CN1473846A - Method for producing rabbit alexin by high density heterotrophically cultivating transgenosis chlorella - Google Patents
Method for producing rabbit alexin by high density heterotrophically cultivating transgenosis chlorella Download PDFInfo
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Abstract
The method of producing rabbit alexin via high density heterotrophically cultivating transgenic chlorella includes adding into bioreactor pH 5.0-7.0 culture medium comprising KNO3, yeast powder, glucose, water and small amount of inorganic salt and trace elements; inoculating chlorella strain with transferred rabbit alexin gene in the amount of 5-15 % of working volume; culture at temperature 25-30 deg.c pH 8.5 and dissolved oxygen over 20 % until highest density of transgenic chlorella cell density; and purifying the cultured matter to obtain pure rabbit alexin. The said process has greatly raised chlorella culturing density in the case of unchanged NP-1 expression amount.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of high Density Heterotrophic transgenosis chlorella and produce the alexinic method of rabbit.
Background technology
People are longer to the research history of chlorella, only are the work of just carrying out in recent years but chlorella is studied as novel expression system.Up to the present, the report of relevant foreign gene energy stably express in chlorella is few.Chinese Academy of Sciences heredity imports chlorella as waiting with rabbit alexin NP-1 gene with the Sun Yong of developmental biology institute, make this gene as a kind of composing type albumen obtained stable expression (Sun Yongru, Fu Rongzhao, Cao Guangcheng, etc.The plant expression carrier plasmid of phylaxin gene.Chinese patent .C12N15/63,1126760,1996-07-17; Sun Yongru, Chen Ying, Ma Jiangsheng, etc.The high-efficient biological reactor of transgenosis chlorella.Chinese patent .C12N15/79,1203277,1998-12-30).This is the first report of foreign protein successful expression in chlorella.
Alexin has important potential using value clinically, but does not still have effective preparation method so far.Rabbit alexin (NP-1) is the widest a kind of of antimicrobial spectrum in the alexin.Change the effective ways that NP-1 gene chlorella is expected to become the alexin preparation.In view of the potential using value of NP-1 and the practical value of chlorella, change NP-1 gene chlorella and have broad application prospects in industries such as seawater seed rearing, sea farming, fodder additives, healthcare products and foodstuff additive; In addition, chlorella has tempting application prospect as a kind of novel expression system.
In view of the potential application of changeing NP-1 gene chlorella, it is this transgenosis chlorella one of gordian technique of practical application of successfully marching toward that high-density large-scale is cultivated.Chinese Academy of Sciences heredity and the Sun Yong of developmental biology institute are as waiting employings Knop mixotrophism substratum mixotrophism cultivation commentaries on classics NP-1 gene chlorella in shaking bottle, but culture efficiency is not high, high-cell density only is about 1.5g/L, can't obtain q.s the transgenosis chlorella cell to carry out the applied research of transgenosis chlorella.
The nutrition pattern that little algae is cultivated has three kinds: the light autotrophy is cultivated, mixotrophism is cultivated and heterotrophism is cultivated.It is the effective ways of realizing little algae high-density culture that heterotrophism is cultivated.The cultivation of the little algae of reporting in the document of transgenosis at present adopts the light autotrophy to cultivate mostly, at the bottom of there is cell density in this training method, culturing process is difficult to shortcomings such as amplification.So far, the little algae of heterotrophism cultivation transgenosis does not see bibliographical information.Therefore be necessary to carry out the research of transgenosis chlorella high-density culture.
Realization transgenosis chlorella high-density culture can be set about from three aspects: the one, determine best nutrition pattern, and the 2nd, select suitable medium, the 3rd, set up suitable culture process.Another patent of invention of applicant has provided the substratum that suitable heterotrophism is cultivated changes NP-1 gene chlorella, therefore is necessary the best nutritional pattern and the culture process that change NP-1 gene chlorella are further studied.
Summary of the invention
The objective of the invention is to solve how to make changes NP-1 gene chlorella and improves keeping under the constant situation of unit cell NP-1 expression amount its cell density to have significantly.
To achieve these goals, the suitableeest nutrition pattern that the present inventor confirms to change NP-1 gene chlorella is a heterotrophism, and further provides a kind of high Density Heterotrophic transgenosis chlorella to produce the alexinic method of rabbit, and this method comprises:
A). adding pH is 5.0～7.0 substratum in bio-reactor, and described substratum is by KNO
30.5～3 grams per liters, yeast powder 3～11 grams per liters, glucose 5～30 grams per liters and small amounts of inorganic salt, trace element and water are formed, press 5～15% of working volume and insert the cultivation of commentaries on classics rabbit phylaxin gene chlorella vulgaris, culture temperature is 25～30 ℃, pH is less than 8.5, the control dissolved oxygen finishes when the highest to cultivate to the transgenosis chlorella cell density more than 20%;
B). purifying obtains the rabbit alexin from step a) gained culture.
The high-density culture technology that the present invention sets up is suitable for NP-1 gene stably express in chlorella.Cultivate commentaries on classics NP-1 gene chlorella with the inventive method heterotrophism, every liter of cell density can reach 17～40g/L, change under the little situation at the NP-1 expression amount, the culture density that improves frond greatly can improve the alexinic output of rabbit greatly, thereby lays a good foundation for the successful Application of this transgenic alga.In addition, the inventive method is easy to operate, is suitable for suitability for industrialized production.
Description of drawings
Fig. 1 is the experimental result synoptic diagram of nutrition pattern among the embodiment 1.Among the figure :-△-expression light autotrophy cultured cells density;--expression heterotrophism cultured cells density;-■-expression mixotrophism cultured cells density.
Fig. 2 is an experimental result synoptic diagram of determining the suitableeest saltpetre concentration among the embodiment 2.Among the figure, △ represents 0.1g/L ▲ for 0.5g/L; ◇ is 0.9g/L; ● be 1.3g/L; Zero is 1.7g/L; is 2.1g/L; ■ is contrast.
Fig. 3 adds the experimental result synoptic diagram of concentration for determining the suitableeest saltpetre among the embodiment 2.Among the figure :-● add the cell density of saltpetre (saltpetre concentration maintains 0.9g/L) in-expression culturing process; Do not add the cell density of saltpetre in-zero-culturing process; Add the NP-1 expression amount of saltpetre (saltpetre concentration maintains 0.9g/L) in-■-culturing process; Do not add the NP-1 expression amount of saltpetre in--culturing process.
Fig. 4 adds the experimental result synoptic diagram of concentration for determining the suitableeest glucose among the embodiment 3.Among the figure: solid line is the curve that utilizes the Haldane model-fitting, and the point that looses is measured value.
Fig. 5 is the experimental result synoptic diagram of 5L bio-reactor among the embodiment 4.Among the figure: the cell density of-■-employing heterotrophism culture process of the present invention, 27.8g/L;-● the NP-1 expression amount of-employing heterotrophism culture process of the present invention.
Fig. 6 is the experimental result synoptic diagram of 15L bio-reactor among the embodiment 5.Among the figure: the cell density of-■-employing heterotrophism culture process of the present invention, 34.2g/L;-● the NP-1 expression amount of-employing heterotrophism culture process of the present invention.
Specific embodiments
The present invention relates to a kind of high Density Heterotrophic transgenosis chlorella and produce the alexinic method of rabbit, this method comprises:
A). adding pH is 5.0～7.0 substratum in bio-reactor, described substratum is made up of KNO3 0.5～3 grams per liter, yeast powder 3～11 grams per liters, glucose 5～30 grams per liters and small amounts of inorganic salt, trace element and water, press 5～15% of working volume and insert the cultivation of commentaries on classics rabbit phylaxin gene chlorella vulgaris, culture temperature is 25～30 ℃, pH is less than 8.5, the control dissolved oxygen finishes when the highest to cultivate to the transgenosis chlorella cell density more than 20%;
B). purifying obtains the rabbit alexin from step a) gained culture.
In the used substratum of the present invention, each component of substratum can change within the specific limits and can the cell density of transgenosis chlorella do not had a significant impact.For example, as inorganic nitrogen-sourced KNO
3To enclose be 0.5～3 grams per liter, can be between 3～11 grams per liters as the yeast powder of organic nitrogen source, can be between 5～30 grams per liters as the glucose of carbon source, so the consumption of these components should not be subjected to the restriction of embodiment.As known to those skilled in the art, also should add small amounts of inorganic salt in the substratum, for example sal epsom, sodium-chlor, lime carbonate, dipotassium hydrogen phosphate, potassium primary phosphate etc., and a small amount of trace element is as Mn, Zn, B, I, M, Cu, Co etc.In the present invention, preferable micro-component should be selected from H
3BO
5, ZnSO
47H
2O, MnSO
4H
2O, (NH
4)
6MoO
244H
2O, CuSO
45H
2O.
In a concrete scheme, described substratum is grouped into by following one-tenth basically: KNO
30.5～3 grams per liters, yeast powder 3～11 grams per liters, glucose 5～30 grams per liters, KH
2PO
40.1～0.3 grams per liter, Ca (NO
3)
20.15～0.45 grams per liter, MgSO
47H
2O 0.01～0.1 grams per liter, KCl 0.05～0.15 grams per liter, FeCl
30.005～0.03 grams per liter; Trace element 0.5～3ml, wherein trace element consists of H
3BO
40.061 grams per liter, ZnSO
47H
2O 0.287 grams per liter, MnSO
4H
2O 0.169 grams per liter, (NH
4)
6MoO
244H
2O0.01235 grams per liter, CuSO
45H
2O 0.00249 grams per liter; Water.
In a better scheme, described substratum consist of glucose 15g/L, KNO
30.9g/L, yeast powder 9g/L, KH
2PO
40.13g/L, Ca (NO
3)
20.2g/L, MgSO
47H
2O 0.05g/L, KCl 0.06g/L, FeCl
30.01g/L, H
3BO
40.061mg/L, ZnSO
47H
2O 0.287mg/L, MnSO
4H
2O 0.169mg/L, (NH
4)
6MoO
244H
2O 0.01235mg/L, CuSO
45H
2O 0.00249mg/L.
After becoming culture media composition according to above-mentioned formulated, can be 5.0～7.0 with the pH of described substratum, and 115-120 ℃ of following autoclaving 15～20 minutes.
After adding above-mentioned substratum in the bio-reactor, add 60～80% back sterilizations of water (should be tap water) to working volume.Then, when temperature is reduced to 25～30 ℃, insert transgenosis chlorella by 5～15% of working volume and begin the heterotrophism cultivation.Culture condition is: temperature: 25～30 ℃, the control dissolved oxygen is more than 20%, and pH is less than 8.5.
In order to obtain higher cell density, begin to add feed supplement liquid in the time of can after inoculation, cultivating 36～60h under the above-mentioned condition, added feed supplement liquid afterwards every 6～14 hours.Cultivation finishes to cultivate when commentaries on classics NP-1 gene chlorella cells density is the highest.The concentration of feed supplement liquid is suitable higher, and purpose is to make the nutrient solution volume be unlikely to feed supplement and too big.
In a preferable embodiment, the component of feed supplement is glucose and KNO
3The adding concentration and can be determined of glucose according to the Haldane model.According to the present invention, it is maximum that glucose feed supplement concentration specific growth rate when 6～10g/L reaches.KNO
3The concentration of adding of solution is preferably 0.9g/L, because under this concentration, the cell density that changes rabbit phylaxin gene chlorella is higher, and the NP-1 expression amount is also higher.
The feed supplement mother liquor will separately be sterilized.In the feed supplement process if the rapidly phenomenon of drop of dissolved oxygen occurs, can regulate rotating speed, dissolved oxygen is remained on the certain level, because the not enough phenomenon that can suppress the growth of frustule even the frond self-dissolving occur of dissolved oxygen, thereby the further raising of NP-1 gene chlorella cells density is changeed in influence.When cultivating, should be noted that pH is unsuitable too high because pH too high frustule be difficult for growth.When pH is higher than 8.5, should use for example 10% sulfuric acid adjusting pH.
After cultivating end, can adopt known ordinary method purifies and separates from the gained culture to obtain the rabbit alexin.
To further illustrate the present invention by embodiment below.Yet, it will be appreciated by those skilled in the art that the present invention is not limited to concrete numerical value and the means among these embodiment.
Embodiment 1: heterotrophism is as the suitableeest nutrition pattern
Shake the 100ml Knop mixotrophism substratum of packing in the bottle at 250ml, change NP-1 gene chlorella light autotrophy, heterotrophism and mixotrophism respectively and cultivate.The incident intensity that the light autotrophy is cultivated and mixotrophism is cultivated is 3klx.Inoculum size is 0.207g/L, and temperature is 28 ℃, incubation time 72 hours.As shown in Figure 1, the maximum cell density (△) of light autotrophy is 0.6g/L, and heterotrophic maximum cell density () is 1.52g/L, and mixotrophic maximum cell density (■) is 1.17g/L.The NP-1 expression amount of light autotrophy (representing with single celled antibacterial circle diameter) is 17.4mm, and heterotrophic NP-1 expression amount is 19.4～21.3mm, and mixotrophic NP-1 expression amount is 20.1～21.8mm (seeing the following form 1).Therefore, select heterotrophism as the suitableeest nutrition pattern of changeing NP-1 gene chlorella.
Table 1
Incubation time antibacterial circle diameter (mm)
(h) mixotrophism heterotrophism light autotrophy
23 21.8 20.8
38 21.6 19.6
47 21.6 20.1
58 21.2 19.4
70 20.1 21.3 17.4
Embodiment 2:KNO
3Add determining of concentration
Shake the following substratum of 100ml of packing in the bottle at 250ml,
Nitrogenous source: KNO
3: 0.9 yeast powder: 9
Carbon source: glucose: 15
Inorganic salt: KH
2PO
4: 0.13 Ca (NO
3)
2: 0.2
MgSO
4·7H
2O：0.05 KCl：0.06
FeCl
3：0.01
Trace element (mg/L): H
3BO
4: 0.061 ZnSO
47H
2O:0.287
MnSO
4·H
2O：0.169 (NH
4)
6MoO
24·4H
2O：0.01235
CuSO
45H
2O:0.00249 is just with KNO in the substratum
3Concentration become 0.1g/L, 0.5g/L, 0.9g/L, 1.3g/L, 1.7g/L, 2.1g/L respectively, and not add KNO
3Substratum do contrast.When cell density is low, in substratum, add the growth that whether saltpetre does not influence is changeed NP-1 gene chlorella; From 67h, the saltpetre of interpolation different concns reveals obvious facilitation to the growth table of transgenic alga, and the cell density comparison is according to being significantly improved.Cultivate 96h, be respectively 3.78g/L, 4.86g/L, 5.11g/L, 5.28g/L, 5.41g/L, 5.21g/L, and contrast has only the 3.29g/L (see figure 2) according to saltpetre concentration order corresponding biomass from low to high.The saltpetre of interpolation different concns is listed in the following table 2 influence of NP-1 expression amount in the transgenosis chlorella.
Table 2
KNO
3Starting point concentration (g/L) 0 0.1 0.5 0.9 1.3 1.7 2.1
Antibacterial circle diameter (mm) 21.9 22.2 21.5 21 17 17.9 17.9
By table 2 as seen, when saltpetre concentration during at 0.1g/L～0.9g/L, antibacterial circle diameter changes less, when saltpetre concentration surpasses 0.9g/L, antibacterial circle diameter drops to about 17mm from 21mm, therefore from the cell density and the NP-1 expression amount of transgenic alga, the interpolation concentration of saltpetre is advisable with 0.5-0.9g/L, and that better is 0.9g/L.Under this concentration, the density of frustule is 1.55 times of contrast, and antibacterial circle diameter (being the expression amount of NP-1) is constant substantially.
As can be seen from Figure 3, adding saltpetre in culturing process does not influence and changes NP-1 gene chlorella growth and NP-1 expresses, and to add concentration be 0.9g/L to the best of saltpetre when therefore determining bioreactor culture.
Embodiment 3: glucose is added determining of concentration
Shake the substratum of 100ml shown in embodiment 2 of packing in the bottle at 250ml, KNO3 concentration 0.9g/L in the substratum, the glucose concn that changes simultaneously in the substratum is 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, 20g/L, 30g/L, 40g/L, 50g/L, 60g/L, 70g/L, with the influence of research glucose concn to commentaries on classics NP-1 gene chlorella growth.
Fig. 4 has provided the relation between initial glucose concentration and the commentaries on classics NP-1 gene chlorella specific growth rate.By this figure as seen, when initial glucose concn not simultaneously, the specific growth rate of transgenosis chlorella is also different.The glucose of high density can suppress to change the growth of NP-1 gene chlorella, makes its specific growth rate less.The glucose of high density is because glucose concn when higher to the restraining effect of growth, and the absorption and the energy transformation of glucose are restricted.When initial glucose concn was 70g/L, it is minimum that specific growth rate reaches, and only is 0.0293h
-1When glucose concn was 7g/L, specific growth rate reached maximum value 0.0696h
-1By fitting of a curve, obtain changeing the Haldane equation of NP-1 gene chlorella:
Wherein: μ m=0.088h
-1, K
S=0.98g/L, Ki=39.68g/L; Optimum glucose concn is S
Mi=6.24g/L, corresponding specific growth rate μ
Mi=0.0669h
-1As seen from Figure 4, glucose concn is when 6～10g/L, and the specific growth rate of transgenic alga maintains on the higher level.This shows, cultivate the glucose concn that changes in the NP-1 gene chlorella process at the bio-reactor heterotrophism and should be controlled in 6～10g/L scope.
Embodiment 4:5L bioreactor culture
Add the substratum among the embodiment 2 in the 5L bio-reactor, add tap water and sterilize to 3.5L, insert commentaries on classics NP-1 gene chlorella by 10% of working volume then when temperature is reduced to 28 ℃, the beginning heterotrophism is cultivated.Culture condition: temperature is 28 ℃, and rotating speed is adjusted to the 400r/min that cultivates when finishing gradually from the 200r/min in when inoculation, and air flow quantity is 1: 1, and pH is less than 8.5, and the control dissolved oxygen is more than 40%.Begin to add feed supplement liquid during the 60h of inoculation back, added KNO afterwards every 12 hours
3, making its concentration is 0.9g/L; Added glucose every 8～12 hours, making its concentration is 7～10g/L.As shown in Figure 5, change NP-1 gene chlorella cells density (■) after 131 hours and reach 27.8g/L, cell density descends afterwards, finishes to cultivate.
Embodiment 5:15L bioreactor culture
Add the substratum among the embodiment 2 in the 15L bio-reactor, add tap water and sterilize to 10L, insert commentaries on classics NP-1 gene chlorella by 10% of working volume then when temperature is reduced to 28 ℃, the beginning heterotrophism is cultivated.Culture condition: temperature is 28 ℃, and rotating speed is adjusted to the 450r/min that cultivates when finishing gradually from the 200r/min in when inoculation, and air flow quantity is 1: 1, and pH is less than 8.5, and the control dissolved oxygen is more than 30%.Begin to add feed supplement liquid during the 60h of inoculation back, added KNO afterwards every 12 hours
3, making its concentration is 0.9g/L; Added glucose every 8～12 hours, making its concentration is 7～10g/L, and 133h changes NP-1 gene chlorella cells density (■) and reaches 34.2g/L, finishes to cultivate.
According to the foregoing description as can be seen, high-density cultivation method of the present invention has improved cell density greatly under the constant substantially situation of NP-1 expression amount, for the successful Application of this commentaries on classics NP-1 gene chlorella is laid a good foundation.
Claims (8)
1. a high Density Heterotrophic transgenosis chlorella is produced the alexinic method of rabbit, it is characterized in that this method comprises:
A). adding pH is 5.0～7.0 substratum in bio-reactor, and described substratum is by KNO
30.5～3 grams per liters, yeast powder 3～11 grams per liters, glucose 5～30 grams per liters and small amounts of inorganic salt, trace element and water are formed, press 5～15% of working volume and insert the cultivation of commentaries on classics rabbit phylaxin gene chlorella vulgaris, culture temperature is 25～30 ℃, pH is less than 8.5, the control dissolved oxygen finishes when the highest to cultivate to the transgenosis chlorella cell density more than 20%;
B). purifying obtains the rabbit alexin from step a) gained culture.
2. method according to claim 1 is characterized in that, can be after inoculation feed supplement during 36～60h, afterwards every feed supplement in 6～14 hours.
3. method according to claim 2 is characterized in that, described feed supplement is to add glucose and KNO
3Solution.
4. method according to claim 3 is characterized in that, the feed supplement concentration of described glucose is the 6-10 grams per liter.
5. method according to claim 3 is characterized in that, described KNO
3The concentration of adding of solution is 0.9 grams per liter.
6. method according to claim 1 is characterized in that, when the pH of nutrient solution was higher than 8.5, the sulfuric acid of available 10% volume was regulated.
7. method according to claim 1 is characterized in that described substratum is grouped into by following one-tenth basically: KNO
30.5～3 grams per liters, yeast powder 3～11 grams per liters, glucose 5～30 grams per liters, KH
2PO
40.1～0.3 grams per liter, Ca (NO
3)
20.15～0.45 grams per liter, MgSO
47H
2O 0.01～0.1 grams per liter, KCl 0.05～0.15 grams per liter, FeCl
30.005～0.03 grams per liter; Trace element 0.5～3ml, wherein trace element consists of H
3BO
40.061 grams per liter, ZnSO
47H
2O 0.287 grams per liter, MnSO
4H
2O 0.169 grams per liter, (NH
4)
6MoO
244H
2O 0.01235 grams per liter, CuSO
45H
2O 0.00249 grams per liter; Water.
8. heterotrophism culture process according to claim 1 is characterized in that substratum adopts the tap water preparation.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1309820C (en) * | 2005-08-25 | 2007-04-11 | 北京科技大学 | Culturing method for heterotrophic chlorella growth without irradiation |
| CN102603885A (en) * | 2011-12-26 | 2012-07-25 | 中国科学院遗传与发育生物学研究所 | Alexin and application thereof to preparation of antibacterial medicament |
| CN102643751A (en) * | 2012-04-25 | 2012-08-22 | 同济大学 | Method for quickly separating and purifying chlorella |
| CN104164366A (en) * | 2013-05-16 | 2014-11-26 | 中国石油化工股份有限公司 | Culture method of microalgae |
-
2003
- 2003-08-11 CN CNA031421954A patent/CN1473846A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1309820C (en) * | 2005-08-25 | 2007-04-11 | 北京科技大学 | Culturing method for heterotrophic chlorella growth without irradiation |
| CN102603885A (en) * | 2011-12-26 | 2012-07-25 | 中国科学院遗传与发育生物学研究所 | Alexin and application thereof to preparation of antibacterial medicament |
| CN102603885B (en) * | 2011-12-26 | 2014-06-18 | 保罗生物园科技股份有限公司 | Alexin and application thereof to preparation of antibacterial medicament |
| CN102643751A (en) * | 2012-04-25 | 2012-08-22 | 同济大学 | Method for quickly separating and purifying chlorella |
| CN104164366A (en) * | 2013-05-16 | 2014-11-26 | 中国石油化工股份有限公司 | Culture method of microalgae |
| CN104164366B (en) * | 2013-05-16 | 2017-10-03 | 中国石油化工股份有限公司 | A kind of method of cultivating microalgae |
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