Neural cilia protein NRP-1 affinity peptide and application thereof

Abstract

The invention discloses a nerve cilia protein NRP-1 affinity peptide and application thereof. Particularly, the polypeptide with affinity to the NRP-1 is selected from a sequence as shown in SEQ ID NO: 1-5. In addition, the invention also provides a bioactive substance containing the polypeptide sequence. The polypeptide or the bioactive substance provided by the invention can be used as a single preparation or can be combined with other tumor treatment drugs so as to be used for realizing tumor treatment or detection through targeted combination with NRP-1. The affinity peptide is good in NRP-1 specificity, high in affinity and low in production, purification and storage cost, and a new thought is provided for tumor targeted therapy and anti-vascular therapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a polypeptide having affinity to a target on the surface of a tumor and an application thereof, and specifically relates to a polypeptide having affinity to neuropilin 1 (NRP-1) and an application thereof. Background Art

[0002] Neuropilins are a family of transmembrane glycoprotein receptors of approximately 130-140 kDa in size. They consist of an N-terminal extracellular domain, a transmembrane domain, and a cytoplasmic domain. The extracellular domain mediates the interaction with a variety of growth factors to promote the activation of its cognate receptor tyrosine kinase. They play a vital role in the formation of the nervous and vascular systems during embryonic development.

[0003] Tumors need a lot of nutrients and oxygen during their growth. This strong nutritional demand and extremely fast growth rate will cause the tumor site to continuously induce the formation of new blood vessel networks to provide the oxygen and nutrients needed for its growth. Existing studies have shown that NRP-1 protein is highly expressed on the surface of many types of tumors and can mediate vascular growth factors such as VEGF to promote tumor proliferation, growth and migration.

[0004] Therefore, NRP-1 protein is an excellent target for developing tumor-targeted and therapeutic drugs. Summary of the invention

[0005] The purpose of the present invention is to provide a polypeptide with high affinity to NRP-1 protein, in particular, the polypeptide of the present invention can specifically bind to the extracellular domain of neuropilin, which has the advantages of convenience, high efficiency, practicality, safety, etc. In addition, the present invention also provides the use of the polypeptide in drugs such as tumor diagnosis, targeting and / or treatment.

[0006] Specifically, the embodiments of the present invention can be described as follows: In one aspect, the present invention provides a polypeptide targeting neuropilin NRP-1, the polypeptide being selected from the sequences shown in SEQ ID NO: 1-5. Preferably, the polypeptide of the present invention is SEQ ID NO: 1 (HAFKHHGHRNPP). Preferably, the polypeptide of the present invention is SEQ ID NO: 2 (WHHHWPYLRTAN). Preferably, the polypeptide of the present invention is SEQ ID NO: 3 (HWPFRHHASHAD). Preferably, the polypeptide of the present invention is SEQ ID NO: 4 (TVDLSLSSTTSV). Preferably, the polypeptide of the present invention is SEQ ID NO: 5 (FGPWKHTHHLQR).

[0007] In yet another aspect, the present invention provides a polypeptide targeting neuropilin NRP-1, wherein the polypeptide is selected from the sequence shown in SEQ ID NO: 1.

[0008] In yet another aspect, the present invention provides an isolated polynucleotide encoding the polypeptide of the present invention.

[0009] In still another aspect, the present invention provides a recombinant vector comprising the isolated polynucleotide of the present invention.

[0010] In yet another aspect, the present invention provides a host cell comprising the recombinant vector of the present invention, or the isolated polynucleotide of the present invention is integrated into its genome.

[0011] In yet another aspect, the present invention provides a biologically active substance comprising the polypeptide of the present invention, selected from the group consisting of: an engineered phage, a fusion protein and a conjugate.

[0012] In another aspect, the present invention provides the use of the polypeptides, biologically active substances, isolated polynucleotides, recombinant vectors and / or host cells described in the present invention in the preparation or screening of biomedical materials and / or drugs for tumor treatment by targeting and binding to NRP-1.

[0013] In another aspect, the present invention provides the use of the polypeptide, biologically active substance, isolated polynucleotide, recombinant vector and / or host cell of the present invention in the preparation or screening of a kit or drug for tumor detection by targeting and binding to NRP-1.

[0014] In another aspect, the present invention provides an anti-tumor drug complex: the drug complex comprises the polypeptide and / or bioactive substance described in the present invention, as well as a preparation or drug for tumor treatment and a drug carrier.

[0015] The preparations or drugs for tumor treatment include biological preparations, chemical drugs, radiotherapy drugs, photothermal therapy drugs, photodynamic therapy drugs and cryotherapy drugs, in particular, proteins, peptides, nucleic acids, antibiotics, anti-inflammatory drugs, anti-tumor drugs, neuroprotective agents, chemotherapeutic agents, cytotoxins, radioisotopes, fluorescent markers, luminescent materials, color-developing substances or enzymes.

[0016] The drug carrier for tumor treatment includes nano drug carriers and micro drug carriers, such as any one of liposomes, inorganic particles, organic particles, polymer micelles, polymer vesicles, carbon materials, microorganisms, etc.

[0017] In yet another aspect, the present invention provides a detection / diagnosis reagent or a detection / diagnosis kit, characterized in that it comprises the polypeptide and / or biologically active substance described in the present invention.

[0018] In yet another aspect, the present invention provides a composite material for tumor detection and targeted therapy, characterized in that it comprises the polypeptide and / or biologically active substance described in the present invention, and other biomedical materials for tumor detection and targeted therapy.

[0019] The polypeptide provided by the present invention and the biologically active substance containing the polypeptide sequence can be used as a single preparation for anti-angiogenic treatment of tumors, or form a composite preparation with other tumor therapeutic drugs. At the same time, the polypeptide can also be combined with other materials to form a composite material for targeted treatment and detection of tumors. The polypeptide of the present invention has a strong affinity and good specificity for neuropilin (NRP-1), and provides a new technical solution for the targeted treatment of tumors and the research and development of anti-angiogenic therapeutic agents.

[0020] In particular, compared with the prior art, the present invention has the following outstanding advantages: (1) The polypeptide screened by the present invention can specifically bind to neuropilin 1 (NRP-1) but not to other proteins, and has specificity; (2) The present invention uses phage bio-panning technology to screen small molecule peptides that bind to neuropilin 1 (NRP-1), which is convenient, fast, simple and efficient, and lays a foundation for the development and clinical application of tumor drugs; (3) The neuropilin 1 affinity polypeptide screened by the present invention can be synthesized using a standardized chemical synthesis process. Compared with other types of biological preparations, its production, purification, and storage costs are greatly reduced, and the target drug can be obtained in large quantities in a short time; and (4) The polypeptide provided by the present invention has a specific binding effect on neuropilin 1, and has both therapeutic and tumor targeting effects, providing a new option for further research on tumor targets, new drug screening, vaccines and early diagnosis of new tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The phage input-output ratios of each round of screening from rounds 1 to 4 in Example 1 are shown.

[0022] Figure 2The statistical graph of the results of the phage enzyme-linked immunosorbent assay (ELISA) in Example 2 is shown, wherein WT: wild-type phage, HP: phage containing SEQ ID NO: 1, WN: phage containing SEQ ID NO: 2, HD: phage containing SEQ ID NO: 3, TV: phage containing SEQ ID NO: 4, FR: phage containing SEQ ID NO: 5. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with the embodiments and drawings. The following embodiments are preferred embodiments of the present invention and the present invention is not limited to the following embodiments. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

[0024] Embodiments of the present invention are as follows: Example 1: Screening of affinity peptides for NRP-1 protein using phage biopanning technology 1) NRP-1 protein (NCBI accession number NP_001019799.1) extracellular domain a. Dilute the NRP-1 protein with 0.1 M NaHCO3 to a final concentration of 100 ug / ml, and pipette 150 ul of the diluted NRP-1 protein and add it to a 24-well plate.

[0025] b. Place the 24-well plate in a humidified chamber and coat overnight at 4°C in a shaker.

[0026] 2) Bio-panning of NRP-1 protein affinity peptides using phage library a. Blocking: Take out the 24-well plate coated overnight, aspirate the liquid, add 2 ml of 5% BSA to block overnight, block at room temperature for 2 hours, then add 2 ml of PBST washing solution, wash 5 times, and remove the blocking solution.

[0027] b. Phage library binding: From Ph.D. TM -12 phage random peptide library (from New England Biolabs), aspirate 10ul of liquid and add it to 90ul PBST. Then, add the mixed solution to the blocked and washed well plate, place it on a shaker, and incubate it at room temperature for 1h to allow the phage library and protein to bind.

[0028] c. Washing: Wash the plate 10 times with TBST solution, 5 min each time, to remove phages not bound to the protein.

[0029] d. Elution: Add elution buffer (0.2 M glycine-HCl, pH 2.2) to the washed well plate and incubate on a shaker for 8 min to elute phages with high affinity for NRP-1 protein.

[0030] e. Neutralization: Add 1 M Tris-HCl, pH 9.1, to the eluate so that the final pH of the mixture is 7.4.

[0031] f. Phage library amplification: Add the eluted phage solution to 20 ml LB medium containing logarithmic phase ER2738 bacteria, let it stand for infection for 15 minutes, and culture it at 37 degrees Celsius with shaking for 4 hours.

[0032] g. Phage purification: After amplification, 20 ml of bacterial liquid-phage mixture was sterilized by centrifugation at 12000g. 4 ml of PEG / NaCl solution (20% PEG, 2.5 M NaCl) was added to the supernatant containing phages, and the mixture was allowed to settle on ice overnight. The precipitate obtained by centrifugation was resuspended in PBS to obtain the purified phage liquid.

[0033] h. Second to fourth round of screening: The phages obtained from the previous round of elution were amplified and purified, and used as the phage library for the next round of input, and co-incubated with the NRP-1 protein. The protein treatment and phage addition steps were as described above ag. The second to fourth round of screening steps were performed in sequence. During the screening, the amount of phage input in each round was ensured to be the same, the amount of phage output in each round was recorded, and the phage input amount / output amount in each round was calculated.

[0034] The results of phage input / output in each round are as follows Figure 1 As shown, when the initial phage input in each round is the same, the output of phage increases with the increase in screening rounds, indicating that the screening effectively enriches phages with affinity for NRP-1 protein.

[0035] i. Obtaining NRP-1 affinity polypeptide sequence by phage DNA sequencing: The eluted phage obtained in the third and fourth rounds of screening were diluted in gradients and added to the logarithmic phase Escherichia coli. The mixture was infected at room temperature for 5 minutes. 200ul of phage-bacteria mixture was added to the IPTG / Xgal solid LB plate, and evenly spread with a coating rod, and then inverted and placed in an incubator for culture. After plaques appear on the plate, monoclonal plaques are picked and added to 5ml LB culture medium for overnight amplification culture, and DNA sequencing of the bacterial liquid is performed. Through phage DNA sequencing, the affinity polypeptide sequence displayed on the phage surface can be clearly identified. The high-frequency polypeptide sequences obtained by the fourth round of phage DNA sequencing are shown in Table 1 below.

[0036] Table 1 Statistics of the frequency of affinity peptides obtained in the fourth round of phage library screening

[0037] As can be seen from the above table, in the fourth round of screening, the frequencies of occurrence of five affinity polypeptide sequences were greater than 3.

[0038] Example 2: Phage enzyme-linked immunosorbent assay (ELISA) test to verify the affinity of the peptide to the target protein NRP-1 1) Coating the target protein: Add 50 µL of 2 µg / mL protein diluted with 0.1 M NaHCO3 to a 96-well ELISA plate, place the plate in a wet box, and incubate overnight at 4°C.

[0039] 2) Block the coated wells: Aspirate the protein solution in the wells and add 200 μL of 5% BSA solution into the wells and incubate at 4°C for 1 h.

[0040] 3) Washing: Discard the blocking solution, invert the ELISA plate on filter paper and tap it to drain all the liquid, then add 200 µL of 0.5% PBST washing solution and incubate for 6 min; discard the liquid and tap it on filter paper again to drain all the liquid, and repeat the washing process 6 times.

[0041] 4) Phage incubation: Add 50 µL of 2×109 pfu / mL phage and incubate at room temperature for 1 h.

[0042] 5) Washing: Aspirate the phage solution and repeat the washing procedure (3) for 6 times with 0.5% PBST washing solution.

[0043] 6) Add primary antibody: Add 100 µL of phage pVIII coat protein antibody diluted in PBST to the well plate and incubate at 37°C for 1 h. Then aspirate the antibody and add 200 µL of 0.5% PBST and wash three times according to step (3); 7) Add secondary antibody: Add 100 µL of horseradish peroxidase (HRP)-conjugated secondary antibody diluted in PBST and incubate at 37°C for 1 h. Then aspirate the antibody and add 200 µL of 0.5% PBST and wash three times according to step (3); 8) Color development: Add 100 µL of TMB display substrate and incubate for 10-20 min.

[0044] 9) Stop the reaction: Add 100 µL of 2M H2SO4 (diluted with water) to terminate the reaction, and measure the absorption peak at 450 nm using an ELISA reader.

[0045] ELISA results Figure 2As shown, among the high-frequency peptides screened, the phage displaying the peptide HAFKHHGHRNPP (HP, SEQ ID NO: 1) has the strongest affinity for NRP-1 protein, which is higher than that of the phage displaying other high-frequency peptide sequences and also higher than that of the wild-type phage; in addition, compared with the wild-type phage, the phage displaying the peptides WHHHWPYLRTAN (WN, SEQID NO: 2), HWPFRHHASHAD (HD, SEQ ID NO: 3), TVDLSLSSTTSV (SEQ ID NO: 4) and FGPWKHTHHLQR (SEQ ID NO: 5) also showed higher affinity for NRP-1 protein, among which * p <0.05,** p <0.01,*** p <0.001,**** p <0.0001.

[0046] The gene and protein sequences involved in the present invention are as follows: SEQ ID NO: 1; Name: Amino acid sequence of NRP-1 affinity peptide Source: Artificial Sequence HAFKHHGHRNPP SEQ ID NO: 2; Name: Amino acid sequence of NRP-1 affinity peptide Source: Artificial Sequence WHHHWPYLRTAN SEQ ID NO: 3; Name: Amino acid sequence of NRP-1 affinity peptide Source: Artificial Sequence HWPFRHHASHAD SEQ ID NO: 4; Name: Amino acid sequence of NRP-1 affinity peptide Source: Artificial Sequence TVDLSLSSTTSV SEQ ID NO: 5; Name: Amino acid sequence of NRP-1 affinity peptide Source: Artificial Sequence FGPWKHTHHLQ

Claims

1. A polypeptide having affinity for neuropilin NRP-1, characterized in that: Selected from the sequences shown in SEQ ID NO: 1-5.

2. A polypeptide having affinity for neuropilin NRP-1, characterized in that: Selected from the sequence shown in SEQ ID NO:

1.

3. An isolated polynucleotide, characterized in that Encodes the polypeptide according to claim 1 or 2.

4. A recombinant vector, characterized in that: Comprising the isolated polynucleotide according to claim 3.

5. A cell, characterized in that The invention comprises the recombinant vector according to claim 4, or the isolated polynucleotide according to claim 3 is integrated into its genome.

6. A biologically active substance, characterized in that Comprising the polypeptide according to claim 1 or 2, selected from the following: (1) Engineered phages; (2) fusion proteins; and (3) Conjugate.

7. Use of the polypeptide according to claim 1 or 2, the isolated polynucleotide according to claim 3, the recombinant vector according to claim 4, the host cell according to claim 5 and / or the biologically active substance according to claim 6 in the preparation or screening of biomedical materials and / or drugs for tumor treatment by targeting and binding to NRP-1.

8. Use of the polypeptide according to claim 1 or 2, the isolated polynucleotide according to claim 3, the recombinant vector according to claim 4, the host cell according to claim 5 and / or the biologically active substance according to claim 6 in the preparation or screening of a kit or drug for tumor detection by targeting and binding to NRP-1.

9. An anti-tumor drug complex, characterized in that: The method comprises the polypeptide according to claim 1 or 2, the cell according to claim 5 and / or the biologically active substance according to claim 6, as well as a preparation or drug for tumor treatment and a drug carrier.

10. A detection reagent or a detection kit, characterized in that: Comprising the polypeptide according to claim 1 or 2, the cell according to claim 5 and / or the biologically active substance according to claim 6.

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