Direct purification device for plant crude exosome

Abstract

The utility model provides a direct purification device for crude plant exosomes, which comprises a sealed cylinder body, the upper part of the sealed cylinder body is sealed by an upper cover plate, the lower part of the sealed cylinder body is sealed by a lower cover plate, the sealed cylinder body is fixed in the middle by the upper cover plate and the lower cover plate through upper and lower cover plate fixing bolts, and a circular hole is formed in the middle of the upper cover plate. The upper cover plate is provided with a sample introduction pipe, the sample introduction pipe is provided with a sample introduction valve, the bottom of the sealing cylinder body is filled with the filler, the bottom of the sealing cylinder body is also provided with a sand core baffle plate, the lower cover plate is provided with a sample discharge pipe, the sample discharge pipe is communicated with the bottom of the sand core baffle plate, and the sample discharge pipe is provided with a sample discharge valve. According to the plant exosome purification device, large-size particles and small-size protein impurity components in a plant crude exosome solution can be directly removed, and large-scale purification of plant exosomes is realized.

Description

TECHNICAL FIELD

[0001] The utility model belongs to biological sample separation and purification technical field, especially relate to a direct purification device for plant crude exosome. BACKGROUND

[0002] Exosome refers to small membrane vesicles containing complex RNA and protein, and is mainly derived from multi-vesicular bodies formed by intracellular fusion of micro-particle invagination, and is released into extracellular matrix after fusion of multi-vesicular body outer membrane and cell membrane.

[0003] At present, most of the large-scale purification solutions of exosome adopt traditional purification technology or technology combination, such as ultracentrifugation, tangential flow filtration, exclusion / hydrophobic / ion chromatography and the like, which not only has complex process and long time consumption, but also has expensive equipment and material prices.

[0004] In summary, the existing problems of the prior art are that the traditional device and equipment are difficult to realize low-cost, simple and convenient separation and purification of exosome in the large-scale separation and purification process. UTILITY MODEL CONTENT

[0005] The utility model aims at solving the defects of the prior art, and provides a direct purification device for plant crude exosome, which aims at rapid, simple and low-cost purification of plant exosome components.

[0006] The utility model adopts the following technical scheme:

[0007] A direct purification device for plant crude exosome, which comprises a sealed cylinder body, the upper part of the sealed cylinder body is sealed by an upper cover plate, the lower part is sealed by a lower cover plate, the sealed cylinder body is fixed in the middle part by upper and lower cover plate fixing bolts, a circular hole is formed in the middle part of the upper cover plate, a rotatable piston is arranged, an ultrasonic probe is installed on the piston, a sample inlet tube is arranged on the upper cover plate, a sample inlet valve is installed on the sample inlet tube, a filler is filled in the bottom of the sealed cylinder body, a sand core baffle is also installed in the bottom of the sealed cylinder body, a sample outlet tube is arranged on the lower cover plate, the sample outlet tube is communicated with the bottom of the sand core baffle, and a sample outlet valve is installed on the sample outlet tube.

[0008] Further, the upper cover plate has a convex thread, and the lower cover plate has a concave thread.

[0009] Further, the power of the ultrasonic probe is 100-700W, and the optimal power is 400W.

[0010] Further, the piston is composed of a bolt, an upper piston cover plate, a lower piston cover plate, a rubber sealing ring, a filter cloth and a screw rod, the bottom of the screw rod is connected with the ultrasonic probe, the lower part of the screw rod is connected with the upper piston cover plate, the lower part of the upper piston cover plate is the bolt for connecting the upper piston cover and the lower piston cover plate, the filter cloth and the rubber sealing ring are sequentially arranged between the upper piston cover plate and the lower piston cover plate, the upper piston cover plate and the lower piston cover plate are provided with sieve holes, the other end of the screw rod is provided with a lifting and twisting handle, and a wire winding device is further arranged on the screw rod.

[0011] Further, the rubber sealing ring is arranged in the groove formed between the upper piston cover plate and the lower piston cover plate after being fixed.

[0012] Further, the filter cloth is selected from 0.22-0.45 mu pores.

[0013] Further, the filler is selected from a size exclusion and ion mixed mode microsphere, and the size exclusion limit is 40 nm.

[0014] The device has the advantages that:

[0015] The device provided by the utility model can separate and purify plant exosomes simply and easily, can remove large-size impurities (>0.45 mu) and small-size protein impurities (<50 nm) in crude exosomes in one-step chromatography, the protein removal rate of single chromatography reaches more than 95%, and therefore, exosome solution with higher purity is obtained.

[0016] The device combines the filtering and ultrasonic components, can be more suitable for the application scene of separating and purifying plant exosomes, avoids the aggregation of large-size impurities and exosome components, simultaneously avoids the slow flow rate caused by the accumulation of impurity components on the filter cloth, and avoids the plugging of the filter membrane. DRAWINGS

[0017] Figure 1 It is a device structure schematic view of the utility model;

[0018] Figure 2 It is a top view of the upper and lower piston cover plates and the rubber sealing ring;

[0019] Figure 3 It is a piston structure exploded view;

[0020] Figure 4 It is a ginseng exosome transmission electron microscope view;

[0021] Figure 5 It is a turmeric exosome transmission electron microscope view.

[0022] In the figure: 1-sealing cylinder, 2-sample inlet tube, 3-sample outlet tube, 4-ultrasonic probe, 5-filler, 6-upper cover plate, 7-lower cover plate, 8-piston, 9-upper and lower cover plate fixing bolt, 10-lifting and twisting handle, 11-winder, 12-sand core baffle;

[0023] 801-bolt, 802-piston upper cover plate, 803-piston lower cover plate, 804-rubber sealing ring, 805-filter cloth, 806-screw rod, 8000-sieve hole. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the utility model more clear, the technical scheme in the utility model is described clearly and completely below, obviously, the described embodiment is a part of the embodiment of the utility model, rather than all the embodiments. Based on the embodiment in the utility model, all other embodiments obtained by the person skilled in the art without making creative labor belong to the protection scope of the utility model.

[0025] As Figures 1-3 shown, the utility model relates to a direct purification device for plant crude exosome, including sealing cylinder 1, wherein sealing cylinder 1 is cylindrical, selects toughened glass material quality, ensures the material strength, the sealing property simultaneously can observe the situation inside the cylinder body. The upper portion of sealing cylinder 1 adopts upper cover plate 6 sealing, and the lower portion adopts lower cover plate 7 sealing, and upper cover plate 6 and lower cover plate 7 pass through upper and lower cover plate fixing bolt 9 and fix sealing cylinder 1 in the middle part, and upper cover plate 6 has the protruding screw thread, and lower cover plate 7 has the recessed screw thread, and the screw thread is used for connecting upper and lower cover plate fixing bolt 9. Upper cover plate 6 and lower cover plate 7 select stainless steel material quality, ensure the material strength and the sealing property of container.

[0026] Wherein, the upper cover plate 6 middle part is opened circular hole, is used to support exosome solution flow and sets up rotatable lifting piston 8, and rotatable lifting piston 8 is used to adjust the filling height of filler 5. Ultrasonic probe 4 is installed on rotatable lifting piston 8, and ultrasonic probe 4 rises and falls along with the lifting of piston 8, and ultrasonic probe 4 is used to prevent crude exosome solution from settling and gathering on one hand, and is used to clean filter cloth 805 in piston 8 on the other hand. The power of ultrasonic probe 4 is 100-700W, and the best power is 400W. Upper cover plate 6 has sample inlet tube 2, and sample outlet tube 2 is communicated with the bottom of sand core baffle 12, and sample inlet valve is installed on sample inlet tube 2, and sample inlet valve is connected with the cooperation of sample pump to pump sample exosome.

[0027] The piston 8 can move up and down in the sealed cylinder 1 through screw motion, and is used for compressing the filler 5 at the bottom area of the sealed cylinder 1. The piston 8 is composed of a bolt 801, an upper piston cover plate 802, a lower piston cover plate 803, a rubber sealing ring 804, a filter cloth 805 and a screw rod 806. The bottom of the screw rod 806 is connected with the ultrasonic probe 4, the lower part of the screw rod 806 is connected with the upper piston cover plate 802, the lower part of the upper piston cover plate 802 is the bolt 801 used for connecting the upper piston cover 802 and the lower piston cover plate 803, the filter cloth 805 and the rubber sealing ring 806 are sequentially arranged between the upper piston cover plate 802 and the lower piston cover plate 803, and the rubber sealing ring 806 is arranged in the groove formed between the upper piston cover plate 802 and the lower piston cover plate 803 after being fixed, so as to prevent the exosome solution from flowing into the filler 5 from the bonding position of the outer wall of the sealed cylinder 1. The upper piston cover plate 802 and the lower piston cover plate 803 are provided with sieve holes 8000, and liquid can flow through the sieve holes 8000. The filter cloth 805 can be selected according to actual needs, and the pore size is 0.22-0.45 mu m, so as to remove cell fragments and secretions with large sizes. The other end of the screw rod 806 is provided with a lifting and twisting handle 10, and a wire winder 11 is arranged on the screw rod 806, and the wire winder 11 is mainly used for collecting the connecting line of the ultrasonic probe 4 when the screw rod 806 rotates.

[0028] The filler 5 is filled in the bottom of the sealed cylinder 1, and a sand core baffle 12 is arranged at the bottom of the sealed cylinder 1. The sand core baffle 12 allows liquid to flow through, and retains the filler 5. The filler 5 can be selected as a size exclusion and ion mixing mode microsphere, and the size exclusion limit is 40 nm, so as to effectively control and remove small molecular protein impurities with a size less than 40 nm.

[0029] The lower cover plate 7 is provided with a sample outlet pipe, and a sample outlet valve is arranged on the sample outlet pipe. The sample outlet valve is used for controlling the flow rate of liquid and the outflow of solution.

[0030] The working mode of the utility model is as follows:

[0031] The utility model is simple to build. In the first step, the lifting and twisting handle 10 is rotated to lift the piston 8 to the top. In the second step, the upper and lower cover plate fixing bolts 9 are removed, the upper cover plate 6 of the sealed cylinder 1 of the device is opened, and the filler 5 is poured into the bottom of the sealed cylinder 1. In the third step, the upper cover plate 6 is covered, and the sealing property is maintained. In the fourth step, the lifting and twisting handle 10 is rotated to press the piston to the top of the filler 5, and the solution in the filler 5 is discharged.

[0032] After the crude exosome solution is pumped into the sealed cylinder 1 of the device through the sample inlet tube 2, the dispersion of the exosomes is dispersed by the ultrasonic probe 4 to maintain the dispersion, and the system pressure generated by the sample pump during the preparation of the crude exosome solution is used to make the crude exosome solution flow through the filter cloth 805 between the upper cover plate 802 and the lower cover plate 803 of the piston, so as to remove most of the large-size impurities (plant fibers, cell components, and macromolecular impurities). During the continuous flow of the crude exosome solution through the filler 5, the filler 5 captures only small-molecular-weight protein impurities through size exclusion and ion exchange effects, without intercepting the larger-sized exosome components, so that the exosomes flow out of the sealed cylinder 1 from the filler, and the impurities such as proteins are intercepted by the device, and the components flowing out of the sample outlet tube are the purified exosome solution after impurity removal.

[0033] Example 1

[0034] Further purification of ginseng exosomes:

[0035] Preparation of ginseng crude solution: 500g of washed ginseng was added to a cell disruptor, and a small amount of water was added to disrupt the cells to form a broken cell solution. After the broken cell solution was simply filtered through a screen to remove unbroken ginseng debris, a ginseng crude solution was obtained.

[0036] Construction of an exosome direct purification device:

[0037] After the mixed mode microsphere filler 5 was filled into the sealed cylinder 1 of the device, the sample outlet valve was opened, the lifting knob 10 was twisted to rotate the screw rod 806 to lower the piston 8 to discharge the preservation solution in the filler 5. The peristaltic pump was used to pump pure water into the sample inlet tube 2, and the pure water was allowed to flow through the filler 5 to wash away the preservation solution. After the pump was stopped and the pure water in the device was basically discharged, the sample outlet valve was closed.

[0038] Purification of exosomes: the device sample outlet valve was opened, and the ginseng crude solution was pumped into the sealed cylinder 1 of the device from the device sample inlet tube using a peristaltic pump. The ginseng crude solution first accumulated at the top of the filler 5 and the piston 8, at which time the ultrasonic probe 4 was turned on to prevent the ginseng crude solution from settling and aggregating. As the ginseng crude solution was pumped in, the self-generated pressure in the device increased, the ginseng crude solution flowed through the filter cloth 805, and then flowed through the filler 5 to flow out of the sample outlet valve. The collected sample was the purified ginseng exosomes.

[0039] Example 2

[0040] Further purification of ginger exosomes:

[0041] Preparation of crude ginger exosome solution: 500g of washed ginger was added to a cell disruptor, and a small amount of water was added to disrupt the cells to form a broken cell solution. After the broken cell solution was simply filtered through a screen to remove unbroken ginger debris, a ginseng crude solution was obtained. The crude solution was concentrated and filtered using a hollow fiber column to obtain a crude ginger exosome solution.

[0042] Build exosome direct purification device: after filling the filler 5 mixed mode microspheres into the device, open the sample valve, twist the lifting twist handle 10 to make the piston 8 descend to discharge the preservative liquid in the filler 5. Continue to pump pure water from the sample inlet tube with a peristaltic pump, and let the pure water flow through the filler 5 to wash away the preservative liquid. Stop pumping pure water, and after the pure water in the device is basically discharged, close the sample valve.

[0043] Load and purify exosomes: open the device sample valve, pump the crude ginger exosome liquid into the device sealed cylinder 1 from the device sample inlet with a peristaltic pump. The crude ginger exosome liquid will first accumulate at the top of the filler 5 and the piston 8. At this time, open the ultrasonic probe 4 to prevent the crude ginger exosome liquid from precipitating and aggregating. As the crude ginger exosome liquid is pumped in, the self-generated pressure in the device increases, and the crude ginger exosome liquid flows through the filter cloth 805 and then flows out from the sample valve after passing through the filler 5. Collecting the sample flowing out is the purified ginger exosome.

[0044] Detection of exosome component samples before and after purification:

[0045] The human ginseng exosomes prepared in Example 1 and the ginger exosomes obtained in Example 2 were observed and analyzed by transmission electron microscopy to observe the morphology and particle size of the exosomes. The main steps are as follows:

[0046] 10 μL of the sample obtained by separation and purification in the above examples was taken and dropped onto a copper mesh. After standing for 5-10 min, the excess liquid was absorbed from the edge of the copper mesh with filter paper, 1-2 drops of 0.5% uranyl acetate aqueous solution at pH 4.5 were dropped, and reacted on the copper mesh for 1 min. After negative staining twice, it was dried and observed by transmission electron microscopy. Transmission electron microscopy observed that the morphology of each group of exosomes was complete without damage, and the particle size was between 30-200 nm. See the attached figure for details. Figures 4-5 .

[0047] The human ginseng exosomes prepared in Example 1 and the ginger exosomes obtained in Example 2 were analyzed by NTA to simulate the particle size and distribution of the exosomes. The main steps are as follows:

[0048] 20 μL of the human ginseng exosomes prepared in Example 1 and the ginger exosomes obtained in Example 2 were diluted 1000 times, and 5 mL of the diluted sample was injected into the sample cell for testing.

[0049] Determination of recovery rate:

[0050] 1 mL of the human ginseng exosomes prepared in Example 1 and the ginger exosomes obtained in Example 2 was taken to determine the BCA protein content A. Then the device was used for separation and purification again, and the BCA protein content B of the purified ginger exosomes was determined. The results are shown in Table 1 below.

[0051] Recovery rate = protein content B / protein content A x 100%.

[0052] Table 1 Recovery experiment results

[0053] Sample Protein content B Protein content A Recovery Ginseng exosome 0.6875 mg 0.6730 mg 97.89% Ginger exosome 0.7864 mg 0.7795 mg 99.12%

[0054] Finally, it should be noted that the above examples are intended to illustrate the technical solutions of the present application, rather than limit them. Although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions described in the foregoing examples can be modified, or some technical features can be replaced by equivalent features. These modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A device for direct purification of plant crude exosomes, characterized in that, The sealing cylinder is fixed in the middle part by the upper cover plate and the lower cover plate through the upper and lower cover plate fixing bolts, the upper cover plate has a circular hole in the middle part, a rotatable and liftable piston is arranged, an ultrasonic probe is installed on the piston, the upper cover plate has a sample inlet pipe, a sample inlet valve is installed on the sample inlet pipe, fillers are filled in the bottom of the sealing cylinder, a sand core baffle is also installed in the bottom of the sealing cylinder, the lower cover plate has a sample outlet pipe, the sample outlet pipe is communicated with the bottom of the sand core baffle, and a sample outlet valve is installed on the sample outlet pipe.

2. The device for direct purification of plant crude exosomes according to claim 1, characterized in that, The upper cover plate has a convex thread, and the lower cover plate has a concave thread.

3. The device for direct purification of plant crude exosomes according to claim 1, characterized in that, The power of the ultrasonic probe is 100-700W.

4. The device for direct purification of plant crude exosomes according to claim 3, characterized in that, The power of the ultrasonic probe is 400W.

5. The device for direct purification of plant crude exosomes according to claim 1, characterized in that, The piston is composed of a bolt, a piston upper cover plate, a piston lower cover plate, a rubber sealing ring, filter cloth and a screw rod, the bottom of the screw rod is connected with the ultrasonic probe, the lower part of the screw rod is connected with the piston upper cover plate, the lower part of the piston upper cover plate is a bolt for connecting the piston upper cover plate and the piston lower cover plate, the filter cloth and the rubber sealing ring are sequentially arranged between the piston upper cover plate and the piston lower cover plate, the piston upper cover plate and the piston lower cover plate have screen holes, a lifting and twisting handle is installed on the other end of the screw rod, and a wire winder is also installed on the screw rod.

6. The device for direct purification of plant crude exosomes according to claim 5, characterized in that, The rubber sealing ring is located in the groove formed between the piston upper cover plate and the piston lower cover plate after being fixed.

7. The device for direct purification of plant crude exosomes according to claim 5, characterized in that, The filter cloth has pores of 0.22-0.45μm.

8. The device for direct purification of plant crude exosomes according to claim 5, characterized in that, The fillers are selected as the exclusion, ion mixed mode microspheres, and the exclusion limit is 40nm.

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