CN108588046A - The method that natural myeloperoxidase is prepared from blood of human body neutrophil leucocyte azurophilic granule - Google Patents
The method that natural myeloperoxidase is prepared from blood of human body neutrophil leucocyte azurophilic granule Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/02—Oxidoreductases acting on a peroxide as acceptor (1.11) with H2O2 as acceptor, one oxygen atom of which is incorporated into the product (1.11.2)
- C12Y111/02002—Myeloperoxidase (1.11.2.2)
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Abstract
The present invention relates to biotechnology and field of immunology, a kind of be specifically related to prepare natural myeloperoxidase in the neutrophil leucocyte azurophilic granule from blood of human body method.This method passes through the research to natural myeloperoxidase biochemical characteristic, to increase the separation of cell tissue and the extraction of destination organization and improve primary leukocyte tissue liquid extraction process in a manner of grinding again, in favor of increasing substantially the degree of enrichment and relative purity of myeloperoxidase before chromatography.Then in conjunction with rational chromatographic methods are improved, to reduce the production yield loss of the myeloperoxidase after chromatography, entire purifying process is made to be more applicable for producing in batches.A series of quality testing by high standards shows to be improved largely in yield, purity, activity, specificity and stability via the natural myeloperoxidase antigen of this technique productions, and high quality is more suitable for medicine detection and scientific research.
Description
Technical field
The present invention relates to biotechnologys and field of immunology, concretely relate to a kind of from blood of human body neutrophil leucocyte
The method that natural myeloperoxidase is prepared in azurophilic granule.
Background technology
Anti-neutrophil cytoplasmic antibody (ANCA) is anti-for itself of neutrophil leucocyte and monocyte cytoplasmic components
Body.These antibody largely belong to IgG types, in the indirect immunofluorescence of ethyl alcohol fixed neutrophil leucocyte and monocyte
(IFA) react in have apparent positive reaction, their one of major target class be myeloperoxidase (MPO) antigen (i.e.
It is so-called core week p-ANCA).Myeloperoxidase (MPO) antigen after purification can be used for immune detection, immunochemistry and life
Change in MPO researchs and detects anti-myeloperoxidase (MPO) autoantibody.Anti- myeloperoxidase (MPO) antibody is certain systems
One of the important serologic marker of property vascular diseases, with idiopathic capilary inflammation, patients with crescent nephritis, arteritis nodosa and Qiu Er
Lattice-Astrid Strauss syndrome is closely related.Neutrophils indirect immunofluorescene assay (IIF) and standardized anti-marrow at present
The detection method that the detection of peroxidase (MPO) antibody ELISA is combined is widely used in the detection of the above disease.To marrow mistake
Oxide enzyme (MPO) antibody carries out high sensitivity, and the detection of high accuracy needs the core material marrow peroxidating in diagnostic reagent
Object enzyme (MPO) antigen has the speciality such as high-purity, high activity, high specific, to avoid the vacation being likely to occur in testing result
Positive, false negative.This proposes very high requirement to the preparation of myeloperoxidase (MPO) antigen.
For detecting, the antigen bulk of product development is broadly divided into recombinant expression and natural extraction two comes in existing market
Source.Relative to the recombinant antigen gone out using host cell expression, the native antigen for extracting and purifying from human blood cells has
Standby many advantages, including the amino acid sequence homology of height, natural protein three-level and quaternary conformation, correct people's blood
Modification after cell translation (such as phosphorylation, glycosylation etc.), and antigen receptor needed for complete protein function expression and
Epitope etc..Meanwhile the problem of aggregation and solubility that recombinant antigen is usually constructed with is not present in native antigen, it can more fully
And its specific antibody be combined, to reduce detection otherness.However since the resource of native protein is limited, source is each
It is different, cause extensive, batch extracting, purifying natural antigen tool to acquire a certain degree of difficulty, and expensive.
The method that document introduces natural myeloperoxidase enzyme purification is mainly the chromatography of routine, usually with neutrophilia
The full cell pyrolysis liquid of granulocyte is as initial crude protein liquid.But full cell pyrolysis liquid contains a large amount of cell foreign protein, needs
Complicated follow-up chromatography is wanted to detach, purification step, therefore yield and purity are all severely impacted.
Xu Luo Ling in 1992 etc. describes one kind and cracking human leukocytes with cetyl trimethylammonium bromide solution, and through sulphur
Sour ammonium fractional precipitation, SephadexG-150 columns extraction normal human leukocytes' myeloperoxidase method (《Sichuan physiological science
Magazine》1st phase in 1992, the purifying and identification of Human Leukocyte Myeloperoxidase, author：Xu Luoling, Li Chengliang, Wu Qi, king
Primary precious jade), the MPO antigen RZ values of acquisition are 0.68, and enzymatic activity 29.77u/mg, SDS-PAGE spectrum shows 3 zone, molecular weight
Respectively 59kd, 38kd, 13.5kd.
It reports within 1994 and a kind of is isolated from leucocyte extracting solution with lectin affinity chromatography and gel permeation chromatography
Method (the J Clin Invest.1994 of myeloperoxidase；94(1):437-444.), the method is less efficient, gained MPO
Purity is not high, is not suitable for mass production and medicine detection uses.
MingHuiZhao and C.MartinLockwood in 1996 disclose it is a kind of with orange-A dye ligands chromatography and
Method (the Journal of Immunological of cation exchange chromatography continuous purification myeloperoxidase
Methods.197(1-2):121-130), the acidic extraction object of normal human neutrophil is extracted from identical raw material.Separation
Afterwards, other known micro ANCA antigens (PR3, BPI) are not found, but the pollution of other unknown antigens is unknown.
Therefore, there is an urgent need for develop the preparation side of a kind of high quality and the natural myeloperoxidase suitable for producing in batches for this field
Method, to be used for scientific research and medicine detection.
Invention content
The present invention provides a kind of thermophilic reddish black from blood of human body neutrophil leucocyte to overcome the above-mentioned deficiency of the prior art
The method that natural myeloperoxidase is prepared in particle, the purpose one of the method are separation and mesh by increasing cell tissue
The extraction of mark tissue and the mode ground again, to improve primary leukocyte tissue liquid extraction process, in favor of increasing considerably color
The degree of enrichment and relative purity of myeloperoxidase before spectrum chromatography.The purpose of the method by combining second is that more improved reasonably
Chromatographic methods, it is natural to more efficiently obtain to reduce the production yield loss of the myeloperoxidase after chromatography
Myeloperoxidase makes entire purifying process be more applicable for producing in batches.
To achieve the goals above, the present invention is achieved by the following technical solutions：
The present invention proposes one kind and preparing natural myeloperoxidase from blood of human body neutrophil leucocyte azurophilic granule
Method, the method includes following seven steps：
Step 1: leucocyte in extraction normal human blood；
Step 2: extraction chromatographs initial protein solution：Blood of human body leucocyte discharges neutrality first in destruction step one
Granulocyte azurophilic granule obtains the initial protein solution of chromatography rich in myeloperoxidase antigen by centrifuging；
Step 3: cation-exchange chromatography：Using cation-exchange chromatography post, it is rich in using gradient salinity chromatography
The Primary purification protein sample of myeloperoxidase (MPO) antigen；
Step 4: dye ligands chromatograph：Post separation Primary purification protein sample is chromatographed with dye ligands, obtains being rich in MPO
The two level protein sample of antigen；
Step 5: sieve chromatography：With sieve chromatography post separation two level protein sample, three of the MPO containing high-purity are obtained
Grade purified protein samples；
Step 6: affinity chromatography：With affinity chromatography post separation three-level purified protein samples, remaining a small amount of miscellaneous antigen is removed,
Obtain the natural myeloperoxidase of final finished；
Step 7: stablizing and preserving：The natural myeloperoxidase of final finished is stored in and is preserved in solution.
Further, the specific method of the initial protein solution of extraction chromatography is in step 2：
(1) leucocyte extracted from blood of human body is washed with lysis buffer, by washed leucocyte and cracking
Cell homogeniser is added in buffer solution, and high-pressure homogenization is crushed leucocyte and obtains the full cell homogenates liquid of leucocyte, is rich in this homogenate
Neutrophil leucocyte cytoplasm azurophilic granule；The lysis buffer be include 15~25mM Tris-HCl (pH6.5-7.0), 1.5
The aqueous solution of~2.5mM NaCl and 3.0~4.0mM MgCl2；
(2) the full cell homogenates liquid of leucocyte is centrifuged, takes supernatant, is as rich in the layer of myeloperoxidase antigen
Analyse initial protein solution；The centrifugation condition is：Rotating speed is 1400~1600rpm, centrifugation time is 5~7 minutes.
Further, chromatography buffer used in cation-exchange chromatography is containing 20~50mM Na-Acetate
The aqueous solution of (pH5.4~5.6) and 0.02~0.05%Lubrol, elution buffer used in cation-exchange chromatography be containing
The aqueous solution of 20~50mM Na-Acetate (pH5.4~5.6), 0.02~0.05%Lubrol and 0~2M NaCl.
Further, chromatography buffer used in dye ligands chromatography is containing 20~50mM Na-Acetate (pH5.4
~5.6) and the aqueous solution of 0.02~0.05%Lubrol, dye ligands chromatograph used elution buffer as containing 20~50mM
The aqueous solution of Na-Acetate (PH5.4-5.6), 0.02~0.05%Lubrol and 0~2M NaCl.
Further, elution buffer used in sieve chromatography be containing 15~25mM Hepes-NaOH (pH 7.5~
8.5), the aqueous solution of 0.02~0.05%Lubrol and 0.1~0.5M NaCl.
Further, elution buffer used in affinity chromatography be containing 15~25mM Hepes-NaOH (pH 7.5~
8.5), the aqueous solution of 0.02-0.05%Lubrol and 0.1~0.5M NaCl.
Further, in the step 8 preserve solution be containing 15~25mM Hepes-NaOH (pH 7.5~8.5),
The aqueous solution of 0.02~0.05%Lubrol and 0.1~0.5M NaCl.
Compared with prior art, the beneficial effects of the present invention are：
(1) one kind provided by the invention isolates and purifies natural marrow peroxide from blood of human body neutrophil leucocyte azurophilic granule
The method of compound enzyme, by the research to natural myeloperoxidase (MPO) biochemical characteristic, increase cell tissue separation and
The extraction of destination organization and the mode ground again, improve primary leukocyte tissue liquid extraction process, and then increase considerably color
The degree of enrichment and relative purity of myeloperoxidase before spectrum chromatography.
(2) method of the invention more improves rational chromatographic methods by combining a whole set of, reduces chromatography layer
The production yield loss of myeloperoxidase after analysis makes entirely to purify so as to more efficiently obtain natural myeloperoxidase
Technique is more applicable for producing in batches.
(3) a series of matter for passing through high standards via the natural myeloperoxidase antigen of the method extraction purification of the present invention
Amount detection proves, is greatly improved in yield, purity, activity, specificity and stability, high quality is more suitable
It is detected for scientific research and medicine.
Description of the drawings
Fig. 1 is the myeloperoxidase protein electrophoresis glue result schematic diagram through cation-exchange chromatography；
Fig. 2 is that the myeloperoxidase zymoprotein through cation-exchange chromatography transfers result schematic diagram；
Fig. 3 is the myeloperoxidase cation-exchange chromatography collection of illustrative plates through cation-exchange chromatography；
Fig. 4 is the myeloperoxidase protein electrophoresis glue result schematic diagram of dyed ligand chromatography；
Fig. 5 is that the myeloperoxidase zymoprotein of dyed ligand chromatography transfers result schematic diagram；
Fig. 6 is that the myeloperoxidase of dyed ligand chromatography chromatographs collection of illustrative plates；
Fig. 7 is the myeloperoxidase protein electrophoresis glue result schematic diagram through sieve chromatography；
Fig. 8 is that the myeloperoxidase through sieve chromatography chromatographs collection of illustrative plates；
Fig. 9 is the myeloperoxidase protein electrophoresis glue result schematic diagram after affinitive layer purification；
Figure 10 is the myeloperoxidase UV Absorption spectrogram that embodiment 1 isolates and purifies；
The purity verification result schematic diagram of myeloperoxidase in Figure 11 embodiments 2.
Specific implementation mode
It shows that example illustrates certain embodiments of the present invention, and should not be construed as the model of the limitation present invention
It encloses.Present disclosure can be improved from material, method and reaction condition simultaneously, all these improvement should all
It falls within the spirit and scope of the present invention.
Embodiment 1：The side of natural myeloperoxidase is isolated and purified from blood of human body neutrophil leucocyte azurophilic granule
Method
1, leucocyte extracts
1) anti-coagulants is added into 2.76L blood, gently mixing；
2) test tube is uprightly statically placed in 30~60min in room temperature or 37 DEG C of incubators, waits for red blood cell natural subsidence；It is visible at this time
3 layers of suspension point in test tube, upper layer are faint yellow blood plasma, and bottom is red blood cell, and it is in canescence to have one on being close to red blood cell layer
Leukocytic cream.
3) cell suspension rich in leucocyte being located above red blood cell layer is drawn with capillary, is moved into another test tube；
4) it is added without Ca2+、Mg2+Hank ' s liquid is at from test tube mouth 3cm, mixing, with horizontal centrifuge 2000r/min from
Heart 10min, abandons supernatant, and acquisition enriched for leukemic cells precipitation 5.25g is being washed twice with method.
The natural myeloperoxidase of the present invention is finally to detach to carry from blood of human body neutrophil leucocyte azurophilic granule
It takes out, therefore it is normal human blood that the source of required leucocyte, which includes but not limited in the present embodiment, it can also
It is to collect to obtain from the materials such as blood transfusion leukoreduction filter film.
2, extraction chromatographs initial protein solution
(1) the enriched for leukemic cells precipitation extracted from blood of human body, 4000rpm low speed are washed with 20ml disruption buffers
After centrifugation 30 minutes, extracts supernatant with Suction filtration device and abandon, obtain washed leucocyte；Homogenizer cold water is connected, is opened
It opens homogenizer and 20ml leucocytoclasia buffer solutions is added into homogenate funnel, it, will be washed white thin after adjusting homogenizer pressure
Born of the same parents and cell homogeniser is added with 20ml leucocytoclasia buffer solutions, high-pressure homogenization is crushed leucocyte, and to obtain the full cell of leucocyte even
Slurries are rich in neutrophil leucocyte cytoplasm azurophilic granule in this homogenate；Used disruption buffer is packet in the present embodiment
Include 15mM Tris-HCl (pH6.5), 1.5mM NaCl and 3.0mM MgCl2Aqueous solution.
(2) the full cell homogenates liquid of leucocyte after homogenate is centrifuged 5 minutes under conditions of 1500rpm, centrifuges knot
Supernatant is taken after beam, is as rich in the initial protein solution of chromatography of myeloperoxidase antigen.
The method of cell disruption used in the present invention includes but not limited to the high-pressure homogenization crush method of the present embodiment, further includes
Physical methods such as multigelation method, ultrasonication method, anxious hot quenching method and including autolysis method, the molten method of enzyme and chemical osmosis etc.
Chemistry and biological method.
3, cation-exchange chromatography
20ml chromatography buffers balance remittance cation-exchange chromatography post of the fine jade from SP-6FF (biology is ground in remittance) is taken first, waits putting down
Starting protein solution is carried in cation exchange column chromatography column after the completion of weighing apparatus, is eluted surface using 20ml elution buffers
The antigen of loading cation is eluted out, and is collected eluent and is obtained the Primary purification egg rich in myeloperoxidase (MPO) antigen
White sample.
Chromatography buffer used in cation-exchange chromatography is Na-Acetate containing 50mM (pH5.5) in the present embodiment
With the aqueous solution of 0.02%Lubrol, elution buffer used in cation-exchange chromatography is Na-Acetate containing 50mM
(pH5.5), the aqueous solution of 1.0M NaCl and 0.02%Lubrol.But chromatography used in cation-exchange chromatography in the present invention
Buffer solution and elution buffer including but not limited to disclosed in the present embodiment, as technical solution of the present invention use it is as shown in table 1
Chromatography buffer and elution buffer can also meet the needs of cation-exchange chromatography in technical solution.
Chromatography buffer and elution buffer used in 1 cation-exchange chromatography of table
Result through cation-exchange chromatography as shown in Figure 1, Figure 2 and Figure 3.Fig. 1 is the primary marrow that protein electrophoresis glue is shown
Peroxidase purification effect can clearly on the diagram according to the molecular weight (59kD and14kD) of myeloperoxidase (MPO)
See that the myeloperoxidase (MPO) of purifying, upper part and lower part also have other antigens, it is therefore desirable to further separation；Fig. 2 is
Primary myeloperoxidase (MPO) purification effect of albumen transfer display, according to the molecular weight (59kD of myeloperoxidase (MPO)
And 14kD), it can clearly see that the myeloperoxidase (MPO) of purifying, this albumen transfer while having transferred sample on the diagram
Another major antigen impurity PR3 in product has to purify in the chromatographic step after staying in and reject；Fig. 3 is MPO cation-exchange chromatographies
Collection of illustrative plates, collection of illustrative plates show that one wave crest of generation when the salt gradient concentration of elution buffer reaches 1M, this wave crest represent the marrow of enrichment
Peroxidase.
4, dye ligands chromatograph
The dyeing that 20ml chromatography buffers balance filler is Reactive Yellow 3-Agarose (Sigma) is taken first
The Primary purification protein sample of collection is carried on dye ligands chromatographic column after the completion, uses 15ml by ligand chromatographic column, ready to balance
Elution buffer affords two level purifying protein solution.Chromatography buffer used in dye ligands chromatography is in the present embodiment
The aqueous solution of Na-Acetate containing 50mM (pH5.5), 0.02%Lubrol, the used elution buffer of dye ligands chromatography
To contain the aqueous solution of 50mM Na-Acetate (pH5.5), 2M NaCl and 0.02%Lubrol.But dye ligands layer in the present invention
Chromatography buffer and elution buffer used in analysis are including but not limited to disclosed in the present embodiment, such as technical solution of the present invention
Can also meet the needs of dye ligands chromatograph in technical solution using chromatography buffer as shown in Table 2 and elution buffer.
Chromatography buffer and elution buffer used in 2 dye ligands of table chromatography
The result of dyed ligand chromatography is as shown in Figure 4, Figure 5 and Figure 6.Fig. 4 is the marrow peroxidating that protein electrophoresis glue is shown
Object enzyme purification effect can clearly be seen on the diagram according to the molecular weight (59kD and 14kD) of myeloperoxidase (MPO)
The myeloperoxidase of purifying and the major impurity albumen LF of upper part, other antigen impurity PR3 and BPI are picked substantially
It removes；Fig. 5 is that albumen transfer shows that myeloperoxidase (MPO) purification effect, the transfer of this albumen use PR3 and MPO antibody tests.
It is substantially free of PR3 antigen impurity from can see in the antigen protein sample that this is purified on the transfer marking of tunica fibrosa.Fig. 6
Collection of illustrative plates is chromatographed for MPO dye ligands, collection of illustrative plates shows one wave crest of generation when the salt gradient concentration of elution buffer reaches 2M, this
Wave crest represents the myeloperoxidase of enrichment.
5, sieve chromatography
Take the molecular sieve chromatography 2 minutes that 50ml elution buffers balance filler is Chromdex 200pg (Bo Gelong)
(flow velocity 0.5ml/min) waits for elution buffer liquid level just or will enter molecular sieve column surface, and two level purifying protein is molten
Liquid is loaded to molecular sieve chromatography, and sample is made to flow into column；(elution buffer is added portionwise) is eluted with 50ml elution buffers,
It is fractionated simultaneously with test tube and collects eluent 1ml/ pipes, obtain three-level purifying protein solution.Sieve chromatography is made in the present embodiment
Elution buffer is the aqueous solution of Hepes-NaOH containing 20mM (pH 8.0), 0.02%Lubrol and 0.4M NaCl.But
Elution buffer used in sieve chromatography is including but not limited to disclosed in the present embodiment in the present invention, such as the technology of the present invention
Scheme can also meet the needs of sieve chromatography in technical solution using elution buffer as shown in table 3.
Elution buffer used in 3 sieve chromatography of table
Result through sieve chromatography is as shown in Figure 7 and Figure 8.Fig. 7 is that the myeloperoxidase that protein electrophoresis glue is shown is pure
Purifying can clearly be seen on the diagram according to the molecular weight (~59kD and 14kD) of myeloperoxidase (MPO) by changing effect
Myeloperoxidase, there be other antigens of minute quantity upper part and lower part, includes mainly LF and BPI, these impurity will be in parent
It is removed in chromatography；Fig. 8 is myeloperoxidase sieve chromatography collection of illustrative plates, and collection of illustrative plates display represents enrichment myeloperoxidase
There are one small bifurcated at the top of wave crest, illustrate the molecular weight of the MPO and other antigens in sample very close to this and Fig. 7 protein electrophoresis
The result that glue is shown is consistent, shows that the MPO of elution need to be further purified.
6, affinity chromatography
In order to further reject the foreign protein in three-level purifying protein solution, such as PR3, BPI etc. are molten by three-level purifying protein
Liquid carries out affinity chromatography.Take the affinity column 2 that 50ml elution buffers balance filler is remittance fine jade parent CNBr-4B (biology is ground in remittance)
Minute (flow velocity 0.5ml/min) waits for elution buffer liquid level just or will enter affinity column surface, three-level is purified
Protein solution is loaded to affinity column, and sample is made to flow into column；With the elution of 20ml elution buffers, (elution buffer adds in batches
Enter), it collects eluent (only absorbing proteins rate reaches peak position front and rear part) and obtains the final finished (MPO rich in high concentration
Solution) 2.2ml.In the present embodiment elution buffer used in affinity chromatography be Hepes-NaOH containing 20mM (pH8.0),
The aqueous solution of 0.02%Lubrol and 0.4M NaCl.
But elution buffer used in affinity chromatography is including but not limited to disclosed in the present embodiment in the present invention, such as this
Inventive technique scheme can also meet the needs of affinity chromatography in technical solution using elution buffer as shown in table 4.
Elution buffer used in 4 affinity chromatography of table
Through affinity chromatography, the results are shown in Figure 9.Fig. 9 is the myeloperoxidase that affinitive layer purification rear electrophoresis glue is shown
Purification effect, antigen is clearly seen other antigen impurity such as PR3 from figure, and BPI etc. has been removed.
7, stablize and preserve
The above-mentioned final finished eluted is rich in the MPO of high concentration, and MPO can be stabilized in elution buffer.Therefore,
The eluent through affinity column can directly be preserved.
MPO rich tissue cell precipitation 5.25g are obtained through primary extraction by the method for the present embodiment, by a series of chromatographies
Chromatography method, the final MPO solution for obtaining 2.2ml and being rich in high concentration.The MPO rich in high concentration obtained through chromatography is molten
Liquid measures albumen concentration by ultraviolet absorption method, and measurement A280 values are 0.553 (as shown in Figure 10), correspond to albumen by 1.467
A concentration of 1mg/ml obtains 0.82mg high-purity myeloperoxidase antigens altogether by embodiment 1 known to calculating.
Embodiment 2：The purity verification of myeloperoxidase (MPO) antigen that embodiment 1 is extracted and is purified
Myeloperoxidase (MPO) antigen that the present embodiment extracts embodiment 1 and purifies has carried out the verification of purity, together
When using Yashraj Biotechnology companies of India purify MPO antigens carried out parallel laboratory test.Verification method is through poly-
It is scanned using the ChemiDoc MP imagers of BioRad after acrylamide gel electrophoresis (SDS-PAGE), Coomassie blue stain
Photodensitometry (Densitometry Analysis) is carried out to running gel result afterwards.Testing result is as shown in figure 11, from this egg
White glue result is it will be evident that the MPO antigens of Yashraj Biotechnology companies of India purifying have doubtful small-molecular-weight miscellaneous
The case where matter or protein degradation, occurs, and then by the myeloperoxidase of technical scheme of the present invention extraction purification (MPO) antigen
Have many characteristics, such as higher purity and thermal stability.
Embodiment 3：Embodiment 1 is extracted and the specificity verification of myeloperoxidase (MPO) antigen of purifying
The method that the present embodiment extracts embodiment 1 and purifies MPO antigens has carried out specific verification, with embodiment 1
Obtained MPO antigens are the potency that standard antigen measures antibody in 13 sample serum samples, while with India Yashraj
The MPO antigens of Biotechnology companies purifying carry out parallel contrast experiment.Verification method uses ELISA (enzyme linked immunologicals
Method) method, 13 samples of detection include 7 normal serum samples and 6 positive serum samples.Testing result is as shown in table 5：
The specificity verification of myeloperoxidase (MPO) antigen of 5 present invention extraction of table and purifying
As can be seen from the above table, India is compared via the natural myeloperoxidase of the method for the present invention extraction purification
The myeloperoxidase of Yashraj Biotechnology companies extraction purification, normal serum potency is lower, MPO antibody sun
Property serum titer higher, show the natural MPO purity higher via the method for the present invention extraction purification, active higher, specificity more
It is high.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Any one skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (7)
1. a kind of method for preparing natural myeloperoxidase in neutrophil leucocyte azurophilic granule from blood of human body, feature exist
In the described method comprises the following steps：
Step 1: leucocyte in extraction normal human blood；
Step 2: extraction chromatographs initial protein solution：Blood of human body leucocyte is thin to discharge neutral grain in destruction step one first
Born of the same parents' azurophilic granule obtains the initial protein solution of chromatography rich in myeloperoxidase antigen by centrifuging；
Step 3: cation-exchange chromatography：Using cation-exchange chromatography post, obtain being rich in marrow mistake using gradient salinity chromatography
The Primary purification protein sample of oxide enzyme (MPO) antigen；
Step 4: dye ligands chromatograph：Post separation Primary purification protein sample is chromatographed with dye ligands, obtains being rich in MPO antigens
Two level protein sample；
Step 5: sieve chromatography：With sieve chromatography post separation two level protein sample, the three-level for obtaining the MPO containing high-purity is pure
Change protein sample；
Step 6: affinity chromatography：With affinity chromatography post separation three-level purified protein samples, remaining a small amount of miscellaneous antigen is removed, is obtained
The natural myeloperoxidase of final finished；
Step 7: stablizing and preserving：The natural myeloperoxidase of final finished is stored in and is preserved in solution.
2. one kind according to claim 1 prepares natural marrow peroxidating from blood of human body neutrophil leucocyte azurophilic granule
The method of object enzyme, which is characterized in that the specific method of the initial protein solution of extraction chromatography is in step 2：
(1) leucocyte extracted from blood of human body is washed with lysis buffer, and washed leucocyte and cracking are buffered
Cell homogeniser is added in liquid, and high-pressure homogenization is crushed leucocyte and obtains the full cell homogenates liquid of leucocyte, and neutrality is rich in this homogenate
Cytoplasm of granulocytes azurophilic granule；The lysis buffer be include 15~25mM Tris-HCl (pH6.5-7.0), 1.5~
2.5mM NaCl and 3.0~4.0mM MgCl2Aqueous solution；
(2) the full cell homogenates liquid of leucocyte is centrifuged, supernatant is taken, as at the beginning of the chromatography rich in myeloperoxidase antigen
Beginning protein solution；The centrifugation condition is：Rotating speed is 1400~1600rpm, centrifugation time is 5~7 minutes.
3. one kind according to claim 1 prepares natural marrow peroxidating from blood of human body neutrophil leucocyte azurophilic granule
The method of object enzyme, which is characterized in that chromatography buffer used in cation-exchange chromatography is containing 20~50mM Na-Acetate
The aqueous solution of (pH5.4~5.6) and 0.02~0.05%Lubrol, elution buffer used in cation-exchange chromatography be containing
The aqueous solution of 20~50mM Na-Acetate (pH5.4~5.6), 0.02~0.05%Lubrol and 0~2M NaCl.
4. one kind according to claim 1 prepares natural marrow peroxidating from blood of human body neutrophil leucocyte azurophilic granule
The method of object enzyme, which is characterized in that chromatography buffer used in dye ligands chromatography is containing 20~50mM Na-Acetate
The aqueous solution of (pH5.4~5.6) and 0.02~0.05%Lubrol, the used elution buffer of dye ligands chromatography is containing 20
The aqueous solution of~50mM Na-Acetate (PH5.4-5.6), 0.02~0.05%Lubrol and 0~2M NaCl.
5. one kind according to claim 1 prepares natural marrow peroxidating from blood of human body neutrophil leucocyte azurophilic granule
The method of object enzyme, which is characterized in that elution buffer used in sieve chromatography is containing 15~25mM Hepes-NaOH (pH
7.5~8.5), the aqueous solution of 0.02~0.05%Lubrol and 0.1~0.5M NaCl.
6. one kind according to claim 1 prepares natural marrow peroxidating from blood of human body neutrophil leucocyte azurophilic granule
The method of object enzyme, which is characterized in that elution buffer used in affinity chromatography is containing 15~25mM Hepes-NaOH (pH
7.5~8.5), the aqueous solution of 0.02-0.05%Lubrol and 0.1~0.5M NaCl.
7. one kind according to claim 1 prepares natural marrow peroxidating from blood of human body neutrophil leucocyte azurophilic granule
The method of object enzyme, which is characterized in that in the step 8 preserve solution be containing 15~25mM Hepes-NaOH (pH 7.5~
8.5), the aqueous solution of 0.02~0.05%Lubrol and 0.1~0.5M NaCl.
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