WO2017075899A1 - Application and drug of novel deinococcus radiodurans ppri protein - Google Patents

Abstract

The present invention provides an application of a PprI protein in preparation of a drug for prevention and treatment of radiation damage of higher eukaryotes. The PprI protein can prevent and treat acute radiation damage of human cells and mice, and improve antioxidation and DNA damage repair capabilities.

Description

Use of a new radiation-resistant cocci PprI protein and its drug

This application claims priority to Chinese Patent Application No. 201510737399.6, entitled "A new use and drug for the PrepI-resistant PrpI protein", filed on November 3, 2015, the entire contents of which are hereby incorporated by reference. The citations are incorporated herein by reference.

Technical field

The invention relates to the field of biotechnology, in particular to the use and medicament of the PprI protein.

Background technique

Ionizing radiation in industrial, agricultural, defense, and medical applications, while bringing great benefits to human society, high-dose ionizing radiation from nuclear accidents, tumor radiotherapy, aerospace, and other nuclear emergencies can cause serious human problems. Acute radiation damage (ARIs) and results in extremely high mortality. The effective prevention and control of ARIs is a huge challenge in the field of international radiation damage. It is related to the nuclear safety of the country and has received great attention from governments in Europe and the United States. The treatment and protection of radiation damage has become a hot topic in research in various countries. Therefore, research and development of anti-radiation damage drugs has been greatly valued in modern medical and health care.

At present, anti-radiation damage drugs mainly include ammonia-based radiation protective agents, cytokines, hormonal drugs, Chinese herbal medicines, metallothionein, marine biological ingredients and the like.

Aminoguanidine compounds are a class of radiation protection agents with a long history and good effects. Aminoethylisothiourea (AET) is a derivative in which the thiol group of cysteamine is substituted with a thiol group. The protective effect of AET is longer than that of cysteamine, and it can be taken orally, and the preventive effect is better. The disadvantage is that the side effects of oral or injection administration are large.

IL-1 is the first cytokine to prove to have radiation protection. As early as 1986, it was found that pre-intraperitoneal injection of IL-1 can alleviate the lethal effect of ionizing radiation on mice in a dose-dependent manner. Cytokines with radiation protection also include G-CSF, GM-CSF, IL-11, SCF, PF4 and the like. However, the shortcoming of these cytokines is that the lifespan in the body is extremely short, only a few seconds or a few hours, increasing the dosage or the frequency of administration, which will cause serious toxic side effects.

Natural steroid hormones (such as estradiol) or synthetic non-steroidal hormones (such as diethylstilbestrol, diethylstilbestrol, etc.) show a certain degree of radiation protection in animal experiments, and before and after irradiation Administration has an effect. Injection of estradiol, estriol, etc., can improve the survival rate of mice after exposure, delay the onset of symptoms of radiation sickness, reduce the severity of symptoms, have obvious radiation protection effect on bone marrow hematopoietic cells, and can promote their restore.

After years of research, it has been found that Chinese medicines with clearing heat and detoxifying, promoting blood circulation to remove blood stasis, nourishing blood and nourishing qi, nourishing yin and whitening have different degrees of anti-radiation effects, such as ginseng, ganoderma lucidum, resveratrol, etc. The low toxicity makes it show great advantages and potential in the research of anti-radiation damage drugs. Metallothioneins (MT) are a class of low molecular weight, cysteine-rich, metal-bound non-enzymatic proteins. It has been reported that oral administration of MT can prolong the survival time of one-time high-dose ionizing radiation mice and reduce the damage of the immune system by one-time high dose and multiple small doses of ionizing radiation. After the mice were irradiated with MT-containing protein milk, the white blood cell count, lymphocyte proliferation rate and bone marrow cell DNA content of the mice were significantly higher than those of the simple irradiation group.

In recent years, people have begun to look at marine life in the field of anti-radiation drugs. Wang Yuxi et al. studied the protective effect of scallop polypeptide on thymocytes damaged by ⁶⁰ Co radiation for the first time, and found that scallop polypeptide has radiation protection effect on thymocytes. Further studies have also found that in addition to ionizing radiation, scallop polypeptide also has protective effects on mouse thymocyte fluid damaged by ultraviolet radiation. The anti-radiation mechanism of scallop polypeptide may be related to its anti-oxidation and immunity.

In recent years, research on anti-radiation damage drugs has achieved remarkable results, but there are still many problems, the main problem is the efficacy and toxicity of drugs. Therefore, the development of anti-radiation damage drugs with small side effects and remarkable effects has important practical significance.

Summary of the invention

In view of this, the present invention provides the use and medicament of the PprI protein. The present inventors have found that PprI protein plays an important role in the prevention and treatment of acute radiation damage in human cells and mice, and enhances the ability of anti-oxidation and DNA damage repair.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides the application of the PprI protein in the preparation of a medicament for preventing and treating radiation damage of higher eukaryotes.

Radiation-resistant cocci are the most non-pathogenic bacteria found on Earth to date. Cause It has high-efficiency DNA damage repair ability, and its resistance to extreme environments such as ionizing radiation, ultraviolet light, and drying has attracted the attention of radiation biologists around the world. The specific mechanism by which radiation-resistant cocci are protected from radiation damage remains unclear. Current research indicates that the tight circular chromosome structure and the repair of DNA double-strand breaks play a crucial role in its radiation resistance.

The pprI gene plays a central role in the DNA repair induced by radiation-resistant cocci. Its expression product, PprI, stimulates transcription of recA and other DNA damage repair genes after exposure. Proteomic analysis showed that after ionizing radiation stimulation, PprI protein was up-regulated as a regulatory switch, and 31 proteins were up-regulated. Among them, RecA and PprA have attracted much attention due to their important role in DNA replication and repair. Expression of the PprI protein in E. coli stimulates the transcription of the recA gene in E. coli and enhances the activity of catalase. Furthermore, expression of the pprI gene in yeast enhances the resistance of the yeast strain to extreme environments and increases ethanol production.

There is no doubt that radiation-resistant cocci are a prokaryote. Due to billions of years of biological evolution and differentiation, prokaryotes and higher eukaryotes have formed two distinct biological lines in terms of gene composition, protein expression, regulatory mechanisms, and codon bias. Furthermore, it is worth noting that there is no homologous sequence between the P. radiodurans pprI gene and human and mammalian cells. Therefore, the discovery of radiation-resistant cocci has been limited to prokaryotic cells such as radiation-resistant cocci, Escherichia coli, and lower eukaryotic yeast systems for 60 years.

In the invention patent of application number: 201410153614.3 [Yang Zhanshan, Wu Wei, Qiao Huiping, Wen Ling, Shi Yi, Ren Lili. A DNA molecule and a Pichia pastoris recombinant plasmid and a Pichia pastoris recombinant strain which efficiently expresses the P. falciparum PprI protein. Chinese invention patent, application number: 201410153614.3. 2014], successfully optimized the modified radiation-resistant cocci pprI gene into Pichia strain, to obtain highly expressed and purified PprI protein. However, due to the huge differences between the two major families of prokaryotes and higher eukaryotes, and the fact that PprI protein is only present in radiation-resistant cocci, can the proprine pro-nuclear protein obtained in the above-mentioned patents in yeast be applied to higher eukaryotic human radiation? The prevention and treatment of damage, its effect, these issues have not been reported so far in the relevant literature at home and abroad. The invention relates to the application of PprI prokaryotic protein purified by Pichia pastoris as a radiation protective drug in acute radiation damage of higher eukaryotes. The study found that prokaryotic PprI protein has a very significant predictor of lethal acute radiation damage in human cells and mammals. The role of prevention and treatment, and clarified the mechanism of action. The invention poses a challenge to the classical traditional theory that the two major germplasms of prokaryotic and eukaryotic organisms have great differences, and the first realization of the anti-radiococci prokaryotic PprI protein for the prevention and treatment of acute radiation damage of higher eukaryotes in the field of international radiation protection. A major leap in large breeds.

In some embodiments provided by the present invention, the prevention of radiation damage is to increase cell or body radiation survival.

In other embodiments provided by the present invention, the prevention of radiation damage is to promote repair of DNA or tissue organs damaged by radiation.

The invention also provides the use of PprI protein for preparing an antioxidant drug.

In some embodiments provided by the invention, the antioxidant is to reduce intracellular ROS (reactive oxygen species) levels. When cells are exposed to ionizing radiation or high oxygen partial pressure, the intracellular oxygen concentration increases, leading to ROS production, further damaging proteins, nucleic acids and other important elements in the cell. In the present invention, the PprI protein can increase the radiation resistance and antioxidant capacity of cells by reducing intracellular ROS levels.

In other embodiments provided by the present invention, the antioxidant is to increase SOD, CAT activity, and/or reduce malondialdehyde concentration.

The invention also provides the use of PprI protein in the preparation of Rad51 protein expression promoter. Rad51 protein plays a key role in the regulation of recombinant exchange during DNA repair. PprI protein can promote the expression of Rad51 protein, thus promoting the repair of damaged DNA.

The invention also provides a PprI protein drug, including a PprI protein.

Preferably, the pharmaceutical dosage form is an injection.

In some embodiments provided herein, the medicament further comprises a pharmaceutically acceptable excipient.

The invention provides the use and medicament of the PprI protein. The invention adopts human umbilical vein endothelial cells and BALB/c mice as research objects, and studies the radiation protection effect and mechanism of PprI protein on γ-ray damage. It was found that PprI protein significantly increased the survival rate, antioxidant capacity and DNA damage repair ability of irradiated cells and decreased the apoptosis rate and γH2AX focus number after irradiation; PprI protein reduced the mortality of irradiated mice. The rate of clonal formation of bone marrow cells and the number of white blood cells and platelets in the irradiated mouse were increased. The PprI protein also reduced the radiation pathological damage of various tissues and organs of the irradiated mice and increased the antioxidant capacity of some tissues and organs. These findings PprI protein plays an important role in the prevention and treatment of acute radiation damage in human cells and mice. Prokaryotic PprI protein can be used as a new type of radiation protection drug for the prevention and treatment of human lethal acute radiation damage. Radiation accidents, nuclear emergencies and acute radiation damage caused by tumor radiotherapy have important application value.

DRAWINGS

Figure 1 shows a PCR amplification program chart;

Figure 2 is a diagram showing the construction of a recombinant expression plasmid pHBM905A-6 × His-PprI;

Figure 3 shows SDS-PAGE detection of Pichia pastoris transformant culture supernatant after 3 days of induction with 1% methanol; lane 1: pHBM905A plasmid transformed Pichia pastoris GS115 strain (negative control); lane 2-9: pHBM905A-6× His-PprI recombinant plasmid Pichia pastoris GS115 strain transformants 1-8;

Figure 4 shows the Western blot analysis of the expression product of Pichia pastoris transformants; lane 1: the culture supernatant of the 2nd yeast transformant induced for 2 days; 2 lanes: the culture supernatant of the yeast transformant induced by the 3rd day; 2 Dao: No. 3 yeast transformant induced culture supernatant for 1 day;

Figure 5 shows a peptide mass fingerprinting map of the protein of interest;

Figure 6 is a graph showing the expression of purified PprI fusion protein stained with 12% SDS-PAGE Coomassie brilliant blue; M: marker; lane 1: supernatant after dialysis; lane 2: effluent; lane 3: 300 mM NaCl, 20 mM Imidazole, pH 8.0 elution fraction; 4-5 lanes: 300 mM NaCl, 250 mM Imidazole, pH 8.0 elution fraction;

Figure 7 shows that PprI increases the survival rate of HUVEC after 4Gy γ-irradiation, and cell viability is determined by CCK-8 colorimetry.

Marked, the test was repeated three times (n=3) (P<0.05, t-test);

Figure 8 shows that PprI increases the colony formation rate of cells, and the data is

Mark, data to

Marked, the test was repeated three times (n=3) (P<0.05, t-test);

Figure 9 shows the flow cytometry analysis of cells treated with annexin V-FITC and PI-TRITC for 48 h after irradiation.

Marked, the experiment was repeated three times (n=3) (P<0.05, t-test), of which 1 showed the control group, 2 showed the PBS-treated group, 3 showed the PprI-treated group, and on the right was the HUVEC-exposed apoptosis-marker protein. Western blot analysis showed that the cell extract was detected by 12% SDS-PAGE and immunolabeling of Bcl-2 and Bax antibodies, and β-Actin was used as the internal reference protein.

Figure 10 shows apoptosis images of human umbilical vein endothelial cells; A), B), C), and D) indicate PBS-treated cells (unirradiated), PprI-treated cells (unirradiated), and PBS-treated cells (irradiated 4Gy, respectively) And PprI treatment of cells (irradiation 4Gy);

Figure 11 shows the determination of ROS levels by flow cytometry 24 h after irradiation. The cell irradiation dose was 4 Gy, and the active oxygen level of the untreated group was set to 100%.

Marked, the test was repeated three times (n=3) (P<0.05, t-test);

Figure 12 shows the SOD (Figure 12-1), CAT (Figure 12-2) activity and MDA (Figure 12-3) concentrations in HUVEC after 24 h of irradiation and non-irradiation. The cell irradiation dose is also 4 Gy, the data is

Marked, the test was repeated three times (n=3) (P<0.05, t-test);

Figure 13 shows the change of γH2AX focus in different groups of cells with time after 4Gy γ-ray irradiation.

Marked, the test was repeated three times (n=3) (P<0.05, t-test);

Figure 14 shows the number of γH2AX focal points of human umbilical vein endothelial cells;

Figure 15 shows immunoblot analysis of Rad51 protein exposed to ionizing radiation cells, β-Actin is an internal reference protein;

Figure 16 shows the survival rate within 30 days after irradiation with 6Gy gamma rays in mice;

Figure 17 shows the changes in percentage of white blood cells, platelets, and lymphocytes at 1d, 7d, 14d, 28d, and 35d after irradiation.

Marked, the test was repeated three times (n=3) (P<0.05, t-test);

Figure 18 shows the clone formation rate of mouse bone marrow cells after 0Gy and 4Gy irradiation.

Marked, the test was repeated three times (n=3) (P<0.05, t-test);

Figure 19 shows pathological examination of liver, lung, testis and small intestine of irradiated mice: a. Histopathological changes in the Nacl injection group (100×); b. Liver histopathological changes in the Nacl injection group (400×); c. PPRI injection group liver histopathological changes (100 ×); d. PprI injection group liver histopathological changes (400 ×); e. Nacl injection group lung histopathological changes (100 ×); f. Nacl injection group Pathological changes of lung tissue (400×); pathological changes of lung tissue in g.PprI injection group (100×); pathological changes of lung tissue in h.PprI injection group (400×); i. changes in testicular histopathology of Nacl injection group (100×); j. Nacl injection group testicular histopathological changes (400 ×); Pathological changes of testis in k.PprI injection group (100×); pathological changes of testis in L.PprI injection group (400×); pathological changes of small intestine in m.Nacl injection group (100×); n.Nacl injection group Histopathological changes in the small intestine (400×); o. Histopathological changes in the small intestine of the PprI injection group (100×); pathological changes in the small intestine of the p.PprI injection group (400×);

Figure 20 shows that pprI enhances the antioxidant activity of mice after irradiation; a. Activity of SOD in different tissues;

Marked, the test was repeated three times (n=3) (P<0.05, t-test); b. CAT activity in different tissues;

Marked, the experiment was repeated three times (n=3) (P<0.05, t-test).

detailed description

The invention discloses the use and medicament of the PprI protein, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.

The use of the PprI protein provided by the present invention and the plasmids, vectors, cells, primers, instruments and the like used in the medicament are commercially available.

The present invention is further illustrated below in conjunction with the embodiments:

Example 1 Construction of pHBM-PprI Expression Vector and Purification of PprI Protein

Based on the codon bias of Pichia pastoris, 40 pairs of DNA primers were designed and synthesized, and their sequences are shown in SEQ ID NOs: 1-40. The whole-gene synthesis method was used to optimize the pprI gene (Gene ID:1798483 DR_0167). And by designing primers 5'-gtcacatcatcaccaccatcatgttccatctg ctaacgtttctccac-3' and 5'-ggccattattgggcagcatcttgtggttca-3' to add a 6xHis tag at its N-terminus (see Figure 1), the sequence of the synthesized pprI gene is shown in SEQ ID NO:81. . The sequences of the primers and genes are shown in Table 1.

Table 1 Sequence of primers and genes

The PCR product was purified and cloned into the Cop I and Not I sites of the Pichia pastoris expression vector pHBM-905A (Professor Ma Lixin from Hubei University) to construct the recombinant expression plasmid pHBM905A-PprI (Fig. 2). The plasmid pHBM905A-PprI was linearized with Sal I enzyme and then electrotransformed into Pichia pastoris GS115 strain for amplification and DNA sequence analysis. GS115 cells containing the expression vector were cultured in BMGY medium for 48 h at 28 ° C and then induced with 1% methanol in BMMY medium at 28.5 ° C. After 3 days, 15 μL of the culture supernatant was collected and the target protein was detected by SDS-PAGE electrophoresis, Western blot and MALDI-TOF mass spectrometry. Da. A protein-expressing protein band was observed near the position of the molecular weight of 43 kDa in the culture supernatant of the five positive Pichia transformants compared to the negative control strain (Fig. 3). The results of Western Blot showed that the target protein could specifically bind to the anti-6×His tag antibody, and the reaction intensity increased with the increase of methanol induction time (Fig. 4). Peptide Mass Fingerprinting (PMF) was performed on an Ultraflex II TOF/TOF mass spectrometer and the results were imported into the OMOSSA database of the National Center for Biotechnology Information (NCBI) for analysis ( Figure 5). The results showed that the protein sequence was indeed derived from the DR_0167 protein of Deinococcus radiodurans. After the extension culture, 1 L of the culture supernatant was collected and purified by a Ni-NTA column to obtain 2-5 mg of the target protein in one time (Fig. 6). Therefore, efficient expression and purification of PprI protein was successfully accomplished in yeast (International Standard: obtaining a protein of greater than or equal to 1 mg for high expression).

The His-tagged PprI protein was purified using a Ni-NTA column (Thermo). After rinsing the bed with 25 mM imidazole, the protein of interest was eluted with 20 mM and 250 mM imidazole. The concentration of the PprI protein was determined using a BCA kit (Sigma, St. Louis, MO, USA).

Example 2 PprI protein anti-radiation test

(1) Test method

1. Cell proliferation assay and clonal survival assay:

Cell proliferation test:

HUVECs were purchased from the ScienCell Research Laboratory and cultured in an incubator at 37 ° C, 5% CO ₂ using ECM medium. The exponential growth phase of HUVEC was inoculated into 96-well plates at a density of 5000 cells/well, 100 μL per well. After cell adherent growth, the cells were replaced with fresh medium containing a final concentration of 4 μg/mL PprI protein at 37 ° C, 5%. 6h cultured in a CO ₂ incubator. The ⁶⁰ Co source of the irradiation center of Suzhou University was used to irradiate γ-rays with an absorbed dose of 4 Gy. 96-well plates were taken 24 h after the irradiation, 10 μL of CCK-8 solution was added to each well, and the 96-well plates were incubated in an incubator for 2 h, and the absorbance at 450 nm was measured with a microplate reader. Each concentration was paralleled with 6 wells, and a blank control group was established, and the experiment was repeated 3 times. Cell survival fraction = experimental group absorbance / control absorbance x 100%.

Cloning survival assay: HUVECs in the exponential growth phase were seeded in 60 mm culture dishes at a density of 100 per dish, 500 per dish, 1000 per dish, 2000 per dish, 5000 per dish. After the cells adhered to the wall, they were replaced with fresh medium containing a final concentration of 4 μg/mL PprI protein, and the absorbed dose was 0, 2, 4, 6, and 8 Gy γ-rays, and each group was set up with 3 parallel samples. The medium was further cultured for 10-14 days, and the culture was terminated when a macroscopic clone appeared in the culture dish. The supernatant was discarded, fixed in methanol, and the number of colonies greater than 50 cells was counted after Giemsa staining, and the experiment was repeated three times. Plating efficiency PE = (number of unirradiated cell clones / number of cells inoculated) × 100%, Survival fraction (SF) = number of clones in a dose-irradiated group / (number of cells inoculated in the group × PE) × 100%. According to the results of cell clone formation, the dose-survival curve was fitted by clicking the multi-target model: S=1-(1e ^-D/D0 ) ^N using Sigmaplot10.0 software, and the average lethal dose D ₀ and extrapolation value N were obtained. The quasi-threshold dose D _q = D ₀ × lnN and the survival fraction SF.

2. Apoptosis test:

Cells were seeded in triplicate in 6-well plates and PBS and PprI proteins were added 6 h before 4 Gy irradiation. 48 h after the irradiation, the cells were stained with FITC-labeled Annexin V and PI (KeyGen, Nanjing, China) and detected by a light-shield flow cytometer (Beckman-Coulter, Brea, CA, USA).

3, ROS (reactive oxygen species) formation test: HUVEC in the exponential growth phase was inoculated into a 6-well plate at a density of 2.0 × 10 ⁵ / hole, and the cell was adhered to a final concentration of 4 μg / mL. The fresh medium of the P. cerevisiae PprI protein (in the unirradiated group and the simple irradiation group) was further cultured in an incubator at 37 ° C for 5% CO ₂ for 6 hours. Three parallel samples were set for each group and the experiment was repeated three times. Thereafter, the absorbed dose was irradiated with 4 Gy gamma rays. The medium was aspirated 24 h after the addition, and 1 mL of diluted DCFH-DA was added to each well (diluted DCFH-DA in a serum-free medium at 1:1000 to give a final concentration of 10 μmol/L), and incubated in an incubator for 20 min. Wash three times with serum-free medium to fully remove DCFH-DA that did not enter the cells. The cells were collected and sent to a flow cytometer (Cytomics FC500. Beckman Coulter. USA) for detection.

4, SOD, CAT enzyme activity determination and MDA concentration determination:

Cells were seeded in triplicate in 6-well plates and PBS and PprI proteins were added 6 h before 4 Gy irradiation. At 24 h after the irradiation, the SOD, CAT activity and MDA concentration were determined by T-SOD kit, CAT kit and MDA kit, respectively.

5. Immunofluorescence test:

The cells were seeded on glass slides in 6-well plates for 12 h, and PBS and PprI proteins were added 6 h before 4 Gy irradiation. After the cells were fixed with 3.7% paraformaldehyde for 30 min, 1% Triton X 100 was added to each well for 15 min at 4 ° C, and then 1 ml of 5% BSA was added to each well for 1 h. The slides were removed, and the anti-γH2AX antibody was added dropwise at 4 ° C overnight, and then TRITC-labeled secondary antibody was added to block at room temperature for 1 h, and stained with DAPI at room temperature for 15 min in the dark, and then sent to laser confocal detection.

6, Western blot:

Western blot analysis was performed using a murine anti-Bcl-2 primary antibody, a murine anti-Bax primary antibody, a murine anti-Rad51 primary antibody, a murine anti-β-Actin primary antibody, and a rabbit anti-mouse secondary antibody.

7. Observation of radiation survival rate of mice:

Clean-grade pure male BALB/c mice, 8-10 weeks old, were provided by the Animal Experimental Center of the Medical College of Suzhou University and randomly divided into 2 control groups and 2 experimental groups, with 10 animals in each group. NaCl saline or PprI protein injection was intramuscularly injected 24 h before the irradiation and 24 h after the irradiation. The mice were irradiated with ⁶⁰ Co-γ rays at a dose rate of 2 Gy/min and the absorbed dose was 6 Gy. The mice were observed in a sterile room for 30 days, and the death of the mice was recorded daily.

8, bone marrow cell clone formation test:

BALB/c mice (male, 8 weeks old, Slack, 12 per group) were intraperitoneally injected with NaCl or PprI protein 1 h after irradiation. Mice were irradiated with 4 Gy with a Co-60 source at a dose rate of 2 Gy/min. The unirradiated group also injected NaCl or PprI protein as described above. After 7 days, the mice were sacrificed, and the bone marrow was taken out and resuspended in 1640 medium to adjust the cell density to 2000 cells/ml. The cells were then mixed with 5% agar to form a 0.3% semi-solid agar medium and incubated at 37 ° C, 5% CO ₂ for 14 d, and the number of clones larger than 50 cells was counted.

9, peripheral blood cell count detection:

Different groups of mice were irradiated with 4 Gy as described above. Blood samples were collected from the EDTA tubes at 1, 7, 14, 28, and 35 days, respectively, using a whole blood cytometer for counting analysis.

10. Pathological observation of mouse tissues and organs:

The lung, liver, kidney and testicular tissues of the mice treated with NaCl and PprI protein were fixed in formalin, embedded in paraffin, sectioned on glass slides, and stained with hematoxylin and eosin under light microscope. Observed. The sections were double-blindly observed by a professional pathologist to determine the pathological changes in the radiation histology.

11. Determination of SOD and CAT activity in different tissues of mice:

BALB/c mice (male, 8 weeks old, Slack, 12 per group) were injected with NaCl and PprI proteins, respectively, as described above. The SOD and CAT activities of blood, plasma, liver, kidney, heart and bone marrow were measured 48 h after 4 Gy gamma irradiation.

12. Statistical analysis: The data was tested to conform to the normal distribution and statistical analysis was performed using analysis of variance and T test.

(2) Experimental results

1. PprI protein increases the survival rate of HUVECs

The present invention investigates the anti-radiation effect of pprI on human umbilical vein endothelial cells HUVECs. The results showed that the addition of PprI protein before 4 Gy gamma irradiation significantly increased cell viability compared to the PBS-treated group (Fig. 7).

Applying a clone formation experiment [10. Puck T, Marcus P. Action of x-rays on mammalian cells. J Exp Med. 103, 653-666 (1956).], counting the number of clones of cells after irradiation with different doses and using a multi-target single The model was fitted with a dose-survival curve. The results showed that the survival rate of HUVECs in the PprI protein group was significantly increased after 2 Gy and 4 Gy irradiation compared with the PBS-treated group (Fig. 8). The Do values of the PBS and PprI protein groups were 1.5837 and 1.6597, respectively, and the Dq values were 0.9936 and 1.5430, respectively. The smaller the Do and Dq values, the higher the radiosensitivity of the cells. These results indicate that the PprI protein successfully increased the radiation resistance of HUVECs.

2. PprI protein reduces the radiation apoptotic rate of HUVECs

The present invention simultaneously measures the level of apoptosis of HUVECs after false exposure (unirradiated radiation) or exposure to 4Gy gamma rays. 4Gy ionizing radiation significantly increased the apoptotic rate of cells in the PBS group, while the addition of PprI protein significantly decreased the apoptotic rate. Bcl-2 protein plays a crucial role in the mitochondrial apoptosis pathway, and overexpression of Bcl-2 protein can inhibit cell destruction. The expression of apoptosis-related proteins (Bcl-2, Bax) was detected by Western blot 48 h after ionizing radiation. Compared with the expression level of cells in the PBS-treated group, PprI protein reduced the expression of the pro-apoptotic protein Bax and promoted the expression of the anti-apoptotic protein Bcl-2 (Fig. 9 and Fig. 10). These studies suggest that apoptosis reduced by PprI protein may be involved in the regulation of mitochondrial channels.

3. PprI protein enhances anti-oxidation and DNA damage repair ability

Oxidative stress is a key factor in the growth of DNA damage. When cells are exposed to ionizing radiation or high oxygen partial pressure, the intracellular oxygen concentration increases, leading to ROS production, further damaging proteins, nucleic acids and other important elements in the cell.

The present invention uses the fluorescent probe DCFH-DA to detect intracellular ROS levels in irradiated human umbilical vein endothelial cells. The results of the study indicate that PprI protein can reduce intracellular ROS levels. The present inventors also studied the activity of the intracellular antioxidant proteins SOD and CAT in irradiated human umbilical vein endothelial cells (Fig. 11). SOD can degrade superoxide anion into hydrogen peroxide, and CAT can degrade hydrogen peroxide directly produced by SOD or ionizing radiation. Compared with the PBS group, the SOD and CAT activities of the PprI protein group were significantly increased after 4Gy γ-irradiation. Furthermore, it was found that the degree of intracellular lipid peroxidation in the PprI protein-treated group was significantly decreased (by measuring the intracellular malondialdehyde-MDA concentration), indicating that the PprI protein reduced the degree of systemic damage of the irradiated cells (Fig. 12).

γH2AX is the earliest marker of DNA double-strand breaks. The present invention further investigates the effect of PprI protein on the formation of γH2AX focal number. Compared with the control group, the number of γH2AX focal points in the PprI protein-treated group was significantly decreased at 1, 2, 4, and 8 h after ionizing radiation (Fig. 13 and Fig. 14).

In addition, the RecA protein plays a key role in the repair of homologous recombination of R. radiodurans. The Rad51 protein of the eukaryote is highly homologous to the RecA protein in both structure and function. The expression level of Rad51 in the irradiated cells of the PprI protein-treated group was significantly increased as compared with the PBS-treated group (Fig. 15). The above results indicate that the PprI protein enhances the radiation resistance of the irradiated human umbilical vein endothelium.

4, PprI enhances the survival rate of irradiated mice

Based on the above studies on the prevention and treatment of cell radiation damage, the present invention further investigated the effect of PprI protein on mortality of BALB/c mice after 6Gy lethal dose gamma irradiation. The study found that the saline-injected group, ie, the control group, died within 9 days after exposure, and the survival rate was 10%; while the 24 hPprI protein injection group (injection dose of 800 μg/kg body weight) was in the same condition. Five patients died, and the survival rate was 50%. In the PhrI protein injection group (injection dose of 800 μg/kg body weight), 24 mice died under the same conditions, and the survival rate was 30% (Fig. 16). These results indicate that PprI protein injection has a significant preventive effect on lethal radiation damage in animals.

In addition, the present invention detects peripheral blood cells of mice 1, 7, 14, 28, 35d after exposure. Changes in WBC, PLT, and percentage of lymphocytes. Compared with the saline-treated group, the WBC count and the percentage of lymphocytes in the PprI protein-treated group were significantly increased on the 7th day after irradiation; the PLT count was significantly increased on the 7th and 14th day after the irradiation (Fig. 17). The present invention also observed changes in the formation rate of bone marrow cells in the un-irradiated mice, the saline-treated mice, and the PprI-protein-treated mice on the 7th day after irradiation. Compared with the saline-treated group, the formation rate of mouse bone marrow cells in the PprI protein-treated group was significantly increased (Fig. 18).

5. PprI promotes the repair of tissues and organs of irradiated animals

The present invention observes histopathological changes in the liver, lung, testis and small intestine of irradiated mice.

Liver: 28 days after irradiation, the liver tissue of the saline-treated group showed degeneration and necrosis of hepatocytes, decreased cell number, nuclear fragmentation, hepatic sinusoidal dilatation, hyperemia, central venous wall detachment, and hepatic lobular tissue structure disorder; In the PprI protein-treated group, the mouse hepatocytes were slightly mitotic on the 28th day after irradiation, the Kupffer cells were slightly increased, and the hepatic lobule structure and hepatic sinusoids returned to normal (Fig. 19a-19d).

Lung: 28 days after irradiation, the lung tissue of the saline-treated group showed red blood cell exudation and inflammatory cell infiltration, the alveolar space was significantly thickened, the blood vessel wall was detached and the glass was changed. In the PprI protein-treated group, there was no red blood cell exudation and inflammatory cell infiltration in the lung tissue of the mice on the 28th day after irradiation, and the alveolar septal fibrosis was not obvious, and the lung tissue structure basically returned to normal (Fig. 19e-19h).

Testis: 28 days after irradiation, the testicular tissue of the saline-treated group showed interstitial broadening, atrophy of the seminiferous tubules, a disordered arrangement, and a broken wall. The spermatogonial cells were arranged in a single layer and recovered slowly. In the PprI protein-treated group, mouse testis tissue-supporting cells, spermatogonia and spermatocytes were regenerated on the 28th day after irradiation, and the testicular structure and spermatogonia completely returned to normal (Fig. 19i-19L).

Small intestine: 14 days after irradiation, the small intestine tissue of the saline-treated group showed severe destruction of the small intestine villus, and the gland and submucosal cells were necrotic, dissolved, and disappeared. In the PprI protein-treated group, the lamina propria, submucosa and mucosal epithelial cells of the small intestine of the mice began to proliferate on the 14th day after irradiation, and the basement membrane was thickened and the villus structure was complete (Fig. 19m-19p).

6. PprI enhances the antioxidant capacity of mice

The present invention measures the effect of PprI on the levels of SOD and CAT in different organs and tissues of irradiated mice. Compared with the saline-treated group, the activity of SOD and CAT in the red blood cells, plasma, liver and bone marrow of the PprI protein-treated group was significantly enhanced at 48 h after irradiation, while the kidney and myocardium were fine. There was no difference in SOD and CAT activity between the cells (Fig. 20a-20b). These results indicate that PprI protein enhances the antioxidant protein activity of certain tissues in mice.

to sum up:

The present invention employs a series of biological and molecular biological methods to systematically elucidate the effect of the radiation-resistant PrpI protein on radiation protection of human umbilical vein endothelial cells and mice irradiated with lethal doses. The invention further optimizes the method for efficient expression and purification of PprI protein in Pichia pastoris; and finds that PprI protein has significant preventive and therapeutic effects on acute radiation damage of human cells and mice. In both studies, the PprI protein treatment group had increased radiation cell survival and decreased apoptotic rate. Remarkably, the activity assay showed that the PprI protein increased the expression and regulation of the eukaryotic Rad51 protein, which is a homologous protein of the prokaryotic DNA repair protein RecA. In addition, we observed that PprI protein enhances the antioxidant response of irradiated cells, thereby attenuating radiation-induced DNA damage. In conclusion, the PrpI protein of the present invention has a vital protective and therapeutic effect on lethal radiation damage in human cells and mice, and can be used as a novel and effective pronuclear anti-radiation protein drug for the prevention and treatment of acute radiation damage in humans. This is of great significance and application value for improving the medical emergency response capabilities of nuclear and radiation accidents and nuclear emergencies in the world and reducing the radiation toxicity of patients with tumor radiation.

The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can also make several improvements and retouchings without departing from the principles of the present invention. It should be considered as the scope of protection of the present invention.

Claims (10)

1. The application of PprI protein in the preparation of a medicament for preventing radiation damage of higher eukaryotes.
2. The use according to claim 1, wherein said controlling radiation damage is to increase cell or body radiation survival.
3. The use according to claim 1, wherein said controlling radiation damage is to promote repair of DNA or tissue organs damaged by radiation.
4. The application of PprI protein in the preparation of antioxidant drugs.
5. The use according to claim 4, wherein the antioxidation is to reduce intracellular ROS levels.
6. The use according to claim 4, wherein the antioxidant is to increase SOD, CAT activity, and/or reduce malondialdehyde concentration.
7. Application of PprI protein in the preparation of Rad51 protein expression promoter.
8. A PprI protein drug characterized by comprising a PprI protein.
9. The medicament according to claim 8, wherein the pharmaceutical dosage form is an injection.
10. The medicament according to claim 8, wherein the medicament further comprises a pharmaceutically acceptable adjuvant.

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