CN102617723B - 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 specifically scorpion active polypeptide and preparation method thereof and application.
Background technology
Autoimmune disorder (autoimmune disease) is that body causes autologous tissue to damage caused disease to autoantigen generation immune response, and namely the human immune system attacks the tissue of self.The whole world approximately has 5～8% population to be subject to the approximately threat of 40 various autoimmune diseases, comprises the cell-mediated rheumatoid arthritis of T, multiple sclerosis, systemic lupus erythematous, Behcet's disease, autoimmune thyroid disease 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.For autoimmune disorder, at present without effective therapy, and the Relapse rate outbreak.Commonly use at present the method for antagonism autoimmune disease, the one, utilize steroid to slow down the inflammatory reaction that causes because of the immune system attack tissue, the 2nd, use immunosuppressive drug, the effect of Immunosuppression system, 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 methods for the treatment of.
Original
After the T cell is activated by specific antigenic substance, breed and break up, generally being formed on two class cells different on function, i.e. effect type memory T cell (effector memory, TEM) and central type memory T cell (central memory, TCM).Studies show that, the activation of various autoimmune disease and TEM cell is closely related.Have two kinds of potassium channels on human T cell's film, a kind of is valtage-gated potassium channel Kv1.3, and another kind is medium conductance calcium-activated potassium channel IKCa1.In recent years, research finds that potassium channel Kv1.3 has significant spatial and temporal expression otherness on dissimilar T cell, after the T cell is activated, only has the TEM cell of performance autoimmune function can express significantly 1500～2000 Kv1.3 potassium channels.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, on the T cytolemma, potassium channel Kv1.3 brings new hope [J.Clin.Invest., 2003,111:1703 for the treatment autoimmune disorder; Immunity, 2008,29:602].
At first the organic molecule medicine receives more concern.Screened, separated, synthesized, transformed and identified a large amount of organic molecule drug candidates [Mol.Pharmacol as 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 potassium channel Kv1.3 potassium channel similar with other, thereby cause most probably the stronger toxic side effects of effect, as lower 2～70 times than Kv1.3 passage in organic small-molecule drug effect homology Kv1.1, Kv1.2, Kv1.4, Kv1.5 isoreactivity.The screening design polypeptide becomes new trend.
The strategy treatment mankind's of Chinese traditional medicine " combatting poison with poison " commonly used difficult and complicated cases, venomous animal is first-selected as 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 is to provide the scorpion active polypeptide of stable treatment autoimmune disorder.
The present invention has obtained many polypeptide genes with important prospect in medicine, by computer-aided screening and designing technique [Biophysical Journal, 2004, the 87:105 that has set up by screening China's 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.By genetic engineering technique, obtained three kinds of scorpion active polypeptide, all contain 3 pairs of disulfide linkage in these three kinds of scorpion active polypeptide molecules, strong in vitro stability, be easy to prolonged preservation.According to the constitutional features of scorpion active polypeptide on taxonomy, the three belongs to the α of same hypotype-KTX family, and all acts on potassium channel Kv1.3.
In one embodiment, the invention provides the scorpion bioactive peptide with aminoacid sequence as shown in SEQ ID NO:1, called after ADC-1.
The present invention also provides to have by replace, lack or add the polypeptide of the resulting aminoacid sequence of one or more amino-acid residues in the aminoacid sequence shown in SEQ ID NO:1.
The present invention also provides the DNA molecular of coding scorpion active polypeptide ADC-1.Due 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 as shown in SEQ ID NO:2.
The invention provides simultaneously the carrier of the DNA molecular with coding ADC-1.Wherein, as preferably, described carrier is the pGEX-6p-1 with DNA molecular of the ADC-1 that can encode.More preferably that described carrier is the pGEX-6p-1 with nucleotide sequence as shown in SEQID NO:2.
The present invention also provides the preparation method of described scorpion active polypeptide ADC-1, comprising:
Step 1: obtain the DNA molecular with the aminoacid sequence shown in SEQ ID NO:1 of can encoding;
Step 2: DNA molecular and expression vector that step 1 is obtained merge, and build 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 at present genetic engineering technique to obtain DNA molecular has a variety of, comprises utilizing digestion with restriction enzyme to have the carrier of the described scorpion active polypeptide DNA molecular of coding or take the cDNA of DNA molecular with the described scorpion active polypeptide of coding as the template pcr amplification.
In one embodiment, the cDNA that preparation method's step 1 is specially to have aminoacid sequence shown in coding SEQ ID NO:1 is template, with the upstream primer with nucleotide sequence shown in SEQ ID NO:3 with have the downstream primer amplification of nucleotide sequence shown in SEQ ID NO:4.
The described expression vector of preparation method's step 2 of the present 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 pGEX series.
The described host cell of preparation method's step 2 of the present 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 of the present invention and can be treated in the rat animal level autoimmune disorders such as multiple sclerosis and rheumatoid arthritis effectively in potassium channel Kv1.3.Therefore the present invention also provides the application of described scorpion active polypeptide in preparation treatment or prevention potassium channel Kv1.3 relative disease medicine, and described potassium channel Kv1.3 relative disease comprises autoimmune disorder, is specially multiple sclerosis or rheumatoid arthritis.
Description of drawings
Fig. 1 shows the electrophoresis detection figure of restructuring scorpion active polypeptide ADC-1 and GST amalgamation and expression albumen thereof, and wherein, swimming lane 1 is protein molecular weight Marker; 2 swimming lanes are the whole-cell protein that ADC-1 induces without IPTG; The whole-cell protein that swimming lane 3 is induced through IPTG for ADC-1; Swimming lane 4 is the fusion rotein GST-ADC-1 after affinity chromatography; Swimming lane 5 is the fusion rotein GST-ADC-1 after 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 the chromatographic separation and purification figure of restructuring scorpion active polypeptide ADC-1 and GST protein;
Fig. 3 shows restructuring scorpion active polypeptide ADC-1 mass spectroscopy figure;
Fig. 4 shows that 1nM scorpion active polypeptide ADC-1 is to the inhibition schematic diagram of potassium channel Kv1.3 electric current.
Embodiment
The present invention utilizes genetic engineering technique by screening China's scorpion active polypeptide gene pool, has obtained three kinds of novel scorpion active polypeptide, all has 3 pairs of disulfide linkage in molecule, and is strong in vitro stability, is easy to prolonged preservation.In one embodiment, the invention provides the scorpion active polypeptide called after ADC-1 with aminoacid sequence as shown in SEQ ID NO:1.
The present invention also provides to have by replace, lack or add the polypeptide of the resulting aminoacid sequence of one or more amino-acid residues in the aminoacid sequence shown in SEQ ID NO:1.
The present invention also provides the DNA molecular of the scorpion active polypeptide ADC-1 of the present invention that encodes.Due 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 as shown in SEQ ID N0:2.
The invention provides simultaneously the carrier of the DNA molecular with coded polypeptide ADC-1.In one embodiment, described carrier is the pGEX-6p-1 with DNA molecular of the described scorpion active polypeptide ADC-1 that can encode.More preferably that described carrier is the pGEX-6p-1 with the nucleotide sequence as shown in SEQ ID NO:2.
In order to prepare scorpion active polypeptide of the present invention, the present invention also provides the preparation method of described scorpion active polypeptide, comprising:
Step 1: obtain the DNA molecular with the described scorpion active polypeptide ADC-1 of coding;
Step 2: DNA molecular and expression vector that step 1 is obtained merge, and build 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 at present genetic engineering technique to obtain DNA molecular has a variety of, comprises utilizing digestion with restriction enzyme to have the carrier of the described scorpion active polypeptide DNA molecular of coding or take cDNA with the described scorpion active polypeptide DNA molecular of coding as the template pcr amplification.
In one embodiment, the cDNA that the invention provides to have the described scorpion active polypeptide ADC-1 of coding is template, with the upstream primer with the nucleotide sequence shown in SEQ ID NO:3 with have the downstream primer amplification of the nucleotide sequence shown in SEQ ID NO:4, obtain and have the described scorpion active polypeptide ADC-1DNA of coding.
Described DNA molecular and the expression vector that step 1 is obtained of preparation method's step 2 of the present invention merges, build recombinant expression vector, and transformed host cell is specially step 1 gained DNA molecular by gel electrophoresis recovery and double digestion insertion expression vector, build recombinant expression plasmid, transform intestinal bacteria.
Wherein, expression vector of the present 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 pGEX series.
Transformed host cell of the present 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.Codon and the prokaryotic system of eukaryotic cell preference have difference, therefore, when expressing eukaryotic gene with prokaryotic system, some codons in eukaryotic gene may be rare codons for prokaryotic cell prokaryocyte, thereby cause expression efficiency and expression level very low.Rosetta (DE3) is the derivative bacterium of BL21 of carrying the pRARE2 plasmid, 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 described step 3 of preparation method of the present 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, then cut and chromatographic separation through desalination and concentration, small intestine kinases enzyme, obtain chromatographically pure scorpion active polypeptide.Wherein, the chromatography media that with it be complementary of the chromatography media of described 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 GST affinity chromatography glue.
In specific embodiments, the cDNA that preparation method of the present invention is specially to have the DNA molecular of the described scorpion active polypeptide ADC-1 of coding is template, with the primer according to the design of the nucleotide sequence of encoding mature peptide moiety, obtain the DNA molecular with the described scorpion active polypeptide ADC-1 of coding, the DNA molecular that gel electrophoresis recovery and double digestion obtain and expression vector pGEX-6p-1, connect and build recombinant expression plasmid, conversion intestinal bacteria Rosetta (DE3) acquisition recombinant bacterium.Then the IPTG inducing culture contains the host cell of recombinant expression vector, collects the thalline ultrasonic disruption, gets supernatant GST affinity chromatography and obtains fusion rotein, then cut and chromatographic separation through desalination and concentration, small intestine kinases enzyme, obtains chromatographically pure scorpion active polypeptide ADC-1.
The present invention has identified that by patch clamp technique described scorpion active polypeptide ADC-1 is to the pharmacological activity of potassium channel Kv1.3.Result shows, but described 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 by simulation treatment multiple sclerosis and rheumatoid arthritis on the rat animal level.The result demonstration, after 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 is by to the research of the toxic action of scorpion active polypeptide ADC-1, find scorpion active polypeptide ADC-1 under the dosage that surpasses 50 times of autoimmune disease animal model therapeutic doses to mouse without overt toxicity, show that scorpion active polypeptide ADC-1 toxic side effect is little.
Therefore the present invention also provides the application of described scorpion active polypeptide in preparation treatment or prevention potassium channel Kv1.3 relative disease medicine, and described potassium channel Kv1.3 relative disease comprises autoimmune disorder, is specially multiple sclerosis or rheumatoid arthritis.
Have 3 pairs of disulfide linkage in scorpion active polypeptide ADC-1 molecule of the present invention, good in vitro stability, be easy to prolonged preservation.But patch clamp technique is identified and is shown specific effect potassium channel Kv1.3, and high specificity is the present active strong polypeptide of having found in the world.Experimentation on animals shows, recombinant polypeptide ADC-1 can effectively treat multiple sclerosis and the rheumatoid arthritis of rat, and effect of drugs is remarkable, and little to the laboratory animal toxic side effect.The preparation method of scorpion active polypeptide of the present invention is simple, and is easy to operate, is easy to the highly purified ADC-1 scorpion of production and preparation active polypeptide.
In order further to understand the present invention, below in conjunction with embodiment, scorpion active polypeptide provided by the invention and preparation method thereof and application are elaborated.
Embodiment 1: the obtaining of scorpion active polypeptide ADC-1 gene
Get 40 three ridge globefish scorpion (Chaerilus tricostatus) scorpion tail glands, (Invitrogen) extracts total RNA with Trizol reagent, and extracting method 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 transforms bacillus coli DH 5 alpha, random choose cDNA clone checks order.Result to order-checking is carried out sequential analysis, altogether acquisition may with interactional 178 the new genes of scorpion bioactive peptide of potassium-channel.By 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 respectively the space structure of 84 scorpion bioactive peptide genes encoding polypeptide, then screen the interaction of they and potassium channel Kv1.3 by computer virtual, 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 as 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.The primer of design is:
The ADC-1 upstream primer is the nucleotide sequence shown in SEQ ID NO:3;
The ADC-1 downstream primer is the nucleotide sequence shown in SEQ ID NO:4;
2, DNA amplification molecule:
Take scorpion venom cDNA as template, carry out pcr amplification with the upstream primer with the nucleotide sequence shown in SEQ ID NO:3 and the downstream primer with the nucleotide sequence shown in SEQ IDNO:4.
The pcr amplification reaction system is:
Add sterile purified water to supply system total amount 50 μ l.
The PCR response procedures is:
3, recombinant expression vector builds:
The pcr amplification product gel electrophoresis is reclaimed, with EcoRI and XhoI double digestion, fragment after enzyme is cut is inserted through the expression vector pGEX-6p-1 of EcoRI and XhoI double digestion (available from Pharmacia company), build recombinant expression plasmid, transform bacillus coli DH 5 alpha (Chinese Typical Representative 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, then with the method for pcr amplification, these cultures was identified.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, transform intestinal bacteria Rosetta (DE3) (Chinese Typical Representative 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, are suspended in damping fluid (50mM Tris-Cl, 1.0mM EDTA, pH8.0) ultrasonic disruption thalline, centrifugal collection supernatant.The gained supernatant obtains fusion rotein by 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 high 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 results are shown in Figure 1, and high performance liquid chromatography separation and purification and mass spectrometry results are seen Fig. 2 and Fig. 3.
Embodiment 4: the pharmacological activity analysis of scorpion active polypeptide ADC-1 to 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 condition, potassium channel Kv1.3 recombinant plasmid is used respectively Sofast
TMThe transfection of transfection reagent box, transfectional cell selectivity on the 0.8mg/mLGeneticin substratum is cultivated.Utilize full cell patch pincers with instrument (EPC-10 two channels patch clamp amplifier HEKA, Elektronik, Lambrecht, Germany), the pharmacological activity of restructuring scorpion active polypeptide ADC-1 is measured and analyzed.Applying all of the setting of experiment parameter, the collection of data and stimulation controlled by Pulse software (Elektronik, Lambrecht, Germany).the wave filter of instrument is set to 10kHz (Bessel), electrode impedance is 2-5M Ω, after forming high resistant (1-5G Ω) sealing-in between electrode and cytolemma, carry out fast electric capacity auto-compensation (c-fast), a little after the negative pressure rupture of membranes, carry out slow electric capacity auto-compensation (c-slow), under the command potential of-70mV, giving the 10mV stride from-60mV increases progressively, the wide depolarize impulse stimulation of 80ms step is to+50mV, observe current conditions, polypeptide A DC-1 is passed 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 recording about cell 100 μ m, the results are shown in Figure 4.
ADC-1 is 0.49 ± 0.45nM to the pharmacological activity of potassium channel Kv1.3 as shown in Figure 4, is 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), bacille Calmette-Guerin vaccine, 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 is put to death, take out rapidly spinal cord, with Ultrasonic Cell Disruptor (Sonics; Materials Inc, America) make 50% PBS homogenate, mix with the Fu Shi Freund's complete adjuvant (bacille Calmette-Guerin vaccine 10mg/ml) of equivalent, lash to water-in-oil emulsion with syringe.
EAE induces Wistar rat EAE model: injection 0.4ml GPSCH-CFA emulsion in the two back legs foot of Wistar rat lift, or intradermal injection simultaneously approximately 1 * 10
10Pertussis vaccine.Weigh every day, observes nervous symptoms.Raise and 2 weeks experimental autoimmune encephalomyelitis namely occurred.
Experimental autoimmune encephalomyelitis (EAE) rat is divided into three groups at random: Normal group (negative control group), model control group (model mouse+physiological saline), 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 group and model control group subcutaneous injection equivalent physiological saline, successive administration.After administration 14 days, observe rat and suffer from the experimental autoimmune encephalomyelitis situation, according to the scoring of rat situation, record every the highest clinical score of animal, the average of getting them is average clinical score, the results are shown in Table 1.
Standards of grading are: without any clinical symptom, and 0 minute; Afterbody tension force disappears, visible slight gait, 1 minute; Two rear myasthenia of limbs can recover after passive standing up, 2 minutes; Two rear acroparalysis can not be recovered after passive standing up, 3 minutes; Tetraplegia companion urine, fecal incontinence, 4 minutes; Moribund condition or death, 5 minutes.
The scoring of each experimental group rat in table 1 EAE model
Experimental result shows: there is no in the pharmacological agent situation model control group score the highest (2.82 ± 0.47); After scorpion active polypeptide ADC-1 treatment, the symptom of rat significantly improves, and average clinical score is 1.23 ± 0.35.This shows, 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 Wistar Rats (Wuhan University's Experimental Animal Center) 40, ♀, body weight 150 ± 10g, after adaptability under the Clean Facility condition raised for 1 week, with the pristane (pristane) of 0.2ml/ dosage in the intradermal injection of experimental group rat root of the tail section.Raise and 2 weeks symptoms of rheumatoid arthritis namely occurred.
Rats with arthritis is divided at random: Normal group (negative control group), model negative control group (model mouse+physiological saline), 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 group and model negative control group subcutaneous injection equivalent physiological saline; The model positive controls is pressed 1.75mgkg
-1Dosage subcutaneous injection methotrexate (MTX), every day 1 time, successive administration.
After 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, record every the highest clinical score of animal, the average of getting them is average clinical score, the results are shown in 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 (mL) according to the difference of sufficient sole of the foot volume before and after modeling, Continuous Observation 21 days the results are shown in Table 3.
The swelling degree of the paw of each experimental group rat of table 3
As shown in Table 2, there is no in the pharmacological agent situation model negative control group joint scoring the highest (3.56 ± 0.76); After scorpion active polypeptide ADC-1 treatment, the symptom of rat significantly improves, and scoring is 1.73 ± 0.48, close with methotrexate for treatment group result (1.79 ± 0.69).Table 3 is measured the result demonstration of the swelling degree of the paw of each experimental group rat, and the rat paw edema degree of scorpion active polypeptide ADC-1 administration group is compared remarkable reduction with the model negative control group.Result shows, 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, minutes 2 groups, each 8 of every group of male and female.With scorpion active polypeptide ADC-1 lyophilized powder 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 physiological saline is done contrast.After administration, Continuous Observation is 7 days, estimate administration after medicine to the animal breath maincenter, cardio-pulmonary function, the impact of central nervous system.
Each treated animal is breathed after administration, motion, and heartbeat is normal, without significantly 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 has no obvious change, and without macroscopic pathology, administration group 3 treated animals and physiological saline group are without significant difference.Experimental result shows, scorpion active polypeptide ADC-1 under the dosage that surpasses 50 times of autoimmune disease animal model therapeutic doses to mouse without overt toxicity.
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
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 39
<212> PRT
＜213〉artificial sequence
<400> 1
Gln Ile His Thr Asn Gln Pro Cys Thr Ser Asn Asn Thr Arg Cys Arg
1 5 10 15
Thr Tyr Cys Ile Lys Val His Lys Ile Gln Ser Gly Lys Cys Met Asn
20 25 30
Ser Lys Cys Val Cys His Pro
35
<210> 2
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＜213〉scorpion
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cagatacaca caaaccagcc atgtacttca aataatacac gttgcagaac ttattgtatc 60
aaagtacata agatcaattc tggaaaatgt atgaattcaa aatgtgtttg tcatccttaa 117
<210> 3
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<212> DNA
＜213〉artificial sequence
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gtgaattcga tgacgatgac aagcagatac acacaaacca g 41
<210> 4
<211> 26
<212> DNA
＜213〉artificial sequence
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cagctcgagt taaggatgac aaacac 26
Claims (9)
1. a scorpion active polypeptide, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO:1.
2. the DNA molecular of the described scorpion active polypeptide of coding claim 1, is characterized in that, its nucleotide sequence is as shown in SEQ ID NO:2.
3. the carrier that has the described DNA molecular of claim 2.
4. carrier according to claim 3, is characterized in that, described carrier is pGEX-6p-1.
5. the preparation method of the described scorpion active polypeptide of claim 1, comprise the steps:
Step 1: the DNA molecular that obtains the aminoacid sequence shown in SEQ ID NO:1 of to encode;
Step 2: DNA molecular and expression vector that step 1 is obtained merge, and build 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.
6. preparation method according to claim 5, it is characterized in that, described step 1 be specially the to encode cDNA of aminoacid sequence shown in SEQ ID NO:1 is template, with the downstream primer amplification of upstream primer and the nucleotide sequence shown in SEQ ID NO:4 of the nucleotide sequence shown in SEQ ID NO:3.
7. the application of the described scorpion active polypeptide of claim 1 in preparation treatment or prevention potassium channel Kv1.3 relative disease medicine.
8. use according to claim 7, it is characterized in that, described potassium channel Kv1.3 relative disease is autoimmune disorder.
9. use according to claim 7, it is characterized in that, described autoimmune disorder is multiple sclerosis or rheumatoid arthritis.
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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 |
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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 (2)
| Title |
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| 小分子蛋白酶抑制剂及多肽毒素的研究回顾;戚正武;《科学通报》;20090930;第54卷(第18期);第61页 * |
| 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》.2009,第54卷(第18期),第2734-2745页. |
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