CN108117599A

CN108117599A - The recombination expression and purification process of Ssm6a and its fusion protein used

Info

Publication number: CN108117599A
Application number: CN201810004866.8A
Authority: CN
Inventors: 原桂勇; 殷海兴; 王凯健; 朱冠军; 谭莹莹
Original assignee: Haimen Mike Alan Biology And Medicine Co Ltd
Current assignee: Haimen Mike Alan Biology And Medicine Co Ltd
Priority date: 2018-01-03
Filing date: 2018-01-03
Publication date: 2018-06-05

Abstract

The extensive recombination expression of polypeptide more the present invention relates to the recombination expression of polypeptide and purifying more particularly to amino acid number and rich in disulfide bond and purifying.The present invention provides a kind of fusion protein of molecular chaperones purification tag inscribe cleavage sites toxin polypeptide Ssm6a, and it includes SEQ ID NO:1 and its amino acid sequence of variant.The present invention also provides a kind of methods recombinantly expressed with toxin polypeptide Ssm6a of the purified source in Scolopendra subspinipes venom.

Description

The recombination expression and purification process of Ssm6a and its fusion protein used

Technical field

It is more the present invention relates to the recombination expression of polypeptide and purifying more particularly to amino acid number and more rich in disulfide bond The extensive recombination expression of peptide and purifying.

Background technology

Chronic ache affects the elderly population of the Adult Groups and 50% more than 20%, greatly reduces the life matter of patient Amount, while also various countries are constituted with huge medical burden.Treatment currently for chronic ache mainly includes the use of non-steroidal Anti-inflammatory agent and opioid drug.Although non-steroidal anti-inflammatory drugs worldwide widely uses, analgesia scope is smaller, and deposits In adverse reactions such as relatively common gastrointestinal tracts and lesions of liver and kidney；And although opioid drug analgesic effect is fine, exist into Addiction causes serious adverse reactions and constipation and the stronger drug withdrawal disorders such as unreal and Heart and lungs injury.Meanwhile also exist at present Much without the other kinds of pain of effective medicine, such as neuropathic pain after nervous system injury, chronic visceral Pain, fibromyalgia, cancer pain and the various pain that tolerance is generated to opioid drug.These present situations are all to new analgesia Medicine exploitation proposes stern challenge.

Voltage-gated sodium channels (Nav) Nav1.7 great expressions are in Primary Sensory Neuron and sympathetic ganglia neural Member by controlling the threshold value of film potential, plays a significant role in pain signal initial period.Its selective depressant almost can be with For various types of pain.The best Nav1.7 inhibitors of ion channels of selectivity is derived from Scolopendra subspinipes at present Toxin polypeptide Ssm6a (the Discovery of a of (Scolopendra subspinipes mutilans) venom selective NaV1.7inhibitor from centipede venom with analgesic efficacy exceeding morphine in rodent pain models.PNAS,2013,110(43):17534-17539.Fusion of Ssm6a with a protein scaffold retains selectivity on NaV1.7and improves its therapeutic potential against chronic pain.Chemical Biology & Drug Design,2017,89(6):852-833.), therefore with very great development.Chinese invention patent (Application No. CN201210453532.1 the analgesic activities of the polypeptide) are had reported.

But the analgesic polypeptide common configuration in the zootoxin such as Ssm6a source is complicated, amino acid number is more, rich in two Sulfide linkage causes renaturation in vitro difficulty high.Therefore, such analgesic polypeptide is difficult the genetic recombination table by chemical synthesis or routine It is obtained up to way of purification, relies only on live body extraction and obtain.At present, by artificial synthesized, it can largely prepare Ssm6a's There is not been reported for method.Also it is only to pass through molecular sieve and reversed phase high efficiency from Scolopendra subspinipes venom in above-mentioned Chinese invention patent Liquid chromatogram isolates and purifies the Ssm6a purified on a small quantity.

Meanwhile for the more complicated toxin polypeptide of this class formation, the synthesis technology manually largely prepared (is closed including chemistry Into and recombination expression) generally do not have general applicability.Meanwhile the purification process of this kind of toxin polypeptide generally uses reversed phase high efficiency Liquid chromatogram, pollution is larger, cost is also higher.

For example, a kind of fusion protein of conotoxin M VII A and Trx is disclosed in Chinese patent application 03142248.9 Pronuclear recombination expression and purification process, the omega-conotoxin M VII A being directed to is made of 25 amino acid, wherein including 6 Cys residues formed three pairs of disulfide bond.Although sulphur oxygen is used also in wherein disclosed pronuclear recombination expression and purification process Albumen and enterokinase, but still fail to realize that product directly and correctly expresses the additional renaturation in vitro step, it is necessary to afterwards Active conotoxin could really be obtained.

The content of the invention

Although from Scolopendra subspinipes venom toxin polypeptide Ssm6a have good target spot selectivity, be expected to exploitation into Antalgesic of new generation has preferable application prospect.But on the one hand the toxin polypeptide is difficult to through conventional chemical synthesis and again It is prepared by group expression way (artificial synthesized and purifying).It can not be closed in the prior art on it by chemical synthesis or manually Into and purifying introduction；Another aspect Scolopendra subspinipes venom origin is limited, complicated component, and toxin polypeptide is extracted from the natural origin The technology difficulty of Ssm6a is very big, of high cost.Therefore the utilization of analgesic polypeptide Ssm6a are limited by very large at present, it can not Meet the huge needs for being used to prepare pain therapy drug.

The object of the present invention is to provide a kind of fusion protein comprising molecular chaperones and toxin polypeptide Ssm6a, wherein described Molecular chaperones promotes the correct folding of disulfide bond in toxin polypeptide Ssm6a, has the ripe toxin polypeptide Ssm6a finally obtained Natural bioactive.

In some embodiments, the amino acid sequence of above-mentioned toxin polypeptide Ssm6a is SEQ ID NO:3rd, its natural change The amino acid sequence of body or artificial variants, wherein fusion protein are SEQ IN NO:1 or its variant.

In some embodiments, above-mentioned toxin polypeptide Ssm6a derives from Scolopendra subspinipes venom.

It is a further object of the present invention to provide the nucleotide sequences for encoding above-mentioned fusion protein；Include its carrier；Comprising The host cell of the carrier.

It is a further object of the present invention to provide above-mentioned fusion proteins to prepare for alleviating or treat in the drug of pain Purposes.

It is a further object of the present invention to provide a kind of recombination expression and purified source in the toxin polypeptide of Scolopendra subspinipes venom The method of Ssm6a, includes the following steps：

(i) structure includes the amalgamation and expression gene of the fusion protein of the coding present invention；

(ii) the amalgamation and expression gene obtained in step (i) is inserted into expression plasmid；Convert host cell；In suitable item Under part, in host cell expression；

(iii) isolation and purification toxin polypeptide Ssm6a,

In some embodiments, above-mentioned host cell is preferably Bacillus coli cells, more preferably e. coli bl21 (DE3) cell；Above-mentioned expression plasmid is preferably pET28a.

The above-mentioned Ssm6a recombination expressions and purification process of the present invention can simply expand life according to the amount of required product Production scale.Meanwhile compared with chemical synthesis, method of the invention need not use expensive modified amino acid, and be not required to completely The reverse phase purification process that must be used in most of polypeptide customary preparation methods is used, avoiding acetonitrile, (at present prepared by polypeptide Almost indispensable reagent, it is expensive, toxicity is larger, it is necessary to be recycled) etc. chemical solvents use, toxicity will not be caused The generation of waste liquid is learned, has greatly saved production cost and subsequent Environmental costs.

As it can be seen that the present invention compensates for technological gap of the prior art, a kind of simple economy, environmentally protective complete is provided New Ssm6a recombination expressions and purification process.

It is further illustrated the present invention in following attached drawing and specific embodiment.However, these attached drawings and specific implementation Scheme should not be considered limiting the scope of the present invention, and the change that those skilled in the art are readily apparent that is included within the present invention Spirit and appended claims protection domain in.

Description of the drawings

Fig. 1 is the schematic diagram of the fusion protein of the present invention.

Fig. 2 is expressing fusion protein of the present invention as a result, swimming lane 1 is total protein, and swimming lane 2 is soluble protein, and swimming lane 3 is heavy Shallow lake albumen, wherein fusion protein (thioredoxin -6 × His-tag- enterokinase cleavage sites point-Ssm6a) basic soluble-expression, Gray scale scanning, which is shown, accounts for total protein 30%.

Fig. 3 a are the sds polyacrylamide gel electrophoresis figures of toxin polypeptide Ssm6a prepared by the present invention：With simple target Band；Fig. 3 b are the HPLC test maps of toxin polypeptide Ssm6a prepared by the present invention, and target product is simple spike, and purity reaches 99.3%.

Fig. 4 is the MALDI-TOF Mass Spectrometric Identification collection of illustrative plates of toxin polypeptide Ssm6a prepared by the present invention, measures molecular weight and is 5318.33。

Fig. 5 a be small ubiquitin modified protein expressing fusion protein as a result, swimming lane 1 is total protein, swimming lane 2 is soluble protein, Swimming lane 3 is protein precipitation, and wherein fusion protein (the small ubiquitin modified protein-Ssm6a of 6 × His-tag-) soluble-expression, gray scale is swept It retouches display target fusion protein and accounts for total protein 5%；Fig. 5 b are cut for fusion protein by SUMO protease, HPLC after peptide purification Testing result is shown as mixed and disorderly false folding and condensate, without correct folded product.

Specific embodiment

The present invention will be described in detail with reference to embodiments.All experiment reagents used and instrument and equipment, such as nothing Special instruction is common commercial reagent and equipment.

Unless otherwise defined, whole terms used herein have general technical staff of the technical field of the invention Normally understood meaning, when using the definition of the following term in part, term used in the singular can also include plural number, And vice versa.The definition of part term given herein is merely to description specific embodiment, it is not intended that limitation.

On the one hand, the present invention provides fusion protein (also referred to as " this hairs comprising molecular chaperones and toxin polypeptide Ssm6a Bright fusion protein "), wherein molecular chaperones promotes the correct folding of disulfide bond in toxin polypeptide Ssm6a, make to finally obtain into Ripe toxin polypeptide Ssm6a has natural bioactive, and can increase the expression quantity and solubility of toxin polypeptide Ssm6a.

In some embodiments, fusion protein of the invention includes molecular chaperones, purification tag, inscribe from N-terminal to C-terminal Cleavage sites and toxin polypeptide Ssm6a.

In some embodiments, " molecular chaperones " of the invention be thioredoxin, disulfide bond reduction enzyme and isomerase, NusA, glutathione-S-transferase, Escherichia coli disulfide formation albumin A (DsbA), Escherichia coli disulfide formation albumin A Mutant (DsbAmut), CMP-KDO synzyme (CKS) or maltose-binding protein (MBP).

In preferred embodiments, " molecular chaperones " of the invention is thioredoxin.

In some embodiments, " purification tag " of the invention is 4-10 histidine (His-Tag), glutathione S Transferase (GST) or FLAG.

In preferred embodiments, " purification tag " of the invention is 6-10 histidine (His-Tag).

In a further preferred embodiment, " purification tag " of the invention is 6 histidines.

In some embodiments, " restriction endonuclease " of the invention is fibrin ferment or enterokinase.

In preferred embodiments, " restriction endonuclease " of the invention is enterokinase.

In some embodiments, " toxin polypeptide Ssm6a " of the invention includes one of following amino acid sequences or under State one of amino acid sequence composition:

(1) such as SEQ ID NO:Amino acid sequence shown in 3；

(2) by SEQ ID NO:Nucleotide sequence coded amino acid sequence shown in 4；

(3) with SEQ ID NO:Amino acid sequence shown in 3 has at least 90%, 91%, 92%, 93%, 94%, 95%th, the amino acid sequence of 96%, 97%, 98%, 99% or more homogeneity；

(4) in SEQ ID NO:One or more in amino acid sequence shown in 3 with 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, missing or are inserted The amino acid sequence entered；With

(5) amino acid sequence encoded by following nucleotide sequence, the nucleotide sequence is because of the degeneracy of genetic codon Property and different from SEQ ID NO:Nucleotide sequence shown in 4.

SEQ ID NO:3 amino acid sequence is as follows：

ADNKCENSLRREIACGQCRDKVKTDGYFYECCTSDSTFKKCQDLLH

SEQ ID NO:4 nucleotide sequence is as follows:

GCCGACAACAAATGCGAAAACAGTCTGCGTCGCGAAATTGCCTGCGGTCAGTGTCGCGATAAAGTCAAA ACCGACGGCTACTTTTACGAGTGCTGCACCAGCGACAGCACCTTCAAAAAATGCCAGGACCTGCTGCACTAA

In a preferred embodiment, the amino acid sequence of toxin polypeptide Ssm6a of the invention is in SEQ ID NO:In amino acid sequence shown in 3 at most 5 at have 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, missing or the amino acid sequence of insertion.

In a further preferred embodiment, the amino acid sequence of toxin polypeptide Ssm6a of the invention is in SEQ ID NO:In amino acid sequence shown in 3 at most 5 at have conserved amino acid substitution amino acid sequence.

In preferred embodiments, " toxin polypeptide Ssm6a " of the invention derives from Scolopendra subspinipes (Scolopendra Subspinipes mutilans) venom.

In a further preferred embodiment, the mature protein of " toxin polypeptide Ssm6a " of the invention includes 46 Amino acid, theoretical molecular weight 5317.97, sequence are SEQ ID NO:3, isoelectric point 6.76 includes three pairs of intramolecular disulfide bonds (arrangement mode C1-C5, C2-C4 and C3-C6), IC50 values are 25nM.

The alias of term " Scolopendra subspinipes " is golden head centipede, belongs to the animal kingdom, Arthropoda, chilopoda, Scolopendromorpha, Wu Centipede section, the arthropod of Scolopendra are distributed mainly on China and Japan.

In some embodiments, fusion protein of the invention includes one of following amino acid sequences or by following amino acid One of sequence composition:

(1) such as SEQ ID NO:Amino acid sequence shown in 1；

(2) by SEQ ID NO:Nucleotide sequence coded amino acid sequence shown in 2；

(3) with SEQ ID NO:Amino acid sequence shown in 1 has at least 90%, 91%, 92%, 93%, 94%, 95%th, the amino acid sequence of 96%, 97%, 98%, 99% or more homogeneity；

(4) in SEQ ID NO:One or more in amino acid sequence shown in 1 with 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, missing or are inserted The amino acid sequence entered；With

(5) amino acid sequence encoded by following nucleotide sequence, the nucleotide sequence is because of the degeneracy of genetic codon Property and different from SEQ ID NO:Nucleotide sequence shown in 2.

SEQ ID NO:1 amino acid sequence is as follows：

MAKPIEVTDQNFDETLGQHPLVLVDFWAEWCAPCRMIAPILEEIAKEYEGKLLVAKLDVDENPKTAMRY RVMSIPTVILFKDGQPVEVLVGAQPKRNYQAKIEKHLPATAGSGHHHHHHDDDDKADNKCENSLRREIACGQCRDKV KTDGYFYECCTSDSTFKKCQDLLH

SEQ ID NO:2 nucleotide sequence is as follows：

ATGGCGAAACCGATTGAAGTTACCGATCAGAACTTTGACGAAACGCTGGGCCAACATCCGCTGGTGCTG GTTGATTTCTGGGCGGAATGGTGCGCCCCGTGTCGTATGATTGCACCGATCCTGGAAGAAATCGCTAAAGAATATGA AGGTAAACTGCTGGTCGCAAAACTGGATGTGGACGAAAACCCGAAAACCGCTATGCGTTACCGCGTTATGAGCATTC CGACGGTCATCCTGTTTAAAGATGGCCAGCCGGTCGAAGTGCTGGTTGGTGCGCAGCCGAAACGCAACTACCAAGCC AAAATCGAAAAACATCTGCCGGCAACCGCTGGATCCCATCACCATCACCATCACGATGACGATGACAAAGCCGACAA CAAATGCGAAAACAGTCTGCGTCGCGAAATTGCCTGCGGTCAGTGTCGCGATAAAGTCAAAACCGACGGCTACTTTT ACGAGTGCTGCACCAGCGACAGCACCTTCAAAAAATGCCAGGACCTGCTGCACTAA

In a preferred embodiment, the amino acid sequence of fusion protein of the invention is in SEQID NO:Shown in 1 Amino acid sequence at most 5 at have 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, missing or the amino acid sequence of insertion.

In a further preferred embodiment, the amino acid sequence of fusion protein of the invention is in SEQ ID NO:1 In shown amino acid sequence at most 5 at have conserved amino acid substitution amino acid sequence.

Term " segment " refers to the part in full length nucleotide sequence or amino acid sequence.

Term " variant ", " variant sequence thereof " or " mutant " may be used interchangeably, and refer to and parent nucleotide sequence or ammonia Base acid sequence has the nucleotide sequence or amino acid sequence of a degree of nucleotide/amino acid sequence identity.Variant sequence Row be similar to parental array, but in its nucleotide sequence or amino acid sequence have at least one or several or it is multiple substitution, Missing or insertion so that they are different from parent nucleotide sequence or amino acid sequence.In addition, variant can retain parent's nucleosides The functional character or activity of acid sequence or amino acid sequence, for example, keeping parent nucleotide sequence or amino acid sequence at least 50%th, 60%, 70%, 80%, 90%, 95%, 98% or 99% bioactivity." variant " includes inherently depositing in nature Natural variant, by natural process obtain natural variant and by manual method obtain artificial variants.

It is defined as compared with control/with reference to " percentage (%) amino acid sequence identity " of amino acid sequence by one (and importing room if necessary, any conservative replacement is not intended as identical sequence) is compared in amino acid sequence, to obtain most During big Percent sequence identity, in this amino acid sequence, the ammonia identical with the amino acid residue of control/reference peptide sequence Base acid number of residues divided by its total amino acid residue numbers, obtained business (as a percentage).It can be used this field various Method carries out sequence alignment to measure percent amino acid sequence homogeneity, for example, soft using publicly available computer Part such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software.Those skilled in the art can determine to compare suitable Suitable parameter includes the use of and obtains high specific to required any algorithm to the sequence compared.

When mentioning the percentage of sequence identity in this application, if not specifically stating otherwise, these percentages are opposite It is calculated in the overall length of longer sequence.It is calculated compared with the overall length of longer sequence and is suitable for both nucleotide sequence and peptide sequence.

Term " 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, missing or the insertions of one or more amino acid " is preferably no more than at 10 (i.e., at most At 10), no more than 9 at, no more than 8 at, no more than 7 at, no more than 6 at, no more than 5 at, no more than 4 at, no more than 3 at, At no more than 2, no more than 1 at amino acid 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, missing or insertion.

Term " conservative replacement/displacement " refers to an amino acid through another 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor/displacement in identical category, example If acidic amino acid is through another acidic amino acid substitution/displacement, a basic amino acid substitutes through another basic amino acid/ Displacement or a neutral amino acid are through another neutral amino acid substitution/displacement.The illustrative following Table A institute of conservative replacement/displacement Show:

Table A

Term " fusion protein of the invention ", " (of the invention) molecular chaperones-purification tag-inscribe cleavage sites-poison The fusion protein of plain polypeptide Ssm6a ", " (of the invention) rTrx -6 × His-tag- enterokinase cleavage sites point-poison The fusion protein of plain polypeptide Ssm6a " and/or " SEQ ID NO:1 amino acid sequence " and its variant in the present invention can be mutual Change use.Term " (of the invention) toxin polypeptide Ssm6a ", " toxin polypeptide Ssm6a ", " (of the invention) toxin polypeptide (egg In vain) ", " (of the invention) active peptides (albumen) " and " SEQ ID NO:3 amino acid sequence " and its variant are in the present invention It may be used interchangeably, refer to the biology that can play the toxin polypeptide Ssm6a mature proteins from Scolopendra subspinipes venom The peptide sequence of activity and function.

Term " His-tag (His- labels) " is a kind of purification tag, is made of 4~10 histidines, and metal ion, As nickel ion or zinc ion have high-affinity.Therefore the fusion protein with His-tag can utilize metal ion affinity chromatography It is purified.

On the other hand, the nucleotide sequence the present invention provides the fusion protein of the coding present invention is (also referred to as " of the invention Nucleotide sequence "), it is formed it includes one of following nucleotide sequence or by one of following nucleotide sequence:

(1) such as SEQ ID NO:Nucleotide sequence or its complementary series shown in 2；

(2) nucleotide sequence of the fusion protein of the coding present invention；

(3) nucleotide sequence, under stringent hybridization conditions with the nucleotide sequence hybridization and coding described in (1) or (2) With SEQ ID NO:The amino acid sequences of 2 activity, wherein the stringent hybridization condition is referring to Sambrook et al., 1989, Molecular Cloning:A Laboratory Manual, Nolan C. write, New York:Cold Spring Harbor Laboratory Press；And

(4) its nucleotide sequence is different from SEQ ID NO due to the degeneracy of genetic codon:Nucleotides sequence shown in 2 The nucleotide sequence of row.

In preferred embodiments, nucleotide sequence coded SEQ ID NO of the invention:Amino acid sequence shown in 1.

In a preferred embodiment, the present invention also provides " the cores for being expressed in host cell of the invention Nucleotide sequence ", " nucleotide sequence for being expressed in Bacillus coli cells of the invention ".It is this be used for host cell/ The nucleotide sequence expressed in Bacillus coli cells can be different from the nucleotides sequence of the amino acid sequence of the above-mentioned coding present invention Row, because this nucleotide sequence for being expressed in host cell/Bacillus coli cells is for host cell/large intestine bar Bacterium cell and expression vector have carried out the nucleotide sequence of codon optimization.

On the other hand, the present invention provides the expression vector (also referred to as " table of the invention of the nucleotide sequence comprising the present invention Up to carrier ").

In a preferred embodiment, expression vector of the invention includes above-mentioned be used in host cell/large intestine bar The nucleotide sequence or amino acid sequence (the preferably SEQ ID NO of the coding present invention expressed in bacterium cell:Amino acid shown in 1 Sequence) nucleotide sequence.

In a further preferred embodiment, expression vector of the invention includes SEQ ID NO:Nucleotide shown in 4 Sequence.SEQ ID NO:Nucleotide sequence shown in 4 has carried out codon optimization and corresponding modification, can preferably express this The amino acid sequence of invention.

In a preferred embodiment, expression vector of the invention is autonomously replicating plasmid carrier.

In a further preferred embodiment, expression vector of the invention is pET28a plasmids.

Term " carrier " is the carrier for referring to nucleotide sequence being transferred to target cell.For example, carrier can include can The coding nucleotide sequence expressed in target cell.In the present invention, " carrier " typically refers to that destination gene expression can be guided And available for any nucleic acid construct being transferred to target gene in target cell.

On the other hand, the present invention provides the host cell (also referred to as " host of the invention of the expression vector comprising the present invention Cell ").The host cell of the present invention is for largely expanding the host cell of the expression vector of the present invention, can be any Suitable host cells known in the art, including eukaryotic host cell and prokaryotic host cell.

In a preferred embodiment, which is Escherichia coli (Escherichia coli) cell.

In a further preferred embodiment, which is e. coli bl21 (DE3) cell.

Bacillus coli cells genetic background understands, operating technology is simple, nutritional requirement is not high, large scale fermentation economy.Greatly Enterobacteria BL21 (DE3) cell/bacterial strain is frequently utilized for expression vector (such as pET systems that high efficient expression contains phage t7 promoter Row) nucleotide sequence, wherein T7 phage rna polymerases are located at bacteriophage lambda DE3 areas, which is integrated on the chromosome of BL21.

On the other hand, the present invention provides the fusion protein of the present invention and is preparing for alleviating or treat in the drug of pain Purposes.

On the other hand, the present invention provides a kind of recombination expression and purified source in the toxin polypeptide of Scolopendra subspinipes venom The method (also referred to as " method of the invention ") of Ssm6a, including：

(ii) by the expression plasmid of the amalgamation and expression gene obtained in step (i) the insertion present invention；Convert the place of the present invention Chief cell；Under the conditions of suitable, in host cell expression；

(iii) the toxin polypeptide Ssm6a of the isolation and purification present invention.

In a preferred embodiment, the host cell after the conversion obtained in step (ii) is in culture to OD₆₀₀It reaches To after about 0.6-0.8, IPTG is added in into culture medium, when inducing that about 7-9 is small at a temperature of about 30-37 DEG C, centrifugation Harvest bacterial precipitation object.In a preferred embodiment, toxin polypeptide Ssm6a described in step (iii) isolation and purification has Body is following step (1)-(3)：

(1) in cleavage step (ii) by the present invention amalgamation and expression gene expression for the present invention fusion protein host Lysate comprising fusion protein is carried out affinity chromatography, obtains the semifinished product of fusion protein by cell；

(2) the fusion protein semifinished product endonuclease digestion that will be obtained in step (1), obtains digestion products solution；

(3) the digestion products solution obtained in step (2) is subjected to ion-exchange chromatography, obtains toxin polypeptide Ssm6a's Purify stoste；Optionally it is further processed to obtain the toxin polypeptide Ssm6a highly finished product that purity is about more than 99%.

In a further preferred embodiment, the affinity chromatography in step (1) is the affine layers of Ni-NTAsepharose Analysis.

In a further preferred embodiment, the restriction endonuclease in step (2) is enterokinase, in molar ratio about 1： 550 to 1:650 add in, and digestion when about 5-7 is small at 20-30 DEG C.

In a further preferred embodiment, the ion-exchange chromatography in step (3) (that is, purification step) first uses Q Sepharose Fast Flow columns, pH are about 7.5-9.0, and then using SP sepharose Fast Flow columns, pH is big About 4.5-5.5.

In especially preferred embodiment, the digestion products solution obtained in step (2) is with about 50-100cm/h's Speed is loaded to SP sepharose with the speed of about 50-100cm/h afterwards by Q sepharose Fast Flow columns Fast Flow columns.

In the most preferred embodiment, Q sepharose Fast Flow columns use and flow through pattern；SP sepharose Fast Flow columns use absorption mode, then pass through about 0-500mM NaCl linear gradient elutions after loading.

In a further preferred embodiment, it is dense by ultrafiltration to further include the purifying stoste that will be obtained to method of the invention Contracting changes liquid to the aqueous acetic acid of about pH5.0, and freeze-drying obtains Ssm6a highly finished product.

Term " about/about " refer in the 50% of given value or range, preferably in 25%, more preferably in 10%-1% It is interior；Or refer in the acceptable standard difference of average.

Term " amalgamation and expression gene " refers to for the nucleotide sequence of amalgamation and expression target gene, wherein except purpose base Outside the nucleotide sequence of cause, it can also include increasing the expression quantity of target gene, increase the dissolubility of target gene, promote The destination protein that destination gene expression obtains keeps its natural activity, point for the destination protein that destination gene expression is promoted to obtain It secretes, the various Expression elements convenient for digestion etc..

Term " purifying stoste " refers to solution of the purity of the destination protein obtained after purifying more than 99%.

Term " highly finished product " or " sterling " refer to purify stoste using appropriate method desalination, change liquid, concentration, it is lyophilized after must The destination protein dry powder arrived.

Term " enterokinase recognition site " is aspartic acid-aspartic acid-aspartic acid-aspartic acid-lysine, That is DDDDK five amino acids, the high specificity hydrolysis ability of enterokinase can ensure the toxin polypeptide Ssm6a N-terminals of the present invention Correctness, so as to get toxin polypeptide Ssm6a and natural toxin polypeptide Ssm6a there is identical amino acid sequence.

When term " flowing through pattern " refers to that the feed liquid containing target product passes through chromatographic column, made wherein using appropriate condition Impurity combined with column packing, target product with feed liquid flow out so as to fulfill separating effect column purification mode.

When term " absorption mode " refers to that the feed liquid containing target product passes through chromatographic column, target is made using appropriate condition Product is combined with column packing and eluted by appropriate condition, so as to fulfill separating and being enriched with the column purification of concentrated effect Mode.

In some embodiments, it is excellent containing the connection peptide being made of 5~10 amino acid in fusion protein of the invention It elects 5~10 glycine as and replaces the connection peptide for being connected and forming with serine.The presence of connection peptide can allow molecular chaperones more to have Effect ground promotes toxin polypeptide Ssm6a to fold, enterokinase site preferably exposes.

In some embodiments, the method for the present invention includes fermentation step, shake flask fermentation major control inducing temperature is about 30 DEG C to 37 DEG C, IPTG concentration about 0.05-0.4mM can realize product high efficient expression with this condition；Tank fermentation need to be controlled strictly PH6.8-7.3 processed ensures thalline normal growth, controls about 30 DEG C to 37 DEG C of inducing temperature, dissolved oxygen about 30%-40%, IPTG is dense About 0.05-0.4mM is spent, realizes product high efficient expression.

In some embodiments, the method for the present invention includes fusion protein extraction step, wherein broken wall pH of buffer needs Control is between 7.5-8.5.PH can cause broken wall insufficient less than 7.5, and subsequent products cannot hang Ni-NTA sepharose Affinity column, and degradation is easily generated higher than 8.5 fusion proteins, influence finished product purity.

In some embodiments, the method for the present invention includes purification step (that is, above-mentioned steps (3)), two steps therein The pH value of column purification needs stringent control：Q sepharose Fast Flow columns need to be controlled in 7.5-9.0, SP sepharose Fast Flow columns need to be controlled in 4.5-5.5.PH value is too high or too low cannot all to obtain the pure of final product or obtained final product Degree reduces.Specifically, if Q sepharose Fast Flow columns pH value is less than 7.5, impurity hanging column reduces and makes product Purity reduces, and can be also suspended on pillar higher than 9.0, Ssm6a, without being separated with impurity；If SP sepharose Fast Flow columns pH is less than 4.5, then impurity hanging column increases, and purity reduces, and higher than 5.5, hanging column does not cause that whole production cannot be obtained product Product.

In a preferred embodiment, present invention recombination expression and purified source are more in the toxin of Scolopendra subspinipes venom The method of peptide Ssm6a comprises the following steps:：

(1) structure includes thioredoxin -6 × His-tag- enterokinase cleavage sites point-Ssm6a amalgamation and expression genes；

(2) according to e. coli codon Preference, optimize toxin polypeptide Ssm6a gene orders, obtain artificial synthesized base Because of segment SEQ ID NO:4；

(3) the synthetic gene segment SEQ ID NO that will be obtained in step (2):4 insertion pET28a plasmids, then will Include SEQ ID NO:4 pET28a plasmids conversion competent escherichia coli cell, the Escherichia coli A for conversion of succeeding；

(4) the Escherichia coli A that step (3) is prepared is seeded to LB culture mediums, 37 DEG C of cultures to OD₆₀₀Reach 0.6- 0.8, then add in IPTG, when 30 DEG C of -37 DEG C of culture 7-9 are small after, be harvested by centrifugation bacterial precipitation object, ultrasonication, centrifugation, will To supernatant add in Ni-NTA sepharose affinity columns chromatographed, dialysed or carried out ethanol precipitation afterwards, melted Hop protein；

(5) fusion protein obtained in step (4) is redissolved in enzyme cutting buffering liquid, in molar ratio 1:550 to 1:650 Enterokinase is added in, when digestion 5-7 is small at 20-30 DEG C, obtains digestion products；

(6) digestion products obtained in step (5) are passed through into Q sepharoseFast Flow with 50-100cm/h speed Column obtains flowing through liquid, and adjusting flows through liquid pH as 4.5-5.5, is then loaded to SP sepharose with the speed of 50-100cm/h Fast Flow columns by 0-500mM NaCl linear gradient elutions, collect target peak, obtain purifying stoste；

(7) the purifying stoste obtained in step (6) is concentrated by ultrafiltration and changes liquid to the aqueous acetic acid of pH5.0, freezing It is dried to obtain Ssm6a highly finished product.

1 genetic recombination engineering bacteria of embodiment is built

Design includes the amalgamation and expression sequence (SEQ of thioredoxin -6 × His-tag- enterokinase cleavage sites point-Ssm6a ID NO:1) its coding gene sequence then, is optimized according to the codon preference of Escherichia coli, obtains the volume after final optimization pass Code nucleotide sequence SEQ ID NO:2, commission Suzhou Jin Weizhi bio tech ltd synthesis (synthesis order number：80- 28327346)。

The genetic fragment of synthesis is connected to double through identical enzyme through Nde I and Xho I double digestions (Fermentas companies) Digestion treated pET28a plasmids (Novagen Products, operated according to manufacturer's recommendation).Restructuring after connection PET28a plasmids send Sangon Biotech (Shanghai) Co., Ltd. to carry out sequence verification (sequencing odd numbers：CX170710388- S), confirm that sequence is accurate.

CaCl₂E. coli bl21 (DE3) competent cell is prepared in facture, and then 42 DEG C of thermal shocks are by above-mentioned sequence The restructuring pET28a plasmids of confirmation are transferred to competent cell, are coated on the LB agar mediums containing 50 μ g/ml kanamycins, and 37 DEG C overnight incubation.Picking monoclonal bacterial strain, extracts plasmid, and sequence verification confirms errorless.

2 recombination engineering shake flask fermentation of embodiment

The strain finally obtained in embodiment 1 is inoculated in 100ml LB culture mediums (the 50 μ g/ml containing kanamycins), 220rpm (rev/min), 37 DEG C of overnight incubations are as seed.Amount according to 1% volume ratio is inoculated in the LB culture mediums of 10L, and (containing blocking, that is mould 50 μ g/ml of element) fermented (2L shaking flasks, liquid amount 1L, totally 10 bottles)：220rpm first, 37 DEG C of cultures to OD₆₀₀Reach 0.8, Temperature adjustment adds in IPTG (final concentration 0.2mM) inducible protein expression 8h (hour) to 30 DEG C, and 8000rpm centrifugation 5min (minute) are received Obtain thalline.

3 recombination engineering 15L tanks of embodiment ferment

The strain finally obtained in embodiment 1 is inoculated in 320ml LB culture mediums (the 50 μ g/ml containing kanamycins), 220rpm, 37 DEG C of overnight incubations are as seed.The LB culture mediums that overnight seed is inoculated in 8L by the amount of 4% volume ratio (contain and block that 50 μ g/ml of mycin).37 DEG C of initial temperature, initial speed 200rpm, initial throughput 1:0.5, whole process is by adding concentrated ammonia liquor control PH value processed is 6.8.By increasing rotating speed, throughput and stream plus mending sugar culture-medium (60% glucose sugar, 2% magnesium sulfate) control dissolved oxygen It is maintained at 30%-40%.Work as OD₆₀₀When reaching 20, temperature adjustment adds in IPTG (final concentration 0.5mM) and starts inducible protein table to 30 DEG C Reach, 10 it is small when after fermentation ends, 8000rpm centrifugation 5min harvest thalline.

4 fusion protein of embodiment extracts

The thalline that embodiment 2 and 3 obtains is according to 1：10 be resuspended in brokenly bacterium buffer solution (50mM Tris-Cl, pH 8.0, 300mM NaCl, 20mM imidazoles), for ultrasound fully after broken wall, 15000rpm centrifuges 30min at 4 DEG C, collects supernatant.Supernatant Liquid add in Ni-NTA sepharose affinity medias, 4 DEG C be slowly stirred 1 it is small when, dress column cross clear liquid.Obtained filler adds in broken After bacterium buffer solution for cleaning, elution buffer (50mM Tris-Cl, pH 8.0, the 300mM NaCl, 250mM of 4 times of column volumes are added in Imidazoles) the lower subject fusion proteins of elution.By dialysis process, obtained fusion protein is changed into liquid to enzyme cutting buffering liquid (50mM Tris-Cl, pH 8.0,2mM CaCl₂)。

5 fusion protein of embodiment extracts

Isometric absolute ethyl alcohol is added in the fusion protein solution that embodiment 4 obtains, is sufficiently stirred precipitation, it is heavy to be obtained by filtration Shallow lake albumen.Protein precipitation is redissolved in enzyme cutting buffering liquid same as Example 4.Obtained fusion protein is through SDS-PAGE electricity Swimming detection, finds the basic soluble-expression of fusion protein, and gray scale scanning shows that soluble fusion protein accounts for about the 30% of total protein.

6 fusion protein of embodiment is cut

Enterokinase is added to embodiment 4 and what embodiment 5 obtained is dissolved in the fusion protein solution of enzyme cutting buffering liquid Carry out digestion.Digestion condition is 25 DEG C of temperature, in molar ratio 1:600 add in enterokinase, when cutting 6 is small.

7 active peptides Ssm6a of embodiment is refined

It is carried out using two-step solution chromatography.

Q sepharose Fast Flow column purifications：Using flowing through pattern.The digestion products loading that embodiment 6 obtains is to Q Sepharose Fast Flow columns, pillar are balanced in advance with buffer solution A (50mM Tris-Cl, pH 8.0), loading flow control Between 50-100cm/h, after rinsed with 1 times of column volumes of buffer A, merge to collect and flow through liquid and flushing liquor, merged Collection liquid.

SP sepharose Fast Flow column purifications：Using absorption mode.Q sepharose Fast Flow column purifications Obtained merging collection liquid glacial acetic acid adjusts pH 4.5, and loading to SP sepharose FastFlow columns, pillar is in advance with slow Fliud flushing B (10mM NaAc, pH 4.5) balance, loading flow control between 50-100cm/h, after delayed with 1 times of column volume Fliud flushing B is rinsed, and then with the buffer solution B linear gradient elution containing 0-500mM NaCl, is collected target peak and is obtained toxin polypeptide Ssm6a purifies stoste.

8 active peptides Ssm6a of embodiment is freezed

The toxin polypeptide Ssm6a purifying stoste being refining to obtain in embodiment 7 is concentrated by ultrafiltration instrument concentration, then changes liquid extremely The aqueous acetic acid of pH 5.0, packing, freeze-drying obtain white sterling (white fluffy solid).The sterling is through SDS-PAGE electricity Swimming is detected as single band (referring to Fig. 3 a)；HPLC is detected as simple spike, and purity reaches 99.3% (referring to Fig. 3 b), MALDI- TOF Mass Spectrometric Identifications molecular weight is 5318.33 (referring to Fig. 4), consistent with theoretical molecular weight 5317.97.This shows what is wherein obtained Three pairs of disulfide bond in toxin polypeptide Ssm6a are to form, and otherwise the molecular weight of product will increase than theoretical molecular weight 5317.97 6。

Every liter of shake flask fermentation fermentation liquid energy obtains toxin polypeptide Ssm6a sterling 6mg, and tank every liter of fermentation liquid energy of fermenting obtains poison Plain polypeptide Ssm6a sterlings 100mg.

Small ubiquitin modified protein (SUMO) amalgamation and expression of 9 toxin polypeptide Ssm6a of embodiment

According to melting for the method synthesis small ubiquitin modified protein-toxin polypeptide Ssm6a of 6 × His-tag- that embodiment 1 describes Expressing gene is closed, is connected into pET28a plasmids, converts e. coli bl21 (DE3) cell；It is described according to embodiment 2 or embodiment 3 Method ferment, obtain thalline；According to the method broken wall that embodiment 4 describes, fusion protein is extracted；Ni-NTA The fusion protein that sepharose affinity columns afford is according to molar ratio 1:500 add in SUMO protease, when 20 DEG C of digestions 4 are small； Digestion products are refined according to the method that embodiment 7 describes.

According to the method for the present embodiment, obtained polypeptide is mixed and disorderly false folding or aggregation state, it is impossible to be obtained just The ripe toxin polypeptide Ssm6a really folded (referring to Fig. 5 a and Fig. 5 b).

One embodiment of the present of invention is described in detail above, but the content is only the preferable implementation of the present invention Example, it is impossible to be construed as limiting the practical range of the present invention, those skilled in the art are not departing from the principle of the invention On the premise of to can also modify the present invention, these modifications also belong to protection scope of the present invention.

Sequence table

<110>Haimen mikey Alan's biological medicine Co., Ltd

<120>The recombination expression and purification process of Ssm6a and its fusion protein used

<130> 2018

<160> 4

<170>PatentIn version 3s .5

<210> 1

<211> 169

<212> PRT

<213>Artificial sequence

<400> 1

Met Ala Lys Pro Ile Glu Val Thr Asp Gln Asn Phe Asp Glu Thr Leu

1 5 10 15

Gly Gln His Pro Leu Val Leu Val Asp Phe Trp Ala Glu Trp Cys Ala

20 25 30

Pro Cys Arg Met Ile Ala Pro Ile Leu Glu Glu Ile Ala Lys Glu Tyr

35 40 45

Glu Gly Lys Leu Leu Val Ala Lys Leu Asp Val Asp Glu Asn Pro Lys

50 55 60

Thr Ala Met Arg Tyr Arg Val Met Ser Ile Pro Thr Val Ile Leu Phe

65 70 75 80

Lys Asp Gly Gln Pro Val Glu Val Leu Val Gly Ala Gln Pro Lys Arg

85 90 95

Asn Tyr Gln Ala Lys Ile Glu Lys His Leu Pro Ala Thr Ala Gly Ser

100 105 110

His His His His His His Asp Asp Asp Asp Lys Ala Asp Asn Lys Cys

115 120 125

Glu Asn Ser Leu Arg Arg Glu Ile Ala Cys Gly Gln Cys Arg Asp Lys

130 135 140

Val Lys Thr Asp Gly Tyr Phe Tyr Glu Cys Cys Thr Ser Asp Ser Thr

145 150 155 160

Phe Lys Lys Cys Gln Asp Leu Leu His

165

<210> 2

<211> 510

<212> DNA

<213>Artificial sequence

<400> 2

atggcgaaac cgattgaagt taccgatcag aactttgacg aaacgctggg ccaacatccg 60

ctggtgctgg ttgatttctg ggcggaatgg tgcgccccgt gtcgtatgat tgcaccgatc 120

ctggaagaaa tcgctaaaga atatgaaggt aaactgctgg tcgcaaaact ggatgtggac 180

gaaaacccga aaaccgctat gcgttaccgc gttatgagca ttccgacggt catcctgttt 240

aaagatggcc agccggtcga agtgctggtt ggtgcgcagc cgaaacgcaa ctaccaagcc 300

aaaatcgaaa aacatctgcc ggcaaccgct ggatcccatc accatcacca tcacgatgac 360

gatgacaaag ccgacaacaa atgcgaaaac agtctgcgtc gcgaaattgc ctgcggtcag 420

tgtcgcgata aagtcaaaac cgacggctac ttttacgagt gctgcaccag cgacagcacc 480

ttcaaaaaat gccaggacct gctgcactaa 510

<210> 3

<211> 46

<212> PRT

<213>Artificial sequence

<400> 3

Ala Asp Asn Lys Cys Glu Asn Ser Leu Arg Arg Glu Ile Ala Cys Gly

1 5 10 15

Gln Cys Arg Asp Lys Val Lys Thr Asp Gly Tyr Phe Tyr Glu Cys Cys

20 25 30

Thr Ser Asp Ser Thr Phe Lys Lys Cys Gln Asp Leu Leu His

35 40 45

<210> 4

<211> 141

<212> DNA

<213>Artificial sequence

<400> 4

gccgacaaca aatgcgaaaa cagtctgcgt cgcgaaattg cctgcggtca gtgtcgcgat 60

aaagtcaaaa ccgacggcta cttttacgag tgctgcacca gcgacagcac cttcaaaaaa 120

tgccaggacc tgctgcacta a 141

Claims

1. a kind of fusion protein includes molecular chaperones and toxin polypeptide Ssm6a, it is characterised in that the molecular chaperones promotes toxin The correct folding of disulfide bond in polypeptide Ssm6a makes the ripe toxin polypeptide Ssm6a finally obtained have natural bioactive.

2. fusion protein as described in claim 1, it is characterised in that from N-terminal to C-terminal comprising molecular chaperones, purification tag, interior Enzyme cutting cleavage site and toxin polypeptide Ssm6a.

3. fusion protein as claimed in claim 1 or 2, it is characterised in that the molecular chaperones is thioredoxin；

The purification tag is 6-10 histidine (His-Tag)；Preferably 6 histidines；

The restriction endonuclease is enterokinase；

The toxin polypeptide Ssm6a includes one of following amino acid sequences or is made of one of following amino acid sequences:

(1) such as SEQ ID NO:Amino acid sequence shown in 3；

(2) by SEQ ID NO:Nucleotide sequence coded amino acid sequence shown in 4；

(3) with SEQ ID NO:Amino acid sequence shown in 3 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%th, the amino acid sequence of 97%, 98%, 99% or more homogeneity；

(4) in SEQ ID NO:One or more in amino acid sequence shown in 3 are with 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, missing or insertion Amino acid sequence；With

(5) amino acid sequence encoded by following nucleotide sequence, the nucleotide sequence is due to the degeneracy of genetic codon Different from SEQ ID NO:Nucleotide sequence shown in 4.

4. such as the fusion protein any one of claim 1-3, it is characterised in that the amino acid sequence of the fusion protein It is formed comprising one of following amino acid sequences or by one of following amino acid sequences:

(1) such as SEQ ID NO:Amino acid sequence shown in 1；

(2) by SEQ ID NO:Nucleotide sequence coded amino acid sequence shown in 2；

(3) with SEQ ID NO:Amino acid sequence shown in 1 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%th, the amino acid sequence of 97%, 98%, 99% or more homogeneity；

(4) in SEQ ID NO:One or more in amino acid sequence shown in 1 are with 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, missing or insertion Amino acid sequence；With

(5) amino acid sequence encoded by following nucleotide sequence, the nucleotide sequence is due to the degeneracy of genetic codon Different from SEQ ID NO:Nucleotide sequence shown in 2.

5. the fusion protein as described in claim 3 or 4, it is characterised in that the amino acid sequence bag of the toxin polypeptide Ssm6a Containing with SEQ ID NO:Amino acid sequence shown in 3 has at least 95%, 96%, 97%, 98%, 99% or more homogeneity Amino acid sequence or by with SEQ ID NO:Amino acid sequence shown in 3 has at least 95%, 96%, 97%, 98%, 99% Or more homogeneity amino acid sequence composition；

The amino acid of the fusion protein includes and SEQ ID NO:Amino acid sequence shown in 1 has at least 95%, 96%, 97%th, the amino acid sequence of 98%, 99% or more homogeneity or by with SEQ ID NO:Amino acid sequence shown in 1 has At least 95%, 96%, 97%, 98%, the amino acid sequence of 99% or more homogeneity composition.

6. the fusion protein as described in claim 3 or 4, it is characterised in that the substitution of one or more amino acid, missing Or insertion is the substitution of amino acid, missing or insertion at most 5, wherein the substitution of amino acid is preferably up to 5 at described at most 5 Locate the substitution of conserved amino acid.

7. such as the fusion protein any one of claim 1-6, it is characterised in that the toxin polypeptide Ssm6a is from few Spine centipede venom.

8. encoding the nucleotide sequence of the fusion protein any one of claim 1-7, it includes following nucleotide sequences One of or be made of one of following nucleotide sequence:

(2) nucleotide sequence of the fusion protein of any one of claim 1-7 is encoded；

(3) nucleotide sequence has under stringent hybridization conditions with the nucleotide sequence hybridization described in (1) or (2) and coding SEQ ID NO:The amino acid sequence of 1 activity；And

(4) it is different from SEQ ID NO due to the degeneracy of genetic codon:The nucleotide sequence of nucleotide sequence shown in 2,

The nucleotide sequence optimized encoding SEQ ID of fusion protein any one of wherein described coding claim 1-7 NO:Amino acid sequence shown in 1.

9. include the expression vector of the nucleotide sequence described in claim 8, it is characterised in that the expression vector is autonomous multiple Plasmid vector processed, preferably pET28a plasmids.

10. include the host cell of the expression vector described in claim 9, it is characterised in that the host cell is Escherichia coli Cell, preferably e. coli bl21 (DE3) cell.

11. fusion protein any one of claim 1-7 is preparing for alleviating or treat the use in the drug of pain On the way.

12. a kind of method recombinantly expressed with toxin polypeptide Ssm6a of the purified source in Scolopendra subspinipes venom, including walking as follows Suddenly：

(i) structure includes the amalgamation and expression gene for encoding the fusion protein any one of claim 1-7；

(ii) the amalgamation and expression gene obtained in step (i) is inserted into expression plasmid；Convert host cell；In suitable condition Under, in host cell expression；

(iii) toxin polypeptide Ssm6a described in isolation and purification,

Wherein described host cell is preferably Bacillus coli cells, more preferably e. coli bl21 (DE3) cell；

The expression plasmid is preferably pET28a.

13. method as claimed in claim 12, it is characterised in that the host cell after the conversion obtained in step (ii) is being trained It supports to OD₆₀₀After reaching about 0.6-0.8, IPTG is added in into culture medium, about 7-9 is induced at a temperature of about 30-37 DEG C Hour, host cell sediment is harvested by centrifugation.

14. method as claimed in claim 12, it is characterised in that step (iii) is specially following step (1)-(3)：

(1) by the host cell that amalgamation and expression gene expression is fusion protein in cleavage step (ii), including for obtaining is melted The lysate of hop protein carries out affinity chromatography, obtains the semifinished product of fusion protein；

(2) the fusion protein semifinished product obtained in step (1) is subjected to digestion with restriction endonuclease, obtains digestion products solution；

(3) the digestion products solution obtained in step (2) is subjected to ion-exchange chromatography, obtains the purifying of toxin polypeptide Ssm6a Stoste；The purifying stoste is optionally further processed, obtains the toxin polypeptide Ssm6a highly finished product that purity is more than 99%.

15. method as claimed in claim 14, it is characterised in that：

Affinity chromatography in-step (1) is Ni-NTA sepharose affinity chromatographys；And/or

Restriction endonuclease in-step (2) is enterokinase, in molar ratio about 1：550 to 1:650 add in, and at about 20-30 DEG C Lower digestion is when about 5-7 is small, preferably at about 25 DEG C, in molar ratio about 1:600 add in enterokinase, when cutting about 6 is small； And/or

Ion-exchange chromatography in-step (3) is first using Q sepharose Fast Flow columns, and pH is about 7.5-9.0, so Afterwards using SP sepharose Fast Flow columns, pH is about 4.5-5.5,

The digestion products solution wherein obtained in preferred steps (2) passes through Q sepharose with the speed of about 50-100cm/h Fast Flow columns are loaded to SP sepharose Fast Flow columns with the speed of about 50-100cm/h afterwards；

Optionally obtained purifying stoste is concentrated by ultrafiltration and changes liquid to the aqueous acetic acid of about pH5.0, freeze-drying obtains Ssm6a highly finished product.

16. method as claimed in claim 15, it is characterised in that Q sepharose Fast Flow columns use and flow through pattern；

SP sepharose Fast Flow columns use absorption mode, are washed after loading by about 0-500mM NaCl linear gradients It is de-.