Low-density lipoprotein purification by low-speed centrifugation

Abstract

The present application relates to a low-density lipoprotein low-speed centrifugal separation and purification method, comprising the following steps: using human serum, plasma and the remaining plasma after extracting other proteins as raw materials, adding PEG6000 to precipitate plasma low-density lipoprotein, then adding NaCl to adjust the liquid density and centrifugal, repeating the process, gradually changing the concentration and ratio of PEG6000 and NaCl until the plasma low-density lipoprotein precipitates and floats on the surface of the liquid after centrifugation, thereby separating from other proteins. The present application has the advantages of simple operation, large processing capacity, easy-to-get and cheap and safe reagents, low equipment requirements, high yield, and provides a convenient condition for various studies of plasma low-density lipoprotein.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, in particular to a low-density lipoprotein low-speed centrifugal separation and purification method. BACKGROUND

[0002] Plasma low-density lipoprotein (LDL) is composed of a series of particles with different sizes, densities and chemical compositions, and is heterogeneous. LDL has a core of cholesteryl ester and triacylglycerol, a phospholipid, free cholesterol and an ApoB-100 outer shell. LDL has always been the focus of research on blood lipids and cardiovascular diseases, but the high requirements of separation and purification methods have made many clinical researchers unable to obtain pure LDL and can only stay in statistical clinical data, which is a pity that they cannot enter the level of structural and functional research. In addition, there are few reports on LDL as a biological agent. As a normal component of blood, LDL should have its application value. In the late 1920s, someone proposed that lipids in the body are combined with proteins. Later research has been ongoing, and there have been classifications and names of alpha lipoprotein and beta lipoprotein. In 1948, a method for separating lipoproteins according to density using an ultracentrifuge was developed, and research on lipoproteins and related diseases also began to flourish. So far, the main method for purifying lipoproteins is ultracentrifugation, and some use chromatography and affinity chromatography, all of which have problems such as high equipment or material requirements, small processing capacity or long cycle. Electrophoresis is very effective for the separation of proteins in the order of hundreds of milligrams, and devices for kilograms have also appeared, but electrophoresis requires pretreatment of the material, and the effect is good after reaching a certain purity, and it is not very effective for scales above kilograms.

[0003] Chinese patent document CN109323910A, published on February 12, 2019, discloses a method for preparing high-purity low-density lipoprotein. According to the density, the chylomicron and very low-density lipoprotein in the plasma are removed by centrifugation, the remaining plasma is added with potassium bromide to adjust the density to 1.045 g / mL, and then centrifugation is performed to obtain the conventional low-density lipoprotein located in the uppermost layer. The K + concentration is reduced by dialysis with a phosphate buffered saline solution, and the low-density lipoprotein is dialyzed before and after dialysis. +The results measured by the fully-automatic biochemical analyzer at different concentrations are compared with the results measured by the osmotic pressure instrument. If there is no obvious difference between the dialysis and the osmotic pressure of the phosphate buffer solution, it is considered that the potassium bromide is completely dialyzed, and then high-purity low-density lipoprotein is obtained. Chinese Patent Document CN109580303A, published on April 5, 2019, discloses a preparation method of low-density lipoprotein, which comprises the following steps: adding a macromolecular compound to serum to remove serum lipoprotein except low-density lipoprotein and high-density lipoprotein; then precipitating low-density lipoprotein with metal ions; then dissolving the precipitate with a buffer and metal ions, and removing a small amount of impurity protein such as albumin; and finally purifying low-density lipoprotein. The literature (Wang KQ, Chen YF, Ran BF, Research on Lipoprotein I. New Method for Large-scale Separation of Human Serum Beta Lipoprotein and Its Certain Property Research Acta Biochimica et Biophysica Sinica 1964 (03): 13-24) has long studied the large-scale extraction of LDL, which greatly improves the extraction amount compared with the ultracentrifugation method, but still needs a high-speed centrifuge and low-temperature operation. The low-temperature ethanol method can achieve a large scale, but faces the situation that the organic solvent may affect the structure of lipoprotein, and the temperature needs to be controlled within a certain range at all times, which is quite inconvenient to operate. The above-mentioned lipoprotein separation and purification operations have various methods, which can be summarized as ultracentrifugation method, various precipitation methods, various chromatography methods, etc. Combining these methods to achieve the purpose of purification is not satisfactory in terms of time, instrument and equipment requirements, operation steps, degree of simplicity, scale and final yield. The present application uses a low-speed centrifuge and inexpensive and non-toxic chemical reagents to separate and purify human low-density lipoprotein from various human plasma or serum and other body fluids, and can also be extracted from the remaining plasma after other proteins are extracted. This is an original purification method for lipoprotein using a new idea, and no similar reports have been found. SUMMARY

[0004] The purpose of the present application is to provide a low-density lipoprotein low-speed centrifugation separation and purification method in view of the deficiencies in the prior art.

[0005] The present application is realized by the following scheme:

[0006] The human serum, plasma and remaining plasma after other proteins are extracted are used as raw materials, PEG 6000 is added to precipitate LDL, NaCl is added to adjust the liquid density and centrifuged, and the process is repeated, the concentration and ratio of PEG 6000 and NaCl are gradually changed until the LDL is precipitated and floats on the surface of the liquid after centrifugation to separate from other proteins, which specifically comprises the following steps:

[0007] (a) taking the raw material, adding PEG 6000, stirring and dissolving, and centrifuging with a centrifuge;

[0008] (b) Add PEG 6000 into the supernatant to make the concentration of PEG 6000 reach 6%, stir to dissolve, stand, and pour the supernatant into another container;

[0009] (c) Dissolve the precipitate with normal saline, centrifuge to discard the insoluble substance, add PEG 6000 into the supernatant to make the concentration of PEG 6000 reach 6%, stand, and take the precipitate after standing, repeat the step for two times, dissolve the precipitate with normal saline, and centrifuge to discard the insoluble substance;

[0010] (d) Add 8-20% NaCl solid powder into the supernatant to stir to dissolve, centrifuge to remove the tiny bubbles, add 8-15% PEG 6000 to stir to dissolve, centrifuge, retain the upper floating substance, discard the lower liquid and precipitate, repeat the step for 2-3 times, and finally dissolve the floating substance with normal saline.

[0011] Further, the centrifugal speed of the centrifuge is 3000 rpm.

[0012] Further, the raw material in the step (a) is human serum, plasma, and the remaining plasma after extracting other proteins.

[0013] Further, the concentration of the NaCl solid powder added in the step (d) is 20%, and the concentration of PEG 6000 is 12%.

[0014] The concentration of the PEG 6000 added in the step (a) is 4%.

[0015] Further, the concentration of the PEG 6000 added in the step (b) is 2%.

[0016] Further, the normal saline in the steps (c) and (d) is 10% of the volume of the raw material.

[0017] It should be noted that the concentrations of the added PEG 6000 and NaCl are calculated based on the amount of added normal saline. For example, if 100 mL of normal saline is added, and N grams of a certain substance are added, the concentration is N%, and the concentration of NaCl in normal saline is relatively low, which basically does not affect the final concentration and can be ignored.

[0018] The advantages of the present application are:

[0019] 1. It is a brand new method for separating lipoprotein, which is simple to operate, has large processing capacity, and uses reagents that are easy to obtain, cheap, safe, and low in equipment requirements, and has high yield, which is suitable for various laboratories and biological product factories, and provides a convenient condition for various researches of LDL.

[0020] 2. The high-value products prepared by the method can be used as raw materials for preparing quality control products and starting materials for purifying related apolipoproteins in the methodological verification of blood lipid determination in biochemical tests. The method will provide technical reserves for the popularization and application of the results of LDL pharmaceutical research. BRIEF DESCRIPTION OF DRAWINGS

[0021] FIG. 1 Figure 1 FIG. 2

[0022] FIG. 3 Figure 2 FIG. 4

[0023] FIG. 5 Figure 3 FIG. 6 DETAILED DESCRIPTION

[0024] The present application will be further described in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. In addition, it should be understood that the LDL separation and purification "new method, new invention and new patent" formed by adding non-precipitants and non-density adjusting agents on the basis of the present formulation should be considered invalid, unless there is the introduction of new precipitants or density adjusting agents or the expansion of the present method to other protein purification work. These equivalent forms also fall within the scope defined by the claims attached hereto.

[0025] Example 1

[0026] 1. Materials and methods

[0027] 1.1 Experimental apparatus and reagents

[0028] Polyethylene glycol 6000 (PEG 6000), NaCl (Wuxi Yatai United Chemical Co., Ltd.; (domestic analytical pure); total protein determination reagent, cholesterol determination reagent, triglyceride determination reagent (Japan Suwa Products); high-density lipoprotein cholesterol (HDL) determination reagent, low-density lipoprotein cholesterol (LDL) determination reagent (Japan One Chemical Products); apolipoprotein B determination reagent (British Langdon Products); lipoprotein alpha (LPa) determination reagent (Japan Nitto Bose Products); magnetic stirrer (Beijing Hengao Instruments and Meters Co., Ltd.); centrifuge (TD5A-WS with 4x100mL rotor and tube, Shanghai Luxiang Centrifuge Instruments Co., Ltd.); biochemical instrument (Japan Toshiba TBA-FX8 biochemical instrument).

[0029] 1.2 Verification of the feasibility of the present study using LDL crude extract

[0030] Take 100 mL of crude LDL extract, add 15 g of PEG 6000 and 20 g of NaCl, stir to dissolve, and centrifuge (centrifugation conditions: 3000 rpm, relative centrifugal force (RCF) = 1580 × g, centrifugation time < 30 min, and the following centrifugations shall be performed under these conditions).

[0031] 1.3 Preliminary test of conditions for complete separation of LDL from contaminating proteins

[0032] Take 20 mL of crude extract and add different amounts of NaCl (the reagent concentrations in this article are expressed in grams per 100 mL, specifically as percentages) at PEG 6000 concentrations of 6%, 8%, 10%, and 12%. After centrifugation, observe the precipitation distribution of LDL. Under different conditions, LDL precipitates may appear at the bottom of the centrifuge tube or float on the surface of the solution, while other precipitated proteins will not float on the surface. In this way, LDL can be extracted. Through preliminary experiments, it was determined that PEG 12% and NaCl 20% should be used as the final separation step.

[0033] 2. Steps for preparing high-purity low-density lipoprotein

[0034] 2.1 Sample Collection

[0035] Collect the remaining samples after clinical testing, and select serum or plasma that are negative for pathogen indicators of infectious diseases such as hepatitis A (HAV), hepatitis B (HBV), hepatitis C (HCV), HIV, and leptospirosis (RPR) and have no obvious lipemia.

[0036] 2.2 Separation Steps

[0037] (1) Add PEG 6000 to the sample, add 40 g (4%) of PEG 6000 per liter, stir to dissolve, centrifuge, and the precipitate contains lipoprotein a and chylomicrons, which will be processed separately;

[0038] (2) Add 20g of PEG 6000 to the supernatant to make the PEG 6000 concentration reach 6%, let it stand for 2-4 hours, and pour the supernatant into another container. This solution can be used for the preparation of high-density lipoprotein.

[0039] (3) Dissolve the precipitate in 100 mL of physiological saline, centrifuge to discard the insoluble matter, add 6 g (6%) of PEG6000 to the supernatant and stir to dissolve, let stand for 2 hours and take the precipitate. Repeat this step twice, then dissolve in 100 mL of physiological saline, centrifuge and discard the insoluble matter.

[0040] (4) Supernatant plus NaCl solid powder 20 g (20%) stirring to dissolve, centrifugation to remove the apparent micro-bubbles, then add PEG 6000 12 g (12%) stirring to dissolve, centrifugation, keep the upper floating, discard the lower liquid and precipitate, repeat this step 2-3 times, finally dissolve the floating with normal saline.

[0041] 2.3 Measure the blood lipid index of each step sample, determine the feasibility of the operation, and identify the final sample by electrophoresis and ultracentrifugation

[0042] Electrophoresis and ultracentrifugation were completed by the Inspection Department of the Shanghai Public Health Clinical Center and Shanghai Beijia Biochemical Reagent Co., Ltd.

[0043] 3. Results

[0044] 3.1 Verification results

[0045] Verification results are shown in Figure 1 , and it is determined that most of the cholesterol and triglycerides are distributed in the upper floating, which is Figure 1 the middle and upper grayish-white part, and the rest is relatively small, which verifies our idea that lipoprotein, due to its fat content, has a lower density than non-lipoprotein. In the precipitated state, increasing the density of the solution to a certain extent, the precipitate of lipoprotein will float on the surface of the liquid, thereby separating from non-lipoprotein.

[0046] 3.2 Pre-test of complete separation conditions of LDL

[0047] The results are shown in Figure 2 , from left to right, a total of 12 centrifuge tubes, 1: P6 N4, 2: P8 N4, 3: P8 N6, 4: P8 N8, 5: P8 N10, 6: P8 N12, 7: P8 N14, 8: P8 N16, 9: P8 N18, 10: P8 N20, 11: P10 N20, 12: P12 N20, P = PEG 6000, N = NaCl, the numbers after P and N represent the grams of PEG 6000 and NaCl added per 100 ml of solution. The PEG 6000 concentration selected in the test can precipitate LDL, but with the increase of NaCl concentration, LDL will appear in the upper floating and the bottom precipitate in the solution, and part of it will be redissolved in the solution. When PEG 6000 reaches 12% and NaCl reaches 20%, LDL is basically present in the floating, which fully meets the separation requirements.

[0048] 3.3 Measurement results during the purification process

[0049] The results are shown in Table 1. After 4% PEG6000 treatment, more than 90% of Lpa was removed, and HDL was basically unchanged. After 6% PEG6000 treatment, 95% of HDL was removed. After two 6% PEG6000 treatments, HDL was completely removed, and less than 2% of Lpa remained. After two 12% PEG6000 and 20% NaCl treatments, Lpa accounted for 0, and ApoB accounted for about 94%.

[0050] Table 1

[0051] 1 2 3 4 5 Units Total protein 68.5 64.8 51.0 11.6 4.4 g / L Total cholesterol 4.57 3.90 22.21 20.13 17.85 mmol / L Triglycerides 1.57 1.20 7.68 7.32 6.59 mmol / L HDL 1.16 1.12 0.45 0 0 mmol / L LDL 2.62 2.46 16.45 16.37 15.75 mmol / L Lpa 222 19 183 34 0 mg / L ApoB 0.87 0.66 4.65 4.48 4.13 g / L Volume 1000 1000 125 110 105 Ml Yield 100％ 93.9％ 78.5％ 68.7％ 63.1％

[0052] Note: 1. Mixed serum. 2. Supernatant after 4% PEG6000 treatment of serum. 3. Precipitate after 6% PEG6000 treatment of serum. 4. Sample after two 6% PEG6000 saline treatments. 5. Sample after two 12% PEG6000 and 20% NaCl solution treatments.

[0053] 3.4 Electrophoresis identification

[0054] The results are shown in Table 1. After 4% PEG6000 treatment, more than 90% of Lpa was removed, and HDL was basically unchanged. After 6% PEG6000 treatment, 95% of HDL was removed. After two 6% PEG6000 treatments, HDL was completely removed, and less than 2% of Lpa remained. After two 12% PEG6000 and 20% NaCl treatments, Lpa accounted for 0, and ApoB accounted for about 94%. Figure 3 , Sample 1 is Lpa, sample 2 is HDL, sample 3 is LDL, and the rest are serum samples. The electrophoresis results show that sample 3 has only one staining band slightly ahead of β, which is different from the HDL of sample 2 at α2 position, and slightly behind the Lpa of sample 1. The scanning results show that the content of sample 3 is >95%. In the final sample, the content of ApoB is >90% of the total protein, which is consistent with the literature report (Zhang Xingyi. High-density lipoprotein [J]. Transfusion Therapy and Blood Products. People's Medical Publishing House, 1996, 9: 36-39). The content of the target band is ≥95% after scanning.

[0055] 3.5 Ultracentrifugation identification (identified by Shanghai Beijia Biological Reagent Co., Ltd.)

[0056] The authors believe that the method of low speed centrifugation is completely feasible to extract and prepare LDL. After the addition of 6% PEG 6000, the serum has a very obvious turbidity, and the precipitate forms quickly. If the precipitate is allowed to settle naturally, it will settle in about 1-4 hours, and the precipitate is relatively dense, and if there is enough time, centrifugation can be omitted. The two subsequent 6% PEG 6000 precipitations can also be performed without centrifugation. After the subsequent addition of 20% NaCl and 12% PEG 6000 and centrifugation, it can be found that the orange component at the bottom of the original centrifuge tube disappears, leaving a small amount of light yellow precipitate or no precipitate, and there is a lot of dense floating material on the surface of the liquid. The detection of samples at each step showed that LDL was completely precipitated at a PEG 6000 concentration of 6%, HDL was not precipitated, and a small amount of residual precipitated inclusions was completely removed during the two 6% PEG treatments. The results of ultracentrifugation showed that the density distribution of the proteins in the sample prepared by this method was 1.006-1.063, which is consistent with the literature (Zhang Xingyi. High-density lipoprotein [J]. Blood Transfusion Therapy and Blood Products. People's Medical Publishing House, 1996, 9: 36-39).

[0057] The results of electrophoresis showed that the purity of the obtained sample was >95%, ultracentrifugation showed that the hydration density of the proteins in the sample was between 1.006-1.063, and specific immunoturbidimetry and specific cholesterol measurement revealed that the sample contained high concentrations of LDL, no HDL and Lpa, and the LDL was completely separated.

[0058] Based on the above experimental results, it can be concluded that the sample obtained in this experiment is LDL.

[0059] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the method of the present application, a number of improvements and supplements can also be made, and these improvements and supplements should also be considered as the protection scope of the present application.

Claims

1. A method for low-density lipoprotein separation and purification by low-speed centrifugation, characterized in that, Includes the following steps: (a) Take the raw material, add PEG 6000 to make the PEG 6000 concentration reach 4%, stir to dissolve, and centrifuge; (b) Add PEG 6000 to the supernatant to make the PEG 6000 concentration reach 6%, stir to dissolve, let stand, and pour the supernatant into another container; (c) Dissolve the precipitate with physiological saline, centrifuge to discard the insoluble matter, add PEG 6000 to the supernatant to make the PEG 6000 concentration reach 6%, stir to dissolve, let stand and take the precipitate. Repeat this step twice, then dissolve with physiological saline, centrifuge and discard the insoluble matter. (d) Add 20% NaCl solid powder to the supernatant and stir to dissolve. Centrifuge to remove tiny air bubbles from the surface. Then add PEG6000 to make the PEG6000 concentration 12%. Centrifuge again, retain the upper floating matter, and discard the lower liquid and precipitate. Repeat this step 2-3 times. Finally, dissolve the floating matter with physiological saline. In the above steps, the centrifuge speed is 3000 rpm and the centrifugation time is less than 30 minutes.

2. The separation and purification method according to claim 1, characterized in that, The raw materials in step (a) are human serum, plasma, and plasma byproducts remaining after the extraction of other proteins.

3. The separation and purification method according to claim 1, characterized in that, The concentration of PEG 6000 added in step (b) is 2%.

4. The separation and purification method according to claim 1, characterized in that, In steps (c) and (d), the saline solution is 10% of the raw material volume.

Images