Sea cucumber peptide rAj-HRP30 and its application in anti-tumor

Abstract

This application belongs to the field of biotechnology, and specifically relates to a recombinant sea cucumber peptide rAj-HRP30 and its application in anti-tumor. The recombinant sea cucumber peptide rAj-HRP30 described in this application targets the FGFR receptor, inhibits the growth of tumor cells, induces apoptosis of tumor cells, and destroys the cytoskeleton of tumor cells in a dose-dependent manner, and has higher anti-tumor activity.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, and particularly relates to a recombinant sea cucumber peptide rAj-HRP30 and its application in anti-tumor or the preparation of anti-tumor drugs. Technical Background

[0002] At present, anti-cancer drugs are divided into three major categories, including: conventional chemotherapy drugs, hormone therapy drugs, and molecular targeted drugs. Among these three categories of anti-cancer drugs, chemotherapy drugs and hormone therapy drugs have limited their therapeutic effects due to their strong toxic and side effects, while molecular targeted drugs have become the development trend of the anti-tumor drug market due to their advantages of good efficacy and small side effects, and they already account for 60% of the global anti-tumor drug market. However, it cannot be ignored that most targeted drugs are monoclonal antibody products, which have the characteristics of a single anti-tumor type, high R & D costs, high production costs, and high selling prices, which seriously limit their widespread use by patients. Therefore, searching for new anti-cancer drugs has become the sacred mission of scientific research workers in related fields around the world.

[0003] Due to the limited land resources, the life systems contained in the large marine ecosystem with their rich bioactive substances have attracted the attention of biopharmaceutical R & D personnel. Looking for drugs from the ocean has become a hot spot for searching for new anti-tumor drugs. Apostichopus japonicus belongs to the phylum Echinodermata, class Holothuroidea, order Aspidochirotida, family Stichopodidae, and is an important economic aquaculture variety of sea treasures in Liaoning region. The research on its active ingredients is still in its infancy. Among the many health care effects of sea cucumbers, its anti-cancer effect has attracted extensive attention. In view of this, this application is proposed. Summary of the Invention

[0004] To solve the above technical problems, our research group carried out a series of mutagenesis, transformation, screening and testing on the anti-tumor active peptide rAj-HRP discovered in the cDNA library of Apostichopus japonicus in the early stage. Since the change in the primary structure of a protein will affect its higher-order structure and then affect its function, it is impossible to simply infer the strength of its anti-tumor function through sequence changes. Therefore, our research group constructed mutants with different expression sequences. Through activity comparison and analysis, it was found that rAj-HRP30 of this application not only has more efficient anti-tumor activity, but also has a different action target from the wild type (rAj-HRP): the action target of rAj-HRP is the EGFR receptor, while the action target of rAj-HRP30 is the FGFR receptor. The primary structure of rAj-HRP30 consists of 30 amino acids and is rich in histidine, with a molecular weight of 3.92 kDa. Further research shows that rAj-HRP30 has a dose-dependent inhibitory effect on tumor cells, and this inhibitory effect is achieved by inhibiting tumor cell adhesion, migration, invasion and inducing tumor cell apoptosis. Because its action target is the FGFR receptor and it belongs to a kind of FGFR inhibitor, rAj-HRP30 is expected to be applied in the field of preparing FGFR inhibitor-based targeted anti-tumor drugs.

[0005] Therefore, this application has at least the following objectives:

[0006] The first objective of this application is to provide a novel recombinant sea cucumber peptide rAj-HRP30 and its encoding gene;

[0007] The second objective of this application is to provide a preparation method of the recombinant sea cucumber peptide rAj-HRP30;

[0008] The third objective of this application is to provide the application of the recombinant sea cucumber peptide rAj-HRP30 in anti-tumor or the preparation of anti-tumor drugs;

[0009] The fourth objective of this application is to provide a screening method for anti-tumor active substances based on the recombinant sea cucumber peptide rAj-HRP30.

[0010] To achieve the above objectives, this application specifically provides the following technical solutions:

[0011] This application first provides a recombinant sea cucumber peptide rAj-HRP30, and its amino acid sequence is any of the following:

[0012] 1) Containing the sequence shown in SEQ ID NO.4;

[0013] 2) Meeting the requirements of conservative amino acid substitution and having at least 90% homology with SEQ ID NO.4.

[0014] The present application also provides a nucleotide encoding the recombinant sea cucumber peptide described above.

[0015] Further, the nucleotide sequence is as shown in SEQ ID NO.5.

[0016] The present application also provides a composition comprising the recombinant sea cucumber peptide or nucleotide described above.

[0017] The present application also provides a recombinant vector comprising the nucleotide sequence described above;

[0018] Preferably, the vector is a cloning vector or an expression vector;

[0019] More preferably, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0020] The present application also provides a recombinant cell comprising the recombinant vector described above.

[0021] The present application also provides a method for preparing the recombinant sea cucumber peptide rAj-HRP30 described above, comprising the step of expressing using the recombinant vector described above;

[0022] Preferably, the method further comprises separation and purification steps.

[0023] The present application also provides any one of the following applications of the recombinant sea cucumber peptide rAj-HRP30 described above:

[0024] 1) Application in anti-tumor activity;

[0025] 2) Application in the preparation of anti-tumor drugs;

[0026] Preferably, application in the preparation of anti-tumor drugs targeting FGFR;

[0027] 3) Application in screening for tumor suppression;

[0028] Preferably, application in a tumor inhibitor targeting FGFR;

[0029] 4) Application in inhibiting the growth or proliferation of tumor cells (in vivo or in vitro);

[0030] 5) Application in anti-tumor activity evaluation.

[0031] The present application also provides any one of the following applications of the nucleotide described above:

[0032] 1) Application in anti-tumor activity;

[0033] 2) Application in the preparation of anti-tumor drugs;

[0034] Preferably, it is used in the preparation of anti-tumor drugs targeting FGFR;

[0035] 3) Application in screening for tumor suppression;

[0036] Preferably, it is used in tumor inhibitors targeting FGFR;

[0037] 4) Application in inhibiting the growth or proliferation of tumor cells (in vivo or in vitro);

[0038] 5) Application in anti-tumor activity evaluation.

[0039] This application also provides a method for inhibiting tumor cells in vitro or in vivo, which is characterized by including the step of administering or expressing the above-mentioned recombinant sea cucumber peptide.

[0040] This application has at least the following beneficial technical effects:

[0041] This application discovers that rAj-HRP30 has a dose-dependent inhibitory effect on tumor cell function. This inhibitory effect is achieved by inhibiting tumor cell adhesion, migration, invasion and inducing tumor cell apoptosis. Moreover, its action target is the FGFR receptor, and it is an FGFR inhibitor. rAj-HRP30 is expected to be applied in the field of preparing FGFR inhibitor-based targeted anti-tumor drugs. Moreover, since it is a small peptide that can be recombinantly expressed in prokaryotes, compared with monoclonal antibody-based targeted drugs, it also has advantages such as lower production and preparation costs and easier high-yield realization. Description of the Drawings

[0042] Figure 1 . Tricine SDS-PAGE electrophoresis map of rAj-HRP30 purified peptide. Among them, lane 1 is the small molecule protein Marker; lane 2 is the recombinant transformed bacteria BL21 induced for expression at low temperature overnight; lane 3 is the recombinant transformed bacteria BL21 not induced for expression at low temperature overnight; lane 4 is the rAj-HRP30 purified peptide.

[0043] Figure 2 . Detection of the inhibitory effect of rAj-HRP30 on the proliferation of Panc02 cells by the CCK method. Among them, the abscissa is the concentration of rAj-HRP30, with the unit of μM; the ordinate is the inhibition rate of Panc02 cell proliferation (%).

[0044] Figure 3 . Detection of the effect of rAj-HRP30 on the apoptosis of Panc02 cells by the TUNEL method (Zeiss laser scanning confocal microscope, 630×).

[0045] Figure 4.Effect of rAj-HRP30 on the cytoskeleton of Panc02 cells detected by FITC-phalloidin method (Zeiss laser scanning confocal microscope, 630×).

[0046] Figure 5 .Effect of rAj-HRP30 on the expression of FGFR1 in Panc02 cells. Among them, (A) Western blot result graph; (B) Statistical graph of relative gray value of FGFR1 expression (FGFR1 / GAPDH) (n = 3; compared with the Control group in each rAj-HRP30 group, significant differences are indicated by *, *p < 0.05, **p < 0.01, ***p < 0.001). Specific implementation manners

[0047] The implementation manners of the present application will be described in detail below in conjunction with embodiments. However, those skilled in the art will understand that the following embodiments are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through market purchase.

[0048] Definition of some terms

[0049] Unless otherwise defined hereinafter, the meanings of all technical terms and scientific terms used in the specific implementation manners of the present application are intended to be the same as those generally understood by those skilled in the art. Although the following terms are believed to be well understood by those skilled in the art, the following definitions are still provided to better explain the present application.

[0050] As used in the present application, the indefinite article or definite article used when referring to a singular noun, such as "a" or "an", "the", includes the plural form of the noun.

[0051] As used in the present application, the terms "comprising", "including", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term "consisting of" is considered to be a preferred embodiment of the term "comprising". If in the following text a group is defined as including at least a certain number of embodiments, this should also be understood as disclosing a group preferably consisting only of these embodiments.

[0052] The term "about" in the present application means an accuracy range that those skilled in the art can understand and still ensure the technical effect of the feature being discussed. This term generally means ±10% deviation from the indicated value, preferably ±5%.

[0053] Furthermore, the terms first, second, third, (a), (b), (c), and the like, in the specification and claims, are used to distinguish between similar elements and are not necessarily intended to describe a sequential or chronological order. It is understood that the terms so used are interchangeable under appropriate circumstances, and that the embodiments described herein can be practiced in other sequences than described or illustrated herein.

[0054] The above terms or definitions are provided merely to aid understanding of the present application and should not be construed as having a scope less than that understood by a person skilled in the art.

[0055] The amino acid sequence of the recombinant sea cucumber peptide rAj-HRP30 described in the present application is any one of the following:

[0056] 1) comprising the sequence shown in SEQ ID NO. 4, or the sequence shown in SEQ ID NO. 4;

[0057] 2) It is understood that the activity will not be affected if conservative amino acid substitutions are made. Therefore, sequences that meet the requirements of conservative amino acid substitutions and have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with SEQ ID NO. 4 are also within the scope of protection of this application.

[0058] The nucleotides of the present application encode the recombinant sea cucumber peptide described above. It can be understood that any base sequence that can encode the above amino acid sequence is within the scope of protection of this patent.

[0059] In some specific embodiments, the nucleotide sequence is as shown in SEQ ID NO.5.

[0060] The composition of the present application may contain the recombinant sea cucumber peptide described above, and may also contain the recombinant nucleotide described above.

[0061] The recombinant vector of the present application may include any of the nucleotide sequences described above;

[0062] The type of the vector is not limited. In some embodiments, it can be a cloning vector, and in other embodiments, it can also be an expression vector.

[0063] In some more specific embodiments, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0064] The recombinant cells of the present application are cells containing the above-mentioned recombinant vectors, and the cell types are not limited.

[0065] The preparation method of the recombinant sea cucumber peptide rAj-HRP30 of the present application can be prepared by various conventional methods known in the prior art, as long as the prepared sequence is consistent with the foregoing sequence. For example, it can be chemically synthesized or biologically expressed. For example, in some specific biological expression methods, it includes the step of expressing using the above-mentioned recombinant vector;

[0066] In some embodiments, the method further includes separation and purification steps.

[0067] The application of the recombinant sea cucumber peptide rAj-HRP30 of the present application can be in many aspects. In the case of inhibiting its anti-tumor activity and anti-tumor mechanism, the present application can protect many aspects of applications, such as:

[0068] 1) Application in anti-tumor activity;

[0069] 2) Application in the preparation of anti-tumor drugs;

[0070] Preferably, application in the preparation of anti-tumor drugs targeting FGFR;

[0071] 3) Application in screening tumor inhibitors;

[0072] Preferably, application in tumor inhibitors targeting FGFR;

[0073] 4) Application in inhibiting the growth or proliferation of tumor cells (in vivo or in vitro);

[0074] 5) Application in anti-tumor activity evaluation.

[0075] Similarly, the coding nucleotide of the present application also has any of the following applications:

[0076] 1) Application in anti-tumor activity;

[0077] 2) Application in the preparation of anti-tumor drugs;

[0078] Preferably, application in the preparation of anti-tumor drugs targeting FGFR;

[0079] 3) Application in screening tumor inhibitors;

[0080] Preferably, application in tumor inhibitors targeting FGFR;

[0081] 4) Application in inhibiting the growth or proliferation of tumor cells (in vivo or in vitro);

[0082] 5) Application in anti-tumor activity evaluation.

[0083] The present application may also relate to methods for inhibiting tumor cells in vitro or in vivo, including the step of administering or expressing the above-mentioned recombinant sea cucumber peptides.

[0084] The present application will be described below in conjunction with specific embodiments.

[0085] Example 1: Screening of sea cucumber peptide Aj-HRP30

[0086] In the present application, a series of mutagenesis modifications and screening tests were carried out on the anti-tumor active peptide rAj-HRP previously discovered in the Apostichopus japonicus cDNA library. For this purpose, the mutant sequences were artificially synthesized by the synthesis method, and their anti-tumor cell proliferation activities were tested respectively. In order to effectively shorten the length of the wild-type peptide to avoid future immunogenic risks and at the same time perform recombinant expression and preparation of the mutant sequences (the sequences should not be too short), after analyzing the predicted three-dimensional structure of rAj-HRP, the present application intercepted peptide segments of about 30 amino acids at different positions of Aj-HRP for analysis, obtained multiple sequences and carried out sequence synthesis. Some specific mutant sequences are as follows:

[0087] Aj-HRPN: MALIRCLLAFAFIVCSVKTFAAPIEDEILDAD (SEQ ID NO.1);

[0088] Aj-HRPM: EDEILDADALSRRRHRHGWFHKLFHHHHH (SEQ ID NO.2);

[0089] Aj-HRPMX: VCSVKTFAAPIEDEILDADALSRRRHRHGWF (SEQ ID NO.3);

[0090] Aj-HRP30: RHRHGWFHKLFHHHHHDKHHHGEHHHGEHH (SEQ ID NO.4);

[0091] After analysis, among these mutants, HRPN is an insoluble peptide and cannot be further studied for its function; the results of CCK of the remaining three peptides on Panc02 cells show that the inhibitory ability of 84 μM of HRPM and 86 μM of HRPMX on Panc02 cells reaches the maximum, and the inhibition rates are 49% and 48% respectively, which do not reach the half-inhibition rate. However, HRP30 can achieve 100% inhibition rate on Panc02 cells only at a concentration of 10.60 μM, and its IC 50 (half-inhibitory concentration) is 3.22 μM. It can be seen that HRP30 has a significant tumor cell inhibitory function compared with other mutants.

[0092] The sequence information of the recombinant peptide Aj-HRP30 was analyzed as follows:

[0093] The cDNA sequence (open reading frame) of Aj-HRP30 is 90 bp, and its protein consists of 30 amino acids with a theoretical molecular weight of 3.9 kDa. The specific amino acid sequence is as follows:

[0094] RHRHGWFHKLFHHHHHDKHHHGGHHHGEHH (SEQ ID NO.4)

[0095] The coding nucleic acid sequence is as follows:

[0096] cgccaccgccacggatggtttcataaattgttccatcatcaccaccatgataaacaccaccatggtgaacaccacc atggtgaacacca (SEQ ID NO.5)

[0097] Example 2: Molecular Cloning, Recombinant Expression and Purification of Sea Cucumber Peptide rAj-HRP30

[0098] 1. Molecular Cloning of the rAj-HRP30 Recombinant Peptide Gene

[0099] 1) Obtaining the aj-hrp30 gene: The aj-hrp30 gene was obtained by artificial synthesis. When synthesizing artificially, a stop codon was added to the 3' end of the aj-hrp gene sequence, which made the recombinant protein expressed by this gene not carry any purification tags. At the same time, when synthesizing this sequence gene, an Nde I restriction site was added to the 5' end of the sequence, and an Xho I restriction site was added to the 3' end of the sequence. The artificial gene synthesis was completed by Nanjing Genscript Biotech Co., Ltd.

[0100] 2) Ligation of the target gene DNA fragment with the vector pET23b: Since the designed primers respectively carry Nde I and Xho I restriction sites (see the underlined parts of the primer sequences), and these two restriction sites are also the multiple cloning sites of pET23b, it makes it possible to ligate the target gene DNA fragment with the vector pET23b. The primer sequences are as follows:

[0101] P1: 5'-XXCATATGCGCCACCGCCACGGATGG-3'

[0102] P2: 5'-XXTCTGAGTCAATGGTGTTCACCATGGTG-3'

[0103] 3) Transformation of the ligation product into the cloning bacterium E. coli BL21 by the CaCl2 method: The host bacterium E. coli BL21 was inoculated into LB (Amp- ) Incubate overnight, and transfer a portion of the bacterial solution to 50 mL of LB culture medium the next day, and continue to culture at 37 °C until the OD 600 reaches between 0.2 and 0.3. Place the culture on ice for 10 min, centrifuge at 5000 rpm at 4 °C for 10 min. Discard the supernatant, invert the centrifuge tube for 1 min, add 5 mL of ice-precooled 0.1 M CaCl2 solution to sensitize and suspend the cells, and place on ice for 10 min. Centrifuge at 5000 rpm at 4 °C for 10 min to recover the cells, discard the supernatant, add 2 mL of ice-cold 0.1 M CaCl2 solution to every 25 mL of the original culture, suspend the cells and place on ice for 3 h, and aliquot the cells at 200 μL per portion. Add 10 μL of DNA solution containing 40 ng to 200 μL of competent cells, gently mix, heat shock at 42 °C for 90 s, and then quickly return to ice. After cooling the cells for 1 - 2 min, add 800 μL of LB (Amp - ) medium, and shake the bacteria at 37 °C at 225 rpm for 45 - 90 min. Take 100 μL of the transformation product and spread it on a 90 mm LB (Amp + ) agar plate. Place it at room temperature for 20 - 30 min, then invert the petri dish and culture at 37 °C for 12 - 16 h. After colonies grow on the LB (Amp+) agar plate, the screening and identification of positive transformants can be carried out.

[0104] 4) Screening and identification of positive transformants: Use the T7 universal primer method for screening and identification of positive transformants. After the 90 bp gene fragment of rAj-HRP30 is inserted into the pET23b plasmid, the length of the PCR amplification product fragment of the T7 universal primer should be 283 bp, while the pET23b empty plasmid has no exogenous gene fragment inserted, and its T7 universal primer PCR amplification product is only 193 bp. Therefore, the positive recombinant can be judged based on the results of the T7 universal primer.

[0105] 2. Induced expression of rAj-HRP30 recombinant peptide gene:

[0106] Induce the expression of 1 L of positive recombinant bacteria with IPTG at a final concentration of 1 mM. The induced expression condition is overnight induction at 30 °C.

[0107] 3. Extraction and purification of rAj-HRP30 recombinant peptide

[0108] 1) Harvest bacteria: Aliquot the induced bacterial solution into 50 mL centrifuge tubes, and centrifuge at 4 °C and 4000 rpm for 10 min;

[0109] 2) Ultrasonic disruption: Collect the bacterial cells, resuspend them in 20 mL of pre-cooled 1×Binding Buffer, pour the suspension into a small beaker, and ultrasonically disrupt it on ice for 30 min (30% power, break for 5 s and stop for 5 s);

[0110] 3) Filtration: Aliquot the broken bacterial solution into centrifuge tubes, centrifuge at 4°C and 12,000 rpm for 20 min, collect the supernatant, and filter it through a 0.45 μm filter into a sterilized centrifuge tube for standby;

[0111] 4) Purification of the rAj-HRP30 gene recombinant peptide: Although this recombinant peptide has no purification tag during gene construction, since the rAj-HRP peptide is rich in histidine, nickel ion affinity chromatography is used for purification in the present invention. The purification process is as follows:

[0112] Equilibrate the nickel ion chromatography column with 10 mL of pre-cooled 1× Binding Buffer. After draining, add the filtered supernatant along the tube wall for sample loading. After the supernatant has drained, add 10 mL of pre-cooled 1× Binding Buffer along the tube wall. After draining, add 10 mL of pre-cooled 0.5× Wash Buffer along the tube wall to elute the miscellaneous proteins. Finally, add 5 mL of pre-cooled 1× Elute Buffer to elute the target peptide and store it frozen;

[0113] 5) Purity identification of the rAj-HRP30 gene recombinant peptide: Take an appropriate amount of the target protein, mix it with the small molecular weight loading Buffer at a ratio of 1:1, and obtain an electrophoresis sample after boiling water bath for 5 min, and then perform small molecule Tricine-SDS PAGE electrophoresis.

[0114] 4. Small molecule Tricine-SDS PAGE electrophoresis of the rAj-HRP30 gene recombinant peptide

[0115] Use three-layer Tricine-SDS PAGE electrophoresis to analyze proteins with relatively small molecular weights. The experimental procedure is as follows:

[0116] 1) Sample preparation: Take 1 mL of the sample from each of the above culture solutions, centrifuge for 15 min (12,000 rpm), discard the supernatant, add 100 μL of small molecular weight loading Buffer to the precipitate, shake well, and then perform a boiling water bath for 5 min;

[0117] 2) Gel preparation: Take a set of gel plates, assemble and check for leaks. Prepare the electrophoresis gel according to the formula in Table 1. Use isobutanol to press the middle layer gel and the lower layer gel to keep the surfaces of each layer of gel flat;

[0118] Table 1. Formulation of small molecule Tricine-SDS PAGE electrophoresis gel

[0119]

[0120] 3) Loading: Install the electrophoresis gel tank. Add 1×anode buffer to the outer tank and 1×cathode buffer to the inner tank until the liquid level covers the loading wells. Slowly pull out the comb and add the small molecular weight prestained protein Marker (3 μL / well) and the electrophoresis sample (20 μL / well) into the loading wells;

[0121] 4) Electrophoresis: Perform electrophoresis at 30 V until the sample runs out of the loading well, then use 80 V until the sample enters the lower gel, and finally use 100 V until the electrophoresis ends.

[0122] 5) Quick fixation, staining and decolorization: Fix for 30 min, stain for 1 - 2 h, decolorize on a shaker 3 times (10 min each time, and change the decolorizing solution. Then add pure water and decolorize on a shaker overnight until clear bands appear), and the results are as Figure 1 shown.

[0123] Example 3. Effect of rAj-HRP30 Peptide on the Proliferation of Mouse Pancreatic Ductal Adenocarcinoma Tumor Cells

[0124] 1) After trypsinizing the Panc02 cells cultured to the logarithmic phase, centrifuge at 2000 rpm for 3 min, discard the supernatant, and add an appropriate amount of complete medium to prepare a cell suspension with a suitable density;

[0125] 2) Take a 96-well plate, add 100 μL of cell suspension and gradient concentrations of rAj-HRP30 to each well. Use PBS to make up the added drug volume to 10 μL and culture at 37°C and 5% CO2 for 24 h;

[0126] 3) Add 10 μL of CCK-8 solution to each well and continue to culture for 3 - 4 h;

[0127] 4) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD value of each well at a wavelength of 450 nm;

[0128] 5) Conduct three sets of repeated experiments, take the average value, use Excel to statistically analyze and calculate the inhibition rate of rAj-HRP30 on the proliferation of Panc02 cells. The formula is as follows:

[0129]

[0130] Use GraphPad Prism 8.0 to plot the graph and calculate the half-maximal inhibitory concentration IC 50 .

[0131] The results are as Figure 2 shown. The abscissa is the concentration of rAj-HRP30 (μM); the ordinate is the inhibition rate of Panc02 cell proliferation (%). rAj-HRP30 can inhibit the proliferation of Panc02 tumor cells in a dose-dependent manner in vitro, and its IC 50It is only 3.23 μM, significantly lower than the original active peptide.

[0132] Example 4. Effect of rAj-HRP30 on tumor cell apoptosis

[0133] Experiment on detecting apoptosis of Panc02 cells induced by rAj-HRP30 using the TUNEL method:

[0134] 1) Collect cells;

[0135] 2) Seed cells: Take a clean and sterile 24-well plate. Use forceps to pick up a clean cell slide and soak it in a small dish containing PBS, washing both sides of the slide to make the slide fit. Add 10 μL of PBS to the bottom of the well plate. Use forceps to pick out the cell slide and add it to the well plate, avoiding leaving air bubbles. Slowly add 500 μL of cell suspension along the wall of each well, maintaining the cell density at 60% - 70%. Shake it "crosswise" 6 - 8 times, let it stand for 5 min, and then slowly transfer it to the incubator and culture for 18 h;

[0136] 3) Drug treatment: Replace the culture medium, set up a control group and drug treatment groups (1.6 μM, 3.2 μM, 6.4 μM), and transfer them to the incubator for continued culture for 20 h;

[0137] 4) Fixation: Remove the liquid in each well, add 500 μL of PBS for washing once, add 4% paraformaldehyde fixative, 500 μl per well, and fix for 30 min;

[0138] 5) Permeabilization: Dilute the 30% Triton X-100 solution to 0.3%. Aspirate the fixative, wash once with PBS, add the PBS solution containing 0.3% Triton X-100, 500 μL / well, and incubate at room temperature for 5 min;

[0139] 6) Add TUNEL detection solution: Remove the permeabilization solution, wash each well 2 times with PBS, add 50 μL of TUNEL detection solution to each well, and incubate in the dark for 60 min (37 °C);

[0140] 7) Slide preparation: Remove the detection solution, add PBS for washing 3 times, 45 s each time. Drop 10 - 15 μL of anti-fluorescence quenching mounting medium on the glass slide. Take out the slide, invert it on the glass slide, let the cells contact the quenching agent, and mount the slide;

[0141] 8) Photograph: Observe and photograph the glass slide under a fluorescence inverted microscope.

[0142] The experimental results are as Figure 3As shown in the figure, in the control group, no green fluorescence was observed in the nuclei of Panc02 cells untreated with rAj-HRP30. As the dose of rAj-HRP30 (1.6 μM, 3.2 μM, 6.4 μM) increased, the brightness of the green fluorescence in the nuclei gradually enhanced. Under the action of 6.4 μM rAj-HRP30, the nuclei of Panc02 cells showed bright green fluorescence, indicating that rAj-HRP30 could induce DNA breakage in Panc02 cells in a dose-dependent manner, leading to apoptosis.

[0143] Example 5. Effects of rAj-HRP30 on the cytoskeleton of tumor cells

[0144] 1) Collect cells;

[0145] 2) Seed cells: Take a clean and sterile 24-well plate. Use forceps to pick up a clean cell slide and soak it in a small dish containing PBS, washing both sides of the slide to make it fit. Add 10 μL of PBS to the bottom of the well plate, use forceps to pick out the cell slide and add it to the well plate, avoiding leaving air bubbles. Slowly add 500 μL of cell suspension along the wall of each well, maintaining the cell density at 50% - 60%, gently shake it "crosswise" 6 - 8 times, let it stand for 5 min, and slowly transfer it to the incubator for 18 h;

[0146] 3) Drug treatment: Replace the culture medium, set up a control group and drug treatment groups (1.6 μM, 3.2 μM, 6.4 μM), and transfer them to the incubator for continued culture for 20 h;

[0147] 4) Fixation: Remove the liquid in each well, add 500 μL of PBS to wash 2 times, add 500 μL of fixative to each well, and fix for 30 min;

[0148] 5) Add Alexa Fluor 488 phalloidin working solution: Use a PBS solution containing 1% BSA to dilute the 1000× stock solution to 1× working solution. Use a pipette to aspirate the fixative, wash 2 times with PBS, add 100 μl of 1× working solution to each well, and incubate in the dark for 60 minutes;

[0149] 6) Add Hoechst 33258 staining solution: Aspirate the 1× working solution, add PBS to wash 2 times, add 500 μL of Hoechst 33258 staining solution to each well, and stain in the dark for 5 min;

[0150] 7) Slide preparation: Remove the Hoechst 33258 staining solution, wash 2 times with PBS, add 10 - 15 μL of anti-fluorescence quenching mounting medium on a glass slide, take out the slide, invert it on the glass slide, let the cells contact the quencher, and mount the slide;

[0151] 8) Photograph: Observe and photograph the glass slide under a fluorescence inverted microscope.

[0152] The results showed that in the control group, the outlines of Panc02 cells were clear, the cytoskeletons were intact, and the blue fluorescence in the nuclei was relatively faint. As the dosage of rAj-HRP30 increased, the cells gradually shrank and became round, the microfilaments gradually disintegrated, and the intensity of the blue fluorescence in the nuclei gradually increased. Under the action of 6.4 μM rAj-HRP30, the cytoskeleton of Panc02 cells had been severely damaged, with only a small part remaining, indicating that rAj-HRP30 could disrupt the cytoskeleton structure of Panc02 cells and induce apoptosis, thereby inhibiting cell movements such as the proliferation, adhesion, and migration of Panc02 cells.

[0153] Example 6. Effect of rAj-HRP30 on the expression of FGFR1 in tumor cells

[0154] The anti-tumor mechanism of rAj-HRP30 was studied by Western Blot method. The specific experimental steps were as follows:

[0155] 1) Sample preparation

[0156] ① After treating Panc02 cells, they were evenly inoculated into 4 culture flasks and cultured in a cell incubator under the conditions of 37 °C and 5% CO2.

[0157] ② When the cells in the culture flasks grew to 70%-80%, the culture medium was removed. After mixing 3 mL of complete medium with gradient concentrations of rAj-HRP30 (final concentrations of 0 μM, 1.6 μM, 3.2 μM, 6.4 μM), the cells were cultured for another 24 h.

[0158] ③ All the cells in the culture flasks (including floating cells) were collected with a cell scraper and transferred to a 10 mL Eppendorf tube together with the culture solution. The scraper and the culture flask were washed with 1 mL of PBS, and the residual cells were collected.

[0159] ④ Balanced, centrifuged at 12,000 rpm for 5 min in a 4 °C centrifuge, the supernatant was discarded, and it was placed on ice for standby.

[0160] ⑤ Prepare cell lysate. Add protease inhibitor PMSF to RIPA at a ratio of 100:1, mix well, and then add 400 μL of cell lysate to each tube of precipitate.

[0161] ⑥ Lysed on ice for 30 min, shaken once every 5 min.

[0162] ⑦ Add 100 μL of 5× Loading Buffer to each tube of protein respectively, mix well, and then boil in water bath for 10 min to prepare WB samples, which were stored at -20 °C after cooling.

[0163] 2) SDS-PAGE electrophoresis, cut the gel;

[0164] 3) Membrane transfer

[0165] ① Cut a PVDF membrane and 8 filter papers (4 large and 4 small) of appropriate size. After the PVDF membrane is activated with methanol, soak it in the membrane transfer buffer together with the filter papers for 10 min;

[0166] ② On the graphite electrode side of the semi-dry membrane transfer apparatus, place them in the order of 4 layers of filter paper → PVDF membrane → protein gel → 4 layers of filter paper. Finally, cover with the metal electrode and perform membrane transfer at a speed of 1 kDa / min;

[0167] 4) Blocking: After the membrane transfer is completed, place the PVDF membrane in the blocking buffer and block for 1 h - 2 h (room temperature, 60 rmp);

[0168] 5) Primary antibody incubation: Place the blocked PVDF membrane in the diluted primary antibody and incubate overnight at 4°C;

[0169] 6) Secondary antibody incubation: Take out the PVDF membrane, wash it four times with 1×TBST Buffer (10 min each time), and then place the PVDF membrane in the diluted secondary antibody and incubate for 1 h (room temperature, 60 rmp);

[0170] 7) Luminescence imaging: Take out the PVDF membrane, wash it four times with 1×TBST Buffer (10 min each time), evenly drop the prepared ECL luminescence solution onto the PVDF membrane so that it can cover the whole membrane, and use a chemiluminescence gel imaging system for exposure imaging;

[0171] 8) Gray scale analysis: Use ImageJ to analyze the gray scale value of the WB bands, and use GraphPad Prism 8.0 for significance analysis and drawing.

[0172] The results showed that after the action of rAj-HRP30 with gradient concentrations (1.6 μM, 3.2 μM, 6.4 μM) on Panc02 cells, the relative expression level of FGFR showed a gradually decreasing trend. This indicates that different from the wild peptide rAj-HRP whose action target is the EGFR receptor, rAj-HRP30 can down-regulate the expression of FGFR1 in Panc02 cells and shows a concentration-dependent characteristic, thereby inhibiting the activation of the downstream signaling pathway and inhibiting the proliferation and metastasis of Panc02 cells. rAj-HRP30 is an FGFR inhibitor, which can target and inhibit the expression of FGFR in tumor cells and then play an anti-tumor function.

[0173] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A recombinant sea cucumber peptide rAj-HRP30, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

4.

2. A nucleic acid, characterized in that, Encoding the recombinant sea cucumber peptide described in claim 1.

3. The nucleic acid according to claim 2, wherein The nucleotide sequence is shown in SEQ ID NO.

5.

4. A composition, characterized in that, Containing the recombinant sea cucumber peptide described in claim 1 or the nucleic acid described in any one of claims 2-3.

5. A recombinant vector, characterized in that, Containing the nucleic acid described in any one of claims 2-3.

6. A recombinant cell, characterized in that, Containing the recombinant vector described in claim 5.

7. The preparation method of the recombinant sea cucumber peptide rAj-HRP30 according to claim 1, characterized in that, Containing the step of expression using the recombinant vector described in claim 5.

8. Use of the recombinant sea cucumber peptide rAj-HRP30 described in claim 1 in the preparation of a drug for treating pancreatic ductal adenocarcinoma.

9. Use of the nucleic acid described in any one of claims 2-3 in the preparation of a drug for treating pancreatic ductal adenocarcinoma.

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