Nano antibody for resisting PCNA protein, fusion protein and application of nano antibody and fusion protein

Abstract

The invention discloses an anti-PCNA protein nano antibody, a fusion protein and application thereof, and belongs to the technical field of biology. According to the invention, the nano antibody specifically targeting PCNA is designed by using an artificial intelligence technology, and the nano antibody has good specificity and high affinity, and can be efficiently combined with a PCNA antigen; furthermore, rational design of the nano-antibody is carried out through artificial intelligence, so that a nano-antibody mutant with higher affinity is obtained; furthermore, a nano antibody or a nano antibody mutant with high affinity and good specificity is fused with an RBCC structural domain or a mutant thereof to obtain a fusion protein, and the fusion protein can utilize the specificity of the nano antibody structural domain to bind a target protein and start a protein degradation pathway, so that targeted degradation of the PCNA protein is realized; and p53 in the cells is activated to be phosphorylated so as to avoid ubiquitination degradation by ubiquitin ligase such as MDM2 and the like, so that the method has a very great clinical application value.

Description

Technical Field

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

[0002] 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, ensuring 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 a variety of 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 a variety of 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, thus 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] Nanobody is a single-domain antibody derived from the heavy-chain antibody of camelids. It has the specificity and affinity of traditional antibodies, and at the same time has a smaller molecular weight and higher stability. Nanobodies can penetrate tissues and cells and are more likely to bind to target proteins, so they have broad application prospects 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] TRIMbody is an emerging TPD technology that fuses nanobodies 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. TRIMbody technology combines the high specificity of nanobodies 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 nano-antibodies targeting PCNA. Summary of the invention

[0009] The object of the present invention is to provide a nano antibody, a fusion protein and an application thereof against PCNA protein. In the present invention, a nano antibody targeting PCNA is designed by using artificial intelligence technology, and the nano antibody has good specificity and affinity. The nano antibody is further fused with the RBCC domain or its mutant to obtain a fusion protein, which can efficiently and accurately degrade PCNA, and therefore has good application prospects in the prevention and / or treatment of diseases related to the PCNA target.

[0010] In the first aspect, the present invention provides a nanobody against PCNA protein, comprising a complementary determining region CDR1, a complementary determining region CDR2 and a complementary determining region CDR3; wherein the complementary determining region CDR1 has an amino acid sequence as shown in SEQ ID NO: 1, the complementary determining region CDR2 has an amino acid sequence as shown in SEQ ID NO: 2, and the complementary determining region CDR3 has an amino acid sequence as shown in any one of SEQ ID NOs: 3-6.

[0011] In the present invention, the term "nanobody" is also known as single domain antibody (sdAb), which refers to an antibody composed of only one V region of the H chain, and can also be called VHH antibody. The ability of the nanobody to bind to the antigen and its stability are basically the same as those of the complete antibody. The nanobody is sequentially composed of framework region FR1, complementary determining region CDR1, framework region FR2, complementary determining region CDR2, framework region FR3, complementary determining region CDR3 and framework region FR4. The nanobody can be truncated at the N-terminus or C-terminus so that it only contains a part of FR1 and / or FR4, or lacks one or two of the framework regions, as long as it substantially maintains antigen binding and specificity. Among them, the complementary determining region is mainly responsible for binding to the antigen.

[0012] In some embodiments, the nanobody further includes framework region FR1, framework region FR2, framework region FR3 and framework region FR4; wherein, the framework region FR1 has the amino acid sequence shown in SEQ ID NO:7, the framework region FR2 has the amino acid sequence shown in SEQ ID NO:8, the framework region FR3 has the amino acid sequence shown in SEQ ID NO:9, and the framework region FR4 has the amino acid sequence shown in SEQ ID NO:10.

[0013] It can be understood that the framework region has a high degree of conservation. Therefore, a conventional framework region in the prior art can be selected according to actual use needs as long as it can improve the stability of the nanobody.

[0014] In some embodiments, the nanobody is selected from any one of the following: A1) having the amino acid sequence shown in any one of SEQ ID NO:11-14; A2) an amino acid sequence having one or several amino acid substitutions, deletions or additions compared with the amino acid sequence defined in A1); A3) an amino acid sequence having more than 80% sequence identity compared 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).

[0015] In the present invention, the inventor designed a nanobody specifically targeting PCNA by using artificial intelligence technology. This nanobody has good specificity, high affinity, and can efficiently bind to the PCNA antigen.

[0016] In a second aspect, the present invention provides a nanobody mutant against PCNA protein, and the nanobody mutant is selected from any one of the following: B1) having an amino acid sequence shown in any one of SEQ ID NOs: 15-17; B2) an amino acid sequence having one or several amino acid substitutions, deletions or additions as compared with the amino acid sequence defined in B1); B3) an amino acid sequence having a sequence identity of more than 80% as compared with the amino acid sequence defined in B1) or B2); B4) an amino acid sequence obtained by linking 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); wherein, in B1), the amino acid sequence shown in SEQ ID NO: 15 is obtained by mutating the amino acid sequence shown in SEQ ID NO: 11 at the following site: L102W, the amino acid sequence shown in SEQ ID NO: 16 is obtained by mutating the amino acid sequence shown in SEQ ID NO: 11 at the following site: S101M, and the amino acid sequence shown in SEQ ID NO: 17 is obtained by mutating the amino acid sequence shown in SEQ ID NO: 14 at the following at least one site: H113K.

[0017] In the present invention, the inventors further rationally designed nanobodies through artificial intelligence to obtain nanobody mutants with higher affinity.

[0018] In some embodiments, in the above-mentioned nanobody mutants, in addition to the above-mentioned mutations occurring at one site among the 101st, 102nd, and 113th positions, they may further have conservative substitutions of amino acids at other sites, so that the mutated amino acids, such as the nanobodies shown in SEQ ID NO: 11 or 14, have higher affinity. Preferably, the conservative substitutions of amino acids retain the higher affinity of the nanobodies of the present invention. It is obvious to those skilled in the art that such substitutions can occur in regions other than the above-mentioned sites while still retaining the corresponding affinity. Preferably, the conservative substitution variants have conservative substitutions of amino acids at at least one position. Examples of conservative substitutions are substitutions occurring within the following groups of amino acids: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, and valine), aromatic amino acids (such as phenylalanine, tryptophan, and tyrosine), and small amino acids (such as glycine, alanine, serine, threonine, and methionine).

[0019] In a third aspect, the present invention provides a fusion protein, which is obtained by linking any one of the above-mentioned nanobodies or the nanobody mutant with an RBCC domain or its mutant through a second linker; wherein, the amino acid sequence of the second linker is as shown in SEQ ID NO:29, the amino acid sequence of the RBCC domain is as shown in SEQ ID NO:27, and the amino acid sequence of the RBCC domain mutant is as shown in SEQ ID NO:28.

[0020] In the present invention, the inventors fused a nanobody or a nanobody mutant with high affinity and good specificity with an RBCC domain or its mutant (truncated TRIM family protein) to obtain a fusion protein. This fusion protein can specifically bind to the target protein using its nanobody domain, initiate the protein degradation pathway, achieve targeted degradation of the PCNA protein, and activate p53 in the cell to phosphorylate it to avoid ubiquitination and degradation by ubiquitin ligases such as MDM2, thereby promoting apoptosis to achieve the efficiency of inhibiting cancer cell proliferation, and has great clinical application value.

[0021] It can be understood that the linker peptide 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.

[0022] In some embodiments, the fusion protein is selected from any one of the following: C1) having an amino acid sequence as shown in any one of SEQ ID NOs: 33-41; C2) an amino acid sequence having one or several amino acid substitutions, deletions or additions compared with the amino acid sequence defined in C1); C3) an amino acid sequence having a sequence identity of more than 80% compared with the amino acid sequence defined in C1) or C2); C4) an amino acid sequence obtained by linking a tag or a signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in C1) or C2) or C3).

[0023] The above-mentioned nanobody, the above-mentioned nanobody mutant, 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).

[0024] In the present invention, in A4), B4) or C4), the connection can be directly connected by 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 (nanobody, nanobody mutant or fusion protein), and its purpose is to isolate, purify, detect or trace. The tag can be separated from the target protein (nanobody, nanobody mutant or 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).

[0025] In a fourth aspect, the present invention provides a biological material, which is selected from any one of the following: D1) a nucleic acid molecule encoding any one of the above nanobodies, the above nanobody mutants or any one of the above fusion proteins; D2) a recombinant vector containing the nucleic acid molecule of D1); D3) a recombinant cell containing the nucleic acid molecule of D1) or the recombinant vector of D2).

[0026] In some embodiments, the nucleic acid molecule defined in D1) is selected from any one of the following: E1) a nucleic acid molecule having a nucleotide sequence shown in any one of SEQ ID NOs: 18-24; E2) a nucleic acid molecule that hybridizes with the nucleic acid molecule defined in E1) under stringent conditions and encodes any one of the above nanobodies, the above nanobody mutants or any one of the above fusion proteins; E3) a nucleic acid molecule having a sequence identity of more than 90% with the nucleic acid molecule defined in E1) or E2) and encoding any one of the above nanobodies, the above nanobody mutants or any one of the above fusion proteins.

[0027] The above 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.

[0028] The above recombinant vector provided by the present invention includes a cloning vector and an expression vector. The cloning vector is used to replicate relevant sequences, and the expression vector is used to express relevant genes. Among them, the vectors used when constructing the expression vector can be at least one of pET23a and pcDNA3.1 vectors.

[0029] In some embodiments, the preparation method of the above recombinant cell includes the step of transforming the above recombinant vector into an expression host cell.

[0030] 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 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.

[0031] In a fifth aspect, the present invention provides the use of any one of the above-mentioned nanobodies, the above-mentioned nanobody mutants, any one of the above-mentioned fusion proteins or the above-mentioned biomaterials in any one of the following: F1) Use in the preparation of a product for preventing and / or treating PCNA target-related diseases; F2) 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.

[0032] 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.

[0033] In a sixth aspect, the present invention provides a pharmaceutical composition for preventing and / or treating PCNA target-related diseases, the pharmaceutical composition comprising any one of the above-mentioned nanobodies, the above-mentioned nanobody mutants, any one of the above-mentioned fusion proteins or the above-mentioned biomaterials, and a pharmaceutically acceptable carrier.

[0034] 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 the 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.

[0035] 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.

[0036] In some embodiments, the dosage form of the pharmaceutical composition includes at least one of solid preparations, semi-solid preparations, and liquid preparations.

[0037] 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).

[0038] In the seventh aspect, the present invention provides a method for preparing any one of the above-mentioned nanobodies, the above-mentioned nanobody mutants or any one 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 nanobody, the nanobody mutant or the fusion protein from the culture.

[0039] 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 any one of the above-mentioned nanobodies, the above-mentioned nanobody mutants or any one of the above-mentioned fusion proteins from the culture are all conventional methods in the art.

[0040] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention designs a nanobody specifically targeting PCNA by using artificial intelligence technology. This nanobody has good specificity and high affinity, and can efficiently bind to the PCNA antigen; further, through the rational design of the nanobody by artificial intelligence, a nanobody mutant with higher affinity is obtained; furthermore, by fusing a nanobody or a nanobody mutant with high affinity and good specificity with the RBCC domain or its mutant, a fusion protein is obtained. This fusion protein can specifically bind to the target protein by using its nanobody domain, initiate the protein degradation pathway, achieve the targeted degradation of the PCNA protein, and 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] 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 nanobody (a) and the PCNA antigen (b) in Example 2 of the present invention. In figure (a), lane M is: Marker, and lanes 1-9 are: PN1-9 respectively. In figure (b), lane M is: Marker, and lanes 1-2 are both PCNA; Figure 3 It is an Elisa test result diagram of the affinity between the nanobody and the PCNA antigen in Example 3 of the present invention; Figure 4These are the BLI test result graphs of the affinities of the nanobodies PN2, PN6, PN7, and PN8 with the PCNA antigen in Example 4 of the present invention; Figure 5 These are the graphs of the binding energies and total scoring results of PN2-WT and mutant PN2M with the PCNA antigen calculated by Rosetta (a and b), and the graphs of the binding energies and total scoring results of PN8-WT and mutant PN8M with the PCNA antigen calculated by Rosetta (c and d) in Example 5 of the present invention; Figures 6A - 6B This is the graph of the energy change results before and after the change of the mutation site of mutant PN2L102W calculated by Amber in Example 5 of the present invention; Figure 6C This is the graph of the energy change results before and after the change of the mutation site of the PN8H113K mutant calculated by Amber in Example 5 of the present invention; Figure 7 This is the Elisa test result graph of the affinity between the nanobody mutant and the PCNA antigen in Example 5 of the present invention; Figure 8 These are the Elisa test result graphs of the affinities of the nanobody PN8 and the mutant PN8H113K with the PCNA antigen in Example 5 of the present invention; Figure 9 These are the Elisa test result graphs of the affinities of the nanobody PN2, the mutants PN2L102W, PN2S101M, and PN2S101F with the PCNA antigen in Example 5 of the present invention; Figure 10 This is the WB result graph of the targeted degradation of the PCNA protein by the fusion proteins (RBCC-PN8, RBCC-PN8M, RS80E-PN8, RS80E-PN8M) in Example 6 of the present invention; Figure 11 This is the WB result graph of the targeted degradation of the PCNA protein by the fusion proteins (RBCC-PN2M1, RBCC-PN2M2) in Example 6 of the present invention; Figure 12 This is the WB result graph and the phosphorylation level result graph for verifying the pathway of the targeted degradation of the PCNA protein by the fusion protein in Example 6 of the present invention. Detailed implementation manners

[0042] 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. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0043] For the experimental methods without specific conditions noted in the examples, they are generally 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" by M.R. Green, "Molecular Biology" by Robert·F·Weaver, etc., or according to the experimental methods recommended by the reagent kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial sources without special instructions.

[0044] Example 1 Design of Nanobodies by Artificial Intelligence First, use artificial intelligence technology combined with bioinformatics tools (such as SEPPA, MetMHCpan, etc.) to predict the hot spots of the PCNA antigen epitope, and perform linear fragmentation on it to generate fragments containing at least four consecutive residues. Subsequently, screen for CDR-like fragments matching the above fragments through the AbAg database, and perform structural superposition and optimization on the above CDR-like fragments and epitope fragments to construct CDR fragments that can specifically bind to the PCNA antigen. Then, select appropriate framework region fragments (the amino acid sequences of the CDR fragments and framework region fragments are shown in Table 1 below). Finally, a series of nanobodies against specific epitopes of the PCNA antigen are obtained, and they are named nanobodies PN1-9 respectively.

[0045] Table 1 Amino Acid Sequences of CDR Fragments and Framework Region Fragments

[0046] Among them, exemplarily, the amino acid sequence of nanobody PN2 is as follows: MEVQLEESGGGLVQPGGSLRLSCAAS GFNIKDT YIGWVRQAPGKGEEWVASI YPTSGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA GSLQKGYLEEFDY WGQGTLVTVSS(SEQ ID NO:11); The underlines represent complementary determining regions CDR1, CDR2, and CDR3 in sequence.

[0047] The amino acid sequence of nanobody PN6 is as follows: MEVQLEESGGGLVQPGGSLRLSCAAS GFNIKDT YIGWVRQAPGKGEEWVASI YPTSGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA GSAVLQKGYFPEEFDY WGQGTLVTVSS (SEQ ID NO:12); The underlines represent Complementary Determining Region 1 (CDR1), Complementary Determining Region 2 (CDR2) and Complementary Determining Region 3 (CDR3) in sequence.

[0048] The amino acid sequence of nanobody PN7 is shown below: MEVQLEESGGGLVQPGGSLRLSCAAS GFNIKDT YIGWVRQAPGKGEEWVASI YPTSGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA GSQKKITDYFEEFDY WGQGTLVTVSS (SEQ ID NO:13); The underlines represent Complementary Determining Region 1 (CDR1), Complementary Determining Region 2 (CDR2) and Complementary Determining Region 3 (CDR3) in sequence.

[0049] The amino acid sequence of nanobody PN8 is shown below: MEVQLEESGGGLVQPGGSLRLSCAAS GFNIKDT YIGWVRQAPGKGEEWVASI YPTSGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA GSAVLQKKITDYFHPKKEEFDY WGQGTLVTVSS (SEQ ID NO:14); The underlines represent Complementary Determining Region 1 (CDR1), Complementary Determining Region 2 (CDR2) and Complementary Determining Region 3 (CDR3) in sequence.

[0050] The nucleotide sequences of the genes encoding nanobodies PN2, PN6, PN7, and PN8 are shown in SEQ ID NO:18 - 21 respectively.

[0051] Example 2 Expression and Purification of Nanobody and PCNA Antigen In order to facilitate the separation and purification of the nanobody, in the present invention, the nanobody in Example 1 is displayed on the surface of Escherichia coli through superfolder green fluorescent protein (sfGFP, the nucleotide sequence of the gene encoding this sfGFP protein is shown in SEQ ID NO:25). Specifically, based on the pET23a vector, the gene encoding the above - mentioned nanobody is ligated and fused with the sfGFP gene through a first linker (its nucleotide sequence is shown in SEQ ID NO:26), and an expression vector is constructed by homologous recombination. The element arrangement structure of this expression vector is as Figure 1As shown. Exemplarily, the recombinant plasmids are pET23a-sfGFP-PN2, pET23a-sfGFP-PN6, pET23a-sfGFP-PN7, and pET23a-sfGFP-PN8 respectively.

[0052] The above-constructed recombinant plasmids were separately 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 separately 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 separately 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 strain were directly obtained from the TEN buffer. SDS-PAGE was used to detect and analyze the secretion and expression of nanobodies, and the results are as Figure 2 shown in a.

[0053] From Figure 2 a, it can be seen that nanobodies PN1-9 were all successfully expressed.

[0054] Exemplarily, the purified nanobodies PN2, PN6, PN7, and PN8 obtained by washing the outer membrane were preserved for subsequent experiments.

[0055] Furthermore, the PCNA antigen sequence (the 1st to 261st amino acids in NCBI accession number: P12004) was cloned into the pET23a vector by homologous recombination to obtain the recombinant plasmid pET23a-PCNA (the vector structure is shown in Figure 1 ), and the constructed recombinant plasmids were separately 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 600When it is about 0.6, add IPTG with a final concentration of 0.5 mM, incubate with shaking at 18 °C for 18 hours, centrifuge at 6000 rpm for 10 min to collect the bacterial cells, wash the cells with PBS, resuspend the cells in PBS, add PMSF with a final concentration of 1 mM, and disrupt the cells by high pressure. Centrifuge at 18000 rpm for 30 min, collect the supernatant, filter the supernatant, and perform Ni-NTA purification. Collect the purified protein, detect it by SDS-PAGE, divide the protein into small aliquots, quickly freeze it in liquid nitrogen, and store it at -80 °C. The identification and analysis results of SDS-PAGE are as Figure 2 shown in b. Calculated by http: / / www.expasy.org / , the expected size of PCNA is 29.8 kDa. The results indicate that the PCNA antigen was successfully expressed.

[0056] Example 3 ELISA Experiment of Nanobody This example is used to verify whether the purified nanobodies PN2, PN6, PN7, and PN8 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, plate 100 μL / well for coating the well plate, and let it stand overnight at 4 °C; b) Wash the plate with PBST, block it with 1% BSA at room temperature for 2 h, 100 μL / well; c) Prepare different concentrations of nanobodies PN2, PN6, PN7, and PN8 with 1% BSA, 100 μL / well, and incubate at room temperature for 1 h; d) Incubate with the secondary antibody HRP-conjugated Mouse anti HA-Tag mAb 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.

[0057] The test results are as Figure 3 shown.

[0058] It can be seen from Figure 3 that the nanobodies PN2, PN6, PN7, and PN8 have good affinity with the PCNA antigen.

[0059] Example 4 Biolayer Interferometry (BLI) Experiment of Nanobody This example is used to further verify the binding of the purified nanobodies PN2, PN6, PN7, and PN8 in Example 2 to the purified PCNA antigen in Example 2, and calculate the affinity between the two.

[0060] Specifically, the PCNA antigen was immobilized on the chip, and nanobodies PN2, PN6, PN7, and PN8 with different concentration gradients were sequentially added to analyze their affinity with the antigen protein. The increase in the optical thickness at the tip of the biosensor within 600 seconds was recorded, and the affinity with PCNA was calculated based on the binding and dissociation rates of antibodies at different concentrations. The results are as Figure 4 shown.

[0061] As can be seen from Figure 4 , the nanobodies PN2, PN6, PN7, and PN8 have good affinity with the PCNA antigen.

[0062] Example 5 Affinity improvement experiment of nanobodies To further improve the affinity of nanobodies, the present invention uses artificial intelligence for rational design of nanobodies, mutates the nanobody PN2, and obtains the following mutants: PN2S101F (denoted as 2S101F), PN2S101G (denoted as 2S101G), PN2K104V (denoted as 2K104V), PN2K104R (denoted as 2K104R), PN2L102W (denoted as 2L102W), PN2S101M (denoted as 2S101M), PN2S101N (denoted as 2S101N), PN2K104I (denoted as 2K104I); mutates the nanobody PN8, and obtains the following mutants: PN8H113K (denoted as 8H113K), PN8T109L (denoted as 8T109L).

[0063] Furthermore, the binding energy and total score of PN2-WT and the mutant PN2M with the PCNA antigen were calculated by Rosetta. Similarly, the binding energy and total score of PN8-WT and the mutant PN8M with the PCNA antigen were calculated by Rosetta. The results are as Figure 5 shown.

[0064] As can be seen from Figure 5 , 2S101F, 2S101G, 2K104R, 2L102W, 2S101M, etc. all have higher scores ( Figure 5 b) and lower energy values ( Figure 5 a), 8H113K has the highest score ( Figure 5 d) and the lowest energy value ( Figure 5 c).

[0065] Furthermore, Amber was used to calculate the individual energy change at the mutation site of PN2L102W. It was found that after the 102nd position of PN2L102W was mutated from L to W, the energy value became lower, with better stability and is expected to have lower affinity ( Figure 6A), for the calculation of the complex pairing energy, it was found that compared with the wild-type L, the paired amino acids of the mutant W also had a lower energy value overall ( Figure 6B ).

[0066] Furthermore, by using Amber to calculate the individual energy change at the mutation site, it was found that after the 113th position of PN8H113K was mutated from H to K, the energy value decreased and it had better stability; for the calculation of the complex pairing energy, it was found that compared with the wild-type H, the paired amino acids of the mutant K also had a lower energy value overall ( Figure 6C ).

[0067] Using the method in Example 2, expression vectors of the nanobody mutants PN2M and PN8M were constructed (see Figure 1 ). Among them, the nanobody mutant PN2M includes 2S101F, 2S101G, 2K104V, 2K104R, 2L102W, 2S101M, 2S101N, 2K104I. The nucleotide sequence of 2S101F is obtained by replacing the nucleotides "AGT" at positions 301-303 in SEQ ID NO:18 with "TTC"; the nucleotide sequence of 2S101G is obtained by replacing the nucleotides "AGT" at positions 301-303 in SEQ ID NO:18 with "GGC"; the nucleotide sequence of 2K104V is obtained by replacing the nucleotides "AAA" at positions 310-312 in SEQ ID NO:18 with "GTG"; the nucleotide sequence of 2K104R is obtained by replacing the nucleotides "AAA" at positions 310-312 in SEQ ID NO:18 with "CGC"; the nucleotide sequence of 2L102W is obtained by replacing the nucleotides "CTG" at positions 304-306 in SEQ ID NO:18 with "TGG"; the nucleotide sequence of 2S101M is obtained by replacing the nucleotides "AGT" at positions 301-303 in SEQ ID NO:18 with "ATG"; the nucleotide sequence of 2S101N is obtained by replacing the nucleotides "AGT" at positions 301-303 in SEQ ID NO:18 with "AAT"; the nucleotide sequence of 2K104I is obtained by replacing the nucleotides "AAA" at positions 310-312 in SEQ ID NO:18 with "ATT". The nanobody mutant PN8M includes 8H113K, 8T109L. The nucleotide sequence of 8H113K is obtained by replacing the nucleotides "CAC" at positions 337-339 in SEQ ID NO:21 with "AAA"; the nucleotide sequence of 8T109L is obtained by replacing the nucleotides "ACC" at positions 325-327 in SEQ ID NO:21 with "CTG".

[0068] Then, induce expression and purification to obtain nanobody mutants 2S101F, 2S101G, 2K104V, 2K104R, 2L102W, 2S101M, 2S101N, 2K104I, 8H113K, and 8T109L.

[0069] Using the method in Example 3, perform an ELISA experiment on the affinity of the above PN2 and its mutant PN2M, PN8 and its mutant PN8M with the PCNA antigen. The results are as Figure 7 shown.

[0070] From Figure 7 it can be seen that compared with the affinity of nanobodies PN2 and PN8 with the PCNA antigen, PN2S101F, PN2L102W, PN2S101M, and PN8H113K have better affinity with the PCNA antigen.

[0071] Using the method in Example 4, perform a biomembrane interference technology experiment on the affinity of nanobodies PN2, PN8, and nanobody mutants 2S101F, 2L102W, 2S101M, 8H113K with the PCNA antigen. The results are as Figure 8 、 9 shown.

[0072] From Figure 8 、 9 it can be seen that compared with the affinity of nanobodies PN2 and PN8 with the PCNA antigen, PN2S101F, PN2L102W, PN2S101M, and PN8H113K have better affinity with the PCNA antigen.

[0073] Finally, obtain nanobody mutants 2L102W, 2S101M, and 8H113K with significantly improved affinity, which are respectively named nanobody mutants PN2M1, PN2M2, and PN8M.

[0074] Among them, the amino acid sequence of nanobody mutant PN2M1 is as follows: MEVQLEESGGGLVQPGGSLRLSCAAS GFNIKDT YIGWVRQAPGKGEEWVASI YPTSGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA GSWQKGYLEEFDY WGQGTLVTVSS(SEQ ID NO:15); The underlines represent complementary determining regions CDR1, CDR2, and CDR3 in sequence.

[0075] The amino acid sequence of nanobody mutant PN2M2 is as follows: MEVQLEESGGGLVQPGGSLRLSCAAS GFNIKDT YIGWVRQAPGKGEEWVASI YPTSGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA GMLQKGYLEEFDY WGQGTLVTVSS(SEQ ID NO:16); The underlines represent complementary determining regions CDR1, CDR2, and CDR3 in sequence.

[0076] The amino acid sequence of the nanobody mutant PN8M is shown below: MEVQLEESGGGLVQPGGSLRLSCAAS GFNIKDT YIGWVRQAPGKGEEWVASI YPTSGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA GSAVLQKKITDYFKPKKEEFDY WGQGTLVTVSS(SEQ ID NO:17); The underlines represent complementary determining regions CDR1, CDR2, and CDR3 in sequence.

[0077] The nucleotide sequences of the genes encoding the nanobody mutants PN2M1, PN2M2, and PN8M are shown as SEQ ID NO:22 - 24 respectively.

[0078] Example 6 Experiment on Targeted Degradation of PCNA Protein by Fusion Protein To achieve the targeted degradation of PCNA protein, in the present invention, the above-mentioned nanobodies PN2, PN6, PN7, PN8 and the nanobody mutants PN2M1, PN2M2 and PN8M are respectively fused with the RBCC domain or its mutant RS80E to obtain a fusion protein, and the fusion protein is transformed into HEK 293T cells to detect the targeted degradation efficiency of the fusion protein on PCNA protein. Specifically, based on the pcDNA3.1 vector, the genes encoding the above-mentioned nanobodies PN2, PN6, PN7, PN8 and the nanobody mutants PN2M1, PN2M2 and PN8M are ligated and fused with the gene encoding the RBCC domain (its amino acid sequence is shown in SEQ ID NO: 27, and its nucleotide sequence is shown in SEQ ID NO: 30) or its mutant RS80E (its amino acid sequence is shown in SEQ ID NO: 28, and its nucleotide sequence is shown in SEQ ID NO: 31)) through a second linker (its amino acid sequence is shown in SEQ ID NO: 29, and its nucleotide sequence is shown in SEQ ID NO: 32)), and an expression vector is constructed by homologous recombination. The element arrangement structures of some expression vectors are as shown in Figure 1 shown. The recombinant plasmids are pcDNA3.1-RBCC-PN2, pcDNA3.1-RBCC-PN6, pcDNA3.1-RBCC-PN7, pcDNA3.1-RBCC-PN8, pcDNA3.1-RBCC-PN2M1, pcDNA3.1-RBCC-PN2M2, pcDNA3.1-RBCC-PN8M, pcDNA3.1-RS80E-PN8, pcDNA3.1-RS80E-PN8M.

[0079] Among them, the amino acid sequences of the fusion proteins RBCC-PN2, RBCC-PN6, RBCC-PN7, RBCC-PN8, RBCC-PN2M1, RBCC-PN2M2, RBCC-PN8M, RS80E-PN8, RS80E-PN8M are shown in SEQ ID NO: 33-41 respectively.

[0080] Using the PEI reagent, exemplary, the above-mentioned recombinant plasmids pcDNA3.1-RBCC-PN8, pcDNA3.1-RBCC-PN8M, pcDNA3.1-RS80E-PN8, pcDNA3.1-RS80E-PN8M, pcDNA3.1-RBCC-PN2M1, pcDNA3.1-RBCC-PN2M2 and the GFP plasmid are respectively electrotransfected into well-conditioned HEK 293T cells. After electrotransfection, the electrotransfection efficiency and protein expression are observed through the GFP plasmid.

[0081] The cells transfected for 48 h were harvested and then subjected to Western blot analysis respectively. According to the Western blot results, the efficiency of protein targeted degradation after 48 h of transfection was calculated. The results are shown in Figure 10 and 11 as follows.

[0082] It can be seen from Figure 10 that the fusion proteins containing the nanobody PN8 and the nanobody mutant PN8M have a good degradation effect on the PCNA antigen, and the degradation efficiency of the fusion protein containing the nanobody mutant PN8M on the PCNA antigen is about 60%. Further, when the RBCC domain mutant RS80E is fused with the nanobody mutant PN8M, the degradation efficiency of the PCNA antigen is significantly improved.

[0083] It can be seen from Figure 11 that the fusion proteins containing the nanobody mutants PN2M1 and PN2M2 have a good degradation effect on the PCNA antigen.

[0084] Finally, the degradation pathway of the fusion protein targeting the degradation of PCNA protein was explored.

[0085] 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. Among them, MG132 can inhibit the ubiquitin proteasome pathway, and BafA1 can inhibit the lysosomal pathway. Three copies of the pcDNA3.1-RBCC-PN8M recombinant plasmid and the GFP plasmid were electrotransfected into HEK 293T cells in good condition. After 24 h of transfection, the corresponding MG132 and BafA1 were added to 2 copies of the cells with the pcDNA3.1-RBCC-PN8M recombinant plasmid. After continuous culture, Western blot analysis was performed. The results are shown in Figure 12 as follows.

[0086] It can be seen from Figure 12 that the fusion protein in the present invention targets the degradation of PCNA through the proteasome ubiquitin pathway and the lysosomal pathway simultaneously, and mainly through the lysosomal pathway. Compared with the control group, after the experimental group RS80E-PN8M targets the degradation of PCNA, the phosphorylated p53 level is significantly increased ( Figure 12 b). The results show that the fusion protein in the present invention targets the degradation of PCNA protein through the ubiquitin-proteasome pathway or the autophagy-lysosome pathway.

[0087] In summary, the present invention designs a nanobody specifically targeting PCNA by using artificial intelligence technology. This nanobody has good specificity and high affinity, and can efficiently bind to the PCNA antigen. Further, through the rational design of the nanobody by artificial intelligence, a nanobody mutant with higher affinity is obtained. Furthermore, by fusing the nanobody or nanobody mutant with high affinity and good specificity with the RBCC domain or its mutant, a fusion protein is obtained. This fusion protein can specifically bind to the target protein using its nanobody domain, initiate the protein degradation pathway, and achieve the targeted degradation of the PCNA protein.

[0088] The above embodiments only express 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 present invention patent shall be subject to the appended claims.

Claims

1. A nano antibody against PCNA protein, characterized in that, including complementarity determining region CDR1, complementarity determining region CDR2 and complementarity determining region CDR3; Wherein, the complementary determining region CDR1 has an amino acid sequence as shown in SEQ ID NO: 1, the complementary determining region CDR2 has an amino acid sequence as shown in SEQ ID NO: 2, and the complementary determining region CDR3 has an amino acid sequence as shown in any one of SEQ ID NOs: 3-6.

2. the nano antibody of anti-PCNA protein according to claim 1, is characterized in that, The Nanobody also includes a framework region FR1, a framework region FR2, a framework region FR3 and a framework region FR4; Among them, the framework region FR1 has the amino acid sequence shown in SEQ ID NO:7, the framework region FR2 has the amino acid sequence shown in SEQ ID NO:8, the framework region FR3 has the amino acid sequence shown in SEQ ID NO:9, and the framework region FR4 has the amino acid sequence shown in SEQ ID NO:

10.

3. the nano antibody of anti-PCNA protein according to claim 2, is characterized in that, The Nanobody is selected from any one of the following: A1) has an amino acid sequence as shown in any one of SEQ ID NOs: 11-14; 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).

4. A nano antibody mutant against PCNA protein, characterized in that, The Nanobody mutant is selected from any one of the following: B1) has an amino acid sequence as shown in any one of SEQ ID NOs: 15-17; 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); Among them, in B1), the amino acid sequence as shown in SEQ ID NO:15 is obtained by mutating the amino acid sequence as shown in SEQ ID NO:11 at the following site: L102W, the amino acid sequence as shown in SEQ ID NO:16 is obtained by mutating the amino acid sequence as shown in SEQ ID NO:11 at the following site: S101M, and the amino acid sequence as shown in SEQ ID NO:17 is obtained by mutating the amino acid sequence as shown in SEQ ID NO:14 at least one of the following sites: H113K.

5. A fusion protein, characterized in that The fusion protein is obtained by connecting the Nanobody according to any one of claims 1 to 3 or the Nanobody mutant according to claim 4 to the RBCC domain or its mutant via a second linker; Among them, the amino acid sequence of the second linker is shown in SEQ ID NO:29, the amino acid sequence of the RBCC domain is shown in SEQ ID NO:27, and the amino acid sequence of the RBCC domain mutant is shown in SEQ ID NO:

28.

6. The fusion protein according to claim 5, characterized in that The fusion protein is selected from any one of the following: C1) has an amino acid sequence as shown in any one of SEQ ID NOs: 33-41; C2) an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence defined in C1); C3) an amino acid sequence having a sequence identity of more than 80% with the amino acid sequence defined in C1) or C2); C4) 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 C1) or C2) or C3).

7. A biomaterial, characterized in that: The biological material is selected from any one of the following: D1) A nucleic acid molecule encoding the Nanobody according to any one of claims 1 to 3, the Nanobody mutant according to claim 4, or the fusion protein according to any one of claims 5 to 6; D2) a recombinant vector containing the nucleic acid molecule described in D1); D3) A recombinant cell containing the nucleic acid molecule described in D1) or the recombinant vector described in D2).

8. Use of the Nanobody according to any one of claims 1 to 3, the Nanobody mutant according to claim 4, the fusion protein according to any one of claims 5 to 6, or the biomaterial according to claim 7 in any of the following: F1) Use in the preparation of products for preventing and / or treating PCNA target related diseases; F2) 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 Nanobody according to any one of claims 1-3, the Nanobody mutant according to claim 4, the fusion protein according to any one of claims 5-6, or the biomaterial according to claim 7, and a pharmaceutically acceptable carrier.

10. A method for preparing the Nanobody according to any one of claims 1 to 3, the Nanobody mutant according to claim 4, or the fusion protein according to any one of claims 5 to 6, characterized in that: The steps include: Cultivating the recombinant cell according to claim 7, and obtaining a culture after inducing expression; Said Nanobody, said Nanobody mutant or said fusion protein is isolated from said culture.

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