Method for removing endotoxin from bovine serum albumin
During the separation of bovine serum albumin, the combination of sealing plates and through-hole plates is used to accelerate the passage of protein liquid and mix with affinity ligands. The problem of reduced separation effect of bovine serum albumin and reduced extraction amount is solved through the design of folding plates and rolling components, and efficient bovine serum albumin extraction is achieved.
Patent Information
- Application Number
- CN202411943231.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-03-21
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Prior Art In the process of separation of bovine serum albumin, larger bovine serum albumin is prone to stuck in the mesh of the separating plate, and it is difficult to mix with ligands, resulting in a reduced separation effect and a reduced extraction amount.
By blocking cotton balls at the drain port, the pretreated affinity ligand is filled in the cylinder, the combination of the sealing pressure plate and the through-hole plate is used to achieve pressure acceleration and mixing of the protein liquid, and the design of the folding plate and the rolling assembly is used to avoid blockage of the through-hole plate and improve the extraction efficiency of bovine serum albumin.
It effectively avoids through-hole plate blockage, improves the efficiency of protein liquid passage, increases the contact area between bovine serum albumin and affinity ligands, and improves the separation effect and extraction amount.
Smart Images
Figure CN119661689A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of protein engineering, and in particular relates to a method for removing endotoxin from bovine serum albumin. Background Art
[0002] Bovine serum albumin (BSA) is an important protein widely used in biochemical experiments. It is mainly made from bovine blood, separated from serum and precipitated with ammonium sulfate, and then refined with caprylic acid. Bovine serum albumin (BSA) contains some impurities during the preparation process, such as endotoxins, peptides and non-protein components. Affinity chromatography is usually used to separate bovine serum albumin.
[0003] Affinity chromatography uses specific affinity ligands, such as antibodies or biotin, which can separate BSA from impurities with specific affinity. First, BSA solution is mixed with affinity ligands to form a complex, then unbound impurities are removed, and finally BSA is dissociated from affinity ligands with appropriate eluent. For example, publication number is the Chinese patent of CN11564598 discharge port 8, which mainly separates protein liquid by liquid separation plate, and after the protein liquid liquid separation is finished, the piston block is driven to move downward by pressing the vertical pipe so that the liquid below the piston block is discharged by the liquid outlet pipe. However, the size of the bovine serum albumin in the protein liquid is one step, and larger bovine serum albumin is difficult to be stuck in the mesh of the liquid separation plate when passing through the liquid separation plate, even if larger bovine serum albumin is pressed out from the mesh by the mode of pressurization, and because bovine serum albumin is larger, the mixing difficulty of bovine serum albumin and part is larger and the time is long, which easily causes larger bovine serum albumin to be discharged together with impurities, causing the separation effect of bovine serum albumin to reduce, and the extraction amount is reduced. Summary of the invention
[0004] The purpose of the present invention is to provide a method for removing endotoxin from bovine serum albumin in view of the shortcomings of the prior art, so as to solve the technical problems in the prior art.
[0005] The purpose of the present invention can be achieved by the following technical solution: A method for removing endotoxin from bovine serum albumin, comprising the following specific steps:
[0006] Step S1: first, plug the drainage port with a cotton ball, then fill the pretreated affinity ligand into the cylinder, place the drainage assembly into the cylinder, and then cover the cover plate;
[0007] Step S2: the liquid inlet is connected to the protein liquid after cell disruption, and the protein liquid is transported into the box through the liquid inlet. After the box is filled, the liquid inlet is closed, and the protein liquid falls freely through the through-hole plate;
[0008] Step S3: Press the sealing plate to apply pressure to the protein solution, accelerate the protein solution to pass through the through-hole plate and mix with the affinity ligand, and at the same time, the telescopic rod gradually contracts;
[0009] Step S4: After the protein solution has completely passed through the through-hole plate, the telescopic rod is completely retracted, and the bovine serum albumin with a larger volume in the protein solution will be blocked by the through-hole plate or blocked in the mesh holes on the through-hole plate; at this time, pressing the sealing pressing plate will drive the through-hole plate to move downward synchronously, and the through-hole plate moves downward and presses the telescopic head in the second stop zone, so that the through-hole plate passes through the second stop zone. At the same time, the through-hole plate drives the folding plate to fold through the telescopic connecting rod;
[0010] Step S5: When the folding plate is completely folded, the box is in a sealed state. As the through-hole plate continues to move downward, the air pressure in the cavity between the through-hole plate and the folding plate gradually increases. When the through-hole plate enters the stop zone 1, the telescopic head and the fixed block in the stop zone 1 limit the stop zone 1.
[0011] Step S6: Pull the sealing pressing plate. Since the fixed block and the telescopic head in the stop zone 1 limit the stop zone 1, the sealing pressing plate moves upward to stretch the telescopic rod. At this time, the air pressure in the cavity between the through-hole plate and the folding plate flows through the through-hole plate to the cavity between the sealing pressing plate and the through-hole plate. In addition, the sealing pressing plate draws air upward from the cavity between the through-hole plate and the folding plate, and the larger bovine serum albumin blocked in the mesh holes of the through-hole plate will be drawn out.
[0012] Step S7: As the sealing plate moves upward, the sealing plate drives the crushing plates to move closer to each other through the transmission teeth and the chain, so that the crushing plates crush the larger bovine serum albumin on the through-hole plate;
[0013] Step S8: As the next wave of protein solution enters the sealed pressing plate, the crushed bovine serum albumin and the protein solution pass through the through-hole plate and mix with the affinity ligand.
[0014] As a further optimization or improvement of this solution, step S3 specifically includes the following steps:
[0015] Step S31: As the sealing pressing plate presses the protein liquid and passes through the through-hole plate, the protein liquid passes through the through-hole plate and falls onto the rotating plate, and is dispersed into several thin streams by the diverter balls on the rotating plate;
[0016] Step S32: When the protein liquid in the box is completely drained, the through-hole plate presses the rotating shaft through the telescopic connecting rod, so that the rotating shaft drives the folding plates on both sides to rotate along the rotating shaft;
[0017] Step S33: As the through-hole plate drives the rotating shaft to move downward through the telescopic connecting rod, the rotating plate and the folding plate gradually overlap to form a closed sealing plate. At this time, the interior of the box is in a sealed state.
[0018] As a further optimization or improvement of this solution, a drainage assembly is installed in the cylinder, and the drainage assembly includes a box body and a sealing pressure plate located at the lifting and lowering position of the box body, a liquid inlet is installed on the sealing pressure plate, the sealing pressure plate is connected to the through-hole plate through a telescopic rod, the through-hole plate is connected to the folding plate through a telescopic connecting rod, and the sealing pressure plate is connected to the rolling assembly.
[0019] As a further optimization or improvement of the present solution, the folding plate includes two folding plates and a rotating plate installed on the folding plates. The two folding plates are connected by a rotating shaft, the rotating shaft is connected to the telescopic connecting rod, a sliding groove is provided on the box body, one end of the rotating plate is slidably connected to the sliding groove, and the other end of the rotating plate is rotatably connected to the folding plate.
[0020] As a further optimization or improvement of the present scheme, the crushing assembly includes transmission teeth, gears and crushing plates. The sealing pressure plate is connected to the transmission teeth through a connecting rod. The tensioning teeth are rotatably installed in the box body. The tensioning teeth are connected to the gears through a chain, and the transmission teeth cooperate with the chain transmission. The crushing plate is horizontally slidably installed in the box body, and a rack is installed on the crushing plate, and the rack cooperates with the gear transmission.
[0021] As a further optimization or improvement of this solution, the sealing pressure plate is connected to the box body through a folding baffle.
[0022] As a further optimization or improvement of this solution, stop area one and stop area two are provided in the box body, the telescopic head and the fixed block are installed in the stop area one, and the through-hole plate is located between the telescopic head and the fixed block, and the telescopic head is installed in the stop area two.
[0023] As a further optimization or improvement of this solution, a bottom plate and legs supporting the cylinder are installed at the bottom of the cylinder, a drainage port is opened at the bottom of the bottom plate, and a cotton ball is laid on the drainage port.
[0024] Beneficial effects of the present invention:
[0025] (1) The present invention accelerates the passage of the protein liquid through the through-hole plate by pressing the sealing pressure plate. When the protein liquid completely passes through the through-hole plate, the telescopic rod is completely retracted. At this time, the sealing pressure plate drives the through-hole plate to move downward synchronously. At the same time, the through-hole plate drives the folding plate to fold through the telescopic connecting rod, so that the rotating plate and the folding plate overlap to form a closed sealing plate. As the through-hole plate moves downward, the air pressure in the cavity between the through-hole plate and the folding plate gradually increases. When the through-hole plate enters the stop zone 1, the telescopic head and the fixed block in the stop zone 1 limit the stop zone 1.
[0026] Pull the sealing pressure plate, and the sealing pressure plate moves upward to stretch the telescopic rod. At this time, the air pressure in the cavity between the through-hole plate and the folding plate flows through the through-hole plate to the cavity between the sealing pressure plate and the through-hole plate. In addition, the sealing pressure plate draws the air in the cavity between the through-hole plate and the folding plate upward, and the larger bovine serum albumin blocked in the mesh hole of the through-hole plate is drawn out, avoiding the problem of reduced protein liquid passing efficiency due to blockage of the through-hole plate.
[0027] Specifically, as the sealing plate moves upward, the sealing plate drives the crushing plates to move closer to each other through the transmission teeth and the chain, so that the bovine serum albumin drawn from the through-hole plate is crushed. At this time, the bovine serum albumin can pass through the through-hole plate smoothly without additional pressure. At the same time, the shape of the crushed bovine serum albumin can be quickly mixed with the ligand to form a complex, thereby reducing the possibility of larger bovine serum albumin being discharged together with impurities and increasing the extraction amount of bovine serum albumin.
[0028] (2) The present invention applies pressure to the protein solution by pressing the sealing plate. As the sealing plate presses the protein solution and passes through the through-hole plate, the protein solution passes through the through-hole plate and falls onto the rotating plate. The diverter balls on the rotating plate disperse the protein solution into several thin streams, thereby reducing the direct impact of the protein solution on the affinity ligand. Meanwhile, the protein solution dispersed into several thin streams can increase the contact area with the affinity ligand, thereby accelerating the mixing of bovine serum albumin in the protein solution and the affinity ligand. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The present invention will be further described below in conjunction with the accompanying drawings.
[0030] Figure 1 It is a schematic diagram of the overall structure of the present invention.
[0031] Figure 2 This is a schematic diagram of the installation structure of the drainage component.
[0032] Figure 3 It is a cross-sectional view of the overall structure of the present invention.
[0033] Figure 4 It is a cross-sectional view of the drainage component structure.
[0034] Figure 5 Schematic diagram of the internal structure of the drainage component.
[0035] Figure 6 This is a structural diagram of the stop zone.
[0036] Figure 7 This is a schematic diagram of the structure of stop zone 2.
[0037] Figure 8 The working status of the drainage component is shown in Figure 1. Figure 1 .
[0038] Fig. 9 The working status of the drainage component is shown in Figure 1. Figure 2 .
[0039] Fig.10 The working status of the drainage component is shown in Figure 1. Figure 3 .
[0040] Fig.11The working status of the drainage component is shown in Figure 1. Figure 4 .
[0041] Fig.12 The working status of the drainage component is shown in Figure 1. Figure 5 .
[0042] The following are marked in the figure: 1. Cylinder; 2. Bottom plate; 3. Cover plate; 4. Leg; 5. Drainage assembly; 51. Box; 52. Sealing pressure plate; 53. Telescopic rod; 54. Through-hole plate; 55. Liquid inlet; 56. Telescopic connecting rod; 57. Folding plate; 571. Folding plate; 572. Rotating shaft; 573. Rotating plate; 574. Slide; 575. Diverter ball; 58. Stop zone one; 59. Stop zone two; 510. Telescopic head; 511. Fixed block; 6. Crushing assembly; 61. Connecting rod; 62. Transmission tooth; 63. Tensioning tooth; 64. Chain; 65. Gear; 66. Rack; 67. Crushing plate; 7. Folding baffle; 8. Drainage outlet; 9. Cotton ball. DETAILED DESCRIPTION
[0043] The following will be combined with the drawings in 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.
[0044] See also Figure 1-Figure 12 , a method for removing endotoxin from bovine serum albumin, comprising the following specific steps:
[0045] Step S1: firstly, plug the cotton ball 9 on the liquid discharge port 8, then fill the pre-treated affinity ligand into the cylinder 1, place the liquid discharge assembly 5 into the cylinder 1, and then cover the cover plate 3;
[0046] Step S2: connect the liquid inlet 55 to the protein solution after cell disruption, and transport the protein solution into the box 51 through the liquid inlet 55. After the box 51 is filled, close the liquid inlet 55, and the protein solution falls freely through the through-hole plate 54;
[0047] Step S3: Press the sealing plate 52 to apply pressure to the protein solution, accelerate the protein solution to pass through the through-hole plate 54 and mix with the affinity ligand, and at the same time, the telescopic rod 53 gradually contracts;
[0048] Step S4: After the protein solution has completely passed through the through-hole plate 54, the telescopic rod 53 is completely retracted, and the bovine serum albumin with a larger volume in the protein solution will be blocked by the through-hole plate 54, or blocked in the mesh of the through-hole plate 54; at this time, pressing the sealing pressing plate 52 will drive the through-hole plate 54 to move downward synchronously, and the through-hole plate 54 moves downward and presses the telescopic head 510 in the second stop zone 59, so that the through-hole plate 54 passes through the second stop zone 59. At the same time, the through-hole plate 54 drives the folding plate 57 to fold through the telescopic connecting rod 56;
[0049] Step S5: When the folding plate 57 is completely folded, the box body 51 is in a sealed state. As the through-hole plate 54 continues to move downward, the air pressure in the cavity between the through-hole plate 54 and the folding plate 57 gradually increases. When the through-hole plate 54 enters the stop zone 1 58, the telescopic head 510 and the fixed block 511 in the stop zone 1 58 limit the stop zone 1 58.
[0050] Step S6: Pull the sealing plate 52. Since the fixed block 511 and the telescopic head 510 in the stop zone 58 limit the stop zone 58, the upward movement of the sealing plate 52 will stretch the telescopic rod 53. At this time, the air pressure in the cavity between the through-hole plate 54 and the folding plate 57 flows through the through-hole plate 54 to the cavity between the sealing plate 52 and the through-hole plate 54. In addition, the sealing plate 52 draws the air in the cavity between the through-hole plate 54 and the folding plate 57 upward, and the larger bovine serum albumin blocked in the mesh of the through-hole plate 54 will be drawn out.
[0051] Step S7: As the sealing plate 52 moves upward, the sealing plate 52 drives the crushing plates 67 to move closer to each other through the transmission teeth 62 and the chain 64, so that the crushing plates 67 crush the larger bovine serum albumin on the through-hole plate 54;
[0052] Step S8: As the next wave of protein solution enters the sealing platen 52 , the crushed bovine serum albumin and the protein solution pass through the through-hole plate 54 and mix with the affinity ligand.
[0053] It should be noted that, after the bovine serum albumin in the protein solution is mixed with the affinity ligand, the discharge port 8 is opened to discharge the impurities except the unbound impurities through the discharge port 8, and finally the bovine serum albumin is dissociated from the affinity ligand with an appropriate eluent. The above-mentioned bovine serum albumin separation process is prior art, and the present invention will not be repeated here, which does not affect the integrity of this solution.
[0054] See also Figure 4-Figure 5 , the step S3 specifically comprises the following steps:
[0055] Step S31: As the sealing pressing plate 52 presses the protein liquid and passes through the through-hole plate 54, the protein liquid passes through the through-hole plate 54 and falls onto the rotating plate 573, and is dispersed into several thin streams by the diverter balls 575 on the rotating plate 573;
[0056] Step S32: When the protein liquid in the box body 51 is completely drained, the through-hole plate 54 presses the rotating shaft 572 through the telescopic connecting rod 56, so that the rotating shaft 572 drives the folding plates 571 on both sides to rotate along the rotating shaft 572;
[0057] Step S33: As the through-hole plate 54 drives the rotating shaft 572 to move downward through the telescopic connecting rod 56, the rotating plate 573 and the folding plate 571 gradually overlap to form a closed sealing plate. At this time, the interior of the box body 51 is in a sealed state.
[0058] It should be noted that the sealing pressure plate 52 provides pressure on the protein liquid inside the box body 51, so that it can quickly pass through the through-hole plate 54 to mix with the affinity ligand, thereby improving the separation efficiency of the equipment. However, under the action of the pressure provided by the sealing pressure plate 52, the protein liquid may be dispersed by the affinity ligand through the through-hole plate 54. Therefore, the present invention installs a diverter ball 575 on the rotating plate 573 to disperse the protein liquid into several thin streams through the diverter ball 575, thereby reducing the direct impact of the protein liquid on the affinity ligand. At the same time, the protein liquid dispersed into several thin streams can increase the contact area with the affinity ligand, thereby accelerating the mixing of bovine serum albumin in the protein liquid with the affinity ligand.
[0059] See also Figure 3-Figure 4 A drainage assembly 5 is installed in the cylinder 1, and the drainage assembly 5 includes a box body 51 and a sealing pressure plate 52 located at the box body 51 and raised and lowered. A liquid inlet 55 is installed on the sealing pressure plate 52. The sealing pressure plate 52 is connected to a through-hole plate 54 through a telescopic rod 53. The through-hole plate 54 is connected to a folding plate 57 through a telescopic connecting rod 56. The sealing pressure plate 52 is connected to a rolling assembly 6.
[0060] Specifically, a bottom plate 2 and legs 4 for supporting the cylinder 1 are installed at the bottom of the cylinder 1 , a drainage port 8 is provided at the bottom of the bottom plate 2 , and a cotton ball 9 is laid on the drainage port 8 .
[0061] It should be noted that the cotton ball 9 is used to prevent the affinity ligand from falling from the liquid discharge port 8 .
[0062] See also Figure 5 The folding plate 57 includes two folding plates 571 and a rotating plate 573 installed on the folding plates 571. The two folding plates 571 are connected by a rotating shaft 572. The rotating shaft 572 is connected to the telescopic connecting rod 56. A sliding groove 574 is provided on the box body 51. One end of the rotating plate 573 is slidably connected to the sliding groove 574, and the other end of the rotating plate 573 is rotatably connected to the folding plate 571.
[0063] It should be noted that the folding plate 57 has two states: Figure 8-Figure 9The middle folding plate 57 is in the first state, in which the folding plate 57 is opened as a whole, and the protein solution is dispersed into several thin streams when passing through the diverter ball 575 on the rotating plate 573, thereby reducing the direct impact of the protein solution on the affinity ligand. At the same time, the protein solution dispersed into several thin streams can increase the contact area with the affinity ligand, thereby accelerating the mixing of the bovine serum albumin in the protein solution and the affinity ligand; for example Figure 10-12 The state of the middle folding plate 57 is the second state, at this time, the protein liquid inside the box body 51 is drained, the rotating plate 573 and the folding plate 571 overlap to form a closed sealing plate, and at this time the inside of the box body 51 is in a sealed state.
[0064] See also Figure 5 The crushing assembly 6 includes a transmission tooth 62, a gear 65 and a crushing plate 67. The sealing pressure plate 52 is connected to the transmission tooth 62 through a connecting rod 61. A tensioning tooth 63 is rotatably installed in the box body 51. The tensioning tooth 63 is connected to the gear 65 through a chain 64, and the transmission tooth 62 and the chain 64 are in transmission cooperation. A crushing plate 67 is horizontally slidably installed in the box body 51, and a rack 66 is installed on the crushing plate 67, and the rack 66 and the gear 65 are in transmission cooperation.
[0065] It should be noted that the transmission teeth 62 can be installed on the outside of the chain 64 or on the inside of the chain 64. No matter where the chain 64 is installed, when the sealing pressure plate 52 moves, it can drive the chain 64 to transmit. The tensioning teeth 63 are used to tension the chain 64. When the sealing pressure plate 52 moves, the sealing pressure plate 52 drives the gear 65 to rotate through the transmission teeth 62 and the chain 64, and the gear 65 drives the crushing plate 67 to move relative to each other by meshing with the rack 66.
[0066] See also Figure 8-Figure 12 The sealing pressure plate 52 is connected to the box body 51 through the folding baffle 7.
[0067] It should be noted that the folding baffle 7 can prevent the protein liquid from contacting the rolling assembly 6 to avoid the protein liquid being contaminated by the rolling assembly 6. At the same time, the folding baffle 7 can also cooperate with the folding plate 57 to provide a sealed environment for the interior of the box 51.
[0068] See 5- Figure 7 The box body 51 is provided with a stop area 1 58 and a stop area 2 59 , the stop area 1 58 is installed with a telescopic head 510 and a fixed block 511 , and the through-hole plate 54 is located between the telescopic head 510 and the fixed block 511 , and the stop area 2 59 is installed with the telescopic head 510 .
[0069] It should be noted that the stop zone 1 58 and the stop zone 2 59 are two areas in the box body 51, see Figure 8-Figure 9 At this time, the through-hole plate 54 is located in the stop zone 2 59, and the sealing pressing plate 52 squeezes the protein liquid; see Figure 10-11At this time, the through-hole plate 54 moves from the stop zone 2 59 to the stop zone 1 58, and the protein liquid is completely discharged from the box 51. As the sealing pressing plate 52 moves downward, the sealing pressing plate 52 pressurizes the cavity between the through-hole plate 54 and the folding plate 57; see Figure 1 The bottom plate 2 and the through-hole plate 54 are limited by the fixed block 511 and the telescopic head 510 in the stop zone 58. As the sealing pressure plate 52 moves upward, the telescopic rod 53 is stretched. At this time, the gas in the cavity between the through-hole plate 54 and the folding plate 57 of the sealing pressure plate 52 flows to the cavity between the sealing pressure plate 52 and the through-hole plate 54 through the through-hole plate 54. In addition, the sealing pressure plate 52 draws the air in the cavity between the through-hole plate 54 and the folding plate 57 upward, and the larger bovine serum albumin blocked in the mesh hole of the through-hole plate 54 is drawn out.
[0070] The implementation principle of the present invention is:
[0071] The protein liquid after cell crushing is externally connected to the liquid inlet 55, and the protein liquid is transported into the casing 51 through the liquid inlet 55. After the casing 51 is filled, the liquid inlet 55 is closed, and the sealing pressing plate 52 is pressed to make the sealing pressing plate 52 provide pressure to the protein liquid. As the sealing pressing plate 52 presses the protein liquid and passes through the through-hole plate 54, the protein liquid passing through the through-hole plate 54 falls onto the rotating plate 573, and is dispersed into several thin streams by the diverter balls 575 on the rotating plate 573, accelerating the protein liquid to pass through the through-hole plate 54 and mix with the affinity ligand; at the same time, the telescopic rod 53 is gradually retracted. When the protein liquid has all passed through the through-hole plate 54, the telescopic rod 53 is completely retracted, and the bovine serum albumin with a larger volume in the protein liquid will be blocked by the through-hole plate 54, or blocked. In the mesh on the through-hole plate 54; at this time, pressing the sealing pressure plate 52 will drive the through-hole plate 54 to move downward synchronously, and the through-hole plate 54 moves downward and presses the telescopic head 510 in the stop zone 2 59, so that the through-hole plate 54 passes through the stop zone 2 59. At the same time, the through-hole plate 54 drives the folding plate 57 to fold through the telescopic connecting rod 56, so that the rotating plate 573 overlaps with the folding plate 571 to form a closed sealing plate. When the folding plate 57 is completely folded, the box body 51 is in a sealed state. As the through-hole plate 54 continues to move downward, the air pressure in the cavity between the through-hole plate 54 and the folding plate 57 gradually increases. When the through-hole plate 54 enters the stop zone 1 58, the telescopic head 510 and the fixed block 511 in the stop zone 1 58 limit the stop zone 1 58.
[0072] When the sealing platen 52 is pulled, the fixed block 511 and the telescopic head 510 in the stop zone 58 limit the stop zone 58, and the upward movement of the sealing platen 52 will stretch the telescopic rod 53. At this time, the air pressure in the cavity between the through-hole plate 54 and the folding plate 57 flows through the through-hole plate 54 to the cavity between the sealing platen 52 and the through-hole plate 54. In addition, the sealing platen 52 draws the air in the cavity between the through-hole plate 54 and the folding plate 57 upward, and the larger bovine serum albumin blocked in the mesh hole of the through-hole plate 54 will be drawn out. As the sealing platen 52 moves upward, the sealing platen 52 is driven by the transmission tooth 62 and the chain 64 to drive the gear 65 to rotate. The gear 65 drives the crushing plate 67 to move relatively close to the gear 65 by meshing with the rack 66, so that the crushing plate 67 crushes the larger bovine serum albumin on the through-hole plate 54. As the next wave of protein liquid enters the sealing platen 52, the crushed bovine serum albumin and the protein liquid are mixed with the affinity ligand through the through-hole plate 54.
[0073] After the bovine serum albumin in the protein solution is mixed with the affinity ligand, the discharge port 8 is opened to discharge the impurities except the unbound impurities through the discharge port 8, and finally the bovine serum albumin is dissociated from the affinity ligand with a suitable eluent.
[0074] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.
Claims
1. A method for removing endotoxin from bovine serum albumin, characterized in that: The specific steps include: Step S1: first, plug the liquid discharge port (8) with a cotton ball (9), then fill the pre-treated affinity ligand into the cylinder (1), place the liquid discharge assembly (5) into the cylinder (1), and then cover the cylinder (1) with a cover plate (3); Step S2: the liquid inlet (55) is connected to the protein solution after cell disruption, and the protein solution is transported into the box (51) through the liquid inlet (55). After the box (51) is filled, the liquid inlet (55) is closed, and the protein solution falls freely through the through-hole plate (54); Step S3: Pressing the sealing plate (52) so that the sealing plate (52) provides pressure to the protein solution, accelerating the protein solution to pass through the through-hole plate (54) and mix with the affinity ligand, while the telescopic rod (53) gradually contracts; Step S4: After the protein solution has completely passed through the through-hole plate (54), the telescopic rod (53) is completely retracted, and the bovine serum albumin with a larger volume in the protein solution will be blocked by the through-hole plate (54) or blocked in the mesh holes on the through-hole plate (54); at this time, pressing the sealing pressing plate (52) will drive the through-hole plate (54) to move downward synchronously, and the through-hole plate (54) moves downward and presses the telescopic head (510) in the second stop zone (59), so that the through-hole plate (54) passes through the second stop zone (59). At the same time, the through-hole plate (54) drives the folding plate (57) to fold through the telescopic connecting rod (56); Step S5: When the folding plate (57) is completely folded, the box body (51) is in a sealed state. As the through-hole plate (54) continues to move downward, the air pressure in the cavity between the through-hole plate (54) and the folding plate (57) gradually increases. When the through-hole plate (54) enters the stop zone 1 (58), the telescopic head (510) and the fixed block (511) in the stop zone 1 (58) limit the stop zone 1 (58). Step S6: Pull the sealing pressure plate (52). Since the fixed block (511) and the telescopic head (510) in the stop zone (58) limit the stop zone (58), the upward movement of the sealing pressure plate (52) will stretch the telescopic rod (53). At this time, the air pressure in the cavity between the through-hole plate (54) and the folding plate (57) flows through the through-hole plate (54) to the cavity between the sealing pressure plate (52) and the through-hole plate (54). In addition, the sealing pressure plate (52) draws the air in the cavity between the through-hole plate (54) and the folding plate (57) upward, and the larger bovine serum albumin blocked in the mesh holes on the through-hole plate (54) will be drawn out. Step S7: As the sealing plate (52) moves upward, the sealing plate (52) drives the crushing plates (67) to move closer to each other through the transmission teeth (62) and the chain (64), so that the crushing plates (67) crush the larger bovine serum albumin on the through-hole plate (54); Step S8: As the next wave of protein solution enters the sealing platen (52), the crushed bovine serum albumin and the protein solution pass through the through-hole plate (54) and mix with the affinity ligand.
2. The method for removing endotoxin from bovine serum albumin according to claim 1, characterized in that: The step S3 specifically comprises the following steps: Step S31: As the sealing pressing plate (52) presses the protein liquid and passes through the through-hole plate (54), the protein liquid passes through the through-hole plate (54) and falls onto the rotating plate (573), and is dispersed into several thin streams by the diverter balls (575) on the rotating plate (573); Step S32: When the protein liquid in the box body 51 is completely drained, the through-hole plate (54) presses the rotating shaft (572) through the telescopic connecting rod (56), so that the rotating shaft (572) drives the folding plates (571) on both sides to rotate along the rotating shaft (572); Step S33: As the through-hole plate (54) drives the rotating shaft (572) to move downward via the telescopic connecting rod (56), the rotating plate 573 and the folding plate 571 gradually overlap to form a closed sealing plate. At this time, the interior of the box body 51 is in a sealed state.
3. The method for removing endotoxin from bovine serum albumin according to claim 1, characterized in that: A liquid discharge assembly (5) is installed in the cylinder (1), and the liquid discharge assembly (5) comprises a box body (51) and a sealing pressure plate (52) located on the box body (51) and raised and lowered, a liquid inlet (55) is installed on the sealing pressure plate (52), the sealing pressure plate (52) is connected to a through-hole plate (54) via a telescopic rod (53), the through-hole plate (54) is connected to a folding plate (57) via a telescopic connecting rod (56), and the sealing pressure plate (52) is connected to a rolling assembly (6).
4. The method for removing endotoxin from bovine serum albumin according to claim 3, characterized in that: The folding plate (57) comprises two folding plates (571) and a rotating plate (573) mounted on the folding plates (571); the two folding plates (571) are connected via a rotating shaft (572); the rotating shaft (572) is connected to the telescopic connecting rod (56); a sliding groove (574) is provided on the box body (51); one end of the rotating plate (573) is slidably connected to the sliding groove (574); and the other end of the rotating plate (573) is rotatably connected to the folding plate (571).
5. The method for removing endotoxin from bovine serum albumin according to claim 4, characterized in that: The crushing assembly (6) comprises a transmission tooth (62), a gear (65) and a crushing plate (67); the sealing pressure plate (52) is connected to the transmission tooth (62) via a connecting rod (61); a tensioning tooth (63) is rotatably installed in the box body (51); the tensioning tooth (63) is connected to the gear (65) via a chain (64), and the transmission tooth (62) and the chain (64) are in transmission cooperation; a crushing plate (67) is horizontally slidably installed in the box body (51); a rack (66) is installed on the crushing plate (67), and the rack (66) and the gear (65) are in transmission cooperation.
6. The method for removing endotoxin from bovine serum albumin according to claim 5, characterized in that: The sealing pressure plate (52) is connected to the box body (51) via a folding baffle (7).
7. The method for removing endotoxin from bovine serum albumin according to claim 3, characterized in that: The box body (51) is provided with a stop area 1 (58) and a stop area 2 (59); a telescopic head (510) and a fixed block (511) are installed in the stop area 1 (58); the through-hole plate (54) is located between the telescopic head (510) and the fixed block (511); and the telescopic head (510) is installed in the stop area 2 (59).
8. The method for removing endotoxin from bovine serum albumin according to claim 3, characterized in that: A bottom plate (2) and legs (4) supporting the cylinder (1) are installed at the bottom of the cylinder (1); a liquid discharge port (8) is provided at the bottom of the bottom plate (2); and a cotton ball (9) is laid on the liquid discharge port (8).