CN116891523B - 一种trpm3截短体、包含其的细胞系及其用途 - Google Patents
一种trpm3截短体、包含其的细胞系及其用途 Download PDFInfo
- Publication number
- CN116891523B CN116891523B CN202310592892.8A CN202310592892A CN116891523B CN 116891523 B CN116891523 B CN 116891523B CN 202310592892 A CN202310592892 A CN 202310592892A CN 116891523 B CN116891523 B CN 116891523B
- Authority
- CN
- China
- Prior art keywords
- trpm3
- cell
- truncate
- plasmid
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108060008547 TRPM3 Proteins 0.000 title claims abstract description 98
- 102000003620 TRPM3 Human genes 0.000 title claims abstract description 98
- 210000004027 cell Anatomy 0.000 claims abstract description 72
- 210000000170 cell membrane Anatomy 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 14
- 150000001875 compounds Chemical class 0.000 claims abstract description 12
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 7
- 238000012216 screening Methods 0.000 claims abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 31
- 239000013612 plasmid Substances 0.000 claims description 30
- 102000004169 proteins and genes Human genes 0.000 claims description 26
- 241000701447 unidentified baculovirus Species 0.000 claims description 19
- 239000000556 agonist Substances 0.000 claims description 15
- 210000004962 mammalian cell Anatomy 0.000 claims description 15
- 239000002773 nucleotide Substances 0.000 claims description 14
- 125000003729 nucleotide group Chemical group 0.000 claims description 14
- 239000013598 vector Substances 0.000 claims description 14
- 241000713666 Lentivirus Species 0.000 claims description 11
- 239000003112 inhibitor Substances 0.000 claims description 10
- 108020004707 nucleic acids Proteins 0.000 claims description 10
- 102000039446 nucleic acids Human genes 0.000 claims description 10
- 150000007523 nucleic acids Chemical class 0.000 claims description 10
- 238000003776 cleavage reaction Methods 0.000 claims description 8
- 239000013604 expression vector Substances 0.000 claims description 8
- 238000003259 recombinant expression Methods 0.000 claims description 8
- 230000007017 scission Effects 0.000 claims description 8
- 108091005804 Peptidases Proteins 0.000 claims description 7
- 239000004365 Protease Substances 0.000 claims description 7
- 241000238631 Hexapoda Species 0.000 claims description 6
- 108700004026 gag Genes Proteins 0.000 claims description 5
- 108700004029 pol Genes Proteins 0.000 claims description 5
- 101150098622 gag gene Proteins 0.000 claims description 4
- 101150088264 pol gene Proteins 0.000 claims description 4
- 108700004030 rev Genes Proteins 0.000 claims description 4
- 101150098213 rev gene Proteins 0.000 claims description 4
- 108020001507 fusion proteins Proteins 0.000 claims 8
- 102000037865 fusion proteins Human genes 0.000 claims 8
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 3
- 108091006146 Channels Proteins 0.000 abstract description 40
- 101000844503 Homo sapiens Transient receptor potential cation channel subfamily M member 3 Proteins 0.000 abstract description 21
- 102000050157 human TRPM3 Human genes 0.000 abstract description 18
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 15
- 230000000638 stimulation Effects 0.000 description 15
- 239000003814 drug Substances 0.000 description 12
- KSEXDSJYVSEVGF-UHFFFAOYSA-N 2-(3,4-dihydro-2h-quinolin-1-yl)-n-(5-methyl-1,2-oxazol-3-yl)-2-phenylacetamide Chemical compound O1C(C)=CC(NC(=O)C(N2C3=CC=CC=C3CCC2)C=2C=CC=CC=2)=N1 KSEXDSJYVSEVGF-UHFFFAOYSA-N 0.000 description 11
- 230000008859 change Effects 0.000 description 11
- 229940079593 drug Drugs 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 239000011521 glass Substances 0.000 description 10
- 230000005764 inhibitory process Effects 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 230000004913 activation Effects 0.000 description 9
- 238000012258 culturing Methods 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 238000001890 transfection Methods 0.000 description 8
- XHBZOAYMBBUURD-UHFFFAOYSA-N Ononetin Natural products C1=CC(OC)=CC=C1CC(=O)C1=CC=C(O)C=C1O XHBZOAYMBBUURD-UHFFFAOYSA-N 0.000 description 7
- 208000002193 Pain Diseases 0.000 description 7
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 6
- 239000007995 HEPES buffer Substances 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 6
- 102100038132 Endogenous retrovirus group K member 6 Pro protein Human genes 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 210000003594 spinal ganglia Anatomy 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 102000004310 Ion Channels Human genes 0.000 description 4
- 108090000862 Ion Channels Proteins 0.000 description 4
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 238000007877 drug screening Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 210000003722 extracellular fluid Anatomy 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 108091008847 TRPM Proteins 0.000 description 3
- 102000027545 TRPM Human genes 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229950010131 puromycin Drugs 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108091006027 G proteins Proteins 0.000 description 2
- 102000030782 GTP binding Human genes 0.000 description 2
- 108091000058 GTP-Binding Proteins 0.000 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 229940035676 analgesics Drugs 0.000 description 2
- 239000000730 antalgic agent Substances 0.000 description 2
- HUCVOHYBFXVBRW-UHFFFAOYSA-M caesium hydroxide Chemical compound [OH-].[Cs+] HUCVOHYBFXVBRW-UHFFFAOYSA-M 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000012411 cloning technique Methods 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 239000003596 drug target Substances 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- HKQYGTCOTHHOMP-UHFFFAOYSA-N formononetin Chemical compound C1=CC(OC)=CC=C1C1=COC2=CC(O)=CC=C2C1=O HKQYGTCOTHHOMP-UHFFFAOYSA-N 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 229960002518 gentamicin Drugs 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 210000000929 nociceptor Anatomy 0.000 description 2
- 230000001473 noxious effect Effects 0.000 description 2
- 230000010412 perfusion Effects 0.000 description 2
- DIJBBUIOWGGQOP-QGVNFLHTSA-N pregnenolone sulfate Chemical compound C1C=C2C[C@@H](OS(O)(=O)=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 DIJBBUIOWGGQOP-QGVNFLHTSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 102000042565 transient receptor (TC 1.A.4) family Human genes 0.000 description 2
- 108091053409 transient receptor (TC 1.A.4) family Proteins 0.000 description 2
- 210000000427 trigeminal ganglion Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- LINMATFDVHBYOS-MBJXGIAVSA-N (2s,3r,4s,5r,6r)-2-[(5-bromo-1h-indol-3-yl)oxy]-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C=C12 LINMATFDVHBYOS-MBJXGIAVSA-N 0.000 description 1
- 101150084750 1 gene Proteins 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108091005462 Cation channels Proteins 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 208000000094 Chronic Pain Diseases 0.000 description 1
- 206010012335 Dependence Diseases 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 102000017934 GABA-B receptor Human genes 0.000 description 1
- 108060003377 GABA-B receptor Proteins 0.000 description 1
- 102000034354 Gi proteins Human genes 0.000 description 1
- 108091006101 Gi proteins Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 208000004454 Hyperalgesia Diseases 0.000 description 1
- 208000035154 Hyperesthesia Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000007920 Neurogenic Inflammation Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 101000933967 Pseudomonas phage KPP25 Major capsid protein Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 241000519995 Stachys sylvatica Species 0.000 description 1
- 108060008648 TRPC Proteins 0.000 description 1
- 102000027549 TRPC Human genes 0.000 description 1
- 101150034827 TRPM3 gene Proteins 0.000 description 1
- 108091008846 TRPML Proteins 0.000 description 1
- 102000027544 TRPML Human genes 0.000 description 1
- 108060009332 TRPP Proteins 0.000 description 1
- 102000003563 TRPV Human genes 0.000 description 1
- 108060008564 TRPV Proteins 0.000 description 1
- 230000036982 action potential Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000004115 adherent culture Methods 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000011021 bench scale process Methods 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 230000009460 calcium influx Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- DJKJXRLREATOMF-UHFFFAOYSA-M cesium;methanesulfonate Chemical compound [Cs+].CS([O-])(=O)=O DJKJXRLREATOMF-UHFFFAOYSA-M 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 210000004240 ciliary body Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000006739 dopaminergic cell death Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- RIKPNWPEMPODJD-UHFFFAOYSA-N formononetin Natural products C1=CC(OC)=CC=C1C1=COC2=CC=CC=C2C1=O RIKPNWPEMPODJD-UHFFFAOYSA-N 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 206010061989 glomerulosclerosis Diseases 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000028161 membrane depolarization Effects 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004001 molecular interaction Effects 0.000 description 1
- 210000002200 mouth mucosa Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 208000004296 neuralgia Diseases 0.000 description 1
- 208000021722 neuropathic pain Diseases 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 230000003040 nociceptive effect Effects 0.000 description 1
- 108091008700 nociceptors Proteins 0.000 description 1
- 210000004416 odontoblast Anatomy 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 229940124636 opioid drug Drugs 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 210000000578 peripheral nerve Anatomy 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 230000009038 pharmacological inhibition Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000955 prescription drug Substances 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 210000003583 retinal pigment epithelium Anatomy 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000035807 sensation Effects 0.000 description 1
- 230000020341 sensory perception of pain Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- MFBOGIVSZKQAPD-UHFFFAOYSA-M sodium butyrate Chemical compound [Na+].CCCC([O-])=O MFBOGIVSZKQAPD-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 210000001170 unmyelinated nerve fiber Anatomy 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000009278 visceral effect Effects 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000012403 whole-cell patch-clamp technology Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/24—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a MBP (maltose binding protein)-tag
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14041—Use of virus, viral particle or viral elements as a vector
- C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- General Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Virology (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提供了一种TRPM3截短体、包含其的细胞系及其用途。所述TRPM3截短体包含如SEQ ID NO:5所示的氨基酸序列。本发明还提供了一种细胞系,其包含所述TRPM3截短体。本发明还提供了一种筛选与TRPM3相互作用的化合物的方法,其包括i)在细胞膜上表达所述TRPM3截短体,使所述化合物与所述TRPM3截短体接触,利用全细胞膜片钳记录通道电流;或ii)使所述化合物与所述细胞系接触,利用全细胞膜片钳记录通道电流。本发明提供的TRPM3截短体,不改变保守功能区,相比野生型人源TRPM3而言,其稳定性和产量都有明显的提升,并且全细胞膜片钳可以记录到较大的持续稳定的特异性通道电流。
Description
技术领域
本发明属于生物技术领域,更具体地,本发明涉及一种TRPM3截短体、包含其的细胞系及其用途。
背景技术
疼痛是一个未解决的全球性医学问题,是患者寻求医疗护理的最常见症状[1]。与这种高医疗需求相反,治疗疼痛的可用药物往往不足。事实上,大约一半的慢性疼痛患者反馈现有处方药不足以缓解疼痛,基于阿片类药物的疼痛疗法又有着严重的副作用,包括耐受性和成瘾性。为了解决这种未被满足的临床需求,不断寻求新的疼痛靶点并开发有效但非成瘾性治疗的新型镇痛药显得尤为重要[2,3]。疼痛伤害感受器的细胞***于支配身体的背根神经节(Dorsal root ganglia,DRG)和支配面部的三叉神经节(Trigeminalganglion,TG)中,其神经元的外周神经末梢(分类为小直径无髓鞘C纤维和有髓鞘中直径Aδ纤维)中表达刺激敏感分子,如细胞表面受体和离子通道。它们的激活可导致钠离子和钙离子内流、去极化和动作电位产生,以此调节向中枢神经***发送的信息,其中TRP通道作为有害刺激的主要分子传感器起着至关重要的作用。
TRP通道由四个亚基组成,形成同源或异源通道。每个亚基的一般结构由6个跨膜螺旋拓扑结构(S1-S6)组成,其中S4对应电压传感器样结构域,能够感知细胞内离子浓度的变化[4-6]。通道的孔由S5和S6亚基之间的α螺旋环形成。根据序列相似性,TRP通道家族被细分为TRPC、TRPV、TRPM、TRPP、TRPML和TRPA6个亚家族[7]。一方面瞬时受体电位(TRP)离子通道参与异质性生理和病理过程,包括温度感觉[8]、内脏伤害感受[9]、促炎细胞因子产生[10]、多巴胺能神经元死亡[11]、神经源性炎症[12]、肾小球硬化[13,14]和癌症[15]等,功能非常多样,另一方面,由于长度和N-/C-末端(均为胞质)所含结构域的高度变异性导致TRP离子通道之间的同源性非常之低,通常只有10-25%,因此靶向TRP离子通道设计药物不会有明显的脱靶效应,这使得其成为非常理想的药物靶点。
TRPM3基因位于9q21.12-13,是第9染色体上最大的基因,它跨越870kb,包括30个外显子[16]。与TRP家族的大多数其他成员一样,TRPM3是一个非选择性的阳离子通道。然而,TRPM3蛋白具有几个独特的特征,使它们与这个多样化家族的其他成员区分开来。TRPM3有许多不同剪切体,已提出编码的蛋白质具有至少23个剪接变体[17],一些变体(如TRPM 3α1)对单价阳离子具有选择性,而其他(如TRPM 3α2)对二价阳离子具有高度选择性[18]。TRPM3主要在伤害感受神经元、胰腺β细胞、肾脏和血管肌肉层中表达,也在脑的各个部分、卵巢、***、成牙本质细胞、脂肪细胞、口腔粘膜、睫状体和视网膜色素上皮中有分布[19 -24]。
目前对于TRPM3也有大量的功能研究报道。有文献指出,TRPM3在有害热检测和热相关炎症中发挥作用[25],温度升高会通过激活TRPM3增加胞质Ca2+[26]。TRPM3被认为是治疗诱发性和自发性神经性疼痛的替代药物靶点,参与急性有害热感应和炎症性痛觉过敏的发展[27,28]。近期,已有文献证实了Gi/o、Gs或Gq偶联受体释放的G蛋白βγ亚基对TRPM 3有抑制作用[29,30]。在小鼠背根神经节(DRG)神经元中,G蛋白βγ亚基介导的Gi偶联GABA-B受体对TRPM3的抑制被认为是伤害性反应的调节机制[31],并被认为是阿片类药物在镇痛治疗中的主要作用机制[32]。此外,TRPM3的药理学抑制减轻了小鼠和大鼠各种临床前模型中的疼痛[33-35]也再次强调了TRPM3作为新型镇痛药物靶标的潜力。
目前,国内外未见人源TRPM3蛋白纯化数据以及通道功能检测相关药物筛选应用。为了针对人源TRPM3开发靶向性的药物分子,亟需对TRPM3进行克隆改造以及筛选方法摸索。
发明内容
由于TRPM3的临床药用价值分子需要抑制剂,而目前缺乏可用于药物筛选的改造型人源TRPM3,现有的技术能在HEK293T细胞上转染鼠源的TRPM3α2序列并使用膜片钳记录到电流,但是如果要做TRPM3为靶点的新药筛选使用人源的序列更为合适,参考很多前期的技术我们发现,使用人源的序列转染到HEK293T细胞很难记录到稳定的电流。为解决野生型人源TRPM3难以用于药物筛选的问题,本发明提供了一种TRPM3截短体、包含其的细胞系及其应用。
具体地,本发明第一方面提供了一种TRPM3截短体,其包含如SEQ ID NO:5所示的氨基酸序列。
本发明第一方面所述的TRPM3截短体还连接了6×His标签、MBP蛋白和/或HRV3C蛋白酶切割位点。
优选地,所述连接发生在上述TRPM3截短体的N端,和/或所述6×His标签、所述MBP蛋白和所述HRV3C蛋白酶切割位点之间通过连接子连接。
更优选地,所述MBP蛋白包含如SEQ ID NO:9所示的氨基酸序列,所述TRPM3截短体包含如SEQ ID NO:4所示的氨基酸序列。
本发明第二方面提供了一种细胞系,其包含本发明第一方面所述的TRPM3截短体。
优选地,所述细胞系选用293T细胞。
本发明第三方面提供了一种筛选与TRPM3相互作用的化合物的方法,其包括i)在细胞膜上表达本发明第一方面所述的TRPM3截短体,使所述化合物与所述TRPM3截短体接触,利用全细胞膜片钳记录通道电流;或ii)使所述化合物与本发明第二方面所述的细胞系接触,利用全细胞膜片钳记录通道电流。
优选地,所述化合物为TRPM3激动剂和/或抑制剂。
本发明第四方面提供了一种分离的核酸,其编码本发明第一方面所述的TRPM3截短体。
优选地,所述MBP蛋白包含如SEQ ID NO:10所示的核苷酸序列,所述TRPM3截短体包含如SEQ ID NO:7或8所示的核苷酸序列。
本发明第五方面提供了一种重组表达载体,其包含启动子和本发明第四方面所述的核酸。
优选地,所述重组表达载体的骨架质粒为pEGBacMam载体或PCDH载体。
本发明第六方面提供了一种质粒组合,其包含如本发明第五方面所述的重组表达载体。
优选地,所述质粒组合还包含编码gag基因、pol基因、rev基因的第二质粒,和编码vsvg基因的第三质粒;更优选地,所述第二质粒的骨架质粒为psPAX2载体,所述第三质粒的骨架质粒为pMD2G载体。
其中,gag基因编码病毒主要的结构蛋白;pol基因编码病毒特异性的酶;rev基因编码调节gag和pol基因表达的调节因子。vsvg基因编码病毒包装所需要的包膜蛋白。
本发明第七方面提供了一种重组杆状病毒,其包含本发明第四方面所述的核酸;优选地,所述重组杆状病毒由如本发明第五方面所述的重组表达载体感染昆虫细胞制得。
本发明第八方面提供了一种重组慢病毒,其包含本发明第四方面所述的核酸;优选地,所述重组慢病毒由如本发明第六方面所述的质粒组合转染哺乳动物细胞制得;所述哺乳动物细胞例如为293T细胞。
本发明第九方面提供了一种制备本发明第四方面所述的TRPM3截短体的方法,其包括使用本发明第七方面所述的重组杆状病毒感染哺乳动物细胞,或使用如本发明第八方面所述的重组慢病毒转染哺乳动物细胞。
优选地,所述哺乳动物细胞为293S或293T细胞。
本发明第十方面提供了一种制备本发明第三方面所述的细胞系的方法,其包括使用本发明第八方面所述的重组慢病毒感染哺乳动物细胞。
优选地,所述哺乳动物细胞为293T细胞。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:
本发明创新性地结合蛋白结构预测,对原本在体外表达无功能的野生型人源TRPM3蛋白序列进行截短改造,并且在蛋白的N端设计了MBP蛋白标签序列,一方面便于对异源表达的蛋白进行纯化,另一方面也提高了蛋白的稳定性和表达量。其次,本发明对蛋白的序列进行酶切位点(HRV3C)设计,改造后的序列可应用于核酸编码的小分子药物库、生物分子相互作用实验、膜片钳药物筛选实验等。并且,本发明提供的TRPM3截短体,不改变保守功能区,相比野生型人源TRPM3而言,其稳定性和产量都有明显的提升,并且全细胞膜片钳可以记录到较大的持续稳定的特异性通道电流。
附图说明
图1为通过无缝克隆技术将TRPM3截短体的核苷酸序列、6×His标签的核苷酸序列、MBP蛋白的核苷酸序列和HRV3C蛋白酶切割位点的核苷酸序列构建在pEGBacMam载体上。
图2为通过无缝克隆技术将TRPM3截短体的核苷酸序列构建在PCDH载体上。
图3为野生型人源TRPM3蛋白的SEC图。
图4为野生型人源TRPM3蛋白的SDS-PAGE图。
图5为TRPM3截短体的SEC图。
图6为TRPM3截短体的SDS-PAGE图。
图7为膜片钳记录通道电流给予的电压斜坡刺激图。
图8为10μM CIM0216刺激下野生型人源TRPM3通道电流在+100mV的幅值随时间变化散点图。
图9为10μM CIM0216刺激下野生型人源TRPM3通道电流典型图。
图10为10μM CIM0216刺激下TRPM3截短体通道电流在+100mV的幅值随时间变化散点图。
图11为10μM CIM0216刺激下TRPM3截短体通道电流典型图。
图12为1μM CIM0216和50μM PregS刺激下TRPM3截短体通道电流在+100mV的幅值随时间变化散点图。
图13为1μM CIM0216和50μM PregS刺激下TRPM3截短体通道电流典型图。
图14为不同浓度PregS刺激下TRPM3截短体通道电流在+100mV的幅值随时间变化散点图。
图15为不同浓度PregS刺激下TRPM3截短体通道电流典型图。
图16为PregS对TRPM3截短体通道激活的EC50(半激活浓度)。
图17为不同浓度的Ononetin(简称Ono)抑制TRPM3截短体通道电流(使用50uMPregS激活)在+100mV的幅值随时间变化散点图。
图18为不同浓度的Ononetin抑制TRPM3截短体通道电流(使用50uM PregS激活)典型图。
图19为Ononetin对TRPM3截短体通道(使用50uM PregS激活)抑制的IC50(半抑制浓度)。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
实施例1基因合成及质粒构建
将野生型人源TRPM3的核苷酸序列(SEQ ID NO:6)、MBP蛋白的核苷酸序列(SEQ IDNO:10)、TRPM3截短体的核苷酸序列(SEQ ID NO:8)送由南京擎科生物技术有限公司分别进行合成,携带EcoRI和NotI酶切位点,将上述MBP蛋白序列和TRPM3截短体序列以及6×His标签序列和HRV3C蛋白酶切割位点序列连接重组至pEGBacMam质粒(图1)。
实施例2重组杆状病毒的制备
1.提取重组杆状病毒质粒
通过热激转化,将含有连接有MBP蛋白的TRPM3截短体的核苷酸序列(SEQ IDNO:7)的重组pEGBacMam质粒(图1)导入到大肠杆菌DH10Bac感受态细胞(上海唯地)中,在含有50μg/mL卡那霉素(aladdin)、7μg/mL庆大霉素(aladdin)、10μg/mL四环霉素(aladdin)、100μg/mL Bluo-gal(Thermofish)、40μg/mL IPTG(aladdin)的LB固体培养基中,于37℃培养48-72小时。挑选均匀的白斑至5mL含有三种抗生素(50μg/mL卡那霉素、7μg/mL庆大霉素、10μg/mL四环霉素)的LB液体培养基中,37℃,200rpm条件下培养12-16小时,抽提重组杆状病毒质粒。
2.重组杆状病毒制备
取细胞培养6孔板(Nest),每孔1×106/2mL sf9昆虫细胞,于27℃恒温恒湿培养箱中贴壁培养30min,取100μL Insect Medium(Sf-900TM III SFM)加入10μL转染试剂(Cellfectin),另100μL Insect Medium加入5μg重组杆状病毒质粒,混合后室温下孵育20分钟,将转染复合物均匀滴加到6孔板中,于27℃条件下继续培养72小时,于4℃6000rpm离心15分钟,取上清加入2%的FBS,避光4度保存即为P1代重组杆状病毒。
取P1代重组杆状病毒按照1:100的比例感染2mL细胞密度为5×105/mL sf9昆虫细胞,27℃的条件下贴壁进行培养72小时,于4℃6000rpm离心15分钟,取上清加入2%的FBS,避光4度保存即为P2代重组杆状病毒。
分出密度为1×106/mL的40mL sf9昆虫细胞,培养24小时后,取P2代重组杆状病毒按照1:100的比例感染,27℃,120rpm的条件下进行培养96小时,于4℃6000rpm离心15分钟,上清液用0.22μm过滤装置过滤后,加入2%的FBS,避光4度保存即为P3代重组杆状病毒。
分出密度为1×106/mL的200mL sf9昆虫细胞,培养24小时后,取P3代重组杆状病毒按照1:100的比例感染,27℃,120rpm的条件下进行培96小时,于4℃6000rpm离心15分钟,上清液用0.22μm过滤装置过滤后,加入2%的FBS,避光4度保存即为P4代重组杆状病毒。
实施例3蛋白纯化小试
将HEK-293S细胞传代,浓度为1.5×106/ml,37℃,8%CO2,60%湿度,120rpm条件下培养24h,取P4代重组杆状病毒按照1:10的比例感染哺乳动物细胞,摇床培养12-18h加入丁酸钠,终浓度为10mM。
72小时后,4℃,6000rpm离心15分钟,收集细胞,用Lysis buffer(50mM HEPES,pH7.4,150mM NaCl,1%Protease Inhibitor Cocktail,EDTA-Free,1%GDN,1mM TCEP)重悬细胞沉淀,4℃下翻转溶膜3小时。溶膜结束后,于4℃,40000rpm条件下离心45分钟,上清液与MBP亲和层析填料4℃翻转结合2.5小时,结合上清液过重力柱,用Wash buffer(25mMHEPES,pH7.4,150mM NaCl,0.02%GDN,1mM TCEP)洗脱杂蛋白,用Elute buffer(25mMHEPES,pH7.4,150mM NaCl,0.02%GDN,1mM TCEP,40mM Maltose)洗脱目的蛋白100KDa的超滤管对目的蛋白进行浓缩,浓缩至体积为500μL,进行凝胶过滤层析,所用凝胶柱型号为Superrose 6Increase 10/300GL(cytiva),缓冲液为SEC buffer(25mM HEPES,pH7.4,150mM NaCl,0.02%GDN,1mM TCEP),收集蛋白样品,测试A280浓度,进行SDS PAGE凝胶电泳检测目的蛋白大小与纯度。结果如图5和图6所示,可表达得到高纯度和高产量的TRPM3截短体。
用同样的实验过程表达野生型TRPM3蛋白,结果如图3和图4所示,无法表达得到高纯度和高产量的野生型TRPM3蛋白。
实施例4稳定细胞系构建
将TRPM3截短体的核苷酸序列(SEQ ID NO:8)送由南京擎科生物技术有限公司进行合成,连接重组至慢病毒载体上(图2),进行转化、提取质粒。
1.制备慢病毒
生物安全柜紫外灯照30分钟,把培养基和试剂放置常温放置。从培养箱中取出状态良好的293T细胞,吸出原培养基,PBS清洗之后加入胰酶消化,轻轻拍打培养皿,将培养皿中的细胞转移至15ml离心管内离心,1000rpm,5min。将细胞沉淀重悬到10cm细胞培养皿中,每孔10mL细胞悬液,过夜后培养箱取出细胞,倒置显微镜观察密度达90%左右即可开始转染,吸出原有培养基,沿皿壁缓慢加入10mL opti-MEM,放置于培养箱中,待转染。用上述慢病毒载体和辅助质粒转染HEK293T细胞。其中辅助质粒具体为含有gag基因、pol基因和rev基因的psPAX2载体和含有vsvg基因的pMD2G载体。转染后6小时,弃去opti-MEM,用新鲜的DMEM培养基代替。转染48小时候将培养皿中上清吸出,超速离心后用0.22μM针孔过滤器过滤后保存。
2.转染细胞
在6孔板中种上细胞,汇合度约30%,第二天将收集后的慢病毒感染293T细胞,同时加入polybrene(5mg/mL)辅助转染,转染12小时后去除培养基,用新鲜的DMEM培养基替换。再继续培养24小时后,开始加入1μg/mL的嘌呤霉素筛选阳性细胞,每天更换新鲜的含嘌呤霉素的选择培养基直至细胞不再继续死亡,更换低浓度的嘌呤霉素维持细胞生长,细胞扩增完成后通过FACS检测靶蛋白表达。
实施例5膜片钳电生理检测
利用全细胞膜片钳技术可以记录细胞系中的TRPM3通道电流,为了测试TRPM3截短体的效果,本发明做了野生型人源TRPM3蛋白截短前后,即野生型人源TRPM3蛋白和TRPM3截短体的电流对照。取转染了野生型人源TRPM3蛋白或TRPM3截短体24小时左右的HEK293T细胞,弃去培养液,加入人工配制的细胞外缓冲液,向记录浴槽内持续灌注22℃的细胞外液,使用蠕动泵灌流***控制灌流溶液的流速,控制流速为2mL/min,在显微镜下找到合适的目标细胞,通常选取单个的饱满,边缘清晰,无凹陷的细胞,将提前拉制好的入液阻抗为2-2.5MΩ的玻璃微电极内灌入人工配制的电极内液,通过微操移动玻璃电极,移动到细胞上,同时用注射器控制玻璃电极内的气压,给予负压,通过记录软件观察到测试的电流方波逐渐减小,持续给予负压,使玻璃电极开口与细胞膜建立高阻(GΩ)封接,继续给予玻璃电极负压让细胞膜破裂从而使细胞内与玻璃电极连通形成全细胞记录模式,细胞内液与玻璃电极内液可以交换,连接玻璃电极的放大器(HEKA EPC10放大器,配套patchmaster记录软件)可以控制细胞内电压,设置不同的电压从而能够记录全细胞的通道电流。记录TRPM3时使用的细胞外液配方如下:138mM NaCl,5.4mM KCl,1mM MgCl2,2mM CaCl2,10mM HEPES,10mM D-glucose,去离子水溶解,使用NaOH将pH调整至7.4,测量渗透压约为315mOsm;电极内液配方如下:130mM CsMES(甲磺酸铯),2mM MgCl2,10mM CsCl2,10mM EGTA,10mM HEPES,去离子水溶解,使用CsOH将pH调整至7.4,渗透压约为315mOsm。
在保持细胞电压为-60mV的刺激下,给予细胞一个-100mV~+100mV的电压斜坡(Ramp)刺激,刺激时长为400ms,每5s刺激一次(图7)。
使用50μM的pregnenolone sulfate(孕烯醇酮硫酸盐,后简称PregS)激活通道,持续记录通道电流,记录的采样频率为50KHz,使用低通滤波2KHz滤除高频的干扰噪音。在检测以TRPM3为靶点的药物对其通道电流的影响时,设置不同的药物浓度梯度,使用细胞外液溶解,如有不溶于水的药物,使用DMSO助溶后,DMSO在药物终浓度占比不应超过0.1%,以避免DMSO对记录的细胞造成损伤。在记录电流的同时通过给药装置连续给不同浓度的药物,观察不同浓度对细胞电流的影响,计算通道在+100mV刺激电压下的电流幅值,做出幅值对时间的散点图。为了计算药物对TRPM3的半抑制浓度(IC50),通常设置5-6个浓度梯度,计算不同浓度对电流的抑制率,在软件graphpad中用Hill equation对浓度抑制曲线做拟合,从而计算出半抑制浓度。每测试一个新的化合物的抑制率通常需要用TRPM3的阳性抑制剂对***做一个整体的测试并作为阳性对照,以确保膜片钳记录***没有问题。
1.野生型人源TRPM3蛋白截短前后的通道电流比较
使用10μM CIM0216很难激活野生型人源TRPM3蛋白的通道电流。图8展示了通过全细胞膜片钳记录的HEK293T细胞膜上的野生型人源TRPM3蛋白通道电流的幅值,膜片钳记录的protocol为-100mV~+100mV的电压斜坡(Ramp)刺激,刺激时长为400ms,每5s刺激一次(膜片钳方法如图7所示),图8中每一个点代表ramp刺激在+100mV下的电流幅值。空白对照(control)为不加任何激动剂或者抑制剂的人工细胞外液,且与之后加的小分子统一DMSO的浓度(不超过0.1%)。由图8可以看出野生型人源TRPM3蛋白在+100mV刺激下的电流幅值并不会因为给激动剂而增大。
图9显示的是通过全细胞膜片钳记录的HEK293T细胞膜上的TRPM3通道电流的典型图,展示了不同条件下,一次ramp刺激的完整的电流的图形。由图9可以看出野生型人源TRPM3蛋白在293T细胞上使用膜片钳很难记录到激活的电流,给10uM的激动剂CIM0216的电流曲线和空白对照几乎一样,+100mV下的电流幅值没有增大。
将野生型人源TRPM3蛋白截短后不仅10uM CIM0216能够激活TRPM3,而且1μMCIM0216和50μM PregS都能激活出电流。图12展示的是通过全细胞膜片钳记录的HEK293T细胞膜上的TRPM3(截短后)通道电流的幅值,膜片钳记录的protocol为-100mV~+100mV的电压斜坡(Ramp)刺激,刺激时长为400ms,每5s刺激一次(膜片钳方法如图7所示),图10中每一个点代表ramp刺激在+100mV下的电流幅值。空白对照(control)为不加任何激动剂或者抑制剂的人工细胞外液,且与之后加的小分子统一DMSO的浓度(不超过0.1%)。
图11展示的是通过全细胞膜片钳记录的HEK293T细胞膜上的TRPM3截短体通道电流的典型图,图11显示了不同条件下,一次ramp刺激的完整的电流的图形,序列截短后,能够在10uM的激动剂CIM0216作用下记录到电流的变化。
图12展示了不同激动剂下TRPM3截短体电流幅值的变化,可以看出在序列截短后,两种激动剂均能记录到电流幅值的变化,提示截短之后能够在293T细胞膜上记录到TRPM3的通道开放。
图13展示的是通过全细胞膜片钳记录的HEK293T细胞膜上的TRPM3截短体通道电流的典型图,图13显示了不同条件下,一次ramp刺激的完整的电流的图形,序列截短后,能够在1μM的激动剂CIM0216和50uM的激动剂PregS作用下记录到电流的变化。
2.测试TRPM3激动剂PregS对HEK293T细胞膜上的TRPM3截短体通道激活的EC50(半最大效应浓度)
图14展示了不同浓度的PregS激动剂下TRPM3截短体电流幅值的变化,可以看出在序列截短后,各个浓度的PregS激动剂均能记录到电流幅值的变化,提示截短之后能够在293T细胞膜上记录到TRPM3的通道开放。
图15展示的是通过全细胞膜片钳记录的HEK293T细胞膜上的TRPM3截短体通道电流的典型图,图15显示了不同条件下,一次ramp刺激的完整的电流的图形,序列截短后,能够在不同浓度的激动剂PregS作用下记录到电流的变化。
如图16所示,通过拟合激活率对浓度对数的曲线计算得到EC50=11.13μM。
3.测试TRPM3抑制剂Ononetin(芒柄花酚)对HEK293T细胞膜上的TRPM3截短体通道抑制的IC50(半抑制浓度)
图17展示了在使用50uM PregS激活后,不同浓度的Ononetin抑制剂下TRPM3截短体电流幅值的变化,可以看出在序列截短后,各个浓度的Ononetin抑制剂均能记录到电流幅值的变化,提示截短之后能够在293T细胞膜上记录到TRPM3的通道开放。
图18展示的是通过全细胞膜片钳记录的HEK293T细胞膜上的TRPM3截短体通道电流的典型图,图18显示了不同条件下,一次ramp刺激的完整的电流的图形,序列截短后,在使用50μM PregS激活后,能够在不同浓度的抑制剂Ononetin作用下记录到电流的变化。
如图19所示,通过拟合抑制率对浓度对数的曲线计算得到IC50=126nM。
本申请所使用的序列如下表所示:
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
参考文献
[1]Goldberg,D.S.,&McGee,S.J.(2011).Pain as a global public healthpriority.BMC Public Health,11,770 10.1186/1471-2458-11-770.
[2]Yaksh,T.L.,Woller,S.A.,Ramachandran,R.,&Sorkin,L.S.(2015).Thesearch for novel analgesics:Targets and mechanisms.F1000Prime Rep,7,5610.12703/P7-56.
[3]Yekkirala,A.S.,Roberson,D.P.,Bean,B.P.,&Woolf,C.J.(2017).Breakingbarriers to novel analgesic drug development.Nature Reviews.Drug Discovery,16,545–564.10.1038/nrd.2017.202.
[4]Held,K.,Gruss,F.,Aloi,V.D.,Janssens,A.,Ulens,C.,Voets,T.,Vriens,J.Mutations in the voltage-sensing domain affect the alternative ionpermeation pathway in the TRPM3 channel.J.Physiol.2018,596,2413–2432.
[5]Hofmann,T.,Chubanov,V.,Gudermann,T.,Montell,C.TRPM5is a voltage-modulated and Ca2+-activated monovalent selective cationchannel.Curr.Biol.2003,13,1153–1158.
[6]Voets,T.,Owsianik,G.,Janssens,A.,Talavera,K.,Nilius,B.TRPM8voltagesensor mutants reveal a mechanism for integrating thermal and chemicalstimuli.Nat.Chem.Biol.2007,3,174–182.
[7]Chang,Y.,Schlenstedt,G.,Flockerzi,V.,Beck,A.Properties of theintracellular transient receptor potential(TRP)channel in yeast,Yvc1.FEBSLett.2010,584,2028–2032.
[8]Moqrich,A.,Hwang,S.W.,Earley,T.J.,Petrus,M.J.,Murray,A.N.,Spencer,K.S.R.,Andahazy,M.,Story,G.M.,Patapoutian,A.Impaired thermosensation in micelacking TRPV3,a heat and camphor sensor in the skin.Science 2005,307,1468–1472.
[9]Cenac,N.,Altier,C.,Chapman,K.,Liedtke,W.,Zamponi,G.,Vergnolle,N.Transientreceptor potential vanilloid-4 has a major role in visceralhypersensitivity symptoms.Gastroenterology 2008,135,937–946.
[10]Thilo,F.,Scholze,A.,Liu,D.Y.,Zidek,W.,Tepel,M.Association oftransient receptorpotential canonical type3(TRPC3)channel transcripts withproinflammatory cytokines.Arch.Biochem.Biophys.2008,471,57–62.
[11]Kim,S.R.,Lee,D.Y.,Chung,E.S.,Oh,U.T.,Kim,S.U.,Jin,B.K.Transientreceptorpotential vanilloid subtype1 mediates cell death of mesencephalicdopaminergic neurons in vivoand in vitro.J.Neurosci.2005,25,662–671.
[12]Hutter,M.M.,Wick,E.C.,Day,A.L.,Maa,J.,Zerega,E.C.,Richmond,A.C.,Jordan,T.H.,Grady,E.F.,Mulvihill,S.J.,Bunnett,N.W.,et al.Transient receptorpotential vanilloid(TRPV-1)promotes neurogenic inflammation in the pancreasvia activation of the neurokinin-1receptor(NK-1R).Pancreas 2005,30,260–265.
[13]Reiser,J.,Polu,K.R.,C.C.,Kenlan,P.,Altintas,M.M.,Wei,C.,Faul,C.,Herbert,S.,Villegas,I.,Avila-Casado,C.,et al.TRPC6 is a glomerularslit diaphragm-associatedchannel required for normal renalfunction.Nat.Genet.2005,37,739–744.
[14]Winn,M.P.,Conlon,P.J.,Lynn,K.L.,Farrington,M.K.,Creazzo,T.,Hawkins,A.F.,Daskalakis,N.,Kwan,S.Y.,Ebersviller,S.,Burchette,J.L.,etal.Medicine:A mutation in theTRPC6 cation channel causes familial focalsegmental glomerulosclerosis.Science 2005,308,1801–1804.
[15]Thebault,S.,Flourakis,M.,Vanoverberghe,K.,Vandermoere,F.,Roudbaraki,M.,Lehen’kyi,V.,Slomianny,C.,Beck,B.,Mariot,P.,Bonnal,J.L.,etal.Differential role of transientreceptor potential channels in Ca2+entry andproliferation of prostate cancer epithelial cells.Cancer Res.2006,66,2038–2047.
[16]Humphray,S.J.,Oliver,K.,Hunt,A.R.,Plumb,R.W.,Loveland,J.E.,Howe,K.L.,Andrews,T.D.,Searle,S.,Hunt,S.E.,Scott,C.E.,et al.DNA sequence andanalysis of humanchromosome 9.Nature 2004,429,369–374.
[17]Shiels,A.TRPM3_miR-204:A complex locus for eye development anddisease.Hum.Genom.2020,14,7.
[18]Behrendt,M.Transient receptor potential channels in the contextof nociception andpain—Recent insights into TRPM3 properties andfunction.Biol.Chem.2019,400,917–926.
[19]Grimm,C.,Kraft,R.,Sauerbruch,S.,Schultz,G.,Harteneck,C.Molecularandfunctional characterization of the melastatin-related cation channelTRPM3.J.Biol.Chem.2003,278,21493–21501.
[20]Hoffmann,A.,Grimm,C.,Kraft,R.,Goldbaum,O.,Wrede,A.,Nolte,C.,Hanisch,U.-K.,Richter-Landsberg,C.,Brück,W.,Kettenmann,H.,et al.TRPM3 isexpressed in sphingosine-responsive myelinating oligodendrocytes:TRPM3 in oligodendrocytes.J.Neurochem.2010,114,654–665.
[21]Kunert-Keil,C.,Bisping,F.,Krüger,J.,Brinkmeier,H.Tissue-specificexpression ofTRP channel genes in the mouse and its variation in threedifferent mouse strains.BMC Genom.2006,7,159.
[22]Samuel,W.,Jaworski,C.,Postnikova,O.A.,Kutty,R.K.,Duncan,T.,Tan,L.X.,Poliakov,E.,Lakkaraju,A.,Redmond,T.M.Appropriately differentiated ARPE-19 cells regainphenotype and gene expression profiles similar to those ofnative RPE cells.Mol.Vis.2017,23,60–89.
[23]Son,A.R.,Yang,Y.M.,Hong,J.H.,Lee,S.I.,Shibukawa,Y.,Shin,D.M.OdontoblastTRP channels and thermo/mechanicaltransmission.J.Dent.Res.2009,88,1014–1019.
[24]Wang,H.-P.,Pu,X.-Y.,Wang,X.-H.Distribution profiles of transientreceptor potentialmelastatin-related and vanilloid-related channels inprostatic tissue in rat.Asian J.Androl.2007,9,634–640.
[25]Held,K.,Voets,T.,Vriens,J.TRPM3 in temperature sensing andbeyond.Temperature2015,2,201–213.
[26]Held,K.,Kichko,T.,De Clercq,K.,Klaassen,H.,Van Bree,R.,Vanherck,J.-C.,Marchand,A.,Reeh,P.W.,Chaltin,P.,Voets,T.,et al.Activation of TRPM3 bya potent syntheticligand reveals a role in peptide release.Proc.Natl.Acad.Sci.USA2015,112,E1363–E1372.
[27]Behrendt,M.(2019).Transient receptor potential channels in thecontext of nociceptionand pain—Recent insights into TRPM3 properties andfunction.Biological Chemistry,400(7),917–926.10.1515/hsz-2018-0455.
[28]Vriens,J.,Owsianik,G.,Hofmann,T.,Philipp,S.E.,Stab,J.,Chen,X.,…Voets,T.(2011).TRPM3 is a nociceptor channel involved in the detection ofnoxious heat.Neuron,70(3),482–494.10.1016/j.neuron.2011.02.051.
[29]Alkhatib,O.,da Costa,R.,Gentry,C.,Quallo,T.,Bevan,S.,Andersson,D.A.Promiscuous G-protein-coupled receptor inhibition of transient receptorpotential melastatin 3ion channels by Gβγsubunits.J.Neurosci.2019,39,7840–7852.
[30]Badheka,D.,Yudin,Y.,Borbiro,I.,Hartle,C.M.,Yazici,A.,Mirshahi,T.,Rohacs,T.Inhibition of transient receptor potential melastatin 3 ion channelsby G-proteinβγsubunits.eLife 2017,6,e26147.
[31]Quallo,T.,Alkhatib,O.,Gentry,C.,Andersson,D.A.,Bevan,S.G proteinβγsubunitsinhibit TRPM3 ion channels in sensory neurons.eLife 2017,6,e26138.
[32]Dembla,S.,Behrendt,M.,Mohr,F.,Goecke,C.,Sondermann,J.,Schneider,F.M.,Schmidt,M.,Stab,J.,Enzeroth,R.,Leitner,M.G.,et al.Anti-nociceptiveaction of peripheralmu-opioid receptors by G-beta-gammaprotein-mediatedinhibition of TRPM3 channels.eLife2017,6,e26280.
[33]Jia,S.,Zhang,Y.,&Yu,J.(2017).Antinociceptive effects ofisosakuranetin in a ratmodel of peripheral neuropathy.Pharmacology,100(3–4),201–207.10.1159/000478986.
[34]Krugel,U.,Straub,I.,Beckmann,H.,&Schaefer,M.(2017).PrimidoneinhibitsTRPM3 and attenuates thermal nociception in vivo.Pain,158(5),856–867.10.1097/j.pain.
[35]Straub,I.,Krugel,U.,Mohr,F.,Teichert,J.,Rizun,O.,Konrad,M.,…Schaefer,M.(2013).Flavanones that selectively inhibit TRPM3 attenuate thermalnociception in vivo.Molecular Pharmacology,84(5),736–750.10.1124/mol.113.086843.
Claims (24)
1.一种TRPM3截短体,其特征在于,所述TRPM3截短体的氨基酸序列如SEQ ID NO:5所示。
2.一种包含如权利要求1所述TRPM3截短体的融合蛋白,其特征在于,所述融合蛋白还包括连接在所述TRPM3截短体的6×His标签、MBP蛋白和/或HRV3C蛋白酶切割位点。
3.如权利要求2所述的融合蛋白,其特征在于,所述连接发生在所述TRPM3截短体的N端,和/或所述6×His标签、所述MBP蛋白和所述HRV3C蛋白酶切割位点之间通过连接子连接。
4.如权利要求3所述的融合蛋白,其特征在于,所述MBP蛋白的氨基酸序列如SEQ IDNO:9所示。
5.如权利要求3所述的融合蛋白,其特征在于,连接了6×His标签、MBP蛋白和HRV3C蛋白酶切割位点的TRPM3截短体的氨基酸序列如SEQ ID NO:4所示。
6.一种细胞系,其特征在于,所述细胞系包含如权利要求1所述的TRPM3截短体或如权利要求2-5任一项所述的融合蛋白。
7.如权利要求6所述的细胞系,其特征在于,所述细胞系选用293T细胞。
8.一种筛选与TRPM3相互作用的化合物的方法,其特征在于,所述方法包括
i)在细胞膜上表达如权利要求1所述的TRPM3截短体或如权利要求2-5任一项所述的融合蛋白,使所述化合物与所述TRPM3截短体或所述融合蛋白接触,利用全细胞膜片钳记录通道电流;或
ii)使所述化合物与如权利要求6或7所述的细胞系接触,利用全细胞膜片钳记录通道电流。
9.如权利要求8所述的方法,其特征在于,所述化合物为TRPM3激动剂和/或抑制剂。
10.一种分离的核酸,其特征在于,所述分离的核酸编码如权利要求1所述的TRPM3截短体或如权利要求2-5任一项所述的融合蛋白。
11.如权利要求10所述的核酸,其特征在于,编码所述MBP蛋白的核苷酸序列如SEQ IDNO:10所示,编码所述TRPM3截短体的核苷酸序列如SEQ ID NO:7或8所示。
12.一种重组表达载体,其特征在于,所述重组表达载体包含启动子和如权利要求10或11所述的核酸;所述重组表达载体的骨架质粒为pEGBacMam载体或PCDH载体。
13.一种质粒组合,其特征在于,所述质粒组合包含如权利要求12所述的重组表达载体。
14.如权利要求13所述的质粒组合,其特征在于,所述质粒组合还包含编码gag基因、pol基因、rev基因的第二质粒,和编码vsvg基因的第三质粒。
15.如权利要求14所述的质粒组合,其特征在于,所述第二质粒的骨架质粒为psPAX2载体,所述第三质粒的骨架质粒为pMD2G载体。
16.一种重组杆状病毒,其特征在于,所述重组杆状病毒包含如权利要求10或11所述的核酸。
17.一种重组杆状病毒,其特征在于,所述重组杆状病毒由如权利要求12所述的重组表达载体感染昆虫细胞制得。
18.一种重组慢病毒,其特征在于,所述重组慢病毒包含如权利要求10或11所述的核酸。
19.一种重组慢病毒,其特征在于,所述重组慢病毒由如权利要求13-15任一项所述的质粒组合转染哺乳动物细胞制得。
20.如权利要求19所述的重组慢病毒,其特征在于,所述哺乳动物细胞为293T细胞。
21.一种制备TRPM3截短体的方法,其特征在于,所述方法包括使用如权利要求16或17所述的重组杆状病毒感染哺乳动物细胞,或使用如权利要求18-20任一项所述的重组慢病毒转染哺乳动物细胞。
22.如权利要求21所述的方法,其特征在于,所述哺乳动物细胞为293S或293T细胞。
23.一种制备细胞系的方法,其特征在于,所述方法包括使用如权利要求18-20任一项所述的重组慢病毒转染哺乳动物细胞。
24.如权利要求23所述的方法,其特征在于,所述哺乳动物细胞为293T细胞。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310592892.8A CN116891523B (zh) | 2023-05-24 | 2023-05-24 | 一种trpm3截短体、包含其的细胞系及其用途 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310592892.8A CN116891523B (zh) | 2023-05-24 | 2023-05-24 | 一种trpm3截短体、包含其的细胞系及其用途 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116891523A CN116891523A (zh) | 2023-10-17 |
CN116891523B true CN116891523B (zh) | 2024-05-17 |
Family
ID=88314061
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310592892.8A Active CN116891523B (zh) | 2023-05-24 | 2023-05-24 | 一种trpm3截短体、包含其的细胞系及其用途 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116891523B (zh) |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1937914A (zh) * | 2004-03-30 | 2007-03-28 | 帕拉迪姆医疗有限公司 | 离子通道 |
CN101848933A (zh) * | 2007-10-26 | 2010-09-29 | 塞诺菲-安万特股份有限公司 | Trpc结构域和sestd1结构域的复合物及涉及所述复合物的方法和用途 |
CN101998989A (zh) * | 2007-06-08 | 2011-03-30 | 塞诺米克斯公司 | Trpml3(mcoln3)作为咸味受体的鉴定及在用于鉴定调控钠转运、吸收或***和/或醛固酮和/或血管加压素的产生或释放的味觉(咸味)调节剂和/或治疗剂的测定中的用途 |
CN105940117A (zh) * | 2013-12-12 | 2016-09-14 | 阿尔玛克诊断有限公司 | ***癌分类 |
CN106232110A (zh) * | 2014-04-14 | 2016-12-14 | 弗莱克斯制药股份有限公司 | 离子通道活化剂和使用方法 |
CN111961687A (zh) * | 2019-09-30 | 2020-11-20 | 深圳晶蛋生物医药科技有限公司 | 一种针对高血压的药物靶点表达和纯化方法 |
EP4058470A2 (en) * | 2019-11-15 | 2022-09-21 | The Regents of the University of Colorado, A Body Corporate | Novel peptide compositions and methods of treating neurological injury |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004076632A2 (en) * | 2003-02-21 | 2004-09-10 | The Queen's Medical Center | Methods of screening for trpm5 modulators |
US7344882B2 (en) * | 2003-05-12 | 2008-03-18 | Bristol-Myers Squibb Company | Polynucleotides encoding variants of the TRP channel family member, LTRPC3 |
US20060275812A1 (en) * | 2005-06-01 | 2006-12-07 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | Assay for agonists and antagonists of ion channels and for regulators of genetic expression |
CA2660286A1 (en) * | 2006-08-09 | 2008-02-21 | Homestead Clinical Corporation | Organ-specific proteins and methods of their use |
GB201107467D0 (en) * | 2011-05-05 | 2011-06-15 | Univ Leuven Kath | Novel treatment of pain |
US20210069180A1 (en) * | 2015-05-01 | 2021-03-11 | Griffith University | Methods for treating chronic fatigue syndrome and myalgic encephalomyelitis |
WO2018165034A1 (en) * | 2017-03-06 | 2018-09-13 | President And Fellows Of Harvard College | Methods of use for trp channel antagonist-based combination cancer therapies |
-
2023
- 2023-05-24 CN CN202310592892.8A patent/CN116891523B/zh active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1937914A (zh) * | 2004-03-30 | 2007-03-28 | 帕拉迪姆医疗有限公司 | 离子通道 |
CN101998989A (zh) * | 2007-06-08 | 2011-03-30 | 塞诺米克斯公司 | Trpml3(mcoln3)作为咸味受体的鉴定及在用于鉴定调控钠转运、吸收或***和/或醛固酮和/或血管加压素的产生或释放的味觉(咸味)调节剂和/或治疗剂的测定中的用途 |
CN101848933A (zh) * | 2007-10-26 | 2010-09-29 | 塞诺菲-安万特股份有限公司 | Trpc结构域和sestd1结构域的复合物及涉及所述复合物的方法和用途 |
CN105940117A (zh) * | 2013-12-12 | 2016-09-14 | 阿尔玛克诊断有限公司 | ***癌分类 |
CN106232110A (zh) * | 2014-04-14 | 2016-12-14 | 弗莱克斯制药股份有限公司 | 离子通道活化剂和使用方法 |
CN111961687A (zh) * | 2019-09-30 | 2020-11-20 | 深圳晶蛋生物医药科技有限公司 | 一种针对高血压的药物靶点表达和纯化方法 |
EP4058470A2 (en) * | 2019-11-15 | 2022-09-21 | The Regents of the University of Colorado, A Body Corporate | Novel peptide compositions and methods of treating neurological injury |
Non-Patent Citations (8)
Title |
---|
Expression of the C-Terminal Domain of Phospholipase Cβ3 Inhibits Signaling via Gαq-Coupled Receptors and Transient Receptor Potential Channels;Gerald Thiel、Oliver G. Rössler;《International Journal of Molecular Sciences》;20220824;第23卷(第17期);第9590篇 * |
H2O2-induced Ca2+ influx and its inhibition by N-(p-amylcinnamoyl) anthranilic acid in the beta-cells: involvement of TRPM2 channels;Muhammad R Bari等;《Journal of Cellular and Molecular Medicine》;20090531;第13卷(第9B期);第3260-3267页 * |
TRPM3:一种新型热痛觉感受通道;刘蕾、赵蒙蒙、张秀琳;《生理科学进展》;20220425;第53卷(第02期);第141-146页 * |
TRP通道超家族的基本特征及其与疾病的关系;TRP通道超家族的基本特征及其与疾病的关系;《生命科学》;20200915;第32卷(第09期);第1-6页 * |
XP_054514854.1: transient receptor potential cation channel subfamily M member 3 isoform X4 [Pan troglodytes];GenBank;《GenBank》;20230411;标题、PROTEIN、CDS、ORIGIN部分 * |
瞬时受体电位通道蛋白的跨膜片段分析;苏秋香等;《中国医科大学学报》;20160615;第45卷(第07期);第610-615页 * |
瞬时受体通道TRPM8及其对***癌的影响;盛晓波等;《肿瘤》;20130825;第33卷(第08期);第739-742页 * |
靶向瞬时受体电位通道天然调节剂的研究进展;曹南开等;《药学学报》;20170512;第52卷(第05期);第673-684页 * |
Also Published As
Publication number | Publication date |
---|---|
CN116891523A (zh) | 2023-10-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5492825A (en) | Mammalian inward rectifier potassium channel cDNA, IRK1, corresponding vectors, and transformed cells | |
JP6203215B2 (ja) | プロミニン−1ペプチド断片およびその使用 | |
TW201107471A (en) | Polypeptides selective for αvβ3 integrin conjugated with a variant of human serum albumin (HSA) and pharmaceutical uses thereof | |
WO1997035881A2 (en) | Receptor and transporter antagonists | |
US8029980B2 (en) | Identification and use of agents that modulate oncogenic transcription agent activity | |
CN111978408B (zh) | 靶向IKKβ的短肽及其在炎症性疾病中的应用 | |
Fuchs et al. | Endogenous [3H] flunitrazepam binding in human embryonic kidney cell line 293 | |
US20040058359A1 (en) | Erbin as a negative regulator of Ras-Raf-Erk signaling | |
JP2000083669A (ja) | Cxcr4ケモカイン受容体のヒトスプライス変異体cxcr4b | |
CN116891523B (zh) | 一种trpm3截短体、包含其的细胞系及其用途 | |
TWI354552B (en) | Oxydecahydronaphthalene modulators of hm74 | |
JPH08502737A (ja) | セロトニン作動性レセプター活性を有するポリペプチド(5ht2c)及び利用 | |
EP2873424B1 (en) | Tau protein mediated neurodegenerative disease therapeutic agent | |
Kurrle et al. | Function of flotillins in receptor tyrosine kinase signaling and endocytosis: role of tyrosine phosphorylation and oligomerization | |
CA2378917A1 (en) | Melanin-concentrating hormone receptor | |
US20050191612A1 (en) | Method of screening insulin content enhancer | |
KR100870296B1 (ko) | 융합 단백질 | |
WO2008120263A2 (en) | Prokineticins receptors antagonists, derivatives and uses thereof | |
JP5519702B2 (ja) | 鎮痛を目的とする、t型カルシウムチャンネルの新規拮抗薬である毒素の同定 | |
US20090081227A1 (en) | Human and mammalian stem cell-derived neuron survival factors | |
US20090010909A1 (en) | Novel GPCR and methods of use of the same | |
JP4016280B2 (ja) | 脂質代謝改善物質のスクリーニング方法 | |
Zheng | Function and Regulation of TRPP2 and TRPP3, and identification of pore gates of TRP channels | |
EP1600458A1 (en) | Novel gene relating to fibrotic conditions | |
US20050233304A1 (en) | Method of screening remedy for renal failure |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |