Bacterial outer capsule vesicle separation and purification equipment

Abstract

The invention discloses separation and purification equipment for bacterial outer membrane vesicles, and belongs to the technical field of separation and purification equipment. The separation and purification equipment comprises a separation barrel and further comprises a first separation mechanism, the first separation mechanism is connected into the separation barrel and comprises a motor and a centrifugal barrel, and the motor is fixedly connected with the lower end of the separation barrel; the centrifugal barrel is fixedly connected with the output end of the motor and rotationally connected into the separating barrel, the upper end of the centrifugal barrel is fixedly connected and communicated with a feeding pipe, the feeding pipe rotationally extends out of the upper end of the separating barrel, a plurality of through grooves are formed in the periphery of the centrifugal barrel, first filtering membranes are fixedly connected into the through grooves, and a discharging hole is formed in the lower end of the separating barrel. The BEV separation device has the advantages that BEV separation can be achieved through the first filtering membrane in the first separation mechanism, preliminary separation and obtaining are achieved, materials are separated and purified through the second filtering membrane by means of the second separation mechanism, and the purification rate of the materials is effectively increased.

Description

Technical Field

[0001] The invention relates to the technical field of separation and purification equipment, in particular to a bacterial exocyst separation and purification equipment. Background Art

[0002] Most bacteria release bacterial extracellular vesicles (BEVs) with diameters ranging from 20-400 nm. They contain rich cargo molecules and affect a variety of biological processes, including the transmission of virulence factors, DNA transfer, and the induction of pathogenesis. Therefore, they have great potential for use as vaccines, anticancer drugs, and nanotechnology.

[0003] Outer membrane vesicles are rich in phospholipids. There are many phospholipid components in nature, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and sphingomyelin (SM). Phosphatidylcholine PC and SM are generally located on the outer leaflet of the lipid bilayer, while PE, PS and PI are located in the inner leaflet. BEV has a net negative surface charge as a whole under physiological conditions. Zeta potential (ZP) is a commonly used method to measure the surface potential of BEV. It can be used as an indicator of the surface charge of BEV. The value is usually in the range of -20mV to -40mV, so BEV with a negative surface charge can also be attracted to positively charged nanomembranes or nanoparticles. Therefore, BEV containing negatively charged membrane lipids can be enriched by using positively charged nanomembranes or nanoparticles.

[0004] In the prior art, BEVs are isolated and obtained using a bacterial membrane vesicle isolation kit, but this method can only obtain a small amount of BEVs and is not suitable for large-scale industrial production. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the existing BEV separation and purification method cannot obtain a large amount of BEV.

[0006] To achieve the above purpose, the technical solution provided by the present invention is:

[0007] A bacterial exocyst vesicle separation and purification device comprises a separation cylinder and a first separation mechanism, wherein the first separation mechanism is connected to the separation cylinder, the first separation mechanism comprises a motor, a centrifugal cylinder and a first filter membrane, the motor is fixedly connected to the lower end of the separation cylinder, the centrifugal cylinder is fixedly connected to the output end of the motor, the centrifugal cylinder is rotatably connected to the separation cylinder, the upper end of the centrifugal cylinder is fixedly connected to and communicated with a feed pipe, the feed pipe is rotatably extended out of the upper end of the separation cylinder, a plurality of through grooves are provided on the outer periphery of the centrifugal cylinder, the first filter membrane is fixedly connected to the through grooves, the first filter membrane is a filter membrane with a pore size of 400 nm, and is used to intercept larger bacteria and bacterial fragments, and a discharge hole is provided at the lower end of the separation cylinder.

[0008] Furthermore, a first control valve is fixedly connected to the feed pipe.

[0009] Furthermore, both upper and lower ends of the centrifugal cylinder are fixedly connected with limit rings, the cross-section of the limit ring is set to L-shape, the inner wall of the separation cylinder is symmetrically provided with limit grooves, the limit ring is rotatably adapted in the limit groove, a sealing ring is fixed in the limit groove, and the limit ring is in contact with the sealing ring.

[0010] Furthermore, a transport funnel is fixedly connected to the lower end of the separation cylinder, the upper end of the transport funnel is connected to the discharge hole, a guide hopper is provided inside the transport funnel, the guide hopper is fixedly connected to the lower end of the separation cylinder, the upper end of the guide hopper is open and the lower end is closed.

[0011] Furthermore, the lower end of the transport funnel is connected to a second separation mechanism, the second separation mechanism includes a shell, the upper end of the shell is fixedly connected to a first connecting pipe, a sealing ring is fixedly connected in the first connecting pipe, the lower end of the transport funnel is fixedly connected to a second connecting pipe, the second connecting pipe is threadedly connected in the first connecting pipe, the second connecting pipe abuts against the sealing ring, an installation ring is fixed in the shell, a second filter membrane is fixed on the installation ring, the lower end of the shell is fixedly connected to a liquid collecting pipe, and a second control valve is connected to the liquid collecting pipe.

[0012] Furthermore, a first regulating pipe is fixedly connected to one side of the shell, the first regulating pipe is connected to the shell, one end of the first regulating pipe is fixedly connected to a control pump, and the first regulating pipe is connected to a third control valve.

[0013] Furthermore, a second regulating tube is fixedly connected to the other side of the shell, the second regulating tube is connected to the shell, a fourth control valve is connected to the second regulating tube, a sealing piston is slidingly connected to the inside of the second regulating tube, an annular limiter is fixedly connected to the inside of the second regulating tube, a connecting rod is fixedly connected to one end of the sealing piston, the connecting rod slides out of the second regulating tube, and a paddle is fixedly connected to the connecting rod.

[0014] The beneficial effects achieved by the present invention using the above structure are as follows:

[0015] 1: The material is initially separated and obtained through the first separation mechanism. The diameter of BEV is 20-400nm. Therefore, nano-scale filter holes with corresponding pore sizes are set in the first separation mechanism. The first filter membrane is a filter membrane with a pore size of 400nm, which is used to intercept larger bacteria and bacterial fragments, thereby realizing the first step of separation of BEV;

[0016] 2: The material separated by the first separation mechanism is separated and purified again by a filter membrane through a second separation mechanism, wherein the second filter membrane is a filter membrane with a pore size of 20nm, which filters out impurities such as small molecule proteins, wherein the second filter membrane is a nano-membrane or nano-material with a positive charge, and the bacterial outer membrane vesicles containing negatively charged membrane lipids are enriched by the positively charged nano-membrane or nano-material, and the vesicles of 20-400nm are retained on the second filter membrane, effectively improving the purification rate of the material. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a front view of the present invention.

[0018] Figure 2 It is an exploded view of the present invention.

[0019] Figure 3 It is a schematic diagram of the first separation mechanism of the present invention.

[0020] Figure 4 It is a cross-sectional view of the first separation mechanism connection of the present invention.

[0021] Figure 5 for Figure 4 A is an enlarged view of the middle image.

[0022] Figure 6 It is a cross-sectional view of the second separation mechanism of the present invention.

[0023] Description of reference numerals:

[0024] 1. Separation cylinder, 101. Limiting groove, 2. First separation mechanism, 201. Motor, 202. Centrifugal cylinder, 203. Through groove, 204. First filter membrane, 205. Limiting ring, 206. Sealing ring, 207. Feed pipe, 208. First control valve, 209. Discharge hole, 3. Transport funnel, 301. Guide hopper, 302. Second connecting pipe, 4. Second separation mechanism, 401. Shell, 402. First connecting pipe, 403. Mounting ring, 404. Collecting pipe, 405. Second control valve, 406. First regulating pipe, 407. Third control valve, 408. Control pump, 409. Second regulating pipe, 410. Fourth control valve, 411. Annular limiting member, 412. Sealing piston, 413. Connecting rod, 414. Paddle, 415. Second filter membrane, 416. Sealing ring. DETAILED DESCRIPTION

[0025] like Figure 1-6 As shown, a bacterial exocyst vesicle separation and purification device includes a separation cylinder 1 and a first separation mechanism 2, wherein the first separation mechanism 2 is connected to the separation cylinder 1, and the first separation mechanism 2 includes a motor 201 and a centrifugal cylinder 202, wherein the motor 201 is fixedly connected to the lower end of the separation cylinder 1, and the centrifugal cylinder 202 is fixedly connected to the output end of the motor 201, and the centrifugal cylinder 202 is rotatably connected to the separation cylinder 1, and the upper end of the centrifugal cylinder 202 is fixedly connected and communicated with a feed pipe 207, and a first control valve 208 is fixedly connected to the feed pipe 207, and the feed pipe 207 is rotatably extended out of the upper end of the separation cylinder 1, and a plurality of through grooves 203 are provided on the outer periphery of the centrifugal cylinder 202, and a first filter membrane 204 is fixedly connected in the through groove 203, and the first filter membrane 204 is a filter membrane with a pore size of 400nm, which is used to intercept larger bacteria and bacterial fragments, and a discharge hole 209 is provided at the lower end of the separation cylinder 1.

[0026] like Figure 4 , 5 As shown, in order to ensure the stable rotation of the centrifugal cylinder 202 in the separation cylinder 1, the upper and lower ends of the centrifugal cylinder 202 are fixedly connected with limit rings 205, the cross-section of the limit ring 205 is set to be L-shaped, and the inner wall of the separation cylinder 1 is symmetrically provided with limit grooves 101, the limit ring 205 is rotatably adapted in the limit groove 101, and a sealing ring 206 is fixed in the limit groove 101, and the limit ring 205 is in contact with the sealing ring 206.

[0027] like Figure 2 As shown, in order to introduce the material separated and purified by the first separation mechanism 2 into the second separation mechanism 4, a transport funnel 3 is fixedly connected to the lower end of the separation cylinder 1, and the transport funnel 3 is connected to the discharge hole 209. A guide hopper 301 is provided on the inner side of the transport funnel 3, and the guide hopper 301 is fixedly connected to the lower end of the separation cylinder 1, and the upper end of the guide hopper 301 is set to be open and the lower end is closed.

[0028] like Figure 6As shown, in order to separate and purify the material again and improve the material purification rate, the lower end of the transport funnel 3 is connected to the second separation mechanism 4, and the second separation mechanism 4 includes a shell 401. The upper end of the shell 401 is fixedly connected to the first connecting pipe 402, and the first connecting pipe 402 is fixedly connected with a sealing ring 416. The lower end of the transport funnel 3 is fixedly connected to the second connecting pipe 302, and the second connecting pipe 302 is threadedly connected to the first connecting pipe 402. The second connecting pipe 302 abuts against the sealing ring 416. The shell 401 is fixedly connected with a mounting ring 403, and the mounting ring 403 is fixedly connected with a second filter membrane 415. The lower end of the shell 401 is fixedly connected to a liquid collecting pipe 404, and the liquid collecting pipe 404 is connected with a second control valve 405. A first regulating tube 406 is fixedly connected to one side of the body 401, the first regulating tube 406 is connected to the shell 401, a control pump 408 is fixedly connected to one end of the first regulating tube 406, a third control valve 407 is connected to the first regulating tube 406, a second regulating tube 409 is fixedly connected to the other side of the shell 401, the second regulating tube 409 is connected to the shell 401, a fourth control valve 410 is connected to the second regulating tube 409, a sealing piston 412 is slidably connected to the inside of the second regulating tube 409, an annular limiter 411 is fixedly connected to the inside of the second regulating tube 409, a connecting rod 413 is fixedly connected to one end of the sealing piston 412, the connecting rod 413 slides out of the second regulating tube 409, and a paddle 414 is fixedly connected to the connecting rod 413.

[0029] When the present invention is used, the first control valve 208 is controlled to open the feed pipe 207, and the BEV material is introduced into the centrifugal cylinder 202. The output end of the motor 201 is used to drive the centrifugal cylinder 202 to rotate, and then the material is filtered and separated by the first filter membrane 204 under the action of centrifugal force. The separated material enters the separation cylinder 1 and then passes through the discharge hole 209 into the transport funnel 3, and enters the housing 401 through the transport funnel 3. According to the actual situation, the third control valve 407 can be opened and the control pump 408 can be used to control the pressure in the housing 401, so that the material is filtered by the second filter membrane 415 to obtain separated and purified BEV. The second filter membrane 415 has a pore size of 20nm. The filter membrane filters out impurities such as small molecules and proteins. The second filter membrane 415 is a positively charged nanomembrane or nanomaterial. The positively charged nanomembrane or nanomaterial is used to enrich the bacterial outer membrane vesicles containing negatively charged membrane lipids. The vesicles of 20-400nm are retained on the second filter membrane 415, which effectively improves the purification rate of the material. The fourth control valve 410 can also be opened, and the paddle 414 is manually pushed to drive the sealing piston 412 to slide in the second regulating tube 409 to increase the pressure in the shell 401, and then the second filter membrane 415 is used to achieve filtration and separation, and then the separated and purified BEV is obtained. The filtered liquid in the shell 401 can be collected through the liquid collecting pipe 404.

[0030] In summary: the material is initially separated and obtained through the first separation mechanism 2, and the BEV diameter is 20-400nm. Therefore, the separation of BEV can be achieved by setting a nanofiltration membrane with corresponding pore size in the first separation mechanism 2. The material separated by the first separation mechanism 2 is separated and purified again by the second separation mechanism 4 using the filtration membrane, which effectively improves the purification rate of the material.

[0031] The present invention and its embodiments are described above, and such description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design a structure and embodiment similar to the technical solution without creativity, which should belong to the protection scope of the present invention.

Claims

1. A bacterial exocyst separation and purification device, comprising a separation cartridge, characterized in that: It also includes a first separation mechanism, which is connected to the separation cylinder. The first separation mechanism includes a motor, a centrifugal cylinder and a first filter membrane. The motor is fixed to the lower end of the separation cylinder, the centrifugal cylinder is fixed to the output end of the motor, the centrifugal cylinder is rotatably connected to the separation cylinder, the upper end of the centrifugal cylinder is fixed to and connected with a feed pipe, the feed pipe rotates and extends out of the upper end of the separation cylinder, a plurality of through grooves are provided on the outer periphery of the centrifugal cylinder, the first filter membrane is fixed to the through grooves, the first filter membrane is a filter membrane with a pore size of 400nm, which is used to intercept larger bacteria and bacterial fragments, and a discharge hole is provided at the lower end of the separation cylinder.

2. A bacterial exocyst separation and purification device according to claim 1, characterized in that: The feed pipe is fixedly connected with a first control valve.

3. A bacterial exocyst separation and purification device according to claim 1, characterized in that: The upper and lower ends of the centrifugal cylinder are fixedly connected to limit rings, the cross-section of the limit ring is set to L-shaped, the inner wall of the separation cylinder is symmetrically provided with limit grooves, the limit ring is rotatably adapted in the limit groove, a sealing ring is fixed in the limit groove, and the limit ring is in contact with the sealing ring.

4. A bacterial exocyst separation and purification device according to claim 3, characterized in that: A transport funnel is fixedly connected to the lower end of the separation cylinder, and the upper end of the transport funnel is connected to the discharge hole. A guide hopper is arranged inside the transport funnel, and the guide hopper is fixedly connected to the lower end of the separation cylinder. The upper end of the guide hopper is open and the lower end is closed.

5. A bacterial exocyst separation and purification device according to claim 4, characterized in that: The lower end of the transport funnel is connected to a second separation mechanism, which includes a shell. The upper end of the shell is fixedly connected to a first connecting pipe, and a sealing ring is fixedly connected to the first connecting pipe. The lower end of the transport funnel is fixedly connected to a second connecting pipe, and the second connecting pipe is threadedly connected to the first connecting pipe. The second connecting pipe abuts against the sealing ring. A mounting ring is fixedly connected to the shell, and a second filter membrane is fixedly connected to the mounting ring. The second filter membrane is a filter membrane with a pore size of 20 nm, and the second filter membrane is a nanomembrane or nanomaterial with a positive charge. The lower end of the shell is fixedly connected to a liquid collecting pipe, and a second control valve is connected to the liquid collecting pipe.

6. A bacterial exocyst separation and purification device according to claim 5, characterized in that: A first regulating pipe is fixedly connected to one side of the shell, the first regulating pipe is communicated with the shell, a control pump is fixedly connected to one end of the first regulating pipe, and a third control valve is connected to the first regulating pipe.

7. A bacterial exocyst separation and purification device according to claim 6, characterized in that: A second regulating tube is fixedly connected to the other side of the shell, the second regulating tube is connected to the shell, a fourth control valve is connected to the second regulating tube, a sealing piston is slidingly connected to the inside of the second regulating tube, an annular limiter is fixedly connected to the inside of the second regulating tube, a connecting rod is fixedly connected to one end of the sealing piston, the connecting rod slides out of the second regulating tube, and a paddle is fixedly connected to the connecting rod.

Images