Wheat bionic peptide as well as biosynthesis method and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses a wheat bionic peptide and a biosynthesis method and application thereof, and the method comprises the following steps: carrying out amino acid sequence analysis on a protein sequence of a target wheat peptide, and determining a target nucleotide sequence corresponding to a coded same protein sequence according to codon degeneracy; optimizing the target nucleotide sequence according to password preference of escherichia coli to obtain a wheat bionic peptide nucleotide sequence; treating and combining the obtained wheat bionic peptide nucleotide sequence and a vector plasmid through restriction enzyme to construct a recombinant plasmid; transferring the recombinant plasmid into host bacteria, performing fermentation culture to induce protein expression, separating protein, and purifying to obtain the wheat biomimetic peptide. The wheat biomimetic peptide is obtained in a biosynthesis mode, and compared with plant extraction and chemical synthesis, the cost is reduced, and meanwhile, the safety of the product is guaranteed.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a wheat bionic peptide and a biosynthesis method and application thereof. Background Art

[0002] As part of wheat seeds, wheat germ contains a variety of nutrients such as proteins, fats, vitamins and minerals. Previously, researchers discovered a small peptide from wheat germ with the sequence YDWPGGRN, which can significantly promote wound healing and named it small peptide YDW. Compared with traditional small molecules, peptides have good pharmacological properties and intrinsic properties, representing an excellent starting point for the design of new therapies, and their specificity can be translated into excellent safety, tolerability and efficacy in the human body. Currently, there are more than 60 peptide-related drugs on the market approved by the US Food and Drug Administration (FDA), and it is expected to grow significantly. Currently, about 140 peptide drugs are in clinical trials, and more than 500 therapeutic peptides are in clinical development. Traditionally, the preparation of wheat germ protein and its components adopts the extraction method, that is, after the wheat is crushed, the protein solution is obtained through water extraction, precipitation, ultrafiltration and other processes, and then the purified wheat polypeptide solution is finally obtained by membrane filtration and column chromatography. Research has found that the content of wheat polypeptide, the target product of this project, in wheat does not exceed 0.3‰, which is extremely low; the content of wheat polypeptide is not stable in wheat of different varieties, origins and years, and the process routes of extraction, separation and purification are complicated, and the cost of large-scale production is very high.

[0003] In practice, after the amino acid sequence of the target wheat polypeptide was analyzed and the amino acid sequence of the octapeptide of YDWPGGRN was obtained, the chemical method, namely the peptide solid phase synthesis method, was used to chemically synthesize the octapeptide. The specific method is: first, the 7 kinds of amino acid side chain groups contained in the octapeptide were protected, and then the amino group of the first amino acid at the amino end was connected to the carrier according to the amino acid sequence, and then the second amino acid was connected through carboxyl protection, amidation, carboxyl deprotection and other operations, and the operation was repeated until the octapeptide was synthesized, and then the side chain groups of the octapeptide were deprotected and separated from the carrier, and the target octapeptide was obtained after purification by column chromatography. Although the yield of this method is significantly improved compared with the extraction method, the process route is complicated, and the organic solvents and reagents used are of many types, large quantities, and high prices, which is not suitable for large-scale production. Summary of the invention

[0004] 1. Purpose of the invention The purpose of the present invention is to provide a wheat bionic peptide and a biosynthesis method and application thereof. The present invention obtains the wheat bionic peptide by biosynthesis, which reduces the cost and ensures the safety of the product compared with plant extraction and chemical synthesis.

[0005] (II) Technical solution To solve the above problems, the first aspect of the present invention provides a method for biosynthesis of wheat bionic peptides, wherein the nucleotide sequence of the wheat bionic peptides is composed of nucleotide fragments connected in series as shown in SEQ ID NO. 2, and the method comprises: The amino acid sequence of the target wheat peptide protein sequence is analyzed, and the target nucleotide sequence encoding the same protein sequence is determined according to the codon degeneracy. The amino acid sequence of the target wheat peptide is shown in SEQ ID NO. 1; The target nucleotide sequence is optimized according to the codon preference of Escherichia coli to obtain the wheat biomimetic peptide nucleotide sequence; The obtained wheat biomimetic peptide nucleotide sequence and the vector plasmid are treated with restriction endonucleases and combined to construct a recombinant plasmid; The recombinant plasmid is transferred into a host bacterium, and the protein expression is induced by fermentation culture. The protein is separated and purified to obtain the wheat bionic peptide.

[0006] Furthermore, the wheat biomimetic peptide nucleotide sequence obtained by optimizing the target nucleotide sequence according to the codon preference of Escherichia coli comprises: The target nucleotide sequence is codon optimized according to the codon preference of Escherichia coli to obtain an improved nucleotide sequence, the nucleotide sequence of which is shown in SEQ ID NO. 2; Then, the nucleotide fragment shown in SEQ ID NO. 2 is concatenated 20-50 times to obtain the wheat biomimetic peptide nucleotide sequence: Furthermore, the vector plasmid includes pET28a(+).

[0007] Furthermore, the host bacteria include Escherichia coli BL21 (DE3), C43 (DE3) and / or Rosseta.

[0008] Furthermore, the restriction endonuclease comprises: NdeI and / or HindIII.

[0009] Furthermore, the protein separation comprises: breaking the bacterial cell wall→collecting the inclusion body protein by centrifugation→washing the inclusion body protein→enzymatically hydrolyzing the inclusion body protein.

[0010] Furthermore, during the protein separation process, the pH is controlled at 2-6, the reaction temperature is controlled at 25-40°C, and the reaction time is controlled at 4-10 h.

[0011] Furthermore, the washing solution for washing the inclusion body protein includes a first washing solution and a second washing solution, wherein the first washing solution includes: Tris (trishydroxymethylaminomethane), EDTA (ethylenediaminetetraacetic acid), urea, TritonX-100 (polyethylene glycol octylphenyl ether) and mercaptoethanol; the second washing solution includes: Tris, EDTA and mercaptoethanol.

[0012] Furthermore, in the first washing solution, Tris is 50-200 mM / L, preferably 100 mM / L Tris, EDTA is 2-6 mM / L, preferably 5 mM / L, urea is 1-4 mM / L, preferably 2 mM / L, TritonX-100 (polyethylene glycol octylphenyl ether) is 1-5% (mass volume ratio, g / L), preferably 2%, and mercaptoethanol is 1-7 mM / L, preferably 5 mM / L.

[0013] Furthermore, the Tris content in the second washing solution is 50-200 mM / L, preferably 100 mM / L Tris. EDTA is 2-6 mM / L, preferably 5 mM / L and mercaptoethanol is 2-6 mM / L, preferably 5 mM / L.

[0014] The second aspect of the present invention provides a wheat bionic peptide prepared by the biosynthesis method of wheat bionic peptide as described in any one of the above descriptions, wherein the nucleotide sequence of the wheat bionic peptide is composed of nucleotide fragments as shown in SEQ ID NO.2 connected in series, and the series connection multiple is 20 to 50 times.

[0015] The third aspect of the present invention provides a use of a wheat bionic peptide prepared by any one of the biosynthetic methods of wheat bionic peptide described above in cosmetics, medicines or foods.

[0016] (III) Beneficial effects The above technical scheme of the present invention has the following beneficial technical effects: the present invention provides a wheat bionic peptide and a biosynthesis method and application thereof, wherein the method performs codon optimization on the amino acid sequence of wheat peptide YDW according to the codon preference of Escherichia coli, so that the expression probability of the wheat peptide is increased while the protein sequence remains unchanged, and the specific process is: performing amino acid sequence analysis on the protein sequence of the target wheat peptide, determining the target nucleotide sequence corresponding to the same protein sequence according to the codon degeneracy, optimizing the target nucleotide sequence according to the codon preference of Escherichia coli to obtain the nucleotide sequence of the wheat bionic peptide, and since the protein sequence of the target small peptide YDW is short and difficult to express in Escherichia coli, the nucleotide sequence of the wheat bionic peptide finally determined is designed by tandem repetition expression, which is composed of a nucleotide fragment as shown in SEQ ID NO.2, which is tandemly connected 20-50 times and used as a gene sequence encoding the wheat bionic peptide, and then using the gene sequence to construct a recombinant plasmid vector, which is transferred into a host bacterium, fermented and cultured to induce protein expression, and the protein is separated and purified to obtain the wheat bionic peptide. The present invention is based on the research results of synthetic biology, and adopts advantageous technologies such as genetic engineering bacteria construction technology, microbial high-density fermentation technology, biological separation and extraction technology, and biological enzyme catalysis technology. For the first time, wheat bionic peptides are obtained by biosynthesis. Compared with plant extraction and chemical synthesis, the production cost is greatly reduced. The cost of the biosynthesis method of the present invention is about 1 / 10 of that of chemical synthesis. At present, the cost of small peptides produced by large-scale plant extraction is about 100-500 yuan per gram, while the cost of the biosynthesis of the present invention is 1g: 30 yuan, which greatly reduces the production cost. At the same time, in terms of safety, the reagents used for biosynthesis fermentation and subsequent purification are simple, and chemical synthesis may introduce by-products (missing peptides, etc.). Compared with plant extraction, the biosynthesis cycle is shorter and the purification process is simpler. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the process route for biosynthesis and purification of wheat biomimetic peptides of the present invention; Figure 2 This is the electrophoresis diagram of the wheat biomimetic peptide inclusion body (different plasmids) of the embodiment of the present invention; Figure 3 This is the electrophoresis diagram of the wheat biomimetic peptide inclusion body (different expression strains) of the embodiment of the present invention; Figure 4 This is the protein electrophoresis diagram of the first washing sample of an embodiment of the present invention; Figure 5 It is the second washing sample protein electrophoresis of the embodiment of the present invention; Figure 6 This is an enzymatic electrophoresis diagram of inclusion bodies of an embodiment of the present invention; Figure 7 This is the liquid phase detection result of the wheat bionic peptide of the present invention. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0019] In view of the problem that wheat peptide YDW has significant efficacy but the cost of chemical synthesis is too high, the present invention provides a biosynthetic method of wheat bionic peptide, wherein the nucleotide sequence of the wheat bionic peptide is composed of nucleotide fragments in series as shown in SEQ ID NO. 2, and the method comprises: S1. Perform amino acid sequence analysis on the protein sequence of the target wheat peptide, and determine the target nucleotide sequence (Mhe gene sequence) encoding the same protein sequence according to the codon degeneracy. The amino acid sequence of the target wheat peptide is shown in SEQ ID NO.1 (corresponding to the full text: Tyr Asp Trp Pro Gly Gly Arg Asn). According to the codon degeneracy, the target nucleotide sequence encoding the same protein sequence is determined, that is, the amino acid sequence shown in SEQ ID NO.1 can be modified by replacing, deleting or adding one or more codons, and has the same function.

[0020] S2, optimizing the target nucleotide sequence according to the codon preference of Escherichia coli to obtain the wheat biomimetic peptide nucleotide sequence, specifically comprising: S21, codon optimization is performed on the target nucleotide sequence according to the codon preference of Escherichia coli to obtain an improved nucleotide sequence, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0021] S22, the improved nucleotide sequence is then connected in series to obtain the wheat biomimetic peptide nucleotide sequence. The nucleotide sequence shown in SEQ ID NO. 2 is connected in series 20-50 times. In view of the problem that the target small peptide YDW protein sequence is short and difficult to express in Escherichia coli, the present invention designs 20-50 times of tandem repetition expression. If the tandem multiple is too small, the protein expression amount is small, and if the tandem multiple is too large, the subsequent purification is difficult. Therefore, the preferred tandem repetition expression is 30 times.

[0022] S3, treating the obtained wheat biomimetic peptide nucleotide sequence with a vector plasmid through restriction endonucleases and combining them to construct a recombinant plasmid; the vector plasmid includes pET28a(+). The restriction endonucleases include: NdeI and / or HindIII.

[0023] S4, transfer the recombinant plasmid into the host bacteria, ferment and culture to induce protein expression, separate the protein, purify it to obtain the wheat biomimetic peptide. The host bacteria include Escherichia coli BL21 (DE3), C43 (DE3) and / or Rosseta. The present invention selects three Escherichia coli as expression hosts (BL21 (DE3), C43 (DE3) and Rosseta) and optimizes them. The protein separation process includes: cell wall breaking → centrifugal collection of inclusion body protein → washing inclusion body protein → enzymatic hydrolysis of inclusion body protein. The specific steps of step S4 are as follows: implanting the recombinant plasmid into Escherichia coli (E.coli); heterologously expressing the multimeric inclusion bodies of the polypeptide by fermentation and culture of E.coli; obtaining the inclusion bodies (inclusion body proteins) by cell crushing, centrifugation and washing of the fermented bacteria; directional enzymatic hydrolysis of the inclusion bodies under the catalysis of pepsin to obtain a polypeptide product with a completely consistent amino acid sequence with the original wheat polypeptide; ultrafiltration and nanofiltration of the enzymatic reaction liquid to finally obtain a certain amount of wheat polypeptide biomimetic peptide product in aqueous solution. In the process of separating proteins, the pH is controlled to be 2-6, the reaction temperature is 25-40°C, and the reaction time is 4-10h.

[0024] The second aspect of the present invention provides a wheat bionic peptide prepared by the biosynthesis method of wheat bionic peptide as described in any one of the above descriptions, wherein the nucleotide sequence of the wheat bionic peptide is composed of nucleotide fragments as shown in SEQ ID NO.2 connected in series, and the series connection multiple is 20 to 50 times.

[0025] The third aspect of the present invention provides a use of a wheat bionic peptide prepared by any one of the biosynthetic methods of wheat bionic peptide described above in cosmetics, medicines or foods.

[0026] The present invention is described in detail below with reference to examples and drawings.

[0027] Example Escherichia coli that can express wheat biomimetic peptides is constructed, and wheat biomimetic peptides are obtained in the form of inclusion bodies through protein induction expression. The inclusion bodies are then washed to remove foreign proteins, followed by an enzyme digestion system to enzymatically hydrolyze the inclusion bodies, and finally a certain amount of wheat peptide biomimetic peptide products are obtained through separation and purification. The process route is as follows: Figure 1 shown.

[0028] 1. Construction and expression of wheat biomimetic peptide recombinant bacteria 1. Plasmid construction and verification The amino acid sequence of the small peptide YDW (target wheat peptide) from wheat germ is shown in SEQ ID NO. 1 (SEQ ID NO. 1 is an abbreviation, and the corresponding full sequence is: Tyr Asp Trp Pro Gly Gly Arg Asn); the corresponding improved nucleotide sequence is shown in SEQ ID NO. 2. The nucleotide fragments shown in SEQ ID NO. 2 were concatenated 20, 30 and 50 times respectively to obtain the nucleotide sequence of the wheat biomimetic peptide, and the nucleotide sequences thereof were shown in SEQ ID NO. 3 (20 times in series), SEQ ID NO. 4 (30 times in series) and SEQ ID NO. 5 (50 times in series), respectively, to construct plasmids with 20, 30 and 50 times of tandem repeats. The constructed plasmids pET28a(+)-MheP20, pET28a(+)-MheP30 and pET28a(+)-MheP50 were respectively transferred into the strain TOP10, and several transformants on the transformation plate were picked for colony PCR. After verification, they were stored in a -80℃ refrigerator. In MheP20 (Mhe is the product name, P20 represents its tandem multiple is 20, and the same applies to the rest), the plasmid vector selected is pET-28a(+), and the restriction sites are NdeI and HindIII.

[0029] The constructed plasmids pET28a(+)-MheP20, pET28a(+)-MheP30 and pET28a(+)-MheP50 were respectively transferred into the strain BL21(DE3), and the strains BL21(DE3)-MheP20, BL21(DE3)-MheP30 and BL21(DE3)-MheP50 were induced to express. The inclusion body samples expressed by the three strains were taken for protein electrophoresis. Figure 2 This is the electrophoresis diagram of wheat biomimetic peptide inclusion body precipitation. Figure 2 From left to right in the figure are the electrophoresis results of inclusion body proteins of the above three strains. It can be seen that the three tandem expression effects of 20, 30 and 50 are relatively good.

[0030] 2. Strain Construction Pick TOP10-MheP30 (strain TOP 10 carrying plasmid (pET-28a) MheP30) from the glycerol tube, extract the recombinant plasmid after overnight culture, and then transfer the recombinant plasmid into Escherichia coli BL21 (DE3), C43 (DE3) and Rosseta respectively, pick several transformants on the transformation plate, perform colony PCR, and store them in a -80℃ refrigerator after verification.

[0031] 3. Protein expression Single clones were picked from three plates, and seed solution was obtained by overnight culture. 1 ml of seed solution was inoculated into 100 ml LB medium, and cultured at 37°C and 200 rpm for about 4 h until OD600 (the absorbance of a certain solution at a wavelength of 600 nm) reached 0.6-1.0. Then the inducer IPTG (isopropylthiogalactoside) was added, the temperature was lowered to 28°C, and cultured for 16 h. Centrifugation was performed, the supernatant was discarded, and the bacteria were collected. The bacterial cell wall breaking process includes: resuspending the bacteria with 1×PBS buffer (pH7.5), breaking with an ultrasonic disruptor at 65 W for 30 min, and keeping on ice throughout the process. Finally, centrifuge at 12000 rpm for 10 min, and the supernatant (protein supernatant) and precipitate (inclusion body protein) were stored at -20°C respectively (centrifugation was performed to collect inclusion body protein). Take a small amount of the three cell-breaking precipitates, mix them with the protein loading buffer, heat at 100°C for 10 min, and perform protein electrophoresis. Figure 3 This is the electrophoresis diagram of wheat biomimetic peptide inclusion body precipitation, which verifies the successful expression of wheat biomimetic peptide in Escherichia coli. Figure 3 In the middle (three groups of results in the red box), the leftmost group is the two parallel protein electrophoresis detection results of the small peptide inclusion bodies (wheat biomimetic peptide inclusion bodies) induced by fermentation of strain BL21 (DE3), the middle group is the two parallel protein electrophoresis detection results of the small peptide inclusion bodies induced by fermentation of strain C43 (DE3), and the rightmost group is the two parallel protein electrophoresis detection results of the small peptide inclusion bodies induced by fermentation of strain Rosseta.

[0032] 2. Enzymatic hydrolysis and purification of wheat biomimetic peptide inclusion bodies 1. Inclusion body pretreatment (washing inclusion body protein) The inclusion body proteins obtained by crushing were washed twice with 5 mL of the first washing solution. After each wash, the mixture was centrifuged at 4°C, 10,000 rpm for 20 min, the supernatant was discarded, and the precipitate in the centrifuge tube was collected. The first washing solution contained 100 mM Tris (trishydroxymethylaminomethane), 5 mM EDTA (ethylenediaminetetraacetic acid), 2 M urea, 2% TritonX-100 (polyethylene glycol octylphenyl ether) and 5 mM mercaptoethanol.

[0033] Then wash twice with 5 mL of the second washing solution. After each wash, centrifuge at 4°C, 10,000 rpm for 20 min, discard the supernatant, and collect the precipitate in the centrifuge tube. The second washing solution contains 100 mM Tris, 5 mM EDTA, and 5 mM mercaptoethanol.

[0034] The precipitate after two washes was mixed with protein loading buffer and heated at 100°C for 10 min. The protein electrophoresis results of the first wash sample are shown in Figure 4, proving that the first washing solution can wash away most of the impurities in the inclusion bodies. Figure 4 The band in the red box is the target inclusion body band after the first wash. Figure 2 Compared with 30K in the image, the band in this area is brighter and more obvious than the band outside the frame. The protein electrophoresis results of the second washing sample are shown in Figure 5 , proving that most of the impurities have been removed after the second washing with washing solution. Figure 5 The band in the red box is the target inclusion body band after the second wash. Figure 4 In comparison, the stripes in this area are brighter and have a more obvious contrast with the stripes outside the frame.

[0035] 2. Enzyme digestion of inclusion bodies Prepare enzymatic hydrolysis buffer, including: (0.4% (w / v) pepsin, 20 mM Tris-HCl and 250 mM NaCl, pH2.0). Add the inclusion body protein after the second wash to the enzymatic hydrolysis buffer according to the preset ratio (1-2%, w / v), and finally add pepsin (0.4-0.6%, w / v) to prepare the reaction solution, start enzymatic hydrolysis, control the reaction temperature at 37°C, and stir at about 200 rpm to fully resuspend the inclusion body in the reaction solution. Enzymatic hydrolysis was performed for about 7 hours to obtain a crude aqueous solution of wheat polypeptide biomimetic peptide. Take the enzymatic hydrolysis product and mix it with the protein loading buffer, heat it at 100°C for 10 min, Figure 6 This is the result of inclusion body enzymatic hydrolysis electrophoresis, which proves that the inclusion body has been completely enzymatically hydrolyzed. Figure 6 This is the process of the gradual enzymatic hydrolysis of the inclusion bodies (the default number of points from left to right is 1-7). From the green framed bands, we can see that points 1-5 are samples at five time points in the enzymatic hydrolysis of the inclusion bodies. The bands in the frame gradually fade until 4 and 5 have disappeared. Point 6 is Figure 2 (30K), the bands of inclusion bodies in 3 and 4, point 7 (far right) is the protein electrophoresis result of the wheat eco-mimicking sample (its molecular weight is too small, and no bands will appear in protein electrophoresis), points 6 and 7 are control samples, and the electrophoresis results of samples 4 and 5 do not show the band of sample 6, and are the same as the results of 7, proving that the enzymatic reaction has reached the end point.

[0036] Wheat biomimetic peptide purification The precipitate in the protein hydrolysis reaction solution of the inclusion body was removed by suction filtration, and the supernatant was purified by a 10 k cutoff membrane and a 3 k cutoff membrane in sequence. The wheat biomimetic peptide was obtained after two purifications. Figure 7 As shown, the peak time is about 10 minutes, the peak shape is good, and there are no mixed peaks.

[0037] 3. Pilot production verification of wheat biomimetic peptide in 100 L fermentation tank Strain fermentation and induced expression Pipette the seed solution (Rosseta-MheP30) from the glycerol tube (the seed solution in the "protein expression" step) and inoculate it into the primary culture medium (150 / 500 mlLB) at a thousandth of the inoculation amount, and culture it overnight. Pipette the bacterial solution from the primary culture medium and inoculate it into the secondary culture medium (2 / 5 LLB) at a hundredth of the inoculation amount, and culture it for about 5 hours. When the OD600 reaches 2~3, stop the culture. Inoculate the bacterial solution in the secondary culture medium into the fermentation tank (60 L high-density culture medium) at a tenth of the inoculation amount, add phosphate and antibiotics that have been pre-filtered, connect ammonia water and glycerol to feed, and start fermentation. When the OD600 reaches 20, stop stirring, add the inducer, control the tank temperature to 28℃, start inducing expression, and stop fermentation after 13 hours of induction. During the fermentation, it is necessary to add feed and alkali in time to control the dissolved oxygen and pH within the appropriate range.

[0038] Fermentation product processing The fermentation liquid was centrifuged and the supernatant was removed to obtain bacterial sludge. The bacterial sludge and PBS buffer were mixed at a weight ratio of 1:4 and the cells were ruptured using a homogenizer. The cell rupture conditions were: once at 200 bar, once at 800-900 bar, once at 1000 bar, and once at 1300 bar. The cell rupture solution was centrifuged using a tubular centrifuge at a speed of 16000 rpm, and the supernatant was removed to obtain a cell rupture precipitate.

[0039] Inclusion body hydrolysis and product purification According to the process in the inclusion body pretreatment, the cell precipitate is washed, and the washed inclusion body is added to the configured enzyme digestion system at 1% (w / v). After 6 hours of enzyme digestion, the sample is detected by liquid phase, and its content is 7.69 mg / ml (yield 77%). After passing through an ultrafiltration tube with a molecular weight of 10,000, the small peptide content is 7.42 mg / ml (yield 74%), and after passing through an ultrafiltration tube with a molecular weight of 3,000, the small peptide content is 7.00 mg / ml (yield 70%). At present, the cost of small peptides produced by large-scale plant extraction is about 100-500 yuan per gram, and the cost of biosynthesis of the present invention is 1 g: 30 yuan, which is not only significantly lower than the plant extraction production method, but also significantly lower than the chemical synthesis method, about 1 / 10 of the chemical synthesis, greatly reducing the production cost.

[0040] The invention provides a wheat bionic peptide and a biosynthesis method and application thereof. The method can obtain the wheat bionic peptide at a relatively low cost. First, the amino acid sequence of the wheat peptide YDW is codon-optimized according to the codon preference of Escherichia coli, so that the expression probability of the wheat peptide is increased while the protein sequence remains unchanged. The protein sequence of the target wheat peptide is subjected to amino acid sequence analysis, and the target nucleotide sequence corresponding to the same protein sequence is determined according to the codon degeneracy. The target nucleotide sequence is optimized according to the codon preference of Escherichia coli to obtain the nucleotide sequence of the wheat bionic peptide. In addition, since the protein sequence of the target small peptide YDW is relatively short and difficult to express in Escherichia coli, the final determined The nucleotide sequence of wheat bionic peptide is designed to be expressed by 20-50 times tandem repetition, and a recombinant plasmid is constructed. The plasmid vector is pET-28a(+), the restriction sites are NdeI and HindIII, and three Escherichia coli are selected as expression hosts (BL21(DE3), C43(DE3) and Rosseta), and the optimization is carried out. The invention also optimizes the induction expression conditions of wheat bionic peptide, obtains wheat bionic peptide in the form of inclusion body through protein induction expression, and then washes the inclusion body to remove foreign proteins, and then configures the enzyme cutting system to enzymatically hydrolyze the inclusion body, and finally obtains a certain content of wheat peptide biomimetic peptide products through separation and purification. The invention obtains wheat bionic peptide by biosynthesis for the first time, and compared with plant extraction and chemical synthesis, it reduces the cost while ensuring the safety of the product.

[0041] Obviously, the described embodiments are part of the embodiments of the present invention, but not all of them. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. In the description of the present invention, it should be noted that the terms "first", "second" and "third" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance. In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. It should be understood that the above-mentioned specific embodiments of the present invention are only used to illustrate or explain the principles of the present invention, and do not constitute a limitation on the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the scope of protection of the present invention. In addition, the claims attached to the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the attached claims, or the equivalent forms of such scope and boundaries. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the embodiments here. And the obvious changes or changes derived from this are still within the scope of protection created by the present invention.

Claims

1. A method for biosynthesis of wheat biomimetic peptides, characterized in that: Wheat biomimetic peptide The nucleotide sequence is composed of nucleotide fragments as shown in SEQ ID NO. 2 in series, and the method comprises: The amino acid sequence of the target wheat peptide protein sequence is analyzed, and the target nucleotide sequence encoding the same protein sequence is determined according to the codon degeneracy. The amino acid sequence of the target wheat peptide is shown in SEQ ID NO. 1; The target nucleotide sequence is optimized according to the codon preference of Escherichia coli to obtain the wheat biomimetic peptide nucleotide sequence; The obtained wheat biomimetic peptide nucleotide sequence and the vector plasmid are treated with restriction endonucleases and combined to construct a recombinant plasmid; The recombinant plasmid is transferred into a host bacterium, and the protein expression is induced by fermentation culture. The protein is separated and purified to obtain the wheat bionic peptide.

2. The biosynthesis method of wheat biomimetic peptide according to claim 1, characterized in that: The method of optimizing the target nucleotide sequence according to the codon preference of Escherichia coli to obtain the wheat biomimetic peptide nucleotide sequence comprises: The target nucleotide sequence is codon optimized according to the codon preference of Escherichia coli to obtain an improved nucleotide sequence, the nucleotide sequence of which is shown in SEQ ID NO. 2; Then the nucleotide fragment shown in SEQ ID NO. 2 is concatenated 20-50 times to obtain the wheat biomimetic peptide nucleotide sequence.

3. The biosynthesis method of wheat biomimetic peptide according to claim 1, characterized in that: The vector plasmid includes pET28a(+).

4. The biosynthesis method of wheat biomimetic peptide according to claim 1, characterized in that: The host bacteria include Escherichia coli BL21 (DE3), C43 (DE3) and / or Rosseta.

5. The biosynthetic method of wheat biomimetic peptide according to claim 1, characterized in that: The restriction endonucleases include: NdeI and / or HindIII.

6. The biosynthesis method of wheat biomimetic peptide according to claim 1, characterized in that: The protein separation comprises: breaking the bacterial cell wall→collecting the inclusion body protein by centrifugation→washing the inclusion body protein→enzymatically hydrolyzing the inclusion body protein.

7. The biosynthesis method of wheat biomimetic peptide according to claim 1, characterized in that: During the protein separation process, the pH is controlled at 2-6, the reaction temperature is 25-40°C, and the reaction time is 4-10 h.

8. The wheat biomimetic peptide prepared by the biosynthesis method of the wheat biomimetic peptide according to any one of claims 1 to 7, characterized in that: The nucleotide sequence of the wheat bionic peptide is composed of nucleotide fragments as shown in SEQ ID NO. 2 connected in series, and the number of connected fragments is 20 to 50 times.

9. Use of the wheat biomimetic peptide prepared by the biosynthesis method of the wheat biomimetic peptide according to any one of claims 1 to 7 in cosmetics, medicines or foods.

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