Stomach pentadecapeptide fusion protein for relieving stomach inflammation and promoting ulcer surface healing as well as preparation method and application of stomach pentadecapeptide fusion protein

Abstract

The invention belongs to the technical field of biology, and particularly relates to a stomach pentadecapeptide fusion protein capable of relieving stomach inflammation and promoting ulcer surface healing and a preparation method and application of the stomach pentadecapeptide fusion protein. The stomach pentadecapeptide fusion protein provided by the invention is composed of gamma-glutamine transpeptidase, a trypsin recognition site and stomach pentadecapeptide. The stomach pentadecapeptide fusion protein can realize high-efficiency expression of the BPC157 in recombinant bacteria, and a new method is provided for producing the BPC157 by using a recombinant protein production technology. Moreover, the stomach pentadecapeptide fusion protein can play a significant role in reducing the gastric mucosa injury area, reducing inflammatory factors and promoting ulcer surface healing under a relatively low dosage, provides a new way for improving the wound repair promoting effect of BPC157, and can be used as a new active component for preparing products for relieving stomach inflammation and promoting ulcer surface healing.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a gastric pentadecapeptide fusion protein capable of relieving gastric inflammation and promoting ulcer surface healing, and a preparation method and application thereof. Background Art

[0002] Small molecule peptides are a type of multifunctional compound derived from proteins, which are composed of several to dozens of amino acids. The amino acids are arranged in different orders, have diverse structures, and have different biological activities. Small molecule peptides can participate in a variety of human metabolism and physiological regulation activities, are easy to digest and absorb, and have the effects of promoting immunity, hormone regulation, antibacterial, antiviral, lowering blood pressure, and lowering blood lipids. They are extremely safe to eat and have high medicinal value. They are functional factors with great development prospects in the current international food industry.

[0003] BPC157 is a 15-amino acid peptide fragment in BPC with a molecular weight of 1417 kDa. This small molecule active peptide has all the functions of the complete BPC molecule in promoting wound repair. It is a peptide that can promote wound repair and protect a variety of damaged important organs. It has been proven to be effective in inflammatory gastrointestinal diseases and has potential practical application value. In addition, BPC157 has good stability and can be stored at room temperature. Therefore, BPC157 has important value for the food industry and the healthcare industry.

[0004] However, BPC157 is a small molecule. If it is directly expressed by recombinant bacteria, it is easily hydrolyzed by proteases secreted by some host bacteria. It is not easy to produce it by recombinant protein production technology, which limits its application to a certain extent. In addition, the wound repair effect of BPC157 requires a relatively high dose to produce the ideal effect. New methods need to be developed to improve its wound repair effect, thereby improving its application value. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a gastric pentadecapeptide fusion protein for relieving gastric inflammation and promoting ulcer healing, as well as a preparation method and application thereof. The gastric pentadecapeptide fusion protein provided by the present invention can achieve efficient expression of BPC157 in recombinant bacteria, providing a new method for producing BPC157 using recombinant protein production technology; in addition, the gastric pentadecapeptide fusion protein can effectively protect the gastric mucosa at a relatively low dosage, providing a new way to improve the effect of BPC157 in promoting wound repair.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: The first aspect of the present invention provides a gastric pentadecapeptide fusion protein for relieving gastric inflammation and promoting ulcer healing. The gastric pentadecapeptide fusion protein is composed of γ-glutamine transpeptidase, a trypsin recognition site and gastric pentadecapeptide, and the connection mode is γ-glutamine transpeptidase-trypsin recognition site-gastric pentadecapeptide.

[0007] GGT is a specific enzyme widely distributed in microorganisms, which can be cleaved into two subunits, large and small, and secreted for extracellular expression. The present invention uses a tandem fusion method to genetically fuse BPC157 and GGT in an "end-to-end" manner, and uses the expression element of GGT to efficiently express the fusion protein, which can achieve efficient expression of BPC157 in recombinant bacteria.

[0008] In addition, gastric juice contains a small amount of trypsin. The fusion protein removes the terminator of γ-glutamine transpeptidase (GGT) and adds a trypsin recognition site at the N-terminus of BPC157 to separate GGT and BPC157 in gastric juice. At present, there is no report that GGT has the effect of protecting gastric mucosa. The present invention has found through experimental research that the fusion protein can significantly reduce the area of ​​gastric mucosal damage, reduce inflammatory factors, and promote the healing of ulcer surface at a lower dosage. This effect is significantly better than using BPC157 alone, indicating that GGT and BPC157 have a synergistic effect in protecting gastric mucosa.

[0009] Therefore, the present invention provides a new method for producing BPC157 using recombinant protein production technology, and provides a new way to improve the effect of BPC157 in promoting wound repair, thereby further improving the application value of BPC157 in the food industry and health care industry. The fusion protein can be used as a new active ingredient to prepare drugs or feed additives that relieve gastric inflammation and promote ulcer healing.

[0010] Preferably, the amino acid sequence of the γ-glutamine transpeptidase is shown in SEQ ID No.1.

[0011] SEQ ID No. 1: .

[0012] Under the premise of not affecting the protein structure and activity, the amino acid sequence obtained by substituting, deleting or adding one or more amino acids or terminally modifying the amino acid sequence shown in SEQ ID No. 1 also falls within the protection scope of the present invention.

[0013] Preferably, the amino acid sequence of the gastric pentadecapeptide is as shown in SEQ ID No.2.

[0014] SEQ ID No. 2:GEPPPGKPADDAGLVHHHHHH*.

[0015] Under the premise of not affecting the protein structure and activity, the amino acid sequence obtained by substituting, deleting or adding one or more amino acids or terminally modifying the amino acid sequence shown in SEQ ID No. 2 also falls within the protection scope of the present invention.

[0016] Preferably, the amino acid sequence of the trypsin hydrolysis site is RR.

[0017] Adding the trypsin hydrolysis site to the N-terminus of BPC157 enables the fusion protein to be separated into GGT and BPC157 in gastric juice.

[0018] Preferably, the amino acid sequence of the gastric pentadecapeptide fusion protein is as shown in SEQ ID No.3.

[0019] SEQ ID No.3:*.

[0020] The gastric pentadecapeptide fusion protein can be chemically synthesized by solid phase synthesis, or can be obtained by constructing a recombinant vector containing its coding gene, and then constructing a recombinant strain expressing the gastric pentadecapeptide fusion protein, inducing the expression of the recombinant strain, and then purifying and replacing the obtained protein.

[0021] The second aspect of the present invention provides a gene encoding the above-mentioned pentadecapeptide fusion protein.

[0022] The third aspect of the present invention provides a recombinant vector containing the above encoding gene.

[0023] Preferably, the sequence of the encoding gene is shown as SEQ ID No.4.

[0024]

[0025] Preferably, the expression vector of the recombinant vector is plasmid pKSVT.

[0026] The fourth aspect of the present invention provides a method for constructing the above-mentioned recombinant vector, which specifically comprises the following operations: using Bacillus licheniformis ( B. licheniformis ) 2709 genome as a template, using specific primers UP-GF / UP-GR, Down-GF / Down-GR for PCR amplification; the sequence of the primer UP-GF is shown in SEQ ID No.5, the sequence of the primer UP-GR is shown in SEQ ID No.6, the sequence of the primer Down-GF is shown in SEQ ID No.7, and the sequence of the primer Down-GR is shown in SEQ ID No.8; using restriction endonucleases Sma I and Sac I. Perform double restriction digestion on plasmid pKSVT to obtain linearized vector; recombinantly connect the PCR amplification product and the linearized vector, and transform the connected product into competent cells. E. coli JM109, and then extract the plasmid from the correctly sequenced transformant to obtain the recombinant vector.

[0027] In the design of the above-mentioned specific primers, the present invention first designs primers for amplifying GGT, and then adds the base sequence of BPC157 as a homologous arm to the primers UP-GR and Down-GF. After PCR amplification, the upper and lower homologous sequences carrying the BPC157 gene can be obtained, and then the recombinant plasmid can be obtained by recombination and connection with the linearized vector.

[0028] The fifth aspect of the present invention provides a recombinant strain expressing the above-mentioned pentadecapeptide fusion protein.

[0029] Preferably, the expression host of the recombinant strain is Bacillus licheniformis. Bacillus licheniformis is an industrial production strain of GARS protein, and Bacillus licheniformis has a stronger protein secretion ability than other Bacillus, and is a suitable host for introducing exogenous genes to improve the expression level of the target protein. GGT in Bacillus licheniformis can be cleaved into large and small subunits and secreted for extracellular expression, which can achieve efficient expression of BPC157 in Bacillus licheniformis.

[0030] The sixth aspect of the present invention provides a method for constructing the above-mentioned recombinant strain, which specifically includes the following operations: methylating the above-mentioned recombinant vector, transforming it into a competent state of Bacillus licheniformis, and screening the strain with correct sequencing after verification, which is the recombinant strain.

[0031] Optionally, Bacillus licheniformis 2709 can be used as the expression host of the recombinant strain.

[0032] The seventh aspect of the present invention provides the use of the above-mentioned recombinant vector or recombinant strain in producing the above-mentioned pentadecapeptide fusion protein.

[0033] Preferably, the method for producing the pentadecapeptide fusion protein using the recombinant strain is: activating the recombinant strain and then fermenting it, and the resulting fermentation liquid contains the pentadecapeptide fusion protein.

[0034] Further preferably, the culture medium components of the fermentation culture are: 6.2%~6.6% corn flour, 3.5%~4.5% soybean cake powder, 0.35%~0.45% Na2HPO4, 0.025%~0.035% KH2PO4, and 0.065%~0.075% Gaofeng α-amylase.

[0035] Preferably, the method further comprises purifying and concentrating the pentadecapeptide fusion protein in the fermentation broth.

[0036] Further preferably, the purification method is: centrifuging the fermentation broth, collecting the supernatant, filtering, and performing affinity chromatography using a His-tag chelated Ni column, first eluting the impurities with a 20 mM imidazole aqueous solution as an eluent, and then eluting the gastric pentadecapeptide fusion protein with a 150 mM imidazole aqueous solution as an eluent to obtain a purified gastric pentadecapeptide fusion protein.

[0037] The concentration replacement method is: concentrating the purified gastric pentadecapeptide fusion protein, and performing replacement 2 to 4 times using 10 mM PBS buffer at pH 7.5 as a replacement fluid.

[0038] The eighth aspect of the present invention provides the use of the above-mentioned pentadecapeptide fusion protein in the preparation of products for preventing and treating gastric ulcers.

[0039] Preferably, the gastric ulcer is an acute gastric ulcer.

[0040] Preferably, the product is a medicine or a feed additive.

[0041] The beneficial effects of the present invention are: 1. The present invention utilizes genetic engineering technology to genetically fuse BPC157 and GGT in an "end-to-end" manner, thereby obtaining a fusion protein of GGT and BPC157, overcoming the defect that BPC157 is easily hydrolyzed by proteases secreted by the host bacteria, and enabling efficient expression of BPC157 in recombinant bacteria, thereby achieving large-scale production and wide application of BPC157.

[0042] 2. The present invention can separate the fusion protein into GGT and BPC157 in gastric juice by adding a trypsin hydrolysis site to the N-terminus of BPC157, thereby exerting its efficacy. Experiments have shown that the fusion protein can have a significant protective effect on the gastric mucosa at a relatively low dosage, can prevent the occurrence of gastric ulcers to a certain extent, and significantly reduce the area of ​​gastric ulcer damage; for acute gastric ulcers that have already occurred, the fusion protein can play an effective therapeutic role, greatly reduce inflammatory factors, reduce the area of ​​ulcers, and thus inhibit the development of gastric ulcers. Therefore, the fusion protein provides a new way to improve the effect of BPC157 in promoting wound repair, and can be used to prepare products for the prevention and treatment of gastric ulcers, such as medicines and feed additives, and lays a certain foundation for replacing antibiotics to treat gastric ulcers. At the same time, since there is no report that GGT has the effect of protecting gastric mucosa, the present invention also opens up a new application direction for GGT.

[0043] 3. In the method for constructing a recombinant vector containing the fusion protein encoding gene provided by the present invention, the base sequence of BPC157 is added as a homology arm to the primers UP-GR and Down-GF for amplifying GGT. After PCR amplification, the upper and lower homologous sequences carrying the BPC157 gene can be obtained, which can then be recombined and connected with the linearized vector to obtain the recombinant vector.

[0044] 4. The recombinant strain expressing the above-mentioned gastric pentadecapeptide fusion protein provided by the present invention uses Bacillus licheniformis as the expression host and has a strong protein secretion ability. Its GGT can be cleaved into large and small subunits and secreted for extracellular expression, which can achieve efficient expression of BPC157 in Bacillus licheniformis. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is the result of PCR amplification of the gene encoding the upper and lower homologous sequences of the integration position of GGT-BPC157 in Example 1 of the present invention. M: DNA molecular weight standard, lane 1: nucleic acid band of the upper homologous arm of the integration position of GGT-BPC157; lane 2: nucleic acid band of the lower homologous arm of the integration position of GGT-BPC157; Figure 2 This is the colony PCR verification result of the integration vector pKSVT-GGT-BPC157 in Example 1 of the present invention. M: DNA molecular weight standard, lane 1: integrated plasmid correctly amplified nucleic acid band; lane 2: integrated plasmid correctly amplified nucleic acid band; lane 3: negative control; Figure 3 The recombinant expression strain of Example 2 of the present invention B. licheniformis 2709-BPC157 double exchange colony PCR verification results. M: DNA molecular weight standard, lane 1: correct double exchange amplified nucleic acid band; lane 2: negative control; Figure 4This is a polyacrylamide gel electrophoresis diagram of the fusion protein expressed in Example 3 of the present invention. M: protein marker; Lane 1: fermentation broth supernatant containing the fusion protein; Figure 5 This is the SDS-PAGE electrophoresis diagram of the fusion protein after purification and replacement in Example 4 of the present invention. M: protein marker; lane 1: column effluent after the fermentation supernatant is combined with the nickel column; lanes 2-4: 20 mM imidazole eluate; lane 5: GGT-BPC157 fusion protein eluted with 150 mM imidazole; lane 6: GGT-BPC157 fusion protein after PBS replacement; Figure 6 This is a diagram showing the protective effect of the fusion protein GGT-BPC157 on acute gastric ulcer in rats in Example 5 of the present invention; PC-A in the figure is a positive standard group; Figure 7 This is a diagram showing the therapeutic effect of the fusion protein GGT-BPC157 in Example 6 of the present invention on acute gastric ulcer in rats; in the figure, MA is the BPC treatment group, PC-A is the positive drug A group, and PC-B is the positive drug B group. DETAILED DESCRIPTION

[0046] 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. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0047] BPC157 is a peptide that can promote wound repair and protect a variety of damaged important organs. It has been proven to be effective in inflammatory gastrointestinal diseases and has potential practical application value. However, BPC157 is a small molecule that is easily hydrolyzed by proteases and is not easy to produce through recombinant protein production technology. In addition, the wound repair effect of BPC157 requires a relatively high dose to produce an ideal effect, and new methods to improve its wound repair effect need to be developed.

[0048] The present invention, through experimental research, uses genetic engineering technology to obtain a gastric pentadecapeptide fusion protein that relieves gastric inflammation and promotes ulcer healing. The gastric pentadecapeptide fusion protein is composed of γ-glutamine transpeptidase, trypsin recognition site and gastric pentadecapeptide, and its connection mode is γ-glutamine transpeptidase-trypsin recognition site-gastric pentadecapeptide. As a preferred embodiment of the present invention, the amino acid sequence of γ-glutamine transpeptidase is shown in SEQ ID No.1, the amino acid sequence of gastric pentadecapeptide is shown in SEQ ID No.2, and the amino acid sequence of the trypsin hydrolysis site is RR. The gastric pentadecapeptide fusion protein can use the expression element of γ-glutamine transpeptidase to efficiently express the fusion protein, thereby achieving efficient expression of BPC157 in recombinant bacteria. Moreover, the gastric pentadecapeptide fusion protein is separated into GGT and BPC157 in gastric juice, and there is a synergistic effect between GGT and BPC157, which can significantly reduce the area of ​​gastric mucosal damage, reduce inflammatory factors, and promote ulcer surface healing at a lower dosage, providing a new way to improve the effect of BPC157 in promoting wound repair.

[0049] The embodiments of the present invention also provide a recombinant vector containing the coding gene of the above-mentioned pentadecapeptide fusion protein and a recombinant strain expressing the above-mentioned pentadecapeptide fusion protein, and provide a construction method of the recombinant vector and the recombinant strain.

[0050] The embodiment of the present invention also provides the use of the above-mentioned recombinant vector or recombinant strain in producing the above-mentioned pentadecapeptide fusion protein.

[0051] The embodiment of the present invention also provides the use of the above-mentioned pentadecapeptide fusion protein in the preparation of products for preventing and treating gastric ulcers.

[0052] The scheme of the present invention is described below through specific embodiments.

[0053] The primer synthesis and sequencing used in the following examples were completed by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0054] Unless otherwise specified, the reagents, drugs or instruments used in the following examples are all conventional commercial products obtained from commercial channels. The methods used in the following examples, unless otherwise specified, are all conventional methods in the art or carried out according to the product instructions.

[0055] Example 1 This example provides a method for constructing a genomic integration vector pKSVT-GGT-BPC157, and the specific steps are as follows: Using the genome of Bacillus licheniformis 2709 as a template, the BPC157 sequence was added to the primers UP-GR / Down-GF (sequence shown in Table 1), and the upper and lower homologous sequences of the integration position of GGT-BPC157 were amplified using specific primers UP-GF / UP-GR and Down-GF / Down-GR (sequence shown in Table 1) (PCR amplification system shown in Table 2). The coding gene amplification results were verified by agarose gel electrophoresis. The results are as follows Figure 1 As shown, the PCR product was recovered. Sma I. Digest the integration vector pKSVT in 10 × QuickCut Green Buffer at 30°C for 15 min and then add the fast restriction endonuclease Sac In I, the integration vector pKSVT was further digested at 37°C for 15 min (see Table 3 for the double enzyme digestion reaction system) to obtain a linearized vector, and the product was recovered by gel cutting. Then, the linearized vector was connected to the upper and lower homologous sequences carrying the BPC157 gene using seamless cloning technology (see Table 4 for the recombination reaction system), and the connection product was transformed into competent cells. E. coli JM109, and spread on kanamycin plates, placed in a 37°C incubator and inverted for 12 h, and finally the transformants were picked and verified by colony PCR using plasmid sequencing primers pKSVT-GF / pKSVT-GR (sequence shown in Table 1). The results are as follows Figure 2 As shown, the plasmid with successful integration of the BPC157 gene was obtained by sequencing verification and was named pKSVT-GGT-BPC157.

[0056] Table 1 Primers used in this example

[0057] Table 2 PCR amplification system used in this example

[0058] Table 3 Double enzyme digestion reaction system Components Sample volume 10 × QuickCut Green Buffer 5 µL <![CDATA[QuickCut TM Small]]> 2.5µL <![CDATA[QuickCut TM Bag]]> 2.5µL Plasmids 15 μL <![CDATA[ddH2O]]> Make up to 50 µL Table 4 Recombination reaction system Components Sample volume <![CDATA[Linearized vector a > X µL <![CDATA[Up and down homologous sequences (inserted fragments) a > Y µL Basic Mix 5µL <![CDATA[ddH2O]]> Make up to 10 µL a X / Y is calculated based on the formula: optimal vector mass = [0.02 × number of base pairs] ng (0.03 pmol); optimal insert mass = [0.04 × number of base pairs] ng (0.06 pmol).

[0059] Example 2 This embodiment provides a method for constructing a recombinant strain of Bacillus licheniformis.

[0060] (1) Methylation modification The transformants successfully sequenced in Example 1 were inoculated into 5 ml LB (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L) test tubes, and the extracted plasmids were transformed into competent cells by heat shock. E. coli 135 / pM.Bam, and spread on a plate containing kanamycin resistance and spectinomycin resistance, and incubate inverted in a 37°C incubator for 12 h. Pick the transformant and place it in a 5 ml LB test tube to culture until OD 600 The concentration of arabinose was 0.2-0.3, and a final volume of 0.2% arabinose was added and cultured overnight at 30°C for 18 h to induce plasmid methylation.

[0061] (2) Transformation and screening The above-mentioned recombinant plasmid after methylation modification was extracted and mixed with the competent cell of Bacillus licheniformis 2709. The mixture was then transferred to a pre-cooled electroporation cup, placed on ice for 5 min, and electroporated at 2500 V. Recovery solution (10 g / L peptone, 5 g / L yeast extract powder, 5 g / L sodium chloride, 0.5 mol / L D-sorbitol, 0.38 mol / L D-mannitol) was quickly added. All solutions were transferred to a sterilized 1.5 mL centrifuge tube in a clean bench. After shaking culture at 37°C for 3 h, the cells were centrifuged at 4000 rpm for 5 min to collect the cells, spread them on a plate containing kanamycin resistance, and cultured inverted at 37°C overnight. The plasmid verification primers pKSVT-GF and pKSVT-GR (sequences are shown in Table 1) were used for colony PCR verification, and the correct transformants were picked to 5 mL LB test tube, cultured at 45℃, started single exchange, and used single exchange verification primers BPC-VF and pKSVT-GF, BPC-VR and pKSVT-GR for single exchange verification. Pick the correctly verified colony, put it into a 5 mL LB test tube, cultured at 37℃, and perform genome double exchange: use the spot plate method to preliminarily screen the colonies with plasmid loss, and then use the genome verification primers BPC-F and BPC-VR (sequences in Table 5) for double exchange verification and sequencing. The positive clone with correct sequencing is the constructed recombinant expression strain B. licheniformis 2709-BPC157, the verification diagram is as follows Figure 3 shown.

[0062] Table 5 Primers used in this example

[0063] Example 3 This example provides the expression of the recombinant strain and the SDS-PAGE verification results of the fusion protein.

[0064] 1. Expression of recombinant strains The correctly sequenced transformants in Example 2 were inoculated into 5 mL LB test tubes and cultured at 37°C and 220 r / min for 12-14 h. A 2% inoculum was inoculated into 50 mL LB shake flasks and continued to be cultured at 37°C and 220 r / min until OD 600 When the pH value reaches between 0.8 and 1.0, inoculate 2% of the inoculum into 100 mL of fermentation medium (6.4% corn flour, 4% soybean meal, 0.4% Na2HPO4, 0.03% KH2PO4, 0.07% Gaofeng's α-amylase), culture at 37°C and 220 r / min with shaking, sample 2 mL after 84 h, and centrifuge at 12,000 rpm to obtain the supernatant.

[0065] 2. SDS-PAGE verification of fusion protein Pipette 20 μL of the above supernatant and add 5 μL of SDS-PAGE 5× loading buffer to mix well by pipetting. Boil for 10 min and centrifuge to perform SDS-PAGE gel electrophoresis (the composition is shown in Table 6). The results of polyacrylamide gel electrophoresis are shown in Figure 4 shown.

[0066] Table 6 SDS-PAGE gel formula

[0067] Example 4 This example provides a method for purifying and replacing the fusion protein GGT-BPC157.

[0068] 1. Protein purification by nickel column All the fermentation broths in Example 3 were centrifuged to collect the supernatant, filtered using a 0.22 µm water filter membrane, and His-tag chelated Ni column was selected for affinity chromatography. The purification results are shown in FIG. Figure 5 The specific steps are as follows: (1) First, pour out the 20% ethanol in the affinity column and wash the column three times with ultrapure water after passing through the membrane; (2) Equilibrate the nickel column with 10 column volumes of binding buffer; (3) Slowly stir the filtered fermentation supernatant and nickel ions in an ice bath at 4°C for 2 h to allow them to combine; (4) After the binding is completed, the supernatant and nickel ions are loaded into the resin column and allowed to flow out naturally; (5) Elute the impurities in the fermentation supernatant using 3 column volumes of 20 mM imidazole aqueous solution; (6) Then, use 9 mL of 150 mM imidazole aqueous solution to elute the target protein and collect the effluent; (7) Finally, use binding buffer to thoroughly elute the nickel column, seal the nickel column with 20% ethanol and store it at 4°C.

[0069] 2. Target protein concentration and replacement Pour the purified target protein into a pre-cooled ultrafiltration tube and concentrate it at a maximum speed of 4000 rpm. When it is concentrated to 1 mL, add 9 mL of replacement solution (10 mM PBS, pH 7.5) and continue to concentrate to 1 mL. Repeat the replacement 3 times to finally obtain the replaced fusion protein. The SDS-PAGE electrophoresis is shown in the figure. Figure 5 shown.

[0070] After testing, the purity of the obtained fusion protein was 33.65%.

[0071] Example 5 This example investigates the protective effect of the fusion protein GGT-BPC157 on acute gastric ulcer in rats.

[0072] Experimental animals and grouping: After healthy male, 5-week-old Wistar rats were adaptively raised for one week, the rectal temperature of the rats was measured once a day, and 30 healthy rats with normal body temperature were randomly selected and randomly divided into 4 groups, 6 rats in each group, namely, blank control group, model group, sample protection group, and positive standard group (BPC157 standard, purity 99.15%). Rats in the blank control group and model group were gavaged with an equal amount of saline solution (5 ml / kg); the sample prepared in Example 4 was dissolved in 0.01 M PBS solution to make 70 µg / mL, which was used for the sample protection group and gavaged at a dose of 5 ml / kg per day. The BPC157 standard was dissolved in saline to make a solution of 70 µg / mL, which was used for the positive standard group and gavaged at a dose of 5 ml / kg per day. Rats in each group were given the drug once by gavage daily for 7 consecutive days. During this period, they were fed with standard feed normally. They were fasted for 24 h after the last administration. On the 8th day, except for the blank control group, the other groups were given the drug again. 90 min after the administration, the rats were gavaged with 95% ethanol at a rate of 1 mL / 200 g. The rats were killed 4 h later, and the stomach tissues of the rats were taken for the detection of experimental indicators.

[0073] Determination of gastric tissue inflammatory factor indexes: gastric tissue and pre-cooled PBS (0.01 M, PH = 7.4) were added to a homogenization tube on ice at a weight (g): volume (L) ratio of 1:9. In an ice water bath, the tissue was fully broken and homogenized three times with a handheld homogenizer, each time with an interval of 15 s. The homogenate was aspirated and placed in a centrifuge tube, centrifuged at 4°C, 5000×g for 10 min, and the supernatant was taken into a new centrifuge tube. The changes in the content of related inflammatory factors TNF-α, IL-1β, and IL-6 in each tissue were detected according to the instructions of the ELISA kit. The results are as follows Figure 6As shown in Figures A-C, compared with the blank control group, the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the model group were significantly increased; compared with the model group, pre-administration of fusion protein GGT-BPC157 for 7 days can significantly reduce gastric mucosal damage in rats (P < 0.0001). The results show that the fusion protein GGT-BPC157 has a good anti-inflammatory effect and significantly reduces gastric mucosal inflammatory response.

[0074] Calculation of gastric mucosal ulcer inhibition rate: Spread the gastric tissue of rats in each experimental group on filter paper, expose the gastric mucosa to the field of view, and take pictures for record. Use a dissecting microscope to measure the gastric mucosal damage of each rat. Use a vernier caliper to measure and calculate the ulcer area, using the size and amount of gastric mucosal bleeding and ulcer as indicators. The ulcer length is the maximum diameter d1 of the gastric ulcer, and the width is the widest distance d2 perpendicular to the diameter. The ulcer area is calculated according to the formula S=π×d1 / 2×d2 / 2, and its ulcer inhibition rate is calculated. Gastric ulcer inhibition rate = (gastric ulcer area in the control group - gastric ulcer area in the experimental group) / gastric ulcer area in the control group × 100%. The measurement and calculation results are as follows Figure 6 As shown in Figure D, the gastric ulcer rate of the model group was 100% after 95% ethanol gavage. After 7 days of protection by fusion protein and BPC157, the sample protection group and the positive standard group had a significant preventive effect on acute gastric ulcer caused by ethanol (P < 0.0001). The results showed that 1 mL of anhydrous ethanol could cause acute injury to the gastric mucosa of rats, and the administration of fusion protein GGT-BPC157 could significantly reduce the injury area and protect the gastric mucosa.

[0075] Example 6 This example investigates the therapeutic effect of the fusion protein GGT-BPC157 on acute gastric ulcer in rats.

[0076] Experimental animals and grouping: After healthy male, 5-week-old Wistar rats were adaptively raised for one week, the rectal temperature of the rats was measured once, and 56 rats with normal body temperature were randomly selected and randomly divided into 5 groups, namely, blank control group, model group, BPC treatment group (fusion protein GGT-BPC157 purified and replaced in Example 4), positive drug group A (BPC157 standard product, purity 99.15%), and positive drug group B (omeprazole), with 8 rats in each group. The sample prepared in Example 4 was dissolved in 0.01 M PBS solution to prepare 70 μg / mL for administration to the BPC treatment group; the BPC157 standard product was dissolved in physiological saline to prepare a solution of 70 μg / mL for administration to the positive drug group A; omeprazole was dissolved in physiological saline to prepare a solution of 70 μg / mL for administration to the positive drug group B. The rats in each group were fed adaptively under normal conditions for 1 week. On the 7th day, after fasting for 24 h but not water deprivation, all groups except the blank control group were gavaged with 95% ethanol at a dose of 1 mL / 200 g. On the second day after modeling, 6 rats were randomly selected from each group. Each treatment group was given the corresponding test drug by gavage at a dose of 5 ml / kg for 7 consecutive days, once a day. The blank control group and the model group were given an equal volume of normal saline, and were fed with standard feed normally during this period.

[0077] Detection of gastric tissue inflammatory factor index and calculation of gastric ulcer inhibition rate: After 7 days of oral administration, rats in each group were fasted for 24 hours, killed, and the stomach was removed. The stomach of the rat was then cut open along the greater curvature of the stomach, and blood stains and residues were washed with 0.01 M phosphate solution at 4°C. The method for measuring gastric tissue inflammatory factors and calculating gastric ulcer inhibition rate were the same as in Example 5. The results are shown in Figure 7 As shown, Figures A to C show the changes in inflammatory factors in the gastric tissues of rats in each group; the inflammatory factors in the model group increased significantly, which was significantly different from the blank control group (P < 0.0001); compared with the model group, the inflammatory factors in the BPC treatment group were significantly reduced (P < 0.0001); Figure 7 Figure D in the middle shows the gastric ulcer inhibition rate of each group after oral administration of ethanol. There is a significant difference between the BPC treatment group and the model group (P < 0.0001).

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A gastric pentadecapeptide fusion protein for relieving gastric inflammation and promoting ulcer healing, characterized in that: The gastric pentadecapeptide fusion protein is composed of γ-glutamine transpeptidase, a trypsin recognition site and gastric pentadecapeptide, and the connection mode is γ-glutamine transpeptidase-trypsin recognition site-gastric pentadecapeptide.

2. The gastric pentadecapeptide fusion protein according to claim 1, characterized in that: The amino acid sequence of the γ-glutamine transpeptidase is shown in SEQ ID No. 1; and / or The amino acid sequence of the gastric pentadecapeptide is shown in SEQ ID No. 2; and / or The amino acid sequence of the trypsin hydrolysis site is RR; and / or The amino acid sequence of the gastric pentadecapeptide fusion protein is shown in SEQ ID No.

3.

3. The gene encoding the gastric pentadecapeptide fusion protein according to claim 1 or 2.

4. A recombinant vector containing the coding gene according to claim 3.

5. The recombinant vector according to claim 4, characterized in that The sequence of the coding gene is shown in SEQ ID No.4; and / or The expression vector of the recombinant vector is plasmid pKSVT.

6. The method for constructing a recombinant vector according to claim 4 or 5, characterized in that: Specifically, the following operations are included: using the genome of Bacillus licheniformis 2709 as a template, using specific primers UP-GF / UP-GR and Down-GF / Down-GR for PCR amplification; the sequence of the primer UP-GF is shown in SEQ ID No.5, the sequence of the primer UP-GR is shown in SEQ ID No.6, the sequence of the primer Down-GF is shown in SEQ ID No.7, and the sequence of the primer Down-GR is shown in SEQ ID No.8; using restriction endonucleases Sma I and Sac I. Perform double restriction digestion on plasmid pKSVT to obtain linearized vector; recombinantly connect the PCR amplification product and the linearized vector, and transform the connection product into competent cells. E. coli JM109, and then extract the plasmid from the correctly sequenced transformant to obtain the recombinant vector.

7. A recombinant strain expressing the gastric pentadecapeptide fusion protein according to claim 1 or 2.

8. The recombinant strain according to claim 7, characterized in that The expression host of the recombinant strain is Bacillus licheniformis.

9. Use of the recombinant vector according to claim 4 or 5 or the recombinant strain according to claim 7 or 8 in producing the gastric pentadecapeptide fusion protein according to claim 1.

10. Use of the gastric pentadecapeptide fusion protein according to claim 1 or 2 in the preparation of products for preventing and treating gastric ulcers.

Images