Fusion protein THSGp and its application in the preparation of anti-tumor drugs

Abstract

The present invention belongs to the field of biomedicine and specifically relates to a fusion protein, THSGp, and its use in the preparation of anti-tumor drugs. The amino acid sequence of the fusion protein THSGp is ​​shown in SEQ ID NO. 1. Under weakly acidic conditions, the fusion protein THSGp of the present invention can insert into the cell membrane, thereby anchoring SPRR2A to the cell membrane, thereby facilitating its activity in disrupting and destroying the cell membrane and exerting its anti-tumor effects. This invention will lay a solid theoretical and experimental foundation for the application of SPRR2A in tumor treatment and provide a new strategy for its use in tumor treatment with strong targeting and high efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a fusion protein THSGp and its application in the preparation of anti-tumor drugs. Background Art

[0002] With the rapid development of biotechnology, the application of macromolecular drugs in cancer treatment is becoming increasingly widespread. Protein-based drugs, due to their unique advantages, are gradually becoming an important means of cancer treatment. Protein-based drugs not only possess high targeting and specificity, enabling precise targeting of tumor cells while minimizing damage to normal cells, but also exhibit low immunogenicity and high biological activity.

[0003] In recent years, protein drugs have made significant progress in tumor treatment research. Among them, protein therapeutics with anti-cancer activity have been widely used to fight cancer. In recent years, antimicrobial peptides have attracted much attention in the field of cancer treatment and have become potential candidate drugs. In 2021, Science reported a new intestinal antibacterial protein-proline-rich small protein 2A, referred to as SPRR2A. This protein is rich in proline and contains 5 pairs of intrachain disulfide bonds. It has unique structural and functional properties and can selectively kill Gram-positive bacteria by destroying the bacterial cell membrane. In addition, SPRR2A is secreted by Paneth cells and goblet cells in the intestine, which can regulate the composition of intestinal microbiota, limit the binding of bacteria to the intestinal surface, and protect the intestinal barrier during infection. However, obtaining active SPRR2A through a prokaryotic expression system remains a huge challenge. The applicant previously used Escherichia coli Rosetta-gami (DE3) pLysS as the expression host and successfully obtained the antibacterial fusion protein Trx-SPRR2A with SPRR2A using a prokaryotic expression system. It was found that the fusion protein not only has antibacterial activity but also anti-tumor activity. As a protein with antibacterial and anti-tumor properties, SPRR2A has broad application prospects in the medical field. Future research will further explore its mechanism of action in different diseases and provide a theoretical basis for the development of new antibacterial and anti-tumor drugs. However, SPRR2A lacks selectivity and cannot effectively target tumor cells, which limits its effectiveness in clinical applications. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a fusion protein THSGp and its application in the preparation of anti-tumor drugs.

[0005] The present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a fusion protein THSGp, which is formed by sequentially connecting thioredoxin Trx, a His tag, an SPRR2A protein, a linker and a low pH insertion peptide, and the amino acid sequence is shown in SEQ ID NO.1.

[0007] Based on the characteristics of the acidic tumor microenvironment and the low-pH insertion peptide pHLIP's ability to efficiently insert its C-terminus into the cell membrane under acidic conditions, the present invention designed SPRR2A at the N-terminus of pHLIP. This led to the first prokaryotic expression of the Trx-His-SPRR2A-GGGGSGGGGS-pHLIP fusion protein, or THSGp for short. Trx enhances solubility; the His tag facilitates protein purification; the SPRR2A protein possesses excellent membrane solubility; the linker provides spatial and interdomain flexibility; and pHLIP, under acidic conditions, forms a transmembrane α-helix, efficiently delivering SPRR2A to the tumor cell surface, enabling SPRR2A to exert its membrane solubility and lead to cell death. This protein can insert into the cell membrane under weakly acidic conditions, thereby anchoring SPRR2A to the cell membrane, facilitating its membrane-disrupting and damaging activities and exerting its anti-tumor effects.

[0008] In some specific embodiments, the nucleotide sequence of the gene encoding the fusion protein THSGp is ​​shown as SEQ ID NO.2.

[0009] In a second aspect, the present invention provides the use of the fusion protein THSGp in the preparation of anti-tumor drugs.

[0010] In some embodiments, the tumor is colon cancer.

[0011] In some embodiments, the tumor is liver cancer.

[0012] In some embodiments, the tumor is breast cancer.

[0013] In some specific embodiments, the drug contains the fusion protein THSGp as the only active ingredient.

[0014] In some embodiments, the drug further comprises pharmaceutically acceptable excipients. The excipients include conventional diluents such as water for injection, microcrystalline cellulose, and the like; fillers such as mannitol, sucrose, lactose, polyethylene glycol, Tween 80, sorbitol, menthol, liquid paraffin, petrolatum, stearic acid, glyceryl monostearate, lanolin, mineral oil, dimethyl sulfoxide, and the like; stabilizers such as disodium edetate, sodium thiosulfate, sodium metabisulfite, sodium sulfite, sodium bisulfite, ethanolamine, sodium bicarbonate, sodium acetate, niacinamide, and vitamin C; osmotic pressure regulators such as sodium chloride and glucose; pH regulators such as triethanolamine, sodium hydroxide, and sodium citrate; and preservatives such as chlorobutanol, parabens, ethylparaben, and benzalkonium bromide.

[0015] In some specific embodiments, the content of the fusion protein THSGp in the drug is 1 wt%-99.9 wt%.

[0016] The present invention has the following beneficial effects:

[0017] The present invention constructs a pH-responsive SPRR2A fusion protein THSGp for the first time and expresses it in prokaryotes. Since SPRR2A mainly exerts its effects by acting on the cell membrane, we design SPRR2A at the N-terminus of pHLIP. Under the acidic microenvironment of the tumor, the C-terminus of pHLIP is inserted into the cell membrane, and SPRR2A at its N-terminus will be anchored on the cell membrane, which is conducive to its biological activity. The present invention constructs and expresses 6 fusion proteins of SPRR2A and pHLIP, and finally screens the THSGp fusion protein with good serum stability. On this basis, further anti-tumor research is carried out, and it is confirmed that THSGp has good anti-tumor activity. The present invention will lay a solid theoretical and experimental foundation for the application of SPRR2A in tumor treatment, and provide a new strategy with strong targeting and high efficiency for its application in tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 For pET32a and SPRR2A-pHLIP Lane M: DNA Marker; Lane 1: Gene amplification of linearized vector pET32a; Lane 2: SPRR2A-pHLIP gene amplification.

[0019] Figure 2 For pET32a- SPRR2A-pHLIP-His A: Lane M: DNA Marker; Lanes 1-5: PCR identification using universal primers; b: Lane M: DNA Marker; Lanes 1 and 2: PCR identification using specific primers.

[0020] Figure 3 For the verification of the recombinant plasmid by PCR and sequencing, a: recombinant plasmid pET32a- SPRR2A-GGGGS-pHLIP- His Agarose gel electrophoresis of the whole plasmid PCR product, lane M: DNA Marker; lane 1: pET32a- SPRR2A- GGGGS-pHLIP-His b: Agarose gel electrophoresis of the whole plasmid PCR product of the recombinant plasmid pET32a-SPRR2A-GGGGSGGGGS-pHLIP-His, lane M: DNA Marker, lane 1: pET32a- SPRR2A- GGGGSGGGGS-pHLIP-His The whole plasmid PCR product.

[0021] Figure 4 For linearized vector pET32a and SPRR2A-pHLIP Gene PCR verification, a: pET32a vector linearization, lane M: DNA Marker; lanes 1 and 2: gene amplification of linearized vector pET32a; b: SPRR2A-pHLIP Gene amplification, lane M: DNA Marker, lanes 1 and 2: SPRR2A-pHLIP gene amplification.

[0022] Figure 5 For the recombinant plasmid pET32a- His-SPRR2A-pHLIP Identification, lane M: DNA Marker; lanes 1-5: SPRR2A-pHLIP gene amplification.

[0023] Figure 6 For the recombinant plasmid full plasmid PCR, a: recombinant plasmid pET32a- His-SPRR2A-GGGGS-pHLIP Agarose gel electrophoresis of the whole plasmid PCR product, lane M: DNA Marker; lane 1: pET32a- His-SPRR2A-GGGGS- pHLIP Full plasmid PCR product; b: recombinant plasmid pET32a- His-SPRR2A-GGGGSGGGGS-pHLIP Agarose gel electrophoresis of the whole plasmid PCR product, lane M: DNA Marker, lane 1: pET32a- His-SPRR2A-GGGGSGGGGS- pHLIP The whole plasmid PCR product.

[0024] Figure 7Expression and purification analysis of Trx-SPRR2A-pHLIP-His, a: Expression of Trx-SPRR2A-pHLIP-His, lane M: Protein Marker; lane 1: Transetta (DE3) bacterial lysate containing empty plasmid pET32a; lane 2: Uninduced bacterial lysate, lanes 3, 4, 5: Induced bacterial lysate, supernatant, and precipitate; b: Purification conditions of Trx-SPRR2A-pHLIP-His, lane M : Protein marker; Lane 1: Supernatant of inducible cells; Lanes 2-9: Samples collected using 8 mL of elution buffer with a gradient of imidazole concentrations: 10 mM, 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 150 mM, and 200 mM; c: 15% SDS-PAGE analysis of purified Trx-SPRR2A-pHLIP-His. Lane M is the protein marker, and Lane 1 is the purified Trx-SPRR2A-pHLIP-His recombinant protein. The bands in the dotted box and arrows are the target proteins.

[0025] Figure 8 Expression and purification analysis of Trx-SPRR2A-GGGGS-pHLIP-His, a: Expression of Trx-SPRR2A-GGGGS-pHLIP-His, lane M: protein marker, lane 1: uninduced bacterial lysate, lanes 2, 3, 4: induced bacterial lysate, supernatant and precipitate; b: exploration of the purification conditions of Trx-SPRR2A-GGGGS-pHLIP-His, lane M: protein marker White marker; Lanes 1 and 2: Post-induction bacterial lysate and supernatant; Lanes 3-8: Samples collected using 8 mL of elution buffer with a gradient of imidazole concentrations: 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, and 150 mM; C: Purified Trx-SPRR2A-pHLIP-His; Lane M: Protein marker; Lane 1: Purified Trx-SPRR2A-GGGGS-pHLIP-His recombinant protein. The bands enclosed by the dashed box and arrow are the target proteins.

[0026] Figure 9Expression and purification analysis of Trx-SPRR2A-GGGGSGGGGS-pHLIP-His, a: Expression of Trx-SPRR2A-GGGGSGGGGS-pHLIP-His, lane M: protein marker, lane 1: uninduced bacterial lysate, lanes 2, 3, 4: induced bacterial lysate, supernatant and precipitate; b: exploration of the purification conditions of Trx-SPRR2A-GGGGSGGGGS-pHLIP-His, Lane M: Protein marker; Lanes 1 and 2: Post-induction bacterial lysate and supernatant; Lanes 3-8: Samples collected using 8 mL of elution buffer with a gradient of imidazole concentrations: 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, and 150 mM; c: Purified Trx-SPRR2A-pHLIP-His; Lane M: Protein marker; Lane 1: Purified Trx-SPRR2A-GGGGSGGGGS-pHLIP-His recombinant protein. The bands enclosed by the dashed box and arrow are the target proteins.

[0027] Figure 10 Analysis of the expression and purification of Trx-His-SPRR2A-pHLIP, a: Expression of Trx-His-SPRR2A-pHLIP, lane M: Protein Marker, lane 1: Transetta (DE3) bacterial lysate containing empty plasmid pET32a, lane 2: Uninduced bacterial lysate, lanes 3, 4, 5: Induced bacterial lysate, supernatant and precipitate; b: Exploration of the purification conditions of Trx-His-SPRR2A-pHLIP, lane M Lane A: Protein marker, lane 1: supernatant of inducible cells, lanes 2-8: samples collected using 8 mL of elution buffer with a gradient of imidazole concentrations: 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 150 mM, and 200 mM; c: 15% SDS-PAGE analysis of purified Trx-His-SPRR2A-pHLIP, lane M: Protein marker, lane 1: purified Trx-His-SPRR2A-pHLIP recombinant protein. The bands in the dotted box and arrows are the target proteins.

[0028] Figure 11Expression and purification analysis of Trx-His-SPRR2A-GGGGS-pHLIP, a: Expression of Trx-His-SPRR2A-GGGGS-pHLIP, lane M: protein marker, lane 1: uninduced bacterial lysate, lanes 2, 3, and 4 are induced bacterial lysate, supernatant, and precipitate; b: Exploration of the purification conditions of Trx-His-SPRR2A-GGGGS-pHLIP, lane M: protein marker r, Lanes 1 and 2: Post-induction bacterial lysate and supernatant; Lanes 3-9: Samples collected using 8 mL of elution buffer with a gradient of imidazole concentrations: 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 150 mM, and 200 mM; c: Purified Trx-His-SPRR2A-GGGGS-pHLIP; Lane M: Protein marker; Lane 1: Purified Trx-His-SPRR2A-GGGGS-pHLIP recombinant protein. The bands enclosed by the dashed box and arrow are the target proteins.

[0029] Figure 12 Expression and purification analysis of Trx-His-SPRR2A-GGGGSGGGGS-pHLIP, a: Expression of Trx-His-SPRR2A-GGGGSGGGGS-pHLIP, lane M: protein marker, lane 1: uninduced bacterial lysate, lanes 2, 3, 4: induced bacterial lysate, supernatant and precipitate; b: Exploration of the purification conditions of Trx-His-SPRR2A-GGGGSGGGGS-pHLIP, lane M: protein marker ker, Lanes 1 and 2: Post-induction bacterial lysate and supernatant; Lanes 3-9: Samples collected using 8 mL of elution buffer with a gradient of imidazole concentrations: 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 150 mM, and 200 mM; c: Purified Trx-His-SPRR2A-GGGGSGGGGS-pHLIP; Lane M: Protein marker; Lane 1: Purified Trx-His-SPRR2A-GGGGSGGGGS-pHLIP recombinant protein. The bands in the dotted box and arrows are the target proteins.

[0030] Figure 13The figures are the results of serum stability test, a: Photo of the centrifuge tube after incubation with neutral medium containing 10% serum for 6 h; b: Photo of the centrifuge tube after incubation with acidic medium containing 10% serum for 2 h; c: SDS-PAGE analysis of the supernatant in a; d: SDS-PAGE analysis of the precipitate in a; e: SDS-PAGE analysis of the supernatant in b; f: SDS-PAGE analysis of the precipitate in b; Among them, 1: Trx-SPRR2A-pHLIP-His, 2: Trx-SPRR2A-GGGGS-pHLIP-His, 3: Trx-SPRR2A-GGGGSGGGGS-pHLIP-His, 4: Trx-His-SPRR2A-pHLIP, 5: Trx-His-SPRR2A-GGGGS-pHLIP, 6: Trx-His-SPRR2A-GGGGSGGGGS-pHLIP.

[0031] Figure 14 This is the mass spectrum of the molecular weight of THSGp.

[0032] Figure 15 The figure shows the results of MTT assay for the anti-tumor activity of THSGp at pH 7.4 and pH 6.5. Figures a to f are HCT-8 cells, HCT-116 cells, HepG2 cells, MCF-7 cells, MDA-MB-231 cells, and MDA-MB-468 cells, respectively.

[0033] Figure 16 MTT assay was used to detect the effect of THSGp on the viability of normal cells. a: MCF-10A cells; b: HIEC-6 cells.

[0034] Figure 17 To detect the uptake of FITC-THSGp and FITC-Trx-SPRR2A by tumor cells using an inverted fluorescence microscope, a: MCF-7 cells; b: MDA-MB-231 cells; scale bar: 20 μm.

[0035] Figure 18 These are the crystal violet staining experimental results after tumor cells were treated with different concentrations of THSGp and Trx-SPRR2A, a: crystal violet staining image of MCF-7, b: quantitative analysis result of a, c: crystal violet staining image of MDA-MB-231, d: quantitative analysis result of c.

[0036] Figure 19EdU staining was used to detect tumor cell proliferation after treatment with 0.5 μM THSGp and Trx-SPRR2A. a: EdU staining of MCF-7 cells, scale bar: 100 μm, b: quantitative analysis of a, c: EdU staining of MDA-MB-231 cells, scale bar: 100 μm, d: quantitative analysis of c.

[0037] Figure 20 WGA detection of tumor cell membrane integrity after treatment with 0.5 μM THSGp and Trx-SPRR2A under different pH conditions, a: MCF-7 cells, scale bar is 25 μm; b: MDA-MB-231 cells, scale bar is 25 μm; c: quantitative analysis of a at 12 h; d: quantitative analysis of a at 24 h; e: quantitative analysis of b at 12 h; f: quantitative analysis of b at 24 h.

[0038] Figure 21 SEM detection of tumor cell membrane integrity after treatment with 0.5 μM THSGp and Trx-SPRR2A under different pH conditions, a: MCF-7 cells; b: MDA-MB-231 cells; scale bars: 500× for 100 μm, 2000× for 20 μm.

[0039] Figure 22 PI staining was used to detect the membrane permeability of tumor cells after treatment with 1 μM THSGp and Trx-SPRR2A under different pH conditions. a: MCF-7 cells; b: MDA-MB-231 cells; scale bar: 100 μm. DETAILED DESCRIPTION

[0040] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0041] The present invention discloses a fusion protein THSGp and its application in preparing an anti-tumor drug. The amino acid sequence of the THSGp is ​​shown in SEQ ID NO.1, and the encoding gene sequence thereof is shown in SEQ ID NO.2.

[0042] SEQ ID NO.1: MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHH HHHSSGMSYQQQQCKQPCQPPPVCPTPKCPEPCPPPKCPEPCPPPKCPQPCPPQQCQQKYPPVTPSPPCQSKYPPKSKGGGGSGGGGSAAEQNPIYWARYADWLFTTTPLLLLDLALLVDADEGT.

[0043] In SEQ ID NO. 1, positions 1 to 109 are Trx, positions 117 to 122 are His, positions 126 to 197 are SPRR2A, positions 110 to 116, positions 123 to 125, and positions 198 to 207 are Linker, and positions 208 to 243 are low pH insertion peptide pHLIP.

[0044] SEQ ID NO.2:.

[0045] Example 1: Preparation and characterization of pH-responsive SPRR2A.

[0046] 1: Experimental method.

[0047] 1.1: Construction of recombinant plasmid.

[0048] Six recombinant plasmids were constructed in this example, as shown in Table 1 and described in detail below.

[0049] Table 1: Size and resistance of six recombinant plasmids.

[0050]

[0051] 1.1.1: Recombinant plasmid pET32a- SPRR2A-pHLIP-His 's construction.

[0052] (1) Amplification SPRR2A-pHLIP Primer design for genes.

[0053] Synthesized by Shanghai Sangon Bioengineering Co., Ltd. SPRR2A-pHLIP The gene, whose sequence is shown in SEQ ID NO.3, was constructed into pUC57- SPRR2A-pHLIP On the carrier. SPRR2A-pHLIP The amplification primer design scheme is shown in Table 2.

[0054] SEQ ID NO.3: ATGTCTTACCAGCAGCAGCAGTGCAAACAGCCGTGCCAGCCGCCGCCGGTTTGCCCGACCCCGAAATGCCCGGAACCGTGCCCGCCGCCGAAATGCCCGGAACCGTGCCCGCCGCCGAAATGCCCGCAGCCGTGCCCGCCGCAGCAGTGCCAGCAGAAAT ACCCGCCGGTTACCCGTCTCCGCCGTGCCAGTCTAAATACCCGCCGAAATCTAAAGCTGCTGAACAGAACCCGATCTACTGGGCTCGTTACGCTGACTGGCTGTTCACCACCCCGCTGCTGCTGCTGGACCTGGCTCTGCTGGTTGACGCTGACGAAGGTACC.

[0055] Table 2: Amplification SPRR2A-pHLIP Gene primer sequences.

[0056]

[0057] (2) SPRR2A-pHLIP amplification and purification.

[0058] pUC-57- SPRR2A-pHLIP As a template, use SPRR2A-pHLIP -F and SPRR2A-pHLIP -R for PCR amplification.

[0059] ①PCR reaction system, see Table 3.

[0060] Table 3: pUC-57- SPRR2A-pHLIP Amplification system.

[0061]

[0062] ②PCR reaction procedure, see Table 4.

[0063] Table 4: pUC-57- SPRR2A-pHLIP Amplification system.

[0064]

[0065] Note: Steps 2-4 are repeated 30 times.

[0066] ③Template digestion.

[0067] Add 1 μL of Dpn1 to each PCR product tube, enzymatically digest the DNA template strand, and add 10× buffer in proportion according to appropriate conditions. React at 37°C for 2 h to ensure complete digestion.

[0068] ④PCR product purification.

[0069] The experiments were performed according to the instructions for PCR product purification.

[0070] (3) Design of primers for linear amplification of pET32a vector.

[0071] The pET32a plasmid was linearized and amplified using PCR technology, and primers were designed as shown in Table 5.

[0072] Table 5: Primers for amplification of pET32a genes.

[0073]

[0074] (4) Amplify the linearized pET32a gene.

[0075] ①PCR reaction system, same as Table 3.

[0076] ②PCR reaction procedure, same as Table 4.

[0077] ③ Template digestion and PCR product purification.

[0078] The steps are the same as ③ and ④ in 1.1.1.

[0079] (5) pET32a -SPRR2A-pHLIP-His Seamless cloning of.

[0080] ① Reaction system, see Table 6.

[0081] Table 6: pET32a -SPRR2A-pHLIP-His Seamless cloning system.

[0082]

[0083] ②Reaction conditions.

[0084] According to the reaction system described above, add all ingredients to a 1.5 ml centrifuge tube and mix thoroughly. Next, carry out the reaction in a metal thermostat at 50°C for 20 minutes. Immediately after completion, place the centrifuge tube on ice for 2 minutes to stop the reaction before proceeding with the conversion.

[0085] (6) pET32a- SPRR2A-pHLIP-His Convert to E.coli DH5α.

[0086] (7) Identification of positive clones.

[0087] Select about 10 single colonies on the plate, pick them up with a pipette tip and place them in sterile LB liquid culture medium, take 1 μL of bacterial liquid as a template, add universal primers for PCR, and then use specific primers to identify the SPRR2A-pHLIP Whether the amplification was successful.

[0088] After PCR reaction, the obtained colonies were placed on a shaker at 37°C and 210 rpm for 8 hours for bacterial preservation experiments. At the same time, 1 mL of bacterial culture was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequence analysis.

[0089] (8) Extract the recombinant plasmid.

[0090] 1.1.2 Construction of pET32a- SPRR2A-GGGGS-pHLIP-His and pET32a- SPRR2A-GGGGSGGGGS- pHLIP-His of the recombinant plasmid.

[0091] pET32a- SPRR2A-pHLIP-His As a template, the recombinant plasmid pET32a- SPRR2A-GGGGS- pHLIP-His and pET32a- SPRR2A-GGGGSGGGGS-pHLIP-His , the primer sequences are shown in Table 7.

[0092] Table 7: Obtained recombinant plasmid pET32a- SPRR2A-GGGGS-pHLIP-His and pET32a- SPRR2A- GGGGSGGGGS-pHLIP-His primer sequences.

[0093]

[0094] 1.1.3: Recombinant plasmid pET32a- His-SPRR2A-pHLIP 's construction.

[0095] (1) SPRR2A-pHLIP Gene amplification.

[0096] Target gene SPRR2A-pHLIP A PCR amplification primer design scheme was developed, and the primers are shown in Table 8.

[0097] Table 8: SPRR2A-pHLIP Gene amplification primer sequences.

[0098]

[0099] (2) SPRR2A-pHLIP Amplification and purification of target genes.

[0100] pUC-57- SPRR2A-pHLIP The vector was used as a template and amplified by PCR.

[0101] ③ Template digestion and PCR product purification.

[0102] The steps are the same as ③ and ④ in 1.1.1.

[0103] ④The recombination reaction product is transformed into E.coli Extraction of DH5α and recombinant plasmid.

[0104] The steps are the same as (6) and (8) in 1.1.1.

[0105] (3) Primer design for pET32a vector

[0106] The pET32a gene expression plasmid was linearly amplified using PCR technology, and primers were designed as shown in Table 9.

[0107] Table 9: Primer sequences for pET32a vector amplification.

[0108]

[0109] (4) Linearized amplification and purification of pET32a vector.

[0110] ③ Template digestion and product purification.

[0111] The steps are the same as ③ and ④ in 1.1.1.

[0112] ④Transformation of the recombination reaction product into E.coli DH5α and extraction of the recombinant plasmid.

[0113] The steps are the same as (6) and (8) in 1.1.1.

[0114] 1.1.4: Construction of pET32a- His-SPRR2A-GGGGS-pHLIP and pET32a- His-SPRR2A- GGGGSGGGGS-pHLIP Primer design for recombinant plasmid genes.

[0115] pET32a- His-SPRR2A-pHLIP As a template, the recombinant plasmid pET32a- His- SPRR2A-GGGGS-pHLIP and pET32a-His-SPRR2A-GGGGSGGGGS-pHLIP The primer sequences of the genes are shown in Table 10.

[0116] Table 10: Construction of recombinant plasmid pET32a- SPRR2A-GGGGS-pHLIP-His and pET32a- SPRR2A- GGGGSGGGGS-pHLIP-His primer sequences.

[0117]

[0118] pET32a- SPRR2A-pHLIP-His The plasmid was used as a template for PCR amplification.

[0119] ③ Template digestion and PCR product purification.

[0120] The steps are the same as ③ and ④ in 1.1.1.

[0121] ④Transformation of the recombination reaction product into E.coli DH5α and extraction of the recombinant plasmid.

[0122] The steps are the same as (6) and (8) in 1.1.1.

[0123] 1.2: Heterologous expression and purification of six pH-responsive SPRR2A proteins.

[0124] In this example, six pH-responsive recombinant proteins were successfully expressed and purified, as shown in Table 11.

[0125] Table 11: Six pH-responsive recombinant proteins successfully expressed and purified in this example.

[0126]

[0127] 1.2.1: Expression of recombinant protein.

[0128] After the sequencing results were confirmed to be correct, the above six recombinant plasmids were transformed into Transetta (DE3) competent cells to obtain engineered bacteria capable of heterologously expressing recombinant proteins for subsequent protein expression experiments.

[0129] (1) Pick out a single transformed colony, inoculate it into 10 mL of LB liquid medium, and culture it in a shaker at 210 rpm and 37°C for 12 hours.

[0130] (2) Take the above bacterial solution and inoculate it into 10 mL of LB liquid culture medium containing 100 ng / μL ampicillin at a ratio of 1:100. Place it on a shaker and culture it at 210 rpm and 37°C for 8 hours.

[0131] (3) Inoculate the bacterial solution into 0.4 L of LB liquid medium and culture at 220 rpm and 37°C until the OD600 reaches between 0.6 and 0.8. Add IPTG with a final concentration of 0.5 mM, adjust the rotation speed to 180 rpm, and continue to culture at 18°C ​​for 12 hours.

[0132] (4) Centrifuge the bacterial solution in the conical flask at 4000 rpm for 15 minutes, discard the supernatant, and store the precipitate in a -20℃ refrigerator.

[0133] (5) Mix the bacterial pellet with 10 mM Tris buffer at a mass ratio of 1:12, place it in a beaker, and use a cell disruptor at 375 W power, running for 5 seconds and stopping for 5 seconds, until the bacterial solution becomes transparent.

[0134] (6) Take 40 μL of the broken bacterial solution as a sample, centrifuge the remaining bacterial solution at 12000 rpm and 4°C for 36 minutes, take the supernatant and precipitate as samples, and detect the expression of the recombinant protein by SDS-PAGE.

[0135] 1.2.2: Purification of recombinant protein.

[0136] 1.2.2.1: Exploration of protein purification conditions.

[0137] (1) Equilibrate the nickel column once with 5 column volumes of 1× Charge Buffer.

[0138] (2) Equilibrate the nickel column once with 3 column volumes of 1× Binding Buffer.

[0139] (3) The supernatant obtained by centrifugation after cell disruption is passed through a nickel column and repeated 2 to 3 times to fully bind the target protein.

[0140] (4) Equilibrate the nickel column once with 5 column volumes of 1× Binding Buffer.

[0141] (5) After elution with 6 mL of Wash Buffer containing gradient imidazole, the eluate was collected for electrophoresis analysis.

[0142] (6) Wash the nickel column once with 40 mL of 1× Strip Buffer to remove nickel ions and residual proteins.

[0143] (7) Wash the nickel column 2 to 3 times with 5 column volumes of deionized water.

[0144] (8) Equilibrate the nickel column once with 5 column volumes of 1× Charge Buffer.

[0145] 1.2.2.2: Recombinant protein purification process.

[0146] (1) Equilibrate the nickel column with 5 column volumes of 1× Charge Buffer.

[0147] (2) Equilibrate the nickel column with 1× Binding Buffer.

[0148] (3) The supernatant obtained by centrifugation after cell disruption is passed through a nickel column and repeated 2 to 3 times to ensure that the target protein is fully bound to the nickel column.

[0149] (4) Equilibrate the nickel column again with 5 column volumes of 1× Binding Buffer.

[0150] (5) To remove impurities, use Wash Buffer containing 50 mM imidazole for elution until no blue band appears after CBB detection.

[0151] (6) Elute the His-tagged protein with Wash Buffer containing 150 mM or 200 mM imidazole.

[0152] (7) Wash the nickel column with 5 column volumes of 1× Strip Buffer.

[0153] (8) Rinse the nickel column with deionized water to ensure that any residual substances are completely removed.

[0154] (9) Equilibrate the nickel column again with 5 column volumes of 1× Charge Buffer.

[0155] 1.2.2.3: SDS-PAGE identification of six pH-responsive SPRR2A proteins.

[0156] (1) Prepare SDS-PAGE protein gel: 15% separation gel and 5% stacking gel.

[0157] (2) Mix the purified target protein with 5× Loading Buffer in a 4:1 ratio, place in a metal bath, and treat at 100°C for 6 minutes.

[0158] (3) Spot the prepared protein sample and protein marker and run at a constant voltage of 60V until the band with the smallest molecular weight in the protein marker reaches the bottom of the separation gel.

[0159] (4) Place the protein gel in the staining solution and stain on a shaker at room temperature for 2 hours. Then use a destaining solution to destain on a shaker at room temperature until the bands are clear and the background is clean.

[0160] (5) Scan the protein gel using a scanner.

[0161] 1.2.2.4: Serum stability testing of six pH-responsive SPRR2A proteins.

[0162] (1) The purified target protein was concentrated by 10 kDa ultrafiltration tube and replaced with 10 mM Tris buffer.

[0163] (2) Six pH-responsive SPRR2A proteins were diluted with neutral / acidic culture medium containing 10% serum to a final concentration of 10 μM and incubated at 37°C.

[0164] (3) The stability of six pH-responsive SPRR2A proteins in serum-containing culture medium was observed through serum stability experiments, and the stability was determined by whether the culture medium was turbid.

[0165] (4) Centrifuge the protein and culture medium mixture at 8000 rpm for 10 min, take the precipitate and supernatant for sample preparation, and perform SDS-PAGE analysis.

[0166] 1.2.2.5: Mass spectrometry identification of Trx-His-SPRR2A-GGGGSGGGGS-pHLIP.

[0167] The screened proteins were separated by SDS-PAGE, and the protein bands that matched the predicted molecular weight were cut out and sent to Shanghai Sangon Biotechnology Co., Ltd. for mass spectrometry identification.

[0168] 2: Experimental results.

[0169] 2.1: Construction of recombinant plasmid.

[0170] 2.1.1: Recombinant plasmid pET32a- SPRR2A-pHLIP-His Build.

[0171] (1) PCR amplification of the linearized vector and target gene.

[0172] like Figure 1 As shown, there is a clear band in the range of 5000 to 8000 bp in lane 1, and a brighter band in the range of 250 to 500 bp in lane 2, which is consistent with the theoretical value: the size of the vector pET32a is 5698 bp, and the target gene SPRR2A-pHLIP The size is 324bp. From the above results, we can see that SPRR2A-pHLIP Amplification of the gene as well as the linearized vector pET32a was successful.

[0173] (2) pET32a- SPRR2A-pHLIP-His Identification and sequencing of recombinant plasmids.

[0174] The results are as follows Figure 2 In lanes 1-5 of a, brighter bands appeared in the range of 750bp-1000bp. Figure 2In b, lanes 1 and 2 showed brighter bands in the range of 250bp-500bp, which is consistent with the theoretical value: pET32a- SPRR2A-pHLIP-His The theoretical length of the fragment between the universal primers is 829 bp; the theoretical length of the fragment between the specific primers is 324 bp. SPRR2A-pHLIP The theoretical sequence of pET32a- SPRR2A-pHLIP-His Build successful.

[0175] 2.1.2: Recombinant plasmid pET32a- SPRR2A-GGGGS-pHLIP-His and pET32a- SPRR2A- GGGGSGGGGS-pHLIP-His Build.

[0176] The recombinant plasmids pET32a- SPRR2A-GGGGS-pHLIP-His and pET32a- SPRR2A-GGGGSGGGGS-pHLIP-His.

[0177] For the recombinant plasmid pET32a- SPRR2A-GGGGS-pHLIP-His ,like Figure 3 As shown in a, there is a clear band in the range of 5000 to 8000 bp in lane 1, which is consistent with the theoretical value: pET32a- SPRR2A-GGGGS-pHLIP-His The theoretical value is 6016bp. SPRR2A-GGGGS-pHLIP The theoretical sequence was consistent with that of pET32a- SPRR2A-GGGGS-pHLIP-His .

[0178] For the recombinant plasmid pET32a- SPRR2A-GGGGSGGGGS-pHLIP-His ,like Figure 3 As shown in b, there is an obvious band in the range of 5000 to 8000 bp in lane 1, which is consistent with the theoretical value: the recombinant plasmid pET32a- SPRR2A-GGGGS-pHLIP- His The theoretical value is 6031bp. SPRR2A-GGGGSGGGGS-pHLIP The theoretical sequence was consistent with that of pET32a- SPRR2A-GGGGSGGGGS-pHLIP-His .

[0179] 2.1.3: Recombinant plasmid pET32a- His-SPRR2A-pHLIP Build.

[0180] (1) PCR amplification of the linearized vector and target gene.

[0181] like Figure 4 As shown in a, there are obvious bands in the range of 5000 to 8000 bp in lanes 1 and 2; Figure 4 As shown in b, there are brighter bands in the 250-500bp range in lanes 1 and 2, which is consistent with the theoretical value: the size of the vector pET32a is 5731bp, SPRR2A-pHLIPThe size is 324bp. From the above results, we can see that SPRR2A-pHLIP Amplification of the gene as well as the linearized vector pET32a was successful.

[0182] (2) pET32a- His-SPRR2A-pHLIP identification and sequencing.

[0183] like Figure 5 Lanes 1-5 show brighter bands in the range of 250bp-500bp, which is consistent with the theoretical value: pET32a- His- SPRR2A-pHLIP The theoretical length of the fragment between the specific primers is 324bp. The sequencing results showed that the recombinant plasmid was consistent with the predicted SPRR2A-pHLIP The theoretical sequence of pET32a is consistent with -SPRR2A-pHLIP-His Build successful.

[0184] 2.1.4: Recombinant plasmid pET32a- His-SPRR2A-GGGGS-pHLIP and pET32a- His-SPRR2A- GGGGSGGGGS-pHLIP Build.

[0185] The recombinant plasmids pET32a- His-SPRR2A-GGGGS-pHLIP and pET32a- His-SPRR2A-GGGGSGGGGS-pHLIP .

[0186] For the recombinant plasmid pET32a- His-SPRR2A-GGGGS-pHLIP ,like Figure 6 As shown in a, there is an obvious band in the range of 5000 to 8000 bp in lane 1, which is consistent with the theoretical value: the recombinant plasmid pET32a- His-SPRR2A-GGGGS-pHLIP The theoretical value of the sequence is 6049 bp. SPRR2A-GGGGS-pHLIP The theoretical sequence of pET32a- His-SPRR2A-GGGGS-pHLIP Build successful.

[0187] For the recombinant plasmid pET32a- His-SPRR2A-GGGGSGGGGS-pHLIP ,like Figure 6 As shown in b, there is an obvious band in the 5000bp-8000bp interval in lane 1, which is consistent with the theoretical value: pET32a- His-SPRR2A-GGGGSGGGGS-pHLIP The theoretical value of the sequence is 6064 bp. SPRR2A-GGGGSGGGGS-pHLIP The theoretical sequence of pET32a- His-SPRR2A-GGGGSGGGGS-pHLIP Build successful.

[0188] 2.2: Heterologous expression and purification of six pH-responsive SPRR2A proteins.

[0189] 2.2.1: Heterologous expression and purification of Trx-SPRR2A-pHLIP-His protein.

[0190] For pET32a- SPRR2A-pHLIP-His The host was transformed with the empty plasmid pET32a, and the strain Transetta (DE3) was used. The culture was then expanded. When the OD600 value was between 0.6 and 0.8, IPTG was added to induce expression. The cells were then subjected to SDS-PAGE analysis after ultrasonic treatment. Figure 7 As shown in Figure a, in lanes 3 and 4, bands appear at 25-30 kDa, consistent with the theoretical molecular weight of the Trx-SPRR2A-pHLIP-His protein. Compared to lanes 1 and 2, lane 3 exhibits a distinct band in the 25-30 kDa region. The target band in lane 4 is more pronounced than in lane 5. This result indicates that the protein can be expressed soluble in E. coli.

[0191] Nickel ions can bind to proteins with histidine tags or imidazole on the nickel column, and imidazole forms a competitive binding with nickel ions. The target protein in the supernatant is adsorbed by the nickel column and treated with imidazole solutions of different concentrations. As the imidazole concentration increases, the protein bound to nickel ions can be eluted step by step, thereby achieving the separation of the target protein. Figure 7 As shown in b, different gradients of imidazole solutions were set for elution, and the electrophoresis analysis of each treated imidazole solution was performed to determine the optimal purification conditions: 50mM imidazole eluent can remove impurities, and 200mM imidazole eluent can elute the target protein. Figure 7 As shown in Figure 3, the purified Trx-SPRR2A-pHLIP-His protein exhibits a single, clear band in the 25-30 kDa molecular weight range, which is consistent with the theoretical molecular weight of the protein. This demonstrates that nickel affinity chromatography is effective in isolating the target protein. The yield of Trx-SPRR2A-pHLIP-His, as determined by the BCA assay, was 1.38 mg / L.

[0192] 2.2.2: Heterologous expression and purification of Trx-SPRR2A-GGGGS-pHLIP-His protein.

[0193] like Figure 8 As shown in a, in lanes 2 and 3, bands appear around 25-35 kDa, consistent with the theoretical molecular weight of the Trx-SPRR2A-GGGGS-pHLIP-His protein. Compared with lane 1, lane 2 shows a distinct band in the 25-35 kDa region, indicating successful induced expression of the protein. Compared with lane 4, the target band in lane 3 is more pronounced, indicating that the protein can be expressed soluble in E. coli.

[0194] like Figure 8As shown in b, different gradients of imidazole solutions were set for elution, and the electrophoresis analysis of each treated imidazole solution was performed to determine the optimal purification conditions: 50mM imidazole eluent can remove impurities, and 200mM imidazole eluent can elute the target protein. Figure 8 As shown in Figure 3, the purified Trx-SPRR2A-GGGGS-pHLIP-His protein exhibits a single, clear band within the 25-35 kDa molecular weight range, consistent with its theoretical molecular weight. This demonstrates that nickel affinity chromatography is effective in isolating the target protein. BCA assay yielded 2.42 mg / L of Trx-SPRR2A-GGGGS-pHLIP-His.

[0195] 2.2.3: Heterologous expression and purification of Trx-SPRR2A-GGGGSGGGGS-pHLIP-His protein.

[0196] like Figure 9 As shown in a, in lanes 2 and 3, bands appear around 25-35 kDa, consistent with the theoretical molecular weight of the Trx-SPRR2A-GGGGSGGGGS-pHLIP-His protein. Compared with lane 1, lane 2 shows a distinct band in the 25-35 kDa region, indicating that the protein was successfully induced for expression. Compared with lane 4, the target band in lane 3 is more pronounced, indicating that the protein can be expressed soluble in E. coli.

[0197] like Figure 9 As shown in b, different gradients of imidazole solutions were set for elution, and the electrophoresis analysis of each treated imidazole solution was performed to determine the optimal purification conditions: 50mM imidazole eluent can remove impurities, and 200mM imidazole eluent can elute the target protein. Figure 9 As shown in Figure 3, the purified Trx-SPRR2A-GGGGSGGGGS-pHLIP-His protein exhibits a single, clear band within the 25-35 kDa molecular weight range, consistent with its theoretical molecular weight. This demonstrates that nickel affinity chromatography is effective in isolating the target protein. BCA assay determined the yield of Trx-SPRR2A-GGGGSGGGGS-pHLIP-His to be 2.56 mg / L.

[0198] 2.2.4: Heterologous expression and purification of Trx-His-SPRR2A-pHLIP protein.

[0199] like Figure 10As shown in Figure a, in lanes 3 and 4, bands appear at 25-35 kDa, consistent with the theoretical molecular weight of the Trx-His-SPRR2A-pHLIP protein. Compared to lanes 1 and 2, lane 3 exhibits a distinct band in the 25-30 kDa region. The target band in lane 4 is more pronounced than in lane 5. This result indicates that the protein can be expressed soluble in E. coli.

[0200] like Figure 10 As shown in b, by setting different concentrations of imidazole solution for elution and performing SDS-PAGE analysis on each treated imidazole solution, the optimal purification conditions for the target protein were determined: 50mM imidazole elution solution can remove impurities, and 200mM imidazole elution solution can elute the target protein. Figure 10 As shown in Figure 3, the purified Trx-His-SPRR2A-pHLIP protein exhibits a single, clear band within the 25-35 kDa molecular weight range, consistent with its theoretical molecular weight. This demonstrates that nickel affinity chromatography is effective in isolating the target protein. The yield of Trx-His-SPRR2A-pHLIP, as determined by the BCA assay, was 1.12 mg / L.

[0201] 2.2.5: Heterologous expression and purification of Trx-His-SPRR2A-GGGGS-pHLIP protein.

[0202] like Figure 11 As shown in a, in lanes 2 and 3, bands appear around 25-35 kDa, consistent with the theoretical molecular weight of the Trx-His-SPRR2A-GGGGS-pHLIP protein. Compared with lane 1, lane 2 shows a distinct band in the 25-35 kDa region, indicating that the protein was successfully induced for expression. Compared with lane 4, the target band in lane 3 is more pronounced, indicating that the protein can be expressed soluble in E. coli.

[0203] like Figure 11 As shown in b, different gradients of imidazole solutions were set for elution, and the electrophoresis analysis of each treated imidazole solution was performed to determine the optimal purification conditions: 50mM imidazole eluent can remove impurities, and 200mM imidazole eluent can elute the target protein. Figure 11As shown in Figure 3, the purified Trx-His-SPRR2A-GGGGS-pHLIP protein exhibits a single, clear band within the 25-35 kDa molecular weight range, consistent with its theoretical molecular weight. This demonstrates that nickel affinity chromatography is effective in isolating the target protein. BCA assay determined the yield of Trx-His-SPRR2A-GGGGS-pHLIP to be 1.20 mg / L.

[0204] 2.2.6: Heterologous expression and purification of Trx-His-SPRR2A-GGGGSGGGGS-pHLIP protein.

[0205] like Figure 12 As shown in a, in lanes 2 and 3, bands appear around 25-35 kDa, consistent with the theoretical molecular weight of the Trx-His-SPRR2A-GGGGSGGGGS-pHLIP protein. Compared with lane 1, lane 2 shows a distinct band in the 25-35 kDa region, indicating that the protein was successfully induced for expression. Compared with lane 4, the target band in lane 3 is more pronounced, indicating that the protein can be expressed soluble in E. coli.

[0206] like Figure 12 As shown in b, different gradients of imidazole solutions were set for elution, and the electrophoresis analysis of each treated imidazole solution was performed to determine the optimal purification conditions: 50mM imidazole eluent can remove impurities, and 200mM imidazole eluent can elute the target protein. Figure 12 As shown in Figure 3, the purified Trx-His-SPRR2A-GGGGSGGGGS-pHLIP protein exhibits a clear, single band in the molecular weight range of 25-35 kDa, consistent with the theoretical molecular weight of the protein. This demonstrates that nickel affinity chromatography is effective in isolating the target protein. BCA assay determined the yield of Trx-His-SPRR2A-GGGGSGGGGS-pHLIP to be 6.02 mg / L.

[0207] 2.3: Serum stability testing of six pH-responsive SPRR2A proteins.

[0208] Six pH-responsive SPRR2A proteins were diluted to 10 μM in neutral / acidic medium containing 10% serum and incubated at 37°C. Figure 13As shown in Figure a, after the six pH-responsive SPRR2A proteins were incubated with neutral culture medium containing 10% fetal bovine serum for 6 hours, Trx-SPRR2A-pHLIP-His, Trx-SPRR2A-GGGGS-pHLIP-His and Trx-His-GGGGSGGGGS-SPRR2A-pHLIP all showed obvious precipitation, while Trx-His-SPRR2A-pHLIP, Trx-His-SPRR2A-GGGGS-pHLIP and Trx-His-SPRR2A-GGGGSGGGGS-pHLIP did not show obvious precipitation. Figure 13 As shown in b, after the six pH-responsive SPRR2A proteins were incubated with acidic culture medium containing 10% fetal bovine serum for 2 hours, Trx-SPRR2A-pHLIP-His, Trx-SPRR2A-GGGGS-pHLIP-His, Trx-His-GGGGSGGGGS-SPRR2A-pHLIP, Trx-His-SPRR2A-pHLIP, and Trx-His-SPRR2A-GGGGS-pHLIP all showed obvious precipitation, while Trx-His-SPRR2A-GGGGSGGGGS-pHLIP did not show obvious precipitation.

[0209] After centrifugation of the above six proteins, the supernatant and precipitate were taken for SDS-PAGE detection. Figure 13 As shown in e, the supernatant bands of Trx-SPRR2A-pHLIP-His, Trx-SPRR2A-GGGGS-pHLIP-His, Trx-His-GGGGSGGGGS-SPRR2A-pHLIP and Trx-His-SPRR2A-GGGGS-pHLIP after incubation with acidic medium were significantly weaker than those of Trx-His-SPRR2A-GGGGSGGGGS-pHLIP, as shown in Figure 13 In the gel image of the precipitate of f, these five proteins all have a distinct band, and the molecular weight is consistent with the target band. This indicates that under acidic conditions, these five proteins are less stable than Trx-His-SPRR2A-GGGGSGGGGS-pHLIP. Figure 13As shown in Figures c and d, after incubation in neutral medium, the supernatant bands of Trx-SPRR2A-pHLIP-His, Trx-SPRR2A-GGGGS-pHLIP-His, Trx-His-GGGGSGGGGS-SPRR2A-pHLIP, and Trx-His-SPRR2A-GGGGS-pHLIP were also significantly weaker than those of Trx-His-SPRR2A-GGGGSGGGGS-pHLIP. The precipitated gel images showed a distinct band near the molecular weight of the target band for each of the three proteins. Therefore, we concluded that Trx-His-SPRR2A-GGGGSGGGGS-pHLIP was more stable than the other five proteins, and Trx-His-SPRR2A-GGGGSGGGGS-pHLIP was ultimately selected for subsequent experiments and named THSGp.

[0210] 2.4: Secondary mass spectrometry and molecular weight determination of THSGp.

[0211] In order to verify the amino acid sequence and molecular weight of THSGp, mass spectrometry identification was performed. The protein bands in SDS-PAGE were cut and subjected to mass spectrometry analysis. Table 12 shows the fragments after proteolysis, and the sequence coverage reached 95%, indicating that the protein was correctly expressed. Mass spectrometry analysis confirmed the presence of these peptides. In addition, the molecular weight of the protein solution with a concentration of 0.3 mg / ml was detected by RapifleX MALDI-TOF / TOF mass spectrometer. Figure 14 The results showed that the molecular weight of THSGp was 25.8 kDa, which was consistent with its theoretical molecular weight of 26.8 kDa.

[0212] Table 12: Mass spectrometric identification of THSGp.

[0213]

[0214] Example 2: Evaluation of the anti-tumor activity of THSGp.

[0215] 1: Experimental method.

[0216] 1.1: Cell culture.

[0217] MCF-7 cells, MDA-MB-231 cells, and MDA-MB-468 cells were cultured in DMEM medium containing 10% FBS; HCT-8 cells, HCT-116 cells, and HepG2 cells were cultured in 1640 medium containing 10% FBS; HIEC-6 cells were cultured in Endothelial Cell Medium, and MCF-10A cells were cultured in CM-0525 medium, a special medium for MCF-10A cells. All cells were cultured in a cell culture incubator.

[0218] 1.2: MTT assay for cell viability (%).

[0219] (1) MDA-MB-231 and MCF-7 cells were seeded in 96-well plates at a density of 3×10 3 The cells were cultured in a 37°C cell culture incubator overnight.

[0220] (2) Trx-SPRR2A and THSGp were diluted to 1 μM, 0.5 μM, 0.25 μM, 0.125 μM and 0.0625 μM using culture medium containing 10% FBS at pH 7.4 and pH 6.5.

[0221] (3) Remove the original culture medium and add 100 μL of neutral or acidic culture medium containing different concentrations of Trx-SPRR2A or THSGp, and incubate at 37°C for 24 hours.

[0222] (4) Mix 100 μL of serum-free culture medium and 20 μL of 5 mg / mL MTT solution, then add 120 μL to each well and incubate at 37°C for 3 hours.

[0223] (5) Remove the culture medium containing MTT and add dimethyl sulfoxide.

[0224] (6) After shaking the plate for 6 minutes using the shaking function of the SpectraMax Plus 384 microplate reader, the absorbance at 570 nm was measured to calculate the cell viability.

[0225] 1.3: Cellular internalization behavior of THSGp.

[0226] Trx-SPRR2A and THSGp were labeled with FITC to obtain FITC-Trx-SPRR2A and FITC-THSGp, respectively. The cellular internalization of THSGp was observed using confocal microscopy.

[0227] (1) MDA-MB-231 and MCF-7 cells were seeded in 96-well plates, with 1×10 cells per well. 6 Cells were cultured in a 37°C cell incubator overnight.

[0228] (2) The original culture medium was removed and the cells were cultured with neutral / acidic culture medium without serum; neutral / acidic culture medium containing 0.5 μM FITC-Trx-SPRR2A; and neutral / acidic culture medium containing 0.5 μM FITC-THSGp for 12 hours.

[0229] (3) Discard the original culture medium in the well, wash the cells twice with phosphate buffered saline (PBS), add 4% paraformaldehyde, and incubate at room temperature for 30 minutes to ensure that the cells are fully fixed.

[0230] (4) Discard the 4% paraformaldehyde, wash the cells twice with phosphate buffer, add 50 μL of ready-to-use DAPI, and incubate at room temperature in the dark for 10 minutes.

[0231] (5) Aspirate DAPI, wash the cells twice with phosphate buffer, add 100 μL of PBS, and observe each treatment group using a Zeiss confocal microscope and collect images.

[0232] 1.4: Detect cell viability by crystal violet staining.

[0233] (1) MCF-7 and MDA-MB-231 cells were seeded in 96-well plates, with 5×10 cells per well. 3 Cells were cultured in a cell culture incubator overnight to ensure that the cells were fully attached to the wall.

[0234] (2) Using culture medium containing 10% fetal bovine serum at pH 7.4 and pH 6.5, Trx-SPRR2A and THSGp were diluted to 1 μM, 0.5 μM, 0.25 μM, and 0.125 μM, respectively. These diluted proteins were then incubated with cells at 37°C and 5% CO2 for 24 hours.

[0235] (3) After the incubation, discard the original culture medium and wash the cells twice with phosphate buffer. Then, add 5% crystal violet solution, 50 μL / well, and incubate on a shaker for 20 minutes.

[0236] (4) Discard the crystal violet solution and wash three times with PBS.

[0237] (5) Turn the 96-well plate upside down, remove excess PBS in the wells, and let it dry in preparation for subsequent detection steps.

[0238] (6) Use Cytation 5 to observe and take pictures of the cells to visually evaluate changes in cell morphology and number. Subsequently, methanol solution was added at 200 μL / well to dissolve the crystal violet in the cells, and then incubated on a shaker for 20 minutes to allow the dye to fully dissolve.

[0239] (7) The absorbance at 570 nm was detected using a SpectraMax Plus 384 microplate reader. The relative viability of the cells was calculated based on the absorbance value to quantitatively evaluate the effect of THSGp on cell activity.

[0240] 1.5: EdU detection of cell proliferation.

[0241] (1) MCF-7 and MDA-MB-231 cells were seeded in 12-well plates, with 1×10 cells per well. 6 The cells were cultured in a cell culture incubator overnight to ensure that the cells were fully attached.

[0242] (2) Trx-SPRR2A and THSGp were diluted to 0.5 μM using culture medium at pH 7.4 and pH 6.5 containing 10% fetal bovine serum. The diluted proteins were then incubated with cells at 37°C and 5% CO2 for 24 hours to evaluate the effects of the proteins on cell proliferation.

[0243] (3) EdU cell proliferation detection kit from BBI was used to perform EdU staining and Hoechst 33342 nuclear staining on cells in each group according to the instructions.

[0244] (4) After staining, add phosphate buffer and use Cytation 5 to observe and take cell images to analyze cell proliferation.

[0245] 1.6: Light microscopy and iFluor 488 wheat germ agglutinin (WGA) staining.

[0246] (1) MCF-7 and MDA-MB-231 cells were seeded in 96-well plates, with 1×10 cells per well. 6 The cells were cultured in a 37°C incubator overnight to ensure normal cell growth.

[0247] (2) The culture medium was discarded and the cells were cultured with serum-free neutral / acidic culture medium; neutral / acidic culture medium containing 0.5 μM FITC-Trx-SPRR2A; and neutral / acidic culture medium containing 0.5 μM FITC-THSGp for 12 hours.

[0248] (3) The cells were observed and photographed under a microscope at 0 h, 12 h, and 24 h of incubation.

[0249] (4) After incubation for 12 hours and 24 hours, add 4% paraformaldehyde and incubate at room temperature for 30 minutes to fix the cells.

[0250] (5) Discard the 4% paraformaldehyde, wash gently twice with PBS, add 50 μL of 10 μg / mL WGA to each well, and incubate at room temperature in the dark for 30 minutes.

[0251] (6) Discard the WGA, gently wash twice with PBS, add 50 μL of ready-to-use DAPI, and incubate at room temperature in the dark for 10 minutes.

[0252] (7) Aspirate DAPI, gently wash twice with PBS, add 100 μL of PBS, observe and collect images of each treatment group using ZOE Fluorescent CellImager, and perform quantitative analysis using WGA, setting the fluorescence of the control group as 100%.

[0253] 1.7: Scanning electron microscopy.

[0254] (1) Place the coverslip soaked in alcohol on an alcohol burner, let it cool, and place it in a 24-well plate. Inoculate MCF-7 and MDA-MB-231 cells at a rate of 1×10 cells per well. 6 Cells were cultured overnight in a cell culture incubator.

[0255] (2) The culture medium was discarded and the cells were cultured with serum-free neutral / acidic culture medium; neutral / acidic culture medium containing 0.5 μM FITC-Trx-SPRR2A; and neutral / acidic culture medium containing 0.5 μM FITC-THSGp for 12 hours.

[0256] (3) Discard the original culture medium, wash twice with phosphate buffer, add electron microscopy fixative, fix for 2 hours at room temperature in the dark, and store at 4°C. Then send to Wuhan Sevier Biotechnology Co., Ltd. for SEM examination.

[0257] 1.8: PI staining experiment.

[0258] (1) MCF-7 and MDA-MB-231 cells were seeded into 96-well plates, with approximately 1×10 cells per well. 6 The cells were cultured in a 37°C incubator overnight to ensure normal cell growth.

[0259] (2) The culture medium was discarded and the cells were cultured with serum-free neutral / acidic culture medium; neutral / acidic culture medium containing 0.5 μM FITC-Trx-SPRR2A; and neutral / acidic culture medium containing 0.5 μM FITC-THSGp for 12 hours.

[0260] (3) Discard the original culture medium, add PI to a final concentration of 50 μg / mL, and incubate at room temperature in the dark for 10 minutes;

[0261] (4) Discard the PI, gently wash twice with PBS, add 100 μL of PBS, and observe and collect images using a ZOE Fluorescent CellImager.

[0262] 2: Experimental results.

[0263] 2.1: MTT assay for cell viability.

[0264] like Figure 15 、 Figure 16As shown, the inhibitory effects of THSGp and Trx-SPRR2A on eight cell types were positively correlated with their concentrations. The inhibitory effect of Trx-SPRR2A was not pH-dependent. In contrast, THSGp showed significant pH dependence on tumor cells, but had no significant effect on two normal cells. Under weakly acidic conditions, the activity of THSGp was generally better than that of Trx-SPRR2A. After treatment with 1 μM THSGp under weakly acidic conditions for 24 hours, the survival rates of MCF-7, MDA-MB-231, MDA-MB-468, HCT-8, HCT-116, and HepG2 cells were 41.34%, 45.51%, 46.09%, 56.94%, 53.76%, and 60.04%, respectively. The IC values ​​obtained by MTT assay were 50 As shown in Table 13, THSGp has low toxicity to normal cells, and the IC 50 The values ​​were 12.91μM and 4.383μM, respectively. The toxicity to tumor cells under weak acidic conditions was much higher than that under neutral conditions. Under acidic conditions, THSGp showed significant cytotoxicity to MCF-7 and MDA-MB-231 cells, with an IC 50 The toxicity of THSGp to other tumor cells was relatively low, indicating that MCF-7 and MDA-MB-231 cells were more sensitive to THSGp. Based on the above experimental results, this example selected MCF-7 and MDA-MB-231 cell lines as subsequent experimental subjects to further study the anti-tumor activity and mechanism of THSGp.

[0265] Table 13: IC of different cells treated with THSGp at different pH 50 value.

[0266]

[0267] Note: - indicates not detected.

[0268] 2.2: Cellular internalization behavior of THSGp.

[0269] like Figure 17As shown, at pH 7.4, almost no green fluorescence was observed in both cells treated with Trx-SPRR2A. At pH 6.5, only a small amount of weak green fluorescence was observed in both cells. THSGp had difficulty targeting the cell surface under neutral conditions, exhibiting only weak green fluorescence in tumor cells. This indicates that neither Trx-SPRR2A nor THSGp under neutral conditions effectively entered cells. However, at a weakly acidic pH 6.5, both cells treated with THSGp exhibited strong green fluorescence, indicating that THSGp was extensively internalized. Trx-SPRR2A exhibited poor cellular internalization under both neutral and weakly acidic conditions. In contrast, THSGp exhibited efficient cellular internalization under weakly acidic conditions, primarily due to its pHLIP sequence. The pHLIP sequence forms a transmembrane α-helix under weakly acidic conditions, successfully delivering SPRR2A to the tumor cell surface. This pH-dependent cellular internalization mechanism provides THSGp with a unique advantage in tumor-targeted therapy, enabling it to function effectively under the weakly acidic conditions of the tumor microenvironment.

[0270] 2.3: Crystal violet staining to detect cell viability.

[0271] like Figure 18 As shown in a and c, with the increase of drug concentration, the number of cells in each treatment group decreased significantly compared with the control group, showing a dose-dependent manner. Under acidic conditions, the number of cells in the THSGp-treated group decreased significantly with the increase of drug concentration. Figure 18 Quantitative results in Figures b and d demonstrate that THSGp at various concentrations exhibited robust inhibitory activity against tumor cells, consistent with the results in Figures a and c. In an acidic environment, treatment with 1 μM THSGp for 24 hours resulted in viability rates of 44.47% and 48.09% for MCF-7 and MDA-MB-231 cells, respectively. This demonstrates that THSGp possesses potent antitumor activity that is pH-dependent.

[0272] These results indicate that Trx-SPRR2A itself cannot efficiently enter tumor cells and inhibit cell growth. Under weakly acidic conditions, pHLIP in the THSGp fusion protein can anchor SPRR2A at its N-terminus to the tumor cell surface by forming a transmembrane α-helix, exerting anti-tumor activity.

[0273] 2.4: EdU detection of cell proliferation.

[0274] like Figure 19As shown in a and b, compared with the control group, the red fluorescence intensity of the two cells did not change significantly after treatment with Trx-SPRR2A at pH 7.4 and THSGp at pH 7.4; however, the red fluorescence intensity of the cells treated with THSGp at pH 6.5 was significantly reduced. Figure 19 Quantitative analysis results in Figures b and d show that the EdU-positive cell rates in the Trx-SPRR2A-treated groups were similar at pH 7.4 and pH 6.5, reaching 96.49% and 93.47% for MCF-7 cells, and 97.52% and 94.22% for MDA-MB-231 cells, respectively. Notably, under weakly acidic conditions, THSGp treatment reduced the EdU-positive cell rates in MCF-7 and MDA-MB-231 cells to 68.46% and 67.80%, respectively. These results demonstrate that THSGp has a potent inhibitory effect on tumor cells in an acidic environment.

[0275] 2.5: iFluor 488 wheat germ agglutinin WGA staining was used to observe the integrity of the cell membrane.

[0276] like Figure 20 As shown in a and b, compared with the control group, after 12 hours, the green fluorescence intensity of both cells in the Trx-SPRR2A treatment group did not change significantly under neutral or acidic conditions, and the cell outlines were intact. However, under acidic conditions, the cell membrane of the THSGp treatment group changed significantly compared with the control group, and no complete cell membrane outline appeared. After 24 hours, these changes were more obvious, and the green fluorescence appeared in fragments, indicating that the cell membrane lost its integrity after THSGp treatment and the cell morphology changed significantly. Figure 20 Quantitative analysis of cf showed that WGA fluorescence intensity decreased after treatment with Trx-SPRR2A and THSGp. At both 12 and 24 hours, WGA fluorescence intensity in the THSGp-treated group under acidic conditions was the lowest among all treatment groups, particularly at 24 hours. This suggests that THSGp kills tumor cells by disrupting the cell membrane, thereby exerting its anti-tumor effects.

[0277] 2.6: Observe cell morphology using scanning electron microscopy.

[0278] like Figure 21 As shown, untreated cells showed intact morphology. Trx-SPRR2A-treated cells showed no significant changes in their cell membrane surface under neutral or acidic conditions, maintaining their intact morphology. In contrast, the THSGp-treated group showed significant cell membrane damage under weakly acidic conditions, manifested as wrinkling, damage, and leakage of contents. This suggests that THSGp-treated cells cause cell membrane damage, resulting in incomplete cell membranes and leakage of contents, which is consistent with the above results and suggests that THSGp exerts its anti-tumor effects by damaging the cell membrane.

[0279] 2.7: PI staining to detect cell membrane permeability.

[0280] like Figure 22 As shown in the figure, no obvious red fluorescence was observed in the control group and the Trx-SPRR2A-treated group under neutral conditions. Under neutral conditions, only a small amount of red fluorescence was observed in the THSGp-treated group and the Trx-SPRR2A-treated group under acidic conditions, indicating that only a small amount of PI was able to enter the cells. However, under acidic conditions, a large amount of red fluorescence was observed in the THSGp-treated group, and under an optical microscope, it was seen that the cell morphology was incomplete, the cell membrane was damaged, and cell fragments were obvious. This indicates that THSGp causes cell death by destroying the cell membrane. Therefore, it can be concluded that THSGp kills tumor cells by destroying the cell membrane, thereby exerting its anti-tumor effect.

[0281] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Claims

1. Fusion protein THSGp, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID NO.

1.

2. The use of the fusion protein THSGp according to claim 1 in the preparation of anti-tumor drugs, characterized in that: The tumor is colon cancer, liver cancer or breast cancer.

3. The use of the fusion protein THSGp in the preparation of anti-tumor drugs according to claim 2, characterized in that: The medicine uses the fusion protein THSGp as the only active ingredient.

4. The use of the fusion protein THSGp in the preparation of anti-tumor drugs according to claim 3, characterized in that: The drug also includes pharmaceutically acceptable excipients.

5. The use of the fusion protein THSGp in the preparation of anti-tumor drugs according to claim 4, characterized in that: The auxiliary material is selected from one or more of a diluent, a filler, a stabilizer, an osmotic pressure regulator, a pH regulator and a preservative.

6. The use of the fusion protein THSGp in the preparation of anti-tumor drugs according to claim 5, characterized in that: In the medicine, the content of the fusion protein THSGp is ​​1wt%-99.9wt%.

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