CN115974964A - Effective peptide fragment of silkworm carboxypeptidase inhibitor, silkworm carboxypeptidase inhibitor and application thereof - Google Patents
Effective peptide fragment of silkworm carboxypeptidase inhibitor, silkworm carboxypeptidase inhibitor and application thereof Download PDFInfo
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- CN115974964A CN115974964A CN202211266984.9A CN202211266984A CN115974964A CN 115974964 A CN115974964 A CN 115974964A CN 202211266984 A CN202211266984 A CN 202211266984A CN 115974964 A CN115974964 A CN 115974964A
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Abstract
The invention discloses an effective peptide segment of a silkworm carboxypeptidase inhibitor, the silkworm carboxypeptidase inhibitor and application thereof, wherein the amino acid sequence of the effective peptide segment of the silkworm carboxypeptidase inhibitor is tyrosine-glycine-valine-serine, the amino acid sequence of the silkworm carboxypeptidase inhibitor is shown in SEQ ID NO.2, the effective peptide segment is overlapped with the positions of tak-285 of a small molecule inhibitor, and researches show that the effective peptide segment and the silkworm carboxypeptidase inhibitor can obviously inhibit the proliferation of gastric cancer cells, can be used as the medical application of gastric cancer, provide a new medicament for the treatment of the gastric cancer and provide possibility for the development of insect source substance antitumor medicaments.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to an effective peptide fragment of a silkworm carboxypeptidase inhibitor, the silkworm carboxypeptidase inhibitor and application of the effective peptide fragment of the silkworm carboxypeptidase inhibitor and the silkworm carboxypeptidase inhibitor in treatment of gastric cancer.
Background
Gastric cancer is a common malignant tumor originated from gastric mucosal epithelium, and threatens the health and life of all human beings. Early gastric cancer has a better prognosis after treatment, while late gastric cancer has a poorer prognosis. In the treatment of gastric cancer, the pain caused by the high toxic and side effects of chemotherapeutic drugs is usually accompanied with patients. Research has shown that bioactive substances such as antibacterial peptide, cantharidin and the like contained in various insects can play an anticancer function, and compared with chemotherapeutic drugs, the bioactive substances have smaller toxic and side effects after being prepared into drugs.
The silkworm is used as a model organism, functions of more and more genes are researched after the whole genome sequencing is completed, but genes with medicinal effects are rarely reported, so that the mining of anti-tumor medicines of silkworm-derived substances has important significance for the development of the silkworm.
Disclosure of Invention
In view of the above, an object of the present invention is to provide an effective peptide fragment of a bombyx mori carboxypeptidase inhibitor; the second purpose of the invention is to provide the application of the effective peptide fragment of the silkworm carboxypeptidase inhibitor in preparing the medicine for treating gastric cancer; the third purpose of the invention is to provide a polypeptide containing the effective peptide fragment of the silkworm carboxypeptidase inhibitor; the fourth purpose of the invention is to provide the application of the polypeptide in preparing the medicine for treating gastric cancer; the fifth purpose of the invention is to provide the application of the silkworm carboxypeptidase inhibitor in preparing the gastric cancer related factor expression inhibitor.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the effective peptide segment of the silkworm carboxypeptidase inhibitor has the following amino acid sequence: tyrosine-glycine-valine-serine.
2. The application of the effective peptide fragment of the silkworm carboxypeptidase inhibitor in preparing the medicine for treating the gastric cancer.
Preferably, the drug of the present invention can inhibit gastric cancer cell proliferation.
3. Polypeptide containing the effective peptide segment of the bombyx mori carboxypeptidase inhibitor.
Preferably, the amino acid sequence of the polypeptide is shown as SEQ ID NO. 2.
4. The application of the polypeptide in preparing the medicine for treating gastric cancer.
Preferably, the gastric cancer is differentiated or undifferentiated gastric cancer cells.
Preferably, the gastric cancer is MKN45 cells, SGC7901 cells or HGC27 cells.
Preferably, the gastric cancer treatment is inhibition of gastric cancer cell proliferation.
5. The application of the silkworm carboxypeptidase inhibitor in preparing the gastric cancer related factor expression inhibitor is characterized in that the gastric cancer related factors are encoding SH structural domain genes, growth factor receptor binding protein 2, guanylate exchange factor, raf protein, c-Myc protein or Cyclin CDK2, CDK4, cyclin D1 and Cyclin E1.
The invention has the beneficial effects that: the invention discloses a construction of a silkworm carboxypeptidase inhibitor gene recombinant expression vector and a recombinant protein obtained. The recombinant protein can obviously inhibit the proliferation of two gastric cancer cell lines of MKN45 and SGC7901, and the inhibition rate of the silkworm carboxypeptidase inhibitor addition group with 10 micrograms per milliliter on the gastric cancer cells can reach 25% on the third day. Through detecting related pathway proteins influencing gastric cancer cell proliferation, part of gastric cancer cells can be quickly proliferated by activating downstream proteins such as MAPK/ERK pathway related proteins through autophosphorylation, and after a silkworm carboxypeptidase inhibitor is added for culture, the pathway of promoting protooncogene c-Myc expression by the MAPK/ERK pathway started by EGF/EGFR is inhibited, so that the expression of related cyclin is influenced, and the proliferation of the gastric cancer cells is inhibited. Then, a polypeptide overlapped with the action position of the existing small molecule inhibitor in the receptor is screened by carrying out molecular docking and virtual screening on the bombyx mori carboxypeptidase inhibitor and the epidermal growth factor receptor. After the polypeptide is synthesized, the gastric cancer cell proliferation inhibition activity is measured, and the ability of inhibiting gastric cancer cell proliferation is found, so that the polypeptide is an effective peptide fragment of a silkworm carboxypeptidase inhibitor, can also be used as a candidate drug of gastric cancer, provides a new drug for the treatment of gastric cancer, and provides possibility for the development of an insect-derived substance antitumor drug.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 shows the results of prokaryotic expression, separation and purification of recombinant protein of bombyx mori carboxypeptidase inhibitor;
FIG. 2 shows the measurement of the inhibitory activity of recombinant silkworm carboxypeptidase inhibitors;
FIG. 3 is the determination of the thermal stability and acid-base stability of the recombinant silkworm carboxypeptidase inhibitor;
FIG. 4 is an observation of gastric cancer cells after treatment with silkworm carboxypeptidase inhibitors;
FIG. 5 shows tyrosine autophosphorylation of MKN45 and SGC7901 cell lines;
FIG. 6 shows the expression detection of proteins associated with the MAPK/ERK pathway upstream;
FIG. 7 shows the effect of silkworm carboxypeptidase inhibitors on c-Myc and its related cyclin expression;
FIG. 8 shows the overlapping of the peptide fragment of the bombyx mori carboxypeptidase inhibitor and the small molecule inhibitor tak-285;
FIG. 9 shows that effective polypeptide of silkworm carboxypeptidase inhibitor can inhibit gastric cancer cell proliferation and clone formation.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, in the preferred embodiments, which do not specify specific conditions, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (third edition, sambrook et al), or according to the conditions recommended by the manufacturers.
The gastric cancer cell lines SGC7901 and MKN45 used in the preferred embodiment were purchased from the American Type Culture Collection (ATCC); human kidney embryo immortalized cells 293FT were purchased from Life Technologies. Cells were maintained by passage at the biological research center of the leading-edge interdisciplinary institute of science, university of southwest.
Example 1 expression of recombinant protein of Bombyx mori carboxypeptidase inhibitor
Prokaryotic expression is carried out on the silkworm carboxypeptidase inhibitor, the pet28a is used as a vector to successfully express the carboxypeptidase inhibitor, and the specific method is that a sequence of removing signal peptide of a silkworm carboxypeptidase inhibitor gene CDS shown as SEQ ID NO.1 is connected to a BamHI enzyme cutting site and an XhoI enzyme cutting site of the pet28a to obtain a recombinant prokaryotic expression vector. And transforming the obtained recombinant expression vector into BL21 competence to obtain the engineering bacterium. Carrying out induction expression on the obtained engineering bacteria, adding 0.5mM IPTG, placing in a shaking table at 37 ℃ for 4 hours, collecting supernatant after induction expression, then eluting by using imidazole gradient, and obtaining purer protein under the elution of 100mM and 200mM imidazole, wherein the result is shown in figure 1, namely the recombinant protein of the bombyx mori carboxypeptidase inhibitor, and the amino acid sequence of the protein is shown in SEQ ID NO. 2.
The obtained recombinant protein of the silkworm carboxypeptidase inhibitor is used for preparing an antibody and is verified, and the band has good single specificity and can be used for later experiments. In order to understand the expression condition of the silkworm carboxypeptidase inhibitor in the tissues, the tissues of five-year-old three-day silkworms are taken as templates, fluorescent quantitative PCR is carried out, the high expression of the silkworm carboxypeptidase inhibitor in silk glands is found, meanwhile, western blot is carried out by using an obtained antibody, the result is verified on the protein level, and the silkworm carboxypeptidase inhibitor is specifically expressed in the silk glands.
Example 2 evaluation of Activity of silkworm carboxypeptidase inhibitor
In order to determine whether the recombinant silkworm carboxypeptidase inhibitor has activity, a carboxypeptidase activity inhibition experiment is performed on the recombinant silkworm carboxypeptidase inhibitor. A PBS negative control group, a potato carboxypeptidase inhibitor positive control group and an experimental group are arranged by taking potato carboxypeptidase as a substrate. The results are shown in FIG. 2. The result shows that the recombinant silkworm carboxypeptidase inhibitor and the potato carboxypeptidase inhibitor have the same inhibitory activity on carboxypeptidase.
In order to determine the inhibition stability of the silkworm carboxypeptidase inhibitor, the silkworm carboxypeptidase inhibitor is respectively treated under different temperature and pH environments, and then the inhibition efficiency of the silkworm carboxypeptidase inhibitor on the carboxypeptidase activity is determined, wherein the results of different temperature measurements are shown in figure 3. The results show that the inhibitory activity of the silkworm carboxypeptidase inhibitor on the carboxypeptidase is slightly reduced with the increase of the treatment temperature, but the silkworm carboxypeptidase inhibitor shows high inhibitory efficiency, and the silkworm carboxypeptidase inhibitor has good thermal stability on the inhibition of the carboxypeptidase. The results of pH stability analysis are shown in fig. 3, and show that the inhibitory efficiency of the silkworm carboxypeptidase inhibitor against carboxypeptidase is gradually increased when the silkworm carboxypeptidase inhibitor is treated at pH 2 to 6, the inhibitory efficiency is the best when the silkworm carboxypeptidase inhibitor is treated at pH 6 to 7, and the inhibitory efficiency of the silkworm carboxypeptidase inhibitor against carboxypeptidase is gradually decreased when the pH is higher than 8. In order to understand the expression condition of the silkworm carboxypeptidase inhibitor in the tissues, the tissues of five-year-old three-day silkworms are taken as templates, fluorescent quantitative PCR is carried out, the high expression of the silkworm carboxypeptidase inhibitor in silk glands is found, western blot is carried out at the same time, and the result is verified on the protein level, so that the silkworm carboxypeptidase inhibitor is specifically expressed in the silk glands.
Example 3 evaluation of ability of recombinant protein of silkworm carboxypeptidase inhibitor to inhibit proliferation of gastric cancer tumor
According to the differentiation degree of the tumor, MKN45 cells with medium differentiation degree, SGC7901 cells with low differentiation degree and HGC27 cells without differentiation degree are selected as experimental objects. The cells in the logarithmic growth phase are taken and added into a 96-well plate, two experimental groups of 1 microgram/mL and 10 microgram/mL silkworm carboxypeptidase inhibitor proteins are arranged, the cell morphology is observed, and the result is shown in figure 4. The results show that the cell morphology of each group observed on the first day has no obvious difference, and the cells are arranged loosely and uniformly. Cells were observed again by day five and found to be full and dense in the negative control wells, whereas in wells containing 10 μ g/mL silkworm carboxypeptidase inhibitor, the number of cells was fewer than in the control, with significant gaps between cells. Meanwhile, CCK-8 cell proliferation experiments are carried out, and the growth speed of the cells added with the silkworm carboxypeptidase inhibitor is slower than that of the negative control group along with the increase of time under the condition that the initial cell number of the three cell lines is similar. After data are respectively measured on the third day and the fifth day, significance analysis is carried out, and the silkworm carboxypeptidase inhibitor is found to remarkably inhibit the proliferation of gastric cancer cells. And selecting cells on the third day for proliferation rate calculation, wherein the 10 mu g/mL silkworm carboxypeptidase inhibitor group proliferates to 75% -80% of the negative control group, and both the cells obviously inhibit the proliferation of the gastric cancer cells, and meanwhile, according to the malignancy degree of the three cell lines, the inhibition effect on MKN45 with medium differentiation degree and low malignancy degree is better, and the inhibition effect on HGC27 with undifferentiation and highest malignancy degree is slightly poor. The inhibition rate of the group of 10 mu g/mL is about 25 percent, the inhibition effect of the gastric cancer medicament tegafur of 25 mu g/mL can be achieved, and the inhibition effect is very obvious.
Example 4 molecular mechanism study of recombinant protein of bombyx mori carboxypeptidase inhibitor for inhibiting gastric cancer tumor proliferation
Phosphorylation of EGFR is one of the important markers of tumor cell proliferation, and levels of EGFR phosphorylation were measured in three cell lines of gastric cancer cells, and as a result, as shown in fig. 5, it was found that both MKN45 and SGC7901 were tyrosine-phosphorylated, whereas HGC27 was not tyrosine-phosphorylated. The results of detecting the expression conditions of the related proteins at the upstream of the MAPK/ERK pathway are shown in figure 6, and the results show that the protein product Shc of the gene which encodes the SH structural domain at the beginning, the growth factor receptor binding protein 2 (Grb 2), the guanylate exchange factor (sos) and the key protein Raf protein which initiates the pathway cascade reaction are all down-regulated and expressed by adding the silkworm carboxypeptidase inhibitor and show dose dependence. Raf protein is highly related to MEK1/2 phosphorylation, and the down-regulation expression of Raf protein also strongly suggests that bombyx mori carboxypeptidase inhibitors may be related to MAPK/ERK pathway. The expression of MAPK/ERK pathway related protein is detected, the expression of MEK1/2 and ERK1/2 is down-regulated, phosphorylation is inhibited, and the dose dependence is also shown. The expression of the protooncogene c-Myc was also detected from the signal pathway map, and the results are shown in FIG. 7. The results show that down-regulated expression of MAPK/ERK pathway-associated proteins results in down-regulated expression of c-Myc protein. The down-regulation expression of c-Myc can inhibit the expression of cycle proteins such as CDK2, CDK4, cyclin D1, cyclin E1 and the like, and finally leads to the inhibition of cell proliferation. Through the experiments, the expression of the c-Myc can be reduced by the silkworm carboxypeptidase inhibitor.
Example 5 screening of lead Compounds for gastric cancer therapy based on effective peptide fragments of recombinant proteins of silkworm carboxypeptidase inhibitors
The original ligand on the structure of the receptor 3poz protein, namely the small molecule tak-285, is successfully removed by PyMOL software. And then performing molecular docking on the ligand silkworm carboxypeptidase inhibitor and the treated receptor epidermal growth factor receptor kinase region. After 30 ten thousand simulation docks, the system returns 30 possible docking modes with the highest score, and selects the first-ranked docking mode for analysis. According to the analysis result, as shown in figure 8, the peptide fragment (tyrosine-glycine-valine-serine) of the bombyx mori carboxypeptidase inhibitor overlaps with the small molecule inhibitor tak-285 positions in the ligand active pocket, which indicates that the polypeptide fragment 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 of becoming a lead compound, a CCK-8 experiment and a plate cloning experiment are carried out, and the results are shown in figure 9. The result shows that the effective peptide fragment can obviously inhibit the proliferation of gastric cancer cells although the inhibition ability of the effective peptide fragment is slightly weaker than that of a silkworm carboxypeptidase inhibitor. The plate cloning experiment shows that compared with the clone formed by cells of a control group, the cell clone is obviously reduced and the number is reduced after the effective polypeptide fragment of the silkworm carboxypeptidase inhibitor is added.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitutions or changes made by the person 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 invention is subject to the claims.
Claims (10)
1. The effective peptide fragment of the silkworm carboxypeptidase inhibitor is characterized in that: the amino acid sequence of the effective peptide segment of the silkworm carboxypeptidase inhibitor is as follows: tyrosine-glycine-valine-serine.
2. The use of the effective peptide fragment of the bombyx mori carboxypeptidase inhibitor of claim 1 for the manufacture of a medicament for the treatment of gastric cancer.
3. The use according to claim 2, wherein the medicament is capable of inhibiting gastric cancer cell proliferation.
4. A polypeptide comprising an effective peptide fragment of the bombyx mori carboxypeptidase inhibitor of claim 1.
5. The polypeptide of claim 4, wherein: the amino acid sequence of the polypeptide is shown as SEQ ID NO. 2.
6. Use of the polypeptide of claim 4 or 5 for the manufacture of a medicament for the treatment of gastric cancer.
7. Use according to claim 6, characterized in that: the gastric cancer is differentiated or undifferentiated gastric cancer cells.
8. Use according to claim 6, characterized in that: the gastric cancer is MKN45 cells, SGC7901 cells or HGC27 cells.
9. Use according to claim 6, characterized in that: the treatment of gastric cancer is to inhibit gastric cancer cell proliferation.
10. The use of the silkworm carboxypeptidase inhibitor of claim 4 or 5 for the preparation of an inhibitor of expression of gastric cancer associated factors, wherein: the gastric cancer related factors are coding SH structural domain genes, growth factor receptor binding protein 2, guanylate exchange factors, raf proteins, c-Myc proteins or Cyclin CDK2, CDK4, cyclin D1 and Cyclin E1.
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