Recombinant protein for specific targeted inhibition of STAT3 dimerization, and preparation method and application thereof

Abstract

The invention provides a recombinant protein for specifically inhibiting STAT3 dimerization in a targeted manner as well as a preparation method and application of the recombinant protein, and belongs to the technical field of molecular biology. The amino acid sequence of the recombinant protein is as shown in SEQ ID NO. 1. Researches find that the recombinant protein can inhibit STAT3 dimerization, and can significantly inhibit tumor activity by entering tumor cells to bind STAT3 and inhibit the STAT3 dimerization. Theoretical basis and gene resources are provided for tumor treatment, and more choices are provided for screening drugs for preventing and / or treating tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a recombinant protein that specifically targets and inhibits STAT3 dimerization, and a preparation method and application thereof. Background Art

[0002] Malignant tumors have become a serious threat to human health.

[0003] The Signal Transducer and Activator of Transcription (STAT) protein family has dual functions of signal transduction and transcriptional activation, and participates in important biological processes such as proliferation, differentiation, apoptosis, and immune regulation. Currently, seven STAT family members have been reported in mammalian cells (STAT1-4, STAT5A, STAT5B, and STAT6), among which STAT3 is persistently activated and expressed at high levels in a variety of malignant tumors (including solid tumors, leukemias, and lymphomas). It is a protein with dual functions of signal transduction and transcriptional activation, and promotes the occurrence of human cancer through immunosuppression.

[0004] The biological characteristics of STAT3 are closely related to its protein structure. The STAT3 protein consists of an amino terminus, a coiled-coil region, a linker region, an SH2 tyrosine polypeptide arm, and a transcriptional activation region at the carboxylic acid end. The amino terminus is the most conserved region of STAT3, providing an action site for transcription factors and regulatory proteins and assisting in DNA binding. The SH2 region contains three parallel β sheets for dimerization of the STAT3 protein. The STAT3 protein can be homopolymerized into dimers or heteropolymerized into dimers, for example, STAT3-STAT3, STAT1-STAT3, STAT5-STAT3 dimers, etc. The tyrosine (Tyr705) polypeptide arm is located between the SH2 region and the carboxylic acid end region and can bind to the SH2 region to form a dimer. The carboxylic acid end region can regulate the transcriptional activation of DNA, the phosphorylation of serine (Ser727) in the transcriptional activation region, and the phosphorylation of tyrosine (Tyr705) close to the carboxylic acid end, thereby regulating the activation of the STAT3 protein and regulating the transcription of target genes. The signal regulation of STAT3 can be summarized into the following four stages: activation of membrane receptors, activation of STAT3 protein, dimerization of STAT3 protein and initiation of cell transcription.

[0005] In recent years, the study of inhibitors targeting STAT3 has become a hot topic in the development of anti-tumor drugs. At present, there are two strategies for inhibiting the STAT3 signaling pathway: indirect and direct strategies: the indirect strategy is to block the upstream molecules of the STAT3 signaling pathway and indirectly inhibit the signal transduction function of STAT3, such as inhibiting JAK, Src, Abl, Lyn, etc.; while the mechanism of direct inhibition of STAT3 targets mainly includes three strategies, namely, inhibiting the phosphorylation of STAT3 monomers, inhibiting the formation of STAT3-STAT3 dimers, and inhibiting the binding of STAT3 entering the nucleus to DNA. According to the site of action, it can be mainly divided into STAT3 inhibitors acting on the SH2 domain, DNA binding domain, N-terminal domain, and C-terminal transcriptional activation domain. At present, most STAT3 inhibitors target the SH2 domain of STAT3 protein. Once this region is inhibited, it will lead to the loss of STAT3 function. In the past decade, scientists have developed a large number of peptide and peptidomimetic STAT3 inhibitors, STAT3 small molecule inhibitors, and a variety of active monomers with STAT3 inhibitory activity targeting the SH2 domain, but most inhibitors are still in the preclinical research stage, and there are currently no drugs on the market. The main reasons are as follows: ① STAT3 inhibitors targeting the SH2 domain mainly work by interfering with the phosphorylation of STAT3 protein Y705 and STAT3 dimerization. However, STAT3-STAT3 dimerization is a protein-protein interaction. Due to the large interaction area, it is difficult to block its interaction with small molecules; ② The activity of currently developed small molecule inhibitors is generally at the micromolar level. In the complex human environment, a higher dose is required to achieve the effect of inhibiting STAT3, which undoubtedly increases the possibility of toxicity to normal tissues due to drug off-target effects; ③ The SH2 domain is highly conserved in a variety of tyrosine proteins, and its site is rich in positively charged residues, which requires a high negative charge carried by small molecules. Therefore, inhibitors designed for the SH2 site generally have the disadvantages of low drugability and large adverse reactions. Therefore, starting from the endogenous regulatory mechanism, research on how to inhibit the abnormal transcriptional regulatory activity of STAT3 in tumors has received increasing attention. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a recombinant protein that specifically targets and inhibits STAT3 dimerization, and a preparation method and application thereof. The recombinant protein can enter tumor cells, bind to and inhibit STAT3 dimerization, thereby inhibiting the activity of tumor cells.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a recombinant protein for specifically targeting and inhibiting STAT3 dimerization. The amino acid sequence of the recombinant protein is shown in SEQ ID NO.1.

[0009] The present invention provides a gene encoding the above recombinant protein.

[0010] Preferably, the nucleotide sequence of the gene is shown as SEQ ID NO.2.

[0011] The present invention provides a biological material containing the above-mentioned recombinant protein or gene, which is any one of the following:

[0012] (1) a recombinant expression vector comprising the above-mentioned recombinant protein or gene;

[0013] (2) Recombinant microorganisms containing the above-mentioned recombinant proteins or genes;

[0014] (3) a recombinant microorganism comprising the recombinant expression vector described in (1);

[0015] (4) a recombinant cell comprising the above-mentioned recombinant protein or gene;

[0016] (5) A recombinant cell comprising the recombinant expression vector described in (1).

[0017] The present invention provides a method for preparing the above recombinant protein, comprising the following steps:

[0018] The recombinant expression vector containing the above recombinant protein or gene is transformed into the recombinant microorganism, cultured, the recombinant expression vector is extracted, and then transfected into recombinant cells, expanded and cultured, extracted and purified to obtain the recombinant protein.

[0019] The present invention provides an application of the above-mentioned recombinant protein, gene, biological material or the recombinant protein obtained by the preparation method in specific targeted inhibition of STAT3 dimerization.

[0020] The present invention provides an application of the above-mentioned recombinant protein, gene, biological material or the recombinant protein obtained by the preparation method in the preparation of drugs for preventing and / or treating tumors.

[0021] Preferably, the tumor includes one or both of lung cancer and breast cancer.

[0022] The present invention provides a medicine for preventing and / or treating tumors, and the medicine comprises the above-mentioned recombinant protein, gene or recombinant protein obtained by the preparation method.

[0023] Preferably, the medicament comprises pharmaceutically acceptable excipients.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides a recombinant protein that specifically targets and inhibits STAT3 dimerization, and a preparation method and application thereof. The present invention has found that the recombinant protein can inhibit STAT3 dimerization, enter tumor cells, bind to STAT3 and inhibit its dimerization, thereby significantly inhibiting tumor activity. The present invention provides a theoretical basis and gene resources for treating tumors, and provides a new option for screening drugs for preventing and / or treating tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the construction of GV219 / TAT-SH2-His plasmid.

[0027] Figure 2 Identify the purified recombinant protein by Western blot.

[0028] Figure 3 Immunofluorescence double staining shows the cellular localization of recombinant protein SH2.

[0029] Figure 4 This is the result of SH2 recombinant protein inhibiting STAT3 dimerization.

[0030] Figure 5 The results show that SH2 recombinant protein inhibits the proliferation of human non-small cell lung cancer cells and human breast cancer cells.

[0031] Figure 6 The SH2 recombinant protein inhibits the migration of human non-small cell lung cancer cells and human breast cancer cells.

[0032] Figure 7 The SH2 recombinant protein inhibits the invasion of human non-small cell lung cancer cells and human breast cancer cells.

[0033] Figure 8 The SH2 recombinant protein inhibited the growth of subcutaneous human non-small cell lung cancer cells and human breast cancer cells in nude mice. DETAILED DESCRIPTION

[0034] The present invention provides a recombinant protein for specifically targeting and inhibiting STAT3 dimerization. The amino acid sequence of the recombinant protein is shown in SEQ ID NO.1.

[0035] The present invention provides a gene encoding the above recombinant protein. As one embodiment, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0036] The present invention provides a biological material containing the above-mentioned recombinant protein or gene, which is any one of the following:

[0037] (1) a recombinant expression vector comprising the above-mentioned recombinant protein or gene;

[0038] (2) Recombinant microorganisms containing the above-mentioned recombinant proteins or genes;

[0039] (3) a recombinant microorganism comprising the recombinant expression vector described in (1);

[0040] (4) a recombinant cell comprising the above-mentioned recombinant protein or gene;

[0041] (5) A recombinant cell comprising the recombinant expression vector described in (1).

[0042] In the present invention, the backbone plasmid of the recombinant expression vector includes a GV219 vector. The preparation method of the recombinant expression vector includes linearizing the GV219 vector using XhoI and KpnI to obtain a linearized vector, using primers shown in sequences SEQ ID NO.4 to SEQ ID NO.5, using human mRNA as a template, amplifying the obtained HA-Shf gene sequence and connecting it with the linearized vector to obtain a GV219 / HA-SHF vector, using the GV219 / HA-SHF vector as a template, amplifying the SH2 sequence containing SHF, introducing a TAT sequence and a His tag protein at the N-terminus and C-terminus of the obtained SHF-SH2 sequence, respectively, to obtain a PCR product, digesting the PCR product with Xho I and KpnI to obtain a PCR digestion product, digesting the GV219 plasmid with Xho I and Kpn I, and connecting it with the PCR digestion product to obtain a GV219 / TAT-SH2-His plasmid, that is, a recombinant expression vector. The human mRNA is extracted from human liver cancer cells HepG2. After centrifugation of HepG2 cells growing in the logarithmic phase, the supernatant is removed and 1 mL of total RNA extraction reagent Trizol is added to extract mRNA and then the concentration is measured, wherein the human liver cancer cells HepG2 are cells that highly express SHF. The recombinant microorganism is preferably a bacterium, such as Escherichia coli, and the Escherichia coli is Escherichia coli DH5α. The preparation method of the recombinant microorganism includes transforming the above-mentioned recombinant expression vector into Escherichia coli to obtain a recombinant microorganism. The recombinant cell includes a mammalian cell, such as HEK293 cell.

[0043] The present invention provides a method for preparing the above recombinant protein, comprising the following steps:

[0044] The recombinant expression vector containing the above recombinant protein or gene is transformed into the recombinant microorganism, cultured, the recombinant expression vector is extracted, and then transfected into recombinant cells, expanded and cultured, extracted and purified to obtain the recombinant protein.

[0045] The recombinant protein prepared by the present invention can inhibit STAT3 dimerization by specific targeting, enters tumor cells, binds to STAT3, and thus inhibits STAT3 dimerization. The recombinant protein can also significantly inhibit tumor activity.

[0046] The present invention provides an application of the above-mentioned recombinant protein, gene, biological material or the recombinant protein obtained by the preparation method in specific targeted inhibition of STAT3 dimerization.

[0047] The present invention provides an application of the above-mentioned recombinant protein, gene, biological material or the recombinant protein obtained by the preparation method in the preparation of drugs for preventing and / or treating tumors.

[0048] In the present invention, the tumor includes one or both of lung cancer and breast cancer, the lung cancer includes non-small cell lung cancer, such as non-small cell lung cancer cell A549, and the breast cancer cell includes breast cancer cell MB231.

[0049] The present invention provides a medicine for preventing and / or treating tumors, and the medicine comprises the above-mentioned recombinant protein, gene or recombinant protein obtained by the preparation method.

[0050] In the present invention, the drug includes pharmaceutically acceptable excipients. The excipients include excipients or solvents, etc. The drug of the present invention can use the above recombinant protein, gene or recombinant protein obtained by the preparation method as an active ingredient in combination with other active ingredients that inhibit tumors, or can use the above recombinant protein, gene or recombinant protein obtained by the preparation method as the only active ingredient to inhibit tumors.

[0051] In the present invention, the GV219 vectors were purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd. Amplification used Takara's high-fidelity PCR amplification kit, and the molecular cloning tool enzyme was purchased from Takara. The plasmid extraction kit and gel recovery kit were purchased from Beijing Kangwei Century Biotechnology Co., Ltd. The E. coli competent cell was DH5α competent cell, purchased from Kangwei Century Biotechnology Co., Ltd. Ni-charged MagBeads were purchased from GenScript Biotechnology Co., Ltd.

[0052] The LB medium formula of the present invention is shown in Molecular Cloning. Trizol RNA extraction, plasmid extraction, PCR reaction, endonuclease digestion, DNA fragment recovery, ligation and transformation in the method mentioned in the present invention are all conventional operation methods in the field of genetic engineering research, and specific reference is made to Molecular Cloning.

[0053] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0054] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0055] Example 1

[0056] Cloning of P39 gene

[0057] 1. Design of SH2 gene

[0058] A new recombinant protein was designed to specifically target and inhibit STAT3 dimerization, named SH2 or SH2 recombinant protein.

[0059] The amino acid sequence of the SH2 recombinant protein is as follows:

[0060] MYGRKKRRQRRR WYHGAISRTDAENLLRLCKEASYLVRNSETSKNDFSLSLKSSQGFMHMKLSR TKE HKYVLGQNSPPFSSVPEIVHHYASRKLPIKGAEHMSLLYPV HHHHHH (SEQ ID NO. 1);

[0061] The recombinant protein has a total length of 114 amino acids, of which the underlined 96 amino acids are SHF protein; methionine (M) for translation of the start codon ATG is introduced at the N-terminus, and "YGRKKRRQRRR" (SEQ ID NO.15) is a TAT sequence, which is a protein transduction signal peptide to guide the fusion protein into the cell; "HHHHHH" (SEQ ID NO.16) is introduced at the C-terminus as a His-tagged protein, which is a screening and identification tag.

[0062] The nucleotide sequence encoding the above recombinant protein is as follows:

[0063] ATGTACGGTCGTAAAAAACGTCGTCAGCGTCGCCGT TGGTATCACGGGGCCATCAGCCGAACC

[0064] GACGCCGAGAACCTGCTCCGGCTGTGCAAAGAGGCCAGCTACCTGGTGCGCAACAGTGAGACCAG

[0065] CAAGAATGACTTCTCCCTCTCCCTCAAGAGCAGCCAGGGATTCATGCACATGAAGCTGTCCCGAAC

[0066] CAAGGAACACAAATATGTGCTGGGCCAGAACAGCCCGCCCTTCAGCAGCGTCCCTGAAATTGTGC

[0067] ACCACTATGCCAGCCGCAAGCTACCCATTAAGGGAGCCGAACACATGTCCCTGCTCTACCCTGTG C

[0068] ACCACCACCATCACCACTAG(SEQ ID NO.2);

[0069] The nucleic acid sequence of the recombinant protein includes 345 nucleotides, of which the underlined 288 nucleotides encode SHF protein; ATG is the start codon, TACGGTCGTAAAAAACGTCGTCAGCGTCGCCGT (SEQ ID NO.17) is the nucleotide sequence encoding the TAT sequence, CACCACCACCATCACCAC (SEQ ID NO.18) is the nucleotide sequence encoding the His tag protein, and TAG is the stop codon.

[0070] At the same time, a control group was set up: the recombinant protein in the control group was named SC or SC recombinant protein.

[0071] The amino acid sequence of the SC recombinant protein is as follows:

[0072] MYGRKKRRQRRRSFKKSSNHRDPKMAYAHLLGLELMSYPRPQCKRETSYKMHNSHIFVVNYGL RVKPGSPHFHKAELSWSEVEERNSATSKSIGLYLILQLSDVASLTHHHHHHH (SEQ ID NO. 3).

[0073] 2. Obtaining SH2 recombinant protein and constructing expression vector

[0074] Construction of GV219 / HA-SHF vector: The commercial GV219 vector was linearized with XhoI and KpnI to obtain a linearized vector; Takara's high-fidelity PCR amplification kit was used, using primers (primer sequences containing exchange pairing bases, restriction sites, and partial sequences of the 5' end of the target gene for PCR to fish out the target gene), human mRNA was used as a template, the amplified HA-Shf gene sequence was connected to the linearized vector, and the recombinant clones were selected for restriction digestion and sequencing identification to obtain the GV219 / HA-SHF vector. The human mRNA was extracted from human liver cancer cells HepG2. After centrifugation of HepG2 cells growing in the logarithmic phase, the supernatant was removed and 1 mL of total RNA extraction reagent Trizol was added to extract mRNA and then the concentration was measured. Among them, human liver cancer cells HepG2 are cells that highly express SHF.

[0075] The primer sequences used in the amplification are: F: ACGGGCCCTCTAGACTCGAGCGCCACCATGTACCCTTATGATGTCCCAGACTATGCTATGCAGCAGG AGGGAGGACCC (SEQ ID NO.4); R: TTAAACTTAAGCTTGGTACCCTAAAGAGTCCGGATGGCCACAGGG (SEQ ID NO.5).

[0076] The Shf sequence in the GV219 / HA-SHF vector was verified by sequencing:

[0077]

[0078] The constructed GV219 / HA-SHF vector was used as a template and the SH2 sequence containing SHF was amplified using Takara's high-fidelity PCR amplification kit. The resulting SHF-SH2 sequence introduced a TAT sequence and a His tag protein at the N-terminus and C-terminus, respectively, and endonuclease (Xho I and Kpn I) sites at both ends to obtain a PCR product.

[0079] Purification of target fragment: The PCR product was digested with Xho I and Kpn I and then recovered using the Kangwei Century Kit.

[0080] Vector digestion and recovery: GV219 plasmid was digested with Xho I and Kpn I, and the plasmid digestion product was recovered using the Kangwei Century Kit after 1% agarose gel electrophoresis.

[0081] Ligation: The PCR digestion product and the plasmid digestion product were ligated with Takara's T4 DNA ligase at 4°C overnight to obtain the GV219 / TAT-SH2-His plasmid, which was then transformed into E. coli Dh5α competent cells and spread onto LB solid culture medium containing 100 μg / mL ampicillin and cultured at 37°C overnight.

[0082] Transformant identification: Pick a single colony, culture it overnight in LB liquid medium (containing 100 μg / mL ampicillin), and screen the positive clones by PCR. Use a plasmid extraction kit to extract the positive plasmid GV219 / TAT-SH2-His; wherein, the forward primer of PCR is: CGCAAATG GGCGGTAGGCGTG (SEQ ID NO.7), and the reverse primer is: TTATTAGGAAAGGACAGTGGG (SEQ ID NO.8).

[0083] Plasmid sequencing identification: The positive plasmids were sent to Shanghai Bioengineering for sequencing. The sequencing results were compared by software to verify the correctness of the gene cloning.

[0084] The schematic diagram of the construction of the GV219 / TAT-SH2-His plasmid is shown in Figure 1 .

[0085] Example 2

[0086] Expression and purification of SH2 recombinant protein of Example 1

[0087] 1. Transfection and expression of HEK293 cells

[0088] The GV219 / TAT-SH2-His plasmid prepared in Example 1 was transfected into HEK293 cells using a liposome transfection reagent (purchased from Life Science). Neomycin was added 48 hours after transfection for pressure screening to construct a stable cell line expressing the SH2 gene.

[0089] 2. Isolation, purification and Western blot identification of SH2 recombinant protein

[0090] After the HEK293 stable cell line was expanded and cultured, the cells were collected and the total cell protein was extracted. The protein extract was filtered with a 0.45 μm filter, and the recombinant protein with the His tag was purified using a Ni column. The relevant operation process was carried out according to the operation guide provided by GenScript.

[0091] The specific process is as follows:

[0092] Transfer the resin to the column, and after complete precipitation, wash the nickel affinity chromatography column with double distilled water to prevent Ni 2+ Precipitation; washing with ddH2O to remove air from the matrix; equilibration with 10× column volume of Binding buffer; loading; washing with 10× column volume of Binding buffer and collecting the effluent; elution with Elution buffer and further ultrafiltration and concentration to obtain the recombinant protein.

[0093] The recombinant protein was subjected to SDS-PAGE electrophoresis and transferred to a membrane, and then identified by His monoclonal antibody. The results are as follows: Figure 2 .

[0094] Figure 2 The results showed that the relative molecular mass of the purified SH2 recombinant protein was about 13 kDa, and the present invention successfully prepared the SH2 recombinant protein.

[0095] Example 3

[0096] SH2 recombinant protein can enter tumor cells

[0097] Human non-small cell lung cancer A549 cells in the logarithmic growth phase were selected for digestion and counting, and 1×10 4 The cells were inoculated on a cell slide, and after culturing for 24 hours, 600 μM of the SH2 recombinant protein prepared in Example 2 was added, and fixed with 4% PFA solution at room temperature for 15 minutes for 6 hours; washed 3 times with 0.3% TritonX100-PBS, each time for 10 minutes; blocked with donkey serum at room temperature for 1 hour, incubated with mouse anti-His at 4°C in the dark overnight, washed 3 times with PBS, each time for 10 minutes; incubated with donkey anti-mouse secondary antibody coupled to AlexaFlour594 at room temperature in the dark for 1 hour, washed 3 times with PBS, each time for 10 minutes; used DyLight TMThe samples were stained with 488Phalloidin dye for 20 min, and then covered with Fluoromount (containing 10ug / mL Hoechest) and photographed with a fluorescence microscope. Figure 3 .

[0098] Figure 3 The results showed that SH2 recombinant protein could successfully enter tumor cells.

[0099] Example 4

[0100] SH2 recombinant protein inhibits STAT3 dimerization

[0101] The construction method of the GV219 / STAT3-Myc and GV141 / STAT3-Flag vectors comprises: linearizing the commercial GV219 and GV141 vectors using XhoI and KpnI respectively to obtain linearized GV219 and GV141 vectors; using human mRNA as a template, using Takara's high-fidelity PCR amplification kit, using primers to amplify the obtained STAT3-Myc and STAT3-Flag gene sequences respectively, connecting them to the linearized GV219 and GV141 vectors respectively, selecting recombinant clones for enzyme digestion and sequencing identification to obtain GV219 / STAT3-Myc and GV141 / STAT3-Flag vectors. The human mRNA is extracted from human non-small cell lung cancer cells A549, and the A549 cells growing in the logarithmic phase are centrifuged, the supernatant is removed, 1 mL of total RNA extraction reagent Trizol is added, and the mRNA is extracted and the concentration is measured. Wherein the human non-small cell lung cancer cell A549 is a cell that highly expresses STAT3.

[0102] The primer sequences for amplifying STAT3-Myc are: F: ACGGGCCCTCTAGACTCGAGCGCCACCATGGCCCAATGGAATCAGCTAC (SEQ ID NO.9), R: TTAAACTTAAGCTTGGTACCTCACAGATCCTCTTCTGAGATGAG (SEQ ID NO.10); the primer sequences for amplifying STAT3-Flag are: F: ACGGGCCCTCTAGACTCGAGATGGCCCAATGGAATCAGC (SEQ ID NO.11), R: AGTCACTTAAGCTTGGTACCGACATGGGGGAGGTAGCGCACT (SEQID NO.12).

[0103]

[0104]

[0105] The STAT3 plasmids GV219 / STAT3-Myc and GV141 / STAT3-Flag with two different tag proteins were grouped and transfected into HEK293 cells using liposome transfection reagent. After culturing for 24 hours, 600 μM SH2 recombinant protein was added. After treatment for 24 hours, the proteins were collected and Anti-DDDDK-tag mAb-Magnetic Beads were added. After overnight at 4°C, the proteins on the magnetic beads were collected and the expression of each protein was detected by Western blot. The results are shown in Figure 4 STAT3-Myc pulled down by STAT3-Flag in each group of IP represents STAT3 dimerization. By comparing the gray value ratio of Myc / Flag in the SC group and the SH2 group, it was found that the STAT3-Myc pulled down by the same amount of STAT3-Flag in the SC group was significantly higher than that in the SH2 group. Figure 4 The results showed that SH2 recombinant protein inhibited STAT3 dimerization.

[0106] Example 5

[0107] Recombinant SH2 protein inhibits the proliferation of human non-small cell lung cancer cells and human breast cancer cells in vitro

[0108] CCK-8 method to detect cell growth curve: human non-small cell lung cancer cells A549 and human breast cancer cells MB231 in the logarithmic growth phase were selected, counted after digestion, and cultured with DMEM complete culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. 1500 cells were inoculated in each well of a 96-well plate, and 3 replicate wells were set up in each group. An equal amount of DMEM complete culture medium was added for culture. 600 μM of SC and SH2 recombinant proteins described in Example 1 were added to the SC group and SH2 group, respectively. On days 0, 1, 2, 3, 4, and 5, according to the instructions of the kit, 10 μL of CCK-8 reagent was added to every 100 μL of culture medium, and incubated at 37°C for 2 hours. The OD value of each well at a wavelength of 450 nm was detected using a multifunctional microplate reader, and the OD value on day 0, i.e. 4 hours after inoculation, was taken as 1 to draw a cell growth curve. The results are as follows Figure 5 .

[0109] Figure 5 The results showed that SH2 recombinant protein could significantly inhibit the proliferation of human non-small cell lung cancer cells and human breast cancer cells.

[0110] Example 6

[0111] Recombinant SH2 protein inhibits migration of human non-small cell lung cancer cells A549 and human breast cancer cells MB231 in vitro

[0112] Human non-small cell lung cancer cells A549 and human breast cancer cells MB231 in the logarithmic growth phase were selected, counted after digestion, and diluted with DMEM. The Transwell chamber was placed in a 24-well plate, and 200 μL of the above cells (i.e., 2×10 4 cells, 600 μM of SC and SH2 recombinant proteins described in Example 1 were added to the upper chamber cells of the SC group and the SH2 group, and 650 μL of DMEM medium containing 20% ​​fetal bovine serum was added to the lower chamber. Human non-small cell lung cancer cells A549 and human breast cancer cells MB231 were cultured for 48 h and 42 h at 37°C, respectively. Then the chambers were taken out, washed once with PBS, fixed with 4% PFA for 15 min-20 min; washed 1-2 times with PBS, stained with 10% crystal violet for 15 min; washed several times with ddH2O; dried, scanned the whole well under an inverted microscope, washed with glacial acetic acid, and detected the OD value of each well at a wavelength of 570 nm with a multifunctional microplate reader. The results are as follows Figure 6 .

[0113] Figure 6 The results showed that SH2 recombinant protein inhibited the migration ability of human non-small cell lung cancer cells and human breast cancer cells cultured in vitro.

[0114] Example 7

[0115] Recombinant SH2 protein inhibits the invasion of human non-small cell lung cancer cells A549 and human breast cancer cells MB231 in vitro

[0116] Human non-small cell lung cancer cells A549 and human breast cancer cells MB231 in the logarithmic growth phase were selected, counted after digestion, and diluted with DMEM. The Transwell chamber was placed in a 24-well plate, and a layer of Matrix gel was first added to the upper chamber. After it solidified, 200 μL of the above cells (i.e., 2×10 4 cells, 600 μM of SC and SH2 recombinant proteins described in Example 1 were added to the upper chamber cells of the SC group and the SH2 group, and 650 μL of DMEM medium containing 20% ​​fetal bovine serum was added to the lower chamber. Human non-small cell lung cancer cells A549 and human breast cancer cells MB231 were cultured for 60 h and 64 h at 37°C, respectively. Then the chambers were taken out, washed once with PBS, fixed with 4% PFA for 15 min-20 min; washed 1-2 times with PBS, stained with 10% crystal violet for 15 min; washed several times with ddH2O; dried, scanned the whole well under an inverted microscope, washed with glacial acetic acid, and detected the OD value of each well at a wavelength of 570 nm with a multifunctional microplate reader. The results are as follows Figure 7 .

[0117] Figure 7 The results showed that SH2 recombinant protein inhibited the invasion of human non-small cell lung cancer cells and human breast cancer cells.

[0118] Example 8

[0119] Recombinant SH2 protein inhibits the growth of subcutaneous tumors of human non-small cell lung cancer cells A549 and human breast cancer cells MB231 in nude mice

[0120] 1.0×10 per balb / c nude mouse 6 A subcutaneous injection of human non-small cell lung cancer cells A549 and human breast cancer cells MB231 at a concentration of 100 μL was performed to establish a subcutaneous model of nude mice. After 20 days, after obvious tumor masses were formed subcutaneously, the mice were randomly divided into: (1) SC; (2) SH2, 6 mice in each group, and the SC and SH2 recombinant proteins described in Example 1 were injected intraperitoneally (15 μg / mouse, once every 2 days for 20 days), and the size of the tumor was measured every 2 days. The nude mice were killed on the 36th day of feeding and the size of the tumor was weighed. The results are shown in the figure. Figure 8 .

[0121] Figure 8 The results showed that SH2 significantly inhibited the growth of subcutaneous non-small cell lung cancer and breast cancer cells in nude mice.

[0122] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant protein that specifically targets and inhibits STAT3 dimerization, characterized in that: The amino acid sequence of the recombinant protein is shown in SEQ ID NO.

1.

2. A gene encoding the recombinant protein according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. A biological material containing the recombinant protein according to claim 1 or the gene according to claim 2, characterized in that: Any of the following: (1) A recombinant expression vector comprising the recombinant protein of claim 1 or the gene of claim 2; (2) A recombinant microorganism comprising the recombinant protein of claim 1 or the gene of claim 2; (3) a recombinant microorganism comprising the recombinant expression vector described in (1); (4) A recombinant cell comprising the recombinant protein of claim 1 or the gene of claim 2; (5) A recombinant cell comprising the recombinant expression vector described in (1).

5. The method for preparing the recombinant protein according to claim 1, characterized in that: The following steps are involved: The recombinant expression vector containing the recombinant protein of claim 1 or the gene of claim 2 is transformed into the recombinant microorganism, cultured, the recombinant expression vector is extracted, and then transfected into recombinant cells, cultured amplified, extracted and purified to obtain the recombinant protein.

6. Use of the recombinant protein according to claim 1, the gene according to claim 2 or 3, the biological material according to claim 4 or the recombinant protein obtained by the preparation method according to claim 5 in specific targeted inhibition of STAT3 dimerization.

7. Use of the recombinant protein according to claim 1, the gene according to claim 2 or 3, the biological material according to claim 4 or the recombinant protein obtained by the preparation method according to claim 5 in the preparation of drugs for preventing and / or treating tumors.

8. The use according to claim 7, characterized in that: The tumor includes one or both of lung cancer and breast cancer.

9. A drug for preventing and / or treating tumors, characterized in that: The drug comprises the recombinant protein according to claim 1, the gene according to claim 2 or 3, or the recombinant protein obtained by the preparation method according to claim 5.

10. The drug according to claim 9, characterized in that The drug includes pharmaceutically acceptable excipients.

Images