TW201215676A - Cell line of stably expressing cannabinoid receptor CB2 and application thereof - Google Patents

Abstract

A cell line of stably expressing cannabinoid receptor CB2 is disclosed, which is transfected with a recombinant plasmid that contains a full-lengh CB2 gene, so that it stably expresses cannabinoid receptor CB2 recombinant protein on its cell membrane surface. Moreover, a method of screening potential ligands of CB2 receptor is also disclosed, which is to culture the aforementioned cell line in the medium that contains a sample solution or not for a period, to measure the relative changes of target molecule concentrations, and then to determine that the sample solution contains potential ligands according to the aforementioned changes. Therefore, it is a fast, safe and accurate way to find out potential ligands of CB2 receptor from a large number of samples.

Description

201215676 VI. Description of the invention: Cao [Technical field to which the invention belongs] The present invention relates to a cell strain and its application to screening compounds, in particular to a cell line expressing a cannabinoid receptor recombinant protein and application thereof Screen for potential ligands for cannabinoid receptors. [Prior Art] There are about 1,200 G-protein coupled receptors in the human gene pool (G-Protein-Coupled Receptors; GPCR), and many diseases have been found to be involved in the regulation of G-protein-coupled receptors, so g-protein coupling is affected. The body is often used as one of the modes of pharmacological research. In the G protein-coupled receptor j superfamily, cannabinoid receptors (CB; or CB receptors) are involved in the regulation of the immune system, and are mainly distributed in peripheral tissues, cells, and immune cells. In humans, CB receptors can be divided into at least two types according to their receptor primary structure, ligand binding site and signal transmission system, respectively, 'CB1 receptor and CB2 receptor. Recent studies have confirmed that CB2 receptors are also expressed in normal nerve cells. The human CB2 gene is located at the position of the short arm end 36 on the first pair of chromosomes, and a protein consisting of 360 amino acids can be translated. When the chronic inflammatory disease associated with the central nervous system (CNS) occurs, the expression of CB2 receptors on microglia is also significantly increased. In the past, it was found that drugs invented for the regulation of CB1 receptors have adverse drug side effects such as central nervous system, such as euphoria, confusion of time and space, lethargy, dizziness, and exercise 201215676 Motor discoordination, memory loss (men^ lapses), so that there are restrictions on the application of drugs for regulating CB1 receptors. In contrast, the drug invented for the purpose of regulating the CB2 receptor does not have the above-mentioned adverse drug side effects.

The drugs invented by the vertical δ stomach for the purpose of regulating the CB2 receptor have no adverse side effects of the above-mentioned drugs. However, in fact, in screening for drugs targeting the CB2 receptor, the following difficulties are encountered. First, most of the fascination still uses radioactive elements to calibrate ligands to directly analyze the binding activity of CB2 receptor ligands. However, calibrating ligands with radioactive elements requires expensive consumables and the operator is exposed to potential radiation activity hazards. Although the CB2 selective ligand of the fluorescent substance is calibrated, there is no risk of radiation, but it is not currently on the market. There is only one ligand for the calibration phosphor that is expected to be available soon, namely "near, infrared dye labeled mbc94 (NIRmbc94)", where mbc94 is the bondable CB2 ligand SR144528. The analog (conjUgabie analogue) has a relative affinity of CB2/CB1 of greater than 700, but NIRmbc94 is still not widely available. In addition, many CB2 selective ligands, after being calibrated with fluorescent substances, have a significant reduction in affinity, although they reduce the risk of radioactive element calibration. For this reason, the progress of ligands for CB2 receptors remains at the research stage. Secondly, changes in the protein content involved in the downstream signal transduction pathway of the CB2 receptor can be determined, for example, changes in the activity of mitogen-activated protein kinase (MAPK) To indirectly analyze the binding activity of the CB2 receptor-ligand. However, this method is susceptible to many 201215676 external factors and human error. In addition, the literature has used the human CB2 receptor gene, and the chimeric G protein gene of Gaqi5 • and G«q〇5, which are cotransfection of cells, thereby analyzing CB2. Receptor-ligand binding activity. When the ligand binds to the CB2 receptor protein, the CB2 receptor protein can be coupled to both the chimeric proteins of G«qi5 and Gaq〇5, and further activate the intracellular phospholipase C-inositol inositol-di The morphine calcium ion (phospholipase C_inositol trisphosphate-Ca2+; PLC-IP3-Ca2+) message transmits the φ-path, which causes the concentration of intracellular divalent calcium ions to change, and then measures the high-permeability divalent calcium ion image. To study the functional properties of CB2 receptors and agonists and antagonists. By measuring the change in the concentration of divalent calcium ions, although the operator is prevented from being exposed to the potential risk of radiation activity, the following disadvantages exist: (1) only the change of the image can be observed, and it is difficult to perform quantitative analysis; Co-transfected cell lines are less likely to select stable expression strains, and the plastids transfected into cells are easily lost. In summary, the above screening method for regulating potential ligands of the CB2 receptor has its own limitations and inevitable risks. In view of this, it is urgent to propose a cell line stably expressing the CB2 receptor for screening for a potential ligand for modulating the CB2 receptor. SUMMARY OF THE INVENTION Accordingly, one aspect of the present invention provides a cell line stably expressing a cannabinoid receptor 2 (CB2; or CB2 receptor) recombinant protein, which utilizes a CB2 receptor. The recombinant plastid of the full-length gene was transfected into the cell line to stably express CB2 by the 201215676 recombinant protein on the cell membrane surface. Another aspect of the present invention provides a method for screening a potential ligand for a cannabinoid receptor, wherein the cell strain is cultured in a culture solution with or without a sample solution, and cultured for a period of time. And detecting, comparing and analyzing the difference between the content of the specific strain of the cell strain cultured with or without the sample solution, and judging whether the sample solution contains a ligand specifically binding to the CB2 receptor recombinant protein according to the difference . When the difference between the current numbers is not zero, it is judged that the sample solution contains potential ligands, thereby permitting rapid and safe screening and screening of potential ligands for the cannabinoid receptor CB2 from a large number of samples. According to the above aspect of the present invention, a cell strain stably expressing a recombinant protein of the cannabinoid receptor is proposed. In one embodiment, the cell strain can have a recombinant plastid, and the recombinant plastid has a nucleic acid sequence having the sequence of the sequence number 丨, thereby expressing a cannabinoid receptor such as the sequence of SEQ ID NO: 2 2 (cannabinoid receptor 2; CB2) recombinant protein. According to an embodiment of the present invention, the cell strain described above may be, for example, human embryonic kidney epithelial cell line HEK293 (American Type Culture Collection Center • [American TyPe Culture Collection; ATCC] registration number: ATCC CRL-1573). According to other aspects of the invention, a method of screening for potential ligands for cannabinoid receptors is also provided. In one embodiment, first, a second culturing step is performed in which the above cell strain is cultured in a culture solution containing or not containing a sample solution, and cultured at 37 ° C, 5% carbon dioxide for 1 hour to 2 hours. It is claimed that the cell line exhibits a cannabinoid receptor 2 (CB2) recombinant protein such as the sequence shown in SEQ ID NO: 2. Next, a detecting step is performed to detect the first 201215676 content of the target substance of one of the cell lines cultured through the sample solution, and to detect the second content of the target substance through the cell line cultured without the sample solution The target may be, for example, a cyclic adenosine monophosphate (cAMP) or a mitogen-activated protein kinase (MAPK). Thereafter, a comparison step is performed to obtain a difference between the first content and the second content. Then, an analysis step is performed to determine whether the sample solution contains a potential ligand specifically bound to the CB2 receptor recombinant protein according to the difference, wherein when the difference is not zero, φ judges that the aforementioned sample solution contains a potential ligand. According to an embodiment of the present invention, the above potential ligand inhibits, for example, the first content of cAMP produced by the aforementioned cell strain by the forskolin. According to an embodiment of the invention, the potential ligand described above promotes a first content such as MARK. A cell strain stably expressing a cannabinoid receptor CB2 of the present invention and a method for screening a potential ligand of a cannabinoid receptor, which is a recombinant plastid containing a full-length gene of a CB2 receptor Dyeing to the cell line and stably expressing the CB2 receptor recombinant protein on the cell membrane surface, and culturing the above cell strain in the culture solution with or without the sample solution, and then analyzing the change of the target content to determine whether the sample solution contains or not The CB2 receptor recombinant protein specifically binds to potential ligands, thereby rapidly, safely and correctly screening potential ligands for cannabinoid receptors from a large number of samples. [Embodiment] As described above, the present invention provides a cell line 201215676 which stably expresses a CB2 receptor and uses the same, which is a recombinant plastid containing a full-length gene of a CB2 receptor, and transfected into a human kidney cell strain and on the surface of a cell membrane. Stable expression of the CB2 Recombinant Protein' to utilize this cell line to rapidly, femalely and correctly select potential ligands for cannabinoid receptors from a large number of samples. The term "cannabinoid receptor CB2 (or CB2 receptor)" as used herein refers to a member of the superfamily of G pr〇tein_c〇upled receptors (GPCR). CB2 receptors are mainly distributed in immune cells as well as in peripheral cells and tissues. Recent studies have indicated that performance • CB2 receptors on immune cells can affect the immunomodulation of endogenous ligands. Many literatures have confirmed that CB2 receptors are also expressed in artificially cultured neurons and nervous systems, and are involved in the regulation of chronic pain and neurodegenerative diseases. In the CB2 receptor full-length gene shaving mouse experiment, it was confirmed that the CB2 receptor has an activity of regulating the co-stimulatory factor (c〇_stimulat〇ry fact〇r) on macrophages. As previously indicated, the drug developed by the regulation of the CB1 receptor has adverse drug side effects such as the central nervous system, so that there are many restrictions on the application of drugs for regulating the CB1 receptor. In contrast, the drug invented for the purpose of regulating the CB2 receptor does not have the above-mentioned adverse drug side effects. For example, it is known that the active substances of echinacea, alkylamides and noni fruit extracts, can bind to the CB2 receptor, thereby regulating the immune response. The term "CB2 receptor recombinant protein" as used herein refers to a polymerase chain reaction (PCR) using a specific primer pair from a human cDNA, such as a commercially available human spleen tissue cDNA. • A nucleic acid fragment that increases the full-length gene of the CB2 receptor. The term "CB2 201215676 receptor full-length gene" as used herein refers to an open reading frame (ORF) comprising the full-length gene of the CB2 receptor, that is, when the nucleic acid fragment is transcribed into mRNA and translated into a CB2 receptor, An amino acid sequence comprising a start codon and a stop codon (st〇p codon) can be shown. In one embodiment, the aforementioned CB2 receptor recombinant protein may, for example, be a protein and/or a tag sequence consisting of 36 amino acids. In one example, the tag sequence of the CB2 receptor recombinant protein can be provided by a vector, wherein the tag sequence can be, for example, a tag sequence of other amino acids and several histidines; His. In another illustration, the tag sequence can be, for example, a tag sequence consisting of i tyrosine ( Tyrine) and 6 histidine acids. It is additionally noted herein that the above method of introducing a tag sequence should be well known to those of ordinary skill in the art to which the present invention pertains, and that the aforementioned tag sequence does not affect the CB2 receptor recombinant protein and is described by the following description. The function of ligand specificity. In one embodiment, the aforementioned specific primer pair may be based on, for example, the National Center for Biotechnology Information (National Center for National Information Center).

Biotechnology Information; NCBI) GenBank, the gene sequence of the human cannabinoid receptor CB2 (hCB2) published by Munro et al. in 1993 (number: ΝΜ_001841.2, a total of 1789 bases) For (base pairs; bp)), the sequence design of this primer pair is performed using DNASTAR Expert Sequence Analysis Software, such as EditSeq 5.0 (DNASTAR Inc.), where the primer pair includes the upstream primer and the downstream primer, and the upstream primer 5, the end is designed with a first restriction enzyme cleavage 'and is complementary to the first base to the sixth base of the hCB2 receptor gene sequence, as shown in sequence identification number 3; and the downstream primer 5, the end is set to 201215676 A second restriction enzyme cleavage site is defined and is complementary to the 1066th base to the 1083th base of the hCB2 receptor gene sequence, as shown in SEQ ID NO: 4. In an example, the first restriction enzyme may be, for example, particularly /7 «1, and the second restriction enzyme may be, for example, Jgel. A nucleic acid fragment of hCB2 receptor of about 1083 bp in sequence of sequence identification number 1 was amplified by human CDNA using the above primer. The cited pairs of the above-described examples are shown in Table 1 which will be described later. The term "cell strain" as used herein, in one embodiment, refers to the human embryonic kidney epithelial cell line HEK293, wherein the cell line is purchased from a ginseng such as the American Type Culture Collection [American Type Culture].

Collection; ATCC], HEK293 cell line with accession number ATCC CRL-1573. In this example, the present invention excludes the use of HEK293T as a cell line for the practice of the present invention. The "recombinant plasmid containing a full-length CB2 gene" as used herein means a recombinant plasmid having a full-length CB2 gene. In one embodiment, the recombinant plastid containing the CB2 full-length gene can be subjected to a restriction enzyme, such as a sequence, by a nucleic acid fragment of the hCB2 receptor full-length gene (such as the sequence number 1). „] and/or J for I′ truncation and purification of the nucleic acid fragment of the hCB2 receptor full-length gene, and then ligated and ligated, for example, in a vector treated with the same restriction enzyme and purified, for example, mammalian cell expression The vector is formed to form a recombinant plastid and is sequenced to determine the sequence constructed correctly. It should be noted that the restriction enzymes designed herein differ only in the sequence side depending on the vector to be ligated, and thus the present invention-embodiment The restriction enzyme cleavage sequence is not limited to the above. The above cited examples are shown in Table i: Table 1 201215676 Sequence Identification Number Sequence 5, - CGCGGTACCT CGATTATGGA G -3' 3 4

Kpnl - Ϊ08Ϊ ^ 5,- ACCGGTAC AG CAATCAGAGA GGTC -3' Agel _ It should be further noted that the term "vector" or "expression carrier" as used herein refers to a vector in which a gene is not embedded. The term "heavy plastid" or "expressing plastid" refers to a recombinant plasmid in which the full-length gene of CB2 has been inserted. The "stable expression" system referred to herein is referred to herein. The above-mentioned "recombinant plasmid" is introduced or transfected into the above cell line, and the selected CB2 receptor "cell strain" is stably expressed. The above method of introducing the recombinant plastid into the cell strain is not limited, and any well-known manner can be used, for example, calcium phosphate, diethylaminoethyl-dextran; DEAE-dextran ), #liposome, electroporation, microinjection, retrovirus infection, gene-gun, and the like. Furthermore, since the above method of introducing or transfecting a cell 'should be well known to those of ordinary skill in the art to which the present invention pertains, and by routine experimentation, cells stably expressing the CB2 receptor recombinant protein can be selected. Strain, so I won't go into details here. Since the cell line of the present invention can stably express the CB2 receptor recombinant protein, it can solve the problem that the screening platform of the current CB2 receptor ligand is too high, and the radioactive element calibrates the ligand to expose to the radiation. The photometric calibration ligand has affinity-[S] 12 201215676 The problem of drastic drop in 'the cell line of the selected CB2 receptor is unstable' or it is difficult to exclude the interference of external interference by indirect analysis, and it is difficult to quantify Analysis and other issues. The term "screening a potential ligand for a CB2 receptor" as used herein means a potential ligand which specifically binds to a CB2 receptor by using the above-described cell line stably expressing a recombinant protein of the CB2 receptor. In one embodiment, the present invention cultures the above cell strain in a culture solution containing or not containing a sample solution, and after detecting for a period of time, detects, compares, and analyzes the cell strain cultured through the solution containing the sample φ product. The content of the target is varied to determine whether the sample solution contains a potential ligand that specifically binds to the CB2 receptor recombinant protein. In the foregoing examples, the "target" used to evaluate a potential ligand generally refers to a protein involved in the downstream signaling pathway of the CB2 receptor after the potential ligand is specifically bound to the CB2 receptor. The change in content, such as cyclic aden〇sine monophosphate (cAMP) or mitogen-activated protein kinase (MAPK), determines whether a sample solution contains potential ligands. As used herein, "potential ligand" means a change in the content of the above target. Referring to Figure 1 there is shown a schematic representation of a portion of the signaling pathway involved in CB2 receptors stimulated by cannabinoid derivatives (or potential ligands) in accordance with an embodiment of the present invention. In general, intracellular 105 adenylate cyclase (AC) catalyzes the production of CAMP, while intracellular cAMP accumulation directly regulates the protein kinase A (PKA)-dependent signal transduction pathway. Furthermore, it regulates the expression of interleukin-m 13 201215676 -2 (interleukin-2; IL-2) and inducible nitric oxide synthase (iNOS) in immune cells. When the CB2 receptor on cell membrane 101 is stimulated by a cannabinoid derivative (or a potential ligand) such as extracellular 1〇3, it can be coupled with an inhibitory G protein (Gi) in the cell 105 to inhibit downstream signaling. Pathway adenylate cyclase (AC) activity, which in turn inhibits intracellular cAMP accumulation. Thus, in one embodiment, a cell line stably expressing a CB2 receptor and a potential ligand can be assessed by measuring changes in intracellular cAMP accumulation stimulated by a cannabinoid derivative (or a potential ligand). Sexual ability. Second, CB2 receptors stimulated by cannabinoid derivatives (or potential ligands) such as extracellular 103, coupled with intracellular 105 inhibitory G protein (Gi), can also stimulate mitosis in downstream signaling pathways. The activation of mitogen-activated protein kinase (MAPK) is shown in Figure 1, where the activation of sputum is related to the migration of immune cells. Thus, in another embodiment, stability can be assessed by measuring activation of a phosphorylated intracellular mitogen-activated protein kinase (μαρκ) stimulated by a cannabinoid derivative (or a potential ligand). The ability of a cell line expressing a CB2 receptor to specifically bind to a potential ligand. In addition, the ligand-stimulated CB2 receptor also stimulates intracellular 105 synthesis of ceramide, which causes the cells to produce pro-apoptotic responses, thereby inhibiting the growth of various tumor cells. When assessing the ability of a cell line stably expressing a CB2 receptor to specifically bind to a potential ligand, the above cell strain ' can be cultured in a culture solution containing or not containing a sample solution, and after culture for a while, for example, at 37. 〇, 5% dioxin 201215676 cultured for 1 hour to 20 hours under carbon, detecting, comparing and analyzing the content change of the above target substance through the cell strain containing the sample solution to determine whether the sample solution contains CB2 A potential ligand for the specific binding of a recombinant protein. In an example, the detecting step detects a first content of cAMP (and/or MAPK) of the cell strain cultured through the sample solution, and detects cAMP (through the cell strain not cultured with the sample solution) (and / or MAPK) second content. Thereafter, a comparison step is performed to obtain a difference between the first content and the second content of cAMP (and/or MAPK) at φ. Then, an analysis step is performed to determine whether the sample solution contains a potential ligand specifically bound to the CB2 receptor recombinant protein according to the difference described above, wherein when the difference is not zero, the sample solution is judged Contains potential ligands. In another illustration, when evaluated by cAMP, the above potential ligand can inhibit the content of cAMP produced by the above-mentioned cell strain by, for example, the extract of the coleus extract (f〇rsk〇iin). In another illustration, when evaluated by ΜΑρκ, the above potential ligands of φ promote the content of the activated state MARK, for example, the protein content of the activated state MARK. By the above-described change in the content of cAMp (and/or ΜΑρκ), it can be judged whether or not a sample solution contains a potential ligand specifically bound to the CB2 receptor recombinant protein. Since the present invention is screened using a cell line stably expressing the CB2 receptor, a potential ligand for regulating the cannabinoid receptor CB2 can be rapidly, safely and correctly screened from a large number of samples. The following embodiments are used to illustrate the application of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various changes without departing from the spirit and scope of the present invention. And refinement 0 Example 1: Construction of a recombinant plastid of the cannabinoid receptor CB2 full-length gene and large-scale culture 1. Synthesis of a nucleic acid fragment of the hCB2 receptor full-length gene by polymerase chain reaction

This example is a recombinant plasmid, a transgenic strain and a large number of cultures which construct the hCB2 receptor protein gene. The nucleic acid fragment of the hCB2 receptor was obtained by amplifying the polymerase chain reaction using a primer pair from a commercially available human spleen tissue cDNA (Clontech, California, U.S.A.). In other words, the primer pair and the human spleen tissue cDNA can be added to a commercially available PCR reaction reagent (for example, 1 Ox Easy-A Reaction Buffer, Stratagene, USA) to carry out a polymerase chain reaction. Obtaining a nucleic acid fragment of the hCB2 receptor, wherein the reagent for the PCR reaction is as exemplified in Table 2: _ Table 2

Reagent volume (μΙ〇 upstream primer (2.5 mM) 5 downstream primer (2.5 mM) 5 10x Easy-ATM Reaction Buffer (Stratagene, USA) 5 dNTPs (2.5 mM; Bertec, Taipei, Taiwan) 5 Secondary deionized water 28 DNA polymerase (Easy-ATM High-Fidelity PCR Cloning 1

Enzyeme, Stratagene, USA) 201215676 Human spleen tissue cDNA__]_ Total 50 When a polymerase chain reaction with human spleen tissue cDNA is performed using a primer pair, the reaction conditions can be exemplified by, for example but not limited to, the following: First, at 94 ° C The double-stranded template DNA is subjected to a pre-denaturation reaction for 5 minutes to 10 minutes. Next, the reaction is carried out at 94 ° C for 30 seconds to 1 minute, and the reaction is carried out at 50 ° C to 60 ° C for 30 seconds to 1 minute to carry out the primer-template DNA annealing reaction at 72 ° C for 30 seconds. 2 minutes of DNA extension reaction, which is a cycle, a total of 30 to 40 cycles of the reaction, but in another embodiment, the above primer pair-template DNA adhesion reaction can be, for example, 55 ° C The reaction was carried out at 60 ° C for 30 seconds, and further at 72 ° C for 1 minute to 1.5 minutes, and a total of 35 cycles of the reaction were repeated. Please refer to FIG. 2, which is a diagram of agaric electrophoresis analysis of a nucleic acid fragment of the hCB2 receptor according to an embodiment of the present invention, wherein the first lane represents a DNA ladder with a 100-base pair (DNA ladder, Bertec). Company, Taipei, Taiwan), and Lane 2 represents a nucleic acid fragment containing the full-length gene of the hCB2 receptor, as indicated by arrow 201. From the results of the second lane of Fig. 2, it was found that the nucleic acid fragment of the full-length gene of the hCB2 receptor (about 1083 bp; as indicated by the arrow 201) was indeed specifically amplified from the cDNA of the human spleen tissue by the primer pair. 2. Constructing a recombinant plastid containing the full-length gene of hCB2 receptor and preparing a transformed strain by the above polymerase chain reaction, the obtained hCB2 is subjected to a restriction enzyme action by a nucleic acid fragment of the full-length gene of [S] 17 201215676, for example, Both the illusion i and NEW ENGLAND Biolabs, MA. USA), after cutting and purifying the nucleic acid fragment of the hCB2 receptor full-length gene, can be constructed and ligated by the same restriction enzyme treatment (for example, ρρπΐ and jgel, both are NEW) ENGLAND Biolabs, MA. USA) and purified vector, for example, mammalian cell expression vector pcDNA3.1/V5-His TOPO TA (Invitrogen, USA) vector' to form recombinant plastids to obtain full-length genes containing hCB2 receptor The recombinant plastid of the nucleic acid fragment (or (pcDNA3.1-hCB2). The sequence of the recombinant plastid is also confirmed by PCR and DNA sequencing. However, the above-mentioned restriction enzyme action of the recombinant plastid, construction of recombinant plastid, and ligation The reaction and the like are well known to those of ordinary skill in the art and will not be further described herein. The above recombinant plasmid can be further transformed into a suitable host, such as a large intestine rod. (5. Co/〇 XL-l Blue strain competent cell, as the host for the transfer and preservation of the above recombinant plastid. After that, blue-white screening, ie using isopropyl-/5-D - isopropyl-beta-D thiogalactopyranoside (IPTG; Amresco, USA) induces the lactose operator of recombinant plastids (/operon) and utilizes 5-bromo-4-chloro-3-indole -bD-galactoside (5-bromo-4-chloro-3-indolyl-bD-galactopyranoside; X-gal) develops colonies of successful transformation, and determines the sequence of the bacteria by PCR and DNA sequencing. A large number of cultures can be carried out, but the above-mentioned construction of recombinant plastids, transformation to bacteria, mass culture, extraction of plastid DNA, optical density (OD 260) value, establishment of standard curve, PCR and DNA sequencing, etc. It is well known to those of ordinary skill in the art, and therefore will not be further described herein. 201215676 Please refer to FIG. 3, which is a diagram of agarose electrophoresis analysis of recombinant plastids by restriction enzymes according to an embodiment of the present invention. The first lane indicates the DNA ladder with 1000 base pairs. The DNA ladder (Bertec's 'Taipei, Taiwan'), the second lane indicates the nucleic acid fragment (about 5.5 kb) obtained by the (pcDNA3.1) restriction enzyme, and the third lane indicates that the recombinant plasmid is blocked by the restriction enzyme. A nucleic acid fragment (about 1083 bp) of the obtained hCB2 receptor full-length gene and a vector fragment (about 5.5 kb) were cut. From the results of the third channel of Fig. 3, it can be seen that after the recombinant plastid is subjected to the restriction enzyme, the nucleic acid fragment of the hCB2 receptor full-length gene (as indicated by arrow 301) and the vector can be specifically cut out (pcDNA3.1; As indicated by arrow 303). Example 2: Establishment and evaluation of stable performance strains

1. Preparation of a large amount of recombinant plastid DNA for cell transfection. The second recombinant plastid obtained in Example 1 was transformed into, for example, E. coli XL-1 Blue strain and cultured in large quantities, using a kit containing an anion exchange resin, such as EndoFree. TM Plasmid Maxi Kit (Qiagen Pierce, USA) extracts large amounts of plastid DNA and contains no bacterial endotoxin to facilitate subsequent transfection of cell lines. 2. Transfection of cell line The human embryonic kidney epithelial cell line HEK293 was purchased from, for example, the American Type Culture Collection (ATCC), a HEK293 cell line with accession number ATCC CRL-1573. Dulbecco's Modified Eagle's Medium-low glucose (DMEM; Invitrogen/Gibco, USA) cultured in low concentrations of glucose. The above cell culture solution was further supplemented with 201215676 10% by volume fetal bovine serum (USDA-grade fetal bovine serum; Biological Industries, Isreal) and 1 volume percent of antibiotic (containing penicillin and peculiarin, penicillin-streptomycin;

Invitrogen/Gibco, U.S.A), was cultured in a 37 ° C, 5% CO 2 incubator. HEK293 is grown by attachment and its cell type is irregular. The cells of lxl06/mL were seeded in a 6 cm diameter culture dish, and cultured in a 37 ° C, 5% C 2 incubator for 16 hours to 18 hours to allow the cells to reach φ about 7 to 8 minutes. Next, the fresh medium was replaced and cultured in a 37 ° C, 5% c 2 incubator for 3 hours. Then, about 5 pg of DNA and about 10 μί of transfer agent, such as TurboFect in vitro Transfection Reagent (Fermentas, Canada), first mixed with about 250 pL, 150 mM NaCl solution, and then mixed the two. The reaction solution is uniformly transfected and allowed to stand at room temperature (for example, about 15 ° C to 35 ° C) for 15 minutes to 30 minutes. At this time, the cells to be transfected should be replaced with a culture medium containing no serum and antibiotics to increase the success rate of transfection. Thereafter, the transfection reaction solution was added to the cells to be transfected, and cultured at 37 ° C in a 5% C 2 incubator for about 6 hours. After the reaction was completed, the fresh culture solution was replaced and cultured in a 37eC, 5% CO 2 incubator for 16 hours to 18 hours, and the stably expressing cell strain was screened by G418 sulfate within 24 hours after the cell transfection. 3. Screening the stable expression strain cells within 24 hours after transfection, replace the transfected cells with fresh culture medium' and add 1〇〇pg/mL of G418 sulfate to the culture medium (Calbiochem, USA·; hereinafter referred to as G418) In order to screen successful transfected cell lines, cell lines that have not been successfully transfected will die. Each time the cell passage 201215676 increases the concentration of G418 at 100 pg/mL, after subculture for about one month, the successfully transfected cells in the culture will grow into a colony. At this time, the cells were treated with 0.5x trypsin-ethylenediaminetetraacetic acid (EDTA) solution, and the treated cells were cultured in a 96-well plate, and then subcultured to a 48-well plate. This gradually expanded the stable expression strain for subsequent experiments. 4. Evaluation of the expression of recombinant plastids of hCB2 receptor full-length gene in stable expression strains 4.1 Evaluation of mRNA expression of hCB2 receptor full-length gene in stable expression strains This example uses reverse transcription-PCR (reverse transcription-PCR; The expression of the mRNA of the hCB2 receptor full-length gene in the stable expression cell line of Example 2 was evaluated by RT-PCR. First, the stable expression cell strain of Example 2 was washed with a phosphate buffer solution (PBS). Next, add about 1 mL of PBS and scrape the cells. After collecting the 1.5 mL microcentrifuge tube, add about 600 pL of commercially available RNA extraction reagent (such as TriSolutionTM Reagent; GeneMark, Canada) to destroy the cell membrane, and then add about 200. Commercially available chloroform (chloroform; Sigma, USA) of μι stratifies DNA, protein and RNA, while RNA is present in the aqueous layer. After centrifuging the supernatant, first use about 500 pL of isopropanol (Sigma, USA) to sink total RNA at about -80 °C, and then use about 1 mL of 100% ethanol, about 7 mL of 75%. The ethanol (DEPC-ethanol) of the secondary deionized water (DEPC_ddH20) treated with diethyl pyrocarbonate (DEPC; Sigma, USA) was used to remove impurities from total RNA. Thereafter, after removing the supernatant 201215676 by centrifugation and evaporating the residual ethanol, about 25 pL of DEPC-ddH20 was added to reconstitute the total RNA. The total RNA extracted by the above was confirmed by RNA electrophoresis and measured by optical density (OD) OD260/〇D280 (OD260/OD280 > 1.8) at wavelengths of 260 nm and 280 nm, and the total RNA obtained was followed. RT-PCR, detection of total RNA concentration and expression of nucleic acid fragments of the hCB2 receptor full-length gene in the cell. However, the above-mentioned extraction of total RNA and optical density (O.D. 260) values are well known to those of ordinary skill in the art and will not be described herein. The total RNA extracted as a template is used as a template to carry out a reverse transcription (RT) reaction to obtain cDNA, wherein the reagent for reverse transcription reaction is exemplified in Table 3-1: _Table 3-1 _ Reagent _ volume ( μ!〇 total RNA 7 oligo dT primer (oligo(dT) primer ; GeneMark, Canada) l lOx Easy-ATM Reaction Buffer (Stratagene, USA) 5 • dNTPs (25 mM ; Bertec, Taipei, Taiwan) 1 DEPC- ddH20 3 Total 12 The reagent of Table 3-1 was reacted at 65 ° C for about 5 minutes to separate the RNA secondary structure and immediately placed on ice for about 5 minutes. Then, add Table 3-2. Illustrative reagent for reverse transcription reaction: Table 3-2 [S] 22 201215676 Reagent volume (pL) 5x first-strand buffer (250 mM Tris-HCl (pH 8.3, room temperature), 375 mM KC1, 15 mM MgCl2 4 dithiothreitol (DTT; 0.1 M; Invitrogen, USA) 2 SupertranscriptTM II reverse transcriptase (Invitrogen, USA) 1 Total 7 When performing a reverse transcription reaction, the reaction conditions may be, for example but not limited to, the following Illustrated: first 'react at 40 ° C to 50 ° C for about 30 minutes to 2 The reverse transcription reaction was carried out for an hour, and then an inactivation reaction was carried out at 70 ° C for about 10 minutes to 30 minutes to deactivate the reverse transcriptase, and then its cdna was preserved at 4 to complete its reverse transcription, and the cDNA was obtained. In another embodiment, the reverse transcription reaction may be carried out, for example, at 40 ° C to 45 ° C for about 30 minutes to 丨 hours, and then at 70 C for about 10 minutes to 20 minutes for inactivation. In the above example, the reverse transcription reaction may be carried out, for example, at 42 ° C for about 5 minutes, and then at 70 ° C for about 15 minutes for inactivation. Next, using the cDNA obtained above as a template, using a primer pair Polymerase chain reaction to increase the nucleic acid fragment of hCB2 receptor, wherein the polymerase chain reaction reagent is as shown in Table 4: , ~~ --L4 Table ____ reagent upstream primer (2.5 mM) volume (μ !〇m 23 201215676 Downstream primer (2.5 mM) 1 2x PCR Mastermnix (0.5U SuperTherm DNA 5 polymerase mix, 200 mM dNTPs, 1.5 mM MgCh, lx buffer; Bertec, Taipei, Taiwan) Example 2 cDNA 3 Total 1利用Using the primer pair and the embodiment The cDNA was subjected to polymerase chain anti seasonal 'reaction conditions may, for example, but not limited to the embodiment shown: First, at 94 C 5 minutes to 10 minutes to make double-stranded DNA template pre-separation reaction. Next, the reaction is carried out at 94 ° C for 30 seconds to 1 minute, and the reaction is carried out at 50 ° C to 60 ° C for 30 seconds to 1 minute to carry out primer-template DNA bonding reaction at 72 ° C for 30 seconds to 2 minutes. The DNA is extended to react in this cycle for a total of 30 to 40 cycles of the reaction, but in another embodiment, the above-described primer pair-template DNA binding reaction can be carried out, for example, at 55 ° C to 60 ° C. In seconds, the reaction was further extended at 72 ° C for 1 minute to 1.5 minutes, and a total of 30 cycles of the reaction were repeated. Referring to Figure 4, there is shown agarose electrophoresis analysis of an rt-PCR product according to an embodiment of the present invention, wherein the first lane represents a DNA ladder mark with a 100 base pair progression (j) NA ladder, Bertec Company, Taipei, Taiwan), Lane 2 represents the RT-PCR product of the untransfected cell line (cell control group), and lane 3 represents the RT_pCR product of the cell line transfected only with plastid (pcDNA3.1/V5_His) ( The empty vector control group), and the fourth lane indicates the RT-PCR product of the cell line transfected with the recombinant vector. Figure 4 above shows the nucleic acid fragment amplified by the introduction of each cell line, while the figure below the figure 4 shows the use of glyceric acid _3_disc 24 201215676 acid dehydrogenase (giyceraidehyde-3-phosphate dehydrog Na e; GAPDH) A primer for the amplification of nucleic acid fragments obtained from each successive strain. From the results of the second to fourth lanes of Fig. 4, it was found that GAPDH of each cell line showed a consistent mRNA expression amount (Fig. 4, lower panel), as indicated by arrow 405. From the results of the fourth lane of Fig. 4, it can be seen that the nucleic acid fragment (about 1083 bp) of the hCB2 receptor full-length gene can be specifically amplified from the stable expression cell strain of the second embodiment by using the primer pair, as shown by the arrow 4〇1. Show. From the results of the second and third lanes of Fig. 4, it is known that the nucleic acid of the hCB2 receptor full-length gene can not be increased by the cell control group # (lane 2) or the empty vector control group (lane 3) by the primer pair. The fragment (Fig. 4 above), which represents the stable expression cell line of Example 2, can stably express the mRNA of the hCB2 receptor full-length gene in the cell. 〇4.2 Evaluation of the expression of the recombinant protein of the hCB2 receptor full-length gene in the stable expression strain. This example uses the Western Immunoblot Analysis to evaluate the performance of the hCB2 receptor-recombinant protein in the stable expression cell line of Example 2. First, after the stable expression cell strain of Example 2 is washed with PBS, a solution buffer of about 200 μM is added. After the cells are rapidly scraped off on ice, they are placed in a microcentrifuge tube of, for example, 1.5 μL. Shake for 60 minutes at 4 °C. The cell fluid was shaken vigorously for another 6 seconds and then immediately allowed to stand on ice for 5 minutes. The aforementioned dissolution buffer solution is referred to by Roche R. et al., 2006, in Histochemistry and Cellology, Vol. 126, No. 2, pp. 177-187, entitled "Presence of the cannabinoid receptors, CB1 and CB2, in human omental and subcutaneous. The formulation disclosed in the literature of adipocytes, wherein the solution [S1 25 201215676 solution buffer solution may comprise 50 mM tris (hydroxymethyl) aminomethane; Tris)-HCl (JT Baker, USA) solution 1 mM ethylenediaminetetraacetic acid (EDTA; Merck, Germany) '150 mM NaCl (Showa, Japan), pH 7.4, and add 1 ° / 〇 octylphenol polyoxyethyl group before use Alcohol (Triton X-100; JT Baker, USA) and 1/100 (v/v) anti-protease cocktail (Roche, Sweden). Thereafter, the above cell solution was centrifuged at 4 ° C for about 10 minutes at a centrifugal force of 1000 x x. Then, the supernatant was taken and quantified by protein, and then subjected to subsequent evaluation. However, the above-mentioned protein quantification can be carried out, for example, using the BCA Protein Assay Reagent Kit (Bio-rad lab, USA), and can be carried out according to the manual provided by the manufacturer, and the method of protein quantification is also well known to any person having ordinary knowledge in the technical field. I will not repeat them here. Next, the supernatant obtained above was further evaluated for the expression of the recombinant protein of the hCB2 receptor by polyacrylamide gel electrophoresis and Western knockdown, and the following is described. The supernatant obtained above was subjected to sodium dodecyl sulfate-p〇lyacryiamide gel electrophoresis (SDS-PAGE) using 10 〇/0 of sodium dodecyl sulfate-p〇lyacryiamide gel electrophoresis (SDS-PAGE) for about 2 hours at 120 volts. . After 'with 192 mM glycine

(Glycine) (Bi〇technology Grade, aMReSCO, U.S.A.) was equilibrated with a transfer buffer of 25 mM Tns-HCl solution (J.T. Baker, U.S.A.). The preparation of the above SDS_PAGE and related drugs and related devices should be known to those of ordinary skill in the art to which the present invention pertains, and will not be further described herein. [s] 26 201215676 The aforementioned SDS-PAGE electrophoresis gel can be subsequently analyzed by Western tumbling method. In this embodiment, the protein of the aforementioned SDS-PAGE gel is transferred by using, for example, a wet-breaking tank (for example, Bio-Rad Scientific Instruments Transfer Unit; Amersham Pharmacia biotech, USA) at 250 mA for about 75 minutes. To the transfer film, wherein the transfer film can be, for example, a polyvinylidene difluoride membrane ♦ PVDF membrane; Immobilon TM-P Transfer

Membrane; Millipore, Ireland). Thereafter, the transfer film can be washed with 1 χ PBST (lxPBS containing 0.05 〇/〇 (v/v) Tween-20 (Showa, Japan)) for about 10 minutes, and then added to Blocking solution (20 mM Tris-Base, 0.1% Sodium). Azide, 1% BSA (Sigma, USA), 125 mM NaCl, 0.2% Tween-20 (Showa, Japan), pH 7.4). (: oscillate for about 1 hour. Then 'Remove the Blocking solution, add primary antibody (antibody: 1 χ PBST = 1: 3500 dilution) and transfer to the transfer membrane, and react at 4 ° C until overnight (about 14 hours to 16 hours). The transfer film was washed 6 times with 1X PBST, and after 10 minutes, the secondary antibody was added to the transfer membrane (antibody: Blocking solution = 1:5000 dilution), and shaken at 4 ° C for about 50 minutes. Lx PBST washes the transfer film 6 times for 10 minutes each time. Then, add cold light coloring agent in the dark, such as Enhanced ChemiLuminescence (ECL) Plus Western blotting detection reagent (Western blotting detection reagent; GE Healthcare , UK), the reaction takes about 1 minute to use the negative film (such as Kodak, USA) to develop and fix the color reaction after fixing. The result is shown in Figure 4. The above Western system is β-actin (β -actin) as a house keeping gene.

[SI 27 201215676 The primary antibody used in the above may be, for example, a rabbit anti-CB2 affinity purified polyclonal antibody (Abeam, USA) and a mouse anti-wan-actin monoclonal antibody. Antibody (Chemicon, USA). The secondary antibody used as described above may be, for example, goat anti-rabbit IgG-HRP (Zymed, USA) which binds to wasabi peroxidase, and goat anti-mouse IgG (goat anti- combined with serrata peroxidase). Mouse IgG-HRP ; Chemicon, USA). However, the above-mentioned use of cold light color development, film development and fixing, etc. may be carried out by reference to the manual provided by the manufacturer and is well known to those of ordinary skill in the art, and will not be further described herein. Referring to Figure 5, there is shown the results of Western analysis of the expression of recombinant proteins stably expressing the hCB2 receptor of a cell line according to an embodiment of the present invention, wherein the first channel represents the cell control group, and the second channel represents The empty vector control group' and the third channel represent stable expression cell lines. Figure 5 is a graph showing the expression of a recombinant protein (approximately 46 kDa) that stably expresses the hCB2 receptor of a cell line. The lower panel of Figure 5 shows the expression of β-actin (about 46 kDa) as an internal control. The left side of Figure 5 indicates the relative position of the protein markers. From the results of Fig. 5, it was found that β-actin of each cell line exhibited a consistent protein expression (Fig. 5 lower panel) as indicated by arrow 507. As can be seen from the results of lane 3 of Figure 5, the specific antibody did detect the recombinant protein of the hCB2 receptor (about 46 kDa) as indicated by arrow 501. From the results of lane 1 and lane 2 of Figure 5, it was found that the specific antibody did not detect the hCB2 receptor protein in the cell control group (lane 1) or the empty vector control group (lane 2) (Fig. 5). Above figure) 'Representative Example 2 The stable expression cell line is indeed stable in the cell 28 201215676 to represent the hCB2 receptor recombinant protein. 4.3 Evaluation of Distribution of Recombinant Proteins of hCB2 Receptor in Stable Expression Lines This example uses immunofluorescence microscopy using a conocal microscope to evaluate the distribution of hCB2 receptor recombinant proteins on the cell membrane of stable expression strains. First, the stable expression cell strain of Example 2 was cultured on a coverslip treated with 1% gelatin (gelatin type B; Sigma, USA), and the cells were fixed with 100 〇/〇 methanol (Merck, Germany), and then added. Primary antibody (affinity purified multi-body antibody against rabbit anti-CB2 receptor; Abeam, USA) and secondary antibody with fluorescent dye (eg goat anti-rabbit IgG combined with Alexa fluro® 488; Molecular Probe, Invitrogen, USA) After reacting with the cells, the cells were sealed with 60% glycerol. Then, using a conjugated focusing microscope that excites fluorescence (eg, CARV II Confocal Imager, BD Bioscience, San Jose, CA., USA), with an oil mirror (eg, HCX PL APO 63X/1.32-0.6, BD, USA), A fluorescent light source (for example, a light source having a wavelength of about 495 nm) is used as a fluorescent light source suitable for the secondary antibody to observe the binding of the antibody to the cell surface. Please refer to Annexes 1A to 1D, which show conjugated focused immunofluorescence staining (confocal) of stable expression cell lines according to an embodiment of the present invention.

Immunofluorescence staining), in which Annex 1A and Annex 1B are the stable cell lines of Example 2, Annex 1C and Annex 1D are the HEK293 cell lines of the empty vector control group, and Annex 1B and Annex 1D are conjugated focused immunofluorescence glasses. Photographs are taken, and Annex 1A and Annex 1C are optical photomicrographs corresponding to Annex 1B and Annex 1D, respectively. Compared with the cell line of the empty vector control group of Annex 1D (without fluorescence), it can be seen that the stable cell line of Example 2 was observed by immunofluorescence staining, and the fluorescent m 29 201215676 distribution was observed on the cell membrane. The recombinant protein representing the hCB2 receptor was indeed distributed on the cell membrane of the stable expression cell line of Example 2, as shown in Annex 丨3. Example 3: Evaluation of the activity of the hCB2 receptor recombinant protein of a stable expression cell line. This example evaluates the hCB2 of a stable expression cell line by measuring the change in the product content of the target protein involved in the downstream signaling pathway of the CB2 receptor. Receptor recombinant protein activity. # L Evaluation of ligand-inhibited cAMP accumulation amount In this example, a fixed concentration of forskolin extract (forskolin) and different concentrations of cannabinoid derivatives were added together to the stable expression cell line of Example 2. The aforementioned Coleus extract stimulates the activity of intracellular adenylate cyclase (AC) and increases the accumulation of intracellular cyclic monophosphate* (cAMp). The cannabinoid derivative binds to the CB2 receptor, coupling the CB2 receptor to the inhibitory G protein (Gi), thereby inhibiting the activity of adenylate cyclase and inhibiting the accumulation of cAMP. By detecting the growth and decline of cAMP accumulation in the cells, the CB2 receptor-specific ligand binding ability of the stable cell strain of Example 2 can be analyzed. First, the stable expression cell strain of Example 2 was cultured in a 12-well culture dish at a cell density of about 5×10 5 /ml, and cultured in a 5% CO 2 incubator at 37 ° C for 16 hours to 18 hours, wherein the culture solution contained 600 pg/mL of G418. When the cells are about 7 to 8 minutes full, the cells are first replaced with a serum-free medium, which additionally contains about 100 μM of 3-isobutyl-1-indenylxanthine (3-isobutyl· 1- Methylxanthine; ΙΒΜΧ), reacted at 37 ° C for about 30 minutes to inhibit the activity of endogeneous 201215676 phosphodiesterase. After removing the supernatant, 10 μC of Coleus extract (forskolin; Sigma, U.S.A.) and different concentrations of cannabinoid derivatives were used to evaluate the specific binding ability of the CB2 receptor to the ligand. A cannabinoid derivative such as the above may be 0, ΙΟ5, ΙΟ6, 1〇7, 1〇8, 109μΜ of CP55940 (one of the ligand derivatives of the hCB2 receptor; (-)-CP55940 » 5-(l , l-dimethylheptyl)-2-[(lR,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-phenol; Cayman Chemical, USA) solution, wherein CP55940 is an activator of CB2 receptor ( Agonist) 'The specific affinity for the CB2 receptor is quite high, and its relative affinity for CB1/CB2 is zero or marginal selectivity. Coleus extract (forsk〇lin) and CP55940 were added together to the stable cell line of Example 2, and reacted at 373⁄4 for about 30 minutes. Then, the reaction was terminated by using 300 pL of a 0.1 M HCl solution at room temperature (e.g., about 15 ° C to 35 ° C) for about 1 minute. Then, the mixture was centrifuged at 4 ° C for about 1 minute at a centrifugal force of 1 〇〇〇 xg, and the supernatant was collected. Thereafter, a commercially available cAMP enzyme immunoassay kit, such as Assay DesignsTM Direct Cyclic AMP Enzyme Immunoassay (EIA) (now incorporated into Enzo® Life Science, USA), is used to assemble a cAMP containing a blue coloring substance. (blue conjugate cAMP), competitive binding assay with both accumulated cAMP 'in the cell to analyze the accumulation of cAMP contained in the supernatant". As stated, when the cAMP concentration of the test solution is higher, it represents binding. The less the receptor binding position that the blue dye cAMP can compete with, the lower the absorbance measured at the wavelength of 405 nm after the color reaction. 201215676 . However, 'the higher the concentration of cp5594〇, the negative regulation of CB2 receptor binding to ligand, which inhibits the accumulation of cAMP in cells, so that the lower the concentration of cAMP, the combination of blue dye The more receptor binding sites that cAMP can compete with, the higher the absorbance measured at 405 nm after color reaction. Please refer to FIG. 6 , which is a graph showing inhibition of intracellular CAMP content by stable ligand cell strains stimulated by ligands according to an embodiment of the present invention, wherein the vertical axis indicates cossile extract (forsk〇iin) stimulated caMP Cumulative Φ (Pmole/mL) 'The horizontal axis is CP55940 with different Log concentration (M). From the results of Fig. 6, it can be seen that the higher the concentration of CP55940, the more the cumulative amount of cAMP produced by the forskolin extract is inhibited, and the hCB2 receptor recombinant protein representing the stable expression of the cell strain of Example 2 is It does have the ability to specifically bind to ligands. 2. Evaluation of ligand-stimulated activated state cAMP accumulation In this example, a cannabinoid derivative was added to the stable cell line of Example 2' by detecting intracellular activated state (ph〇Sph〇ryiated • The growth and decline of intracellular mitogen-activated protein kinase (MAPK), and the specific binding ability of the CB2 receptor to the ligand of the stable cell strain of Example 2 can be analyzed. First, the stable cell strain of Example 2 was cultured in a petri dish of about 6 cm in diameter at a cell density of about 5 x 1 〇 5 /ml, and cultured in a 37 ° C, 5% C 2 incubator for 16 hours. 18 hours 'where the culture solution contains

600 pg/mL of G418. After the cells are about 5 to 6 minutes full, add CP55940 to ΙΟΟηΜ for 0, 1, 2, 3, and 4 hours, respectively, after washing twice with lx PBS • Add about 100 pL of the lysis buffer (for example, G〇ld Iysis

[SI 32 201215676 buffer], after rapidly dropping onto the cells on ice, placed in a microcentrifuge tube of, for example, 1.5 mL, and shaken at 4 ° C for 30 minutes. It was centrifuged at 4 ° C for about 10 minutes with a centrifugal force of 10 〇〇〇 Xg. Then, the supernatant was taken and quantified by the protein, and then subjected to Western decantation as in Example 2 4.2. Please refer to Fig. 7, which shows the results of western tumbling analysis of the phosphorylated MAPK content of the stable expression cell line according to an embodiment of the present invention, wherein the upper left and lower left images show the empty vector control group, and the upper right The figure and the lower right panel show stable cell lines. The upper left panel and the upper right panel show the expression of the phosphorylated MAPK of the stable cell line, as indicated by arrow 711. The left lower panel and the lower right panel show the expression of total extracellular signal-regulated kinase (total ERK) as an internal control. The left side of Figure 7 indicates the relative position of the protein markers. From the results of the lower left panel and the lower right panel of Fig. 7, it was found that the total extracellular regulated kinase of each cell line exhibited a consistent protein expression amount. From the upper left panel and the upper right panel of Fig. 7, the specific antibody can detect the expression of activated MAPK, and with the increase of CP55940 treatment time, the content of activated MAPK of the stable cell strain of Example 2 also follows. The increase is as shown by the trend of the content change 711a, and the hCB2 receptor recombinant protein representing the performance of the stable expression cell line of the second embodiment does have a specific binding ability to the ligand. In addition, it should be added that the present invention exemplifies a specific primer pair, hCB2 receptor gene, vector, host cell, cell strain, transfection method, fluorescent dye and its analysis method to obtain the above stable performance hCB2. A method for screening a cell line of a recombinant protein and a potential ligand thereof, but the present invention [s] 33 201215676 is not limited thereto. Anyone skilled in the art to which the present invention pertains may also use other primer pairs, hCB2 receptor genes, vectors, host cells, cell lines, transfection methods, fluorescent stains, without departing from the spirit and scope of the present invention. And other assays to obtain the above-described cell strain stably expressing the hCB2 receptor recombinant protein and thereby screening potential ligands. According to several embodiments of the present invention described above, the cell line of the present invention which stably expresses the cannabinoid receptor CB2 and the method thereof for screening potential ligands for the cannabinoid receptor have the advantage of utilizing the CB2 receptor. The recombinant plastid of the full-length gene is transfected into the cell line and stably expresses the CB2 receptor recombinant protein on the surface of the cell membrane, and the above cell strain is cultured in the culture solution containing or not containing the sample/glutamine, and then the target content is analyzed. The change is made to determine whether the sample solution contains a potential ligand that specifically binds to the CB2 receptor recombinant protein', thereby rapidly, safely and correctly screening out potential ligands for the cannabinoid receptor from a large number of samples. The present invention has been disclosed in the above-described embodiments, and it is not intended to limit the invention, and any one of ordinary skill in the art to which the invention pertains can make various changes and modifications without departing from the spirit and scope of the invention. The scope of protection of the present invention is therefore defined by the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; A derivative of the CB2 receptor stimulated by a derivative (or potential ligand) is involved in a partial signaling pathway [S] 34 201215676 Schematic. Fig. 2 is a diagram showing the agarose electrophoresis analysis of the nucleic acid fragment of the hCB2 receptor according to an embodiment of the present invention. Fig. 3 is a diagram showing the agarose electrophoresis analysis of recombinant plastids by restriction enzymes according to an embodiment of the present invention. Figure 4 is a graph showing the agarose electrophoresis analysis of the rt-PCR product according to an embodiment of the present invention. Fig. 5 is a graph showing the results of Western knocking analysis showing the expression of a recombinant protein of the hCB2 receptor which stably expresses a cell line according to an embodiment of the present invention. Fig. 6 is a graph showing inhibition of intracellular c AMP content by stable expression of a cell line stimulated by a ligand according to an embodiment of the present invention. Fig. 7 is a view showing the results of Western knocking analysis of the phosphorylated MAPK content of a stable expression cell line according to an embodiment of the present invention. Heart Attachment 1A to Annex id are photographs showing conjugated focused immunofluorescence staining of stable expression cell lines according to the embodiment of the present invention. [Main element symbol description] 101 · Cell membrane 711a: Content change 103: Extracellular 105: Cell 201/301/303/401/405/501/507/711 : Arrow [S] 35 201215676 Sequence Listing &lt;110&gt; National Pingtung University of Science and Technology

&lt;120&gt; Cell line stably expressing cannabinoid receptor CB2 and its application &lt;130&gt; None &lt;160&gt; 4 &lt;210&gt; 1 &lt;211&gt; 367 &lt;212&gt; PRT

&lt;213> Human Cannabinoid Receptor CB2 (Python Cell) &lt;400〉 1

Met Glu Glu Cys Trp Val Thr Glu lie Ala Asn Gly Ser Lys Asp Gly Leu Asp Ser Asn 15 10 15 20

Pro Met Lys Asp Tyr Met lie Leu Ser Gly Pro Gin Lys Thr Ala Val Ala Val Leu Cys 21 25 30 35 40

Thr Leu Leu Gly Leu Leu Ser Ala Leu Glu Asn Val Ala Val Leu Tyr Leu lie Leu Ser 41 45 50 55 60

Ser His Gin Leu Arg Arg Lys Pro Ser Tyr Leu Phe lie Gly Ser Leu Ala Gly Ala Asp 61 65 70 75 80

Phe Leu Ala Ser Val Val Phe Ala Cys Ser Phe Val Asn Phe His Val Phe His Gly Val 81 85 90 95 100

Asp Ser Lys Ala Val Phe Leu Leu Lys lie Gly Ser Val Thr Met Thr Phe Thr Ala Ser 101 105 110 115 120

Val Gly Ser Leu Leu Leu Thr Ala lie Asp Arg Tyr Leu Cys Leu Arg Tyr Pro Pro Ser 121 125 130 135 140

Tyr Lys Ala Leu Leu Thr Arg Gly Arg Ala Leu Val Thr Leu Gly lie Met Trp Val Leu 141 145 150 155 160

Ser Ala Leu Val Ser Tyr Leu Pro Leu Met Gly Trp Thr Cys Cys Pro Arg Pro Cys Ser 161 165 170 175 180

Glu Leu Phe Pro Leu lie Pro Asn Asp Tyr Leu Leu Ser Trp Leu Leu Phe lie Ala Phe 181 185 190 195 200

Leu Phe Ser Gly lie lie Tyr Thr Tyr Gly His Val Leu Trp Lys Ala His Gin His Val 201 205 210 215 220

Ala Ser Leu Ser Gly His Gin Asp Arg Gin Val Pro Gly Met Ala Arg Met Arg Leu Asp 221 225 230 235 240

Val Arg Leu Ala Lys Thr Leu Gly Leu Val Leu Ala Val Leu Leu lie Cys Trp Phe Pro 241 245 250 255 260

Val Leu Ala Leu Met Ala His Ser Leu Ala Thr Thr Leu Ser Asp Gin Val Lys Lys Ala 261 265 270 275 280

Phe Ala Phe Cys Ser Met Leu Cys Leu lie Asn Ser Met Val Asn Pro Val lie Tyr Ala 281 285 290 295 300

Leu Arg Ser Gly Glu lie Arg Ser Ser Ala His His Cys Leu Ala His Trp Lys Lys Cys 301 305 310 315 320

Val Arg Gly Leu Gly Ser Glu Ala Lys Glu Glu Ala Pro Arg Ser Ser Val Thr Glu Thr 321 325 330 335 340 201215676

Glu Ala Asp Gly Lys lie Thr Pro Trp Pro Asp Ser Arg Asp Leu Asp Leu Ser Asp Cys 341 345 350 355 360 , Tyr His His His His His His 361 365 &lt;210&gt; 2 &lt;211> 1083 &lt;212&gt; DNA &lt;213&gt;Artificial sequence &lt;400〉 2

atggaggaat gctgggtgac agagatagcc aatggctcca aggatggctt ggattccaac 60 cctatgaagg attacatgat cctgagtggt ccccagaaga cagctgttgc tgtgttgtgc 120 actcttctgg gcctgctaag tgccctggag aacgtggctg tgctctatct gatcctgtcc 180 tcccaccaac tccgccggaa gccctcatac ctgttcattg gcagcttggc tggggctgac 240 ttcctggcca gtgtggtctt tgcatgcagc tttgtgaatt tccatgtttt ccatggtgtg 300 gattccaagg ctgtcttcct gctgaagatt ggcagcgtga ctatgacctt cacagcctct 360 gtgggtagcc tcctgctgac cgccattgac cgatacctct gcctgcgcta tccaccttcc 420 tacaaagctc tgctcacccg tggaagggca ctggtgaccc tgggcatcat gtgggtcctc 480 tcagcactag tctcctacct gcccctcatg ggatggactt gctgtcccag gccctgctct 540 gagcttttcc cactgatccc caatgactac ctgctgagct ggctcctgtt catcgccttc 600 ctcttttccg gaatcatcta cacctatggg catgttctct ggaaggccca tcagcatgtg 660 gccagcttgt ctggccacca ggacaggcag gtgccaggaa tggcccgaat gaggctggat 720 gtgaggttgg ccaagaccct agggctagtg ttggctgtgc tcctcatctg ttggttccca 780 gtgctggccc tcatggccca cagcctggcc actacgctca gtgaccaggt caagaaggcc 840 tttgctttct gctccatgct gtgcctcatc aactccatgg tcaaccctgt catctatgct 900 ctacggagtg gagagatccg ctcctctgcc catcactgcc tggctcactg gaagaagtgt 960 gtgaggggcc ttgggtcaga ggcaaaagaa gaagccccga gatcctcagt caccgagaca 1020 gaggctgatg ggaaaatcac tccgtggcca gattccagag atctagacct ctctgattgc 1080 tac 1083 [5] 2 201215676 &lt; 210 &gt; 3 &lt; 211 &gt; 21 &lt; 212 &gt; DNA &lt;213&gt;Artificialsequence&lt;400&gt; 3 cgcggtacct cgattatgga g

&lt;210> 4 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;400&gt; 4 accggtacag caatcagaga ggtc

Claims (1)

201215676 VII. Patent application scope: 1. A cell strain stably expressing a cannabinoid receptor, wherein the cell strain has a recombinant plastid, and the recombinant plastid has a nucleic acid sequence having the sequence of sequence identification number 1 , thereby A cannabinoid receptor 2 (CB2) recombinant protein which exhibits a sequence as shown in SEQ ID NO: 2 2. A cell line stably expressing a cannabinoid receptor according to claim 1 of the scope of the patent application, wherein This cell line is a human embryonic kidney epithelial cell line HEK293 (derived from the American Type Culture Collection; ATCC, accession number: ATCC CRL-1573). 3. A method for screening a potential ligand for a cannabinoid receptor, comprising at least: performing a culture step of culturing a cell strain in a culture solution containing or not containing a sample solution at 37 ° C, 5 Incubating for 1 hour to 20 hours under % carbon dioxide, wherein the cell strain comprises a cell strain according to any one of claim 1 to claim 2, and the cell line exhibits as shown in sequence identification number 2. a recombinant protein such as a sequence of cannabinoid receptor 2 (CB2); performing a detection step to detect a first content of one of the target cells cultured through the cell containing the sample solution, and detecting the culture through the solution containing the sample a second content of the target of the cell strain, wherein the target is cyclic adenosine monophosphate (201215676 cAMP) or mitogen-activated protein kinase (ΜΑΡΚ); ^ performing a comparison step to obtain a difference between the first content and the second content; and performing an analysis step to determine whether the sample solution contains the difference based on the difference One specific binding of the recombinant protein potential CB2 receptor ligand (potential ligand), wherein when the difference is not zero, it is determined that the sample solution containing the potential ligand. 4. A method of screening a potential ligand for a cannabinoid receptor according to claim 3, wherein the potential coordination system inhibits the cAMP produced by the cell strain stimulated by forskolin The first content. 5. A method of screening a potential ligand for a cannabinoid receptor according to claim 3, wherein the potential coordination system promotes the first content of the φ MARK. 6. The method of screening a potential ligand for a cannabinoid receptor according to claim 3, wherein the first content and the second content of the MARK are a protein expression amount.