CN102993289A - Scolopendra mutilans polypeptide toxin kappa-SLPTX-Ssm4a and gene and application thereof - Google Patents
Scolopendra mutilans polypeptide toxin kappa-SLPTX-Ssm4a and gene and application thereof Download PDFInfo
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Abstract
The invention relates to scolopendra mutilans polypeptide toxin kappa-SLPTX-Ssm4a and gene and application thereof, belonging to the technical field of biomedicine. The scolopendra mutilans polypeptide toxin kappa-SLPTX-Ssm4a consists of amino acid sequence of SEQ ID NO:1; gene of encoded scolopendra mutilans polypeptide toxin kappa-SLPTX-Ssm4a consists of nucleotide sequence of SEQ ID NO:1; and the scolopendra mutilans polypeptide toxin kappa-SLPTX-Ssm4a selectively restrains potassium channel subtype Kv1.3 on T cell, thus being a specific potassium channel inhibitor. The scolopendra mutilans polypeptide toxin kappa-SLPTX-Ssm4a is applied in medicine for preparing and curing or preventing self immunological diseases related to T cell.
Description
Technical field:
The present invention relates to a kind of few sour jujube centipede polypeptide toxin kappa-SLPTX-Ssm4a and gene and application, belong to field of biomedicine technology.
Background technology:
Potassium channel is a kind of important transmembrane protein on cytolemma, has very important physiological significance.Potassium channel is distributed in the cells such as skeletal muscle, nerve, heart, blood vessel, body of gland widely.To find at present most species, a kind of ionic channel that effect is the most complicated.Potassium ion distribution of a great variety is also very extensive, and wherein potassium-channel hypotype Kv1.3 mainly is distributed in the surface of T cell, is the key of effector T cell continuous activation.
The T cell is to be formed by intrathymic lymphoid stem cell differentiation, is that in lymphocyte, quantity is maximum, the class cell that function is the most complicated.The T cell is lymphocytic main ingredient, it has the various biological function, and as the direct killing target cell, auxiliary or inhibition B cell produces antibody, to specific antigens and former responsing reaction and the generation cytokine of mitogenesis, resist disease in health and infect and tumour formation.It is a kind of and the closely-related autoimmune disorder of T cell that psoriatic is commonly called as " psoriasis ", and improving autoimmunity is the psoriatic a kind of important method for the treatment of.Kv1.3, as an important factor of T cell persistence activation, plays the part of vital role on the physiological function of T cell, has confirmed that at present Kv1.3 and numerous disease are closely related, and Kv1.3 has also become the popular target spot of drug development.For example rheumatoid arthritis, psoriatic, hepatitis, multiple sclerosis, obesity, transplant rejection and inflammatory neuropathy are closely related for the autoimmune disease of Kv1.3 mediation.In the inhibitor of Kv1.3, flavonoid can be used for treating lupus erythematosus, relevant rheumatism; Shk toxin analogue is for prevention of obesity and type ii diabetes.
Centipede is a kind of traditional Chinese medicine, warm in nature, and flavor is hot, poisonous.The function of there is breath wind antispastic, dispersing pathogen accumulation, removing obstruction in channels to relieve pain.Contain abundant polypeptide composition in centipede poison gland, verified at present these polypeptide compositions mostly are neurotoxin.Polypeptide can optionally suppress sodium channel, and 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 kappa-SLPTX-Ssm4a and gene and application.
Few sour jujube centipede polypeptide toxin kappa-SLPTX-Ssm4a 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 TDDESSNKCAKTKRRENVCRVCGNRSGNDEYYSECCESDYRYERCLDLLR N, this polypeptide comprises that 51 amino-acid residue molecular weight are 6042.57Da, iso-electric point is 5.3, it is 1-5 that molecule contains three pairs of its matching methods of disulfide linkage, 2-4,3-6.
The gene clone of few sour jujube centipede polypeptide toxin kappa-SLPTX-Ssm4a comprises:
The total RNA of few sour jujube centipede poison gland extracts, the mRNA purifying, and mRNA reverse transcription and cDNA library build, and the 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 ＇ ATTCTACAGGCCGAGGCGGCCGACATG 3 ＇.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 563 Nucleotide, comprised signal peptide, precursor peptide and mature peptide from 5 ' end to 3 ' terminal sequence gene order, wherein underscore is partly mature peptide.
The application of few sour jujube centipede polypeptide toxin kappa-SLPTX-Ssm4a of the present invention in preparing the Immunological diseases medicine relevant to the T cell.
Few sour jujube centipede polypeptide toxin kappa-SLPTX-Ssm4a of the present invention, potassium-channel on rat dorsal root ganglion is had to obvious restraining effect, this toxin can optionally act on potassium channel hypotype Kv1.3, the psoriatic of mouse and hepatitis B model show that this polypeptide has good result for the treatment of, application that can be in treatment psoriatic and hepatitis B medicine.
The accompanying drawing explanation:
The separation and purification that Fig. 1-A to Fig. 1-D is polypeptide toxin kappa-SLPTX-Ssm4a.Wherein: Fig. 1-A toxin is through the separation graph of G50 molecular sieve, and wherein arrow is depicted as the purpose peak.Fig. 1-B purpose toxin is the reverse hplc collection of illustrative plates for the first time, and wherein arrow is desired polypeptides.Fig. 1-C molecules of interest is reverse hplc for the second time.The Mass Spectrometric Identification figure of Fig. 1-D molecules of interest.
Fig. 2-A to Fig. 2-C is that polypeptide toxin kappa-SLPTX-Ssm4a is to the potassium channel action effect.Wherein: Fig. 2-A 5 μ M toxin kappa-SLPTX-Ssm4a suppress the potassium current on rat dorsal root ganglion fully.Fig. 2-B 1 μ M toxin kappa-SLPTX-Ssm4a can selectivity suppress potassium channel hypotype Kv1.3 electric current.Fig. 2-C detoxifying function is in the dense effect curve of potassium channel hypotype Kv1.3.Potassium current on the complete suppressor T cell of Fig. 2-D 100 nM kappa-SLPTX-Ssm4a.Fig. 2-E toxin kappa-SLPTX-Ssm4a acts on the dense effect curve of potassium current on the T cell.
Fig. 3-A to Fig. 3-F is that polypeptide toxin is to the therapeutic action of mouse psoriasis model.Wherein: Fig. 3- A 1,10, and 100 nM kappa-SLPTX-Ssm4a reduce the amount of TNF-α significantly.Fig. 3- B 1,10, and 100 nM kappa-SLPTX-Ssm4a reduce the amount of IFN-γ significantly.Fig. 3- C 1,10, and 100 nM kappa-SLPTX-Ssm4a reduce the amount of IL-2 significantly.Fig. 3- D 1,10, and 100 nM kappa-SLPTX-Ssm4a reduce the amount of IL-8 significantly.Fig. 3- E 1,10, and 100 nM kappa-SLPTX-Ssm4a reduce the amount of IL-17 significantly.Fig. 3- F 1,10, and 100 nM kappa-SLPTX-Ssm4a reduce the amount of IL-22 significantly.
Fig. 4-A to Fig. 4-E is that polypeptide toxin is to the therapeutic action of mouse hepatitis model.Wherein: Fig. 4-A canavalin(e) makes ALT content significantly increase with respect to control group, and kappa-SLPTX-Ssm4a obviously reduces ALT content, and canavalin(e) and kappa-SLPTX-Ssm4a reduce ALT content after combining use equally.Fig. 4-B canavalin(e) makes AST content significantly increase with respect to control group, and kappa-SLPTX-Ssm4a obviously reduces AST content, and canavalin(e) and kappa-SLPTX-Ssm4a reduce AST content after combining use equally.Fig. 4-C canavalin(e) makes TBIL content significantly increase with respect to control group, and kappa-SLPTX-Ssm4a obviously reduces TBIL content, and canavalin(e) and kappa-SLPTX-Ssm4a reduce TBIL content after combining use equally.Fig. 4-D canavalin(e) makes DBIL content significantly increase with respect to control group, and kappa-SLPTX-Ssm4a obviously reduces DBIL content, and canavalin(e) and kappa-SLPTX-Ssm4a reduce DBIL content after combining use equally.Fig. 4-E canavalin(e) makes IBIL content significantly increase with respect to control group, and kappa-SLPTX-Ssm4a obviously reduces IBIL content, and canavalin(e) and kappa-SLPTX-Ssm4a reduce IBIL content after combining use equally.
Embodiment
1, the separation and purification of kappa-SLPTX-Ssm4a
The separation and purification of kappa-SLPTX-Ssm4a is divided into three steps: (1) molecular sieve; Few sour jujube centipede venom of 4mL (absorption value under 280 nm is 200 OD) adopts self-chambering post (filler is Sephadex G-50,2.6 100 cm) to separate.Adopt 0.1 M 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 280 nm with the every pipe solution of UV spectrophotometer measuring, draws elution curve.(2) find out the elution peak at desired polypeptides place, further adopt the 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(250 x 4.6 mm) the chromatographic column separation.Elute soln is acetonitrile (containing 0.1%TFA), and flow velocity is 3ml/min, and detecting wavelength is 280/215 nm.(3) will after the elution peak freeze-drying of purpose sample in second step, adopt again the reverse hplc separation and purification.Adopt Phenomenex C18(250 x 4.6 mm) the chromatographic column separation.Elute soln is acetonitrile (containing 0.1% TFA), and flow velocity is 0.7ml/min, and detecting wavelength is 280/215 nm.
As shown in Fig. 1-A to Fig. 1-C, few thick poisons of sour jujube centipede are crossed molecular sieving and are separated by reverse hplc, wherein arrow be denoted as ，Gai peak, purpose peak through reverse hplc for the second time for the second time reverse hplc separate and produce a single main peak.
The evaluation of lps molecule quality is all used U.S. Bruker company to produce ProFLEXTMIII type mass spectrograph, and adopt MALDI-TOF to be detected: reflective-mode detects the lps molecule amount; Laser repetition rate 5.00 Hz; N2 optical maser wavelength 337nm; Ion source acceleration voltage 1 (IS/1) 20 Kv; The control of instrument and the processing of data are completed by control software and the Sun workstation of the exploitation of Brucker company.The saturated solution for 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 on mass spectral:mass spectrographic sample disc, detection molecules amount after drying at room temperature.By external standard method or marker method, proofreaied and correct.
Result is as Fig. 1-D, and the molecular weight of few sour jujube centipede toxin kappa-SLPTX-Ssm4a is in 6042.3 Da molecules, to have to contain three pairs of disulfide linkage.
2, kappa-SLPTX-Ssm4a aminoacid sequence and gene order
2.1 reach 99.9% kappa-SLPTX-Ssm4a 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 kappa-SLPTX-Ssm4a.
2.2 the purifying of few sour jujube centipede poison gland mRNA:
Few sour jujube centipede poison gland mRNA separation and purification adopts the PolyATtract mRNA Isolation Systems test kit of U.S. PROMEGA company.
A. get the total RNA 500 μ g of few sour jujube centipede poison gland 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 l 20 * SSC solution of people 3 μ l, mix, the placement room temperature is cooling, is called A liquid.
B. the washing of magnetic bead (SA-PMP): magnetic bead is flicked and mixes, to magnetic frame absorption 30 seconds, abandon supernatant, add 0. 5 * SSC, 0. 3m1, to magnetic frame absorption 30 seconds, finally add 0.1 ml 0. 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. l ml DEPC aqueous suspension, to magnetic frame, adsorb 30 seconds, supernatant is moved to new test tube, then add 0. 15 m1 DEPC water Eddy diffusions, 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 3 M sodium acetates, pH 5. 2, the equal-volume Virahol, and 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 library builds
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 0.5ml, aseptic centrifuge tube adds 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, adds 2 μ l deionized waters and makes cumulative volume reach 5 μ l.
2. the reagent mixed 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. add following reagent 2.0 μ l 5 * the first chain bufferings, 1.0 μ l 20 mM dithiothreitol (DTT), 1.0 μ l 10 mM dNTP mixtures, 1.0 μ l PowerScript ThermoScript II in centrifuge tube.
5. reagent of short duration centrifugal in the mixing centrifuge tube, be incubated 1 hour at 42 ℃.
6. centrifuge tube is placed in and ends the synthetic of the first chain on ice.
7. get cDNA first chain of 2 μ l synthesizeds from centrifuge tube standby.
B. adopt long end polymerase chain reaction (LD-PCR) method second chain that increases
1.95 ℃ preheating PCR instrument.
2. 2 μ l cDNA the first chains (mRNA reverse transcription), 80 μ l deionized waters, 10 μ l 10 * Advantage 2 PCR bufferings, 2 μ l 50 * dNTP mixtures, 2 μ l 5 ' PCR primers, 2 μ l CDS III/3 ' PCR primers and 2 μ l Escherichia coli polymerase centrifuge tubes are reacted.
3. in the 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. the Wizard of PROMEGA company for the PCR product
sV Gel and PCR Clean-Up System test kit carries out extracting and reclaiming, and step is as follows:
1. the cDNA two strands that will obtain by PCR adds isopyknic film binding buffer to put upside down to mix, then will mix liquid and proceed to the centrifugal purification post, and standing 5 minutes of room temperature, fully be combined DNA with pellosil.Centrifugal 1 minute of 16,000 g, outwell the waste liquid in collection tube.
2., in the centrifugal purification post, centrifugal 1 minute of 16,000 g, outwell the waste liquid in collection tube to add the elutriant (containing ethanol) of 700 μ l.
3. repeating step 2.
4. 16,000 g centrifugal 5 minutes.
5. the centrifugal purification post is placed in to new centrifuge tube.
6. add 30 μ l ultrapure waters, at room temperature standing 5 minutes.
7. 16,000 g centrifugal 1 minute, and 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, be inoculated in 3m1 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 50 m1 LB nutrient solutions, 37 ℃ of vibrations 2 hours.Work as OD
600value reaches at 0.35 o'clock, the results bacterial cultures.
2. bacterium is transferred in aseptic, disposable, ice-cold 50 m1 polypropylene tube, on ice, 10 min put in side, make culture be cooled to 0 ℃.
3. in 4 ℃ with centrifugal 10 min of 4100 r/min, to reclaim cell.
4. pour out nutrient solution, pipe is inverted to l min so that last trace nutrient solution flows to end.
5. the 0.1mol/L CaCl of every 50ml initial incubation liquid and 30 ml precoolings
2-MgCl
2solution (80 mmol/L MgCl
2, 20 mmol/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 l min so that last trace nutrient solution flows to end.
8. every 2m1 ice-cold 0.1 mol/L CaCl for 50 m1 initial incubation things
2resuspended every part of cell precipitation, standby after packing.
2.4. the conversion that enzyme is cut, connected and connects product:
1. 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 in Eppendorf tube, full dose is 5 μ l.
2. add 5 μ l(equivalent) the ligase enzyme buffer mixture.
3. 16 ℃ are reacted 2 hours.
4. full dose (10 μ l) is added in 100 μ l DH5 α competent cells, in ice, places 30 minutes.
5. after 42 ℃ of 90 seconds of heating, 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. 5 m1 LB liquid nutrient medium washing bacterium colonies for each LB plate, add 30% glycerine frozen.The cDNA built is approximately containing 1 * 10
6individual independent clone.
2.5, few sour jujube centipede toxin kappa-SLPTX-Ssm4a 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 ＇ ATTCTACAGGCCGAGGCGGCCGACATG 3 ＇.
The 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 the purpose band, with the DNA purification kit, carry out purifying.The purpose fragment of purifying is connected in the pMD19-T carrier, obtains the connection product.Transform previously prepared good bacillus coli DH 5 alpha competent cell by connecting product.Finally, get appropriate converted product and be applied on the LB flat board containing Amp, through 37 ℃ of single bacterium colonies of cultivating 16 H-shapeds one-tenth, be the positive colony containing the purpose fragment.
Picking list bacterium colony detects the size of Insert Fragment with the M13 primer.The positive colony of selecting containing the purpose fragment send the order-checking of DNA sequencing company.
The aminoacid sequence of few sour jujube centipede toxin kappa-SLPTX-Ssm4a 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 kappa-SLPTX-Ssm4a to potassium channel on the DRG neurone
Select the SD rat that birth ± 4 week, body weight are about 140～200 g, after anesthesia, disconnected neck is put to death, and chooses rapidly vertebra and it is cut into to 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 be attached on the canalis spinalis inwall, expose the Dorsal root nerve fiber of canalis spinalis and rib intersection.In thoracic vertebrae and lumbar portion, select 10～15 preferably dorsal root ganglion put into the culture dish that the interim nutrient solution of 2 mL is housed.After isolating neuroganglion and nerve fiber, with Wei Nasi, shear deganglionate outer floss and axon, put into and fill the approximately culture dish of the interim nutrient solution of 0.5 mL.Remove interim nutrient solution, with Wei Nasi, cut the neuroganglion of separation is shredded.Then will shred rear neurocyte and proceed in the vial that contains 15 mL Digestive systems, under 34 ℃, in the environment of 110 rpm with Digestive system enzymolysis 20～30 min.Take out vial piping and druming cell every 8～10 min during enzymolysis agglomerating to prevent cell; Enzymolysis stops adding trypsin inhibitor in backward Digestive system, enzymolysis reaction.Solution after enzymolysis is proceeded to centrifugal in centrifuge tube (1000 rpm, 2-5 min), 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 2 mL nutrient solutions, puts into 37 ℃ of constant incubator (5% CO
2, 95% air) in, cultivate after 3～4 hours for record current.
Extracellular fluid must be placed under room temperature and change again the nutrient solution in culture dish after balance before experiment, to prevent the acute variation of solution temperature.To prevent while changing solution that cell from coming off from the culture dish bottom.Select under inverted microscope 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 the 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.The glass electrode initial resistance is that 1.5～2.5 M Ω are relatively good.After between electrode and cytolemma, forming giga ohm (G Ω) sealing-in of high resistance, mend the fast electric capacity of electrode.Then by cell clamp built in-60 mV, 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.Cell clamp is made as to-80 mV after forming full cell record pattern, cytotostatic 4～6 min start record current.System resistance (Rs) remains within the scope of 5～10 M Ω and preferably can remain unchanged in experimentation, and the Cascade System resnstance transformer is generally between 30～60%.
4, the effect of few sour jujube centipede toxin kappa-SLPTX-Ssm4a to potassium channel hypotype Kv1.3.
Cell transfecting:
(1) transfection the day before yesterday, with containing antibiotic nutrient solution, not cultivating and change in culture dish containing the microbiotic nutrient solution.
(2) reach 80-90% when cell density, abandon nutrient solution, the PBS rinsing once, adds 2 mL serum-free mediums (Opti-MEM).
(3) configuration A liquid: standing 5 min under 240 ul serum free mediums+10 ul lipofectamine 2000 per well (cumulative volume 250 ul) room temperatures.
(4) configuration solution B: add 4 ngDNA 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) the DNA-liposome is mixed rear even, be added dropwise in cell gently.
After (7) 6 hours, change the full substratum that contains serum.
Extracellular fluid must be placed under room temperature and change again the nutrient solution in culture dish after balance before experiment, to prevent the acute variation of solution temperature.To prevent while changing solution that cell from coming off from the culture dish bottom.Select under inverted microscope 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 the 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.The glass electrode initial resistance is that 1.5～2.5 M Ω are relatively good.After between electrode and cytolemma, forming giga ohm (G Ω) sealing-in of high resistance, mend the fast electric capacity of electrode.Then by cell clamp built in-60 mV, 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.Cell clamp is made as to-80 mV after forming full cell record pattern, cytotostatic 4～6 min start record current.System resistance (Rs) remains within the scope of 5～10 M Ω and preferably can remain unchanged in experimentation, and Cascade System resistance (Rseries compensation) compensates generally between 30～60%.
5, few sour jujube centipede toxin kappa-SLPTX-Ssm4a is to psoriatic treatment.
Toxin kappa-SLPTX-Ssm4a adopts PBS to be dissolved into 1,10,100 nM concentration, and with the T cell, hatch 1 hour in advance.Hatch after 1 hour and adopt anti-CD3
+/ CD28
+the antibody activated T cell.After t cell activation 16 hours, adopt immunoenzyme connection adsorption method to detect TNF α in the T cell, hIL-2, IL-8, IL-17, the content of IL-22 and IFN-γ.
6, few sour jujube centipede toxin kappa-SLPTX-Ssm4a is to the hepatitis treatment effect.
At large mouse tail vein injection 15mg/kg canavalin(e) of 6-8 week, mouse forms oxyhepatitis model, the kappa-SLPTX-Ssm4a of injection 5mg/kg before half an hour before the injection scalpel legumin.Laboratory animal is divided into 4 groups, is respectively: injection scalpel soybean protein group, control group, injection kappa-SLPTX-Ssm4a group and while injection scalpel soybean protein and kappa-SLPTX-Ssm4a group.After injection scalpel soybean protein 4 hours, the liver of getting mouse detects AST and ALT content.Detect TNF-α in mice serum, interleukin-2 (IL-2) and Gan Rao Su – γ (INF-γ) content after 8 hours.
SEQUENCE LISTING
<110 > Kunming Institute of Zoology, Chinese Academy of Sciences
<120 > few sour jujube centipede polypeptide toxin kappa-SLPTX-Ssm4a and gene and application
<130> 1
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 51
<212> PRT
<213> Scolopendra subspinipes mutilans
<400> 1
Thr Asp Asp Glu Ser Ser Asn Lys Cys Ala Lys Thr Lys Arg Arg Glu
1 5 10 15
Asn Val Cys Arg Val Cys Gly Asn Arg Ser Gly Asn Asp Glu Tyr Tyr
20 25 30
Ser Glu Cys Cys Glu Ser Asp Tyr Arg Tyr Glu Arg Cys Leu Asp Leu
35 40 45
Leu Arg Asn
50
<210> 2
<211> 563
<212> DNA
<213> Scolopendra subspinipes mutilans
<400> 2
atgaattctt cgatagctat tttgctagta atggctctaa ttatgttttc tttggacaaa 60
tcttattcga ctgatgatga atcaagcaat aaatgcgcga aaacaaaacg tcgtgaaaac 120
gtttgccgag tatgtggtaa ccgatcgggc aatgatgaat attattccga atgttgcgaa 180
tctgattata gatatgaaag gtgtctggat ttgctgcgaa attgacttca tagcattctc 240
tactataaaa ccagcgaaat gcttttttga atccatgatt ttttatgtta ttaattttct 300
aaaattttga cagtaaacaa tttgtcaaaa agtattgttt ttttgttaaa tttaagaatt 360
atataaatgg aaaattttat aaaattgtaa atttctttta aatttacaat gggtttttgt 420
tttgttttat ataaatataa tttcgtttgt aattttatta taatattgct gataaccttt 480
tgttagtact aaaggattag gctgtgattt ttattcaaaa taaattgctc tcaataaaaa 540
aaaaaaaaaa aaaagaaaaa aaa 563
Claims (4)
1. few sour jujube centipede polypeptide toxin kappa-SLPTX-Ssm4a, is characterized in that being comprised of the aminoacid sequence shown in SEQ ID NO:1.
2. the gene of the few sour jujube centipede polypeptide toxin kappa-SLPTX-Ssm4a of coding, is characterized in that being comprised of the nucleotide sequence shown in SEQ ID NO:1.
3. the polypeptide toxin kappa-SLPTX-Ssm4a shown in claim 1 is the specific inhibitor of potassium channel Kv1.3 on a kind of T cell, the application in preparation self property Immunological diseases medicine.
4. self the property Immunological diseases in claim 3 comprise: rheumatoid arthritis, psoriatic, hepatitis, multiple sclerosis, obesity, transplant rejection and inflammatory neuropathy.
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|---|---|---|---|
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN116715743A (en) * | 2023-07-24 | 2023-09-08 | 东北林业大学 | Centipede polypeptide SLP_Ssm1a and encoding gene and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107987144A (en) * | 2017-12-14 | 2018-05-04 | 中国科学院昆明动物研究所 | A kind of centipede polypeptide SLP_SsTx and its encoding gene and application |
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| CN116715743A (en) * | 2023-07-24 | 2023-09-08 | 东北林业大学 | Centipede polypeptide SLP_Ssm1a and encoding gene and application thereof |
| CN116715743B (en) * | 2023-07-24 | 2024-01-26 | 东北林业大学 | Centipede polypeptide SLP_Ssm1a and encoding gene and application thereof |
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Inventor after: Lai Ren Inventor after: Rong Mingqiang Inventor after: Yang Shilong Inventor after: Xiao Yao Inventor after: Kang Di Inventor before: Lai Ren Inventor before: Rong Mingqiang Inventor before: Yang Shilong Inventor before: Xiao Yao Inventor before: Kang Di |