CN111855984A - Method for evaluating biological safety by using intestinal flora of mice - Google Patents
Method for evaluating biological safety by using intestinal flora of mice Download PDFInfo
- Publication number
- CN111855984A CN111855984A CN202010766965.7A CN202010766965A CN111855984A CN 111855984 A CN111855984 A CN 111855984A CN 202010766965 A CN202010766965 A CN 202010766965A CN 111855984 A CN111855984 A CN 111855984A
- Authority
- CN
- China
- Prior art keywords
- mouse
- mice
- intestinal flora
- deep
- sea
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
- G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Urology & Nephrology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Toxicology (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for evaluating biological safety by using intestinal flora of mice. The method uses mouse intestinal flora as biological model, and adds the separated mixed phage in deep sea sediment for continuous treatment for 3 days. Taking a fecal sample of the mouse after 1 week, carrying out 16S rRNA gene sequencing, determining physiological indexes of the mouse, and comparing the physiological indexes with a control sample to determine the influence of the deep-sea bacteriophage on the intestinal flora structure and the health of the mouse. Wherein, compared with a control group, if three repetitions in the treatment group have significant changes of bacterial abundance and mouse physiological indexes, the group of deep sea phages has biological safety problem. The invention provides a method for evaluating the biological safety of deep-sea phages.
Description
Technical Field
The invention belongs to the field of biosafety assessment, and particularly relates to a method for assessing biosafety of deep-sea phage by using mouse intestinal flora.
Background
Viruses are ubiquitous in marine ecosystems, are the largest in number, and are highly diverse in gene. The depth of virus-host interactions in the global oceans is very deep, and viruses can infect a variety of organisms, from simple green algae to large eukaryotes. Marine viruses have a major impact on nutrient and energy cycles, biogeochemistry, population dynamics, genetic exchanges and evolution in the marine environment. Although most marine viruses are non-culturable, most of them can be associated by calculation with a dominant, ecologically relevant microbial host, i.e. most are bacteriophages (phages). Due to the wide host range, deep sea phages may have the ability to infect human gut microbiota.
Human intestinal microorganisms contain billions of microorganisms and are one of the most important factors affecting human health. Over the past decade, due to the rapid development of technology, there has been a great understanding of the composition, function and role of the gut microbiota in human health and disease. The intestinal microorganisms can ferment indigestible polysaccharides, thereby producing metabolites such as Short Chain Fatty Acids (SCFAs), which play an important role in the metabolism, immunity, development and behavior of the host. Changes in the composition of the intestinal flora directly participate in the pathogenesis of various pathological states such as obesity, circulatory diseases, Inflammatory Bowel Diseases (IBDs), colorectal cancer (CRC) and autism.
In recent years, as deep sea research has been conducted, a large number of deep sea samples are brought to land every year. However, the effect of deep-sea phages on human health is not clear. The human intestinal flora may include some potential hosts for phages originating from deep sea. Once bacteria in the gut are infected with deep sea phage, gut flora balance will be disrupted, possibly leading to disease. Thus, there is a need to evaluate the biosafety of deep-sea phages.
Disclosure of Invention
The invention provides a method for evaluating the biological safety of deep-sea phage by using a mouse intestinal flora as a biological model, which is realized by 16S rRNA gene sequencing and mouse physiological index determination. And (4) determining whether the deep sea bacteriophage has biosafety problems by comparing the results of the experimental group and the treatment group.
The purpose of the invention is realized by the following technical scheme:
a method for evaluating biological safety by utilizing intestinal flora of mice comprises the following steps:
1) dividing the mice into an experimental group and a control group, wherein the male and female groups are half, and performing intragastric lavage on the mice of the treatment group by using a deep-sea phage suspension and sterile water in the control group; continuously feeding for 5-9 days in an aseptic environment after 2-5 days of continuous gavage, respectively taking two groups of mouse excrement samples for total DNA extraction, and taking mouse blood and intestinal tissue samples for measurement of physiological indexes;
2) and (3) carrying out gene sequencing on the extracted total DNA, analyzing the sequencing result, and counting and analyzing the measurement result of the physiological indexes of the mouse.
The invention takes the intestinal microbial flora of the model organism mouse as a model for evaluating the biological safety of the deep-sea bacteriophage, and researches the influence of the deep-sea bacteriophage on the human health.
In the step 1), the mice are ICR mice with the size of 8 weeks.
The experimental group and the control group are 4-8 in each group. Most preferably, 6 of each of the experimental and control groups are present.
Continuously feeding for 7 days in a sterile environment after continuous gavage for 3 days.
The measurement of the physiological index includes: body weight, blood routine, ALT (glutamate pyruvate transaminase), GLU (fasting plasma glucose), tuft cell morphology and number, IL-25 expression level. The measured physiological indexes of the mice comprise: body weight, blood routine, ALT, GLU, tuft cell morphology and number, IL-25 expression level.
The deep sea bacteriophage used is a mixed bacteriophage isolated from samples of deep sea sediments from different locations.
In the step 2), the sequencing method adopted by the gene sequencing is 16S rRNA gene sequencing.
Compared with the prior art, the invention has the following advantages:
the method of the invention uses the intestinal flora of the mice as a biological model, adds the mixed phage separated from the deep sea sediment into the biological model, and continuously processes the biological model for 3 days. Taking a fecal sample of the mouse after 1 week, carrying out 16S rRNA gene sequencing, determining physiological indexes of the mouse, and comparing the physiological indexes with a control sample to determine the influence of the deep-sea bacteriophage on the intestinal flora structure and the health of the mouse. Wherein, compared with a control group, if three repetitions in the treatment group have significant changes of bacterial abundance and mouse physiological indexes, the group of deep sea phages has biological safety problem. The invention provides a method for evaluating the biological safety of deep-sea phages.
The invention can evaluate the biological safety of deep-sea bacteriophage and has the advantages of high accuracy and obvious effect.
Drawings
FIG. 1 shows the results of gene sequencing of 16S rRNA of the intestinal flora structure of the mouse in example 1.
FIG. 2 shows the results of gene sequencing of 16S rRNA of mouse intestinal bacteria altered by the influence of the phage in the middle-deep sea of example 1.
FIG. 3 is a graph showing the physiological indices of mice changed by the influence of the phage in the middle deep sea of example 1.
FIG. 3-1 is a graph showing the number of mouse blood cells changed by the influence of the bacteriophage in example 1 in the middle deep sea
FIG. 3-2 is a graph showing the ratio of blood cells of mice changed by the influence of the bacteriophage in the middle deep sea of example 1
FIGS. 3 to 3 show ALT (glutamic-pyruvic transaminase) altered by the influence of phage of middle and deep sea in example 1
FIGS. 3 to 4 are GLU (blood glucose) levels altered by the influence of deep sea phages in example 1
FIGS. 3 to 5 show the body weight of mice changed by the influence of the phage in the middle-deep sea of example 1
FIGS. 3-6 show the number of intestinal tuft cells in mice altered by the influence of phage in the middle and deep sea of example 1
FIGS. 3 to 7 are graphs showing the level of IL-25 expression in mice altered by the influence of the phage in the middle-deep sea of example 1
FIG. 4 shows the results of gene sequencing of 16S rRNA of the intestinal flora structure of the mouse in example 2.
FIG. 5 shows the results of gene sequencing of 16S rRNA of mouse intestinal bacteria altered by the influence of phage in deep sea of example 2.
FIG. 6 is a graph showing the physiological indices of mice changed by the influence of the deep sea phage of example 2.
FIG. 6-1 is a graph showing the number of mouse blood cells changed by the influence of the bacteriophage in example 2 in the deep sea
FIG. 6-2 is a graph showing the ratio of blood cells of mice changed by the influence of the bacteriophage in deep sea of example 2
FIGS. 6-3 show ALT (glutamic-pyruvic transaminase) altered by the effect of phage in example 2 in the middle and deep sea
FIGS. 6 to 4 are GLU (blood glucose) levels altered by the influence of deep sea phages in example 2
FIGS. 6 to 5 show the body weight of mice changed by the influence of the phage in the middle-deep sea of example 2
FIGS. 6-6 show the number of intestinal tuft cells in mice altered by the influence of phage in the middle and deep sea of example 2
FIGS. 6-7 are graphs showing the level of mouse IL-25 expression altered by the influence of the phage in the middle-deep sea of example 2
Wherein V represents a treatment group, N represents a control group, C represents a female, X represents a male, 0 represents before treatment, and 1 represents after treatment; WBC for white blood cells, RBC for white blood cells, Hb for hemoglobin, Hct for hematocrit, MCV for red blood cell volume, MCH for mean red blood cell hemoglobin amount, MCHC for mean red blood cell hemoglobin concentration, PLT for platelets, NEUT for neutrophils, LYMPH for lymphocytes, MONO for monocytes, EO for eosinophils, BASO for basophils.
Detailed Description
The invention is described in further detail with reference to the following drawings, but the drawings and examples are not intended to limit the technical solutions of the invention, and all changes and equivalents that are made based on the teachings of the invention fall within the scope of the invention.
The test reagents used in the following examples were purchased from conventional Biochemical reagent companies unless otherwise specified; 16S rRNA gene sequencing was performed by sequencing Inc.
The deep-sea bacteriophage used in the invention is obtained by separating deep-sea sediments, and samples of the deep-sea sediments are taken from the middle ridges of the sea mountains and the pacific ocean.
Example 1:
(1) isolation of phages in the ridge sediments in the pacific:
1. a10 g sample of the pacific spine sediment (sample information see Table 1-1) was weighed into a 50mL centrifuge tube and 5mL of SM solution (25mM Tris-HCl,200mM NaCl,20mM MgCl)2pH 7.5), and shaking for resuspension for 20 min;
centrifuging at 2.4 deg.C and 5000 Xg for 10 min;
3. adding SM solution into the precipitate, and repeating for 5 times;
4. mixing the 5 collected supernatants, filtering with 0.45 μm filter membrane, and filtering with 0.22 μm filter membrane for sterilization;
5. adding PEG-6000 with final concentration of 10% into the solution, and precipitating overnight;
centrifuging at 6.4 deg.C and 40000 Xg for 2 h;
7. resuspending the precipitate with sterile water;
8. 10. mu.L of the virus solution was stained with tungsten phosphate and observed by a transmission electron microscope.
TABLE 1-1 Pacific mid-ridge deposit sample information
| Sample name | Latitude and longitude | Depth (m) |
| DP_114 | E 45.8 S 37.3 | 3,921 |
(2) Treatment of mice with deep-sea phage:
1. taking 6 female mice and 6 male mice (8 weeks), randomly dividing into two groups, wherein each group comprises 3 female mice and 3 male mice;
2. one group was gavaged with 100. mu.L of phages isolated from deep sea sediments, and the other group was gavaged with sterile water as a control group, and after 3 days of continuous treatment, the group was kept for 4 days in a sterile environment.
(3) Extraction of Total DNA of intestinal flora in mice (MP Fast DNA)TMFecal DNA extraction kit):
1. weighing 0.5g of mouse feces, and adding the mouse feces into a sample treatment tube;
2. add 825 μ L of Sodium Phosphate Buffer and 275 μ L of PLS into the tube and shake for 15 s;
3.14000 Xg, centrifuging for 5min, and removing the supernatant;
4. 978. mu.L of Sodium Phosphate Buffer and 122. mu.L of MT Buffer were added to the tube, and the mixture was shaken at 6m/s for 40 s;
5.14000 Xg, centrifuging for 15min, collecting supernatant, and adding into another sterile 2mL centrifuge tube;
6. adding 250 μ L PPS into the tube, mixing by turning upside down gently, standing at 4 deg.C for 10min, and centrifuging at 14000 × g for 2 min;
7. transferring the supernatant into a sterile 15mL centrifugal tube, adding 1mL Binding matrix solution into the tube, and uniformly mixing for 3 min;
8. placing the centrifuge tube on a shelf, standing for 3min to combine the DNA with the medium;
9. transferring the mixed solution to another sterile 2mL centrifugal tube, centrifuging for 2min at 14000 Xg, and discarding the supernatant;
10. adding 1mL of Wash Buffer # 1 into the tube, and uniformly mixing;
11. transfer the mixture to SPINTMCentrifuging at 14000 Xg for 1min on a Filter, and discarding the filtrate;
12. adding 500 mu L of Wash Buffer # 2 to the Spin Filter, centrifuging for 2min at 14000 Xg, and removing the filtrate;
13.14000 Xg, standing for 5min, and volatilizing;
14. the Spin Filter was transferred to a Catch Tube, 60. mu.L of TES Solution was added, the Solution was centrifuged at 14000 Xg for 2min, the Spin Filter was discarded, and the DNA sample was stored at-80 ℃.
(4) Mouse blood routine assay:
1. add 20ul of anticoagulant (EDTA) to the EP tube;
2. taking about 200ul of blood from the fundus of the mouse, adding the blood into a tube, and uniformly mixing;
3. detection was performed using a Sysmex XT-2000i Whole blood cell analyzer.
(5) Mouse biochemical indicators (ALT, GLU) assay:
1. blood was taken from the fundus of the mouse in EP tubes;
placed in a 2.4 degree freezer overnight, serum was separated (approximately 200 ul);
3. detection was performed using a fully automated biochemical analyzer, Roche cobas c 311.
(6) Detecting the number and the shape of the cells of the mouse intestinal canal:
1. taking a mouse intestinal tissue sample about 2cm, cleaning the mouse intestinal tissue sample by using 4-degree precooled PBS, transferring the mouse intestinal tissue sample into 10mL of 4% paraformaldehyde, and fixing the mouse intestinal tissue sample on ice for 1 h;
2. washing the sample with precooled PBS, transferring the sample into 10mL of 2% sucrose solution, and standing the sample in a refrigerator with 4 ℃ overnight;
3. manufacturing a mould by using tinfoil, and adding a freezing embedding medium (OTC) into the mould;
4. washing the sample with PBS, transferring to a mold, allowing the observation part to approach the bottom, and standing in a-20 deg.C refrigerator for 1 h;
5. slicing on a cryomicrotome, each slice having a thickness of 10 mm;
6. washing with TBST (10mM Tris-HCl; 150mM NaCl, 0.1% Triton X-100, pH 7.4) for 10 min;
7. washing with PBS for 10min, and repeating the washing;
8. surrounding the tissue, immunohistochemical pencils were placed in a frame, and blocking solution (3% BSA, 0.3% Triton X-100, 2% serum, 0.06% NaN3, in PBS) was added and incubated for 1h at room temperature;
9. blocking solution was aspirated, primary antibody (DCLK1) was added and incubated overnight at 4 ℃;
10. absorbing primary antibody, adding secondary antibody, and incubating at room temperature for 2 h;
11. washing with PBS for 10min, and repeating twice;
12. staining with DAPI in dark for 3 min;
13. washing with PBS for 10 min;
14. observed under a fluorescent microscope.
(7) Detection of IL-25 expression level:
1. taking a mouse intestinal tissue sample, and extracting total RNA by using a Kit (TransZol Up Plus RNA Kit);
2. using a kit (
III RT Supermix for qPCR) to prepare a cDNA template;
3. clean PCR tubes were removed and 5ul 2 XHieffTM qPCR was added
Green Master Mix, 0.2ul of upstream primer of IL25, 0.2ul of downstream primer, 1ul of cDNA and 3.6ul of ddH2O, blow-beating and mixing uniformly, and then measuring on a real-time fluorescence quantitative PCR instrument, wherein the program is as follows:
95℃30s；
95 ℃ for 5s, 60 ℃ for 30s,40 cycles.
(8) And (3) detecting the weight of the mouse:
1. weighing before gavage and before sacrifice, respectively;
the results show that: as shown in the attached figures 1 and 2, under the influence of phage in deep sea sediments of ridge DP _144 in the Pacific, the abundance of bacteria of 23 genera in the intestinal flora of mice is changed; meanwhile, as shown in figure 3, the weight of the treated mice is increased more obviously, various physiological indexes including the number of leucocytes are changed, the number of tuft cells in intestinal tissues is increased, the expression level of IL25 is up-regulated, and the mice are proved to have inflammatory symptoms, and the deep-sea phage at the site has the biological safety problem.
Example 2
(1) Separation of phage in the sea mountain sediment:
1. 10g of Haishan sediment sample is weighed into a 50mL centrifuge tube, and 5mL of SM solution is added(25mM Tris-HCl,200mM NaCl,20mM MgCl2pH 7.5), and shaking for resuspension for 20 min;
centrifuging at 2.4 deg.C and 5000 Xg for 10 min;
3. adding SM solution into the precipitate, and repeating for 5 times;
4. mixing the 5 collected supernatants, filtering with 0.45 μm filter membrane, and filtering with 0.22 μm filter membrane for sterilization;
5. adding PEG-6000 with final concentration of 10% into the solution, and precipitating overnight;
centrifuging at 6.4 deg.C and 40000 Xg for 2 h;
7. resuspending the precipitate with sterile water;
8. 10. mu.L of the virus solution was stained with tungsten phosphate and observed by a transmission electron microscope.
TABLE 2-1 Haishan sediment sample information
| Sample name | Latitude and longitude | Depth (m) |
| DP_138 | E 49.6 S 37.5 | 1,511 |
(2) Treatment of mice with deep-sea phage:
1. taking 6 female mice and 6 male mice (8 weeks), randomly dividing into two groups, wherein each group comprises 3 female mice and 3 male mice;
2. one group was gavaged with 100. mu.L of phages isolated from deep sea sediments, and the other group was gavaged with sterile water as a control group, and after 3 days of continuous treatment, the group was kept for 4 days in a sterile environment.
(3) Extraction of Total DNA of intestinal flora in mice (MP Fast DNA)TMFecal DNA extraction kit):
1. weighing 0.5g of mouse feces, and adding the mouse feces into a sample treatment tube;
2. add 825 μ L of Sodium Phosphate Buffer and 275 μ L of PLS into the tube and shake for 15 s;
3.14000 Xg, centrifuging for 5min, and removing the supernatant;
4. 978. mu.L of Sodium Phosphate Buffer and 122. mu.L of MT Buffer were added to the tube, and the mixture was shaken at 6m/s for 40 s;
5.14000 Xg, centrifuging for 15min, collecting supernatant, and adding into another sterile 2mL centrifuge tube;
6. adding 250 μ L PPS into the tube, mixing by turning upside down gently, standing at 4 deg.C for 10min, and centrifuging at 14000 × g for 2 min;
7. transferring the supernatant into a sterile 15mL centrifugal tube, adding 1mL Binding matrix solution into the tube, and uniformly mixing for 3 min;
8. placing the centrifuge tube on a shelf, standing for 3min to combine the DNA with the medium;
9. transferring the mixed solution to another sterile 2mL centrifugal tube, centrifuging for 2min at 14000 Xg, and discarding the supernatant;
10. adding 1mL of Wash Buffer # 1 into the tube, and uniformly mixing;
11. transfer the mixture to SPINTMCentrifuging at 14000 Xg for 1min on a Filter, and discarding the filtrate;
12. adding 500 mu L of Wash Buffer # 2 to the Spin Filter, centrifuging for 2min at 14000 Xg, and removing the filtrate;
13.14000 Xg, standing for 5min, and volatilizing;
14. the Spin Filter was transferred to a Catch Tube, 60. mu.L of TES Solution was added, the Solution was centrifuged at 14000 Xg for 2min, the Spin Filter was discarded, and the DNA sample was stored at-80 ℃.
(4) Mouse blood routine assay:
1. add 20ul of anticoagulant (EDTA) to the EP tube;
2. taking about 200ul of blood from the fundus of the mouse, adding the blood into a tube, and uniformly mixing;
3. detection was performed using a Sysmex XT-2000i Whole blood cell analyzer.
(5) Mouse biochemical indicators (ALT, GLU) assay:
1. blood was taken from the fundus of the mouse in EP tubes;
placed in a 2.4 degree freezer overnight, serum was separated (approximately 200 ul);
3. detection was performed using a fully automated biochemical analyzer, Roche cobas c 311.
(6) Detecting the number and the shape of the cells of the mouse intestinal canal:
1. taking a mouse intestinal tissue sample about 2cm, cleaning the mouse intestinal tissue sample by using 4-degree precooled PBS, transferring the mouse intestinal tissue sample into 10mL of 4% paraformaldehyde, and fixing the mouse intestinal tissue sample on ice for 1 h;
2. washing the sample with precooled PBS, transferring the sample into 10mL of 2% sucrose solution, and standing the sample in a refrigerator with 4 ℃ overnight;
3. manufacturing a mould by using tinfoil, and adding a freezing embedding medium (OTC) into the mould;
4. washing the sample with PBS, transferring to a mold, allowing the observation part to approach the bottom, and standing in a-20 deg.C refrigerator for 1 h;
5. slicing on a cryomicrotome, each slice having a thickness of 10 mm;
6. washing with TBST (10mM Tris-HCl; 150mM NaCl, 0.1% Triton X-100, pH 7.4) for 10 min;
7. washing with PBS for 10min, and repeating the washing;
8. surrounding the tissue, immunohistochemical pencils were placed in a frame, and blocking solution (3% BSA, 0.3% Triton X-100, 2% serum, 0.06% NaN3, in PBS) was added and incubated for 1h at room temperature;
9. blocking solution was aspirated, primary antibody (DCLK1) was added and incubated overnight at 4 ℃;
10. absorbing primary antibody, adding secondary antibody, and incubating at room temperature for 2 h;
11. washing with PBS for 10min, and repeating twice;
12. staining with DAPI in dark for 3 min;
13. washing with PBS for 10 min;
14. observed under a fluorescent microscope.
(7) Detection of IL-25 expression level:
1. taking a mouse intestinal tissue sample, and extracting total RNA by using a Kit (TransZol Up Plus RNA Kit);
2. using a kit (
III RT Supermix for qPCR) to prepare a cDNA template;
3. clean PCR tubes were removed and 5ul 2 XHieffTM qPCR was added
Green Master Mix, 0.2ul of upstream primer of IL25, 0.2ul of downstream primer, 1ul of cDNA and 3.6ul of ddH2O, blow-beating and mixing uniformly, and then measuring on a real-time fluorescence quantitative PCR instrument, wherein the program is as follows:
95℃30s；
95 ℃ for 5s, 60 ℃ for 30s,40 cycles.
(8) And (3) detecting the weight of the mouse:
1. weighing before gavage and before sacrifice, respectively;
the results show that: as shown in fig. 4 and 5, after phage treatment in the deep sea sediments of the marine mountain DP _138, the abundance of bacteria of 4 genera in the mouse intestinal flora was changed; meanwhile, as shown in figure 6, various physiological indexes of the mouse are not obviously changed, and the fact that the deep-sea bacteriophage at the site has no biosafety problem is proved.
The whole process is characterized as shown in the attached figures 1-6, and in conclusion: the method for evaluating the biological safety of the deep-sea bacteriophage by using the mouse intestinal flora as a model can accurately and stably screen out the deep-sea bacteriophage with biological safety problems, mark the position information of the sampling point, and need special treatment when performing scientific research, exploration, mining and other activities on the site in the future.
Claims (7)
1. A method for evaluating biological safety by utilizing intestinal flora of mice is characterized by comprising the following steps:
1) dividing the mice into an experimental group and a control group, wherein the male and female groups are half, and performing intragastric lavage on the mice of the treatment group by using a deep-sea phage suspension and sterile water in the control group; continuously feeding for 5-9 days in an aseptic environment after 2-5 days of continuous gavage, respectively taking two groups of mouse excrement samples for total DNA extraction, and taking mouse blood and intestinal tissue samples for measurement of physiological indexes;
2) and (3) carrying out gene sequencing on the extracted total DNA, analyzing the sequencing result, and counting and analyzing the measurement result of the physiological indexes of the mouse.
2. The method for biosafety assessment by using intestinal flora of mice according to claim 1, wherein the mice in step 1) are ICR mice 8 weeks old.
3. The method for evaluating the biological safety by utilizing the intestinal flora of the mouse as claimed in claim 1, wherein in the step 1), 4 to 8 mice are used in each group of the experimental group and the control group.
4. The method for evaluating biosafety by using the intestinal flora of mice as claimed in claim 1, wherein in the step 1), the feeding is continued in a sterile environment for 7 days after the continuous gavage for 3 days.
5. The method for biosafety assessment by using mouse intestinal flora according to claim 1, wherein the measurement of the physiological index in step 1) comprises: body weight, blood routine, ALT, GLU, tuft cell morphology and number, IL-25 expression level.
6. The method for biosafety assessment by using mouse intestinal flora according to claim 1, wherein in step 1), the deep sea phage is a mixed phage separated from deep sea sediment samples from different locations.
7. The method for biosafety assessment by using mouse intestinal flora according to claim 1, wherein in the step 2), the sequencing method adopted by gene sequencing is 16S rRNA gene sequencing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010766965.7A CN111855984A (en) | 2020-08-03 | 2020-08-03 | Method for evaluating biological safety by using intestinal flora of mice |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010766965.7A CN111855984A (en) | 2020-08-03 | 2020-08-03 | Method for evaluating biological safety by using intestinal flora of mice |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111855984A true CN111855984A (en) | 2020-10-30 |
Family
ID=72952675
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010766965.7A Pending CN111855984A (en) | 2020-08-03 | 2020-08-03 | Method for evaluating biological safety by using intestinal flora of mice |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111855984A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115316343A (en) * | 2022-09-09 | 2022-11-11 | 复旦大学附属中山医院 | Kit for constructing humanized flora mouse model |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1603796A (en) * | 2004-11-04 | 2005-04-06 | 上海交通大学 | Detection method for overall structure change of intestinal flora based on molecule mark |
| CN103773887A (en) * | 2014-02-19 | 2014-05-07 | 大连大学 | Preparation method of mice intestinal flora alteration ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus sequence-based Polymerase Chain Reaction) fingerprint spectrum |
| CN103898015A (en) * | 2014-03-21 | 2014-07-02 | 大连理工大学 | Vibrio alginolyticus bacteriophage and application thereof to prevention of sea cucumber disease |
| KR20160057081A (en) * | 2014-11-13 | 2016-05-23 | 강동우 | The Change in Gut Micro-Biome in Malnourished Adolescent Mice And its Effect on Psychological Behavior |
| CN109745336A (en) * | 2019-01-28 | 2019-05-14 | 兰州大学 | Codonopsis pilosula polysaccharide is as the application for adjusting human body intestinal canal flora drug |
-
2020
- 2020-08-03 CN CN202010766965.7A patent/CN111855984A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1603796A (en) * | 2004-11-04 | 2005-04-06 | 上海交通大学 | Detection method for overall structure change of intestinal flora based on molecule mark |
| CN103773887A (en) * | 2014-02-19 | 2014-05-07 | 大连大学 | Preparation method of mice intestinal flora alteration ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus sequence-based Polymerase Chain Reaction) fingerprint spectrum |
| CN103898015A (en) * | 2014-03-21 | 2014-07-02 | 大连理工大学 | Vibrio alginolyticus bacteriophage and application thereof to prevention of sea cucumber disease |
| KR20160057081A (en) * | 2014-11-13 | 2016-05-23 | 강동우 | The Change in Gut Micro-Biome in Malnourished Adolescent Mice And its Effect on Psychological Behavior |
| CN109745336A (en) * | 2019-01-28 | 2019-05-14 | 兰州大学 | Codonopsis pilosula polysaccharide is as the application for adjusting human body intestinal canal flora drug |
Non-Patent Citations (3)
| Title |
|---|
| HE TIANLIANG 等: "Deep-Sea Hydrothermal Vent Viruses Compensate for Microbial Metabolism in Virus-Host Interactions", 《MBIO》 * |
| HSU BRYAN B 等: "Dynamic Modulation of the Gut Microbiota and Metabolome by Bacteriophages in a Mouse Model", 《CELL HOST & MICROBE》 * |
| WANG YIQIAN 等: "Genome Analysis of Deep-Sea Thermophilic Phage D6E", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115316343A (en) * | 2022-09-09 | 2022-11-11 | 复旦大学附属中山医院 | Kit for constructing humanized flora mouse model |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105154564B (en) | The unified analysis method estimated based on environment DNA technology fish DNA abundance | |
| CN103805696B (en) | A kind of microRNA molecule mark of diagnostics classes rheumatic arthritis and detection kit thereof | |
| CN104040349A (en) | Methods for monitoring responsiveness to anti-SMAD7 therapy | |
| CN104651513B (en) | A kind of detection method of gout serum miRNAs biomarkers and its expression quantity | |
| CN112094907A (en) | Peripheral red blood cell micronucleus DNA and uses thereof | |
| CN108845129B (en) | Application of biomarker of active tuberculosis diseases | |
| CN111855984A (en) | Method for evaluating biological safety by using intestinal flora of mice | |
| JP2017510248A (en) | Ruterial and its separation and culture method | |
| CN110878350B (en) | Kit for sepsis diagnosis and prognosis evaluation | |
| RU2715633C2 (en) | Method for identification in immune repertoire of cdr3 sites associated with diseases | |
| CN115803448A (en) | Micronucleus DNA from peripheral red blood cells and uses thereof | |
| CN114214456A (en) | Method for differential diagnosis of EBV infected cell subtype and application thereof | |
| Piewbang et al. | Epizootic reptilian ferlavirus infection in individual and multiple snake colonies with additional evidence of the virus in the male genital tract | |
| US20210396753A1 (en) | Blood-based signatures for diagnosis and sub-typing of inflammatory bowel disease subsets | |
| CN114686593B (en) | Exosome SmallRNA related to breast cancer and application thereof | |
| CN115011710B (en) | Intragastric flora marker related to prognosis of gastric cancer and application thereof | |
| EP2818558B1 (en) | Method for assessing the viability of viruses with lymphotropism | |
| CN105567804B (en) | Application and kit of the NR_073415.2 in sense organ transplant patient's cytomegalovirus infection | |
| CN105567805B (en) | A kind of kit of sense organ transplant patient cytomegalovirus infection | |
| US20180051252A1 (en) | Luterial and method for isolating and culturing the same | |
| CN112852962B (en) | Kit for detecting relative expression quantity of MLL-SEPT6 fusion gene | |
| CN109694912A (en) | The nucleic acid compositions and its kit and detection method of application, the detection methylation of methylation sites | |
| CN105713961B (en) | System discovery, mark and the functional study of mycobacterium tuberculosis non-coding RNA | |
| CN112852811B (en) | lncRNA molecule and application thereof | |
| Hosoki et al. | Angioimmunoblastic T-cell lymphoma developed with lymphocytic pleural effusion |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201030 |
|
| RJ01 | Rejection of invention patent application after publication |