CN107217013B

CN107217013B - Deinococcus T1R5 and application thereof in radiation protection

Info

Publication number: CN107217013B
Application number: CN201710043119.0A
Authority: CN
Inventors: 霍向东; 许琴; 史深; 张丽娟; 张志东; 朱静; 王玮; 王岩军; 谢玉清; 石玉瑚
Original assignee: Institute Of Microbial Applications Xinjiang Academy Of Agricultural Sciences (china Xinjiang-Armenia Bioengineering Research And Development Center)
Current assignee: Institute Of Microbial Applications Xinjiang Academy Of Agricultural Sciences (china Xinjiang-Armenia Bioengineering Research And Development Center)
Priority date: 2017-01-19
Filing date: 2017-01-19
Publication date: 2020-08-07
Anticipated expiration: 2037-01-19
Also published as: CN107217013A

Abstract

The invention discloses deinococcus T1R5 and application thereof in radiation protection, and a deinococcus new species is separated and screened from soil samples in arid regions of Xinjiang and major saline-alkali regionsDeinococcus sp.) T1R5CGMCC No.12800, wherein the strain can tolerate 0-1% (W/V) NaCl, the optimal growth temperature is 30 ℃, the maximum growth temperature is 42 ℃, the strain can survive under the radiation of gamma rays with the dosage of 20KGry, and the strain has obvious radiation resistance.

Description

Deinococcus T1R5 and application thereof in radiation protection

Technical Field

The invention relates to the technical field of microbial strains and application thereof, in particular to a novel radiation-resistant bacterium and the technical field of application thereof in preparation of a radiation protection preparation.

Background

The deinococcus is pink, non-spore and non-pathogenic gram-positive micrococcus, the bacterial colony is convex, the surface is smooth, the diameter of the thallus is measured to be between 10 and 25 mu m after separation and purification, and the division mode is single, paired or multi-plane division. Due to the morphology of deinococcus and Micrococcus (A)Micrococcus) Similarly, it was named at the beginning of the discoveryMicrococcus radio- durans. After 30 years, scientists reclassified it as a new one with the closest relationship to itDeinococ- caceaeMedicine and foodDeinococcusGenus (deinococcus). The currently known genus deinococcusDeinococcus9 species are involved, and the forms thereof are different. Deinococcus is one of the most radiation resistant organisms found on earth, which are resistant to ionizing radiation, ultraviolet rays and H₂O₂And DNA and protein damage caused by other factors has extremely strong tolerance and resistance. The unique biological properties, physiological mechanisms and genetic mechanisms of deinococcus have become of increasing interest to researchers.

Currently, more than 2000 nuclear tests are performed in 8 countries worldwide. The nuclear tests cause radioactive pollution of different degrees and different properties to each test site and peripheral areas, and have certain influence on the health and survival of people in local areas. As 12-31 days in 2014, 437 nuclear power reactors in operation are in common in the world, and in addition, more than 100 nuclear power stations are being built, the nuclear technology utilizes the life which gradually goes to people from the military field, and the negative effects of the nuclear technology are increasingly prominent while the nuclear technology benefits human beings. With the concern of nuclear warfare and nuclear terrorism caused by the tension of the world nuclear safety situation, the protection and treatment of radiation injury are receiving more and more attention. More than 300 million cancer patients in China need postoperative radiotherapy every year, the radiotherapy can kill cancer cells and simultaneously cause acute radiation damage to surrounding normal tissues and organs and even the whole body, and the side reaction of the radiotherapy seriously limits the wide application of radiotherapy in tumor treatment.

Currently, the treatment of radiation injury mainly adopts drug therapy, related drugs comprise sulfur-containing compounds, hormones, cytokines, Chinese herbal medicines and the like, and the sulfur-containing compounds generally have the problem of large side effect; the use of hormone drugs has an influence on the sexual organs and the reproductive system, and the radiation protection effect of cytokine drugs such as interleukins and colony stimulating factor drugs is influenced by the administration time and produces different effects according to the administration time; the Chinese herbal medicine anti-radiation medicine such as phenols, polysaccharides, natural flavonoids and the like has the characteristics of undefined active ingredients, low toxicity and the like. The above drugs have the disadvantages of slow treatment effect, large side effect and the like on the whole. Radiation-resistant microorganisms are an extreme microbial resource capable of surviving high radiation doses, and have an ultra-strong DNA damage repair capacity and an ability to scavenge various free radicals in cells, by which the radiation-resistant microorganisms can survive and proliferate in a high radiation environment.

Although there are many reports of bioremediation using microorganisms, so far, the application of radiation-resistant microorganisms in prevention and treatment of relevant injury and disease caused by radiation and preparation of relevant drugs has not been reported. Therefore, the radiation-resistant characteristics of the radiation-resistant microorganisms need to be further explored, resistant strains with prevention and treatment effects are provided for radiation injury and diseases, and the method has practical significance for the technical field of biological prevention and treatment.

Disclosure of Invention

Aiming at the technical current situation that research on deinococcus in the aspect of radiation protection preparations is less documented in the prior art and the technical current situation that new strains are separated and screened to have stronger tolerance to gamma rays, the invention aims to provide new resistant strains with relatively high tolerance and stable performance for treating radiation injury and diseases by microorganisms, and provides new resistant strainsA new strain deinococcus T1R5 and its application in radiation protection are provided. The invention separates and screens a deinococcus sp new species in arid area soil samples in Xinjiang and major saline-alkali areasDeinococcus sp.) T1R5CGMCC No.12800, the separated strain is used for preparing a radiation protection preparation, and the radiation protection preparation is applied to a radiation animal, can obviously improve the survival rate of the irradiated animal, and has wide application value in the technical field of application of radiation-resistant microbial strains.

The invention adopts the main technical scheme that:

the deinococcus new species T1R5 which can be applied to the radiation protection aspect is obtained by screening and separating soil samples in arid regions of Xinjiang and major saline-alkali regions. The radiation protection preparation prepared by the separated and screened bacterial strain with the number of T1R5 can survive under the radiation of gamma rays with the dose of 20KGry when being applied to the radiation animals such as Kunming mice and the like, can obviously improve the survival rate of the radiation animals, and has very great application prospect when being prepared by the novel deinococcus cocci strain T1R5 provided by the invention.

The invention specifically provides a novel deinococcus species (Deinococcus sp.) T1R5CGMCC No. 12800. Separating and sampling from soil samples of arid regions in Xinjiang and major saline-alkali areas to screen out a strain of bacteria deinococcus (Deinococcus sp.) T1R5, the bacterium can survive under gamma-ray radiation with the dose of 20KGry, and has higher radiation resistance.

According to the particularity of the geographic environment of Xinjiang and the characteristics of an enrichment place of microbial resources in the extreme environment of Xinjiang, the invention cultures microbial strains in arid soil samples of Xinjiang and major saline-alkali areas, separates and screens a batch of excellent strains, and separates and screens a deinococcus bacterium (T1R 5) from the strainsDeinococcus sp.) The microbial strain belongs to the genus deinococcus, can tolerate NaCl with the concentration of 0-1 percent, has the optimal growth temperature of 30 ℃ and the highest growth temperature of 42 ℃, can grow on 1/5TGY culture medium, and has a convex, round, smooth, opaque and pink colony. The strains are subjected to reference of Bergey's Manual of bacteria identification and general Manual of bacteria identificationThe T1R5 strain is determined to be a member of the deinococcus by morphological, physiological and biochemical tests, but has different characteristics from the common deinococcus member strains and has the characteristics of a plurality of new strains.

The gene sequencing of the said strain, SEQUENCE reference to SEQUENCE L ISTING provided after the attachment, 16SRNA homology analysis, phylogenetic analysis, B L AST homology comparison to determine the species with the nearest genetic relationship with the experimental strain, obtaining the SEQUENCE of the related species from the database, and combining with the database of the bacterial biodiversity research center to perform cluster analysis and phylogenetic tree construction, the T1R5 strain belongs to deinococcus (A. coli)Deinococcus sp.) The highest homology of the strain T1R5 and the model strain of the genus is 97.54% ((Deinococcus desertiVCD115^T) The homology with other strains in the genus is less than 97.0 percent, and the strain is preliminarily determined to be deinococcus (deinococcus)Deinococcus sp.) The new species, which has the characteristic of typicality of the new species, is tentatively named as deinococcus from the taxonomic point of view (Deinococcus sp.) T1R 5. The strain has been deposited in the international collection of microorganisms under the Budapest treaty before the filing date: china general microbiological culture Collection center (CGMCC); address: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101; the preservation date is 2016, 07 and 20 days, the preservation number of the strain is CGMCC No.12800, and the temporary name is deinococcus after microbiological identification (Deinococcus sp.）T1R5。

Deinococcus (A)Deinococcus sp.) Physiological and biochemical properties of T1R 5: gram staining is positive, aerobic, non-motile, and negative in catalase, urease, hydrolyzed cellulose, starch, tween 20, tween 40, tween 80, hydrogen sulfide, nitrate and methyl red tests; positive in curd, milk peptonization, gelatin test, indole and Proskauer test; only D-mannose is utilized in GN III, amygdalin, aesculin, melibiose, sucrose, trehalose, glycogen, and D-lyxose can be utilized in API 50CH system for fermentation to produce acid; gelatin, sucrose, galactose, arabinose and D-mannose on API 20NE test paper are positive, and the reaction is different from the physiological reaction of common deinococcus member strainsBiochemical characteristics, characteristic of some new species.

Meanwhile, the invention further provides deinococcus sp (deinococcus sp.) (Deinococcus sp.) The method for preparing the radiation protection preparation by using T1R5CGMCC No.12800 comprises the following specific steps:

inoculating activated fresh slant strain T1R5 into seed test tube containing 5ml 1/5TGY liquid culture medium, culturing at 30 deg.C, shaking at 200rpm for 36h, inoculating into 1/5TGY liquid fermentation bottle at 2%, culturing at 30 deg.C, shaking at 220rpm for 72h, centrifuging the culture at 8000rpm for 3min, discarding supernatant, collecting thallus, washing thallus with physiological saline, centrifuging for three times, collecting thallus, diluting with physiological saline to viable count concentration of 1 × 10¹⁰cfu/ml, ultrasonic crushing by adopting 4 ℃ ice water bath, the power is 400w, 90 times, each time exceeds 5 seconds, stopping for 5 seconds to obtain cell crushing liquid, filling to obtain the radiation protection preparation, and keeping at 4 ℃ for later use.

Further, the present invention provides radioprotective formulations prepared by using deinococcus bacterium (deinococcus: (a))Deinococcussp.) T1R5CGMCCNo.12800, which is obtained by the method for preparing the radiation protection preparation, can survive under the gamma-ray radiation with the dose of 20KGry, obviously improves the survival rate of the radiated animals and has wide application value.

By implementing the specific technical scheme provided by the invention, the following beneficial effects can be achieved by implementing the content of the invention:

(1) the deinococcus (A) provided by the inventionDeinococcus sp.) T1R5CGMCC No.12800 is a typical new strain, can tolerate 0-1% (W/V) NaCl, can grow on 1/5TGY culture medium, has the optimum growth temperature of 30 ℃ and the maximum growth temperature of 42 ℃, can survive under the radiation of gamma rays with the dose of 20KGry, has higher radiation resistance, and has the characteristics of simple culture condition and quick propagation.

(2) Deinococcus isolated and screened by the invention (Deinococcus sp.) The radiation protection preparation obtained by fermentation technology of T1R5CGMCC No.12800 is applied to a mouse with low radiation dose, and the mouse is irradiated for 6Gy cumulatively at a dose rate of 0.86Gy/min and then treated by QiqiCoccus (A)Deinococcus sp.) The T1R5 radioprotectant injection treatment group was raised for one month, no mice died, and the survival rate was 100%. The mice of the control group die after being irradiated for 2 weeks until being fed for one month, the total survival rate is 83.4 percent, and the protection index reaches 1.2; in an 8Gy irradiation experiment, the mice in the group treated by the injection of the radioresistant bacteria T1R5 die after 2 weeks of radiation until the mice are raised for one month, and the total survival rate is 83.4%; and the mice in the control group die after being radiated for 1 week, and the total survival rate reaches 58.8 percent and the protection index reaches 1.4 after the mice are raised for one month.

(3) Deinococcus isolated and screened by the invention (Deinococcus sp.) The radiation protection preparation obtained by T1R5CGMCC No.12800 through fermentation technology is applied to a mouse with high radiation dose, and the mouse is irradiated by 6Gy through radiation at a dose rate of 1.20Gy/min and cumulative irradiation of 6Gy, and then is irradiated by deinococcus (deinococcus)Deinococcus sp.) The mice die continuously 2 weeks after the T1R5 radiation protection preparation injection treatment group radiates, the survival rate is 66.7%, the mice die continuously on the day after the control group radiates, the total survival rate is 11.2% after the control group is raised for one month, and the protection index reaches 5.95; under the same dosage rate condition, in a 10Gy irradiation experiment, the mice of the radiation-resistant bacteria T1R5 injection treatment group die after 1 week of radiation until the mice are raised for one month, the total survival rate is 38.9%, while the mice of the control group die after 1 week of radiation until the mice are raised for one month, the total survival rate is 25%, and the protection index reaches 1.56.

(4) The deinococcus sp (deinococcus sp.) provided by the inventionDeinococcus sp.) After the T1R5 radiation protection preparation is injected, the radiation survival rate of mice can be obviously improved, and especially under the condition of high dose rate and low dose, the protection effect is more obvious.

Drawings

FIG. 1 shows the strain deinococcus (A)Deinococcus sp.) Phylogenetic dendrogram of T1R5CGMCC No. 12800.

FIG. 2 shows the strain deinococcus (A)Deinococcus sp.) The radiation resistance curve chart of T1R5CGMCC No.12800 and related strains shows that the horizontal axis represents radiation dose and the vertical axis represents survival rate.

FIG. 3 shows the strain deinococcus (A)Deinococcus sp.) T1R5CGMCC No.12800 has protection effect on Kunming mouse irradiation under the condition of dosage rate of 0.86Gy/min, wherein the horizontal axis represents radiation dosage and the vertical axis represents survival rate.

FIG. 4 shows the strain deinococcus (A), (B), (CDeinococcus sp.) The strain T1R5 has the protection effect on Kunming mouse irradiation under the dosage rate of 1.20Gy/min of T1R5CGMCC No.12800, the horizontal axis represents the radiation dosage, and the vertical axis represents the survival rate.

Detailed Description

The present invention will be described below by way of examples, but the present invention is not limited to the following examples. All raw and auxiliary materials selected for use in the present invention, as well as methods for culturing the selected bacterial species, are well known and used in the art, and all percentages referred to herein are by weight unless otherwise indicated.

The following basic culture media are adopted in the embodiment of the invention:

TGY culture medium composed of peptone 5 g, yeast extract 3g, glucose 2g, distilled water 1000m L, and pH = 7.0.

The first embodiment is as follows: deinococcus (A)Deinococcus sp.) Isolation, screening and identification of T1R5

1. Separation and purification: separated from soil samples of arid regions in Xinjiang and major saline-alkali areas. 100g of a soil sample is weighed and placed⁶⁰Irradiating to a dose of 10KGy under a Co radiation source, adding 5.0g of a sample to be irradiated into a culture solution containing 50 ml of 1/5TGY, carrying out enrichment culture at 30 ℃ and 150 r/min for 48 h, sucking 1ml of the enrichment solution, carrying out gradient dilution, coating on a TGY solid plate, carrying out constant temperature culture at 28 ℃, picking single colonies after the single colonies grow out from the plate, carrying out streaking separation and purifying.

By the deinococcus provided above (Deinococcus sp.) T1R5, 100g of soil sample is weighed and placed⁶⁰Irradiating to a dose of 10KGy under a Co radiation source, adding 5.0g of a sample to be irradiated into a culture solution containing 50 ml of 1/5TGY, carrying out enrichment culture at 30 ℃ and 150 r/min for 48 h, sucking 1ml of the enrichment solution, coating the enrichment solution on a TGY solid plate after gradient dilution, carrying out constant temperature culture at 28 ℃, selecting bacterial colonies after the bacterial colonies grow out, and respectively carrying out streak inoculation on corresponding plates until the bacterial colonies with different shapes, sizes, colors and the like are obtained until the bacterial colonies grow out until the bacterial colonies are inoculated on the corresponding plates until the bacterial colonies grow outAnd (4) no impurity colony.

The T1R5 strain was cultured in 1/5TGY medium at 30 ℃ for 5 days, and the colony was convex, round, smooth, opaque and pink. The optimal growth temperature of the strain is 30 ℃, the maximum growth temperature is 42 ℃, the strain can tolerate 0-1% (W/V) NaCl, the strain is classified and identified according to Bergey's Manual of bacteria identification and general Manual of bacteria identification, and the initial conclusion is that the strain is deinococcus (R) ((R))Deinococcus sp.）。

Deinococcus (A)Deinococcus sp.) Physiological and biochemical properties of T1R 5: gram staining is positive, aerobic, non-motile, and negative in catalase, urease, hydrolyzed cellulose, starch, tween 20, tween 40, tween 80, hydrogen sulfide, nitrate and methyl red tests; positive in curd, milk peptonization, gelatin test, indole and Proskauer test; only D-mannose is utilized in GN III, amygdalin, aesculin, melibiose, sucrose, trehalose, glycogen, and D-lyxose can be utilized in API 50CH system for fermentation to produce acid; gelatin, sucrose, galactose, arabinose, D-mannose reacted positive on API 20NE paper.

Strain deinococcus (A)Deinococcus sp.) T1R5 has been deposited at the Budapest treaty on the International Collection of microorganisms: china general microbiological culture Collection center (CGMCC). Address: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101. the preservation date is 2016, 07, 20 days, and the preservation number of the strain is CGMCC No. 12800. Temporarily named deinococcus after microbiological identification (Deinococcus sp.）T1R5。

The deinococcus (A) provided by the inventionDeinococcus sp.) T1R5CGMCC No.12800 can be cultured in solid culture medium or liquid culture medium suitable for proliferation, and can be preserved for more than 3 months per passage by conventional solid slant culture and low-temperature preservation method, and the strain preserved for a long time can be preserved for more than 1 year by dry freezing method or preserved for a long time by glycerinum tube.

2. Classifying and identifying: by observing deinococcus (Deinococcus sp.) T1R5 colony shape and growthPhysical and biochemical characteristics, extracting total DNA of the strain, performing PCR amplification by using a bacterial ITS PCR amplification universal primer, performing gel cutting purification and sequencing on a PCR product, performing ITS B L AST comparison, and performing cluster analysis and phylogenetic tree construction.

By extracting deinococcus (A), (B), (C), (Deinococcus sp.) The total DNA of T1R5 is PCR amplified by using the universal primer for bacterial ITS PCR amplification, PCR products are purified and sequenced by cutting gel, and the SEQUENCE of the PCR products is referred to SEQUENCE L ISTING provided in the attached text.

Obtaining the sequence of related species from the database, combining the standard model bacteria sequence of the bacterial biodiversity research center database, using MEGA 5.0 software package and using the adjacency method (Neighbor-Joining method) to perform clustering analysis and phylogenetic tree construction, wherein the phylogenetic tree is shown in figure 1, and the strains belong to deinococcus (deinococcus) known by the phylogenetic treeDeinococcus sp.) The highest homology of the strain T1R5 and the model strain of the genus has been 97.54% ((Deinococcus desertiVCD115^T) The homology with other strains in the genus is less than 97.0 percent, and the strain is preliminarily determined to be deinococcus (deinococcus)Deinococcus sp.) New species, tentatively named deinococcus (Deinococcus sp.）T1R5。

By the above-mentioned species deinococcus (Deinococcus sp.) The strain T1R5CGMCC No.12800 is characterized in that the strain morphology, culture characteristic observation, physiological and biochemical index determination and molecular level identification are carried out by observing the strain morphology, observing the strain culture characteristic, determining the growth temperature, testing the tolerance and the like, and comparing with common deinococcus, according to the methods of Bergey's bacteria identification handbook and common bacteria identification handbook, although the strain with the number T1R5 has some common attributes compared with the common deinococcus, the strain with the number T1R5 has obvious physiological and biochemical characteristic difference and molecular level difference with the common deinococcus, which shows that the strain T1R5 is a typical new strain, and the strain with the number T1R5 is comprehensively identified as the deinococcus from the strain classification angle (the) (i.Deinococcus sp.）。

Example two: deinococcus (A)Deinococcus sp.) Radiation resistance of T1R5CGMCC No.12800

From freshly cultured deinococcus (Deinococcus sp.) Removing a ring of lawn from T1R5 slant, inoculating to 1/5TGY liquid culture medium, culturing at 30 deg.C and 150rpm for 4 days with shaking, centrifuging at 5000R/min to collect thallus, washing with 0.05 mol/L potassium phosphate buffer solution with pH of 7.2 for three times, and adjusting to OD₆₀₀About 1.0, taking 5ml of the bacterial suspension, filling the bacterial suspension into a 15 × 150mm sterile test tube, and placing the sterile test tube at a dosage rate of 100 Gry/min⁶⁰Co gamma ray is irradiated in different doses, 1ml of irradiated bacteria liquid is diluted in gradient mode, 0.2 m L diluted liquid is coated onto 1/5TGY agar medium plate and cultured at 30 deg.c, the colony number is observed and recorded, the survival rate is calculated and the likeD.radiodurans、D. desertiVCD115^TColi DH5 α as control strain by⁶⁰Co gamma ray irradiation treatment strain T1R5 and contrast bacteriaD.radioduransSurvival of different strains at different doses was calculated after d.deserti and e.coli DH5 α.

The results are shown in FIG. 2, the gamma-ray radiation resistance of the strain T1R5D. desertiVCD115T were very similar and all survived at 20KGry doses, slightly lower than the bacteriaD.radioduransRadiation resistance of (1).

Example three: deinococcus (A)Deinococcus sp.) Method for preparing radiation protection preparation from T1R5

Using deinococcus (Deinococcus sp.) The specific steps of preparing the radiation protection preparation by using T1R5CGMCC No.12800 are as follows:

inoculating activated fresh slant strain T1R5 into seed test tube containing 5ml 1/5TGY liquid culture medium, culturing at 30 deg.C, shaking at 200rpm for 36h, inoculating into 1/5TGY liquid fermentation bottle at 2%, culturing at 30 deg.C, shaking at 220rpm for 72h, centrifuging the culture at 8000rpm for 3min, discarding supernatant, collecting thallus, washing thallus with physiological saline, centrifuging for three times, collecting thallus, diluting with physiological saline to viable count concentration of 1 × 10¹⁰cfu/ml, ultrasonic crushing with 4 deg.C ice-water bath and power400w for 90 times, each time for over 5 seconds, stopping for 5 seconds to obtain cell disruption solution, filling to obtain the radiation protection preparation, and keeping at 4 ℃ for later use.

Example four: deinococcus (A)Deinococcus sp.) Application of T1R5CGMCC No.12800 in preparing radiation protection preparation in radiation protection

The experimental animals are Kunming mice with the average weight of 15-22 g, purchased from the research center of experimental animals in the autonomous region of Uygur autonomous region of Xinjiang, and randomly selected by male and female. After feeding the purchased Kunming mice for 3 days in a laboratory, the injection test is started, the test group is injected with Deinococcus sp T1R5 radioprotection preparation, and the control group is injected with normal saline.

(1) Deinococcus (A)Deinococcus sp.) Effect of T1R5 radioprotective preparation on low radiation dose Kunming mice:

use of deinococcus (Deinococcus sp.) T1R5 radioprotective preparation is prepared by injecting Kunming mouse intraperitoneally with 0.5ml per mouse every 7 days for three times, and injecting control group with equal amount of normal saline. Irradiation was performed 42 days after the first injection, and irradiation was performed⁶⁰Co irradiation is carried out, the dose is respectively 6Gy and 8Gy, the dose rate is 0.86Gy/min, each irradiation dose is set to be 3 times, and 18 mice are treated each time. In one month after the irradiation, the mice were strictly fed according to the program, and the number of deaths of the mice was recorded and analyzed.

The test results are shown in figure 3, and the mice are irradiated with 6Gy cumulative radiation at a dose rate of 0.86Gy/min and then treated with deinococcus bacterium (deinococcus sp.) (Deinococcus sp.) The T1R5 radioprotectant injection treatment group was raised for one month, no mice died, and the survival rate was 100%. The mice of the control group die after being irradiated for 2 weeks until being fed for one month, the total survival rate is 83.4 percent, and the protection index reaches 1.2; in an 8Gy irradiation experiment, the mice in the group treated by the injection of the radioresistant bacteria T1R5 die after 2 weeks of radiation until the mice are raised for one month, and the total survival rate is 83.4%; and the mice in the control group die after being radiated for 1 week, and the total survival rate reaches 58.8 percent and the protection index reaches 1.4 after the mice are raised for one month.

Therefore, the deinococcus (deinococcus) provided by the inventionDeinococcus sp.）T1After the R5 radioprotective preparation is injected, the radiation survival rate of mice can be obviously improved.

(2) Deinococcus (A)Deinococcus sp.) Effect of T1R5 radioprotective preparation on high radiation dose kunming mice:

use of deinococcus (Deinococcus sp.) T1R5 radioprotective preparation is injected into Kunming mouse, 0.5ml is injected into abdominal cavity of each mouse, and is injected once every 7 days for three times, and control group is injected with normal saline with same amount. Irradiation is carried out 42 days after the first injection, 60Co irradiation is adopted, the dose is respectively 6Gy and 10Gy, the dose rate is 1.20Gy/min, 3 repetitions are set for each irradiation dose, and 18 mice are treated each time. In one month after the irradiation, the mice were strictly fed according to the program, and the number of deaths of the mice was recorded and analyzed.

The test results are shown in figure 4, and the mice are irradiated by deinococcus after irradiation of 6Gy by radiation with dosage rate of 1.20Gy/minDeinococcus sp.) The mice die continuously 2 weeks after the T1R5 radiation protection preparation injection treatment group radiates, the survival rate is 66.7%, the mice die continuously on the day after the control group radiates, the total survival rate is 11.2% after the control group is raised for one month, and the protection index reaches 5.95; under the same dosage rate condition, in a 10Gy irradiation experiment, the mice of the radiation-resistant bacteria T1R5 injection treatment group die after 1 week of radiation until the mice are raised for one month, the total survival rate is 38.9%, while the mice of the control group die after 1 week of radiation until the mice are raised for one month, the total survival rate is 25%, and the protection index reaches 1.56.

Therefore, the deinococcus (deinococcus) provided by the inventionDeinococcus sp.) After the T1R5 radiation protection preparation is injected, the radiation survival rate of mice can be obviously improved, and especially under the condition of high dose rate and low dose, the protection effect is more obvious.

Deinococcus provided by the above series of examples (Deinococcus sp.) T1R5CGMCC No.12800 is a typical new strain, can tolerate 0-1% (W/V) NaCl, has the optimum growth temperature of 30 ℃ and the maximum growth temperature of 42 ℃, can survive under the radiation of gamma rays with the dosage of 20KGry, has higher radiation resistance, and has the advantages of simple culture condition, high reproduction rate and the likeFast reproduction. Deinococcus (A) and (B)Deinococcus sp.) The radiation protection preparation obtained by the fermentation technology of T1R5CGMCC No.12800 is applied to radiated animals, can obviously improve the survival rate of the radiated animals, and has wide application value in the technical field of microbial strain application.

The above examples are merely illustrative for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications can be made while remaining within the scope of the present invention.

SEQUENCE LISTING

<110> institute of microorganism application of Sinkiang academy of agricultural sciences (Xinjiang-Yameiya bioengineering research and development center, China)

<120> deinococcus radiodurans T1R5 and application thereof in radiation protection

<130>T1R5

<160>1

<170>PatentIn version 3.3

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<211>1467

<212>DNA

<213>T1R5

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<222>(1)..(1467)

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acaatggatg gtacaacgcg cagccagccc gcgagggtga gcgaatcgct gaaagccatc 1260

cccagttcag atcggagtct gcaactcgac tccgtgaagt tggaatcgct agtaatcgtg 1320

ggtcagcata ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccatg 1380

ggagtacgtt gcagttgaaa ccgccgggag ctgtaaggca ggcgtctaga ctgtggcgca 1440

tgactggggt gaagtcgtaa caaggta 1467

Claims

1. A Deinococcus (Deinococcus sp.) T1R5 is characterized in that the strain preservation number of the Deinococcus (Deinococcussp.) T1R5 is CGMCC No. 12800.

2. A method for preparing a radiation protection preparation by utilizing Deinococcus sp T1R5 is characterized by comprising the following steps:

(1) inoculating activated fresh Deinococcus sp (Deinococcus sp.) T1R5 into a seed test tube filled with 5ml 1/5TGY liquid culture medium, culturing at 30 ℃, carrying out shake culture at 200rpm for 36h, then inoculating into a 1/5TGY liquid fermentation bottle according to the inoculation amount of 2%, carrying out culture at 30 ℃, and carrying out shake culture at 220rpm for 72 h; centrifuging the culture at 8000rpm for 3min, removing supernatant, and collecting thallus;

(2) washing the thallus with normal saline, centrifuging for three times, collecting thallus, diluting with normal saline to viable count concentration of 1 × 10¹⁰cfu/ml, ultrasonically crushing by adopting 4 ℃ ice water bath with the power of 400w for 90 times, exceeding 5 seconds each time, stopping for 5 seconds to obtain cell crushing liquid, filling to obtain the radiation protection preparation, and keeping at 4 ℃ for later use;

the strain preservation number of the Deinococcus sp T1R5 is CGMCC No. 12800.

3. An application of a radiation protection preparation prepared by Deinococcus sp T1R5 in radiation protection is characterized in that the strain preservation number of the Deinococcus sp T1R5 is CGMCC No. 12800.