CN101422600A - Use of scorpion source immunoloregulation polypeptide in preparing medicine for treating multiple sclerosis disease - Google Patents

Abstract

The invention discloses an application of scorpion source activity polypeptide BmKTX in preparing medicaments for curing or preventing multiple sclerosis. The scorpion source activity polypeptide BmKTX is produced and reconstructed by the gene project. The polypeptide medicament is characterized by: (1) strong particularity; the IC50 value of the scorpion source activity polypeptide BmKTX function medicament target potassium channel Kv1.3 is 102pM, and the scorpion source activity polypeptide BmKTX function medicament is the existing known polypeptide medicament with strong activity in the international world; (2) obvious medicament effect; the animal experiments prove that the recombination of the BmKTX polypeptide can cure multiple sclerosis of rats effectively; and (3) small toxic side effects; in animal experiments, the scorpion source activity polypeptide BmKTX does not cause obvious toxicity and side effects on experimental animals. The invention is simple and feasible in method and convenient in operation.

Description

The application of scorpion source immunoloregulation polypeptide in the medicine of treatment multiple sclerosis

Technical field

The invention belongs to biological technical field, more specifically relate to the purposes of a kind of scorpion source activity polypeptide BmKTX in the medicine of treatment or prevention multiple sclerosis.

Background technology

Autoimmune disease (autoimmune disease) 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 the population of 5-8% to be subjected to the threat of about 40 various autoimmune diseases approximately.These diseases relate to one or more tissues of whole body and organ, have a strong impact on health and life.At autoimmune disease, 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 serious adverse, and all can only slow down the development speed of the state of an illness, non-radical curing of disease.In autoimmune disease, comprise rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (sle), Behcet disease, autoimmunity thyroid and sick type i diabetes etc. based on the cell-mediated autoimmune disease of T.This class disease is affecting millions of people's life, and at the pathogeny of this human refractory disease, researching and developing novel immunosuppressive drug has become one of field of global keen competition.

In recent years, with the T cell of disease association on potassium channel Kv1.3 be accredited as new target drone [J.Clin.Invest., 2003, the 111:1703 of treatment autoimmune disease; Trends in Pharmacological Sciences, 2004,25:280; Curr Pharm Des.2006,12 (18): 2199], for grabbing following huge pharmaceutical market, big in the world drugmaker and scientific research institution of university are just starting one to take turns with potassium channel Kv1.3 are being the new drug development upsurge of target now.The organic molecule medicine is because of the cheap first-selection that becomes research and development.Up to the present, a large amount of organic molecule drug candidates [Trends inPharmacological Sciences has been screened, has separated, has synthesized, transformed and identified in mechanisms such as Merck company, Australian Walter and Eliza Hall drug research technique center, U.S.'s University of California, 2004,25:280; Mol.Pharmacol., 2004,65:1364; Journal of Medicinal Chemistr, 2006,49 (4): 1433; Mol.Pharmacol., 2005,68:1254].These organic molecule drug effects are with source channels Kv1.1, Kv1.2, and Kv1.4, Kv1.5 isoreactivity are than the low 2-70 of Kv.1.3 passage doubly.The different potassium channel poor selectivity of this effect, potential toxicity seriously become the pathogenic shortcoming of small-molecule drug research and development, and this mainly is that the amino acid residue sequence in organic molecule drug candidate effect Kv1.1-Kv1.7 target zone is almost completely consistent.Distinctly with the organic molecule target spot be, the target region of polypeptide effect presents abundant sequence polymorphism, and this has just determined the polypeptide drugs research and development to be the inevitable course.

Under situation is obstructed in the research and development of targeting passage Kv1.3 small-molecule drug, in the world eye is focused on potassium channel Kv1.3 specificity zootoxin peptide inhibitor.The human difficult miscellaneous diseases of strategy treatment of Chinese traditional medicine " treating the poisonous disease with poisonous drugs " commonly used, venomous animal is first-selected as scorpion, Serpentis, Aranea, Bufo siccus, Scolopendra etc.Modern study has shown in the venom of these venomous animals and has been rich in the zootoxin galanin peptide, ion channel on these polypeptid specificity function cells films.Now, target-specific animal polypeptide phallotoxins has become the resource (Nat.Rev.Drug.Discov.2003 2:63) of globalization research and development/treatment relevant disease important drugs.When potassium channel Kv1.3 progressively is realized is in the effective drug targets process of treatment autoimmune disease, and the screening of regulating new drug based on the autoimmune of zootoxin galanin peptide just becomes an important research direction.In the natural toxin of various venomous animals, the inhibitor of the Kv1.3 passage of most of high-affinity all comes from scorpion source activity peptide (the Trends Pharmacol.Sci.2004 25:280 of scorpion source activity glandular secretion, Curr.Pharm.Des.2006 12:2199), have the structure general character of 3-4 in this class peptide molecule to disulfide bond.U.S.'s University of California and Thelma Hopkins hospital are being the lead drug [Curr Med Chem.2004,11:3041, Mol.Pharmacol., 2005,67 (4): 1369] of molecular skeleton with actinocongestin polypeptide ShK at cooperative research and development just.ShK is 35 residue polypeptide of 3 pairs of disulfide bond being separated to from sea anemone, is just modifying by the chemiluminescent polypeptide synthetic method in nearly 2 years and the analog of more than 0 kind of ShK polypeptide of Synthetic 2.In 2006, found that successfully ShK analog (ShK-L5) can treat rheumatic arthritis and type i diabetes (PNAS preferably, 2006,103:17414), Centre National de la Recherche Scientifique is in recent years with plain polypeptide OSK1 of passage Kv1.3 specificity scorpion source activity and AOSK1[Biochem.J., 2005,385:95; Mol.Pharmacol., 2006,69:354] be molecular skeleton, design and chemosynthesis 38 residue similar polypeptides of 3 pairs of disulfide bond of kind more than 30, quicken novel immunosuppressive polypeptide medicament research and development.

Compare with organic molecule, guide's polypeptide drugs of targeting potassium channel Kv1.3 have high specificity.IC50 value as the AOSK1 of Centre National de la Recherche Scientifique research and development be 3pM, and it is 16pM that the U.S. researches and develops ShK polypeptide IC50 value, and reports that at present the IC50 value of best organic molecule lead drug is 3nM.As seen, guide's polypeptide drugs have improved 1000 times than the specificity of organic molecule lead drug.Simultaneously, the selectivity of guide's polypeptide drugs also is greatly improved.Therefore, potential novel immunosuppressive polypeptide medicament research and development has become the new direction of the cell-mediated autoimmune disease of treatment T.

Summary of the invention

The objective of the invention is to be to provide a kind of scorpion source activity polypeptide BmKTX application in the medicine of preparation treatment or prevention multiple sclerosis.

Potassium channel Kv1.3 has been accredited as the target of prevention and the cell-mediated autoimmune disease of treatment T, and under the organic molecule medicament research and development seriously baffled situation, countries such as the U.S., France, Australia were were researching and developing the immunosuppressive polypeptide medicine at present.Main purpose of the present invention has been to find the application of a kind of scorpion source activity polypeptide in the medicine of conduct preparation treatment or prevention autoimmune disease.

To achieve these goals, the present invention adopts following technical measures:

Prepared genetic engineering scorpion source activity polypeptide BmKTX.

To contain scorpion source activity polypeptide BmKTX expression of gene plasmid, transformed into escherichia coli DE3 (available from Novagen company).To the escherichia coli IPTG (available from Merck company) that transforms.Collect thalline behind the inducing culture, the ultrasonic disruption antibacterial also obtains fusion rotein by the GST affinity chromatograph.The fusion rotein solution that collection obtains obtains reorganization scorpion source activity polypeptide BmKTX (Biochemistry 199736:13473) again through desalination and concentration, little enterokinase (EK) enzyme action, chromatographic isolation.

Identify the pharmacological activity of scorpion source activity polypeptide BmKTX by patch clamp technique to potassium channel Kv1.3.

But reorganization scorpion source activity polypeptide BmKTX specificity suppresses potassium channel Kv1.3 electric current, and its IC50 value is 100pM, but pharmacology data shows the target potassium channel Kv1.3 of scorpion source activity polypeptide BmKTX specific effect autoimmune disease.

The purposes of scorpion source activity polypeptide BmKTX in the medicine of conduct preparation treatment or prevention multiple sclerosis.

Choose inbred line female Wistar rats (age in 6-8 week, body weight 150 ± 10g), Cavia porcellus (300-400g is available from Wuhan University's Experimental Animal Center).Main agents: Fu Shi Freund's complete adjuvant (Gibcol/BRL), bacillus calmette-guerin vaccine, pertussis vaccine (Shanghai institute of Biological Products), Cavia porcellus MBP (Sigma).The preparation of GPSCH-CFA (full spinal cord homogenate-Fu Shi Freund's complete adjuvant mixed emulsion): after Cavia porcellus execution, take out spinal cord rapidly, with Ultrasonic Cell Disruptor (Sonics; Materials Inc America) makes 50% PBS homogenate, mixes with the Fu Shi Freund's complete adjuvant (bacillus calmette-guerin vaccine 10mg/ml) of equivalent, lashes 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 the Wistar rat lift, or intradermal injection about 1 * 10 simultaneously ¹⁰Pertussis vaccine.Weigh every day, observes nervous symptoms.Raise and 2 weeks experimental autoimmune encephalomyelitis promptly occurred.Then the experimental autoimmune encephalomyelitis rat is divided at random: 10 of model mouse+normal saline (model negative control group); 10 of model mouse+polypeptide drugs; Other establishes 10 of normal control groups (negative control group).The administration group is by 150 μ gkg ^-1The dosage subcutaneous injection, every morning 1 time; Model negative control group and negative control group subcutaneous injection equivalent normal saline; Successive administration.After the administration 21 days, observe rat and suffer from the experimental autoimmune encephalomyelitis situation.Standards of grading are 0 minute: do not have any clinical symptoms; 1 minute: afterbody tension force disappeared; 2 minutes: the back myasthenia of limbs; 3 minutes: hind leg was paralysed fully; 4 minutes: quadriplegia or moribund condition.

Experimental result shows: do not having under the Drug therapy situation model negative control group score the highest (35 minutes/10); After the polypeptide drugs BmKTX treatment, symptom of rats significantly improves, and must be divided into 14 fens/10, this shows, scorpion source activity BmKTX polypeptide can be treated multiple sclerosis effectively.

As seen, the present invention has following characteristics: (1) high specificity.The IC50 value of scorpion source activity polypeptide BmKTX drugs with function target potassium channel Kv1.3 is 102pM, is the active strong polypeptide drugs of having found in the world at present; (2) effect of drugs is remarkable.Zoopery shows that reorganization BmKTX polypeptide can effectively be treated multiple sclerosis.(3) toxic and side effects is little.In the zoopery, scorpion source activity polypeptide BmKTX does not cause tangible toxicity and side effect to laboratory animal.

Description of drawings

Fig. 1 analyzes sketch map for the electrophoresis detection of reorganization scorpion source activity polypeptide BmKTX and amalgamation and expression Protein G ST-BmKTX thereof

1 is that rBmKTX is without the inductive whole-cell protein of IPTG; 2 is that rBmKTX is through the inductive whole-cell protein of IPTG; 3 is the fusion rotein GST-BmKTX after the desalination and concentration by ultrafiltration; 4 is the product of fusion rotein GST-BmKTX behind the EK enzyme action; 5 is the rBmKTX polypeptide that obtains through the HPLC separation and purification.

Fig. 2 is reorganization scorpion source activity polypeptide BmKTX and the proteinic chromatographic separation and purification sketch map of GST

Fig. 3 is the inhibition sketch map of variable concentrations BmKTX polypeptide to potassium channel Kv1.3 electric current

Fig. 4 suppresses the concentration dependent sketch map of potassium channel Kv1.3 electric current for recombinant polypeptide BmKTX

The specific embodiment

Embodiment 1: expression and the purification of reorganization scorpion source activity polypeptide BmKTX

To contain scorpion source activity polypeptide BmKTX expression carrier plasmid pGEX-6p-1-BmKTX (the gene order structure that has been inserted coding BmKTX by the expression vector pGEX-6p-1 available from peace agate West Asia company forms Biochem.J.2000 346:805-809) transformed into escherichia coli DE3.To collecting thalline behind the escherichia coli IPTG inducing culture that transforms, the ultrasonic disruption antibacterial also obtains fusion rotein by the GST affinity chromatograph.The fusion rotein solution that collection obtains obtains reorganization scorpion source activity polypeptide BmKTX again through desalination and concentration, little enterokinase (EK) enzyme action, chromatographic isolation.

By high pressure liquid chromatography the BmKTX polypeptide is further separated, remove GST protein, obtain chromatographically pure BmKTX polypeptide (Fig. 2), the BmKTX polypeptide that different phase is obtained and carried out PAGE electrophoresis detection (Fig. 1) with gst fusion protein.

Embodiment 2: reorganization scorpion source activity polypeptide BmKTX is to the pharmacological activity analysis of potassium channel Kv1.3 target

The COS-7 cell is cultivated containing under 37 ℃ of the DMEM culture medium of 10% hyclone, the 5% CO2 condition, with potassium channel Kv1.3 recombiant plasmid Sofast ^TMThe transfection of transfection reagent box, transfectional cell selectivity on 0.8mg/ml Geneticin culture medium is cultivated.(Lambrecht Germany), measures and analyzes the pharmacological activity of reorganization scorpion source activity polypeptide BmKTX for EPC-10 dual pathways 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 by Pulse software.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 the cell membrane, carry out fast electric capacity and compensate (c-fast) automatically, a little behind the negative pressure rupture of membranes, carry out slow electric capacity and compensate (c-slow) automatically, under the command potential of-70mV, give the 10mV stride increases progressively, the 80ms step is wide depolarization impulse stimulation to+50mV from-60mV, observe current conditions, (China) filling system is realized accurately perfusion to the BmKTX polypeptide for INBIO Inc, Wuhan by MPS-2.After scorpion source activity polypeptide BmKTX dissolving, spray administration through DAD drug-supplying system (ALA), delivery tube is most advanced and sophisticated apart from writing down about cell 100 μ m.BmKTX is 102pM (Fig. 3 and Fig. 4) to the pharmacological activity of potassium channel Kv1.3.

Embodiment 3: the application of scorpion source activity polypeptide BmKTX in the medicine of conduct preparation treatment or prevention multiple sclerosis.

Choose inbred line female Wistar rats (age in 6-8 week, body weight 150 ± 10g), Cavia porcellus (300-400g is available from Wuhan University's Experimental Animal Center).Main agents: Fu Shi Freund's complete adjuvant (Gibcol/BRL), bacillus calmette-guerin vaccine, pertussis vaccine (Shanghai institute of Biological Products), Cavia porcellus MBP (Sigma).The preparation of GPSCH-CFA (full spinal cord homogenate-Fu Shi Freund's complete adjuvant mixed emulsion): after Cavia porcellus execution, take out spinal cord rapidly, with Ultrasonic Cell Disruptor (Sonics; MaterialsInc America) makes 50% PBS homogenate, mixes with the Fu Shi Freund's complete adjuvant (bacillus calmette-guerin vaccine 10mg/ml) of equivalent, lashes 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 the Wistar rat lift, or intradermal injection about 1 * 10 simultaneously ¹⁰Pertussis vaccine.Weigh every day, observes nervous symptoms.Raise and 2 weeks experimental autoimmune encephalomyelitis promptly occurred.Then the experimental autoimmune encephalomyelitis rat is divided at random: 10 of model mouse+normal saline (model negative control group); 10 of model mouse+polypeptide drugs; Other establishes 10 of normal control groups (negative control group).The administration group is by 150 μ gkg ^-1The dosage subcutaneous injection, every morning 1 time; Model negative control group and negative control group subcutaneous injection equivalent normal saline; Successive administration.After the administration 21 days, observe rat and suffer from the experimental autoimmune encephalomyelitis situation.Standards of grading are 0 minute: do not have any clinical symptoms; 1 minute: afterbody tension force disappeared; 2 minutes: the back myasthenia of limbs; 3 minutes: hind leg was paralysed fully; 4 minutes: quadriplegia or moribund condition.Marking result such as following table:

	The model negative control group	Negative control group	The BmKTX polypeptide
				Score	35/10	0/10	14/10

Experimental result shows: do not having under the Drug therapy situation model negative control group score the highest (35 minutes/10); After the polypeptide drugs BmKTX treatment, symptom of rats significantly improves, and must be divided into 14 fens/10, this shows, scorpion source activity BmKTX polypeptide can be treated multiple sclerosis effectively.

Claims (1)

1, the application of a kind of scorpion source activity polypeptide BmKTX in the medicine of preparation treatment or prevention multiple sclerosis.

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