CN114107353A - Plasmid for efficiently expressing polypeptide toxin and preparation method and application thereof - Google Patents
Plasmid for efficiently expressing polypeptide toxin and preparation method and application thereof Download PDFInfo
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- CN114107353A CN114107353A CN202111270816.2A CN202111270816A CN114107353A CN 114107353 A CN114107353 A CN 114107353A CN 202111270816 A CN202111270816 A CN 202111270816A CN 114107353 A CN114107353 A CN 114107353A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
The plasmid for efficiently expressing the polypeptide toxin is obtained by cloning a coding gene of the polypeptide toxin into a plasmid vector pET-26b-DsbC, wherein the plasmid vector pET-26b-DsbC carries His, pelB/ompT and DsbC labels, and the DsbC label and pelB/ompT signal peptide are combined to act, so that the polypeptide to be expressed is transferred into periplasm of cells which is more beneficial to protein folding and disulfide bond formation, the soluble expression of the polypeptide toxin is improved, the problem that the polypeptide toxin is not expressed or the expression quantity is low is solved, and the high-yield expression of the polypeptide toxin is realized. The invention also provides a preparation method of the plasmid, which is used for preparing the plasmid for expressing the spider toxin and then culturing and expressing the spider toxin by using genetic engineering bacteria, the prepared spider toxin has high yield and low production cost, and the preparation method has huge application prospect in the production field of polypeptide toxin.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a plasmid for efficiently expressing polypeptide toxin and a preparation method and application thereof.
Background
Currently, animal polypeptide toxins are hot spots in medical research, not only as tool reagents to study the physiological effects of molecular targets (such as ion channels and receptors), but also as drugs to study or treat human diseases. LGTX-F2 is derived from langerhans spider, and both wild type and mutant thereof can effectively promote the enzyme activities of blood coagulation factors FXa, FXIIa, thrombin and Kallikrein and accelerate the coagulation of blood. The amount of polypeptide obtained by the traditional method from animal venom directly or by chemical synthesis is small, and the complex renaturation process is needed for the polypeptide with disulfide bonds, which results in high cost.
Disclosure of Invention
The invention aims to provide a plasmid for efficiently expressing polypeptide toxin and a preparation method and application thereof aiming at the defects in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a preparation method of a plasmid for efficiently expressing polypeptide toxin, which comprises the following steps:
step S1, synthesizing the nucleotide sequence of the coding gene of the polypeptide toxin;
s2, cloning the coding gene of the polypeptide toxin obtained in the step S1 into a plasmid vector pET-26b-DsbC, wherein the coding gene of the polypeptide toxin and the plasmid vector pET-26b-DsbC are subjected to double enzyme digestion by NcoI and Xho I, a target fragment is recovered, a gene fragment of the polypeptide toxin and the pET-26b-DsbC vector fragment are connected by T4 ligase, and the obtained connector is a recombinant plasmid;
and step S3, transforming the recombinant plasmid obtained in the step S2 into escherichia coli DH5 alpha competent cells, and selecting a single clone and extracting a plasmid with a correct sequence to obtain a plasmid for expressing the polypeptide toxin.
Further, the polypeptide toxin contains a plurality of disulfide bonds.
Further, in step S2, the plasmid vector pET-26b-DsbC is obtained by inserting a DsbC tag protein-encoding gene into the N-terminal cloning site of pET-26 b.
The invention also provides a plasmid for efficiently expressing the polypeptide toxin, which is obtained by adopting the preparation method.
The invention also provides a preparation method of the spider toxin omega-LGTX-F2, which comprises the following steps:
step S1, adopting the preparation method of the plasmid for efficiently expressing the polypeptide toxin to prepare a plasmid for expressing the spider toxin omega-LGTX-F2;
step S2, introducing the plasmid expressing spider toxin omega-LGTX-F2 obtained in step S1 into a host expression system;
step S3, inoculating and culturing the host obtained in the step S2, and inducing the host by IPTG to express an antibody to obtain a recombinant protein omega-LGTX-F2-pET-26 b-DsbC;
step S4, extracting and purifying the recombinant protein omega-LGTX-F2-pET-26 b-DsbC obtained in the step S3;
and step S5, cutting off the pET-26b-DsbC from the high-purity recombinant protein omega-LGTX-F2-pET-26 b-DsbC obtained in the step S4 by using TEV enzyme to obtain spider toxin omega-LGTX-F2.
Further, in step S1, the nucleotide sequence of the encoding gene of spider toxin omega-LGTX-F2 is shown as SEQ ID NO. 1, and the amino acid sequence thereof is shown as SEQ ID NO. 2.
Further, in step S2, the expression system of the host includes a prokaryotic expression system, and the prokaryotic expression system includes escherichia coli BL 21.
Further, in step S3, the colony of Escherichia coli BL21 is inoculated into a kanamycin-containing lysis broth culture medium for antibody expression, the inoculation amount of Escherichia coli BL21(DE3) is 1-3%, and the concentration of inducer IPTG is 0.1-1 mmol/L; the induction temperature is as follows: 28-37 ℃; the induction time is as follows: 12-16 h.
Further, in step S4, the chromatography column used in the purification process is a His-tag protein purification column.
The invention also provides spider toxin omega-LGTX-F2 which is prepared by the method.
The technical scheme provided by the invention has the beneficial effects that:
(1) the plasmid for efficiently expressing the polypeptide toxin is obtained by cloning a coding gene of the polypeptide toxin into a plasmid vector pET-26b-DsbC, wherein the plasmid vector pET-26b-DsbC carries His, pelB/ompT and DsbC labels. The preparation method of the plasmid is a novel prokaryotic expression vector pET-26b-DsbC constructed by combining disulfide oxidoreductase DsbC with pET-26b, the DsbC is added into the N end of the vector pET-26b to be used as a fusion tag, and is combined with pelB/ompT signal peptide on the vector pET-26b to transfer the polypeptide to be expressed into the periplasm of cells which is more beneficial to protein folding and disulfide bond formation, so that the soluble expression of polypeptide toxin is further improved, the problem that the target protein is not expressed or the expression quantity is low is solved, and the high-yield expression of the polypeptide toxin is realized. The DsbC tag used in the present invention is present in the periplasm of E.coli cells and is a protein having disulfide isomerase activity. It contains two subunits, and forms a fixed V-shape, hydrophobic and uncharged residues are distributed on the surface of the V-shape, which plays an important role in substrate recognition and isomerase activity, correctly paired disulfide bonds are usually embedded in the protein, and wrong protein is exposed. By utilizing the characteristics, the mispaired disulfide bonds can be screened, the isomerase activity can catalyze the mispaired disulfide bonds to form correct conformation, and the correct conformation is reflected as a better expression effect. And the signal peptide pelB/ompT carried by the pET-26b is positioned at the N end of the secretory protein, so that the expressed toxin can be effectively guided to pass through the plasma membrane of escherichia coli and be secreted to the periplasm space, and the periplasm space comprises a series of enzymes, so that an oxidation environment is provided, the formation of a disulfide bond is facilitated, the folding of the protein is promoted, and the yield of the target protein is improved.
(2) The invention also provides a method for preparing and producing the polypeptide toxin by utilizing the plasmid pET-26b-DsbC for efficiently expressing the polypeptide toxin, and the specific application is that the invention provides a method for preparing the spider toxin omega-LGTX-F2. The spider toxin contains 4 pairs of disulfide bonds, gene engineering bacteria are constructed by a conventional means of gene engineering to express the spider toxin, the spider toxin with the correct conformation is difficult to completely express, and the obtained spider toxin with the correct conformation is often presented in the form of an inclusion body, so that the solubility is poor, and the yield is low. The preparation method comprises the steps of inserting a gene for coding the spider toxin omega-LGTX-F2 into a multiple cloning site of a prokaryotic expression plasmid vector pET-26b-DsbC to enable the gene to be positioned at the downstream of a DsbC signal peptide and a coding gene thereof to obtain a recombinant expression vector, then transforming the recombinant expression vector into escherichia coli to obtain the recombinant escherichia coli, culturing the recombinant escherichia coli, expressing, separating and purifying, and removing pET-26b-DsbC by enzyme digestion to obtain the spider toxin omega-LGTX-F2. The spider toxin omega-LGTX-F2 obtained by the preparation method has high yield, and the problems of protein aggregation containing disulfide bonds and low yield are perfectly solved by using the expression of the plasmid pET-26 b-DsbC.
The method is simple to prepare, the production period of the spider toxin omega-LGTX-F2 is shortened, the yield of the prepared spider toxin omega-LGTX-F2 is high, the production cost is low, the method is suitable for large-scale production, and the preparation method has a huge application prospect in the field of production of polypeptide toxins.
Drawings
FIG. 1 is a diagram of the structure of plasmid pET-26 b-DsbC;
FIG. 2 is a diagram of the structure of plasmid pET-26 b;
FIG. 3 is a schematic diagram of the structure of a plasmid expressing ω -LGTX-F2 constructed using the pET-26b-DsbC expression plasmid;
FIG. 4 is a diagram showing the result of electrophoresis of the recombinant protein ω -LGTX-F2-pET-26b-DsbC prepared in example 2 after cleavage with TEV enzyme;
FIG. 5 is a diagram showing the result of electrophoresis of the recombinant protein ω -LGTX-F2-pET-32a prepared in comparative example 1 after cleavage with TEV enzyme.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be further described with reference to the accompanying drawings and examples.
Example 1
Construction of pET-26b-DsbC expression plasmid
The DsbC gene without the signal peptide was inserted into the N-terminal cloning site of pET-26b vector to obtain pET-26b-DsbC expression plasmid, whose structure diagram is shown in FIG. 1, containing T7 promoter, Lac operon, ribosome binding site, DsbC coding region, pelB/ompT, MSC, His coding region, TEV site and T7 terminator.
In previous studies, the applicant found that the polypeptide toxin omega-LGTX-F2 is prokaryotic expressed by using a vector pET-32a, and the protein of the polypeptide toxin omega-LGTX-F2 presents the form of an inclusion body, so that the recombinant protein cannot be obtained by normal means. The polypeptide has 4 pairs of disulfide bonds, and can form a correct conformation through specific folding processing to be successfully expressed. In view of the multifaceted properties of their polypeptide toxins, it was investigated whether they could be successfully produced by secretory expression, thus transporting the recombinant proteins to the periplasmic space of cells for expression which is more conducive to production and folding. The applicant constructs pET-26b-DsbC expression plasmid shown in figure 1, and the selected pET-26b prokaryotic expression vector shown in figure 2 carries a signal peptide pelB/ompT sequence which comprises 22-24 amino acids, has the function of periplasm positioning of cells and is beneficial to the output of protein. And the carrier does not have other special labels, so that the DsbC label which is beneficial to folding and processing of the disulfide bond is embedded into the N end of the carrier, and the condition that partial functions of the carrier are lost due to great influence on other parts of the carrier is avoided. In order to prove the expression capacity of the newly constructed pET-26b-DsbC prokaryotic expression vector, the newly constructed pET-26b-DsbC prokaryotic expression vector is utilized to express the polypeptide toxin omega-LGTX-F2, the recombinant protein is successfully produced through secretory expression, and the yield can reach 2.5mg/L through a series of purification processes.
Example 2
Spider toxin omega-LGTX-F2 was prepared by genetic engineering using pET-26b-DsbC expression plasmid prepared in example 1
(1) Preparation of plasmid expressing omega-LGTX-F2
The expressed gene series was optimized according to the codon usage bias of E.coli, and the specific recognition coding sequence ENLYFQG was introduced at the N-terminus of ω -LGTX-F2 in order that TEV enzyme could efficiently cleave the recombinant protein to remove the fusion tag.
The omega-LGTX-F2 gene (Shanghai bioengineering company) is synthesized by a chemical method, and is connected into a prokaryotic expression vector pET-26b-DsbC (purchased from Novagen company) which is cut by NcoI and Xho I and added with DsbC label after double enzyme cutting, and DNA sequencing is carried out on the recombinant plasmid.
(2) Introducing a plasmid expressing spider toxin omega-LGTX-F2 into a host expression system
The successfully constructed plasmid expressing the spider toxin omega-LGTX-F2 is transferred into competent cells of an escherichia coli BL21(DE3) strain (purchased from Biotech company of Beijing Ongzhike), inverted and cultured overnight at 37 ℃, a single colony is picked to 1mL of culture medium containing corresponding resistance on the next day, and the culture medium is enriched at 220rpm at 37 ℃ to obtain the genetically engineered bacteria.
(3) Inoculating and culturing the genetic engineering bacteria, and inducing with IPTG to perform antibody expression to obtain recombinant protein omega-LGTX-F2-pET-26 b-DsbC
The strain is prepared according to the following steps of 1: inoculating 100 proportion into 10mL LB culture medium (containing kanamycin 100 mug/mL), shaking at 37 deg.C for overnight, transferring into LB culture medium proportionally, shaking at 37 deg.C and 220rpm for large-scale shaking culture until OD600 value is about 0.8, adding IPTG with final concentration of 1mmol/L, shaking culture at 28 deg.C and 100rpm for 12-16 hours.
And (3) centrifuging the overnight-induced and expressed thallus at 5000rpm for 8min, enriching the thallus, discarding the supernatant, blowing and rinsing the thallus by ultrapure water twice, and centrifuging at 5000rpm and discarding the supernatant.
The method is beneficial to blowing and hitting the resuspended thallus by the 1xBinding Buffer prepared in advance, sterile granules are needed to be blown and hit, and the volume of the enriched 1L bacterial liquid thallus is about 100ml of 1xBinding Buffer for breaking the thallus. And (3) placing the resuspended thalli into a pre-precooled high-pressure homogenizer for bacteria breaking, selecting 30MPa for the high-pressure homogenizer, and continuously breaking the thalli for 3 times. And (3) centrifuging the crushed thallus at 12000rpm for 20min, and repeating for 2 times to obtain a supernatant containing the recombinant protein omega-LGTX-F2-pET-26 b-DsbC.
(4) Extracting and purifying the obtained omega-LGTX-F2 recombinant protein
And (3) loading the omega-LGTX-F2 recombinant protein supernatant solution obtained in the step (a) into an affinity chromatography column containing His Bind Resin, uniformly dripping, and purifying to obtain high-concentration fusion protein. Before passing through the column, 5-10 times of 1xBinding Buffer is used for balancing the affinity chromatography column. And eluting the fusion protein by imidazole eluent with gradually increasing concentration, desalting the eluent by adding the eluent into an ultrafiltration tube (with the molecular weight cutoff of 10kDa), centrifuging at 4 ℃ and 5000rpm for 30 minutes, and collecting the desalted omega-LGTX-F2 recombinant protein in the ultrafiltration tube. The collected omega-LGTX-F2 recombinant protein was subjected to concentration determination by Nano Drop and split for 12% SDS-PAGE identification and enzyme digestion.
(5) The obtained high-purity recombinant protein omega-LGTX-F2-pET-26 b-DsbC is cut off by TEV enzyme to obtain spider toxin omega-LGTX-F2.
The separated and purified recombinant protein omega-LGTX-F2-pET-26 b-DsbC is cut by using recombinant TEV enzyme and is cut at 16 ℃ overnight. SDS-PAGE electrophoresis results show that the plasmid can effectively express omega-LGTX-F2, enzyme digestion is complete, and spider toxin omega-LGTX-F2 is obtained.
Comparative example 1
The spider toxin omega-LGTX-F2 was prepared by genetic engineering means using a commercially available pET-32a expression plasmid.
The procedure of the preparation method is the same as that of example 2.
FIG. 3 is a schematic diagram of the structure of a plasmid expressing ω -LGTX-F2 constructed using the pET-26b-DsbC expression plasmid. The DsbC tag in the figure has disulfide isomerase activity and can catalyze mismatched disulfide isomerization and re-pairing. When the pelB signal peptide is expressed in protein, the target gene can be positioned in the periplasm of cells, so that the protein production is facilitated. The His label is a poly His label and can be used for separating and purifying recombinant protein by nickel ion affinity chromatography; the arrow indicates the cutting site of the recombinant TEV protease, and the enzyme specifically recognizes the Glu-Asn-Leu-Tyr-Phe-Gln-Gly amino acid sequence and has high specificity and high activity cutting.
As shown in FIG. 4, it is a diagram showing the result of the electrophoresis of the recombinant protein ω -LGTX-F2-pET-26b-DsbC prepared in example 2 after cleavage with TEV enzyme. M: protein Marker, 1: the recombinant protein omega-LGTX-F2-pET-26 b-DsbC which is not cut by TEV enzyme, 2: the product pET-26b-DsbC of the recombinant protein omega-LGTX-F2-pET-26 b-DsbC cut by TEV enzyme, and the arrow indicates the corresponding band after TEV enzyme cutting.
FIG. 5 is a diagram showing the result of the electrophoresis of the recombinant protein ω -LGTX-F2-pET-32a prepared in comparative example 1 after cleavage with TEV enzyme. M: protein Marker, 1: recombinant protein omega-LGTX-F2-pET-32 a, 2 which is not cut by TEV enzyme, and recombinant protein omega-LGTX-F2-pET-32 a product pET-32a cut by TEV enzyme. The corresponding bands after TEV enzyme cleavage are indicated by arrows.
The difference in expression level between the two is judged by the depth of the recombinant protein band in FIG. 4 and FIG. 5, and secondly, the toxin is not completely insoluble, but mostly forms inclusion bodies, and lane 2 in FIG. 4 still has a certain band of interest, but the yield after purification is low. The difference in the yields of the target proteins of example 2 and comparative example 1 is shown in table 1:
table 1:
as shown in Table 1, the yield of spider toxin omega-LGTX-F2 prepared by using pET-26b-DsbC expression plasmid through genetic engineering means is improved by more than 3 times, and further proves that the pET-26b-DsbC expression plasmid is favorable for protein folding and disulfide bond formation in periplasm of cells, and meanwhile, pelB/ompT in the pET-26b-DsbC expression plasmid is favorable for periplasm positioning of cells, so that the output yield of the protein is improved.
The features of the embodiments and embodiments described herein above may be combined with each other without conflict.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Paeder biomedical Co., Suzhou Ltd
<120> plasmid for efficiently expressing polypeptide toxin and preparation method and application thereof
<141> 2021-10-12
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 195
<212> DNA
<213> spider toxin omega-LGTX-F2
<400> 1
gctgaagctt gcaccccgcg cctgcatgat tgctctcatg atcgccattc ttgctgccgc 60
ggcgaactgt ttaaagatgt ttgttattgt ttttatccgg aaggtgaaga taaaaccgaa 120
gtttgtagct gtcagcagcc gaaaagccat aaatatattg aaaaagttgt tgataaaacc 180
aaaaccctgg tgggt 195
<210> 2
<211> 65
<212> PRT
<213> spider toxin omega-LGTX-F2
<400> 2
Ala Gly Ala Cys Thr Pro Ala Leu His Ala Cys Ser His Ala Ala His
1 5 10 15
Ser Cys Cys Ala Gly Gly Leu Pro Leu Ala Val Cys Thr Cys Pro Thr
20 25 30
Pro Gly Gly Gly Ala Leu Thr Gly Val Cys Ser Cys Gly Gly Pro Leu
35 40 45
Ser His Leu Thr Ile Gly Leu Val Val Ala Leu Thr Leu Thr Leu Val
50 55 60
Gly
65
Claims (10)
1. A preparation method of a plasmid for efficiently expressing polypeptide toxin is characterized in that: the method comprises the following steps:
s1, synthesizing the nucleotide sequence of the coding gene of the polypeptide toxin;
s2, cloning the coding gene of the polypeptide toxin obtained in the step S1 into a plasmid vector pET-26b-DsbC, wherein the coding gene of the polypeptide toxin and the plasmid vector pET-26b-DsbC are subjected to double enzyme digestion by NcoI and Xho I, a target fragment is recovered, a gene fragment of the polypeptide toxin and the pET-26b-DsbC vector fragment are connected by T4 ligase, and the obtained connector is a recombinant plasmid;
s3, transforming the recombinant plasmid obtained in the step S2 into escherichia coli DH5 alpha competent cells, and selecting a single clone and extracting a plasmid with a correct sequence to obtain a plasmid for expressing the polypeptide toxin.
2. The method of claim 1, wherein the plasmid for efficient expression of the polypeptide toxin comprises: in step S2, the plasmid vector pET-26b-DsbC is obtained by inserting a DsbC tag protein coding gene into the N-terminal cloning site of pET-26 b.
3. The method of claim 2, wherein the plasmid for efficient expression of the polypeptide toxin comprises: the polypeptide toxin contains a plurality of disulfide bonds.
4. A plasmid for efficiently expressing a polypeptide toxin, which is characterized in that: prepared by the process according to any one of claims 1 to 3.
5. A method for preparing spider toxin omega-LGTX-F2, which is characterized by comprising the following steps: the method comprises the following steps:
s1, preparing a plasmid for expressing the spider toxin omega-LGTX-F2 by adopting the preparation method of the plasmid for efficiently expressing the polypeptide toxin according to claim 4;
s2, introducing the spider toxin omega-LGTX-F2 expression plasmid obtained in the step S1 into a host expression system;
s3, inoculating and culturing the host obtained in the step S2, and inducing with IPTG to perform antibody expression to obtain a recombinant protein omega-LGTX-F2-pET-26 b-DsbC;
s4, extracting and purifying the recombinant protein omega-LGTX-F2-pET-26 b-DsbC obtained in the step S3;
s5, cutting off the pET-26b-DsbC by TEV enzyme from the high-purity recombinant protein omega-LGTX-F2-pET-26 b-DsbC obtained in the step S4 to obtain spider toxin omega-LGTX-F2.
6. The method of claim 5, wherein: in step S1, the nucleotide sequence of the encoding gene of spider toxin omega-LGTX-F2 is shown as SEQ ID NO. 1, and the amino acid sequence thereof is shown as SEQ ID NO. 2.
7. The method of claim 5, wherein: in step S2, the expression system of the host comprises a prokaryotic expression system, and the prokaryotic expression system comprises Escherichia coli BL 21.
8. The method of claim 7, wherein: in the step S3, the bacterial colony of the escherichia coli BL21 is inoculated in a lysis broth culture medium containing kanamycin for antibody expression, the inoculation amount of the escherichia coli BL21(DE3) is 1-3%, and the concentration of an inducer IPTG is 0.1-1 mmol/L; the induction temperature is as follows: 27-28 ℃; the induction time is as follows: 12-16 h.
9. The method of claim 5, wherein: in step S4, the chromatography column used in the purification process is a His-tag protein purification column.
10. A spider toxin omega-LGTX-F2, characterized by: prepared by the preparation method as described in claims 5-9.
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| CN116284308A (en) * | 2023-03-27 | 2023-06-23 | 成都佩德生物医药有限公司 | Polypeptide LGTX-F2 mutant and application thereof |
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| CN116236819A (en) * | 2023-05-09 | 2023-06-09 | 成都佩德生物医药有限公司 | Method for purifying polypeptide toxins in batches and composite double-layer chromatographic column |
| CN116236819B (en) * | 2023-05-09 | 2023-08-04 | 成都佩德生物医药有限公司 | Method for purifying polypeptide toxins in batches and composite double-layer chromatographic column |
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