CN117777246A - Polypeptide targeting discoid structural domain receptor 1 and application thereof - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a polypeptide targeting discoid structural domain receptor 1 and application thereof, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

Description

Polypeptide targeting discoid structural domain receptor 1 and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a polypeptide targeting discoid structural domain receptor 1 and application thereof.

Background

Disc domain receptor 1 (Discoidin domain receptor, DDR 1), a protein found in cell membranes and cytoplasm, is a member of the Receptor Tyrosine Kinases (RTKs) and plays an important regulatory role in the interaction between extracellular matrix and cells, and is widely expressed in various tissues and cell types, including the kidney, liver, lung, heart, central nervous system, etc. The discovery of DDR1 protein receptor molecules can be traced back to the 90 s of the 20 th century, when researchers discovered this protein when screening and identifying collagen receptors. In recent years, more and more research on disc domain receptor 1 (DDR 1) has been focused, and a great deal of research has revealed that DDR1 plays an important role in cell differentiation, proliferation, adhesion and migration, and also has the effect of promoting malignant transformation of cells and invasion and metastasis of tumors. Expression of DDR1 has been previously reported by the international top journal Nature to help establish a physical barrier around tumors, preventing T cells from infiltrating into the tumor, thus impeding the killing of tumor cells.

DDR1 is taken as a novel RTKs protein and becomes one of potential cancer treatment targets. The activity of DDR1 proteins can be activated by binding to extracellular matrix components (e.g., collagen), thereby initiating a series of downstream signaling pathways and regulating related cellular functions. With further research on the function and regulatory mechanism of DDR1 protein, the importance of the protein in cell biology and disease research is further known. Research on DDR1 protein not only helps to reveal the mechanism of cell signal transmission, but also can provide new ideas and methods for diagnosis and treatment of related diseases. In view of the high expression of DDR1 associated with many diseases, through multiple rounds of screening of polypeptide phage libraries, the specific binding sequence of DDR1 is searched, and after the obtained specific binding polypeptide is used for modifying liposome and exosome, related therapeutic drugs are wrapped for targeted treatment of various diseases associated with the high expression of DDR1.

Disclosure of Invention

According to the invention, through a high-capacity dodecapeptide phage library, polypeptides combined with DDR1 are screened, 129 phage clones are randomly selected for preliminary verification through phage ELISA after three rounds of screening, 50 positive clones subjected to preliminary verification are subjected to sequencing analysis, the sequences are translated into amino acids and then sequenced and subjected to multi-sequence comparison, 10 different binding peptide sequences are obtained, and DBP124 polypeptides are highly enriched. Based on the found polypeptide fragment DBP124 with high binding capacity to DDR1, the binding condition of the polypeptide fragment DBP124 to DDR1 is fully verified.

In a first aspect, the invention provides a polypeptide targeting discotic domain receptor 1, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

The sequence of SEQ ID NO.1 is specifically as follows:

TGSLPHIGYYHMGGGSAHARVPFYSHSPHLPLKLMMKPD

the amino acid sequence of the polypeptide targeting the discoid structural domain receptor 1 provided by the invention can be a sequence shown as SEQ ID NO.1 or an amino acid sequence obtained by adding one or more amino acids which do not influence the function of the polypeptide targeting the discoid structural domain receptor 1 at two ends of the sequence shown as SEQ ID NO. 1.

Further, the invention also provides a derivative polypeptide, which is obtained by generating the polypeptide targeting the discoid domain receptor 1 through enzymolysis or adding a tag sequence to the N end or the C end of the polypeptide targeting the discoid domain receptor 1.

Further, the above derivative polypeptide can be obtained by adding one or more amino acids capable of being cleaved by enzymolysis in vivo or in vitro to the two ends of the polypeptide targeting discoid domain receptor 1, and the polypeptide sequence targeting discoid domain receptor 1 can be obtained after the cleavage by enzymolysis, so that the above derivative polypeptide has the same function as the polypeptide targeting discoid domain receptor 1.

Further, the tag sequence of the present invention is not particularly limited without affecting the function of the polypeptide targeting discoid domain receptor 1, and may be selected from at least one of His6 tag, GST tag, EGFP tag, MBP tag, nus tag, HA tag, igG tag, FLAG tag, c Myc tag and ProfinityeXact tag, which are commonly used in the art.

Furthermore, the polypeptide targeting the discoid domain receptor 1 provided by the invention can be modified based on the amino acid sequence, and the modifications comprise: coupling or fusion with an antibody, carrier, ligand, albumin, fc fragment, amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, biotinylation, fluorophore modification, polyethylene glycol modification, and immobilization modification.

Further, the present invention also provides a fusion protein or immunoconjugate comprising said discotic domain receptor 1 targeting polypeptide or said derivative polypeptide.

Further, the fusion protein or the immunoconjugate is obtained by coupling or fusing the polypeptide targeting the discotic domain receptor 1 or the derivative polypeptide with an antibody, a carrier, a ligand, albumin or an Fc fragment.

Furthermore, the obtaining of the polypeptide targeting the discoid structural domain receptor 1 and the derivatives thereof is carried out by adopting a method known in the prior art, and the chemical synthesis is carried out by using a polypeptide automatic synthesizer; or deducing a nucleotide sequence by the short peptide sequence, and then cloning the nucleotide sequence into a vector for biosynthesis; or from existing organisms in large quantities.

In a second aspect, the invention provides a nucleic acid having a nucleotide sequence as shown in SEQ ID NO. 2.

The sequence of SEQ ID NO.2 is specifically as follows:

ACTGGTAGTTTGCCTCATATTGGTTATTATCATATGGGTGGAGGTTCGGCCCATGCCCGGGTACCTTTCTATTCTCACTCTCCTCATTTGCCGCTGAAGTTGATGATGAAGCCGGATGGTGGAGGT

in a third aspect, the invention provides a vector comprising said nucleic acid.

In a fourth aspect the invention provides a host cell or host bacterium of the vector.

In a fifth aspect, the invention provides the use of said polypeptide, or said nucleic acid, in the manufacture of a tumor targeted medicament.

In a sixth aspect the invention provides said polypeptide, or said nucleic acid, for use in the visual localization of a tumor after labelling of a fluorescent dye.

The seventh aspect of the invention provides the polypeptide, or the nucleic acid is used for constructing a targeted therapeutic means for DDR1 high expression diseases.

In an eighth aspect the invention provides a pharmaceutical composition comprising said polypeptide or said nucleic acid.

Further, the pharmaceutical composition contains more than one pharmaceutically acceptable carrier.

In a ninth aspect, the invention provides a detection reagent comprising said polypeptide.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention provides a polypeptide with high affinity with human murine DDR1, which can be used for tumor imaging and targeted therapy by combining with the wide expression of DDR1 in glioma.

2. After the specific binding polypeptide obtained by the invention is used for modifying liposome and exosome, related therapeutic drugs are wrapped for targeted treatment of various diseases accompanied with DDR1 high expression.

3. The invention provides a targeting scheme for diagnosis and treatment strategies developed around DDR1, and lays a foundation for research in the related field.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings which are required in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are some embodiments of the invention and that other drawings may be obtained from these drawings without inventive effort for a person skilled in the art.

FIG. 1A shows the expression level of a glioma cell line DDR1 detected by Western-blot, and B shows the expression condition of DDR1 in a cell membrane detected by flow cytometry;

FIG. 2 shows the cell expression of DDR1 detected by immunofluorescence;

FIG. 3 is a Coomassie brilliant blue stain to verify DDR1 protein purity;

FIG. 4 shows that 129 monoclonal phages were selected after three rounds of screening to verify their binding capacity to human DDR1, and 50 monoclonal with Ratio > 5 were selected;

FIG. 5 shows the binding capacity test of 50 positive phage clones to human DDR1 (B), and the binding capacity test of 50 positive phage clones to murine DDR1 (C);

FIG. 6 shows that 7 sequences (D and E) are obtained by selecting and sequencing clones with strong binding force from 50 positive phages, and then the 7 positive sequences are quantified to the same titer to detect the binding capacity of the 7 positive sequences with human-derived and murine-derived DDR1, and D5 is a negative control;

FIG. 7 shows protein purification by selecting the binding front four polypeptide sequences (DBP 10/DBP105/DBP118/DBP 124) and the control polypeptide (P35) in the phage library, fusing them with EGFP tag and purifying;

FIG. 8A is an ELISA assay for four binding polypeptides of DBP10/DBP105/DBP118/DBP124 with human DDR1, B is an ELISA assay for four binding polypeptides of DBP10/DBP105/DBP118/DBP124 with murine DDR 1;

FIG. 9A shows the Biacore binding ability assay of DBP124-EGFP polypeptide to human DDR1, and B shows the Biacore binding ability assay of DBP124-EGFP polypeptide to murine DDR 1;

FIG. 10A is a cell ELISA of DBP124-EGFP polypeptide and U87 cell line, and B is a cell ELISA of DBP124-EGFP polypeptide and U251 cell line;

FIG. 11 is a cell ELISA quantification result (C and D) of DBP124-EGFP polypeptide and U87 and U251 cell lines;

FIG. 12 is a Coomassie brilliant blue stain to verify DBP124-PE38 protein purity;

FIG. 13 shows the ability of glioma cell line CCK8 to detect DBP124-PE38 killing;

FIG. 14 is a graph showing the therapeutic targeting ability of GL261 to detect DBP124-PE38 in subcutaneous neoplasia.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

Screening and verification of DDR1 binding polypeptides comprises the following steps:

1) Screening of dodecapeptide phage library and preliminary validation of positive clones by ELISA

Screening phage display dodecapeptide library by immune tube method, wherein the selected phage display library capacity is 2×10, based on Ph.D. Phage Display Libraries (NEB#E8100S, E8101S, E8110S, E8111L, E8120S) ⁹ . The screening steps are as follows: a) Coating target protein DDR1 on an immune tube according to the concentration of 20 mug/mL, wherein the principle of screening by an immune tube method is to coat the protein on the surface of the immune tube with high adsorption force, and carrying out enrichment screening for 3 rounds by adding a phage display antibody library into the immune tube and incubating, washing and eluting with antigen protein adsorbed on the surface of the immune tube; b) The third round of phage is eluted by using 0.5% PBST, 129 monoclonal antibodies are randomly picked for ELISA verification after the eluent is coated with the plate, BSA is coated at the same time as a control, and ELISA reading is 5 times greater than corresponding BSA reading and reading is greater than 0.5 is taken as a positive standard; c) Sequencing the positive monoclonal identified by phage ELISA for 2 times to determine sequence information, extracting the sequence to obtain a binding polypeptide protein sequence, and comparing and analyzing the sequence to obtain the distribution frequency of the positive sequence. The result is a plurality of DDR1 binding peptide sequences in which the DBP124 polypeptide is highly enriched.

2) Purified expression of binding polypeptide fusion proteins

The binding polypeptide gene sequence was cloned into pcoldEGFP vector while fusion expressing the hemagglutinin tag (hemagglutinin HA tag) for subsequent detection. The expression purification steps are as follows: a) To prevent inclusion body formation and protein degradation, induction was performed at 16 ℃ using IPTG at a concentration of 0.2 mM; b) Performing a large amount of induction expression according to the pre-experiment induction conditions, and performing bacteria breaking under the working condition of 1000W of a high-pressure bacteria breaker; c) Centrifuging 17000g at 4 ℃ for 30 minutes, taking supernatant and incubating with Ni filler at 4 ℃ for 1 hour; g) After Ni column purification, molecular sieve separation was performed, AKATA parameters were set at 0.5mL flow rate/min, and collected every 1 mL. The polypeptide EGFP fusion protein is obtained through the experiment, and can be used in the subsequent verification process.

3) ELISA assay for binding polypeptides

This experiment was used to verify whether expression of purified polypeptide EGFP fusion proteins in vitro could directly interact with in vitro purified antigen proteins. The method comprises the following steps: a) Diluting antigen protein into 5 mug/ml with PBS, plating 100 mug/well, coating the well plate, and standing at 4 ℃ overnight; b) Blocking with 3% BSA/PBS for 2h at room temperature, 200. Mu.l/well; c) Preparing polypeptide EGFP fusion proteins with different concentrations by 1% BSA/PBST, diluting in a double ratio, and incubating for 1h at room temperature; d) Incubation of secondary antibody anti-HAHRP (1: 3000 1h at room temperature; e) TMB color development; e) Stopping the reaction by using a stopping solution; g) The absorbance was measured at 450nm using a microplate reader and a binding curve was prepared based on the absorbance. The results show that DBP124-EGFP in the 4 DDR1 small fragment binding peptides obtained through the purification has the strongest binding capacity with DDR1 proteins from two species.

4) Surface plasmon resonance experiments (surface plasmon resonance, SPR)

This experiment was used to further verify antigen binding to the polypeptide, chemically synthesized DBP124 polypeptide was used for SPR binding analysis, and the equilibrium constants of the two were calculated. Purified antigen proteins were immobilized on a chip, different concentrations of DBP124 binding peptide were added sequentially to analyze affinity with the antigen proteins, the reaction signal was recorded for 360 seconds, kinetic curves were made, and each relevant parameter was calculated. The detection of the binding of DBP124 polypeptide to DDR1 human and murine DDR1 proteins further shows that the obtained DBP124 polypeptide has good binding capacity to DDR1.

Example 2

Western blot experiments to detect the expression level of the target protein of the cell line.

The method comprises the following steps: a) Protein extraction: extracting protein from cells, lysing the cells with SDS-Tris, centrifuging 12000g for 3 min, taking supernatant for quantification, adding Loading buffer for 5 min at 100 ℃, and preserving at-40 ℃; b) SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and proteins are separated according to molecular weight; c) And (3) film transfer: transferring the separated proteins by electrophoresis onto a polymer membrane, the polymer membrane used being a polyacrylamide gel membrane; d) Blocking at room temperature for 1h with 5% BSA/TBST, preventing non-specific antibodies from binding to the polymer membrane; e) 3% BSA/TBST configuration anti-DDR1 mab rabit (1: 1000 After overnight incubation at 4 ℃ the membranes were washed 3 times with TBST for 5 minutes each; f) Secondary antibody 3% bsa/TBST anti-rabit HRP (1: 5000 After 1h at room temperature, the membrane was washed 3 times with TBST for 5 minutes each; g) ECL developer development. The results show that DDR1 is expressed in 5 brain glioma cells such as U87/U251 in different degrees, and the DDR1 expression level of the U251 cell line is highest.

Example 3

Flow cytometry is used to detect the expression level of a protein of interest on a cell membrane.

The method comprises the following steps: a) Washing twice with PBS; EDTA (ph=9) digestion for 2 minutes in an oven at 37 ℃; b) Adding a proper amount of PBS, centrifuging for 4 minutes at 300g, and centrifuging after PBS re-suspending and precipitating; c) Fixing 0.25% PFA for 5 min, centrifuging, suspending the precipitated PBS, and centrifuging again; d) Blocking for 1 hour with 3% BSA PBS; e) 1% BSA/PBS was conjugated with Anti-DDR1 mab rabit (1: 200 Incubation for 1 hour with primary anti-resuspension, centrifugation PBS for 3 times; f) 1% bsa/PBS with fluorescent secondary antibody Anti-rabit 488 (1: 1000 1 hour, and 3 times of centrifugal PBS; g) And (3) flow cytometry analysis. The cell line expression conditions are verified, which shows that DDR1 is expressed in U87/U251 brain glioma cells to different degrees.

Example 4

Immunofluorescence techniques are used to detect expression of proteins of interest in common cell lines.

The method comprises the following steps: a) Cells were washed 3 times with 1×pbs for 3 minutes each in culture plates; b) Fixing with 4% paraformaldehyde for 15 min, and washing with 1×PBS for 3 times each for 3 min; c) Dropwise adding 4% donkey serum/PBS, and sealing for 1h at room temperature; d) Blocking solution was washed off with PBS, and a sufficient amount of diluted Anti-DDR1 mab rabit (1: 1000 Primary antibody and placed in a wet box, incubated overnight at 4 ℃; e) PBST was washed 3 times for 3 minutes each with the addition of diluted fluorescent secondary antibody-rabit 488 (1: 5000 After 1h incubation, 3 washes with PBST for 3 minutes each; f) Counterstaining the core: dripping DAPI, incubating for 5 minutes in dark, and carrying out nuclear staining on the specimen, and washing off redundant DAPI by PBST for 5 minutes multiplied by 4 times; g) The acquired images were observed under a fluorescence microscope.

Screening of western blot experiments, flow cytometry and immunofluorescence experiments of cell membrane proteins showed that DDR1 was expressed in 5 glioma cells such as U87/U251, wherein the DDR1 expression level of the U251 cell line was the highest (FIG. 1A), flow cytometry showed that DDR1 was expressed in the cell membrane of glioma cell line (FIG. 1B), and immunofluorescence technique was used to verify the cell membrane expression of DDR1 on glioma (FIG. 2).

Example 5

DDR1 protein purity verification

The extra-membrane fragment of DDR1 is the primary target for the screening of the binding polypeptide phage of the invention. In order to ensure the accuracy and specificity of screening, the invention purchases commercial high-purity DDR1 protein, and subsequently the invention verifies the purity of the protein through coomassie brilliant blue staining, so that the protein can be used in the subsequent screening process, and the experimental result is shown in figure 3.

Example 6

Cell ELISA experiments to detect the affinity of polypeptides to a cell line expressing a protein of interest.

The method comprises the following steps: a) 3000 cells per well were plated in 96-well plates overnight; b) Polypeptide EGFP fusion protein PBST is diluted in a gradient mode according to set concentration, and incubated for 2 hours; c) Washing with PBST for 3 times each for 10 minutes; d) Saphire readings. The experiment uses EGFP as a control, and the comparison shows that the D124-EGFP has higher affinity to glioma cells, so as to verify the targeting capability of the D124 polypeptide to glioma cell lines.

Example 7

This example provides for the screening identification of DDR1 small fragment binding peptides.

The invention firstly carries out screening of phage dodecapeptide library on DDR1 fusion protein, after three rounds of screening, about 129 clones are selected from the library obtained in the third round, and then phage ELISA is carried out twice for preliminary verification, 50 positive clones are preliminarily identified (figure 4), sequencing is carried out, and the sequencing result shows that 50 clones are normal. The 50 positive phages obtained were then subjected to ELISA for human and murine DDR1 phages, and were found to have a better binding capacity as well (B, C in fig. 5). Binding polypeptide sequences can be obtained from sequencing results according to sequences before and after the small fragment binding peptide, and 26 different binding polypeptide sequences are obtained by sequencing after translating 50 sequences into amino acids and performing multi-sequence alignment. Phage ELISA experiments were performed by quantifying 7 phages with high ELISA absorbance ratios to the same titer, and found that DBP10, DBP105, DBP118, DBP124 bound strongly to DDR1 proteins from two species (D, E in FIG. 6), where D5 was the negative control.

Example 8

According to the invention, 4 binding polypeptides with better binding force and an irrelevant polypeptide (P35) in a phage library are selected as a reference, are fused with EGFP labels and then purified, and polypeptide EGFP fusion proteins with higher purity are obtained after purification (figure 7).

Example 9

This example provides affinity detection of DDR1 and polypeptide EGFP fusion proteins.

The binding of polypeptide EGFP fusion protein to DDR1 proteins of human and mouse sources was further detected by ELISA, and the result shows that DBP124-EGFP binding capacity of 4 DDR1 small fragment binding peptides obtained by the purification is strongest with DDR1 proteins of two species sources (FIG. 8A. B).

Example 10

This example provides Biacore affinity detection of DDR1 polypeptide EGFP.

In order to further determine the binding capacity of the DBP124 polypeptide determined in the ELISA results of the present invention, the affinity constant of the DBP124 polypeptide to DDR1 protein was further examined using Biacore, confirming that the DBP124 polypeptide binds to both DDR1 human and murine DDR1 proteins (FIG. 9), with KD of 2.02X10, respectively ^-8 M and 3.64×10 ^-9 M, further demonstrate that DBP124 polypeptide obtained by the invention has good binding capacity with DDR1.

Example 11

In order to detect the binding capacity of the DBP124 polypeptide and glioma cells, cell ELISA experiments are carried out by screening DDR1 expression cell lines U87 and U251, and the affinity of the DBP124 polypeptide and DDR1 protein is further detected, so that the DBP124 polypeptide has better binding capacity with the DDR1 expression cell lines (figures 10-11).

Example 12

DDR1 binding polypeptide DBP124 and pseudomonas aeruginosa exotoxin PE38 protein are fused and purified to obtain polypeptide PE38 fusion protein with higher purity (figure 12).

Example 13

In order to further determine the targeting delivery capability of the DBP124 polypeptide in the results, PE38 is used as a control to detect the targeting effect of DBP124-PE38 on DDR1 expression cell lines, CCK8 experiments are carried out to detect the delivery effect, and the experimental results show that the DBP124 polypeptide remarkably enhances the cytotoxicity of PE38 (figure 13) and has better targeting delivery capability on PE 38.

Example 14

In order to further explore the targeting killing capability of DBP124-PE38 in vivo, the invention carries out subcutaneous tumorigenesis of glioma cell line GL261 on a C57 mouse, and the targeting killing effect on tumors is detected by tail vein injection, and experimental results show that DBP124-PE38 remarkably prolongs the median survival time of the mouse (figure 14), and has better targeting treatment capability on subcutaneous tumors.

While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A polypeptide targeting discoid structural domain receptor 1, which is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

2. A nucleic acid encoding the polypeptide of claim 1, wherein the nucleotide sequence of the nucleic acid is set forth in SEQ ID No. 2.

3. A vector comprising the nucleic acid of claim 2.

4. A host cell or host bacterium comprising the vector of claim 3.

5. Use of the polypeptide of claim 1, or the nucleic acid of claim 2, for the preparation of a tumor targeted drug.

6. The polypeptide of claim 1, or the nucleic acid of claim 2, for visual localization of a tumor after labeling with a fluorescent dye.

7. The polypeptide of claim 1, or the nucleic acid of claim 2, for use in the construction of a targeted therapeutic for DDR1 high expression diseases.

8. A pharmaceutical composition comprising the polypeptide of claim 1 or the nucleic acid of claim 2.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers.

10. A detection reagent comprising the polypeptide of claim 1.