CN104592372A - Preparation method of nostoc flagelliforme glycoprotein by suspension culture - Google Patents
Preparation method of nostoc flagelliforme glycoprotein by suspension culture Download PDFInfo
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Abstract
The invention discloses a preparation method of nostoc flagelliforme glycoprotein by suspension culture. The method comprises the following steps: selecting nostoc flagelliforme cell strains and carrying out suspension culture of nostoc flagelliforme; filtering to remove fronds; extracting the nostoc flagelliforme glycoprotein; coarsely purifying the nostoc flagelliforme glycoprotein; and finely purifying the nostoc flagelliforme glycoprotein. The preparation method of the nostoc flagelliforme glycoprotein by suspension culture, disclosed by the invention, is simple and convenient to operate and high in extraction rate; the extracted glycoprotein is good in quality and high in medicinal value, has a good medical healthcare efficiency, and has the activity of resisting tumors, resisting oxidation, resisting viruses and improving the immunity; the glycoprotein relates to directional homing of cells, action mechanisms of glycoproiein toxin and hormone, can be used for selectively gathering medicines at the focus, so that the medicine effect is improved, the normal tissue growth and the functions of an immune system are not affected and adverse reaction is reduced; and the nostoc flagelliforme glycoprotein has extremely high medical value.
Description
Technical field
The present invention relates to biological technical field, a kind of suspension culture is delivered vegetables the preparation method of glycoprotein specifically.
Background technology
Deliver vegetables (
nostoc flagelliforme), having another name called hair-like nostoc, is a kind of Lu Sheng marine alga with very high edibleness and pharmaceutical use that well can grow in arid area.Deliver vegetables primary growth in the surface such as calcareous soil or arid grassland, China mainly distribute northwestern arid, semiarid desert steppe area.
The nutritive value of delivering vegetables is very high, and especially the content of protein is higher, every kilogram deliver vegetables in containing 200 ~ 234.1 grams, protein, also containing the multiple mineral element such as calcium, iron and 20 various trace elements.Edible deliver vegetables to rickets, hypertension, postpartum the illness such as anemia have therapeutic action, also have defaecation diuresis, clearing heat and detoxicating, effects such as healing wound, and human body blood fat and cholesterol level can be made to reduce.
Glycoprotein is the associated proteins that a class is formed by connecting with covalent linkage by carbohydrate homopolypeptide or protein.There is enhancing immunoloregulation function, function of tumor inhibition, the aging speed of reducing blood-fat, hypoglycemic, anti-oxidant, anti-fatigue, delaying human body organ, and there is the functions such as immunization, molecule, cell recognition.Research shows, the active glucoprotein in delivering vegetables has good medicinal health care function, has antitumor, anti-oxidant, antiviral, raising immunizing power isoreactivity.At present, wild hair-like seaweed resource is deficient, limits the acquisition of nutritive ingredient of delivering vegetables under certain condition.Therefore, high real in necessary from the deliver vegetables method of glycoprotein of middle extraction of delivering vegetables of a kind of simple to operate, extraction yield is studied.
Summary of the invention
Object of the present invention is: provide a kind of simple, fast, the method for glycoprotein of delivering vegetables from the middle extraction of delivering vegetables of artificial suspension culture that extraction yield is high, improve the utility value of delivering vegetables.
The present invention is for solving the problems of the technologies described above, and the technical scheme adopted is: a kind of suspension culture is delivered vegetables the preparation method of glycoprotein, and preparation process is:
Step one, aseptically, get the hair weeds cells kind after activation, be placed in liquid nutrient medium and carry out cultivation 18 ~ 22 days, then, the nutrient solution of delivering vegetables obtained is carried out centrifugation 8 ~ 12min with the rotating speed of 4000r/min, the upper strata hair weeds cells obtained after collecting centrifugation, in the fresh liquid substratum of transposition in culturing bottle, culturing bottle being placed on temperature is in the constant temperature biochemical cultivation case of 25 DEG C, and in controlling box, intensity of illumination is 4000Lux, cultivates 28 ~ 32 days;
Step 2, adopt filter paper to filter the culture of step one, get filtrate, obtain removing the nutrient solution of frond;
Step 3, be the ratio of 9:1 by volume, getting nutrient solution that step 2 obtains and NaCl volumetric molar concentration is that the vat liquor of 0 ~ 0.25mol/L is placed in Erlenmeyer flask, after fully shaking up mixing, transposition carries out supersound process 15 ~ 25min in ultrasonic oscillation device, then, the mixed solution obtained being placed in temperature is carry out heating 1 ~ 6h in the water-bath of 30 ~ 80 DEG C, afterwards, filter mixed solution, gained filtrate is glycoprotein mixing solutions of delivering vegetables;
Step 4, the glycoprotein mixing solutions of delivering vegetables step 3 obtained are placed in rotatory evaporator to carry out reduction vaporization and concentrates, and temperature when controlling concentrating under reduced pressure is 45 ~ 55 DEG C, until liquor capacity is 1/4 of original volume, namely obtains glycoprotein sample liquid; Be the ratio of 1:4 by volume, get glycoprotein sample liquid and Sevage reagent, the mixing solutions that gel precipitation is contained in lower floor is obtained after abundant concussion mixing, the metaprotein of upper and lower two-layer intersection in removing mixing solutions, afterwards, under 4 DEG C of conditions, leave standstill 10 ~ 20h, get the upper liquid in mixing solutions, for subsequent use;
Step 5: add dehydrated alcohol in the upper liquid that points 5 times obtain to step 4, the mass concentration at every turn adding ethanol in rear mixing solutions is made to be respectively 50%, 60%, 70%, 80% and 90%, and each dehydrated alcohol add rear all need mixing solutions to be placed in 4 DEG C of conditions under staticly settle 10 ~ 15h, the lower sediment thing obtained after collecting each leaving standstill, for subsequent use after merging;
Step 6: throw out step 5 collected adopts acetone and ether respectively to wash 2 ~ 3 times respectively, then, the ratio adding 1 ~ 3mL distilled water according to every gram of throw out is redissolved, afterwards, dialysis tubing is adopted to carry out dialysis desalting process 68 ~ 76h to the solution after redissolution, then, lyophilize is carried out to the solution after process, obtains glycoprotein crude product of delivering vegetables;
Step 7, ratio according to 0.2g:5mL, what step 6 obtained delivers vegetables glycoprotein dissolving crude product in deionized water, then with the film that aperture is 0.45 μm, solution is filtered, afterwards, the filtrate obtained is splined on DEAE-52 cellulose anion exchange column and carries out separation and purification, and be the NaHCO of 0.05 mol/L, 0.1mol/L and 0.5mol/L successively by volumetric molar concentration
3solution carries out interim wash-out to exchange column, the time of each wash-out is 120min, afterwards, ultraviolet absorption method is adopted to be that the go out peak position of 280nm place to glycoprotein solution of delivering vegetables is detected at wavelength, and collect the glycoprotein solution of delivering vegetables of its first absorption peak, the second absorption peak and the 4th absorption peak respectively, afterwards, respectively lyophilize is carried out to the glycoprotein solution of delivering vegetables collected, namely obtain the glycoprotein sterling of delivering vegetables of different molecular weight.
Liquid nutrient medium in described step one is BG-11 substratum.
The NaCl solution of to be volumetric molar concentration the be 0.08mol/L of the vat liquor in described step 3.
In described step 3, the power of ultrasonic oscillation device is 500W.
Dialysis tubing used in described step 4 is 3500Da dialysis tubing.
The invention has the beneficial effects as follows:
1, the invention provides a kind of easy and simple to handle, preparation method that extraction yield is high suspension culture delivers vegetables glycoprotein, adopt suspension culture hair weeds cells, in conjunction with the obtained glycoprotein mixing solutions of delivering vegetables of the Assisted Extraction such as sonic oscillation, heating in water bath mode, then carry out the steps such as reduction vaporization, Sevage method deproteinated, membrane filtration, exchange column separation and purification wash-out and obtained glycoprotein sterling of delivering vegetables efficiently, fast.The glycoprotein quality better extracted, pharmaceutical use are high, have good medicinal health care function, have antitumor, anti-oxidant, antiviral, raising immunizing power isoreactivity.And this glycoprotein relates to, and the orientation of cell is gone back to the nest, glycoprotein toxin and mechanism of hormone, focus on focus with can making drug selectivity, improve drug effect, and do not affect the growth of healthy tissues and immune function, reduce untoward reaction, there is high medical value.
2, the invention provides a kind of suspension culture to deliver vegetables the preparation method of glycoprotein, once, and long-time leaving standstill effectively can remove free protein to Sevage method deproteinated, avoids the glycoprotein loss that deproteinated number of times too much causes; The filter membrane of 3500Da is selected in ultrafiltration, while desalination, also can remove other materials such as oligopeptide and amino acid; Use NaHCO
3the replaceable electronegative glycoprotein going out to be adsorbed by Mierocrystalline cellulose cationic groups of eluant solution, and use the NaHCO of 0.05mol/L, 0.1mol/L and 0.5mol/L respectively
3solution carries out interim wash-out, along with the rising of elute soln concentration, and HCO
3 -the pH of concentration and solution raises, and this is faster, favourable to wash-out glycoprotein.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described in detail:
In the leaching process of delivering vegetables glycoprotein: first deliver vegetables by the BG-11 substratum suspension culture of preparation, cross elimination frond, then the Nacl vat liquor of 0 ~ 0.25mol/L is configured, be mixed in Erlenmeyer flask with the nutrient solution removing frond according to the ratio of 1:9, first ultrasonic-assisted extraction 20min is carried out after shaking up, be placed in water-bath hot water extraction 1 ~ 6h at 30 DEG C ~ 80 DEG C again, last filter paper filtering, supernatant liquor is glycoprotein mixing solutions; Take Sevage method and ethanol precipitation method subsequently, with organic solvent washing, dialysis drying obtains thick sterling; For thick sterling filtering membrane, upper DEAE-52 cellulose anion exchange column carries out separation and purification, uses NaHCO
3solution carries out wash-out, and what adopt ultraviolet absorption method detection glycoprotein at 280nm place goes out peak position, lyophilize, obtains the glycoprotein sterling of delivering vegetables of different molecular weight.
Described suspension culture is delivered vegetables the preparation method of glycoprotein, comprises the following steps:
Step one, suspension culture are delivered vegetables
Cultivate in BG-11 liquid nutrient medium from picking hair weeds cells solid medium of delivering vegetables under aseptic condition, every 20d is one-period; After initial cultivation 20d, the centrifugal 10min of 4000r/min, then collecting cell is suspended in the BG-11 substratum of fresh sterile, in biochemical cultivation case, carry out enlarged culturing under 25 DEG C and 4000lx intensity of illumination condition;
Step 2, excessively elimination frond
To deliver vegetables nutrient solution through filter paper filtering, and frond is stayed on filter paper, obtains the nutrient solution removing frond;
The extraction of step 3, glycoprotein of delivering vegetables
Steps A, nutrient solution step 2 obtained and Nacl concentration are that the vat liquor of 0.08mol/L is mixed in Erlenmeyer flask in the ratio of 9:1 and shakes up;
Step B, by the mixed solution of Erlenmeyer flask in steps A at ultrasonic power be 500W condition under, carry out ultrasonic-assisted extraction 20min;
Step C, again the mixed solution in step B is placed in water-bath, lixiviate 4.27h at temperature is 72 ~ 77 DEG C;
Step D, finally use the solution after filter paper filtering step C lixiviate, the supernatant liquor obtained is glycoprotein mixing solutions of delivering vegetables;
The thick purifying of step 4, glycoprotein of delivering vegetables
Step is 1.: the glycoprotein solution decompression of delivering vegetables step 3 obtained is concentrated into 1/4th of original volume, obtain glycoprotein sample liquid, be the floating preteins that 1:4 removes in glycoprotein sample by Sevage method by glycoprotein sample liquid and Sevage reagent (Sevage reagent is mixed in the ratio of 5:1 by chloroform and propyl carbinol) ratio, deproteinated once, 4 DEG C of hold over night, abandon lower floor's floating preteins layer;
Step is 2.: collect step 1. in upper strata glycoprotein solution, add dehydrated alcohol successively, respectively when alcohol concn is 50%, 60%, 70%, 80%, 90% in 4 DEG C of precipitation 12h, the precipitation obtained under finally collecting each alcohol concn;
Step is 3.: by step 2. in precipitation acetone, the ether collected respectively wash 2 times, then redissolve to 8 ~ 12mL with distilled water, then to dialyse 72h desalination with 3500Da dialysis tubing, lyophilize, just obtains the thick sterling of glycoprotein of delivering vegetables;
Consummateization of step 5, glycoprotein of delivering vegetables
Get the thick sterling of glycoprotein of delivering vegetables that step 4 obtains to dissolve in deionized water in the ratio of mass volume ratio 0.2g:5mL, cross sample thief liquid after 0.45 μm of filter membrane, DEAE-52 cellulose anion exchange column in sample liquid is carried out separation and purification, after loading, uses 0.05 respectively, 0.1,0.5mol/LNaHCO
3carry out interim wash-out, each concentration wash-out 120min, what adopt ultraviolet absorption method detection glycoprotein at 280nm place goes out peak position, collect the first absorption peak, the second absorption peak and the 4th absorption peak containing glycoprotein fraction respectively, lyophilize, obtains the consummate product of glycoprotein of delivering vegetables of different molecular weight.
embodiment 1
Suspension culture is delivered vegetables the preparation method of glycoprotein, comprises the following steps:
Step one, suspension culture are delivered vegetables
From after picking hair weeds cells solid medium of delivering vegetables carries out cultivation 18d in BG-11 liquid nutrient medium under aseptic condition, by the nutrient solution of delivering vegetables that obtains with 4000r/min rotating speed centrifugation 8min, then collecting cell is suspended in the BG-11 substratum of fresh sterile, in biochemical cultivation case, carry out enlarged culturing 28 days under 25 DEG C and 4000lx intensity of illumination condition;
Step 2, excessively elimination frond
To deliver vegetables nutrient solution through filter paper filtering, and frond is stayed on filter paper, obtains the nutrient solution removing frond;
The extraction of step 3, glycoprotein of delivering vegetables
Steps A, nutrient solution step 2 obtained and concentration are that the vat liquor of the Nacl of 0.05mol/L is mixed in Erlenmeyer flask in the ratio of 9:1 and shakes up;
Step B, by the mixed solution of Erlenmeyer flask in steps A at ultrasonic power be 500W condition under, carry out ultrasonic-assisted extraction 15min;
Step C, again the mixed solution in step B is placed in water-bath, lixiviate 6h at temperature is 30 DEG C;
Step D, finally use the solution after filter paper filtering step C lixiviate, the supernatant liquor obtained is glycoprotein mixing solutions of delivering vegetables;
The thick purifying of step 4, glycoprotein of delivering vegetables
Step is 1.: the glycoprotein solution of delivering vegetables step 3 obtained is 45 DEG C in temperature, pressure is be evaporated to 1/4th of original volume under the condition of 1Mpa, obtain glycoprotein sample liquid, be the ratio of 1:4 by volume, get glycoprotein sample liquid and Sevage reagent (Sevage reagent is mixed in the ratio of 5:1 by chloroform and propyl carbinol), the floating preteins in removing glycoprotein sample, deproteinated once, 4 DEG C of standing 10h, abandon lower floor's floating preteins layer;
Step is 2.: collect step 1. in upper strata glycoprotein solution, point add dehydrated alcohol wherein 5 times, respectively when alcohol concn is 50%, 60%, 70%, 80%, 90% in 4 DEG C of precipitation 10h, and the lower sediment obtained under collecting 5 alcohol concn;
Step is 3.: by step 2. in precipitation acetone, the ether collected respectively wash 2 times, then redissolve to 8mL with distilled water, then to dialyse 68h desalination with 3500Da dialysis tubing, lyophilize, just obtains the thick sterling of glycoprotein of delivering vegetables;
Consummateization of step 5, glycoprotein of delivering vegetables
Get the thick sterling of glycoprotein of delivering vegetables that step 4 obtains to dissolve in deionized water in the ratio of mass volume ratio 0.2g:5mL, cross sample thief liquid after 0.45 μm of filter membrane, DEAE-52 cellulose anion exchange column in sample liquid is carried out separation and purification, after loading, uses the NaHCO of 0.05mol/L, 0.1mol/L and 0.5mol/L respectively
3carry out interim wash-out, each concentration wash-out 120min, what adopt ultraviolet absorption method detection glycoprotein at 280nm place goes out peak position, collect the first absorption peak, the second absorption peak and the 4th absorption peak containing glycoprotein fraction respectively, lyophilize, obtains the consummate product of glycoprotein of delivering vegetables of different molecular weight.
embodiment 2
Suspension culture is delivered vegetables the preparation method of glycoprotein, comprises the following steps:
Step one, suspension culture are delivered vegetables
Cultivate in BG-11 liquid nutrient medium from picking hair weeds cells solid medium of delivering vegetables under aseptic condition, every 20d is one-period; After initial cultivation 20d, by the nutrient solution of delivering vegetables that obtains with 4000r/min rotating speed centrifugation 10min, then collecting cell is suspended in the BG-11 substratum of fresh sterile, in biochemical cultivation case, carry out enlarged culturing 30 days under 25 DEG C and 4000lx intensity of illumination condition;
Step 2, excessively elimination frond
To deliver vegetables nutrient solution through filter paper filtering, and frond is stayed on filter paper, obtains the nutrient solution removing frond;
The extraction of step 3, glycoprotein of delivering vegetables
Steps A, nutrient solution step 2 obtained and concentration are that the Nacl vat liquor of 0.08mol/L is mixed in Erlenmeyer flask in the ratio of 9:1 and shakes up;
Step B, by the mixed solution of Erlenmeyer flask in steps A at ultrasonic power be 500W condition under, carry out ultrasonic-assisted extraction 20min;
Step C, again the mixed solution in step B is placed in water-bath, lixiviate 4.27h at temperature is 75 DEG C;
Step D, finally use the solution after filter paper filtering step C lixiviate, the supernatant liquor obtained is glycoprotein mixing solutions of delivering vegetables;
The thick purifying of step 4, glycoprotein of delivering vegetables
Step is 1.: the glycoprotein solution of delivering vegetables step 3 obtained is 50 DEG C in temperature, pressure is be evaporated to 1/4th of original volume under the condition of 1Mpa, obtain glycoprotein sample liquid, be the ratio of 1:4 by volume, mixed with Sevage reagent (Sevage reagent is mixed with the ratio of propyl carbinol in 5:1 by chloroform) by glycoprotein sample liquid by Sevage method, the floating preteins in removing glycoprotein sample, deproteinated once, 4 DEG C leave standstill 15 h and spend the night, abandon lower floor's floating preteins layer;
Step is 2.: collect step 1. in upper strata glycoprotein solution, point add dehydrated alcohol wherein 5 times, and respectively when alcohol concn is 50%, 60%, 70%, 80%, 90% in 4 DEG C of precipitation 12h, and the lower sediment obtained under collecting 5 alcohol concn;
Step is 3.: by step 2. in precipitation acetone, the ether collected respectively wash 2 times, then redissolve to 12mL with distilled water, then to dialyse 72h desalination with 3500Da dialysis tubing, lyophilize, just obtains the thick sterling of glycoprotein of delivering vegetables;
Consummateization of step 5, glycoprotein of delivering vegetables
Get the thick sterling of glycoprotein of delivering vegetables that step 4 obtains to dissolve in deionized water in the ratio of mass volume ratio 0.2g:5mL, cross sample thief liquid after 0.45 μm of filter membrane, DEAE-52 cellulose anion exchange column in sample liquid is carried out separation and purification, after loading, uses the NaHCO of 0.05mol/L, 0.1mol/L and 0.5mol/L respectively
3carry out interim wash-out, each concentration wash-out 120min, what adopt ultraviolet absorption method detection glycoprotein at 280nm place goes out peak position, collect the first absorption peak, the second absorption peak and the 4th absorption peak containing glycoprotein fraction respectively, lyophilize, obtains the consummate product of glycoprotein of delivering vegetables of different molecular weight.
embodiment 3
Suspension culture is delivered vegetables the preparation method of glycoprotein, comprises the following steps:
Step one, suspension culture are delivered vegetables
From after picking hair weeds cells solid medium of delivering vegetables carries out cultivation 32d in BG-11 liquid nutrient medium under aseptic condition, by the nutrient solution of delivering vegetables that obtains with 4000r/min rotating speed centrifugation 12min, then collecting cell is suspended in the BG-11 substratum of fresh sterile, in biochemical cultivation case, carry out enlarged culturing 32 days under 25 DEG C and 4000lx intensity of illumination condition;
Step 2, excessively elimination frond
To deliver vegetables nutrient solution through filter paper filtering, and frond is stayed on filter paper, obtains the nutrient solution removing frond;
The extraction of step 3, glycoprotein of delivering vegetables
Steps A, nutrient solution step 2 obtained and concentration are that the Nacl vat liquor of 0.25mol/L is mixed in Erlenmeyer flask in the ratio of 9:1 and shakes up;
Step B, by the mixed solution of Erlenmeyer flask in steps A at ultrasonic power be 500W condition under, carry out ultrasonic-assisted extraction 25min;
Step C, again the mixed solution in step B is placed in water-bath, lixiviate 1h at temperature is 80 DEG C;
Step D, finally use the solution after filter paper filtering step C lixiviate, the supernatant liquor obtained is glycoprotein mixing solutions of delivering vegetables;
The thick purifying of step 4, glycoprotein of delivering vegetables
Step is 1.: the glycoprotein solution of delivering vegetables step 3 obtained is 55 DEG C in temperature, pressure is be evaporated to 1/4th of original volume under the condition of 1Mpa, obtain glycoprotein sample liquid, be the ratio of 1:4 by volume, mixed with Sevage reagent (Sevage reagent is mixed with the ratio of propyl carbinol in 5:1 by chloroform) by glycoprotein sample liquid by Sevage method, the floating preteins in removing glycoprotein sample, deproteinated once, 4 DEG C of standing 20h spend the night, and abandon lower floor's floating preteins layer;
Step is 2.: collect step 1. in upper strata glycoprotein solution, point add dehydrated alcohol wherein 5 times, and respectively when alcohol concn is 50%, 60%, 70%, 80%, 90% in 4 DEG C of precipitation 15h, and the lower sediment obtained under collecting 5 alcohol concn;
Step is 3.: by step 2. in precipitation acetone, the ether collected respectively wash 2 times, then redissolve to 10mL with distilled water, then to dialyse 72h desalination with 3500Da dialysis tubing, lyophilize, just obtains the thick sterling of glycoprotein of delivering vegetables;
Consummateization of step 5, glycoprotein of delivering vegetables
Get the thick sterling of glycoprotein of delivering vegetables that step 4 obtains to dissolve in deionized water in the ratio of mass volume ratio 0.2g:5mL, cross sample thief liquid after 0.45 μm of filter membrane, DEAE-52 cellulose anion exchange column in sample liquid is carried out separation and purification, after loading, uses the NaHCO of 0.05mol/L, 0.1mol/L and 0.5mol/L respectively
3carry out interim wash-out, each concentration wash-out 120min, what adopt ultraviolet absorption method detection glycoprotein at 280nm place goes out peak position, collect the first absorption peak, the second absorption peak and the 4th absorption peak containing glycoprotein fraction respectively, lyophilize, obtains the consummate product of glycoprotein of delivering vegetables of different molecular weight.
Claims (5)
1. suspension culture is delivered vegetables a preparation method for glycoprotein, and it is characterized in that, preparation process is:
Step one, aseptically, get the hair weeds cells kind after activation, be placed in liquid nutrient medium and carry out cultivation 18 ~ 22 days, then, the nutrient solution of delivering vegetables obtained is carried out centrifugation 8 ~ 12min with the rotating speed of 4000r/min, the upper strata hair weeds cells obtained after collecting centrifugation, in the fresh liquid substratum of transposition in culturing bottle, culturing bottle being placed on temperature is in the constant temperature biochemical cultivation case of 25 DEG C, and in controlling box, intensity of illumination is 4000Lux, cultivates 28 ~ 32 days;
Step 2, adopt filter paper to filter the culture of step one, get filtrate, obtain removing the nutrient solution of frond;
Step 3, be the ratio of 9:1 by volume, getting nutrient solution that step 2 obtains and NaCl volumetric molar concentration is that the vat liquor of 0 ~ 0.25mol/L is placed in Erlenmeyer flask, after fully shaking up mixing, transposition carries out supersound process 15 ~ 25min in ultrasonic oscillation device, then, the mixed solution obtained being placed in temperature is carry out heating 1 ~ 6h in the water-bath of 30 ~ 80 DEG C, afterwards, filter mixed solution, gained filtrate is glycoprotein mixing solutions of delivering vegetables;
Step 4, the glycoprotein mixing solutions of delivering vegetables step 3 obtained are placed in rotatory evaporator to carry out reduction vaporization and concentrates, and temperature when controlling concentrating under reduced pressure is 45 ~ 55 DEG C, until liquor capacity is 1/4 of original volume, namely obtains glycoprotein sample liquid; Be the ratio of 1:4 by volume, get glycoprotein sample liquid and Sevage reagent, the mixing solutions that gel precipitation is contained in lower floor is obtained after abundant concussion mixing, the metaprotein of upper and lower two-layer intersection in removing mixing solutions, afterwards, under 4 DEG C of conditions, leave standstill 10 ~ 20h, get the upper liquid in mixing solutions, for subsequent use;
Step 5: add dehydrated alcohol in the upper liquid that points 5 times obtain to step 4, the mass concentration at every turn adding ethanol in rear mixing solutions is made to be respectively 50%, 60%, 70%, 80% and 90%, and each dehydrated alcohol add rear all need mixing solutions to be placed in 4 DEG C of conditions under staticly settle 10 ~ 15h, the lower sediment thing obtained after collecting each leaving standstill, for subsequent use after merging;
Step 6: throw out step 5 collected adopts acetone and ether respectively to wash 2 ~ 3 times respectively, then, the ratio adding 1 ~ 3mL distilled water according to every gram of throw out is redissolved, afterwards, dialysis tubing is adopted to carry out dialysis desalting process 68 ~ 76h to the solution after redissolution, then, lyophilize is carried out to the solution after process, obtains glycoprotein crude product of delivering vegetables;
Step 7, ratio according to 0.2g:5mL, what step 6 obtained delivers vegetables glycoprotein dissolving crude product in deionized water, then with the film that aperture is 0.45 μm, solution is filtered, afterwards, the filtrate obtained is splined on DEAE-52 cellulose anion exchange column and carries out separation and purification, and be the NaHCO of 0.05 mol/L, 0.1mol/L and 0.5mol/L successively by volumetric molar concentration
3solution carries out interim wash-out to exchange column, the time of each wash-out is 120min, afterwards, ultraviolet absorption method is adopted to be that the go out peak position of 280nm place to glycoprotein solution of delivering vegetables is detected at wavelength, and collect the glycoprotein solution of delivering vegetables of its first absorption peak, the second absorption peak and the 4th absorption peak respectively, afterwards, respectively lyophilize is carried out to the glycoprotein solution of delivering vegetables collected, namely obtain the glycoprotein sterling of delivering vegetables of different molecular weight.
2. a kind of suspension culture according to claim 1 is delivered vegetables the preparation method of glycoprotein, it is characterized in that: the liquid nutrient medium in described step one is BG-11 substratum.
3. a kind of suspension culture according to claim 1 is delivered vegetables the preparation method of glycoprotein, it is characterized in that: the NaCl solution of to be volumetric molar concentration the be 0.08mol/L of the vat liquor in described step 3.
4. a kind of suspension culture according to claim 1 is delivered vegetables the preparation method of glycoprotein, it is characterized in that: in described step 3, the power of ultrasonic oscillation device is 500W.
5. a kind of suspension culture according to claim 1 is delivered vegetables the preparation method of glycoprotein, it is characterized in that: dialysis tubing used in described step 4 is 3500Da dialysis tubing.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113397032A (en) * | 2021-05-24 | 2021-09-17 | 江苏省农业科学院 | Nutrient delivery system based on burdock glycoprotein and preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710280A (en) * | 2013-11-12 | 2014-04-09 | 天津科技大学 | Large-scale cultivation method of nostoc flagelliforme cells |
| CN103923201A (en) * | 2014-04-01 | 2014-07-16 | 宁波大学 | Preparation method of hippocampus active glycoprotein |
| CN103976411A (en) * | 2014-04-30 | 2014-08-13 | 河南科技大学 | Manufacturing method of nostoc flagelliforme product |
| CN104119427A (en) * | 2013-04-24 | 2014-10-29 | 启东盖天力药业有限公司 | Trametes robiniophia Murr. proteoglycan protein, and preparation method and use thereof |
| CN104120057A (en) * | 2014-06-30 | 2014-10-29 | 河南科技大学 | Nostoc flagelliforme wine and preparation method thereof |
-
2015
- 2015-01-19 CN CN201510025002.0A patent/CN104592372A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104119427A (en) * | 2013-04-24 | 2014-10-29 | 启东盖天力药业有限公司 | Trametes robiniophia Murr. proteoglycan protein, and preparation method and use thereof |
| CN103710280A (en) * | 2013-11-12 | 2014-04-09 | 天津科技大学 | Large-scale cultivation method of nostoc flagelliforme cells |
| CN103923201A (en) * | 2014-04-01 | 2014-07-16 | 宁波大学 | Preparation method of hippocampus active glycoprotein |
| CN103976411A (en) * | 2014-04-30 | 2014-08-13 | 河南科技大学 | Manufacturing method of nostoc flagelliforme product |
| CN104120057A (en) * | 2014-06-30 | 2014-10-29 | 河南科技大学 | Nostoc flagelliforme wine and preparation method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113397032A (en) * | 2021-05-24 | 2021-09-17 | 江苏省农业科学院 | Nutrient delivery system based on burdock glycoprotein and preparation method and application thereof |
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