A method for isolating and purifying natural tissue factor lipids

Abstract

The application relates to the field of biochemical technology and discloses a method for separating and purifying natural tissue factor lipids, which comprises the following steps: S1, selecting fresh animal tissue, cutting the fresh animal tissue into pieces, soaking the pieces in an EDTA solution, drying the pieces, and crushing the pieces to obtain tissue powder; S2, adding the tissue powder into a vitamin C aqueous solution, homogenizing, centrifuging, and filtering to obtain a filtrate; S3, adding a condensed amine aqueous solution into the filtrate, centrifuging, and collecting a precipitate to obtain a crude natural tissue factor lipid product; and S4, washing, dissolving, and filtering the crude natural tissue factor lipid product to obtain a natural tissue factor lipid solution, i.e. a thromboplastin solution. Through the technical scheme, the problems of low extraction efficiency, low purity, and easy loss of activity of tissue factor in the related art are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biochemistry, in particular to a method for separating and purifying natural tissue factor lipids. BACKGROUND

[0002] Natural tissue factor lipids, i.e. thromboplastin, is a lipoprotein complex with procoagulant properties or activity, which is normally abundant in tissue fluid, brain, lung, placental tissue, amniotic fluid. As a key factor in the coagulation cascade, thromboplastin plays a crucial role in the hemostatic process. However, the traditional method of extracting thromboplastin has problems such as low efficiency, low purity, and easy loss of activity, which limits its wide application in clinical and scientific research. Therefore, it is particularly important to develop an efficient and stable thromboplastin separation and purification technology. SUMMARY

[0003] The present application provides a method for separating and purifying natural tissue factor lipids, which solves the problems of low extraction efficiency, low purity and easy loss of activity in related technologies.

[0004] The technical scheme of the present application is as follows:

[0005] The present application provides a method for separating and purifying natural tissue factor lipids, which includes the following steps:

[0006] S1, selecting fresh animal tissue, cutting it into pieces, and then soaking it in an EDTA solution, drying and crushing to obtain tissue powder;

[0007] S2, adding the tissue powder to a vitamin C aqueous solution, homogenizing, centrifuging, and filtering to obtain a filtrate;

[0008] S3, adding a polybrene aqueous solution to the filtrate, centrifuging, and collecting the precipitate to obtain a crude natural tissue factor lipid product;

[0009] S4, washing, dissolving and filtering the crude natural tissue factor lipid product to obtain a high-purity natural tissue factor lipid solution, i.e. a thromboplastin solution.

[0010] As a further technical scheme, the animal tissue includes one or more of brain, lung and placenta.

[0011] As a further technical scheme, the EDTA solution is an EDTA physiological saline solution, and the concentration of the EDTA physiological saline solution is 15-20 mM.

[0012] In the present application, after cutting fresh animal tissue into pieces, EDTA physiological saline solution is added for soaking to chelate metal ions and prevent enzyme denaturation, thereby improving the purity and purification efficiency of thromboplastin.

[0013] As a further technical solution, the drying method is freeze-drying.

[0014] As a further technical solution, the preservation method of the tissue powder is nitrogen filling sealing, and the preservation temperature of the tissue powder is-15~-20 DEG C.

[0015] As a further technical solution, the ratio of the tissue powder to the vitamin C aqueous solution is 1g:10~100mL.

[0016] As a further technical solution, the mass concentration of the vitamin C aqueous solution is 0.1%~0.2%.

[0017] As a further technical solution, the rotation speed of the homogenate is 12000rpm, and the homogenate time is 1~5min.

[0018] As a further technical solution, the rotation speed of the centrifugation is 5000rpm, and the centrifugation time is 10min.

[0019] As a further technical solution, in step S2, the filtration is performed by using a 0.22mu m membrane.

[0020] As a further technical solution, the mass concentration of the polybrene aqueous solution is 0.5%~1%.

[0021] As a further technical solution, the mass ratio of the polybrene aqueous solution to the filtrate is 0.05~1:1.

[0022] As a further technical solution, the adding speed of the polybrene aqueous solution is 1mL / min.

[0023] In the present application, by controlling the adding speed of the polybrene aqueous solution, the precipitation can be avoided too fast, so that the impurities are wrapped in, and the purity of the thromboplastin is improved.

[0024] As a further technical solution, the temperature of the vitamin C aqueous solution is-5~5 DEG C.

[0025] As a further technical solution, in step S4, the filtration is performed by using an ultrafiltration technology.

[0026] As a further technical solution, the ultrafiltration technology is performed by using an ultrafiltration membrane with a molecular weight cut-off of 10KD.

[0027] In the present application, by using the ultrafiltration technology, the impurity content is reduced, and the purity of the thromboplastin is further improved.

[0028] As a further technical solution, in step S4, the natural tissue factor lipoid crude product is dissolved in a protective solution after being washed, and a natural tissue factor lipoid solution, i.e., a thromboplastin solution, is obtained after filtration.

[0029] As a further technical solution, the protective solution comprises 1% HEPES, 0.3% calcium chloride, and 0.05% sodium azide, and the pH value of the protective solution is 7.3.

[0030] As a further technical solution, the solvent for the washing is physiological saline, and the number of times of the washing is 1-3 times.

[0031] As a further technical solution, the thromboplastin is stored in a liquid or lyophilized form.

[0032] The working principle and beneficial effects of the present application are as follows:

[0033] In the present application, the vitamin C aqueous solution is added for extraction, the antioxidant property of vitamin C is used to protect the activity of thromboplastin, and the condensate amine aqueous solution is added to specifically combine with the thromboplastin to form a large amount of flocculent precipitate, thereby improving the extraction efficiency and purity of the thromboplastin. The thromboplastin produced by the present application is composed of natural glycosylated complete tissue factor protein and natural phospholipid, the combination of which is more firm, and the thromboplastin has higher activity and stability. As a drug development raw material, the thromboplastin has higher affinity to the human body, and potential adverse effects caused by artificial synthetic raw materials are avoided. Moreover, the whole process is simple to operate and easy to scale up, thereby providing reliable technical support for the wide application of the thromboplastin. BRIEF DESCRIPTION OF DRAWINGS

[0034] The present application will be further described in detail below in combination with the drawings and specific embodiments.

[0035] Figure 1 FIG. 2 is a stability comparison chart of the purified thromboplastin prepared in Example 1 with the thromboplastin before purification and commercially available synthetic lipoid;

[0036] Figure 2 FIG. 3 is a thin layer chromatogram of the purified thromboplastin prepared in Example 1 and commercially available synthetic lipoid;

[0037] In the figure, 1 is commercially available synthetic lipoid, and 2 is the purified thromboplastin of the present application. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without any creative labor are involved in the protection scope of the present application.

[0039] Example 1

[0040] A method for separating and purifying natural tissue factor lipids, comprising the following steps:

[0041] S1, select 10 g of fresh rabbit brain and quickly cut it into pieces, soak it in 15 mM EDTA physiological saline, freeze-dry the soaked tissue, then crush it through a 60 mesh sieve to obtain tissue powder, put the powder into a plastic bag, seal it with nitrogen, and store it at -20°C for standby;

[0042] S2, take the frozen tissue powder and put it into a high-speed homogenizer, add 0.1 wt% vitamin C solution at -5°C according to the ratio of 1 g:10 mL, homogenize at 12000 rpm for 1 min, centrifuge at 5000 rpm for 10 min at 5°C, collect the supernatant and filter it through a 0.22 μm filter membrane to obtain the filtrate;

[0043] S3, add 0.5 wt% polybrene solution to the filtrate at a rate of 1 mL / min, the mass ratio of polybrene solution to filtrate is 1:9, after standing and layering, collect the precipitate by centrifugation to obtain the crude natural tissue factor lipids;

[0044] S4, wash the crude natural tissue factor lipids with physiological saline once, dissolve the washed crude product in a protective solution (1% HEPES, 0.3% calcium chloride, 0.05% sodium azide, pH 7.3), filter through a 10KD ultrafiltration membrane to remove residual polybrene and small molecular impurities, obtain thromboplastin solution, concentrate the thromboplastin solution and then dispense it, and store it in liquid form.

[0045] The stability of the purified thromboplastin prepared in this example is compared with that of the thromboplastin before purification and the commercially available synthetic lipids, as shown in Figure 1 . Figure 2 is a thin layer chromatogram of the purified thromboplastin and the commercially available synthetic lipids.

[0046] Example 2

[0047] A method for separating and purifying natural tissue factor lipids, comprising the following steps:

[0048] S1, select 10 g of fresh rabbit brain and quickly cut it into pieces, soak it in 18 mM EDTA physiological saline, freeze-dry the soaked tissue, then crush it through a 60 mesh sieve to obtain tissue powder, put the powder into a plastic bag, seal it with nitrogen, and store it at -15°C for standby;

[0049] S2, take the frozen tissue powder into a high-speed homogenizer, add 0 ℃ 0.15wt% vitamin C aqueous solution according to the solid-liquid ratio of 1g:50mL, high-speed homogenize for 3min at 12000rpm, centrifuge at 5000rpm for 10min at 5℃, collect the supernatant and filter through a 0.22μm filter membrane to obtain the filtrate;

[0050] S3, add 0.75wt% polybrene aqueous solution to the filtrate at a speed of 1mL / min, the mass ratio of polybrene aqueous solution to filtrate is 1:4, after standing and layering, collect the precipitate by centrifugation to obtain the crude natural tissue factor lipidate;

[0051] S4, wash the crude natural tissue factor lipidate with normal saline for 2 times, dissolve the washed crude product in a protective solution (1% HEPES, 0.3% calcium chloride, 0.05% sodium azide, pH value is 7.3), remove the residual polybrene and small molecular impurities by filtering through a 10KD ultrafiltration membrane to obtain a thromboplastin solution, concentrate, freeze-dry and then sub-pack the thromboplastin solution, and store it in freeze-dried form.

[0052] Example 3

[0053] A method for separating and purifying natural tissue factor lipidate, comprising the following steps:

[0054] S1, select 10g fresh rabbit brain, quickly cut and shred, soak in 20mM EDTA normal saline, freeze-dry the soaked tissue, then crush it through a 60 mesh sieve to obtain tissue powder, put the powder tissue into a plastic bag, seal it with nitrogen, and store it at -20℃ for standby;

[0055] S2, take the frozen tissue powder into a high-speed homogenizer, add 0 ℃ 0.15wt% vitamin C aqueous solution according to the solid-liquid ratio of 1g:50mL, high-speed homogenize for 3min at 12000rpm, centrifuge at 5000rpm for 10min at 5℃, collect the supernatant and filter through a 0.22μm filter membrane to obtain the filtrate;

[0056] S3, add 0.75wt% polybrene aqueous solution to the filtrate at a speed of 1mL / min, the mass ratio of polybrene aqueous solution to filtrate is 1:4, after standing and layering, collect the precipitate by centrifugation to obtain the crude natural tissue factor lipidate;

[0057] S4, wash the crude natural tissue factor lipidate with normal saline for 2 times, dissolve the washed crude product in a protective solution (1% HEPES, 0.3% calcium chloride, 0.05% sodium azide, pH value is 7.3), remove the residual polybrene and small molecular impurities by filtering through a 10KD ultrafiltration membrane to obtain a thromboplastin solution, concentrate, freeze-dry and then sub-pack the thromboplastin solution, and store it in freeze-dried form.

[0058] The purified thromboplastin prepared in Example 1 was compared with the thromboplastin before purification and the commercially available synthetic lipoid in stability, i.e. the coagulation time of each sample was determined during storage at 37°C, and the experimental results are shown in Table 1 and Figure 1

[0059] Table 1 Stability comparison results

[0060]

[0061] As shown in Table 1, the stability of the thromboplastin prepared in the present application is higher than that of the thromboplastin before purification and the commercially available synthetic lipoid, which indicates that the purification improves the purity and stability of the thromboplastin.

[0062] The above is only the preferred embodiment of the present application, and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.​

Claims

1. A method for separating and purifying lipid compounds of natural tissue factors, characterized in that, Includes the following steps: S1. Select fresh animal tissue, cut it into pieces, soak it in EDTA solution, dry it, and pulverize it to obtain tissue powder; S2. Add the tissue powder to a vitamin C aqueous solution, homogenize, centrifuge, and filter to obtain the filtrate; S3. Add coagulated amine aqueous solution to the filtrate, and collect the precipitate after centrifugation to obtain crude natural tissue factor lipids. S4. The crude product of natural tissue factor lipids is washed, dissolved and filtered to obtain a solution of natural tissue factor lipids, namely, a thromboplastin solution. The animal tissue is one or more of the following: brain, lung, and placenta.

2. The method for separating and purifying lipid compounds of natural tissue factors according to claim 1, characterized in that, The EDTA solution is a physiological saline solution of EDTA, and the concentration of the physiological saline solution of EDTA is 15~20mM.

3. The method for separating and purifying lipid compounds of natural tissue factors according to claim 1, characterized in that, The ratio of the tissue powder to the vitamin C aqueous solution is 1g:10~100mL.

4. The method for separating and purifying lipid compounds of natural tissue factors according to claim 1, characterized in that, The mass concentration of the vitamin C aqueous solution is 0.1% to 0.2%.

5. The method for separating and purifying lipid compounds of natural tissue factors according to claim 1, characterized in that, The mass concentration of the condensed amine aqueous solution is 0.5% to 1%.

6. The method for separating and purifying lipid compounds of natural tissue factors according to claim 1, characterized in that, The mass ratio of the coagulated amine aqueous solution to the filtrate is 0.05~1:

1.

7. The method for separating and purifying lipid compounds of natural tissue factors according to claim 1, characterized in that, The temperature of the vitamin C aqueous solution is -5~5℃.

8. The method for separating and purifying lipid compounds of natural tissue factors according to claim 1, characterized in that, The filtration uses a nanofiltration membrane, and the separation uses ultrafiltration technology.

9. The method for separating and purifying lipid compounds of natural tissue factors according to claim 1, characterized in that, The thromboplastin is stored in liquid or lyophilized form.

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