CN106404480A - Method for extracting myoglobin in pig hearts and human total antioxidant status kit containing myoglobin - Google Patents
Method for extracting myoglobin in pig hearts and human total antioxidant status kit containing myoglobin Download PDFInfo
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Abstract
The invention provides a method for extracting myoglobin in pig hearts and a human total antioxidant status kit containing the myoglobin. The method comprises the following steps: a buffer is added into fresh pig hearts, homogenate and centrifugation are carried out, ammonium sulfate is added into a crude extract, stirring, precipitation, and centrifugation are carried out, and a supernatant is reserved; ammonium sulfate is added into the supernatant till a saturation state, precipitation is carried out for more than 2 hours, centrifugation is carried out, and the supernatant is removed; an ammonium sulfate containing buffer is used for redissolving the precipitate to an original volume, and filtering is carried out for waiting sampling; a hydrophobic filling material is filled into a column for sampling, balance is carried out till proteins do not flow out, the ammonium sulfate containing buffer is used for washing out, and target protein is collected according to an eluting peak. The method has high output and low extraction cost, obtained myoglobin has good stability and can be applied to a TAS kit, in order to detect concentration of total antioxidant substances in serum, urine and other components in body; at the same time the raw materials have abundant sources and are easy to obtain, and batch production requirements of the kit are satisfied.
Description
Technical field
The present invention relates to biochemistry, detectable field and in particular to be a kind of to adopt ammonium sulfate precipitation, hydrophobic layer
Analysis two-step method rapid extraction in the minds of fresh pig obtains high-purity Myoglobin, makes dry powder by spray drying technology, and will
It is applied to prepare people's total antioxidant status (TAS) test kit, the technology of detection people's total antioxidant status；It is specifically a boar
Extract in the heart the method for Myoglobin and the people's total antioxidant status test kit containing it.
Background technology
Active oxygen (Reactive oxygen species, ROS) mainly includes hydroxyl radical free radical, superoxide radical and mistake
Hydrogen oxide.Active oxygen can be produced in the normal physiological metabolism process of body, simultaneously some envirment factors such as ultra-vioket radiation, suction
Cigarette, pollution etc. also can induced activity oxygen generation.Free radical can cause DNA damage thus leading to mutation moreover it is possible to make protein, core
The macromolecules cross-linkings such as acid and then impact cell normal function, as metabolic intermediate, its strong respond can for free radical simultaneously
So that the many kinds of substance in cell is aoxidized, induced oxidation stress, participate in presenile dementia, the disease of many diseases such as parkinson disease
Reason process.
Research human organism's oxidation resistance is mainly passed through to analyze certain specific antioxidant content of body and (or) freedom at present
The change of base metabolite, such as lipid peroxide (LPO), superoxide dismutase (SOD), Glutathione (GSH), Vitamin C
Acid, Vitamin E etc..And can not be accurately anti-by way of single antioxidant content change in this component by vitro detection
Film projector body antioxidant levels.
Total antioxidant status (Total Antioxidant Status, TAS) reflection body maintains oxidation antioxidation state
The ability of balance.Document report shows, the total antioxidant status of body and diabetes, bronchial asthma, atherosclerosiss, wind
Multiple disease such as wet arthritis, hepatic injury, presenile dementia, cancer has close relationship, therefore measures body blood plasma, blood
Clearly, total antioxidant concentration in the composition such as urine, has very important biology and clinical meaning, is conducive to more comprehensively
Ground assessment body antioxidant status, grasp the defence capability of body, provide foundation for implementing antioxidation auxiliary treatment, to effective
Ground prevention disease and the deterioration controlling relevant disease.
The associated proteins that Myoglobin (Myoglobin, MB) is made up of a peptide chain and a prosthetic heme group, are muscle
The protein of interior storage oxygen.The distinctive structure of MB makes it be very beneficial for the storage of oxygen and transport, but exactly this oxygen-enriched shape
State makes it be highly prone to the attack of free radical, oxidized.The MB of the state of oxidation can form activated state by coupling catalysed ABTS
ABTS, i.e. ABTS+, ABTS+In blueness, there is absworption peak at 600nm, and internal antioxidant can suppress this reaction
Produce, judge the oxidation resistance of body by the size that it suppresses.
The prothetic group of Myoglobin is haemachrome, and it forms a ring by four pyroles subunits, and ring center is a ferrous iron
Ion, the particularity of this structure of matter and complexity make to express the restructuring Myoglobin of acquisition at present by genetic engineering mode
Have such problems as to yield poorly, stability poor, limit its application in TAS test kit.There is document report at present, two can be adopted
Secondary column chromatography extracts to the Myoglobin in the animal tissues such as pig myocardium, sardine muscle, but the method is loaded down with trivial details, extracts
Inefficient；Also it has been reported that the Stability of Myoglobin extracting from wild ox cardiac muscle, equus cardiac muscle is good, but shortcoming is former
Material is difficult to obtain, high cost is it is impossible to meet the batch production demand of test kit.
Content of the invention
The present invention, carries it is proposed that a kind of method extracting Myoglobin in Cor Sus domestica, the method yield height for above-mentioned deficiency
Take low cost, the Myoglobin of acquisition has good stability, can be applied in TAS test kit, to detect body serum, urine
Total antioxidant concentration in the compositions such as liquid, raw material sources are abundant simultaneously, are readily available, and can meet the batch life of test kit
Product demand.
For solving above-mentioned technical problem, the technical solution used in the present invention is：The side of Myoglobin is extracted in a kind of Cor Sus domestica
Method, key step includes：
1), after fresh Cor Sus domestica being removed fat and connective tissue, add disruption buffer, quickly even with tissue refiner
Slurry, and with High speed refrigerated centrifuge centrifugation, remove precipitation, obtain crude extract；
2) add ammonium sulfate, stirring in crude extract, after precipitation 0.5-1.5h under room temperature, be centrifuged with High speed refrigerated centrifuge,
Retain supernatant；
3) add ammonium sulfate to saturation in supernatant, precipitate more than 2h under room temperature, and use High speed refrigerated centrifuge
Centrifugation, supernatant discarded, obtain precipitation；
4) buffer of precipitation liquid containing ammonium sulfate redissolves that (original volume herein is to original volume：Broken buffering in step (1)
The volume of liquid), filter and treat loading；
5), after dewatering filling dress post, with the buffer balance of liquid containing ammonium sulfate, balance and terminate rear loading, after loading finishes, continue
Continuous balance flows out to no albumen, then with the buffer solution elution containing ammonium sulfate, collects destination protein according to eluting peak；
6) collect the destination protein obtaining after SDS-PAGE detects purity, add protective agent to pass through spray drying technology
Prepare dry powder.
The present invention mainly adopts ammonium sulfate precipitation and hydrophobic chromatography method to extract Myoglobin, and ammonium sulfate precipitation has adaptation
Property wide, to albumen not damaged, the low advantage of cost of material；Same hydrophobic chromatography is a step purification, and sharp separation, when having process
Between short, low production cost the advantages of, the two is used in combination, quickly, simply can extract from Cor Sus domestica and obtain highly purified flesh
Lactoferrin.
By above-mentioned purification process, the high-purity Myoglobin of acquisition, it is carried out with the analysis display such as yield, purity：
1000g Cor Sus domestica can extract the acquisition natural Myoglobin of 2g, the response rate more than 50%, find through SDS-PAGE analysis, extracted
Myoglobin purity up to more than 95%.The method has that raw material is easy to get, low cost, method are simple, without complex device,
The advantages of economical and practical, extraction yield is high, can achieve batch production.
Above-mentioned buffer used is phosphate buffer, Tris (trishydroxymethylaminomethane) buffer, HEPES (4- hydroxyl second
Base piperazine ethanesulfonic acid) buffer, MOP (3- (N- morpholinyl) propane sulfonic acid) buffer, one or more of borate buffer solution,
Concentration is 1mM～1000mM, and pH scope is between 6～9.5.Wherein the buffer used in each step can not
Unanimously, but in order to operate simply general whole process to use same buffer；
Above-mentioned ammonium sulfate mass concentration used is：
1) it is 40-60% in crude extract, this step passes through salting out, allow the strong protein of hydrophobicity form precipitation, from
The heart is removed；
2) redissolve precipitation, be 30-40% in level pad, under this concentration conditions, Myoglobin has certain dredging
Aqueouss, can combine with dewatering filling；
3) elution buffer is 15-25%, and under this concentration conditions, Myoglobin hydrophobicity weakens, can be with dewatering filling
Dissociation, thus be eluted.
Above-mentioned dewatering filling used is one of phenyl, butyl, octyl group, isopropyl.For commercially available prod, such as Xi'an perseverance
Become the dewatering filling of the phenyl class that the production code member that bio tech ltd sells is 13-0132- (01～06), production code member
Dewatering filling of butyl-like of 13-0231- (01～06) for 13-0132- (01～06) etc..
Protective agent used by above-mentioned spray drying be one of trehalose, corn dextrin, corn starch, Radix Acaciae senegalis or
Several, calculated with the volume of destination protein collection liquid, the mass concentration that protective agent uses is 0.1%～10%, protectant plus
Enter to protect Myoglobin that degeneration, gathering, precipitation do not occur in being spray-dried high-temperature process, can effectively increase spray simultaneously
Mist is dried heating surface area, is conducive to the formation of spray dried powder.
The present invention also provides a kind of people's total antioxidant status (TAS) test kit containing above-mentioned Myoglobin, this test kit
It is made up of reagent 1 and reagent 2：
Reagent 1 is：
Buffer 5～1000mM (mmol/L)
2,2 '-diazonium-bis--(3- ethylbenzothiazoline sulfonate) (ABTS, 2,2- azino-two (3- ethyl-benzothiazole-
6- sulfonic acid) di-ammonium salts) 10～1000 μM (10-6mol/L)
Myoglobin 1～100g/L
Preservative 0.01～2g/L；
Reagent 2 is：
Hydrogen peroxide (H2O2) 2～100mM.
Buffer in mentioned reagent box of the present invention is phosphate buffer, Tris (trishydroxymethylaminomethane) buffering
Liquid, HEPES (4- hydroxyethyl piperazine ethanesulfonic acid) buffer, MOP (3- (N- morpholinyl) propane sulfonic acid) buffer, borate buffer solution
One or more of, and pH scope is between 5.5～8.5.
Preservative in mentioned reagent box of the present invention is azido compound, thimerosal, PC series one of preservative or
Several.
TAS test kit in the present invention, can be used for detecting and assess body serum, blood plasma, urine, saliva, cell cracking
The total antioxidant status of the compositions such as liquid.
The test kit of the present invention, because Myoglobin has good stability, can be configured to liquid reagent, at 37 DEG C
Under the conditions of can stablize 15d, can stablize under the conditions of 4 DEG C 1 year about, test kit performance indications are held essentially constant；Simultaneously
Myoglobin derives from Cor Sus domestica, and extraction process is simple, extract yield height, low production cost, solves from equus cardiac muscle, bone
The high cost problem that bone flesh extracts, effectively reduces the production cost of test kit, produces good economic benefit.
Advantages of the present invention and beneficial effect：
1st, the present invention passes through ammonium sulfate precipitation, hydrophobic chromatography effect directly extraction Myoglobin, former material in the minds of fresh pig
Material is easy to get, extraction step is simple, extraction ratio is high, low production cost, easily realize industrialization.
2nd, pass through said extracted method, the natural Myoglobin that purity reaches more than 95% can be obtained, have good stability, can answer
For 15d in TAS test kit, can be stablized under the conditions of 37 DEG C of TAS test kit, 1 year about can be stablized under the conditions of 4 DEG C.
3rd, pass through the TAS test kit in the present invention, various aspects of performance meets requirement, can be used for detecting body serum, urine
Deng total antioxidant concentration in composition, assess total antioxidant status.
Brief description
The SDS-PAGE collection of illustrative plates of Fig. 1 embodiment of the present invention 1.
The SDS-PAGE collection of illustrative plates of Fig. 2 embodiment of the present invention 2.
Specific embodiment：
The present invention is expanded on further with reference to instantiation, but is not limitation of the present invention, in the spirit of the present invention
With any modification made in the range of right protection, the protection category of the present invention all will be included.
Example 1：Myoglobin is extracted from Cor Sus domestica
1), after fresh for 500g Cor Sus domestica being removed fat and connective tissue, add 1000ml 50mM phosphate buffer
PH8.0, is quickly homogenized after 5min with tissue refiner, and High speed refrigerated centrifuge 12000rpm is centrifuged 40min, removes precipitation, obtains
Obtain crude extract；
2) ammonium sulfate solids accounting for runic liquid quality 40%, gentle agitation, to be dissolved complete, room temperature are added in crude extract
After lower precipitation 1h, it is centrifuged 40min with High speed refrigerated centrifuge 12000rpm, discards precipitation, retain supernatant；
3) ammonium sulfate solids are continuously added in supernatant to saturation, gentle agitation, to be dissolved complete, room temperature is sunk
After the 2h of shallow lake, it is centrifuged 40min, supernatant discarded with High speed refrigerated centrifuge 12000rpm, obtains precipitation；
4) precipitation is redissolved to 1000ml with the buffer containing 30% ammonium sulfate, with 0.45 μm of membrane filtration so that clarifying；
5) 200ml phenyl hydrophobic filler is loaded in chromatographic column, with the 100mM phosphoric acid buffer of 3000ml 30% ammonium sulfate
Liquid pH8.0 balances, and balance terminates rear loading, after loading finishes, continues balance and flows out to no albumen, then with containing 15% sulphuric acid
The 100mM phosphate buffer pH8.0 eluting of ammonium, collects destination protein according to eluting peak；
6) detect the purity of destination protein in collection liquid by SDS-PAGE, electrophoresis result shows that purity is not less than 95%
(Fig. 1), collection liquid is added 2% Radix Acaciae senegalis according to volume, dry powder is prepared by spray drying technology, is wherein spray-dried
Condition is 150 DEG C of inlet temperature, spray velocity 100ml/h.
Example 2：Myoglobin is extracted from Cor Sus domestica
1), after fresh for 1000g Cor Sus domestica being removed fat and connective tissue, add 2000ml 120mM Tris buffer
PH8.8, is quickly homogenized after 10min with tissue refiner, and High speed refrigerated centrifuge 12000rpm is centrifuged 40min, removes precipitation, obtains
Obtain crude extract；
2) add 50% ammonium sulfate solids in crude extract, gentle agitation, to be dissolved completely, with high after precipitation 1h under room temperature
Fast refrigerated centrifuger 12000rpm is centrifuged 40min, retains supernatant；
3) add ammonium sulfate solids to saturation, gentle agitation in supernatant, wait to be completely dissolved, under room temperature, precipitate 2h
Afterwards, it is centrifuged 40min, supernatant discarded with High speed refrigerated centrifuge 12000rpm, obtain precipitation；
4) precipitation is redissolved to 2000ml with 35% ammonium sulfate 150mM Tris pH of buffer 8.5, after 0.45 μm of membrane filtration,
Prepare loading；
5) by 500ml octyl group dewatering filling, load in chromatographic column, with the 150mM Tris buffer containing 35% ammonium sulfate
PH8.5 balances, and balance terminates rear loading, after loading finishes, continues balance and flows out to no albumen, then with containing 25% ammonium sulfate
150mM Tris pH of buffer 8.5 eluting, according to eluting peak collect destination protein；
6) detect the purity of destination protein in collection liquid by SDS-PAGE, electrophoresis result shows that purity is not less than 95%
(Fig. 2), collection liquid is pressed volume and add 5% trehalose, dry powder is prepared by spray drying technology, wherein spray drying condition is
170 DEG C of inlet temperature, spray velocity 250ml//h.
Example 3：Myoglobin is extracted from Cor Sus domestica
1), after fresh for 800g Cor Sus domestica being removed fat and connective tissue, 1400ml, 250mM. borate buffer are added
In pH7.2, quickly it is homogenized after 25min with tissue refiner, High speed refrigerated centrifuge 8000rpm is centrifuged 40min, remove precipitation,
Obtain crude extract；
2) add 60% ammonium sulfate solids, gentle agitation in crude extract, under room temperature, after precipitation 1h, use high speed frozen centrifugation
Machine 8000rpm is centrifuged 40min, retains supernatant；
3) add ammonium sulfate solids to saturation in supernatant, under room temperature, after precipitation 2h, use High speed refrigerated centrifuge
7500rpm is centrifuged 50min, supernatant discarded, obtains precipitation；
4) precipitation is redissolved to 1400ml with 40% saturated ammonium sulfate buffer, and 0.8 μm of membrane filtration treats loading；
5) 600ml butyl dewatering filling is filled after post, put down with the 200mM borate buffer solution pH7.2 containing 40% ammonium sulfate
Weighing apparatus 3000ml, balance terminates rear loading, after loading finishes, continues balance and flows out to no albumen, then with containing 20% ammonium sulfate
200mM borate buffer solution pH7.2 eluting, collects destination protein according to eluting peak；
6) detect the purity of destination protein in collection liquid by SDS-PAGE, electrophoresis result shows that purity is not less than 95%
(Fig. 2), 4% corn dextrin will be added in collection liquid, dry powder is prepared by spray drying technology, spray drying condition is import
120 DEG C of temperature, spray velocity 80ml/h.
The spectrogram that after the Myoglobin SDS-PAGE electrophoresis obtaining, strip analysis obtain, as shown in accompanying drawing 1-2, shows band
Clearly, purity is high, and in Cor Sus domestica, the molecular size range of Myoglobin is about in 16.7KDa.
Embodiment 4：The preparation of TAS test kit
The Myoglobin extracting acquisition in case study on implementation 1,2,3 is prepared TAS test kit.
Reagent 1 is：
100mM Tris buffer, pH7.4
200μM ABTS
20g/L Myoglobin
1g/L sodium azide
Reagent 2 is：
30mM H2O2
Test kit testing conditions are：RateA method, R1:R2:S=250:50:5, dominant wavelength 600nm, commplementary wave length 405nm, read
Several point 22-32, reaction is upwards.
Embodiment 5：The preparation of TAS test kit
The Myoglobin extracting acquisition in case study on implementation 1,2,3 is prepared TAS test kit.
Reagent 1 is：
100mM phosphate buffer, pH6.8
200μM ABTS
10g/L Myoglobin
1g/L thimerosal
Reagent 2 is：
30mM H2O2
Test kit testing conditions are：Two point rate assay, R1:R2:S=210:80:10, dominant wavelength 600nm, commplementary wave length
405nm, reading point 25-35, reaction is upwards.
Embodiment 6：TAS test kit performance detection
The performance indications of the TAS test kit of configuration in embodiment 4 are detected.
1) range of linearity measures：Antioxidant is represented for measuring the range of linearity of TAS test kit with Vitamin E.Will
A series of Vitamin E (Vitamin E, VE) multiple of normal saline dilution, the TAS kit measurement prepared by the present invention is not
With the concentration of VE under extension rate, each point is surveyed 3 times, with its mean value calculation linear coefficient.Data is shown in Table 1：
Table 1：Under different extension rates, the measurement result of antioxidant (VE) concentration
| Extension rate | 1 | 0.9 | 0.8 | 0.7 | 0.6 | 0.5 | 0.4 | 0.3 | 0.2 | 0.1 | 0 |
| Measure concentration 1 (mM) | 2.56 | 2.21 | 2.03 | 1.78 | 1.55 | 1.19 | 0.95 | 0.72 | 0.50 | 0.26 | 0.07 |
| Measure concentration 2 (mM) | 2.53 | 2.26 | 2.05 | 1.76 | 1.51 | 1.26 | 0.96 | 0.71 | 0.51 | 0.26 | 0.08 |
| Measure concentration 3 (mM) | 2.56 | 2.31 | 2.04 | 1.70 | 1.58 | 1.29 | 1.05 | 0.78 | 0.54 | 0.23 | 0.07 |
| Mean concentration (mM) | 2.55 | 2.26 | 2.04 | 1.75 | 1.55 | 1.25 | 0.99 | 0.74 | 0.52 | 0.25 | 0.08 |
Mensure concentration in table 1 and extension rate are carried out statistical analysis, obtaining dependent linear equation is：
Y=2.502X+0.0201, R2=0.9987, be derived from test kit of the present invention the detection range of linearity be 0～
2.5mM, meets 0～2.2mM of description requirement.
2) precision measures：Using the Ve under TAS test kit respectively two variable concentrations of continuous detecting height, detect number of times
For 20 times, calculate CV value, in order to represent precision.Data such as table 2, table 3：
Table 2：The continuous detecting result of high concentration antioxidant (VE)
According to table 2, through statistical analysis, the average measured value of high concentration VE is 2.237mM, and standard deviation is 0.0615mM, and CV is
2.75%, the coefficient of variation meets description and requires 5%, and detection precision is high.
Table 3：The continuous detecting result of low concentration antioxidant (VE)
| Detection number of times | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Concentration (mM) | 0.75 | 0.73 | 0.72 | 0.74 | 0.74 | 0.73 | 0.77 | 0.76 | 0.73 | 0.73 |
| Detection number of times | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| Concentration (mM) | 0.69 | 0.68 | 0.73 | 0.79 | 0.76 | 0.71 | 0.79 | 0.7 | 0.72 | 0.72 |
According to table 2, through statistical analysis, the average measured value of low concentration VE is 0.7345mM, and standard deviation is 0.02947mM, CV
For 4.01%, the coefficient of variation meets description and requires 5%, and detection precision is high.
Embodiment 7：TAS test kit performance detection
The performance indications of the TAS test kit of configuration in embodiment 5 are detected.
1) range of linearity measures：Represent antioxidant with Vitamin E for measuring the range of linearity of TAS test kit.Will
A series of Vitamin E (Vitamin E, VE) multiple of normal saline dilution, the TAS kit measurement prepared by the present invention is not
With the concentration of VE under extension rate, each point is surveyed 3 times, with its mean value calculation linear coefficient.Data is shown in Table 1：
Table 1：Under different extension rates, the measurement result of antioxidant (VE) concentration
| Extension rate | 1 | 0.9 | 0.8 | 0.7 | 0.6 | 0.5 | 0.4 | 0.3 | 0.2 | 0.1 | 0 |
| Measure concentration 1 (mM) | 2.55 | 2.23 | 2.02 | 1.78 | 1.53 | 1.22 | 0.96 | 0.74 | 0.52 | 0.25 | 0.06 |
| Measure concentration 2 (mM) | 2.56 | 2.24 | 2.01 | 1.79 | 1.55 | 1.23 | 0.97 | 0.76 | 0.50 | 0.26 | 0.07 |
| Measure concentration 3 (mM) | 2.54 | 2.22 | 1.99 | 1.77 | 1.51 | 1.22 | 0.97 | 0.73 | 0.49 | 0.23 | 0.06 |
| Mean concentration (mM) | 2.55 | 2.23 | 2.01 | 1.78 | 1.53 | 1.22 | 0.97 | 0.74 | 0.50 | 0.25 | 0.06 |
Mensure concentration in table 1 and extension rate are carried out statistical analysis, obtaining dependent linear equation is：
Y=2.501X+0.008, R2=0.9986, be derived from test kit of the present invention the detection range of linearity be 0～
2.5mM, meets 0～2.2mM of description requirement.
2) precision measures：Using the Ve under TAS test kit respectively two variable concentrations of continuous detecting height, detect number of times
For 20 times, calculate CV value, in order to represent precision.Data such as table 2, table 3：
Table 2：The continuous detecting result of high concentration antioxidant (VE)
According to table 2, through statistical analysis, the average measured value of high concentration Ve is 2.235mM, and standard deviation is 0.0476mM, and CV is
2.13%, the coefficient of variation meets description and requires 5%, and detection precision is high.
Table 3：The continuous detecting result of low concentration antioxidant (VE)
| Detection number of times | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Concentration (mM) | 0.78 | 0.73 | 0.74 | 0.69 | 0.72 | 0.76 | 0.73 | 0.74 | 0.78 | 0.7 |
| Detection number of times | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| Concentration (mM) | 0.76 | 0.75 | 0.73 | 0.74 | 0.69 | 0.76 | 0.78 | 0.78 | 0.76 | 0.71 |
According to table 2, through statistical analysis, the average measured value of low concentration Ve is 0.7415mM, and standard deviation is 0.02925mM, CV
For 3.94%, the coefficient of variation meets description and requires 5%, and detection precision is high.
To sum up, according to the TAS test kit of the inventive method preparation, scope linearly good to the detection of antioxidant is wide,
The coefficient of variation is low, and precision is high, can be satisfied with the detection of body antioxidant status.
Claims (10)
1. in a kind of Cor Sus domestica extract Myoglobin method it is characterised in that：By ammonium sulfate precipitation, hydrophobic chromatography two-step method from
Rapid extraction Myoglobin in the minds of fresh pig, obtains highly purified Myoglobin.
2. in Cor Sus domestica according to claim 1 extract Myoglobin method it is characterised in that：Its main extraction step bag
Include：
1), after fresh Cor Sus domestica being removed fat and connective tissue, add disruption buffer, be quickly homogenized with tissue refiner, and
With High speed refrigerated centrifuge centrifugation, remove precipitation, obtain crude extract；
2) add ammonium sulfate, stirring in crude extract, after precipitation 0.5-1.5h under room temperature, with High speed refrigerated centrifuge centrifugation, retain
Supernatant；
3) add ammonium sulfate to saturation in supernatant, precipitate more than 2h under room temperature, and be centrifuged with High speed refrigerated centrifuge,
Supernatant discarded, obtains precipitation；
4) buffer of precipitation liquid containing ammonium sulfate redissolves to original volume, filters and treats loading；
5), after dewatering filling dress post, with the buffer balance of liquid containing ammonium sulfate, balance terminates rear loading, after loading finishes, continues flat
Weigh and flow out to no albumen, then with the buffer solution elution containing ammonium sulfate, destination protein is collected according to eluting peak；
6) collect the destination protein obtaining after SDS-PAGE detects purity, add protective agent to prepare by spray drying technology
Dry powder.
3. in Cor Sus domestica according to claim 2 extract Myoglobin method it is characterised in that：Buffer used is phosphoric acid
One or more of buffer, Tris buffer, HEPES buffer solution, mop buffer liquid, borate buffer solution, concentration is
1mM～1000mM, and pH scope is 6～9.5.
4. in Cor Sus domestica according to claim 2 extract Myoglobin method it is characterised in that：Sulfur in described crude extract
Sour ammonium concentration is 40～60%, and in redissolution precipitation, level pad, ammonium sulfate concentrations are 30～40%, ammonium sulfate concentrations during eluting
For 15～25%.
5. in Cor Sus domestica according to claim 2 extract Myoglobin method it is characterised in that：Dewatering filling used is benzene
One of base, butyl, octyl group, isopropyl.
6. in Cor Sus domestica according to claim 2 extract Myoglobin method it is characterised in that：Described spray drying institute
With protective agent be trehalose, one or more of corn dextrin, corn starch, Radix Acaciae senegalis, use quality concentration is
0.1%～10%.
7. a kind of containing claim 1 method preparation Myoglobin people's total antioxidant status test kit it is characterised in that：Should
Test kit is made up of reagent 1 and reagent 2, specially：
Reagent 1 is：
Reagent 2 is：
Hydrogen peroxide 2～100mM.
8. people's total antioxidant status test kit according to claim 7 it is characterised in that：Described buffer delays for phosphoric acid
Rush one or more of liquid, Tris buffer, HEPES buffer solution, mop buffer liquid, borate buffer solution, and pH scope exists
5.5～8.5.
9. people's total antioxidant status test kit according to claim 7 it is characterised in that：Preservative used is Azide
One or more of compound, thimerosal, PC series preservative.
10. people's total antioxidant status test kit according to claim 7 it is characterised in that：Described test kit can be used for
Detection and assessment body serum, blood plasma, urine, saliva, the total antioxidant status in cell pyrolysis liquid composition.
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| CN114773454A (en) * | 2022-04-27 | 2022-07-22 | 广州蕊特生物科技有限公司 | Extraction and purification method for extracting myoglobin from horse heart |
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| CN108918415A (en) * | 2018-07-19 | 2018-11-30 | 美康生物科技股份有限公司 | People's total antioxidant status detection kit |
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