A tumor acidity response functional polypeptide capable of targeting activation of p53 and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a tumor acidity response functional polypeptide capable of targeting and activating p53 and application thereof. The amino acid sequence of the polypeptide is shown as SEQ ID NO. 1. The application first designs and expresses the tumor acidity response functional polypeptide Trx-AAN-pHLIP-NLS-PMI (TApNP) capable of targeting and activating the tumor suppressor protein p53, and comprehensively evaluates the physical and chemical characteristics, intracellular internalization, nuclear uptake and anti-tumor activity of the polypeptide. The results show that in a weak acid environment, the TApNP can deliver PMI into cells and guide it to the cell nucleus, and effectively inhibit the proliferation of tumor cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a tumor acidity response functional polypeptide capable of targeting and activating p53 and application thereof. BACKGROUND

[0002] Protein therapy has many advantages in tumor treatment, but there are various difficulties in delivering pharmacologically active proteins or polypeptides to specific tissues or cells, including instability in blood circulation, enzyme degradation, short half-life, immunogenicity, and difficulty in penetrating the cell membrane, etc.

[0003] Compared with genome editing applications that modify DNA, direct delivery of functional proteins has better specificity, higher safety and wider applicability. In order to reach the intracellular target, in addition to ensuring the stability of the protein, some difficulties need to be overcome: (1) penetrating the hydrophobic, negatively charged cell membrane and the dense actin network inside the cell; (2) escaping from the endosome to avoid lysosomal degradation; (3) moving to its intracellular target in the reducing cytoplasm; (4) according to the mode of action of the protein, if necessary, entering the target organelle where it functions, such as the nucleus or mitochondria.

[0004] Physical techniques such as electroporation, microinjection, and ultrasound perforation can overcome these difficulties, and researchers have developed some engineered nanodevices, such as nanoneedles, nanowires or nanotubes, for intracellular delivery of various drugs including proteins and nucleic acids. Although studies have shown that these physical methods are effective, they are limited to in vitro or ex vivo applications and are difficult to achieve in vivo drug delivery, so intracellular delivery of proteins remains an important challenge to be overcome. SUMMARY

[0005] To solve the above technical problems, the application provides a tumor acidity response functional polypeptide capable of targeting and activating p53 and application thereof.

[0006] In a first aspect, the application provides a tumor acidity response functional polypeptide capable of targeting and activating p53, the amino acid sequence of the polypeptide being shown as SEQ ID NO. 1.

[0007] In a second aspect, the application provides a gene encoding the tumor acidity response functional polypeptide capable of targeting and activating p53, the nucleotide sequence of the gene being shown as SEQ ID NO. 2.

[0008] In a third aspect, the application provides a recombinant plasmid containing the gene.

[0009] Further, the preparation method of the recombinant plasmid comprises the following steps:

[0010] 1) the target gene AAN-pHLIP-PMI with the sequence as shown in SEQ ID NO. 5 is synthesized;

[0011] 2) the target gene AAN-pHLIP-PMI is inserted into the pET32a plasmid by the seamless cloning technology to obtain the pET32a-AAN-pHLIP-PMI recombinant plasmid, which is transformed into E. coli DH5a, and the positive clone is identified, the colony PCR identification is determined to be correct, and the pET32a-AAN-pHLIP-PMI recombinant plasmid is extracted;

[0012] 3) the pET32a-AAN-pHLIP-NLS-PMI recombinant plasmid is obtained by full plasmid PCR using the primer with the sequence as shown in SEQ ID NO. 10-11 and the pET32a-AAN-pHLIP-PMI recombinant plasmid as a template.

[0013] In a fourth aspect, the application provides a genetically engineered bacterium containing the recombinant plasmid.

[0014] Further, the construction method of the genetically engineered bacterium is to transform the recombinant plasmid into E. coli BL21 (DE3) competent cells for protein expression, so as to obtain an engineered bacterium for heterologous expression of the polypeptide.

[0015] In a fifth aspect, the application provides application of the polypeptide in preparation of a medicine for treating cancer, and the cancer includes colon cancer.

[0016] Further, the medicine can inhibit the growth and proliferation of colon cancer cells under weak acidic conditions.

[0017] In a sixth aspect, the application provides a medicine for treating colon cancer, and the effective component is the polypeptide.

[0018] Further, the medicine for treating colon cancer further includes a pharmaceutically acceptable carrier.

[0019] Compared with the prior art, the application has the following beneficial effects:

[0020] The application first designs and expresses a tumor acidity response function polypeptide Trx-AAN-pHLIP-NLS-PMI (TApNP) which can target and activate the tumor suppressor protein p53, and comprehensively evaluates the physical and chemical characteristics, intracellular internalization, nuclear uptake and anti-tumor activity of the polypeptide. The results show that in a weak acidic environment, the TApNP can deliver the PMI to the cell and guide it to the cell nucleus, and effectively inhibit the proliferation of tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1PCR identification results of the recombinant plasmid pET32a-AAN-pHLIP-PMI.

[0022] Figure 2 PCR identification results of the pET32a-AAN-pHLIP-NLS-PMI full plasmid

[0023] Figure 3 12% SDS-PAGE analysis of TApP expression, a is the result of inducing expression of TApP at 16℃, 25℃, 37℃, lane M represents 14-120 kDa protein Marker; T, S, P represent the whole bacteria liquid, supernatant and precipitate after IPTG induction and ultrasonic crushing, respectively; b is the result of inducing expression without adding IPTG and adding IPTG at 16℃, lane W represents the supernatant after lysis of bacteria liquid without adding IPTG, and the arrow points to the band of the target protein TApP.

[0024] Figure 4 15% SDS-PAGE analysis of TApNP expression, a is the result of inducing expression of TApNP at 16℃, 25℃, 37℃. Lane M represents 10-190 kDa protein Marker, T, S, P represent the whole bacteria liquid, supernatant and precipitate after IPTG induction and ultrasonic crushing, respectively; b is the result of inducing expression without adding IPTG and adding IPTG at 25℃, lane W represents the supernatant after lysis of bacteria liquid without adding IPTG, and the band of the target protein TApNP is in the dotted line box.

[0025] Figure 5 15% SDS-PAGE (a) and Western blot (b) detection of purified TApP and TApNP, lane M represents 10-190 kDa Marker.

[0026] Figure 6 FITC-TApP and FITC-TApNP uptake by HCT116 cells detected by DeltaVision (scale: 20 μm).

[0027] Figure 7 Flow cytometry detection of FITC-TApP and FITC-TApNP uptake by HCT116 cells.

[0028] Figure 8 Relative fluorescence intensity of FITC in nuclear protein of each treatment group (compared with the control group).

[0029] Figure 9MTT assay was used to detect the cell viability. The relative viability of HCT116 cells treated with different concentrations of PMI, TApP and TApNP at pH 7.4 (a) and pH 6.5 (b) for 48 h was detected by MTT assay.

[0030] Figure 10 Crystal violet staining was used to detect the cell viability. The relative viability of HCT116 cells treated with different concentrations of PMI, TApP and TApNP at pH 7.4 (a) and pH 6.5 (b) for 48 h was detected by crystal violet staining. a, cell images after crystal violet staining (scale bar: 200 μm); b, quantitative analysis of crystal violet staining at pH 7.4; c, quantitative analysis of crystal violet staining at pH 6.5.

[0031] Figure 11 Calcein-AM / PI staining was used to detect the cell viability. The relative viability of HCT116 cells treated with 25 μM of PMI, TApP and TApNP at pH 7.4 (a) and pH 6.5 (b) for 48 h was detected by Calcein-AM / PI staining. a, HCT116 cell images after Calcein-AM / PI staining (scale bar: 100 μm); b, quantitative analysis of viable cell ratio of Calcein-AM / PI staining images.

[0032] Figure 12 EdU staining was used to detect the cell proliferation. a, images of cells after EdU staining (scale bar: 100 μm); b, quantitative analysis of proliferative cell ratio of EdU staining images. DETAILED DESCRIPTION

[0033] The present application will be described in detail below with reference to the accompanying drawings and specific examples, but should not be understood as limiting the present application. If not specifically stated, 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, if not specifically stated, can be obtained from commercial channels.

[0034] The amino acid sequence of the tumor acidity-responsive functional polypeptide TApNP capable of targeting and activating p53 provided by the present application is shown in SEQ ID NO. 1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 2. The amino acid sequence of the polypeptide TApP without nuclear localization signal peptide (NLS) is shown in SEQ ID NO. 3, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 4.

[0035] Example 1: Preparation and characterization of TApP and TApNP

[0036] 1. Experimental methods

[0037] 1.1 Construction of recombinant plasmid pET32a-AAN-pHLIP-PMI

[0038] 1.1.1 Synthesis of target gene AAN-pHLIP-PMI

[0039] The target gene AAN-pHLIP-PMI (SEQ ID NO. 5) was synthesized by Sangon Biotech Co., Ltd., which was inserted into the Sma I site of pUC57 plasmid to obtain the pUC57-AAN-pHLIP-PMI recombinant plasmid.

[0040] 1.1.2 Design of primers for amplifying AAN-pHLIP-PMI gene

[0041] Primers AAN-pHLIP-PMI-F (SEQ ID NO. 6) and AAN-pHLIP-PMI-R (SEQ ID NO. 7) were designed for PCR amplification of the AAN-pHLIP-PMI target fragment.

[0042] 1.1.3 Amplification and purification of target gene AAN-pHLIP-PMI

[0043] Four groups of target gene AAN-pHLIP-PMI were PCR amplified using pUC57-AAN-pHLIP-PMI recombinant plasmid as the template.

[0044] (3) Template digestion

[0045] 1 μL Dpn1 was added to each PCR product, and 10x buffer was added in proportion, and reacted at 37°C for 2 h to digest the template.

[0046] (4) Purification of PCR product

[0047] SanPrep column PCR product purification kit was used to recover the DNA fragment to obtain the purified recovered DNA solution.

[0048] 1.1.4 Extraction of pET32a vector plasmid

[0049] SanPrep column plasmid DNA mini-extraction kit was used to extract the pET32a vector plasmid:

[0050] 1.1.5 Design of primers for linearizing and amplifying pET32a vector

[0051] Primers pET32a-F (SEQ ID NO. 8) and pET32a-R (SEQ ID NO. 9) were designed for linearizing and amplifying pET32a vector by PCR method.

[0052] 1.1.6 Linearization amplification of vector pET32a

[0053] PCR amplification of 4 groups of linearized vectors with pET32a as template, the system is 50 μL.

[0054] (3) Template digestion and PCR product purification

[0055] Use Dpn 1 to digest the template, the steps are the same as 1.1.3(3).

[0056] (4) PCR product purification

[0057] Use the kit to recover the DNA fragment, the steps are the same as 1.1.3(4).

[0058] 1.1.7 Construction of pET32a-AAN-pHLIP-PMI recombinant plasmid by seamless cloning

[0059] Use the ready-to-use seamless cloning kit reaction to obtain pET32a-AAN-pHLIP-PMI recombinant plasmid.

[0060] (1) Seamless cloning reaction system (calculated according to the molar ratio of target fragment to linear vector as 3:1), as shown in Table 1.

[0061] Table 1 Seamless cloning reaction system

[0062] Reagents Volume 2x Seamless cloning Master Mix 10.0 μL AAN-pHLIP-PMI 0.1 μL pET32a 1.1 μL ddH2O 8.8 μL Total 20.0 μL

[0063] (2) Seamless cloning reaction conditions

[0064] Mix the reagents in the EP tube according to the above system, mix well, and react in a constant temperature water bath at 50℃ for 20 min. Immediately after the reaction is completed, transform it.

[0065] 1.1.8 Transformation into E. coli DH5α

[0066] Transform the extracted pET32a-AAN-pHLIP-PMI recombinant plasmid into E. coli DH5α competent cells for plasmid cloning:

[0067] 1.1.9 Identification of positive clones

[0068] Randomly select 10 single colony bacteria on the plate, pick them with a white gun head and add them to 40 μL LB medium, take 1 μL of them as a template for colony PCR to identify whether the target gene AAN-pHLIP-PMI exists. The primers used are T7 Primer and T7 Terminator Primer (universal primers).

[0069] The colony PCR product was collected and detected by nucleic acid gel electrophoresis to determine whether the band was correct. The bacterial solution was inoculated into LB medium and cultured at 37°C, 200 rpm overnight. The bacterial glycerol was preserved at -20°C, and part of the bacterial solution was sent to Sangon Biotech Company for sequencing.

[0070] 1.1.10 Extraction of recombinant plasmid

[0071] After confirming that the colony PCR identification and sequencing results were correct, the recombinant plasmid pET32a-AAN-pHLIP-PMI was extracted using a kit, and the extraction steps were the same as those in 1.1.4.

[0072] 1.1.11 Transformation into E. coli BL21 (DE3)

[0073] The extracted pET32a-AAN-pHLIP-PMI recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells for protein expression, and the transformation steps were the same as those in 1.1.8, thereby obtaining the engineered bacteria for heterologous expression of Trx-AAN-pHLIP-PMI (TApP).

[0074] 1.2 Preparation of TApNP

[0075] 1.2.1 Primer design for amplifying pET32a-AAN-pHLIP-NLS-PMI plasmid

[0076] pET32a-AAN-pHLIP-NLS-PMI-F: the sequence is shown in SEQ ID NO. 10;

[0077] pET32a-AAN-pHLIP-NLS-PMI-R: the sequence is shown in SEQ ID NO. 11.

[0078] 1.2.2 Obtaining plasmid pET32a-AAN-pHLIP-NLS-PMI by whole plasmid PCR

[0079] The plasmid pET32a-AAN-pHLIP-NLS-PMI was obtained by whole plasmid PCR using the pET32a-AAN-pHLIP-PMI plasmid as the template.

[0080] 1.2.3 Template digestion

[0081] The template was digested using Dpn 1, and the steps were the same as those in 1.1.3 (3).

[0082] 1.2.4 Identification of positive clones

[0083] The product obtained by whole plasmid PCR was subjected to nucleic acid gel electrophoresis to detect whether the band position was correct.

[0084] 1.2.5 Transformation into E. coli DH5a

[0085] The extracted pET32a-AAN-pHLIP-NLS-PMI recombinant plasmid was transformed into E. coli DH5a competent cells for plasmid cloning, and the transformation step was the same as 1.1.8. Single colonies were randomly picked and added to LB medium, which was cultured at 37°C and 200 rpm overnight. The bacterial glycerol was preserved at -20°C, and part of the bacterial solution was sent to Sangon Biotech Company for sequencing.

[0086] 1.2.6 Extraction of recombinant plasmid

[0087] The colony PCR identification was determined to be correct by sequencing, and the recombinant plasmid pET32a-AAN-pHLIP-NLS-PMI was extracted using a kit, and the extraction step was the same as 1.1.4.

[0088] 1.2.7 Transformation into E. coli BL21(DE3)

[0089] The extracted pET32a-AAN-pHLIP-NLS-PMI recombinant plasmid was transformed into E. coli BL21(DE3) competent cells for protein expression, and the transformation step was the same as 1.1.11, thereby obtaining the engineered bacteria for heterologous expression of Trx-AAN-pHLIP-NLS-PMI (TApNP).

[0090] 1.3 Heterologous expression of TApP and TApNP proteins

[0091] (1) 100 μL of the engineered bacteria for heterologous expression of TapP and TApNP were inoculated into 3 tubes of 10 mL LB medium (containing 100 ng / μL Amp), numbered ①-③, and cultured at 37°C and 200 rpm for 8 h;

[0092] (2) The bacterial solution in tubes ①-③ was inoculated into 3 bottles of 0.4 L LB liquid medium (Amp, 100 ng / μL), labeled, and cultured at 37°C and 250 rpm;

[0093] (3) When the OD600 was 0.6 to 0.8, the ① medium was cooled to 16°C and then 0.5 mM IPTG was added, and the culture was incubated at 16°C and 180 rpm for 12 h; the ② medium was cooled to 25°C and then an equal amount of IPTG was added, and the culture was incubated at 25°C and 180 rpm for 12 h; the ③ medium was directly added with an equal amount of IPTG, and the culture was incubated at 37°C and 180 rpm for 12 h;

[0094] (4) The ①-③ bacterial solution was centrifuged at 8000 rpm for 15 min, and the ①-③ bacterial bodies were collected and weighed;

[0095] (5) Resuspend the bacteria in 20 mM Tris buffer at a ratio of 1:12 (W / V), and perform ultrasonic disruption (set the power at 50%, work for 5 s, and pause for 7 s) until the bacterial solution is transparent;

[0096] (6) Take 100 μL of the bacterial solution after ultrasonic disruption, centrifuge part of the bacterial solution at 12000 rpm at 4°C for 30 min, take the supernatant, resuspend the precipitate in 20 mM Tris buffer at a ratio of 1:12 (W / V), and take the sample;

[0097] (7) Perform SDS-PAGE on the samples of whole bacteria, supernatant, and precipitate to detect the expression of TApP or TApNP under different temperature conditions.

[0098] 1.4 Purification of TApP and TApNP proteins

[0099] The reagents used are shown in Table 2:

[0100] Table 2 Reagents used for protein purification

[0101]

[0102] (1) The column material is combined with nickel ions, and 40 mL of 1xcharge buffer is passed through the column 3 times;

[0103] (2) Equilibrium, pass 40 mL of 1xbinding buffer through the column;

[0104] (3) Protein column, pass the supernatant after cell disruption centrifugation through the column, and repeat the column 2 times;

[0105] (4) Remove impurities, pass the column with 20 mM Tris buffer until CBB does not turn blue;

[0106] (5) Remove impurities, pass the column with 40 mM imidazole for TApP (use 60 mM imidazole for TApNP) in wash buffer until CBB does not turn blue;

[0107] (6) Elute TApP, pass the column with 50 mL of 150 mM imidazole in wash buffer (use 200 mM imidazole for TApNP), and collect the eluted solution;

[0108] (7) Regenerate the column material, pass the column with 20 mL of 1xstrip buffer to wash away the nickel ions and residual proteins;

[0109] (8) Clean the column material, pass the column with 100 mL of deionized water;

[0110] (9) Seal the column and add 20 mL of 1x charge buffer.

[0111] (10) Concentrate TApP and TApNP by ultrafiltration centrifuge tube and replace into 20 mM Tris buffer, then filter sterilization by 0.22 μm filter membrane in the biological safety cabinet, and determine the concentration of TApP and TApNP by BCA method.

[0112] 1.5 SDS-PAGE and WB identification of TApP and TApNP

[0113] 1.5.1 SDS-PAGE detection

[0114] (1) Prepare 12% or 15% separation gel and 5% concentration gel;

[0115] (2) Add 1 / 4 volume of 5x loading buffer to the protein sample, mix and heat in a 100°C metal bath for 6 min;

[0116] (3) Load protein marker and sample into the well, constant voltage 60V before sample enters the gel, then adjust to 120V after bromophenol blue reaches the separation gel, stop electrophoresis when the smallest band of protein marker migrates to 1-2 cm from the front; (4) Stain the gel in coomassie brilliant blue staining solution for 3 h, discard the staining solution, and remove the floating color in the destaining solution, replace several times until the bands are clear;

[0117] (5) Scan the gel with a canon scanner, and determine the protein purity by Image J gray scale analysis.

[0118] 1.5.2 Western blot identification

[0119] (1) First, electrophorese the protein according to the above method, and place the gel in the electrotransfer buffer;

[0120] (2) Soak the PVDF membrane in methanol for 30 s, wash with ultrapure water for 2 min each time, and also place in the electrotransfer liquid;

[0121] (3) Install the electrotransfer tank, with the cathode (black side) as the bottom, and place the filter paper, gel, PVDF membrane, filter paper and two pieces of sponge in order, with no air bubbles between the filter paper, gel and PVDF membrane, cover with the anode (white side) and clamp tightly, install into the tank, with the black side of the clamp opposite the black side of the tank;

[0122] (4) Place the electrotransfer tank in an ice water bath, and electrotransfer at a constant current of 300 mA for 90 min;

[0123] (5) After electroporation, remove the PVDF membrane, mark the front side with the adhesive side, soak in methanol for 30 seconds and blow dry, soak in methanol again for 30 seconds, wash with water for 2 minutes, soak in Ponceau S for 5 minutes, observe whether the strip is intact, and clean with TBST 3 times for 7 minutes each time.

[0124] (7) Block with TBST blocking solution containing 5% skim milk at room temperature for 1 hour, and wash with TBST 3 times for 7 minutes each time;

[0125] (9) Dilute the primary antibody with TBST at a ratio of 1:1000, incubate the PVDF membrane with the primary antibody at 4°C overnight, and wash with TBST 3 times for 7 min each time;

[0126] (10) Dilute the HRP-labeled secondary antibody with TBST at a ratio of 1:1000, incubate the PVDF membrane at room temperature for 2 hours, and wash with TBST 3 times for 7 minutes each time;

[0127] (11) Protein bands can be observed by taking pictures on a multifunctional imager using the ultrasensitive ECL chemiluminescence kit.

[0128] 2 Results and Analysis

[0129] 2.1 Construction of recombinant plasmids

[0130] 2.1.1 Construction of recombinant plasmid pET32a-AAN-pHLIP-PMI

[0131] The colony PCR identification results of the recombinant plasmid pET32a-AAN-pHLIP-PMI are as follows: Figure 1 As shown, a distinct band appears around 750 bp, consistent with the theoretical size (700 bp) of the universal primer-intergenic fragment (Trx-AAN-pHLIP-PMI) in the pET32a-AAN-pHLIP-PMI recombinant plasmid. Two bacterial cultures corresponding to lanes 6 and 8 were sequenced by Sangon Biotech. The sequencing results were consistent with the theoretical sequence of the AAN-pHLIP-PMI gene, indicating that the recombinant plasmid pET32a-AAN-pHLIP-PMI was successfully constructed using seamless cloning.

[0132] 2.1.2 Construction of recombinant plasmid pET32a-AAN-pHLIP-NLS-PMI

[0133] After obtaining the recombinant plasmid pET32a-AAN-pHLIP-NLS-PMI through whole-plasmid PCR, the product was subjected to agarose gel electrophoresis, and images were obtained using a nucleic acid gel imaging system. Figure 2 As shown, a distinct band appears between 5000bp and 8000bp, which is consistent with the theoretical value (5928bp) of the pET32a-AAN-pHLIP-NLS-PMI recombinant plasmid.

[0134] The obtained recombinant plasmid was transformed into E. coli DH5α, and three single colonies were randomly selected, preserved, and sequenced. The sequencing results were identical to the AAN-pHLIP-NLS-PMI gene sequence, indicating that the pET32a-AAN-pHLIP-NLS-PMI recombinant plasmid was successfully constructed.

[0135] 2.2 Heterologous expression of TApP and TApNP

[0136] 2.2.1 Expression of TApP

[0137] like Figure 3 As shown in Figure a, compared with 25℃ and 37℃, the expression level of TApP in the supernatant after low-temperature induction at 16℃ was the highest (arrow). Therefore, TApP expression was induced overnight at 16℃. In Figure b, compared with the supernatant of the lysate without IPTG inducer, a clear band appeared in the supernatant with IPTG inducer at 14-25kDa (arrow), which is consistent with the theoretical molecular weight of TApP, indicating that TApP was successfully expressed. Moreover, the corresponding band in the precipitate was not obvious, proving that the target protein was mainly expressed in soluble form at this temperature.

[0138] 2.2.2 Expression of TApNP

[0139] like Figure 4 As shown in Figure a, compared with 16℃ and 37℃, the expression level of TApNP in the supernatant after low-temperature induction at 25℃ was the highest (dashed box). Therefore, TApNP expression was induced overnight at 25℃. In Figure b, compared with the supernatant of the lysis buffer without IPTG inducer, a clear band appeared in the supernatant with IPTG inducer at 15-20kDa (dashed box), which is consistent with the theoretical molecular weight of TApNP, indicating that TApNP was successfully expressed. Moreover, the amount of target protein in the supernatant was greater than that in the precipitate, proving that the target protein was mainly expressed in soluble form at this temperature.

[0140] 2.3 Purification of TApP and TApNP

[0141] like Figure 5 As shown in Figure a, single bands appeared in the 15-20 kDa range for both purified TapP and TapNP, consistent with the theoretical molecular weights (TApP: 18.3 kDa, TapNP: 19.2 kDa). Gray-scale analysis revealed purities of 94.00% and 91.26%, respectively, indicating successful purification of TapP and TapNP. In addition to SDS-PAGE, we also analyzed TapP and TapNP by Western blotting using rabbit anti-His-tag polyclonal antibody as the primary antibody. Figure 5The Western Blot results shown in Figure b again confirmed the successful expression and purification of TApP and TApNP with His-tag. The protein concentration was detected by BCA method, and the yield of TApP and TApNP was calculated to be 3.87 mg / L and 10.21 mg / L, respectively.

[0142] Example 2: Study on the intracellular internalization and nuclear localization of TApNP

[0143] 1. Experimental method

[0144] 1.1 Cell culture

[0145] HCT116 cells were cultured in 1640 medium (10% FBS, 1% penicillin-streptomycin mixture) in a constant temperature incubator at 37°C and 5% CO2. The cells were observed daily using an inverted microscope, and the medium was replaced in time according to the observation results. When the cell density reached 80%, the cells were digested and subcultured using 0.25% trypsin.

[0146] 1.2 DeltaVision detection of the intracellular internalization behavior of TApNP

[0147] (1) First, sterilize the cell climbing sheet and dry it in a 12-well plate, then 6 HCT116 cells were plated in the 12-well plate at a density of 1 x 10

[0148] (2) FITC-labeled PMI, TApP and TapNP were obtained, and the three FITC-labeled proteins (FITC-PMI, FITC-TApP or FITC-TApNP) were diluted to 1 μM with RPMI-1640 medium (without FBS) at pH 7.4 or 6.5, respectively.

[0149] (3) Discard the original medium in the wells, and add ① RPMI-1640 medium (without FBS) at pH 7.4; ② RPMI-1640 medium (without FBS) at pH 6.5; ③ pH 7.4 medium containing 1 μM FITC-PMI; ④ pH 6.5 medium containing 1 μM FITC-PMI; ⑤ pH 7.4 medium containing 1 μM FITC-TApP; ⑥ pH 6.5 medium containing 1 μM FITC-TApP; ⑦ pH 7.4 medium containing 1 μM FITC-TApNP and ⑧ pH 6.5 medium containing 1 μM FITC-TApNP, and incubate in the cell incubator for 12 h.

[0150] (4) Discard the original medium in the wells, and add 4% paraformaldehyde to fix the cells at room temperature for 30 min.

[0151] (5) Slowly wash twice with PBS, add DAPI to cover the cells, and incubate at room temperature for 10 min.

[0152] (6) Aspirate the DAPI and wash twice with PBS. Remove the coverslips from the wells, aspirate excess liquid, and drop 10 μL of antifade mounting medium onto a glass slide, place the coverslip cell-side down onto it, and seal with nail polish.

[0153] (7) Use DeltaVision to collect images.

[0154] 1.3 Flow cytometry analysis of TApNP cellular internalization behavior

[0155] (1) Seed HCT116 cells at a density of 2 x 105cells per well in a 6-well plate and incubate in a CO2incubator overnight. 6

[0156] (2) Dilute FITC-PMI, FITC-TApP, and FITC-TApNP to 1 μM in RPMI-1640 medium (without FBS) at pH 7.4 or 6.5, respectively, and incubate in a cell incubator for 3 h with RPMI-1640 medium (without FBS) at pH 7.4 or 6.5 as negative controls.

[0157] (3) Discard the original medium in the wells, wash twice with PBS, and digest the cells before placing them in a 4 mL EP tube and centrifuging at 2000 rpm for 5 min.

[0158] (4) Discard the supernatant and wash the cells twice with PBS.

[0159] (5) Resuspend the cells with 600 μL of PBS and filter them, and analyze and record the fluorescence intensity with a flow cytometer.

[0160] 1.4 Nucleus uptake of TApNP

[0161] (1) Seed the cells in a 60 mm cell culture dish and incubate in a CO2incubator for 12 h.

[0162] (2) Dilute FITC-PMI, FITC-TApP, and FITC-TApNP to 1 μM in RPMI-1640 medium (without FBS) at pH 7.4 or 6.5, respectively, and incubate in a cell incubator for 12 h with RPMI-1640 medium (without FBS) at pH 7.4 or 6.5 as negative controls.

[0163] ​(3) Discard the original culture medium in the hole, wash twice with PBS, scrape the adherent cells with a cell scraper, add 1 mL PBS to the cells and move them to a centrifuge tube, centrifuge at 3000 rpm for 10 min at 4°C.

[0164] (4) Extract the nuclear protein using the nuclear protein extraction kit: discard the supernatant, add 450 μL of prepared pre-cooled Hypotonic Buffer (7 mL Buffer mixed with 35 μL phosphatase inhibitor, 70 μL PMSF and 7 μL DTT) to each tube of cells, suspend the precipitate, ice bath for 10 min, shake well; centrifuge the suspension at 3000 rpm for 5 min at 4°C, immediately discard the supernatant, add 400 μL of Hypotonic Buffer and shake for 30 s, then centrifuge at 5000 rpm for 5 min at 4°C, discard the supernatant, and the precipitate is the cell nucleus; add 200 μL of Lysis Buffer (2 mL Buffer mixed with 10 μL phosphatase inhibitor, 20 μL PMSF and 2 μL DTT) to the precipitate, suspend the precipitate, ice bath for 20 min, centrifuge at 15000 rpm for 10 min at 4°C, carefully aspirate the supernatant nuclear protein extract.

[0165] (5) Measure the protein concentration using the BCA protein concentration determination kit, add 30 μg of protein per well to the 96-well plate, supplement with PBS to 100 μL, shake well and then use the enzyme marker to detect the fluorescence intensity of FITC (excitation wavelength: 490 nm, emission wavelength: 525 nm). Then, take the fluorescence intensity of the negative control as the reference to calculate the relative fluorescence intensity of the remaining treatment groups.

[0166] 2 Results and analysis

[0167] 2.1 DeltaVision detection of the intracellular internalization behavior of TApNP

[0168] As Figure 6As shown, under both neutral and acidic conditions, the PMI-treated groups exhibited only minimal and weak fluorescence, indicating that free PMI could not effectively enter cells under either pH condition. Similarly, TapP and TapNP also showed only weak fluorescence signals at pH 7.4, indicating that TapP and TapNP were also difficult to internalize under neutral conditions. However, at pH 6.5, the TapP and TapNP-treated groups showed abundant and strong green fluorescence signals, confirming that both entered cells in large quantities under acidic conditions. Furthermore, co-localization of FITC and DAPI was detected in the TapNP-treated group, indicating that it could enter the cell nucleus under the guidance of NLS. These results indicate that free PMI itself is difficult to effectively enter cells, while under weakly acidic conditions, TapP and TapNP containing pHLIP can be effectively internalized and enter cells, and TapNP can enter the nucleus of tumor cells.

[0169] 2.2 Flow cytometry analysis of TApNP internalization behavior

[0170] like Figure 7 As shown, only weak fluorescence intensity was detected in the PMI treatment group under both neutral and acidic conditions. The fluorescence intensity of the TApP and TApNP treatment groups was slightly enhanced compared to the PMI treatment group at pH 7.4, while in an acidic environment, the fluorescence intensity of both the TApP and TApNP treatment groups was significantly stronger than that of the PMI group. Quantitative analysis of the average fluorescence intensity showed that, compared to the other treatment groups, the average fluorescence intensity of the TApP and TApNP treatment groups in an acidic environment was significantly stronger, with the TApNP treatment group exhibiting the strongest fluorescence intensity, approximately twice that of the TApP treatment group. These results are consistent with Delta Vision detection results, indicating that free PMI itself cannot effectively enter cells, while under acidic conditions, TApP and TApNP containing pHLIP can effectively internalize and enter cells.

[0171] 2.3 Nuclear uptake of TApNP

[0172] like Figure 8As shown, compared with the negative control, the fluorescence intensity of the PMI treatment group was slightly enhanced under neutral and acidic conditions, indicating that free PMI was difficult to effectively enter the nucleus under the two pH conditions. At pH 7.4, the fluorescence intensity of the TApP and TApNP treatment groups was slightly higher than that of the PMI group, indicating that under neutral conditions, TApP and TApNP were also difficult to be taken up by the nucleus, which was related to their weak intracellular internalization behavior under neutral conditions. At pH 6.5, the fluorescence intensity of the TApP and TApNP treatment groups was significantly enhanced, among which the fluorescence intensity of the TApNP group was the strongest, about 1.5 times that of the TApP group, indicating that the NLS in TApNP could guide PMI to efficiently enter the nucleus. The above results show that under weak acidic conditions, pHLIP can mediate TApNP to effectively enter the cell, and then under the guidance of NLS, it can efficiently enter the nucleus.

[0173] Example 3: Evaluation of the anti-tumor activity of TApNP

[0174] 1 Experimental method

[0175] 1.1 MTT detection of cell viability

[0176] (1) HCT116 cells were plated in a 96-well plate at a density of 3x10 3 cells per well, and incubated in a CO2 incubator overnight.

[0177] (2) Prepare 1640 culture solution containing 10% FBS and protein (PMI, TApP or TApNP) at a concentration of 50 μM at pH 7.4 or pH 6.5, then gradient dilute the three proteins to 25 μM, 12.5 μM, 6.25 μM and 3.125 μM with 10% FBS-containing pH 7.4 or pH 6.5 1640 culture solution, respectively.

[0178] (3) Discard the original culture medium in the wells, and add 100 μL of the pH 7.4 or pH 6.5 1640 culture solution containing different concentrations of proteins prepared in the previous step, with 5 replicates for each concentration, and 10% FBS-containing pH 7.4 or pH 6.5 1640 culture solution as a negative control, and incubate in a CO2 incubator for 48 h.

[0179] (4) Discard the liquid in the wells, add 100 μL of FBS-free 1640 culture medium and 20 μL of MTT solution to each well, and incubate in a CO2 incubator for 4 h.

[0180] (5) Discard the liquid in the wells, add 150 μL of DMSO to each well, and shake for 10 min.

[0181] (6) Detect the absorbance at 570 nm with a microplate reader, and calculate the relative viability of the cells.

[0182] 1.2 Crystal violet staining to detect cell viability

[0183] (1) HCT116 cells were plated in 96-well plates at a density of 3 x 10 3 cells per well and incubated in a C02 incubator overnight.

[0184] (2) 1640 medium containing 10% FBS and proteins (PMI, TApP or TApNP) at concentrations of 25 mM, 12.5 mM and 6.25 mM was prepared at pH 7.4 or pH 6.5.

[0185] (3) The original medium in the wells was discarded, and 100 pL of the 1640 medium containing different concentrations of proteins and no proteins prepared in the previous step at pH 7.4 or pH 6.5 was added, and incubated in a C02 incubator for 48 h.

[0186] (4) The liquid in the wells was discarded, washed twice with PBS, and 50 pL of crystal violet solution was added to each well, and incubated at room temperature for 20 min.

[0187] (5) The crystal violet solution was aspirated, and washed with PBS for 3 times.

[0188] (6) The 96-well plate was inverted on filter paper and dried overnight at room temperature.

[0189] (7) An inverted microscope was used to capture images, and after completion, 200 pL of methanol was added to each well to dissolve the crystal violet, and shaken for 15 min.

[0190] (8) The absorbance at 570 nm was detected by a microplate reader, and the absorbance value of the negative control was taken as 100%, and the relative viability of the cells was calculated.

[0191] 1.3 Calcein-AM / PI staining to detect cell viability

[0192] Cell viability was detected using a live / dead cell staining kit from BestBio:

[0193] (1) HCT116 cells were plated in 24-well plates at a density of 2 x 10 4 cells per well and incubated in a C02 incubator overnight.

[0194] (2) 1640 medium containing 10% FBS and proteins (PMI, TApP or TApNP) at a concentration of 25 mM was prepared at pH 7.4 or pH 6.5.

[0195] (3) The original medium in the wells was discarded, and 500 pL of the 1640 medium containing 25 mM of proteins and no proteins prepared in the previous step at pH 7.4 or pH 6.5 was added, and incubated in a C02 incubator for 48 h.

[0196] (4) First dilute the staining solution A and B by 10 times with reagent C in the live / dead cell staining kit (Beyotime). Add 10 μL of the diluted staining solution A and 1 μL of the diluted staining solution B to 1 mL of 1640 medium without FBS, mix well to obtain the Calcein-AM staining solution and the PI staining solution, respectively.

[0197] (5) Centrifuge the 24-well plate at 1000 rpm for 5 min before discarding the original culture solution to avoid loss of dead cells, and centrifuge before discarding the liquid in the hole in the subsequent steps.

[0198] (6) Wash twice with PBS, add 50 μL of Calcein-AM staining solution to each well, incubate at room temperature for 30 min in the dark, centrifuge and discard the staining solution. Then add 50 μL of PI staining solution, incubate at room temperature for 5 min in the dark, centrifuge and discard the staining solution.

[0199] (7) Wash twice with PBS and use DeltaVision to collect images. Calcein-AM stains live cells green and PI stains dead cells red, so the ratio of live cells to total cells can be calculated.

[0200] 1.4 EdU staining to detect cell proliferation

[0201] Use EdU cell proliferation detection kit to detect cell proliferation:

[0202] (1) Seed HCT116 cells in a 24-well plate at a density of 2 x 10 4 cells per well, and incubate in a CO2 incubator overnight.

[0203] (2) Prepare 1640 medium containing 10% FBS and 25 μM protein (PMI, TApP or TApNP) at pH 7.4 or pH 6.5.

[0204] (3) Discard the original culture medium in the hole, and add 500 μL of 1640 medium containing 25 μM protein and 500 μL of 1640 medium without protein prepared in the previous step, and incubate in a CO2 incubator for 48 h.

[0205] (4) Dilute EdU with 10% FBS RPMI-1640 cell culture medium at a ratio of 1:500 to obtain 2x EdU working solution, add 500 μL per well after preheating at 37°C, and continue to incubate in a CO2 incubator for 3 h.

[0206] (5) Discard the liquid in the well, wash with PBS for 2 times, add 300 μL 4% paraformaldehyde to each well for fixation at room temperature for 15 min.

[0207] (6) Discard the fixing solution in the well, add 300 μL of 2 mg / mL glycine to each well, incubate at room temperature for 5 min to neutralize the excess fixing solution therein.

[0208] (7) Discard the glycine in the well, wash with PBS for 1 time, add 300 μL of 0.5% Trition X-100 to each well, incubate at room temperature for 10 min to permeabilize the cells, then discard the permeabilization solution in the well, wash with PBS for 1 time.

[0209] (8) Dissolve Click Additive in 10.4 mL of deionized water to prepare Click Additive Solution. Add 200 μL of CuSO4, 10 μL of Azide 555 and 500 μL of Click Additive Solution to 4.3 mL of Click Reaction Buffer to prepare Click reaction solution.

[0210] (9) Discard the PBS washing solution in the well, add 200 μL of Click reaction solution to each well, incubate in the dark for 30 min, then discard the reaction solution in the well, wash with PBS for 3 times.

[0211] (10) Dilute 1000x Hoechst 33342 with PBS at a ratio of 1:1000 to prepare cell nucleus staining solution.

[0212] (11) Discard the PBS washing solution in the well, add 200 μL of cell nucleus staining solution to each well, incubate at room temperature in the dark for 10 min.

[0213] (12) Discard the staining solution in the well, wash with PBS for 3 times, and collect images using DeltaVision. The cell nucleus is blue and the EdU positive cells are red, and the percentage of EdU positive cells is calculated accordingly.

[0214] 2 Results

[0215] 2.1 MTT detection of cell viability

[0216] As Figure 9As shown, under both pH conditions, different concentrations of PMI exhibited only a weak inhibitory effect on the growth of HCT116 cells, and there was no obvious pH dependence. The cell survival rate of the 50 μM PMI treatment group was 81.59%. In contrast, the inhibition of cell viability by TapP and TapNP showed a pH and concentration dependence. At pH 7.4, both proteins had weak inhibitory effects on tumor cells, with cell survival rates of 80.04% and 74.73% after treatment with 50 μM TapP and TapNP, respectively. However, under pH 6.5, the activities of TapP and TapNP at all concentrations were superior to PMI, and TapNP showed the strongest activity at the same protein concentration. The cell survival rate of the 50 μM TapNP treatment group was 35.45%, which was superior to TapP's 41.28%. The results indicate that under acidic conditions, pHLIP can effectively deliver PMI into cells, and NLS can guide PMI into the nucleus, thereby inhibiting tumor cell growth.

[0217] 2.2 Crystal violet staining to detect cell viability

[0218] like Figure 10 As shown, under neutral conditions, none of the three proteins significantly inhibited the growth of HCT116 cells; however, under weakly acidic conditions, with increasing protein concentration, PMI, TapP, and TapNP all led to varying degrees of reduction in tumor cell numbers, with TapP and TapNP showing more pronounced effects. Quantitative analysis results indicated that at pH 6.5, TapNP exhibited the strongest tumor cell inhibitory ability under all three tested concentration conditions, with a cell survival rate of 48.49% in the 25 μM TapNP treatment group. These results demonstrate that TapNP, containing both pHLIP and NLS, possesses the strongest inhibitory ability on tumor cell growth, exhibiting a clear concentration- and pH-dependent effect.

[0219] 2.3 Calcein-AM / PI staining to detect cell viability

[0220] like Figure 11As shown, abundant green fluorescence and weak red fluorescence were observed in the PMI treatment group at both pH levels, as well as in the TAPP and TAPNP treatment groups at pH 7.4. However, under acidic conditions, abundant and strong red fluorescence appeared in both the TAPP and TAPNP treatment groups. Quantitative analysis indicated that cell viability was slightly reduced in the PMI treatment group under both pH conditions; while both TAPP and TAPNP exhibited pH-dependent antitumor activity, with TAPNP showing the strongest growth-inhibiting activity under acidic conditions. After 48 hours of treatment with 25 μM TAPNP, the cell viability of HCT116 cells was 46.39%. These results suggest that TAPNP, containing both pHLIP and NLS sequences, exhibits the strongest killing ability against tumor cells under weakly acidic conditions.

[0221] 2.4 EdU staining to detect cell proliferation

[0222] like Figure 12 As shown, abundant red fluorescence was observed in the PMI treatment groups at both pH levels and in all treatment groups under pH 7.4 conditions; however, red fluorescence was significantly reduced in the TApP and TApNP treatment groups under weakly acidic conditions, indicating that TApP and TApNP can significantly inhibit HCT116 cell proliferation. Quantitative analysis showed that after 48 hours of treatment with the three proteins, the number of proliferating cells decreased compared to the control group; and under pH 6.5 conditions, the TApNP treatment group exhibited the strongest tumor cell proliferation inhibitory activity, with the EdU-positive cell rate reaching 13.96% after 48 hours of treatment with 25 μM TApNP. These results indicate that TApNP can effectively inhibit tumor cell proliferation under weakly acidic conditions.

[0223] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A tumor acidity-responsive polypeptide that can target and activate p53, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. A gene encoding the tumor acidity-responsive polypeptide that can target and activate p53 as described in claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A recombinant plasmid containing the gene described in claim 2.

4. The method for preparing the recombinant plasmid according to claim 3, characterized in that, Includes the following steps: 1) The target gene with the synthesized sequence shown in SEQ ID NO.5 AAN-pHLIP-PMI ; 2) Using seamless cloning technology to transfer the target gene AAN-pHLIP-PMI Inserting pET32a plasmid yields pET32a- AAN- pHLIP-PMI Recombinant plasmids were then transformed into... E. coli DH5α was identified as a positive clone, confirming that the colony PCR identification and sequencing results were accurate, and pET32a was extracted. -AAN-pHLIP-PMI Recombinant plasmids; 3) pET32a -AAN-pHLIP-PMI Using the recombinant plasmid as a template, and primers with sequences shown in SEQ ID NO.10-11, pET32a- was obtained by whole-plasmid PCR. AAN-pHLIP-NLS-PMI Recombinant plasmid.

5. Genetically engineered bacteria containing the recombinant plasmid as described in claim 3.

6. The method for constructing the genetically engineered bacteria according to claim 5, characterized in that, The recombinant plasmid was transformed into... E. coli BL21(DE3) competent cells are used for protein expression, thereby obtaining engineered bacteria that heterologously express the polypeptide of claim 1.

7. The use of the polypeptide of claim 1 in the preparation of a medicament for treating cancer, characterized in that, The cancer in question is colon cancer.

8. The application according to claim 7, characterized in that, The drug can inhibit the growth and proliferation of colon cancer cells under weakly acidic conditions.

9. A drug for treating colon cancer, characterized in that, The active ingredient is the polypeptide described in claim 1.

10. A medicament for treating colon cancer according to claim 9, characterized in that, It also includes pharmaceutically acceptable carriers.

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