CN102977201B - Scoropendra subspinipes mutilans polypeptide toxin mu-SLPTX-Ssm6a and gene and application thereof - Google Patents
Scoropendra subspinipes mutilans polypeptide toxin mu-SLPTX-Ssm6a and gene and application thereof Download PDFInfo
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Abstract
The invention relates to scoropendra subspinipes mutilans polypeptide toxin mu-SLPTX-Ssm6a and gene and application of the scoropendra subspinipes mutilans polypeptide toxin mu-SLPTX-Ssm6a, and belongs to the technical field of biomedicine. The scoropendra subspinipes mutilans polypeptide toxin mu-SLPTX-Ssm6a is composed of amino acid sequence shown in SEQ ID NO: 1. Gene of coding the scoropendra subspinipes mutilans polypeptide toxin mu-SLPTX-Ssm6a is composed of nucleotide sequence shown in the SEQ ID NO: 1. The scoropendra subspinipes mutilans polypeptide toxin mu-SLPTX-Ssm6a is sodium channel inhibitor and is applied in preparation of analgesia medicine. The scoropendra subspinipes mutilans polypeptide toxin mu-SLPTX-Ssm6a has an obvious inbibitional effect on a sodium channel current on dorsal root ganglion of a rat, and is the sodium channel inhibitor. The scoropendra subspinipes mutilans polypeptide toxin mu-SLPTX-Ssm6a is capable of selectively acting on sodium channel subtype Nav 1.7. An analgesia model of a mouse shows that the polypeptide has a good analgesic effect and can be applied in the preparation of the analgesia medicine.
Description
Technical field:
The present invention relates to a kind of few sour jujube centipede polypeptide toxin mu-SLPTX-Ssm6a and gene and application, belong to field of biomedicine technology.
Background technology:
Voltage-gated sodium channel is distributed widely in the excitability tissue of muscle, neurone, body of gland, cardiac muscle, is a kind of important cross-film structural protein, and it is movable closely related with the specific function such as excitement, contraction, secretion and cynapse transmission of cell.Hypotype noticeable critical function in sodium channel is exactly relevant with the process of nociception transmission, neuropathic pain.Except tetraodotoxin non-sensitive type sodium channel Nav1.8 and 1.9 is the important participant of this process, Nav1.7 is also one of key factor wherein.On minor diameter peripheral sensory neurone, Nav1.7 can set Nav1.8 and Nav1.9 activates open threshold value.In causing scorching pain model animal, find that Nav1.7 expression level is obviously raised.The Nav1.7 mutant relevant to lupus erythematosus activates by facilitation, and the inactivation time of the Nav1.7 mutant relevant to the extreme pain disorder of paroxysmal is extended.Particularly, when Nav1.7 can not produce function, patient can lose the ability of experiencing the pain sensation, but other functions such as the sense of heat, pressure sensation are acted normally.The application prospect therefore, for the specific blockage agent of sodium channel with very important analgesic.
Centipede is a kind of traditional Chinese medicine, warm in nature, and taste is pungent, poisonous.The function of there is breath wind antispastic, dispersing pathogen accumulation, removing obstruction in channels to relieve pain.In centipede poison gland, contain abundant polypeptide composition, verified at present these polypeptide compositions mostly are neurotoxin.Polypeptide can optionally suppress sodium channel, potassium channel or calcium channel, so the composition in centipede venom has a wide range of applications future, as exploitation becomes analgesic and treatment autoimmune disease medicine.
Summary of the invention:
The object of the present invention is to provide a kind of few sour jujube centipede polypeptide toxin mu-SLPTX-Ssm6a and gene and application.
Few sour jujube centipede polypeptide toxin mu-SLPTX-Ssm6a provided by the invention, by molecular sieve and reverse high performance liquid chromatography separation and purification, to obtain from the thick poison of few sour jujube centipede, its aminoacid sequence (SEQ ID NO:1) is ADNKCENSLRREIACGQCRDKVKTDGYFYECCTSDSTFKKCQDLLH, this polypeptide comprises that 46 amino-acid residue molecular weight are 5318.97, iso-electric point is 6.76, and it is C1-C5, C2-C6 and C3-C4 that molecule contains three pairs of disulfide linkage arrangement modes.
The gene clone of few sour jujube centipede polypeptide toxin mu-SLPTX-Ssm6a comprises:
The total RNA of few sour jujube centipede poison gland extracts, mRNA purifying, and mRNA reverse transcription and cDNA library build, and design primer utilizes the few sour jujube centipede calcium channel activator polypeptide gene of PCR method screening.Amplimer length is 17 Nucleotide, its sequence is 5 '-GCGAAAAATGGAAATCT-3 ', another amplimer of PCR is 3 ' PCR Primer primer in the SMART TM cDNA Library Construction Kit of CLONTECH company, and its sequence is 5 ＇ ATTCTACAGGCCGAGGCGGCCGACATG3 ＇.The positive monoclonal that obtains carries out gene nucleotide series mensuration.
Gene sequencing result show the to encode gene (SEQ ID NO:2) of few sour jujube centipede calcium channel activator is comprised of 426 Nucleotide, from 5 ' end to 3 ' terminal sequence gene order, comprised signal peptide, precursor peptide and mature peptide, wherein underscore is partly mature peptide.
tacattatgagaatttggtcgttaaccagaatccttacttttatcgtaatttttaatttt 60
M R I W S L T R I L T F I V I F N F 18
gctgaagcgggtggagagtgcatgaaaaaatgtgacagtccagatatgatacgcgaaata 120
A E A G G E C M K K C D S P D M I R E I 38
tttacgcgctgcacaatggttaaacgtgatacacagttctctgagaattcaggacatctt 180
F T R C T M V K R D T Q F S E N S G H L 58
atacctaaacgctccgttgttgctgataataaatgtaattcattacgtcgtgaaatagct 240
I P K R S V V↓
A D N K C N S L R R E I A 78
tgcggacaatgccgtgataaagtgaaaactgatgggtatttttacgaatgttgcacatct 300
C G Q C R D K V K T D G Y F Y E C C T S 98
gattctacatttaaaaagtgtcaggatttgctgcacaaatgaccttcatggtgttctatg 360
D S T F K K C Q D L L H↓K- 118
ctataaaaccagtgaaatgtgatttaattttctaaaattttcatgctgttttattttatt 420
gacactaaacaactattgtttcataaactaactattgtgaatattgctgataaccttttg 480
tttactactaaaggattaggctgtgatttttattcaaaataaattactttcaataactaa 540
aaaaaaaaaaaaaaaaaaaaaaaaaaa 567
Few sour jujube centipede polypeptide toxin mu-SLPTX-Ssm6a of the present invention applies in preparing analgesic.
Few sour jujube centipede polypeptide toxin mu-SLPTX-Ssm6a of the present invention, has the restraining effect of highly significant to the sodium Channel Current on rat dorsal root ganglion, be a kind of sodium channel inhibitor; This toxin can optionally act on sodium-ion channel hypotype Nav1.7, and the analgesic model of mouse shows that this polypeptide has good analgesic effect, application that can be in preparing analgesic.
Accompanying drawing explanation:
Figure 1A to Fig. 1 C is the separation and purification of polypeptide toxin mu-SLPTX-Ssm6a.Wherein: Figure 1A: toxin adopts the separation and purification of C18 reverse hplc after G50 molecular sieve, and wherein arrow is depicted as object peak.Figure 1B: desired polypeptides further adopts reverse hplc separated.Fig. 1 C: the Mass Spectrometric Identification figure of molecules of interest.
Fig. 2 A to Fig. 2 C is the effect of mu-SLPTX-Ssm6a to rat drg neuron.Wherein: Fig. 2 A:1 μ M mu-SLPTX-Ssm6a all suppresses TTX-S sodium current on rat drg neuron.Fig. 2 B:mu-SLPTX-Ssm6a suppresses the dense effect curve of TTX-S sodium current on rat drg neuron, its IC
50value can suppress TTX-S sodium current fast for 23nM. Fig. 2 C:1 μ M mu-SLPTX-Ssm6a, and extracellular fluid also can the restraining effect of wash-out mu-SLPTX-Ssm6a to TTX-S sodium channel.Fig. 2 D:mu-SLPTX-Ssm6a make TTX-S sodium channel I-V curve turn right drift but do not change activation voltage and reversal voltage.Fig. 2 E:mu-SLPTX-Ssm6a makes the drift of turning right of the electric lead curve of TTX-S sodium channel.Fig. 2 F:mu-SLPTX-Ssm6a does not change the stable state inactivation curve of TTX-S sodium channel.
Fig. 3 A to Fig. 3 C is that mu-SLPTX-Ssm6a optionally acts on Nav1.7.Wherein: Fig. 3 A:2 μ M mu-SLPTX-Ssm6a suppresses Nav1.1 electric current 25%.Fig. 3 B:1 μ M mu-SLPTX-Ssm6a suppresses Nav1.2 electric current approximately 31%.Fig. 3 C:1 μ M mu-SLPTX-Ssm6a suppresses Nav1.6 electric current approximately 25%.Fig. 3 D:50nM mu-SLPTX-Ssm6a suppresses Nav1.7 electric current approximately 63%.The dense effect curve of Fig. 3 E:mu-SLPTX-Ssm6a to the effect of sodium ion different subtype.
Fig. 4 A to Fig. 4 C is that mu-SLPTX-Ssm6a is to sodium ion different subtype kinetics function.Wherein: the electric lead curve that Fig. 4 A:mu-SLPTX-Ssm6a makes Nav1.1 is toward the direction of depolarization about 10.7mV that drifts about, but do not change stable state inactivation curve.Fig. 4 B:mu-SLPTX-Ssm6a makes the electric lead curve of Nav1.2 toward approximately+12.9 of direction of depolarization drift, but does not change stable state inactivation curve.The electric lead curve that Fig. 4 C:mu-SLPTX-Ssm6a makes Nav1.6 is toward the direction of depolarization about 9.55mV that drifts about, but do not change stable state inactivation curve.The electric lead curve that Fig. 4 D:mu-SLPTX-Ssm6a makes Nav1.7 is toward the direction of depolarization about 13.5mV that drifts about, but do not change stable state inactivation curve.
Fig. 5 A to Fig. 5 C is the analgesic effect of mu-SLPTX-Ssm6 to different pain models.Wherein: Fig. 5 A:1nM, the mu-SLPTX-Ssm6 of 10nM and 100nM obviously reduces formalin and causes neuropathic pain.Fig. 5 B:1nM, the mu-SLPTX-Ssm6 of 10nM and 100nM obviously reduces formalin and causes inflammatory pain.Fig. 5 C:1nM, the mu-SLPTX-Ssm6 of 10nM and 100nM obviously reduces the pain that acetic acid causes.Fig. 5 D:1nM, the mu-SLPTX-Ssm6 of 10nM and 100nM obviously reduces the pain that thermal conductance causes.
Embodiment
1, the separation and purification of mu-SLPTX-Ssm6a
The separation and purification of ω-SLPTX-Ssm1a is divided into three steps: (1) molecular sieve; Few sour jujube centipede venom of 4mL (absorption value under 280nm is 200OD) adopts self-chambering post (filler is Sephadex G-50,2.6100cm) separation.Adopt 0.1M PBS (PH=6.0) wash-out, flow velocity is 0.3mL/min, within every ten minutes, changes collection tube.Solution after collection is the absorption value at 280nm with the every pipe solution of UV spectrophotometer measuring, draws elution curve.(2) find out the elution peak at desired polypeptides place, further adopt reverse hplc separation and purification.To after the elution peak freeze-drying of molecular sieve, again be dissolved in the deionized water of 2ml, adopt Phenomenex C18 (250x4.6mm) chromatographic column separated.Elute soln is acetonitrile (containing 0.1%TFA), and flow velocity is 3ml/min, and detection wavelength is 280/215nm.(3) will after the elution peak freeze-drying of object sample in second step, adopt again reverse hplc separation and purification.Adopt Phenomenex C18 (250x4.6mm) chromatographic column separated.Elute soln is acetonitrile (containing 0.1%TFA), and flow velocity is 0.7ml/min, and detection wavelength is 280/215nm.
As shown in Figure 1A to Fig. 1 C, it is separated by reverse hplc that few thick poisons of sour jujube centipede are crossed molecular sieving, and wherein (arrow is denoted as object peak, and this peak is through a single main peak of the reverse hplc separated generation of reverse hplc for the second time for the second time.
The evaluation of lps molecule quality is all used U.S. Bruker company to produce ProFLEXTMIII type mass spectrograph, adopts MALDI-TOF to detect: reflective-mode detects lps molecule amount; Laser repetition rate 5.00Hz; N2 optical maser wavelength 337nm; Ion source acceleration voltage 1 (IS/1) 20Kv; Control software and sun station that the control of instrument and the processing of data are developed by Brucker company complete.The saturated solution of preparing matrix CCA (α-cyano-4-hydroxyc-innamic acid) with 0.1%TFA-50% acetonitrile-49.9% deionized water mixed solution, then getting 1 μ l sample mixes with the saturated solution of 1 μ l CCA, get again 0.5 μ l mixed solution point sample in mass spectrographic sample disc, detection molecules amount after drying at room temperature.By external standard method or marker method, proofread and correct.
Result is as Fig. 1 C, and the molecular weight of few sour jujube centipede toxin mu-SLPTX-Ssm6a is in 5318.4Da molecule, to have to contain three pairs of disulfide linkage.
2, mu-SLPTX-Ssm6a aminoacid sequence and gene order
2.1 reach 99.9% mu-SLPTX-Ssm6a employing Edman degradation principles through Mass Spectrometric Identification purity, utilize full-automatic Protein Sequencer to record partial sequence, more finally determine complete aminoacid sequence according to the gene order of mu-SLPTX-Ssm6a.
The purifying of 2.2 few sour jujube centipede poison gland mRNA:
Few sour jujube centipede poison gland mRNA separation and purification adopts U.S. PROMEGA company
mRNA Isolation Systems test kit.
A. get the total RNA500 μ of few sour jujube centipede poison gland g and be dissolved in 500 μ l DEPC water, put into 65 ℃ of water-baths 10 minutes, add Oligo (dT) probe and the 13 μ l20 * SSC solution of people 3 μ l, mix, placement room temperature is cooling, is called A liquid.
B. the washing of magnetic bead (SA-PMP): magnetic bead is flicked and mixed, to magnetic frame absorption 30 seconds, abandon supernatant, add 0.5 * SSC0.3ml, to magnetic frame absorption 30 seconds, finally add 0.1ml0.5 * SSC and suspend, be referred to as B liquid.
C. A liquid is added in B liquid, room temperature was placed 10 minutes, to magnetic frame absorption 30 seconds, abandon supernatant, with 0.1 * SSC washing 4 times, finally abandon supernatant, add 0.1ml DEPC aqueous suspension, to magnetic frame, adsorb 30 seconds, supernatant is moved to new test tube, then add 0.15ml DEPC water Eddy diffusion, to magnetic frame absorption 30 seconds, moving supernatant to above-mentioned test tube, is the south China tree toad skin mRNA of purifying in supernatant.
D. add 1/10 volume 3M sodium acetate, pH5.2, equal-volume Virahol, in-70 ℃ of placements 30 minutes, 4 ℃, centrifugal 10 minutes of 12000rpm, abandoned supernatant, is precipitated and dissolved in 10 μ l DEPC water.
3.3 few sour jujube centipede venom gland cDNA libraries build
Adopt the CreatorTM SMART TM cDNA Library Construction Kit of CLONTECH company Construction of Plasmid cDNA Library test kit.
A.cDNA the first chain synthesizes (mRNA reverse transcription):
1. at the aseptic centrifuge tube of 0.5ml, add the few sour jujube centipede poison gland mRNA of 1 μ l, 1 μ l SMART IV oligonucleotide, 1 μ l CDS III/3 ' PCR primer, add 2 μ l deionized waters and make cumulative volume reach 5 μ l.
2. the reagent mixing in centrifuge tube is also of short duration centrifugal, and 72 ℃ are incubated 2 minutes.
3. centrifuge tube is hatched on ice 2 minutes.
4. in centrifuge tube, add following reagent 2.0 μ l5 * the first chain bufferings, 1.0 μ l20mM dithiothreitol (DTT), 1.0 μ l10mM dNTP mixtures, 1.0 μ l PowerScript ThermoScript II.
5. mix reagent of short duration centrifugal in centrifuge tube, 42 ℃ of insulations 1 hour.
6. centrifuge tube is placed in and ends the synthetic of the first chain on ice.
7. from centrifuge tube, get cDNA first chain of 2 μ l synthesizeds standby.
B. adopt long end polymerase chain reaction (LD-PCR) method second chain that increases
1.95 ℃ of preheating PCR instrument.
2. 2 μ l cDNA the first chains (mRNA reverse transcription), 80 μ l deionized waters, 10 μ l10 * Advantage2PCR buffering, 2 μ l50 * dNTP mixtures, 2 μ l5 ' PCR primers, 2 μ l CDS III/3 ' PCR primers and 2 μ l Escherichia coli polymerase centrifuge tubes are reacted.
3. in PCR instrument, by following program, increase:
(1) 95 ℃ of 20 second
(2) 22 circulations:
95 ℃ of 5 second
68 ℃ 6 minutes
4. after loop ends, cDNA two strands synthetic in centrifuge tube is carried out to extracting.
C.PCR product PROMEGA company
sV Gel and PCR Clean-Up System test kit carries out extracting and reclaiming, and step is as follows:
1. add isopyknic film binding buffer to put upside down the cDNA two strands obtaining by PCR and mix, then will mix liquid and proceed to centrifugal purification post, standing 5 minutes of room temperature, is fully combined DNA with pellosil.Centrifugal 1 minute of 16,000g, outwells the waste liquid in collection tube.
2., in centrifugal purification post, centrifugal 1 minute of 16,000g, outwells the waste liquid in collection tube to add the elutriant (containing ethanol) of 700 μ l.
3. repeating step 2.
Centrifugal 5 minutes of 4.16,000g.
5. centrifugal purification post is placed in to new centrifuge tube.
6. add 30 μ l ultrapure waters, at room temperature standing 5 minutes.
Centrifugal 1 minute of 7.16,000g, pipe end solution is the cDNA two strands of purified mistake.
D. the preparation of bacillus coli DH 5 alpha competent cell:
1. the single DH5 α of picking bacterium colony, is inoculated in 3ml containing in the LB substratum of penbritin, 37 ℃ of overnight incubation, get next day above-mentioned bacterium liquid in proportion 1:100 be inoculated in 50ml LB nutrient solution, 37 ℃ of vibrations 2 hours.Work as OD
600value reaches at 0.35 o'clock, results bacterial cultures.
2. bacterium is transferred in aseptic, disposable, an ice-cold 50ml polypropylene tube, on ice, 10min puts in side, makes culture be cooled to 0 ℃.
3. in 4 ℃ with the centrifugal 10min of 4100r/min, to reclaim cell.
4. pour out nutrient solution, pipe is inverted to 1min so that last trace nutrient solution flows to end.
5. the 0.1mol/L CaCl of every 50ml initial incubation liquid and 30ml precooling
2-MgCl
2solution (80mmol/L MgCl
2, 20mmol/L CaCl
2) resuspended every part of cell precipitation.
6. in 4 ℃ with the centrifugal 10min of 4100r/min, to reclaim cell.
7. pour out nutrient solution, pipe is inverted to 1min so that last trace nutrient solution flows to end.
8. the ice-cold 0.1mol/L CaCl of 2ml for every 50ml initial incubation thing
2resuspended every part of cell precipitation, standby after packing.
2.4. the conversion that enzyme is cut, connected and connects product:
1. in Eppendorf tube, add 1 μ l Takara pMD19-T carrier, the double-stranded solution of the few sour jujube centipede poison gland cDNA of 4 μ l, full dose is 5 μ l.
2. the ligase enzyme buffer mixture that adds 5 μ l (equivalent).
3.16 ℃ are reacted 2 hours.
4. full dose (10 μ l) is added in 100 μ l DH5 α competent cells, places 30 minutes in ice.
5.42 ℃ are heated after 90 seconds, then in ice, place 1 minute.
6. add 37 ℃ of LB substratum 890 μ l of bathing of temperature, 37 ℃ of slow shaking culture 60 minutes.
7. get 200 μ l and coat on the LB substratum that contains X-Gal, IPTG, Amp 37 ℃ and cultivate 16 hours, form single bacterium colony.
8. 5ml LB liquid nutrient medium washing bacterium colony for each LB plate, adds 30% glycerine frozen.The cDNA building is approximately containing 1 * 10
6individual independent clone.
2.5, few sour jujube centipede toxin mu-SLPTX-Ssm6a gene clone screening:
Amplimer length is that 17 its sequences of Nucleotide are 5 '-GCGAAAAATGGAAATCT-3 ', and another amplimer of PCR is the SMART of CLONTECH company
tM3 ' PCR Primer primer in cDNA Library Construction Kit, its sequence is 5 ＇ ATTCTACAGGCCGAGGCGGCCGACATG3 ＇.
PCR reaction is carried out under the following conditions: 94 ℃ of 30 second, 56 ℃ of 30 second and 72 ℃ of 45 second, 30 circulations.
Amplified production is detected with 1% agarose gel electrophoresis, cut object band, with DNA purification kit, carry out purifying.The object fragment of purifying is connected in pMD19-T carrier, obtains connection product.By connecting product, transform previously prepared good bacillus coli DH 5 alpha competent cell.Finally, get appropriate converted product and be applied on the LB flat board containing Amp, through 37 ℃ of single bacterium colonies of cultivating 16h formation, be the positive colony containing object fragment.
Picking list bacterium colony detects the size of Insert Fragment with M13 primer.The positive colony of selecting containing object fragment send the order-checking of DNA sequencing company.
The aminoacid sequence of few sour jujube centipede toxin mu-SLPTX-Ssm6a and gene order are respectively as shown in SEQ ID NO:1 and SEQ ID NO:2.
3. lack the activity of sour jujube centipede toxin mu-SLPTX-Ssm6a to calcium channel on DRG neurone
Select the SD rat that birth ± 4 week, body weight are about 140～200g, after anesthesia, disconnected neck is put to death, and chooses rapidly vertebra and is cut into 2～4 sections, and canalis spinalis is cut off along the direction vertical with rib, then in filling the beaker of a small amount of nutrient solution, soaks canalis spinalis; Carefully tear the colourless mucous membrane being attached on canalis spinalis inwall, expose the Dorsal root nerve fiber of canalis spinalis and rib intersection.In thoracic vertebrae and lumbar portion, select 10～15 good dorsal root ganglion and put into the culture dish that the interim nutrient solution of 2mL is housed.Isolate after neuroganglion and nerve fiber, with Wei Nasi, shear floss and the axon outside deganglionate, put into the culture dish that fills the interim nutrient solution of about 0.5mL.Remove interim nutrient solution, with Wei Nasi, cut separated neuroganglion is shredded.Then by shredding rear neurocyte, proceed in the vial that contains 15mL Digestive system, at 34 ℃, in the environment of 110rpm with Digestive system enzymolysis 20～30min.During enzymolysis, every 8～10min, take out vial piping and druming cell agglomerating to prevent cell; Enzymolysis stops in backward Digestive system, adding trypsin inhibitor, enzymolysis reaction.Solution after enzymolysis is proceeded to centrifugal in centrifuge tube (1000rpm, 2-5min), remove supernatant.With containing the nutrient solution re-suspended cell of 10% calf serum, the cell after resuspended is divided into 3～4 wares, and every ware adds 2mL nutrient solution, puts into 37 ℃ of constant incubator (5%CO
2, 95% air) in, cultivate after 3～4 hours for record current.
Before experiment, extracellular fluid must be placed under room temperature and after balance, change again the nutrient solution in culture dish, to prevent the acute variation of solution temperature.While changing solution, to prevent that cell from coming off from culture dish bottom.Under inverted microscope, select that cytolemma is comparatively smooth, the uniform cell of tenuigenin, under 20～25 ℃ of conditions of room temperature, carry out patch clamp experiments.Selecting 100 μ l borosilicate glass capillary tube is glass electrode material, glass electrode is drawing instrument (PC-10, Narishige) above through two steps, draw and form, the very hot polishing rear electrode of vitreous electricity tip aperture is 1.5～3.0 μ m, fills with intracellular fluid after having drawn in glass electrode.Glass electrode initial resistance is that 1.5～2.5M Ω is relatively good.After forming giga ohm (G Ω) sealing-in of high resistance between electrode and cytolemma, mend the fast electric capacity of electrode.Then by cell clamp built in-60mV, give a short and strong negative pressure, the cytolemma of clamping down in electrode is broken rapidly, then is compensated the slow electric capacity of cell.Form cell clamp is made as-80mV after full cell record pattern, cytotostatic 4～6min starts record current.System resistance (Rs) remains within the scope of 5～10M Ω and preferably can remain unchanged in experimentation, and Cascade System resnstance transformer is generally between 30～60%.
4, the effect of few sour jujube centipede toxin mu-SLPTX-Ssm6a to sodium channel hypotype.
Cell transfecting:
(1) transfection the day before yesterday, with not cultivating and change in culture dish containing microbiotic nutrient solution containing antibiotic nutrient solution.
(2) when cell density reaches 80-90%, abandon nutrient solution, PBS rinsing once, adds 2mL serum-free medium (Opti-MEM).
(3) configuration A liquid: standing 5min under 240ul serum free medium+10ul lipofectamine2000per well (cumulative volume 250ul) room temperature.
(4) configuration solution B: add 4ngDNA in 250 μ L serum-free mediums, the standing 5min of room temperature.
(5) A liquid and B liquid are mixed to standing 20min under room temperature.
(6) DNA-liposome is mixed rear even, be added dropwise in cell gently.
After (7) 6 hours, change the full substratum that contains serum.
Active detection:
Before experiment, extracellular fluid must be placed under room temperature and after balance, change again the nutrient solution in culture dish, to prevent the acute variation of solution temperature.While changing solution, to prevent that cell from coming off from culture dish bottom.Under inverted microscope, select that cytolemma is comparatively smooth, the uniform cell of tenuigenin, under 20～25 ℃ of conditions of room temperature, carry out patch clamp experiments.Selecting 100 μ l borosilicate glass capillary tube is glass electrode material, glass electrode is drawing instrument (PC-10, Narishige) above through two steps, draw and form, the very hot polishing rear electrode of vitreous electricity tip aperture is 1.5～3.0 μ m, fills with intracellular fluid after having drawn in glass electrode.Glass electrode initial resistance is that 1.5～2.5M Ω is relatively good.After forming giga ohm (G Ω) sealing-in of high resistance between electrode and cytolemma, mend the fast electric capacity of electrode.Then by cell clamp built in-60mV, give a short and strong negative pressure, the cytolemma of clamping down in electrode is broken rapidly, then is compensated the slow electric capacity of cell.Form cell clamp is made as-80mV after full cell record pattern, cytotostatic 4～6min starts record current.System resistance (Rs) remains within the scope of 5～10M Ω and preferably can remain unchanged in experimentation, and Cascade System resistance (Rseries compensation) compensates generally between 30～60%.
5, the analgesic effect of few sour jujube centipede toxin mu-SLPTX-Ssm6a
Formalin analgesic experiment:
Female kunming mice (18～22g), is divided into four groups (n=10).Mu-SLPTX-Ssm6a lyophilized powder is dissolved in stroke-physiological saline solution, 30 minutes abdominal injection modes administration before experiment starts, the physiological saline of control group injection same volume.In the sole place of mouse injection 20mL concentration, be 0.92% formalin, adopt shooting to record mouse and always lick the pin time.
Writhing method analgesic experiment:
Kunming mice (18～22g), is divided into four groups (n=10).Mu-SLPTX-Ssm6a lyophilized powder is dissolved in stroke-physiological saline solution, adopt the administration of abdominal injection mode, after few sour jujube centipede toxin mu-SLPTX-Ssm6a injection 30min, adopt abdominal injection mode to the acetic acid (10ml/kg body weight) of injected in mice 0.6%, the writhing number of times of 30min after counting injected in mice acetic acid.
Tail-flick method analgesic experiment:
Kunming mice (18～22g), is divided into four groups (n=10).With mouse tail pain threshold detector, filtering out Basic Pain Threshold comes for test the mouse of 3～7s, the infrared beam of mouse tail pain threshold detector impinges upon 1/3 place apart from mousetail root, the threshold of pain time refers to that from mouse, accepting infrared beam is irradiated to the timed interval between mouse generation yaw, tests out the Basic Pain Threshold of mouse before the few sour jujube centipede toxin mu-SLPTX-Ssm6a of injection.Few sour jujube centipede toxin mu-SLPTX-Ssm6a lyophilized powder is dissolved in stroke-physiological saline solution, adopt the administration of abdominal injection mode, respectively the threshold of pain of 10min, 30min, 60min, 180min, 360min mensuration mouse after few sour jujube centipede toxin mu-SLPTX-Ssm6a injection.For fear of the tissue injury that causes mouse, if threshold of pain is greater than 10s after mouse administration, the threshold of pain of this mouse is just designated as 10s.Control group gives the stroke-physiological saline solution of same volume.
SEQUENCE LISTING
<110> Kunming Institute of Zoology, Chinese Academy of Sciences
The few sour jujube centipede polypeptide toxin mu-SLPTX-Ssm6a of <120> and gene and application
<130> 1
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 336
<212> DNA
<213> Scolopendra subspinipes mutilans
<400> 1
atgagaattt ggtcgttaac cagaatcctt acttttatcg taatttttaa ttttgctgaa 60
gcgggtggag agtgcatgaa aaaatgtgac agtccagata tgatacgcga aatatttacg 120
cgctgcacaa tggttaaacg tgatacacag ttctctgaga attcaggaca tcttatacct 180
aaacgctccg ttgttgctga taataaatgt aattcattac gtcgtgaaat agcttgcgga 240
caatgccgtg ataaagtgaa aactgatggg tatttttacg aatgttgcac atctgattct 300
acatttaaaa agtgtcagga tttgctgcac aaaatg 336
<210> 2
<211> 45
<212> PRT
<213> Scolopendra subspinipes mutilans
<400> 2
Ala Asp Asn Lys Cys Glu Asn Ser Leu Arg Arg Glu Ile Ala Cys Gly
1 5 10 15
Gln Cys Arg Asp Lys Val Lys Thr Asp Gly Tyr Phe Tyr Glu Cys Cys
20 25 30
Thr Ser Asp Ser Thr Phe Lys Lys Cys Gln Leu Leu His
35 40 45
Claims (2)
1. lack sour jujube centipede polypeptide toxin mu-SLPTX-Ssm6a, its mature peptide aminoacid sequence consists of ADNKCENSLRREIACGQCRDKVKTDGYFYECCTSDSTFKKCQDLLH.
2. the application of the said few sour jujube centipede polypeptide toxin mu-SLPTX-Ssm6a of claim 1 in preparing analgesic.
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| CN103724411B (en) * | 2013-06-21 | 2014-12-10 | 江苏教育学院 | Anti-insect toxin, coding sequence of anti-insect toxin as well as preparation and application of recombinant toxin |
| CN105085646A (en) * | 2015-07-14 | 2015-11-25 | 中国科学院昆明动物研究所 | Centipede analgesic peptide SLP_RhTx and gene and application thereof |
| CN105732789A (en) * | 2016-03-03 | 2016-07-06 | 中国科学院昆明动物研究所 | Haemadipsa sylvestris analgesic peptide mh2620 and gene and application thereof |
| CN108117599A (en) * | 2018-01-03 | 2018-06-05 | 海门迈克艾伦生物医药有限公司 | The recombination expression and purification process of Ssm6a and its fusion protein used |
| CN110713530B (en) * | 2018-07-12 | 2023-03-24 | 湖南师范大学 | Application of sodium channel blocker mu-TRTX-Ca 1a as analgesic drug |
| CN112552388B (en) * | 2020-12-30 | 2022-07-01 | 湖南师范大学 | Scolopendra mutilans polypeptide toxins rpTx-1, rpTx-2 and rpTx-3 as well as coding gene and application thereof |
| CN116769007B (en) * | 2023-07-24 | 2024-02-20 | 东北林业大学 | Centipede analgesic polypeptide PvTx and encoding gene and application thereof |
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| Chemical Punch Packed in Venoms Makes Centipedes Excellent Predators;Shilong Yang et al.;《Molecular & Cellular Proteomics》;20120517;640-650 * |
| novel centipede toxinμ-SLPTX-Ssm6a targeting the voltage sensor of sodium channel;容明强等;《生命的分子机器及其调控网络——2012年全国生物化学与分子生物学学术大会摘要集》;20120824;85 * |
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