CN104356211B - One kind application Triton X 114 and allantoin remove endotoxic method in recombinant protein solution - Google Patents
One kind application Triton X 114 and allantoin remove endotoxic method in recombinant protein solution Download PDFInfo
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- CN104356211B CN104356211B CN201410485475.4A CN201410485475A CN104356211B CN 104356211 B CN104356211 B CN 104356211B CN 201410485475 A CN201410485475 A CN 201410485475A CN 104356211 B CN104356211 B CN 104356211B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
- C12N2720/00011—Details
- C12N2720/00051—Methods of production or purification of viral material
Abstract
Endotoxic method in recombinant protein solution is removed the invention discloses one kind application Triton X 114 and allantoin, is comprised the following steps：1ml Triton X 114 are added in 100ml VP2 recombinant protein solution, it is fully miscible to obtain mixture A, by mixture A ice baths 30min；10min, stirring are placed in mixture A water-baths under the conditions of 37 DEG C；Under the conditions of 25 DEG C, the mixture A stirred is centrifuged into 15min under 12000g, upper strata aqueous phase is removed；It will be used to removing endotoxic allantoin and be applied directly in upper strata aqueous phase, and obtain mixture B, mixture B is shaken into 20min at room temperature；Under the conditions of 25 DEG C, the mixture B after concussion is centrifuged into 5min under 5000g, bottom sediment is removed, retains supernatant solution, supernatant is to remove endotoxic VP2 recombinant proteins solution.This method has that treating capacity is big, process cycle is short, production cost is low, removes endotoxin thoroughly, endotoxin content can be controlled into the advantage in livestock and poultry clinical practice critical field, and meet the demand of large-scale production.
Description
Technical field
The invention belongs to the purified technology of protein in veterinary biologics field, more particularly to one kind application Triton
X-114 and allantoin remove endotoxic method in recombinant protein solution.
Background technology
Endotoxin be a kind of composition on gram-negative bacteria cell wall lipopolysaccharides, mainly by the specific chains of O-, core
Polysaccharide, the parts of lipoid A tri- composition, its major toxicity composition is lipoid A.Endotoxin is only when bacterial death dissolves or manually square
Just discharged after method destruction bacterium cell.Circulated when containing endotoxin or by the biological products of contaminated with endotoxins into poultry, fowl body
After system, when reaching finite concentration, poultry, fowl body can be caused to produce a series of allergy, such as high fever, shock.
With the development of molecular biology technology, recombinant protein biological products are gradually exploited.Due to most of weights
Histone matter is using Bacillus coli expression, and it belongs to Gram-negative bacteria, needs to break thalline in purifying protein
Wall, while release recombinant protein, lipopolysaccharides is also discharged into buffer solution therewith, causes contaminated with endotoxins.
Due to endotoxin under strong acid, highly basic and high temperature all extremely stable, and property pole heterogeneity, traditional various methods
Part endotoxin can only be removed by such as extracting, adsorb and chromatographing, and not reach the requirement of livestock and poultry clinical practice, it is difficult to which finding one kind has
Endotoxic method in the removal recombinant protein solution of effect.
The method reported at present has Triton X-114 cloud point methods, is carried out using the Triton X-114 both sexes features having
The method of endotoxin removal, but find that this method also can only drop endotoxic content from 10,000,000 EU/ml through overtesting
As little as 100,000 EU/ml or so, do not reach the standard of livestock and poultry clinical practice.
The content of the invention
In view of this, it is an object of the invention to propose that a kind of application Triton X-114 and allantoin remove restructuring egg
Endotoxic method in white matter solution, by first adding Triton X-114, rear addition allantoin removes recombinant protein solution
In endotoxic method, compare and Triton X-114 be used alone, can make endotoxin content reduce by 1000 times, well solve
Endotoxin content does not reach the problem of livestock and poultry clinical practice standards.
In some illustrative embodiments, one kind application Triton X-114 and allantoin are removed in recombinant protein solution
Endotoxic method, comprises the following steps：
In 100mlVP2 recombinant protein solution add Triton X-1141ml, fully it is miscible after obtain mixture A,
By the mixture A ice baths 30min；By mixture A, 10min, stirring are placed in water-bath under the conditions of 37 DEG C；, will under the conditions of 25 DEG C
The mixture A stirred centrifuges 15min under 12000 turns/min, removes upper strata aqueous phase；It will be used to removing endotoxic
Allantoin is applied directly in the upper strata aqueous phase, obtains mixture B, and the mixture B is shaken into 20min at room temperature；25℃
Under the conditions of, the mixture B after concussion is centrifuged into 5min under 5000 turns/min, bottom sediment is removed, retains supernatant solution,
The supernatant is to remove endotoxic VP2 recombinant proteins solution.
Compared with prior art, the present invention has advantages below：This method has that treating capacity is big, process cycle is short, production
Cost is low, remove endotoxin thoroughly, endotoxin content can be controlled into the advantage in livestock and poultry clinical practice critical field, and accords with
Close the demand of large-scale production.
For above-mentioned and related purpose, one or more embodiments include will be explained in below and in claim
In the feature that particularly points out.
Embodiment
Preferred embodiment below, the present invention is described in detail, to enable those skilled in the art to put into practice them.
The present invention is described in detail below.
In some illustrative embodiments, one kind application Triton X-114 and allantoin are removed in recombinant protein solution
Endotoxic method, comprises the following steps：TritonX-1141ml is added in 100mlVP2 recombinant protein solution, it is fully mixed
Mixture A is obtained after molten, by the mixture A ice baths 30min；By mixture A, 10min is placed in water-bath under the conditions of 37 DEG C, is stirred
Mix；Under the conditions of 25 DEG C, the mixture A stirred is centrifuged into 15min under 12000g, upper strata aqueous phase is removed；It will be used for
Remove endotoxic allantoin to be applied directly in the upper strata aqueous phase, obtain mixture B, the mixture B is shaken at room temperature
Swing 20min；Under the conditions of 25 DEG C, the mixture B after concussion is centrifuged into 5min under 5000g, removed in bottom sediment, reservation
Clear solution, the supernatant is to remove endotoxic VP2 recombinant proteins solution.
Wherein, allantoin is purchased from Sigma companies (P101415527)；Triton X-114, it is limited purchased from Suo Laibao science and technology
Company.
In some illustrative embodiments, the VP2 recombinant proteins solution is led to by chicken infectivity bursa of Fabricius virus VP 2
Cross technique for gene engineering acquisition.
In some illustrative embodiments, the allantoin addition is 5-50mg/ml.
In some illustrative embodiments, the allantoin addition is 5mg/ml, 10mg/ml, 30mg/ml, 50mg/
ml。
In some illustrative embodiments, it is characterised in that：The allantoin is solid-state.
In some illustrative embodiments, it is characterised in that：The TritonX-114 is stoste.
The allantoin addition of embodiment 1 is 10mg/ml
Triton X-1141ml are added in 100mlVP2 recombinant protein solution, is placed on magnetic stirring apparatus and stirs
Uniformly, mixture A is obtained, by the mixture A ice baths 30min；By mixture A, 10min is placed in water-bath under the conditions of 37 DEG C, is stirred
Mix；Under the conditions of 25 DEG C, the mixture A stirred is centrifuged into 15min under 12000g, upper strata aqueous phase is removed；By 10mg/
Ml is applied directly in the upper strata aqueous phase for removing endotoxic allantoin, mixture B is obtained, by the mixture B in room
The lower concussion 20min of temperature；Under the conditions of 25 DEG C, the mixture B after concussion is centrifuged into 5min under 5000g, bottom sediment is removed,
Retain supernatant solution, the supernatant is to remove endotoxic VP2 recombinant proteins solution.
Wherein, glass apparatus used is soaked 24 hours with 0.5M NaOH solutions, after pure water 3-5 times, in 250
Toasted two hours at DEG C；Centrifuge (3K15) is purchased from Sigma companies.
The allantoin addition of embodiment 2 is 5mg/ml
Triton X-1141ml are added in 100mlVP2 recombinant protein solution, is placed on magnetic stirring apparatus and stirs
Uniformly, mixture A is obtained, by the mixture A ice baths 30min；By mixture A, 10min is placed in water-bath under the conditions of 37 DEG C, is stirred
Mix；Under the conditions of 25 DEG C, the mixture A stirred is centrifuged into 15min under 12000g, upper strata aqueous phase is removed；By 5mg/
Ml is applied directly in the upper strata aqueous phase for removing endotoxic allantoin, mixture B is obtained, by the mixture B in room
The lower concussion 20min of temperature；Under the conditions of 25 DEG C, the mixture B after concussion is centrifuged into 5min under 5000g, bottom sediment is removed,
Retain supernatant solution, the supernatant is to remove endotoxic VP2 recombinant proteins solution.
The allantoin addition of embodiment 3 is 30mg/ml
Triton X-1141ml are added in 100mlVP2 recombinant protein solution, is placed on magnetic stirring apparatus and stirs
Uniformly, mixture A is obtained, by the mixture A ice baths 30min；By mixture A, 10min is placed in water-bath under the conditions of 37 DEG C, is stirred
Mix；Under the conditions of 25 DEG C, the mixture A stirred is centrifuged into 15min under 12000g, upper strata aqueous phase is removed；By 30mg/
Ml is applied directly in the upper strata aqueous phase for removing endotoxic allantoin, mixture B is obtained, by the mixture B in room
The lower concussion 20min of temperature；Under the conditions of 25 DEG C, the mixture B after concussion is centrifuged into 5min under 5000g, bottom sediment is removed,
Retain supernatant solution, the supernatant is to remove endotoxic VP2 recombinant proteins solution.
The allantoin addition of embodiment 4 is 50mg/ml
Triton X-1141ml are added in 100mlVP2 recombinant protein solution, is placed on magnetic stirring apparatus and stirs
Uniformly, mixture A is obtained, by the mixture A ice baths 30min；By mixture A, 10min is placed in water-bath under the conditions of 37 DEG C, is stirred
Mix；Under the conditions of 25 DEG C, the mixture A stirred is centrifuged into 15min under 12000g, upper strata aqueous phase is removed；By 50mg/
Ml is applied directly in the upper strata aqueous phase for removing endotoxic allantoin, mixture B is obtained, by the mixture B in room
The lower concussion 20min of temperature；Under the conditions of 25 DEG C, the mixture B after concussion is centrifuged into 5min under 5000g, bottom sediment is removed,
Retain supernatant solution, the supernatant is to remove endotoxic VP2 recombinant proteins solution.
Using TAL gel semiquantitative method, endotoxin in the VP2 recombinant protein solution of the gained of embodiment 1~4 is determined
Content.
1. a pair sensitivity of the limulus reagent is checked：Use tachypleus amebocyte lysate box (sensitivity is 0.25EU/ml) and endotoxin standard
(10EU/ml), is operated (λ=0.25EU/ml) by shop instruction.
Wherein, TAL use Zhanjiang Bo Kang Marine Bio Co., Ltd. produce tachypleus amebocyte lysate box (sensitivity is 0.25EU/
ml)。
Check and the results are shown in Table 1.
The sensitivity of the limulus reagent of table 1 is checked
Check result and illustrate that the sensitivity of TAL used is consistent with sign value, can be used according to sign value.
2. the dilution of pair VP2 recombinant protein solution and application TAL detect endotoxin content.
(1) 20 μ l VP2 solution are taken to be added in the 21 flint glass F tubules equipped with 780 μ l endotoxin-free waters for injection, vortex
Vibration mixes 15min.(1:40 times of dilutions)
(2) the μ l solution of area 100 is added to the flint glass F tubule of 900 μ l endotoxin-frees water for injection 22 from 21 flint glass F tubules
Middle vortex vibration mixes 1min.(1:400 times of dilutions)
(3) the μ l solution of area 100 is added to the flint glass F tubule of 900 μ l endotoxin-frees water for injection 23 from 22 flint glass F tubules
Middle vortex vibration mixes 1min.(1:4000 times of dilutions)
(4) the μ l solution of area 100 is added to the flint glass F tubule of 900 μ l endotoxin-frees water for injection 24 from 23 flint glass F tubules
Middle vortex vibration mixes 1min.(1:40000 times of dilutions)
(5) the μ l solution of area 100 is added to the flint glass F tubule of 900 μ l endotoxin-frees water for injection 25 from 24 flint glass F tubules
Middle vortex vibration mixes 1min.(1:400000 times of dilutions)
(6) the μ l solution of area 100 is added to the flint glass F tubule of 900 μ l endotoxin-frees water for injection 26 from 25 flint glass F tubules
Middle vortex vibration mixes 1min.(1:4000000 times of dilutions)
(7) the μ l solution of area 100 is added to the flint glass F tubule of 900 μ l endotoxin-frees water for injection 27 from 26 flint glass F tubules
Middle vortex vibration mixes 1min.(1:40000000 times of dilutions)
(8) the μ l solution of area 100 is added to the flint glass F tubule of 900 μ l endotoxin-frees water for injection 28 from 27 flint glass F tubules
Middle vortex vibration mixes 1min.(1:400000000 times of dilutions)
(9) positive control and each pipe of negative control separately are set, respectively takes 100 μ l solution to be added to 100 μ l's from No. 21-28 pipe
In TAL, mix 10 seconds, be inverted observation result after 37 DEG C of water-bath 60min, gel flows down as feminine gender, marked as 29, does not flow down
For the positive, numbering is 210.
This TAL λ=0.25, therefore endotoxic concentration should be the extension rate of each pipe again during reading numerical values divided by 4 are
Corresponding numerical value (EU/ml).It the results are shown in Table 2.
The VP2 recombinant proteins solution of table 2 removes the detection of endotoxin content before endotoxin
Removed 3. pair embodiment 1 carries out Triton X-114 with allantoin use in conjunction after VP2 recombinant protein solution
Endotoxin is detected.
(1) take 20 μ l to remove endotoxic VP2 recombinant proteins solution to be added to equipped with 780 μ l endotoxin-free waters for injection
1 flint glass F tubule in, vortex vibration mix 15min.(1:40 times of dilutions)
(2) the μ l solution of area 100 is added in the flint glass F tubule of 900 μ l endotoxin-frees water for injection 2 from 1 flint glass F tubule
Vortex vibration mixes 1min.(1:400 times of dilutions)
(3) the μ l solution of area 100 is added in the flint glass F tubule of 900 μ l endotoxin-frees water for injection 3 from 2 flint glass F tubules
Vortex vibration mixes 1min.(1:4000 times of dilutions)
(4) the μ l solution of area 100 is added in the flint glass F tubule of 900 μ l endotoxin-frees water for injection 4 from 3 flint glass F tubules
Vortex vibration mixes 1min.(1:40000 times of dilutions)
(5) the μ l solution of area 100 is added in the flint glass F tubule of 900 μ l endotoxin-frees water for injection 5 from 4 flint glass F tubules
Vortex vibration mixes 1min.(1:400000 times of dilutions)
(6) the μ l solution of area 100 is added in the flint glass F tubule of 900 μ l endotoxin-frees water for injection 6 from 5 flint glass F tubules
Vortex vibration mixes 1min.(1:4000000 times of dilutions)
(7) the μ l solution of area 100 is added in the flint glass F tubule of 900 μ l endotoxin-frees water for injection 7 from 6 flint glass F tubules
Vortex vibration mixes 1min.(1:40000000 times of dilutions)
(8) the μ l solution of area 100 is added in the flint glass F tubule of 900 μ l endotoxin-frees water for injection 8 from 7 flint glass F tubules
Vortex vibration mixes 1min.(1:400000000 times of dilutions)
(9) positive control and each pipe of negative control separately are set, the horseshoe crab for respectively taking 100 μ l solution to be added to 100 μ l is managed from No. 1-8
In reagent, mix 10 seconds, be inverted observation result after 37 DEG C of water-bath 60min, gel flows down as feminine gender, marked as 9, does not flow down as sun
Property, numbering is 10.
Method progress Triton X-114 same as Example 1 are carried out to embodiment 2~4 to go with allantoin use in conjunction
Except the endotoxin detection after VP2 recombinant protein solution.It the results are shown in Table 3.
The VP2 recombinant proteins solution of table 3 removes the detection of endotoxin content before endotoxin
As shown in table 2, the VP2 recombinant protein solution endotoxin contents of preparation are between 10,000,000-1 hundred million EU/ml, such as table
Shown in 3, when allantoin addition is 5mg, endotoxin is removed by use in conjunction Triton X-114 and allantoin, can be by VP2
The endotoxin content of recombinant protein solution is reduced between 10-1000EU/ml.Allantoin addition is 10mg, 30mg, 50mg
When, endotoxin is removed by use in conjunction Triton X-114 and allantoin, the endotoxin of VP2 recombinant protein solution can be contained
Amount is reduced between 10-100EU/ml, has reached livestock and poultry clinical practice standard.
Experimental result shows that the addition induced by endotoxin of increase allantoin, which is removed, to be had no significant effect, and is taken into consideration and is produced into
This, preferably the addition of allantoin is 10mg/ml.
Embodiment 5 first adds allantoin, then adds TritonX-114
10mg/ml allantoin is added in 100mlVP2 recombinant protein solution, is placed on magnetic stirring apparatus and stirs
Uniformly, mixture A is obtained, by the mixture A ice baths 30min；By mixture A, 10min is placed in water-bath under the conditions of 37 DEG C, is stirred
Mix；Under the conditions of 25 DEG C, the mixture A stirred is centrifuged into 15min under 12000g, upper strata aqueous phase is removed；Will
Triton X-1141ml are applied directly in the upper strata aqueous phase, obtain mixture B, and the mixture B is shaken at room temperature
20min；Under the conditions of 25 DEG C, the mixture B after concussion is centrifuged into 5min under 5000g, bottom sediment is removed, retains supernatant
Solution, the supernatant is to remove endotoxic VP2 recombinant proteins solution.
Embodiment 5 is carried out to detect endotoxin identical method with embodiment 1, egg is recombinated to removing the VP2 after endotoxin
White matter solution is detected.It the results are shown in Table 4.
Embodiment 6 adds allantoin, TritonX-114 simultaneously
Triton X-1141ml, 10mg/ml allantoin are added in 100mlVP2 recombinant protein solution, is placed into
Stirred on magnetic stirring apparatus, obtain mixture A, by the mixture A ice baths 30min；By mixture A under the conditions of 37 DEG C
10min, stirring are placed in water-bath；Under the conditions of 25 DEG C, the mixture A stirred is centrifuged into 15min under 12000g, removed
Upper strata aqueous phase；20min is shaken at room temperature；Under the conditions of 25 DEG C, 5min is centrifuged under 5000g, bottom sediment is removed, retains supernatant
Solution, the supernatant is to remove endotoxic VP2 recombinant proteins solution.
Embodiment 6 is carried out to detect endotoxin identical method with embodiment 1, egg is recombinated to removing the VP2 after endotoxin
White matter solution is detected.It the results are shown in Table 4.
The TritonX-114 of table 4 and the influence of allantoin order of addition induced by endotoxin removal effect
As shown in table 4, the method for embodiment 1, is better than embodiment 5 and embodiment 6 on endotoxic effect is removed
Method.
Embodiment 1 is preferably method, and the VP2 recombinant proteins solution and the endotoxic VP2 of removal to embodiment 1 are recombinated
Protein solution carries out immunodiffusion test：
(1) making sheet：Take solution A 24ml, second liquid 76ml to be put in triangular flask to mix, plus 0.8g or 1g agaroses, 8g chlorinations
Sodium, triangular flask boils in a water bath melts agarose etc., then adds 1% thimerosal 1ml, diameter 90mm plate is taken, to plate
In plus the above-mentioned colloids of 20ml, it is to be solidified after be put in general refrigerator 4 DEG C and save backup.
Wherein, solution A is Na2HPO4·12H2O 3.85g, plus distilled water is to 1000ml, fully dissolving is saved backup；Second liquid
For KH2PO41.36g, plus distilled water is to 1000ml, fully dissolving is saved backup.
(2) punching and sample-adding：Punched or punched with quincunx card punch by hexagon-shaped pattern with diameter 4mm card punch.
Centre bore is 3mm with outer perimeter holes distance.Centre bore fills it up with 0.04~0.05ml infections chicken cloacal bursa virus positive serums, surrounding
Fill it up with various concentrations expression product to be checked and infections chicken cloacal bursa virus standard agp antigen in hole.It is put in mistake in 37 DEG C of incubators
At night, through 24~48h, take out, observe milky precipitation line.
Wherein, during infections chicken cloacal bursa virus positive serum, infections chicken cloacal bursa virus standard agp antigen are purchased from
Veterinary medicament supervision institute of state.
Testing result is shown in Table 5.
The immunodiffusion test testing result of table 5
As shown in the results, the antigen titre of VP2 recombinant proteins solution no significant difference before and after endotoxin removal, explanation
The method has no significant effect to the antigen active and antigen titre of VP2 recombinant proteins, can be applied to actual production.
Claims (6)
1. one kind application Triton X-114 and allantoin remove endotoxic method in recombinant protein solution, its feature exists
In：Comprise the following steps：
Triton X-114 1ml are added in 100ml VP2 recombinant protein solution, it is fully miscible to obtain mixture A, by institute
State mixture A ice baths 30min；By mixture A, 10min, stirring are placed in water-bath under the conditions of 37 DEG C；Under the conditions of 25 DEG C, it will stir
The uniform mixture A centrifuges 15min under 12000g, removes upper strata aqueous phase；It will be used to removing endotoxic allantoin straight
Connect and be added in the upper strata aqueous phase, obtain mixture B, the mixture B is shaken into 20min at room temperature；, will under the conditions of 25 DEG C
The mixture B after concussion centrifuges 5min under 5000g, removes bottom sediment, retains supernatant solution, and the supernatant is
Remove endotoxic VP2 recombinant proteins solution.
2. according to the method described in claim 1, it is characterised in that：The VP2 recombinant proteins solution is by avian infectious Fa Shi
Bursal disease virus VP2 is obtained by technique for gene engineering.
3. according to the method described in claim 1, it is characterised in that：The allantoin addition is 5-50mg/ml.
4. method according to claim 3, it is characterised in that：The allantoin addition be 5mg/ml, 10mg/ml,
30mg/ml or 50mg/ml.
5. according to the method described in claim 1, it is characterised in that：The allantoin is solid-state.
6. according to the method described in claim 1, it is characterised in that：The TritonX-114 is stoste.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1529711A (en) * | 2001-06-28 | 2004-09-15 | ά | Methods and reagents for detecting endotoxin |
| WO2013180647A1 (en) * | 2012-05-31 | 2013-12-05 | Agency For Science, Technology And Research | Chromatographic purification of immunoglobulin g preparations with particles having multimodal functionalities |
| CN103849631A (en) * | 2012-11-28 | 2014-06-11 | 普莱柯生物工程股份有限公司 | Infectious bursal disease virus subunit antigen-containing vaccine composition, preparation method and application thereof |
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2014
- 2014-09-22 CN CN201410485475.4A patent/CN104356211B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1529711A (en) * | 2001-06-28 | 2004-09-15 | ά | Methods and reagents for detecting endotoxin |
| WO2013180647A1 (en) * | 2012-05-31 | 2013-12-05 | Agency For Science, Technology And Research | Chromatographic purification of immunoglobulin g preparations with particles having multimodal functionalities |
| CN103849631A (en) * | 2012-11-28 | 2014-06-11 | 普莱柯生物工程股份有限公司 | Infectious bursal disease virus subunit antigen-containing vaccine composition, preparation method and application thereof |
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Denomination of invention: A method for removing endotoxin from recombinant protein solution by Triton X-114 and allantoin Effective date of registration: 20211222 Granted publication date: 20170829 Pledgee: Shandong Zhucheng rural commercial bank Limited by Share Ltd. Pledgor: SHANDONG SINDER TECHNOLOGY Co.,Ltd. Registration number: Y2021980015708 |
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Denomination of invention: A Method of Removing Endotoxin from Recombinant Protein Solution by Triton X-114 and Allantoin Effective date of registration: 20230608 Granted publication date: 20170829 Pledgee: Shandong Zhucheng rural commercial bank Limited by Share Ltd. Pledgor: SHANDONG SINDER TECHNOLOGY Co.,Ltd. Registration number: Y2023980043376 |
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