A kind of method for extracting bovine serum albumin
Technical field
The present invention relates to protein extracting process field, more particularly to a kind of method for extracting bovine serum albumin.
Background technology
Livestock and poultry blood is one of important byproduct of livestock and poultry, includes abundant nutriment and various bioactivators,
Haemocyanin is protein most abundant in blood plasma, is the carrier of aliphatic acid in blood, and each protein molecular can carry seven
Individual fatty acid molecule, these fatty acid molecules are combined in the gap of albumen, and their afterbodys rich in carbon are imbedded in the inside safety
The hydrone of surrounding is avoided on ground, and aliphatic acid is the composition for building lipid, and lipid constitutes cell peripheral and intracellular all
Biomembrane, aliphatic acid is the carrier of the important sources of energy, aliphatic acid or liposoluble vitamin again, when somagenic need energy
Or when needing construction material, fat cell is just discharged into aliphatic acid in blood, and aliphatic acid is obtained by haemocyanin, and transported
The position of needs is sent to, haemocyanin can equally carry many other water insoluble molecules, just because of haemocyanin is
So generally it is present in blood and is easily purified, so it turns into one of protein that scientist studies earliest, ox
Haemocyanin is widely used in terms of animal cell culture, diagnostic reagent, food emulsification, can carried as a kind of function factor
High T lymphocytes in human body quantity and antioxidation activity, with great medical science Development volue.
The preparation method of albumin is many in cow's serum, is mainly carried using salting out method, organic solvent precipitation method and heat shock method
Take, after salting out method ammonium sulfate precipitation, refine and obtain through octanoic acid processing;Organic solvent precipitation method is used for human blood system earliest
The production of product, using the low-k property and protein of ethanol under certain temperature, pH, ionic strength and concentration conditions
Different solubility separated plasma protein in different concentration ethanol, can produce a variety of plasma proteins;Heat shock method is to take
Ox blood after sodium citrate anti-freezing centrifugation with blood plasma is obtained, and heating adds Sodium Caprylate, adjusts after pH value plus ethanol, then through filtering, taking off
Salt, concentration, it is lyophilized after albumin in cow's serum, it is still, existing to be used to prepare the ox obtained by the method for bovine serum albumin(BSA)
Seralbumin yield or purity are all than relatively low, and such as fat of the impurity containing higher concentration in the bovine serum albumin(BSA) extracted
Fat acid, protease, nuclease and endotoxin, this kind of impurity limit further applying for bovine serum albumin(BSA).
The content of the invention
The present invention is in order to solve the above technical problems, there is provided a kind of method for extracting bovine serum albumin.
Realized particular by following technical scheme:
A kind of method for extracting bovine serum albumin, comprises the following steps:
(1) fresh bovine blood is collected at ox vein, haemocyte and blood plasma are separated into through blood separator, is added in blood plasma
Enter 20~30% protein deposition agent of plasma purity, separate, obtain solid protein material and solution material;
(2) solid protein material is handled:Solid protein material is added water stirring, protein solution is obtained, solid protein material quality is added
12~18% anti-coagulants, stirring shakes up, and centrifuges, and takes supernatant, and it is 4.5~5 to add hydrochloric acid regulation supernatant pH value, is added
The dehydrating agent of supernatant quality 4~8%, is mixed thoroughly, is stood, centrifuging and taking precipitation, and 1 must be expected by drying;
(3) solution material is handled:By solution material, dispersant, oxidant by 1.2~1.8: 0.1~0.2: 0.35~0.45
Mass ratio is mixed, and obtains compound, is carried out oxidation processes, is added 0.12~0.15% metal agent of mixture quality, is centrifuged,
Supernatant is taken, the protein precipitation agent of supernatant quality 10~15% is added, 12~18h is stood, centrifuging and taking precipitation is dried
Material 2;
(4) material 1 is mixed with material 2, obtains compound, compound is first washed with ethanol 2~3 times, then 2~3 are washed with ether
It is secondary, drying.
Described protein deposition agent, be 12~18% by content sulfosalisylic aqueous acid and content be 10~20%
Trichloroacetic acid solution be mixed to prepare by 0.8~1.2: 1.2~1.8 mass ratio.
Described anti-coagulants, be by sodium citrate and concentration for 0.012~0.18% salt solution by 1: 0.3~0.6 matter
Amount is than mixing.
Described dehydrating agent, is to mix mannitol and glycerine by 0.2~0.3: 0.12~0.18 mass ratio.
Described dispersant is one kind in lauryl sodium sulfate, polyacrylamide and methyl anyl alcohol.
Described oxidant, it by concentration is 22~28ml/L ozone to be, thiolase is by 1.2~1.6: 0.25~0.35
Mass ratio is mixed.
Described oxidation processes, are that material is heated into 34~36 DEG C, the reaction time is 2.5~3.5h.
The mass ratio of described metal agent, wherein zinc ion, lead ion and iron ion is 0.8~1.2: 0.1~0.2:
0.36~0.48.
Described protein precipitation agent, be by mass mixings such as methanol and ethanol, with hydrochloric acid conditioning solution pH value be 4.5~
5, mix thoroughly.
In summary, the beneficial effects of the present invention are:It is effective to improve cow's serum egg using the extracting method of the present invention
White recovery rate, the extraction rate reached of bovine serum albumin is to 95.6%, and purity significantly improves cow's serum egg up to more than 99.6%
White quality, effectively meets demand of the existing market to bovine serum albumin, effectively the medical value of lifting bovine serum albumin.
Wherein, the separation of blood plasma and haemocyte is first carried out to blood, reduces the difficulty extracted, first pass through sulfosalicylic acid and
Protein deposition agent is made by suitable concn proportioning in trichloroacetic acid, passes through the combination between the positive and negative charge of institute's band so that blood
Protein in slurry largely settles down, then the anti-coagulants being made up of sodium citrate and salt solution, forms soluble chelating thing,
Increase solution osmotic pressure, it is to avoid fibrinogen formation fibrin, it is to avoid blood plasma solidification, meanwhile, also destroy blood
Fibrinogen in slurry, the purity for raising bovine serum albumin provides basis;When handling solution material, wherein also containing portion
Point protein and substantial amounts of impurity, therefore, by solution material carry out oxidation processes, by regulate and control oxidation be by temperature, with
And using suitable oxidant so that the aliphatic acid in blood plasma obtains oxidation Decomposition to greatest extent, improves bovine serum albumin
Purity, then by situation of the specific metal to the specific destruction of enzyme, it is effective to remove in blood plasma using the metal of proper ratio
The impurity such as original protease, nuclease, further increase the purity of bovine serum albumin.
Embodiment
The embodiment to the present invention is described in further detail below, but the invention is not limited in these realities
Mode is applied, any improvement or replacement on the present embodiment essence spirit still falls within the claims in the present invention claimed
Scope.
Embodiment 1
A kind of method for extracting bovine serum albumin, comprises the following steps:
(1) fresh bovine blood is collected at ox vein, haemocyte and blood plasma are separated into through blood separator, is added in blood plasma
Enter 20% protein deposition agent of plasma purity, separate, obtain solid protein material and solution material;
Described protein deposition agent, is the sulfosalisylic aqueous acid for being 12% by content and three chloroethenes that content is 10%
Aqueous acid is mixed to prepare by 0.8: 1.2 mass ratio;
(2) solid protein material is handled:Solid protein material is added water stirring, protein solution is obtained, solid protein material quality is added
12% anti-coagulants, stirring shakes up, and centrifuges, and takes supernatant, and it is 4.5 to add hydrochloric acid regulation supernatant pH value, adds supernatant
The dehydrating agent of quality 4%, is mixed thoroughly, is stood, centrifuging and taking precipitation, and 1 must be expected by drying;
Described anti-coagulants, is to mix sodium citrate and concentration by 1: 0.3 mass ratio for 0.012% salt solution;
Described dehydrating agent, is to mix mannitol and glycerine by 0.2: 0.12 mass ratio;
(3) solution material is handled:Solution material, lauryl sodium sulfate, oxidant are mixed by 1.2: 0.1: 0.35 mass ratio
Close, obtain compound, by mixed material heating to 34 DEG C, the reaction time is 2.5h, add 0.12% metal of mixture quality
Agent, centrifugation, takes supernatant, adds the protein precipitation agent of supernatant quality 10%, stands 12h, and centrifuging and taking precipitation is dried
Material 2;
Described oxidant, is to mix concentration by 1.2: 0.25 mass ratio for 22ml/L ozone, thiolase;
The mass ratio of described metal agent, wherein zinc ion, lead ion and iron ion is 0.8: 0.1: 0.36;
Described protein precipitation agent, it, by mass mixings such as methanol and ethanol, is 4.5 with hydrochloric acid conditioning solution pH value to be,
Mix thoroughly;
(4) material 1 is mixed with material 2, obtains compound, compound is first washed with ethanol 2~3 times, then 2~3 are washed with ether
It is secondary, drying.
Embodiment 2
A kind of method for extracting bovine serum albumin, comprises the following steps:
(1) fresh bovine blood is collected at ox vein, haemocyte and blood plasma are separated into through blood separator, is added in blood plasma
Enter 30% protein deposition agent of plasma purity, separate, obtain solid protein material and solution material;
Described protein deposition agent, is the sulfosalisylic aqueous acid for being 18% by content and three chloroethenes that content is 20%
Aqueous acid is mixed to prepare by 1.2: 1.8 mass ratio;
(2) solid protein material is handled:Solid protein material is added water stirring, protein solution is obtained, solid protein material quality is added
18% anti-coagulants, stirring shakes up, and centrifuges, and takes supernatant, and it is 5 to add hydrochloric acid regulation supernatant pH value, adds supernatant matter
The dehydrating agent of amount 8%, is mixed thoroughly, is stood, centrifuging and taking precipitation, and 1 must be expected by drying;
Described anti-coagulants, is to mix sodium citrate and concentration by 1: 0.6 mass ratio for 0.18% salt solution;
Described dehydrating agent, is to mix mannitol and glycerine by 0.3: 0.18 mass ratio;
(3) solution material is handled:Solution material, polyacrylamide, oxidant are mixed by 1.8: 0.2: 0.45 mass ratio, obtained
Compound, by mixed material heating to 36 DEG C, the reaction time is 3.5h, adds 0.15% metal agent of mixture quality, from
The heart, takes supernatant, adds the protein precipitation agent of supernatant quality 15%, stands 18h, and centrifuging and taking is precipitated, and 2 must be expected by drying;
Described oxidant, is to mix concentration by 1.6: 0.35 mass ratio for 28ml/L ozone, thiolase;
The mass ratio of described metal agent, wherein zinc ion, lead ion and iron ion is 1.2: 0.2: 0.48;
Described protein precipitation agent, is, by mass mixings such as methanol and ethanol, to be 5 with hydrochloric acid conditioning solution pH value, mix
It is even;
(4) material 1 is mixed with material 2, obtains compound, compound is first washed with ethanol 2~3 times, then 2~3 are washed with ether
It is secondary, drying.
Embodiment 3
A kind of method for extracting bovine serum albumin, comprises the following steps:
(1) fresh bovine blood is collected at ox vein, haemocyte and blood plasma are separated into through blood separator, is added in blood plasma
Enter 25% protein deposition agent of plasma purity, separate, obtain solid protein material and solution material;
Described protein deposition agent, is the sulfosalisylic aqueous acid for being 15% by content and three chloroethenes that content is 15%
Aqueous acid is mixed to prepare by 1: 1.5 mass ratio;
(2) solid protein material is handled:Solid protein material is added water stirring, protein solution is obtained, solid protein material quality is added
15% anti-coagulants, stirring shakes up, and centrifuges, and takes supernatant, and it is 4.8 to add hydrochloric acid regulation supernatant pH value, adds supernatant
The dehydrating agent of quality 6%, is mixed thoroughly, is stood, centrifuging and taking precipitation, and 1 must be expected by drying;
Described anti-coagulants, is to mix sodium citrate and concentration by 1: 0.5 mass ratio for 0.015% salt solution;
Described dehydrating agent, is to mix mannitol and glycerine by 0.25: 0.15 mass ratio;
(3) solution material is handled:Solution material, methyl anyl alcohol, oxidant are mixed by 1.5: 0.15: 0.4 mass ratio, obtain mixed
Material is closed, by mixed material heating to 35 DEG C, the reaction time is 3h, adds 0.13% metal agent of mixture quality, centrifugation takes
Supernatant, adds the protein precipitation agent of supernatant quality 13%, stands 15h, and centrifuging and taking is precipitated, and 2 must be expected by drying;
Described oxidant, is to mix concentration by 1.4: 0.3 mass ratio for 5ml/L ozone, thiolase;
The mass ratio of described metal agent, wherein zinc ion, lead ion and iron ion is 1: 0.15: 0.42;
Described protein precipitation agent, it, by mass mixings such as methanol and ethanol, is 4.8 with hydrochloric acid conditioning solution pH value to be,
Mix thoroughly;
(4) material 1 is mixed with material 2, obtains compound, compound is first washed with ethanol 2~3 times, then 2~3 are washed with ether
It is secondary, drying.
Embodiment 4
A kind of method for extracting bovine serum albumin, comprises the following steps:
(1) fresh bovine blood is collected at ox vein, haemocyte and blood plasma are separated into through blood separator, is added in blood plasma
Enter 20% protein deposition agent of plasma purity, separate, obtain solid protein material and solution material;
Described protein deposition agent, is the sulfosalisylic aqueous acid for being 18% by content and three chloroethenes that content is 20%
Aqueous acid is mixed to prepare by 1: 1.2 mass ratio;
(2) solid protein material is handled:Solid protein material is added water stirring, protein solution is obtained, solid protein material quality is added
12% anti-coagulants, stirring shakes up, and centrifuges, and takes supernatant, and it is 5 to add hydrochloric acid regulation supernatant pH value, adds supernatant matter
The dehydrating agent of amount 4%, is mixed thoroughly, is stood, centrifuging and taking precipitation, and 1 must be expected by drying;
Described anti-coagulants, is to mix sodium citrate and concentration by 1: 0.3 mass ratio for 0.18% salt solution;
Described dehydrating agent, is to mix mannitol and glycerine by 0.2: 0.15 mass ratio;
(3) solution material is handled:Solution material, polyacrylamide, oxidant are mixed by 1.2: 0.1: 0.45 mass ratio, obtained
Compound, by mixed material heating to 34 DEG C, the reaction time is 3.5h, adds 0.12% metal agent of mixture quality, from
The heart, takes supernatant, adds the protein precipitation agent of supernatant quality 15%, stands 12h, and centrifuging and taking is precipitated, and 2 must be expected by drying;
Described oxidant, is to mix concentration by 1.6: 0.35 mass ratio for 22ml/L ozone, thiolase;
The mass ratio of described metal agent, wherein zinc ion, lead ion and iron ion is 1.2: 0.1: 0.4;
Described protein precipitation agent, is, by mass mixings such as methanol and ethanol, to be 5 with hydrochloric acid conditioning solution pH value, mix
It is even;
(4) material 1 is mixed with material 2, obtains compound, compound is first washed with ethanol 2~3 times, then 2~3 are washed with ether
It is secondary, drying.
Embodiment 5
A kind of method for extracting bovine serum albumin, comprises the following steps:
(1) fresh bovine blood is collected at ox vein, haemocyte and blood plasma are separated into through blood separator, is added in blood plasma
Enter 20% protein deposition agent of plasma purity, separate, obtain solid protein material and solution material;
Described protein deposition agent, is the sulfosalisylic aqueous acid for being 12% by content and three chloroethenes that content is 20%
Aqueous acid is mixed to prepare by 1.2: 1.2 mass ratio;
(2) solid protein material is handled:Solid protein material is added water stirring, protein solution is obtained, solid protein material quality is added
18% anti-coagulants, stirring shakes up, and centrifuges, and takes supernatant, and it is 5 to add hydrochloric acid regulation supernatant pH value, adds supernatant matter
The dehydrating agent of amount 4%, is mixed thoroughly, is stood, centrifuging and taking precipitation, and 1 must be expected by drying;
Described anti-coagulants, is to mix sodium citrate and concentration by 1: 0.6 mass ratio for 0.18% salt solution;
Described dehydrating agent, is to mix mannitol and glycerine by 0.3: 0.12 mass ratio;
(3) solution material is handled:Solution material, lauryl sodium sulfate, oxidant are mixed by 1.5: 0.2: 0.35 mass ratio
Close, obtain compound, by mixed material heating to 34 DEG C, the reaction time is 3.5h, add 0.12% metal of mixture quality
Agent, centrifugation, takes supernatant, adds the protein precipitation agent of supernatant quality 15%, stands 12h, and centrifuging and taking precipitation is dried
Material 2;
Described oxidant, is to mix concentration by 1.2: 0.35 mass ratio for 22ml/L ozone, thiolase;
The mass ratio of described metal agent, wherein zinc ion, lead ion and iron ion is 1.2: 0.1: 0.48;
Described protein precipitation agent, it, by mass mixings such as methanol and ethanol, is 4.5 with hydrochloric acid conditioning solution pH value to be,
Mix thoroughly;
(4) material 1 is mixed with material 2, obtains compound, compound is first washed with ethanol 2~3 times, then 2~3 are washed with ether
It is secondary, drying.
Experimental example
The present invention is counted to the recovery rate and purity of bovine serum albumin respectively, with tradition to bovine serum albumin
Extracting method is as control, as a result as shown in table 1:
Table 1
Project |
The inventive method |
Conventional method |
Average recoveries % |
95.6 |
93.2 |
Purity % |
99.6 |
99.3 |