CN1179975C - Scorpion peptide for treating arrhythmia and its preparing process and application - Google Patents

Abstract

The present invention discloses an antiarrhythmic scorpion peptide, a preparation method and application thereof. The amino acid sequence of the active peptide is GYIRGSNGCLWGNEGCNKECKGFGYCWTWGLACWCEGLTWKSESNTCG. The method for preparing the antiarrhythmic scorpion peptide comprises the procedures of designing a PCR primer, using PCR to amplify antiarrhythmic peptide gene segments in the cDNA library of buthus martensii scorpion venom, and using an expression vector to convert the gene segments into escherichia coli for expression. The antiarrhythmic scorpion peptide is characterized in that the designed PCR upstream primer contains the enzyme cutting sites of small intestine kinase, the antiarrhythmic peptide gene segments which are amplified by PCR are converted into escherichia coli BL21 to obtain recombinant escherichia coli BL21(pGEX/BmKIM) by restriction enzyme cutting after being cloned into an expression vector pGEX-5x-1, the recombinant escherichia coli BL21(pGEX/BmKIM) is purified by the affinity columns of glutathione transferase after being induced by IPTG, the obtained fusion protein is expressed by the enzyme cutting of the small intestine kinase, and soluble antiarrhythmic peptide is obtained after the fusion protein is desalted. The present invention has effective antiarrhythmic activity, and the obtained recombinant peptide is soluble and has high yield. Therefore, the antiarrhythmic scorpion peptide can be widely used for preparing antiarrhythmic peptide medicines.

Description

BmKIM and its production and application

Technical field

The present invention relates to a kind of BmKIM, and the method that produces BmKIM.Specifically, the present invention relates to a kind of BmKIM of efficient bio-active, adopt the method for genetically engineered and biological chemistry means generation BmKIM, the application of the BmKIM of generation in the preparation anti-arrhythmic.

Background technology

Buthus martensii Karscs is the widest class scorpion kind of China's distribution, because of having complicated composition and character, its scorpion venom produces multiple physiology, pharmacologically active, become the main source of scorpion class medicinal material, antitumor, treatment rheumatism, anti-epileptic and cardiovascular disorder are had important smelting treatment effect.But because the scorpion venom complicated component, many composition roles even opposite, and effective constituent often content is low, these have all limited the therapeutic action of scorpion venom undoubtedly.From scorpion venom, find out and separate or produce effective composition and then become very necessary by engineered method.The biochemical institute in Shenyang Pharmaceutical University and Shanghai all separates from scorpion venom and has obtained antiepilepsy peptide, yet, from scorpion venom, do not find as yet so far a kind of can the cardiopathic composition of treatment.Recently, (Zhang JH such as Chinese patent 00112016.6 and Zhang Jinghai, Hua ZC, Xu Z, Zheng WJ, Zhu DX, Prep BiochemBiotechnol 2001 Feb 31:49-57) found the gene of buthus martensii Karscs anti-neuroexcitation peptide and adopt the pET28a carrier in e. coli bl21, to express and produce anti-neuroexcitation peptide of scorpion, yet, make that the nervous excitation peptide of expressing generation must be a fusion recombinant protein that contains a plurality of additional set propylhomoserins (His) owing to do not contain the proteolytic enzyme restriction enzyme site that the Histidine in the expression vector and anti-neuroexcitation peptide are separated in the PCR primer of its design.These extra Histidines can influence the structure of recombinant peptide so that its relevant function.It is reported that this recombinant peptide only can make prolong 20% the latent period of mouse nervous excitation outbreak, and output is not high, and most of inclusion body that forms, soluble.The activity of the product anti-neuroexcitation effect that the method that promptly adopts Zhang Jinghai to describe is expressed is not high, and uses limited.

Summary of the invention

One object of the present invention is to provide a kind of BmKIM, has efficient bio-active

Another object of the present invention is to provide a kind of method of producing the efficient bio-active BmKIM, and the BmKIM that adopts method of the present invention to produce not only has the effect of anti-neuroexcitation, and tangible antiarrhythmic activity is arranged.

Of the present invention also have a purpose to be to provide the application of a kind of BmKIM in anti-arrhythmic.

A further object of the present invention is to provide a kind of application of BmKIM in anti-arrhythmic that produces with gene engineering method.

A kind of BmKIM is characterized in that, the aminoacid sequence of this bioactive peptide is as follows:

DGYIRGSNGCLWGNEGCNKECKGFGYCWTWGLACWCEGLTWKSESNTCG。

BmKIM provided by the invention and the mature amino acid sequence one of deriving from BmKIM cDNA to, and do not contain influential bioactive additional set propylhomoserin fragment and other aminoacid sequence.BmKIM is solvable state, does not form inclusion body, and circular dichroism spectrum shows that the natural Buthotoxin polypeptide of its secondary structure and other is similar, the function of promptly this Toplink actual response arrhythmia-resistant scorpion gene.Recombinant peptide provided by the invention not only has antiepileptic effect but also tangible antiarrhythmic effect is arranged.

A kind of method of producing BmKIM comprises design PCR primer, with the PCR BmKIM gene fragment that increases from scorpion of Buthus martensii venom cDNA storehouse, and is transformed into expression in escherichia coli by expression vector.It is characterized in that: the PCR primer of design contains small intestine kinases restriction enzyme site, with the BmKIM gene fragment clone of pcr amplification in the pGEX-5x-1 expression vector, be transformed into and obtain recombination bacillus coli BL21 (pGEX/BmKIM) in the e. coli bl21, after IPTG induces, cut the fusion rotein of expression with small intestine kinases enzyme, obtain soluble BmKIM through desalination again.

From scorpion venom gland cDNA library (the Xian-chun Zeng that makes up, Wen-Xin Li, Shun-Yi Zhu, Fang Peng, Toxicon 2000 38:893-899) screen the cDNA sequence similar to the anti-neuroexcitation peptide gene in, in deduced amino acid, there are three amino acid whose differences in its signal peptide district, other zone is identical, claims that this cDNA sequence is the BmKIM gene:

ATG?AAA?CTA?TTT?CTT?TTA?CTA?GTT?TTC?TTT?GCT?TCA?ATG?CTA?ATT

M K L F L L L V F F A S M L I

GAT?GGC?TTA?GTT?AAT?GCT

GGA?AGT?AAC?GGA

D G L V N A D G Y I R G S N G

TGC?TTA?TGG?GGA?AAT?GAA?GGT?TGC?AAT?AAA?GAA?TGC?AAA?GGC?TTC

C L W G N E G C N K E C K G F

GGT?TAT?TGC?TGG?ACC?TGG?GGA?CTT?GCA?TGC?TGG?TGT?GAA?GGC?CTT

G Y C W T W G L A C W C E G L

ACA?TGG?AAA GGC?AAA?AAG?TAA

T W K S E S N T C G end

Above-mentioned is the cDNA sequence of BmKIM, the part of underscore is the signal peptide district, dash area is that Buthotoxin polypeptide is processed in the sophisticated process in vivo according to passing through the cut part of regular meeting, the zone of adding some points is the design of primers district, adds the surplus matrix and shows the amino acid different with anti-neuroexcitation peptide of scorpion.According to BmKIM gene order design upstream, downstream primer, BmKIM gene skewer is gone into expression vector pGEX-5x-1 plasmid, make up recombinant expression vector, transformed into escherichia coli BL21 obtains recombination bacillus coli BL21 (pGEX/BmKIM).Induce through IPTG, express Thiadiazolidine isomerase-BmKIM fusion rotein, cut with small intestine kinases enzyme behind gsh affinity chromatography column purification, enzyme is cut product through SephadexG-50 purifying and desalination, obtains soluble and activated reorganization BmKIM.

A kind of method of production BmKIM of optimization is characterized in that:

The PCR upstream primer

A1,5 '-GCC GGATCCCC GATGACGATGACAAGGATGGATATATAAGA-3 ', this primer contain BamHI restriction enzyme digestion sites and small intestine kinases restriction enzyme site;

The PCR downstream primer

A2,5 '-GC CCTCGAGThis primer of TCAACCGCATGTATTACTTTCAG-3 ' contains the XohI restriction enzyme digestion sites.

Cut rear clone to pGEX-5x-1 through the pcr amplification products therefrom through BamHI and XohI enzyme with this primer, obtain recombination bacillus coli BL21 (pGEX/BmKIM) through conversion.The IPTG abduction delivering carries out enzyme with the small intestine kinases to the fusion rotein of expressing and cuts, and obtains the BmKIM of solubility.

Mature peptide section according to the BmKIM gene, promptly do not comprise the signal peptide section and add last three amino acid whose gene fragment design primers that can be cut man-hour in vivo, be BmKIM cDNA 64-79nt place design PCR upstream primer A1,5 '-GCC GGATCCCC GATGACGATGACAAGGATGGATATATAAGA-3 ' contains BamHI restriction enzyme digestion sites and small intestine kinases restriction enzyme site; According to BmKIM cDNA 226-246nt place design downstream primer A2,5 '-GCC CTCGAGTCAACCGCATGTATTACTTTCAG-3 ' contains the XohI restriction enzyme digestion sites.Owing to contain small intestine kinases restriction enzyme site gene order in the PCR upstream primer, contained small intestine kinases restriction enzyme site in this fusion rotein Thiadiazolidine isomerase-BmKIM that makes expression of recombinant e. coli obtain, thereby just cut by small intestine kinases enzyme and can separate Thiadiazolidine isomerase and BmKIM, to obtain not contain the BmKIM of additional amino acid.

A kind of method of production BmKIM of optimization also comprises:

1. with the Thiadiazolidine isomerase affinity column expressed protein is carried out purifying.

Recombination bacillus coli BL21 (pGEX/BmKIM) is through IPTG abduction delivering recombination fusion protein: Thiadiazolidine isomerase-BmKIM, utilize the Thiadiazolidine isomerase affinity column to carry out purifying, can effectively fusion rotein be separated with other foreign protein, fusion rotein hangs on the pillar.

2. small intestine kinases enzyme is cut the BmKIM that fusion rotein obtains having antiarrhythmic activity.

The fusion rotein that is hung on the pillar is directly cut through small intestine kinases enzyme, and the Thiadiazolidine isomerase in the fusion rotein is separated with BmKIM, and Thiadiazolidine isomerase is stayed on the pillar, and BmKIM flows out from affinity column.At last, enzyme is cut product obtains biologically active by Sephadex G-50 desalination BmKIM.

The application of BmKIM in the preparation anti-arrhythmic.

The application of BmKIM in the preparation anti-arrhythmic that utilizes gene engineering method to produce.

BmKIM provided by the invention and the BmKIM of producing by method of the present invention can be used to prepare antiarrhythmic drug.This BmKIM can with pharmaceutically acceptable carrier, for example; Vehicle such as glucose, lactose, N.F,USP MANNITOL, glycine, water etc.; Weighting agent such as starch, sucrose etc.; Lubricant such as talcum powder, calcium stearate, polyoxyethylene glycol etc.; Absorption enhancer such as Sodium desoxycholate, ox sulphur glycocholate, N-Methyl pyrrolidone, EDTA, Tween-80, SDS, Brij-78 etc.; Tackifier such as methylcellulose gum, polyacrylamide, hyaluronic acid sodium etc. or with other the treatment antiarrhythmic drug mix use.The BmKIM that the present invention produces can composition form by injection, eye conjunctiva absorb, snuffing is gone into, the mode of skin absorption, drop rectum with drug is used with aqua, fixing agent or other formulation.The BmKIM that the present invention produces can mix with one or more carriers, and the conventional production method according to pharmaceutical field is made into required formulation then.This pharmaceutical composition contains weight ratio can be the activeconstituents of 0.2%-97%.

Because the resulting different recombinant peptides of different production methods are also inevitable difference to some extent on function.In accordance with the present production process, resulting recombinant protein has following advantage and effect:

Do not contain the extra fragments of forming by a plurality of Hi s amino acid or other amino acid in A, the recombinant peptide, with the mature peptide of inferring by cDNA one to.

B, resulting recombinant peptide are soluble, non-inclusion body state and output height, and output is 2mg BmKIM/L culture.

C, prove that by the big white mouse epilepsy model resulting recombinant peptide on big white mouse is apparent, on the electroencephalogram, still on the electromicroscopic photograph of brain highly effective anti-epileptic (nervous excitation) effect is arranged all, can prolong the epileptic seizures time 47%.

D, the resulting recombinant peptide of the present invention not only have anti-epileptic (nervous excitation) to act on but also have fairly obvious antiarrhythmic effect, can increase the consumption 50%-100% that brings out the required napelline of irregular pulse.

Description of drawings

Fig. 1 is SDS-PAGE protein electrophorese figure.First classify the total protein that does not have the inductive recombination bacillus coli as, and second classifies the total protein of the recombination bacillus coli that IPTG induced as, and the 3rd classifies protein labeling as, be respectively 31,20,16,14,6.3,3.5KD α, the 4th classifies the fusion rotein Thiadiazolidine isomerase-BmKIM that obtains by Thiadiazolidine isomerase affinity chromatography column purification as, and the 5th classifies Thiadiazolidine isomerase as, the 6th classifies the fusion rotein after small intestine kinases enzyme is cut as, and the 7th classifies the BmKIM of purifying as.

Fig. 2 is the circular dichroism spectrogram of reorganization BmKIM and other two kinds of Buthotoxin polypeptides.BmKIM is a BmKIM, and AaHIT2 is a kind of α type Buthotoxin polypeptide, and CssII is a kind of β type Buthotoxin polypeptide.

Fig. 3 is the sodium channel current figure of full cell voltage patch clamp record.A is contrast, and B is the ventricle cellular sodium channel current behind the adding reorganization BmKIM.

Fig. 4 is the electroencephalogram of recombinant peptide effect epileptic rat.A figure is injected to insane dose electroencephalogram again behind the intracerebral injection recombinant peptide; B figure is injected to insane dose electroencephalogram again behind the intracerebral injection physiological saline

Fig. 5 is recombinant peptide effect epileptic rat hippocampus ultrastructure Electronic Speculum figure.A figure is injected to insane dose rat hippocampal ultrastructure Electronic Speculum figure again behind the intracerebral injection recombinant peptide; B figure is injected to insane dose rat brain electrograph again behind the intracerebral injection physiological saline.

Embodiment

Embodiment 1: expression, the enzyme of reorganization BmKIM are cut and purifying

With 1: 100 ratio inoculation arrhythmia-resistant scorpion peptide recombined colibacillus BL21 (pGEX/BmKIM), 37 ℃ were cultured to OD in containing the LB liquid nutrient medium of penbritin ₆₀₀0.8 in time, add IPTG (final concentration is 1.0mM) culture induced, and add the pH value to 8.5 that NaOH transfers substratum, then with culture 28 ℃ of cultivations 4 hours to carry out the expression of goal gene.50 times of cultures after concentrated the inducing, ultrasonic wave broken cell (80HZ, 30 seconds/time, to culture become limpid till), centrifugal 5 minutes of 12000rpm, gained supernatant join in the GST affinity chromatography glue that 26 ℃ of effects made fusion rotein (GST-BmKIM) fully combine with GST affinity chromatography glue in 1 hour behind the thorough mixing.Tris-Cl buffered soln (1.0mM with the EDTA that contains 50mM, pH8.0) wash GST affinity chromatography glue repeatedly and remove foreign protein, add small intestine kinase solution (24ug/mlGST affinity chromatography glue) then, effect is 10 minutes behind the mixing, and the fusion rotein Thiadiazolidine isomerase-BmKIM that hangs on the affinity column is directly cut to separate GST and reorganization BmKIM.Add and the isopyknic double distilled water of GST affinity chromatography glue in GST affinity chromatography glue, collection effusive liquid from GST affinity chromatography glue also detects the yield of reorganization BmKIM by 15% SDS-PAGE protein electrophorese, see accompanying drawing 1.Contain reorganization BmKIM solution through Sephadex G-50 desalination and remove the GST that may sneak on a small quantity with what collect.The mechanism of gel-filtration and condition: 100ml Sephadex G-50, use the double distilled water wash-out, flow velocity is 0.4ml/ minute.Preserved by the content of reorganization BmKIM in the 15%SDS-PAGE protein electrophorese detection collection liquid and with its lyophilize.

Embodiment 2: reorganization BmKIM amino acid analysis

Hydrolysis reorganization BmKIM according to a conventional method, with the component of 121-MB Beckman amino acidanalyser according to the methods analyst peptide of analysis of amino acid, hydrolytic process: reagent A: in the vacuum, 6N HCl handled 24 hours for 110 ℃.Reagent B:4N methylsulfonic acid, 0.2% 3-(2-Padil) indoles, carried out 24,48,72 hours in a vacuum by 110 ℃.Sample is dissolved in the solution that 1.5ml contains 40%n-propyl alcohol and 0.1% trifluoroacetic acid, and every pipe packing 0.1ml dries up with drying nitrogen, add reagent A, B after, container seals in a vacuum, collects the hydrolysis content.The result is as follows:

The shared mole of amino acid %

Aspartic acid (Asp+Asn) 11.4

Threonine 5.0

Serine 6.5

L-glutamic acid (Glu+Gln) 6.6

Proline(Pro) 1.8

Glycine 17.8

L-Ala 3.5

1/2 halfcystine 13.1

Xie Ansuan 0.0

Methionine(Met) 0.0

Isoleucine 3.4

Junket amino 6.5

Phenylalanine 1.6

Methionin 7.8

Histidine 0.0

Tryptophane 8.3

Arginine 1.8

Shell propylhomoserin 4.9

Embodiment 3: reorganization BmKIM N-terminal determined amino acid sequence

Sample uses Applied Biosystems 476A sequenator to carry out Edman to decompose, the PTH amino acid that obtains with the analysis of AppliedBiosystems Model120APTH-analyser.The result shows that ten amino-acid sequences of N-terminal are " DGYIRGSNGC ".

Embodiment 4: the circular dichroism spectrum of reorganization BmKIM is measured

The sample of getting 0.1-0.3mg/ml is put into the quartz specimen groove of 2mm,, carries out circular dichroism spectrum in the 250-180nm wavelength region and measures at 25 ℃ with the Jasco-715 chromatographic instrument.Accompanying drawing 2 shows that the secondary structure of the BmKIM of recombinating is similar to the secondary structure of other Buthotoxin polypeptide.

Embodiment 5: the active detection of reorganization BmKIM peptide

Detect with the method for full cell patch pincers activity recombinant peptide.Separating myocardium cell at first: at 37 ℃ of cardiac perfusions, treat the heart back adding 0.2mM Ca that stops to beat to rabbit with no calcium tyrode's solution ²⁺, 0.04% collagenase I perfusion 8 minutes.Ventricle is shredded, in fresh Tai Shi solution incubation 5-10 minute, add 0.05% bSA and CaCl ₂To final concentration be 1.0mM.Isolating ventricular muscle cell suspension is sucked in the sample cell of patch clamp, the outer liquid (mM) of patch clamp operation: NaCl 30, choline chloride 60 110, and KCl5.4, CaCl2 0.1, and MgCl2 1.0, and NaH2PO4 0.33, and HEPES 10 usefulness NaOH transfer pH to 7.3.Interior liquid (mM): CsCl 120, and CaCl2 1.0, and MgCl2 5.0, and Na2ATP 5.0, and EGTA 11, and HEPES 10, and glucose 11 is transferred pH to 7.3 with CsOH.Clamp down on voltage and be-80mV, test voltage be-70mV-+30mV, and it is 5mv that the step increases, and 15-20 ℃ is carried out.Application EPC-9 amplifier and Pulse/Pulsefit software (HEKAelektronik, Germany).Voltage stimulates ventricular muscle cell also to write down sodium channel current, adds the reorganization BmKIM then, writes down sodium channel current again, if the obvious minimizing of sodium channel current then represent that this recombinant peptide has activity, sees accompanying drawing 3.

Embodiment 6: the anti-epileptic experiment

Get 12 of SD big white mouse, be divided into control group and experimental group at random, 6 every group.Weigh abdominal injection vetanarcol (45mg/Kg) anesthesia.The reorganization BmKIM of purifying is through the administration of rat encephalic hippocampus, and dosage is the 30ug/Kg body weight, and blank is a physiological saline.2 hours after rats by intraperitoneal injection to insane dose of pentetrazole 50mg/Kg body weight, observes rat shape be state and write down electroencephalogram.After 24 hours, punching, brain perfusion stationary liquid: the phosphoric acid buffer that contains 2% Paraformaldehyde 96,0.1% glutaraldehyde.Take out the hippocampal tissue electron microscopic observation.The result shows, the time ratio control group of having injected one group of rat outbreak epilepsy of recombinant peptide has prolonged 47%, and the anomaly peak in the electroencephalogram obviously reduces, and electromicroscopic photograph shows also that cerebral hippocampal is organized and also obtained protection and see accompanying drawing 4,5.

Embodiment 7: the anti-arrhythmia test

Napelline is a kind of toxin that causes irregular pulse and can be till death, also is a kind of means of manufacturing irregular pulse model commonly used.By experiment one, the check BmKIM is anti-to change the ability that napelline acts on till death; By experiment two, the anti-napelline of check BmKIM institute is to ARR efficient.Experiment one: get 12 of small white mouses, be divided into control group and experimental group at random, 6 every group.Weigh experimental group tail vein injection reorganization BmKIM 5ug/20g small white mouse 0.2ml, control group tail vein injection 0.2ml physiological saline.After one hour,, observe the reaction of small white mouse to all mice by intraperitoneal injection napelline 20ug/20g small white mouses.Under the room temperature, in 10 minutes, control group can be spat out white foams and dead, though the struggle situation also can appear in experimental group, finally all can survive.Show that recombinant peptide has the effect of obvious anti-napelline.Experiment two: get 12 of big white mouse, be divided into control group and experimental group at random, 6 every group.Weigh, the reorganization BmKIM of purifying is through rat leg intravenously administrable, and dosage is the 50ug/kg body weight, and blank is a physiological saline.1 hour after rat jugular vein perfusion napelline caused the irregular pulse animal model in 4ug/ minute.Observe electrocardiogram(ECG, recording room is (VE) early, chamber speed (VT), and chamber (VF) time of occurrence that declines, thus calculate required napelline consumption, test-results is as follows:

Napelline consumption (ug/Kg rat)

Medicament chamber early chamber speed chamber declines

Physiological saline 38 ± 4 67 ± 5 90 ± 10

BmKIM 75 ± 5* 100 ± 8* 148 ± 14*

* P＜0.05vs physiological saline

Compare with control group, the napelline consumption required to rat ventricular obviously increases, and illustrates that the reorganization BmKIM has obvious antiarrhythmic effect.

Embodiment 8

Ampulla: activeconstituents 2mg

NaCl 9mg

Preparation method: activeconstituents and sodium-chlor are dissolved in 1 liter of injection water, filter gained solution, in the ampoule of under aseptic condition, packing into.

Embodiment 9

Nasal spray: activeconstituents 80mg

NaCl 8mg

EDTA 1mg

Borate buffer (pH6.5) 10mg

Spheron MD 30/70 10mg

Double distilled water is settled to 2ml

The preparation method: a kind of composition of each adding in the double distilled water of proper volume until dissolving fully, and then adds another kind of composition.After being settled to 2ml, this solution is filtered on sterilizing filter, separate in the bottle of packing into and according to suitable dosage.

Embodiment 10

Suppository: activeconstituents 10mg

Cholesterol 2%

Semi-synthetic fatty acid ester 1.5g

Double distilled water 10%

The preparation method: recombinant peptide after cholesterol absorption, with the semi-synthetic fatty acid ester 1.5g of fused below 70 ℃ mixing, is made suppository with an amount of dissolved in distilled water.

Embodiment 11

Eye drops: activeconstituents 4mg

EDTA 0.5mg

Sodium desoxycholate 1mg

Hyaluronic acid sodium 1mg

Nipagin A 0.03mg

Borate buffer solution (pH7.4) 5mg

Preparation method: with borate buffer lytic activity composition, add dissolved EDTA and Sodium desoxycholate, constantly stir adding hyaluronic acid sodium and nipagin A down, fully mixing.

Embodiment 12

Liposomal formulation: activeconstituents 10mg

Yelkin TTS 240mg

Cholesterol 120mg

Double hexadecyl acid ester 18mg

The preparation method: with Z ether dissolving cholesterol, Yelkin TTS, and add double hexadecyl acid ester, add activeconstituents again, supersound process (strength of current is 0.1-0.5A) makes it to become stable w/o type emulsion.Remove Z ether at 20 ℃ of-25 ℃ of following reduction vaporizations.Add 5ml distilled water and continue the Z ether that reduction vaporization is removed remnants.Promptly make electronegative liposome turbid liquor.

Claims (4)

1, a kind of BmKIM is characterized in that, the aminoacid sequence of this bioactive peptide is as follows:

DGYIRGSNGCLWGNEGCNKECKGFGYCWTWGLACWCEGLTWKSESNTCG。

2, a kind of preparation method who realizes the BmKIM of claim 1, comprise the following steps: to design the PCR primer, with the PCR antiarrhythmia peptide gene fragment that from scorpion of Buthus martensii venom cDNA storehouse, increases, and be transformed into expression in escherichia coli by expression vector, the PCR upstream primer of design contains small intestine kinases restriction enzyme site, antiarrhythmia peptide gene fragment process digestion with restriction enzyme with pcr amplification, be cloned in the pGEX-5x-1 expression vector, be transformed into and obtain recombination bacillus coli BL21 (pGEX/BmKIM) in the e. coli bl21, after IPTG induces, through Thiadiazolidine isomerase affinity chromatography column purification, cut the fusion rotein of expressing gained with small intestine kinases enzyme, and desalination and obtain soluble antiarrhythmia peptide.

3, the preparation method of BmKIM according to claim 2 is characterized in that: the PCR upstream primer

A1，5’-GCCGGATCCCCGATGACGATGACAAGGATGGATATATAAGA-3’；

The PCR downstream primer

A2，5’-GCCCTCGAGTCAACCGCATGTATTACTTTCAG-3’。

4, the application of the described BmKIM of claim 1 in the preparation anti-arrhythmic.

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