Binder for targeted combination with PCNA protein, fusion protein and application thereof

Abstract

The invention discloses a binder in targeted binding with PCNA protein, fusion protein and application of the binder and the fusion protein, and belongs to the technical field of biology. According to the present invention, the binder specifically targeting PCNA is designed by using the artificial intelligence technology, and the binder has characteristics of good specificity and high affinity, and can efficiently bind to the PCNA antigen; further, a binder with high affinity and good specificity is fused with an RBCC structural domain or a mutant thereof, or the binder is directly fused with an Fc fragment to obtain a fusion protein, and the fusion protein can utilize the binder structural domain specificity to bind a target protein and start a protein degradation pathway, so that targeted degradation of the PCNA protein is realized; and p53 in cells is activated to be phosphorylated so as to avoid ubiquitination degradation by ubiquitin ligase such as MDM2 and the like, so that cell apoptosis is promoted to achieve the efficiency of inhibiting cancer cell amplification, and great clinical application value is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a binder, a fusion protein that specifically binds to PCNA protein, and their applications. Background Art

[0002] The Proliferating Cell Nuclear Antigen (PCNA) gene encodes a nuclear protein that is mainly involved in DNA replication and repair processes. PCNA forms a trimeric ring structure, slides around the DNA strand, and serves as a loading factor for DNA polymerase to ensure the efficiency and accuracy of DNA replication. PCNA plays a key role in cell proliferation, and its expression level is closely related to the cell proliferation rate. PCNA acts as a sliding clamp during the initiation and elongation of DNA replication, and ensures the efficiency and accuracy of DNA replication by interacting with various replication and repair proteins. PCNA is also involved in multiple DNA repair pathways, including Base Excision Repair (BER), Nucleotide Excision Repair (NER), and Mismatch Repair (MMR). These repair mechanisms play a key role in maintaining genomic stability and preventing the accumulation of mutations.

[0003] PCNA is highly expressed in various cancers, and its level is positively correlated with the proliferation rate and malignancy of tumor cells. Due to the key role of PCNA in DNA replication and repair, its abnormal expression or dysfunction may lead to genomic instability, thereby promoting the development and progression of cancer. Therefore, PCNA has become an important target for cancer research and treatment.

[0004] Existing PCNA inhibitors mainly inhibit DNA replication and repair by blocking its interaction with DNA polymerase or other repair proteins. However, the research on PCNA degraders is relatively less. The existing PCNA inhibitors have limited effects and may be accompanied by a relatively high risk of side effects. Therefore, it is necessary to develop more efficient and specific PCNA-targeted degradation technologies.

[0005] A binder is a type of small molecule, peptide, or protein that has high specificity and high affinity and can bind to a specific target protein. Therefore, binders have shown broad application potential in biomedical research and treatment.

[0006] Targeted protein degradation (TPD) technology uses the ubiquitin proteasome system and lysosomal degradation system in cells to achieve specific and efficient degradation of disease-related proteins, thereby achieving the effect of disease treatment. Compared with traditional small molecule inhibitors, TPD has more advantages: event-driven, low dosage, only catalyst amount is needed to work; it can also target some undruggable proteins.

[0007] TRIM away is an emerging TPD technology that fuses binder with TRIM21, a member of the TRIM (Tripartite motif-containing) protein family. TRIM21 is an E3 ubiquitin ligase that can specifically recognize and bind to target proteins, promote their ubiquitination and ultimately degradation in the proteasome. TRIM-binder technology combines the high specificity of binder with the efficient degradation function of TRIM21, providing a more efficient and precise means of PCNA degradation, which is expected to improve the therapeutic effect of PCNA-related cancers.

[0008] However, there are no reports on the research of binder targeting PCNA. Summary of the invention

[0009] The purpose of the present invention is to provide a binder, fusion protein and application thereof for targeting and binding PCNA protein, so as to solve the problem that the prior art lacks a binder with strong specificity, high affinity and high efficiency in binding PCNA antigen.

[0010] In the first aspect, the present invention provides a binder that targets and binds to PCNA protein, and the binder is selected from any one of the following: A1) having an amino acid sequence as shown in any one of SEQ ID NOs: 1-6; A2) an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence defined in A1); A3) an amino acid sequence having more than 80% sequence identity compared to the amino acid sequence defined in A1) or A2); A4) an amino acid sequence obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in A1) or A2) or A3).

[0011] In the present invention, the inventors use artificial intelligence technology to design a binder that specifically targets and binds to PCNA. The binder has good specificity and high affinity and can efficiently bind to PCNA antigen.

[0012] In a second aspect, the present invention provides a fusion protein, which is obtained by connecting the above-mentioned binder with an RBCC domain or a mutant thereof through a third linker, or by fusing the above-mentioned binder with an Fc fragment; wherein, the amino acid sequence of the third linker is as shown in SEQ ID NO: 18, the amino acid sequence of the RBCC domain is as shown in SEQ ID NO: 16, and the amino acid sequence of the RBCC domain mutant is as shown in SEQ ID NO: 17.

[0013] In the present invention, the inventors obtained a fusion protein by fusing a binder with high affinity and good specificity with an RBCC domain or a mutant thereof (truncated TRIM family protein), or directly fusing the binder with an Fc fragment. The fusion protein can specifically bind to the target protein using its binder domain, initiate the protein degradation pathway, achieve targeted degradation of the PCNA protein, activate the intracellular p53 to phosphorylate it to avoid ubiquitination and degradation by ubiquitin ligases such as MDM2, and promote apoptosis to achieve the efficiency of inhibiting cancer cell proliferation, which has great clinical application value.

[0014] It can be understood that the linker peptide used in the fusion process can be selected from conventional linker peptides in the prior art according to actual usage needs, as long as it can enable better fusion of the two proteins.

[0015] In some embodiments, the fusion protein is selected from any one of the following: B1) having an amino acid sequence as shown in any one of SEQ ID NO: 22-31; B2) an amino acid sequence having one or several amino acid substitutions, deletions or additions compared with the amino acid sequence defined in B1); B3) an amino acid sequence having more than 80% sequence identity compared with the amino acid sequence defined in B1) or B2); B4) an amino acid sequence obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in B1) or B2) or B3).

[0016] The above-mentioned binder and the above-mentioned fusion protein provided by the present invention can be natural, recombinant or synthetic active polypeptides, and the active polypeptides can be natural purified products, chemically synthesized products, or products produced using recombinant techniques from prokaryotic hosts (such as Escherichia coli) or eukaryotic hosts (such as yeast, higher plants).

[0017] In the present invention, in A4) and B4), the connection can be directly connected through a peptide bond or through a linker, and the connection method is a conventional method in the art. Among them, the tags include but are not limited to: GST (glutathione S-transferase) tag protein, Trx (thioredoxin) tag protein, His tag protein (His-tag), Flag tag protein, LacZ tag protein, GFP (green fluorescent protein), sfGFP (superfolder green fluorescent protein), HA tag (hemagglutinin tag). Those skilled in the art can select appropriate tag proteins according to actual usage needs. The use of the tag does not change the function of the target protein (binder, fusion protein), and its purpose is to separate, purify, detect or trace. The tag can be separated from the target protein (binder, fusion protein) by chemical cleavage methods or enzymatic methods known in the art (such as introducing a protease cleavage site and using TEV protease to cleave and remove the tag).

[0018] In a third aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned binder or any one of the above-mentioned fusion proteins.

[0019] In some embodiments, the nucleic acid molecule is selected from any one of the following: C1) a nucleic acid molecule having a nucleotide sequence shown in any one of SEQ ID NO: 7-12; C2) a nucleic acid molecule that hybridizes with the nucleic acid molecule defined in C1) under stringent conditions and encodes the above-mentioned binder; C3) a nucleic acid molecule having a sequence identity of more than 90% with the nucleic acid molecule defined in C1) or C2) and encoding the above-mentioned binder.

[0020] The above-mentioned nucleic acid molecule provided by the present invention can be DNA, such as cDNA, genomic DNA or recombinant DNA; it can also be RNA, such as mRNA or hnRNA, etc.; and this nucleic acid molecule can generally be obtained by PCR amplification or artificial synthesis methods.

[0021] In a fourth aspect, the present invention provides a recombinant vector comprising any one of the above-mentioned nucleic acid molecules.

[0022] The above-mentioned recombinant vector provided by the present invention includes a cloning vector and an expression vector. The cloning vector is used to replicate related sequences, and the expression vector is used to express related genes. Among them, the vectors used when constructing the expression vector can be at least one of pET23a and pcDNA3.1 vectors.

[0023] In a fifth aspect, the present invention provides a recombinant cell comprising any one of the above-mentioned nucleic acid molecules or the above-mentioned recombinant vector.

[0024] In some embodiments, the preparation method of the above-mentioned recombinant cell includes the step of transforming the above-mentioned recombinant vector into an expression host cell.

[0025] In the present invention, the expression host cell is a conventional host cell in the art, as long as it can meet the requirements that the recombinant vector can stably replicate by itself and the genes carried by it can be effectively expressed. It can be a prokaryotic cell or a eukaryotic cell, such as Escherichia coli, yeast, etc. Escherichia coli can be, for example, E. coli BL21(DE3), Rosetta(DE3), BL21(DE3)plysS. In the present invention, the Escherichia coli expression host E. coli BL21(DE3) is preferably used.

[0026] In a sixth aspect, the present invention provides the use of the above-mentioned binder, any one of the above-mentioned fusion proteins, any one of the above-mentioned nucleic acid molecules, the above-mentioned recombinant vector or the above-mentioned recombinant cell in any one of the following: D1) Use in the preparation of a product for preventing and / or treating PCNA target-related diseases; D2) Use in the preparation of a product for screening, diagnosing or assisting in the diagnosis of PCNA target-related diseases; wherein, the PCNA target-related disease is a PCNA-positive tumor.

[0027] In the present invention, a PCNA-positive tumor refers to a tumor in which PCNA expression is detected in tumor cells. Such tumors can be, for example, common cancers such as lung cancer, gastric cancer, liver cancer, breast cancer, prostate cancer, colorectal cancer, esophageal cancer, etc.

[0028] In a seventh aspect, the present invention provides a pharmaceutical composition for preventing and / or treating PCNA target-related diseases. The pharmaceutical composition includes the above-mentioned binder, any one of the above-mentioned fusion proteins, any one of the above-mentioned nucleic acid molecules, the above-mentioned recombinant vector or the above-mentioned recombinant cell, and a pharmaceutically acceptable carrier.

[0029] In the present invention, the term "pharmaceutically acceptable carrier" refers to excipients widely used in the field of drug production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide methods to enable the active ingredient to dissolve at a desired rate after the subject receives the administration, or to promote the effective absorption of the active ingredient after the subject receives the administration of the composition. Pharmaceutical excipients can be inert fillers or provide certain functions, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition.

[0030] The pharmaceutical composition provided by the present invention can be prepared by any method known to those skilled in the art according to the disclosed content. For example, it includes, but is not limited to, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or freeze-drying processes.

[0031] In some embodiments, the dosage form of the pharmaceutical composition includes at least one of solid preparations, semi-solid preparations and liquid preparations.

[0032] The pharmaceutical composition provided by the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra - arterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be in a controlled - release or sustained - release dosage form (such as liposomes or microspheres).

[0033] In the eighth aspect, the present invention provides a method for preparing the above - mentioned binder and any of the above - mentioned fusion proteins, comprising the following steps: culturing the above - mentioned recombinant cells, and obtaining a culture after induced expression; separating the binder or the fusion protein from the culture.

[0034] In the present invention, there are no special requirements for the culture method, culture conditions and culture medium, as long as the normal growth of the recombinant cells is ensured. And the methods for separating the above - mentioned binder or fusion protein from the culture are all conventional methods in the art.

[0035] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention designs a binder specifically targeting PCNA by using artificial intelligence technology. This binder has good specificity and high affinity, and can efficiently bind to the PCNA antigen. Further, by fusing the binder with high affinity and good specificity with the RBCC domain or its mutant, or directly fusing the binder with the Fc fragment, a fusion protein is obtained. This fusion protein can specifically bind to the target protein using its binder domain, initiate the protein degradation pathway, achieve the targeted degradation of the PCNA protein, activate the intracellular p53 and phosphorylate it to avoid ubiquitination and degradation by ubiquitin ligases such as MDM2, and promote apoptosis to achieve the efficiency of inhibiting cancer cell proliferation, having great clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a schematic diagram of the element arrangement structure of the recombinant expression vector constructed in the present invention; Figure 2 It is a SDS - PAGE detection result diagram of the binder in Example 2 of the present invention. Among them, lane M is Marker, and lanes 1 - 6 are PB1 - sfGFP, PB2 - sfGFP, PB3 - sfGFP, PB4 - sfGFP, PB5 - sfGFP, PB6 - sfGFP respectively, and lanes 7 - 8 are PB25 - sfGFP, PB52 - sfGFP; Figure 3 It is a SDS - PAGE detection result diagram of the PCNA antigen in Example 2 of the present invention; Figures 4-5Respectively, it is the ELISA test result diagram of the affinity between binder and PCNA antigen in Example 3 of the present invention; Figure 6 Respectively, it is the BLI test result diagram of the affinity between binder and PCNA antigen in Example 4 of the present invention. Among them, Figure (a) and (b) are structural simulation diagrams, and Figure (c) is the affinity result diagram; Figure 7 It is the WB result diagram of the fusion proteins (RBCC-PB2, RBCC-PB25) targeting the degradation of PCNA protein in Example 5 of the present invention; Figure 8 It is the WB result diagram and phosphorylation level result diagram of the fusion protein (RS80E-PB2) targeting the degradation of PCNA protein in Example 5 of the present invention; Figure 9 It is the WB result diagram of the fusion proteins (PB127-Fc, PB127-GFP-Fc) targeting the degradation of PCNA protein in Example 5 of the present invention; Figure 10 It is the WB result diagram for verifying the pathway of the fusion protein targeting the degradation of PCNA protein in Example 5 of the present invention. Detailed implementation manners

[0037] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0038] For the experimental methods without specific conditions indicated in the embodiments, they are usually carried out according to the conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in "Molecular Cloning: A Laboratory Manual" written by M.R. Green, "Molecular Biology" written by Robert·F·Weaver, etc., or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the embodiments can be obtained from commercial channels without special instructions.

[0039] Example 1 Artificial intelligence design of binder First, using artificial intelligence technology in combination with tools such as ProteinMPNN, RFdiffusion, AlphaFold3, and HADDOCK, a series of binders targeting specific epitopes of PCNA were designed and named binder PB1-6, PB25, PB52, PB127, and PB127-GFP respectively.

[0040] Among them, exemplarily, the amino acid sequence of binder PB2 is shown as follows: MKKYVIEEELSDEELLEKFKEIVELMKEEPVDITVTGAHALEIFRAVTELAKLLGVKVNIDVTLTESSAKYEPLEKKMLEYVRETVEAEKKKEELA (SEQ ID NO:1); The amino acid sequence of binder PB5 is shown as follows: KSVVYELTYEDKSGNKVKVTVTYDEETGEITLTQERTSKRVVNGKLVEVKETFLWRLSSSGTAEEALATIKKAYQRGAKTPVKVSFVKEIS (SEQ ID NO:2); The amino acid sequence of binder PB6 is shown as follows: MMKNVDVDIKYDKDGTIKVTIKSEDTTIEIEEKVKTKEEREAVLDIVFEVMRRLLKGEDIEEIKKKMEEELKKIK (SEQ ID NO:3); The amino acid sequence of binder PB25 is shown as follows: MKKYVIEEELSDEELLEKFKEIVELMKEEPVDITVTGAHALEIFRAVTELAKLLGVKVNIDVTLTESSAKYEPLEKKMLEYVRETVEAEKKKEELAGGGGSGGGGSGGGGSKSVVYELTYEDKSGNKVKVTVTYDEETGEITLTQERTSKRVVNGKLVEVKETFLWRLSSSGTAEEALATIKKAYQRGAKTPVKVSFVKEIS (SEQ ID NO:4); The amino acid sequence of binder PB52 is shown as follows: KSVVYELTYEDKSGNKVKVTVTYDEETGEITLTQERTSKRVVNGKLVEVKETFLWRLSSSGTAEEALATIKKAYQRGAKTPVKVSFVKEISGGGGSGGGGSGGGGSMKKYVIEEELSDEELLEKFKEIVELMKEEPVDITVTGAHALEIFRAVTELAKLLGVKVNIDVTLTESSAKYEPLEKKMLEYVRETVEAEKKKEELA (SEQ ID NO:5); The amino acid sequence of binder PB127 is shown as follows: SEKEEELKKLIEEAIKLEKEGKKEEAKKKLEEALELAKELGYDATAEAVQQKLDKL (SEQ ID NO:6).

[0041] The nucleotide sequences of the genes encoding binders PB2, PB5, PB6, PB25, PB52, and PB127 are shown in SEQ ID NO:7-12, respectively.

[0042] Example 2 Expression and Purification of Binder and PCNA Antigen To facilitate the separation and purification of the binder, in the present invention, the binder in Example 1 was displayed on the surface of Escherichia coli through superfolder green fluorescent protein (sfGFP, the nucleotide sequence of the gene encoding the sfGFP protein is shown in SEQ ID NO:13). Specifically, based on the pET23a vector, the genes encoding the above-mentioned binders PB2, PB5, PB6, and PB127 were ligated and fused with the sfGFP gene through a first linker (its nucleotide sequence is shown in SEQ ID NO:14), and the genes encoding the above-mentioned binders PB25 and PB52 were ligated and fused with the sfGFP gene through a second linker (its nucleotide sequence is shown in SEQ ID NO:15), and an expression vector was constructed by homologous recombination. The element arrangement structure of this expression vector is as Figure 1 shown. Exemplarily, the recombinant plasmids are pET23a-PB2-sfGFP, pET23a-PB5-sfGFP, pET23a-PB6-sfGFP, pET23a-PB25-sfGFP, pET23a-PB52-sfGFP, and pET23a-PB127-sfGFP.

[0043] The constructed recombinant plasmids were respectively transformed into Escherichia coli competent cell strain BL21(DE3), and cultured statically overnight at 37°C to obtain a series of recombinant strains. Then, single colonies were respectively picked and inoculated into 100 mL of LB liquid medium (the concentration of ampicillin was 50 μg / mL), and cultured with shaking at 37°C. When the OD 600 was about 0.6, IPTG with a final concentration of 0.5 mM was added, and the culture was induced with shaking at 18°C for 18 hours. After the culture was completed, the cells and the culture supernatant were respectively collected by centrifugation at 12,000 rpm and 4°C. For the cells, they were washed with TEN buffer (50 mM Tris-HCl, 5 mM EDTA, 50 mM NaCl, pH 8.0) at 4°C for more than 4 hours, and the proteins secreted and expressed by the strains were directly obtained from the TEN buffer. SDS-PAGE was used to detect and analyze the secretion and expression of the binder, and the results were as Figure 2 shown.

[0044] As can be seen from Figure 2 , the binders PB1-6, PB25, and PB52 were all successfully expressed.

[0045] Exemplarily, the purified binders PB1-6, PB25, PB52, and PB127-GFP obtained by washing the outer membrane were preserved for subsequent experiments.

[0046] Furthermore, the PCNA antigen sequence (amino acids 1-261 in NCBI accession number: P12004) was cloned into the pET23a vector by homologous recombination to obtain the recombinant plasmid pET23a-PCNA. The constructed recombinant plasmids were respectively transformed into Escherichia coli competent cell strain BL21(DE3), single colonies were picked and inoculated into 100 mL of LB liquid medium (the concentration of ampicillin was 50 μg / mL), and cultured statically overnight at 37°C. When the OD 600 was about 0.6, IPTG with a final concentration of 0.5 mM was added, and the culture was induced with shaking at 18°C for 18 hours. The cells were collected by centrifugation at 6,000 rpm for 10 min, washed with PBS, and then the cell pellet was resuspended in PBS, and PMSF with a final concentration of 1 mM was added, followed by high-pressure crushing. The supernatant was collected by centrifugation at 18,000 rpm for 30 min, and after filtration, Ni-NTA purification was carried out. The purified protein was collected and detected by SDS-PAGE. The protein was divided into small portions, snap-frozen in liquid nitrogen, and stored at -80°C. The identification and analysis results of SDS-PAGE were as Figure 3 shown. Calculated by http: / / www.expasy.org / , the expected size of PCNA was 29.8 kDa, and the results indicated that the PCNA antigen was successfully expressed.

[0047] ELISA Experiment of Binder in Example 3 Exemplarily, this example is used to verify whether the purified binders PB1-6 and PB127-GFP in Example 2 can directly interact with the purified PCNA antigen in Example 2. Specifically, the steps are as follows: a) Dilute the PCNA antigen with 1×ELSIA coating buffer to 1 μg / mL, and plate 100 μL per well to coat the well plate, then leave it overnight at 4°C; b) Wash the plate with PBST, and block it with 1% BSA at room temperature for 2 h, 100 μL per well; c) Prepare binders PB1-6 and PB127-GFP with different concentrations using 1% BSA, 100 μL per well, and incubate at room temperature for 1 h; d) Incubate with HRP-conjugated Mouse anti HA-Tag mAb secondary antibody at room temperature for 1 h; e) Develop color with TMB and terminate the reaction with the termination solution; f) Measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay reader.

[0048] The test results are shown in Figure 4 and 5 respectively.

[0049] It can be seen from Figure 4 and 5 that binders PB2, PB5, PB6, and PB127-GFP have good affinity with the PCNA antigen, and the affinity of PB127-GFP with PCNA is about 20 times higher than that of PB127.

[0050] Biolayer Interferometry (BLI) Experiment of Binder in Example 4 Exemplarily, this example is used to further verify the binding of the purified binders PB2, PB5, PB25, and PB52 in Example 2 to the purified PCNA antigen in Example 2, and calculate the affinity between the two.

[0051] Specifically, the PCNA antigen is immobilized on the chip, and different concentration gradients of binders PB2, PB5, PB25, and PB52 are sequentially added to analyze the affinity with the antigen protein. Record the increase in the optical thickness at the tip of the biosensor within 600 seconds, and calculate the affinity with PCNA according to the binding and dissociation rates of different concentrations of the binder. The results are shown in Figure 6 respectively.

[0052] It can be seen from Figure 6It can be seen that binders PB2, PB5, PB25, and PB52 have good affinity with the PCNA antigen, and binder PB25 has better affinity with the PCNA antigen.

[0053] Example 5 Experiment on Targeted Degradation of PCNA Protein by Fusion Protein In order to achieve the targeted degradation of PCNA protein, in the present invention, the above-mentioned binders PB2, PB5, PB6, PB25, PB52, and PB127 are respectively fused with the RBCC domain or its mutant RS80E to obtain fusion proteins, and the fusion proteins are transformed into HEK 293T cells to detect the targeted degradation efficiency of the fusion proteins on PCNA protein.

[0054] Specifically, based on pcDNA3.1 as the basic vector, the genes encoding the above-mentioned binders PB2, PB5, PB6, PB25, PB52, and PB127 are ligated and fused with the gene encoding the RBCC domain (its amino acid sequence is shown in SEQ ID NO:16, and the nucleotide sequence is shown in SEQ ID NO:19) or its mutant RS80E (its amino acid sequence is shown in SEQ ID NO:17, and the nucleotide sequence is shown in SEQ ID NO:20)) through a third linker (its amino acid sequence is shown in SEQ ID NO:18, and the nucleotide sequence is shown in SEQ ID NO:21), and an expression vector is constructed by homologous recombination. The element arrangement structure of the expression vector is as Figure 1 shown. The recombinant plasmids are respectively pcDNA3.1-RBCC-PB2, pcDNA3.1-RBCC-PB5, pcDNA3.1-RBCC-PB6, pcDNA3.1-RBCC-PB25, pcDNA3.1-RBCC-PB52, pcDNA3.1-RBCC-PB127, pcDNA3.1-RS80E-PB2, pcDNA3.1-RS80E-PB25, pcDNA3.1-PB127-Fc, and pcDNA3.1-PB127-GFP-Fc.

[0055] Among them, the amino acid sequences of the fusion proteins RBCC-PB2, RBCC-PB5, RBCC-PB6, RBCC-PB25, RBCC-PB52, RBCC-PB127, RS80E-PB2, RS80E-PB25, PB127-Fc, and PB127-GFP-Fc are shown in SEQ ID NO:22-31 respectively.

[0056] The nucleotide sequences of the genes encoding the fusion proteins PB127-Fc and PB127-GFP-Fc are shown in SEQ ID NO: 32-33 respectively.

[0057] First, exemplarily, transfection was carried out using the PEI reagent. The recombinant plasmids pcDNA3.1-RBCC-PB2, pcDNA3.1-RBCC-PB25 and the GFP plasmid were respectively electrotransfected into well-conditioned HEK 293T cells. After electrotransfection, the electrotransfection efficiency and protein expression were observed through the GFP plasmid.

[0058] Cells electrotransfected for 48 h were selected and then subjected to Western blot detection respectively. According to the Western blot results, the efficiency of protein targeted degradation was calculated 48 h after transfection. The results are as Figure 7 shown.

[0059] From Figure 7 it can be seen that the fusion protein RBCC-PB2 has the most obvious degradation effect, and the efficiency of protein targeted degradation is about 70% at 48 h.

[0060] According to the above method, exemplarily, the above recombinant plasmids pcDNA3.1-RS80E-PB2 and the GFP plasmid were respectively electrotransfected into well-conditioned HEK 293T cells. After electrotransfection, the electrotransfection efficiency and protein expression were observed through the GFP plasmid.

[0061] Cells electrotransfected for 48 h were selected and then subjected to Western blot detection respectively. According to the Western blot results, the efficiency of protein targeted degradation was calculated 48 h after transfection. The results are as Figure 8 shown.

[0062] From Figure 8 it can be seen that the fusion protein RS80E-PB2 also has an obvious degradation effect, and the efficiency of protein targeted degradation is about 80% at 48 h. Moreover, compared with the control group, the phosphorylated p53 level is significantly increased after the experimental group RS80E-PB2 targets and degrades PCNA.

[0063] Similarly, according to the above method, exemplarily, the above recombinant plasmids pcDNA3.1-PB127-Fc, pcDNA3.1-PB127-GFP-Fc, Trim21 and the GFP plasmid were respectively electrotransfected into well-conditioned HEK 293T cells. After electrotransfection, the electrotransfection efficiency and protein expression were observed through the GFP plasmid.

[0064] Cells transfected with electric transfection for 48 h were selected and then subjected to Western blot detection respectively. According to the Western blot results, the efficiency of protein targeted degradation after 48 h of transfection was calculated. The results are as Figure 9 shown.

[0065] As can be seen from Figure 9 , after transfection with two different plasmids, obvious protein targeted degradation effects were observed.

[0066] Finally, the degradation pathway of the fusion protein targeting and degrading PCNA protein was explored.

[0067] Specifically, the MG132 proteasome inhibitor with a final concentration of 15 μM or the BafA1 autophagy inhibitor with a final concentration of 200 nM was selected for use. Among them, MG132 can inhibit the ubiquitin proteasome pathway, and BafA1 can inhibit the lysosomal pathway; 3 copies of the pcDNA3.1-RS80E-PB2 recombinant plasmid and the GFP plasmid were transfected into HEK 293T cells in good condition. After 24 h of transfection, the corresponding MG132 and BafA1 were added to 2 portions of the cells carrying the pcDNA3.1-RS80E-PB2 recombinant plasmid. After continuous culture, Western blot detection was performed. The results are as Figure 10 shown.

[0068] As can be seen from Figure 10 , the fusion protein in the present invention targets and degrades PCNA protein through the ubiquitin-proteasome pathway.

[0069] In summary, the present invention designs a binder specifically targeting PCNA by using artificial intelligence technology. This binder has good specificity, high affinity, and can efficiently bind to the PCNA antigen; further, by fusing the binder with high affinity and good specificity with the RBCC domain or its mutant, or directly fusing the binder with the Fc fragment, a fusion protein is obtained. This fusion protein can specifically bind to the target protein using its binder domain, initiate the protein degradation pathway, and achieve the targeted degradation of PCNA protein.

[0070] The above embodiments only represent the implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. A binder targeting PCNA protein, characterized in that: The binder is selected from any one of the following: A1) has an amino acid sequence as shown in any one of SEQ ID NOs: 1-6; A2) an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence defined in A1); A3) an amino acid sequence having a sequence identity of more than 80% with the amino acid sequence defined in A1) or A2); A4) An amino acid sequence obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in A1) or A2) or A3).

2. A fusion protein, characterized in that: The fusion protein is obtained by connecting the binder according to claim 1 with the RBCC domain or a mutant thereof through a third linker, or by fusing the binder according to claim 1 with an Fc fragment; Among them, the amino acid sequence of the third linker is shown in SEQ ID NO:18, the amino acid sequence of the RBCC domain is shown in SEQ ID NO:16, and the amino acid sequence of the RBCC domain mutant is shown in SEQ ID NO:

17.

3. The fusion protein according to claim 2, characterized in that The fusion protein is selected from any one of the following: B1) has an amino acid sequence as shown in any one of SEQ ID NOs: 22-31; B2) an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence defined in B1); B3) an amino acid sequence having a sequence identity of more than 80% with the amino acid sequence defined in B1) or B2); B4) An amino acid sequence obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in B1) or B2) or B3).

4. A nucleic acid molecule encoding the binder according to claim 1 or the fusion protein according to any one of claims 2 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that The nucleic acid molecule is selected from any one of the following: C1) a nucleic acid molecule having a nucleotide sequence as shown in any one of SEQ ID NOs: 7-12; C2) a nucleic acid molecule that hybridizes with the nucleic acid molecule defined in C1) and encodes the binder according to claim 1; C3) A nucleic acid molecule having a sequence identity of 90% or more with the nucleic acid molecule defined in C1) or C2) and encoding the binder according to claim 1.

6. A recombinant vector, characterized in that: Comprising the nucleic acid molecule according to any one of claims 4-5.

7. A recombinant cell, characterized in that Comprising the nucleic acid molecule according to any one of claims 4-5 or the recombinant vector according to claim 6.

8. Use of the binder according to claim 1, the fusion protein according to any one of claims 2-3, the nucleic acid molecule according to any one of claims 4-5, the recombinant vector according to claim 6 or the recombinant cell according to claim 7 in any of the following: D1) Application in the preparation of products for preventing and / or treating PCNA target related diseases; D2) Application in the preparation of products for screening, diagnosis or auxiliary diagnosis of PCNA target related diseases; in, The PCNA target-related disease is a PCNA-positive tumor.

9. A pharmaceutical composition for preventing and / or treating PCNA target site related diseases, characterized in that: The pharmaceutical composition comprises the binder according to claim 1, the fusion protein according to any one of claims 2-3, the nucleic acid molecule according to any one of claims 4-5, the recombinant vector according to claim 6 or the recombinant cell according to claim 7, and a pharmaceutically acceptable carrier.

10. A method for preparing the binder according to claim 1 and the fusion protein according to any one of claims 2 to 3, characterized in that: The steps include: Cultivating the recombinant cell according to claim 7, and obtaining a culture after inducing expression; The binder or the fusion protein is isolated from the culture.

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