CN109234281A - The method for improving cellular anti-oxidant capacity using human lactoferrin gene rice is turned - Google Patents
The method for improving cellular anti-oxidant capacity using human lactoferrin gene rice is turned Download PDFInfo
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- CN109234281A CN109234281A CN201811091626.2A CN201811091626A CN109234281A CN 109234281 A CN109234281 A CN 109234281A CN 201811091626 A CN201811091626 A CN 201811091626A CN 109234281 A CN109234281 A CN 109234281A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
Abstract
The present invention relates to a kind of methods for improving cellular anti-oxidant capacity using human lactoferrin gene rice, since human lactoferrin has certain Scavenging activity to DPPH, ABTS and hydroxyl radical free radical, the present invention is the following steps are included: turning human lactoferrin Protein Separation and detection, turning human lactoferrin to radicals scavenging and the identification for turning human lactoferrin to human macrophage.The solution of the present invention provides good proof for the lactoferrin anti-oxidation function turned in human lactoferrin gene rice, method in the present invention is applicable not only to human macrophage, while this method is also applied for turning human lactoferrin gene rice to the antioxidation of other cells of the mankind.
Description
Technical field
The present invention relates to technical field of bioengineering, improve more particularly to a kind of application human lactoferrin gene rice thin
The method of born of the same parents' oxidation resistance.
Background technique
Transgenosis has very big potentiality as a kind of new technology, both effectiveness and safe for GM food
Property identification be GM food development an emphasis.For turning human lactoferrin gene rice, which solve common days
Lactoferrin content is low in right dairy products and is not easy to the problem of saving, but with regard to its application method and actual functional effect and
Speech, which still needs the relevant technologies, to be explored and is identified, the technology and index system of this respect belong to GM food evaluation
Required important evidence, by taking the anti-oxidation function of lactoferrin as an example, but so far, the technology and foundation of this respect are still
Compare shortage.Hu Zhi can significantly improve the immunity of mouse with equal researchs discovery lactoferrin.But the above correlative study master
It concentrates on the lactoferrin being separated in cow's milk or turns lactoferrin, and it is free to the removing for turning human lactoferrin in rice
Base activity is but without any report.
Summary of the invention
In order to solve the above technical problems, the present invention adopts the following technical scheme:
A kind of method that utilization turns human lactoferrin gene rice raising cellular anti-oxidant function, comprising the following steps:
The separation of step (1) albumen is with reference to (Wu Jing joyous etc., 2013)[1]Method, Protein Detection: separating and purifies and turns people
Lactoferrin measures the concentration for turning human lactoferrin after isolating and purifying using SDS-PAGE method and saves backup；
Step (2): radicals scavenging: A. removes DPPH free radical: the protein solution of various concentration in step 1 is added to
It shakes up, stands in LDP, measure light absorption value, the DPPH scavenging capacity of sample is calculated by clearance rate formula；
B. ABTS is removed+Free radical: taking the protein solution in step 1, and ABTS is added+Mixed liquor is uniformly mixed, and is kept away at room temperature
Light is stood, and then measures light absorption value, using deionized water as blank control, the ABTS of sample is calculated by formula+It removes and lives
Property；
C. scavenging hydroxyl: taking the protein solution in step 1, and FeSO is added4H is added after mixing in solution2O2Solution mixes
It stands, is added after salicylic acid mixes in water-bath after even, centrifugation after having reacted takes supernatant to measure light absorption value, uses after centrifugation
Distilled water passes through the clearance rate of the hydroxy radical of sample at formula calculating as blank control group；
Step (3): preferably, in step (2) A, the formula of the DPPH scavenging capacity are as follows: clearance rate (%)=[(D
Blank-D sample)/D blank] x100, in formula: D sample is the light absorption value of sample solution；D blank is the light absorption value of blank solution.
In any of the above-described scheme preferably, in step (2) B, the ABTS+The formula of scavenging capacity are as follows: ABTS+ is clear
Except rate (%)=(sample/0.7 1-D) x100, in formula: D sample is the light absorption value of sample solution；D blank is the suction of blank solution
Light value.
In any of the above-described scheme preferably, in step (2) C, the formula of the Scavenging activity on hydroxyl free radical are as follows: remove
Rate (%)=[(D blank-D sample)/D blank] x100, in formula: D sample is the light absorption value of sample solution；D blank is that blank is molten
The light absorption value of liquid.
In any of the above-described scheme preferably, in step (3), the human macrophage is to be based on by DEME culture
37 DEG C, 5%CO2Under the conditions of cultivate, when cell grows to 80%, with 0.25% trypsase EDTA vitellophag, after passage,
The human macrophage of logarithmic growth.
In any of the above-described scheme preferably, in step (1), the SDS-PAGE method select 10% separation gel and
It 5% spacer gel and is developed the color by dying method with coomassie brilliant blue to single mark band.
In any of the above-described scheme preferably, in step (2) B, the ABTS+Mixed liquor the preparation method comprises the following steps: will
Constant volume remixes 0.0384gABTS and 0.0134g potassium peroxydisulfate to 10mL respectively, by mixed liquor avoid light place at room temperature
10~18h forms ABTS+Free radical stock solution, to the ABTS+Free radical stock solution is diluted with phosphate buffer, in 734nm
It is lower light absorption value is transferred to 700 ± 0.02 to obtain ABTS+Mixed liquor, the pre- stand-by heat at 30 DEG C.
In any of the above-described scheme preferably, in step (3), the human macrophage is changed to other cell strain systems
It is equally applicable.
In any of the above-described scheme preferably, in step (2) A, the condition for measuring light absorption value is 515nm wavelength, step
(2) in B, the condition of light absorption value is measured as 734nm wavelength, in step (2) C, the condition for measuring light absorption value is 510nm wavelength.
In any of the above-described scheme preferably, in step (3) A, the condition for measuring absorption peak is 490nm wavelength, step
Suddenly in (3) B, the condition for measuring light absorption value is 570nm wavelength.
Turning human lactoferrin has certain Scavenging activity, the solution of the present invention pair to DPPH, ABTS and hydroxyl radical free radical
Good proof is provided in turning the anti-oxidant of human lactoferrin gene rice, the method in the present invention is applicable not only to people's macrophage
Cell, while this identification method is also applied for turning human lactoferrin gene rice to the antioxidation of other cells of the mankind.
Specific embodiment
Embodiment 1
1 material
Human macrophage CBR131739 is purchased from middle section purchased from neat (Shanghai) bioengineering Co., Ltd, macrophage strain is matched
Institute's Shanghai school of life and health sciences, No. GenBank are as follows: AAA59511.1；DEME (high sugar) culture medium is purchased from Ji Tai company, fetal calf serum
It is purchased from Ji Tai company；DPPH (1,1- diphenyl -2- picryl hydrazine), ABTS [2,2- connection (the 3- ethyls-benzothiazole -6- sulphur of nitrogen-two
Acid)], PBS, penicillin, streptomysin, lipopolysaccharides, Griess kit-EDTA and 0.25% trypsase be purchased from Sigma company.
2 method of protein detection
It separates and purifies and turn human lactoferrin, turn the dense of human lactoferrin after isolating and purifying using the measurement of SDS-PAGE method
It spends and saves backup, with 10% separation gel and 5% spacer gel, electrophoresis is carried out to the protein sample being collected into, coomassie is bright
Blue decoration method colour developing protein band.
3 radicals scavengings experiment
3.1 remove DPPH free radical
Each 10ml of protein sample for choosing various concentration is added separately to shake in the LDP solution that 3ml concentration is 5mmol/L
Even, then 25 DEG C of standing 30min measure light absorption value at 515nm, and the DPPH that sample can be calculated with following formula, which is removed, to live
Property:
Clearance rate (%)=[(D blank-D sample)/D blank] x100.
In formula: D sample is the light absorption value of sample solution；D blank is the light absorption value of blank solution.
Table 1: various concentration rHLF removes the influence of DPPH to macrophage
| DPPH clearance rate (%) | RHLF concentration |
| 20.24 | 0.125 |
| 33.42 | 0.25 |
| 39 | 0.5 |
| 43 | 1 |
| 56 | 2 |
| 77.2 | 4 |
We as the concentration of rHLF is ascending, it can be seen that while being sequentially increased from table 1, in macrophage
DPPH clearance rate also increases therewith.
3.2 remove ABTS+Free radical
ABTS+The preparation of mixed liquor: isometric 7mmol/LABTS aqueous solution and 2.50mmol/L potassium peroxydisulfate is water-soluble
Liquid mixing, then 10~18h of avoid light place at room temperature, forms ABTS+Free radical stock solution.It is slow with phosphoric acid to working solution
Light absorption value is transferred to 700 ± 0.02 formation ABTS at 734nm by fliud flushing dilution+Mixed liquor, pre- stand-by heat under the conditions of 30 DEG C.
Take 0.5ml solution that 1mlABTS is added+Mixed liquor is uniformly mixed, and is protected from light at 25 DEG C after standing 30min and is detected under wavelength 734nm
Absorbance, and make blank control with deionized water.
ABTS+Clearance rate (%)=(sample/0.7 1-D) x100.D sample in formula is the light absorption value of sample solution.
Table 2: various concentration rHLF removes ABTS to macrophage+Influence
| ABTS+Clearance rate (%) | RHLF concentration |
| 36.1 | 0.125 |
| 58.23 | 0.25 |
| 60.22 | 0.5 |
| 71.34 | 1 |
| 76.52 | 2 |
| 82.12 | 4 |
We as the concentration of rHLF is ascending, it can be seen that while being sequentially increased from table 2, in macrophage
ABTS+Clearance rate also increases therewith.
3.3 scavenging hydroxyls activity
6mmol/LFeSO is added in 1mL sample liquid46mmol/LH is added after mixing in solution 1ml2O2Solution 1ml, after mixing
10min is stood, 6mmol/L salicylic acid 1ml is added.37 DEG C of water-bath 30min, 3000r/min centrifugation 10min, take after mixing
Supernatant measures light absorption value at 510nm, using distilled water as blank control group, calculates clearance rate.
Clearance rate (%)=[(D blank-D sample)/D blank] x100.
Table 3: influence of the various concentration rHLF to macrophage scavenging hydroxyl
| Scavenging action to hydroxyl free radical (%) | RHLF concentration |
| 7.13 | 0.125 |
| 11.21 | 0.25 |
| 24.33 | 0.5 |
| 35.42 | 1 |
| 39.83 | 2 |
| 41.25 | 4 |
We are, it can be seen that hydroxyl while being sequentially increased as the concentration of rHLF is ascending, in macrophage from table 3
Free radical scavenging activity also increases therewith.
Embodiment 2
1 material
Human marrow mesenchymal stem cell derives from Beijing China ox biology laboratory, culture medium are as follows: 10.0%FBS, 2mmol
L-1L-Glutamine, 100UmL-1Streptomysin, 100UmL·The α MEM culture medium of penicillin, each culture hole Goat Placenta mention
Object is taken to be added to final concentration of 0.4 μ g/mL, condition of culture are as follows: 37 DEG C, 5%CO2It is cultivated in cell incubator.Fetal calf serum purchase
Yu Jitai company；DPPH (1,1- diphenyl -2- picryl hydrazine), ABTS [2,2- connection (the 3- ethyls-benzothiazole -6- sulphur of nitrogen-two
Acid)], PBS, penicillin, streptomysin, lipopolysaccharides, Griess kit-EDTA and 0.25% trypsase be purchased from Sigma company.
2 method of protein detection
It separates and purifies and turn human lactoferrin, turn the dense of human lactoferrin after isolating and purifying using the measurement of SDS-PAGE method
It spends and saves backup, with 10% separation gel and 5% spacer gel, electrophoresis is carried out to the protein sample being collected into, coomassie is bright
Blue decoration method colour developing protein band.
3 radicals scavengings experiment
3.1 remove DPPH free radical
Each 10ml of protein sample for choosing various concentration is added separately to shake in the LDP solution that 3ml concentration is 5mmol/L
Even, then 25 DEG C of standing 30min measure light absorption value at 515nm, are control with ascorbic acid, can be calculated with following formula
The DPPH scavenging capacity of sample:
Clearance rate (%)=[(D blank-D sample)/D blank] x100.
In formula: D sample is the light absorption value of sample solution；D blank is the light absorption value of blank solution.
Table 4: influence of the various concentration rHLF to medulla mesenchyma cell clearance DPPH
| DPPH clearance rate (%) | RHLF concentration |
| 18.06 | 0.125 |
| 32.40 | 0.25 |
| 40.13 | 0.5 |
| 42.58 | 1 |
| 57.33 | 2 |
| 79.87 | 4 |
We as the concentration of rHLF is ascending, it can be seen that while being sequentially increased from table 4, medulla mesenchyma cell
In DPPH clearance rate also increase therewith.
3.2 remove ABTS+Free radical
ABTS+The preparation of mixed liquor: isometric 7mmol/LABTS aqueous solution and 245mmol/L potassium peroxydisulfate is water-soluble
Mixed liquor 10~18h of avoid light place at room temperature is formed ABTS by liquid mixing+Free radical stock solution.To working solution phosphorus
Acid buffer dilution, is transferred to 700 ± 002 for light absorption value at 734nm and obtains ABTS+Mixed liquor, the pre- stand-by heat at 30 DEG C.It takes
1mlABTS is added in 0.5ml solution+Mixed liquor is uniformly mixed, and is protected from light to measure under wavelength 734nm after standing 30min at room temperature and be inhaled
Light value, using deionized water as blank control.
ABTS+Clearance rate (%)=(sample/0.7 1-D) x100.D sample in formula is the light absorption value of sample solution.
Table 5: influence of the various concentration rHLF to medulla mesenchyma cell clearance ABTS+
| ABTS+Clearance rate (%) | RHLF concentration |
| 34.26 | 0.125 |
| 57.43 | 0.25 |
| 61.83 | 0.5 |
| 70.39 | 1 |
| 77.56 | 2 |
| 83.55 | 4 |
We as the concentration of rHLF is ascending, it can be seen that while being sequentially increased from table 5, medulla mesenchyma cell
In ABTS+Clearance rate also increases therewith.
3.3 scavenging hydroxyls activity
6mmol/LFeSO is added in 1mL sample liquid46mmol/LH is added after mixing in solution 1ml2O2Solution 1ml, after mixing
10min is stood, 6mmol/L salicylic acid 1ml is added.37 DEG C of water-bath 30min, 3000r/min centrifugation 10min, take after mixing
Supernatant measures light absorption value at 510nm, using distilled water as blank control group, calculates clearance rate.
Clearance rate (%)=[(D blank-D sample)/D blank] x100.
Table 6: influence of the various concentration rHLF to medulla mesenchyma cell clearance hydroxy radical
| Hydroxy radical (%) | RHLF concentration |
| 8.56 | 0.125 |
| 12.35 | 0.25 |
| 24.12 | 0.5 |
| 39.21 | 1 |
| 42.32 | 2 |
| 45.05 | 4 |
We as the concentration of rHLF is ascending, it can be seen that while being sequentially increased from table 6, medulla mesenchyma cell
In Scavenging action to hydroxyl free radical also increase therewith.
Embodiment 3
1 material
It is removed from liquid nitrogen primary human cardiac microvascular endothelial cells (HCMEC) cryopreservation tube, 37 DEG C of water baths melt rapidly,
It reaches in the coated culture bottle of left-handed poly-D-lysine, is put into certain suitable Endothelial cell culture base, 37 DEG C, 5% dioxy
Change and cultivated in carbon incubator, 2~3d changes liquid, and 4~5d is passed on 1 time, and with trypsin digestion cell, experiment uses the 5th generation cell；DPPH
(1,1- diphenyl -2- picryl hydrazine), ABTS [2,2- connection nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid)], PBS, penicillin, chain
Mycin, lipopolysaccharides, Griess kit-EDTA and 0.25% trypsase are purchased from Sigma company.
2 method of protein detection
It separates and purifies and turn human lactoferrin, turn the dense of human lactoferrin after isolating and purifying using the measurement of SDS-PAGE method
It spends and saves backup, with 10% separation gel and 5% spacer gel, electrophoresis is carried out to the protein sample being collected into, coomassie is bright
Blue decoration method colour developing protein band.
3 radicals scavengings experiment
3.1 remove DPPH free radical
Each 10ml of protein sample for choosing various concentration is added separately to shake in the LDP solution that 3ml concentration is 5mmol/L
Even, then 25 DEG C of standing 30min measure light absorption value at 515nm, are control with ascorbic acid, can be calculated with following formula
The DPPH scavenging capacity of sample:
Clearance rate (%)=[(D blank-D sample)/D blank] x100.
In formula: D sample is the light absorption value of sample solution；D blank is the light absorption value of blank solution.
Table 7: various concentration rHLF removes the influence of DPPH to vascular endothelial cell
| DPPH clearance rate (%) | RHLF concentration |
| 16.53 | 0.125 |
| 29.47 | 0.25 |
| 41.22 | 0.5 |
| 44.64 | 1 |
| 59.73 | 2 |
| 81.75 | 4 |
We as the concentration of rHLF is ascending, it can be seen that while being sequentially increased from table 7, in vascular endothelial cell
DPPH clearance rate also increase therewith.
3.2 remove ABTS+Free radical
ABTS+The preparation of mixed liquor: isometric 7mmol/LABTS aqueous solution and 245mmol/L potassium peroxydisulfate is water-soluble
Mixed liquor 10~18h of avoid light place at room temperature is formed ABTS by liquid mixing+Free radical stock solution.To working solution phosphorus
Acid buffer dilution, is transferred to 700 ± 002 for light absorption value at 734nm and obtains ABTS+Mixed liquor, the pre- stand-by heat at 30 DEG C.It takes
1mlABTS is added in 0.5ml solution+Mixed liquor is uniformly mixed, and is protected from light to measure under wavelength 734nm after standing 30min at room temperature and be inhaled
Light value, using deionized water as blank control.
ABTS+ clearance rate (%)=(sample/0.7 1-D) x100.D sample in formula is the light absorption value of sample solution.
Table 8: various concentration rHLF removes ABTS to vascular endothelial cell+Influence
| ABTS+Clearance rate (%) | RHLF concentration |
| 37.16 | 0.125 |
| 53.09 | 0.25 |
| 61.67 | 0.5 |
| 72.64 | 1 |
| 77.89 | 2 |
| 83.52 | 4 |
We as the concentration of rHLF is ascending, it can be seen that while being sequentially increased from table 8, in vascular endothelial cell
ABTS+Clearance rate also increases therewith.
3.3 scavenging hydroxyls activity
6mmol/LFeSO is added in 1mL sample liquid46mmol/LH is added after mixing in solution 1ml2O2Solution 1ml, after mixing
10min is stood, 6mmol/L salicylic acid 1ml is added.37 DEG C of water-bath 30min, 3000r/min centrifugation 10min, take after mixing
Supernatant measures light absorption value at 510nm, using distilled water as blank control group, calculates clearance rate.
Clearance rate (%)=[(D blank-D sample)/D blank] x100.
Table 9: influence of the various concentration rHLF to vascular endothelial cell scavenging hydroxyl
| Scavenging action to hydroxyl free radical (%) | RHLF concentration |
| 8.98 | 0.125 |
| 11.85 | 0.25 |
| 25.33 | 0.5 |
| 38.81 | 1 |
| 43.62 | 2 |
| 48.77 | 4 |
We as the concentration of rHLF is ascending, it can be seen that while being sequentially increased from table 9, in vascular endothelial cell
Scavenging action to hydroxyl free radical also increase therewith.
Embodiment described above only describe the preferred embodiments of the invention, not to model of the invention
It encloses and is defined, without departing from the spirit of the design of the present invention, those of ordinary skill in the art are to technical side of the invention
The various changes and improvements that case is made should all be fallen into the protection scope that claims of the present invention determines.
Claims (10)
1. a kind of application turns the method that human lactoferrin gene rice improves cellular anti-oxidant capacity, comprising the following steps:
Step (1): Protein Separation and detection: with saturated ammonium sulfate salt precipitating, weak cation exchange chromatographic technique and molecular sieve layer
Analysis is separated and is purified to human lactoferrin, and is measured using SDS-PAGE method and turned the dense of human lactoferrin after isolating and purifying
It spends and saves backup；
Step (2): radicals scavenging: A. removes DPPH free radical: the protein solution of various concentration in step (1) is added to
It shakes up, stands in LDP, measure light absorption value；
B. ABTS is removed+Free radical: taking the protein solution in step (1), and ABTS is added+Mixed liquor is uniformly mixed, and is protected from light at room temperature
It stands, then measures light absorption value, using deionized water as blank control, the ABTS of sample is calculated by formula+Scavenging capacity；
C. scavenging hydroxyl: taking the protein solution in step (1), and FeSO is added4H is added after mixing in solution2O2Solution mixes
After stand, be added salicylic acid, water-bath after mixing, react after centrifugation, taken after centrifugation supernatant measure light absorption value, with distill
Water passes through the clearance rate of the hydroxy radical of sample at formula calculating as blank control group.
2. application according to claim 1 turns the method that human lactoferrin gene rice improves cellular anti-oxidant capacity,
It is characterized in that, in step (2) A, the formula of the DPPH scavenging capacity are as follows: clearance rate (%)=[(D blank-D sample)/D is empty
It is white] x100, in formula: D sample is the light absorption value of sample solution；D blank is the light absorption value of blank solution.
3. application according to claim 1 turns the method that human lactoferrin gene rice improves cellular anti-oxidant capacity,
It is characterized in that, in step (2) B, the ABTS+The formula of scavenging capacity are as follows: ABTS+Clearance rate (%)=(sample/0.7 1-D)
X100, in formula: D sample is the light absorption value of sample solution；D blank is the light absorption value of blank solution.
4. identification according to claim 1 turns human lactoferrin gene rice antioxidation method, which is characterized in that step
(2) in C, the formula of the Scavenging activity on hydroxyl free radical are as follows: clearance rate (%)=[(D blank-D sample)/D blank] x100, formula
In: D sample is the light absorption value of sample solution；D blank is the light absorption value of blank solution.
5. identification according to claim 1 turns human lactoferrin gene rice antioxidation method, which is characterized in that step
(3) in, the human macrophage is to be based on 37 DEG C by DEME culture, 5%CO2Under the conditions of cultivate.
6. identification according to claim 1 turns human lactoferrin gene rice antioxidation method, which is characterized in that step
(1) in, the SDS-PAGE method selects 10% separation gel and 5% spacer gel and by dying method with coomassie brilliant blue to list
Mark band develops the color.
7. application according to claim 1 turns the method that human lactoferrin gene rice improves cellular anti-oxidant capacity,
It is characterized in that, in step (2) B, the ABTS+Mixed liquor the preparation method comprises the following steps: by 0.0384gABTS and 0.0134g persulfuric acid
Constant volume remixes potassium to 10mL respectively, by mixed liquor 10~18h of avoid light place at room temperature, forms ABTS+Free radical storage
Standby liquid, to the ABTS+Free radical stock solution is diluted with phosphate buffer, and light absorption value is transferred to 700 ± 002 at 734nm and is obtained
To ABTS+Mixed liquor, the pre- stand-by heat at 30 DEG C.
8. application according to claim 1 turns the method that human lactoferrin gene rice improves cellular anti-oxidant capacity,
It is characterized in that, in step (3), the human macrophage replaces with other cell strain systems.
9. identification according to claim 1 turns human lactoferrin gene rice antioxidation method, which is characterized in that step
(2) in A, the condition of light absorption value is measured as 515nm wavelength, in step (2) B, the condition for measuring light absorption value is 734nm wavelength, step
Suddenly in (2) C, the condition for measuring light absorption value is 510nm wavelength.
10. application according to claim 1 turns the method that human lactoferrin gene rice improves cellular anti-oxidant capacity,
It is characterized in that, in step (3) A, measures the condition of absorption peak as 490nm wavelength, in step (3) B, measure the condition of light absorption value
For 570nm wavelength.
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