WO2020094005A1 - 重组Martentoxin的制备、表征及应用 - Google Patents

重组Martentoxin的制备、表征及应用 Download PDF

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WO2020094005A1
WO2020094005A1 PCT/CN2019/115709 CN2019115709W WO2020094005A1 WO 2020094005 A1 WO2020094005 A1 WO 2020094005A1 CN 2019115709 W CN2019115709 W CN 2019115709W WO 2020094005 A1 WO2020094005 A1 WO 2020094005A1
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martx
polypeptide
recombinant
sequence
fusion protein
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French (fr)
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曹春阳
刘新莲
陶杰
吉永华
蓝文贤
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中国科学院上海有机化学研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43522—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from scorpions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to the preparation, characterization and application of recombinant Martentoxin, and belongs to the field of polypeptide toxins and biotechnology engineering. Specifically, the present invention relates to a recombinant preparation technology and characterization of physicochemical properties of the toxin-Martentoxin isolated and purified from the venom of Buthus martensi Karsch (BmK), and activation of the recombinant toxin in large-conductance calcium ion and voltage The application of potassium ion (BK) channel.
  • venom of poisonous organisms such as snakes, centipedes, scorpions, spiders, anemones, conical snails, etc. contains a large number of pharmacologically active peptides.
  • venom derivatives containing intramolecular disulfide bonds have been approved by the FDA for the treatment of hypertension, pain and diabetes [Venoms: venoms: venoms as a source of development for human therapeutics [M] .City: RoyalSociety of Chemistry , 2015].
  • More toxin molecules are in clinical research, they target diseases such as epilepsy, cancer and autoimmunity [Curr.Pharm.Des.2007,13,2927-2934; Toxins2010,2,2851-2871; ExpertOpi.Biol. Ther. 2011,11,1469-1484].
  • the scorpion venom of Buthus martensi Karsch (BmK) is rich in a variety of naturally active peptides, most of which can act on ion channels. So far, the scorpion venom has obtained 14 short-chain potassium channel blockers (BmTX1-3, BmKTX, BmP01-3, BmP05, BmP09, BmKK1-4, and Martentoxin) by isolation and purification 13482; J. Biol. Chem. 2004, 279, 34562-34569; Eur. J. Biochem. 1997, 245, 457-64].
  • This Martentoxin (referred to as MarTX for short) consists of 37 amino acids with three pairs of disulfide bonds. It is a short-chain polypeptide toxin that specifically acts on the BK channel [J. Neurochem. 2003, 84, 325-335], which can significantly inhibit nerves Type ( ⁇ + ⁇ 4) BK channel current.
  • MarTX toxin has a small molecular weight and contains many pairs of disulfide bonds, it is easy to cause inclusion body expression. Therefore, it is difficult to obtain a large amount of soluble, high-purity protein using this method.
  • the object of the present invention is to provide a method for preparing MarTX toxin with high yield, high purity and high activity.
  • fusion protein having the structure shown in Formula I from the N-terminus to the C-terminus:
  • P1 is the purification label element
  • MBP maltose binding protein element
  • P3 is a protease recognition site element
  • P4 is a MarTX polypeptide element
  • the P1 is a 6 ⁇ His sequence.
  • the fusion protein may not include the P1 element.
  • sequence of the P2 is SEQ ID NO: 4.
  • sequence of P2 is an amino acid sequence obtained by replacing, deleting, changing, inserting or adding one or more amino acids on the basis of SEQ ID NO: 4.
  • the protease is selected from the group consisting of HRV 3C protease, Thrombin enzyme, Factor Xa, Enterkinase (Enterkinase), TEV enzyme, Prescission, TAGZyme, SUMO protease, or a combination thereof.
  • the protease is Thrombin enzyme.
  • sequence of P4 is SEQ ID NO: 2.
  • sequence of P4 is an amino acid sequence obtained by replacing, deleting, changing, inserting or adding one or more amino acids on the basis of SEQ ID NO: 2.
  • 1 to 30 amino acid residues may be added to the N-terminus or C-terminus of the sequence of Formula I, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues .
  • between P1 and P2, between P2 and P3, or between P3 and P4, 1 to 30 amino acid residues may be added, preferably 1 to 10 amino acid residues, more Preferably 1 to 5 amino acid residues.
  • the order from the N-terminal to the C-terminal elements further includes: P2-P1-P3-P4, P4-P3-P2-P1, P2-P3- P4-P1 or P4-P3-P1-P2.
  • an isolated polynucleotide encoding the fusion protein of the first aspect of the invention.
  • the polynucleotide includes a nucleic acid construct of formula II from 5 'to 3',
  • Z1, Z2, Z3 and Z4 respectively encode P1, P2, P3 and P4 in Formula I in the first aspect of the present invention
  • sequence of Z2 is SEQ ID NO: 3.
  • sequence of Z4 is SEQ ID NO: 1.
  • sequence of the elements from 5 'to 3' in the sequence of the polynucleotide further includes: Z2-Z1-Z3-Z4, Z4-Z3-Z2-Z1, Z2-Z3 -Z4-Z1 or Z4-Z3-Z1-Z2.
  • a vector comprising the polynucleotide of the second aspect of the present invention.
  • the vector is pETDuet-MarTX-1
  • the pETDuet-MarTX-1 is obtained by inserting the sequence SEQ ID NO: 3 into the vector pETDuet-1.
  • a host cell containing the vector or genome of the third aspect of the present invention integrated with the polynucleotide of the second aspect of the present invention.
  • the host cell is E. coli Origami B (DE3).
  • a method for producing the fusion protein of the first aspect of the present invention comprising the steps of:
  • the host cell according to the fourth aspect of the present invention is cultured to express the fusion protein according to the first aspect of the present invention.
  • the protease is selected from the group consisting of HRV 3C protease, Thrombin enzyme, Factor Xa, Enterkinase, TEV enzyme, Prescission, TAZyme, SUMO protease, or a combination thereof.
  • the protease is Thrombin enzyme.
  • the MarTX polypeptide purified by the method has a yield of ⁇ 2 mg, preferably ⁇ 3 mg, and more preferably ⁇ 4 mg per liter of expressed cells.
  • a ( ⁇ + ⁇ 4) BK channel current inhibitor drug screening method including the steps of:
  • the half-effect concentration (EC50) value of the MarTX polypeptide blocking ( ⁇ + ⁇ 4) BK channel current is C0, and the inhibitor drug candidate blocking ( ⁇ + ⁇ 4 )
  • the half effect concentration (EC50) value of the BK channel current is C1
  • the inhibitor drug candidate is an ( ⁇ + ⁇ 4) BK channel inhibitor superior to MarTX polypeptide.
  • Figure 1 shows the physical map and multiple cloning sites of the expression vector pETDuet-1.
  • Figure 2 shows the MarTX gene PCR process and agarose gel electrophoresis.
  • A PCR primers. The BamH I site of the upstream primer (Primer-UP) and the Xho I site of the downstream primer (Primer-DO) have been marked with a horizontal line.
  • B Composition of PCR reaction system.
  • C PCR reaction program.
  • D Agarose gel electrophoresis after completion of the reaction.
  • Figure 3 shows a double-digested agarose gel electrophoresis diagram of the expression vector pETDuet-1 and Martentoxin gene.
  • M represents DNA marker (DL5000), from bottom to top is 100bp, 250bp, 500bp, 750bp, 1000bp, 1500bp, 2000bp, 3000bp, 5000bp.
  • Lane 1 is the double digestion result of pETDuet-1 vector by restriction enzymes EcoR and Not I
  • lane 2 is the double digestion result of EcoR I and Not I of the Martentoxin gene fragment.
  • Figure 4 shows a schematic diagram of the recombinant expression vector pETDuet-1-MarTX.
  • Figure 5 shows the chromatograms of pGEX-4T-3-MarTX recombinant plasmid and purified gel:
  • A Schematic diagram of pGEX-4T-3-MarTX expression plasmid;
  • B MarTX Superdex 75 peak diagram.
  • Figure 6 shows the chromatogram of pGEX-6P-1-MarTX recombinant plasmid and purified gel.
  • A Schematic diagram of pGEX-6P-1-MarTX expression plasmid.
  • B MarTX Superdex 75 peak map.
  • Figure 7 shows the chromatogram of pSMT3-MarTX recombinant plasmid and purified gel.
  • A Schematic diagram of pSMT3-MarTX expression plasmid.
  • B MarTX Superdex 75 peak map.
  • Figure 8 shows the purification of pETDuet-1-MarTX recombinant plasmid expression toxin.
  • A Purification results of nickel column. The fusion protein has been marked with a red line.
  • B Amylose Resin column purification results after digestion.
  • C Gel chromatography column Superdex 75 ultraviolet absorption peak diagram.
  • D SDS-PAGE detection after purification of recombinant MarTX.
  • Figure 9 shows the one-dimensional hydrogen spectrum of the recombinant MarTX toxin.
  • Figure 10 shows the circular dichroism of recombinant MarTX toxin.
  • Figure 11 shows MALDI-TOF mass spectrometry identification of recombinant MarTX toxins.
  • Figure 12 shows a superposition of the 20 optimal solution structures of the recombinant MarTX toxin (partial superposition of secondary structures). The N-terminus and C-terminus of the polypeptide have been marked.
  • Figure 13 shows a Laplace diagram of the 20 optimal solution conformations of recombinant MarTX toxin.
  • Figure 14 shows the superimposed structure of recombinant MarTX toxin and natural toxin.
  • Red indicates recombinant MarTX
  • green indicates the reported MarTX structure (PDB: 1M2S). The disulfide bond and the N-terminal and C-terminal have been marked.
  • Figure 15 shows the inhibitory effect of recombinant MarTX toxin on ( ⁇ + ⁇ 4) BK channel current.
  • (A) The whole-cell current of recombinant MarTX toxin on ( ⁇ + ⁇ 4) BK channel HEK293T, the clamping potential is -80mV, and the current is induced by + 80mV voltage stimulation and 300nM internal calcium.
  • the inventor unexpectedly developed a method for purification and preparation of MarTX polypeptide. Specifically, in the construction of the recombinant plasmid of MarTX, at the 5 'end of the gene fragment corresponding to the target protein, the gene sequence encoding the His tag and encoding the MBP tag is added, and the constructed recombinant plasmid is transformed into a vector strain E. coli Origami B (DE3), and culture and induce expression.
  • E. coli Origami B E. coli Origami B
  • Experimental data on the characterization of the purified MarTX polypeptide shows that the target protein obtained by the method has higher expression level, purity and activity than the target protein obtained in the comparative embodiment. On this basis, the present invention has been completed.
  • MarTX protein As used herein, “Martentoxin protein”, “MarTX toxin” and “MarTX polypeptide” “recombinant MarTX toxin” are used interchangeably and refer to the Martentoxin protein, which is composed of 37 amino acids, has three pairs of disulfide bonds, is a specific Short-chain peptide toxins that act sexually on BK channels can significantly inhibit neuronal ( ⁇ + ⁇ 4) BK channel currents.
  • genes provided in the examples of the present invention are derived from Scorpio scorpioides, they are derived from other similar species (especially scorpions belonging to the same family or genus as Scorpio scorpioides). Ground, the sequence is shown in SEQ ID NO: 1)
  • the gene sequence of MarTX with certain homology (conservative) is also included in the scope of the present invention, as long as a person skilled in the art reads this application The information provided can easily isolate this sequence from other species (especially scorpions).
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the form of DNA includes: DNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • Polynucleotides encoding mature polypeptides include: coding sequences encoding only mature polypeptides; mature polypeptide coding sequences and various additional coding sequences; mature polypeptide coding sequences (and optional additional coding sequences) and non-coding sequences.
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide including additional coding and / or non-coding sequences.
  • the present invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs and derivatives of polyglycosides or polypeptides having the same amino acid sequence as the present invention.
  • This polynucleotide variant may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants.
  • allelic variant is a form of substitution of a polynucleotide. It may be a substitution, deletion, or insertion of one or more nucleotides, but it will not substantially change the function of the polypeptide it encodes. .
  • the invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60 ° C; or (2) There are denaturants, such as 50% (v / v) methylphthalamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) Only the identity between the two sequences is at least 90%, More preferably, the hybridization occurs only when it exceeds 95%.
  • MarTX gene of the present invention is preferably derived from Scorpion scorpion, other species (especially scorpions) are highly homologous (e.g., have more than 80%, such as 85%, 90%, 95 % Or even 98% sequence identity) are also within the scope of the present invention.
  • Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.
  • the full-length MarTX nucleotide sequence of the present invention or a fragment thereof can generally be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available DNA library or cDNA prepared according to conventional methods known to those skilled in the art
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • the relevant sequence can be obtained in large quantities by the recombination method. It is usually cloned into a vector, and then transferred into cells, and then the relevant sequences are isolated from the proliferated host cells by conventional methods.
  • synthetic methods can be used to synthesize the relevant sequences, especially when the length of the fragments is short.
  • a long sequence can be obtained by synthesizing multiple small fragments and then connecting them.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the invention by chemical synthesis.
  • the present invention relates to a MarTX polypeptide and its variants for treating epilepsy.
  • the amino acid sequence of the polypeptide is shown in SEQ ID NO: 2.
  • the polypeptide of the present invention can effectively treat and / or prevent epilepsy.
  • the present invention also includes 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, and most preferably 98 % Or more, such as 99%) homologous polypeptides or proteins with the same or similar functions.
  • the "same or similar function” mainly refers to: “alleviates the symptoms of epilepsy”.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide.
  • the polypeptide of the present invention may be a naturally purified product, or a chemically synthesized product, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated or may be non-glycosylated.
  • the polypeptide of the present invention may or may not include the starting methionine residue.
  • the present invention also includes MarTX polypeptide fragments and analogs having MarTX polypeptide activity.
  • fragment and “analog” refer to a polypeptide that substantially retains the same biological function or activity of the natural MarTX polypeptide of the present invention.
  • the polypeptide fragment, derivative or analog of the present invention may be: (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues
  • the group may or may not be encoded by the genetic code; or (ii) a polypeptide having a substitution group in one or more amino acid residues; or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, For example, a polypeptide formed by fusion of polyethylene glycol; or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or Fusion protein).
  • These fragments, derivatives and analogs are within the scope of those skilled in the art according to the definitions herein.
  • the polypeptide variant is the amino acid sequence shown in SEQ ID NO .: 2, after several (usually 1-10, preferably 1-8, more preferably 1-4) , Optimally 1-2) derived, substituted or deleted at least one amino acid derived derivative sequence, and the C-terminal and / or N-terminal to add one or several (usually within 10, preferably within 5 , More preferably within 3) amino acids.
  • SEQ ID NO .: 2 amino acid sequence shown in SEQ ID NO .: 2
  • Optimally 1-2) derived, substituted or deleted at least one amino acid derived derivative sequence and the C-terminal and / or N-terminal to add one or several (usually within 10, preferably within 5 , More preferably within 3) amino acids.
  • the function of the protein is usually not changed.
  • Adding one or several (such as 1-3) amino acids at the C-terminal and / or N-terminal is usually Does not change the function of the protein.
  • substitution Ala Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu (L) Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Leu; Val; Ile; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe; Thr
  • the invention also includes analogs of the claimed proteins.
  • the difference between these analogues and the natural SEQ ID NO: 2 can be the difference in amino acid sequence, the difference in the modification form that does not affect the sequence, or both.
  • Analogues of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagen, but also by site-directed mutagenesis or other known molecular biology techniques. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the protein of the present invention is not limited to the representative proteins listed above.
  • Modified (usually without changing the primary structure) forms include: in vivo or in vitro chemically derived forms of proteins such as acetate or carboxylation. Modifications also include glycosylation, such as those that are modified during protein synthesis and processing. This modification can be accomplished by exposing the protein to an enzyme that performs glycosylation (such as mammalian glycosylation or deglycosylation enzymes). Modified forms also include sequences with phosphorylated amino acid residues (eg phosphotyrosine, phosphoserine, phosphothreonine).
  • the present invention provides a fusion protein, characterized in that the fusion protein has the structure shown in Formula I from the N-terminus to the C-terminus:
  • P1 is the purification label element
  • MBP maltose binding protein element
  • P3 is a protease recognition site element
  • P4 is a MarTX polypeptide element
  • the fusion protein may not include the purification tag element of P1, and only the maltose binding protein element of P2 may be used for purification of Amylose Resin chromatography column. That is, the difference between including or not including the P1 element is only the difference in purification efficiency, but whether or not the P1 element is included or not, the expression level of the fusion protein of the present invention in the host cell of the present invention and the conformation of the resulting protein The stability is significantly better than other forms of recombinant MarTX toxins already available in the prior art.
  • the protease is selected from the group consisting of HRV 3C protease, Thrombin enzyme, Factor Xa, Enterkinase, TEV enzyme, Prescission, TAGZyme, SUMO protease, or a combination thereof.
  • the protease is Thrombin enzyme.
  • the sequence of the fusion protein further includes: P2-P1-P3-P4, P4-P3-P2-P1, P2-P3-P4-P1, or P4-P3-P1-P2. That is, the sequence between the sequence of His tag, MBP tag, and MarTX polypeptide can be flexibly changed, as long as the cleavage site corresponding to the P3 element is close to the MarTX polypeptide and between the MarTX polypeptide and the MBP tag, so that The MarTX polypeptide after protease digestion does not need to include the MBP tag described in P2.
  • the resulting MarTX polypeptide may contain a His tag because the His tag is only 6 and His residues, and its presence will not affect the activity and structure of the MarTX polypeptide.
  • the method for purifying and preparing MarTX polypeptide provided by the present invention includes the steps of:
  • the expression of the fusion protein is performed in the vector strain E. coli Origami B (DE3), and the conditions for culturing and inducing expression thereof are all the conventional conditions for culturing and expressing the protein of the strain.
  • the P1 is a His tag, which is used for affinity chromatography of the fusion protein with a nickel column.
  • Thrombin cleavage site in the P2 and MarTX polypeptide sequences, 6U / mL Thrombin enzyme is used for overnight digestion, and the protein solution is placed in a 3.5kDa dialysis bag Stir overnight.
  • the P2 is an MBP tag, which is used to purify the fusion protease cleavage product with an Amylose Resin chromatography column.
  • the cut P1-P2 is bound to the affinity chromatography column, and the target protein MarTX polypeptide is flowed through. The flow-through components were collected to obtain MarTX polypeptide without tag.
  • a gel chromatography column is further included in the purification step, that is, molecular sieve.
  • the purification step collect all elution peaks with a retention volume of 110 mL, which is the unlabeled MarTX peptide protein peak.
  • the MarTX polypeptide with relatively pure purity can be obtained.
  • the recombinant MarTX toxin obtained by the present invention has a higher yield, and 2L cells can obtain about 3mg of polypeptide. Compared with other existing MarTX toxin expression and purification technologies, the yield is increased by nearly 10 times .
  • the MarTX polypeptide prepared by the method of the present invention has high purity, correct physical and chemical properties in all aspects, and has electrophysiological activity. Due to the low yield of natural MarTX toxins and difficulty in obtaining them, it is of great significance to use the recombinant MarTX toxins obtained by this patent to replace the natural MarTX toxins to conduct studies on the pharmacological mechanism of BK channels.
  • the recombinant expression method of the present invention overcomes the defect that MarTX toxin has a small molecular weight of the polypeptide and contains many pairs of disulfide bonds which can easily lead to inclusion body expression, making it difficult to obtain a large amount of soluble, high-purity protein.
  • the method of the present invention realizes the green manufacturing of MarTX polypeptides, can provide new ideas for the production and preparation of similar polypeptide drugs, and becomes a research hotspot in the field of drug synthesis and fine chemicals.
  • the MarTX (Martentoxin) plasmid was provided by Ji Yonghua's research group of Shanghai University. It contains 37 amino acids. It is predicted by ProtParam online software. The relative molecular weight is about 4.065 kDa, and the theoretical pI value is 8.65.
  • the gene fragment is located between the BamH I and Sma I cloning sites of the pGEX-4T-3 vector, and there is an enterokinase cleavage site between the fusion-expressed GST tag and the target polypeptide (Figure 5).
  • E. coli DH5 ⁇ , BL21 (DE3) and Origami B (DE3) strains were all purchased from Novagen, and were prepared by our laboratory.
  • First design PCR primers add BamH I and Xho I cleavage sites and protective bases at the 5 'and 3' ends of the MarTX gene fragment, and perform PCR reaction with the pGEX-4T-3-MarTX recombinant plasmid as a template. Identification by 1% agarose gel electrophoresis. The electropherogram showed that the target band was bright and single, and the PCR was successful ( Figure 2). Subsequently, the tapping and recycling work of the target gene fragment is carried out.
  • the solid petri dish is inverted in a 37 ° C incubator overnight, and generally cultivated for 14-16h.
  • the pSMT3-MarTX recombinant plasmid insert the toxin gene fragment between BamH I and Xho I, which is the same as the cloning site of the pGEX-6P-1-MarTX recombinant plasmid. Therefore, the pSMT3-MarTX recombinant plasmid was successfully constructed by the same procedure: PCR reaction, double digestion of pSMT3 vector and gene fragments (BamH I and Xho I endonucleases), ligation reaction, transformation, and sequencing identification.
  • MCS1 and MCS2 There are two multiple cloning sites MCS1 and MCS2 in the pETDuet-1 plasmid.
  • the MBP tag encoding the maltose binding protein was constructed between the Nco I and EcoR I sites of MCS1, and six His-tags were designed at the N-terminus of the MBP tag.
  • the 5 'end of the MarTX gene fragment was added to the Thrombin cleavage site and the EcoR I cloning site by PCR reaction, and the Not I cloning site was added to the 3' end.
  • the primer design is shown in Table 2.
  • E. coli Origami B (DE3) expression strain was transferred into E. coli Origami B (DE3) expression strain for expression.
  • E. coli Origami B (DE3) is used as the expression strain.
  • the pGEX-4T-3-MarTX recombinant plasmid was transferred into E.coli BL21 (DE3) host cells for expression; for the pGEX-6P-1-MarTX recombinant plasmid, it was transferred into E.coli BL21 (DE3) Expression in the expression strain; for the pSMT3-MarTX recombinant plasmid, the plasmid was transferred into E. coli BL21 (DE3) competent cells for expression.
  • fusion protein expressed by pETDuet-1-MarTX recombinant plasmid that is, MBP tag recombinant MarTX polypeptide
  • add 15mL of broken bacteria BufferA per liter of bacteria see Table 2 for details of the composition
  • protease inhibitor PMSF to the final concentration 0.1mM.
  • the fusion protein expressed by the recombinant plasmid pGEX-4T-3-MarTX it has a GST tag, so the protein is first combined with the GST column during purification, and then most of the contaminated protein is removed by gradient elution with a reduced glutathione solution. Afterwards, GST tags were removed by enterokinase digestion at room temperature, and then purified by a second GST column and a gel chromatography column to obtain MarTX polypeptide.
  • the fusion protein expressed by the recombinant plasmid pGEX-6P-1-MarTX it has a GST tag.
  • the protein is first combined with the GST column, and then a reduced glutathione solution is used for gradient elution to remove most of the contaminated proteins. Afterwards, the peptide was separated from the GST tag by 3C enzyme digestion at 4 ° C, and then purified by a second GST column and a gel chromatography column to obtain MarTX polypeptide.
  • the protein was first combined with a nickel column during purification, and then a gradient elution with an imidazole salt solution was used to remove most of the contaminated protein. After that, it was digested with UlpI enzyme at 4 ° C, followed by a second nickel column purification and a gel chromatography column purification to obtain MarTX polypeptide.
  • Recombinant MarTX concentration is 0.42mM, 10% D 2 O is added to Buffer of 25mM NaH 2 PO4, 100mM NaCl, pH 6.80, one-dimensional hydrogen spectrum is collected on 600MHz nuclear magnetic spectrometer, sampling temperature is 20 °C, experimental spectrum See Figure 9.
  • the nuclear magnetic signal of the recombinant MarTX toxin obtained is relatively divergent, and there are obvious methyl characteristic peaks near the high field region, and the amide hydrogen and aromatic ring hydrogen of the toxin are widely distributed in the low field region of 6.5-10.5ppm, indicating that the polypeptide has not aggregated , The space structure is well folded.
  • the recombinant toxin of the present invention contains the ⁇ -helix and ⁇ -sheet secondary structural elements of the published structure, indicating that the folding is normal.
  • Buffer conditions of the purified toxin sample were exchanged into ddH 2 O, and the molecular weight of the recombinant MarTX was 4200.5444, as determined by MALDI-TOF, see FIG. 11.
  • the recombinant MarTX has two more GS amino acids at the N-terminus, and the theoretical molecular weight is 4208.80, so the measured molecular weight is roughly equivalent to the theoretical value.
  • Two-dimensional NMR spectra were collected from the recombinant MarTX samples. The experiment was conducted on an Aligent 600MHz spectrometer. The sample concentration was 1 mM, the sampling temperature was 20 ° C, and the sampling buffer was 25 mM NaH 2 PO 4 , 100 mM NaCl, 10% D 2 O, pH 6.80. The collected two-dimensional homonuclear NMR spectrum is shown in Table 4.
  • Example 5 Inhibition of ( ⁇ + ⁇ 4) BK channel current by recombinant MarTX toxin
  • the literature [Biophys.J.2008,94,3706-3713] indicates that MarTX toxin interacts with the extracellular loop region of the B4 channel ⁇ 4 auxiliary subunit, thereby inhibiting the ( ⁇ + ⁇ 4) BK channel current.
  • the invention examines the electrophysiological activity of recombinant MarTX toxin on ( ⁇ + ⁇ 4) BK channel through patch clamp experiment.
  • HEK293T Human embryonic kidney cells
  • DMEM Gibco, USA
  • FBS heat-inactivated FBS
  • the growth conditions of the cells in the Petri dish were 5% CO 2 and a constant temperature of 37 ° C. Constant humidity 95%.
  • Transient transfection technology was used to transform human ( ⁇ + ⁇ 4) BK channel plasmids (containing the gene sequences of hSlo ⁇ (U23767) and ⁇ 4 (KCNMB4; AF207992)) into HEK293 cells for expression by lipofection.
  • Example 6 Determination of the concentration of half effect of BK channel blocked by recombinant MarTX toxin
  • the 10 ⁇ M recombinant MarTX toxin can effectively inhibit the current generated by the ( ⁇ + ⁇ 4) BK channel, and has a dose-dependent characteristic, see FIG. 15B.
  • the dose-effect curve showed that the half-effect concentration (EC 50 ) of the recombinant MarTX toxin blocking ( ⁇ + ⁇ 4) BK channel was 0.32 ⁇ 0.012 ⁇ M.
  • the recombinant MarTX toxin obtained by the prokaryotic expression purification of the present invention can effectively block the ( ⁇ + ⁇ 4) BK channel current, and can be used as an inhibitor of the neuronal BK channel for the function of the neuronal BK channel.
  • Related epilepsy and other diseases research can effectively block the ( ⁇ + ⁇ 4) BK channel current, and can be used as an inhibitor of the neuronal BK channel for the function of the neuronal BK channel.
  • the MarTX gene is integrated into the vector plasmids pGEX-4T-3 (which expresses GST tags) and pGEX-6P-1 (which expresses GST tags, respectively) ) And pSMT3 (which express His tags and SUMO tags), and expressed in E. coli BL21 (DE3) respectively.
  • the three groups of comparative examples have lower protein yields or lower purity than the purification results of the fusion protein of the present invention.
  • both pGEX-4T-3-MarTX and pGEX-6P-1-MarTX contain GST tags.
  • the molecular weight of GST tags is about 26 kDa, while the target toxin is only about 4 kDa.
  • protease may be The enzyme-cutting site and the peptide are wrapped in it, so that the protease cannot realize the enzyme-cutting function, resulting in a very small amount of the target polypeptide after enzyme-cutting.
  • pSMT3-MarTX its expression is already very small, that is, it has already failed in the expression step.
  • MarTX toxin has a small molecular weight and is not easy to cut with the fusion label. And the toxin contains three pairs of disulfide bonds, which is not easy to reconstruct correctly.
  • the disulfide bond protein expression system is conducive to the correct folding of disulfide bonds.
  • Origami B (DE3) belongs to the Escherichia coli clonal strain and contains trxB / gor gene mutation. Mutating these two genes at the same time allows the strain to more efficiently generate disulfide bonds in the cytoplasm, which helps to form active proteins containing disulfide bonds.
  • the inventor finally developed the MarTX polypeptide preparation method of the present invention, using the recombinant plasmid of the present invention, and using the E. coli Origami B (DE3) strain, successfully obtained MarTX toxin samples with high yield and purity overcome the shortcomings of MarTX toxin due to the small molecular weight of the peptide and the easy inclusion of multiple pairs of disulfide bonds leading to the expression of inclusion bodies, making it difficult to obtain large amounts of soluble, high-purity proteins .
  • the technical solution of the present invention can obtain about 3 mg of polypeptide in 2L cells, and the output is increased by nearly 10 times, thereby achieving efficient and green manufacturing of MarTX polypeptide.

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Abstract

提供一种重组Martentoxin(简称MarTX毒素)的制备方法，包括将Martentoxin基因和麦芽糖结合蛋白与载体pETDuent-1连接得到重组表达载体pETDuent-1-MarTX，将该重组表达载体转化至E.coli Origami B(DE3)，筛选得到高效表达菌株，通过诱导表达、亲和层析纯化获得了具有生物活性，可显著抑制大电导钙离子和电压激活的钾离子(BK)通道电流的重组MarTX毒素。还提供了所述MarTX毒素在(α+β4)BK通道电流抑制剂的药物筛选中的应用。

Description

重组Martentoxin的制备、表征及应用 技术领域
本发明涉及重组Martentoxin的制备、表征及应用，属于多肽毒素和生物技术工程领域。具体地，本发明涉及一种从东亚钳蝎(Buthus martensi Karsch,BmK)毒液中分离纯化得到的毒素—Martentoxin的重组制备技术和理化性质的表征，以及该重组毒素在大电导钙离子和电压激活的钾离子(BK)通道中的应用。
背景技术
研究表明，蛇、蜈蚣、蝎子、蜘蛛、海葵、锥螺等有毒生物的毒液中含有大量具有药理活性的多肽。目前有多种含分子内二硫键的毒液衍生物通过FDA批准，治疗高血压、疼痛及糖尿病[Venoms to drugs:venom as a source for the development of human therapeutics[M].City:Royal Society of Chemistry,2015]。更多毒素分子正处于临床研究中，它们靶向癫痫、癌症和自身免疫等疾病[Curr.Pharm.Des.2007,13,2927-2934；Toxins 2010,2,2851-2871；Expert Opi.Biol.Ther.2011,11,1469-1484]。
东亚钳蝎(Buthus martensi Karsch,BmK)的蝎毒中富含多种具有天然活性的多肽，其中大部分可作用于离子通道。迄今为止，该蝎毒通过分离纯化已获得14种短链钾离子通道阻断剂(BmTX1-3、BmKTX、BmP01-3、BmP05、BmP09、BmKK1-4和Martentoxin)[Biochemistry 1997,36,13473-13482；J.Biol.Chem.2004,279,34562-34569；Eur.J.Biochem.1997,245,457-64]。这种Martentoxin(简称MarTX)由37个氨基酸构成，具有三对二硫键，是一种特异性作用于BK通道的短链多肽毒素[J.Neurochem.2003,84,325-335]，可显著抑制神经型(α+β4)BK通道电流。
目前，人工获取多肽的方法主要有三种：重组表达法、内源提取法和化学合成法。其中，内源提取法是最古老也最直接的方法，但提取出的多肽种类有限，产量较低。应用最多的化学合成法为Fmoc(9-芴甲氧羰基)固相合成法。该方法涉及线性肽链的合成、碘氧化体系、半胱氨酸的保护与保护基的脱除、以及变复性等诸多有机体系，步骤繁琐复杂，二硫键易错配，产率低下，且未能实现多肽类药物的绿色制造。而对于重组表达法，由于MarTX毒素分子量较小，且含多对二硫键易导致包涵体表达，因此运用该方法较难获得大量的、可溶的、高纯度蛋白。
因此，本领域迫切需要开发一种MarTX毒素的制备方法，以获得高产量、高纯度和高活性的MarTX毒素生物活性蛋白。
发明内容
本发明的目的就是提供一种高产量、高纯度和高活性的MarTX毒素的制备方法。
在本发明的第一方面，提供了一种融合蛋白，所述融合蛋白从N端到C端具有式I所示结构：
P1-P2-P3-P4   (式I)
式中，
P1为纯化标签元件；
P2为麦芽糖结合蛋白元件(MBP)；
P3为蛋白酶识别位点元件；
P4为MarTX多肽元件；
“-”表示连接上述元件的肽键。
在另一优选例中，所述P1为6×His序列。
在另一优选例中，所述融合蛋白可不包括P1元件。
在另一优选例中，所述P2的序列为SEQ ID NO:4.
在另一优选例中，所述P2的序列为在SEQ ID NO:4的基础上进行一个或多个氨基酸的替换、缺失、改变、插入或增加，所得到的氨基酸序列。
在另一优选例中，所述蛋白酶选自下组：HRV 3C蛋白酶、Thrombin酶、Factor Xa、肠激酶(Enterkinase)、TEV酶、Prescission、TAGZyme、SUMO蛋白酶，或其组合。
在另一优选例中，所述蛋白酶为Thrombin酶。
在另一优选例中，所述P4的序列为SEQ ID NO:2。
在另一优选例中，所述P4的序列为在SEQ ID NO:2的基础上进行一个或多个氨基酸的替换、缺失、改变、插入或增加，所得到的氨基酸序列。
在另一优选例中，所述式I的序列的N端或C端可添加1至30个氨基酸残基，较佳地1至10个氨基酸残基，更佳地1至5个氨基酸残基。
在另一优选例中，所述P1和P2之间、P2和P3之间，或P3和P4之间，可添加1至30个氨基酸残基，较佳地1至10个氨基酸残基，更佳地1至5个氨基酸残基。
在另一优选例中，所述融合蛋白的序列中，从N端到C端各元件之间的排序还包括：P2-P1-P3-P4、P4-P3-P2-P1、P2-P3-P4-P1或P4-P3-P1-P2。
在本发明的第二方面，提供了一种分离的多核苷酸，所述多核苷酸编码本发明第一方面所述的融合蛋白。
在另一优选例中，所述多核苷酸包括从5’到3’的式II的核酸构建物，
Z1-Z2-Z3-Z4   (式II)
式中，Z1、Z2、Z3和Z4分别编码本发明第一方面中式I中的P1、P2、P3和P4；
其中，所述Z2的序列为SEQ ID NO:3。
在另一优选例中，所述Z4的序列为SEQ ID NO:1。
在另一优选例中，所述多核苷酸的序列中，从5’到3’各元件之间的排序还包括：Z2-Z1-Z3-Z4、Z4-Z3-Z2-Z1、Z2-Z3-Z4-Z1或Z4-Z3-Z1-Z2。
在本发明的第三方面，提供了一种载体，所述载体含有本发明第二方面所述的多核苷酸。
在另一优选例中，所述载体为pETDuet-MarTX-1，所述pETDuet-MarTX-1为在载体pETDuet-1中插入序列SEQ ID NO:3所得。
在本发明的第四方面，提供了一种宿主细胞，所述宿主细胞含有本发明第三方面所述的载体或基因组中整合有本发明第二方面所述的多核苷酸。
在另一优选例中，所述宿主细胞为E.coli Origami B(DE3)。
在本发明第五方面，提供了一种产生本发明第一方面所述的融合蛋白的方法，包括步骤：
在适合表达的条件下，培养本发明第四方面所述的宿主细胞，从而表达出本发明第一方面所述的融合蛋白。
在本发明第六方面，提供了一种制备MarTX多肽的方法，包括步骤：
(i)用蛋白酶对本发明第一方面所述的融合蛋白酶切，从而获得酶切产物，该酶切产物对应于MarTX多肽；和
(ii)从酶切产物中分离或纯化出所述的MarTX多肽。
在另一优选例中，所述蛋白酶选自下组：HRV 3C蛋白酶、Thrombin酶、Factor Xa、肠激酶(Enterkinase)、TEV酶、Prescission、TAGZyme、SUMO蛋白酶，或其组合。
在另一优选例中，所述蛋白酶为Thrombin酶。
在另一优选例中，所述方法纯化得到的MarTX多肽，从每升表达菌体所得的产量为≥2mg，较佳地≥3mg，更佳地≥4mg。
在本发明第七方面，提供了一种(α+β4)BK通道电流抑制剂的药物筛选方法，包括步骤：
(i)使用本发明第六方面所述的方法，纯化制备MarTX多肽；
(ii)以MarTX多肽作为阳性对照，对(α+β4)BK通道电流抑制剂候选药物进行筛选。
在另一优选例中，所述步骤(ii)中，MarTX多肽阻断(α+β4)BK通道电流的半数效应浓度(EC50)值为C0，所述抑制剂候选药物阻断(α+β4)BK通道电流的半数效应浓度(EC50)值为C1，
当C1≤C0时，则所述抑制剂候选药物是优于MarTX多肽的(α+β4)BK通道抑制剂。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了表达载体pETDuet-1的物理图谱和多克隆位点。
图2显示了MarTX基因PCR过程及琼脂糖凝胶电泳。(A)PCR引物。上游引物(Primer-UP)的BamH I位点和下游引物(Primer-DO)的Xho I位点已用横线标 出。(B)PCR反应体系组成。(C)PCR反应程序。(D)反应完成后琼脂糖凝胶电泳图。
图3显示了表达载体pETDuet-1和Martentoxin基因的双酶切琼脂糖凝胶电泳图。
图中M表示DNA marker(DL5000)，从下至上为100bp，250bp，500bp，750bp，1000bp，1500bp，2000bp，3000bp，5000bp。泳道1为pETDuet-1载体经限制性内切酶EcoR I和Not I双酶切结果，泳道2为Martentoxin基因片段的EcoR I和Not I双酶切结果。
图4显示了重组表达载体pETDuet-1-MarTX图谱示意图。
图5显示了pGEX-4T-3-MarTX重组质粒及纯化凝胶色谱图：(A)pGEX-4T-3-MarTX表达质粒示意图；(B)MarTX Superdex 75峰图。
图6显示了pGEX-6P-1-MarTX重组质粒及纯化凝胶色谱图。(A)pGEX-6P-1-MarTX表达质粒示意图。(B)MarTX Superdex 75峰图。
图7显示了pSMT3-MarTX重组质粒及纯化凝胶色谱图。(A)pSMT3-MarTX表达质粒示意图。(B)MarTX Superdex 75峰图。
图8显示了pETDuet-1-MarTX重组质粒表达毒素的纯化。
(A)镍柱纯化结果。融合蛋白已用红线标出。(B)酶切后Amylose Resin层析柱纯化结果。(C)凝胶色谱柱Superdex 75紫外吸收峰图。(D)重组MarTX纯化后的SDS-PAGE检测。
图9显示了重组MarTX毒素的一维氢谱。
图10显示了重组MarTX毒素的圆二色谱。
图11显示了重组MarTX毒素的MALDI-TOF质谱鉴定。
图12显示了重组MarTX毒素的20个最优溶液结构叠加图(二级结构部分叠加)。多肽的N端、C端已标出。
图13显示了重组MarTX毒素20个最优溶液构象的拉氏图。
图14显示了重组MarTX毒素与天然毒素的结构叠加图。
红色表示重组MarTX，绿色表示已报道的MarTX结构(PDB:1M2S)。二硫键以及N端、C端已标出。
图15显示了重组MarTX毒素对(α+β4)BK通道电流的抑制效应。
(A)重组MarTX毒素对(α+β4)BK通道HEK293T的全细胞电流，钳制电位为-80mV，电流由+80mV的电压刺激和300nM的内钙诱发。(B)重组MarTX毒素抑制(α+β4)BK通道的剂量依赖曲线，I f表示各浓度毒素作用下的残余电流百分 比，曲线由Hill方程拟合。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，意外地开发出一种纯化制备MarTX多肽的方法。具体地，在MarTX的重组质粒的构建中，在目标蛋白所对应的基因片段的5’端，加入编码His标签以及编码MBP标签的基因序列，并且将构建得到的重组质粒转化至载体菌株E.coli Origami B(DE3)，并进行培养和诱导表达。对纯化得到的MarTX多肽的表征实验数据表明，用所述方法得到的目标蛋白，相比于对比实施方案所得的目标蛋白，具有更高的表达量、纯度和活性。在此基础上完成了本发明。
术语
Martentoxin蛋白及其编码序列
如本文所用，“Martentoxin蛋白”、“MarTX毒素”和“MarTX多肽”“重组MarTX毒素”可互换使用，指Martentoxin蛋白，其由37个氨基酸构成，具有三对二硫键，是一种特异性作用于BK通道的短链多肽毒素，可显著抑制神经型(α+β4)BK通道电流。
应理解，尽管本发明的实例中提供的基因来源于东亚钳蝎，但是来源于其它类似的物种(尤其是与东亚钳蝎属于同一科或属的蝎类)的、与本发明的序列(优选地，序列如SEQ ID NO:1所示)具有一定同源性(保守性)的MarTX的基因序列，也包括在本发明的范围内，只要本领域技术人员在阅读了本申请后根据本申请提供的信息可以方便地从其它物种(尤其是蝎类)中分离得到该序列。
本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括：DNA、基因组DNA或人工合成的DNA，DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NO:1所示的编码区序列相同或者是简并的变异体。
编码成熟多肽的多核苷酸包括:只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是 还包括附加编码和/或非编码序列的多核苷酸。本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多苷或多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的多肽的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酞胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。
应理解，虽然本发明的MarTX基因优选来自东亚钳蝎，但是来自其它物种(尤其是蝎类)的与东亚钳蝎MarTX基因高度同源(如具有80％以上，如85％,90％,95％甚至98％序列相同性)的其它基因也在本发明考虑的范围之内。比对序列相同性的方法和工具也是本领域周知的，例如BLAST。
本发明的MarTX核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的DNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或 如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明涉及一种用于治疗癫痫的MarTX多肽及其变体，在本发明的一个优选例中，所述多肽的氨基酸序列如SEQ ID NO:2所示。本发明的多肽能够有效治疗和/或预防癫痫。
本发明还包括与本发明的SEQ ID NO:2所示序列具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％)同源性的具有相同或相似功能的多肽或蛋白。
所述“相同或相似功能”主要是指：“缓解癫痫的症状”。
本发明的多肽可以是重组多肽、天然多肽、合成多肽。本发明的多肽可以是天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主，本发明的多肽可以是糖基化的，或可以是非糖基化的。本发明的多肽还可包括或不包括起始的甲硫氨酸残基。
本发明还包括具有MarTX多肽活性的MarTX多肽片段和类似物。如本文所用，术语“片段”和“类似物”是指基本上保持本发明的天然MarTX多肽相同的生物学功能或活性的多肽。
本发明的多肽片段、衍生物或类似物可以是：(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的；或(ii)在一个或多个氨基酸残基中具有取代基团的多肽；或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽；或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或融合蛋白)。根据本文的定义这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明中，所述的多肽变体是如SEQ ID NO.:2所示的氨基酸序列，经过若干个(通常为1-10个，较佳地1-8个，更佳地1-4个，最佳地1-2个)取代、缺失或添加至少一个氨基酸所得的衍生序列，以及在C末端和/或N末端添加一个或数个(通常为10个以内，较佳地为5个以内，更佳地为3个以内)氨基酸。例如，在 所述蛋白中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能，在C末端和/或N末端添加一个或数个(如1-3个)氨基酸通常也不会改变蛋白质的功能。这些保守性变异最好根据表1进行替换而产生。
表1
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还包括所要求保护的蛋白的类似物。这些类似物与天然SEQ ID NO:2差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些蛋白的类似物包括天然或诱导的遗传变异体。诱导变异体 可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分子生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的蛋白并不限于上述列举的代表性的蛋白。
修饰(通常不改变一级结构)形式包括：体内或体外蛋白的化学衍生形式如乙酸化或羧基化。修饰还包括糖基化，如那些在蛋白质合成和加工中进行糖基化修饰。这种修饰可以通过将蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。
本发明融合蛋白
本发明提供了一种融合蛋白，其特征在于，所述融合蛋白从N端到C端具有式I所示结构：
P1-P2-P3-P4   (式I)
式中，
P1为纯化标签元件；
P2为麦芽糖结合蛋白元件(MBP)；
P3为蛋白酶识别位点元件；
P4为MarTX多肽元件；
“-”表示连接上述元件的肽键。
在一个优选的实施方式中，所述的融合蛋白可不包括所述P1的纯化标签元件，仅利用所述P2的麦芽糖结合蛋白元件也可进行Amylose Resin层析柱的纯化。即包括或不包括所述P1元件的差别仅在于纯化的效率有所差异，但无论包括或不包括所述P1元件，本发明的融合蛋白在本发明宿主细胞中的表达量以及所得蛋白的构象稳定性均显著优于现有技术中已有的重组MarTX毒素的其它形式。
在一个优选的实施方案中，所述蛋白酶选自下组：HRV 3C蛋白酶、Thrombin酶、Factor Xa、肠激酶(Enterkinase)、TEV酶、Prescission、TAGZyme、SUMO 蛋白酶，或其组合。在另一优选例中，所述蛋白酶为Thrombin酶。
在其它优选的实施方式中，所述融合蛋白的序列还包括：P2-P1-P3-P4、P4-P3-P2-P1、P2-P3-P4-P1或P4-P3-P1-P2。即，His标签、MBP标签以及MarTX多肽的序列之间的顺序可灵活变动，只需保证所述P3元件所对应的酶切位点紧邻MarTX多肽，并位于MarTX多肽与MBP标签之间，使得经过蛋白酶酶切以后的MarTX多肽中，不包含P2所述的MBP标签即可。而最终所得的MarTX多肽是可以包含His标签的，因为His标签仅为6和His残基，其存在不会影响MarTX多肽的活性和结构。
本发明制备方法
本发明提供的纯化制备MarTX多肽的方法，包括步骤：
(i)用蛋白酶对本发明第一方面所述的融合蛋白酶切，从而获得酶切产物，该酶切产物对应于MarTX多肽；和
(ii)从酶切产物中分离或纯化出所述的MarTX多肽。
在本发明中，对于所述融合蛋白的表达，是在载体菌株E.coli Origami B(DE3)中进行的，其培养和诱导表达的条件，均为该菌株的培养和表达蛋白的常规条件。
在一个优选的实施方式中，所述的P1为His标签，其用于将所述融合蛋白用镍柱进行亲和层析。
在一个优选的实施方式中，所述的P2与MarTX多肽序列中存在一段Thrombin的酶切位点，使用6U/mL的Thrombin酶进行过夜酶切，酶切同时使蛋白溶液在3.5kDa透析袋中搅拌过夜。
在一个优选的实施方式中，所述的P2为MBP标签，其用于将融合蛋白酶切产物用Amylose Resin层析柱进行纯化。在这一步骤中，是将切下来的P1-P2结合在亲和层析柱上，而将目标蛋白MarTX多肽穿流。收集穿流成分，从而获得不带标签的MarTX多肽。
在一个优选的实施方式中，还包括凝胶色谱柱进行纯化的步骤，即分子筛。在这一步骤中，收集保留体积为110mL所有的洗脱峰，即为不带标签的MarTX多肽蛋白峰。经这一步骤纯化后，可获得纯度较纯的MarTX多肽。
本发明的主要优点包括：
1)相比其他表达质粒和纯化方法，本发明获得的重组MarTX毒素产量较高，2L菌体可得到约3mg多肽，相比于现有的其它MarTX毒素表达纯化技术，产量提高了近10倍。
2)本发明方法制备所得的MarTX多肽，纯度高、各方面理化性质正确，同时具有电生理活性。由于天然MarTX毒素产量低，获得困难，因此可运用该专利获得的重组MarTX毒素替代天然MarTX毒素进行对BK通道药理机制等的研究，具有重大意义。
3)本发明的重组表达法，克服了MarTX毒素由于多肽分子量较小，且含多对二硫键易导致包涵体表达，从而较难获得大量的、可溶的、高纯度蛋白的缺陷。
4)本发明方法实现了MarTX多肽的绿色制造，可为类似多肽药物的生产制备提供新思路，成为药物合成和精细化学品领域的研究热点。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
如无特别说明，实施例所用的材料和试剂均为市售产品。
实施例1：MarTX重组质粒构建
MarTX(Martentoxin)质粒由上海大学吉永华课题组提供，共37个氨基酸，通过ProtParam在线软件对MarTX进行预测，相对分子量约为4.065kDa，理论pI值为8.65。基因片段位于pGEX-4T-3载体的BamH I和Sma I克隆位点之间，融合表达的GST标签与目的多肽之间存在肠激酶酶切位点(图5)。
宿主菌E.coli DH5α、BL21(DE3)和Origami B(DE3)菌种均购自Novagen公司，后均由本实验室自主制备。
1.1 pGEX-6P-1-MarTX重组质粒的构建
a)PCR反应
首先设计PCR引物，在MarTX基因片段的5’端和3’端添加BamH I和Xho I酶切位点和保护碱基，以pGEX-4T-3-MarTX重组质粒为模版进行PCR反应，完成后通过1％的琼脂糖凝胶电泳鉴定。电泳图显示目的条带明亮单一，PCR成功(图2)。随后进行目的基因片段的割胶回收工作。
b)pGEX-6P-1载体和基因片段的双酶切、连接反应
分别取25μL PCR回收产物和pGEX-6P-1载体溶液，加入Takara公司的3μL10×K Buffer，BamH I和Xho I内切酶各1μL组成30μL双酶切体系，于37℃水浴锅中酶切2小时，之后进行1％琼脂糖凝胶电泳鉴定并割胶回收。
在200μL灭菌EP管中加入双酶切后的载体0.5μL，MarTX切后片段4.5μL，加入T4连接酶1μL，ddH 2O 3μL组成10μL连接体系，置于16℃金属浴中连接过夜。
c)转化、测序鉴定
连接完成后，转入扩增菌株E.coli DH5α感受态中，转化步骤如下：
1)取出-80℃保存的高效感受态细胞DH5α100μL，置于冰上缓慢解冻10min；
2)取5-10μL连接反应液加至感受态细胞中，轻轻吹打混匀，置于冰上30min；
3)42℃水浴热激90s，迅速置于冰上2-3min；
4)加入800μL LB培养液至管中，于37℃190rpm振荡培养40min；
5)3000rpm离心10min，弃去大部分培养液，使细胞在余下的培养液中悬浮，接种至含相应抗生素的固体LB培养基上，涂布均匀；
6)该固体培养皿于37℃培养箱倒置过夜，一般培养14-16h。
次日挑取大小均一的菌落进行培养，菌液浑浊后送上海铂尚生物技术有限公司进行测序鉴定。经过以上步骤我们成功构建了pGEX-6P-1-MarTX重组质粒。
1.2 pSMT3-MarTX重组质粒的构建
对于pSMT3-MarTX重组质粒，将毒素的基因片段插入BamH I和Xho I之间，这与pGEX-6P-1-MarTX重组质粒的克隆位点相同。因此，通过相同的操作步骤： PCR反应、pSMT3载体和基因片段的双酶切(BamH I和Xho I内切酶)、连接反应以及转化、测序鉴定成功构建了pSMT3-MarTX重组质粒。
1.3 pETDuet-1-MarTX重组质粒的构建
pETDuet-1质粒中存在两个多克隆位点MCS1和MCS2。将编码麦芽糖结合蛋白的MBP标签构建于MCS1的Nco I和EcoR I位点之间，同时于MBP标签的N端设计6个His-tag。首先通过PCR反应将MarTX基因片段的5’端加入Thrombin酶切位点和EcoR I克隆位点，于3’端加入Not I克隆位点，引物设计如表2所示。
表2 pETDuet-1-MarTX重组质粒的引物设计
Figure PCTCN2019115709-appb-000001
按照1.1的步骤，以pGEX-4T-3-MarTX重组质粒为模版完成PCR反应，然后进行pETDuet-1质粒和MarTX基因片段的双酶切(EcoR I和Not I内切酶)、连接反应以及转化、测序鉴定，成功构建了pETDuet-1-MarTX重组质粒。
实施例2：重组MarTX毒素的原核表达
对于pETDuet-1-MarTX重组质粒，将其转入E.coli Origami B(DE3)表达菌株中进行表达。本发明选用E.coli Origami B(DE3)作为表达菌株。
步骤如下：
制备E.coli Origami B(DE3)感受态细胞，利用热休克法(42℃热激90秒)将测序正确的重组表达载体pETDuet-1-MarTX转化至Origami B(DE3)中，得到含有重组质粒的表达菌株。高效原核表达的步骤如下：
1)从平板上挑取表达菌株单克隆接种至含5mL LB培养基(含氨苄抗生素)的试管中，37℃、220rpm培养过夜；
2)次日将5mL摇浑的菌液接入100mL(含氨苄抗生素)中瓶LB培养液中， 37℃、220rpm振荡培养3h；
3)取其中20mL转接入1L(含氨苄抗生素)LB培养液中37℃220rpm培养至OD 600达0.6-0.8左右时，用冰水浴冷却，加入IPTG至终浓度为0.5mM，于18℃220rpm诱导表达；
4)表达20h后，18℃、5000rpm离心15min收集菌体弃上清，-80℃保存待用。
同样地，将pGEX-4T-3-MarTX重组质粒转入E.coli BL21(DE3)宿主细胞中进行表达；对于pGEX-6P-1-MarTX重组质粒，将其转入E.coli BL21(DE3)表达菌株中进行表达；对于pSMT3-MarTX重组质粒，将该质粒转入E.coli BL21(DE3)感受态细胞中进行表达。
实施例3：重组MarTX毒素的纯化制备
3.1纯化步骤
对于pETDuet-1-MarTX重组质粒表达的融合蛋白，即MBP标签重组MarTX多肽，每升菌体加入15mL破菌Buffer A(成分详见表2)，重悬均匀后加入蛋白酶抑制剂PMSF至终浓度为0.1mM。用高压破菌仪降温至4℃左右破菌，压力设置1000bar左右，重复破菌3次。将破菌液收集至离心管中，4℃16000rpm离心50min后，收集含重组融合蛋白的上清。
具体纯化方法步骤如下：
1)处于Buffer A环境中的融合蛋白先与镍柱结合，然后用Buffer B咪唑盐溶液进行梯度洗脱除去大部分杂蛋白，完成第一根亲和柱纯化并进行SDS-PAGE电泳检测，参见附图8A；
2)收集目的融合蛋白(约46.8kDa)于18℃、2L Buffer C中进行透析酶切，按照6U/mL加入Thrombin酶，于3.5kDa透析袋中搅拌酶切过夜；
3)随后用恒流泵将酶切混合物加载至Amylose Resin层析柱上，收集穿流成分(FL)和Buffer D洗脱成分。His-MBP-tag经Buffer E洗脱除去，完成第二根亲和柱纯化并进行SDS-PAGE电泳检测，参见附图8B；
4)然后将FL成分和Buffer D洗涤液(通常含有少量重组MarTX毒素)浓缩至2mL进行下一步凝胶色谱柱纯化；
5)凝胶色谱柱Superdex 75提前用Buffer F平衡，然后将样品加载到柱子上，用Buffer F进行洗脱。收集保留体积为110mL左右的洗脱峰即为重组MarTX毒素样品，之前的吸收峰为MBP-tag或未切开的融合蛋白。紫外吸收峰图参见附图8C；
6)SDS-PAGE检测纯化效果,参见附图8D。纯化过程中的相关缓冲液组分如表3所示。
表3重组MarTX毒素纯化过程中的相关缓冲液
Figure PCTCN2019115709-appb-000002
对实施例1中构建的其它重组质粒所表达的重组蛋白，采用类似相应的纯化方式。
对于pGEX-4T-3-MarTX重组质粒表达的融合蛋白，带有GST标签，因此纯化时先将蛋白与GST柱结合，然后用还原型谷胱甘肽溶液进行梯度洗脱除去大部分杂蛋白。之后于室温下进行肠激酶酶切去除GST标签，再进行第二次GST柱和一次凝胶色谱柱纯化，从而得到MarTX多肽。
对于pGEX-6P-1-MarTX重组质粒表达的融合蛋白，带有GST标签，纯化时先将蛋白与GST柱结合，然后用还原型谷胱甘肽溶液进行梯度洗脱除去大部分杂蛋白。之后于4℃下进行3C酶酶切将多肽与GST标签分开，再进行第二次GST柱和一次凝胶色谱柱纯化，从而得到MarTX多肽。
对于pSMT3-MarTX重组质粒表达的融合蛋白，带有His标签，因此纯化时先将蛋白与镍柱结合，然后用咪唑盐溶液进行梯度洗脱除去大部分杂蛋白。之 后于4℃下进行UlpI酶酶切，再进行第二次镍柱纯化和一次凝胶色谱柱纯化，从而得到MarTX多肽。
3.2纯化结果
对于pGEX-4T-3-MarTX重组质粒表达的融合蛋白，经过不同诱导温度和时间的摸索(18℃诱导20h或28℃诱导5h)，以及尝试了不同厂家的肠激酶，都未能获得足够量的MarTX进行后续的实验研究，2L菌体最多只能得到约0.3mg多肽，表达量太低(图5)。因此需要进一步优化重组质粒，尝试将其基因片段构建于其他载体中，并对表达纯化方法进行改进。
对于pGEX-6P-1-MarTX重组质粒表达的融合蛋白，我们发现3C酶不易将融合蛋白的GST标签与毒素切开，酶切后得到的MarTX仍较少，实验失败(图6)。
对于pSMT3-MarTX重组质粒表达的融合蛋白，经过第一次镍柱纯化、4℃条件下UlpI酶酶切、第二次镍柱纯化和凝胶色谱柱纯化，该重组质粒也未能得到目的毒素，实验再一次失败(图7)。
然而，对于pETDuet-1-MarTX重组质粒表达的融合蛋白，经以上表达及纯化后，获得了产量和纯度均较高的重组MarTX毒素，2L LB培养基的菌体可得到约3mg多肽，参见附图8C。相比上海大学提供的pGEX-4T-3-MarTX表达纯化方案，2L菌体可得到约3mg多肽，产量提高了近10倍，满足了后续实验毒素的样品需求。
实施例4：重组MarTX毒素的表征
为了进一步确认得到的重组MarTX毒素是否正确，对重组毒素的分子量以及二级结构进行了验证，同时利用一维氢谱考察了毒素在溶液中的折叠情况。
4.1重组MarTX毒素的 1H NMR
重组MarTX浓度为0.42mM，于25mM NaH 2PO4，100mM NaCl，pH 6.80的Buffer中加入10％的D 2O，在600MHz核磁谱仪上采集一维氢谱，采样温度为20℃，实验谱图参见附图9。
获得的重组MarTX毒素的核磁信号比较发散，在高场区附近有明显的甲基特征峰，且毒素的酰胺氢和芳环氢于6.5-10.5ppm的低场区分布广泛，说明多肽未发生聚集，空间结构折叠良好。
4.2重组MarTX毒素的圆二色谱
为了进一步对毒素的二级结构进行验证，测定了重组MarTX的圆二色谱(CD光谱)。将毒素的Buffer条件交换至ddH 2O中，样品浓度为0.5mg/mL，实验结果参见附图10。
由此可知，本发明的重组毒素包含已发表结构的α螺旋和β折叠二级结构元件，说明折叠正常。
4.3重组MarTX毒素的质量测定
将纯化后的毒素样品Buffer条件交换至ddH 2O中，经MALDI-TOF测定，重组MarTX的分子量为4200.5444，参见附图11。重组MarTX经Thrombin酶切后其N端多GS两个氨基酸，理论分子量为4208.80，因此测定的分子量与理论值大致相当。
4.4重组MarTX毒素的溶液结构解析
对重组MarTX样品进行了二维NMR谱图采集。实验于Aligent 600MHz谱仪上进行，样品浓度为1mM，采样温度为20℃，采样Buffer为25mM NaH 2PO 4，100mM NaCl，10％D 2O，pH 6.80。采集的二维同核NMR谱图如表4所示。
表4重组MarTX毒素谱图采集及参数设置
Experiment Number of increments SW(Hz) Carrier frequency(ppm)
DQF_COSY 1000(H)×256(H) 6906(H)×6906(H) 4.82(H)×4.82(H)
TOCSY 1024(H)×256(H) 9579(H)×6010(H) 4.82(H)×4.82(H)
NOESY 1024(H)×256(H) 12019(H)×9597(H) 4.82(H)×4.82(H)
对以上谱图进行信号归属，然后进行结构计算和精修得到100个溶液结构。从100个精修结构中按照能量排序选出20个能量最低的结构作为重组MarTX毒素的代表性构象，对20个最优溶液结构进行叠加，结果表明除了毒素中比较灵活的loop外，其余二级结构部分相对收敛，参见附图12。
运用PROCHECK_NMR软件对重组MarTX毒素的20个最优溶液构象进行评估，参见附图13。Ramachandran图分析结果显示其
Figure PCTCN2019115709-appb-000003
角和ψ角都处于可接受区域，20个最优构象平均有91.8％的残基处在最允许区域，4.4％的残基处在次允许区域，没有残基处于不允许区域，表明结构合理。
将得到的结构与已报道的天然MarTX(PDB：1M2S)溶液结构进行叠加，RMSD值为
Figure PCTCN2019115709-appb-000004
参见附图14。表明核磁解析的重组MarTX毒素结构与从东亚钳蝎毒液中提取的天然毒素结构一致，说明通过本发明的原核表达体系成功制备得到了MarTX毒素，可用于下一步对毒素活性的考察中。
实施例5：重组MarTX毒素对(α+β4)BK通道电流的抑制
文献[Biophys.J.2008,94,3706-3713]表明MarTX毒素与BK通道β4辅助亚基的胞外loop区相互作用，从而抑制了(α+β4)BK通道电流。本发明通过膜片钳实验考查了重组MarTX毒素对(α+β4)BK通道的电生理活性。
实验步骤：人类胚胎肾细胞(HEK293T)在含10％热灭活的FBS的DMEM(Gibco公司，美国)培养液中培养，细胞在培养皿中的生长条件为5％CO 2、恒温37℃、恒湿95％。采用瞬时转染技术，通过脂质体转染法将人源(α+β4)BK通道质粒(含hSloα(U23767)、β4(KCNMB4；AF207992)的基因序列)转入HEK293细胞中表达。
在室温(21℃-25℃)下利用EPC-9放大器(HEKA Eletronik，德国)进行全细胞电压膜片钳实验。实验中细胞外液为NaCl 135mM，KCl 5mM，MgCl 2 1.2mM，CdCl 2 2.5mM，HEPES 5mM，glucose 10mM(以NaOH调节pH至7.4)；电极内液为NaCl 10mM，KCl 117mM，MgSO 4 2mM，HEPES 10mM，MgATP 2mM，EGTA 1mM(以KOH调节pH至7.2)。
数据处理：数据通过PulseFit 8.5(HEKA Eletronik，Germany)和Origin 8.5(Northampton公司，美国)进行分析。实验结果用平均值±标准误差(Mean±S.E.M)表示，n为实验细胞个数。Fraction of current为加入药物(重组MarTX毒素)后BK通道的稳态电流与未加入药物时对照电流的比值。
实验结果：经以上膜片钳实验测定发现，利用本发明发展的原核重组表达系统，获得的重组MarTX毒素具有电生理活性，参见附图15A。10μM重组MarTX毒素可显著抑制(α+β4)BK通道电流，I f(Fraction of current)＝0.33±0.06，n＝6，与文献报道的药理学性质相符。
实施例6：重组MarTX毒素阻断(α+β4)BK通道半数效应浓度的测定
10μM重组MarTX毒素可有效抑制由(α+β4)BK通道产生的电流，且具有剂量依赖性特点，参见附图15B。量效曲线显示的重组MarTX毒素阻断(α+β4)BK通道的半数效应浓度(EC 50)为0.32±0.012μM。
由此可得，经本发明原核表达纯化得到的重组MarTX毒素可实现对(α+β4)BK通道电流的有效阻断，可作为神经型BK通道的抑制剂，用于与神经型BK通道功能相关的癫痫等疾病的研究。
讨论
在本发明的实施例中，有三组进行平行实验的对比例，具体地，分别将MarTX基因整合至载体质粒pGEX-4T-3(其表达GST标签)、pGEX-6P-1(其表达GST标签)和pSMT3(其表达His标签和SUMO标签)中，并分别在E.coli BL21(DE3)中进行表达。
根据其分别的表达和纯化的结果(参见实施例3)，三组对比例相较于本发明融合蛋白的纯化结果，得到的蛋白产量低或纯度低。
分析其原因，对于pGEX-4T-3-MarTX和pGEX-6P-1-MarTX，均含有GST标签，GST标签分子量约26kDa，而目的毒素只有约4kDa，并且由于GST标签的结构特征，可能将蛋白酶酶切位点及多肽包裹其中，使蛋白酶无法实现酶切功能，从而导致酶切后的目的多肽量极少。对于pSMT3-MarTX，其表达量就已经很少，即在表达步骤就已经失败。
总结对比例效果较差的原因，本发明人推测可能因为MarTX毒素分子量较小，不易与融合标签切开。且毒素中含有三对二硫键，不易重构正确。而二硫键蛋白表达系统有利于二硫键的正确折叠。Origami B(DE3)属于大肠杆菌克隆型菌种，含有trxB/gor基因突变。同时突变这两个基因使得该菌株能够更加高效地在细胞质内生成二硫键，有助于含二硫键的活性蛋白形成。
因此，经过了大量的尝试和摸索，本发明人最终开发出了本发明的MarTX多肽制备方法，使用本发明所述的重组质粒，并使用所述E.coli Origami B(DE3)菌株，成功获得产量和纯度均较高的MarTX毒素样品，克服了MarTX毒素由于多肽分子量较小，且含多对二硫键易导致包涵体表达，从而较难获得大量的、可溶的、高纯度蛋白的缺陷。本发明的技术方案相比现有技术中已有的MarTX 毒素的表达纯化方案，2L菌体可得到约3mg多肽，产量提高了近10倍，实现了MarTX多肽的高效绿色制造。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (14)

  1. 一种融合蛋白，其特征在于，所述融合蛋白从N端到C端具有式I所示结构：
    P1-P2-P3-P4     (式I)
    式中，
    P1为纯化标签元件；
    P2为麦芽糖结合蛋白元件(MBP)；
    P3为蛋白酶识别位点元件；
    P4为MarTX多肽元件；
    “-”表示连接上述元件的肽键。
  2. 如权利要求1所述的融合蛋白，其特征在于，所述P2的序列为SEQ ID NO:4。
  3. 如权利要求1所述的融合蛋白，其特征在于，所述P4的序列为SEQ ID NO:2。
  4. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码权利要求1至3中任一项所述的融合蛋白。
  5. 如权利要求4所述的多核苷酸，其特征在于，所述多核苷酸包括从5’到3’的式II的核酸构建物，
    Z1-Z2-Z3-Z4     (式II)
    式中，Z1、Z2、Z3和Z4分别编码本发明第一方面中式I中的P1、P2、P3和P4；
    其中，所述Z2的序列为SEQ ID NO:3。
  6. 如权利要求5所述的多核苷酸，其特征在于，所述Z4的序列为SEQ ID NO:1。
  7. 一种载体，其特征在于，所述载体含有权利要求4所述的多核苷酸。
  8. 如权利要求7所述的载体，其特征在于，所述载体为pETDuet-MarTX-1，所述pETDuet-MarTX-1为在载体pETDuet-1中插入序列SEQ ID NO:3所得。
  9. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求7所述的载体或基因组中整合有权利要求4所述的多核苷酸。
  10. 如权利要求9所述的宿主细胞，其特征在于，所述宿主细胞为E.coli Origami B(DE3)。
  11. 一种产生权利要求1至3中任一项所述的融合蛋白的方法，其特征在于，包括步骤：
    在适合表达的条件下，培养权利要求9所述的宿主细胞，从而表达出权利要求1所述的融合蛋白。
  12. 一种制备MarTX多肽的方法，其特征在于，包括步骤：
    (i)用蛋白酶对权利要求1所述的融合蛋白酶切，从而获得酶切产物，该酶切产物对应于MarTX多肽；和
    (ii)从酶切产物中分离或纯化出所述的MarTX多肽。
  13. 一种(α+β4)BK通道电流抑制剂的药物筛选方法，其特征在于，包括步骤：
    (i)使用权利要求12所述的方法，纯化制备MarTX多肽；
    (ii)以MarTX多肽作为阳性对照，对(α+β4)BK通道电流抑制剂候选药物进行筛选。
  14. 如权利要求13所述的药物筛选方法，其特征在于，所述步骤(ii)中，MarTX多肽阻断(α+β4)BK通道电流的半数效应浓度(EC50)值为C0，所述抑制剂候选药物阻断(α+β4)BK通道电流的半数效应浓度(EC50)值为C1，
    当C1≤C0时，则所述抑制剂候选药物是优于MarTX多肽的(α+β4)BK通道抑制剂。
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