阅读说明：本技术 一种脂蛋白纯化方法 (lipoprotein purification method ) 是由 李长路 于 2019-08-21 设计创作，主要内容包括：本发明公开了一种脂蛋白纯化方法,其特征在于：包括以下步骤：1)在血清中加入硫酸右旋糖酐溶液和氯化钙溶液,混匀,2～8℃保存12～24小时后离心；2)取步骤1)中的沉淀,加入沉淀溶解液溶解后离心,取上清,得到低密度脂蛋白；3)取步骤1)中的上清,加入硫酸右旋糖酐溶液和氯化钙溶液,混匀,2～8℃保存2～5小时后离心,取沉淀；4)取步骤3)中的沉淀,加入沉淀溶解液溶解后离心,取上清,得到高密度脂蛋白；其中,沉淀溶解液中含有：三氨基甲烷、草酸钾、十二烷基琥珀酸、脱氧胆酸钠和壬基酚聚氧乙烯醚。可以简单、高效地提取血清中的低密度脂蛋白和高密度脂蛋白。(the invention discloses a lipoprotein purification method, which is characterized by comprising the following steps: the method comprises the following steps: 1) adding a dextran sulfate solution and a calcium chloride solution into serum, uniformly mixing, storing at 2-8 ℃ for 12-24 hours, and centrifuging; 2) taking the precipitate in the step 1), adding a precipitate dissolving solution for dissolving, centrifuging, and taking the supernatant to obtain low-density lipoprotein; 3) taking the supernatant obtained in the step 1), adding a dextran sulfate solution and a calcium chloride solution, uniformly mixing, storing at the temperature of 2-8 ℃ for 2-5 hours, centrifuging, and taking a precipitate; 4) taking the precipitate in the step 3), adding a precipitate dissolving solution for dissolving, centrifuging, and taking the supernatant to obtain high-density lipoprotein; wherein the precipitation dissolving solution contains: triaminomethane, potassium oxalate, dodecyl succinic acid, sodium deoxycholate and nonylphenol polyoxyethylene ether. Can simply and efficiently extract low-density lipoprotein and high-density lipoprotein in serum.)

1. A method for purifying lipoproteins, comprising: the method comprises the following steps:

1) Adding a dextran sulfate solution and a calcium chloride solution into serum, uniformly mixing, storing at 2-8 ℃ for 12-24 hours, and centrifuging;

2) taking the precipitate in the step 1), adding a precipitate dissolving solution for dissolving, centrifuging, and taking the supernatant to obtain low-density lipoprotein;

3) Taking the supernatant obtained in the step 1), adding a dextran sulfate solution and a calcium chloride solution, uniformly mixing, storing at the temperature of 2-8 ℃ for 2-5 hours, centrifuging, and taking a precipitate;

4) Taking the precipitate in the step 3), adding a precipitate dissolving solution for dissolving, centrifuging, and taking the supernatant to obtain high-density lipoprotein; wherein the precipitation dissolving solution contains: triaminomethane, potassium oxalate, dodecyl succinic acid, sodium deoxycholate and nonylphenol polyoxyethylene ether.

2. The purification process according to claim 1, characterized in that: the concentration of each component in the precipitation solution is as follows: 30 mu mol/L of triaminomethane, 0.06mol/L of potassium oxalate, 1g/L of dodecyl succinic acid, 4g/L of sodium deoxycholate and 8g/L of nonylphenol polyoxyethylene ether.

3. The purification process according to claim 1 or 2, characterized in that: the mass concentration of the dextran sulfate is 4-6%.

4. The purification process according to claim 3, characterized in that: the concentration of the calcium chloride is 1.5 mol/L-2.5 mol/L.

5. the purification method according to claim 4, characterized in that: in the step 1), the volume ratio of the serum to the dextran sulfate solution to the calcium chloride solution is 1000: (5-10): (20-40).

6. The purification method according to claim 5, characterized in that: in the step 1), the centrifugal speed is 3000-4000 r/min, and the centrifugal time is 20-40 min.

7. the purification process according to claim 1 or 2, characterized in that: the amount of the precipitation dissolving solution added in the step 2) is 25-35 mL based on 1L of serum in the step 1); further, in the step 2), the centrifugal speed is 8000-12000 r/min, and the centrifugal time is 15-25 min.

8. the purification method according to claim 4, characterized in that: in the step 3), the volume ratio of the supernatant to the dextran sulfate solution to the calcium chloride solution is 1000: (75-85): (20-40).

9. The purification method according to claim 8, characterized in that: in the step 3), the centrifugal speed is 3500-4500 r/min, and the centrifugal time is 25-35 min.

10. The purification process according to claim 1 or 2, characterized in that: taking 1L of serum in the step 1), and adding 15-25 mL of precipitation dissolving solution in the step 4); further, in the step 4), the centrifugal speed is 12000-15000 r/min, and the centrifugal time is 15-25 min.

Technical Field

the invention relates to the technical field of biology, and particularly relates to a lipoprotein purification method.

Background

Lipoproteins are complexes composed of lipids and proteins. There is no covalent bonding between the lipids and proteins within the lipoproteins, most often through hydrophobic bond interactions between the non-polar portion of the lipid and the protein components. Blood lipids are insoluble in water and must be combined with apolipoproteins to form lipoproteins in order to be dissolved in the blood and transported to tissues for metabolism. Plasma lipoproteins can be classified into four major groups by the ultracentrifugation technique, depending on the size of the lipoprotein particles and their density classification, i.e. on the floating behavior within a specific salt density: chylomicron (CM): d <0.95 g/ml; very Low Density Lipoprotein (VLDL): d is 0.95-1.006 g/ml; low Density Lipoprotein (LDL): d is 1.006-1.063 g/ml; high Density Lipoprotein (HDL): d is 1.063-1.21 g/ml.

the currently used methods for separating and purifying lipoproteins mainly include ultracentrifugation, electrophoresis and precipitation. The ultracentrifugation method is a method of separating and purifying various lipoproteins under the action of a strong centrifugal force according to the difference of specific gravities (densities) of the lipoproteins in plasma. The ultracentrifugation method is an effective technique for separating pure lipoprotein, and is widely applied to the research of lipoprotein apolipoprotein metabolism at present. The electrophoresis separation method is to separate different lipoproteins with different charges due to different protein contents, and to judge and confirm the differences in the electrophoretic mobility of plasma lipoproteins. The precipitation separation method is characterized in that lipoprotein and polyanion are combined to form compound precipitate under the conditions of different polyanions, different metal ions with 2 valences and different pH values by utilizing different compositions and physicochemical properties of the lipoprotein, so as to achieve the aim of separating various lipoproteins.

However, the purification methods in the prior art still have the problems of long time consumption, high cost and low yield, and therefore, it is very necessary to provide a new method for purifying lipoproteins.

Disclosure of Invention

The present invention aims to provide a method for purifying lipoproteins, which can efficiently separate low-density lipoproteins from high-density lipoproteins.

The technical scheme adopted by the invention is as follows:

a method for purifying lipoproteins, comprising: the method comprises the following steps:

1) Adding a dextran sulfate solution and a calcium chloride solution into serum, uniformly mixing, storing at 2-8 ℃ for 12-24 hours, and centrifuging;

2) taking the precipitate in the step 1), adding a precipitate dissolving solution for dissolving, centrifuging, and taking the supernatant to obtain low-density lipoprotein;

3) Taking the supernatant obtained in the step 1), adding a dextran sulfate solution and a calcium chloride solution, mixing uniformly, and storing at 2-8 DEG C

Centrifuging after 5 hours, and taking a precipitate;

4) Taking the precipitate in the step 3), adding a precipitate dissolving solution for dissolving, centrifuging, and taking the supernatant to obtain high-density lipoprotein;

wherein the precipitation dissolving solution contains: triaminomethane, potassium oxalate, dodecyl succinic acid, sodium deoxycholate and nonylphenol polyoxyethylene ether.

Further, the concentration of each component in the precipitation dissolving solution is as follows: 30 mu mol/L of triaminomethane, 0.06mol/L of potassium oxalate, 1g/L of dodecyl succinic acid, 4g/L of sodium deoxycholate and 8g/L of nonylphenol polyoxyethylene ether.

further, the mass concentration of the dextran sulfate is 4-6%.

Furthermore, the concentration of the calcium chloride is 1.5 mol/L-2.5 mol/L.

further, the volume ratio of the serum, the dextran sulfate solution and the calcium chloride solution in the step 1) is 1000: (5-10): (20-40).

Further, in the step 1), the centrifugal speed is 3000-4000 r/min, and the centrifugal time is 20-40 min.

Further, the amount of the precipitation dissolving solution added in the step 2) is 25-35 mL based on 1L of serum in the step 1); further, in the step 2), the centrifugal speed is 8000-12000 r/min, and the centrifugal time is 15-25 min.

further, in the step 3), the volume ratio of the supernatant to the dextran sulfate solution to the calcium chloride solution is 1000: (75-85): (20-40).

Further, the centrifugation speed in the step 3) is 3500-4500 r/min, and the centrifugation time is 25-35 min.

further, the amount of the precipitation dissolving solution added in the step 4) is 15-25 mL based on 1L of serum in the step 1); further, in the step 4), the centrifugal speed is 12000-15000 r/min, and the centrifugal time is 15-25 min.

The invention has the beneficial effects that:

the invention discloses a method for purifying lipoprotein, which can simply and efficiently extract low-density lipoprotein and high-density lipoprotein in serum.

Drawings

FIG. 1 is an LDL electropherogram in which 1-Marker, 2-example 1, 3-example 2, 4-example 3, 5-control 1, 6-control 2, and 7-control 3;

FIG. 2 is a chart of HDL electrophoresis, wherein 1-Marker, 2-example 1, 3-example 2, 4-example 3, 5-control 1, 6-control 2, and 7-control 3.

Detailed Description

The present invention will be described in further detail with reference to examples. It will also be understood that the following examples are included merely for purposes of further illustrating the invention and are not to be construed as limiting the scope of the invention, as the invention extends to insubstantial modifications and adaptations of the invention following in the light of the principles set forth herein. The specific process parameters and the like of the following examples are also only one example of suitable ranges, and the skilled person can make a selection within the suitable ranges through the description herein, and are not limited to the specific data of the following examples.

The concentrations of the respective components in the precipitation solutions used in the following examples were: 30 mu mol/L of triaminomethane, 0.06mol/L of potassium oxalate, 1g/L of dodecyl succinic acid, 4g/L of sodium deoxycholate and 8g/L of nonylphenol polyoxyethylene ether, and the pH value is 7.5.

The dextran sulfate used in the following examples had a mass concentration of 5 wt% and the calcium chloride solution had a concentration of 2 mol/L.