CN101392252B - Lychas mucronatus Kv1.3 blocker gene, preparation method and application - Google Patents

Abstract

The invention discloses a gene of a Lychas mucronatus Kv1.3 blocking agent Lm8, a preparation method and an application thereof. The method comprises the steps: firstly, a cDNA library of poison glands of the Lychas mucronatus is constructed; a positive clone of the gene of the Lychas mucronatus Kv1.3 blocking agent Lm8 is selected from the cDNA library of the poison glands of the Lychas mucronatus; sequencing is carried out and a sequence analysis is carried out for analyzing coding characteristics of the Lychas mucronatus Kv1.3 blocking agent Lm8 to determine the amino acid sequence of the blocking agent Lm8; a genetic engineering method is adopted for constructing a recombinant Escherichia coli of the Lychas mucronatus Kv1.3 blocking agent Lm8 to express and purify a recombinant Lm8 polypeptide with high purity rate; and a patch clamp verifies the function of the recombinant Lm8 polypeptide for high-effectively blocking a voltage gate mKv1.3. The polypeptide of the Lychas mucronatus Kv1.3 blocking agent can be applied to the preparation of drugs for the treatment or prevention of autoimmune diseases.

Description

A kind of Lychas mucronatus Kv1.3 blocker gene and preparation method and application

Technical field

The invention belongs to biological technical field, the present invention relates to a kind of Lychas mucronatus Kv1.3 blocker gene, the separation method of this gene and this Lychas mucronatus Kv1.3 blocker of production.Specifically, the present invention relates to a kind of separation of Lychas mucronatus Kv1.3 blocker gene; Relate to a kind of Lychas mucronatus Kv1.3 blocker recombination bacillus coli and construction process thereof; The preparation method who also relates to a kind of Lychas mucronatus Kv1.3 blocker also relates to the purposes of Kv1.3 blocker in autoimmune disease simultaneously.

Background technology

Scorpion (scorpion) is a kind of traditional rare Chinese medicine of China, just puts down in writing the medicinal history of scorpion before more than 2000 year.Ming Dynasty's Compendium of Material Medica more write up scorpion cures mainly multiple diseases such as " all urticarias, apoplexy, hemiplegy hemiplegia, facial hemiparalysis, language are puckery, brothers take out sincere; the pediatric epilepsy scared tetany ", have antitumor, desinsection; Antibacterial and antibiotic effect, its effect is mainly derived from scorpion tail poison gland excretory venom.The staple of venom is one type and has a multiple bioactive micromolecule polypeptide by what 10-100 amino acid was formed; They can selectivity, the membranin on the specific and cytolemma interacts; Thereby change cell to the penetrating ability of ionic, regulate various kinds of cell metabolic process and various physiological processes such as emiocytosis, hormonal action and signal transduction such as cell volume, pH, membrane potential, irritability.Therefore, scorpion toxin has important theoretical research meaning and application and development value.But because the scorpion venom complicated component, structural similitude is difficult to separate, many composition roles even opposite, and effective constituent often content is low, these have all limited the research and the application of scorpion venom undoubtedly.The physiological function of necessary employing modern molecular biology technique research scorpion venom monomer component.

Ionic channel (ion channel) is the special hydrophilic protein matter microchannel of a type on the cytolemma; Because the generation of the function of ionic channel, textural anomaly and numerous disease and develop relevantly, so the correlative study of ionic channel has become one of emphasis of various countries' scientists study.Potassium-channel is an ionic channel important on the cytolemma; It is the only way which must be passed of ion turnover cell; Adjusting for cell function has a very important role; And because its dysfunction can cause multiple neuropsychiatric disease, so the structure of potassium-channel and functional study also are the forward position focuses of present molecular neurobiology.In recent years, valtage-gated potassium-channel Kv1.3 has become an effective target of treating numerous diseases.Research to potassium channel Kv1.3 shows; The activation of Kv1.3 control human T-lymphocyte; Narrow spectrum Kv1.3 inhibitors of ion channels will exert an influence to the lymphocytic activation of T; This type suppressor factor becomes new immunosuppressive factor probably, can be used for preventing and treating graft-rejection and treatment autoimmune disease.

The patent that relates to scorpion venom gland toxin gene at present has 7: the 1. application of Scorpio toxin BmKAS in pharmacy; This invention relates to the application of Scorpio toxin BmKAS in the anti-periphery injury of preparation medicine, and the application of Scorpio toxin BmKAS in the anti-hyperalgesic of preparation; 2. a kind of recombinant baculovirus that contains divalent insect-resistant gene, this invention relate to Scorpio insect-specific neurotoxin gene BmKIT and a kind of chitinase genes of insects (Chi), obtain to contain the recombinant baculovirus AcNPV-BmKIT-Chi of divalent insect-resistant gene; 3. arrhythmia-resistant scorpion peptide recombined colibacillus and construction process thereof, this invention has related to a kind of BmKIM BmKIM gene recombined escherichia coli, is used for the recombinant peptide that mass production is solvable, have antiarrhythmic activity; 4. BmKIM, this disclosure of the Invention a kind of BmKIM BmKIM, the resulting reorganization of the present invention BmKIM polypeptide has efficient antiarrhythmic activity, and solubility is good, output is high; 5. a kind of scorpion chloride channel neurotoxin gene-rBmKCTa of synthetic; This patent relates to chloride channel neurotoxin BmKCT gene; The rBmK CTa of the improvement of employing genetically engineered gained is inhibited to neurogliocyte, can be used for preparing through suppressing the medicine of the treatable disease of neurogliocyte; 6. a kind of purposes of transitional scorpion toxin BmK abT; The purposes of the transitional scorpion toxin BmK abT that the invention discloses; It can be used as the sodium channel modulator of a uniqueness; The research sodium channel is formed, the probe of 26S Proteasome Structure and Function; The modulator and the medicine of adjustable and treatment sodium channel relative disease can prepare the modulator or the medicine of regulating or treating hyperkalemic paralysis, congenital myotonia and other skeletal muscle disease, the 3rd type long QT interval disease (LQT3), primary ventricular fibrillation and other heart diseases; 7. a kind of recombinant scorpion toxin and solubility expression and purification process, this invention relate to a kind of recombinant scorpion toxin rBmKIT and solubility expression and purification process.

Although 1. 2. 3. 4. 5. 6. 7. patent all be content about the Scorpio toxin; The scorpion toxin gene BmKAS that patent relates to, BmKIT, BmKIM, BmKCT, BmKabT and the present invention obtain comes from Lychas mucronatus (Lychas mucronatus; Lm) the Lm8 gene is diverse toxin gene, and 1. 2. 3. 4. 5. 6. 7. patented invention all do not relate to the function of potassium-channel blocker.

Summary of the invention

One object of the present invention is to provide a kind of Lychas mucronatus Kv1.3 blocker gene, and this gene is little, easy handling.

Another object of the present invention also is to provide a kind of construction process of Lychas mucronatus Kv1.3 blocker recombination bacillus coli, and method is easy, and is safe, low production cost, and purifying is simple and product purity is high.

The present invention has a purpose to provide a kind of efficient bio-active of the Lychas mucronatus Kv1.3 blocker of producing with gene engineering method, the application of Lychas mucronatus Kv1.3 blocker in the medicine of preparation treatment or prevention autoimmunization disease again.

To achieve these goals, of the present invention being contemplated that: 1. make up a high quality Lychas mucronatus poison gland tissue cDNA library; 2. be the basis with above-mentioned 1. poison gland tissue cDNA library, screened clone's 8 (numberings) through the random sequencing method, sequential analysis is a kind of potassium-channel toxin gene; Called after Lm8, the preservation of this bacterium, depositary institution: Chinese typical culture collection center; Preservation address: China. Wuhan. Wuhan University; Preservation date: on August 29th, 2007, deposit number: CCTCC NO:M207131, classification name: Escherichia coli DH5 α/Lm8/pSPORT1; 3. made up the genetic engineering bacterium of expressing and producing reorganization scorpion toxin Lm8; Depositary institution: Chinese typical culture collection center; Address: China. Wuhan. Wuhan University; Preservation date: on August 29th, 2007, deposit number: CCTCCNO:M207132, classification name: Escherichia coli Rossetta ^TM(DE3)/Lm8/pGEX-6p-1; 4. adopt engineered method, obtained highly purified reorganization scorpion toxin Lm8, purity reaches 95%; 5. through the patch clamp method, measured the reorganization scorpion toxin Lm8 that 4. obtains efficient blocking-up effectiveness to voltage gated k+ channel blocker Kv1.3, it half imitates that to suppress dosage IC50 be 26.40 ± 1.62nM.Lychas mucronatus toxin Lm8 has significant application value in the medicine of preparation treatment or prevention autoimmune disease.

One object of the present invention is to provide a kind of Lychas mucronatus Kv1.3 blocker gene.At first make up high quality Lychas mucronatus poison gland tissue cDNA library.Comprise that its poison gland is used for the separation of total RNA; The scorpion tail gland of getting 500mg is ground into fine powder in liquid nitrogen, through the total RNA of scorpion venom gland of Trizol method preparation; The total RNA of the scorpion venom gland of preparation adopts its quality of denaturing formaldehyde detected through gel electrophoresis.

Be 3mg through the total RNA amount of the scorpion venom gland of method for preparing, adopt PolyA Tract mRNA separation system (available from U.S. Promega) to separate and purified mRNA.The scorpion venom gland mRNA that ultraviolet spectrophotometer is measured its acquisition measures about 10 μ g; With 5 μ gmRNA as initial amount; First chain and second chain that carry out cDNA are synthetic; Continue being connected of double-stranded cDNA and SalI adapter, NotI digests double-stranded cDNA, and excessive SalI adapter and the enzyme removed in the double-stranded cDNA molecule are cut small segment; Double-stranded cDNA is connected and conversion the steps such as evaluation of poison gland cell cdna library with pSPORT1 carrier (available from American I nvitrogen).It is 1.5*10 that the result has obtained an abundance ⁶Individual clone's/μ gcDNA.The positive insertion rate in the scorpion venom gland cDNA library of adopting this method to make up reaches more than 95%, and the toxin gene fraction of coverage of Lychas mucronatus reaches more than 95%.

From the Lychas mucronatus poison gland cell cdna library that builds, select 100 clone's at random, send company's order-checking.Sequential analysis shows that clone's 8 is a new Lychas mucronatus potassium-channel toxin gene, called after Lm8.A kind of isolated polypeptide gene, its sequence are the nucleotide sequence shown in the SEQ ID NO:1: ggaaggaaaatgaacaaagtttgctttgtcgtcgttcttgttctcttcgtggctct ggctgcatatgtgtcacctatcgaaggtgtaccaacaggaggatgcccactttcgg attcgctgtgtgccaaatattgcaagtcccacaaatttggcaaaaccggaagatgc accggaccaaacaagatgaaatgtaaatgtctcgtgtaataaaatgtaagcgaata aagatttgtgaatgaaaaaaaaaaaaa (Fig. 1).

Another object of the present invention also is to provide a kind of Lychas mucronatus Kv1.3 blocker polypeptide.A kind of isolating protein, its sequence are the aminoacid sequence shown in the SEQ ID NO:2.65 amino-acid residues of the prosoma organization form coding of Lm8 (SEQ ID NO:2); Form by two parts; Be signal peptide (25 residues) and mature peptide (40 residues): MNKVCFVVVLVLFVALAAYVSPIEGVPTGGCPLSDSLCAKYGKSHKFGKTGRCTGP NKMKCKCLV (SEQ ID NO:2); Signal peptide sequence is preceding 25 amino-acid residue MNKVCFVVVLVLFVALAAYVSPIEG, and its mature peptide is made up of 40 amino acid: VPTGGCPLSDSLCAKYCKSHKFGKTGRCTGPNKMKCKCLV (Fig. 1).

Another object of the present invention is to provide a kind of construction process (synoptic diagram that makes up recombinant expression vector is seen Fig. 2) of Lychas mucronatus Kv1.3 blocker recombination bacillus coli.Comprise design and synthetic PCR upstream primer (8-FP:5-GCGGGATCCGATGACGATGACAAGGTACCAACAGGAGGATGC-3) and downstream primer (8-RP:5-GCCCTCGAGTTACACGAGACATTTACA-3); With from Lychas mucronatus poison gland cDNA storehouse, the increase mature peptide encode fragment of Kv1.3 blocker gene of round pcr, and be transformed into recipient bacterium intestinal bacteria Rossetta through expression vector ^TM(DE3) (available from U.S. NOVAGEN).It is characterized in that; The PCR primer of design contains small intestine kinases restriction enzyme site (gatgacgatgacaag:DDDDK); The Kv1.3 blocker mature peptide encode fragment of pcr amplification is cut through restriction enzyme (BamHI and XhoI) enzyme; Reclaim behind the purifying and be connected with pGEX-6p-1 (available from the U.S. Novagen) plasmid that the XhoI enzyme is cut through BamHI; Connect among the product transformed into escherichia coli competence DH5a (available from China typical culture collection center), PCR method and double digestion method are identified positive colony (Fig. 3 and Fig. 4).Further order-checking conclusive evidence of positive colony Kv1.3 blocker mature peptide encode fragment is cloned in the expression vector.Extract the correct sub-plasmid Lm8/pGEX-6p-1 of recombinant clone of reorganization order-checking, be transformed into intestinal bacteria Rossetta ^TM(DE3) obtain recombination bacillus coli Rossetta in ^TM(DE3)/and Lm8/pGEX-6p-1, be used to produce soluble and activated reorganization Lychas mucronatus Kv1.3 blocker polypeptide.

Another object of the present invention is to provide a kind of gene engineering method of producing Lychas mucronatus Kv1.3 blocker polypeptide.With recombination bacillus coli Rossetta ^TM(DE3)/Lm8/pGEX-6p-1 induces through the IPTG of 0.1mM; High expression level Thiadiazolidine isomerase-Lm8 fusion rotein (GST-Lm8) (Fig. 5); Obtained highly purified fusion rotein GST-Lm8 (Fig. 5) through gsh affinity chromatography column purification, cut spend the night (Fig. 5) with 28 ℃ of enzymes of small intestine kinases after the centrifugal desalination of ultra worry, enzyme is cut product and through high-efficient liquid phase chromatogram HPLC Lm8 and GST branch is opened (Fig. 6); The manual Lm8 protein peak of collecting; Freezing freeze-drying (40 ℃) has obtained the good reorganization Lychas mucronatus Kv1.3 blocker polypeptide Lm8 of high purity and solubility, and final yield reaches 1mg/L.Mass spectroscopy reorganization apicule wolf Kv1.3 blocker polypeptide Lm8 and expection molecular weight size consistent (Fig. 7).In accordance with the present production process, resulting recombinant peptide has following characteristics: do not contain unnecessary amino-acid residue in the recombinant peptide, and consistent with the mature peptide of inferring by cDNA; Resulting recombinant peptide is soluble non-inclusion body state, and output is high, and output is the 1mg/L culture.

A further object of the invention provides the efficient restraining effect of a kind of Lychas mucronatus Kv1.3 blocker of producing with gene engineering method to voltage gated k+ channel blocker Kv1.3.Comprise the structure (Fig. 8) of stablizing overexpression people voltage gated k+ channel blocker Kv1.3 green fluorescent protein clone Cos7/mKv1.3; Adopt the method for full cell patch pincers; Find that reorganization Lychas mucronatus toxin polypeptide Lm8 has tangible blocking effect to being expressed in Cos7 clone Kv1.3 electric current, and have dose-dependently (Fig. 9).Through concentration rely on curve determination reorganization Lychas mucronatus toxin polypeptide Lm8 be 26.40 ± 1.62nM (Figure 10) to the inhibition concentration IC50 of partly imitating of Kv1.3 electric current.The Lychas mucronatus Kv1.3 blocker that adopts method of the present invention to produce has the effect of the valtage-gated potassium-channel Kv1.3 of efficient inhibition, in the medicine of preparation treatment or prevention autoimmune disease (like asthma, muscle gelling, lupus erythematosus, scleroderma, rheumatoid arthritis etc.), has important use to be worth.

The present invention is a kind of Lychas mucronatus Kv1.3 blocker gene and preparation method and application.Obtained a brand-new potassium ion voltage-gated channel Kv1.3 blocker gene.And, having made up a kind of Lychas mucronatus Kv1.3 blocker recombination bacillus coli, its method is easy, and is safe, low production cost, purifying is simple and product purity is high.Genetically engineered is produced the Kv1.3 blocker on a large scale, is used to prevent and treat autoimmune disease.

Description of drawings

Fig. 1 Lychas mucronatus Lm8 gene and amino acid thereof.

The corresponding amino sequence of cDNA sequence below for inferring; Signal peptide amino acid is with single underscore mark; Double underline partly is the PolyA tailing signal.

Fig. 2 makes up recombinant expression vector Lm8-pGEX-6p-1 synoptic diagram.

Extracting pGEX-6p-1 plasmid; Pcr amplification Kv1.3 blocker gene; BamHI and XhoI enzyme are cut the pcr amplification product of pGEX-6p-1 and Kv1.3 blocker gene; The plasmid that enzyme is cut is connected with segmental, obtains the sub-plasmid Lm8-pGEX-6p-1 of recombinant clone.

Fig. 3 PCR method is identified reorganization Lm8-pGEX-6p-1 positive colony.

M is DNA Marker; 1 is the PCR negative control; 2 is the PCR positive control; The positive transformant of 3-5.

Fig. 4 enzyme blanking method is identified reorganization Lm8-pGEX-6p-1 positive colony.

M is DNA Marker; 1 is the positive control of Lm8 gene fragment; 2 is BamHI and XhoI double digestion recombinant plasmid Lm8-pGEX-6p-1; 3 is BamHI single endonuclease digestion recombinant plasmid Lm8-pGEX-6p-1.

The recombinate SDS-PAGE of expression and purification of GST-Lm8, Lm8 of Fig. 5.

A:1 is standard protein molecular weight Marker; 2 is recombination bacillus coli Rossetta ^TM(DE3)/Lm8/pGEX-6p-1 induces without IPTG; 3 is recombination bacillus coli Rossetta ^TM(DE3)/Lm8/pGEX-6p-1 induces through IPTG; 4 is recombination bacillus coli Rossetta ^TM(DE3)/pGEX-6p-1 induces without IPTG; 5 is recombination bacillus coli Rossetta ^TM(DE3)/pGEX-6p-1 induces through IPTG.

B:1 is standard protein molecular weight Marker; 2 for to have obtained highly purified fusion rotein GST-Lm8 through gsh affinity chromatography column purification; 3 is GST albumen; 4 is the Lm8 albumen of HPLC purifying; 5 cut the product that spends the night for 28 ℃ of enzymes of small intestine kinases.

Fig. 6 HPLC separates GST and Lm8 albumen.

The Lm8 albumen of arrow indication purifying.

Fig. 7 proteic mass spectroscopy of Lm8 of recombinating.

Fig. 8 stablizes overexpression people voltage gated k+ channel blocker Kv1.3 green fluorescent protein clone Cos7/mKv1.3.

Fig. 9 recombinates Lm8 albumen to the concentration of Kv1.3 electric current blocking-up effectiveness.

Figure 10 Lm8 albumen of recombinating relies on curve to the concentration of Kv1.3 electric current.

Embodiment

Following embodiment can make those skilled in the art more fully understand the present invention, but does not limit the present invention in any way.

Embodiment 1: the scorpion tail gland that 500mg is 1. got in the extraction of the total RNA of scorpion venom gland (Trizol LS single stage method: Trizol LS is available from beautiful Invitrogen) is ground into fine powder in liquid nitrogen; Add 10ml TRIZOL reagent mixing; Room temperature (20-25 ℃, below identical) was placed 5 minutes; 2. add the 2ml chloroform then and mixed 15 seconds, room temperature was placed 2-3 minute, centrifugal 15 minutes of 4 ℃ of following 12000g; 3. the 1 times of volume Virahol of addition of fetching water, room temperature was placed 10 minutes, centrifugal 10 minutes of 4 ℃ of following 12000g the RNA deposition; 4. deposition is used 5ml75% washing with alcohol, centrifugal 5 minutes of 7500g; 5. be dissolved in DEPC-treated water after the RNA deposition drying, 55-60 ℃ is incubated 10 minutes with thorough dissolving RNA.Whole process is carried out with reference to TRlZOL (Total RNA Isolation) Reagent Kit recommend method.

The separation and purification of embodiment 2:mRNA

Adopt PolyA Tract mRNA separation system (Promega; USA) separation and purified mRNA; Its principle of work is based on Oligo (dT) and the complementary pairing characteristic that mRNA 3 ' holds poly (A) tail, with biotin labeling Oligo (dT), holds the annealing of poly (A) to form crossbred through it and mRNA 3 '; Catch and wash vitamin H Oligo (dT)/mRNA crossbred with indicating affinity plain magnetic bead and magnetic separation rack then, use the ddH of no RNA enzyme at last ₂O elutes it, reaches the purpose of separating mRNA from total RNA.1. the preparation of sample: RNA is joined among the GTC of the 800 μ l that contain 32 μ l beta-mercaptoethanols.2. the annealing of probe: get 250pM concentration Oligo (dT) 5 μ l, adding distil water to 50 μ l; The dilution buffer (dilution buffer has added 32 μ l beta-mercaptoethanols) that adds 1.6ml preheating (70 ℃), with the RNA mixing, 70 ℃ of incubations 5 minutes.3. the activation of magnetic bead: the magnetic bead SA-PMPS (available from U.S. Promega company) that gets 1.2ml is in the centrifuge tube of 1.5ml; (Trisodium Citrate of the sodium-chlor of 0.15 mol and 0.015 mol, pH7.0) resuspended SA-PMPS is with magnet stand absorption magnetic bead, original volume 0.5 * SSC washing SA-PMPS3 time with 0.5 * SSC.4. obtaining of mRNA: the RNA of 70 ℃ of incubations is mixed with SA-PMPS, and room temperature is placed to put in 5 minutes and is adsorbed magnetic bead on the magnet stand, abandons supernatant; The 0.5XSSC suspension magnetic bead of 2ml, SSC is removed in washing repetition 2 times for the last time as far as possible; The ddH that adds no RNA enzyme ₂O to magnetic bead, mixing gently, centrifugal then (12000g * 3 minute) or magnet stand absorption magnetic bead; Get supernatant, obtain mRNA.Concentration and purity through electrophoresis and ultraviolet determination mRNA.5. the deposition of mRNA: add the absolute ethyl alcohol of glycogen and 2.5 times of volumes among the mRNA that will 4. obtain, deposition is spent the night, and mRNA will be used for the synthetic of cDNA.

3: the first chain cDNA of embodiment are synthetic

1. in 1.5ml Ep pipe, add 2 μ l Not I Primer-adapter (American I NVITOGEN company) and 6 μ l mRNA (containing 3 μ g mRNA); 70 ℃ of incubation 10min; Be put in rapidly on ice; After centrifugal, add following ingredients: 4 μ l 5X first strand buffer (American I NVITOGEN company); 2 μ l 0.1MDTT; 1 μ l 10mM dNTPs; 1 μ l DEPCH ₂O.Centrifugal behind the mixing gently, put to 2min for 37 ℃; 2. add 5 μ l reversed transcriptive enzymes, get 2 μ l behind the mixing, add 1 μ l [α- ³²P] dCTP (4 μ Ci) (spike pipe).With above-mentioned reactive component (sample hose) while 37 ℃ of incubation 1h, put into termination reaction on ice then; 3. for the spike pipe, add 43 μ l 20mM EDTA and 5 μ l yeast tRNA successively, behind the mixing; Get respectively two part 10 μ l o'clock on two filter membranes, wash 3 times each 5min for 1 part with 10%TCA; 95% ethanol is washed 1 time, and after the dry air, putting into the 1.5ml scintillation solution (is 1 ^#Sample); After other 1 part of dry air, putting into the 1.5ml scintillation solution (is 2 ^#Sample).30 μ l traced fluids add 1.5 μ l 7.5M NH in addition ₄OAc and 90 μ l absolute ethyl alcohols (20 ℃), behind the mixing immediately 14, the centrifugal 20min of 000rpm; Abandon supernatant, add 0.5ml 70% absolute ethyl alcohol (20 ℃), 14; The centrifugal 2min of 000rpm abandons supernatant, and 37 ℃ of dry 10min let ethanol volatilize; Be dissolved in 10 μ l TEN solution, add 10 μ l 2X sample loading buffers, get 10 μ l and be used for alkaline gel electrophoresis.With [α- ³²P] dCTP mark λ DNA HindIII fragment makes molecular weight marker; 4. room temperature held 15min behind the mixing adds 2 μ l 0.2M EDTA termination reactions.Get 6 μ l reaction solutions and 6ml 2X alkalescence electrophoretic buffer mixing, behind the electrophoresis 5h, soak 20min, until the bromjophenol blue flavescence with 7%TCA.Blot (about 8h) with toilet paper then, carry out radioautograph; 5. for sample hose, be used for the synthetic of second chain.

4: the second chain cDNA of embodiment are synthetic

1. in sample hose, add following ingredients on ice successively; 2. gently behind the mixing, 16 ℃ of incubation 2h; 3. add 2 μ l (10units) T4 archaeal dna polymerases, continue 16 ℃ of reaction 5min; 4. change on ice, add 10 μ l0.5M EDTA; 5. add equal-volume (150 μ l) phenol/chloroform/primary isoamyl alcohol (25/24/1), thoroughly behind the vortex, under the room temperature 14, the centrifugal 5min of 000rpm.Change water (140 μ l) over to another 1.5ml Ep pipe; 6. the 7.5M NH that adds 70 μ l ₄OAc and 0.5ML absolute ethyl alcohol (20 ℃), behind the vortex, under the room temperature 14, the centrifugal 20min of 000rpm; 7. abandon supernatant, add 0.5ml 70%7 alcohol (20 ℃), the same centrifugal 2min.Abandon supernatant, 37 ℃ of dry 10min.

Embodiment 5: double-stranded cDNA is connected with Sal I adapter

1. use 25 μ l DEPCddH ₂The cDNA sample of O dissolving embodiment 4, according to the form below adds successively then; 2. mixing gently, 16 ℃ of reactions spend the night (about 20h).3. use (25/24/1) extracting of phenol/chloroform/primary isoamyl alcohol and NH ₄Behind the Oac/ ethanol sedimentation, 37 ℃ of dry 10min.

Embodiment 6:Not I digests double-stranded cDNA

1. the sample with embodiment 5 is dissolved in 41 μ l, and according to the form below adds successively then; 2. behind the mixing, 37 ℃ of incubation 2h; 3. use phenol/chloroform/primary isoamyl alcohol (25/24/1) extracting once, use 7.5M NH then ₄The Oac/ ethanol sedimentation, 37 ℃ of dry 10min.4. be dissolved in 70 μ l TEN, get 1 μ l and be used for quantitatively, all the other-20 ℃ of preservations are subsequent use.

Embodiment 7: excessive Sal I adapter and the enzyme removed in the double-stranded cDNA molecule are cut small segment

(Amersham USA) removes excessive Sal I adapter and enzyme and cuts small segment with nucleon extraction and purification kit.1. room temperature low suspension resin is got 600 μ l then and is added in the centrifugal post, and the centrifugal 10s of 2000rpm removes liquid.Central authorities add the above-mentioned cDNA solution of 40 μ l at resin.The same centrifugal; 2. collect elutriant and be used for ligation.

Embodiment 8: double-stranded cDNA is connected and conversion with the pSPORT1 carrier

1. in 1.5ml Ep pipe, add following ingredients successively; 2. react 16h under the room temperature; 3. in 2. reaction solution, add following ingredients successively: 5.0 μ l yeast tRNA, 12.5 μ l 7.5M NH40ac, 70 μ l absolute ethyl alcohols (20 ℃).14000 centrifugal 20min immediately behind the vortex mixing; 4. deposition is dissolved in 4 μ l with 70% ethanol (20 ℃) washing after 37 ℃ of dryings; 5. get 2 μ l electric shock and transform 50 μ l E.coli K12 MC1061.

Embodiment 9: the preliminary evaluation of poison gland cell cdna library

Identify the quality in library, forward primer: 5 ' TCGACCCACGCGTCCG 3 ' (pressing SalI adapter sequences Design) with PCR method; Reverse primer: 5 ' GAGCGGCCGCCCT15 3 ' (pressing the sequences Design of NotI primer-adapter).

Embodiment 10: random sequencing Policy Filtering cDNA library

From the Lychas mucronatus poison gland cell cdna library that builds, select 100 clone's at random, serve the order-checking of Hai Sanbo company.Sequence typing software is BioEdit v4.5.8 (Tom Hall; 1999); Homology comparison and signal peptide cutting site forecasting software be respectively CLUSTAL X 1.8 (Thompson et al., 1997) and PC/GENE (Intelligenetics Inc., Switzerland).Sequential analysis shows that clone's 8 is a brand-new toxin gene, called after Lm8.Its sequence is the nucleotide sequence shown in the SEQ ID NO:1: ggaaggaaaatgaacaaagtttgctttgtcgtcgttcttgttctcttcgtggctct ggctgcatatgtgtcacctatcgaaggtgtaccaacaggaggatgcccactttcgg attcgctgtgtgccaaatattgcaagtcccacaaatttggcaaaaccggaagatgc accggaccaaacaagatgaaatgtaaatgtctcgtgtaataaaatgtaagcgaata aagatttgtgaatgaaaaaaaaaaaaa.This gene contains a complete ORFs, and two tailing signal aataaa are arranged.65 amino-acid residues of the prosoma organization form coding of Lm8; Form by two parts, i.e. signal peptide (25 residues) and mature peptide (40 residues): MNKVCFVVVLVLFVALAAYVSPIEGVPTGGCPLSDSLCAKYCKSHKFGKTGRCTGP NKMKCKCLV (SEQ ID NO:2).Signal peptide sequence is preceding 25 amino-acid residue MNKVCFVVVLVLFVALAAYVSPIEG, and its ripe toxin is made up of 40 amino acid: VPTGGCPLSDSLCAKYCKSHKFGKTGRCTGPNKMKCKCLV (Fig. 1).

Embodiment 11: design primer and pcr amplification Lychas mucronatus Kv1.3 blocker gene

The Lychas mucronatus Kv1.3 blocker gene (Lm8 gene) sequences Design PCR forward and the reverse primer that are provided according to SEQID NO:1: 8-FP (8-FP:5-GCG GGATCCGATGACGATGACAAGGTACCAACAGGAGGATGC-3) and 8-RP (8-RP:5-GCC CTCGAGTTACACGAGACATTTACA-3), be template from scorpion venom glandular cell cDNA storehouse, to screen Lm8 gene (SEQID NO:1) clone that is obtained, carry out the PCR reaction, the mature peptide region sequence of the Lychas mucronatus Kv1.3 blocker gene that increases in a large number.The PCR reaction conditions: each 1 μ l of 1 μ l Taq polysaccharase (1U), four kinds of (VITAMIN B4, guanine, cytosine(Cyt), thymus pyrimidine) deoxymononucleotide geometric ratio mixed solutions of 0.5 μ l (10mmol/L), 16.5 μ l aseptic double-distilled waters, 10 times of PCR damping fluids of 2.5 μ l, 1.5 μ l magnesium chlorides (25mmol/L), A1 (10 μ mol/L) and A2 (10 μ mol/L) primer and Lm8 gene template, TV is 25 μ l.The PCR reaction process: 60 seconds, 55 ℃ renaturation of 300 seconds, 94 ℃ sex change of 94 ℃ of preparatory sex change are extended 60 seconds, 35 times, 72 ℃ last extensions 300 seconds of circulation for 60 seconds, 72 ℃.

The double digestion of embodiment 12:Lm8 and pGEX-6p-1 be connected

Gained PCR product among the embodiment 1 is passed through phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting, absolute ethyl alcohol (2.5 times of volumes) post precipitation is with 50 μ l aqua sterilisa dissolution precipitations.With restriction enzyme BamHI and XhoI (Takara Company products) the PCR product that reclaims and expression vector pGEX-6p-1 plasmid being carried out enzyme cuts.Each 1 μ l of endonuclease reaction: BamHI (14U/ μ l) and XhoI (20U/ μ l), 10 times of damping fluid 2.5 μ l,, PCR product or pGEX-6p-1 plasmid 50-100ng, adding sterilized water to TV is 25 μ l.37 ℃ of water-baths 5 hours, enzyme are cut product through phenol: chloroform: the primary isoamyl alcohol extracting, absolute ethyl alcohol (2.5 times of volumes) post precipitation is used T ₄Dna ligase is connected the PCR product with expression vector pGEX-6p-1.Ligation: T ₄Dna ligase (1U/ μ l) 1 μ l, the mol ratio of PCR product and expression vector pGEX-6p-1 is 3: 1, and the DNA total amount is 0.1 μ g, 5 times of ligase enzyme reaction buffer 4 μ l, adding sterilized water to TV is 20 μ l, places 24 hours for 16 ℃.

Embodiment 13: escherichia coli DH5a and Rossetta ^TM(DE3) preparation of competent cell

The preparation of DH5 α competent cell: the single bacterium colony of picking DH5 α on streak plate, be inoculated in 5ml LB nutrient solution, 37 ℃, overnight cultures in the 250rpm shaking table; Transfer in 5ml LB substratum with 1% amount, grow to OD ₆₀₀To 0.4～0.6, get bacterium liquid 1ml in the 1.5ml of precooling Eppendorf pipe, ice bath 5～10 minutes, 4 ℃ 12, centrifugal 20～30 seconds of 000rpm collects thalline, is inverted 1 minute, and ice bath is 10 minutes again; Deposition is resuspended in the 0.1M CaCl of 1ml precooling ₂In, ice bath 20～40 minutes (can omit), 4 ℃ 12, centrifugal 20～30 seconds of 000rpm collects thalline, thalline is resuspended in the CaCl of 150 μ l precoolings ₂In, ice bath 2～7 hours, 4 ℃ of refrigerators are preserved, as are placed on-70 ℃ and then can preserve 6 months.

E.coli Rossetta ^TM(DE3) preparation of competent cell is same as the preparation of bacillus coli DH 5 alpha competent cell.

Embodiment 14: connect the conversion of product and the evaluation of positive colony

20 μ l ligation liquid among the embodiment 12 are added to the DH5 α competent cell of 100 μ l, mixing, ice bath 30 minutes, 42 ℃ of water-baths 90 seconds (can not shake), ice bath is 2 minutes again; Add equal-volume 2 * LB nutrient solution, 37 ℃ of shaking tables (120rpm) incubation 1 hour; Shake up bacterium liquid, get 200 μ l and coat LB/AP ⁺On the agar plate, treat to be inverted in after bacterium liquid blots 37 ℃ and cultivated observations 12～16 hours.

LB/AP ⁺Picking list bacterium colony is 10 on the agar plate, contains in the LB liquid nutrient medium of ammonia benzyl 37 ℃ of joltings 4 hours in 500 μ l, gets 2 μ l bacterium liquid as template, carries out PCR with the forward and reverse primer among the embodiment 11.Positive colony of PCR screening further uses the restriction enzyme digestion reaction to identify.Clone's of the positive result of both is served Hai Sanbo company and is carried out sequencing analysis.Sequencing primer is the universal sequencing primer thing pGEX5 ' primer to the pGEX-6p-1 plasmid.

Embodiment 15: recombinant expression plasmid Lm8/pGEX-6p-1 extracts and genetic engineering bacterium Rossetta ^TM(DE3) (Lm8/pGEX-6p-1) preparation

Positive colony of proving conclusively checking order among the embodiment 14 is according to alkaline lysis method of extracting recombinant expression plasmid Lm8/pGEX-6p-1 (method is seen " molecular cloning " second edition).The Lm8/pGEX-6p-1 plasmid that extracts is changed over to the intestinal bacteria competence Rossetta of embodiment 13 preparations according to the method for transformation among the embodiment 14 ^TM(DE3) in the cell.Flat board is LB/AP ⁺Agar plate.Picking mono-clonal obtains genetic engineering bacterium Rossetta ^TM(DE3) (Lm8/pGEX-6p-1), be stored in Chinese typical culture collection center.Recombination bacillus coli Escherichia coli Rossetta ^TM(DE3)/and Lm8/pGEX-6p-1, CCTCC NO:M207132.

Embodiment 16: expression and the affinity chromatography of reorganization GST-Lm8

Son (recombination bacillus coli Rossetta is cloned in ratio inoculation with 1: 100 in containing the LB liquid nutrient medium of penbritin ^TM(DE3)/Lm8/pGEX-6p-1), 37 ℃ are cultured to OD ₆₀₀0.8 in time, add IPTG (final concentration is 0.1mM) culture induced, and then culture cultivated 4 hours to carry out the expression (see figure 5) of goal gene at 28 ℃.50 times of cultures after concentrated the inducing; UW broken cell (80HZ; 30 seconds/inferior; To culture become limpid till), centrifugal 15 minutes of 12000rpm, gained supernatant join in the GST affinity chromatography glue that 26 ℃ of effects made fusion rotein GST-Lm8 fully combine with GST affinity chromatography glue in 1 hour behind the thorough mixing.(1.0mM pH8.0) washes GST affinity chromatography glue repeatedly and removes foreign protein with the Tris-Cl buffered soln of the EDTA that contains 50mM.The GSH solution of using 10mM then is according to 50ml/L culture wash-out fusion rotein GST-Lm8.

Embodiment 17: reorganization proteic concentrating and desalinating of GST-Lm8 and small intestine kinases enzyme are cut

With the fusion rotein GST-Lm8 of wash-out among the embodiment 16 10KDa through 15ml ultra consider pipe (Millipore, Centricon, USA) centrifugal concentrating and desalinating, centrifugal speed is 3500rpm/min, temperature is 4 ℃.The GST-Lm8 fusion rotein of concentrating and desalinating carries out little enteropeptidase (Biowisdom, China) digestion then.Small intestine kinases endonuclease reaction system is following: small intestine kinases 10U, and 10 times of damping fluid 100 μ l, GST-Lm8 fusion rotein 10mg, adding sterilized water to TV is 1000 μ l.37 ℃ of water-baths 12 hours.The albumen result that enzyme is cut digestion can detect (see figure 6) in the Tricine-SDS-PAGE electrophoresis.

Embodiment 18:HPLC separates rLm8 albumen and mass spectroscopy rLm8 albumen

The GST-Lm8 fusion rotein of digestion among the embodiment 17 is passed through HPLC (U.S.'s Anji human relations Company products), Lm8 albumen and GST albumen sepn.The parameter of HPLC is set to: separate pillar C18 column (EliteHPLC, China, 10 * 250mm, 5 μ m), and flow velocity 5ml/min, liquid phase is CH3CN (10%to 80%) elutriant that contains 0.1%TFA, ultraviolet detection is arranged on the 230nm place.Manual collection Lm8 protein peak, and lyophilize (40 ℃).SDS-PAGE protein electrophorese through the Tris-Tricine damping fluid detects the reorganization Lm8 albumen of collecting in the liquid, and measures content with the Bradford method.Highly purified rLm8 albumen is through MALDI-TOF-MS (Voyager-DESTR; Applied Biosystems) measuring its molecular weight is 4241.05 (see figure 7)s; Consistent with theoretical derivation, show success acquisition the on all four recombinant protein of highly purified and natural Lm8 albumen.

Embodiment 19: the Cos7 clone of stablizing the green fluorescent protein of overexpression mKv1.3

The voltage gated k+ channel blocker Kv1.3 of mouse is cloned among the eucaryon green fluorescence expression vector pIRES2-EGFP, has made up recombinant eukaryon expression vector pIRES2-EGFP-mKv1.3.Correct positive colony of order-checking conclusive evidence is used for extracting in a large number the pIRES2-EGFP-mKv1.3 plasmid.

Plasmid pIRES2-EGFP-mKv1.3 transfection Cos7 cell uses the transfection test box available from Guangzhou sun horse company.The righttest DNA concentration of transfection is about 5-30 μ g/mL.In order to obtain best transfection efficiency, cell density should be 60-70%.For 24 orifice plates, ideal conditions is 18-24h before transfection, every hole inoculation 8 * 10 ⁴-2.0 * 10 ⁵Individual cell.0.6 being diluted in 30 μ L, μ g DNA plasmid do not contain among the DMEM of serum and microbiotic, gently mixing; 1-2 μ L Suo Hua-SofastTM is diluted among the 30 μ LDMEM, gently mixing; 30 μ L Suo Hua-SofastTM dilute droplets are added in the DNA diluent, while drip mixing; Incubated at room 15-20min; 60 μ L Suo Hua-SofastTM/DNA mixtures are added in every hole and shake gently and make uniform mixing; Place 37 ℃ of CO ₂After incubator is hatched 24-48h, observe and analysis report gene transfection efficient.Sucking-off nutrient solution and throw out are washed monolayer once with PBS more then, and every hole adds heat the in advance perfect medium of (37 ℃) of 2mL.After cultivating 72h on the non-selective substratum; Institute's metastatic gene is given full expression to, use trypsin digestion and cell again, with 1: 10 concentration switching; After adding non-selective culture medium culturing 24h; Change the selective medium that contains proper concn G418 (800 μ g/mL) again, this substratum 2-3 in week every 2-4d must change 1 time so that remove the dead cell remains and make drug-resistant colonies be able to growth.The stably transfected cell line that screening obtains can be able to identify (see figure 8) through egfp expression.With the cultivation of going down to posterity of the Cos7 cell colony of strong expression green fluorescent protein, be used for next step patch clamp test.

Embodiment 20: reorganization Lm8 albumen is to the efficient blocking effect of mKv1.3 electric current

Adopt the two step drawn glass electrodes of microelectrode puller (PP-83, Japanese photoelectricity), carry out polished finish again, select the tip neat, opening diameter is about the electrode of 0.5-2 μ m.Detect with the method for full cell patch pincers activity recombinant peptide Lm8.Draw that cell is positioned in the sample pool among the embodiment 19, give a baby a bath on the third day after its birth time, add extracellular fluid again, in order to measuring with extracellular fluid.EPC-10 magnifying glass record, Pulse/Pulsefit (HEKA elektronik, Germany) software operation are adopted in the patch clamp test.The outer liquid (mM) of patch clamp operation: NaCl 125, and KCl 5, MgSO ₄1.2, CaCl ₂1.0, Na ₂HPO ₄1.6, NaH ₂PO ₄0.4 Glucose 10.5 and HEPES 32.5 transfer to pH 7.4 with 1mM NaOH.Interior liquid (mM): KF 145, MgCl ₂2, EGTA 10and HEPES 10 transfers to pH 7.2 with 1mM KOH.Clamp down on voltage and be-80mV, TV be-80mV---+50mV, and it is 10mv that the step increases, frequency of stimulation 0.5Hz, 25 ℃ are carried out.The voltage irritation cell also writes down the mKv1.3 electric current, adds reorganization apicule wolf Lm8 polypeptide through drug delivery system, writes down the mKv1.3 change in current again.The result shows that reorganization apicule wolf Lm8 polypeptide has blocking effect (see figure 9) efficiently to the mKv1.3 electric current, and it is 26.40 ± 1.62nM (see figure 10) that its half effect suppresses dosage.

SEQUENCE LISTING

< 110>Wuhan University

< 120>a kind of Lychas mucronatus Kv1.3 blocker gene and preparation method and application

< 130>a kind of Lychas mucronatus Kv1.3 blocker gene and preparation method and application

<160>2

<170>PatentIn version 3.1

<210>1

<211>251

<212>DNA

<213>Lychas mucronatus

<400>1

ggaaggaaaa tgaacaaagt ttgctttgtc gtcgttcttg ttctcttcgt ggctctggct 60

gcatatgtgt cacctatcga aggtgtacca acaggaggat gcccactttc ggattcgctg 120

tgtgccaaat attgcaagtc ccacaaattt ggcaaaaccg gaagatgcac cggaccaaac 180

aagatgaaat gtaaatgtct cgtgtaataa aatgtaagcg aataaagatt tgtgaatgaa 240

aaaaaaaaaa a 251

<210>2

<211>65

<212>PRT

<213>Lychas mucronatus

<400>2

Met Asn Lys Val Cys Phe Val Val Val Leu Val Leu Phe Val Ala Leu

1 5 10 15

Ala Ala Tyr Val Ser Pro Ile Glu Gly Val Pro Thr Gly Gly Cys Pro

20 25 30

Leu Ser Asp Ser Leu Cys Ala Lys Tyr Cys Lys Ser His Lys Phe Gly

35 40 45

Lys Thr Gly Arg Cys Thr Gly Pro Asn Lys Met Lys Cys Lys Cys Leu

50 55 60

Val

65

Claims (3)

1. a Lychas mucronatus Kv1.3 blocker gene recombination bacillus coli is characterized in that: Lychas mucronatus Kv1.3 blocker gene recombination bacillus coli Escherichia coli Rossetta ^TM(DE3)/and Lm8/pGEX-6p-1, CCTCC NO:M207132.

2. isolated polypeptide, its gene order is the 10th to the 207th a nucleotide sequence of the nucleotide sequence shown in the SEQ ID NO:1.

3. isolating protein, its sequence is the aminoacid sequence shown in the SEQ ID NO:2.

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