A nanobody against pcna protein, fusion protein and application thereof

Abstract

The application discloses a kind of anti-PCNA protein nanobody, fusion protein and its application, belong to biotechnology field.The application is designed by using artificial intelligence technology to specifically target the nanobody of PCNA, the nanobody has good specificity, high affinity, can efficiently combine PCNA antigen;Further, the higher affinity nanobody mutant of the rational design of nanobody is obtained by artificial intelligence;Further, by the fusion of nanobody or nanobody mutant with high affinity and good specificity and RBCC domain or its mutant, the fusion protein is obtained, the fusion protein can utilize its nanobody domain specific binding target protein, start protein degradation pathway, realize the targeted degradation of PCNA protein, and activate intracellular p53 to make it phosphorylated to avoid ubiquitination degradation by MDM2 such as ubiquitin ligase, has very big clinical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a nanobody against PCNA protein, a fusion protein and application thereof. BACKGROUND

[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 acts 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 DNA replication initiation and elongation, interacts with various replication and repair proteins to ensure the efficiency and accuracy of DNA replication. PCNA is also involved in various 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 genome stability and preventing mutation accumulation.

[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 block its interaction with DNA polymerase or other repair proteins to inhibit DNA replication and repair. However, there are relatively few studies on PCNA degrading agents. Existing PCNA inhibitors have limited effect and may be associated with higher risk of side effects, so it is necessary to develop more efficient and specific PCNA targeting degradation technology.

[0005] Nanobody is a single-domain antibody derived from heavy chain antibodies of Camelidae, which has the specificity and affinity of traditional antibodies, while having smaller molecular weight and higher stability. Nanobody can penetrate tissues and cells, and is more easily combined with target proteins, so it has wide application prospects in biomedical research and treatment.

[0006] Targeted protein degradation (TPD) technology utilizes 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, lower drug dosage, only a catalytic dose is needed to work; it can also target some undruggable proteins.

[0007] TRIMbody is an emerging TPD technology that fuses a nanobody 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 degrade them in the proteasome. The TRIMbody technology combines the high specificity of nanobodies and the efficient degradation function of TRIM21, providing a more efficient and precise PCNA degradation method, which is expected to improve the treatment effect of PCNA-related cancers.

[0008] However, there is currently no research report on nanobodies targeting PCNA. SUMMARY

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

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

[0011] In the present application, the term "nanobody" also referred to as single domain antibody (sdAb) refers to an antibody consisting of only one H chain V region, which can also be referred to as VHH antibody. The ability of nanobody to bind to antigen and its stability are basically consistent with those of complete antibody. Nanobody is composed of framework region FR1, complementarity determining region CDR1, framework region FR2, complementarity determining region CDR2, framework region FR3, complementarity determining region CDR3 and framework region FR4 in turn. Nanobody can be truncated at the N-terminus or C-terminus to make it only contain part of FR1 and / or FR4, or lack one or two of the framework regions, as long as the antigen binding and specificity are essentially maintained. Among them, the complementarity determining region is mainly responsible for binding to the antigen.

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

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

[0014] In some embodiments, the nanobody is selected from any one of the following: A1) has an amino acid sequence as shown in any one of SEQ ID NO: 11-14; A2) has an amino acid sequence with one or several substitutions, deletions or additions of amino acids compared with the amino acid sequence defined in A1); A3) has an amino acid sequence with 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 application, the inventors designed a nanobody specifically targeting PCNA using artificial intelligence technology, which has good specificity, high affinity and can efficiently bind to PCNA antigen.

[0016] In a second aspect, the present application provides a Nanobody mutant against PCNA protein, the Nanobody mutant is selected from any one of the following: B1) having an amino acid sequence as shown in any one of SEQ ID NO: 15-17; B2) having an amino acid sequence with substitution, deletion or addition of one or several amino acids compared to the amino acid sequence defined in B1); B3) having an amino acid sequence with more than 80% sequence identity compared to the amino acid sequence defined in B1) or B2); B4) an amino acid sequence obtained by connecting a tag or 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 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 at least one of the following sites: H113K.

[0017] In the present application, the inventors further use artificial intelligence to rationally design Nanobody mutants with higher affinity.

[0018] In some embodiments, in addition to the mutation at one of the 101st, 102nd and 113th sites in the above-mentioned Nanobody mutants, the Nanobody mutants can further have conservative substitution of amino acids at other sites, so that the mutated Nanobody with an amino acid sequence as shown in SEQ ID NO: 11 or 14 has higher affinity. Preferably, the conservative substitution of amino acids retains the higher affinity of the Nanobody of the present application. It is obvious to those skilled in the art that such substitution can occur in regions other than the above-mentioned sites while still retaining the corresponding affinity. Preferably, the conservative substitution variant has conservative substitution of amino acids at at least one position. Examples of conservative substitution are substitution 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, 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 application provides a fusion protein, which is obtained by connecting any of the above-mentioned nanobodies or the above-mentioned nanobody mutants with the RBCC domain or the mutant thereof through a second linker; wherein the amino acid sequence of the second linker is shown as SEQ ID NO: 29, the amino acid sequence of the RBCC domain is shown as SEQ ID NO: 27, and the amino acid sequence of the RBCC domain mutant is shown as SEQ ID NO: 28.

[0020] In the present application, the inventors fuse the nanobodies or the nanobody mutants with high affinity and good specificity with the RBCC domain or the mutant thereof (a truncated TRIM family protein) to obtain a fusion protein, which can specifically bind to a target protein through the nanobody domain thereof, start a protein degradation pathway, realize the targeted degradation of the PCNA protein, activate the p53 in the cell to avoid ubiquitination and degradation by the MDM2 ubiquitin ligase, promote apoptosis to inhibit the expansion efficiency of cancer cells, and has great clinical application value.

[0021] It can be understood that the connecting peptide can be a conventional connecting peptide in the prior art according to actual needs, as long as the two proteins can be well fused.

[0022] In some embodiments, the fusion protein is selected from any one of the following: C1) has an amino acid sequence shown as any one of SEQ ID NO: 33-41; C2) has an amino acid sequence with substitution, deletion or addition of one or more amino acids compared with the amino acid sequence defined in C1); C3) has an amino acid sequence with more than 80% sequence identity compared with the amino acid sequence defined in C1) or C2); and C4) is an amino acid sequence obtained by connecting a tag or a signal peptide to the N-terminus and / or the C-terminus of the amino acid sequence defined in C1), C2) or C3).

[0023] The above-mentioned nanobodies, the above-mentioned nanobody mutants and the above-mentioned fusion proteins provided by the present application can be natural, recombinant or synthetic active polypeptides, which can be natural purified products, chemically synthesized products, or products produced from prokaryotic hosts (such as E. coli) or eukaryotic hosts (such as yeast, higher plants) using recombinant technology.

[0024] In the A4), B4) or C4) of the present application, the connection can be directly connected by a peptide bond or connected through a linker, and the method of connection is a method conventional in the art. Among them, the tag includes but is 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 (super-fold green fluorescent protein), HA tag (hemagglutinin tag). Those skilled in the art can select a suitable tag protein according to the actual use 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 track. The tag can be separated from the target protein (nanobody, nanobody mutant or fusion protein) by chemical cleavage or enzymatic method known in the art (such as introducing a protease cleavage site to remove the tag using TEV protease).

[0025] In a fourth aspect, the present application provides a biological material selected from any one of the following: D1) a nucleic acid molecule encoding any of the above-mentioned nanobodies, the above-mentioned nanobody mutants or the above-mentioned 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 as shown in any one of SEQ ID NOs: 18-24; E2) a nucleic acid molecule hybridizing to the nucleic acid molecule defined in E1) under stringent conditions and encoding any of the above-mentioned nanobodies, the above-mentioned nanobody mutants or the above-mentioned fusion proteins; E3) a nucleic acid molecule having 90% or more sequence identity to the nucleic acid molecule defined in E1) or E2) and encoding any of the above-mentioned nanobodies, the above-mentioned nanobody mutants or the above-mentioned fusion proteins.

[0027] The above-mentioned nucleic acid molecule provided by the present application can be DNA, such as cDNA, genomic DNA or recombinant DNA; or RNA, such as mRNA or hnRNA, etc.; and the nucleic acid molecule can generally be obtained by PCR amplification or artificial synthesis method.

[0028] The above-mentioned recombinant vector provided by the present application includes a cloning vector for replicating the relevant sequence and an expression vector for expressing the relevant gene, wherein the vector used when constructing the expression vector can be at least one of pET23a, pcDNA3.1 vector.

[0029] In some embodiments, the method for preparing the above-mentioned recombinant cell comprises the step of transforming the above-mentioned recombinant vector into an expression host cell.

[0030] In the present application, the expression host cell is a host cell that is conventional in the art, as long as the recombinant vector can be stably replicated by itself and the gene carried thereby can be effectively expressed, which can be a prokaryotic cell or a eukaryotic cell, such as E. coli, yeast, etc. The E. coli can be, for example, E. coli BL21(DE3), Rosetta(DE3), BL21(DE3)plysS. In the present application, the preferred E. coli expression host is E. coli BL21(DE3).

[0031] In a fifth aspect, the present application 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 biological materials in any one of the following: F1) the preparation of a product for preventing and / or treating a PCNA target point related disease; F2) the preparation of a product for screening, diagnosing or assisting in the diagnosis of a PCNA target point related disease; wherein the PCNA target point related disease is a PCNA positive tumor.

[0032] In the present application, the PCNA positive tumor refers to a tumor in which PCNA expression is detected in tumor cells. The tumor can be, for example, a common cancer such as lung cancer, gastric cancer, liver cancer, breast cancer, prostate cancer, colorectal cancer, esophageal cancer, etc.

[0033] In a sixth aspect, the present application provides a pharmaceutical composition for preventing and / or treating a PCNA target point related disease, which comprises any one of the above-mentioned nanobodies, the above-mentioned nanobody mutants, any one of the above-mentioned fusion proteins or the above-mentioned biological materials, and a pharmaceutically acceptable carrier.

[0034] In the present application, the term "pharmaceutically acceptable carrier" refers to an excipient widely used in the field of drug production. The excipient is mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method so that the active ingredient is dissolved at the desired rate after the subject receives the administration, or promotes the active ingredient to be effectively absorbed after the subject receives the administration of the composition. The pharmaceutical excipient can be an inert filler, or provide a certain function, such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient of the composition.

[0035] The pharmaceutical composition provided by the present application can be prepared according to the disclosure using any method known to those skilled in the art. For example, including but not limited to conventional mixing, dissolving, granulating, emulsifying, pulverizing, encapsulating, entrapping or lyophilizing processes.

[0036] In some embodiments, the dosage form of the pharmaceutical composition comprises at least one of a solid preparation, a semi-solid preparation, a liquid preparation.

[0037] The pharmaceutical compositions of the present application 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 compositions of the present application can also be in a controlled or sustained release dosage form (e.g. liposomes or microspheres).

[0038] In a seventh aspect, the present application provides a method for preparing the nanobody of any one of the above, the nanobody mutant of the above or the fusion protein of any one of the above, comprising the following steps: culturing the recombinant cell, obtaining a culture after inducing expression; and separating the nanobody, the nanobody mutant or the fusion protein from the culture.

[0039] In the present application, the culture method, culture condition and culture medium are not particularly required, as long as the normal growth of the recombinant cell is ensured. The method for separating the nanobody of any one of the above, the nanobody mutant of the above or the fusion protein of any one of the above from the culture is a conventional method in the art.

[0040] The present application has the following beneficial effects: Different from the prior art, the present application designs a nanobody specifically targeting PCNA by using artificial intelligence technology. The nanobody has good specificity and high affinity, and can efficiently bind to the PCNA antigen. Further, the nanobody mutant with higher affinity is obtained by using artificial intelligence to rationally design the nanobody. Furthermore, the fusion protein is obtained by fusing the nanobody or the nanobody mutant with high affinity and good specificity with the RBCC domain or the mutant thereof. The fusion protein can specifically bind to the target protein by using the nanobody domain, start the protein degradation pathway, realize the targeted degradation of the PCNA protein, activate the p53 in the cell to make it phosphorylated to avoid ubiquitination degradation by MDM2 and other ubiquitin ligases, promote cell apoptosis to inhibit the expansion efficiency of cancer cells, and has great clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 It is a schematic diagram of the element arrangement structure of the recombinant expression vector constructed in the present application;

[0042] 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 application, wherein lane M in figure (a) is Marker, and lanes 1-9 are PN1-9 respectively, and wherein lane M in figure (b) is Marker, and lanes 1-2 are both PCNA;

[0043] Figure 3 It is an Elisa test result diagram of the affinity of the nanobody and the PCNA antigen in Example 3 of the present application;

[0044] Figure 4 Figures of BLI test results of the affinity of the nanobodies PN2, PN6, PN7, PN8 in the embodiment 4 of the present application to PCNA antigen, respectively;

[0045] Figure 5 Figures of the binding energy and total score results of the PN2-WT and mutant PN2M to PCNA antigen calculated by Rosetta (a and b) and the binding energy and total score results of the PN8-WT and mutant PN8M to PCNA antigen calculated by Rosetta (c and d) in the embodiment 5 of the present application;

[0046] Figures 6A-6B Figures of the energy change results of the mutant PN2L102W before and after the mutation site change calculated by Amber in the embodiment 5 of the present application;

[0047] Figure 6C Figure of the energy change results of the PN8H113K mutant before and after the mutation site change calculated by Amber in the embodiment 5 of the present application;

[0048] Figure 7 Figure of the Elisa test results of the affinity of the nanobody mutant to PCNA antigen in the embodiment 5 of the present application;

[0049] Figure 8 Figures of the Elisa test results of the affinity of the nanobody PN8, mutant PN8H113K to PCNA antigen in the embodiment 5 of the present application, respectively;

[0050] Figure 9 Figures of the Elisa test results of the affinity of the nanobody PN2, mutant PN2L102W, PN2S101M, PN2S101F to PCNA antigen in the embodiment 5 of the present application, respectively;

[0051] Figure 10 Figure of the WB results of the fusion protein (RBCC-PN8, RBCC-PN8M, RS79E-PN8, RS79E-PN8M) targeting degradation of PCNA protein in the embodiment 6 of the present application;

[0052] Figure 11 Figure of the WB results of the fusion protein (RBCC-PN2M1, RBCC-PN2M2) targeting degradation of PCNA protein in the embodiment 6 of the present application;

[0053] Figure 12 Figure of the WB results of the fusion protein targeting degradation of PCNA protein pathway and the phosphorylation level results in the embodiment 6 of the present application. DETAILED DESCRIPTION

[0054] The technical solutions in the embodiments of the present application will be clearly and completely described in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0055] The experimental methods not specified in the embodiments 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, or according to the experimental methods suggested by the manufacturers of reagent kits and instrument equipment. The reagents and biological materials used in the embodiments can be obtained from commercial channels if not otherwise specified.

[0056] Embodiment 1 Artificial intelligence designed nanobody

[0057] First, the hot spot epitope of the PCNA antigen is predicted by using artificial intelligence technology combined with bioinformatics tools (such as SEPPA, MetMHCpan, etc.), and linear fragmentation is performed to generate fragments containing at least four consecutive residues. Then, the CDR-like fragments matched with the above fragments are screened through the AbAg database, and the above CDR-like fragments are subjected to structure superposition and optimization with the epitope fragments to construct CDR fragments capable of specifically binding to the PCNA antigen, and then appropriate framework region fragments (the amino acid sequences of the CDR fragments and the framework region fragments are shown in Table 1 below) are selected. Finally, a series of nanobodies against specific epitopes of the PCNA antigen are obtained, which are named nanobodies PN1-9, respectively.

[0058] Table 1 Amino acid sequences of CDR fragments and framework region fragments

[0059]

[0060] Exemplarily, the amino acid sequence of the nanobody PN2 is as follows:

[0061] MEVQLEESGGGLVQPGGSLRLSCAAS GFNIKDT YPTSGY GSLQKGYLEEFDY GFNIKDT YPTSGY GSAVLQKGYFPEEFDY GFNIKDT YPTSGY GSQKKITDYFEEFDY GFNIKDT YPTSGY GSAVLQKKITDYFHPKKEEFDY YIGWVRQAPGKGEEWVASI Figure 1 TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA Figure 2 WGQGTLVTVSS (SEQ ID NO: 11);

[0062] The underlined sequences represent, in order, complementarity determining regions CDR1, CDR2 and CDR3.

[0063] The amino acid sequence of Nanobody PN6 is as follows:

[0064] MEVQLEESGGGLVQPGGSLRLSCAAS Figure 2 YIGWVRQAPGKGEEWVASI Figure 1 TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA Figure 2 WGQGTLVTVSS (SEQ ID NO: 12);

[0065] The underlined sequences represent, in order, complementarity determining regions CDR1, CDR2 and CDR3.

[0066] The amino acid sequence of Nanobody PN7 is as follows:

[0067] MEVQLEESGGGLVQPGGSLRLSCAAS Figure 3 YIGWVRQAPGKGEEWVASI Figure 3 TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA Figure 4 WGQGTLVTVSS (SEQ ID NO: 13);

[0068] The underlined sequences represent, in order, complementarity determining regions CDR1, CDR2 and CDR3.

[0069] The amino acid sequence of Nanobody PN8 is as follows:

[0070] MEVQLEESGGGLVQPGGSLRLSCAAS Figure 4 YIGWVRQAPGKGEEWVASI Figure 5 TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA Figure 5 WGQGTLVTVSS (SEQ ID NO: 14);

[0071] The underlined sequences represent, in order, complementarity determining regions CDR1, CDR2 and CDR3.

[0072] The nucleotide sequences of the genes encoding Nanobodies PN2, PN6, PN7, PN8 are as shown in SEQ ID NOs: 18-21, respectively.

[0073] Example 2 Expression and purification of Nanobodies, PCNA antigens

[0074] In order to facilitate the separation and purification of the nanobodies, in the present application, the nanobodies in Example 1 are displayed on the surface of E. coli by super-fold green fluorescent protein (sfGFP, the nucleotide sequence of the gene encoding the sfGFP protein is shown as SEQ ID NO: 25). Specifically, the gene encoding the above-mentioned nanobodies is connected and fused with the sfGFP gene through a first linker (the nucleotide sequence of which is shown as SEQ ID NO: 26) by taking pET23a as a basic vector, and an expression vector is constructed by means of homologous recombination, the element arrangement structure of the expression vector is shown as Figure 5 For example, the recombinant plasmids are respectively pET23a-sfGFP-PN2, pET23a-sfGFP-PN6, pET23a-sfGFP-PN7, and pET23a-sfGFP-PN8.

[0075] The above-constructed recombinant plasmids are respectively transformed into E. coli competent cells BL21 (DE3) strains, and are incubated at 37°C overnight to obtain a series of recombinant strains. Then, single colonies are respectively picked and inoculated into 100 mL of LB liquid medium (the concentration of ampicillin is 50 μg / mL), and are incubated at 37°C with shaking. When the OD 600 is about 0.6, 0.5 mM of IPTG is added, and the culture is induced at 18°C for 18 hours with shaking. After the culture is completed, the bacterial cells and the culture medium supernatant are respectively collected by centrifugation at 12000 rpm at 4°C. The bacterial cells are 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 protein secreted and expressed by the strain is directly obtained from the TEN buffer. The secreted and expressed nanobodies are detected and analyzed by SDS-PAGE, and the results are shown in Figure 5 a.

[0076] As can be seen from Figure 5 a, the nanobodies PN1-9 are all successfully expressed.

[0077] For example, the purified nanobodies PN2, PN6, PN7, and PN8 obtained by washing the outer membrane are stored for subsequent experiments.

[0078] Further, the PCNA antigen sequence (the 1-261 amino acids in NCBI accession number: P12004) is cloned into the pET23a vector by means of homologous recombination to obtain the recombinant plasmid pET23a-PCNA (the vector structure is shown as Figure 5), the constructed recombinant plasmid was transformed into E. coli competent cell BL21 (DE3) strain, respectively, a single colony was picked and inoculated into 100 mL LB liquid medium (the concentration of ampicillin was 50 μg / mL), and the culture was incubated at 37°C overnight. When the OD 600 was about 0.6, 0.5 mM IPTG was added, and the culture was induced at 18°C for 18 hours. The bacterial cells were collected by centrifugation at 6000 rpm for 10 min, washed with PBS, resuspended in PBS, and added with 1 mM PMSF. The bacterial cells were broken by high pressure. The supernatant was collected by centrifugation at 18000 rpm for 30 min. The purified protein was detected by SDS-PAGE, and the protein was divided into small portions, frozen by liquid nitrogen, and stored at -80°C. The SDS-PAGE identification analysis results are shown in Figure 6A b. The expected size of PCNA is 29.8 kDa calculated by http: / / www.expasy.org / , and the results show that the PCNA antigen is successfully expressed.

[0079] Example 3. ELISA experiment of nanobody

[0080] This example is used to verify whether the purified nanobodies PN2, PN6, PN7, and PN8 in Example 2 can directly interact with the PCNA antigen purified in Example 2. Specifically, the steps are as follows:

[0081] a) The PCNA antigen was diluted with 1x ELSIA coating solution to 1 μg / mL, and 100 μL / well was plated to coat the well plate, which was incubated at 4°C overnight;

[0082] b) The well plate was washed with PBST, and 100 μL / well of 1% BSA was used for room temperature blocking for 2 hours;

[0083] c) Different concentrations of nanobodies PN2, PN6, PN7, and PN8 were prepared using 1% BSA, 100 μL / well, and incubated at room temperature for 1 hour;

[0084] d) HRP-conjugated Mouse anti HA-Tag mAb secondary antibody was incubated at room temperature for 1 hour;

[0085] e) TMB color development, and the reaction was terminated by a stop solution;

[0086] f) The absorbance value was measured at 450 nm by an enzyme-labeled instrument.

[0087] The test results are shown in Figure 6B .

[0088] From Figure 6CAs can be seen, nanobodies PN2, PN6, PN7, and PN8 have good affinity for PCNA antigen.

[0089] Example 4: Experiment with Nanobody Biomembrane Interference (BLI) Technology

[0090] This embodiment is used to further verify the binding of the purified nanobodies PN2, PN6, PN7, and PN8 from Example 2 to the purified PCNA antigen from Example 2, and to calculate the affinity between the two.

[0091] Specifically, the PCNA antigen was immobilized on a chip, and nanobodies of different concentration gradients (PN2, PN6, PN7, and PN8) were sequentially added to analyze their affinity for the antigen protein. The increase in optical thickness at the biosensor tip was recorded over 600 seconds, and the affinity for PCNA was calculated based on the binding and dissociation rates of the antibodies at different concentrations. The results are as follows: Figure 1 As shown.

[0092] from Figure 7 As can be seen, nanobodies PN2, PN6, PN7, and PN8 have good affinity for PCNA antigen.

[0093] Example 5: Experiment to enhance the affinity of nanobodies

[0094] To further enhance the affinity of nanobodies, this invention employs artificial intelligence for the rational design of nanobodies. The nanobodies PN2 are mutated to obtain 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), and PN2K104I (denoted as 2K104I). Similarly, the nanobodies PN8 are mutated to obtain the following mutants: PN8H113K (denoted as 8H113K) and PN8T109L (denoted as 8T109L).

[0095] Furthermore, the binding energies and total scores of PN2-WT and the mutant PN2M with the PCNA antigen were calculated using Rosetta. Similarly, the binding energies and total scores of PN8-WT and the mutant PN8M with the PCNA antigen were calculated using Rosetta. The results are as follows: Figure 7 As shown.

[0096] from Figure 8 As can be seen, 2S101F, 2S101G, 2K104R, 2L102W, and 2S101M all have higher scores. Figure 8b) and lower energy value (E) GFNIKDT YPTSGY GSWQKGYLEEFDY GFNIKDT YPTSGY GMLQKGYLEEFDY GFNIKDT YPTSGY GSAVLQKKITDYFKPKKEEFDY a), PN8H113K has the highest score (S) Figure 1 d) and the lowest energy value (E) Figure 10 c).

[0097] Further using Amber to calculate the energy change of the mutation site of PN2L102W alone, it is found that after the 102nd position of PN2L102W is mutated from L to W, the energy value is lower, with better stability, and is expected to have lower affinity (E Figure 10 ) For complex pairing energy calculation, it is found that the total pairing amino acids of W after mutation compared with L before mutation also have lower energy value (E Figure 11 ).

[0098] Further using Amber to calculate the energy change of the mutation site of PN8H113K alone, it is found that after the 113th position of PN8H113K is mutated from H to K, the energy value is lower, with better stability; for complex pairing energy calculation, it is found that the total pairing amino acids of K after mutation compared with H before mutation also have lower energy value (E Figure 12 ).

[0099] Using the method in Example 2, the expression vectors of nanobody mutants PN2M and PN8M are constructed (see Figure 12). Among them, the nucleotide sequence of 2S101F is that the nucleotides "AGT" at positions 301-303 in SEQ ID NO: 18 are replaced with "TTC"; the nucleotide sequence of 2S101G is that the nucleotides "AGT" at positions 301-303 in SEQ ID NO: 18 are replaced with "GGC"; the nucleotide sequence of 2K104V is that the nucleotides "AAA" at positions 310-312 in SEQ ID NO: 18 are replaced with "GTG"; the nucleotide sequence of 2K104R is that the nucleotides "AAA" at positions 310-312 in SEQ ID NO: 18 are replaced with "CGC"; the nucleotide sequence of 2L102W is that the nucleotides "CTG" at positions 304-306 in SEQ ID NO: 18 are replaced with "TGG"; the nucleotide sequence of 2S101M is that the nucleotides "AGT" at positions 301-303 in SEQ ID NO: 18 are replaced with "ATG"; the nucleotide sequence of 2S101N is that the nucleotides "AGT" at positions 301-303 in SEQ ID NO: 18 are replaced with "AAT"; the nucleotide sequence of 2K104I is that the nucleotides "AAA" at positions 310-312 in SEQ ID NO: 18 are replaced with "ATT". The nucleotide sequence of 8H113K is that the nucleotides "CAC" at positions 337-339 in SEQ ID NO: 21 are replaced with "AAA"; the nucleotide sequence of 8T109L is that the nucleotides "ACC" at positions 325-327 in SEQ ID NO: 21 are replaced with "CTG".

[0100] Then, expression and purification were induced to obtain the nanobody mutants 2S101F, 2S101G, 2K104V, 2K104R, 2L102W, 2S101M, 2S101N, 2K104I, 8H113K, 8T109L.

[0101] Using the method in Example 3, the affinities of the above-mentioned PN2 and its mutants PN2M, PN8 and its mutants PN8M to the PCNA antigen were subjected to ELISA experiments, and the results are shown in Figure 12

[0102] As can be seen from ​ , compared with the affinities of the nanobodies PN2 and PN8 to the PCNA antigen, the affinities of PN2S101F, PN2L102W, PN2S101M and PN8H113K to the PCNA antigen are better. ​

[0103] The affinities of the nanobodies PN2, PN8, and nanobody mutants 2S101F, 2L102W, 2S101M, and 8H113K to the PCNA antigen were determined by bio-layer interferometry using the method described in Example 4, and the results are shown in Table 2. ​ , 9

[0104] As can be seen from Table 2, the nanobodies PN2S101F, PN2L102W, PN2S101M, and PN8H113K have better affinities to the PCNA antigen than the nanobodies PN2 and PN8. ​ , 9

[0105] Finally, the nanobody mutants 2L102W, 2S101M, and 8H113K, which have significantly improved affinities, were obtained and named as nanobody mutants PN2M1, PN2M2, and PN8M, respectively.

[0106] The amino acid sequence of the nanobody mutant PN2M1 is shown below:

[0107] MEVQLEESGGGLVQPGGSLRLSCAAS ​ YIGWVRQAPGKGEEWVASI ​ TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA ​ WGQGTLVTVSS (SEQ ID NO: 15);

[0108] The underlined regions represent the complementarity determining regions CDR1, CDR2, and CDR3, respectively.

[0109] The amino acid sequence of the nanobody mutant PN2M2 is shown below:

[0110] MEVQLEESGGGLVQPGGSLRLSCAAS ​ YIGWVRQAPGKGEEWVASI ​ TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA ​ WGQGTLVTVSS (SEQ ID NO: 16);

[0111] The underlined regions represent the complementarity determining regions CDR1, CDR2, and CDR3, respectively.

[0112] The amino acid sequence of the nanobody mutant PN8M is shown below:

[0113] ​​MEVQLEESGGGLVQPGGSLRLSCAAS ​ YIGWVRQAPGKGEEWVASI ​ TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA ​ WGQGTLVTVSS (SEQ ID NO: 17);

[0114] The underlined represents the complementarity determining regions CDR1, CDR2 and CDR3 in turn.

[0115] The nucleotide sequences of the genes encoding the nanobody mutants PN2M1, PN2M2 and PN8M are shown in SEQ ID NOs: 22-24, respectively.

[0116] Example 6 Experiment of fusion protein targeting degradation of PCNA protein

[0117] In order to achieve the targeted degradation of PCNA protein, in the present application, the above-mentioned nanobodies PN2, PN6, PN7, PN8 and nanobody mutants PN2M1, PN2M2 and PN8M are respectively fused with RBCC domain or its mutant RS79E 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. Specifically, taking pcDNA3.1 as a basic vector, the genes encoding the above-mentioned nanobodies PN2, PN6, PN7, PN8 and nanobody mutants PN2M1, PN2M2 and PN8M are connected with the gene encoding RBCC domain (the amino acid sequence is shown in SEQ ID NO: 27, and the nucleotide sequence is shown in SEQ ID NO: 30) or its mutant RS79E (the amino acid sequence is shown in SEQ ID NO: 28, and the nucleotide sequence is shown in SEQ ID NO: 31) through a second linker (the amino acid sequence is shown in SEQ ID NO: 29, and the nucleotide sequence is shown in SEQ ID NO: 32) for fusion, and the expression vector is constructed by homologous recombination, and the element arrangement structure of part of the expression vector is shown in ​ 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-RS79E-PN8, pcDNA3.1-RS79E-PN8M, respectively.

[0118] The amino acid sequences of the fusion proteins RBCC-PN2, RBCC-PN6, RBCC-PN7, RBCC-PN8, RBCC-PN2M1, RBCC-PN2M2, RBCC-PN8M, RS79E-PN8 and RS79E-PN8M are shown in SEQ ID NOs: 33-41, respectively.

[0119] The above-mentioned recombinant plasmids pcDNA3.1-RBCC-PN8, pcDNA3.1-RBCC-PN8M, pcDNA3.1-RS79E-PN8, pcDNA3.1-RS79E-PN8M, pcDNA3.1-RBCC-PN2M1, pcDNA3.1-RBCC-PN2M2 and the GFP plasmid were respectively electroporated into well-conditioned HEK 293T cells using a PEI reagent. After electroporation, the electroporation efficiency and protein expression were observed by the GFP plasmid.

[0120] The transfected cells at 48 h were collected, and then Western blot detection was performed. According to the Western blot results, the efficiency of protein targeted degradation at 48 h after transfection was calculated, and the results are shown in ​ and 11 .

[0121] As can be seen from ​ , the fusion proteins containing the nanobody PN8 and the nanobody mutant PN8M have good degradation effect on the PCNA antigen, and the fusion protein containing the nanobody mutant PN8M has a degradation efficiency of about 60% on the PCNA antigen; further, when the RBCC domain mutant RS79E is fused with the nanobody mutant PN8M, the degradation efficiency on the PCNA antigen is significantly improved.

[0122] As can be seen from ​ , the fusion proteins containing the nanobody mutants PN2M1 and PN2M2 have good degradation effect on the PCNA antigen.

[0123] Finally, the degradation pathway of the fusion protein targeted degradation of the PCNA protein was explored.

[0124] Specifically, 3 parts of pcDNA3.1-RBCC-PN8M recombinant plasmid and GFP plasmid are electroporated into well-conditioned HEK 293T cells, and 24 hours after transfection, corresponding MG132 and BafA1 are added to 2 parts of cells with pcDNA3.1-RBCC-PN8M recombinant plasmid, and Western blot detection is performed after continued culture, and the results are shown in ​ .

[0125] As can be seen from ​ , the fusion protein in the present application targets the degradation of PCNA through the ubiquitin-proteasome pathway and the lysosome pathway at the same time, and the lysosome pathway is dominant, and compared with the control group, the level of phosphorylated p53 is obviously improved after the experimental group RS79E-PN8M targets the degradation of PCNA. ​ b). The results show that the fusion protein in the present application targets the degradation of PCNA protein through the ubiquitin-proteasome pathway or the autophagy-lysosome pathway.

[0126] To sum up, the present application designs a nano antibody specifically targeting PCNA by using artificial intelligence technology, which has good specificity, high affinity and can efficiently bind to PCNA antigen; further, the nano antibody is rationally designed by artificial intelligence to obtain a nano antibody mutant with higher affinity; further, the nano antibody or nano antibody mutant with high affinity and good specificity is fused with the RBCC domain or its mutant to obtain a fusion protein, which can specifically bind to the target protein through its nano antibody domain, start the protein degradation pathway and realize the targeted degradation of PCNA protein.

[0127] The above-described embodiments only express the embodiments of the present application, which are described in detail and specifically, but should not be understood as limiting the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the present application patent should be subject to the appended claims.

Claims

1. A nanobody against PCNA protein, characterized in that, Including complementarity determination region CDR1, complementarity determination region CDR2 and complementarity determination region CDR3; The amino acid sequence of the complementarity-determining region CDR1 is shown in SEQ ID NO:1, the amino acid sequence of the complementarity-determining region CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of the complementarity-determining region CDR3 is shown in any one of SEQ ID NO:3-6.

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

10.

3. The anti-PCNA protein nanobody according to claim 2, characterized in that, The nanobody is selected from any of the following: A1) The amino acid sequence is shown in any one of SEQ ID NO:11-14; A4) An amino acid sequence obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in A1).

4. A nanobody mutant against PCNA protein, characterized in that, The nanobody mutant is selected from any one of the following: B1) The amino acid sequence is shown in any one of SEQ ID NO:15-17; B4) An amino acid sequence obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in B1). 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 at least the following site: H113K.

5. A fusion protein, characterized in that, The fusion protein is obtained by linking the nanobody of any one of claims 1-3 or the nanobody mutant of claim 4 with the RBCC domain or a mutant thereof through a second connector; 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. The amino acid sequence of the fusion protein is shown in any one of SEQ ID NO: 37, 38, 39, and 41.

6. A biomaterial, characterized in that, The biomaterial is selected from any one of the following: D1) A nucleic acid molecule encoding the nanobody of any one of claims 1-3, the nanobody mutant of claim 4, or the fusion protein of claim 5; D2) A recombinant vector containing the nucleic acid molecules described in D1); D3) Recombinant cells containing the nucleic acid molecule described in D1) or the recombinant vector described in D2).

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises any one of the nanobodies of claims 1-3, the nanobodies mutant of claim 4, the fusion protein of claim 5, or the biomaterial of claim 6, and a pharmaceutically acceptable carrier.

8. A method for preparing a nanobody according to any one of claims 1-3, a nanobody mutant according to claim 4, or a fusion protein according to claim 5, characterized in that, Includes the following steps: The recombinant cells of claim 6 are cultured and induced to express the desired expression to obtain a culture. Isolate the nanobody, the nanobody mutant, or the fusion protein from the culture.

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