CN105017412B - A method of the separating high-purity bovine serum albumin(BSA) from cow's serum - Google Patents
A method of the separating high-purity bovine serum albumin(BSA) from cow's serum Download PDFInfo
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Abstract
The invention belongs to Protein purification techniques field, a kind of method for specifically disclosing separating high-purity bovine serum albumin(BSA) from cow's serum.Cow's serum after dilution is successively passed through that ammonium sulfate precipitation, sieve chromatography, anion-exchange chromatography, pathogenic microorganism inactivate, sieve chromatography, ultrafiltration concentration, filtration sterilization finally obtain high-purity bovine serum albumin by the present invention.The purity of bovine serum albumin(BSA) is higher than 99.5%, moreover, without containing small-molecule substance, such as sodium chloride, ammonium sulfate, amino acid etc. in bovine serum albumin(BSA)；In addition, being free of live virus in bovine serum albumin(BSA).
Description
Technical field
The present invention relates to Protein purification techniques fields, and in particular, to one kind separating high-purity cow's serum from cow's serum
The method of albumin.
Background technique
Bovine serum albumin(BSA)（BSA）, also known as fifth component（Cohn Fraction V）And the main component of bovine blood,
Comprising 583 amino acid residues, molecular weight is 66.430 kDa, and isoelectric point is about 4.7.The name（Fifth component）Rule is derived from
The Cohn seralbumin partition method of original adoption cold ethanol precipitation method.According to CohnShi method, in the 5th ethanol fraction
In have found seralbumin.So-called bovine serum albumin(BSA) fifth component is a kind of to mainly contain bovine serum albumin(BSA) in fact
Mixture, rather than pure bovine serum albumin(BSA).Albumin can be with a variety of cations, anion and other small-molecule substance knots
It closes.Albumin in blood, which mainly serves, maintains osmotic pressure, pH buffer function, carrier function and trophism.It is thin in animal
In born of the same parents' free serum culture, addition albumin can play the role of physiology and mechanical protection and carrier function.Bovine serum albumin(BSA) is in life
Change and be widely used in experiment, such as in western blot and prepares the closed reagent in elisa plate.BSA generally as
Stabilizer is used in the preservation solution and reaction solution of restriction enzyme or modification enzyme because some enzymes it is unstable at low concentrations or
Activity is low.After BSA is added, it may play the role of " protecting " or " carrier ", many enzymes can make its activity big after adding BSA
Amplitude improves.Bovine serum albumin(BSA) is a kind of protein with the very multi-functional feature such as strong-hydrophobicity and hydrophily, other eggs
White matter does not have such feature, will if bovine serum albumin(BSA) is impure, such as containing other oroteins or small-molecule substance
It will affect its function.
Separation obtain in pure primarily discrete the removings blood plasma of sero-abluminous purpose other more than 200 kinds of foreign proteins with
Polluter that may be present obtains apyrogeneity, nonreactive antigen-antibody reaction, free of contamination albumin etc..Currently, seralbumin
The report majority of purifying is isolated and purified about source of people is sero-abluminous：The method of early stage such as, the chlorination nanofarad of Denis
(1847), the grade ammonium sulfate salting-out crystallisation (1894 of sodium sulphate method (1890) and Hermmarsten of Hofmeister
Year).China's early application ammonium sulfate salting-out process produces human serum albumins, but since ammonium sulfate production process is cumbersome, later
Using cold ethanol precipitation method.It so far is industrially still the main method for producing human serum albumins.Utilize cold ethanol precipitation method
There are also deficiencies：Ethyl alcohol is a kind of non-specific precipitation agent, although significant to human serum albumins production effect, product is pure
Degree is not very high；Ethyl alcohol as organic solvent is also possible to make protein aggregates or denaturation；In non-enclosed environment
Organic solvent is also larger to staff's harm, while remaining on ethyl alcohol in product and being difficult to remove.Also someone uses PEG precipitating instead thus
Method to avoid protein aggregation or denaturation, and is not easy to remove from albumin solution, and industrial production has certain difficulty.For solution
Certainly the small-molecule chemicals reagent such as ammonium sulfate, ethyl alcohol, PEG remains in seralbumin and seralbumin purity is not high asks
Topic, just using the albumin of chromatography production.But it is difficult to effectively inhibit or except pyrogen removal or external source using chromatography merely
Virus etc. seriously affects the safety of product.To make up chromatographic deficiency, chromatography is developed in the recent period and has mutually been tied with cold ethanol precipitation
The method of conjunction, the two has complementary advantages, using certain effect after production.
But the bovine serum albumin(BSA) of these methods purifying can have the following disadvantages：1）It is remained in seralbumin a large amount of
Small-molecule substance or chemical reagent, such as sodium chloride, ammonium sulfate, PEG, ethyl alcohol, amino acid, vitamin, microelement etc.；
2）Seralbumin purity is not high enough；3）The microorganisms such as exogenous virus have not had removal in serum, influence sero-abluminous peace
Quan Xing.
Summary of the invention
The purpose of the invention is to overcome the deficiencies of the prior art and provide one kind separating high-purity ox blood from cow's serum
The method of pure albumen.
To achieve the goals above, the present invention is achieved by the following technical programs：
A method of the separating high-purity bovine serum albumin(BSA) from cow's serum includes the following steps：
S1. physiological saline is added into cow's serum or PBS solution dilutes cow's serum；Add into the cow's serum after dilution
Enter moderate amount of sulfuric acid ammonium, supernatant is collected by centrifugation in 2 ~ 8 DEG C of standing 30min or more；
S2. the PBS solution of 2 ~ 3 times of volumes is added into supernatant, carries out hydrophobic chromatography after mixing, is washed using PBS solution
It is de-, collect eluent；
S3. eluent is subjected to sieve chromatography, collects the protein peak of not saliferous and other small molecules；The chromatography is filled out
Material is G10-G75 glucan；
S4. bovine serum albumin(BSA) is further purified using anion-exchange chromatography, by 0.1 ~ 1.5M concentration sodium chloride into
Row gradient elution collects consummate bovine serum albumin(BSA)；
S5. the inactivator that nonprotein denaturation class is added inactivates pathogenic microorganism, carries out molecular sieve layer later
Analysis collects bovine serum albumin(BSA) using ultrapure water elution；The sieve chromatography filler is the glucan of G25 or G50；
S6. using aperture be 30K film packet be concentrated by ultrafiltration, make bovine serum albumin(BSA) ultimate density reach 5mg/ml with
On；Finally degerming is filtered using 0.2 micron of filter membrane.
Kinds of protein complicated composition in cow's serum, therefore to by bovine serum albumin(BSA), therefrom high-purity is separated,
It must exclude the doping of other foreign proteins.Traditional technology is using blanket chlorination nanofarad, sodium sulphate method and sulfuric acid mostly
Ammonium salt fractionation crystallisation can not be arranged at all although these blanket methods can also be separated to bovine serum albumin(BSA)
Except the doping of other foreign proteins is not high to get the purity of the bovine serum albumin(BSA) arrived.The present invention by for many years to cow's serum at
The analysis and research being grouped as are distinguished according to the bioactivity of seralbumin and other foreign proteins or miscellaneous material, develop one kind
The method of high-purity separation albumin.Although the hydrophobic chromatography that the present invention uses, sieve chromatography, ion-exchange chromatography are all
For the conventional technical means of separation and purification of protein, still, the sequencing of various chromatographies is that can not arbitrarily adjust change,
And we have discovered that the resolution ratio and current-carrying capacity of hydrophobic chromatography are better than ion exchange when separating albumin, therefore should
Combination is optimum combination.
Preferably, the additional amount of physiological saline or PBS solution described in S1 is 1 ~ 3 times of cow's serum volume.
The effect of ammonium sulfate is to make protein denaturation, the hydrophobic grouping in exposure albumen, so as to according to hydrophobic property difference
It is separated, it is preferable that the dosage of ammonium sulfate described in S1 is that 10 ~ 30g ammonium sulfate is added in every 100 milliliters of dilute serums.
It is highly preferred that ammonium sulfate described in S1 is added by the amount that 15 ~ 25g ammonium sulfate is added in every 100 milliliters of dilute serums.
Most preferably, ammonium sulfate described in S1 is added by the amount that 20 ~ 25g ammonium sulfate is added in every 100 milliliters of dilute serums.
Into the cow's serum after dilution be added ammonium sulfate after, preferably 2 ~ 8 DEG C stand 1 hour more than be centrifuged again, receipts
Collect supernatant；The optimum condition of the centrifugation is 5000 ~ 8000 revs/min, is centrifuged 30 minutes to 1 hour.
Hydrophobic chromatography is sent out using some hydrophobic molecules in the hydrophobic ligands and mobile phase being coupled on stationary phase carrier
Raw invertibity in conjunction with and separated.This method is based on the hydrophobic difference of protein, and in high level salt solution, protein can be with
Hydrophobic aglucon combines, and other foreign protein is then without such property, using such property, point that can be preliminary by protein
From.Preferably, the filler of hydrophobic chromatography described in S2 is phenyl-glucan or the other dewatering fillings of butyl-glucan.Hydrophobic chromatography
When, foreign protein, which is present in, to be penetrated in liquid, is eluted using PBS solution, is collected eluent, is mainly contained the white egg of serum in eluent
It is white.
The present invention carry out sieve chromatography purpose be desalination and removal small-molecule substance, for example, ammonium sulfate, sodium chloride,
The substances such as amino acid, vitamin, small protein.
Preferably, the filler of anion-exchange chromatography described in S4 is DEAE- glucan.
Preferably, the inactivator of the denaturation of nonprotein described in S5 class is beta-propiolactone.Using beta-propiolactone to the micro- life of cause of disease
The optimum condition that object is inactivated is 37 DEG C and inactivates 24 hours.Beta-propiolactone according to its in the solution final concentration of 5 ‰ plus
Enter amount addition.
Compared with prior art, the present invention has the advantages that：
The purity of bovine serum albumin(BSA) is higher than 99.5% in the bovine serum albumin(BSA) being prepared through the invention, moreover, ox
Without containing small-molecule substance, such as sodium chloride, ammonium sulfate, amino acid etc. in seralbumin；In addition, in bovine serum albumin(BSA) not
Containing live virus.
Detailed description of the invention
Fig. 1 is that cow's serum phenyl-glucan hydrophobic chromatography separates sero-abluminous effect picture in embodiment 1；Wherein, blue
Color curve is separation situation of change of Proteins in Serum during hydrophobic chromatography；Red curve is conductance in solution（It is various
Ion concentration）Situation of change；Black arrow is bovine serum albumin white peak.
Fig. 2 is the effect that bovine serum albumin solution screens out salt and small-molecule substance by G25 dextran molecule in embodiment 1
Fruit figure；Wherein, blue curve is albumen change in protein situation during sieve chromatography in bovine serum albumin solution；It is red
Color curve is conductance in solution（Various ion concentrations）Situation of change；Black arrow is bovine serum albumin white peak；Red arrow is
Small-molecule substance protein peak.
Fig. 3 is that bovine serum albumin(BSA) passes through the further consummate effect of DEAE- glucan ion-exchange chromatography in embodiment 1
Figure；Wherein, blue curve is that the thick pure sample of bovine serum albumin(BSA) passes through ion-exchange chromatography Separation of Proteins situation；Red curve
For conductance in solution（Various ion concentrations）Situation of change；Black arrow is bovine serum albumin white peak.
Fig. 4 is that consummate bovine serum albumin solution passes through G50 dextran molecule sieve chromatography desalination and small molecule in embodiment 1
The effect picture of substance；Wherein, blue curve is Separation of Proteins situation in consummate rear bovine serum albumin solution；Red curve is
Conductance in solution（Various ion concentrations）Situation of change；Black arrow is bovine serum albumin white peak；Red arrow is small molecule object
Matter protein peak.
Fig. 5 is that smart bovine serum albumin(BSA) and cow's serum after purification carries out the comparison of SDS-PAGE electrophoretic effects in embodiment 1
Figure；M：Standard protein Marker；2：Bovine serum albumin(BSA) after consummate；3：The molecular weight of the corresponding protein of arrow.
Fig. 6 is that cow's serum butyl-glucan hydrophobic chromatography separates sero-abluminous effect picture in embodiment 2；Wherein, blue
Color curve is separation situation of change of Proteins in Serum during hydrophobic chromatography；Red curve is conductance in solution（It is various
Ion concentration）Situation of change；Black arrow is bovine serum albumin white peak.
Fig. 7 is the effect that bovine serum albumin solution screens out salt and small-molecule substance by G50 dextran molecule in embodiment 2
Fruit figure；Wherein, blue curve is albumen change in protein situation during sieve chromatography in bovine serum albumin solution；It is red
Color curve is conductance in solution（Various ion concentrations）Situation of change；Black arrow is bovine serum albumin white peak；Red arrow is
Small-molecule substance protein peak.
Fig. 8 is that bovine serum albumin(BSA) passes through the further consummate effect of DEAE- glucan ion-exchange chromatography in embodiment 2
Figure；Wherein, blue curve is that the thick pure sample of bovine serum albumin(BSA) passes through ion-exchange chromatography Separation of Proteins situation；Red curve
For conductance in solution（Various ion concentrations）Situation of change；Black arrow is bovine serum albumin white peak.
Fig. 9 is that consummate bovine serum albumin solution passes through G25 dextran molecule sieve chromatography desalination and small molecule in embodiment 2
The effect picture of substance；Wherein, blue curve is Separation of Proteins situation in consummate rear bovine serum albumin solution；Red curve is
Conductance in solution（Various ion concentrations）Situation of change；Black arrow is bovine serum albumin white peak；Red arrow is small molecule object
Matter protein peak.
Figure 10 is that smart bovine serum albumin(BSA) and cow's serum after purification carries out the comparison of SDS-PAGE electrophoretic effects in embodiment 2
Figure；M：Standard protein Marker；2：Bovine serum albumin(BSA) after consummate；3：The molecular weight of the corresponding protein of arrow.
Specific embodiment
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
Embodiment 1
A method of the separating high-purity bovine serum albumin(BSA) from cow's serum, specific step is as follows：
S1. 50mL physiological saline is added into 50mL cow's serum to mix, 20g ammonium sulfate is then added and mixes, is put into 2 ~ 8
Static 2 hours in DEG C refrigerator, then 8000 revs/min, it is centrifuged 30 minutes, collects supernatant.
S2. the PBS solution of 300 mL is added into supernatant, after mixing, direct loading carries out hydrophobic chromatography, and chromatography is filled out
Material is phenyl-glucan（GE company）, filler is eluted using normal saline solution, collects physiological saline eluting peak.Hydrophobic chromatography
It separates sero-abluminous effect picture and sees Fig. 1.
S3. it then is removed salt and small-molecule substance using molecular sieve, chromatographic stuffing is G25 glucan（GE company）,
Collect the protein peak of not saliferous and other small molecules.Seen by the effect picture that G25 dextran molecule screens out salt and small-molecule substance
Fig. 2.
S4. anion-exchange chromatography is then carried out, chromatographic stuffing is DEAE- glucan（GE company）, the direct loading of sample
Gradient elution is carried out using 0.5M sodium chloride afterwards, collects bovine serum albumin white peak.DEAE- glucan ion-exchange chromatography is further
Consummate effect picture is shown in Fig. 3.
S5. beta-propiolactone is added, in the solution final concentration of 5 ‰, 37 DEG C inactivate 24 hours；Then molecule is carried out
Sieve chromatography uses G50 glucan（GE company）Chromatographic stuffing carries out desalination and removal small-molecule substance, this process uses ultrapure water
Bovine serum albumin(BSA) is collected in elution.G50 dextran molecule screens out salt and the effect picture of small-molecule substance is shown in Fig. 4.
S6. it is then concentrated by ultrafiltration using the film packet that aperture is 30K, reaches final bovine serum albumin(BSA) concentration
5mg/mL or more；Finally degerming is filtered up to high-purity bovine serum albumin using 0.2 micron of filter membrane；Bovine serum albumin
The purity of white middle bovine serum albumin(BSA) is 99.7%.Bovine serum albumin(BSA) and cow's serum after will be consummate carry out SDS-PAGE electrophoresis
Effect compares, and as a result sees Fig. 5.
Embodiment 2
S1. 100mL physiological saline is added into 50mL cow's serum to mix, 40g ammonium sulfate is then added and mixes, is put into 2 ~ 8
Static 2 hours in DEG C refrigerator, then 8000 revs/min, it is centrifuged 30 minutes, collects supernatant.
S2. 300mL physiological saline is added into supernatant, after mixing, direct loading carries out hydrophobic chromatography, chromatographic stuffing
For butyl-glucan（GE company）, filler is eluted using the PBS solution of 0.1M, collects the PBS eluting peak of 0.1M.Hydrophobic chromatography
It separates sero-abluminous effect picture and sees Fig. 6.
S3. it then is removed salt and small-molecule substance using molecular sieve, chromatographic stuffing is G50 glucan（GE company）,
Collect the protein peak of not saliferous and other small molecules.G50 dextran molecule screens out salt and the effect picture of small-molecule substance is shown in Fig. 7.
S4. ion-exchange chromatography is then carried out, chromatographic stuffing is DEAE- glucan（GE company）, after the direct loading of sample
Gradient elution is carried out using 0.5M sodium chloride, collects bovine serum albumin white peak.DEAE- glucan ion-exchange chromatography is further smart
Pure effect picture is shown in Fig. 8.
S5. beta-propiolactone is added, in the solution final concentration of 5 ‰, 37 DEG C inactivate 24 hours.Then molecule is carried out
Sieve chromatography carries out desalination and removal small-molecule substance using G25 dextran chromatography filler, this process uses ultrapure water elution, receives
Collect bovine serum albumin(BSA).G25 dextran molecule screens out salt and the effect picture of small-molecule substance is shown in Fig. 9.
S6. it is then concentrated by ultrafiltration using the film packet that aperture is 30K, is reached using final bovine serum albumin(BSA) concentration
5mg/ml or more.Finally degerming is filtered up to high-purity bovine serum albumin using 0.2 micron of filter membrane.Bovine serum albumin
The purity of white middle bovine serum albumin(BSA) is 99.6%.Bovine serum albumin(BSA) and cow's serum after will be consummate carry out SDS-PAGE electrophoresis
Effect compares, the result is shown in Figure 10.
Embodiment 3
A method of the separating high-purity bovine serum albumin(BSA) from cow's serum, specific step is as follows：
S1. 50mL physiological saline is added into 50mL cow's serum to mix, 30g ammonium sulfate is then added and mixes, is put into 2 ~ 8
Static 3 hours in DEG C refrigerator, then 8000 revs/min, it is centrifuged 60 minutes, collects supernatant.
S2. the PBS solution of 250 mL is added into supernatant, after mixing, direct loading carries out hydrophobic chromatography, and chromatography is filled out
Material is phenyl-glucan（GE company）, filler is eluted using normal saline solution, collects physiological saline eluting peak.
S3. it then is removed salt and small-molecule substance using molecular sieve, chromatographic stuffing is G25 glucan（GE company）,
Collect the protein peak of not saliferous and other small molecules.
S4. anion-exchange chromatography is then carried out, chromatographic stuffing is DEAE- glucan（GE company）, the direct loading of sample
Gradient elution is carried out using 0.2M sodium chloride afterwards, collects bovine serum albumin white peak.
S5. beta-propiolactone is added, in the solution final concentration of 5 ‰, 37 DEG C inactivate 24 hours；Then molecule is carried out
Sieve chromatography uses G50 glucan（GE company）Chromatographic stuffing carries out desalination and removal small-molecule substance, this process uses ultrapure water
Bovine serum albumin(BSA) is collected in elution.
S6. it is then concentrated by ultrafiltration using the film packet that aperture is 30K, reaches final bovine serum albumin(BSA) concentration
5mg/mL or more；Finally degerming is filtered up to high-purity bovine serum albumin using 0.2 micron of filter membrane.
Comparative example 1
A method of the separating high-purity bovine serum albumin(BSA) from cow's serum, basic operation are unique different with embodiment 1
Be to remove S3 step, in the bovine serum albumin(BSA) finally obtained the purity of bovine serum albumin(BSA) be 80%.
Claims (7)
1. a kind of method of the separating high-purity bovine serum albumin(BSA) from cow's serum, which is characterized in that include the following steps：
S1. physiological saline or PBS solution are added into cow's serum, cow's serum is diluted；It is added into the cow's serum after dilution suitable
Ammonium sulfate is measured, supernatant is collected by centrifugation in 2~8 DEG C of standing 30min or more；
S2. the PBS solution of 2~3 times of volumes is added into supernatant, carries out hydrophobic chromatography after mixing, is eluted using PBS solution,
Collect eluent；
S3. eluent is subjected to sieve chromatography, collects the protein peak of not saliferous and other small molecules；The chromatographic stuffing is
G10~G75 glucan；
S4. bovine serum albumin(BSA) is further purified using anion-exchange chromatography, ladder is carried out by 0.1~1.5M concentration sodium chloride
Degree elution, collects consummate bovine serum albumin(BSA)；
S5. the inactivator that nonprotein denaturation class is added inactivates pathogenic microorganism, carries out sieve chromatography later, uses
Ultrapure water elution collects bovine serum albumin(BSA)；The sieve chromatography filler is the glucan of G25 or G50；
S6. it is concentrated by ultrafiltration using the film packet that aperture is 30K, bovine serum albumin(BSA) ultimate density is made to reach 5mg/ml or more；
Finally degerming is filtered using 0.2 micron of filter membrane；
Ammonium sulfate described in S1 is added by the amount that 10~30g ammonium sulfate is added in every 100 milliliters of dilute serums.
2. the method according to claim 1, wherein the additional amount of physiological saline or PBS solution described in S1 is ox
1~3 times of serum volume.
3. according to the method described in claim 2, it is characterized in that, ammonium sulfate described in S1 is added as in every 100 milliliters of dilute serums
The amount for entering 15~25g ammonium sulfate is added.
4. according to the method described in claim 3, it is characterized in that, ammonium sulfate described in S1 is added as in every 100 milliliters of dilute serums
The amount for entering 20~25g ammonium sulfate is added.
5. the method according to claim 1, wherein the filler of hydrophobic chromatography described in S2 be phenyl-glucan or
Butyl-glucan.
6. the method according to claim 1, wherein the filler of anion-exchange chromatography described in S4 is the Portugal DEAE-
Glycan.
7. the method according to claim 1, wherein the inactivator of the denaturation class of nonprotein described in S5 is in β-the third
Ester.
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| CN105384791B (en) * | 2015-12-09 | 2016-07-06 | 青海北极牦牛生物科技有限公司 | A kind of albuminous production technology of high-purity Serum of Yaks |
| CN106749626B (en) * | 2017-01-24 | 2020-05-19 | 西藏森威医疗科技有限公司 | Method for extracting albumin and globulin from bovine serum and method for utilizing bovine serum |
| CN107383185A (en) * | 2017-07-31 | 2017-11-24 | 天津市正江现代生物技术有限公司 | A kind of extracting method of high-purity bovine serum albumin |
| CN107365376A (en) * | 2017-07-31 | 2017-11-21 | 天津市正江现代生物技术有限公司 | A kind of extracting method of deer haemocyanin |
| CN108191971B (en) * | 2018-01-02 | 2022-02-22 | 汇海(苏州)生物技术有限公司 | Preparation method of recombinant human metallothionein III alpha fragment pure product |
| CN108546294A (en) * | 2018-07-19 | 2018-09-18 | 北京博莱得利生物技术有限责任公司 | The preparation method and products thereof of dog albumin |
| CN109627322A (en) * | 2019-01-07 | 2019-04-16 | 中国科学院过程工程研究所 | A method of human serum albumins is isolated and purified from V supernatant of Cohn component |
| CN109734796B (en) * | 2019-02-01 | 2022-04-15 | 广州蕊特生物科技有限公司 | Process for separating albumin from haemolytic serum |
| CN109776673B (en) * | 2019-02-20 | 2022-03-29 | 天津理工大学 | High-purity bovine serum albumin purification process suitable for pilot scale production |
| CN110339209B (en) * | 2019-07-18 | 2023-06-06 | 上海泰坦科技股份有限公司 | Preparation method of calf serum for removing thyroxine binding protein |
| CN112083158A (en) * | 2020-08-29 | 2020-12-15 | 赵峻岭 | Preparation method of bovine serum for confining liquid |
| CN113968905A (en) * | 2021-10-27 | 2022-01-25 | 哈尔滨国生生物科技股份有限公司 | Bovine serum albumin and application thereof |
| CN114276437A (en) * | 2021-12-04 | 2022-04-05 | 南京岚煜生物科技有限公司 | Preparation method of compound high-value reference substance for five immune terms |
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