Effective peptide segment of carboxypeptidase inhibitor of silkworm, carboxypeptidase inhibitor of silkworm and application thereof

Abstract

The present invention discloses an effective peptide segment of a carboxypeptidase inhibitor of Bombyx mori, the carboxypeptidase inhibitor of Bombyx mori and its application. The amino acid sequence of the effective peptide segment of the carboxypeptidase inhibitor of Bombyx mori is tyrosine-glycine-valine-serine. The amino acid sequence of the carboxypeptidase inhibitor of Bombyx mori is as shown in SEQ ID NO.2. The effective peptide segment overlaps with the position of the small molecule inhibitor tak-285. It is found that both the effective peptide segment and the carboxypeptidase inhibitor of Bombyx mori can significantly inhibit the proliferation of gastric cancer cells, and can be used as a medical use for gastric cancer, providing a new drug for the treatment of gastric cancer and providing the possibility for the development of anti-tumor drugs from insect-derived substances.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an effective peptide segment of a silkworm carboxypeptidase inhibitor, a silkworm carboxypeptidase inhibitor, and further to applications of the effective peptide segment of the silkworm carboxypeptidase inhibitor and the silkworm carboxypeptidase inhibitor in treating gastric cancer. Background Art

[0002] Gastric cancer is a common malignant tumor originating from the gastric mucosal epithelium, threatening human health and life. Early-stage gastric cancer has a good prognosis after treatment, while late-stage gastric cancer has a poor prognosis. During gastric cancer treatment, patients often suffer from the high toxicity and side effects of chemotherapy drugs. Studies have shown that bioactive substances such as antimicrobial peptides and cantharidin, found in various insects, can exert anti-cancer effects and, compared to chemotherapy drugs, have fewer toxic side effects.

[0003] As a model organism, the functions of more and more genes in the silkworm have been studied after the completion of whole genome sequencing, but genes with medicinal effects are rarely reported. Therefore, the discovery of anti-tumor drugs derived from silkworm-derived substances is of great significance to the development of silkworms. Summary of the Invention

[0004] In view of this, one of the objects of the present invention is to provide an effective peptide segment of a silkworm carboxypeptidase inhibitor; a second object of the present invention is to provide the use of the effective peptide segment of the silkworm carboxypeptidase inhibitor in the preparation of a drug for treating gastric cancer; a third object of the present invention is to provide a polypeptide containing the effective peptide segment of the silkworm carboxypeptidase inhibitor; a fourth object of the present invention is to provide the use of the polypeptide in the preparation of a drug for treating gastric cancer; and a fifth object of the present invention is to provide the use of the silkworm carboxypeptidase inhibitor in the preparation of a gastric cancer-related factor expression inhibitor.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] 1. An effective peptide segment of a silkworm carboxypeptidase inhibitor, wherein the amino acid sequence of the effective peptide segment of the silkworm carboxypeptidase inhibitor is as follows: tyrosine-glycine-valine-serine.

[0007] 2. Use of the effective peptide fragment of the silkworm carboxypeptidase inhibitor in the preparation of drugs for treating gastric cancer.

[0008] Preferably, the drug of the present invention can inhibit the proliferation of gastric cancer cells.

[0009] 3. A polypeptide containing the effective peptide segment of the silkworm carboxypeptidase inhibitor.

[0010] Preferably, the amino acid sequence of the polypeptide is shown in SEQ ID NO.2.

[0011] 4. Use of the polypeptide in the preparation of a medicament for treating gastric cancer.

[0012] Preferably in the present invention, the gastric cancer is differentiated or undifferentiated gastric cancer cells.

[0013] Preferably in the present invention, the gastric cancer is MKN45 cells, SGC7901 cells or HGC27 cells.

[0014] Preferably in the present invention, the treatment of gastric cancer is to inhibit the proliferation of gastric cancer cells.

[0015] 5. Use of the silkworm carboxypeptidase inhibitor in the preparation of an inhibitor for the expression of gastric cancer-related factors, where the gastric cancer-related factors are genes encoding SH domains, growth factor receptor-binding protein 2, guanine nucleotide exchange factor, Raf protein, c-Myc protein or cyclin CDK2, CDK4, Cyclin D1 and Cyclin E1.

[0016] The beneficial effects of the present invention are as follows: The present invention discloses the construction of a recombinant expression vector for the silkworm carboxypeptidase inhibitor gene and the obtaining of a recombinant protein. This recombinant protein can significantly inhibit the proliferation of two gastric cancer cell lines, MKN45 and SGC7901. On the third day, the inhibition rate of the gastric cancer cells in the group added with 10 micrograms per milliliter of the silkworm carboxypeptidase inhibitor can reach 25%. By detecting the related pathway proteins affecting the proliferation of gastric cancer cells, it is found that some gastric cancer cells can rapidly proliferate by autophosphorylating and activating downstream proteins such as MAPK / ERK pathway-related proteins. After adding the silkworm carboxypeptidase inhibitor for culturing, the pathway that promotes the expression of the proto-oncogene c-Myc by the MAPK / ERK pathway initiated by EGF / EGFR is inhibited, thereby affecting the expression of related cyclins and inhibiting the proliferation of gastric cancer cells. Subsequently, through molecular docking and virtual screening of the silkworm carboxypeptidase inhibitor and the epidermal growth factor receptor, we screened a polypeptide whose binding position in the receptor overlaps with that of the existing small molecule inhibitor. After synthesizing the polypeptide, its inhibitory activity against the proliferation of gastric cancer cells was measured, and it was found that it has the ability to inhibit the proliferation of gastric cancer cells. Therefore, this polypeptide is an effective peptide segment of the silkworm carboxypeptidase inhibitor and can also be used as a candidate drug for gastric cancer, providing a new drug for the treatment of gastric cancer and the possibility for the development of anti-tumor drugs from insect-derived substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] To make the objectives, technical solutions and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0018] Figure 1 Results of the prokaryotic expression and isolation and purification of the recombinant protein of the silkworm carboxypeptidase inhibitor;

[0019] Figure 2 Determination of the inhibitory activity of the recombinant silkworm carboxypeptidase inhibitor;

[0020] Figure 3 For the determination of the thermal stability and acid-base stability of recombinant silkworm carboxypeptidase inhibitor;

[0021] Figure 4 For the observation of gastric cancer cells after treatment with silkworm carboxypeptidase inhibitor;

[0022] Figure 5 For the tyrosine autophosphorylation of MKN45 and SGC7901 cell lines;

[0023] Figure 6 For the detection of the expression of upstream related proteins in the MAPK / ERK pathway;

[0024] Figure 7 For the effect of silkworm carboxypeptidase inhibitor on the expression of c-Myc and its related cyclins;

[0025] Figure 8 The peptide segment of silkworm carboxypeptidase inhibitor overlaps with the binding position of the small molecule inhibitor tak-285;

[0026] Figure 9 The effective polypeptide of silkworm carboxypeptidase inhibitor inhibits the proliferation and colony formation of gastric cancer cells. Detailed implementation mode

[0027] Next, the preferred embodiments of the present invention will be described in detail in conjunction with the accompanying drawings. For the experimental methods without specific conditions indicated in the preferred embodiments, they are usually carried out under conventional conditions, such as the conditions described in the Molecular Cloning Experiment Guide (Third Edition, written by J. Sambrook et al.), or according to the conditions recommended by the manufacturer.

[0028] The gastric cancer cell lines SGC7901 and MKN45 used in the preferred embodiments were purchased from the American Type Culture Collection (ATCC); the human immortalized kidney embryo cells 293FT were purchased from Life Technologies Corporation. The cells were passaged and preserved by the Biology Research Center of the Frontier Interdisciplinary Research Institute of Southwest University.

[0029] Example 1. Expression of recombinant protein of silkworm carboxypeptidase inhibitor

[0030] The prokaryotic expression of the carboxypeptidase inhibitor of Bombyx mori was carried out, and the carboxypeptidase inhibitor was successfully expressed using pet28a as a vector. The specific method was to ligate the sequence of the Bombyx mori carboxypeptidase inhibitor gene CDS without the signal peptide shown in SEQ ID NO.1 into the BamHI and XhoI restriction sites of pet28a to obtain a recombinant prokaryotic expression vector. The obtained recombinant expression vector was transformed into BL21 competent cells to obtain engineering bacteria. The obtained engineering bacteria were induced to express, 0.5 mM IPTG was added, and then they were placed in a shaker at 37 °C for 4 hours. After induction, the supernatant was collected, and then elution was carried out using an imidazole gradient. Relatively pure protein was obtained under the elution of 100 mM and 200 mM imidazole. The results are as Figure 1 shown, which is the recombinant protein of the carboxypeptidase inhibitor of Bombyx mori, and the amino acid sequence of this protein is shown in SEQ ID NO.2.

[0031] The obtained recombinant protein of the carboxypeptidase inhibitor of Bombyx mori was used to prepare antibodies and verified. The bands are single and the specificity is good, which can be used for later experiments. In order to understand the expression of the carboxypeptidase inhibitor of Bombyx mori in tissues, we used the tissues of the fifth instar Bombyx mori on the third day as templates for fluorescence quantitative PCR and found that it was highly expressed in the silk gland. At the same time, Western blot was carried out using the obtained antibodies to verify this result at the protein level. The carboxypeptidase inhibitor of Bombyx mori was specifically expressed in the silk gland.

[0032] Example 2: Activity evaluation of the carboxypeptidase inhibitor of Bombyx mori

[0033] In order to understand whether the recombinant carboxypeptidase inhibitor of Bombyx mori has activity, a carboxypeptidase activity inhibition experiment was carried out on it. Using potato carboxypeptidase as a substrate, a PBS negative control group, a potato carboxypeptidase inhibitor positive control group and an experimental group were set up. The results are as Figure 2 shown. The results show that the recombinant carboxypeptidase inhibitor of Bombyx mori and the potato carboxypeptidase inhibitor have the same inhibitory activity on carboxypeptidase.

[0034] In order to determine the inhibitory stability of the carboxypeptidase inhibitor of Bombyx mori, the carboxypeptidase inhibitor of Bombyx mori was treated in different temperature and pH environments respectively, and then its inhibitory efficiency on carboxypeptidase activity was measured. The measurement results at different temperatures are as Figure 3 shown. The results show that with the increase of the treatment temperature, the inhibitory activity of the carboxypeptidase inhibitor of Bombyx mori on carboxypeptidase decreased slightly, but all showed a high inhibitory efficiency. The carboxypeptidase inhibitor of Bombyx mori has good thermal stability for the inhibition of carboxypeptidase. The results of pH stability analysis are as Figure 3As shown, the results show that when the pH of the treatment with Bombyx mori carboxypeptidase inhibitor is 2 - 6, the inhibitory efficiency of Bombyx mori carboxypeptidase inhibitor on carboxypeptidase gradually increases. When the pH is 6 - 7, the inhibitory efficiency is the best. However, after the pH is higher than 8, the inhibitory efficiency of Bombyx mori carboxypeptidase inhibitor on carboxypeptidase gradually decreases. In order to understand the expression of Bombyx mori carboxypeptidase inhibitor in tissues, tissues of the fifth instar and third day of Bombyx mori were used as templates for fluorescence quantitative PCR. It was found that it was highly expressed in the silk gland. At the same time, Western blot was performed to verify this result at the protein level, and Bombyx mori carboxypeptidase inhibitor was specifically expressed in the silk gland.

[0035] Example 3. Evaluation of the ability of the recombinant protein of Bombyx mori carboxypeptidase inhibitor to inhibit the proliferation of gastric cancer tumors

[0036] According to the degree of tumor differentiation, MKN45 cells with medium differentiation, SGC7901 cells with low differentiation, and undifferentiated HGC27 cells were selected as experimental subjects. Logarithmic growth phase cells were taken and added to 96-well plates. Two experimental groups of 1 μg / mL and 10 μg / mL of Bombyx mori carboxypeptidase inhibitor protein were set up, and the cell morphology was observed. The results are as Figure 4 shown. The results show that there were no obvious differences in the cell morphology of each group on the first day, and the cells were arranged loosely and evenly. When observing the cells again on the fifth day, it was found that in the negative control wells, the cells had grown densely, while in the wells containing 10 μg / mL of Bombyx mori carboxypeptidase inhibitor, the number of cells was less than that of the control group, and there were obvious gaps between the cells. During the same period, a CCK-8 cell proliferation experiment was carried out. It was found that when the initial cell numbers of the three cell lines were similar, with the increase of time, the growth rate of the cells added with Bombyx mori carboxypeptidase inhibitor was slower than that of the negative control group. After measuring the data on the third day and the fifth day respectively and performing a significance analysis, it was found that Bombyx mori carboxypeptidase inhibitor significantly inhibited the proliferation of gastric cancer cells. The cells on the third day were selected for calculating the proliferation rate. The proliferation in the 10 μg / mL Bombyx mori carboxypeptidase inhibitor group was 75% - 80% of that of the negative control group, and both significantly inhibited the proliferation of gastric cancer cells. At the same time, according to the malignancy degree of the three cell lines, for MKN45 with medium differentiation and low malignancy degree, the inhibitory effect was better, and for HGC27 with undifferentiated and the highest malignancy degree, the inhibitory effect was slightly worse. The inhibition rate of the 10 μg / mL group was about 25%, which could achieve the inhibitory effect of 25 μg / mL of the gastric cancer drug tegafur, and the inhibitory effect was very obvious.

[0037] Example 4. Exploration of the molecular mechanism of the recombinant protein of Bombyx mori carboxypeptidase inhibitor in inhibiting the proliferation of gastric cancer tumors

[0038] The phosphorylation of EGFR is one of the important markers of tumor cell proliferation. The phosphorylation levels of EGFR in three cell lines of gastric cancer cells were detected. The results are as Figure 5As shown, it was found that tyrosine phosphorylation occurred in both MKN45 and SGC7901, while tyrosine phosphorylation did not occur in HGC27. The results of detecting the expression of proteins related to the upstream of the MAPK / ERK pathway are as Figure 6 shown. It was found that from the protein product Shc of the gene encoding the SH domain at the beginning, growth factor receptor-bound protein 2 (Grb2), guanine nucleotide exchange factor (sos), to the key protein Raf protein that triggers the pathway cascade reaction, the expression was down-regulated due to the addition of the silkworm carboxypeptidase inhibitor, and showed a dose-dependent manner. The phosphorylation of Raf protein is highly correlated with that of MEK1 / 2. The down-regulated expression of Raf protein also strongly suggests that the silkworm carboxypeptidase inhibitor may be related to the MAPK / ERK pathway. The results of detecting the expression of proteins related to the MAPK / ERK pathway showed that the expression of MEK1 / 2 and ERK1 / 2 was down-regulated and phosphorylation was inhibited, also showing a dose-dependent manner. According to the signal pathway diagram, the expression of the proto-oncogene c-Myc was also detected, and the results are as Figure 7 shown. The results showed that the down-regulated expression of proteins related to the MAPK / ERK pathway led to the down-regulated expression of c-Myc protein. The down-regulated expression of c-Myc can inhibit the expression of cyclins such as CDK2, CDK4, Cyclin D1 and Cyclin E1, and finally lead to the inhibition of cell proliferation. Through the above experiments, it was found that the silkworm carboxypeptidase inhibitor can down-regulate the expression of c-Myc.

[0039] Example 5: Obtaining a lead compound for gastric cancer treatment based on the screening of effective peptide segments of the recombinant protein of silkworm carboxypeptidase inhibitor

[0040] Using the PyMOL software, the original ligand, the small molecule tak-285, was successfully removed from the structure of the receptor 3poz protein. Subsequently, the ligand silkworm carboxypeptidase inhibitor and the processed receptor epidermal growth factor receptor kinase region were subjected to molecular docking. After 300,000 times of simulated docking, the system returned 30 possible docking modes with the highest scores, and the docking mode ranked first was selected for analysis. The analysis found that the results are as Figure 8 shown. In the ligand active pocket, the peptide segment (tyrosine-glycine-valine-serine) of the silkworm carboxypeptidase inhibitor overlapped with the position of the small molecule inhibitor tak-285, suggesting that this polypeptide segment may inhibit the activity of the epidermal growth factor receptor. In order to detect whether the effective peptide segment of the silkworm carboxypeptidase inhibitor has the potential to become a lead compound, CCK-8 experiments and plate cloning experiments were carried out, and the results are as Figure 9 shown. It was found that although the inhibitory ability of the effective peptide segment was slightly weaker than that of the silkworm carboxypeptidase inhibitor, it could still significantly inhibit the proliferation of gastric cancer cells. Through the plate cloning experiment, it was found that compared with the clones formed by the control group cells, the cell clones became significantly smaller and the number decreased after the addition of the effective polypeptide fragment of the silkworm carboxypeptidase inhibitor.

[0041] The above-described embodiments are merely preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. Use of an effective peptide segment of a carboxypeptidase inhibitor from Bombyx mori in the preparation of a medicament for treating gastric cancer, characterized in that: The amino acid sequence of the effective peptide segment of the carboxypeptidase inhibitor of Bombyx mori is as follows: tyrosine-glycine-valine-serine.

2. Use of a polypeptide in the preparation of a medicament for treating gastric cancer, characterized in that: The amino acid sequence of the polypeptide is shown as SEQ ID NO.2.

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