CN1405319A - Method for producing human or animal metallothionein product using liquid submerged fermented transgenic engineered bacteria - Google Patents
Method for producing human or animal metallothionein product using liquid submerged fermented transgenic engineered bacteria Download PDFInfo
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- CN1405319A CN1405319A CN 01128543 CN01128543A CN1405319A CN 1405319 A CN1405319 A CN 1405319A CN 01128543 CN01128543 CN 01128543 CN 01128543 A CN01128543 A CN 01128543A CN 1405319 A CN1405319 A CN 1405319A
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Abstract
The invention refers to a method of producing human or animal metal sulfur albumen (MT) goods by liquid's deep-layer fermenting gene-transferring engineering bacterium. It largely cultivates reformed engineering bacterium expressing human or animal MT by deep-layer fermenting, and by rough filtering, centrifugal washing, makes active bacterium frozen and drought foods expressing aim MT or by polyelectrolyte complexing of reformed engineering bacterium centrifugally washed makes active bacterium microcapsule expressing aim MT.
Description
Technical field
The present invention relates to a kind of liquid submerged fermented transgenic engineered bacteria and produce the method for human or animal metallothionein product using.
Background technology
From reports such as nineteen fifty-seven Margoshes and Vellee are isolated metallothionein(MT) (hereinafter to be referred as MT) from the horse kidney of accumulating cadmium since; a large amount of papers is to structure, physics and chemistry and biological characteristics, separation and purification and the detection method of MT; genomic constitution and regulation and control; and done research and discussed in medical treatment, health care, food, makeup, enriching precious metal with at numerous areas such as aspect the environment protection; shown the tempting prospect of MT, be subjected to the extensive concern of countries in the world correlation theory research and application facet.
At present, domestic patent about the MT aspect also is not a lot, relates to production method still less, and the patent relevant with MT goods formulation almost still belongs to blank.
In view of MT is a kind of stress protein, directly production method can induce certain general domesticated animal (as rabbit) can produce MT by certain heavy metal species (as Zn++), adopts the separation from this animal viscera of corresponding physics and chemistry technology, purifying then and obtains corresponding articles.But there are two weakness in this kind approach: 1, induce certain animal can only obtain this kind animal corresponding M T; 2, feeding animals is subject to the restriction of natural condition and scale.
Data shows that the coordination position of structure, aminoacid sequence, metal ion and the halfcystine of the Mammals MT that had studied is identical, thereby shows its conservative property in organic evolution.But be in the MT of Different Evolutionary level biology, must have its structural otherness, animal kingdom is no exception, and as the MT of giant salamander (Megalobatrachus davidinus), its structure just is different from mammiferous MT.The different structure of MT provides basic substance for its different biological functions or function difference, also will provide multiple new approach for its utilization.Yet not all animal (as rare or animals on the brink of extinction) can both be extracted MT in a large number by obtaining internal organs.
Summary of the invention
The objective of the invention is to provide a kind of liquid submerged fermented transgenic engineered bacteria to produce the method for human or animal metallothionein product using, processing method by the fermentation recombinant bacterial strain, obtain purpose MT in a large number, make: 1, expressed purpose MT is people or any animal, be not subjected to the restriction of people or certain animal viscera source difficulty, all can carry out suitability for industrialized production; 2, production process is not subjected to the restriction of natural condition, and is easy to mass-producing; 3, by seed selection or improvement fermentation condition, can improve constantly output, reduce production costs recombinant bacterial strain; 4, the existing report of the biology of viable bacteria MT even pharmacology function can be expected, it is applied to medicine and protective foods is fully possible.Viable bacteria MT goods also will provide the instrument that can add utilization for the pollution to environment of some heavy metal of enrichment, recovery and removing of noble metal.
The objective of the invention is to realize with the following methods.The method that this liquid submerged fermented transgenic engineered bacteria is produced human or animal metallothionein product using is that transgenic engineered bacteria is passed through liquid submerged fermentation, the recombinant bacterial strain of a large amount of culture expression human or animal MT, and then remove slag, centrifuge washing, produce the recombinant bacterial strain viable bacteria freeze-dried products of expressing purpose MT by coarse filtration; Or with the recombinant bacterial strain of centrifuge washing through polyelectrolyte complex, produce and contain the recombinant bacterial strain viable bacteria microcapsule of expressing purpose MT.Recombinant bacterial strain can be the microorganism that contains protokaryon, eucaryon.The MT mono-clonal of its expression can be the people, also can be any animal.The active bacteria formulation formulation of expressing human or the corresponding MT of certain animal is freeze-dried products or microcapsule.
The concrete steps of production method of the present invention can comprise:
1, the fermentation of recombinant bacterial strain
1) substratum
By recipient bacterium the nutritional condition requirement is decided.Prokaryotic micro-organisms such as intestinal bacteria (E.coli) are basic medium with the LB substratum; Eukaryotic microorganisms such as yeast saccharomyces cerevisiae (S.cerevisiae) are basic medium with the YPD substratum.
Bacteria culture medium (weight %)
Yeast powder (or extract): 0.2-1.2 peptone: 0.5-1.5
Other are water for NaCL:0.2-1.2
PH：6.8-7.2
Microzyme culture medium (weight %)
Glucose: 1.5-2.5 yeast extract: 0.5-1.5
Other are water for Tryptones: 1.5-2.5
PH：4.5-5.5
Its feature 1) be to add the selective pressure composition in slant strains and level liquid seed culture medium, this composition is decided on certain antibiotics resistance gene that vector plasmid had, and corresponding microbiotic addition is 5～300 μ g/ml;
Its feature 2) be to add heavy metal ion (as Zn++, Cu++ etc.) in level liquid seed culture medium and fermention medium, addition is 0.01～0.5%.2) fermentation condition
Inoculum size: 1～10%
The leavening temperature of growing microorganism is 20～45 ℃; 20～37 ℃ of purpose MT synthesis phase leavening temperatures; Dissolved oxygen is 10～60%
Fermented incubation time: 5～70h
Stirring velocity: 50～800r/min
Air flow (liquid V/ gas v): 1: 0.2～3
Glucose flow feeding time and concentration: the beginning after 3～6 hours of fermenting, control final concentration 0.05～0.5mol/L
Inductive condition: inductor is promptly decided as interpolation isopropyl-(IPTG) IPTG final concentration 0.1～10mmol/L by the character decision of the promotor of recombinant bacterial strain on the character of vector plasmid promotor; And for example alternating temperature is cultivated: 25～45 ℃; Or interpolation heavy metal ion (as Cu++, Ag++ etc.), final concentration 0.1～10mmol/L.The inducing culture time: before the logarithmic growth, mid-term, beginning after fermenting about 4～48 hours, inducing culture 3～30h.2, the production technique of viable bacteria MT freeze-dried products
1) the fermented liquid Plate Filtration is removed slag, collect filtrate.
2) 4000～4500r/min collected thalline in centrifugal 20～30 minutes.
3) with Sterile Saline (NaCL 0.85%) centrifugal (the rotating speed time is the same) washing 3 times, collect thalline.
4) bacteria containing amount adds protective material (final concentration 5～15% skim-milks+3～8% sucrose on demand
+0.05～
0.5% potassiumiodide), packing ,-35～-45 ℃ of vacuum freezedryings become goods.
5) preservation-5 ℃.3, the production technique of viable bacteria MT microencapsulated product
1) the fermented liquid Plate Filtration is removed slag, collect filtrate.
2) 4000～4500r/min collected thalline in centrifugal 20～30 minutes.
3) with Sterile Saline (NaCL 0.85%) centrifugal (the rotating speed time is the same), wash 3 times, get wet thallus.
4) 1 part of wet thallus (W)+2 part (V) aseptic deionized water+2 parts (V) 4% sterile sodium alginate solution fully is mixed into bacteria suspension.
5) the suitable pressure of this suspension, aperture splash into 20 parts of aseptic CaCL of (V) 0.05mol/L
2In the solution, become the 2.7-4.0mm diameter sphere, placed 1 hour.
6) CaCL that inclines
2Solution, 40 parts of (V) aseptic deionized waters wash 1 time.
7) add 20 parts of aseptic CaCL of (V) 0.05mol/L again
2Solution, 4 ℃ of balances 10～12 hours.
8) CaCL that inclines
2Solution injects 10 parts of (V) Sterile Salines (NaCL 0.85%) solution (or aseptic LB liquid nutrient medium), and is aseptic subpackaged.
9) deposit preservation for 4 ℃.Annotate:
1, the production of above-mentioned lyophilized powder and microencapsulated product is all carried out in constant temperature clean room (below 20 ℃, clean 100 grades).
2, the used aseptic NaCl solution of above-mentioned technology, aseptic deionized water and aseptic CaCl
2Solution etc. are all earlier through high pressure (1.1kg/cm
2, 30min) sterilization.Sterile sodium alginate solution, earlier through 100 ℃ of tyndallizations or use 0.22～1.0 μ m filtering with microporous membrane degerming, but also autoclaving.Advantage of the present invention and effect
1. that the recombinant bacterial strain of the present invention's use is expressed is the MT of people or any animal, is not subjected to the restriction of people or certain animal (especially rare or animals on the brink of extinction) internal organs source difficulty, can make it carry out mass-producing, batch production production.
2. liquid submerged fermentation recombinant bacterial strain, thalline specific growth rate height, MT is fast in endobacillary biosynthesizing, and is with short production cycle, and all fermenting processs were generally all finished in tens of hours, and production cost is lower, and production process is not subjected to the restriction of natural condition.
3. use active bacteria formulation, production technique is simplified, and has avoided needed number of chemical reagent or organic solvent in scale operation MT monomer separation, the purge process, and the full scale production process does not cause any pollution to environment.
4. medicine and the protective foods that is used for oral administration for MT provides a kind of novel form; For the enrichment of the micro-noble metal of some occasion (as Ag etc.) or eliminate some heavy metal (as Hg, Cd etc.) and pollute that provide can be for the new tool of utilization.
It below is non-limiting examples of the present invention
1, recombinant bacterial strain
The MT recombinant bacterial strain uses rabbit MT recombinant bacterial strain.Recipient bacterium is E.coli:M
15[PREP
4] carrier: plasmid PQE-30, inductor: IPTG, selective pressure: kantlex.
2, slant strains is cultivated
LB solid medium (weight %)
Yeast powder: 0.5
Peptone: 1.0
NaCl：0.5
Agar: 1.8
30 ℃ of cultivation 24h are stand-by
3, liquid seeds is cultivated
Liquid seed culture medium (weight %)
Yeast powder: 0.5
Peptone: 1.0
ZnSO
4·7H
2O：0.03
Kantlex: 7.5mg/150ml (millipore filtration degerming)
PH：7.0
The liquid seeds culture condition
500ml triangular flask substratum loading amount: 150ml
Culture temperature: 30 ℃
Shaking speed: 240r/min
It is stand-by to cultivate 24h
4, liquid submerged fermentation
1) fermention medium (weight %)
Yeast powder: 1.0
Peptone: 2.0
NaCL：1.0
K
2HPO
4：0.04
KH
2PO
4：0.02
ZnSO
4·7H
2O：0.03
Glucose: 1.0
Vegetables oil: 0.13～0.15
PH：7.0
2) fermentation condition:
The 3L fermentor tank, substratum loading amount 2L, inoculum size 5%.Stirring velocity 240～600r/min, air flow (liquid V/ gas V) 1: 1～2 is regulated stirring velocity and air flow dissolved oxygen is maintained about 30%.37 ± 1 ℃ of growing microorganism leavening temperatures.The beginning of fermenting after 2 hours is per hour measured reducing sugar, acetate and PH each 1 time, ferments to begin to add glucose in 3 hours and (control its final concentration 0.1～0.15mol/L).Add ammoniacal liquor and regulate PH7～7.2.Cultivate about 8 hours bacteriums and enter logarithmic phase, (control its final concentration 1～1.2mmol/L), reduce to 30 ± 1 ℃ with leavening temperature this moment, carries out gentle inducing culture about 6 hours, finishes fermentation to add IPTG.
Measuring final viable count is 6～8 * 10
9Individual/ml, rabbit Zn-MT accounts for about 25% of bacterial protein content.
3, rabbit Zn-MT viable bacteria freeze-dried products produces
1) will ferment through the filtration of sheet frame canvas, collect filtrate.
2), collect thalline with centrifugal 30 minutes of filtrate 4000r/min.
3) with stroke-physiological saline solution (NaCL 0.85%) centrifugal (the rotating speed time is the same), wash 3 times, get wet thallus 100g and (calculate total viable count and be about 14000 * 10
9Individual).
4) 100g wet thallus+600ml sterilized water+aseptic potassiumiodide 0.35g of the aseptic sucrose 35g+ of aseptic skim-milk 70g+ is stirred into bacteria suspension.
5) aseptic subpackaged ampoule, the 1ml/ bottle.
6) ℃ vacuum freezedrying-40, sealing by fusing.
7) the sampling observation viable count on average 〉=10 * 10
9Individual/ml, freeze-drying survival rate about 67%.
4, rabbit Zn-MT viable bacteria microcapsule produces
1) fermented liquid is filtered through the sheet frame canvas, collect filtrate.
2), collect thalline with centrifugal 30 minutes of filtrate 4000r/min.
3) with stroke-physiological saline solution (NaCL 0.85%) centrifugal (the rotating speed time is the same) washing 3 times,
Wet thallus 100g (calculates total viable count and is about 14000 * 10
9Individual).
4) 100g wet thallus+200ml aseptic deionized water+200ml 4% sterile sodium alginate solution fully is mixed into bacteria suspension.
5) bacteria suspension impouring bottom is connected in the groove of some vertebra shape pipes, vertebra shape pipe outlet internal diameter is 0.3～0.5mm, allows bacteria suspension splash into by vertebra shape pipe and fills the aseptic CaCL of 2000ml 0.05mol/L
2In the basin of solution, bacteria suspension solidifies and is 2.7～3.0mm diameter sphere microcapsule.Placed 1 hour for 20～22 ℃.
6) CaCL that inclines
2Solution is with 4000ml left and right sides aseptic deionized water flushing microcapsule.
7) add the aseptic CaCL of 2000ml 0.05mol/L again
2Solution, 4 ℃ of balances 12 hours.
8) CaCL that inclines
2Solution injects the aseptic LB liquid nutrient medium of 1000ml, and is aseptic subpackaged.
9) put 4 ℃ of preservations.
10) sampling observation microcapsule viable count 〉=15 * 10
9Individual/g, encystation surviving rate about 63%.
Claims (6)
1, a kind of liquid submerged fermented transgenic engineered bacteria is produced the method for human or animal metallothionein product using, it is characterized in that it being that transgenic engineered bacteria is passed through liquid submerged fermentation, the recombinant bacterial strain of a large amount of culture expression human or animal MT, and then remove slag, centrifuge washing, produce the recombinant bacterial strain viable bacteria freeze-dried products of expressing purpose MT by coarse filtration; Or with the recombinant bacterial strain of centrifuge washing through polyelectrolyte complex, produce and contain the recombinant bacterial strain viable bacteria microcapsule of expressing purpose MT.
2, liquid submerged fermented transgenic engineered bacteria according to claim 1 is produced the method for human or animal metallothionein product using, it is characterized in that recombinant bacterial strain can be the microorganism that contains protokaryon, eucaryon, the MT mono-clonal of its expression can be the people, also can be any animal.
3, liquid submerged fermented transgenic engineered bacteria according to claim 1 is produced the method for human or animal metallothionein product using, it is characterized in that the concrete steps of production method can comprise:
(1), the fermentation of recombinant bacterial strain
1) substratum
By recipient bacterium the nutritional condition requirement is decided: prokaryotic micro-organisms such as intestinal bacteria (E.coli) are basic medium with the LB substratum; Eukaryotic microorganisms such as yeast saccharomyces cerevisiae (S.cerevisiae) are basic medium with the YPD substratum;
Bacteria culture medium (weight %)
Yeast powder (or extract): 0.2-1.2 peptone: 0.5-1.5
Other are water for NaCL:0.2-1.2
PH：6.8-7.2
Microzyme culture medium (weight %)
Glucose: 1.5-2.5 yeast extract: 0.5-1.5
Other are water for Tryptones: 1.5-2.5
PH：4.5-5.5
2) fermentation condition
Inoculum size: 1～10% leavening temperature: 20～45 ℃
Fermented incubation time: 5～70h stirring velocity: 50～800r/min
Air flow (liquid V/ gas v): 1: 0.2～3
Glucose flow feeding time and concentration: the beginning after 3～6 hours of fermenting, control
Final concentration 0.05～0.5mol/L
Inductive condition: the character on the vector plasmid promotor is decided.As add IPTG,
Final concentration 0.1～10mmol/L; Alternating temperature: 25～45 ℃ or interpolation heavy metal ion
(Cu++, Zn++ etc.), final concentration 0.1～10mmol/L.
The inducing culture time: before the logarithmic growth, mid-term, opened after fermenting about 4～48 hours
Beginning, inducing culture 3～30h.
(2), the production technique of viable bacteria MT freeze-dried products
1) the fermented liquid Plate Filtration is removed slag, collect filtrate.
2) 4000～4500r/min collected thalline in centrifugal 20～30 minutes.
3) with Sterile Saline (NaCL 0.85%) centrifugal (the rotating speed time is the same) washing 3 times, collect thalline.
4) bacteria containing amount adds protective material (final concentration 5～15% skim-milks+3～8% sucrose+0.05～0.5% potassiumiodide) on demand, packing, and-35～-45 ℃ of vacuum freezedryings become goods.
5) preservation-5 ℃.
(3), the production technique of viable bacteria MT microencapsulated product
1) the fermented liquid Plate Filtration is removed slag, collect filtrate.
2) 4000～4500r/min collected thalline in centrifugal 20～30 minutes.
3) with Sterile Saline (NaCL 0.85%) centrifugal (the rotating speed time is the same), wash 3 times, get wet thallus.
4) 1 part of wet thallus (W)+2 part (V) aseptic deionized water+2 parts (V) 4% sterile sodium alginate solution fully is mixed into bacteria suspension.
5) the suitable pressure of this suspension, aperture splash into 20 parts of aseptic CaCL of (V) 0.05mol/L
2In the solution, become the 2.7-4.0mm diameter sphere, placed 1 hour.
6) CaCL that inclines
2Solution, 40 parts of (V) aseptic deionized waters wash 1 time.
7) add 20 parts of aseptic CaCL of (V) 0.05mol/L again
2Solution, 4 ℃ of balances 10～12 hours.
8) CaCL that inclines
2Solution injects 10 parts of (V) Sterile Salines (NaCL 0.85%) solution (or aseptic LB liquid nutrient medium), and is aseptic subpackaged.
9) deposit preservation for 4 ℃.
4, liquid submerged fermented transgenic engineered bacteria according to claim 3 is produced the method for human or animal metallothionein product using, it is characterized in that it being in slant strains and level liquid seed culture medium, to add the selective pressure composition, this composition is decided on certain antibiotics resistance gene that vector plasmid had, and corresponding microbiotic addition is 5～300 μ g/ml; Be to add heavy metal ion such as Zn++, Cu++ etc. in level liquid seed culture medium and fermention medium, addition is 0.01～0.5%.
5, liquid submerged fermented transgenic engineered bacteria according to claim 3 is produced the method for human or animal metallothionein product using, it is characterized in that the production of viable bacteria MT freeze-dried products and microencapsulated product, all at the constant temperature clean room: below 20 ℃, carry out in clean 100 grades.
6, liquid submerged fermented transgenic engineered bacteria according to claim 3 is produced the method for human or animal metallothionein product using, it is characterized in that used aseptic NaCl solution, aseptic deionized water and aseptic CaCl
2Solution etc. are all earlier through high pressure (1.1kg/cm
2, 30min) sterilization, sterile sodium alginate solution, earlier through 100 ℃ of tyndallizations or use 0.22～1.0 μ m filtering with microporous membrane degerming, but also autoclaving.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01128543 CN1405319A (en) | 2001-08-13 | 2001-08-13 | Method for producing human or animal metallothionein product using liquid submerged fermented transgenic engineered bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01128543 CN1405319A (en) | 2001-08-13 | 2001-08-13 | Method for producing human or animal metallothionein product using liquid submerged fermented transgenic engineered bacteria |
Publications (1)
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|---|---|
| CN1405319A true CN1405319A (en) | 2003-03-26 |
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ID=4668389
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100348179C (en) * | 2005-11-01 | 2007-11-14 | 浙江大学 | Prepn process of microcapsule with included anticancer medicine |
| CN103525713A (en) * | 2013-09-17 | 2014-01-22 | 东华理工大学 | Gene-recombinant saccharomyces cerevisiae for efficiently adsorbing uranium contained in water solution and construction method thereof |
| CN108404860A (en) * | 2018-05-18 | 2018-08-17 | 刘凡领 | A kind of preparation method of inorganic heavy metal ion sorbing material |
-
2001
- 2001-08-13 CN CN 01128543 patent/CN1405319A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100348179C (en) * | 2005-11-01 | 2007-11-14 | 浙江大学 | Prepn process of microcapsule with included anticancer medicine |
| CN103525713A (en) * | 2013-09-17 | 2014-01-22 | 东华理工大学 | Gene-recombinant saccharomyces cerevisiae for efficiently adsorbing uranium contained in water solution and construction method thereof |
| CN108404860A (en) * | 2018-05-18 | 2018-08-17 | 刘凡领 | A kind of preparation method of inorganic heavy metal ion sorbing material |
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