CN1824674A - Method of extracting lacto ferritin from transgene lactoferritin paddy rice - Google Patents
Method of extracting lacto ferritin from transgene lactoferritin paddy rice Download PDFInfo
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- CN1824674A CN1824674A CN 200610050073 CN200610050073A CN1824674A CN 1824674 A CN1824674 A CN 1824674A CN 200610050073 CN200610050073 CN 200610050073 CN 200610050073 A CN200610050073 A CN 200610050073A CN 1824674 A CN1824674 A CN 1824674A
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Abstract
The present invention discloses a method for extracting lactoferrin from transgenic lactoferrin rice. Said method includes the following steps: 1), adding water or buffer solution in transgenic lactoferrin rice seed, mixing them, pulverizing to form wet powder; or directly pulverizing transgenic lactoferrin rice seed, then adding water or buffer solution to form wet powder; the buffer solution contains NaCl, whose concentration is less than 0.1 mol/L and whose pH value is 6-9, the temperature of the above-mentioned pulverization process is below 70deg.C, the material-adding ratio of water or buffer solution and transgenic lactoferrin rice seed is 1-10ml/g; and 2), mixing and shaking the above-mentioned wet powder, then making centrifugal separation for 10min, and separating precipitate and supernatant.
Description
Technical field
The present invention relates to a kind of method of from transgenosis lactoferrin paddy rice, extracting lactoferrin.
Background technology
Lactoferrin matter (LF) is one of main active substance in the breast milk.It is a kind of protein with various biological function (B.Lo ¨ nnerdal, S.Iyer, Lactoferrin (1995) Molecular structure andbiological function, Annu.Rev.Nutr.15:93-110).It not only participates in the transhipment of iron, and have antimicrobial, anti-oxidant, anticancer, regulate function such as immunity system, be considered to a kind of novel antibiotic cancer therapy drug and the food and feed additive of great exploitation potential for its.
LF can promote the germicidal action and the phagolysis of neutrophilic leukocyte or scavenger cell, and the breeding of NK cell activity and lymphocyte, neutrophilic leukocyte is had regulating effect.In the ordinary course of things, contain LF 0.30～0.5 μ g/mL in the serum; But in case infect, LF will be discharged in the blood from the activatory neutrophilic granulocyte, and discharging amount rises at ordinary times 20 times, so LF can be regarded as anti-inflammatory factors.
The LF of people and Niu has been found the effect that suppresses lipid peroxidation (Shinmoto, etc.), therefore can play the effect of antioxidant as food and feed additive.
LF also has direct sterilizing ability, but concrete sterilization mechanism is also not fully aware of at present.Reports such as Ellison, LF can adhere to bacterial membrane and make bacterium death by the permeability that changes film.Report LF such as Ellison can cause the release of lipopolysaccharides in the Gram-negative bacteria adventitia, thereby change the permeability of after birth.
LF also has mycocidal ability, studies the more of report for the mycocandida effect.These antibacteriums and fungi activity be not only by capturing the iron ion in the pathogenic agent microenvironment, but also by destroying cytolemma with its N one end in conjunction with the cell walls of bacterium and fungi, cause the leakage of entocyte to kill them.
A kind of lactoferrin that can strengthen the human body sclerotin of New Zealand scientist new discovery in milk, this discovery make milk become a kind of osteoporotic weapon of antagonism that possesses.The phenomenon explanation that the osteogenesis that the direct injection lactoferrin can cause is obviously accelerated, this method makes the fracture patient early recovery applicable to the treatment fracture.
Lactoferrin mainly extracted from the animal Ruzhong in the past, but the cost height yields poorly.People's lactoferrin for want of raw materials, mass production is impossible.But plant transgenic technology makes and utilizes the plant production lactoferrin to become possibility.Utilize the Rice Production lactoferrin that existing report (2003 J.Pediatr.Gastroenterol.Nutr.36:190-199 such as Suzuki) is arranged.But lactoferrin content is lower in the plant, has only gross weight usually below 1%.Can directly eat although contain the plant of lactoferrin, extract the high density lactoferrin and can utilize lactoferrin more widely.
Nandi etc. (2002) report extracts lactoferrin (Nabdi et al., Plant Science 163:713-722) with the PBS damping fluid of the NaCl that contains 0.35M from transgenic paddy rice seed.But so the NaCl of concentration can influence the practical application of lactoferrin.For example, the lactoferrin that will contain high density NaCl can cause the high NaCl content of product directly as foodstuff additive or the various beverages that become as preparation of raw material, does not meet actual needs.And adopt the desalination step, promptly increase the loss that cost may cause part output (lactoferrin) again.
Summary of the invention
At the deficiencies in the prior art part, the invention provides the method for from transgenosis lactoferrin paddy rice, extracting lactoferrin that a kind of cost is low, output is high.
The present invention is to realize by such technical scheme for reaching above purpose: a kind of method of extracting lactoferrin from transgenosis lactoferrin paddy rice is provided, may further comprise the steps successively:
1), in transgenosis lactoferrin rice paddy seed, add entry or damping fluid, pulverizes after the mixing, form wet powder;
Perhaps directly transgenosis lactoferrin rice paddy seed is pulverized, added entry or damping fluid after the pulverizing and mix the formation wet powder;
It is 6～9 damping fluid that above-mentioned damping fluid is concentration＜0.1mol/L of NaCl and pH value, and above-mentioned crushing process temperature all remains on below 70 ℃, and the feed ratio of water or damping fluid and transgenosis lactoferrin rice paddy seed is 1～10 a milliliter/gram.
2), above-mentioned wet powder is mixed concussion; 5000g is after centrifugal 10 minutes again, precipitation separation and supernatant solution.
As a kind of improvement of from transgenosis lactoferrin paddy rice, extracting the method for lactoferrin of the present invention: in the precipitation of gained, add earlier entry or damping fluid, mix the back and form wet powder, described damping fluid is that the concentration＜0.1mol/L of NaCl and pH value are 6～9 damping fluid, and described water or damping fluid and sedimentary feed ratio are 1～10 milliliter/gram; Repeating step 2 again) 1～3 time.
As the further improvements in methods of from transgenosis lactoferrin paddy rice, extracting lactoferrin of the present invention: mix concussion 2～3 hours step 2).
In the invention process, the contriver has made following simultaneous test:
Test 1, different N aCl concentration extracting solution are to the influence of lactoferrin extracted amount (only the concentration difference of NaCl in the damping fluid, all the other are all with reference to step of the present invention):
Get the lactoferrin rice paddy seeds of two parts of each 2 grams, respectively in the containing 0.3M NaCl and do not contain in PBS (pH7.5) damping fluid of NaCl and pulverize of 5mL, with 250 rev/mins frequency oscillations 2 hours, 5000g after centrifugal 10 minutes again, precipitation separation and supernatant solution.Lactoferrin in the supernatant solution has been carried out the SDS-PAGE analysis, and be standard protein with ox BSA protein, the lactoferrin total amount in the sample of SDS-PAGE being analyzed by the densitometric scan technology is calculated.Utilize PBS to contain 0.3M NaCl extracting solution, the extracted amount of lactoferrin is 3.1mg/mL; Utilize PBS not contain NaCl and then be 3.4mg/mL.
Test 2, the different pH of extracting solution are to the influence of lactoferrin extracted amount (only pH of buffer difference, all the other are all with reference to step of the present invention):
After the lactoferrin rice paddy seed is pulverized, add the damping fluid of the different pH of 10mL in every gram powder.With 250 rev/mins frequency oscillations 2 hours, 5000g after centrifugal 10 minutes again, precipitation separation and supernatant solution.Lactoferrin in the supernatant solution has been carried out the SDS-PAGE analysis, and be standard protein with ox BSA protein, the lactoferrin total amount in the sample of SDS-PAGE being analyzed by the densitometric scan technology is calculated, and concrete outcome sees Table 1.
The lactoferrin amount of the damping fluid gained of table 1, different pH
| Extracting solution | Lactoferrin amount (mg/mL) |
| 0.01M NaAc(pH4.0) | 0.14 |
| 0.01M NaAc(pH5.0) | 0.22 |
| 0.01M NaAc(pH6.0) | 0.29 |
| 0.01M Tris-HCl(pH7.0) | 0.35 |
| 0.01M Tris-HCl(pH8.0) | 0.30 |
| 0.01M ris-HCl(pH9.0) | 0.31 |
Learn that from above-mentioned data the appropriate pH value of extracting solution is 6～9.
With the method for from transgenosis lactoferrin paddy rice, extracting lactoferrin of the present invention, can make lactoferrin concentration account for more than 40% of gross protein (being supernatant solution); And can guarantee does not have salt or only contains low concentration of salt in the final lactoferrin that extracts.Therefore the lactoferrin that extracts with the inventive method is applicable to synthetic beverage, makes the lactoferrin that contains activated vegetalitas source in the beverage; Also be applicable to and be used as foodstuff additive or fodder additives, can play the effect of strengthening functional food performance and feeding quality.Utilize method of the present invention, can from transgenic paddy rice seed, extract lactoferrin more quickly and easily, thereby reduce production costs; Also owing to need not or reduce the usage quantity of salt in a large number, therefore can save the increase of the cost that desalination causes and the loss of output.
Description of drawings
Fig. 1 is the gel electrophoresis of protein figure that the present invention extracts the lactoferrin that obtains.
Embodiment
In following examples the molecular biology that uses and biochemical method be known technology, the documents of publishing in the John Wiley and Sons company that Ausubel writes such as Current Protocols in MolecularBiology all have detailed explanation.
The acquisition of the transgenic paddy rice seed of embodiment 1, expression lactoferrin:
It is synthetic that human lactoferrin plasmagene (hLF) is given birth to the worker by Shanghai, and promotor is a paddy rice Gt promotor.Gt and hLF are cloned among the plasmid pCAM1300 and obtain pCAM1300-GT-hLF.PCAM1300-GT-hLF changes the rice varieties show water 11 and the Taibei 309 over to by Agrobacterium.HLF can be expressed in rice paddy seed by promotor Gt1 control.In 40 transgenic lines (being 2801～2840) that obtain, two transgenic lines wherein, lactoferrin 2824 and lactoferrin 2831, lactoferrin that can the expression ratio higher level is used as material of the present invention.
Certainly, all transgenosis lactoferrin rice paddy seeds all can be used as material used in the present invention.
The extraction of human lactoferrin matter in embodiment 2, the rice paddy seed is carried out following steps: (water of indication is meant the water that does not contain NaCl in the present embodiment) successively
Method A:
Step 1, in the rice paddy seed of embodiment 1 gained, add entry or damping fluid, pulverize after the mixing, form wet powder; Perhaps directly the rice paddy seed of embodiment 1 gained is pulverized, added entry or damping fluid after the pulverizing and mix the formation wet powder; It is 6～7.5 damping fluid that used damping fluid is concentration＜0.1mol/L of NaCl and pH value, and above-mentioned crushing process temperature all remains on below 70 ℃.The feed ratio of water or damping fluid and this rice paddy seed is 1 a milliliter/gram.
Step 2, with above-mentioned wet powder with 250 rev/mins frequency compounding concussion 2～3 hours, 5000g after centrifugal 10 minutes again, precipitation separation and supernatant solution.
Step 3, the precipitation of collecting the above-mentioned steps gained, in the precipitation of gained, add earlier entry or damping fluid, mix the back and form wet powder, described damping fluid is that the concentration＜0.1mol/L of NaCl and pH value are 6～7.5 damping fluid, water or damping fluid and sedimentary feed ratio be 5 milliliters/restrain; Repeating step 2 again) 1～3 time.
Collect all supernatant solution and be gross protein.
Method B:
The feed ratio of water or damping fluid and this rice paddy seed changes 5 milliliters/gram in the step 1; Water or damping fluid and this sedimentary feed ratio change 1 milliliter/gram in the step 3; All the other are all with method A.Collect all supernatant solution and be gross protein.
Method C:
The feed ratio of water or damping fluid and this rice paddy seed changes 10 milliliters/gram in the step 1; Water or damping fluid and this sedimentary feed ratio change 10 milliliters/gram in the step 3; All the other are all with method A.Collect all supernatant solution and be gross protein.
Adopt aforesaid method, the gross protein weight of gained be lactoferrin more than 40%.
Embodiment 3, lactoferrin content and purity check:
The lactoferrin that extracts can be analyzed content and purity with SDS-PAG.According to the universal method of SDS-PAGE (the R James Kingston is etc. hard-cover molecular biology experiment guide [M] for Ao Sibai, F Brunt. Beijing: Science Press, 1998,334-338).The lactoferrin sample that embodiment 2 extracts is analyzed.Major protein band on the Western analysis revealed SDS-PAGE glue is a lactoferrin.The densitometric scan analysis finds that it accounts for 40～60% of total protein content, specifically as shown in Figure 1:
When selecting for use lactoferrin 2824 to carry out embodiment 2, the lactoferrin that 1 representative is extracted from lactoferrin paddy rice 2824;
When selecting for use lactoferrin 2831 to carry out embodiment 2, the lactoferrin that 2 representatives are extracted from lactoferrin paddy rice 2831;
3,4,5 representatives is negative control;
When selecting for use lactoferrin 2803 to carry out embodiment 2, the lactoferrin that 6 representatives are extracted from lactoferrin paddy rice 2803;
7 the representative be standard protein.
The application that embodiment 4, lactoferrin extract product:
The lactoferrin of said extracted can be used as foodstuff additive or fodder additives, can strengthen the performance and the quality of the fodder of functional food; Can also become various beverages as preparation of raw material.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (3)
1, a kind of method of extracting lactoferrin from transgenosis lactoferrin paddy rice is characterized in that may further comprise the steps:
1), in transgenosis lactoferrin rice paddy seed, add entry or damping fluid, pulverizes after the mixing, form wet powder;
Perhaps directly transgenosis lactoferrin rice paddy seed is pulverized, added entry or damping fluid after the pulverizing and mix the formation wet powder;
It is 6～9 damping fluid that described damping fluid is concentration＜0.1mol/L of NaCl and pH value, and above-mentioned crushing process temperature all remains on below 70 ℃, and the feed ratio of described water or damping fluid and transgenosis lactoferrin rice paddy seed is 1～10 a milliliter/gram;
2), above-mentioned wet powder is mixed concussion, 5000g after centrifugal 10 minutes again, precipitation separation and supernatant solution.
2, the method for from transgenosis lactoferrin paddy rice, extracting lactoferrin according to claim 1, it is characterized in that: in the precipitation of gained, add earlier entry or damping fluid, mix the back and form wet powder, described damping fluid is that the concentration＜0.1mol/L of NaCl and pH value are 6～9 damping fluid, and described water or damping fluid and sedimentary feed ratio are 1～10 milliliter/gram; Repeating step 2 again) 1～3 time.
3, the method for extracting lactoferrin from transgenosis lactoferrin paddy rice according to claim 1 and 2 is characterized in that: mix concussion 2～3 hours described step 2).
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104109204A (en) * | 2013-04-16 | 2014-10-22 | 武汉禾元生物科技有限公司 | Method for separating and purifying recombinant human lactoferrins from paddy rice seeds |
| CN105238858A (en) * | 2015-09-25 | 2016-01-13 | 浙江大学 | Method for specificity identification and flanking sequence detection of transgenic recombinant human lactoferrin rice strain G281 |
| WO2020088207A1 (en) * | 2018-11-02 | 2020-05-07 | 武汉禾元生物科技股份有限公司 | Method for separating and purifying low-iron saturation recombinant human lactoferrin from genetically engineered rice seeds |
-
2006
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104109204A (en) * | 2013-04-16 | 2014-10-22 | 武汉禾元生物科技有限公司 | Method for separating and purifying recombinant human lactoferrins from paddy rice seeds |
| WO2014169760A1 (en) * | 2013-04-16 | 2014-10-23 | 武汉禾元生物科技有限公司 | Method for separating and purifying recombined human lactoferrin from rice seeds |
| CN104109204B (en) * | 2013-04-16 | 2017-11-07 | 武汉禾元生物科技股份有限公司 | A kind of method that restructuring lactoferrin is isolated and purified from rice paddy seed |
| CN105238858A (en) * | 2015-09-25 | 2016-01-13 | 浙江大学 | Method for specificity identification and flanking sequence detection of transgenic recombinant human lactoferrin rice strain G281 |
| CN105238858B (en) * | 2015-09-25 | 2019-02-05 | 浙江大学 | It is a kind of for detecting the flanking sequence and its specificity identification method that turn restructuring lactoferrin rice strain G281 |
| WO2020088207A1 (en) * | 2018-11-02 | 2020-05-07 | 武汉禾元生物科技股份有限公司 | Method for separating and purifying low-iron saturation recombinant human lactoferrin from genetically engineered rice seeds |
| CN111138524A (en) * | 2018-11-02 | 2020-05-12 | 武汉禾元生物科技股份有限公司 | Method for separating and purifying low-iron saturation recombinant human lactoferrin from genetically engineered rice seeds |
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