CN1212332C - Preparation method of ribonucleic acid - Google Patents
Preparation method of ribonucleic acid Download PDFInfo
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- CN1212332C CN1212332C CN 03137647 CN03137647A CN1212332C CN 1212332 C CN1212332 C CN 1212332C CN 03137647 CN03137647 CN 03137647 CN 03137647 A CN03137647 A CN 03137647A CN 1212332 C CN1212332 C CN 1212332C
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Abstract
The present invention relates to a preparing method of ribonucleic acid. The the prior art is improved in multiple aspects by the method. Consequently, the production efficiency of RNA is improved, the quality and the yield of products can be ensured, the production cost is reduced obviously, and the economic benefit is improved.
Description
[invention field]
The present invention relates to a kind of preparation method of Yeast Nucleic Acid.
[background technology]
At present, the preparation method of existing multiple Yeast Nucleic Acid (RNA) is comprising ethanol precipitation, Guanidinium hydrochloride and stain remover partition method, phenol method and industrial yeast method.Generally be the liver organization starting material of mouse, sheep, pig, ox, tortoise to be smashed to pieces make homogenate, at first remove DNA and nuclear material, then under suitable acidity condition, RNA and protein separation with centrifugation.Be further purified RNA at last.
Write at Li Liangtao, Li Mingye, " up-to-date biochemical drug technology of preparing ", the general preparation method of RNA has described in branch press of Chinese Medicine section in 3 months calendar year 2001s, also described the manufacture method of liver kytoplasm RNA, comprising homogenate; Extract, remove albumen; Precipitate, remove glycogen; Complexing, washing; Steps such as preparation.
In " animal biochemical pharmacy ", internal organs biochemical pharmacy information center of the Ministry of Commerce writes at the station, the People's Health Publisher, and the 192-195 page or leaf discloses pork liver kytoplasm RNA extracting method in publishing in June, 1980, i.e. sodium laurylsulfonate (SDS)/phenol/chloroform method.This method is summarized as follows:
1, get fresh liver, clean with 0.9%NaCl solution, mincer rubs, and adds the damping fluid of SSC pH=7, and add-on 1: 1.6 by weight, temperature are no more than 15 ℃, and last colloid barreling grinds evenly, and centrifugal again 30 minutes, natural filtration obtained supernatant liquor then.
2. described supernatant liquor added SDS pH=9.0 solution in 1: 1 by volume, stirred 15 minutes, added 90% phenol of this supernatant liquor volume 1/2 again, stirred 15 minutes, added the chloroform of this supernatant liquor volume 1/2 again, stirred the centrifugal then supernatant liquor that obtains 15 minutes.
3. get the supernatant liquor that preceding step obtains and add its volume 1/3 phenol, stirred 15 minutes, add its volume 1/3 chloroform again, stirred 15 minutes, the centrifuging and taking supernatant liquor adds the equal-volume chloroform toward supernatant liquor, stirs the centrifugal supernatant liquor that obtains.
4. get the supernatant liquor that preceding step obtains, add the 20%Kac of its volume 1/10, add 95% ethanol again, make alcohol concn reach 70%, precipitation is no more than 20 ℃, sedimentation time 12 hours to 5 days.
5. the throw out that obtains of preceding step after weighing, with the dissolving of 0.1MNaAc solution, adds the phenol of dissolving liquid long-pending 1/2 through centrifugal again, stirred 15 minutes, and added and the isopyknic chloroform of phenol, stir the centrifuging and taking supernatant liquor, supernatant liquor adds the equal-volume chloroform, stirs, the centrifugation supernatant liquor again.
6. getting the preceding step supernatant liquor, to add its volume ratio be 1: 1 2.5M K
2HPO
4It is 1: 1 ethylene glycol monomethyl ether that solution, centrifuging and taking supernatant liquor, supernatant liquor add its volume ratio, stirs the centrifugal supernatant liquor that obtains.
7. the supernatant liquor of getting preceding step adds and send the freezer precipitation more than 12 hours after equal-volume 0.2MNaAc solution and 1/2 volume 1%CTAB solution stir a little, and centrifugal again abandoning supernatant must precipitate.
8. the precipitation of getting preceding step adds ethanol again and makes alcohol concn reach 70% with 0.1M NaAc dissolving, stirs the centrifuging and taking precipitation.Answer triplicate as this step 1.
9. the precipitated rna of washes clean is weighed, seal, send freezer or refrigerator to preserve after the sealing with a small amount of dehydrated alcohol.
But there is following defective in the production method of this RNA:
A. the production cycle long, the every batch of product needs three day time just can finish; B. production efficiency is low, and can only feed intake 20 kilograms of bright livers of per tour do not adapt to the demand of producing and selling far away under the general condition; C. the organic solvent consumption is big, and phenol, chloroform consumption are big, has both increased the cost of product, is unfavorable for environmental protection again, and the yield of phenol, chloroform increases, the also corresponding raising of the degree of pollution; D. labour intensity is big: because reagent dosage is many, cause the operational volume in the production process to increase the also corresponding enhancing of its labour intensity.Therefore, still need at present a kind of can the production efficiency height, production cost is low, helps environment, the economical and practical RNA production method that helps operating.
[summary of the invention]
[problem that invention will solve]
The objective of the invention is to propose a kind of production method of Yeast Nucleic Acid,, can also guarantee the quality and the yield of product, reduce cost, increase economic efficiency so that when improving RNA production efficiency.
[technical scheme]
The inventor develops the production method of a kind of Yeast Nucleic Acid that satisfies the object of the invention through number of research projects.
A kind of production method of Yeast Nucleic Acid is characterized in that this method comprises the steps:
1. homogenate: get 100 weight part animal liver, wash, drain, rub pork liver with mincer, pork liver and SSC (pH7.0) solution is in volume/weight in proportion 1: 0.8-2.2 mixes, colloidal mill homogenate, supernatant liquor is got in the feed liquid centrifugation, filters the filtrate that obtains the 70-210 parts by volume;
2. get the filtrate of step (1), add 7-21 parts by volume SDS solution, stir, add 23-100 parts by volume 80-90% phenol solution again, restir adds 23-100 parts by volume chloroform again, stirs, and supernatant liquor is reclaimed in the feed liquid centrifugation;
3. get the supernatant liquor of step (2), add 5-20 parts by volume 20% potassium acetate solution, mixing adds 80-420 parts by volume ethanol again, to alcohol concn be 70%, mixing, natural subsidence, centrifugation obtain throw out;
4. with the throw out of 12 parts by volume sodium acetates redissolution liquid dissolving steps (3), add 2-11 parts by volume 80% phenol again, stir, add 2-11 parts by volume chloroform again, stir, centrifugation is again reclaimed and is got supernatant liquor;
5. get the supernatant liquor of the step (4) of 18 parts by volume, add 1.8-12 parts by volume 80% phenol, stir, add 1.8-12 parts by volume chloroform again, stir, centrifugation is reclaimed and is got supernatant liquor;
6. get step (5) supernatant liquor, add 2-14 parts by volume chloroform, stir, centrifugation is reclaimed and is got supernatant liquor;
7. toward the supernatant liquor of step (6), adding 95% ethanol to alcohol concn is 70%, mixing, and natural sedimentation, the supernatant liquor that inclines, centrifugation gets the RNA crude product;
8. with 13 parts by volume K
2HPO
4Solution dissolves above-mentioned RNA crude product and obtains lysate, adds the 5-10 ethylene glycol monomethyl ether then, the centrifugal supernatant liquor that obtains;
9. get step (8) supernatant liquor, add 5-8 parts by volume sodium acetate solution again, mixing, and then add 2-4.5 parts by volume adding CTAB solution, and mixing, precipitation, centrifugal, obtain throw out;
10. get the throw out of step (9), with the sodium acetate solution dissolving of 10 parts by volume, adding 95% ethanol to concentration again is 70%, abandoning supernatant, and precipitation and centrifugal separation repeats this step 3-4 time, obtains RNA product of the present invention like this.
Described SSC solution (pH=7.0) is after adding purified water to 1 liter with 5.85 gram sodium-chlor, 14.7 gram Trisodium Citrates, 5 gram bentonite, is adjusted to Glacial acetic acid that pH=7.0 prepares again.
Described SDS solution (pH=9.0) is after adding pure water to 1 liter with 50 gram SDS, 133 milliliters of TEA, 4.0 gram EDTA, is adjusted to Glacial acetic acid that pH=9.0 prepares again.
Described 80% phenol solution is dissolved to 800 milliliters of phenol and 1 gram oxine 1 liter with pure water and obtains.
Described 20% potassium acetate solution obtains with 1 liter of pure water dissolving, 200 gram potassium acetates.
It is after restraining EDTA and add purified water to 1 liter with 13.6 gram sodium acetates, 5.0 gram SDS, 13.3 milliliters of TEA, 0.8 that described sodium acetate redissolves liquid, is adjusted to Glacial acetic acid that pH=7.0 prepares again.
Described sodium acetate solution is after adding purified water to 1 liter with 13.6 gram sodium acetates, 0.8 gram EDTA, is adjusted to Glacial acetic acid that pH=7.0 prepares again, and this solution is cooled to room temperature 121 ℃ of sterilizations 30 minutes down.
Described K
2HPO
4Solution is with water for injection dissolving 296.7 gram K
2HPO
4.H
2O and 0.8 gram EDTA are adjusted to phosphoric acid that pH=8.0 prepares again.
Described CTBA solution is with water for injection dissolving 30 gram CTBA, and water transfers to 1 liter and prepares again.
The method according to this invention, described employing centrifugation are under rotating speed 1000-3000 rev/min condition centrifugal 10-30 minute.
Preferably, used bright liver is healthy, removes the liver of pork liver, beef liver and other animal thereof of fatty liver muscle.It more preferably is pork liver.
Preferably bright liver is cleaned with 0.9% sodium chloride solution or pure water.
[beneficial effect]
The present invention has done many improvement to prior art:
After removing albumen at first for the first time, increased alcohol precipitation one time, made volume-diminished when removing albumen for the second time nearly 20 times.Preferably, in the homogenate process, reduced the SSC add-on, by being reduced to 1: 1.2 (by volume) in original 1: 1.6.The add-on of SDS was reduced to 1: 1/10 (by volume) by original 1: 1 when removing albumen for the first time, but the total amount of its SDS does not become.
Remove albumen for five times and change four times into and remove albumen by existing the employing, and in will make with extra care in the past twice removes albumen and be advanced in the raw product purifying of clean room between this helps making with extra care.
Shortened the time of alcohol precipitation, be no less than 12 hours, be reduced to about 2 hours, shortened the treatment time so greatly from prior art is sedimentary.
Compared with prior art, consumption of ethanol reduces by 40%.
Directly use K when removing glycogen
2HPO
4The dissolving with 0.1MNaAc (0.15L/ kilogram) has been removed in dissolving, and K
2HPO
4Concentration changes to 1.3M by original 2.5M, and the consumption of ethylene glycol monomethyl ether also was reduced to 1: 1/2 (by volume) by original 1: 1 simultaneously.Add 1%CTAB solution in proportion at 1: 1/2 by prior art during complexing, be improved to 1: 1/4 3%CTAB of adding, the concentration of NaAc solution also is improved to 0.33M by the 0.2M of prior art simultaneously.The complex-precipitation time was improved to more than 12 hours 30 minutes by prior art.In a word, these improvement cause the production cycle to shorten, and foreshorten to 2 days by 3 days, and the consumption of organic solvent reduces significantly, and phenol drops to 25% of prior art consumption; The chloroform consumption drops to 15%; The alcohol consumption descends 20～30%.Increase output, charging capacity rises to 165 kilograms from 20 kilograms of prior art every days, so production cost obviously reduces.
[embodiment]
Embodiment 1
Get health, remove the bright liver double centner of fatty liver muscle, clean with 0.9% sodium chloride solution, rub with mincer, add 120 liters of SSC solution (pH=7.0), temperature is no more than 15 ℃, stirs, last colloidal mill homogenate, homogenate adopts whizzer (2890 rev/mins) centrifugation 30 minutes, and centrifugal back obtains 120 liters of supernatant liquors with 100 order nylon yarn natural filtrations.
Then, in described supernatant liquor, add 12 intensification degree and be no more than 15 ℃, the SDS solution of pH=9.0, cumulative volume reaches 132 liters, stir, add 44 liter of 80% phenol (temperature is no more than 30 ℃) again, stir after 15 minutes, add 44 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, get 120 liters of supernatant liquors.
The supernatant liquor of step adds 12 liter of 20% liquor kalii acetici forward, add 400 liter of 95% ethanolic soln then, make that alcohol concn reaches 70% in this solution, after letting alone natural subsidence, the lower sediment thing obtains throw out with whizzer (2890 rev/mins) centrifugation 20 minutes.
Then, with 12 liters of described sodium acetates redissolution liquid dissolution precipitation things, get 15.6 liters of lysates, in this lysate, add 5.2 liter of 80% phenol again, stirred 5 minutes, add 5.2 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, obtain 15 liters of supernatant liquors.
Then, what step obtained in front 15 goes up in the clear liquid and to add 5 liter of 80% phenol, stirs 15 minutes, adds 5 liters of chloroforms again, stirs 15 minutes, adopts whizzer (2890 rev/mins) centrifugation 20 minutes then, obtains 14 liters of supernatant liquors.
Add 7 liters of chloroforms again in the supernatant liquor that step obtains in front, stirred 5 minutes, adopt whizzer (2890 rev/mins) centrifugation 15 minutes then, reclaim supernatant liquor, in the supernatant liquor that reclaims, add 95% ethanol, reach 70%, let alone the solution sedimentation until alcohol concn, lower floor's material after the sedimentation obtains rough RNA like this with whizzer (2890 rev/mins) centrifugation 20 minutes.
Then, use 13 liters of dipotassium hydrogen phosphate solutions to dissolve described crude rna, obtain 16 liters of lysates, add 8 liters of ethylene glycol monomethyl ether in lysate, adopt whizzer (1400 rev/mins) centrifugation 30 minutes then, separation obtains 23 and goes up clear liquid.Add 12 liters of 0.33M sodium acetate solutions toward this supernatant liquor again, add 8 liters of 3%CTAB solution again, after the natural subsidence, centrifugation goes out throw out after stirring a little.
The 0.1M sodium acetate solution of following with 10 liters is dissolved in the resulting precipitation of preceding step, after the stirring and dissolving, adds 95% ethanolic soln again, make alcohol concn reach 70%, let alone the solution sedimentation, lower floor's material adopts whizzer centrifugation, this step triplicate then.Obtain pure rna product of the present invention like this.
RNA total amount (calculating): 150.2 grams with RNA
Quality product: the Yeast Nucleic Acid quality standard that meets National Drug Administration's promulgation.
Embodiment 2
Get health, remove the bright liver double centner of fatty liver muscle, clean with 0.9% sodium chloride solution, rub with mincer, add 100 liters of SSC solution (pH=7.0), temperature is no more than 15 ℃, stirs, last colloidal mill homogenate, homogenate adopts whizzer (2890 rev/mins) centrifugation 30 minutes, and centrifugal back obtains 90 liters of supernatant liquors with 100 order nylon yarn natural filtrations.
Then, in described supernatant liquor, add 9 intensification degree and be no more than 15 ℃, the SDS solution of pH=9.0, cumulative volume reaches 100 liters, stir, add 33 liter of 80% phenol (temperature is no more than 30 ℃) again, stir after 15 minutes, add 33 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, get 80 liters of supernatant liquors.
The supernatant liquor of step adds 8 liter of 20% liquor kalii acetici forward, add 190 liter of 95% ethanolic soln then, make that alcohol concn reaches 70% in this solution, after letting alone natural subsidence, the lower sediment thing obtains throw out with whizzer (2890 rev/mins) centrifugation 30 minutes.
Then, with 12 liters of described sodium acetates redissolution liquid dissolution precipitation things, get 13 liters of lysates, in this lysate, add 4.3 liter of 80% phenol and 4.3 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, obtain 15 liters of supernatant liquors.
Then, what step obtained in front 18 goes up in the clear liquid and to add 5 liter of 80% phenol, stirs 15 minutes, adds 5 liters of chloroforms again, stirs 15 minutes, adopts whizzer (2890 rev/mins) centrifugation 10 minutes then, obtains 14 liters of supernatant liquors.
Add 7 liters of chloroforms again in the supernatant liquor that step obtains in front, stirred 5 minutes, adopt whizzer (2890 rev/mins) centrifugation 10 minutes then, reclaim supernatant liquor, in the supernatant liquor that reclaims, add 95% ethanol, reach 70%, let alone the solution sedimentation until alcohol concn, lower floor's material after the sedimentation obtains rough RNA like this with whizzer (2890 rev/mins) centrifugation 10 minutes.
Then, use 13 liters of dipotassium hydrogen phosphate solutions to dissolve described crude rna, obtain 16 liters of lysates, add 8 liters of ethylene glycol monomethyl ether in lysate, adopt whizzer (1400 rev/mins) centrifugation 10 minutes then, separation obtains 14 and goes up clear liquid.Add 7 liters of 0.33M sodium acetate solutions toward this supernatant liquor again, add 3.5 liters of 3%CTAB solution again, after the natural subsidence, centrifugation goes out throw out after stirring a little.
The 0.1M sodium acetate solution of following with 10 liters is dissolved in the resulting precipitation of preceding step, after the stirring and dissolving, adds 95% ethanolic soln again, make alcohol concn reach 70%, let alone the solution sedimentation, lower floor's material adopts whizzer centrifugation, this step triplicate then.Obtain pure rna product of the present invention like this.
RNA total amount (calculating): 150.6 grams with RNA
Quality product: the Yeast Nucleic Acid quality standard that meets National Drug Administration's promulgation.
Embodiment 3
Get health, remove the bright liver double centner of fatty liver muscle, clean with 0.9% sodium chloride solution, rub with mincer, add 140 liters of SSC solution (pH=7.0), temperature is no more than 15 ℃, stirs, last colloidal mill homogenate, homogenate adopts whizzer (2890 rev/mins) centrifugation 30 minutes, and centrifugal back obtains 130 liters of supernatant liquors with 100 order nylon yarn natural filtrations.
Then, in described supernatant liquor, add 13 intensification degree and be no more than 15 ℃, the SDS solution of pH=9.0, cumulative volume reaches 140 liters, stir, add 47 liter of 80% phenol (temperature is no more than 30 ℃) again, stir after 15 minutes, add 47 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, get 120 liters of supernatant liquors.
The supernatant liquor of step adds 12 liter of 20% liquor kalii acetici forward, add 290 liter of 95% ethanolic soln then, make that alcohol concn reaches 70% in this solution, after letting alone natural subsidence, the lower sediment thing obtains throw out with whizzer (2890 rev/mins) centrifugation 30 minutes.
Then, with 12 liters of described sodium acetates redissolution liquid dissolution precipitation things, get 15 liters of lysates, in this lysate, add 5.0 liter of 80% phenol and 5.0 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, obtain 16 liters of supernatant liquors.
Then, what step obtained in front 18 goes up in the clear liquid and to add 5.3 liter of 80% phenol, stirs 15 minutes, adds 5.3 liters of chloroforms again, stirs 15 minutes, adopts whizzer (2890 rev/mins) centrifugation 10 minutes then, obtains 15 liters of supernatant liquors.
Add 7.5 liters of chloroforms again in the supernatant liquor that step obtains in front, stirred 5 minutes, adopt whizzer (2890 rev/mins) centrifugation 10 minutes then, reclaim supernatant liquor, in the supernatant liquor that reclaims, add 95% ethanol, reach 70%, let alone the solution sedimentation until alcohol concn, lower floor's material after the sedimentation obtains rough RNA like this with whizzer (2890 rev/mins) centrifugation 10 minutes.
Then, use 13 liters of dipotassium hydrogen phosphate solutions to dissolve described crude rna, obtain 16.5 liters of lysates, add 8 liters of ethylene glycol monomethyl ether in lysate, adopt whizzer (1400 rev/mins) centrifugation 10 minutes then, separation obtains 14 and goes up clear liquid.Add 7 liters of 0.33M sodium acetate solutions toward this supernatant liquor again, add 3.5 liters of 3%CTAB solution again, after the natural subsidence, centrifugation goes out throw out after stirring a little.
The 0.1M sodium acetate solution of following with 10 liters is dissolved in the resulting precipitation of preceding step, after the stirring and dissolving, adds 95% ethanolic soln again, make alcohol concn reach 70%, let alone the solution sedimentation, lower floor's material adopts whizzer centrifugation, this step triplicate then.Obtain pure rna product of the present invention like this.
RNA total amount (calculating): 149.8 grams with RNA
Quality product: the Yeast Nucleic Acid quality standard that meets National Drug Administration's promulgation.
Embodiment 4
Get health, remove the bright liver double centner of fatty liver muscle, clean with 0.9% sodium chloride solution, rub with mincer, add 160 liters of SSC solution (pH=7.0), temperature is no more than 15 ℃, stirs, last colloidal mill homogenate, homogenate adopts whizzer (2890 rev/mins) centrifugation 30 minutes, and centrifugal back obtains 145 liters of supernatant liquors with 100 order nylon yarn natural filtrations.
Then, in described supernatant liquor, add 14.5 intensification degree and be no more than 15 ℃, the SDS solution of pH=9.0, cumulative volume reaches 160 liters, stir, add 53 liter of 80% phenol (temperature is no more than 30 ℃) again, stir after 15 minutes, add 53 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, get 130 liters of supernatant liquors.
The supernatant liquor of step adds 13 liter of 20% liquor kalii acetici forward, add 320 liter of 95% ethanolic soln then, make that alcohol concn reaches 70% in this solution, after letting alone natural subsidence, the lower sediment thing obtains throw out with whizzer (2890 rev/mins) centrifugation 30 minutes.
Then, with 12 liters of described sodium acetates redissolution liquid dissolution precipitation things, get 15 liters of lysates, in this lysate, add 5.0 liter of 80% phenol and 5.0 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, obtain 16 liters of supernatant liquors.
Then, what step obtained in front 18 goes up in the clear liquid and to add 5.3 liter of 80% phenol, stirs 15 minutes, adds 5.3 liters of chloroforms again, stirs 15 minutes, adopts whizzer (2890 rev/mins) centrifugation 10 minutes then, obtains 15 liters of supernatant liquors.
Add 7.5 liters of chloroforms again in the supernatant liquor that step obtains in front, stirred 5 minutes, adopt whizzer (2890 rev/mins) centrifugation 10 minutes then, reclaim supernatant liquor, in the supernatant liquor that reclaims, add 95% ethanol, reach 70%, let alone the solution sedimentation until alcohol concn, lower floor's material after the sedimentation obtains rough RNA like this with whizzer (2890 rev/mins) centrifugation 10 minutes.
Then, use 13 liters of dipotassium hydrogen phosphate solutions to dissolve described crude rna, obtain 16.5 liters of lysates, add 8 liters of ethylene glycol monomethyl ether in lysate, adopt whizzer (1400 rev/mins) centrifugation 10 minutes then, separation obtains 14 and goes up clear liquid.Add 7 liters of 0.33M sodium acetate solutions toward this supernatant liquor again, add 3.5 liters of 3%CTAB solution again, after the natural subsidence, centrifugation goes out throw out after stirring a little.
The 0.1M sodium acetate solution of following with 10 liters is dissolved in the resulting precipitation of preceding step, after the stirring and dissolving, adds 95% ethanolic soln again, make alcohol concn reach 70%, let alone the solution sedimentation, lower floor's material adopts whizzer centrifugation, this step triplicate then.Obtain pure rna product of the present invention like this.
RNA total amount (calculating): 150.3 grams with RNA
Quality product: the Yeast Nucleic Acid quality standard that meets National Drug Administration's promulgation.
Embodiment 5
Get health, remove the bright liver double centner of fatty liver muscle, clean with 0.9% sodium chloride solution, rub with mincer, add 180 liters of SSC solution (pH=7.0), temperature is no more than 15 ℃, stirs, last colloidal mill homogenate, homogenate adopts whizzer (2890 rev/mins) centrifugation 30 minutes, and centrifugal back obtains 165 liters of supernatant liquors with 100 order nylon yarn natural filtrations.
Then, in described supernatant liquor, add 16.5 intensification degree and be no more than 15 ℃, the SDS solution of pH=9.0, cumulative volume reaches 185 liters, stir, add 60 liter of 80% phenol (temperature is no more than 30 ℃) again, stir after 15 minutes, add 60 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, get 140 liters of supernatant liquors.
The supernatant liquor of step adds 14 liter of 20% liquor kalii acetici forward, add 340 liter of 95% ethanolic soln then, make that alcohol concn reaches 70% in this solution, after letting alone natural subsidence, the lower sediment thing obtains throw out with whizzer (2890 rev/mins) centrifugation 30 minutes.
Then, with 12 liters of described sodium acetates redissolution liquid dissolution precipitation things, get 16 liters of lysates, in this lysate, add 5.3 liter of 80% phenol and 5.3 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, obtain 18 liters of supernatant liquors.
Then, what step obtained in front 18 goes up in the clear liquid and to add 6.0 liter of 80% phenol, stirs 15 minutes, adds 6.0 liters of chloroforms again, stirs 15 minutes, adopts whizzer (2890 rev/mins) centrifugation 10 minutes then, obtains 16 liters of supernatant liquors.
Add 8.0 liters of chloroforms again in the supernatant liquor that step obtains in front, stirred 5 minutes, adopt whizzer (2890 rev/mins) centrifugation 10 minutes then, reclaim supernatant liquor, in the supernatant liquor that reclaims, add 95% ethanol, reach 70%, let alone the solution sedimentation until alcohol concn, lower floor's material after the sedimentation obtains rough RNA like this with whizzer (2890 rev/mins) centrifugation 10 minutes.
Then, use 13 liters of dipotassium hydrogen phosphate solutions to dissolve described crude rna, obtain 16.0 liters of lysates, add 8 liters of ethylene glycol monomethyl ether in lysate, adopt whizzer (1400 rev/mins) centrifugation 10 minutes then, separation obtains 14 and goes up clear liquid.Add 7 liters of 0.33M sodium acetate solutions toward this supernatant liquor again, add 3.5 liters of 3%CTAB solution again, after the natural subsidence, centrifugation goes out throw out after stirring a little.
The 0.1M sodium acetate solution of following with 10 liters is dissolved in the resulting precipitation of preceding step, after the stirring and dissolving, adds 95% ethanolic soln again, make alcohol concn reach 70%, let alone the solution sedimentation, lower floor's material adopts whizzer centrifugation, this step triplicate then.Obtain pure rna product of the present invention like this.
RNA total amount (calculating): 149.6 grams with RNA
Quality product: the Yeast Nucleic Acid quality standard that meets National Drug Administration's promulgation.
Embodiment 6
Get health, remove the bright liver double centner of fatty liver muscle, clean with 0.9% sodium chloride solution, rub with mincer, add 200 liters of SSC solution (pH=7.0), temperature is no more than 15 ℃, stirs, last colloidal mill homogenate, homogenate adopts whizzer (2890 rev/mins) centrifugation 30 minutes, and centrifugal back obtains 190 liters of supernatant liquors with 100 order nylon yarn natural filtrations.
Then, in described supernatant liquor, add 19.0 intensification degree and be no more than 15 ℃, the SDS solution of pH=9.0, cumulative volume reaches 204 liters, stir, add 68 liter of 80% phenol (temperature is no more than 30 ℃) again, stir after 15 minutes, add 68 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, get 160 liters of supernatant liquors.
The supernatant liquor of step adds 16 liter of 20% liquor kalii acetici forward, add 400 liter of 95% ethanolic soln then, make that alcohol concn reaches 70% in this solution, after letting alone natural subsidence, the lower sediment thing obtains throw out with whizzer (2890 rev/mins) centrifugation 30 minutes.
Then, with 12 liters of described sodium acetates redissolution liquid dissolution precipitation things, get 15 liters of lysates, in this lysate, add 5.0 liter of 80% phenol and 5.0 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, obtain 16 liters of supernatant liquors.
Then, what step obtained in front 18 goes up in the clear liquid and to add 5.3 liter of 80% phenol, stirs 15 minutes, adds 5.3 liters of chloroforms again, stirs 15 minutes, adopts whizzer (2890 rev/mins) centrifugation 10 minutes then, obtains 15 liters of supernatant liquors.
Add 7.5 liters of chloroforms again in the supernatant liquor that step obtains in front, stirred 5 minutes, adopt whizzer (2890 rev/mins) centrifugation 10 minutes then, reclaim supernatant liquor, in the supernatant liquor that reclaims, add 95% ethanol, reach 70%, let alone the solution sedimentation until alcohol concn, lower floor's material after the sedimentation obtains rough RNA like this with whizzer (2890 rev/mins) centrifugation 10 minutes.
Then, use 13 liters of dipotassium hydrogen phosphate solutions to dissolve described crude rna, obtain 17.0 liters of lysates, add 8.5 liters of ethylene glycol monomethyl ether in lysate, adopt whizzer (1400 rev/mins) centrifugation 10 minutes then, separation obtains 14 and goes up clear liquid.Add 7 liters of 0.33M sodium acetate solutions toward this supernatant liquor again, add 3.5 liters of 3%CTAB solution again, after the natural subsidence, centrifugation goes out throw out after stirring a little.
The 0.1M sodium acetate solution of following with 10 liters is dissolved in the resulting precipitation of preceding step, after the stirring and dissolving, adds 95% ethanolic soln again, make alcohol concn reach 70%, let alone the solution sedimentation, lower floor's material adopts whizzer centrifugation, this step triplicate then.Obtain pure rna product of the present invention like this.
RNA total amount (calculating): 150.5 grams with RNA
Quality product: the Yeast Nucleic Acid quality standard that meets National Drug Administration's promulgation.
Embodiment 7
Get health, remove the bright liver double centner of fatty liver muscle, clean with 0.9% sodium chloride solution, rub with mincer, add 120 liters of SSC solution (pH=7.0), temperature is no more than 15 ℃, stirs, last colloidal mill homogenate, homogenate adopts whizzer (2890 rev/mins) centrifugation 30 minutes, and centrifugal back obtains 120 liters of supernatant liquors with 100 order nylon yarn natural filtrations.
Then, in described supernatant liquor, add 12.0 intensification degree and be no more than 15 ℃, the SDS solution of pH=9.0, cumulative volume reaches 132 liters, stir, add 33 liter of 80% phenol (temperature is no more than 30 ℃) again, stir after 15 minutes, add 33 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, get 95 liters of supernatant liquors.
The supernatant liquor of step adds 9.5 liter of 20% liquor kalii acetici forward, add 230 liter of 95% ethanolic soln then, make that alcohol concn reaches 70% in this solution, after letting alone natural subsidence, the lower sediment thing obtains throw out with whizzer (2890 rev/mins) centrifugation 30 minutes.
Then, with 12 liters of described sodium acetates redissolution liquid dissolution precipitation things, get 14 liters of lysates, in this lysate, add 3.5 liter of 80% phenol and 3.5 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, obtain 15 liters of supernatant liquors.
Then, what step obtained in front 18 goes up in the clear liquid and to add 3.8 liter of 80% phenol, stirs 15 minutes, adds 3.8 liters of chloroforms again, stirs 15 minutes, adopts whizzer (2890 rev/mins) centrifugation 10 minutes then, obtains 16 liters of supernatant liquors.
Add 4.0 liters of chloroforms again in the supernatant liquor that step obtains in front, stirred 5 minutes, adopt whizzer (2890 rev/mins) centrifugation 10 minutes then, reclaim supernatant liquor, in the supernatant liquor that reclaims, add 95% ethanol, reach 70%, let alone the solution sedimentation until alcohol concn, lower floor's material after the sedimentation obtains rough RNA like this with whizzer (2890 rev/mins) centrifugation 10 minutes.
Then, use 13 liters of dipotassium hydrogen phosphate solutions to dissolve described crude rna, obtain 14.0 liters of lysates, add 7.0 liters of ethylene glycol monomethyl ether in lysate, adopt whizzer (1400 rev/mins) centrifugation 10 minutes then, separation obtains 12 and goes up clear liquid.Add 6 liters of 0.33M sodium acetate solutions toward this supernatant liquor again, add 3.0 liters of 3%CTAB solution again, after the natural subsidence, centrifugation goes out throw out after stirring a little.
The 0.1M sodium acetate solution of following with 10 liters is dissolved in the resulting precipitation of preceding step, after the stirring and dissolving, adds 95% ethanolic soln again, make alcohol concn reach 70%, let alone the solution sedimentation, lower floor's material adopts whizzer centrifugation, this step triplicate then.Obtain pure rna product of the present invention like this.
RNA total amount (calculating): 150.2 grams with RNA
Quality product: the Yeast Nucleic Acid quality standard that meets National Drug Administration's promulgation.
Embodiment 8
Get health, remove the bright liver double centner of fatty liver muscle, clean with 0.9% sodium chloride solution, rub with mincer, add 120 liters of SSC solution (pH=7.0), temperature is no more than 15 ℃, stirs, last colloidal mill homogenate, homogenate adopts whizzer (2890 rev/mins) centrifugation 30 minutes, and centrifugal back obtains 115 liters of supernatant liquors with 100 order nylon yarn natural filtrations.
Then, in described supernatant liquor, add 11.5 intensification degree and be no more than 15 ℃, the SDS solution of pH=9.0, cumulative volume reaches 125 liters, stir, add 62.5 liter of 80% phenol (temperature is no more than 30 ℃) again, stir after 15 minutes, add 62.5 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, get 120 liters of supernatant liquors.
The supernatant liquor of step adds 12.0 liter of 20% liquor kalii acetici forward, add 300 liter of 95% ethanolic soln then, make that alcohol concn reaches 70% in this solution, after letting alone natural subsidence, the lower sediment thing obtains throw out with whizzer (2890 rev/mins) centrifugation 30 minutes.
Then, with 12 liters of described sodium acetates redissolution liquid dissolution precipitation things, get 14 liters of lysates, in this lysate, add 7.0 liter of 80% phenol and 7.0 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, obtain 16 liters of supernatant liquors.
Then, what step obtained in front 18 goes up in the clear liquid and to add 8.0 liter of 80% phenol, stirs 15 minutes, adds 8.0 liters of chloroforms again, stirs 15 minutes, adopts whizzer (2890 rev/mins) centrifugation 10 minutes then, obtains 18 liters of supernatant liquors.
Add 9.0 liters of chloroforms again in the supernatant liquor that step obtains in front, stirred 5 minutes, adopt whizzer (2890 rev/mins) centrifugation 10 minutes then, reclaim supernatant liquor, in the supernatant liquor that reclaims, add 95% ethanol, reach 70%, let alone the solution sedimentation until alcohol concn, lower floor's material after the sedimentation obtains rough RNA like this with whizzer (2890 rev/mins) centrifugation 10 minutes.
Then, use 13 liters of dipotassium hydrogen phosphate solutions to dissolve described crude rna, obtain 15.0 liters of lysates, add 7.5 liters of ethylene glycol monomethyl ether in lysate, adopt whizzer (1400 rev/mins) centrifugation 10 minutes then, separation obtains 14.0 and goes up clear liquid.Add 7.0 liters of 0.33M sodium acetate solutions toward this supernatant liquor again, add 3.5 liters of 3%CTAB solution again, after the natural subsidence, centrifugation goes out throw out after stirring a little.
The 0.1M sodium acetate solution of following with 10 liters is dissolved in the resulting precipitation of preceding step, after the stirring and dissolving, adds 95% ethanolic soln again, make alcohol concn reach 70%, let alone the solution sedimentation, lower floor's material adopts whizzer centrifugation, this step triplicate then.Obtain pure rna product of the present invention like this.
RNA total amount (calculating): 150.1 grams with RNA
Quality product: the Yeast Nucleic Acid quality standard that meets National Drug Administration's promulgation.
Embodiment 9
Get health, remove the bright liver double centner of fatty liver muscle, clean with 0.9% sodium chloride solution, rub with mincer, add 120 liters of SSC solution (pH=7.0), temperature is no more than 15 ℃, stirs, last colloidal mill homogenate, homogenate adopts whizzer (2890 rev/mins) centrifugation 30 minutes, and centrifugal back obtains 110 liters of supernatant liquors with 100 order nylon yarn natural filtrations.
Then, in described supernatant liquor, add 11.0 intensification degree and be no more than 15 ℃, the SDS solution of pH=9.0, cumulative volume reaches 120 liters, stir, add 80 liter of 80% phenol (temperature is no more than 30 ℃) again, stir after 15 minutes, add 80 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, get 130 liters of supernatant liquors.
The supernatant liquor of step adds 13.0 liter of 20% liquor kalii acetici forward, add 310 liter of 95% ethanolic soln then, make that alcohol concn reaches 70% in this solution, after letting alone natural subsidence, the lower sediment thing obtains throw out with whizzer (2890 rev/mins) centrifugation 30 minutes.
Then, with 12 liters of described sodium acetates redissolution liquid dissolution precipitation things, get 14 liters of lysates, in this lysate, add 9.0 liter of 80% phenol and 9.0 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, obtain 15 liters of supernatant liquors.
Then, what step obtained in front 10 goes up in the clear liquid and to add 10.0 liter of 80% phenol, stirs 15 minutes, adds 17.0 liters of chloroforms again, stirs 15 minutes, adopts whizzer (2890 rev/mins) centrifugation 10 minutes then, obtains 12 liters of supernatant liquors.
Add 12.0 liters of chloroforms again in the supernatant liquor that step obtains in front, stirred 5 minutes, adopt whizzer (2890 rev/mins) centrifugation 10 minutes then, reclaim supernatant liquor, in the supernatant liquor that reclaims, add 95% ethanol, reach 70%, let alone the solution sedimentation until alcohol concn, lower floor's material after the sedimentation obtains rough RNA like this with whizzer (2890 rev/mins) centrifugation 10 minutes.
Then, use 13 liters of dipotassium hydrogen phosphate solutions to dissolve described crude rna, obtain 16.0 liters of lysates, add 8.0 liters of ethylene glycol monomethyl ether in lysate, adopt whizzer (1400 rev/mins) centrifugation 10 minutes then, separation obtains 14.0 and goes up clear liquid.Add 7.0 liters of 0.33M sodium acetate solutions toward this supernatant liquor again, add 3.5 liters of 3%CTAB solution again, after the natural subsidence, centrifugation goes out throw out after stirring a little.
The 0.1M sodium acetate solution of following with 10 liters is dissolved in the resulting precipitation of preceding step, after the stirring and dissolving, adds 95% ethanolic soln again, make alcohol concn reach 70%, let alone the solution sedimentation, lower floor's material adopts whizzer centrifugation, this step triplicate then.Obtain pure rna product of the present invention like this.
RNA total amount (calculating): 150.0 grams with RNA
Quality product: the Yeast Nucleic Acid quality standard that meets National Drug Administration's promulgation.
Embodiment 10
Get health, remove the bright liver double centner of fatty liver muscle, clean with 0.9% sodium chloride solution, rub with mincer, add 120 liters of SSC solution (pH=7.0), temperature is no more than 15 ℃, stirs, last colloidal mill homogenate, homogenate adopts whizzer (2890 rev/mins) centrifugation 30 minutes, and centrifugal back obtains 120 liters of supernatant liquors with 100 order nylon yarn natural filtrations.
Then, in described supernatant liquor, add 12.0 intensification degree and be no more than 15 ℃, the SDS solution of pH=9.0, cumulative volume reaches 130 liters, stir, add 44 liter of 80% phenol (temperature is no more than 30 ℃) again, stir after 15 minutes, add 44 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, get 105 liters of supernatant liquors.
The supernatant liquor of step adds 10.5 liter of 20% liquor kalii acetici forward, add 260 liter of 95% ethanolic soln then, make that alcohol concn reaches 70% in this solution, after letting alone natural subsidence, the lower sediment thing obtains throw out with whizzer (2890 rev/mins) centrifugation 30 minutes.
Then, with 12 liters of described sodium acetates redissolution liquid dissolution precipitation things, get 14 liters of lysates, in this lysate, add 4.6 liter of 80% phenol and 4.6 liters of chloroforms again, stirred 15 minutes, and adopted whizzer (2890 rev/mins) centrifugation 30 minutes, obtain 16 liters of supernatant liquors.
Then, what step obtained in front 10 goes up in the clear liquid and to add 5.3 liter of 80% phenol, stirs 15 minutes, adds 5.3 liters of chloroforms again, stirs 15 minutes, adopts whizzer (2890 rev/mins) centrifugation 10 minutes then, obtains 15 liters of supernatant liquors.
Add 7.5 liters of chloroforms again in the supernatant liquor that step obtains in front, stirred 5 minutes, adopt whizzer (2890 rev/mins) centrifugation 10 minutes then, reclaim supernatant liquor, in the supernatant liquor that reclaims, add 95% ethanol, reach 70%, let alone the solution sedimentation until alcohol concn, lower floor's material after the sedimentation obtains rough RNA like this with whizzer (2890 rev/mins) centrifugation 10 minutes.
Then, use 13 liters of dipotassium hydrogen phosphate solutions to dissolve described crude rna, obtain 16.0 liters of lysates, add 8.0 liters of ethylene glycol monomethyl ether in lysate, adopt whizzer (1400 rev/mins) centrifugation 10 minutes then, separation obtains 12.0 and goes up clear liquid.Add 6.0 liters of 0.33M sodium acetate solutions toward this supernatant liquor again, add 3.0 liters of 3%CTAB solution again, after the natural subsidence, centrifugation goes out throw out after stirring a little.
The 0.1M sodium acetate solution of following with 10 liters is dissolved in the resulting precipitation of preceding step, after the stirring and dissolving, adds 95% ethanolic soln again, make alcohol concn reach 70%, let alone the solution sedimentation, lower floor's material adopts whizzer centrifugation, this step triplicate then.Obtain pure rna product of the present invention like this.
RNA total amount (calculating): 150.2 grams with RNA
Quality product: the Yeast Nucleic Acid quality standard that meets National Drug Administration's promulgation.
Claims (5)
1, a kind of production method of Yeast Nucleic Acid is characterized in that this method comprises the steps:
(1) homogenate: get the bright liver of 100 weight parts, wash, drain, rub bright liver with mincer, bright liver and SSC pH7.0 solution are in volume/weight in proportion 1: 0.8-2.2 mixes, colloidal mill homogenate, supernatant liquor is got in the feed liquid centrifugation, filters the filtrate that obtains the 70-210 parts by volume;
(2) get the filtrate of step (1), add 7-21 parts by volume SDS solution, stir, add 23-100 parts by volume 80-90% phenol solution again, restir adds 23-100 parts by volume chloroform again, stirs, and supernatant liquor is reclaimed in the feed liquid centrifugation;
(3) get the supernatant liquor of step (2), add 5-20 parts by volume 20% potassium acetate solution, mixing adds 80-420 parts by volume ethanol again, to alcohol concn be 70%, mixing, natural subsidence, centrifugation obtains throw out;
(4) with the throw out of 12 parts by volume sodium acetates redissolution liquid dissolving steps (3), add 2-11 parts by volume 80% phenol again, stir, add 2-11 parts by volume chloroform again, stir, centrifugation is again reclaimed and is got supernatant liquor;
(5) get the supernatant liquor of the step (4) of 18 parts by volume, add 1.8-12 parts by volume 80% phenol, stir, add 1.8-12 parts by volume chloroform again, stir, centrifugation is reclaimed and is got supernatant liquor;
(6) get step (5) supernatant liquor, add 2-14 parts by volume chloroform, stir, centrifugation is reclaimed and is got supernatant liquor;
(7) toward the supernatant liquor of step (6), adding 95% ethanol to alcohol concn is 70%, mixing, and natural sedimentation, the supernatant liquor that inclines, centrifugation gets the RNA crude product;
(8) with 13 parts by volume K
2HPO
4Solution dissolves above-mentioned RNA crude product and obtains lysate, adds 5-10 parts by volume ethylene glycol monomethyl ether then, the centrifugal supernatant liquor that obtains;
(9) get step (8) supernatant liquor, add 5-8 parts by volume sodium acetate solution again, mixing, and then add 2-4.5 parts by volume adding CTAB solution, and mixing, precipitation, centrifugal, obtain throw out;
(10) get the throw out of step (9), with the sodium acetate solution dissolving of 10 parts by volume, adding 95% ethanol to concentration again is 70%, abandoning supernatant, and precipitation and centrifugal separation repeats this step 3-4 time, obtains the RNA product like this,
Wherein: described SSC pH=7.0 solution is after adding purified water to 1 liter with 5.85 gram sodium-chlor, 14.7 gram Trisodium Citrates, 5 gram bentonite, is adjusted to Glacial acetic acid that pH=7.0 prepares again;
Described SDS pH=9.0 solution is after adding pure water to 1 liter with 50 gram SDS, 133 milliliters of TEA, 4.0 gram EDTA, is adjusted to Glacial acetic acid that pH=9.0 prepares again;
Described 80% phenol solution is dissolved to 800 milliliters of phenol and 1 gram oxine 1 liter with pure water and obtains;
It is after restraining EDTA and add purified water to 1 liter with 13.6 gram sodium acetates, 5.0 gram SDS, 13.3 milliliters of TEA, 0.8 that described sodium acetate redissolves liquid, is adjusted to Glacial acetic acid that pH=7.0 prepares again;
Described sodium acetate solution is after adding purified water to 1 liter with 13.6 gram sodium acetates, 0.8 gram EDTA, is adjusted to Glacial acetic acid that pH=7.0 prepares again;
Described K
2HPO
4Solution is with water for injection dissolving 296.7 gram K
2HPO
4.H
2O and 0.8 gram EDTA are adjusted to phosphoric acid that pH=8.0 prepares again;
Described CTBA solution is with water for injection dissolving 30 gram CTBA, and water transfers to 1 liter and prepares again.
2, method according to claim 1 is characterized in that described centrifugation is under rotating speed 1000-3000 rev/min condition centrifugal 10-30 minute.
3, method according to claim 1 is characterized in that used bright liver is healthy, removes the liver of pork liver, beef liver and other animal thereof of fatty liver muscle.
4, method according to claim 1 is characterized in that used bright liver is a pork liver.
5, method according to claim 1 is characterized in that described bright liver is clean with 0.9% sodium chloride solution or pure water.
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| CN 03137647 CN1212332C (en) | 2003-06-19 | 2003-06-19 | Preparation method of ribonucleic acid |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03137647 CN1212332C (en) | 2003-06-19 | 2003-06-19 | Preparation method of ribonucleic acid |
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| CN1212332C true CN1212332C (en) | 2005-07-27 |
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