Recombinant ginseng tetradecapeptide as well as preparation method and application of fermented product of recombinant ginseng tetradecapeptide

Abstract

The invention discloses a recombinant ginseng tetradecapeptide and a preparation method and application of a fermented product of the recombinant ginseng tetradecapeptide. The recombinant ginseng tetradecapeptide comprises an amino acid sequence formed by repeating an amino acid sequence of natural ginseng tetradecapeptide as a repeating unit; the amino acid sequence of the natural ginseng tetradecapeptide is SEQ ID NO: 1: ETVEIIDSEGGGDA, and the number of times of repetition is 7-20. According to the invention, natural ginseng tetradecapeptide is repeated for a certain number of times and then is subjected to fermentation expression by using an engineering strain, a fermentation product with high safety and direct feeding potential can be prepared, and the prepared recombinant ginseng tetradecapeptide and the fermentation product thereof can effectively inhibit epinephrine from decomposing fat and regulate inflammatory factors.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a preparation method and application of a recombinant ginseng tetradecapeptide and a fermentation product thereof. Background Art

[0002] There are about 114 million diabetes patients in my country, accounting for about 27% of the world's diabetes patients. my country has become the country with the largest number of diabetes patients in the world. In recent years, the prevalence of diabetes in adults in China has increased significantly, reaching 10.4%, and the incidence is getting younger and younger, and the prevalence in rural populations has increased rapidly. Diabetes can cause damage to the retina, kidneys, nervous system and cardiovascular and cerebrovascular systems. It is the main cause of blindness, renal failure, cardiovascular and cerebrovascular accidents and amputations in my country, and the disease burden is heavy. Among people over 18 years old in my country, about 11.2% have type 2 diabetes. Among the diabetic population, type 2 diabetes patients can account for more than 90%. Diabetic patients need to take hypoglycemic drugs for a long time. The hypoglycemic drugs currently on the market include insulin injection preparations and oral hypoglycemic drugs. Oral hypoglycemic drugs are mainly chemical drugs. This type of drug mainly repairs and improves the function of pancreatic β cells and promotes their secretion of insulin by increasing insulin sensitivity and delaying the metabolism of carbohydrates in the small intestine, but most of them have certain side effects. Therefore, the development of safe and effective hypoglycemic drugs has become a hot topic.

[0003] Peptide hypoglycemic drugs have the advantages of good activity, simple structure, high stability, good absorption, low immunogenicity, and few side effects. Hypoglycemic peptides are divided into two categories: endogenous hypoglycemic peptides and exogenous hypoglycemic peptides. Most peptides are endogenous peptides or other natural peptides with clear structures and clear mechanisms of action. Compared with general small molecule drugs, peptide drugs have higher activity, smaller dosages, and lower toxic side effects. The metabolites are amino acids, which have almost no immunogenicity, and can be chemically synthesized. The product purity is high and the quality is controllable.

[0004] Existing peptide hypoglycemic drugs have problems such as high chemical synthesis cost and difficult biosynthesis and purification. The use of recognized safe strains such as brewer's yeast can directly produce highly safe fermented products containing ginseng peptides. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a preparation method and application of a recombinant ginseng 14 peptide and its fermented products in view of the deficiencies of the above-mentioned prior art. The present invention repeats the natural ginseng 14 peptide for a certain number of times and then ferments and expresses it, which can increase the expression amount and slow down the degradation of short peptides. The recombinant ginseng 14 peptide is a small molecule polypeptide, which has the advantages of easy absorption, higher activity, smaller dosage, and lower toxic side effects. It can effectively inhibit adrenaline from decomposing fat, assist in stabilizing blood sugar and regulating inflammatory factors, help insulin to play a better role, and has the potential for direct feeding.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a recombinant ginseng tetradecapeptide, characterized in that it includes an amino acid sequence composed of the amino acid sequence of natural ginseng tetradecapeptide as a repeating unit; the amino acid sequence of the natural ginseng tetradecapeptide is SEQ ID NO: 1: ETVEIIDSEGGGDA, and the number of repetitions is 7 to 20 times.

[0007] Furthermore, the present invention provides a gene encoding the above-mentioned recombinant ginseng tetradecapeptide.

[0008] Furthermore, the present invention provides an expression vector containing the above gene.

[0009] The above-mentioned expression vector is characterized in that the expression vector is selected from one of PYES2, pMG36e, PHY300-PLK and PHT43, and the toxin-producing and antibacterial genes are removed.

[0010] Furthermore, the present invention provides a host cell containing the above expression vector.

[0011] The above-mentioned host cell is characterized in that the host cell is selected from any one of Saccharomyces cerevisiae, lactic acid bacteria and Bacillus subtilis.

[0012] Furthermore, the present invention provides a method for preparing the above-mentioned recombinant ginseng tetradecapeptide, characterized in that it includes a gene encoding the recombinant ginseng tetradecapeptide, constructing an expression vector containing the gene of the recombinant ginseng tetradecapeptide, transferring the expression vector into a host cell and culturing it in a culture medium, inducing expression and then purifying the polypeptide to obtain the recombinant ginseng tetradecapeptide and a fermented product containing the recombinant ginseng tetradecapeptide.

[0013] The above method is characterized in that the polypeptide purification method is selected from any one of salting-out method, ultrafiltration method, affinity chromatography and gel filtration chromatography.

[0014] Furthermore, the present invention provides an application of the above-mentioned recombinant ginseng tetradecapeptide in the preparation of a drug for the management of diabetes.

[0015] Furthermore, the present invention provides an application of the above-mentioned recombinant ginseng tetradecapeptide in the preparation of a fermented product with higher safety and direct feeding potential.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] 1. The recombinant ginseng 14-peptide of the present invention is fermented and expressed by repeating the natural ginseng 14-peptide for a certain number of times, which can increase the expression amount and slow down the degradation of short peptides.

[0018] 2. Compared with chemical synthesis and plant extraction methods, the recombinant ginseng fourteen peptide of the present invention has lower cost and is expressed using a safer strain, so safety is guaranteed to a certain extent.

[0019] 3. The recombinant ginseng tetradecapeptide prepared by the present invention contains a hydrophilic FLAG tag, which increases the expression amount and can obtain the target polypeptide after affinity purification, thereby significantly improving the purification efficiency.

[0020] 4. The recombinant ginseng tetradecapeptide prepared by the present invention is a small molecule polypeptide, which has the advantages of easy absorption, higher activity, smaller dosage and lower toxic side effects.

[0021] 5. The recombinant ginseng tetradecapeptide prepared by the present invention and its fermented product can effectively inhibit the decomposition of fat by adrenaline, assist in stabilizing blood sugar and regulating inflammatory factors, and help insulin to play a better role.

[0022] 6. The recombinant ginseng tetradecapeptide prepared by the present invention and its fermented product are expressed by fermentation of a strain with higher safety and have the potential for direct feeding.

[0023] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the original plasmid of PYES2.

[0025] Figure 2 This is the recombinant plasmid of the ginseng tetradecapeptide PYES2-7GSP14αF of Example 1 of the present invention.

[0026] Figure 3 This is the glucan gel electrophoresis diagram of the target gene (GSP) in Example 1 of the present invention.

[0027] Figure 4 This is a graph verifying protein expression in Example 1 of the present invention.

[0028] Figure 5 This is a diagram of protein expression and purification in Example 1 of the present invention.

[0029] Figure 6 This is a diagram of Western Blotting verification of protein expression and purification in Example 1 of the present invention.

[0030] Figure 7 This is a graph showing that the recombinant ginseng tetradecapeptide of Example 3 of the present invention inhibits adrenaline from decomposing fat.

[0031] Figure 8 This is a diagram of the recombinant ginseng tetradecapeptide regulating cellular inflammatory factors according to Example 4 of the present invention. DETAILED DESCRIPTION

[0032] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, 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, unless otherwise specified, can be obtained from commercial sources.

[0033] The present invention repeats the natural ginseng 14 peptides for 7 to 20 times. The amino acid sequence of the natural ginseng 14 peptides is shown in SEQ ID NO: 1. In simple terms, the natural ginseng 14 peptides are connected head to tail according to specific experimental requirements. For example, repeating the natural ginseng 14 peptides twice is to connect the amino acid fragments shown in SEQ ID NO: 1 head to tail to form a 2-repeated amino acid fragment of ETVEIIDSEGGGDAETVEIIDSEGGGDA. Other repetition times can be deduced by analogy.

[0034] In a specific embodiment, the natural ginseng tetradecapeptide is repeated 7 times, and the amino acid sequence repeated 7 times is as shown in SEQ ID NO: 2. The gene encoding the recombinant ginseng tetradecapeptide repeated 7 times is codon-optimized for host cell expression, and BamHI and EcoRI restriction sites are added at both ends during the design process to facilitate later gene manipulation, and an α signal peptide and a FLAG tag are added at the N-terminus. After the above optimization, the gene encoding the recombinant ginseng tetradecapeptide is obtained, and its nucleotide sequence is as shown in SEQ ID NO: 3, positions 1-6 are BamHI restriction endonuclease restriction sites, positions 7-31 are essential nucleotide sequences of the original plasmid, positions 32-286 are nucleotide sequences of α signal peptides, positions 287-310 are nucleotide sequences of FLAG tags, positions 311-604 are nucleotide sequences corresponding to the natural ginseng tetradecapeptide repeated 7 times, positions 605-607 are terminators, and positions 608-613 are EcoRI restriction endonuclease restriction sites. When other repeat numbers are involved, only positions 311-604 need to be replaced with the specific required nucleotide sequence.

[0035] The present invention provides an expression vector containing the gene, which may contain regulatory sequences (such as transcription and translation start and stop codons) that are specific to the type of host (e.g., bacteria, fungi, plants or animals) into which the vector is to be introduced, as appropriate and taking into account whether the vector is DNA-based or RNA-based.

[0036] In a specific embodiment, the expression vector is PYES2, and its plasmid map is as follows: Figure 1 shown.

[0037] The present invention also provides a host cell comprising the expression vector, wherein the host cell refers to a cell into which an exogenous nucleic acid has been introduced, including the offspring of such a cell. The host cell includes transformants and transformed cells, which include primary transformed cells and offspring derived therefrom, regardless of the number of passages. The offspring may not be completely identical to the parent cell in terms of nucleic acid content and may contain mutations, and the host cell is selected from any one of the safer strains of Saccharomyces cerevisiae, lactic acid bacteria and Bacillus subtilis.

[0038] In a specific embodiment, the host cell is Saccharomyces cerevisiae BY4741.

[0039] The present invention also provides a method for preparing the recombinant ginseng tetradecapeptide and its fermented product, which comprises the following steps: using the above-mentioned host cell for expression, the fermented liquid after fermentation is concentrated as the fermented product, and then separating and purifying to obtain the recombinant ginseng tetradecapeptide. The host cell expression refers to culturing the host cell, and the culture medium and culture conditions are well known to those skilled in the art.

[0040] In a specific embodiment, the host cell is Saccharomyces cerevisiae. After obtaining the genetically engineered bacteria of Saccharomyces cerevisiae, the specific culture conditions are as follows: the genetically engineered bacteria of Saccharomyces cerevisiae are inoculated into SD-URA liquid culture medium, using raffinose as the carbon source and YNB as the nitrogen source, and cultured at 30°C and 220rpm for 40 hours. After the seed liquid is expanded, it is inoculated into a 1L shake flask (500mL of 1% yeast powder, 2% peptone, and 4% galactose liquid culture medium) at a 10% inoculation amount. The culture temperature is 29°C, expression is induced, and the culture time is 48 hours. During this period, a phosphate buffer is used to control the pH to 5.5-6.5.

[0041] The present invention does not impose any limitation on the expression mode, and it can be confirmed as needed. For example, the expression is induced expression, and the inducer for induced expression is galactose.

[0042] In a specific embodiment, galactose is added for induction culture, the temperature during the induction stage is 29° C. After 48 hours of induction, the bacterial solution is centrifuged and precipitated and then collected by suction filtration.

[0043] The present application does not impose any limitation on the separation and purification method, which can be determined according to different hosts, plasmids and culture media, for example, salting-out method, ultrafiltration method, affinity chromatography method or gel filtration chromatography method can be used.

[0044] Example 1: Expression of recombinant ginseng tetradecapeptide

[0045] The recombinant ginseng tetradecapeptide gene of the present invention was chemically synthesized (nucleotide sequence as shown in SEQ ID NO:3, positions 311-604). During synthesis, BamHI and EcoRI recognition sites were added to the 5' and 3' ends respectively, and after being digested by restriction endonucleases BamHI and EcoRI, it was cloned into the expression vector PYES2 (nucleotide sequence as shown in SEQ ID NO:4, plasmid map as shown in Figure 2 ), using Saccharomyces cerevisiae BY4741 as the expression host bacteria, the obtained cloned plasmid was transformed into BY4741 by the lithium acetate transformation method; the plate was spread on the nutrient-deficient medium SD-URA solid plate, and inverted and cultured at 30°C overnight to obtain Saccharomyces cerevisiae transformants; the Saccharomyces cerevisiae transformants were identified by the bacterial liquid PCR method, and the strain with the target size band and successfully matched for testing was the desired Saccharomyces cerevisiae engineering strain.

[0046] The genetically engineered yeast strain obtained above was inoculated into SD-URA medium, using raffinose as the carbon source and YNB as the nitrogen source, and cultured at 30°C and 220rpm for 40h, then inoculated into a 500mL shake flask with an initial volume of 300mL at a 10% inoculation amount, and the medium was replaced with a liquid medium containing 1% yeast powder, 2% peptone, and 4% galactose. The culture temperature was 29°C, and the expression was induced for 48h. During this period, phosphate buffer was used to control the pH at 5.5-6.5. The fermentation broth obtained was concentrated to obtain the fermentation product, and the supernatant obtained was collected by centrifugation. After ultrafiltration, the protein expression was verified by SDS-PAGE and Western Blotting. PCR, SDS-PAGE and Western Blotting verification are as follows Figures 3 to 5 shown.

[0047] Figure 3 The target gene was analyzed by dextran gel electrophoresis. PCR was performed on the bacterial solution of the transformants. The rightmost side of the electrophoresis graph is the positive control (PCR using chemically synthesized plasmid as template), the leftmost side is DNA 5000MARKER, and the middle side is PCR using plasmids extracted from the bacterial solution of different transformants as templates. Basically, obvious bands appeared and were the same size as the positive control, indicating that the transformation was successful.

[0048] Figure 4 This is the result of using FLAG antibody test paper to detect the fermentation broth and purified protein solution. The FLAG antibody test paper uses the colloidal gold method to detect the FLAG tag, which has strong specificity and extremely high sensitivity when combined with the FALG tag. It can be clearly seen that the fermentation broth supernatant contains the target protein, but the concentration is low. After preliminary ultrafiltration through an ultrafiltration tube, it is tested again with the label test paper to determine the expression of the target protein.

[0049] Figure 5This is the SDS-PAGE electrophoresis diagram after affinity purification of the protein, with MARKER on the far left, followed by flow-through 1, flow-through 2, column wash 1, column wash 2, column wash 3, FLAG elution, and column balance. The recombinant ginseng 14 peptide with FLAG tag has an obvious band at 10-15kD in the elution lane, which is slightly larger than the expected molecular weight. This may be caused by electrophoresis errors, and further verification is needed to determine whether this band is the target band.

[0050] Figure 6 Western Blotting verification of FLAG tag, the first one on the right is MARKER, Figure 6 It is obvious that the protein containing the FLAG tag is located at a molecular weight of 15kD, which preliminarily verifies that the target protein is successfully expressed.

[0051] Example 2: Purification of recombinant ginseng tetradecapeptide

[0052] (1) The supernatant collected by centrifugation in Example 1 was filtered out using a 0.22 μm filter membrane for affinity in the next step;

[0053] (2) Take an appropriate amount of FLAG affinity filler and load it into the column, drain out the protective solution, and wash the equilibrated column material with 5 volumes of TBS for later use;

[0054] (3) Add the supernatant after filtration and sterilization to the column, allow the supernatant to slowly flow through the column, and load the sample multiple times to ensure protein binding;

[0055] (4) Wash away unbound proteins with 10 column volumes of TBS. If necessary, collect the flow-through for subsequent analysis.

[0056] (5) Finally, 5 column volumes of 1× FLAG elution buffer (0.5 mg / mL) were used to elute the target protein bound to the FLAG antibody;

[0057] (6) Regenerate the gel with 5 column volumes of acidic eluent, then wash the equilibrated gel with 10 column volumes of TBS, add preservation solution and store at 4°C;

[0058] (7) Ultrafiltration desalting: G25 desalting column desalting, i.e. using 25 mL of G25 filler, the operation process is similar to the gel filtration chromatography step, 6.5 mL of sample is loaded each time, about 8 mL is collected, and desalting is completed 10 minutes after loading;

[0059] (8) The desalted solution was placed in a -20°C refrigerator for pre-freezing for 4 h, and then transferred to a vacuum freeze dryer for freeze-drying. After 48 h, the freeze-dried protein was collected and the freeze-dried protein sample was stored in a -80°C refrigerator for later use.

[0060] Note:

[0061] TBS: 50mM Tris, 150mM NaCl, pH7.4;

[0062] Acidic eluent: 0.1 M Tris-HCl, pH 2.5-3.5;

[0063] FLAG eluent: 1× FLAG protein powder was dissolved in TBS to make a 0.5 mg / mL solution;

[0064] Storage solution: 50% glycerol, 50% TBS, 0.01% sodium azide.

[0065] Example 3: Efficacy of recombinant ginseng tetradecapeptide in inhibiting adrenaline from decomposing fat

[0066] Porcine adipocytes were cultured at 1×10 4 The cells were inoculated in a 96-well plate containing recombinant ginseng tetradeoxypeptide and adrenaline to detect the effect of recombinant ginseng tetradeoxypeptide on adrenaline decomposition of fat cells (producing glycerol), and a control group was set up.

[0067] The brief description is as follows: cells in logarithmic growth phase were digested and centrifuged, washed twice with PBS, treated with trypsin, centrifuged, resuspended and counted, and diluted to a final concentration of 1×10 5 / mL, add 100μL of cell suspension to each well, incubate overnight at 37℃ in a 5% CO2 incubator, aspirate the original culture medium, add 100μL of MEM culture medium containing 0.1mg / ml epinephrine and 0.2mg / mL, 0.4mg / mL, 0.6mg / mL, 0.8mg / mL, and 1mg / mL recombinant ginseng tetradecapeptide, respectively, add 100μL of MEM culture medium containing 0.1mg / ml epinephrine to the control group, set 6 replicate wells for each concentration, take the supernatant after culturing for 24 hours, centrifuge and use a glycerol detection kit to detect the glycerol content in each well.

[0068] Calculation formula:

[0069]

[0070] The results of recombinant ginseng 14-peptide inhibiting adrenaline from decomposing pig fat cells are shown in Figure 7 The results show that the recombinant ginseng tetradecapeptide of the present invention can significantly inhibit the hydrolysis of adrenaline on fat cells, and the promoting effect becomes more obvious as the concentration increases.

[0071] Example 4: Regulatory effect of recombinant ginseng tetradecapeptide on cellular inflammatory factors

[0072] HepG2 cells were cultured at 1×10 4 The cells were inoculated in a 96-well plate containing ginseng tetradecapeptide, and the expression levels of inflammatory cytokines (TNF-a and IL-6) were detected. A control group was set up.

[0073] The brief description is as follows: cells in logarithmic growth phase were digested and centrifuged, washed twice with PBS, treated with trypsin, centrifuged, resuspended and counted, and diluted to a final concentration of 1×10 5 / mL, 100uL of cell suspension was added to each well, incubated overnight at 37°C in a 5% CO2 incubator, the original culture medium was aspirated, and MEM culture medium containing 18mM glucosamine was first added for induction for 16 hours to induce insulin resistance in HepG2 cells. Then the culture medium was aspirated, washed twice with PBS, and 100μL of MEM culture medium containing 0.2mg / mL, 0.4mg / mL, 0.6mg / mL, 0.8mg / mL, and 1mg / mL recombinant ginseng tetradecapeptide was added respectively. The control group was not induced to induce insulin resistance, and the cells were cultured with complete culture medium. Six replicate wells were set for each concentration. After 24 hours of culture, the mRNA and protein expression levels of inflammatory factors were analyzed by RT-qPCR and protein immunoblotting.

[0074] The results of the regulatory effect of recombinant ginseng tetradecapeptide on inflammatory cytokines in IR-HepG2 cells are shown in Figure 8 The results showed that the recombinant ginseng tetradecapeptide of the present invention can significantly reduce the mRNA and protein expressions of inflammatory factors in IR-HepG2 cells.

[0075] Note: *P<0.05, **P<0.01, ***P<0.001.

[0076] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A recombinant ginseng tetradecapeptide, characterized in that: It comprises an amino acid sequence repeatedly constructed by using the amino acid sequence of natural ginseng tetradecapeptide as a repeating unit; the amino acid sequence of the natural ginseng tetradecapeptide is SEQ ID NO: 1: ETVEIIDSEGGGDA, and the number of repetitions is 7 to 20 times.

2. A gene encoding the recombinant ginseng tetradecapeptide as claimed in claim 1.

3. An expression vector containing the gene as claimed in claim 2.

4. The expression vector according to claim 3, characterized in that The expression vector is selected from one of PYES2, pMG36e, PHY300-PLK and PHT43, and toxin-producing and antibacterial genes are removed.

5. A host cell containing the expression vector according to claim 3.

6. The host cell according to claim 5, characterized in that The host cell is selected from any one of Saccharomyces cerevisiae, lactic acid bacteria and Bacillus subtilis.

7. A method for preparing the recombinant ginseng tetradecapeptide as claimed in claim 1, characterized in that: The method comprises a gene encoding a recombinant ginseng tetradecapeptide, constructing an expression vector containing the gene of the recombinant ginseng tetradecapeptide, transferring the expression vector into a host cell and culturing the cell in a culture medium, inducing expression and then purifying the polypeptide to obtain the recombinant ginseng tetradecapeptide and a fermented product containing the recombinant ginseng tetradecapeptide.

8. The method according to claim 7, characterized in that The polypeptide purification method is selected from any one of salting-out, ultrafiltration, affinity chromatography and gel filtration chromatography.

9. Use of the recombinant ginseng tetradecapeptide as claimed in claim 1 in the preparation of a drug for the management of diabetes.

10. Use of the recombinant ginseng tetradecapeptide as claimed in claim 1 in the preparation of a fermented product with high safety and direct feeding potential.

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