Eukaryotic recombinant expression vector for expressing soybean peptide Vglycin and NK fusion protein and application of eukaryotic recombinant expression vector in preparation of fusion protein

Abstract

The invention relates to the technical field of bioengineering, in particular to an eukaryotic recombinant expression vector for expressing soybean peptide Vglycin and NK fusion protein and application of the eukaryotic recombinant expression vector in preparation of the fusion protein. According to the invention, pPIC9K-NK-3Vglycin-His is constructed, and on the basis of the pPIC9K-NK-3Vglycin-His, the fusion protein of the bioactive soybean peptides Vglycin and NK is expressed and purified. Wherein the alpha factor secretion signal peptide on the pPIC9K vector can enable the recombinant protein to be secreted outside the cell; the alcohol oxidase AOX1 gene promoter can strictly regulate and control the expression of a target gene; the His-tag is convenient to purify; the introduction of NK greatly prolongs the length of the fusion expression protein. The method for producing the bioactive fusion protein has the advantages of rapidness, high efficiency, low cost, high yield, high activity and the like.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, in particular to a eukaryotic recombinant expression vector for expressing soybean peptide Vglycin and NK fusion protein and application thereof in preparing the fusion protein. Background Art

[0002] Vglycin, a soybean peptide extracted from peas, is a 37-amino acid polypeptide with a molecular weight of 3786.4 Da. Its amino acid sequence, shown in SEQ ID NO. 5, contains six cysteine residues, which pair up to form three disulfide bonds: C3-C20, C7-C22, and C15-C32. The positions of these six cysteine residues are highly conserved. The C3-C20 and C7-C22 disulfide bonds form the backbone structure, while C15-C32 is interspersed within the backbone, forming a compact protein structure. Vglycin exhibits strong anti-pepsin, trypsin, and Glu-C protease activity. Vglycin has biological activities such as regulating glucose and lipid metabolism.

[0003] The His-tag, also known as a polyhistidine tag, is a short peptide consisting of six histidine chains (His-His-His-His-His-His, SEQ ID NO. 11). It is typically attached to the N- or C-terminus of a protein and can be used for the isolation and purification of recombinant proteins. Histidine forms coordination bonds with immobilized transition metal ions, enabling separation via nickel-column affinity chromatography. During the chromatography process, the His-tag is fused to the N- or C-terminus of the target protein, allowing it to be captured by nickel ions immobilized on various resins, thereby achieving purification of the target protein.

[0004] Nattokinase (NK) is an alkaline serine protease secreted by natto, a traditional Japanese fermented food. It boasts a long half-life, strong specificity, minimal side effects, and oral administration. The gene encoding nattokinase (aprN) begins with GTG and has an 1146bp open reading frame encoding 381 amino acids, including a 29-amino acid signal peptide, a 77-amino acid propeptide, and a 275-amino acid mature peptide, with a molecular weight of 27.7kDa. Inserting nattokinase downstream of the signal peptide significantly extends the length of the fusion protein, facilitating subsequent protein detection and purification.

[0005] Traditional bio-extraction and purification of the bioactive soy peptides Vglycin and nattokinase NK is complex, tedious, and produces low yields. Each step in the chemical synthesis of peptides requires the removal of byproducts. Consequently, as the amino acid sequence length increases, the chemical synthesis becomes more complex, resulting in more byproducts, complex purification, and increased costs. Recombinant expression of these proteins or peptides using genetic engineering techniques is the most cost-effective approach. Therefore, exploring better recombinant expression systems for the expression and purification of bioactive soy peptides holds immense theoretical and economic value. Summary of the Invention

[0006] The purpose of the present invention is to provide a eukaryotic recombinant expression vector for expressing soybean peptide Vglycin and NK fusion protein and its application in preparing fusion protein, so as to solve the problems existing in the above-mentioned prior art.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a eukaryotic recombinant expression vector for expressing a soybean peptide Vglycin and nattokinase NK fusion protein. The eukaryotic recombinant expression vector comprises a nattokinase NK gene, a soybean peptide Vglycin gene, and a 6His tag inserted into a multiple cloning site of the eukaryotic expression vector; the nucleotide sequence of the nattokinase NK gene is shown in SEQ ID NO.3; and the nucleotide sequence of the soybean peptide Vglycin gene is shown in SEQ ID NO.5.

[0009] Preferably, the copy number of the soybean peptide Vglycin gene is 3 copies;

[0010] The soybean peptide Vglycin genes are connected by an acid-sensitive site; the nucleotide sequence of the acid-sensitive site is GATCCG.

[0011] Preferably, the multiple cloning sites are EcoRI and NotI.

[0012] Preferably, the copy number of the 6His tag is 3 copies.

[0013] Preferably, the eukaryotic expression vector is pPIC9K-His.

[0014] The present invention provides a recombinant engineering bacterium comprising the above-mentioned eukaryotic recombinant expression vector.

[0015] The present invention provides the use of the above-mentioned eukaryotic recombinant expression vector or the above-mentioned recombinant engineering bacteria in the preparation of soybean peptide Vglycin and nattokinase NK fusion protein.

[0016] The present invention provides a method for preparing a soybean peptide Vglycin and nattokinase NK fusion protein, comprising the following steps:

[0017] The above-mentioned recombinant engineered bacteria are induced to culture, and the culture obtained by the induced culture is centrifuged, purified and eluted to obtain the soybean peptide Vglycin and nattokinase NK fusion protein.

[0018] Preferably, the culture medium used in the induction culture is BMMY culture medium; the temperature of the induction culture is 30°C, the rotation speed is 220 rpm, and the time is 72 hours;

[0019] And / or, the centrifugation temperature is 4°C, the speed is 5000 rpm, and the time is 5 min;

[0020] And / or, the purification comprises the step of purification using a Ni-NTA column;

[0021] And / or, the elution includes elution with a low-concentration imidazole buffer and elution with a high-concentration imidazole buffer.

[0022] Preferably, before the induction culture, seed liquid culture and activation culture are also included.

[0023] Further preferably, in the present invention, the Bradford method is used to perform quantitative analysis on the recombinant protein sample when estimating the yield.

[0024] Further preferably, in the present invention, when performing biological activity identification, the glucose uptake capacity of HepG2 cells in a basal state or under specific treatment is detected.

[0025] The present invention discloses the following technical effects:

[0026] This invention utilizes modern biotechnology to construct a eukaryotic recombinant expression vector, pPIC9K-NK-3Vglycin-His, for the expression and purification of a fusion protein of the bioactive soybean peptide Vglycin and nattokinase NK. In this fusion expression technique, the alpha factor secretion signal peptide on the pPIC9K vector enables extracellular secretion of the recombinant protein; the alcohol oxidase AOX1 gene promoter strictly regulates target gene expression; the His-tag facilitates purification of the recombinant protein; and the introduction of NK significantly extends the length of the fusion protein. This invention is a fast and efficient method for producing a fusion protein of the bioactive soybean peptide Vglycin and nattokinase NK, with low cost, high yield, and high activity, and has a promising market prospect.

[0027] At the same time, the present invention also provides an efficient and stable fusion expression and separation and purification technology for the bioactive soybean peptide Vglycin and nattokinase NK in eukaryotic cells, including the construction of a pPIC9K-NK-3Vglycin-His recombinant expression plasmid, linearization of the recombinant expression plasmid by single enzyme digestion, efficient expression in the Pichia pastoris GS115 strain, and purification of the soybean peptide Vglycin and nattokinase NK fusion protein. The present invention achieves efficient recombinant expression and rapid separation and purification of bioactive soybean peptides in a eukaryotic expression system through fusion expression technology, thereby achieving the production of bioactive peptides from soybeans and greatly reducing production costs. The soybean peptide Vglycin has multiple functions and has huge market demand. The use of this technology to recombinantly express and purify soybean peptides has the advantages of high yield, high activity, simple process, low production cost, etc., and has high industrial development value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 Designed a map for the recombinant plasmid pPIC9K-NK-3Vglycin-His;

[0030] Figure 2 Schematic diagram of double enzyme digestion identification of recombinant expression plasmid pPIC9K-NK-3Vglycin-His (left) and linearization diagram of recombinant plasmid pPIC9K-NK-3Vglycin-His (right); wherein, M in the left figure is a marker, and lane 1 is the recombinant expression plasmid pPIC9K-NK-3Vglycin-His; M in the right figure is a marker, and lane SaC I is the single enzyme digested pPIC9K-NK-3Vglycin-His recombinant expression plasmid, and Vector is the uncut pPIC9K-NK-3Vglycin-His recombinant expression plasmid;

[0031] Figure 3 Schematic diagram of the identification of pPIC9K-NK-3Vglycin-His Pichia pastoris engineering bacteria Figure 1 Wherein, M is a marker, lanes 1 to 20 are recombinant engineered bacteria samples 1 to 20, and N is a negative control group of GS115 strain that was not transformed with any plasmid;

[0032] Figure 4Schematic diagram of the identification of pPIC9K-NK-3Vglycin-His Pichia pastoris engineering bacteria Figure 2 Wherein, M is a marker, lanes 1 to 20 are recombinant engineered bacteria samples 1 to 20, and N is a negative control group of GS115 strain that was not transformed with any plasmid;

[0033] Figure 5 This is the sequence diagram of the sequencing results of the PCR products of the engineered bacteria;

[0034] Figure 6 Western blotting was used to detect the expression of the fusion protein before concentration. Lane P is the recombinant protein His-VDAC1 with a histidine tag, with a molecular weight of approximately 33 kDa, as a positive control. Lanes 1 / 4 / 5 / 7 / 18 / 20 are the culture supernatant samples of the corresponding numbered engineered bacteria. Lane N is a blank negative control group of the GS115 strain that was not transformed with any plasmid.

[0035] Figure 7 After concentration, Western blotting was used to detect the expression of the fusion protein. Lane P is the recombinant protein His-VDAC1 with a histidine tag, with a molecular weight of approximately 33 kDa, as a positive control. Lanes 1 / 4 / 5 / 7 / 18 / 20 are the supernatant samples of the corresponding numbered engineered bacteria culture. N is the blank negative control group of the GS115 strain that was not transformed with any plasmid.

[0036] Figure 8 is the linear regression equation of the standard protein;

[0037] Figure 9 is the protein content in the fermentation broth;

[0038] Figure 10 This is the image taken under a fluorescence microscope at a wavelength of 488nm;

[0039] Figure 11 Glucose absorption activity. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Since many bioactive soybean peptides derived from higher organisms have complex spatial structures and contain many disulfide bonds, Pichia pastoris has the subcellular structure of eukaryotic organisms and has the functions of post-translational modifications such as glycosylation, acetylation, and protein phosphorylation. Compared with the prokaryotic expression system, it can form correct disulfide bond pairing and spatial conformation folding.

[0046] Example 1 Construction of recombinant expression plasmid pPIC9K-NK-3Vglycin-His

[0047] (1) Test materials

[0048] Plasmids and strains: The recombinant expression plasmid pPIC9K-NK-3Vglycin-His and Escherichia coli competent DH5α were purchased from Beijing Qingke Biotechnology Co., Ltd. and Coolaber Biotechnology Co., Ltd., respectively.

[0049] Reagents: Restriction enzymes EcoR I and Not I were purchased from Takara Biotechnology Co., Ltd., and ampicillin was purchased from New England Biolabs.

[0050] Among them, the recombinant expression plasmid pPIC9K-NK-3Vglycin-His is composed of the nucleotide sequence of the recombinant plasmid pPIC9K-His, the acid-sensitive site Asp-Pro, the nattokinase NK gene, and the Vglycin gene in series;

[0051] (2) The construction method is as follows: using pPIC9K as the basic plasmid, using EcoR I and Not I as restriction sites, the bean kinase NK gene, 3 copies of the soybean peptide Vglycin gene and 3×6His are sequentially inserted from 5' to 3', and the soybean peptide Vglycin genes are connected in series with an acid-sensitive restriction site; wherein the amino acid sequence of the 3×6His tag is as shown in SEQ ID NO.1, specifically HHHHHHHHHHHHHHHHHH; the nucleotide sequence of 3×6His is as shown in SEQ ID NO.2, specifically CATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCAT; the nucleotide sequence of the bean kinase NK gene is as shown in SEQ ID NO.As shown in Figure 3, specifically GCGCAATCTGTTCCTTATGGCATTTCTCAAATTAAAGCGCCGGCTCTTCACTCTCAAGGCTACACAGGCTCTAACGTAAAAGTAGCTGTTATCGACAGCGGAATTGACTCTTCTCATCCTGACTTAAACGTCAGAGGCGGAGCAAGCTTCGTTCCTTCTGAAACAAACCCATACCAGGACGGCAGTTCTCACGGTACGCATGCCGCCGGTACGATTGCCGCTCTTAATAACTCAATCGGTGTTCTGGGCGTAGCGCCAAGCGCATCATTATATGCAGTAAAAGTGCTTGATTCAACAGGAAGCGGCCAATATAGCTGGATTATTAACGGCATTGAGTGGGCCATTTCCAACAATATGGATGTTATCAACATGAGCCTTGGCGGACCTACTGGTTCTACAGCGCTGAAAACAGTAGTTGATAAAGCGGTTTCCAGCGGTATCGTCGTTGCTGCCGCAGCCGGAAACGAAGGTTCATCCGGAAGCACAAGCACAGTCGGCTACCCTGCAAAATATCCTTCTACTATTGCAGTAGGTGCGGTAAACAGCAGCAACCAAAGAGCTTCATTCTCCAGCGTAGGTTCTGAGCTTGATGTAATGGCTCCTGGCGTGTCCATCCAAAGCACACTTCCTGGAGGCACTTACGGCGCTTATAACGGAACGTCCATGGCGACTCCTCACGTTGCCGGAGCAGCAGCGCTAATTCTTTCTAAGCACCCGACTTGGACAAACGCGCAAGTCCGTGATCGTTTAGAAAGCACTGCAACATATCTTGGAAACTCTTTCTACTATGGAAAAGGGTTAATCAACGTACAAGCAGCTGCACAA; The amino acid sequence of nattokinase NK encoded by the nattokinase NK gene is as shown in SEQ ID NO.4, specifically AQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHAAGTIAALNNSIGVLGVAPSASLYAVKVLDSTGSGQYSWIINGIEWAISNNMDVINMSLGGPTGSTALKTVVDKAVSSGIVVAAAAGNEGSSGSTSTVGYPAKYPSTIAVGAVNSSNQRASFSSVGSELDVMAPGVSIQSTLPGGTYGAYNGTSMATPHVAGAAALILSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQ; the nucleotide sequence of the soybean peptide Vglycin gene is shown in SEQ ID The amino acid sequence of the soybean peptide Vglycin encoded by the soybean peptide Vglycin gene is shown in SEQ ID NO. 6, specifically VSCNGVCSPFEMPPCGSSACRCIPYGLVVGNCRHPSG; the amino acid sequence of the acid-sensitive site is shown as Asp-Pro, and the nucleotide sequence is shown as GATCCG; the map of the recombinant expression plasmid pPIC9K-NK-3Vglycin-His (pPIC9K-NK-3Vg-His) is shown in SEQ ID NO. 7. Figure 1 As shown, in the open reading frame of the pPIC9K vector, from 5' to 3', there are: AOX promoter, alpha factor secretion signal peptide, nattokinase protein NK gene, 3 copies of soybean peptide Vglycin gene (soybean peptide Vglycin genes are connected in series with acid-sensitive enzyme cleavage sites) and 3 copies of 6×His tag.

[0052] (2) Implementation Plan

[0053] The constructed recombinant expression plasmid pPIC9K-NK-3Vglycin-His was transformed into competent Escherichia coli DH5α. After ampicillin resistance screening, positive clones were picked, and the plasmid was extracted in large quantities. After purification, restriction endonucleases EcoRI and Not I were added and digested at 37°C for 2 h. The digestion product was electrophoresed on 1% agarose gel. After gel recovery and purification, it was sent to Qingke Biotechnology Co., Ltd. for DNA sequencing, and the correct recombinant plasmid pPIC9K-NK-3Vglycin-His was obtained.

[0054] The results of double digestion with restriction enzymes EcoR I and Not I are as follows: Figure 2 As shown in the left panel, lane M is a molecular weight marker, and lane 1 shows the pPIC9K-NK-3Vglycin-His recombinant plasmid digested with double enzymes. The linearized DNA band should run slower than the intact helical DNA plasmid, consistent with theoretical results.

[0055] Example 2 Single enzyme linearization of the recombinant expression plasmid pPIC9K-NK-3Vglycin-His

[0056] (1) Test materials

[0057] Plasmids and strains: pPIC9K vector and Pichia pastoris competent GS115 were purchased from Beijing Qingke Biotechnology Co., Ltd. and Coolaber Biotechnology Co., Ltd., respectively.

[0058] Reagents: Restriction endonuclease Sac I was purchased from NEB.

[0059] The construction method of the recombinant expression plasmid pPIC9K-NK-3Vglycin-His is the same as that in Example 1.

[0060] (2) Implementation Plan

[0061] The recombinant expression plasmid pPIC9K-NK-3Vglycin-His was purified and then digested with restriction endonuclease SaC I at 37°C for 3 h. The digested product was electrophoresed on 1% agarose gel. The linearized band should run slower than the intact helical DNA plasmid, which is consistent with the theory ( Figure 2 (Right figure in the figure) The large fragment was recovered by gel excision to obtain the linearized plasmid pPIC9K-NK-3Vglycin-His.

[0062] Example 3 Screening of Monoclonal Yeast Engineering Strains with High Expression of Fusion Proteins

[0063] (1) Test materials

[0064] Primers and strains: Pichia pastoris competent GS115 was purchased from Coolaber Biotechnology Co., Ltd.; primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0065] Reagents: Taq Mix Buffer was purchased from Takara Biotechnology Co., Ltd.

[0066] (2) Implementation Plan:

[0067] A. Consult the map information to obtain universal primers for the pPIC9K vector. The primer sequences used for PCR amplification are as follows:

[0068] 5'AOX-F: GACTGGTTCCAATTGACAAGC, SEQ ID NO.7;

[0069] 3'AOX-R: GGCAAATGGCATTCTGACAT, SEQ ID NO. 8.

[0070] B. Using the synthesized NK-Vglycin coding sequence as a template, design forward and reverse primers with EcoR I and Not I restriction sites, respectively. The primer sequences for amplifying NK-Vglycin (underlined positions indicate restriction sites) are as follows:

[0071] Vglycin-F (EcoR I): CCGGAATTCGTGTCCTGCAACGGCGTG, SEQ ID NO.9;

[0072] Vglycin-R(Not I):AAATAT GCGGCCGC TTAATGATGATGATGATGATGATGATGATGATGATGATGATGATGATGATGATGATGCTTATCATCATCATCGCCGGACGGATGAC, SEQ ID NO.10;

[0073]

[0074] C. Screening of engineered yeast strains expressing fusion proteins

[0075] The linear plasmid pPIC9K-NK-3Vglycin-His obtained in Example 2 was electroporated into Pichia pastoris competent cells GS115 to obtain recombinant engineered bacteria. That is, the DNA of the linear plasmid pPIC9K-NK-3Vglycin-His was transferred into Pichia pastoris GS115 competent cells by electroporation, so that it was integrated into the genome of Pichia pastoris competent cells GS115 by homologous recombination. After screening by histidine plate culture, single colonies of the recombinant engineered bacteria were picked, and the genomic DNA of the recombinant engineered bacteria was extracted and used as a template for amplification using primers 5'AOX-F and 3'AOX-F. The electrophoresis results showed that the numbers 1 / 4 / 5 / 7 / 18 / 20 had a single clear band of approximately 1727 bp, which was different from the blank control group of the GS115 strain without the plasmid, which was consistent with the expected results ( Figure 3 In addition, PCR identification using primers Vglycin-F (EcoR I) and 3'AOX-R (Not I) showed a single clear band of approximately 1360 bp at a position close to 1500 bp, which was consistent with the expected result ( Figure 4 The PCR product was purified and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were completely consistent with the designed gene fragment ( Figure 5 ), indicating that the expression vector was successfully introduced into Pichia pastoris and integrated into the GS115 genome. A total of 6 pPIC9K-NK-3VG-His-expressing clone strains, numbered 1 / 4 / 5 / 7 / 18 / 20, were screened for expanded induced culture.

[0076] Example 4 Induced expression, culture and purification of Vglycin and Nattokinase NK fusion protein

[0077] (1) Test materials

[0078] A. Induced expression culture of fusion protein

[0079] The recombinant strain of genetically engineered bacteria constructed in Example 3 was inoculated into YPD liquid medium at a ratio of 1% and cultured at 30°C with shaking overnight; the activated fresh bacterial liquid was inoculated into 25 mL of BMGY medium at a ratio of 1% and cultured at 30°C and 220 rpm overnight until the OD 600=0.8~1.0; centrifuge at 4000rpm for 5min at room temperature, and collect the bacteria under sterile conditions; resuspend the bacteria in 100mL BMMY medium, and induce culture at 30℃ and 220rpm. The induction time is zero hour at this time; thereafter, filter-sterilized methanol is added every 24h to a final concentration of 1%, and the induction time is 72h. Collect the sample after 72h of induction, centrifuge at 8000rpm for 15min, and collect the fermentation supernatant as the polypeptide sample. The presence of the target polypeptide in the fermentation supernatant of the engineered bacteria was detected by 15% SDS-PAGE electrophoresis and Western blotting. Anti-His antibody was used to detect that the supernatant of the No. 1 / 5 / 7 / 18 engineered bacteria had obvious characteristic bands at around 55kDa, and the molecular weight was consistent with the theoretical value ( Figure 6 ), which verified that the fusion protein was successfully expressed in Pichia pastoris GS115, suggesting that VG may be successfully expressed in Pichia pastoris GS115 strain, and that subsequent large-scale induction culture and concentrated extraction of Vglycin can be considered.

[0080] B. Concentration and purification of recombinant protein

[0081] The supernatant sample, in which the soybean peptide Vglycin expression was detected, was added with PMSF (phenylmethylsulfonyl fluoride) at a volume ratio of 1:100 and applied to a Ni-NTA column for purification. The supernatant sample was slowly dripped to allow the protein to better bind to the column. After loading, contaminants were eluted using 5 column volumes of low-concentration imidazole buffer (50mM NaH2PO4, 500mM NaCl, 20mM imidazole, pH 8.0). The protein sample eluted with high-concentration imidazole buffer (50mM NaH2PO4, 500mM NaCl, 250mM imidazole, pH 8.0) was then eluted and collected. The Vglycin and nattokinase NK fusion protein was further purified using fast protein liquid chromatography (FPLC) from the Ni-NTA elution. Specific operation: The sample is loaded into the system through the injection pump. After stabilization, the salt concentration is set to increase from 0% to 100% linear gradient elution, and the elution flow rate is controlled at 0.5-2mL / min. The sample is collected step by step according to the UV absorption peak (such as A280). The sample is separated by 15% SDS-PAGE electrophoresis and the purification effect is detected by Western blotting ( Figure 7 ), it was observed that after purification, the crude extract of the recombinant polypeptide had a single band ( Figure 7 ), it is possible to consider concentrating and extracting VG after subsequent large-scale induction culture.

[0082] Example 5 Estimation of target yield of recombinantly expressed Vglycin

[0083] (1) Reagents: BCA protein quantification kit was purchased from Guangzhou Jiebaisi Biotechnology Co., Ltd.

[0084] (2) Implementation Plan

[0085] The recombinant protein samples were quantitatively analyzed using the Bradford method. Using bovine serum albumin (BSA) as the standard protein, a series of standard solutions with a concentration gradient of 0 to 1000 μg / mL were prepared. After each standard was thoroughly mixed with the Bradford working solution, the absorbance value was measured at a wavelength of 540 nm using a microplate reader ( Figure 8 ), with the standard protein concentration as the horizontal axis (x) and the corresponding absorbance value as the vertical axis (y), a standard curve was drawn to obtain the linear regression equation y = 0.7123x + 0.2282 (R 2 =0.999), the absorbance value was substituted into the linear regression equation to calculate the protein content of the sample solution. Figure 7 The protein content in the fermentation broth of the corresponding strain No. 5 reached 4.52 mg / L ( Figure 9 ). Thus, the method provided by the present invention can be used for concentrated extraction of soybean peptide Vglycin and nattokinase NK fusion protein after large-scale induction culture.

[0086] Example 6 Verification of biological activity of fusion expressed NK-Vglycin protein

[0087] (1) Test materials

[0088] Cells: HepG2 human hepatocellular carcinoma cell line was purchased from Takara Biotechnology Co., Ltd. and is commonly used to study glucose metabolism in hepatocytes in vitro;

[0089] Probe: Glucose Uptake Fluorescence Assay Kit with 2-NBDG was purchased from Beyotime Biotechnology Co., Ltd.

[0090] (2) Implementation Plan

[0091] The glucose uptake capacity of HepG2 cells was detected under basal state or specific treatment.

[0092] Cultured HepG2 cells were seeded into 12-well plates. When the cell abundance was close to 60%, recombinant protein soybean peptide Vglycin and nattokinase NK fusion protein (NK+VG), nattokinase NK (NK), and soybean peptide Vglycin (VG) were added for 24 h, with the concentration of each being 100 nM. Then, 100 μM 2-NDBG was added for 2 h. After washing with PBS three times, the cells were immediately imaged under a fluorescence microscope at a wavelength of 488 nm ( Figure 10). Blank control group (Control): The 2-NDBG fluorescence intensity of HepG2 cells under non-stimulated conditions was approximately 22AU (imageJ analysis). Uniform intracellular fluorescence signals were observed under a fluorescence microscope, reflecting the basal glucose uptake. After treatment with the purified NK+VG fusion protein and naturally extracted soybean peptide Vglycin for 30 minutes, the fluorescence intensity significantly increased to 26-30RFU, indicating enhanced glucose absorption activity ( Figure 11 After treatment with NK under the same conditions, the fluorescence intensity remained at 18-22 AU, which was comparable to that of the negative control group.

[0093] The above results indicate that the exogenously recombinantly expressed extracted soybean peptide Vglycin and nattokinase NK fusion protein can improve the glucose uptake ability and has the same biological activity as the naturally extracted soybean peptide Vglycin.

[0094] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A eukaryotic recombinant expression vector for expressing a fusion protein of soybean peptide Vglycin and nattokinase NK, characterized in that: The eukaryotic recombinant expression vector is a eukaryotic expression vector in which the nattokinase NK gene, the soybean peptide Vglycin gene and the 6His tag are inserted into the multiple cloning site of the eukaryotic expression vector; the nucleotide sequence of the nattokinase NK gene is shown in SEQ ID NO.3; the nucleotide sequence of the soybean peptide Vglycin gene is shown in SEQ ID NO.

5.

2. The eukaryotic recombinant expression vector according to claim 1, characterized in that The copy number of the soybean peptide Vglycin gene is 3 copies; The soybean peptide Vglycin genes are connected by an acid-sensitive site; the nucleotide sequence of the acid-sensitive site is GATCCG.

3. The eukaryotic recombinant expression vector according to claim 1, characterized in that The multiple cloning sites are EcoRI and NotI.

4. The eukaryotic recombinant expression vector according to claim 1, characterized in that The copy number of the 6His tag is 3 copies.

5. The eukaryotic recombinant expression vector according to claim 4, characterized in that The eukaryotic expression vector is pPIC9K.

6. A recombinant engineered bacterium comprising the eukaryotic recombinant expression vector according to any one of claims 1 to 5.

7. Use of the eukaryotic recombinant expression vector according to any one of claims 1 to 5 or the recombinant engineered bacteria according to claim 6 in the preparation of a fusion protein of soybean peptide Vglycin and nattokinase NK.

8. A method for preparing a fusion protein of soybean peptide Vglycin and nattokinase NK, characterized in that: The following steps are involved: The recombinant engineered bacteria according to claim 6 are induced to culture, and the culture obtained by the induced culture is centrifuged, purified and eluted to obtain the soybean peptide Vglycin and nattokinase NK fusion protein.

9. The preparation method according to claim 8, characterized in that The culture medium used in the induction culture is BMMY culture medium; the temperature of the induction culture is 30°C, the rotation speed is 220 rpm, and the time is 72 hours; And / or, the centrifugation temperature is 4°C, the speed is 5000 rpm, and the time is 5 min; And / or, the purification comprises the step of purification using a Ni-NTA column; And / or, the elution includes elution with a low-concentration imidazole buffer and elution with a high-concentration imidazole buffer.

10. The preparation method according to claim 8, characterized in that Before the induction culture, the process also includes seed liquid culture and activation culture.

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