CN101264062A - Production technology for preparing freeze-drying genetic engineering bacterium competence cell and protective agent formula - Google Patents
Production technology for preparing freeze-drying genetic engineering bacterium competence cell and protective agent formula Download PDFInfo
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- CN101264062A CN101264062A CNA2007100643256A CN200710064325A CN101264062A CN 101264062 A CN101264062 A CN 101264062A CN A2007100643256 A CNA2007100643256 A CN A2007100643256A CN 200710064325 A CN200710064325 A CN 200710064325A CN 101264062 A CN101264062 A CN 101264062A
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Abstract
The invention discloses a manufacturing technique for preparing frozen-out gene engineering bacteria competent cells. The invention also relates to a prescription of protecting agent, which is wide in application by taking as the host bacteria for transferring outer DNA in researching, developing, producing and checking of biotechnology. The protecting agent is one of the most fundamental matching consumption preparations. The invention utilizes vacuum freeze drying technology to deal with the poor gene engineering bacteria competent cells, and can save the cells steadily at a wide temperature range of 20 DEG C below zero to 4 DEG C for long time while keeping high transformation efficiency. The invention enables the cells to store and long-distance transport conveniently. The invention relates to a manufacturing technique for preparing frozen-out competent cells, quality inspection regulation and the frozen-out protecting agent, comprising culture conditions of gene engineering bacteria, technological processes of freezing and drying and the composition and matching of the protecting agent. The protecting agent is formed by water and one or arbitrary combination of the following materials: gelatin, degreasing milk, dextran, trehalose, sucrose, sorbitol or mannitol.
Description
Technical field
The present invention relates to a kind of genetic engineering host bacterium viable bacteria freeze dried powder, all have a wide range of applications, belong to one of supporting consumption preparation the most basic in the biotechnology in biotechnology research, exploitation, production and in detecting.
Background technology
Genetic engineering is a brand-new biotechnology science that is born in 1970's on the basis of molecular biology and molecular genetics comprehensive development.Its key character is exactly an exogenous genetic material---and nucleic acid molecules can be bred in different host's biologies, can cross over natural species barrier, gene from any biology is placed in the new biology, and this biology can have no sibship with original biology, and this ability has also determined it all can being applied in scientific domain widely.
In general, the concrete experimentation of genetic engineering is with the hereditary material of artificial method with needed a certain donor biology---the DNA macromole extracts, after cutting with the proper implements enzyme under the condition that exsomatizes, it with couple together as the dna molecular of carrier, import in the recipient cell of a certain easier growth, breeding with carrier then, to allow exogenous genetic material " settle down " therein, duplicate normally and express, thereby obtain a kind of brand-new technology of new species.In brief, all operation all will be through the process of setting out on basis the most: obtain genes of interest → be connected into carrier → conversions and enter amplification amplification in the genetic engineering host bacterium → other concrete experiment.Analyze its concrete process one by one, just can have cognitive clearly the used concrete research method of genetic engineering and its required matched reagent.Genes of interest is ever-changing because of experimental requirements and purpose, the method that obtains genes of interest also is divided into direct separation and pcr amplification method, associated separate nucleic acid test kit and pcr amplification matched reagent become the main product of some biotech firms, wherein are no lack of some world-famous enterprises, as the German QIAGEN company in the world of becoming famous with the separate nucleic acid test kit; The celebrated companies such as ABI, TAKARA with the PCR series of products.Mention carrier; cloning vehicle that some are classical and serial expression vector are by some external major company research and development and obtain patent protection; market checking through recent decades; substantially formed the monopolization on certain meaning; pET series expression vector as Novagen company; other also comprises some pUC that application-specific is arranged series cloning vehicle, shuttle expression carrier, suicide plasmid carrier etc.As for the generalized theory of genetic engineering host bacterium, it should be a kind of active somatic cell, a kind of definite DNA small fragment (connector of carrier and genes of interest) is bred therein, realize that so a small amount of DNA sample " copy " goes out a large amount of DNA, and be not pollute any other DNA sequence, purified dna molecular group in a large number, and then the realization molecular cloning, or directly produce a certain amount of purpose product (can be that albumen also can be some secondary metabolites that produce by biotransformation).Become the first-selection of genetic engineering host bacterium owing to himself characteristics through the escherichia coli fungus strain of heredity domestication.
As the host bacterium, escherichia coli have following advantage: 1. genetic background and physiological property research are quite thorough, a lot of different Drug resistance, Different Nutrition dependent form and the selective application of the strain of different correcting mutant types have been arranged, can select different strains according to different carriers.2. although the space of Bacillus coli cells is little, fertility is strong, in the nutritional condition abundance, when condition of culture is fit to, can finish a generation in 20 minutes, and it is very fast to grow.Through the certain culture condition optimizing, can realize high density fermentation, and the large scale fermentation cost is low, have huge productive potentialities.3. the general serious karyonide system of gene expression dose is high, and the expression of some exogenous gene in escherichia coli can reach 5%～70% of total protein, and downstream purification technology is simple, make things convenient for automatization to control, and is easy to industrialization.Also have some unsatisfactory parts as the expression system escherichia coli owing to some limiting factors, therefore other organism more in recent years, as: bacillus cereus, yeast, filamentous fungi or zooblast etc. more and more come into one's own as the host bacterium of expressing, and use also more and more widely.But, no matter use which kind of expressed receptor system all will be with the host bacterium of escherichia coli as initial gene amplification, also to want to buy with the corresponding carrier of these expression systems and to be built into shuttle vector so that earlier in escherichia coli after the amplification again by transforming or transfection enters in other the acceptor systems.In sum, in view of the prepotency of himself and traditional application experience, escherichia coli have become most basic instrument bacterial strain in the genetic engineering experimental implementation process.
Competence be escherichia coli be in can high efficiency acquisition exogenous gene a kind of state, its acquisition is the condition of culture by control bacillus coli gene engineering bacteria, make its population growth to a certain state, it is early stage to be typically chosen in logarithmic growth, utilization contains the reagent cryogenic conditions processing down of some bivalent metal ion, cause colibacillary cell wall to synthesize defective, make the external source genes of interest be convenient to enter escherichia coli, can also keep a kind of cell state of high growth of cell and metabolic activity simultaneously.Because the cell wall defective of competent cell but also will maintain a kind of state of high transformation efficiency, therefore a little less than being highly brittle, conventional manufacture method is essential uses-80 ℃ of refrigerators to preserve, and transports with dry ice, and preserves after 2 months its biological activity decline obviously.At present, domestic most agent and retail trader do not provide-80 ℃ of cold storage establishments, and the fixed point of dry ice preparation and transport and bring great inconvenience to salesman.In addition, some R﹠D institutions are because condition restriction also can cause the instability of product even biological activity to completely lose in the preservation process, and these have all had a strong impact on this product quality stability and marketing is used.
Freeze Drying Technique was invented by British Wollaston early than 1813, be meant that specifically a sample solution freezes at low temperatures, sublimation drying under vacuum condition is removed ice crystal then, treat to carry out adsorption stripping and dry again after distillation finishes, remove the drying means of part bound water.The drying of sample product is carried out in the temperature below 0 ℃ basically in actual mechanical process, promptly under the state that product freezes, carry out, up to the later stage, in order further to reduce the remaining water content of dry products, just allow product rise to temperature more than 0 ℃, but generally be no more than 40 ℃.Whole drying is to carry out under lower temperature, and in left ice shelf when freezing of material itself, so its dry back constancy of volume, loose porous.Therefore lyophilization has following advantage: 1. lyophilization is carried out at low temperatures, for the material particularly suitable of many thermal sensitivitys.Degeneration can not take place or lose biologos as protein, microorganism and so on.2. when dry at low temperatures, some the volatile ingredient losses in the material are very little, are fit to some chemical productss, medicine and food drying.3. in freezing dry process, the effect of microbial growth and enzyme can't be carried out, and therefore can protect different Drug resistance, Different Nutrition dependent form and the selective application of the strain of different correcting mutant types, can select different strains according to different carriers.2. although the space of Bacillus coli cells is little, fertility is strong, in the nutritional condition abundance, when condition of culture is fit to, can finish a generation in 20 minutes, and it is very fast to grow.Through the certain culture condition optimizing, can realize high density fermentation, and the large scale fermentation cost is low, have huge productive potentialities.3. the general serious karyonide system of gene expression dose is high, and the expression of some exogenous gene in escherichia coli can reach 5%～70% of total protein, and downstream purification technology is simple, make things convenient for automatization to control, and is easy to industrialization.Also have some unsatisfactory parts as the expression system escherichia coli owing to some limiting factors, therefore other organism more in recent years, as: bacillus cereus, yeast, filamentous fungi or zooblast etc. more and more come into one's own as the host bacterium of expressing, and use also more and more widely.But, no matter use which kind of expressed receptor system all will be with the host bacterium of escherichia coli as initial gene amplification, also to want to buy with the corresponding carrier of these expression systems and to be built into shuttle vector so that earlier in escherichia coli after the amplification again by transforming or transfection enters in other the acceptor systems.In sum, in view of the prepotency of himself and traditional application experience, escherichia coli have become most basic instrument bacterial strain in the genetic engineering experimental implementation process.
Competence be escherichia coli be in can high efficiency acquisition exogenous gene a kind of state, its acquisition is the condition of culture by control bacillus coli gene engineering bacteria, make its population growth to a certain state, it is early stage to be typically chosen in logarithmic growth, utilization contains the reagent cryogenic conditions processing down of some bivalent metal ion, cause colibacillary cell wall to synthesize defective, make the external source genes of interest be convenient to enter escherichia coli, can also keep a kind of cell state of high growth of cell and metabolic activity simultaneously.Because the cell wall defective of competent cell but also will maintain a kind of state of high transformation efficiency, therefore a little less than being highly brittle, conventional manufacture method is essential uses-80 ℃ of refrigerators to preserve, and transports with dry ice, and preserves after 2 months its biological activity decline obviously.At present, domestic most agent and retail trader do not provide-80 ℃ of cold storage establishments, and the fixed point of dry ice preparation and transport and bring great inconvenience to salesman.In addition, some R﹠D institutions are because condition restriction also can cause the instability of product even biological activity to completely lose in the preservation process, and these have all had a strong impact on this product quality stability and marketing is used.
Freeze Drying Technique was invented by British Wollaston early than 1813, be meant that specifically a sample solution freezes at low temperatures, sublimation drying under vacuum condition is removed ice crystal then, treat to carry out adsorption stripping and dry again after distillation finishes, remove the drying means of part bound water.The drying of sample product is carried out in the temperature below 0 ℃ basically in actual mechanical process, promptly under the state that product freezes, carry out, up to the later stage, in order further to reduce the remaining water content of dry products, just allow product rise to temperature more than 0 ℃, but generally be no more than 40 ℃.Whole drying is to carry out under lower temperature, and in left ice shelf when freezing of material itself, so its dry back constancy of volume, loose porous.Therefore lyophilization has following advantage: 1. lyophilization is carried out at low temperatures, for the material particularly suitable of many thermal sensitivitys.Degeneration can not take place or lose biologos as protein, microorganism and so on.2. when dry at low temperatures, some the volatile ingredient losses in the material are very little, are fit to some chemical productss, medicine and food drying.3. in freezing dry process, the effect of microbial growth and enzyme can't be carried out, and therefore can keep original character.4. owing to carry out drying under the state that freezes, so volume is almost constant, has kept original structure, concentration phenomena can not take place.5. dried matter is loose porous, is spongy, adds that dissolving almost recovers original character rapidly and fully immediately behind the water.6. because drying is carried out under vacuum, oxygen is few, so the material of some easy oxidations has obtained protection.7. drying can be got rid of the above moisture of 95-99%, makes dry back product energy long preservation and unlikely going bad.Sample after lyophilization, lucifuge long term store at room temperature, when needing to use, the redissolution buffer of adding distil water or normal saline or other formulated is made suspension, can return to the state before the lyophilizing.Therefore, lyophilization is widely used in medical industry, food industry, scientific research and other departments at present.Shsckell test in 1909 is carried out the lyophilizing preservation with this method to antitoxin, strain, rabies virus and other biological product, has obtained better effects.In World War II, the wilderness demand of blood products has been stimulated the development of Freeze Drying Technique greatly, from then on this technology has entered the commercial Application stage.After this, the develop rapidly of refrigeration and vacuum equipment provides strong material conditions for fast-developing Freeze Drying Technique.Enter eighties of last century; the rapid development of science and technology and the people provide powerful power to the demand of health care for the develop rapidly of medicine Freeze Drying Technique, have obtained huge achievement at aspects such as lyophilizing damage and protection mechanism, freeze-dry process, freezer dryers.Because freeze-drying possesses low temperature, drying, anoxybiotic requirement simultaneously, meets very much the ideal conditions of culture presevation, be the most effective method for preserving of generally acknowledging at present.
The present invention is in view of wretched insufficiency and domestic and international great demand in engineering bacteria competent cell production of present large intestine bar gene bacterium and the application, in conjunction with self-condition, the preparation method and the Freeze Drying Technique of competent cell are organically combined, break through tradition, adopt unique freeze drying process to handle the colibacillus engineering strain, be prepared into freeze dried competent cell, be applicable to the efficient chemical conversion of foreign DNA, particularly when genes of interest content low or purchase when building gene library use with the obvious advantage.In addition; the present invention relates to optimize the protection liquid of preparation; can be under high temperature, dehydration, abnormal conditions such as freezing stabilizing cell membrane; prevent protein denaturation; not only can effectively protect the activity of biomacromolecule and organism in the lyophilization process; and can effectively suppress being separated and crystalline formation of lyophilized products in the storage process, and overcome the shortcoming of common competent cell to storage and traffic condition sensitivity, under the temperature conditions of wide region, still can keep high transformation efficiency.A large amount of experimental results show that plasmids such as using pUC18 detects, and transformation efficiency can reach 10
8T/ μ g preserved 1 year under-20 ℃ of conditions, and transformation efficiency still can reach 10
7More than the T/ μ g.Compare with product in the market, have more advance and advantage, mainly contain following advantage:
1 is safe: the preparation technology of vacuum, low temperature, low pressure makes this product long shelf-life, the survival rate height, and aberration rate is low.
2 solubilities are good: the redissolution buffer that adds optimization can absorb water rapidly and be reduced into the preceding state of lyophilization, has shortened the strain recovery time.
3 dehydrations are thorough: can tolerate long-distance transport and long preservation.
4 outward appearances are good: dry back forms loose structure, and color is constant substantially.
5 batches stable: the industrialized amplification mode of production, difference is low between batch, the repeatability height.
Summary of the invention
The object of the present invention is to provide a kind of good stability, genetic engineering bacterium competence cell freeze-dried preparation that transformation efficiency is high, said preparation can be preserved 6～12 months so that the solid form of freeze dried powder is stable in-25 ℃～4 ℃ wide temperature ranges.For reaching goal of the invention, the present invention also provides the method for large-scale production lyophilization competent cell, the prescription that production technology and a kind of freeze drying protectant thereof are amplified in pilot scale.
The preparation of lyophilization competent cell of the present invention can realize by following technical scheme:
The preparation of 1 seed
The competent cell that is used for genetic engineering operation of preparation provided by the invention can be gram-positive cell also can be negative cells, comprise Escherichia sp., Klebsiella sp., Samonella sp., Bacillus sp., Streptococcus sp., Shigella sp., Staphylococcus sp., Pseudomoniae sp. etc. and by the deutero-mutation of above kind, subspecies comprise: Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis, Samonella typhimurium, Streptococcusaureus, Staphylococcus mutans, Staphylococcus pneumoniae, the preferred kind of Pseudomoniae syringae. the present invention is colon bacillus (Escherichi), all kinds of DH5 that comprise of Escherichia coli kind particularly, DH5 α, DH10, DH10B, DH101, RR1, JV30, DH11S, DM1, DH10B/p3, DH5 α MCR, DH5 α 5 ' IQ, DH5 α 5 ', SCS1, Stab2, DH12S, DH5 α-E, DH10BAC, XL1-Blue MRF, XL2-Blue MR, SUREStrain, SURE 2Strain, XL1-Blue, XL2-Blue, AG1, JM109, JM110/SCS110, NM522, TOPPStrains, XL1-Red, BL21Strains, the bacterium that derives of TK B1Strain and above bacterium.
The culture medium of genetic engineering bacterium is selected conventional Luria Broth prescription for use, should add some auxotrophic restricted compositions as one sees fit according to the kind feature of strain in addition, should add the amino acid nutrient composition of corresponding disappearance as some aminoacid deficiencies, be beneficial to thalli growth.In addition, also need add 20mM MgCL
2, 0.05～1% glucose also should add certain density PEG4000 in the SOB culture medium.The cultivation temperature of competent cell is from 15 ℃～37 ℃, and further optimizing temperature range is 20 ℃～30 ℃, and optimal growth temperature is 23 ℃.
Aseptic condition is activation dry powder strain Ecoli.DH5 α down, and inoculation LB solid medium (10g trptone, 10g NaCl, 5gyeast extract, 15g agar pH7.01L) is cultivated 24hr for 37 ℃.A few strain monoclonals of picking are inoculated 25ml SOB culture medium (2%trptone, 10mM NaCl, 0.5%yeast extract, 2.5mM KCl, 10mM MgCl respectively
2, 10mM MgSO
4) 37 ℃ of 275rpm shaking tables cultivate, the dense OD550 of monitoring bacterium is greatly about 0.5 o'clock, and the ratio that adds 4ml glycerol by 6ml bacterium liquid mixes cultured bacterium liquid with glycerol, is distributed into 1ml/ behind the mixing and manages-80 ℃ of preservations.
The activation of 2 competent cells and cultivation
The population growth of cell is controlled by temperature, ventilation and the incubation time of control seed culture state and amplification culture.Be typically chosen in logarithmic growth stable phase in latter stage early harvest cell, because the cellular metabolism of this moment is vigorous, growth is steady and injury resistance that lyophilization is caused is the strongest.Amplification culture can adopt the mode of shake-flask culture or fermentor cultivation according to the actual demand of producing, and typical shake-flask culture is to control ventilation by control shaking table revolution.Shake bottle and can select 1/5 loading amount and fermentation tank can be selected 4/5 the loading amount of adding.The revolution that shakes bottle is controlled at 150～400rpm, can be controlled in 200～300rpm after the optimization.The incubation time of cell can be finished by the monitoring dissolved oxygen, cell is through transferred species amplification culture a period of time, detect OD550 between 0.3～1.0 by detecting the UV, visible light analyser, preferably between 0.5～0.8, further optimize between 0.65～0.75 to the most desirable.
The strain of-80 ℃ of preservations places ice-water bath to melt out, and changes over to by 0.3% transferred species amount and shakes amplification culture in the bottle greatly, and culture medium is for adding the SOB of 0.001%PEG 4000.20 ℃～30 ℃, the 275rpm shaking table is cultivated about 20hr, and the dense OD550 of monitoring bacterium is greatly about 0.65～0.75.Cultured cell was placed cooled on ice 15～30 minutes, 4 ℃ of centrifugal 10 minutes collection thalline of 4000rpm.Bacterial sediment is resuspended in FSB buffer (10mM KAc pH7.5, the 10mM CaCl of pre-cooling
2.H
2O, 45mMMnCl
2.4H
2O, 100mM KCl
2, 3mM chlorination six cobaltammines, 10% glycerol is regulated pH6.4 with 0.1N HCl) in, add FSB buffer according to 1/3～1/5 ratio of original cultivation bacterium liquid.Outstanding even cell was placed 20 minutes on ice, cooling.4000rpm4 ℃ of centrifugal 10 minutes collection thalline abandoned supernatant.
The lyophilization of 3 competent cells
. precipitation is resuspended in the preferred freeze drying protectant, and wherein protectant consumption is to cultivate 1/8～1/25 of initial volume, and preferred proportion is 1/12～1/20.Freeze drying protectant is made by the material and the water that are selected from gelatin, skim milk, dextran, trehalose, sucrose, sorbitol or mannitol one of them or combination in any, and the weight proportion between each component is as follows:
Gelatin: 0.001%～2% or skim milk: 0.5%～50%
Dextran: 0.1%～20%
Trehalose or sucrose: 0.1%～20%
Sorbitol or mannitol: 0.1%～20%
All the other are water
More preferably following prescription:
Gelatin: 0.01%～1% or skim milk: 10%～20%
Dextran: 3%～10%
Trehalose or sucrose: 3%～8%
Sorbitol or mannitol: 3%～8%
All the other are water
Dextran belongs to medicinal dextran-10 in the prescription, dextran-20, and dextran-40, water are the pharmaceutical injection water.Frozen-dried protective liquid of the present invention can prepare by the following method; get gelatin, dextran, skim milk, trehalose, sucrose, sorbitol or the mannitol of formula ratio or their combination in any; the water for injection that adds formula ratio mixes them, and dissolving becomes frozen-dried protective liquid.
Preferred formulated method is as follows:
1 takes by weighing an amount of gelatin, and room temperature was placed 5 minutes, stirred under 40 ℃ of water bath condition, and it is uniformly dispersed, and handles 30 minutes with activated carbon adsorption under 90 ℃.
2 take by weighing dextran, trehalose, sucrose, sorbitol or mannitol or their combination in any, and each is an amount of, and heating is dissolved the back and added 90 ℃ of following adsorption treatment of active carbon of 0.8% 30 minutes.
The above-mentioned mixed solution of 3 usefulness, 0.45 μ M filtering with microporous membrane is used the water for injection standardize solution behind the mix homogeneously, 115 ℃ of autoclavings 20 minutes.
Suspension/pipe by 150 μ l cells and frozen-dried protective liquid is divided in the bottle; should can adopt sterile gauze to cover bottleneck pre-freeze under the aseptic condition before the lyophilizing, put 5 hours for-80 ℃; or directly be put in advance on the freeze dryer grillage that is chilled to-50 ℃, placed 10 hours.Adjust freeze dryer grillage temperature to-45 ℃, put into the strain pipe of pre-freeze, close to the doorstep, open vacuum pump, the beginning lyophilizing.Put 2hr for-45 ℃ and be warming up to 10 ℃ (slow intensifications that preferably can be controlled).Lyophilizing is sealed after finishing.-20 ℃ of refrigerators are preserved, and detect the back and use.
4 freeze dried bacterial strain recovery and detections
Be placed on ice being kept at-20 ℃ of lyophilizing competent cells in the refrigerator, add 100 μ l redissolution buffer CM Buffer (10mM KAc pH7.0,100mM KCl, 45mM MnCl
2.4H
2O, 10mM CaCl
2.2H
2O, 3mM chlorination six cobaltammines, 10% glycerol, 5% sucrose is regulated pH6.4 with 0.1N HCl) soft rotation, mix homogeneously, ice bath left standstill 5 minutes.
Add target DNA (should note used DNA volume surpass competent cell suspension volume 1/10), gently revolve mixing, ice bath left standstill 30 minutes.60-90 second is placed in 42 ℃ of water-baths, transfers to immediately in the ice bath, places 2-3 minute, does not shake centrifuge tube.Add the aseptic SOC or the LB culture medium (not containing antibiotic) of 37 ℃ of preheatings of 900 μ l, mixing is placed on 37 ℃ of shaking table shaken cultivation 1 hour (150 rev/mins).Mixing is drawn competent cell that 50-100 μ l transformed and is added to and contains on corresponding antibiotic SOB or the LB solid agar culture medium, with aseptic elbow glass rod cell evenly is coated with out gently.Blank is for adding the aquesterilisa of equal volume.Place room temperature to be absorbed flat board, be inverted flat board, cultivate more than 16 hours for 37 ℃, observe counting, calculate transformation efficiency according to following formula until liquid.
Transformation efficiency(CFU/μg)：
For example use positive control as detecting, to get 50pg plasmid pUC19 with plasmid DNA pUC19, and after reactant mixture dilutes 100 times with SOC, get 100 μ l and be coated with flat board, if 100 transformant clones are arranged on the conversion flat board, transformation efficiency is 2 * 10 as shown in the formula calculating so
9
5 stability tests
The lyophilizing competent cell of the present invention's preparation can be stablized the storage regular period and not lose its activity of conversion in wide temperature range.Active be meant at 4 ℃～-180 ℃,, be preferably in to preserve under-20 ℃ of conditions and actively still can keep 60%～100% of initial activity in 150 days～450 days, preferably keep 70%～100% initial activity preferably at-20 ℃～-80 ℃ in suitable temperature range.
According to the competent cell of the production technology and the freeze drying protectant formulation of embodiment 1, preserved 12 months-20 ℃ of conditions.From begin to preserve 0,1,2,3,4,5,6,8,10 and got 5～10 competent cells in 12 months respectively and detect activity of conversion, get in addition three with the equivalent sterilized water as blank.Simultaneously, because therefore moisture can add the mensuration of one group of moisture: detect moisture according to dry weight-loss method in the state-promulgated pharmacopoeia appendix from the stability of another angle reflection lyophilized formulations.
Table 1 is listed the different time sections sampling inspection results.These results show: competent cell prepared in accordance with the present invention is stable, preserves under-20 ℃ of conditions and still can keep high transformation efficiency in 1 year.
| Storage time (moon) | Transformation efficiency | Blank | Product appearance | Moisture |
| 0 | 4.2×10 8 | Qualified | Qualified | Qualified |
| 1 | 4.6×10 8 | Qualified | Qualified | Qualified |
| 2 | 1.8×10 8 | Qualified | Qualified | Qualified |
| 3 | 3.0×10 8 | Qualified | Qualified | Qualified |
| 4 | 2.5×10 8 | Qualified | Qualified | Qualified |
| 5 | 1.5×10 8 | Qualified | Qualified | Qualified |
| 6 | 8.8×10 7 | Qualified | Qualified | Qualified |
| 8 | 7.2×10 7 | Qualified | Qualified | Qualified |
| 10 | 8.5×10 7 | Qualified | Qualified | Qualified |
| 12 | 6.4×10 7 | Qualified | Qualified | Qualified |
Preserve stability of formulation for-20 ℃ under table 1 embodiment 1 process conditions
Table 2 is listed the competent cell according to the production technology of embodiment 2 and freeze drying protectant formulation ,-20 ℃ of condition different time sections sampling inspection results.
| Storage time (moon) | Transformation efficiency | Blank | Product appearance | Moisture |
| 0 | 6.7×10 8 | Qualified | Qualified | Qualified |
| 1 | 7.4×10 8 | Qualified | Qualified | Qualified |
| 2 | 8.3×10 8 | Qualified | Qualified | Qualified |
| 3 | 2.0×10 8 | Qualified | Qualified | Qualified |
| 4 | 3.5×10 8 | Qualified | Qualified | Qualified |
| 5 | 6.2×10 8 | Qualified | Qualified | Qualified |
| 6 | 3.8×10 8 | Qualified | Qualified | Qualified |
| 8 | 9.1×10 7 | Qualified | Qualified | Qualified |
| 10 | 6.7×10 7 | Qualified | Qualified | Qualified |
| 12 | 7.8×10 7 | Qualified | Qualified | Qualified |
Preserve stability of formulation for-20 ℃ under table 2 embodiment 2 process conditions
Fairly comprehensive consideration of the present invention in the competence preparation process the various important factor in order of cell culture as: culture medium proportioning, need the ratio of temperature, dissolved oxygen, time, use FSB Buffer and the freeze drying protectant of the various ions that add and nutrient substance, cultivation, the prescription of freeze drying protectant etc., and preferred above all influence factors.Sterile working, low temperature, vacuum, drying in the process of producing product of the present invention, active component is not destroyed, and is difficult for taking place oxidation.The product formulation outward appearance is loose porous, solubility good, and water content is low, and period of storage is long, and stability is improved, and is convenient to long term store, transportation and sale, also is convenient to guarantee product quality.Preparation of the present invention compared with prior art has stability by force, and raw material is easy to get, and advantage easy to use has more advance.
The specific embodiment
With following embodiment explanation the present invention, but to limit it absolutely not.Be the competent cell of the high transformation efficiency that obtains having good stability, must be careful conscientious, accurately strict in preparation process, take a short cut, all can directly influence experimental result with sordid container, unpurified water or outmoded reagent.The used all ingredients purity of the present invention all requires more than the analytical pure, unless do different explanations, all reagent described here all can be bought from the suppliers of any sale chemistry, biochemistry, biotechnology or pharmaceutical purpose reagent and obtain.
Embodiment 1: 100 of specifications
The preparation of 1 seed
Aseptic condition is activation dry powder strain Ecoli.DH5 α down, and inoculation LB solid medium (10g trptone, 10g NaCl, 5gyeast extract, 15g agar pH7.01L) is cultivated 24hr for 37 ℃.A few strain monoclonals of picking are inoculated 25ml SOB culture medium (2%trptone, 10mM NaCl, 0.5%yeast extract, 2.5mM KCl, 10mM MgCl respectively
2, 10mM MgSO
4) 37 ℃ of 275rpm shaking tables cultivate, the dense OD550 of monitoring bacterium is greatly about 0.5 o'clock, and the ratio that adds 4ml glycerol by 6ml bacterium liquid mixes cultured bacterium liquid with glycerol, is distributed into 1ml/ behind the mixing and manages-80 ℃ of preservations.
The activation of 2 competent cells and cultivation
The strain of-80 ℃ of preservations places ice-water bath to melt out, and gets 0.45ml kind liquid and changes big 2L over to and shake greatly in the bottle, and amplification culture, culture medium are the SOB that 300ml adds 0.001%PEG 4000.23 ℃ of 275rpm shaking tables are cultivated about 20hr, and the dense OD550 of monitoring bacterium is greatly about 0.72.Cultured cell was placed cooled on ice 20 minutes, 4 ℃ of centrifugal 10 minutes collection thalline of 4000rpm.Bacterial sediment is resuspended in FSB buffer (10mM KAc pH7.5, the 10mM CaCl of 60ml pre-cooling
2.H
2O, 45mMMnCl
2.4H
2O, 100mM KCl
2, 3mM chlorination six cobaltammines, 10% glycerol is regulated pH6.4 with 0.1N HCl) in, outstanding even cell was placed 20 minutes on ice, cooling.4 ℃ of centrifugal 10 minutes collection thalline of 4000rpm are abandoned supernatant.
The lyophilization of 3 competent cells
Precipitation is resuspended in the following optimization of the 20ml freeze drying protectant, and the weight proportion between each component is as follows:
Gelatin: 0.1%
Dextran: 8%
Trehalose: 5%
Sorbitol: 5%
All the other are water
Suspension/pipe by 150 μ l cells and frozen-dried protective liquid is divided in the bottle, should for example can adopt sterile gauze to cover bottleneck pre-freeze under the aseptic condition before the lyophilizing, puts pre-freeze 5 hours for-80 ℃.Adjust freeze dryer grillage temperature to-45 ℃, put into the strain pipe of pre-freeze, close to the doorstep, open vacuum pump, when vacuum is lower than 20, begin lyophilizing.Put 2hr for-45 ℃ and be warming up to 10 ℃ (slow intensifications that preferably can be controlled).Lyophilizing is sealed after finishing.-20 ℃ of refrigerators are preserved, and detect the back and use.
4 freeze dried bacterial strain recovery and detections
Be placed on ice being kept at-20 ℃ of lyophilizing competent cells in the refrigerator, add 100 μ l redissolution buffer CM Buffer (10mM KAc pH7.0,100mM KCl, 45mM MnCl
2.4H
2O, 10mM CaCl
2.2H
2O, 3mM 3mM chlorination six cobaltammines, 10% glycerol, 5% sucrose is regulated pH6.4 with 0.1N HCl) soft rotation, mix homogeneously, ice bath left standstill 5 minutes.Add 50pg plasmid DNA pUC18 and gently revolve mixing, ice bath left standstill 30 minutes.42 ℃ of water-baths were placed 90 seconds, transferred to immediately in the ice bath, placed 3 minutes, did not shake centrifuge tube.Add the aseptic SOC or the LB culture medium (not containing antibiotic) of 37 ℃ of preheatings of 900 μ l, mixing is placed on 37 ℃ of shaking table shaken cultivation 1 hour (150 rev/mins).Mixing is drawn the competent cell that 100 μ l have transformed and is added on the LB solid agar culture medium that contains ampicillin, with aseptic elbow glass rod cell evenly is coated with out gently.Place room temperature to be absorbed flat board, be inverted flat board, cultivate more than 16 hours for 37 ℃, observe counting, calculate transformation efficiency until liquid.
Detecting moisture according to dry weight-loss method in the state-promulgated pharmacopoeia appendix is controlled at below 3%.
Embodiment 2: 1000 of specifications
The preparation of 1 seed
Aseptic condition is activation dry powder strain Ecoli.DH5 α down, and inoculation LB solid medium (10g trptone, 10g NaCl, 5gyeast extract, 15g agar pH7.01L) is cultivated 24hr for 37 ℃.A few strain monoclonals of difference picking, inoculation 25ml SOB culture medium (2%trptone, 10mM NaCl, 0.5%yeast extract, 2.5mM KCl, 10mM MgCl2,10mM MgSO4) 37 ℃ of 275rpm shaking tables are cultivated, and the dense OD550 of monitoring bacterium is greatly about 0.5 o'clock, and the ratio that adds 4ml glycerol in 6ml bacterium liquid mixes cultured bacterium liquid with glycerol, be distributed into the 1ml/ pipe behind the mixing ,-80 ℃ of preservations.
The activation of 2 competent cells and cultivation
The strain of-80 ℃ of preservations places ice-water bath to melt out, and gets 0.45ml * 10 kind of liquid and changes big 2L * 10 over to and shake greatly in the bottle, and amplification culture, culture medium are the SOB that every bottle of 300ml adds 0.001%PEG 4000.23 ℃ of 275rpm shaking tables are cultivated about 20hr, and the dense OD550 of monitoring bacterium is greatly about 0.68.Cultured cell was placed cooled on ice 20 minutes, 4 ℃ of centrifugal 10 minutes collection thalline of 4000rpm.Bacterial sediment is resuspended in FSB buffer (10mM KAc pH7.5, the 10mM CaCl of 600ml pre-cooling
2H
2O, 45mM MnCl
24H
2O, 100mM KCl
2, 3mM chlorination six cobaltammines, 10% glycerol is regulated pH6.4 with 0.1N HCl) in, outstanding even cell was placed 20 minutes on ice, cooling.4 ℃ of centrifugal 10 minutes collection thalline of 4000rpm are abandoned supernatant.
The lyophilization of 3 competent cells
Precipitation is resuspended in the following optimization of the 160ml freeze drying protectant, and the weight proportion between each component is as follows:
Gelatin: 0.05%
Dextran: 6%
Trehalose: 5%
Mannitol: 5%
All the other are water
Suspension/pipe by 150 μ l cells and frozen-dried protective liquid is divided in the bottle, should for example can adopt sterile gauze to cover bottleneck pre-freeze under the aseptic condition before the lyophilizing, directly is put in advance on the freeze dryer grillage that is chilled to-50 ℃, places pre-freeze 10 hours.Adjust freeze dryer grillage temperature to-45 ℃, put into the strain pipe of pre-freeze, close to the doorstep, open vacuum pump, when vacuum is lower than 20, begin lyophilizing.Put 2hr for-45 ℃ and be warming up to 10 ℃ (slow intensifications that preferably can be controlled).Lyophilizing is sealed after finishing.-20 ℃ of refrigerators are preserved, and detect the back and use.
4 freeze dried bacterial strain recovery and detections
Be placed on ice being kept at-20 ℃ of lyophilizing competent cells in the refrigerator, add 100 μ l redissolution buffer CM Buffer (10mM KAc pH7.0,100mM KCl, 45mM MnCl
24H
2O, 10mM CaCl
22H
2O, 3mM 3mM chlorination six cobaltammines, 10% glycerol, 5% sucrose is regulated pH6.4 with 0.1N HCl) soft rotation, mix homogeneously, ice bath left standstill 5 minutes.Add 50pg plasmid DNA pUC18 and gently revolve mixing, ice bath left standstill 30 minutes.42 ℃ of water-baths were placed 90 seconds, transferred to immediately in the ice bath, placed 3 minutes, did not shake centrifuge tube.Add the aseptic SOC or the LB culture medium (not containing antibiotic) of 37 ℃ of preheatings of 900 μ l, mixing is placed on 37 ℃ of shaking table shaken cultivation 1 hour (150 rev/mins).Mixing is drawn the competent cell that 100 μ l have transformed and is added on the LB solid agar culture medium that contains ampicillin, with aseptic elbow glass rod cell evenly is coated with out gently.Place room temperature to be absorbed flat board, be inverted flat board, cultivate more than 16 hours for 37 ℃, observe counting, calculate transformation efficiency until liquid.
Detecting moisture according to dry weight-loss method in the state-promulgated pharmacopoeia appendix is controlled at below 3%.
Claims (10)
1. a freeze dried competence powder is characterized in that Freeze Drying Technique is combined with the preparation technology of genetic engineering bacterium competence cell, obtains a kind of genetic engineering bacterium competence cell preparation method of freeze dried competence powder.
2. the preparation of claim 1, be freeze dried powder, wherein said this production of articles process sterile working, low temperature, vacuum, drying, the gained preparation can be with solid form, still keep high transformation efficiency one period stable preservation the in-20 ℃～4 ℃ wide temperature ranges, be convenient to store and transportation.
3. the preparation of claim 1 makes its preservation activity of conversion loss in a year under-20 ℃ of conditions be lower than 30% when it is characterized in that making preservable injectable powder by liquid solution.
4. the preparation of claim 1; be made up of acceptable freeze drying protectant pharmaceutically, it is characterized in that: freeze drying protectant is wherein made by the material and the water that are selected from gelatin, skim milk, dextran, trehalose, sucrose, sorbitol or mannitol one of them or combination in any.
Gelatin: 0.001%～2% or skim milk: 0.5%～50%
Dextran: 0.1%～20%
Trehalose or sucrose: 0.1%～20%
Sorbitol or mannitol: 0.1%～20%
All the other are water
5. the preparation of claim 1, freeze drying protectant is wherein made by the material and the water that are selected from gelatin, skim milk, dextran, trehalose, sucrose, sorbitol or mannitol one of them or combination in any.
Gelatin: 0.01%～1% or skim milk: 10%～20%
Dextran: 3%～10%
Trehalose or sucrose: 3%～8%
Sorbitol or mannitol: 3%～8%
All the other are water
6. the preparation of claim 1, the weight proportion between each component of freeze drying protectant wherein is as follows:
Gelatin: 0.1%
Dextran: 8%
Trehalose: 5%
Sorbitol: 5%
All the other are water
7. the preparation of claim 1, the weight proportion between each component of freeze drying protectant wherein is as follows:
Gelatin: 0.05%
Dextran: 6%
Trehalose: 5%
Mannitol: 5%
All the other are water
8. protectant preparation method of claim 4 is characterized in that, the process following steps:
A takes by weighing an amount of gelatin 1g, and room temperature was placed 5 minutes, stirred under 40 ℃ of water bath condition, and it is uniformly dispersed, and handles 30 minutes with activated carbon adsorption under 90 ℃.
B takes by weighing dextran, trehalose, sucrose, sorbitol or mannitol or their combination in any, and each is an amount of, and heating is dissolved the back and added 90 ℃ of following adsorption treatment of active carbon of 0.8% 30 minutes.
C uses the water for injection standardize solution with the above-mentioned mixed solution of 0.45 μ M filtering with microporous membrane behind the mix homogeneously, 115 ℃ of autoclavings 20 minutes.Packing is standby under the aseptic condition of cooling back.
9. the production technology of the preparation of claim 1, the population growth of cell is controlled by temperature, ventilation and the incubation time of control seed culture state and amplification culture.The revolution that shakes bottle is controlled at 200～300rpm; The incubation time of cell can be finished by the monitoring dissolved oxygen, and cell detects OD550 through transferred species amplification culture a period of time between 0.5～0.8 by detecting the UV, visible light analyser; The cultivation temperature scope of competent cell is 20 ℃～30 ℃.
10. the production technology of the preparation of claim 1, the revolution that shakes bottle is controlled at 275rpm; Cell detects OD550 through transferred species amplification culture a period of time between 0.65～0.75 by detecting the UV, visible light analyser; The cultivation temperature of competent cell is 23 ℃.
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| CNA2007100643256A CN101264062A (en) | 2007-03-12 | 2007-03-12 | Production technology for preparing freeze-drying genetic engineering bacterium competence cell and protective agent formula |
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|---|---|---|---|
| CNA2007100643256A CN101264062A (en) | 2007-03-12 | 2007-03-12 | Production technology for preparing freeze-drying genetic engineering bacterium competence cell and protective agent formula |
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