Application of IGF2BP1 fragment in preparation of inhibition tool for screening and blocking interaction between IGF2BP1 and SOX2 protein

Abstract

The invention discloses an application of an IGF2BP1 fragment in preparation of an inhibition tool for screening and blocking the interaction between IGF2BP1 and SOX2 protein, and the IGF2BP1 fragment is an amino acid sequence from 161st to 578th of the IGF2BP1, and is shown as SEQ ID NO.01. The invention also discloses an application of the IGF2BP1 fragment in preparation of an inhibition tool for screening and blocking the interaction between IGF2BP1 and SOX2 protein. According to the present invention, the 161-578 amino acid fragment (IGF2BP1 [delta] C (161-578)) of the IGF2BP1 protein is the minimum functional structure domain of the IGF2BP1 protein directly interacting with the SOX2 protein, and the fragment sequence provides the clear targeting site for the subsequent accurate blocking of the IGF2BP1-SOX2 interaction through the dual verification of the bimolecular fluorescence complementation and the co-immunoprecipitation;

Description

Application of IGF2BP1 fragment in the preparation of inhibitory tools for screening the interaction between IGF2BP1 and SOX2 protein Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of the IGF2BP1 fragment in the preparation of inhibitory tools for screening and blocking the interaction between IGF2BP1 and SOX2 proteins. Background Technology

[0002] Bimolecular fluorescence complementation (BFF) is a direct and rapid technique for determining the localization and interaction of target proteins in living cells. This technique cleverly fuses two complementary fragments of a fluorescent protein molecule with the target protein for expression. If the fluorescent protein activity is restored, it indicates that the two target proteins have interacted. Immunoprecipitation is a classic method for studying protein-protein interactions, based on the specific interaction between antibodies and antigens. This technique is based on the principle that if protein X interacts with protein Y in solution or in cells, the complex can be precipitated by antibodies against either protein. Both methods are widely used in experiments identifying protein-protein interactions.

[0003] SOX2 protein is a potent transcription factor that promotes multiple aspects of stem cell stemness maintenance and self-renewal, as well as various malignant processes in tumors (such as tumorigenesis, tumor cell proliferation, migration, invasion and metastasis, maintenance of tumor stem cell characteristics, and chemotherapy resistance). IGF2BP1 and SOX2 proteins not only play important roles in tissue and organ development, but they are also closely related to the malignant progression of tumors, with IGF2BP1 participating in RNA m6A methylation modification. IGF2BP1 and SOX2 exhibit functional overlap in the malignant progression of tumors.

[0004] Existing research shows that SOX2 is highly expressed in various tumors and promotes tumor stem cell self-renewal and tumor progression by regulating downstream target genes. For example, in esophageal squamous cell carcinoma, SOX2 maintains tumor stemness and enhances chemotherapy resistance; in hepatocellular carcinoma, SOX2 synergizes with metastasis-associated transcription factors to promote tumor invasion and distant metastasis. Similarly, IGF2BP1, as an RNA-binding protein, stabilizes oncogene mRNA by recognizing m6A modification sites, playing a pro-cancer role in solid tumors such as liver cancer, breast cancer, and colon cancer. Studies have shown that upregulation of IGF2BP1 can enhance tumor cell proliferation and migration, exhibiting "fetal protein" characteristics during embryonic development and tumorigenesis, and reproducing in adult tumor tissues.

[0005] Furthermore, the literature reports indirect associations between IGF2BP1 and SOX2 in the tumor microenvironment. For example, in endometrial cancer, lncRNA linc01194 promotes SOX2 expression by binding to IGF2BP1, thereby driving tumor progression; in hepatocellular carcinoma, IGF2BP1 stabilizes oncogene mRNAs such as MYC and co-regulates cell cycle and stemness markers with SOX2. However, these studies mainly focus on the post-transcriptional regulation of SOX2 mRNA by IGF2BP1 (such as m6A-mediated enhanced stability), while evidence for direct interaction between IGF2BP1 and SOX2 proteins is limited. Only preliminary reports suggest that the two may form a complex through the KH domain, but precise location and functional verification of the binding domain are lacking.

[0006] In cancer treatment strategies, blocking protein-protein interactions (PPIs) has become a hot topic, but the development of inhibitors targeting the IGF2BP1-SOX2 axis has lagged behind. Existing PPI inhibitors are mostly small molecules or antibody-based drugs, lacking sufficient affinity and specificity, and struggling to penetrate tumor cells. Short peptide aptamers, as an emerging tool, offer advantages such as high affinity, ease of synthesis, and low immunogenicity; however, screening for specific short peptides targeting the IGF2BP1-SOX2 binding interface still faces challenges, including the lack of efficient primary screening platforms and high false-positive rates.

[0007] Therefore, screening for small molecule drugs that target the binding domain of the IGF2BP1 protein that interacts with the SOX2 protein is of great significance for establishing therapeutic targets for esophageal squamous cell carcinoma. Accurate identification of this binding domain and screening for high-affinity short peptide aptamers will provide a foundation for designing novel PPI inhibitors and promote the development of precision oncology. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of the IGF2BP1 fragment in the preparation of inhibitory tools for screening and blocking the interaction between IGF2BP1 and SOX2 protein.

[0009] The technical solution of the present invention is as follows:

[0010] The application of the IGF2BP1 fragment in the preparation of inhibitory tools for screening the interaction between IGF2BP1 and SOX2 protein, wherein the IGF2BP1 fragment is the amino acid sequence of IGF2BP1 from position 161 to 578, as shown in SEQ ID NO.01.

[0011] In a preferred embodiment of the present invention, the nucleotide sequence of the IGF2BP1 fragment is shown in SEQ ID NO. 02.

[0012] An expression vector loaded with a nucleotide sequence encoding an IGF2BP1 fragment, wherein the IGF2BP1 fragment is the amino acid sequence of IGF2BP1 from position 161 to 578, as shown in SEQ ID NO.01.

[0013] In a preferred embodiment of the present invention, the nucleotide sequence is as shown in SEQ ID NO.02.

[0014] In a preferred embodiment of the invention, it is based on pBiFc-VN173.

[0015] The above expression vector was used in the preparation of an inhibitory tool that blocks the interaction between IGF2BP1 and SOX2 proteins.

[0016] A method for screening inhibitory tools that block the interaction between IGF2BP1 and SOX2 proteins, comprising the following steps:

[0017] (1) The nucleotide sequence encoding the IGF2BP1 fragment is directionally inserted into the first vector to obtain the first expression vector. The IGF2BP1 fragment is the amino acid sequence of IGF2BP1 from position 161 to 578, as shown in SEQ ID NO.01.

[0018] (2) Insert the candidate peptides into the second vector to obtain the second expression vector;

[0019] (3) The first expression vector obtained in step (1) and the second expression vector obtained in step (2) were co-transfected into HEK293T cells, and the fluorescence signal generated by bimolecular fluorescence complementarity was observed.

[0020] (4) The candidate peptides that produce fluorescent signals are verified by immunoprecipitation to confirm their specific binding to the IGF2BP1 fragment.

[0021] In a preferred embodiment of the present invention, the nucleotide sequence is as shown in SEQ ID NO.02.

[0022] In a preferred embodiment of the present invention, the first carrier is pBiFc-VN173.

[0023] In a preferred embodiment of the present invention, the second carrier is pBiFc-VC155.

[0024] The beneficial effects of this invention are:

[0025] 1. This invention clarifies that the amino acid fragment at positions 161–578 of the IGF2BP1 protein (i.e., IGF2BP1ΔC(161-578)) is the smallest functional domain that directly interacts with the SOX2 protein. The sequence of this fragment is verified by both bimolecular fluorescence complementation and immunoprecipitation, providing a clear target site for subsequent precise blocking of the IGF2BP1-SOX2 interaction.

[0026] 2. This invention provides a novel application of this domain in the preparation of screening tools that inhibit the interaction between IGF2BP1 and SOX2, enabling those skilled in the art to directly use this fragment as a fixed target for high-throughput screening, avoiding non-specific interference caused by using full-length proteins, and greatly improving the specificity and success rate of screening.

[0027] 3. This invention establishes a complete method for screening inhibitory tools based on the combination of bimolecular fluorescence complementation technology and immunoprecipitation technology. The method has clear and reproducible steps and can be implemented using only conventional molecular biology methods without the need for expensive instruments or reagents.

[0028] 4. Using this invention, four core sequences of only 11 amino acids each were successfully obtained from the authorized restricted peptide aptamer library (ZL201410339641.X). After these short peptides are expressed in TrxA-restricted form, they can specifically bind to the 161–578 fragment of IGF2BP1, thereby effectively competitively blocking the full-length protein-protein interaction between IGF2BP1 and SOX2.

[0029] 5. This invention is versatile; by simply replacing other known binding domains, it can be extended to the rapid screening of other important protein-protein interaction inhibitors, demonstrating significant industrial application value and broad technical applicability. Attached Figure Description

[0030] Figure 1 is a schematic diagram of the cloning of the full-length IGF2BP1 and the corresponding truncated mutants in Example 1 of the present invention, as well as the construction of the bimolecular fluorescent complementary vector. Wherein: A: Design of full-length and truncated mutants of IGF2BP1; B: Samples 1, 2, 3, and 4 represent the gel electrophoresis of PCR products of IGF2BP1ΔC (479-578), IGF2BP1ΔC (399-578), IGF2BP1ΔC (271-578), and IGF2BP1ΔC (161-578), respectively; C: Samples 1, 2, 3, 4, and 5 represent pBiFc-VC155-IGF2BP1ΔC (161-578), pBiFc-VC155-IGF2BP1ΔC (271-578), pBiFc-VC155-IGF2BP1ΔC (399-578), pBiFc-VC155-IGF2BP1ΔC (479-578), and pBiFc-VC155- IGF2BP1 expression vector was identified by gel electrophoresis after enzyme digestion.

[0031] Figure 2 is a schematic diagram of the binding domains of IGF2BP1 and SOX2 proteins determined by the bimolecular fluorescence complementation experiment in Example 1 of the present invention (red fluorescence is the internal control), including the fluorescence image after co-transfection of full-length and truncated SOX2 and full-length and truncated IGF2BP1 expression vectors into HEK293T cells.

[0032] Figure 3 is a schematic diagram of the interaction between the truncated mutants of IGF2BP1 and the SOX2 protein verified by the immunoprecipitation experiment in Example 2 of this invention. Western blot results showed that lane 1 (Control) as a blank control showed neither FLAG nor IGF2BP1 bands; FLAG antibody detected bands around 35KD in all four experimental groups; HA antibody detected bands of approximately 35KD, 55KD, 60KD, and 70KD in lanes 2-5, respectively. This indicates that each truncated mutant of IGF2BP1 can be precipitated by the FLAG-tagged SOX2 protein, demonstrating an interaction between each truncated mutant of IGF2BP1 and the SOX2 protein. It was determined that the domain in which the interaction occurs between IGF2BP1 and SOX2 protein is located in IGF2BP1ΔC (161-578), i.e., between amino acid sites 1-160 of the IGF2BP1 protein.

[0033] Figure 4 shows the candidate peptide aptamers that target the IGF2BP1ΔC (161-578) domain obtained from the peptide aptamer library in Example 3 of the present invention.

[0034] Figure 5 shows the identification of candidate peptide aptamers that target and bind to the IGF2BP1ΔC (161-578) domain in Example 3 of the present invention. Detailed Implementation

[0035] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0036] Example 1: Determination of the interaction binding domains between IGF2BP1 and SOX2 proteins using bimolecular fluorescence complementation technique.

[0037] (1) Construction of pBiFc-VC155-IGF2BP1 expression vector: Using cDNA reverse transcribed from RNA of the human esophageal squamous cell carcinoma line KYSE450 as a template, high-fidelity PCR amplification of IGF2BP1 cDNA was performed using primers as shown in Table 1. After digestion of the amplification product with restriction endonucleases SalI and KpnI, it was inserted into the pBiFcVC155 vector (with a FLAG tag, purchased from Addgene) to form the pBiFc-VC155-IGF2BP1 expression vector. The amplification primer sequences are shown in Table 1. Enzyme digestion identification and sequencing results showed that the expected IGF2BP1 target expression vector was obtained and can be used for dual fluorescence complementary vectors (Figure 1, Table 2).

[0038] Table 1: Primers used for PCR amplification

[0039] Target fragment primer sequences (5'-3') pBiFc-VC155-IGF2BP1 Forward: 5'-acgcgtcgacatgaacaagctttacatcggcaacc-3' (SEQ ID NO.03) Reverse: 5'-ggggtacctcacttcctccgtgcctgg-3' (SEQ ID NO.04) pBiFc-VC155-IGF2BP1ΔC (479-578) Forward: 5'-acgcgtcgacatgaacaagctttacatcggcaacc-3' (SEQ ID NO.05) Reverse: 5'-ggggtaccagttctcctccttgagtttgcc-3' (SEQ ID NO.05) NO.06) pBiFc-VC155-IGF2BP1ΔC (399-578) Forward:5'-acgcgtcgacatgaacaagctttacatcggcaacc-3' (SEQ ID NO.07) Reverse:5'-ggggtaccggagctatagggagcagcccc -3' (SEQ ID NO.08) pBiFc-VC155-IGF2BP1ΔC (271-578) Forward:5'-acgcgtcgacatgaacaagctttacatcggcaacc-3' (SEQ ID NO.09) Reverse:5'-ggggtaccgccgttttggtgtccttagcc-3' (SEQ ID NO.10) pBiFc-VC155-IGF2BP1ΔC (161-578) Forward:5'-acgcgtcgacatgaacaagctttacatcggcaacc-3' (SEQ ID NO.11) Reverse:5'-ggggtaccgtgctatctgctcatcgggga -3' (SEQ ID NO.12) surface

[0040] Table 2: Full-length and truncated mutants of IGF2BP1 obtained by PCR

[0041]

[0042] The reaction conditions for high-fidelity PCR amplification in this step are as follows:

[0043] (2) Construction of IGF2BP1 truncated protein expression vectors: Using pBiFc-VC155-IGF2BP1 as a template, high-fidelity PCR amplification of truncated IGF2BP1 was performed using the primer pairs in Table 1 above. After digesting the amplification products with restriction endonucleases SalI and KpnI, they were inserted into the pBiFcVC155 vector to form pBiFc-VC155-IGF2BP1ΔC (479-578), pBiFc-VC155-IGF2BP1ΔC (399-578), pBiFc-VC155-IGF2BP1ΔC (271-578) and pBiFc-VC155-IGF2BP1ΔC (161-578) expression vectors. The results of enzyme digestion identification and sequencing showed that the expected IGF2BP1 target expression vectors that can be used for dual fluorescence complementary expression were obtained (Figure 1, Table 2).

[0044] The reaction conditions for high-fidelity PCR amplification in this step are as follows:

[0045]

[0046] (3) Using an endotoxin-free plasmid extraction kit, extract the correctly sequenced expression vectors for bimolecular fluorescence complementation and the pDsRED2-C1 vector (purchased from Clontech). Using Lipofectamine 2000 transfection reagent, 200 ng of the expression vectors pBiFc-VN173-SOX2, pBiFc-VN173-SOX2ΔN (1-167), pBiFc-VN173-SOX2ΔN (1-237), pBiFc-VN173-SOX2ΔN (1-257), and pBiFc-VN173-SOX2ΔN (1-277), and 200 ng of the expression vectors pBiFc-VC155-IGF2BP1, pBiFc-VC155-IGF2BP1ΔC (479-578), and pBiFc-VC155-IGF2BP1ΔC (479-578) were transfected. The expression vectors IGF2BP1ΔC (399-578), pBiFc-VC155-IGF2BP1ΔC (271-578), and pBiFc-VC155-IGF2BP1ΔC (161-578), along with 50 ng of pDsRED2-C1 (using pDsRed2-C1 transfection as an internal control), were used to form 25 different combinations, which were co-transfected into 12-well HEK293T cells. The 25 combinations are as follows:

[0047] pBiFc-VN173-SOX2 and pBiFc-VC155-IGF2BP1

[0048] pBiFc-VN173-Sox2 and pBiFc-VC155-IGF2BP1ΔC (479-578).

[0049] pBiFc-VN173-SOX2 and pBiFc-VC155-IGF2BP1ΔC (399-578).

[0050] pBiFc-VN173-SOX2 and pBiFc-VC155-IGF2BP1ΔC (271-578).

[0051] pBiFc-VN173-SOX2 and pBiFc-VC155-IGF2BP1ΔC(161-578).

[0052] pBiFc-VN173-SOX2ΔN(1-167) and pBiFc-VC155-IGF2BP1,

[0053] pBiFc-VN173-SOX2ΔN (1-167) and pBiFc-VC155-IGF2BP1ΔC (479-578).

[0054] pBiFc-VN173-SOX2ΔN (1-167) and pBiFc-VC155-IGF2BP1ΔC (399-578).

[0055] pBiFc-VN173-SOX2ΔN (1-167) and pBiFc-VC155-IGF2BP1ΔC (271-578).

[0056] pBiFc-VN173-SOX2ΔN (1-167) and pBiFc-VC155-IGF2BP1ΔC (161-578).

[0057] pBiFc-VN173-SOX2 ΔN (1-237) and pBiFc-VC155-IGF2BP1,

[0058] pBiFc-VN173-SOX2 ΔN (1-237) and pBiFc-VC155- IGF2BP1ΔC (479-578).

[0059] pBiFc-VN173-SOX2 ΔN (1-237) and pBiFc-VC155- IGF2BP1ΔC (399-578).

[0060] pBiFc-VN173-SOX2 ΔN (1-237) and pBiFc-VC155-IGF2BP1ΔC (271-578).

[0061] pBiFc-VN173-SOX2ΔN (1-237) and pBiFc-VC155-IGF2BP1ΔC (161-578).

[0062] pBiFc-VN173-SOX2ΔN(1-257) and pBiFc-VC155-IGF2BP1,

[0063] pBiFc-VN173-SOX2ΔN (1-257) and pBiFc-VC155-IGF2BP1ΔC (479-578).

[0064] pBiFc-VN173-SOX2ΔN (1-257) and pBiFc-VC155-IGF2BP1ΔC (399-578).

[0065] pBiFc-VN173-SOX2ΔN (1-257) and pBiFc-VC155-IGF2BP1ΔC (271-578).

[0066] pBiFc-VN173-SOX2ΔN (1-257) and pBiFc-VC155-IGF2BP1ΔC (161-578).

[0067] pBiFc-VN173-SOX2ΔN(1-277) and pBiFc-VC155-IGF2BP1,

[0068] pBiFc-VN173-SOX2ΔN (1-277) and pBiFc-VC155-IGF2BP1ΔC (479-578).

[0069] pBiFc-VN173-SOX2ΔN (1-277) and pBiFc-VC155-IGF2BP1ΔC (399-578).

[0070] pBiFc-VN173-SOX2ΔN (1-277) and pBiFc-VC155-IGF2BP1ΔC (271-578).

[0071] pBiFc-VN173-SOX2ΔN (1-277) and pBiFc-VC155-IGF2BP1ΔC (161-578);

[0072] For the above pBiFc-VN173-SOX2, pBiFc-VN173-SOX2ΔN (1-167), pBiFc-VN173-SOX2 ΔN (1-237), pBiFc-VN173-SOX2 ΔN (1-257) and pBiFc-VN173-SOX2ΔN (1-277), please refer to CN106519006A (ZL201611108011.7).

[0073] (4) After transfected HEK293T cells were cultured in a cell culture incubator for 24 h, fluorescence was observed under a fluorescence microscope. Preliminary results showed that the interaction region between IGF2BP1 protein and SOX2 protein was located at IGF2BP1ΔC (161-578) and SOX2ΔN (1-257), that is, amino acids 1-160 of IGF2BP1 protein and amino acids 278-317 of SOX2 protein are the regions where the two proteins bind to each other (Figure 2).

[0074] Example 2: Immunoprecipitation technique to verify the interaction binding domains of IGF2BP1 and SOX2 proteins.

[0075] (1) Transfection: according to the following four combinations:

[0076] pBiFc-VN173-SOX2 and pBiFc-VC155-IGF2BP1ΔC (479-578).

[0077] pBiFc-VN173-SOX2 and pBiFc-VC155-IGF2BP1ΔC (399-578).

[0078] pBiFc-VN173-SOX2 and pBiFc-VC155-IGF2BP1ΔC (271-578).

[0079] pBiFc-VN173-SOX2 and pBiFc-VC155-IGF2BP1ΔC(161-578).

[0080] The plasmid was transfected into HEK293T cells, and the cell proteins could be extracted for immunoprecipitation after 48 h.

[0081] (2) Immunoprecipitation: Thermo Scientific ChIP-Grade Protein A / G reagent was used to perform standard operations according to the instructions to harvest the protein. Since SOX2 and Flag tag are fused and expressed in pBiFc-VN173-SOX2, Flag tag antibody was used for immunoprecipitation.

[0082] (3) Western blot verification of the interaction between SOX2 and truncated IGF2BP1: The samples of immunoprecipitation were run by SDS electrophoresis, and the transfer membrane was incubated with Flag primary antibody (mouse source) and HA primary antibody (rabbit source) respectively, and identified with diluted secondary antibody (goat anti-rabbit) and secondary antibody (goat anti-mouse).

[0083] (4) Immunoprecipitation was used to verify the correctness of the interaction binding domain between IGF2BP1 and SOX2 proteins: The results showed that the Control group, as a blank control, had neither FLAG nor IGF2BP1 bands; FLAG antibody detected bands near 35 kD in all four experimental groups; HA antibody detected bands near 35 kD, 55 kD, 60 kD, and 70 kD in the four experimental groups, respectively (Figure 3). This indicates that each truncated IGF2BP1 mutant can be precipitated by SOX2 protein with the FLAG tag, indicating that there is an interaction between each truncated IGF2BP1 mutant and SOX2 protein. It was determined that the interaction domain between IGF2BP1 and SOX2 protein exists in IGF2BP1ΔC (161-578), that is, between amino acid sites 1-160 of the IGF2BP1 protein.

[0084] The amino acid sequence of the above IGF2BP1ΔC (161-578) is shown in SEQ ID NO.01: MNKLYIGNLNESVTPADLEKVFAEHKISYSGQFLVKSGYAFVDCPDEHWAMKAIETFSGKVELQGKRLEIEHSVPKKQRSRKIQIRNIPPQLRWEVLDSLLAQYGTVENCEQVNTESETAVVNVTYSNREQTRQAIMKLNGHQLENHALKVSYIPDEQIA;

[0085] Its nucleotide sequence is as SEQ ID NO.02 shows: atgaacaagctttacatcggcaacctcaacgagagcgtgacccccgcggacttggagaaagtgtttgcggagcacaagatctcctacagcggccagttcttggtcaaatccggc tacgccttcgtggactgcccggacgagcactgggcgatgaaggccatcgaaactttctccgggaaagtagaattacaaggaaaacgcttagagattgaacattcggtgcccaaaaaacaaag gagccggaaaattcaaatccgaaatattccaccccagctccgatgggaagtactggacagcctgctggctcagtatggtacagtagagaactgtgagcaagtgaacaccgagagtgagacgg cagtggtgaatgtcacctattccaaccgggagcagaccaggcaagccatcatgaagctgaatggccaccagttggagaaccatgccctgaaggtctcctacatccccgatgagcagatagca.

[0086] Example 3 Screening and identification of candidate peptide aptamers that specifically bind to the IGF2BP1ΔC (161-578) domain

[0087] (1) Construction of pBiFcVN173-IGF2BP1ΔC(161-578) expression vector: The open reading frame of IGF2BP1ΔC(161-578) with specific sticky ends obtained by high-fidelity PCR and enzyme digestion was directionally inserted into pBiFc-VN173 (purchased from Addgene) to obtain the pBiFcVN173-IGF2BP1ΔC(161-578) expression vector.

[0088] (2) Screening of specific peptide aptamers that bind to the IGF2BP1ΔC (161-578) domain: By random selection, 30 peptide aptamer expression vectors were randomly selected from the peptide aptamer library (CN104088019A, ZL201410339641.X) and co-transfected with pBiFcVN173-IGF2BP1ΔC (161-578) into HEK293T cells, and the appearance of fluorescence was observed (Figure 4).

[0089] (3) Identification method for IGF2BP1ΔC (161-578) peptide aptamers, specifically including the following steps:

[0090] a. Using the IGF2BP1ΔC(161-578) domain as a template, high-fidelity PCR amplification of IGF2BP1ΔC(161-578) was performed through primer design.

[0091] b. IGF2BP1ΔC(161-578) was transferred into the pBiFc-VN173 expression vector using an enzyme digestion and ligation method to obtain the pBiFc-VN173-IGF2BP1ΔC(161-578) expression vector.

[0092] c. Using a co-transfection method, pBiFcVN173-IGF2BP1ΔC (161-578) and pBiFcVC155-P38, pBiFcVC155-P140, pBiFcVC155-P144, and pBiFcVC155-P148 were transfected into HEK293T cells in groups. Immunoprecipitation was performed using Flag antibody, and the results were identified using HA antibody. The results are shown in Figure 5.

[0093] The results of this embodiment are as follows: Four short peptides with a core sequence of only 11 amino acids were successfully obtained from the above peptide aptamer library, as detailed below:

[0094] P38: YGSLRLVSEVG (SEQ ID NO. 13),

[0095] P140: VFGLTFNCFLM (SEQ ID NO. 14),

[0096] P144: LLCFILCFFIL (SEQ ID NO. 15),

[0097] P148:AVFSILFFPLL (SEQ ID NO. 16),

[0098] As shown in Figures 4 and 5, the above short peptides, when expressed in TrxA-bound form, can specifically bind to the 161–578 fragment of IGF2BP1, thereby effectively and competitively blocking the full-length protein-protein interaction between IGF2BP1 and SOX2.

[0099] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. The application of the IGF2BP1 fragment in the preparation of inhibitory tools for screening and blocking the interaction between IGF2BP1 and SOX2 protein, characterized in that: The IGF2BP1 fragment is the amino acid sequence of IGF2BP1 from position 161 to 578, as shown in SEQ ID NO.

01.

2. The use as described in claim 1, characterized in that: The nucleotide sequence of the IGF2BP1 fragment is shown in SEQ ID NO.

02.

3. An expression vector, characterized in that: The nucleotide sequence encoding the IGF2BP1 fragment is shown in SEQ ID NO.

01.

4. An expression vector as described in claim 3, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

02.

5. An expression vector as described in claim 3 or 4, characterized in that: It is based on pBiFc-VN173.

6. The use of the expression vector according to claims 3 to 6 in the preparation of an inhibitory tool that blocks the interaction between IGF2BP1 and SOX2 protein.

7. A method for screening inhibitory tools that block the interaction between IGF2BP1 and SOX2 proteins, characterized in that: The procedure includes the following steps: (1) Directing the insertion of the nucleotide sequence encoding the IGF2BP1 fragment into the first vector to obtain the first expression vector, wherein the IGF2BP1 fragment is the amino acid sequence of IGF2BP1 from position 161 to 578, as shown in SEQ ID NO.01; (2) Inserting the candidate peptides into the second vector to obtain the second expression vector; (3) Co-transfecting the first expression vector obtained in step (1) and the second expression vector obtained in step (2) into HEK293T cells and observing the fluorescence signal generated by bimolecular fluorescence complementarity; (4) Performing immunoprecipitation verification on the candidate peptides that generate fluorescence signals to confirm their specific binding to the IGF2BP1 fragment.

8. The method as described in claim 7, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

02.

9. The method as described in claim 7 or 8, characterized in that: The first carrier is pBiFc-VN173.

10. The method as described in claim 7 or 8, characterized in that: The second carrier is pBiFc-VC155.

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