CN102617723A - Scorpion venom active peptides, preparation method thereof and application - Google Patents
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Abstract
The invention belongs to the technical field of biology, and discloses scorpion venom active peptides, a preparation method thereof and application. The scorpion venom active peptides have an amino acid sequence showed in SEQIDNO:1. The preparation method includes obtaining DNA (deoxyribonucleic acid) molecules with coded scorpion venom active peptides; fusing the DNA molecules with an expression vector; building and recombining the expression vector; converting the expression vector into host cells; inducing expression fusion protein; and separating purely expressed fusion protein. The invention further provides the application of the scorpion venom active peptides in terms of preparation of medicines for curing or preventing relevant diseases of potassium channel Kv1.3.
Description
Technical field
The invention belongs to biological technical field, relate to scorpion active polypeptide and preparation method thereof and application specifically.
Background technology
Autoimmune disorder (autoimmune disease) is that body causes autologous tissue to damage caused disease to autoantigen generation immunoreation, and promptly the human immune system attacks the tissue of self.The whole world has 5～8% population to receive the threat of about 40 various autoimmune property diseases approximately, comprises the cell-mediated rheumatoid arthritis of T, multiple sclerosis, systemic lupus erythematous, Behcet's disease, AITD and type i diabetes etc.The joint part of immune system attack sufferer in rheumatoid arthritis, multiple sclerosis are that the refreshing marrow sheath (myelin sheaths) of neurocyte is under attack.These diseases relate to one or more tissues of whole body and organ, have a strong impact on HUMAN HEALTH and life.To autoimmune disorder, do not have effective therapy at present, and the state of an illness is shown effect repeatedly.The method of antagonism autoimmune disease commonly used at present; The one, utilize steroid to slow down the inflammatory reaction that is caused because of the immune system attack tissue; Two are to use immunosuppressive drug, suppress immune effect, but these two kinds of methods all can cause severe side effect; And all can only slow down the tempo of the state of an illness, can not radical curing of disease.In order to change the backward situation of autoimmune disorder pharmacological agent, be badly in need of exploring new disease prevention and treat-ment.
After original
T cell is activated by specific antigenic substance; Breed and break up; Generally be formed on two types of different on function cells; Be effect type memory T cell (effector memory; TEM) and the central type memory T cell (central memory, TCM).Research shows that the activation of various autoimmune property disease and TEM cell is closely related.On human T cell's film, have two kinds of potassium channels, a kind of is valtage-gated potassium channel Kv1.3, and another kind is medium conductance calcium-activated potassium channel IKCa1.In recent years, discover that potassium channel Kv1.3 has significant spatial and temporal expression otherness on dissimilar T cells, after the T cell is activated, have only the TEM cell of performance autoimmune function can express 1500～2000 Kv1.3 potassium channels significantly.This potassium channel Kv1.3 high expression level situation is confirmed in the patient's who suffers from different autoimmune disorders TEM cell in succession.Therefore, potassium channel Kv1.3 brings new hope [J.Clin.Invest., 2003,111:1703 for the treatment autoimmune disorder on the T cytolemma; Immunity, 2008,29:602].
The organic molecule medicine at first receives more concern.Screened, separated, synthesized, transformed and identified a large amount of organic molecule drug candidates [Mol.Pharmacol like mechanisms such as Merck company, Australian Walter and Eliza Hall Center For Research And Development of Pharmaceutical, California, USA universities; 2004,65:1364; Journal of Medicinal Chemistry, 2006,49 (4): 1433; Mol.Pharmacol, 2005,68:1254].But the organic molecule drug candidate almost all acts on the similar potassium channel with other of potassium channel Kv1.3; Thereby cause the stronger toxic side effects of effect most probably, lower 2～70 times like organic small-molecule drug effect homology Kv1.1, Kv1.2, Kv1.4, Kv1.5 isoreactivity than Kv1.3 passage.Screening design polypeptide becomes new trend.
The human difficult and complicated cases of strategy treatment of Chinese traditional medicine " combatting poison with poison " commonly used, venomous animal is first-selected like scorpion, snake, spider, toad, centipede etc.Modern study has shown in the venom of these venomous animals and has been rich in the active animal polypeptide, but ionic channel on these polypeptide specific effect cytolemma.Now, these active animal polypeptide have become the important drugs resource [Nat.Rev.Drug.Discov.2003 2:63] of global competition research and development.
Summary of the invention
Technical problem to be solved by this invention provides the scorpion active polypeptide of stable treatment autoimmune disorder.
The present invention has obtained many polypeptide genes with important prospect in medicine, through computer-aided screening and designing technique [Biophysical Journal, 2004, the 87:105 that has set up through screening China scorpion active polypeptide gene pool; Proteins, 2008,70:744; Journal ofProteome Research, 2010,9:3118], the new gene of scorpion bioactive peptide of virtual screening to 3 a possibility specific effect potassium channel Kv1.3.Through genetic engineering technique, obtained three kinds of scorpion active polypeptide, these three kinds of scorpion active polypeptide intramolecularly all contain 3 pairs of disulfide linkage, and are strong in vitro stability, are easy to prolonged preservation.According to the constitutional features of scorpion active polypeptide on taxonomy, the three belongs to the α-KTX family of same hypotype, and all acts on potassium channel Kv1.3.
In one embodiment, the invention provides scorpion bioactive peptide, called after ADC-1 with aminoacid sequence shown in SEQ ID NO:1.
The present invention also provides to have through in the aminoacid sequence shown in the SEQ ID NO:1, replacing, lack or add the polypeptide of the resulting aminoacid sequence of one or more amino-acid residues.
The present invention also provides the dna molecular of coding scorpion active polypeptide ADC-1.Owing to the degeneracy of codon, can there be the nucleotide sequence of a variety of specific polypeptide of the present invention of can encoding.In one embodiment, its nucleotide sequence is shown in SEQ ID NO:2.
The invention provides the carrier of dna molecular simultaneously with coding ADC-1.Wherein, as preferably, said carrier is the pGEX-6p-1 with dna molecular of the ADC-1 that can encode.More preferably be that said carrier is the pGEX-6p-1 with nucleotide sequence shown in SEQID NO:2.
The present invention also provides the preparation method of said scorpion active polypeptide ADC-1, comprising:
Step 1: obtain dna molecular with the aminoacid sequence shown in the SEQ ID NO:1 of can encoding;
Step 2: dna molecular and expression vector that step 1 obtained are merged, make up recombinant expression vector, and transformed host cell;
Step 3: induce the host cell expression fusion rotein that contains recombinant expression vector, the fusion rotein that separation and purification is expressed.
The method of utilizing genetic engineering technique to obtain dna molecular at present has a variety of, comprises utilizing digestion with restriction enzyme to have the carrier of the said scorpion active polypeptide dna molecular of coding or being the template pcr amplification with the cDNA of dna molecular with the said scorpion active polypeptide of coding.
In one embodiment; The cDNA that preparing method's step 1 is specially to have aminoacid sequence shown in the coding SEQ ID NO:1 is a template, with upstream primer with nucleotide sequence shown in the SEQ ID NO:3 and the downstream primer amplification with nucleotide sequence shown in the SEQ ID NO:4.
The said expression vector of preparing method's step 2 according to the invention comprises that pGEX series, pET are serial, pQ E series and pMAL series.In a preferred embodiment, expression vector is the pGEX-6p-1 in the pGEX series.
The said host cell of preparing method's step 2 according to the invention is the escherichia coli expression bacterial strain, comprises Rosetta series and BL21 series bacterial strain.In a preferred embodiment, host cell is Rosetta (DE3) bacterial strain.
Experiment shows, but scorpion active polypeptide ADC-1 specific effect according to the invention and can be treated autoimmune disorders such as multiple sclerosis and rheumatoid arthritis in the rat animal level effectively in potassium channel Kv1.3.Therefore the present invention also provides the application of said scorpion active polypeptide in preparation treatment or prevention potassium channel Kv1.3 relative disease medicine, and said potassium channel Kv1.3 relative disease comprises autoimmune disorder, is specially multiple sclerosis or rheumatoid arthritis.
Description of drawings
Fig. 1 shows reorganization scorpion active polypeptide ADC-1 and the proteic electrophoresis detection figure of GST amalgamation and expression thereof, and wherein, swimming lane 1 is protein molecular weight Marker; 2 swimming lanes are that ADC-1 is without IPTG inductive whole-cell protein; Swimming lane 3 is that ADC-1 is through IPTG inductive whole-cell protein; Swimming lane 4 is the fusion rotein GST-ADC-1 after the affinity chromatography; Swimming lane 5 is the fusion rotein GST-ADC-1 after the desalination and concentration by ultrafiltration; Swimming lane 6 is the product of fusion rotein GST-ADC-1 after the EK enzyme is cut; Swimming lane 7 is through the ADC-1 of HPLC separation and purification polypeptide;
Fig. 2 shows reorganization scorpion active polypeptide ADC-1 and the proteinic chromatographic separation and purification figure of GST;
Fig. 3 shows reorganization scorpion active polypeptide ADC-1 mass spectroscopy figure;
Fig. 4 shows the inhibition synoptic diagram of 1nM scorpion active polypeptide ADC-1 to potassium channel Kv1.3 electric current.
Embodiment
The present invention utilizes genetic engineering technique through screening China scorpion active polypeptide gene pool, has obtained three kinds of novel scorpion active polypeptide, and intramolecularly all has 3 pairs of disulfide linkage, and is strong in vitro stability, is easy to prolonged preservation.In one embodiment, the invention provides scorpion active polypeptide called after ADC-1 with aminoacid sequence shown in SEQ ID NO:1.
The present invention also provides to have through in the aminoacid sequence shown in the SEQ ID NO:1, replacing, lack or add the polypeptide of the resulting aminoacid sequence of one or more amino-acid residues.
The present invention also provides the dna molecular of the scorpion active polypeptide ADC-1 according to the invention that encodes.Owing to the degeneracy of codon, can there be the nucleotide sequence of a variety of specific polypeptide of the present invention of can encoding.In one embodiment, its nucleotide sequence is shown in SEQ ID N0:2.
The invention provides the carrier of dna molecular simultaneously with coded polypeptide ADC-1.In one embodiment, said carrier is the pGEX-6p-1 with dna molecular of the said scorpion active polypeptide ADC-1 that can encode.More preferably be that said carrier is the pGEX-6p-1 with the nucleotide sequence shown in SEQ ID NO:2.
In order to prepare scorpion active polypeptide according to the invention, the present invention also provides the preparation method of said scorpion active polypeptide, comprising:
Step 1: obtain dna molecular with the said scorpion active polypeptide ADC-1 of coding;
Step 2: dna molecular and expression vector that step 1 obtained are merged, make up recombinant expression vector, and transformed host cell;
Step 3: induce the host cell expression fusion rotein that contains recombinant expression vector, the fusion rotein that separation and purification is expressed.
The method of utilizing genetic engineering technique to obtain dna molecular at present has a variety of, comprises utilizing digestion with restriction enzyme to have the carrier of the said scorpion active polypeptide dna molecular of coding or being the template pcr amplification with cDNA with the said scorpion active polypeptide dna molecular of coding.
In one embodiment; The cDNA that the invention provides to have the said scorpion active polypeptide ADC-1 of coding is a template; Usefulness has the upstream primer and the downstream primer amplification with the nucleotide sequence shown in the SEQ ID NO:4 of the nucleotide sequence shown in the SEQ ID NO:3, obtains to have the said scorpion active polypeptide ADC-1DNA of coding.
Preparing method's step 2 according to the invention is said to merge dna molecular and expression vector that step 1 obtained; Make up recombinant expression vector; And transformed host cell is specially step 1 gained dna molecular through gel electrophoresis recovery and double digestion insertion expression vector; Make up recombinant expression plasmid, transformed into escherichia coli.
Wherein, expression vector according to the invention comprise that pGEX series, pET are serial, pQ E series and pMAL series.In a preferred embodiment, expression vector is the pGEX-6p-1 in the pGEX series.
Transformed host cell according to the invention is the escherichia coli expression bacterial strain, comprises Rosetta series and BL21 series bacterial strain.In a preferred embodiment, host cell is Rosetta (DE3) bacterial strain.The codon of eukaryotic cell preference has different with prokaryotic system; Therefore; When expressing eukaryotic gene with prokaryotic system, some codons in the eukaryotic gene possibly be rare codons for prokaryotic cell prokaryocyte, thereby cause expression efficiency and expression level very low.Rosetta (DE3) is the BL21 that carries the pRARE2 plasmid bacterium that derives, and can replenish seven kinds of rare codons (AUA, AGG that intestinal bacteria lack; AGA, CUA, CCC; GGA and CGG) corresponding tRNA, improve the especially expression level of eukaryotic gene in prokaryotic system of foreign gene.
The said step 3 of preparation method according to the invention is specially the host cell that the IPTG inducing culture contains recombinant expression vector; Collect the thalline ultrasonic disruption; Get the supernatant affinity chromatography and obtain fusion rotein, cut and chromatographic separation through desalination and concentration, small intestine kinases enzyme again, obtain chromatographically pure scorpion active polypeptide.Wherein, the chromatography media that with it be complementary of the chromatography media of said affinity chromatography for selecting according to the selectivity label of expression vector.In a preferred embodiment, expression vector is pGEX-6p-1, and the chromatography media of affinity chromatography is a GST affinity chromatography glue.
In specific embodiments; The cDNA that preparation method according to the invention is specially with the dna molecular with the said scorpion active polypeptide ADC-1 of coding is a template; Use nucleotide sequence designed primer, obtain dna molecular, dna molecular that gel electrophoresis recovery and double digestion obtain and expression vector pGEX-6p-1 with the said scorpion active polypeptide ADC-1 of coding according to the encoding mature peptide moiety; Connect and make up recombinant expression plasmid, transformed into escherichia coli Rosetta (DE3) acquisition reorganization bacterium.The IPTG inducing culture contains the host cell of recombinant expression vector then, collects the thalline ultrasonic disruption, gets supernatant GST affinity chromatography and obtains fusion rotein, cuts and chromatographic separation through desalination and concentration, small intestine kinases enzyme again, obtains chromatographically pure scorpion active polypeptide ADC-1.
The present invention has identified the pharmacological activity of said scorpion active polypeptide ADC-1 to potassium channel Kv1.3 through patch clamp technique.The result shows; But said scorpion active polypeptide ADC-1 specificity suppresses potassium channel Kv1.3 electric current; The peptide concentration (IC50 value) that suppresses the electric current half is 0.49 ± 0.45nM, but shows the target potassium channel Kv1.3 of scorpion active polypeptide ADC-1 specific effect autoimmune disease.
The present invention detects scorpion active polypeptide ADC-1 medication effect through simulation treatment multiple sclerosis and rheumatoid arthritis on the rat animal level.The result shows that after the scorpion active polypeptide ADC-1 treatment, the symptom of rat multiple sclerosis and rheumatoid arthritis significantly improves, and shows that scorpion active polypeptide ADC-1 can treat multiple sclerosis and rheumatoid arthritis effectively.
The present invention finds that through the toxic action research to scorpion active polypeptide ADC-1 scorpion active polypeptide ADC-1 does not have overt toxicity to mouse under the dosage that surpasses 50 times of autoimmune disease animal model therapeutic doses, show that scorpion active polypeptide ADC-1 toxic side effect is little.
Therefore the present invention also provides the application of said scorpion active polypeptide in preparation treatment or prevention potassium channel Kv1.3 relative disease medicine, and said potassium channel Kv1.3 relative disease comprises autoimmune disorder, is specially multiple sclerosis or rheumatoid arthritis.
Scorpion active polypeptide ADC-1 intramolecularly according to the invention has 3 pairs of disulfide linkage, and is good in vitro stability, is easy to prolonged preservation.But patch clamp technique identifies and shows specific effect potassium channel Kv1.3 that high specificity is the active strong polypeptide of having found in the world at present.Experimentation on animals shows that recombinant polypeptide ADC-1 can effectively treat multiple sclerosis of rats and rheumatoid arthritis, and effect of drugs is remarkable, and little to the laboratory animal toxic side effect.The preparation method of scorpion active polypeptide according to the invention is simple, and is easy to operate, is easy to produce and prepare highly purified ADC-1 scorpion active polypeptide.
In order further to understand the present invention, scorpion active polypeptide provided by the invention and preparation method thereof and application are elaborated below in conjunction with embodiment.
Embodiment 1: the obtaining of scorpion active polypeptide ADC-1 gene
Get 40 three ridge globefish scorpions (Chaerilus tricostatus) scorpion tail gland, (Invitrogen) extracts total RNA with Trizol reagent, and process for extracting is with reference to Trizol test kit specification sheets.With Poly A Tract mRNA Isolation System (Promega) test kit purified mRNA, and adopt Superscript Plasmid System cDNA library construction kit (Gibco/BRL) test kit construction cDNA library.CDNA is cloned in the pSPORT1 carrier, and transformed into escherichia coli DH5 α, random choose cDNA clone checks order.Result to order-checking carries out sequential analysis, altogether acquisition maybe with interactional 178 the new genes of scorpion bioactive peptide of potassium-channel.Through computer-aided screening and designing technique [Biophysical Journal, 2004, the 87:105 that sets up; Proteins, 2008,70:744; Journal of Proteome Research; 2010; 9:3118], predict the space structure of 84 scorpion bioactive peptide genes encoding polypeptide respectively, then the interaction through computer virtual screening they and potassium channel Kv1.3; Obtained to have the cDNA gene of coding scorpion active polypeptide ADC-1DNA molecule, coded mature peptide has the scorpion active polypeptide ADC-1 of aminoacid sequence shown in SEQ ID NO:1.
Embodiment 2: the dna molecular of amplification coding scorpion active polypeptide ADC-1
1, design of primers:
Nucleotide sequence with encoding mature peptide moiety in the cDNA gene order of scorpion active polypeptide ADC-1 designs primer as the template sequence that PCR reacts.Designed primer is:
The ADC-1 upstream primer is the nucleotide sequence shown in the SEQ ID NO:3;
The ADC-1 downstream primer is the nucleotide sequence shown in the SEQ ID NO:4;
2, DNA amplification molecule:
With scorpion venom cDNA is template, carries out pcr amplification with upstream primer with the nucleotide sequence shown in the SEQ ID NO:3 and the downstream primer with the nucleotide sequence shown in the SEQ IDNO:4.
The pcr amplification reaction system is:
Add sterile purified water and supply system total amount 50 μ l.
The PCR response procedures is:
3, recombinant expression vector makes up:
The pcr amplification product gel electrophoresis is reclaimed; With EcoRI and XhoI double digestion; Fragment after enzyme cut is inserted through the expression vector pGEX-6p-1 of EcoRI and XhoI double digestion (available from Pharmacia company); Make up recombinant expression plasmid, transformed into escherichia coli DH5 α (Chinese typical culture collection center).Picking list bacterium colony from the LB flat board that contains penbritin was cultivated 5 hours for 37 ℃ in containing the LB liquid nutrient medium of penbritin, with the method for pcr amplification these cultures was identified then.The pcr amplification template is with cultivating the bacterium liquid that obtains, and the primer of amplification, reaction conditions and reaction process are with the dna molecular amplification procedure.Choose PCR and identify that correct bacterial classification checks order, obtain the correct bacterial classification that contains recombinant expression vector of sequence, extract plasmid, transformed into escherichia coli Rosetta (DE3) (Chinese typical culture collection center).
Embodiment 3: expression and the purifying of scorpion active polypeptide ADC-1
Extract the plasmid transformation escherichia coli Rosetta (DE3) of the correct pGEX-6p-1-ADC-1 bacterial classification that contains recombinant expression vector of sequencing sequence.The intestinal bacteria that IPTG (Huamei Bio-Engrg Co.) inducing culture transforms are collected thalline, be suspended in damping fluid (50mM Tris-Cl, 1.0mM EDTA, pH8.0) in, ultrasonic disruption thalline, centrifugal collection supernatant.The gained supernatant obtains fusion rotein through the GST affinity chromatography.The fusion rotein solution desalination and concentration that collection obtains, little enteropeptidase (EK) enzyme are cut, and obtain scorpion active polypeptide ADC-1.Utilize performance liquid chromatography that the ADC-1 polypeptide is further separated, remove GST protein, obtain chromatographically pure ADC-1 polypeptide.ADC-1 polypeptide theoretical molecular is 4443.1Da, shows that through mass spectroscopy its molecular weight is 4439.7Da, and is consistent with the molecular weight of inferring according to aminoacid sequence.Wherein, the ADC-1 polypeptide that different steps is obtained and carried out the PAGE electrophoresis detection with gst fusion protein, the result sees Fig. 1, performance liquid chromatography separation and purification and mass spectrometry results are seen Fig. 2 and Fig. 3.
Embodiment 4: scorpion active polypeptide ADC-1 is to the pharmacological activity analysis of potassium channel Kv1.3
The HEK-293T cell is being contained 37 ℃ of DMEM substratum, the 5%CO of 10% foetal calf serum
2Cultivate under the condition, potassium channel Kv1.3 recombinant plasmid is used Sofast respectively
TMThe transfection of transfection reagent box, transfectional cell selectivity on the 0.8mg/mLGeneticin substratum is cultivated.(Lambrecht Germany), measures and analyzes the pharmacological activity of reorganization scorpion active polypeptide ADC-1 for EPC-10 two channels patch clamp amplifier HEKA, Elektronik with instrument to utilize full cell patch pincers.The setting of experiment parameter, the collection of data and stimulation apply that all (Elektronik, Lambrecht Germany) control through Pulse software.The wave filter of instrument is set to 10kHz (Bessel), and electrode impedance is 2-5M Ω, after forming high resistant (1-5G Ω) sealing-in between electrode and the cytolemma; Carry out fast electric capacity and compensate (c-fast) automatically, behind the negative pressure rupture of membranes, carry out slow electric capacity and compensate (c-slow) automatically a little; Under the command potential of-70mV; Give the 10mV stride increases progressively, the 80ms step is wide depolarize impulse stimulation to+50mV from-60mV, observe current conditions, with polypeptide A DC-1 through MPS-2 (INBIO Inc; Wuhan, China) filling system is realized accurately perfusion.After 3 kinds of scorpion active polypeptide dissolvings, spray administration through DAD drug delivery system (ALA), delivery tube is most advanced and sophisticated apart from writing down about cell 100 μ m, and the result sees Fig. 4.
Can know that by Fig. 4 ADC-1 is 0.49 ± 0.45nM to the pharmacological activity of potassium channel Kv1.3, be the active strong polypeptide of having found in the world at present.
Embodiment 5: the effect experiment of scorpion active polypeptide ADC-1 treatment multiple sclerosis
Laboratory animal: choose inbred lines female Wistar rats (age in 6-8 week, body weight 150 ± 10g), cavy (300-400g is available from Wuhan University's Experimental Animal Center).
Main agents: Fu Shi Freund's complete adjuvant (Gibcol/BRL), BCG-CWS, pertussis vaccine (Shanghai institute of Biological Products), cavy MBP (Sigma).
The preparation of full spinal cord homogenate-Fu Shi Freund's complete adjuvant mixed emulsion (GPSCH-CFA): after cavy execution; Take out spinal cord rapidly; With Ultrasonic Cell Disruptor (Sonics & Materials Inc; America) process 50% PBS homogenate, mix, lash to water-in-oil emulsion with syringe with the Fu Shi Freund's complete adjuvant (BCG-CWS 10mg/ml) of equivalent.
EAE induces Wistar rat EAE model: injection 0.4ml GPSCH-CFA emulsion in the two back legs foot of the Wistar rat lift, or intradermal injection about 1 * 10 simultaneously
10Pertussis vaccine.Weigh every day, observes nervous symptoms.Raise and 2 weeks EAE promptly occurred.
EAE (EAE) rat is divided into three groups at random: normal control group (negative control group), model control group (model mouse+saline water), administration group (model mouse+ADC-1 polypeptide), 10 every group.The administration group is pressed 100 μ gkg with polypeptide A DC-1
-1The dosage subcutaneous injection, every morning 1 time, normal control group and model control group subcutaneous injection equivalent saline water, successive administration.After the administration 14 days, observe rat and suffer from the EAE situation, according to the scoring of rat situation, write down every the highest clinical score of animal, the average of getting them is average clinical score, and the result sees table 1.
Standards of grading are: do not have any clinical symptom, 0 minute; Afterbody tension force disappears, and visible slight gait is clumsy, 1 minute; Two backs myasthenia of limbs can recover 2 fens after passive the standing up; Two backs acroparalysis can not be recovered 3 fens after passive the standing up; Tetraplegia companion urine, fecal incontinence, 4 minutes; Moribund condition or death, 5 minutes.
The scoring of each experimental group rat in the table 1 EAE model
Experimental result shows: do not having under the pharmacological agent situation model control group score the highest (2.82 ± 0.47); After the scorpion active polypeptide ADC-1 treatment, symptom of rats significantly improves, and average clinical score is 1.23 ± 0.35.This shows that scorpion active polypeptide ADC-1 can treat multiple sclerosis effectively.
Embodiment 6: the effect experiment of scorpion active polypeptide ADC-1 treatment rheumatoid arthritis
Choose no-special pathogen (SPF) level inbred lines Wistar rat (Wuhan University's Experimental Animal Center) 40; ♀; Body weight 150 ± 10g, after flexibility raised for 1 week under the cleaning level envrionment conditions, with the pristane (pristane) of 0.2ml/ dosage in the intradermal injection of experimental group rat root of the tail portion.Raise and 2 weeks symptoms of rheumatoid arthritis promptly occurred.
Rats with arthritis is divided at random: normal control group (negative control group), model negative control group (model mouse+saline water), model positive controls (model mouse+methotrexate), administration group (model mouse+Wistar polypeptide), 10 every group.The administration group is by 100 μ gkg
-1The dosage subcutaneous injection, every morning 1 time, successive administration; Normal control group and model negative control group subcutaneous injection equivalent saline water; The model positive controls is pressed 1.75mgkg
-1Dosage subcutaneous injection methotrexate (MTX), every day 1 time, successive administration.
After the administration 21 days, observe rat and suffer from arthritic conditions.Observe rat and suffer from the rheumatoid arthritis situation, according to the scoring of rat situation, write down every the highest clinical score of animal, the average of getting them is average clinical score, and the result sees table 2.Standards of grading are: redness and swelling of joints of rat, 1 minute; Two redness and swelling of joints of rat, 2 minutes; Each redness and swelling of joints of rat, 3 minutes; The serious sacroiliitis of the whole limbs of rat, 4 minutes.
The arthritis score of each experimental group rat of table 2
From testing the 8th day, the next day survey 1 rat hindleg sole of the foot volume and record, and calculate swelling degree (mL) according to the difference of sufficient sole of the foot volume before and after modeling, observed continuously 21 days, the result sees table 3.
The swelling degree of the paw of each experimental group rat of table 3
Can know do not having under the pharmacological agent situation by table 2, model negative control group joint scoring the highest (3.56 ± 0.76); After the scorpion active polypeptide ADC-1 treatment, symptom of rats significantly improves, and scoring is 1.73 ± 0.48, close with methotrexate for treatment group result (1.79 ± 0.69).The result that table 3 is measured the swelling degree of the paw of each experimental group rat shows that the rat paw edema degree of scorpion active polypeptide ADC-1 administration group is compared remarkable reduction with the model negative control group.The result shows that scorpion active polypeptide ADC-1 can treat rheumatoid arthritis effectively.
Embodiment 7: the toxic action research of scorpion active polypeptide ADC-1
Choose the 18-20g kunming mice, divide 2 groups, each 8 of every group of male and female.Scorpion active polypeptide ADC-1 lyophilized powder is used physiological saline solution, with the dosage of 5mg/kg (animal model dosage 50 times) the 1st group of mouse carried out the once abdominal cavity injection polypeptide solution, the 2nd group of injection equivalent saline water is done contrast.Observed 7 days continuously after the administration, estimate administration after medicine to the animal breath maincenter, cardio-pulmonary function, the influence of cns.
Each treated animal is breathed after administration, motion, and heartbeat is normal, does not have tangible abnormal response.Administration was carried out gross anatomy with all animals after 7 days, observed the volume of main organs (heart, liver, spleen, lung, kidney), color, and quality is not seen obvious change, does not have macroscopic pathology, administration group 3 treated animals and saline water group do not have significant difference.Experimental result shows that scorpion active polypeptide ADC-1 does not have overt toxicity to mouse under the dosage that surpasses 50 times of autoimmune disease animal model therapeutic doses.
The explanation of above embodiment just is used for helping to understand method of the present invention and core concept thereof.Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of claim of the present invention.
SEQUENCE LISTING
< 110>Wuhan University
< 120>the scorpion active immne is regulated polypeptide and preparation method thereof and application
< 130>the scorpion active immne is regulated polypeptide and preparation method thereof and application
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Thr Tyr Cys Ile Lys Val His Lys Ile Gln Ser Gly Lys Cys Met Asn
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Claims (11)
1. a scorpion active polypeptide is characterized in that, has the aminoacid sequence shown in SEQ ID NO:1.
2. a scorpion active polypeptide is characterized in that, has through in the aminoacid sequence shown in the SEQ ID NO:1, replacing, lack or adding the resulting aminoacid sequence of one or more amino-acid residues.
3. the dna molecular of coding claim 1 or 2 said scorpion active polypeptide.
4. the dna molecular of the said scorpion active polypeptide of coding claim 1 is characterized in that having the nucleotide sequence shown in SEQ ID NO:2.
5. the carrier that has the said dna molecular of claim 3.
6. according to the said carrier of claim 5, it is characterized in that said carrier is pGEX-6p-1.
7. the preparation method of the said scorpion active polypeptide of claim 1 comprises the steps:
Step 1: obtain dna molecular with the aminoacid sequence shown in the SEQ ID NO:1 of can encoding;
Step 2: dna molecular and expression vector that step 1 obtained are merged, make up recombinant expression vector, and transformed host cell;
Step 3: induce the host cell expression fusion rotein that contains recombinant expression vector, the fusion rotein that separation and purification is expressed.
8. according to the said preparation method of claim 7; It is characterized in that; The cDNA that said step 1 is specially to have aminoacid sequence shown in the coding SEQ ID NO:1 is a template, with upstream primer with the nucleotide sequence shown in the SEQ ID NO:3 and the downstream primer amplification with the nucleotide sequence shown in the SEQ ID NO:4.
9. the application of the said scorpion active polypeptide of claim 1 in preparation treatment or prevention potassium channel Kv1.3 relative disease medicine.
10. according to the said application of claim 9, it is characterized in that said potassium channel Kv1.3 relative disease is an autoimmune disorder.
11., it is characterized in that said autoimmune disorder is multiple sclerosis or rheumatoid arthritis according to the said application of claim 10.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210103416 CN102617723B (en) | 2012-04-10 | 2012-04-10 | Scorpion venom active peptides, preparation method thereof and application |
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| CN107987145A (en) * | 2017-12-18 | 2018-05-04 | 武汉大学 | Scorpion active peptides ADP-7 and its application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101063103A (en) * | 2007-04-26 | 2007-10-31 | 武汉大学 | Hainan sporic scorpion antibiotic and preparation method and application |
| CN102127160A (en) * | 2010-12-14 | 2011-07-20 | 武汉摩尔生物科技有限公司 | Scorpion active polypeptides as well as preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101063103A (en) * | 2007-04-26 | 2007-10-31 | 武汉大学 | Hainan sporic scorpion antibiotic and preparation method and application |
| CN102127160A (en) * | 2010-12-14 | 2011-07-20 | 武汉摩尔生物科技有限公司 | Scorpion active polypeptides as well as preparation method and application thereof |
Non-Patent Citations (1)
| Title |
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| 戚正武: "小分子蛋白酶抑制剂及多肽毒素的研究回顾", 《科学通报》, vol. 54, no. 18, 30 September 2009 (2009-09-30), pages 2734 - 2745 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107987145A (en) * | 2017-12-18 | 2018-05-04 | 武汉大学 | Scorpion active peptides ADP-7 and its application |
| CN107987145B (en) * | 2017-12-18 | 2021-04-16 | 武汉大学 | Scorpion active polypeptide ADP-7 and application thereof |
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