CN117209585A - Efficient extraction method of human serum natural lipoprotein (a) - Google Patents
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Abstract
The application relates to the technical field of blood lipoproteins, in particular to a high-efficiency extraction method of human serum natural lipoproteins (a), which comprises the following steps: 1) Adsorbing the crude product containing the target protein by macroporous adsorption resin aiming at a normal human sample, and eluting the target protein by using buffer solution; 2) Then loading the sample into an anion exchange column, and eluting after balancing and impurity washing; 3) The method comprises the steps of further purifying the ion column eluent by using an apolipoprotein a (Apo-a) antibody coupled filler, and adding a protective agent and a stabilizing agent into the final eluent to obtain the stable and clear human serum natural lipoprotein (a). The method of the application only needs three steps of operation, can efficiently separate and purify Lp (a) with low cost, and the prepared product has high purity, high yield and low cost.
Description
Technical Field
The application relates to the technical field of blood lipoproteins, in particular to a high-efficiency extraction method of human serum natural lipoproteins (a).
Background
Lipoprotein- (a) (Lp (a)) is a specific blood lipoprotein, consisting essentially of apolipoprotein (a) (Apo-a) and a similar Low Density Lipoprotein (LDL) particle. Wherein, the similar low density lipoprotein particles mainly comprise triglyceride, phospholipid, cholesterol and other lipids, and the lipid and lipoprotein shells are combined together through hydrophobic interaction. The highly glycosylated apolipoprotein in the component is the unique antigenic component of Lp (a) and the amino acid sequence has homology to plasmin. LP (a) prevents intravascular clot dissolution and promotes atherosclerosis formation, and its sustained elevation in level is closely related to angina, myocardial infarction, cerebral hemorrhage. The normal level of Lp (a) in serum is about 100-200 mg/L, exceeding 300 mg/L, and the probability of atherosclerosis is 2 times that of normal persons. Lp (a) may also be involved in wound healing and tissue repair, combining with some bacteria, with some anti-infective effects.
Currently, the mainstream measurement method of Lp (a) is an immunoassay method, and in the measurement process, lp (a) having a certain purity and concentration is required to be used as a calibrator to establish a standard curve. In addition, the research on the separation and purification method of Lp (a) can lay a foundation for the research and prevention and treatment of atherosclerosis and the detection method of Lp (a).
Lp (a) is known to bind several plasma components and extracellular matrix components, such as fibrinogen, fibrin, plasminogen, monocytes, fibronectin, etc., and to influence purification results when preparing Lp (a) antigen. In addition, the Lp (a) density interval is mostly distributed between 1.05-1.12 g/mL, overlaps with the density of LDL and HDL, has about 40-80 ten thousand molecular weight, has the difference among individuals, and has great separation and purification difficulty.
The common separation method of Lp (a) is to use ultracentrifugation, precipitation separation and column chromatography to obtain protein with a certain purity. Ultracentrifugation refers to density gradient centrifugation, which is a method of separation and purification during the process of ultracentrifugation according to the target differences between lipoproteins and other components. The precipitation separation mainly achieves the purpose of separation by increasing the ionic strength or changing the pH and other conditions to precipitate the lipoprotein according to the difference of solubility isoelectric points of the lipoprotein and other components. Column chromatography commonly used in combination in the Lp (a) separation process is mainly molecular sieve, and the purpose of separation is achieved according to the molecular weight.
The existing separation method has high equipment requirement, small treatment capacity, long time consumption and high cost. Therefore, there is an urgent need in the art to develop a simple and efficient Lp (a) extraction method, and to standardize the measurement of the power-assisted Lp (a) and to study biology.
Disclosure of Invention
Aiming at the defects that the separation and purification of Lp (a) in the prior art needs to simultaneously use operations such as ultracentrifugation, precipitation separation, column chromatography and the like, which have long time, expensive instrument and equipment, high cost, low recovery rate and the like, the application provides a high-efficiency extraction method of human serum natural lipoprotein (a).
In order to achieve the above purpose, the present application provides the following technical solutions:
a high-efficiency extraction method of human serum natural lipoprotein (a), comprising the following steps:
(1) Adsorbing the crude product containing the target protein by macroporous adsorption resin aiming at a normal human sample, and eluting the target protein by using buffer solution;
(2) Then loading the sample into an anion exchange column, and eluting after balancing and impurity washing;
(3) The method comprises the steps of further purifying the ion column eluent by using an apolipoprotein a (Apo-a) antibody coupled filler, and adding a protective agent and a stabilizing agent into the final eluent to obtain the stable and clear human serum natural lipoprotein (a).
Wherein in step (1), the sample comprises serum or plasma. Preferably, the sample is serum.
Wherein in the step (1), the macroporous adsorption resin is at least one of D-101 type, HPD-100 type, D1400 type, H103 type and ADS-8 type. Preferably, the macroporous adsorbent resin is of the D-101 type.
Wherein in the step (1), the buffer solution is any one of Tris, HEPES, MOPS, CHES and MOPSO, and the pH value is 7.5-8.8; the salt ion contains a certain concentration of salt ion, wherein the salt ion is NaCl or KCl, and the concentration is 50-150 mM. Preferably, the buffer component is Tris and the salt ion is NaCl.
Wherein, in the step (2), the anion exchange column is any one of QFF, QHP, DEAE, capto Q and QAE. Preferably, the anion exchange filler is a lower cost qff.
Wherein in the step (2), the balance buffer is 10-50 mM Tris-HCl, the pH is 7.5-8.5, and the NaCl concentration is 150 mM.
Wherein, in the step (2), the impurity washing buffer solution is 10-50 mM Tris-HCl, the pH value is 7.5-8.5, and the NaCl concentration is 200 mM.
Wherein in the step (2), the elution buffer is 10-50 mM Tris-HCl, the pH is 7.5-8.5, and the NaCl concentration is 400 mM.
Wherein in the step (3), the filler is any one of Epoxy pre-Activated filler (Epoxy Purose 4 FF), NHS pre-Activated filler (NHS-Activated loads 4 FF) and CNBr pre-Activated filler (CNBr-Activated Crystarose 4 FF). Preferably, the filler is an epoxy pre-activated filler.
In the step (3), the protective agent is at least one of glycerol, ethylene glycol, sucrose, glycine, arginine and trehalose, and the concentration of the protective agent is 5-20%. Preferably, the protective agent is glycerol.
Wherein in the step (3), the stabilizer is at least one of EDTA, EGTA, oxidized glutathione and 2-hydroxyethyl disulfide; the concentration of the stabilizer is 0.5-2 mM. Preferably, the stabilizer is 2-hydroxyethyl disulfide.
Further, the method of the application specifically comprises the steps of centrifuging a sample to remove impurities, loading the supernatant into a glass column filled with D-101 type resin macroporous adsorption resin, adsorbing lipoprotein mixed components, and eluting the mixed components containing human serum natural lipoprotein (a) by using a buffer solution;
loading the eluted components into a gravity column or a pre-packed column containing QFF packing, and obtaining eluent containing the target protein by balancing, washing impurities and eluting;
the method comprises the steps of coupling an apolipoprotein a (Apo-a) antibody to Epoxy pre-activated filler (Epoxy Purose 4 FF), further purifying the ion column eluent, and adding a protective agent and a stabilizing agent to obtain the stable human serum natural lipoprotein (a).
Compared with the prior art, the application has the beneficial effects that:
(1) The efficient extraction method of the human serum natural lipoprotein (a) only needs two steps of operation, and can efficiently separate and purify Lp (a) with low cost. The method comprises the steps of firstly adsorbing a component containing lipoprotein by using macroporous adsorption resin, eluting a mixed component containing Lp (a) by using buffer solution, loading an eluted sample into anion exchange chromatographic packing, and purifying by using an affinity column coupled with an antibody after eluting, thereby obtaining the Lp (a) with high product purity, high yield and low cost.
(2) The application does not use an ultracentrifugation method, does not use expensive instruments, shortens the time and reduces the cost. The sample is adsorbed and eluted by macroporous adsorption resin, and the Lp (a) with high purity, high yield and clarification can be obtained by eluting by using an ion exchange column and an affinity column coupled with the antibody, and the recovery rate reaches 80%.
(3) The high-efficiency extraction method of the human serum natural lipoprotein (a) utilizes macroporous adsorption resin to adsorb the lipoprotein, utilizes the charge difference of the protein, uses an ion exchange column and an affinity column coupled with an antibody of the apolipoprotein a, can obtain the target protein through three simple operations, solves the complex operation that the purification of Lp (a) in the prior art can obtain the target protein only by simultaneously using ultracentrifugation, precipitation separation, chromatography purification and a large amount of metal ions, has simple operation and saves time and cost.
Drawings
FIG. 1 shows the SDS-PAGE test during purification of Lp (a);
FIG. 2 shows the results of the freeze-thaw stability of Lp (a);
FIG. 3 shows the results of the acceleration stability of Lp (a) at 37 ℃.
Detailed Description
The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application. The following will illustrate a simple and efficient extraction method of Lp (a) native lipoprotein a in the present application by specific examples, which are given for illustrative purposes only and are not intended to limit the scope of the present application, by extracting Lp (a) from serum samples in two steps.
A high-efficiency extraction method of human serum natural lipoprotein (a), comprising the following steps:
(1) Pretreating serum sample with macroporous adsorbent resin to obtain crude extract of Lp (a)
The macroporous adsorption resin used was D-101 type and was packed into a gravity empty column, the column volume was 30 mL and the diameter was 3 cm. The gravity column was equilibrated with 90 mL buffer A (10 mM Tris-HCl,50 mM NaCl,pH 8.0). After centrifugation of 100 mL serum samples at 8000 rpm, the supernatant was loaded onto a equilibrated gravity column, the column volume was equilibrated for 3 further times using buffer A, and the crude extract containing Lp (a) was eluted using buffer B (10 mM Tris-HCl,150 mM NaCl,pH 8.0).
The concentration of Lp (a) in the fractions eluted by the gravity column packed with the adsorption resin was measured by using an Lp (a) latex turbidimetric reagent (nine-intensity bio-lipoprotein (a) measuring kit, latex immunonephelometry, cat# TGS 9151Z), and the specific content is shown in Table 1.
Table 1 Lp (a) results of concentration test during preparation
| Component (A) | Serum | Macroporous resin elution | Ion exchange column elution | Antibody affinity column elution |
| Concentration (mg/L) | 185.7 | 155.4 | 455.8 | 510.6 |
The purity of Lp (a) protein was checked by SDS-PAGE, and the results are shown in FIG. 1.
(2) Purifying the Lp (a) crude extract by an anion exchange column
In order to further increase the purity of Lp (a), the crude extract eluted from the adsorption column was further purified using an anion exchange column. Any one of anion exchange columns such as QFF, QHP, DEAE, capto Q, QAE and the like can be selected. In this example a QFF gravity column was used with a column volume of 10 mL.
The anion exchange column QFF was equilibrated with 30 mL buffer B, and the crude extract was slowly loaded into the equilibrated QFF column and equilibrated with 30 mL buffer B. The wash was then performed using 50mL buffer C (10 mM Tris-HCl,200 mM NaCl,pH 8.0). After washing, the final step was eluted with 30 mL buffer D (10 mM Tris-HCl,400 mM NaCl,pH 8.0) to obtain a clear and transparent fraction of purified Lp (a).
The concentration of Lp (a) in the eluted fraction of the ion exchange column was measured using an Lp (a) latex turbidimetric reagent, and the specific contents are shown in Table 1. The purity of Lp (a) protein was checked by SDS-PAGE, and the results are shown in FIG. 1.
(3) Further purification of the protein of interest by antibodies conjugated to apolipoprotein a
An apolipoprotein a antibody (Huatuo organism, cat# HT 1620075), one of the components of lipoprotein (a), was coupled to an Epoxy preactivated packing (Epoxy Purose 4FF, qian Chun Biotech Co., ltd.) according to the coupling method of the specification and packed into a gravity column. The anion eluted fractions were loaded onto a gravity column containing the conjugated antibodies using 50mL of equilibration buffer (10 mM Na 2 HPO 4 The pH was 7.4, the NaCl concentration was 150 mM), the mixture was equilibrated, eluted with 20 mL elution buffer (0.1M Glycine,pH 3.0), and neutralized with 5 mL neutralization buffer (1 mM Tris-HCl, pH 8.0), to finally obtain lipoprotein (a) having a higher purity.
The concentration of Lp (a) in the eluted fraction of the ion exchange column was measured using an Lp (a) latex turbidimetric reagent, and the specific contents are shown in Table 1. The purity of Lp (a) protein was checked by SDS-PAGE, and the results are shown in FIG. 1. According to the results, lp (a) in serum can be simply and efficiently enriched and purified through three steps of purification, the content of the impurity protein is low, the purity is high, and the research on a calibrator and the biochemical function of the calibrator can be satisfied.
(4) And adding a protective agent and a stabilizing agent into the Lp (a) eluting component to prepare the Lp (a) liquid component which can be stably stored.
Any one of glycerol, ethylene glycol, sucrose, glycine, arginine and trehalose can be added as a protective agent to the Lp (a) component eluted from the affinity column exchange column, in this example, glycerol with a final concentration of 10% is added as a protective agent, the sample can be frozen at-20 ℃, the freeze-thawing stability of Lp (a) is tested by using the Lp (a) latex reagent, and the concentration deviation of freeze thawing is less than 5% for five times, and the results are shown in fig. 2 and table 2.
Table 2 Lp (a) freeze-thaw stability bias data
In addition, it is necessary to add a stabilizer to further improve the stability of Lp (a). The stabilizer can be one of EDTA, EGTA, oxidized glutathione and 2-hydroxyethyl disulfide. In this example, 2-hydroxyethyl disulfide was added as a stabilizer at a final concentration of 1 mM.
The stability of the finally obtained Lp (a) component was examined using an Lp (a) latex reagent, and two concentration points were selected as evaluation concentrations, and the stability was evaluated by accelerating for 7 days at 37 ℃, which showed that the Lp (a) concentration deviation was less than 5%, and the results are shown in fig. 3 and table 3.
TABLE 3 Lp (a) acceleration stability data at 37℃
Although embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the application, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A method for efficiently extracting human serum natural lipoprotein (a), which is characterized by comprising the following steps:
(1) Adsorbing the crude product containing the target protein by macroporous adsorption resin aiming at a normal human sample, and eluting the target protein by using buffer solution;
(2) Then loading the sample into an anion exchange column, and eluting after balancing and impurity washing;
(3) The method comprises the steps of further purifying the ion column eluent by using an apolipoprotein a (Apo-a) antibody coupled filler, and adding a protective agent and a stabilizing agent into the final eluent to obtain the stable and clear human serum natural lipoprotein (a).
2. The efficient extraction method of human serum natural lipoproteins (a) according to claim 1, characterized in that: in the step (1), the sample includes serum or plasma.
3. The efficient extraction method of human serum natural lipoproteins (a) according to claim 1, characterized in that: in the step (1), the macroporous adsorption resin is at least one of D-101 type, HPD-100 type, D1400 type, H103 type and ADS-8 type.
4. The efficient extraction method of human serum natural lipoproteins (a) according to claim 1, characterized in that: in the step (1), the buffer solution is any one of Tris, HEPES, MOPS, CHES and MOPSO, and the pH value is 7.5-8.8; the salt ion contains a certain concentration of salt ion, wherein the salt ion is NaCl or KCl, and the concentration is 50-150 mM.
5. The efficient extraction method of human serum natural lipoproteins (a) according to claim 1, characterized in that: in the step (2), the anion exchange column is any one of QFF, QHP, DEAE, capto Q and QAE.
6. The efficient extraction method of human serum natural lipoproteins (a) according to claim 1, characterized in that: in the step (2), the balance buffer is 10-50 mM Tris-HCl, the pH is 7.5-8.5, and the NaCl concentration is 150 mM.
7. The efficient extraction method of human serum natural lipoproteins (a) according to claim 1, characterized in that: in the step (2), the impurity washing buffer solution is 10-50 mM Tris-HCl, the pH is 7.5-8.5, and the NaCl concentration is 200 mM; in the step (2), the elution buffer is 10-50 mM Tris-HCl, the pH is 7.5-8.5, and the NaCl concentration is 400 mM.
8. The efficient extraction method of human serum natural lipoproteins (a) according to claim 1, characterized in that: in the step (3), the filler for antibody coupling is any one of epoxy preactivated filler, NHS preactivated filler and CNBr preactivated filler.
9. The efficient extraction method of human serum natural lipoproteins (a) according to claim 1, characterized in that: in the step (3), the protective agent is at least one of glycerol, ethylene glycol, sucrose, glycine, arginine and trehalose, and the concentration of the protective agent is 5-20%;
the stabilizer is at least one of EDTA, EGTA, oxidized glutathione and 2-hydroxyethyl disulfide; the concentration of the stabilizer is 0.5-2 mM.
10. The efficient extraction method of human serum natural lipoproteins (a) according to claim 1, characterized in that: centrifuging the sample to remove impurities, loading the supernatant into a glass column filled with D-101 type resin macroporous adsorption resin, adsorbing lipoprotein mixed components, and eluting the mixed components containing the human serum natural lipoprotein (a) by using a buffer solution;
loading the eluted components into a gravity column or a pre-packed column containing QFF packing, and obtaining eluent containing target protein components through balancing, impurity washing and elution;
the method comprises the steps of further purifying the ion column eluent by using an apolipoprotein a (Apo-a) antibody coupled filler, and adding a protective agent and a stabilizing agent into the final eluent to obtain the stable and clear human serum natural lipoprotein (a).
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| CN101082045A (en) * | 2007-01-22 | 2007-12-05 | 耿永健 | Preparation method of apolipoprotein-J |
| US20110087008A1 (en) * | 2007-08-17 | 2011-04-14 | Csl Behring Gmbh | Methods for purification of alpha-1-antitrypsin andapolipoprotein a-1 |
| CN109053877A (en) * | 2018-10-09 | 2018-12-21 | 广东菲鹏生物有限公司 | The method and apolipoprotein B of apolipoprotein B are extracted from serum |
| CN111153985A (en) * | 2020-01-20 | 2020-05-15 | 宁波赛珀生物技术有限公司 | Separation and purification method of serum apolipoprotein A-II |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101082045A (en) * | 2007-01-22 | 2007-12-05 | 耿永健 | Preparation method of apolipoprotein-J |
| US20110087008A1 (en) * | 2007-08-17 | 2011-04-14 | Csl Behring Gmbh | Methods for purification of alpha-1-antitrypsin andapolipoprotein a-1 |
| CN109053877A (en) * | 2018-10-09 | 2018-12-21 | 广东菲鹏生物有限公司 | The method and apolipoprotein B of apolipoprotein B are extracted from serum |
| CN111153985A (en) * | 2020-01-20 | 2020-05-15 | 宁波赛珀生物技术有限公司 | Separation and purification method of serum apolipoprotein A-II |
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