CN103993057B - The derivational expression method of Research of Recombinant Human Endostatin - Google Patents
The derivational expression method of Research of Recombinant Human Endostatin Download PDFInfo
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Abstract
The derivational expression method of the Research of Recombinant Human Endostatin of the present invention, by engineering bacterium fermentation to fermentation liquid OD600Value is 10 15, is added thereto to derivant IPTG, carbon source material and nitrogen source, and making the concentration of IPTG in fermentation liquid is 0.2 0.4mmol/L, at 30 37 DEG C, cultivate 2.5 3.5h, then be added thereto to the derivant IPTG of final concentration of 0.1 0.3mmol/L, continue to cultivate 3 5h,.The derivational expression method of the Research of Recombinant Human Endostatin of the present invention, can meet the needs of the large-scale production of Research of Recombinant Human Endostatin, realizes efficient abduction delivering under higher cell concentration.
Description
Technical field
The invention belongs to the extraction purification field of Research of Recombinant Human Endostatin, be specifically related to a kind of restructuring
The derivational expression method of Human endostatin.
Background technology
Human endostatin, is that the tumor-blood-vessel growth that effect is the strongest at present, experiment effect is best presses down
Preparation.Experiment shows, Endostatin can produce inhibitory action to the blood vessel of vascular endothelial cell, growth,
The growth of tumor and transfer can be played good inhibiting effect.From the point of view of its mechanism of action, people's blood vessel
Endostatin is to make tumor lack nutrient substance and oxygen by the blood supply of tumor tissues in suppression human body
Gas and stop growing so that progressively atrophy until death, the change commonly used compared to current clinic
Treat medicine, there is the advantage without obvious toxic-side effects, thus receive the extensive concern of medical circle.
At present, Human endostatin mainly first passes through gene recombination technology and obtains carrier's Ink vessel transfusing
The engineering bacteria of skin chalone gene, more extracted for engineering bacterium expression target protein purification is prepared.
During the extraction purification of Research of Recombinant Human Endostatin, the fermentation of engineering bacteria and induction engineering bacteria table
Can intelligent's endostatin research, to obtain higher product yield and preferable the playing of product purity
Pivotal role.
Chinese patent literature CN154667A discloses a kind of pQE30/en pPIC9K/en in escherichia coli
Technology for high-efficiency expression, converts recombiant plasmid pBendo in expression type Host Strains E.coli BL21 (DE3),
Filter out positive recombinant bacterium E.coli BL21-Endo, be inoculated in 2ml, containing 60-90 μ g/mL Kan's
In LB culture fluid, 36-38 DEG C, 200-250rpm shaken cultivation 2-3h, make bacterium solution OD600nmAt 0.4-0.6,
Adding concentration in bacterium solution is the isopropyl-β-D-thiogalactoside of 0.8-1.0mol/L, at 36-38 DEG C
Under, 180-200rpm continues the expression of shaken cultivation 3-5h inducing endothelial chalone, and expression reaches thalline
The 38% of total protein.
According to said method in the large-scale production of industry, then need to expend substantial amounts of culture medium,
Culture fluid, to keep the cell concentration of fermented liquid to be in reduced levels, and improves cell concentration, and
Synchronize to improve derivant consumption, the most not only can be owing to the engineering bacteria of Research of Recombinant Human Endostatin be to induction
The toleration of agent rises rapidly, sensitivity is remarkably decreased, and then inducing recombinant human inevitably occurs
The phenomenon that the expression of endostatin research albumen declines, it is impossible to obtain higher abduction delivering amount, with
Time, under the cell concentration of high concentration, it is also desirable to culture medium that concentration is higher, culture fluid, and thalline exists
Under this environment, necessarily growth is very fast, the easy pre-mature exhaustion of nutrient, in turn result in thalline cross presenility and from
Molten, it is impossible to obtain preferable expression.The most any situation, all means weight in large-scale production
The production efficiency of group Human endostatin is extremely low, and substantial amounts of raw material can be caused to expend.Therefore, above-mentioned
Although method expression can reach 38%, but, this expression is only capable of in a small amount of culture fluid,
The OD of fermentation liquid600nmRealizing under conditions of being worth between 0.4-0.6, in other words, said method is only
Can realize in small test, the efficient abduction delivering under low engineering bacteria concentration, and then obtain denier
Product, it is impossible to be applicable to industrialization.
Summary of the invention
The technical problem to be solved is Research of Recombinant Human Endostatin of the prior art induction
It is relatively low that expression is not suitable for industrial mass production, production efficiency, and then provides one to meet
The large-scale production of Research of Recombinant Human Endostatin needs, realizes efficiently induction under higher cell concentration
Expression, the derivational expression method of the Research of Recombinant Human Endostatin that fermentation expression amount is high.
The derivational expression method of the Research of Recombinant Human Endostatin of the present invention, comprises the following steps: by engineering
Bacterium is fermented to fermentation liquid OD600Value for 10-15, be added thereto to derivant IPTG, carbon source material with
And nitrogen source, making the concentration of IPTG in fermentation liquid is 0.2-0.4mmol/L, at 30-37 DEG C,
Cultivation 2.5-3.5h, then it is added thereto to the derivant IPTG of final concentration of 0.1-0.3mmol/L, continue
Continuous cultivation 3-5h,.
It should be noted that described carbon source and nitrogen source, its selection is the most unique, it is possible to provide thalline
Carbon needed in sweat, the material of nitrogen.In the present invention, described carbon source material is glucose；
Described nitrogen source is one or more in yeast extract, tryptone and caseinhydrolysate.
Preferably, the concentration of described glucose is 12-15g/L；The concentration of described yeast extract is
4-6g/L；The concentration of described tryptone is 10-20g/L；The concentration of described caseinhydrolysate is 10-20
g/L.Most preferably, described carbon source material is the glucose of 13g/L；Described nitrogen source is 5g/L's
Yeast extract.
It is 0.3mmol/L that described derivant IPTG first time addition makes the concentration of IPTG in fermentation liquid
Amount, second time adds the derivant IPTG of final concentration of 0.2mmol/L.
Preferably, by described engineering bacterium fermentation to fermentation liquid OD600After value is 12.5, it is added thereto to lure
Lead agent IPTG.
Described engineering bacterium fermentation specifically includes following steps: takes engineering bacteria and expands in LB culture medium
After, by the inoculum concentration of the 5-8% of fermentation volume, it is seeded in fermentation medium, then ferments to OD600
Value is 10-15.Wherein, described LB culture medium includes: tryptone 10g/L, yeast extract 5g/L,
Sodium chloride 5g/L.
Described fermentation medium includes: 5-15g/L tryptone, 15-25g/L yeast extract,
7.5-17.5g/L hydrolase protein, 1-9g/L KH2PO4, 10-20g/L glucose.
The technique scheme of the present invention, has the advantage that compared to existing technology
(1) derivational expression method of Research of Recombinant Human Endostatin of the present invention, at engineering bacteria
Ferment to OD600After value is 12.5, it is added thereto to derivant IPTG, at 30-37 DEG C, cultivates
After 2.5-3.5h, then it is added thereto to derivant IPTG.Derivant of the prior art is disposably added
Add and changed into adding at twice, can effectively avoid owing to thalline is to the rising of derivant toleration, sensitivity
Property decline and the expression of Research of Recombinant Human Endostatin that causes declines.Especially, IPTG induction
Agent is fermented with 0.2-0.4mmol/L fermentation liquid and 0.1-0.3mmol/L in above-mentioned two opportunity respectively
The consumption of liquid adds, and its inducing effect is ideal, is combined in after adding derivant for the first time meanwhile
Add carbon source material and nitrogen source, can avoid using high-concentration culturing base, culture fluid at the fermentation initial stage
And the thalline caused crosses presenility, supplement the nutrient needed for thalline the most in time, it is ensured that twice lure
Lead agent addition and all can realize good inducing effect.Our experiments show that, use said method, can be
OD600Under the value high cell density for 10-15, it is achieved the summary table of inducing recombinant human's endostatin research
The amount of reaching reaches more than 30%, meets the large-scale production requirement of Research of Recombinant Human Endostatin.
(2) derivational expression method of Research of Recombinant Human Endostatin of the present invention, the institute of employing
The culture medium stating fermentation includes: 5-15g/L tryptone, 15-25g/L yeast extract, 7.5-17.5g/L
Hydrolase protein, 1-9g/LKH2PO4, 10-20g/L glucose.Above-mentioned fermentation medium can be preferable
Ensure the material that thalline is required when fermentation, especially, add derivant for the first time in sweat
Time the carbon source material that adds and nitrogen source cooperate, it is achieved that preferably fermentation and abduction delivering effect
Really.
Detailed description of the invention
Embodiment 1
The derivational expression method of the Research of Recombinant Human Endostatin described in the present embodiment, first takes engineering bacteria
In LB culture medium after amplification, by the inoculum concentration of the 6% of fermentation volume, being seeded to specification is 50L
Fermentation tank in fermentation medium in, fermentating liquid volume is 44L, ferments to OD600Value is 12.5,
Then, be added thereto to derivant IPTG, 13g/L of 3.75g carbon source material glucose and
The nitrogen source yeast extract of 5g/L, at 30 DEG C, cultivates 2.5h, then is added thereto to 2.4g
Derivant IPTG, continue cultivate 4h,.
Wherein, described fermentation medium is by 10g/L tryptone, 20g/L yeast extract, 12.5g/L
Hydrolase protein, 1g/L KH2PO4, 10g/L glucose composition.
Embodiment 2
The derivational expression method of the Research of Recombinant Human Endostatin described in the present embodiment, first takes engineering bacteria
In LB culture medium after amplification, by the inoculum concentration of the 5% of fermentation volume, being seeded to specification is 50L
Fermentation tank in fermentation medium in, fermentating liquid volume is 44L, ferments to OD600Value is 10,
Then, be added thereto to derivant IPTG, 15g/L of 2.49g carbon source material glucose and
The nitrogen source yeast extract of 6g/L, at 34 DEG C, cultivates 3h, then is added thereto to 3.75g
Derivant IPTG, continue cultivate 3h,.
Wherein, described fermentation medium is by 5g/L tryptone, 25g/L yeast extract, 7.5g/L
Hydrolase protein, 9g/L KH2PO4, 20g/L glucose composition.
Embodiment 3
The derivational expression method of the Research of Recombinant Human Endostatin described in the present embodiment, first takes engineering bacteria
In LB culture medium after amplification, by the inoculum concentration of the 8% of fermentation volume, being seeded to specification is 50L
Fermentation tank in fermentation medium in, fermentating liquid volume is 44L, ferments to OD600Value is 15,
Then, be added thereto to derivant IPTG, 12g/L of 4.99g carbon source material glucose and
The nitrogen source yeast extract of 4g/L, at 37 DEG C, cultivates 3.5h, then is added thereto to 1.25g
Derivant IPTG, continue cultivate 5h,.
Wherein, described fermentation medium is by 15g/L tryptone, 15g/L yeast extract, 17.5g/L
Hydrolase protein, 5g/L KH2PO4, 15g/L glucose composition.
Embodiment 4
The derivational expression method of the Research of Recombinant Human Endostatin described in the present embodiment, first takes engineering bacteria
In LB culture medium after amplification, by the inoculum concentration of the 6% of fermentation volume, being seeded to specification is 50L
Fermentation tank in fermentation medium in, ferment to OD600Value is 12.5, then, is added thereto to
The carbon source material glucose of derivant IPTG, 13g/L and the nitrogen source tryptone of 15g/L,
Making the concentration of IPTG in fermentation liquid is 0.3mmol/L, at 33 DEG C, and cultivation 2.5h, more wherein
Add the derivant IPTG of final concentration of 0.2mmol/L, continue to cultivate 5h,.
Wherein, described fermentation medium is by 5g/L tryptone, 25g/L yeast extract, 7.5g/L
Hydrolase protein, 9g/L KH2PO4, 18g/L glucose composition.
Embodiment 5
The derivational expression method of the Research of Recombinant Human Endostatin described in the present embodiment, first takes engineering bacteria
In LB culture medium after amplification, by the inoculum concentration of the 6% of fermentation volume, being seeded to specification is 50L
Fermentation tank in fermentation medium in, ferment to OD600Value is 12.5, then, is added thereto to
The carbon source material glucose of derivant IPTG, 13g/L and the nitrogen source hydrolysis cheese egg of 20g/L
In vain, making the concentration of IPTG in fermentation liquid is 0.2mmol/L, at 33 DEG C, and cultivation 2.5h, then to
Wherein it is added thereto to the derivant IPTG of final concentration of 0.1mmol/L, continues to cultivate 5h,.
Wherein, described fermentation medium is by 5g/L tryptone, 25g/L yeast extract, 7.5g/L
Hydrolase protein, 9g/L KH2PO4, 18g/L glucose composition.
Embodiment 6
The derivational expression method of the Research of Recombinant Human Endostatin described in the present embodiment, first takes engineering bacteria
In LB culture medium after amplification, by the inoculum concentration of the 6% of fermentation volume, being seeded to specification is 50L
Fermentation tank in fermentation medium in, ferment to OD600Value is 12.5, then, is added thereto to
The carbon source material glucose of derivant IPTG, 13g/L, and the nitrogen source tryptone of 10g/L,
The nitrogen source caseinhydrolysate of 15g/L, making the concentration of IPTG in fermentation liquid is 0.4mmol/L,
At 32 DEG C, cultivation 2.5h, then it is added thereto to the derivant IPTG of final concentration of 0.3mmol/L,
Continue to cultivate 5h,.
Wherein, described fermentation medium is by 5g/L tryptone, 25g/L yeast extract, 7.5g/L
Hydrolase protein, 9g/L KH2PO4, 18g/L glucose composition.
Embodiment 7
The derivational expression method of the Research of Recombinant Human Endostatin described in the present embodiment, first by engineering bacteria
Ferment in the medium to OD600Value is 12.5, then, is added thereto to derivant IPTG, 13g/L
Carbon source material glucose and the nitrogen source yeast extract of 4g/L, the nitrogen source thing of 20g/L
The nitrogen source caseinhydrolysate of matter tryptone and 10g/L, makes the concentration of IPTG in fermentation liquid be
0.4mmol/L, at 30 DEG C, cultivates 2.5h, then is added thereto to luring of final concentration of 0.2mmol/L
Lead agent IPTG, continue to cultivate 5h,.
Wherein, component and each amounts of components of described fermentation medium are the most same as in Example 1.
Comparative example 1
The derivational expression method of the Research of Recombinant Human Endostatin described in this comparative example, first takes engineering bacteria
In LB culture medium after amplification, by the inoculum concentration of the 6% of fermentation volume, being seeded to specification is 50L
Fermentation tank in fermentation medium in, fermentating liquid volume is 44L, ferments to OD600Value is 12.5,
Then, then, the disposable derivant IPTG adding 6.15g, at 30 DEG C, cultivates 5h, i.e.
Can.
Described fermentation medium includes: 20g/L tryptone, 25g/L yeast extract, 12.5g/L
Hydrolase protein, 9g/L KH2PO4, 23g/L glucose.
Comparative example 2
The derivational expression method of the Research of Recombinant Human Endostatin described in this comparative example, first takes engineering bacteria
In LB culture medium after amplification, by the inoculum concentration of the 6% of fermentation volume, being seeded to specification is 50L
Fermentation tank in fermentation medium in, fermentating liquid volume is 44L, ferments to OD600Value is 12.5,
Then, the most disposably add 6.15g derivant IPTG, 13g/L carbon source material glucose,
And the nitrogen source yeast extract of 5g/L, at 30 DEG C, cultivate 5h,.
Component and each amounts of components of described fermentation medium are the most same as in Example 1.
Comparative example 3
The derivational expression method of the Research of Recombinant Human Endostatin described in this comparative example, first takes engineering bacteria
In LB culture medium after amplification, by the inoculum concentration of the 6% of fermentation volume, being seeded to specification is 50L
Fermentation tank in fermentation medium in, fermentating liquid volume is 44L, ferments to OD600Value is 12.5,
Then, it is added thereto to the derivant IPTG of 3.75g, at 30 DEG C, cultivation 2.5h, more wherein
Add the derivant IPTG of 2.4g, continue to cultivate 5h,.
Component and each amounts of components of described fermentation medium are the most same as in Example 1.
Comparative example 4
The derivational expression method of the Research of Recombinant Human Endostatin described in this comparative example, first takes engineering bacteria
In LB culture medium after amplification, by the inoculum concentration of the 6% of fermentation volume, being seeded to specification is 50L
Fermentation tank in fermentation medium in, fermentating liquid volume is 44L, ferments to OD600Value is 12.5,
Then, it is added thereto to the derivant IPTG of 3.75g, at 30 DEG C, cultivates 1.5h, add wherein
Enter the carbon source material glucose of 13g/L and the nitrogen source yeast extract of 5g/L, cultivate 1h,
It is added thereto to the derivant IPTG of 2.4g again, continues to cultivate 5h,.
Component and each amounts of components of described fermentation medium are the most same as in Example 1.
Effect experimental examples
For the technique effect of the present invention being described, to the recombinant human in embodiment 1-7 and comparative example 1-4
The final fermentation liquid of the derivational expression method of endothelial tube chalone carries out the mensuration of fermentation expression amount:
(1) experimental technique:
With fermentation time situation of change, thalli growth situation is evaluated by observing biomass in sweat,
And after fermentation expression terminates, use the polyacrylamide gel electrophoresis in 2010 editions Chinese Pharmacopoeias
(SDS-PAGE) in the fermentation liquid after measuring abduction delivering, target protein Research of Recombinant Human Endostatin accounts for
The amount of total protein, is designated as fermentation expression amount.
(2) experimental result:
The abduction delivering test result of table 1 embodiment 1-7 and comparative example 1-4
Comparative example 1 is disposably added thereto to all carbon source materials, nitrogen source, and derivant,
Thalli growth is too fast, after inoculation the i.e. senilism self-dissolving of 6h, and its target protein expressed only has 17%,
Comparative example 2 does not adds derivant by several times, but supplements carbon source material, nitrogen source in the later stage, though bacterium
Bulk-growth is all right, but target protein expression is the highest.Comparative example 3 adds derivant by several times, but
Non-supplementary carbon source material, nitrogen source, 8h is i.e. because of carbon source material, nitrogen source deficiency after inoculation
And apoptosis.Comparative example 4 adds derivant, also supplementary carbon source material, nitrogen source by several times, but it is mended
The opportunity filling carbon source material and nitrogen source adds derivant and after a period of time of fermenting for first time, bacterium
Though body well-grown, but target protein expression is the most relatively low.The results showed: use the present invention's
Method induction thalline express Research of Recombinant Human Endostatin, fermentation expression amount more than 30%, the highest can
Reach 42%.
Therefore, the derivational expression method of the Research of Recombinant Human Endostatin of the present invention, may be adapted to extensive
Produce Research of Recombinant Human Endostatin, under fermentation liquid height cell concentration, it is achieved higher abduction delivering
Amount.
Obviously, above-described embodiment is only for clearly demonstrating example, and not to embodiment party
The restriction of formula.For those of ordinary skill in the field, the most also may be used
To make other changes in different forms.Here without also all of embodiment being given
With exhaustive.And the obvious change thus extended out or variation are still in the guarantor of the invention
Protect among scope.
Claims (7)
1. the derivational expression method of Research of Recombinant Human Endostatin, it is characterised in that comprise the following steps:
By engineering bacterium fermentation to fermentation liquid OD600Value for 10-15, be added thereto to derivant IPTG,
Carbon source material and nitrogen source, making the concentration of IPTG in fermentation liquid is 0.2-0.4mmol/L,
At 30-37 DEG C, to recombinant 2.5-3.5h, then it is added thereto to final concentration of 0.1-0.3mmol/L
Derivant IPTG, continue cultivate 3-5h,.
The derivational expression method of Research of Recombinant Human Endostatin the most according to claim 1, its feature exists
In, described carbon source material is glucose；Described nitrogen source is yeast extract, tryptone and water
Solve one or more in casein.
The derivational expression method of Research of Recombinant Human Endostatin the most according to claim 2, its feature exists
In, the concentration of described glucose is 12-15g/L；The concentration of described yeast extract is 4-6g/L；Institute
The concentration stating tryptone is 10-20g/L；The concentration of described caseinhydrolysate is 10-20g/L.
4. according to the derivational expression method of the arbitrary described Research of Recombinant Human Endostatin of claim 1-3,
It is characterized in that, described derivant IPTG adds for the first time makes the concentration of IPTG in fermentation liquid be
The amount of 0.3mmol/L, second time adds the derivant IPTG of final concentration of 0.2mmol/L.
The derivational expression method of Research of Recombinant Human Endostatin the most according to claim 4, its feature
Being, the fermentation of described engineering bacteria specifically includes following steps: takes engineering bacteria and expands in LB culture medium
After increasing, by the inoculum concentration of the 5-8% of fermentation volume, it is seeded in fermentation medium, then ferments to OD600
Value is 10-15.
6. according to the abduction delivering side of the arbitrary described Research of Recombinant Human Endostatin of claim 1-3 or 5
Method, it is characterised in that by described engineering bacterium fermentation to fermentation liquid OD600After value is 12.5, wherein
Add derivant IPTG.
The derivational expression method of Research of Recombinant Human Endostatin the most according to claim 6, its feature
Being, the culture medium of described fermentation includes: 5-15g/L tryptone, 15-25g/L yeast extract,
7.5-17.5g/L hydrolase protein, 1-9g/L KH2PO4, 10-20g/L glucose.
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