CN116355932A - Recombinant vector and method for preparing mu-conotoxin - Google Patents
Recombinant vector and method for preparing mu-conotoxin Download PDFInfo
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- 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
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract
The invention relates to the technical field of biology, and discloses a recombinant vector and a method for preparing mu-conotoxin. The recombinant vector provided by the invention can smoothly adopt a common biological expression system such as escherichia coli in the field to carry out exogenous expression production, and the obtained expression product can be subjected to relatively simple purification, cutting and separation treatment to obtain the high-purity mu-conotoxin, so that the high-efficiency batch production of the mu-conotoxin is realized, and the recombinant vector has a wide application prospect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a recombinant vector and a method for preparing mu-conotoxin.
Background
Conotoxin peptides are a class of neurotoxin secreted by conomollusks and are rich in active disulfide bonds, and widely act on targets including ion channels, G-protein coupled receptors, transporters, enzymes, and the like. According to their site of action on the neuromuscular system, they are largely classified into 4 classes: (1) Alpha-conotoxins acting at the acetylcholine receptor (AchR); (2) Mu-conotoxin acting in the active phase and on sodium ion channels; (3) inhibiting omega-conotoxins of cav2.2 channels; (4) Delta-conotoxins acting in the inactive phase and acting in sodium channels.
In recent years, it has been found that conotoxins can be used as clinical drugs or novel drug-targeting compounds, and that mu-conotoxins can also be used in cosmetics as anti-wrinkle factors. How to obtain enough high-purity conotoxin becomes a key factor for restricting the application of the conotoxin in the fields of medicine, cosmetics and the like.
At present, natural extraction and chemical synthesis are main obtaining ways of conotoxin, but natural extraction efficiency is low, cost of chemically synthesizing polypeptide is high, and conotoxin is rich in disulfide bonds, and artificial synthesis is difficult, so that the expression of conotoxin peptide by a microbial cell factory provides a new way for mass production of conotoxin. However, on the one hand, because conotoxins are usually short peptides with amino acid residues of not more than 50 and are rich in disulfide bonds, the activity of the conotoxins is greatly affected by secondary structures, and on the other hand, the conotoxins themselves have toxicity, so that the conotoxins are easy to adversely affect host cells in the expression production process by using a microbial cell factory, or can be degraded by the host cells as harmful substances, and the conotoxins cannot be smoothly produced by using conventional biological expression modes.
Disclosure of Invention
The invention aims to solve the problems that high-purity conotoxin is difficult to produce in a large scale in the prior art, and provides a recombinant vector and a method for preparing mu-conotoxin. The recombinant vector provided by the invention can code the fusion protein of GST protein tag and mu-conotoxin, and compared with the mu-conotoxin, the fusion protein has obviously reduced toxicity to cells, so that the recombinant vector can be smoothly produced by adopting common biological expression host cells in the field such as escherichia coli and the like. On the other hand, the enterokinase recognition sequence is also inserted into the recombinant vector provided by the invention, so that the subsequent GST protein tag and the cutting of mu-conotoxin and the recovery of mu-conotoxin are more convenient.
In order to achieve the above object, in one aspect, the present invention provides a recombinant vector for biologically expressing a mu-conotoxin fusion protein, which comprises, in order, a GST tag encoding gene, an enterokinase recognition sequence encoding gene, and a mu-conotoxin encoding gene.
In a second aspect, the present invention provides a mu-conotoxin fusion protein expressed from the recombinant vector as described in the first aspect above.
In a third aspect, the invention provides a method for preparing mu-conotoxin, which comprises introducing the recombinant vector of the first aspect into an expression system for biological expression.
Through the technical scheme, the invention has the following beneficial effects:
(1) The invention solves the problems that the conotoxin has small molecular weight and certain toxicity by means of fusion expression of the GST tag and the conotoxin, so that the production is difficult to carry out by means of biological expression. Meanwhile, through a mode of introducing enterokinase recognition sequences between the GST tag and the conotoxin, the fusion protein is cut in the subsequent process, so that the conotoxin short peptide without other redundant amino acids can be prepared simply and conveniently.
(2) After being introduced into the existing conventional microorganism expression host (such as escherichia coli and the like), the recombinant vector provided by the invention can successfully and efficiently express the mu-conotoxin with correct amino acid sequence and secondary structure. The high-purity mu-conotoxin can be obtained by simple purification, cutting and separation treatment of the expression product, so that the high-efficiency mass production of the mu-conotoxin is realized, and the method is very suitable for popularization and application.
Drawings
FIG. 1 is a plasmid map of recombinant vector Pex-N-GST-enterokinase-CnIIIC constructed in example 1.
FIG. 2 is a graph showing the result of agarose gel electrophoresis verification of PCR products of the constructed recombinant vector in example 1.
FIGS. 3 (a) - (c) are graphs showing SDS-PAGE results of GST-enterokinase-CnIIIC fusion protein crude protein solution, cleavage product solution, GST-enterokinase solution, recovered enterokinase solution and separated CnIIIC solution in example 2.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In the synthesis of conotoxins in organisms of the genus conotoxin, a polypeptide precursor having from 70 to 120 amino acid residues is usually first synthesized by translation and can be divided into three parts: the signal peptide (pre region), the precursor peptide (pro region) and the mature peptide region are processed to separate the mature peptide region and thereby form the biologically active conotoxin. Therefore, in the art, when the biological expression of conotoxin is performed by using a biological method such as genetic engineering, it is generally desired to directly express the mature peptide of conotoxin by a host cell, so that the conotoxin can be obtained more efficiently. However, studies have shown that when conotoxins are produced by means of biological expression, on the one hand, the toxicity of the conotoxins themselves tends to adversely affect the normal growth and metabolism of the host cell. On the other hand, conotoxins (mature peptides) are usually short peptides of very small molecular weight, whose number of amino acid residues is generally not more than 50, and more particularly 20-30 amino acid residues for mu-conotoxins, but whose secondary structure is rather complex, usually rich in disulfide bonds, and whose post-translational processing is important for obtaining active conotoxins. Therefore, it is very difficult to biologically express conotoxin (mature peptide) by using common engineering bacteria such as escherichia coli. Some researchers have also achieved biological expression of conotoxins by using tandem expression or fusion expression of the conotoxin with other proteins. However, the tandem expression is a product obtained by repeatedly tandem connection of the mature peptide amino acid sequences of the conotoxin, and the operation such as chemical cutting and secondary purification is needed after the purification of the expression product. In addition, although the tandem expression solves the toxicity problem of the conotoxin to the host cell, the problems of whether the posttranslational modification is correct, whether the chemical cleavage affects the structure of the conotoxin and whether the secondary structure of the product after the chemical cleavage is correct and the like still exist, so that the activity of the product has great uncertainty. Similarly, fusion expression of other proteins with conotoxins has similar problems as tandem expression, making the production of biological expression of conotoxins (mature peptides) challenging.
The inventor of the invention discovers that when mu-conotoxin is connected with a specific protein label for (fusion) expression, on one hand, the toxicity of the mu-conotoxin to host cells can be reduced, the influence on the growth metabolism of the host cells is avoided, and on the other hand, the mu-conotoxin can also undergo a correct post-translational modification process, so that the mu-conotoxin with correct secondary structure and biological activity is obtained. Meanwhile, when the expression vector is constructed, a specific enzyme cutting site sequence (such as enterokinase recognition sequence) is inserted between the mu-conotoxin encoding gene and the protein tag encoding gene, so that the expression and modification of the mu-conotoxin are not influenced, and the later protein tag cutting and mu-conotoxin separation operation flow is simplified, so that the biological expression production efficiency of the mu-conotoxin is further improved.
Based on the above findings, the present invention provides, in one aspect, a recombinant vector for biologically expressing mu-conotoxin, which comprises, in order, a GST tag encoding gene, an enterokinase recognition sequence encoding gene, and a mu-conotoxin encoding gene.
The inventor of the present invention found that when the above elements are used in the recombinant vector, mu-conotoxin having correct primary and secondary structures can be obtained by smoothly expressing the recombinant vector in a common host cell such as E.coli. However, when the elements are replaced (for example, GST tag and/or enterokinase recognition sequence is replaced with other protein tag or cleavage sequence), the expressed short peptide amino acid sequence may be correct, but the secondary structure (for example, the number of disulfide bonds, etc.) may be wrong, resulting in the problem that the obtained mu-conotoxin is inactive.
According to a preferred embodiment of the present invention, the amino acid sequence of the GST tag is as set forth in SEQ ID NO: 1.
Any gene sequence capable of expressing the GST tag as described above may be suitable for use in the recombinant vector of the present invention, and in order to enhance expression efficiency, according to a preferred embodiment of the present invention, the nucleotide sequence of the GST tag encoding gene is as shown in SEQ ID NO: 2.
In order to facilitate the cutting of GST tags and mu-conotoxins in the expression products, thereby obtaining mu-conotoxins without redundant amino acid sequences, the coding gene of an enterokinase recognition sequence is inserted between the GST tags and the mu-conotoxin sequences when the recombinant vector is constructed, so that the GST tags in the expression products can be easily and conveniently cut off through the action of enterokinase, and the structure of the mu-conotoxins is not damaged. In order to ensure that the cut mu-conotoxin does not contain redundant amino acids, according to a preferred embodiment of the invention, the enterokinase recognition sequence has an amino acid sequence as set forth in SEQ ID NO: 3.
DDDDK(SEQ ID NO：3)
Any gene sequence capable of encoding an enterokinase recognition sequence as described above, wherein the nucleotide sequence of the enterokinase recognition sequence encoding gene is set forth in SEQ ID NO: 4.
GACGACGACGACAAG(SEQ ID NO：4)
The recombinant vector provided by the invention can be used for expressing any mu-conotoxin and obtaining the short peptide with activity. According to a preferred embodiment of the present invention, wherein the μ -conotoxin is CnIIIC having the amino acid sequence shown in SEQ ID NO: shown at 5.
QGCCNGPKGCSSKWCRDHARCC(SEQ ID NO：5)
Any gene sequence capable of encoding a mu-conotoxin as described above may be suitable for use in the recombinant vector of the present invention. According to a preferred embodiment of the present invention, the nucleotide sequence of the mu-conotoxin encoding gene is as set forth in SEQ ID NO: shown at 6.
In order to further increase the expression efficiency, the expression amount, and the like of the recombinant vector provided by the present invention, according to a preferred embodiment of the present invention, the recombinant vector may further contain at least one genetic element selected from the group consisting of a promoter, an operator, and a terminator. Such as the T7 promoter, the Tac promoter, the LacI lactose operon, etc.
Any starting vector commonly used in the field for constructing a recombinant vector so as to realize exogenous protein expression production by using engineering bacteria host cells can be used for constructing the recombinant vector provided by the invention. According to a preferred embodiment of the present invention, wherein the recombinant vector is selected from at least one of pEX plasmid, pGH plasmid, pET plasmid, pLST plasmid, pTST plasmid, pEST plasmid, pBAD plasmid, pTC plasmid, pTG plasmid, pTFN plasmid and pLAS plasmid as a starting vector. In the present invention, the selection of the departure vector is not particularly limited, and the selection of the departure vector may be performed according to the expression characteristics of the selected expression system.
According to a preferred embodiment of the present invention, the recombinant vector is selected from pEX plasmid as a starting vector, wherein GST tag coding gene, enterokinase recognition sequence coding gene and mu-conotoxin coding gene are inserted in sequence. Preferably, the nucleotide sequence of the recombinant vector is shown as SEQ ID NO:7 (the plasmid map is shown in FIG. 1).
The construction method of the recombinant vector also belongs to the content of the invention. Any recombinant vector construction method commonly used in the art may be suitable for use in the present invention. For example, GST tag encoding gene, enterokinase recognition sequence encoding gene and mu-conotoxin encoding gene may be synthesized separately and then integrated into a starting vector (e.g., pEK plasmid, etc.). Alternatively, a starting vector containing a GST tag may be directly selected, and the enterokinase recognition sequence encoding gene and the mu-conotoxin encoding gene may be integrated therein. The invention is not particularly limited to a specific construction method, as long as the GST tag encoding gene, enterokinase recognition sequence encoding gene and mu-conotoxin encoding gene in the prepared recombinant vector can be sequentially arranged and expressed.
Because the enterokinase recognition sequence coding gene and the mu-conotoxin coding gene selected in the recombinant vector provided by the invention are short in sequence length, in order to simplify the operation and improve the connection efficiency of the starting vector and the exogenous gene, according to a preferred embodiment of the invention, the enterokinase recognition sequence coding gene and the mu-conotoxin coding gene can be directly spliced into one exogenous gene sequence in a long fragment form, different enzyme digestion sites are respectively arranged at the N end of the enterokinase recognition sequence coding gene and the C end of the mu-conotoxin coding gene, and the enterokinase recognition sequence coding gene and the mu-conotoxin coding gene are connected with the starting vector (such as pEK plasmid containing GST tag coding gene and the like) containing GST tags, which is subjected to double enzyme digestion treatment, so that the enterokinase recognition sequence coding gene and the mu-conotoxin coding gene are integrated into the recombinant vector. The starting support containing the GST tag may be a commercially available related product or may be prepared by itself according to the prior art.
In a second aspect, the invention provides a method for preparing a mu-conotoxin, which method comprises introducing the recombinant vector of the first aspect into an expression system for biological expression.
Any expression system commonly used in the art for expression of foreign proteins may be suitable for use in the methods provided herein. According to a preferred embodiment of the invention, wherein the expression system is selected from a prokaryotic expression system and/or a eukaryotic expression system.
According to a preferred embodiment of the invention, wherein the prokaryotic expression system is selected from E.coli and/or B.subtilis.
Preferably, the prokaryotic expression system is E.coli. More preferably E.coli DE3.
According to a preferred embodiment of the invention, wherein the eukaryotic expression system is selected from pichia pastoris and/or saccharomyces cerevisiae.
The expression product obtained by the method is a fusion protein containing the GST tag, an enterokinase recognition sequence and mu-conotoxin. Thus, in order to obtain a mu-conotoxin of high purity and free of redundant amino acids, according to a preferred embodiment of the present invention, the method further comprises the steps of sequentially purifying, cleaving and isolating the expression product.
Because GST protein tag is inserted into the recombinant vector provided by the invention, the tag can be directly utilized for purifying the expression product. According to a preferred embodiment of the present invention, wherein the purification is performed in such a way that the GST-tagged protein is purified. The purification of the expression product obtained by the method provided by the invention can be carried out by a person skilled in the art by any means commonly used in the art for protein purification using protein tags, for example, a commercially available or self-prepared GST protein affinity chromatography column can be used for purification of the expression product.
The purpose of the cleavage is to cleave the mu-conotoxin in the expression product from the GST tag attached thereto, thereby obtaining a mu-conotoxin short peptide free of redundant amino acids. Because the enterokinase recognition sequence is inserted between the mu-conotoxin encoding gene and the GST tag encoding gene in the recombinant vector provided by the invention, the cleavage treatment mode is to cleave the purified product by enterokinase, so that the GST tag in the purified product is cleaved. The amount of enterokinase used in the present invention is not particularly limited as long as GST tag in the purified product can be excised efficiently. Since enterokinase does not affect the structure and function of mu-conotoxin, enterokinase can be used in a moderate excess during cleavage in order to ensure cleavage effect.
Since enterokinase is introduced into the purified product in the cleavage step, the cleaved enterokinase recognition sequence is also present in the cleaved system. Thus, in order to obtain high purity mu-conotoxin, the cleavage product also needs to be isolated. Any means capable of separating mu-conotoxin in a system containing the above substances can be applied to the method (e.g., ion exchange chromatography, high performance liquid chromatography, etc.) provided by the present invention. According to a preferred embodiment of the present invention, wherein the separation is performed by ion exchange chromatography to remove enterokinase introduced in the cleavage step, thereby obtaining mu-conotoxin of high purity.
The present invention will be described in detail by examples. It should be understood that the following examples are illustrative only and are not intended to limit the invention.
In the examples below, reagents and materials were used as commercially available products from regular biological or chemical reagent suppliers, unless otherwise specified.
Example 1
This example is used to illustrate the construction of the recombinant vector provided by the present invention.
(1) enterokinase-CnIIIC gene synthesis
The following sequences were synthesized by the biological technology limited company of borreliaceae, beijing:
wherein GACGACGACGACAAG is enterokinase recognition sequence (enterokinase) coding gene (SEQ ID NO: 4), CAAGGGTGCTGCAACGGCCCGAAAGGCTGCAGCAGCAAATGGTGCCGCGATCATGCGCGCTGCTGTTGA (SEQ ID NO: 6) is mu-conotoxin (CnIIIC) coding gene. The 5' end is HindIII enzyme cutting siteAAGCTT) The 3' section is NotI enzyme cutting site #GCGGCCGC)
(2) Construction of Pex-N-GST-enterokinase-CnIIIC recombinant vector
The pEX-N-GST plasmid (purchased from vast plasmid platform) containing GST tag coding gene and the enterasinase-CnIIIC fragment synthesized in the step (1) were digested with HindIII and NotI (purchased from NEB corporation), respectively, and the digested products were electrophoresed using 1% agarose gel, and fragments of about 5200bp (pEX-N-GST plasmid after digestion) and 90bp (enterasinase-CnIIIC fragment after digestion) were recovered by gel digestion. After ligation of the recovered product with T4 ligase, the ligation product was transformed into E.coli DH 5. Alpha. Competent cells (purchased from full gold Co.), coated with LB plates (containing 100. Mu.g/mL ampicillin), and cultured overnight. The next day positive monoclonal was picked, colony PCR was verified using T7 and T7ter primers (both synthesized by Boxing Corp.) and the PCR product was sequenced by 1% agarose gel electrophoresis (results shown in FIG. 2), screening for approximately 1000bp monoclonal, and verifying that the sequence was correct, namely the Pex-N-GST-enterokinase-CnIIIC recombinant vector (plasmid map shown in FIG. 1)
T7 primer: TAACGACTCACTTAGAGG (SEQ ID NO: 9)
T7ter primer: TGCTAGTTATTGCTCAGCGG (SEQ ID NO: 10)
Example 2
This example is useful for illustrating the biological expression of mu-conotoxin using the recombinant vectors provided by the present invention.
The enterokinase used in this example was purchased from Biyun Biotechnology Co., ltd (product code number P4237) and had an amino acid sequence of IVGGSDSREGAWPWVVALYFDDQQVCGASLVSRDWLVSAAHCVYGRNMEPSKWKAVLGLHMASNLTSPQIETRLIDQIVINPHYNKRRKNNDIAMMHLEMKVNYTDYIQPICLPEENQVFPPGRICSIAGWGALIYQGSTADVLQEADVPLLSNEKCQQQMPEYNITENMVCAGYEAGGVDSCQGDSGGPLMCQENNRWLLAGVTSFGYQCALPNRPGVYARVPRFTEWIQSFLH (SEQ ID NO: 11).
Construction of BL21 (DE 3) -pEX-N-GST-enterokinase-CnIIIC strain and protein induced expression
The Pex-N-GST-enterokinase-CnIIIC recombinant vector obtained in example 1 was transformed into competent cells of escherichia coli expression bacterium BL21 (DE 3) (purchased from whole gold company), coated with LB plates (containing 100 μg/mL ampicillin), and cultured overnight. The positive monoclonal is selected the next day, colony PCR verification is carried out by using T7 and T7ter primers, the PCR product is subjected to 1% agarose gel electrophoresis, and the monoclonal with the band of about 1000bp is selected, namely BL21 (DE 3) -pEX-N-GST-enterokinase-CnIIIC expression strain.
Positive monoclonal was picked and inoculated into 30mL LB liquid medium containing 100. Mu.g/mL ampicillin, and the strain was activated overnight at 37 ℃. The next day 1mL of the activated bacterial liquid is taken, the volume ratio of 1:1000 is transferred into 1L of LB liquid medium containing 100 mug/mL of ampicillin, and the culture is carried out at 37 ℃ until the OD 600 Inducible expression was performed by adding inducer IPTG at a final concentration of 0.5mM (conditions of 25 ℃,17±1 h) =0.65±0.15.
Centrifuging the strain solution after induced expression at 8000rpm, collecting the expressed strain, and adding PBS buffer (140 mM NaCl,2.7mM KCl,10mM Na) 2 HPO 4 ，1.8mM KH 2 PO 4 pH 7.3), and the OD of the resuspension 600 The value was about 60, using a high pressure homogenizer (U.S. PHD company, model D-3L) is crushed for 3-4 cycles, and then centrifuged at 10000rpm for 20min at 4 ℃, and the supernatant is collected to obtain GST-enterokinase-CnIIIC fusion protein crude protein liquid.
Purification and cleavage of the expression product
The crude GST-enterokinase-CnIIIC fusion protein solution was filtered through a 0.22 μm filter and then purified by GST column (GE pre-packed column, 5mL GSTrap HP) wherein the binding buffer was PBS (140mM NaCl,2.7mM KCl,10mM Na2HPO4,1.8mM KH2PO4), pH7.3; the elution buffer was 50mM Tris-HCl containing 10mM reduced glutathione, pH8.0.
The specific procedures of purification and cleavage are as follows:
(1) Balancing the GST column with a binding buffer, balancing 5 column volumes;
(2) Loading the filtered crude protein liquid, and washing with a binding buffer solution until an ultraviolet absorption peak is stable (the dosage of the binding buffer solution is about 3-5 column volumes), wherein GST-enterokinase-CnIIIC fusion proteins in the crude protein liquid are all adsorbed on the column through GST labels;
(3) Enterokinase excision buffer (50 mM Tris-HCl,0.05M NaCl,50% glycerol, ph=8) was prepared and then the column was washed with 10 column volumes of enterokinase excision buffer;
(4) According to 5mL GSTrap pre-packed column combined with 40mg GST tag protein, 30 mu L (300 units) enterokinase solution and 4.97mL enterokinase excision buffer solution are mixed, the mixture is loaded into the column, the upper end and the lower end of the column are respectively blocked by plugs, and the column is incubated for 20+/-4 hours at 25 ℃;
(5) Removing the column plug, flushing the column with enterokinase excision buffer solution with 3 times of column volume, and collecting effluent liquid to obtain the cutting product solution. The cleavage product solution only contains CnIIIC and enterokinase after cleavage of GST tag, and GST tag and enterokinase recognition sequence (GST-enterokinase) are still combined on the column;
(6) Washing and recovery of GST column: the GST-enterase remaining on the column was washed with 10 column volumes of elution buffer, the eluted GST-enterase solution was collected, then washed with 5 column volumes of binding buffer and stored in 20% ethanol.
Isolation of (III) mu-conotoxin CnIIIC
Since the isoelectric point of the selected enterokinase is 5.2, and the isoelectric point of CnIIIC is 8.69, when ph=8, the two are different in charging property, cnIIIC is positively charged, and enterokinase is negatively charged. Thus, the separation of CnIIIC from the cleavage product solution was performed by anion exchange chromatography.
A GE anion exchange pre-cartridge (HiTrap Q HP 5 mL) was used, in which the binding buffer: 50mM Tris-HCl,50mM NaCl,pH =8; elution buffer: 50mM Tris-HCl,1M NaCl, pH=8.
The specific separation treatment process is as follows:
(1) Balancing the anion exchange column with 5 column volumes of binding buffer;
(2) Loading the sample by adopting a cutting product solution, and collecting a loading flow through liquid (namely a separated CnIIIC solution);
(3) Enterokinase elution and recovery: washing 5-10 column volumes with a binding buffer solution after loading is finished until an ultraviolet absorption peak is stabilized, then washing enterokinase bound to the column with an elution buffer solution, and collecting an elution product (recovering enterokinase solution) until the ultraviolet absorption peak is reduced to the vicinity of a base line;
(4) Washing and recovering the ion exchange column: the column volumes were washed 5 times with binding buffer, filled with 20% ethanol and stored.
(IV) detection and liquid quality identification of CnIIIC samples
SDS-PAGE detection was performed on the GST-enterokinase-CnIIIC fusion protein crude protein solution, cleavage product solution, GST-enterokinase solution, recovered enterokinase solution and separated CnIIIC solution, respectively, and the results are shown in FIGS. 3 (a) - (c). The result in the figure shows that the recombinant vector expression product provided by the invention can obtain the mu-conotoxin short peptide CnIIIC with higher purity after GST column purification (purification), enterokinase cleavage GST tag (cleavage) and anion exchange chromatography purification (separation) treatment.
The obtained CnIIIC solution was subjected to liquid chromatography (entrusted to Beijing blue lotus Baiao Biotechnology Co., ltd.) and the assay result showed that the molecular weight of the sample for examination was 2391.89Da (which corresponds to the theoretical molecular weight of CnIIIC of 2.39 kDa) and had 3 pairs of disulfide bonds (which corresponds to the number of CnIIIC theoretical disulfide bonds), thereby demonstrating that the accurate conotoxin short peptide CnIIIC could be obtained by the E.coli expression system and purification method constructed in examples 1-2 above.
Example 3
This example is used to illustrate the detection of the biological activity of mu-conotoxin expressed by the method of the invention.
Construction of expression rat Na using pcDNA3.1 vector (Invitrogen) and HEK293 cells (ATCC CRL-1573) using whole cell patch clamp method v 1.4 HEK293 cells (SCN4A_RAT, uniprot No. P15390) (construction methods refer to pcDNA3.1 vector description and H.Chen, et al, (2000). Modulation of cloned skeletal muscle sodium channels by the scorpion toxins Lqh II, lqh III, and Lqh. Alpha. IT.,439 (4), 423-432.Doi:10.1007/s 004249900181), the conotoxin peptides obtained in example 2 were tested for biological activity. For specific methods reference is made to the above-mentioned h.chen, et al, (2000) and chaine, mohamed, et al, (1994) Sodium channel mutations in paramyotonia congenita uncouple inactivation from activation.neuron,12 (2), 281-294.Doi:10.1016/0896-6273 (94) 90271-2. CnIIIC pair expressing rat Na using patch clamp amplifier (Axon, axopatch-200B model) and Axon pCLAMP pattern analysis software v 1.4 Na of HEK293 cells v 1.4 influence of the electric signal of the channel is detected, and the detection process adopts the following reagents and parameters:
electrode inner liquid: 35mM NaCl,105mM CsF,10mM EGTA,10mMHEPES,pH7.4.
Extracellular fluid (control): 150mM NaCl,2mM KCl,1.5mM CaCl 2 ，1mM MgCl 2 10mM HEPES, pH7.4, containing 1mg/mL BSA.
Extracellular fluid (experimental group): control extracellular fluid + CnIIIC solution isolated in example 2, final concentration of CnIIIC was 1 μm.
Detection conditions: electrode impedance is 1-2MΩ, temperature is 20+ -1deg.C, clamping voltage is-80 mV, and stimulating voltage is-20 mV.
The control and experimental groups were used for 5 replicates, respectively, and the average was taken. The results showed that Na when using the extracellular fluid of the experimental group v The electrical signal of the 1.4 channel is obviously reduced, and the inhibition rate reaches 94.2+/-0.8 percent (inhibition rate= (electrical signal of control group-electrical signal of experimental group)/electrical signal of control group multiplied by 100 percent). This demonstrates that the mu-conotoxin peptide CnIIIC obtained in example 2 has biological activity.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (10)
1. The recombinant vector for biologically expressing the mu-conotoxin is characterized by sequentially comprising a GST tag coding gene, an enterokinase recognition sequence coding gene and a mu-conotoxin coding gene.
2. The recombinant vector of claim 1, wherein the GST tag has an amino acid sequence as set forth in SEQ ID NO:1 is shown in the specification;
and/or, the amino acid sequence of the enterokinase recognition sequence is shown as SEQ ID NO:3 is shown in the figure;
and/or, the amino acid sequence of the mu-conotoxin is shown as SEQ ID NO: shown at 5.
3. The recombinant vector according to claim 2, wherein the nucleotide sequence of the GST-tag encoding gene is as set forth in SEQ ID NO:2 is shown in the figure;
and/or, the nucleotide sequence of the enterokinase recognition sequence encoding gene is shown as SEQ ID NO:4 is shown in the figure;
and/or, the nucleotide sequence of the mu-conotoxin encoding gene is shown as SEQ ID NO: shown at 6.
4. The recombinant vector according to claim 1, wherein the recombinant vector is selected from at least one of pEX plasmid, pGH plasmid, pET plasmid, pLST plasmid, pTST plasmid, pEST plasmid, pBAD plasmid, pTC plasmid, pTG plasmid, pTFN plasmid and pLAS plasmid as an origin vector.
5. The recombinant vector according to any one of claims 1-4, wherein the nucleotide sequence of the recombinant vector is set forth in SEQ ID NO: shown at 7.
6. A method for preparing a μ -conotoxin, comprising introducing the recombinant vector of any of claims 1-4 into an expression system for biological expression.
7. The method of claim 6, wherein the expression system is selected from a prokaryotic expression system and/or a eukaryotic expression system.
8. The method of claim 7, wherein the prokaryotic expression system is selected from escherichia coli and/or bacillus subtilis;
and/or the eukaryotic expression system is selected from Saccharomyces cerevisiae and/or Pichia pastoris.
9. The method of claim 6, wherein the method further comprises the step of sequentially purifying, cleaving, and isolating the expression product.
10. The method of claim 9, wherein the purification is performed in a manner such that the GST tag protein is purified;
and/or the cleavage is performed by cleavage of the purified product using enterokinase such that the GST tag in the purified product is cleaved;
and/or the separation treatment mode is ion exchange chromatography to remove enterokinase introduced in the cutting step, so as to obtain the purified mu-conotoxin.
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