-nAChRs
[0105]
[0106] From the results in Table 1, it can be seen that nicotine and anhalamine have the strongest activation effects on α4β2-nAChRs, followed by nornicotine and neonicotine. The activation effects of anatabine, N-methylneonicotine, and cotinine (a nicotine metabolite) are further reduced, and anabasine has almost no activation effect on α4β2-nAChRs. These results can reflect indirectly the strength of the production of euphoria and dependence by these substances in activating the α4β2-nAChRs receptor in nerve cells in vivo.
[0107] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as within the scope described in this specification.
[0108] The above-described embodiments merely represent several implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.
Claims
1. A recombinant cell, characterized in that, The recombinant cell overexpresses the nicotinic acetylcholine receptor α4 subunit and the nicotinic acetylcholine receptor β2 subunit.
2. The recombinant cell according to claim 1, wherein, The amino acid sequence of the nicotinic acetylcholine receptor α4 subunit is as shown in SEQ ID NO:1, and / or, the amino acid sequence of the nicotinic acetylcholine receptor β2 subunit is as shown in SEQ ID NO:2; Optionally, the recombinant cell contains a nucleotide sequence encoding the nicotinic acetylcholine receptor α4 subunit and a nucleotide sequence encoding the nicotinic acetylcholine receptor β2 subunit; Optionally, the nucleotide sequence encoding the nicotinic acetylcholine receptor α4 subunit is as shown in SEQ ID NO:3, and / or, the nucleotide sequence encoding the nicotinic acetylcholine receptor β2 subunit is as shown in SEQ ID NO:
4.
3. The recombinant cell according to claim 1 or 2, characterized in that, The host cell of the recombinant cell is a mammalian cell; Optionally, the mammalian cell includes one or more of 293 cells, PC12 cells, and N2A cells.
4. The recombinant cell according to any one of claims 1 to 3, characterized in that, The recombinant cell contains a recombinant expression vector, and the recombinant expression vector carries at least one of a nucleotide sequence encoding the nicotinic acetylcholine receptor α4 subunit and a nucleotide sequence encoding the nicotinic acetylcholine receptor β2 subunit; Optionally, the expression vector used for the recombinant expression vector includes one or both of pCMV-3×Flag-Puro plasmid and pCDNA3.1-Hyg plasmid.
5. The recombinant cell according to any one of claims 1 to 4, characterized in that, The expression vector used for the recombinant expression vector is provided with a selection marker, and the selection marker includes an antibiotic resistance gene. Optionally, the antibiotic resistance gene includes one or both of G418 resistance gene and hygromycin resistance gene.
6. A method for constructing a recombinant cell, characterized in that, It includes the following steps: transferring the nucleotide encoding the nicotinic acetylcholine receptor α4 subunit and the nucleotide encoding the nicotinic acetylcholine receptor β2 subunit into a host cell to obtain the recombinant cell according to any one of claims 1 to 5, and the recombinant cell overexpresses the nicotinic acetylcholine receptor α4 subunit and the nicotinic acetylcholine receptor β2 subunit.
7. The construction method according to claim 6, characterized in that, It includes the following steps: transferring a recombinant expression vector carrying at least one of a nucleotide encoding the nicotinic acetylcholine receptor α4 subunit and a nucleotide encoding the nicotinic acetylcholine receptor β2 subunit into the host cell to obtain the recombinant cell.
8. Use of the recombinant cell according to any one of claims 1 to 5 or the recombinant cell constructed by the construction method according to any one of claims 6 to 7 in characterizing the activity of activating nicotinic acetylcholine receptors in at least one of tobacco alkaloids, tobacco extracts, and tobacco smoke.
9. A method for characterizing the activity of activating nicotinic acetylcholine receptors by at least one of tobacco alkaloids, tobacco extracts and tobacco smoke, characterized in that, Characterizing the activity of activating nicotinic acetylcholine receptors in at least one of tobacco alkaloids, tobacco extracts, and tobacco smoke using the recombinant cell according to any one of claims 1 to 5 or the recombinant cell constructed by the construction method according to any one of claims 6 to 7; Optionally, the method includes the following steps: In the presence of a buffer, contacting the starved recombinant cell with a calcium ion fluorescent probe to prepare a cell containing the calcium ion fluorescent probe; and, Contacting at least one of the tobacco alkaloids, tobacco extracts, and tobacco smoke to be tested with the cell containing the calcium ion fluorescent probe, and measuring the fluorescence intensity.
10. The method according to claim 9, characterized in that, The method further comprises the following steps: establishing a cumulative dose curve of at least one of the tobacco alkaloids, tobacco extracts, and tobacco smoke to be measured, and calculating the median effective concentration.