CN117229381A - Recombinant ginseng hypoglycemic peptide and preparation method and application thereof - Google Patents
Recombinant ginseng hypoglycemic peptide and preparation method and application thereof Download PDFInfo
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Abstract
The application discloses a recombinant ginseng hypoglycemic peptide, a preparation method and application thereof, wherein the recombinant ginseng hypoglycemic peptide is formed by repeating a short amino acid sequence of a natural ginseng hypoglycemic peptide as a repeating unit; the short amino acid sequence is ETVEIIDSEGGGDA, and the repetition number is 2-15. The recombinant ginseng hypoglycemic peptide of the application is prepared by repeating the natural ginseng hypoglycemic peptide for a certain times and then carrying out fermentation expression, thus being capable of effectively relieving the problem of degrading short peptide and remarkably improving the yield, and the prepared recombinant ginseng hypoglycemic peptide is capable of effectively reducing blood sugar.
Description
Technical Field
The application belongs to the technical field of genetic engineering, and particularly relates to a recombinant ginseng hypoglycemic peptide, and a preparation method and application thereof.
Background
Diabetes is called a health killer and poses a great threat to human health. At present, diabetes mellitus can only be relieved by hypoglycemic drugs, and has serious drug dependence. The long-term hyperglycemia of diabetics can cause a series of complications and even loss of life. Of the people older than 18 years in our country, type II diabetics have about 11.2%. In the diabetic population, type II diabetics can account for over 90%. Clinically, drugs such as insulins, insulinotropic agents, glucosidase inhibitors, insulin sensitizers and the like are commonly used for treatment.
The peptide hypoglycemic agent has obvious advantages, and provides a new direction and a new thought for the research and development of hypoglycemic agents in the future. Firstly, peptides are mostly endogenous peptides or other natural peptides, the structure is clear, and the action mechanism is clear; secondly, compared with the general micromolecular medicaments, the polypeptide medicaments have higher activity, smaller medicament dosage and lower toxic and side effects, and the metabolic products are amino acids; thirdly, compared with protein, the peptide has little immunogenicity, can be chemically synthesized, and has high purity and controllable quality; fourth, peptides tend to circumvent gastrointestinal digestion, overcoming the disadvantage of protein molecules being destroyed by digestive enzymes and thus not being orally available.
The existing hypoglycemic drugs are easy to be degraded by digestive enzymes after being orally taken, and the use cost is high; and the short amino acid sequence is easy to degrade, denature and the like. If the composition and distribution of the short amino acids are increased repeatedly, the degradation of the short amino acids can be effectively relieved, so that the stability and the drug effect of the short amino acids are improved.
Disclosure of Invention
The application aims to solve the technical problems of the prior art and provides a recombinant ginseng hypoglycemic peptide and a preparation method and application thereof. The recombinant ginseng hypoglycemic peptide of the application is obtained by repeating the natural ginseng hypoglycemic peptide for a certain times and then carrying out fermentation expression, thus being capable of effectively relieving the problem of degrading short peptide and remarkably improving the yield, and the recombinant ginseng hypoglycemic peptide is capable of effectively reducing blood sugar.
In order to solve the technical problems, the application adopts the following technical scheme: a recombinant ginseng hypoglycemic peptide is characterized by being formed by repeating a short amino acid sequence of a natural ginseng hypoglycemic peptide as a repeating unit; the short amino acid sequence is ETVEIIDSEGGGDA (SEQ ID NO: 1), and the repetition number is 2-15.
In addition, the application also provides a gene for encoding the recombinant ginseng hypoglycemic peptide.
Further, the present application provides an expression vector containing the above gene.
Further, the present application provides a host cell containing the above expression vector.
The host cell is characterized in that the host cell is selected from any one of pichia pastoris, saccharomyces cerevisiae, escherichia coli and bacillus subtilis.
Further, the application provides a method for preparing the recombinant ginseng hypoglycemic peptide, which is characterized by comprising the steps of encoding the recombinant ginseng hypoglycemic peptide, constructing an expression vector containing the recombinant ginseng hypoglycemic peptide gene, transferring the expression vector into a host cell, culturing the host cell transferred into the expression vector in a culture medium, and purifying polypeptide after induced expression to obtain the recombinant ginseng hypoglycemic peptide.
The method is characterized in that the purification method is selected from any one of salting out method, ultrafiltration method, affinity chromatography and gel filtration chromatography.
Further, the application provides application of the recombinant ginseng hypoglycemic peptide in preparation of a medicament for treating diabetes.
Further, the application provides application of the recombinant ginseng hypoglycemic peptide in preparation of medicaments for treating insulin resistance diseases.
Compared with the prior art, the application has the following advantages:
1. the recombinant ginseng hypoglycemic peptide of the application is obtained by repeating the natural ginseng hypoglycemic peptide for a certain times and then carrying out fermentation expression, thus being capable of effectively relieving the problem of easy degradation of short peptide.
2. Compared with the extraction of natural ginseng products, the recombinant ginseng hypoglycemic peptide can obtain higher yield, and compared with the extraction of natural products, the recombinant ginseng hypoglycemic peptide avoids the extraction operation of strong acid and strong alkali organic matters, and ensures the safety of products.
3. The recombinant ginseng hypoglycemic peptide prepared by the application is obtained by expression of escherichia coli, the expression yield is high, the target polypeptide can be obtained after one-step affinity purification, other redundant operations are not needed, and the purification efficiency can be effectively improved.
4. The recombinant ginseng hypoglycemic peptide prepared by the application is a small molecular polypeptide, and has the advantages of easy absorption, higher activity, smaller dosage and lower toxic and side effects.
5. The recombinant ginseng hypoglycemic peptide prepared by the application can effectively reduce blood sugar.
6. The recombinant ginseng hypoglycemic peptide prepared by the application can be added into health care products after simple treatment, and can be used as health care food.
The technical scheme of the application is further described in detail below with reference to the accompanying drawings and the examples.
Drawings
FIG. 1 shows pTYB11 original plasmid.
FIG. 2 is a recombinant plasmid of the ginseng hypoglycemic peptide pTYB11-ADGPRFT according to the embodiment 1 of the present application.
FIG. 3 is a diagram showing the electrophoresis of a target Gene (GSP) dextran gel according to example 1 of the present application.
FIG. 4 shows an electrophoresis pattern of protein expression in example 1 of the present application.
FIG. 5 is a Western Blotting-verifying protein expression electrophoresis pattern of example 1 of the present application.
FIG. 6 is a graph showing that recombinant ginseng hypoglycemic peptide according to example 4 promotes absorption of glucose by HepG2 cells.
FIG. 7 is a graph showing that the recombinant ginseng hypoglycemic peptide according to example 5 of the present application promotes the absorption of glucose by IR-HepG2 cells.
Detailed Description
The present application will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the application. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
The application repeats the natural ginseng hypoglycemic peptide for 2-15 times. The amino acid sequence of the natural ginseng hypoglycemic peptide is shown as SEQ ID NO. 1. In short, according to specific experimental requirements, the natural ginseng hypoglycemic peptide is connected end to end, for example, the natural ginseng hypoglycemic peptide is repeated 2 times, namely, the amino acid fragments shown in SEQ ID NO.1 are connected end to end, so that a 2-repeated amino acid fragment of ETVEIIDSEGGGDAETVEIIDSEGGGDA is formed. Other repetition times may be similarly used.
In a specific embodiment, the natural ginseng hypoglycemic peptide has the amino acid sequence shown as SEQ ID NO.2, wherein the repeated times of the natural ginseng hypoglycemic peptide are 4 times and the repeated times of the natural ginseng hypoglycemic peptide have the amino acid sequence shown as SEQ ID NO.2. The gene for coding the recombinant ginseng hypoglycemic peptide with the repetition number of 4 is subjected to codon optimization aiming at host cell expression, sac II and EcoR I restriction enzyme cutting sites are respectively added at two ends in the design process to facilitate later gene operation, the gene for coding the recombinant ginseng hypoglycemic peptide is obtained through the optimization, the nucleotide sequence of the gene is shown as SEQ ID NO.3, 1 st to 6 th are Sac II restriction endonuclease cutting sites, 7 th to 115 th original plasmid essential nucleotide sequences, 116 th to 283 th are nucleotide sequences corresponding to the natural ginseng hypoglycemic peptide repeated 4 times, 284 th to 286 th are terminators, and 287 nd to 292 th are EcoR I restriction endonuclease cutting sites. When other repetition numbers are involved, it is only necessary to replace positions 116 to 283 with the specifically required nucleotide sequence.
The present application provides an expression vector containing the gene, which may contain regulatory sequences such as transcription and translation initiation and termination codons, which are specific for the type of host (e.g., bacterial, fungal, plant or animal) to be introduced into the vector, and which take into account whether the vector is DNA-based or RNA-based, as appropriate.
In a specific embodiment, the expression vector is pTYB11, the plasmid map of which is shown in fig. 1.
The application also provides a host cell comprising the expression vector, which refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include transformants and transformed cells, including primary transformed cells and progeny derived therefrom, regardless of the number of passages. The offspring may not be exactly identical in nucleic acid content to the parent cell but can contain mutations, and the host cell can be selected from any of Pichia pastoris, saccharomyces cerevisiae, E.coli, and B.subtilis.
In a specific embodiment, the host cell is E.coli BL21 (DE 3).
The application also provides a method for preparing the recombinant ginseng hypoglycemic peptide, which comprises the following steps: the host cell is used for expression, and then the expression is separated and purified to obtain the recombinant DNA. The expression of the host cell means that the host cell is cultured, and the culture medium and the culture conditions are well known to those skilled in the art.
In a specific embodiment, the host cell is escherichia coli, and specific culture conditions are as follows after obtaining escherichia coli genetically engineered bacteria: inoculating the escherichia coli genetic engineering bacteria into an LB (LB) culture medium, culturing for 16 hours at 37 ℃ and 220rpm, inoculating the seed solution into a 500mL shake flask according to 1% of inoculum size after expanding culture, wherein the culture temperature is 37 ℃, and adding IPTG (isopropyl-beta-D-thiogalactoside) for induction expression when the seed solution grows to a logarithmic growth phase, wherein the culture temperature is 25 ℃ and the culture time is 20 hours.
The expression pattern is not limited in any way, and the application can be confirmed as required, for example, the expression is induced expression, and the inducer is IPTG for the induced expression.
In a specific embodiment, IPTG is added for induction culture, the temperature of the induction stage is 25 ℃, bacterial liquid is centrifugally taken out to precipitate after 20 hours of induction, phosphate buffer solution is used for resuspension, and ultrasonic crushing is used.
The method for separation and purification is not limited in any way, and may be determined according to the implementation, and for example, salting out, ultrafiltration, affinity chromatography or gel filtration chromatography may be used.
Example 1: expression of recombinant ginseng hypoglycemic peptide
The application chemically synthesizes the recombined ginseng hypoglycemic peptide gene (the nucleotide sequence is shown as the 116 th-283 th positions of SEQ ID NO. 2). In the synthesis process, sac II and EcoR I recognition sites are respectively added at the 5 'end and the 3' end, the cloning is carried out to an expression vector pTYB11 (the nucleotide sequence is shown as SEQ ID NO.4, the plasmid map is shown as figure 2) after restriction enzyme Sac II and EcoR I are digested, and the obtained cloning plasmid is transformed into BL21 (DE 3) by a heat shock method by taking escherichia coli BL21 (DE 3) as an expression host bacterium. Coating the plates on LB solid plates added with ampicillin sodium, and inversely culturing overnight at 37 ℃ to obtain the escherichia coli genetic engineering bacteria.
Inoculating the obtained escherichia coli genetic engineering bacteria into an LB culture medium, inoculating the escherichia coli genetic engineering bacteria into a 500mL shake flask with an initial volume of 100mL according to an inoculum size of 1% when culturing for 16 hours, adding IPTG to perform induction culture when the culture temperature is 37 ℃ and OD600 = 0.6-0.8, performing induction at the induction stage temperature of 25 ℃ for 20 hours, and centrifugally collecting sediment. And carrying out ultrasonic disruption on the sediment, and carrying out protein expression condition verification. PCR, SDS-PAGE and Western Blotting assays are shown in FIGS. 3-5.
FIG. 3 shows the agarose gel electrophoresis of the target gene, PCR was performed on the recombinant plasmid, and a distinct band appears on the right side of the electrophoresis pattern, about 286bp, similar to the size of the target gene fragment.
FIG. 4 shows SDS-PAGE, and the recombinant ginseng hypoglycemic peptide with CBD-intein label has obvious band at about 60kD molecular weight in SDS-PAGE, and the molecular weight is matched with the expected molecular weight, and the successful expression of the target protein is verified.
FIG. 5 shows Western Blotting verification of CBD tag, and FIG. 5 shows that CBD tag is located at 60kD molecular weight, and the target protein is combined, so that the target protein is verified to be expressed successfully.
Example 2: purification of recombinant ginseng hypoglycemic peptide
(1) After the sediment centrifugally collected in the embodiment 1 is resuspended, the supernatant is ultrafiltered to 50 percent of the initial volume after ultrasonic crushing, 3 to 5 times of pure water is added, and then the mixture is ultrafiltered and concentrated to 5 percent of the initial volume;
(2) Taking a proper amount of filler for column packing, completely draining the protective liquid, and washing the balance column material with a Binding Buffer with 5 times of the volume for standby;
(3) Centrifuging the protein crushing liquid, taking supernatant, filtering by a 0.45 mu M filter membrane, adding the supernatant into the central position of the column, and slowly flowing through the column;
(4) Washing off unbound proteins with a Washing Buffer of 10 column volumes, and if necessary, collecting the flow-through for subsequent analysis;
(5) Then, the column is quickly washed by using a clear Buffer (30 mM-50 mM DTT is added before use) with 3 times of column volume, a sample outlet is closed, and the column is placed at 4 ℃ for 16-40 hours (the Cleavage reaction duration influences the Cleavage efficiency, so that the incubation time can be properly prolonged);
(6) Finally eluting the target protein fused with the intein-chitin binding domain by using an absorption Buffer with a volume of 3 times of the column volume;
(7) The chitin column should be cleared of intein-chitin binding domain fusion proteins and uncleaved proteins of interest after use;
(8) Cleaning procedure: washing the column with Stripping Solution with 3 times of column volume, soaking for 30 min-1 h, washing the resin with Stripping Solution with 7 times of column volume, washing the column with water with 20 times of column volume, and balancing the column with 20% ethanol solution with 5 times of column volume;
(9) Ultrafiltration and desalination; 25mL of G25 packing is adopted for desalting by a G25 desalting column, the operation process is similar to that of gel filtration chromatography, each time of loading 6.5mL, about 8mL is collected, and desalting can be completed after 10min of loading;
(10) Concentrating to 20-30% of the initial volume, pre-freezing in a refrigerator at-20deg.C for 4 hr, lyophilizing in a vacuum lyophilizing machine for 48 hr, collecting lyophilized protein, and storing the lyophilized protein sample in a refrigerator at-80deg.C for later use.
Note that:
Binding/Washing Buffer:20mM HEPES(pH8.0),500mM NaCl,0.1mM EDTA,0.1％～0.5％ Triton X-100；
cleavage Buffer, 20mM HEPES (pH 8.0), 500mM NaCl,0.1mM EDTA,50mM DTT (added before use);
Elution Buffer:20mM HEPES(pH8.0),500mM NaCl,0.1mM EDTA；
Stripping Solution:0.5M NaOH。
example 3 application of recombinant Ginseng hypoglycemic peptide in diabetes treatment drug
HepG2 cells were grown at 1X 10 4 The cells were inoculated into 96-well plates containing ginseng hypoglycemic peptide, and the effect of the cells on glucose absorption was examined, and a control group was set.
The following is a brief description: taking cells in logarithmic growth phase, digesting and centrifuging, washing with PBS for 3 times, treating with pancreatin containing EDTA, centrifuging, re-suspending, and diluting to final concentration of 1×10 5 mu.L of each well of cell suspension was added at 5% CO per mL 2 Incubated overnight at 37℃in an incubator, the original medium was aspirated, 100. Mu.L of MEM medium containing 20mM,40mM,80mM and 100mM of ginseng hypoglycemic peptide was added, 100. Mu.L of MEM medium containing 1mM of insulin was added to the control group, 6 wells were set for each concentration, and after culturing for 24 hours, the supernatant was centrifuged and the glucose content of each well was measured using a glucose oxidase test kit.
The calculation formula is as follows:
c standard: the concentration of the standard substance is 5.55mmol/L
The results of the promotion of glucose uptake by HepG2 cells by recombinant ginseng hypoglycemic peptide are shown in fig. 6. The result shows that the recombinant ginseng hypoglycemic peptide can obviously promote HepG2 cells to absorb glucose, the 40mM recombinant ginseng hypoglycemic peptide has the promotion effect equivalent to that of 1mM insulin, and the promotion effect is more obvious along with the increase of concentration.
Example 4 application of recombinant Ginseng hypoglycemic peptide in insulin resistance therapeutic drug
HepG2 cells were grown at 1X 10 4 The cells were inoculated into 96-well plates containing ginseng hypoglycemic peptide, and the effect of the cells on glucose absorption was examined, and a control group was set.
The following is a brief description: taking cells in logarithmic growth phase, digesting and centrifuging, washing with PBS for 3 times, treating with pancreatin containing EDTA, centrifuging, re-suspending, and diluting to final concentration of 1×10 5 Each mL, 100uL per well was added to the cell suspension at 5% CO 2 Incubation overnight at 37℃in incubator, aspiration of the original medium, first addition of MEM medium containing 18mM glucosamine for 16 hours, induction of insulin resistance in HepG2 cells, then aspiration of the medium, washing 2 times with PBS, addition of 100. Mu.L of MEM medium containing 20mM,40mM,80mM,100mM of ginseng hypoglycemic peptide, respectively, no insulin resistance induction in control group, cell culture with complete medium, 6 replicate wells at each concentration, centrifugation of supernatant after 24 hours, and detection of glucose content in each well using glucose oxidase test kit.
The calculation formula is as follows:
c standard: the concentration of the standard substance is 5.55mmol/L
The results of the promotion of glucose uptake by IR-HepG2 cells by recombinant ginseng hypoglycemic peptide are shown in FIG. 7. The result shows that the recombinant ginseng hypoglycemic peptide can obviously promote the IR-HepG2 cells to absorb glucose, and the promotion effect is more obvious along with the increase of the concentration.
Note P <0.05, P <0.01, P <0.001, compared to model set.
The foregoing description is only a preferred embodiment of the present application, and is not intended to limit the present application, and any simple modification, variation and equivalent structural changes of the above embodiment according to the technical matter of the present application still fall within the scope of the technical solution of the present application.
Claims (9)
1. A recombinant ginseng hypoglycemic peptide is characterized by being formed by repeating a short amino acid sequence of a natural ginseng hypoglycemic peptide as a repeating unit; the short amino acid sequence is ETVEIIDSEGGGDA (SEQ ID NO: 1), and the repetition number is 2-15.
2. A gene encoding the recombinant ginseng hypoglycemic peptide according to claim 1.
3. An expression vector comprising the gene of claim 2.
4. A host cell comprising the expression vector of claim 3.
5. The host cell of claim 4, wherein the host cell is selected from any one of Pichia pastoris, saccharomyces cerevisiae, E.coli and B.subtilis.
6. A method for preparing the recombinant ginseng hypoglycemic peptide according to claim 1, which is characterized by comprising the steps of constructing an expression vector containing the gene of the recombinant ginseng hypoglycemic peptide by encoding the gene of the recombinant ginseng hypoglycemic peptide, transferring the expression vector into a host cell, culturing the host cell transferred into the expression vector in a culture medium, and purifying the polypeptide after induced expression to obtain the recombinant ginseng hypoglycemic peptide.
7. The method according to claim 6, wherein the purification method is selected from any one of salting out method, ultrafiltration method, affinity chromatography method and gel filtration chromatography method.
8. Use of the recombinant ginseng hypoglycemic peptide according to claim 1 for preparing a medicament for treating diabetes.
9. Use of the recombinant ginseng hypoglycemic peptide according to claim 1 in the preparation of a medicament for treating insulin resistance diseases.
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