Method for separating and purifying natural tissue factor lipids

Abstract

The invention relates to the technical field of biochemistry, and provides a method for separating and purifying natural tissue factor lipids, which comprises the following steps: S1, selecting fresh animal tissues, dicing, soaking in an EDTA (Ethylene Diamine Tetraacetic Acid) solution, drying, and crushing to obtain tissue powder; s2, adding the tissue powder into a vitamin C aqueous solution, homogenizing, centrifuging and filtering to obtain filtrate; s3, adding a polybrene aqueous solution into the filtrate, centrifuging, and collecting precipitate to obtain a natural tissue factor lipid crude product; and S4, washing, dissolving and filtering the natural tissue factor lipid compound crude product to obtain a high-purity natural tissue factor lipid compound solution, namely a thromboplastin solution. Through the technical scheme, the problems of low tissue factor extraction efficiency, low purity and easy activity loss in related technologies are solved.

Description

Technical Field

[0001] The present invention relates to the field of biochemical technology, and in particular to a method for separating and purifying natural tissue factor liposomes. Background Art

[0002] Natural tissue factor lipid, namely thromboplastin, is a lipoprotein complex with procoagulant properties or activity. Under normal circumstances, it is abundant in tissue fluid, brain, lung, placental tissue, and amniotic fluid. As a key factor in the coagulation cascade reaction, thromboplastin plays a vital role in the hemostasis 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 of the invention

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

[0004] The technical solution of the present invention is as follows: The present invention provides a method for separating and purifying natural tissue factor liposomes, comprising the following steps: S1. Select fresh animal tissue, cut it into pieces, soak it in EDTA solution, dry it, and grind it into tissue powder; S2, adding the tissue powder to the vitamin C aqueous solution, homogenizing, centrifuging, and filtering to obtain a filtrate; S3, adding polycondensate aqueous solution to the filtrate, collecting the precipitate after centrifugation to obtain a crude product of natural tissue factor lipidate; S4. Washing, dissolving and filtering the crude natural tissue factor lipidate product to obtain a high-purity natural tissue factor lipidate solution, namely, a thromboplastin solution.

[0005] As a further technical solution, the animal tissue includes one or more of brain, lung, and placenta.

[0006] As a further technical solution, the EDTA solution is a physiological saline solution of EDTA, and the concentration of the physiological saline solution of EDTA is 15-20 mM.

[0007] In the present invention, fresh animal tissue is cut into pieces and then soaked in EDTA physiological saline solution to chelate metal ions, prevent enzyme denaturation, and improve the purity and purification efficiency of thromboplastin.

[0008] As a further technical solution, the drying method is freeze-drying.

[0009] As a further technical solution, the tissue powder is stored by nitrogen-filled sealing, and the storage temperature of the tissue powder is -15~-20°C.

[0010] As a further technical solution, the solid-liquid ratio of the tissue powder and the vitamin C aqueous solution is 1g:10~100mL.

[0011] As a further technical solution, the mass concentration of the vitamin C aqueous solution is 0.1%~0.2%.

[0012] As a further technical solution, the rotation speed of the homogenization is 12000 rpm, and the homogenization time is 1 to 5 minutes.

[0013] As a further technical solution, the centrifugal rotation speed is 5000 rpm and the centrifugal time is 10 min.

[0014] As a further technical solution, in step S2, the filtration is performed using a 0.22 μm film.

[0015] As a further technical solution, the mass concentration of the coagulant amine aqueous solution is 0.5%~1%.

[0016] As a further technical solution, the mass ratio of the aqueous solution of coagulant amine to the filtrate is 0.05~1:1.

[0017] As a further technical solution, the addition rate of the polycondensate aqueous solution is 1 mL / min.

[0018] In the present invention, by controlling the adding speed of the aqueous solution of coagulant amine, it is possible to avoid excessive precipitation, which would cause impurities to be encapsulated, thereby improving the purity of thromboplastin.

[0019] As a further technical solution, the temperature of the vitamin C aqueous solution is -5~5°C.

[0020] As a further technical solution, in step S4, the filtration adopts ultrafiltration technology.

[0021] As a further technical solution, the ultrafiltration technology is to use an ultrafiltration membrane with a molecular weight cutoff of 10KD.

[0022] In the present invention, the ultrafiltration technology is adopted to reduce the impurity content and further improve the purity of thromboplastin.

[0023] As a further technical solution, in step S4, the crude natural tissue factor lipidate is washed, dissolved in a protective solution, and filtered to obtain a high-purity natural tissue factor lipidate solution, namely, a thromboplastin solution.

[0024] As a further technical solution, the protective solution includes 1% HEPES, 0.3% calcium chloride, and 0.05% sodium azide, and the pH value of the protective solution is 7.3.

[0025] As a further technical solution, the washing solvent is physiological saline, and the washing times are 1 to 3 times.

[0026] As a further technical solution, the high-purity thromboplastin is stored in liquid or freeze-dried form.

[0027] The working principle and beneficial effects of the present invention are: In the present invention, by adding a vitamin C aqueous solution for extraction, the antioxidant property of vitamin C is utilized to protect the activity of thromboplastin, and a polycondensate aqueous solution is added to specifically bind to thromboplastin to form a large amount of flocculent precipitation, thereby improving the extraction efficiency and purity of thromboplastin. The thromboplastin produced by the present invention is composed of natural glycosylated complete tissue factor protein and natural phospholipids, the two are more firmly combined, have higher activity and stability, and have greater affinity to the human body as a raw material for drug development, thereby avoiding potential adverse effects caused by artificial synthetic raw materials. Moreover, the whole process is simple to operate and easy to mass produce, thereby providing reliable technical support for the wide application of thromboplastin. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0029] Figure 1 This is a stability comparison chart of the purified thromboplastin prepared in Example 1, the thromboplastin before purification, and the commercially available synthetic lipid product; Figure 2 The thin layer chromatogram of the purified thromboplastin in Example 1 and the commercially available synthetic lipid; In the figure: 1 is a commercially available synthetic lipid, and 2 is the purified thromboplastin of the present invention. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1 A method for separating and purifying natural tissue factor liposomes comprises the following steps: S1. Select 10 g of fresh rabbit brain and cut it into pieces quickly, soak it in physiological saline containing 15 mM EDTA, freeze-dry the soaked tissue, and then grind it through a 60-mesh sieve to obtain tissue powder. Put the powdered tissue into a plastic bag, seal it with nitrogen, and store it at -20°C for later use; S2, taking the frozen tissue powder and putting it into a high-speed homogenizer, adding a -5°C 0.1wt% vitamin C aqueous solution according to a solid-liquid ratio of 1g:10mL, homogenizing at 12000rpm for 1min, centrifuging at 5000rpm for 10min at 5°C, collecting the supernatant and filtering through a 0.22μm filter membrane to obtain a filtrate; S3, adding 0.5 wt% aqueous solution of polycondensate to the filtrate at a rate of 1 mL / min, the mass ratio of the aqueous solution of polycondensate to the filtrate being 1:9, allowing to stand for stratification, and collecting the precipitate by centrifugation to obtain a crude product of natural tissue factor lipidate; S4. Wash the crude product of natural tissue factor lipidate once with physiological saline, dissolve the washed crude product in protective solution (1% HEPES, 0.3% calcium chloride, 0.05% sodium azide, pH 7.3), filter through a 10KD ultrafiltration membrane to remove residual coagulation amine and small molecule impurities to obtain a high-purity thromboplastin solution, concentrate the high-purity thromboplastin solution, and then package it and store it in liquid form.

[0032] The stability comparison of the purified thromboplastin prepared in this example with the thromboplastin before purification and the commercially available synthetic lipid Figure 1 As shown; Figure 2 The figure shows the thin layer chromatogram of purified thromboplastin and commercially available synthetic lipid.

[0033] Example 2 A method for separating and purifying natural tissue factor liposomes comprises the following steps: S1. Select 10 g of fresh rabbit brain and cut it into pieces quickly, soak it in 18 mM EDTA saline, freeze-dry the soaked tissue, and then grind it through a 60-mesh sieve to obtain tissue powder. Put the powdered tissue into a plastic bag, fill it with nitrogen, and store it at -15°C for later use; S2, taking the frozen tissue powder and putting it into a high-speed homogenizer, adding 0.15wt% vitamin C aqueous solution at 0°C according to the solid-liquid ratio of 1g:50mL, high-speed homogenization at 12000rpm for 3min, centrifuging at 5000rpm for 10min at 5°C, collecting the supernatant and filtering through a 0.22μm filter membrane to obtain a filtrate; S3, adding 0.75wt% polycondensate aqueous solution to the filtrate at a rate of 1mL / min, the mass ratio of polycondensate aqueous solution to the filtrate being 1:4, allowing to stand for stratification, and collecting the precipitate by centrifugation to obtain a crude product of natural tissue factor lipidate; S4. Wash the crude product of natural tissue factor lipidate twice with physiological saline, dissolve the washed crude product in protective solution (1% HEPES, 0.3% calcium chloride, 0.05% sodium azide, pH 7.3), filter through a 10KD ultrafiltration membrane to remove residual polycondensing amines and small molecule impurities to obtain a high-purity thromboplastin solution, concentrate the high-purity thromboplastin solution, lyophilize it, and then package it and store it in a lyophilized form.

[0034] Example 3 A method for separating and purifying natural tissue factor liposomes comprises the following steps: S1. Select 10 g of fresh rabbit brain and cut it into pieces quickly, soak it in 20 mM EDTA saline, freeze-dry the soaked tissue, and then grind it through a 60-mesh sieve to obtain tissue powder. Put the powdered tissue into a plastic bag, seal it with nitrogen, and store it at -20°C for later use; S2, taking the frozen tissue powder and putting it into a high-speed homogenizer, adding 0.2wt% vitamin C aqueous solution at 0°C according to the solid-liquid ratio of 1g:100mL, high-speed homogenization at 12000rpm for 5min, centrifuging at 5000rpm for 10min at 5°C, collecting the supernatant and filtering it through a 0.22μm filter membrane to obtain a filtrate; S3, adding 1 wt% aqueous solution of polycondensate to the filtrate at a rate of 1 mL / min, the mass ratio of the aqueous solution of polycondensate to the filtrate being 1:1, allowing to stand for stratification, and collecting the precipitate by centrifugation to obtain a crude product of natural tissue factor lipidate; S4. Wash the crude product of natural tissue factor lipidate with physiological saline for three times, dissolve the washed crude product in protective solution (1% HEPES, 0.3% calcium chloride, 0.05% sodium azide, pH 7.3), filter through a 10KD ultrafiltration membrane to remove residual polycondensing amines and small molecule impurities to obtain a high-purity thromboplastin solution, concentrate and freeze-dry the high-purity thromboplastin solution, and then package it and store it in a freeze-dried form.

[0035] The stability of the purified thromboplastin prepared in Example 1 was compared with that of the purified thromboplastin and the commercially available synthetic lipid. The coagulation time of each sample was measured during storage at 37°C. The experimental results are shown in Tables 1 and Figure 1 shown.

[0036] Table 1 Stability comparison results

[0037] As shown in Table 1, the stability of the thromboplastin prepared in the present invention is higher than that of the thromboplastin before purification and the commercially available synthetic lipid, indicating that the purity and stability of the thromboplastin are improved after purification.

[0038] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for separating and purifying natural tissue factor liposomes, characterized in that: The following steps are involved: S1. Select fresh animal tissue, cut it into pieces, soak it in EDTA solution, dry it, and grind it into tissue powder; S2, adding the tissue powder to the vitamin C aqueous solution, homogenizing, centrifuging, and filtering to obtain a filtrate; S3, adding polycondensate aqueous solution to the filtrate, collecting the precipitate after centrifugation to obtain a crude product of natural tissue factor lipidate; S4. Washing, dissolving and filtering the crude natural tissue factor lipidate product to obtain a high-purity natural tissue factor lipidate solution, namely, a thromboplastin solution.

2. The method for separating and purifying natural tissue factor liposomes according to claim 1, characterized in that: The animal tissues include one or more of brain, lung, and placenta.

3. The method for separating and purifying natural tissue factor lipoate 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 15-20 mM.

4. The method for separating and purifying natural tissue factor lipids according to claim 1, characterized in that: The solid-liquid ratio of the tissue powder and the vitamin C aqueous solution is 1g:10~100mL.

5. The method for separating and purifying natural tissue factor lipids according to claim 1, characterized in that: The mass concentration of the vitamin C aqueous solution is 0.1% to 0.2%.

6. The method for separating and purifying natural tissue factor liposomes according to claim 1, characterized in that: The mass concentration of the coagulant amine aqueous solution is 0.5% to 1%.

7. The method for separating and purifying natural tissue factor lipids according to claim 1, characterized in that: The mass ratio of the coagulant amine aqueous solution to the filtrate is 0.05-1:

1.

8. The method for separating and purifying natural tissue factor lipids according to claim 1, characterized in that: The temperature of the vitamin C aqueous solution is -5~5°C.

9. The method for separating and purifying natural tissue factor lipids according to claim 1, characterized in that: The filtration adopts nanofiltration membrane, and the separation adopts ultrafiltration technology.

10. The method for separating and purifying natural tissue factor liposomes according to claim 1, characterized in that: The high-purity thromboplastin is stored in liquid or lyophilized form.

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