CN102633875A - Renaturation liquid for recombining chicken alpha interferon inclusion body as well as preparation method and application thereof - Google Patents
Renaturation liquid for recombining chicken alpha interferon inclusion body as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a renaturation liquid for recombining a chicken alpha interferon inclusion body as well as a preparation method and an application thereof. The renaturation liquid mainly comprises arginine, EDTA (Ethylene Diamine Tetraacetic Acid), oxidized glutathione, Tris and glycerol, wherein the concentration of each component is as follows: 0.2-0.8 M of arginine, 0.5-2 mM of EDTA, 0.3-0.9 mM of toxidized glutathione, 60-100 mM of Tris and 15-30% (v/v) of glycerol; and the pH (Potential of Hydrogen) of the renaturation liquid is 8.0-8.5. The renaturation liquid is prepared by dissolving in distilled water and utilizing HCl to adjust the pH value; the renaturation liquid has an application prospect on the aspect of recombining the chicken alpha interferon inclusion body; and the renaturation liquid has a reasonable proportion for components to carry out a three-step washing and purifying and drip sample injection primary renaturation process method on the inclusion body to better separate a bacterial protein with the inclusion body, so that the renaturation rate of the protein is greatly improved.
Description
Technical field
The present invention relates to a kind of renaturation solution and preparation method thereof and application, especially a kind of renaturation solution that is used for the recombined chicken alpha interferon inclusion body and preparation method thereof and application.
Background technology
Interferon, rabbit (English interferon is abbreviated as IFN) is one type of high reactivity by emiocytosis, multifunctional cytokine, and it mainly acts on is to suppress virus multiplication and antitumor, improves the health anti-virus ability.The chicken interferon molecular weight is that the protein of 17～34KD has the protein general character, to thermally-stabilised, can preserve for a long time for 2-8 ℃; But 20 ℃ of its activity of prolonged preservation, 56 ℃ then are destroyed, and pH2～10 scope internal interference elements are not destroyed; Be prone to by the nucleicacidase neutralization, can be by the proteolytic enzyme deactivation.The genetically engineered chicken alpha-interferon is biological products nontoxic, harmless, noresidue, to suppressing duplicating with enhance immunity power aspect of virus certain effect is arranged; Intestinal bacteria are the expression systems that are widely used for expressing at present foreign protein the most; Yet intestinal bacteria great expression recombinant protein usually causes its aggregate and forms albumen precipitation-inclusion body; Inclusion body protein does not have biological activity, needs complicated dissolving, renaturation and purifying to obtain the product of function.
Protein renaturation has two kinds of methods usually at present.The one, dilution method, dilution is dissolved with proteic sex change liquid, and when denaturing agent concentration was enough low in the solution, albumen began renaturation; The 2nd, dialysis method is removed denaturing agent with means such as dialysis, ultrafiltration.Dilution method operating liquid amount is big, and protein concn is little simultaneously, for follow-up purifying protein has increased difficulty, has also increased production cost simultaneously; Dialysis method is higher to equipment requirements, has increased the input of equipment, and running time while is longer, possibly cause protein precipitation.At present, the renaturation yield of inclusion body probably is 15-25%, and most of valuable target protein is because can not renaturation and can not be utilized.The reason that renaturation yield is lower mainly is the cofactor that in the renaturation process of recombinant protein, lacks some protein folding, or environment is uncomfortable and can't form correct secondary key etc.Therefore, seek a kind of effective renaturation solution and protein renaturation method, make it can improve the protein renaturation rate and can effectively reduce the renaturation cost again, have very important production practice meaning for the gene recombinant protein drug manufacture.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of renaturation solution that is used for the recombined chicken alpha interferon inclusion body.
Another technical problem to be solved by this invention is to provide the above-mentioned preparation method who is used for the renaturation solution of recombined chicken alpha interferon inclusion body.
Another technical problem to be solved by this invention is to provide the above-mentioned preparation method who is used for the renaturation solution of recombined chicken alpha interferon inclusion body to use.
For solving the problems of the technologies described above, technical scheme of the present invention is:
A kind of renaturation solution that is used for the recombined chicken alpha interferon inclusion body; Mainly be made up of l-arginine, EDTA, Sleep-promoting factor B, Tris and glycerine, wherein each concentration of component is respectively: 0.2～0.8M l-arginine, 0.5～2mM EDTA, 0.3～0.9mM Sleep-promoting factor B, 60～100mM Tris, glycerol content 15～30% (v/v).
Preferably, the above-mentioned renaturation solution that is used for the recombined chicken alpha interferon inclusion body, said renaturation solution pH is 8.0～8.5.
Preferably; The above-mentioned renaturation solution that is used for the recombined chicken alpha interferon inclusion body; Wherein each concentration of component is respectively: 0.4～0.6M l-arginine, 0.5～1.5mM EDTA, 0.5～0.8mM Sleep-promoting factor B, 70～85mM Tris, glycerol content 15～25% (v/v), renaturation solution pH is 8.0～8.3.
Preferably, the above-mentioned renaturation solution that is used for the recombined chicken alpha interferon inclusion body, wherein each concentration of component is respectively: 0.2M l-arginine, 0.5mM EDTA, 0.3mM Sleep-promoting factor B, 60mM Tr i s, glycerol content 15% (v/v), renaturation solution pH is 8.0.
Preferably, the above-mentioned renaturation solution that is used for the recombined chicken alpha interferon inclusion body, wherein each concentration of component is respectively: 0.5M l-arginine, 1.5mM EDTA, 0.6mM Sleep-promoting factor B, 75mM Tris, glycerol content 20% (v/v), renaturation solution pH is 8.2.
The above-mentioned preparation method who is used for the renaturation solution of recombined chicken alpha interferon inclusion body, concrete preparation process is following:
(1) accurately takes by weighing each component by above-mentioned each component ratio relation;
(2) with the fully dissolving in zero(ppm) water of each component;
(3) regulate the pH value with HCl.
Preferably, the above-mentioned preparation method who is used for the renaturation solution of recombined chicken alpha interferon inclusion body, said step is regulated pH8.0～8.5 with HCl in (3).
The above-mentioned application of renaturation solution aspect the recombined chicken alpha interferon inclusion body that is used for the recombined chicken alpha interferon inclusion body, the concrete grammar step is:
(1) extract inclusion body protein: centrifugal collection thalline, and, obtain chicken alpha-interferon inclusion body protein matter with high speed frozen centrifugation after the thalline ultrasonication;
(2) purifying inclusion body protein matter: at first inclusion body is resuspended in the first washings high speed frozen centrifugation by mass volume ratio 1: 10～1: 40; By mass volume ratio 1: 10～1: 40 inclusion body is resuspended in the second washings high speed frozen centrifugation then; By mass volume ratio 1: 20～1: 50 inclusion body is resuspended in the 3rd washings high speed frozen centrifugation at last, the set of dispense of said first washings is than being NaCl137mmol/L, KCl 2.7mmol/L, Na
2HPO
410mmol/L, KH
2PO
42mmol/L, 0.2%～1.4% tritonX-100, pH 8.0～8.5; Second washings is 1～3mol/L urea; The 3rd washings is 0.6～2mol/L NaCl;
(3) dissolving and renaturation inclusion body protein matter: by mass volume ratio be 1: 10～1: 50 the centrifugal back of step (2) gained deposition is resuspended with denaturing agent, room temperature is placed to deposition and dissolves back high speed frozen centrifugation gradually, the collection supernatant; Under the 1-10 ℃ of condition; Supernatant is dropwise joined in the renaturation solution according to the invention; Volume ratio is 1: 50～1: 100, stirs 12～24h simultaneously, and the set of dispense of said denaturing agent is than being 1～2mM EDTA, 20～50mM Tris, 6～10mM DTT, pH 8.0～8.5 in per 5～7M Guanidinium hydrochloride.
The above-mentioned application of renaturation solution aspect the recombined chicken alpha interferon inclusion body that is used for the recombined chicken alpha interferon inclusion body; Centrifugal collection thalline condition is 4 ℃, 4000～6000rpm/min, 10～20min in the said step (1); With thalline ultrasonication condition is 400 watts of power, work 3 seconds, intermittently 7 seconds, 15～30 minutes working hours, and the freezing centrifugal condition of high speed is 4 ℃, 8000～10000rpm/min, 10～20min; Inclusion body is resuspended in behind first washings, second washings and the 3rd washings freezing centrifugal condition of high speed and is 4 ℃, 8000～10000rpm/min, 10～20min in the step (2); Step (3) high speed frozen centrifugation condition is 4 ℃, 8000～10000rpm/min, 10～20min, under 4 ℃ of conditions supernatant is dropwise joined in the renaturation solution according to the invention simultaneously.
Preferably, the above-mentioned application of renaturation solution aspect the recombined chicken alpha interferon inclusion body that is used for the recombined chicken alpha interferon inclusion body, thalline is intestinal bacteria in the said step (1).
Preferably; The set of dispense of denaturing agent is than being 1.5mMEDTA, 30～40mM Tris, 8mM DTT, pH 8.5 in every 6M Guanidinium hydrochloride in the above-mentioned application of renaturation solution aspect the recombined chicken alpha interferon inclusion body that is used for the recombined chicken alpha interferon inclusion body, said step (3).
The invention has the beneficial effects as follows:
The above-mentioned renaturation solution that is used for the recombined chicken alpha interferon inclusion body is through the rational proportion to each component; Make inclusion body protein pass through the process method of three step washing purifying and a renaturation of drop application of sample, tropina is well separated with inclusion body, proteinic renaturation yield is improved greatly; Renaturation step and required equipment have been simplified simultaneously; Practice thrift the time, thereby improved the yld of target protein greatly, reduced the production cost of recombined chicken alpha interferon; The preparation method of this renaturation solution is simple, is fit to requirements of large-scale industrial production.
Embodiment
Below in conjunction with specific embodiment technical scheme according to the invention is further described.
Embodiment 1
A kind of renaturation solution that is used for the recombined chicken alpha interferon inclusion body; Mainly form by l-arginine, EDTA, Sleep-promoting factor B, Tris and glycerine; Wherein each concentration of component is respectively: 0.2M l-arginine, 0.5mMEDTA, 0.3mM Sleep-promoting factor B, 60mM Tris, glycerol content 15% (V/V), renaturation solution pH is 8.0.
The above-mentioned preparation method who is used for the renaturation solution of recombined chicken alpha interferon inclusion body, concrete preparation process is following:
(1) accurately takes by weighing each component by above-mentioned each component ratio relation;
(2) with the fully dissolving in zero(ppm) water of each component;
(3) regulate pH8.0 with 0.1mol/L HCl.
The above-mentioned application of renaturation solution aspect the recombined chicken alpha interferon inclusion body that is used for the recombined chicken alpha interferon inclusion body, the concrete grammar step is:
(1) extracts inclusion body protein: 4 ℃, 5000r pm/min, the centrifugal collection coli somatic of 15min; The thalline weight in wet base is 74g/L; And under 400 watts of power, work 3 seconds, intermittently 7 seconds, 25 minutes working hours condition with the 10g wet thallus ultrasonic be resuspended in the PBS damping fluid broken; 4 ℃, 8000rpm/min, 20min high speed frozen centrifugation obtain chicken alpha-interferon inclusion body protein matter;
(2) purifying inclusion body protein matter: at first inclusion body is resuspended in the first washings high speed frozen centrifugation by mass volume ratio 1: 10; By mass volume ratio 1: 10 inclusion body is resuspended in the second washings high speed frozen centrifugation then; By mass volume ratio 1: 20 inclusion body is resuspended in the 3rd washings high speed frozen centrifugation at last; The freezing centrifugal condition of said high speed is 4 ℃, 8000rpm/min, 20min, and wherein the set of dispense of first washings is than being NaCl 137mmol/L, KCl 2.7mmol/L, Na
2HPO
410mmol/L, KH
2PO
42mmol/L, 0.8% tritonX-100, pH 8.5; Second washings is a 1.5mol/L urea; The 3rd washings is 1mol/L NaCl;
(3) dissolving and renaturation inclusion body protein matter: by mass volume ratio be 1: 50 the centrifugal back of step (2) gained deposition is resuspended with denaturing agent, room temperature is placed to deposition dissolve gradually after, 4 ℃, 8000rpm/min, 20min high speed frozen centrifugation, collection supernatant; Under 4 ℃ of conditions, supernatant is dropwise joined in the renaturation solution of above-mentioned preparation, volume ratio is 1: 50, stirs 12 simultaneously, and the set of dispense of said denaturing agent is than being 7M Guanidinium hydrochloride, 2mM EDTA, 50mM Tris, 6mM DTT, pH 8.0;
(4) results renaturation recombined chicken alpha interferon albumen is measured through SDS-PAGE, and purity reaches 93%, and renaturation yield is 42%.
Embodiment 2
A kind of renaturation solution that is used for the recombined chicken alpha interferon inclusion body; Mainly form by l-arginine, EDTA, Sleep-promoting factor B, Tris and glycerine; Wherein each concentration of component is respectively: 0.5M l-arginine, 1.5mMEDTA, 0.6mM Sleep-promoting factor B, 75mM Tris, glycerol content 20% (V/V), renaturation solution pH is 8.2.
The above-mentioned preparation method who is used for the renaturation solution of recombined chicken alpha interferon inclusion body, concrete preparation process is following:
(1) accurately takes by weighing each component by above-mentioned each component ratio relation;
(2) with the fully dissolving in zero(ppm) water of each component;
(3) regulate pH8.2 with 0.1mol/L HCl.
The above-mentioned application of renaturation solution aspect the recombined chicken alpha interferon inclusion body that is used for the recombined chicken alpha interferon inclusion body, the concrete grammar step is:
(1) extracts inclusion body protein: 4 ℃, 6000r pm/min, the centrifugal collection coli somatic of 20min; The thalline weight in wet base is 77g/L; And under 400 watts of power, work 3 seconds, intermittently 7 seconds, 30 minutes working hours condition with the 10g wet thallus ultrasonic be resuspended in the PBS damping fluid broken; 4 ℃, 9000rpm/min, 15min high speed frozen centrifugation obtain chicken alpha-interferon inclusion body protein matter;
(2) purifying inclusion body protein matter: at first inclusion body is resuspended in the first washings high speed frozen centrifugation by mass volume ratio 1: 30; By mass volume ratio 1: 30 inclusion body is resuspended in the second washings high speed frozen centrifugation then; By mass volume ratio 1: 40 inclusion body is resuspended in the 3rd washings high speed frozen centrifugation at last; The freezing centrifugal condition of said high speed is 4 ℃, 10000r pm/min, 15min, and wherein the set of dispense of first washings is than being NaCl 137mmol/L, KCl 2.7mmol/L, Na
2HPO
410mmol/L, KH
2PO
42mmol/L, 1.2% tritonX-100, pH 8.2; Second washings is a 2mol/L urea; The 3rd washings is 1.5mol/L NaCl;
(3) dissolving and renaturation inclusion body protein matter: by mass volume ratio be 1: 35 the centrifugal back of step (2) gained deposition is resuspended with denaturing agent, room temperature is placed to deposition dissolve gradually after, 4 ℃, 8500rpm/min, 15min high speed frozen centrifugation, collection supernatant; Under 4 ℃ of conditions, supernatant is dropwise joined in the renaturation solution of above-mentioned preparation, volume ratio is 1: 80, stirs 16h simultaneously, and the set of dispense of said denaturing agent is than being 1.5mM EDTA, 30～40mM Tris, 8mM DTT, pH 8.5 in every 6M Guanidinium hydrochloride;
(4) results renaturation recombined chicken alpha interferon albumen is measured through SDS-PAGE, and purity reaches 95%, and renaturation yield is 53%.
Embodiment 3
A kind of renaturation solution that is used for the recombined chicken alpha interferon inclusion body; Mainly form by l-arginine, EDTA, Sleep-promoting factor B, Tris and glycerine; Wherein each concentration of component is respectively: 0.8M l-arginine, 2mMEDTA, 0.9mM Sleep-promoting factor B, 100mM Tris, glycerol content 30% (V/V), renaturation solution pH is 8.5.
The above-mentioned preparation method who is used for the renaturation solution of recombined chicken alpha interferon inclusion body, concrete preparation process is following:
(1) accurately takes by weighing each component by above-mentioned each component ratio relation;
(2) with the fully dissolving in zero(ppm) water of each component;
(3) regulate pH8.5 with 0.1mol/L HCl.
The above-mentioned application of renaturation solution aspect the recombined chicken alpha interferon inclusion body that is used for the recombined chicken alpha interferon inclusion body, the concrete grammar step is:
(1) extracts inclusion body protein: 4 ℃, 4000rpm/min, the centrifugal collection coli somatic of 10min; The thalline weight in wet base is 72g/L; And under 400 watts of power, work 3 seconds, intermittently 7 seconds, 15 minutes working hours condition with the 10g wet thallus ultrasonic be resuspended in the PBS damping fluid broken; 4 ℃, 8000rpm/min, 10min high speed frozen centrifugation obtain chicken alpha-interferon inclusion body protein matter;
(2) purifying inclusion body protein matter: at first inclusion body is resuspended in the first washings high speed frozen centrifugation by mass volume ratio 1: 10; By mass volume ratio 1: 10 inclusion body is resuspended in the second washings high speed frozen centrifugation then; By mass volume ratio 1: 20 inclusion body is resuspended in the 3rd washings high speed frozen centrifugation at last; The freezing centrifugal condition of said high speed is 4 ℃, 8000rpm/min, 10min, and wherein the set of dispense of first washings is than being NaCl 137mmol/L, KCl 2.7mmol/L, Na
2HPO
410mmol/L, KH
2PO
42mmol/L, 0.2% tritonX-100, pH 8.0; Second washings is a 1mol/L urea; The 3rd washings is 0.6mol/L NaCl;
(3) dissolving and renaturation inclusion body protein matter: by mass volume ratio be 1: 50 the centrifugal back of step (2) gained deposition is resuspended with denaturing agent, room temperature is placed to deposition dissolve gradually after, 4 ℃, 10000rpm/min, 20min high speed frozen centrifugation, collection supernatant; Under 10 ℃ of conditions, supernatant is dropwise joined in the renaturation solution of above-mentioned preparation, volume ratio is 1: 100, stirs 24h simultaneously, and the set of dispense of said denaturing agent is than being 2mM EDTA, 50mM Tris, 10mM DTT, pH 8.5 in every 7M Guanidinium hydrochloride;
(4) results renaturation recombined chicken alpha interferon albumen is measured through SDS-PAGE, and purity reaches 90%, and renaturation yield is 39%.
Embodiment 4
A kind of renaturation solution that is used for the recombined chicken alpha interferon inclusion body; Mainly form by l-arginine, EDTA, Sleep-promoting factor B, Tris and glycerine; Wherein each concentration of component is respectively: 0.4M l-arginine, 0.8mMEDTA, 0.8mM Sleep-promoting factor B, 70mM Tris, glycerol content 20% (V/V), renaturation solution pH is 8.1.
The above-mentioned preparation method who is used for the renaturation solution of recombined chicken alpha interferon inclusion body, concrete preparation process is following:
(1) accurately takes by weighing each component by above-mentioned each component ratio relation;
(2) with the fully dissolving in zero(ppm) water of each component;
(3) regulate pH8.1 with 0.1mol/L HCl.
The above-mentioned application of renaturation solution aspect the recombined chicken alpha interferon inclusion body that is used for the recombined chicken alpha interferon inclusion body, the concrete grammar step is:
(1) extracts inclusion body protein: 4 ℃, 6000rpm/min, the centrifugal collection coli somatic of 20min; The thalline weight in wet base is 74g/L; And under 400 watts of power, work 3 seconds, intermittently 7 seconds, 30 minutes working hours condition with the 10g wet thallus ultrasonic be resuspended in the PBS damping fluid broken; 4 ℃, 10000rpm/min, 20min high speed frozen centrifugation obtain chicken alpha-interferon inclusion body protein matter;
(2) purifying inclusion body protein matter: at first inclusion body is resuspended in the first washings high speed frozen centrifugation by mass volume ratio 1: 40; By mass volume ratio 1: 40 inclusion body is resuspended in the second washings high speed frozen centrifugation then; By mass volume ratio 1: 50 inclusion body is resuspended in the 3rd washings high speed frozen centrifugation at last; The freezing centrifugal condition of said high speed is 4 ℃, 10000 rpm/min, 10～20min, and wherein the set of dispense of first washings is than being NaCl 137mmol/L, KCl 2.7mmol/L, Na
2HPO
410mmol/L, KH
2PO
42mmo l/L, 1.4% tritonX-100, pH 8.5; Second washings is a 3mol/L urea; The 3rd washings is 2mol/L NaCl;
(3) dissolving and renaturation inclusion body protein matter: by mass volume ratio be 1: 10 the centrifugal back of step (2) gained deposition is resuspended with denaturing agent, room temperature is placed to deposition dissolve gradually after, 4 ℃, 8000rpm/min, 10min high speed frozen centrifugation, collection supernatant; Under 1 ℃ of condition, supernatant is dropwise joined in the renaturation solution of above-mentioned preparation, volume ratio is 1: 50, stirs 12h simultaneously, and the set of dispense of said denaturing agent is than being 1mM EDTA, 20mM Tris, 6mM DTT, pH 8.0 in every 5M Guanidinium hydrochloride;
(4) results renaturation recombined chicken alpha interferon albumen is measured through SDS-PAGE, and purity reaches 91%, and renaturation yield is 41%.
Embodiment 5
A kind of renaturation solution that is used for the recombined chicken alpha interferon inclusion body; Mainly form by l-arginine, EDTA, Sleep-promoting factor B, Tris and glycerine; Wherein each concentration of component is respectively: 0.6M l-arginine, 1.5mMEDTA, 0.5mM Sleep-promoting factor B, 85mM Tris, glycerol content 25% (V/V), renaturation solution pH is 8.3.
The above-mentioned preparation method who is used for the renaturation solution of recombined chicken alpha interferon inclusion body, concrete preparation process is following:
(1) accurately takes by weighing each component by above-mentioned each component ratio relation;
(2) with the fully dissolving in zero(ppm) water of each component;
(3) regulate pH8.3 with 0.1mol/L HCl.
The above-mentioned application of renaturation solution aspect the recombined chicken alpha interferon inclusion body that is used for the recombined chicken alpha interferon inclusion body, the concrete grammar step is:
(1) extracts inclusion body protein: 4 ℃, 4500r pm/min, the centrifugal collection coli somatic of 17min; The thalline weight in wet base is 75g/L; And under 400 watts of power, work 3 seconds, intermittently 7 seconds, 22 minutes working hours condition with the 10g wet thallus ultrasonic be resuspended in the PBS damping fluid broken; 4 ℃, 8500rpm/min, 13min high speed frozen centrifugation obtain chicken alpha-interferon inclusion body protein matter;
(2) purifying inclusion body protein matter: at first inclusion body is resuspended in the first washings high speed frozen centrifugation by mass volume ratio 1: 25; By mass volume ratio 1: 30 inclusion body is resuspended in the second washings high speed frozen centrifugation then; By mass volume ratio 1: 35 inclusion body is resuspended in the 3rd washings high speed frozen centrifugation at last; The freezing centrifugal condition of said high speed is 4 ℃, 9000rpm/min, 15min, and wherein the set of dispense of first washings is than being NaC l 137mmol/L, KCl 2.7mmol/L, Na
2HPO
410mmol/L, KH
2PO
42mmol/L, 1.0% tritonX-100, pH 8.4; Second washings is a 2mol/L urea; The 3rd washings is 1.2mol/L NaCl;
(3) dissolving and renaturation inclusion body protein matter: by mass volume ratio be 1: 40 the centrifugal back of step (2) gained deposition is resuspended with denaturing agent, room temperature is placed to deposition dissolve gradually after, 4 ℃, 8500rpm/min, 16min high speed frozen centrifugation, collection supernatant; Under 7 ℃ of conditions, supernatant is dropwise joined in the renaturation solution of above-mentioned preparation, volume ratio is 1: 80, stirs 20h simultaneously, and the set of dispense of said denaturing agent is than being 2mM EDTA, 35mM Tris, 7mM DTT, pH 8.2 in every 6M Guanidinium hydrochloride;
(4) results renaturation recombined chicken alpha interferon albumen is measured through SDS-PAGE, and purity reaches 94%, and renaturation yield is 47%.
The inclusion body protein of one of the foregoing description 1-5 is through cultivating recombination bacillus coli, abduction delivering recombinant protein under 37 ℃, 200rpm/min condition, thus form inclusion body.
Said each amounts of components of one of embodiment 1-5 is increased or reduces according to same ratio, and the gained proportion relation all belongs to protection scope of the present invention.
The measuring method of renaturation yield in the various embodiments of the present invention is done brief account at present.
The total protein content measuring method is: adopt the Bradford method to measure total protein content.
(1) Coomassie brilliant blue G250 (Braford) analytic liquid preparation
Take by weighing 0.100g Xylene Brilliant Cyanine G G-250, be dissolved in 50mL 95% ethanol, add the phosphoric acid of 100mL85% again; Stir, with the solution with water constant volume to 1000mL, with twice of the continuous suction filtration of B; Remove insoluble particles and can be stored in brown bottle, 4 ℃ of preservations.
(2) drafting of typical curve
Get 6 test tubes, clean oven dry, be numbered 1,2,3,4,5,6, add 0mL, 0.15mL, 0.30mL, 0.45mL, 0.60mL and 0.75mL standard bovine serum albumen solution (concentration is 0.1mg/mL) respectively, supply 1.0mL with zero(ppm) water respectively, shake up; Every test tube adds 5mL Xylene Brilliant Cyanine G G-250 analytic liquid then, mixing, and room temperature leaves standstill 2min; Not add proteic first test tube of standard is blank; Measuring the light absorption value of solution in each pipe in the 595nm place, is X-coordinate with the protein concn, and their corresponding absorbance is an ordinate zou; The drawing standard curve sees the following form 1
Table 1 protein determination data form
Get the 1mL sample solution, add 5mL Coomassie brilliant blue G-250 staining fluid, mixing; Room temperature leaves standstill 2min, measures the solution absorbency value in the 595nm place, if measure the light absorption value scope not between 0.2～0.8; Reply solution dilutes or concentrates, and could guarantee the accuracy of observed value.Getting 1mL zero(ppm) water replaces sample solution as blank.With the absorbance * extension rate of institute's test sample article, in the protein contnt calculation formula that the substitution typical curve obtains as a result, thereby calculate the protein concn of sample.
Renaturation yield is exactly that renaturation finishes, and centrifugal ratio of going the post precipitation soluble proteins to account for the preceding inclusion body protein of renaturation can draw through aforesaid method.
Above-mentioned detailed description of this a kind of renaturation solution that is used for the recombined chicken alpha interferon inclusion body and preparation method thereof being carried out with reference to embodiment; Be illustrative rather than determinate; Can enumerate out several embodiment according to institute's limited range; Therefore in the variation and the modification that do not break away under the general plotting of the present invention, should belong within protection scope of the present invention.
Claims (10)
1. renaturation solution that is used for the recombined chicken alpha interferon inclusion body; It is characterized in that: mainly be made up of l-arginine, EDTA, Sleep-promoting factor B, Tris and glycerine, wherein each concentration of component is respectively: 0.2～0.8M l-arginine, 0.5～2mM EDTA, 0.3～0.9mM Sleep-promoting factor B, 60～100mM Tris, glycerol content 15～30% (v/v).
2. the renaturation solution that is used for the recombined chicken alpha interferon inclusion body according to claim 1 is characterized in that: said renaturation solution pH is 8.0～8.5.
3. the renaturation solution that is used for the recombined chicken alpha interferon inclusion body according to claim 1 and 2; It is characterized in that: wherein each concentration of component is respectively: 0.4～0.6M l-arginine, 0.5～1.5mM EDTA, 0.5～0.8mM Sleep-promoting factor B, 70～85mM Tris, glycerol content 15～25% (v/v), renaturation solution pH is 8.0～8.3.
4. the renaturation solution that is used for the recombined chicken alpha interferon inclusion body according to claim 1 and 2; It is characterized in that: wherein each concentration of component is respectively: 0.2M l-arginine, 0.5mM EDTA, 0.3mM Sleep-promoting factor B, 60mM Tris, glycerol content 15% (v/v), renaturation solution pH is 8.0.
5. the renaturation solution that is used for the recombined chicken alpha interferon inclusion body according to claim 3; It is characterized in that: wherein each concentration of component is respectively: 0.5M l-arginine, 1.5mM EDTA, 0.6mM Sleep-promoting factor B, 75mM Tris, glycerol content 20% (v/v), renaturation solution pH is 8.2.
6. the described preparation method who is used for the renaturation solution of recombined chicken alpha interferon inclusion body of one of claim 1-5 is characterized in that: concrete preparation process is following:
(1) accurately takes by weighing each component by above-mentioned each component ratio relation;
(2) with the fully dissolving in zero(ppm) water of each component;
(3) regulate the pH value with HCl.
7. the described application of renaturation solution aspect the recombined chicken alpha interferon inclusion body that is used for the recombined chicken alpha interferon inclusion body of one of claim 1-5, it is characterized in that: the concrete grammar step is:
(1) extract inclusion body protein: centrifugal collection thalline, and, obtain chicken alpha-interferon inclusion body protein matter with high speed frozen centrifugation after the thalline ultrasonication;
(2) purifying inclusion body protein matter: at first inclusion body is resuspended in the first washings high speed frozen centrifugation by mass volume ratio 1: 10～1: 40; By mass volume ratio 1: 10～1: 40 inclusion body is resuspended in the second washings high speed frozen centrifugation then; By mass volume ratio 1: 20～1: 50 inclusion body is resuspended in the 3rd washings high speed frozen centrifugation at last, the set of dispense of said first washings is than being NaCl137mmol/L, KCl 2.7mmo l/L, Na
2HPO
410mmol/L, KH
2PO
42mmol/L, 0.2%～1.4% tritonX-100, pH 8.0～8.5; Second washings is 1～3mol/L urea; The 3rd washings is 0.6～2mol/L NaCl;
(3) dissolving and renaturation inclusion body protein matter: by mass volume ratio be 1: 10～1: 50 the centrifugal back of step (2) gained deposition is resuspended with denaturing agent, room temperature is placed to deposition and dissolves back high speed frozen centrifugation gradually, the collection supernatant; Under the 1-10 ℃ of condition; Supernatant is dropwise joined in the renaturation solution according to the invention; Volume ratio is 1: 50～1: 100, stirs 12～24h simultaneously, and the set of dispense of said denaturing agent is than being 1～2mM EDTA, 20～50mM Tris, 6～10mM DTT, pH 8.0～8.5 in per 5～7M Guanidinium hydrochloride.
8. the application of renaturation solution aspect the recombined chicken alpha interferon inclusion body that is used for the recombined chicken alpha interferon inclusion body according to claim 7; It is characterized in that: centrifugal collection thalline condition is 4 ℃, 4000～6000rpm/min, 10～20min in the said step (1); With thalline ultrasonication condition is 400 watts of power, work 3 seconds, intermittently 7 seconds, 15～30 minutes working hours, and the freezing centrifugal condition of high speed is 4 ℃, 8000～10000rpm/min, 10～20min; Inclusion body is resuspended in behind first washings, second washings and the 3rd washings freezing centrifugal condition of high speed and is 4 ℃, 8000～10000rpm/min, 10～20min in the step (2); Step (3) high speed frozen centrifugation condition is 4 ℃, 8000～10000rpm/min, 10～20min, under 4 ℃ of conditions supernatant is dropwise joined in the renaturation solution according to the invention simultaneously.
According to claim 7 or the 8 described renaturation solutions that are used for the recombined chicken alpha interferon inclusion body in the application aspect the recombined chicken alpha interferon inclusion body, it is characterized in that: thalline is intestinal bacteria in the said step (1).
10. the application of renaturation solution aspect the recombined chicken alpha interferon inclusion body that is used for the recombined chicken alpha interferon inclusion body according to claim 7 is characterized in that: the set of dispense of denaturing agent is than being 1.5mM EDTA, 30～40mM Tris, 8mM DTT, pH 8.5 in every 6M Guanidinium hydrochloride in the said step (3).
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| CN102851340A (en) * | 2012-08-30 | 2013-01-02 | 郑州后羿制药有限公司 | Preparation method for efficient chicken gene engineering chicken interferon alpha |
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| CN106008668A (en) * | 2016-06-29 | 2016-10-12 | 山东省生物制品研究所 | Renaturation solution capable of enhancing protocaryon inclusion body expression renaturation rate, and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102851340A (en) * | 2012-08-30 | 2013-01-02 | 郑州后羿制药有限公司 | Preparation method for efficient chicken gene engineering chicken interferon alpha |
| CN104292325A (en) * | 2014-07-28 | 2015-01-21 | 哈德逊（天津）生物技术有限责任公司 | Method and reagent for preparing soluble interleukin recombinant protein from inclusion body |
| CN104292325B (en) * | 2014-07-28 | 2018-08-03 | 哈德逊（天津）生物技术有限责任公司 | A kind of method and reagent preparing soluble interleukin-6 recombinant protein from inclusion body |
| CN104628832A (en) * | 2015-02-16 | 2015-05-20 | 天津生机集团股份有限公司 | Method for purifying regrouped chicken colibacillosis outer membrane protein inclusion body and carrying out renaturation by utilizing high-efficiency renaturation liquid |
| CN106008668A (en) * | 2016-06-29 | 2016-10-12 | 山东省生物制品研究所 | Renaturation solution capable of enhancing protocaryon inclusion body expression renaturation rate, and preparation method and application thereof |
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