Thioredoxin DrTrx2 from deinococcus radiodurans and application thereof

Abstract

The invention relates to application of thioredoxin DrTrx2 coming from deinococcus radiodurans and having an anti-radiation function in preparation of drugs, the drugs are used for improving the anti-radiation capacity of organisms, and the amino acid sequence of the thioredoxin DrTrx2 is shown as SEQ ID NO.3. The thioredoxin DrTrx2 has the anti-radiation function, is most sensitive when responding to heavy ion radiation, and has the anti-radiation function. And the single protein has an anti-radiation function and can be used as a radiation-resistant functional component.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to the use of heterologous expression proteins. Background Art

[0002] With economic and social development, the gradual increase in human lifespan, and environmental changes, diseases such as cancer have become a serious threat to people's health and property safety, hindering further socioeconomic development. In addition to chemotherapy and treatment, carbon heavy ion radiation can generate a Bragg peak, precisely releasing energy to the tumor site and exerting powerful tumor-killing capabilities. Heavy ion radiation also has relatively low toxicity due to its rapid energy drop after passing through the tumor. The superior therapeutic effect of heavy ion radiation on tumors has made it a frontier and hot topic in cancer radiotherapy research. However, many tumors are resistant to heavy ion radiation, and protecting surrounding tissues remains a clinical challenge. Therefore, improving the protection of healthy tissues, overcoming radiation tolerance, and enhancing radiosensitization remain bottlenecks in cancer treatment. Furthermore, with rising living standards and the expansion of human activities, the demand for radiation products, including ultraviolet and environmental radiation, is increasing. However, to date, no functional protein products with a clear mechanism of action have been developed.

[0003] Deinococcus radiodurans is a widely used model organism for studying resistance to heavy ion radiation. It can withstand heavy ion radiation doses of 10,000 to 15,000 grays (10k-15kGy), which is 1,500 times the dose that kills a human and 15 times the dose that singes a cockroach. Previous research focused on the mechanisms of heavy ion resistance at the DNA level. It was discovered that D. radiodurans can completely reconstruct its genome from broken chromosome fragments within a few hours, partially explaining its ability to tolerate high doses of radiation. In addition to its strong resistance to ion radiation, the bacterium also exhibits strong tolerance to other extreme environments, such as oxidants and extreme dryness. This is attributed to its abundant presence of functional proteins such as catalase, peroxidase, peroxide oxidoreductase, and superoxide dismutase. However, how this redox capacity contributes to its radiation resistance remains unclear. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for heterologously expressing the redox protein DrTrx2 derived from Deinococcus radiodurans.

[0005] According to one aspect of the present invention, there is provided a use of a thioredoxin protein DrTrx2 from Deinococcus radiodurans having an anti-radiation function in the preparation of a drug, wherein the drug is used to improve the radiation resistance of an organism. The amino acid sequence of the thioredoxin protein DrTrx2 is shown in SEQ ID NO.3.

[0006] The use described in the present invention, wherein the drug is an agent that can increase the expression level of DrTrx2 protein in target cells.

[0007] The use described in the present invention, wherein the agent for increasing the expression of DrTrx2 protein in target cells is a gene expressing DrTrx2 protein introduced into the target cells and expressed, and the nucleotide sequence of the gene expressing DrTrx2 protein is shown in SEQ ID NO.1.

[0008] According to another aspect of the present invention, there is provided a use of a functional core region of thioredoxin DrTrx2 in preparing a drug for improving an organism's radiation resistance, wherein the functional core region of thioredoxin DrTrx2 comprises Cys64 and Cys67.

[0009] According to another aspect of the present invention, a method for detecting the sensitivity of tumor tissue to radiation is provided, characterized in that the content of the peptide shown in SEQ ID NO. 6 in the tumor tissue is detected.

[0010] VVNSETPVVVDFHAQWCGPCK (SEQ ID NO. 6) can represent human thioredoxin. By detecting the content of this peptide in tumor tissue, the radiation sensitivity of cancer tissue can be predicted, thereby assisting in the radiotherapy of tumors.

[0011] The present invention discovers the role of thioredoxin DrTrx2 and provides a new use thereof. Thioredoxin DrTrx2 has an anti-radiation function. Thioredoxin DrTrx2 is most sensitive when responding to heavy ion radiation. Moreover, a single protein has an anti-radiation function and can be used as an effective ingredient for radiation resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 .Carbon heavy ions 12 C 6+ Redox proteome analysis of Deinococcus radiodurans

[0013] A. Iodo TMT labeling at different doses 12 C 6+ Flowchart of the complete protein of Deinococcus radiodurans cells;

[0014] B. Proteomic mass spectrometry data results;

[0015] C. Log2 of the abundance ratio of oxidized peptides under 80Gy and 0Gy radiation conditions and histogram of the corresponding peptide quantity distribution and Gaussian fitting;

[0016] D. Log2 of the abundance ratio of oxidized peptides under 160Gy and 0Gy radiation conditions and histogram of the corresponding peptide quantity distribution and Gaussian fitting;

[0017] E. Log2 of the ratio of the abundance of reduced peptides under 80Gy and 0Gy radiation conditions and histogram of the corresponding peptide quantity distribution and Gaussian fitting;

[0018] F. Log2 of the ratio of the abundance of reduced peptides under 160Gy and 0Gy radiation conditions and histogram of the corresponding peptide quantity distribution and Gaussian fitting.

[0019] Figure 2 Identification and detection of all cysteine-containing peptides labeled with iodoTMT

[0020] A. Distribution of the number of peptides of different lengths;

[0021] B. Box plot display 12 C 6+ Quantification and median values ​​of the redox proteome of irradiated Deinococcus radiodurans;

[0022] C. Heat map showing different doses 12 C 6+ Correlations between the oxidation state and oxidized and reduced state peptides in groups or between groups after irradiation of Deinococcus radiodurans.

[0023] Figure 3 Quantitative oxidation-reduction proteomics reveals 12 C 6+ Changes in the redox state of DrTrx2 protein and its sensitivity after radiation

[0024] A. Different doses 12 C 6+ Changes in the redox state of all proteins in Deinococcus radiodurans cells after irradiation;

[0025] B. Classification results of proteins based on changes in redox state in A;

[0026] C. Analyze the biological process of the protein in B whose reduced state level gradually increases with increasing radiation dose;

[0027] D. Analyze the ratio of proteins in the two biological process categories in C;

[0028] E. Paired radar plot analysis of the fluctuation of the reduced state of proteins involved in the redox process in C. The distance of the protein from the origin represents the reduced state level.

[0029] Figure 4 .DrTrx2 coding sequence

[0030] Figure 5.DrTrx2 gene 3' end fused with a Flag tag DNA sequence

[0031] Figure 6 .DrTrx2 gene encoding amino acid sequence

[0032] Figure 7 The amino acid sequence of the protein encoded by DrTrx2 after a Flag tag is fused to its 3' end

[0033] Figure 8 .Construction of recombinant expression vector of DrTrx2 gene

[0034] A. Plasmid map of pRADK vector;

[0035] B. Agarose gel electrophoresis detection of the DrTrx2-Flag tag fusion construct cloned from the genome of Deinococcus radiodurans;

[0036] C. Agarose gel electrophoresis detection of pRADK-DrTrx2-Flag fusion expression vector plasmid;

[0037] D. Double enzyme digestion and agarose gel electrophoresis detection of pRADK-DrTrx2-Flag expression vector.

[0038] Figure 9 . 60 Co irradiation confirms the universality of DrTrx2 protein in resisting heavy ion radiation

[0039] A. Anti-Flag antibody was used to detect the expression of DrTrx2 protein in the constructed strain Deino-DrTrx2-Flag. The Deino-Flag strain was used as an experimental control.

[0040] B. Growth curves of Deino-DrTrx2-Flag and Deino-Flag strains;

[0041] C. Different doses 60 Lethality curves of Deino-DrTrx2-Flag and Deino-Flag strains after Co irradiation, where D10 represents the radiation dose that causes 90% lethality;

[0042] D. Different doses 60 Survival rates of Deino-DrTrx2-Flag and Deino-Flag strains after Co irradiation;

[0043] E. Different doses 60 The proportion of the reduced form of DrTrx2 after Co irradiation (left) and the total amount of DrTrx2 protein expression (right).

[0044] Figure 10Protein hybridization detection of different doses 60 The abundance of Co-irradiated DrTrx2 protein. The NEM-labeled DrTrx2 protein band represents the reduced form.

[0045] Figure 11 UV irradiation confirmed the radiation resistance of the reduced active site of DrTrx2

[0046] A. Diagram of the DrTrx2 protein structure and the cysteine ​​mutation sites in the active site and zinc finger structure;

[0047] B. Based on different doses 12 C 6+ Changes in the redox state of the Cys27 peptide fragment in the treated DrTrx2 enzyme-cleaved peptide fragment;

[0048] C. Expression levels of wild-type and different cysteine ​​mutant DrTrx2 in recombinant E. coli strains after UV irradiation for different times;

[0049] D. DH5α-pRADK, DH5α-DrTrx2, DH5α-DrTrx2 after UV irradiation at different times C7,10S ,DH5α-DrTrx2 C64,67S and DH5α-DrTrx2 C7,10,64,67S lethality curve of the strain;

[0050] E. Survival rate of recombinant expression strains based on D.

[0051] Figure 12 .Construction and expression of eukaryotic expression vector of DrTrx2 gene

[0052] A. Colony PCR detection of DrTrx2 and DrTrx2 C7,10S 、DrTrx2 C64,67S and DrTrx2 C7,10,64,67S Agarose gel electrophoresis detection of constructed fragments;

[0053] B. Western blot detection of DrTrx2 and DrTrx2 C7,10S 、DrTrx2 C64,67S and DrTrx2 C7,10,64,67S Expressed in HIEC-6 cells.

[0054] Figure 13 Cell survival rate of human small intestinal epithelial cells HIEC-6 12-36 hours after γ-ray irradiation

[0055] Figure 14 .Comparison of amino acid sequence similarity between DrTrx2 and human homologous thioredoxin. DETAILED DESCRIPTION

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0057] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0058] The present invention can be more easily understood through the following examples, but the present invention is not limited to these examples.

[0059] Example 1 Redox Proteome Identification 12 C 6+ Differential proteins of Deinococcus radiodurans irradiated by heavy ions

[0060] 1. Culture of Deinococcus radiodurans and 12 C 6+ Heavy ion radiation

[0061] Deinococcus radiodurans was purchased from the China Center for Comprehensive Culture Collection of Microorganisms. The purchased strain (CGMCC1.3828) was cultured in TGY medium containing 1% tryptone, 0.5% yeast extract, and 0.5% glucose overnight at 30°C. The next day, a certain volume of the cultured bacterial suspension was placed in fresh TGY medium to an initial bacterial concentration of 0.1 OD. 600 / mL and continue to grow and culture. The strain was cultured to the mid-logarithmic growth phase (10OD 600 / mL), centrifuged at 10,000 rpm for 3 min to collect the cells. Washed the cells with 0.9% Phosphate Buffered Saline (PBS) and repeated three times. The collected cells were resuspended in 0.1M Na2HPO4-NaH2PO4 (pH 7.0) buffer and used for 12 C 6+ radiation.

[0062] Using 32keV / μm 12 C 6+The collected bacteria were irradiated with a beam at a rate of 30 Gy / min. The doses used were 0, 80 and 160 Gy. The irradiated cells were lysed with HES buffer and broken by ultrasonic vibration. The HES buffer lysis solution includes 100 mM HEPES, 1 mM EDTA, 0.1 mM Neocuproine, 0.1% SDS and protease inhibitor cocktail. The broken cells were centrifuged at 14,800 rpm and 4°C for 15 min to remove cell debris, and the supernatant was collected. The whole cell protein concentration was determined using a BCA kit (23227, Thermo Fisher Scientific)

[0063] 2. Based on carbon heavy ions 12 C 6+ Preparation of Redox Proteome Samples from Irradiated Deinococcus radiodurans

[0064] 100 μg of total cell protein was incubated with 20 mM N-Ethylmaleimide (NEM) at room temperature for 30 minutes. NEM blocks reduced cysteine ​​and quantifies the proportion of oxidized cysteine. Samples without NEM blocking were used to determine both oxidized and reduced cysteine ​​content. Protein samples with and without NEM blocking were incubated with a mixture of tris(2-carboxyethyl)phosphine (TCEP) at a final concentration of 5 mM at 50°C and 900 rpm for 1 hour to reduce oxidized disulfide bonds in the protein.

[0065] Take 10 μL of mass spectrometry grade methanol and add it to the Iodoacetyl Tandem MassTag 6 (iodoTMT 6 Dissolve the reagent in a TCEP (90102, Thermo Fisher Scientific) tube, vortex, centrifuge, and protect from light. Add iodoTMT reagent to a final concentration of 4 mM to the TCEP-reduced sample and incubate at 37°C in the dark for 1 hour. The labeling includes labeling the protein sample blocked with NEM at 0 Gy with iodoTMT6-126; iodoTMT 6 Protein samples without NEM blocking under -127 labeling conditions at 0Gy; iodoTMT 6 -128 labeled protein sample after 80 Gy irradiation and blocking with NEM; iodoTMT 6 -129 labeled protein sample without NEM blocking after 80Gy irradiation; iodoTMT 6 -130 labeled protein sample after 160Gy irradiation and blocking with NEM; iodoTMT 6-131 labeled protein samples without NEM blocking after 160Gy irradiation. Add dithiothreitol (DTT) to the labeled protein at a final concentration of 20mM and incubate at 37°C in the dark for 15 minutes to terminate the reaction. Mix each labeled sample in a separate tube ( Figure 1 A).

[0066] Denature and digest the labeled sample on a 30KD FASP column. Wet the column membrane with 30 μL of equilibration solution, which consists of 8 M Urea and 50 mM NH4HCO3. Apply the labeled sample to the equilibrated FASP column and centrifuge at 12,000 rpm for approximately 35 minutes at room temperature until all liquid is removed. Proteins are now deposited on the FASP column membrane. Add 200 μL of wash solution to the FASP column membrane and centrifuge at 12,000 rpm for approximately 35 minutes at room temperature, repeating this process twice. The wash solution consists of 8 M Urea and 50 mM NH4HCO3 to denature the proteins. Then, add replacement solution to remove urea. Centrifuge at 12,000 rpm for approximately 35 minutes at room temperature, repeating this process twice. The replacement solution consists of 50 mM NH4HCO3. Finally, add 12 ng / μL of Trypsin to the FASP column and incubate at 37°C for 12-14 hours. After enzymatic hydrolysis, the protein peptides were transferred to PCR tubes and vacuum dried.

[0067] A C18 desalting column is used to remove salt from the enzymatically hydrolyzed peptides. The C18 desalting column is first activated with 20 μL of methanol, which is then centrifuged to remove the methanol, and this is repeated. 20 μL of wash solution is added to the C18 desalting column. The wash solution comprises 80% acetonitrile and 0.5% formic acid. The wash solution is removed by centrifugation, and this is repeated. Then, 20 μL of equilibration solution is added to the C18 desalting column, which is then centrifuged to remove the equilibration solution, and this is repeated. The equilibration solution comprises 2% acetonitrile and 0.1% formic acid. 20 μL of equilibration solution is added to the vacuum-dried enzymatically hydrolyzed peptides, and the column is gently tapped to completely dissolve the peptides. The column is then transferred to the equilibrated C18 desalting column. The salt is removed by centrifugation, and the protein is now bound to the C18 filler. 20 μL of equilibration solution is added to the peptide-bound C18 desalting column to wash the column, and after centrifugation, 20 μL of wash solution is added to elute the peptides. The peptides are then eluted by centrifugation, and the sample is retained. Elute once more with 20 μL of wash buffer, vacuum dry the eluted liquid, and store it in a -80°C freezer until mass spectrometry analysis. Add 750 μL of TBS buffer to the vacuum-dried protein peptides to dissolve the peptides. The TBS buffer contains 0.15 M NaCl and 25 mM Tris-HCl, pH 7.5.

[0068] Enrich the iodoTMT-labeled peptides using iodoTMT resin. Pipette 250 μL of resin and centrifuge to remove the buffer. Wash the iodoTMT resin with 1 mL of TBS buffer, centrifuge to remove the TBS, and repeat the wash twice. Resuspend the resin in 250 μL of TBS buffer. Mix the 250 μL of resin with 750 μL of dissolved peptides and incubate overnight on a shaker at 4°C. Wash the peptide-bound resin with 1 mL of TBS buffer containing 2 M urea to reduce nonspecific binding, centrifuging and repeating twice. Then wash the resin with 1 mL of TBS buffer and 1 mL of deionized water, centrifuging and repeating twice. Finally, add 1 mL of TMT eluent to the resin, centrifuge, and collect the TMT-labeled peptides. Repeat the elution process three times, centrifuging at maximum speed for the final elution. Collect all eluents and evaporate the peptide solution to dryness in a vacuum centrifuge at 45°C. 25 μL of a reagent containing 5% acetonitrile and 0.1% formic acid was added to dissolve the dried peptides, and 5 μL of the sample was taken for ultra-performance liquid chromatography-mass spectrometry analysis.

[0069] 3. LC-MS / MS Analysis of Redox Proteome Samples

[0070] The ultra-high performance liquid chromatography (UPLC) system was an EASY-nLC 1200 (UPLC) system. The reversed-phase chromatographic column used was an octadecyl silica bonded column (bed length 15 mm, column inner diameter 150 μm, filler particle size 1.9 μm). The sample was separated and eluted on a C18 silica column at a flow rate of 600 nL / min and a nonlinear gradient of 180 min. The separated peptides were ionized under high voltage conditions of 2 kV and sorted by an Orbitrap Exploris 480 mass spectrometer using data-dependent scanning mode for data acquisition. The mass spectrometer has an ultra-high-resolution mass analyzer with a resolution of 120,000 for data acquisition and a mass analysis range of 375-1,500 m / z. The automatic gain control (AGC) of the mass analyzer was 300%, and the maximum injection time was 50 ms. Based on the ion scan range, the top 20 most intense precursor ions were selected for secondary mass spectrometry fragmentation using higher-energy collisional dissociation (HCD) mode. The ion scanning range of the secondary mass spectrometer is 200-2,000 m / z, and the mass resolution is 15,000. The standard secondary mass spectrometer fragmentation energy is 37%, and fragmented ions with 2-7 positive charges are selected for sequence analysis. The raw mass spectrometry data obtained from the redox proteome experiment were parsed using the Andromeda engine using MaxQuant software version 1.6.17.0 for database search. The spectra were searched in the database using a search engine tool to obtain quantitative information on the abundance of peptides for each sample. The database is the protein sequence of Deinococcus radiodurans derived from UniProt.

[0071] The database search parameters were as follows: 20 ppm mass tolerance for both primary and secondary mass spectrometry; a maximum of 2 missed sites for trypsin digestion; a minimum peptide length of 7 amino acids; an Orbitrap mass analyzer; fixed modifications including N-ethylmaleimide on cysteines; and variable modifications including N-terminal acetylation, carbamidomethylation on cysteines, and oxidation of methionines. The peptide spectrum matching false discovery rate (FDR) was ≤5%.

[0072] 4. Analysis of the enrichment efficiency of cysteine-containing peptides and the labeling efficiency of iodoTMT

[0073] In the label-free identification, the identification efficiency of cysteine-containing peptides was 6%. In contrast, the enrichment efficiency of cysteine-containing peptides after iodoTMT labeling and anti-TMT resin enrichment was 34%. Among the 3382 peptides identified, 1150 contained cysteine, corresponding to 605 proteins. Among them, the peptides labeled by iodoTMT accounted for 98.63% of the number of cysteine-containing peptides identified ( Figure 1 B, Figure 2 A).

[0074] 5. 12 C 6+ Screening of proteins with significant changes in oxidized and reduced states in Deinococcus radiodurans cells after heavy ion irradiation

[0075] 0Gy, 80Gy, and 160Gy are different 12 C 6+ The signal intensities of the same redox state peptides under the conditions of radiation dose showed a high correlation, providing a theoretical basis for the analysis of redox state changes ( Figure 2 C) Different 12 C 6+ The abundance of peptides containing oxidized cysteine ​​did not change significantly under the radiation dose, but the abundance of both oxidized and reduced peptides gradually increased with the increase of radiation dose ( Figure 2 B). The distribution of the log2 of the ratio of the abundance of the oxidized and reduced states under 80 Gy and 160 Gy irradiation to the abundance of the oxidized and reduced states under 0 Gy conditions shows that the log2 value of the maximum number of peptides (i.e., the average value) deviates from 0 to varying degrees ( Figure 1C, D, E, F). The average values ​​of the log2 of the ratio of the oxidation state abundance of 80 Gy and 160 Gy to 0 Gy were -0.238 and -0.153, respectively. The standard deviations SD of the Gaussian fitting curves were basically consistent, 0.472 and 0.475, respectively. The average values ​​indicate that the oxidation state level of both 80 Gy and 160 Gy radiation decreased compared with 0 Gy, but the change was not significant. The results are consistent with the abundance trend of oxidized cysteine ​​peptides ( Figure 2 B) The standard deviations of the Gaussian fits of the log2 distributions of the ratios of the reduced-state abundance at 80 Gy and 160 Gy relative to 0 Gy were 0.195 and 0.272, respectively, but overall these were lower than the standard deviations of the oxidized-state abundance. The standard deviation was higher at 160 Gy than at 80 Gy, indicating a wider range of reduced-state variation after 160 Gy. The mean values ​​of the log2 distributions were 0.616 and 0.898 for 160 Gy and 80 Gy, respectively, indicating that the variation in the reduced-state was greater than that in the oxidized-state and that the variation in the reduced-state increased with increasing radiation dose.

[0076] 6. Identification of 0Gy, 80Gy, and 160Gy 12 C 6+ Proteins with increased levels of reduction in Deinococcus radiodurans after irradiation

[0077] By analyzing the changes in the reduced state of all peptides, it was found that the reduced state levels of most peptides gradually increased with the increase of radiation dose ( Figure 3 A). The peptides were divided into three categories. The first category contained 35 peptides whose reduction levels were basically the same at 80 Gy and 0 Gy, but increased rapidly at 160 Gy. The second category contained 114 peptides whose reduction levels increased rapidly at 80 Gy, but remained basically stable at 160 Gy. However, in the third category, the reduction levels of 198 peptides continued to increase ( Figure 3 B)

[0078] 7. The reduced state of DrTrx2 plays an important role in resisting heavy ion radiation

[0079] GO cluster analysis of the third category of proteins revealed that 66.16% and 22.72% of the proteins were enriched in metabolism-related signaling pathways and redox processes, respectively ( Figure 3 C, D). The correlation between the two processes is reflected in the fact that many metabolites produced by redox reactions have antioxidant capacity. In addition, changes in redox state affect the synthesis of some anti-radiation metabolites. By analyzing the reduction state levels of 45 proteins in the redox process, it was found that the reduction level of thioredoxin 2 (DrTrx2) increased most significantly at both 80Gy and 160Gy doses ( Figure 3E). This indicates that the reduced state of DrTrx2 plays an important role in resisting heavy ion radiation.

[0080] Example 2: DrTrx2 single protein significantly enhances the radiation resistance of Deinococcus radiodurans and non-radiation-resistant Escherichia coli

[0081] 1. Construction of recombinant DrTrx2 expression vector

[0082] The recombinant DrTrx2 protein gene of the present invention was cloned from the genome of Deinococcus radiodurans by PCR, and the sequence was the same as that of GenBank No. NZ_CP038664.1 (SEQ ID NO.1, Figure 4 ). A Flag tag coding sequence (SEQ ID NO.2, Figure 5 The amino acid sequence of DrTrx2 protein is as follows Figure 6 (SEQ ID NO.3), the amino acid sequence of the protein encoded by DrTrx2 after the Flag tag was fused to the 3' end is as follows Figure 7 (SEQ ID NO.4). The fusion gene was inserted into the downstream of the strong expression promoter of the pRADK shuttle vector groESL to obtain a recombinant DrTrx2 expression vector, named pRADK-DrTrx2-Flag. The pRADK-Flag empty vector was used as an experimental control. The constructed fusion expression vector was successfully constructed by enzyme digestion and agarose gel electrophoresis. Figure 8 ), and the accurate expression of DrTrx2 gene was confirmed by Western blotting ( Figure 9 A)

[0083] 1atgagtgacatcctgacctgtacccactgccaggccaaaaaccgcgtcggtgctgtgccc

[0084] 61gccggacaggtgccgagctgcgcccgctgcggcgccgcgctgccctggctgcacgacggc

[0085] 121accgacgcgaccttcgagcaggaccttcagacaagcgtgccggtgctggtggacttctgg

[0086] 181gcgccgtggtgcggcccctgccgcgtgatggggccggttctcgaagacctcgcccgcgac

[0087] 241ctgcccggcaaggtgcgggtggtgaaggtcaacgtggacgagaacccgcgcaccgccgcc

[0088] 301cgtttcgaggtccgcagcattcccacgctgctgatgttcaaggacggggaagaggtggac

[0089] 361cagatggtcggcgtaacccagaaggcggcgctgcgggcgcgggtggaacacctcaaccag

[0090] 421ctttcctga(SEQ ID NO.1)

[0091] 1atgagtgacatcctgacctgtacccactgccaggccaaaaaccgcgtcggtgctgtgccc

[0092] 61gccggacaggtgccgagctgcgcccgctgcggcgccgcgctgccctggctgcacgacggc

[0093] 121accgacgcgaccttcgagcaggaccttcagacaagcgtgccggtgctggtggacttctgg

[0094] 181gcgccgtggtgcggcccctgccgcgtgatggggccggttctcgaagacctcgcccgcgac

[0095] 241ctgcccggcaaggtgcgggtggtgaaggtcaacgtggacgagaacccgcgcaccgccgcc

[0096] 301cgtttcgaggtccgcagcattcccacgctgctgatgttcaaggacggggaagaggtggac

[0097] 361cagatggtcggcgtaacccagaaggcggcgctgcgggcgcgggtggaacacctcaaccag

[0098] 421ctttccGATTACAAGGATGACGACGATAAG(SEQ ID NO.2)

[0099] MSDILTCTHCQAKNRVGAVPAGQVPSCARCGAALPWLHDG

[0100] TDATFEQDLQTSVPVLVDFWAPWCGPCRVMGPVLEDLARD

[0101] LPGKVRVVKVNVDENPRTAARFEVRSIPTLLMFKDGEEVDQMVGVTQKAALRARVEHLNQLS(SEQ IDNO.3)

[0102] MSDILTCTHCQAKNRVGAVPAGQVPSCARCGAALPWLHDG

[0103] TDATFEQDLQTSVPVLVDFWAPWCGPCRVMGPVLEDLARD

[0104] LPGKVRVVKVNVDENPRTAARFEVRSIPTLLMFKDGEEVDQMVGVTQKAALRARVEHLNQLSDYKDDDDK(SEQ ID NO.4)

[0105] 2. Construction of recombinant DrTrx2 radiation-resistant strain

[0106] The fusion expression vectors pRADK-DrTrx2-Flag and pRADK-Flag were respectively introduced into radioresistant Deinococcus by the CaCl2 transformation method. The radioresistant Deinococcus expressing DrTrx2-Flag and Flag were named Deino-DrTrx2-Flag and Deino-Flag strains, respectively. The transformed strains were placed in TGY solid culture medium containing 4μg / mL chloramphenicol to screen positive transformants. The TGY culture medium contains tryptone 1%, yeast extract 0.5%, and glucose 0.5%. The culture was statically cultured at 30°C for 3 days. The growing positive transformants Deino-DrTrx2-Flag and Deino-Flag strains were placed in TGY liquid culture medium containing 4μg / mL chloramphenicol and cultured under the same environment. Their growth was monitored and it was found that the growth curves were basically the same. When the culture was 30h, the bacterial growth density reached a maximum value (10OD 600 / mL), and the growth density remained basically unchanged during the 30-40 h culture process ( Figure 9 B) The growth curve shows that the expression of the exogenous gene does not affect the growth of the transformant strain.

[0107] 3. Recombination of DrTrx2 Radiation-resistant Sphaeroides60 Co heavy ion irradiation treatment

[0108] The Deino-DrTrx2-Flag and Deino-Flag strains were cultured and grown to 10OD 600 / mL and collect and enrich the bacteria to 500OD 600 Each tube was irradiated with a dose of 0, 80, 400, 1000, 2000, 5000, 10000 and 20000 Gy at a radiation rate of 4.2 kGy / h. 60 The bacteria were collected by Co heavy ion irradiation. The irradiated cells were -1 to 10 -4 The cells were diluted in a gradient range of 100 μL, and 100 μL of the cells were spread on TGY medium, placed in a static culture at 30° C. for 3 days, and the growth phenotype was observed and the grown colonies were counted.

[0109] 4. Overexpression of DrTrx2 in Deinococcus radiodurans significantly improves its resistance 60 Co heavy ion radiation capability

[0110] The Deino-DrTrx2-Flag and Deino-Flag strains were 90% lethal when 60 Co radiation dose (D 10 ) were 5.128 and 2.454 kGy, respectively, indicating that overexpression of DrTrx2 protein can improve the radiation resistance of Deinococcus radiodurans and has universality to different radiation irradiation ( Figure 9 C).

[0111] The mortality rate curve shows that 60 When the Co radiation dose was 2 kGy, Deino-DrTrx2-Flag and Deino-Flag showed survival rates of 82.93% and 10.49%, respectively, indicating that overexpression of DrTrx2 single protein increased radiation resistance by nearly 8 times; when the dose was increased to 5 kGy, the strains showed survival rates of 4.12% and 0.2%, respectively, indicating that overexpression of DrTrx2 single protein increased radiation resistance by nearly 18 times. The results confirmed that DrTrx2 protein itself can improve the radiation resistance of radioresistant bacteria, and as the dose increases, the radiation resistance also increases from 8 times to 18 times ( Figure 9 D)

[0112] 5. Analysis of DrTrx2 expression levels and the role of its reduced state in radiation resistance

[0113] NEM labels the reduced cysteine ​​in DrTrx2 protein, and the molecular weight migration characterizes the reduced form of DrTrx2. DrTrx2 without NEM labeling represents the total amount of protein expression ( Figure 10The proportion of reduced DrTrx2 in the total amount of expression was defined as the reduction level of DrTrx2 protein. By analyzing the relationship between DrTrx2 protein expression and reduction level and survival curve, it was found that protein expression was more important in resisting radiation than the reduced state ( Figure 9 E)

[0114] when 60 When the Co dose ranged from 0 to 2000Gy, the expression level of DrTrx2 protein was very high and did not change significantly, but the reduced state ratio gradually increased. This shows that the change in the reduced state of DrTrx2 within this radiation dose range can resist the damage caused by ion radiation and did not change the survival curve. From 2000Gy to 20000Gy, the reduced state ratio of DrTrx2 gradually increased, but the expression level of DrTrx2 protein decreased significantly, and it will further decrease with the increase in dose; at the same time, it was found that the survival rate of radioresistant Deinococcus also decreased rapidly. These results indicate that within this radiation dose range, the expression level of DrTrx2 protein may have a greater effect on resisting radiation. At 2000Gy, both the expression level of DrTrx2 protein and the reduced state suddenly dropped sharply, which 60 The survival curve of Co response has a turning point here.

[0115] Example 3: DrTrx2 achieves heavy ion radiation resistance through the cysteine ​​residue in the active site

[0116] 1. Construction of a Radiation-Sensitive Escherichia coli Recombinant Strain Containing a DrTrx2 Cysteine ​​Mutant

[0117] Based on the importance of the reduced state of DrTrx2 in resisting heavy ion irradiation, the changes in the redox state of cysteines in the protein in response to heavy ions were analyzed. The amino acid sequence and structure of the DrTrx2 protein showed that all cysteines were located in the catalytic active site or zinc finger structure ( Figure 11 A). The zinc finger structure is located at the N-terminus of the protein, including four cysteines Cys7, Cys10, Cys27 and Cys30; the catalytic site includes two cysteines Cys64 and Cys67, located at the C-terminus of the DrTrx2 protein. 12 C 6 After irradiation, the peptides were cleaved by enzymes to analyze the changes in the reduced state of the catalytic site or zinc finger structure peptides ( Figure 11 B) We identified a peptide containing Cys27, and the reduced state of the peptide gradually increased with increasing radiation dose, indicating that the increase in the reduced state of DrTrx2 when it resists heavy ion radiation is related to cysteine.

[0118] The cysteine ​​(Cys) in the catalytic site and zinc finger structure in the expression vector DrTrx2-Flag-pRADK was mutated to serine (Ser). C64,67S -Flag-pRADK, zinc finger cysteine ​​mutant DrTrx2 C7,10S -Flag-pRADK, and mutant DrTrx2 C7,10,64,67S -Flag-pRADK.

[0119] The constructed mutant plasmids were introduced into radiation-sensitive Escherichia coli DH5α, and the transformants were named DH5α-DrTrx2, DH5α-DrTrx2 C7,10S 、DH5α-DrTrx2 C64,67S , and DH5α-DrTrx2 C7,10,64,67S The transformant DH5α was plated on a Luria-Bertani (LB) medium containing 50 μg / mL ampicillin and screened at 37° C. The LB medium contained 1% tryptone, 0.5% yeast extract, and 1% NaCl.

[0120] 2. Ultraviolet irradiation demonstrates that DrTrx2 protects cells against heavy ion radiation through the cysteine ​​residue in its active site

[0121] The transformant cells were inoculated into LB medium and cultured to 1OD 600 / mL. Take 10mL of the cells and place them in a culture dish, and irradiate them with UV for 0, 20, 300 and 1800 seconds respectively. Take 500μL of the irradiated cells, add 1mL of 2X lysis buffer and vortex and dissolve at room temperature for 1min. The 2X lysis buffer includes 4% SDS, 20% glycerol, 0.2% bromophenol blue, 50mM Tris-HCl, pH6.8. After the whole cell lysate is separated by 15% SDS-PAGE gel, protein immunoblotting is used to detect the protein expression of different mutants and different radiation times ( Figure 11 C). The primary antibody for protein hybridization was an anti-Flag tag antibody (F1804, Sigma-Aldrich), and the secondary antibody was a goat anti-mouse antibody. Hybridization results showed that the protein expression level of the cysteine ​​mutant was slightly lower than that of the wild-type; however, UV irradiation at different times did not significantly change the protein content, indicating that UV irradiation within this time range did not damage the protein.

[0122] The irradiated cells were spread in LB medium containing ampicillin according to gradient dilution and grown at 37°C. The number of growing clones was counted and the expression of DH5α-DrTrx2 and DH5α-DrTrx2 after UV irradiation was analyzed. C7,10S 、DH5α-DrTrx2 C64,67S and DH5α-DrTrx2 C7,10,64,67S Survival rate ( Figure 11 D, E). The survival rate of the DH5α-pRADK control strain after 300 seconds of UV irradiation was only 2.45%, but the survival rate of the DrTrx2-introduced strain DH5α-DrTrx2 increased to 10.44%, a 4.2-fold increase compared to the control. These results not only confirm the sensitivity of E. coli to UV radiation but also reveal the radiation resistance function of DrTrx2. DH5α-DrTrx2 transformed strain with active site mutant protein C64,67S The survival rate of the strain DH5α-DrTrx2 was 6.97%, which was 33% lower than that of the wild-type protein transformed strain. These results indicate that the cysteine ​​in the active site of DrTrx2 plays a key role in radiation resistance. Compared with the active site mutation, the transformed strain DrTrx2 with zinc finger structure mutation under the same radiation conditions C7,10S The survival rate was 9.66%, which was slightly different from that of the wild-type protein. These results indicate that the zinc finger structure does not have anti-radiation function. In addition, the mutant DH5α-DrTrx2 with cysteine ​​residues in the active site and zinc finger structure C7,10,64,67S The growth phenotype of DH5α-DrTrx2 C64,67S Similarly, the importance of the active site in radiation resistance is further confirmed. The UV radiation survival phenotype confirms that DrTrx2 achieves radiation resistance through the cysteine ​​in the catalytic active center.

[0123] Example 4 Application of Thioredoxin DrTrx2 to Significantly Improve Radiation Resistance of Mammalian Cells in Protecting Cells and Organisms

[0124] 1. Construction of recombinant DrTrx2 eukaryotic expression vector

[0125] DrTrx2 and DrTrx2 were amplified from the prokaryotic expression vector using the subcloning method. C7,10S 、DrTrx2 C64,67S and DrTrx2 C7,10,64,67S By introducing restriction sites HindIII and BamHI, the eukaryotic expression plasmids pCDNA3.1-DrTrx2 and pCDNA3.1-DrTrx2 based on pCDNA3.1 were constructed using double enzyme digestion method. C7,10S , pCDNA3.1-DrTrx2 C64,67Sand pCDNA3.1-DrTrx2 C7,10,64,67S The constructed plasmid was transformed into DH5α, and single clones were picked for colony PCR. The results showed that the target gene was inserted into the plasmid ( Figure 12 A). The correctly sequenced plasmid was further transfected into normal human intestinal epithelial cells HIEC-6. 48 hours after transfection, the cells were harvested and lysed with RIPA lysis buffer to obtain TCL. The expression of the target gene was detected by WB. The results showed that DrTrx2, DrTrx2 C7,10S 、DrTrx2 C64,67S and DrTrx2 C7,10,64,67S Can correctly express ( Figure 12 B).

[0126] 2. DrTrx2 significantly enhances the resistance of normal human intestinal epithelial cells to 60 Co heavy ion radiation capability

[0127] Normal human intestinal epithelial cells HIEC-6 were plated in 6-well plates and transfected with pCDNA3.1-DrTrx2 and pCDNA3.1-DrTrx2 respectively when the confluence reached 80%. C7,10S , pCDNA3.1-DrTrx2 C64,67S and pCDNA3.1-DrTrx2 C7 ,10,64,67S 8 hours after transfection, the normal culture medium was replaced, and 24 hours after transfection, the cells were digested and seeded into 96-well plates at a density of 2000 cells per well and cultured overnight. 60 Gamma irradiation was performed using a Co-γ irradiation device with an absorbed dose of 4 Gy at a distance of 4 m. HIEC-6 cell culture plates were mounted on a custom plastic tray. During irradiation, the cells rotated with the tray to ensure uniform radiation absorption. At 12, 24, and 36 hours after irradiation, 100 μL of culture medium containing 10% CCK-8 was added to each well and incubated for 1 hour. The OD value of each well was measured using an enzyme-linked immunosorbent assay (ELISA).

[0128] The results are as follows Figure 13 As shown in the figure, the survival rate of the control cells was only 22.67% 36 hours after irradiation, but after overexpressing DrTrx2, the cell survival rate increased to 64.33%, which was 2.8 times higher than that of the control group. This indicates the radiation sensitivity of HIEC-6 cells and also confirms the anti-radiation function of DrTrx2 in mammalian cells. C64,67SWhen the cells were exposed to the same radiation, the survival rate was 44.33%, which was 31.09% lower than that of cells expressing wild-type DrTrx2 protein. This result indicates that the cysteine ​​in the active site of DrTrx2 plays a key role in radiation resistance. Compared with the active site mutation, the zinc finger structure mutant protein DrTrx2 expressed under the same radiation conditions C7,10S The cell survival rate was 62%, which was not significantly different from that of cells expressing wild-type DrTrx2 protein. These results indicate that the zinc finger structure does not have anti-radiation function. In addition, the cell survival rate of cells expressing proteins with mutations in both the active site and the zinc finger structure cysteine ​​site was similar to that of cells with mutations in the active site alone, further confirming the importance of the active site in resisting radiation. The survival rate test results of the normal human epithelial cells HIEC-6 subjected to gamma ray radiation confirmed that DrTrx2 achieves its anti-radiation function through the cysteine ​​in the catalytic active center.

[0129] The same study was conducted on the human epithelial Hela cell line and obtained similar results to HIEC-6 cells. After overexpressing DrTrx2, the cell survival rate increased to 60.15%, which was 2.65 times higher than the control group.

[0130] Both the human epidermis and intestinal epithelium are radiation-sensitive target organs. The human epidermis is also a key area for beauty. DrTrx2's radiation-resistant and protective effects could pave the way for the development of related protective products.

[0131] Example 5 Radiation resistance of human thioredoxin homologous to DrTrx2 and its detection technology

[0132] DrTrx2 is a thioredoxin protein, and its sequence similarity to its homologous human thioredoxin protein is 63% ( Figure 14 ), more importantly, the WCGPC sequence containing two cysteine ​​catalytic site regions is completely conserved in the thioredoxin sequences of the two species.

[0133] The human thioredoxin gene encoding Trx (sequence shown in SEQ ID NO. 5) was amplified from our prokaryotic expression vector using a subcloning method. By introducing the restriction sites HindIII and BamHI, a double-enzyme digestion method was used to construct the eukaryotic expression plasmid pCDNA3.1-hTrx based on pCDNA3.1. The constructed plasmid was transformed into DH5α cells, and single colonies were selected for colony PCR and sequencing to verify the correct target gene sequence. The correct plasmid was transfected into the human HIEC-6 cell line. Cells were harvested 48 hours after transfection and subjected to western blot analysis using the same method as in Example 4, confirming correct expression.

[0134] SEQ ID NO.5, i.e., human thioredoxin is as follows:

[0135] atggctcagcgacttcttctgaggaggttcctggcctctgtca

[0136] tctccaggaagccctctcagggtcagtggccacccctcacttccagagccctgcagaccc

[0137] cacaatgcagtcctggtggcctgactgtaacacccaacccagccggacaatatacacca

[0138] cgaggatctccttgacaacctttaatatccaggatggacctgactttcaagaccgagtgg

[0139] tcaacagtgagacaccagtggttgtggatttccacgcacagtggtgtggaccctgcaaga

[0140] tcctggggccgaggttagagaagatggtggccaagcagcacgggaaggtggtgatggcca

[0141] aggtggatattgatgaccacacagacctcgccattgagtatgaggtgtcagcggtgccca

[0142] ctgtgctggccatgaagaatggggacgtggtggacaagtttgtgggcatcaaggatgagg

[0143] atcagttggaggccttcctgaagaagctgattggctga

[0144] Human HIEC-6 cells were plated in 6-well plates and transfected with pCDNA3.1-DrTrx2 and pCDNA3.1-hTrx respectively when the confluence reached 80%. Normal culture medium was replaced 8 hours after transfection, and cells were digested 24 hours after transfection and seeded into 96-well plates at a density of 2000 cells per well and cultured overnight. 60Gamma irradiation was performed using a Co-γ irradiation device, with an absorbed dose of 4 Gy at a distance of 4 m. HIEC-6 cell culture plates were mounted on a custom plastic tray, and the cells rotated with the tray during irradiation to ensure uniform radiation absorption. At 12, 24, and 36 hours after irradiation, 100 μL of culture medium containing 10% CCK-8 was added to each well and incubated for 1 hour. The OD value of each well was measured using an enzyme-labeled analyzer. We obtained results similar to those in the Example. After overexpressing DrTrx2, the cell survival rate was 2.9 times that of the control group; after overexpressing hTrx, the cell survival rate was 2.37 times that of the control group. Adding either DrTrx2 or hTrx has been shown to enhance radiation resistance in mammalian cells.

[0145] The opposite of protective effects is the prediction of tumor site radiosensitivity and radioresistance. The tryptic peptide VVNSETPVVVDFHAQWCGPCK (SEQ ID NO. 6) from human containing the catalytic core WCGPC sequence with two cysteine ​​residues can represent human thioredoxin.

[0146] HIEC-6 cells overexpressing human thioredoxin were lysed using either RIPA lysis buffer or 8M urea lysis buffer to obtain TCLs, similar to those in Example 4. Using a similar method as in Example 4, 100 μg of total cell protein was denatured with 5 mM DTT at 45°C for 30 minutes, cooled to room temperature, and incubated in the dark with 10 mM IAA for 30 minutes at room temperature. The alkylated sample was then treated with 12 ng / μL Trypsin and digested in a 37°C incubator for 12-14 hours. After enzymatic digestion, the protein peptides were subjected to a C18 desalting column to remove salts from the enzymatic peptides. The C18 desalting column was first activated with 20 μL of methanol and centrifuged to remove the methanol. This was repeated once. 20 μL of wash buffer, consisting of 80% acetonitrile and 0.5% formic acid, was added to the C18 desalting column. The wash buffer was removed by centrifugation and repeated once. 20 μL of equilibration buffer was then added to the C18 desalting column and centrifuged to remove the equilibration buffer. This was repeated once. The equilibration solution includes 2% acetonitrile and 0.1% formic acid. 20 μL of equilibration solution is added to the vacuum-dried enzymatic peptide segment, and the peptide segment is thoroughly dissolved by flicking, and then transferred to the equilibrated C18 desalting column. The salt is removed by centrifugation, and the protein is now bound to the C18 filler. 20 μL of equilibration solution is added to the C18 desalting column bound to the peptide segment to wash the column, and after centrifugation, 20 μL of washing solution is added to elute the column, and the peptide segment is eluted by centrifugation, and the sample is retained. Elute once again with 20 μL of washing solution, vacuum dry the eluted liquid, store it in a -80°C refrigerator, and wait for mass spectrometry detection. The ultra-high performance liquid chromatography is an EASY-nLC 1200 (UPLC) system. The reverse chromatographic column used is an octadecyl silica bonded column (column bed length 15, column inner diameter 150 μm, filler particle size 1.9 μm). The sample was separated and eluted on a C18 silica gel column at a flow rate of 600 nL / min and a nonlinear gradient for 180 min. The separated peptides were ionized at 2 kV and sorted on an Orbitrap Exploris 480 mass spectrometer, where data were acquired using a data-dependent scan mode. The mass spectrometer features an ultra-high-resolution mass analyzer with a resolution of 120,000 and a mass analysis range of 375-1,500 m / z. The mass analyzer's automatic gain control (AGC) was set to 300%, and the maximum injection time was 50 ms. Using selected ion reaction monitoring (SRM) mode targeting the precursor ion of the alkylated VVNSETPVVVDFHAQWCGPCK (SEQ ID NO. 6) peptide, we were able to effectively detect and quantitatively compare the characteristic peptide VVNSETPVVVDFHAQWCGPCK representing human Trx in total cell proteome samples.When Trx levels are high, cancer tissue has poor sensitivity to radiation; when Trx levels are low, cancer tissue has good sensitivity to radiation; if cysteine ​​disappears due to mutation, the tumor tissue where it is located will be more sensitive to radiation, thereby knowing the tumor tissue's anti-radiation sensitivity in advance and achieving the prediction goal.

[0147] Similar to the detection of human Trx protein levels, we also performed quantitative PCR analysis using the now mature gene sequence containing the WCGPCK region and obtained the same results, achieving the goal of predicting the radiation sensitivity of tumor tissue.

Claims

1. Use of thioredoxin DrTrx2 from Deinococcus radiodurans in the preparation of a medicament for improving an organism's radiation resistance. The amino acid sequence of the thioredoxin DrTrx2 is shown in SEQ ID NO.

3.

2. The use according to claim 1, wherein the drug is an agent that can increase the expression level of DrTrx2 protein in target cells.

3. The use according to claim 2, wherein the agent for increasing the expression of DrTrx2 protein in target cells is a gene expressing DrTrx2 protein introduced into the target cells and expressed, and the nucleotide sequence of the gene expressing DrTrx2 protein is as shown in SEQ ID NO.1 or SEQ ID NO.

5.

4. Use of the functional core region of thioredoxin DrTrx2 in the preparation of a drug for improving the radiation resistance of an organism, wherein the functional core region of thioredoxin DrTrx2 comprises Cys64 and Cys67.

5. A method for detecting the sensitivity of tumor tissue to radiation, characterized in that The content of the peptide shown in SEQ ID NO. 6 in the tumor tissue was detected.

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