Extraction method of virus supernatant for constructing plasmids

Abstract

The invention discloses a virus supernatant extraction method for constructing plasmids, and relates to the field of supernatant extraction, and the method comprises the following steps: S1, culturing a virus supernatant, culturing tool cells by using a culture dish, transfecting assembled virus elements into the tool cells, and culturing in an incubator at 37 DEG C for 24-48 hours; s2, liquid supernatant is extracted, the liquid supernatant in the culture dish is extracted through a needle tube assembly, the needle tube assembly comprises a needle tube body and a suction tube matched with the needle tube body in an inserted mode, the suction tube is inserted into the needle tube body and used for sucking the liquid supernatant, the suction tube is in a circular ring shape, and the outer diameter of the suction tube is gradually decreased in the direction away from the needle tube body; one end, far away from the needle tube body, of the suction tube is in smooth transition; s3, filtering the supernate, removing the suction pipe from the insertion ring, and then mounting the filtering device on the insertion ring; and S4, measuring supernate, aligning the needle tube body provided with the filtering device to the scale centrifuge tube, and injecting the supernate in the needle tube into the scale centrifuge tube to finish the collection and extraction work of the supernate. The invention aims to reduce the harm of virus liquid to laboratory personnel and ensure the safety of the laboratory personnel.

Description

Technical Field

[0001] This application relates to the field of supernatant extraction, and more particularly to a method for extracting viral supernatant for constructing plasmids. Background Technology

[0002] The applications of viral fluids in the medical field are mainly reflected in virology research, vaccine development, gene therapy, and antiviral treatment. Viral fluids typically refer to liquids containing viral particles, commonly used in laboratories for vaccine preparation, studying viral characteristics and transmission mechanisms, or for clinical treatment. Viral vectors, especially adenoviruses and lentiviruses, can be used to culture cell lines, study the interaction between viruses and host cells, and help scientists better understand the biological characteristics of viruses. By loading the target gene into viral particles and then transfecting them into tool cells to produce viral fluid, which is then used to infect target cells, a stable cell line containing the target gene can be successfully constructed for further scientific research experiments. Researchers can simulate the viral infection process in a controlled environment, providing important data support for antiviral strategies and vaccine development.

[0003] In the prior art, a method for extracting supernatant includes the following steps:

[0004] S1; Incubate the virus supernatant in a petri dish.

[0005] S2; Use a syringe to extract the supernatant from the culture dish. The syringe consists of a syringe body and a needle. The needle is inserted into the supernatant, and the needle body moves in a piston-like motion to generate negative pressure, drawing the supernatant from the needle into the syringe body.

[0006] S3; Remove the needle and then install the gyroscope-shaped filter device onto the needle body. The filter device includes a filter body with a filter chamber extending through it, and a filter plate is installed inside the filter chamber. When the supernatant in the syringe body is discharged, it is filtered through the filter plate.

[0007] S4; Finally, inject the filtered supernatant into a graduated centrifuge tube to complete the collection and extraction of the supernatant.

[0008] Regarding the aforementioned technologies, existing needles typically use circular, pointed metal objects. The inventors believe that viral supernatants are highly toxic and infectious. Whether using needles to extract the supernatant or removing them to replace filters, needles can easily cause injury to laboratory personnel, potentially leading to viral harm. Therefore, improvements are urgently needed. Summary of the Invention

[0009] To reduce harm to laboratory personnel and ensure their safety, this application provides a method for extracting viral supernatant for constructing plasmids.

[0010] This application provides a method for extracting viral supernatant for constructing plasmids, comprising the following steps:

[0011] S1, culture virus supernatant, culture tool cells in culture dish, transfect the assembled virus elements into tool cells, and incubate in a 37°C incubator for 24-48 hours;

[0012] S2, Extract the supernatant. Use a syringe assembly to extract the supernatant from the culture dish. The syringe assembly includes a syringe body and a suction tube that are inserted into the syringe body. The suction tube is inserted into the syringe body for aspirating the supernatant. The suction tube is annular in shape, and its outer diameter gradually decreases in the direction away from the syringe body. The end of the suction tube away from the syringe body has a smooth transition.

[0013] S3, filter the supernatant. After removing the suction tube from the connector ring, install the filter device onto the connector ring.

[0014] S4. Measure the supernatant, align the syringe body equipped with the filter with the graduated centrifuge tube, and inject the supernatant from the syringe into the graduated centrifuge tube to complete the collection and extraction of the supernatant.

[0015] By adopting the above technical solution and using a conical pipette, on the one hand, the risk of injury to laboratory personnel can be reduced, the risk of viral infection can be decreased, and the safety of supernatant extraction can be improved. On the other hand, compared with the existing pipettes with spikes, the pipette with a smooth transition design can firstly reduce the slippage of the pipette in the culture dish, improving the stability of supernatant extraction; secondly, it can allow the end of the pipette to better fit the culture dish, improving the convenience of supernatant extraction; and finally, it can also reduce the risk of abrasions to laboratory personnel and improve the safety of supernatant extraction.

[0016] Preferably, the supernatant extraction step includes the following steps:

[0017] S2.1 Assemble the syringe assembly. Place the suction tube on the mounting frame, which has multiple insertion holes for inserting the suction tube. Insert the syringe body and the suction tube into the insertion holes and remove the suction tube from the insertion holes.

[0018] S2.2, Extract the supernatant. Insert the suction tube into the culture dish and, with the suction of the syringe body, draw the supernatant into the syringe body.

[0019] S2.3, disassemble the syringe assembly, and finally remove the aspiration tube to complete the aspiration of the supernatant.

[0020] By adopting the above technical solution and using a mounting bracket to prevent the suction tube from being blocked, the suction tube can be placed and installed more conveniently, improving the ease of use of the suction tube and reducing the contact between laboratory personnel and the suction tube to a certain extent, thereby improving the safety of the supernatant extraction process.

[0021] Preferably, the filtration device includes a filter body and a filter element, the filter body is inserted into the syringe body, the filter element is disposed on the filter body for filtering the supernatant, and the mounting bracket has a plurality of mounting holes for inserting and installing the filter body.

[0022] By adopting the above technical solution, the filter device can be more easily connected and matched with the syringe body, improving the convenience of replacing the suction tube and the filter device.

[0023] Preferably, the mounting bracket is rotatably connected to multiple disassembly plates, and the mounting bracket is provided with multiple positioning rotating posts. The positioning rotating posts are located next to the insertion hole or mounting hole. The disassembly plate is provided with a positioning rotating hole that is inserted and engaged with the positioning rotating post. The disassembly plate is provided with a disassembly hole that penetrates one side of the disassembly plate. The suction tube sidewall is provided with a suction slot that is inserted and engaged with the disassembly hole. The filter body sidewall is provided with a filter slot that is inserted and engaged with the disassembly hole.

[0024] By adopting the above technical solution, when in use, the suction tube and the filter body are inserted into the disassembly hole, which can fix the suction tube and the filter body. This allows for more stable installation and disassembly of the suction tube and the filter body, improves the convenience of using the suction tube and the filter body, reduces injury to laboratory personnel, improves the safety of supernatant extraction, and allows for easier disposal of the suction tube and the filter body by removing the disassembly plate when disassembling them.

[0025] Preferably, the filter body is annular, the outer diameter of the filter body increases first and then decreases along its length, and a filter cavity for installing filter elements is provided inside the filter body. The filter cavity is cylindrical and is inserted into the needle tube body.

[0026] By adopting the above technical solution and using a cylindrical filter chamber, the residue of supernatant in the filter body can be reduced, enabling more efficient extraction of supernatant and improving the utilization rate of extracted supernatant.

[0027] Preferably, the filter sheet is composed of multiple filter membranes, and the pore size of the filter membranes gradually decreases in the direction away from the needle body.

[0028] By adopting the above technical solution and using multilayer membranes, each membrane can filter impurities of different particle sizes, thereby allowing the supernatant passing through the membranes to be filtered sequentially, improving the filtration efficiency of the supernatant and the convenience of supernatant extraction.

[0029] Preferably, the filter chamber is provided with a plurality of filter screens, the inner diameter of the filter screens gradually decreases in the direction away from the needle body, and the filter membrane is disposed on the filter screens.

[0030] By adopting the above technical solution and using an arc-shaped filter screen to fix the filter membrane, not only can the stability of the filter membrane be improved, but also the contact area between the filter membrane and the supernatant can be increased, thereby enabling the supernatant to be filtered more efficiently and improving the convenience of supernatant extraction.

[0031] Preferably, the syringe body is provided with a disassembly slide, which slides along the length of the syringe body. A snap-fit ​​piece is provided at one end of the disassembly slide near the suction tube. The snap-fit ​​piece has an insertion slot, which engages with the suction slot and the filter slot.

[0032] By adopting the above technical solution, after the suction tube and the filter body are inserted into the snap-fit ​​piece, the installation and removal of the suction tube and the filter body can be realized more conveniently by pushing the disassembly slide, reducing the contact between laboratory personnel and the suction tube and the filter body, and improving the safety of virus supernatant extraction.

[0033] Preferably, a push plate is slidably connected to the bottom of the insertion slot. The push plate is used to push the suction tube and filter body away from the snap-fit ​​piece. A connecting plate is slidably connected inside the disassembly slide. The connecting plate is connected to the push plate and slides in the same direction as the push plate. A sliding button is slidably provided on the disassembly slide. The sliding button is connected to the connecting plate and slides in the same direction as the push plate.

[0034] By adopting the above technical solution, when in use, pressing the sliding button will cause the push plate to slide, thereby pushing the suction tube or filter body out of the insertion slot, thus improving the convenience of disassembling the suction tube and the filter body.

[0035] Preferably, the disassembly slide is provided with several elastic elements for driving the connecting plate away from the needle body.

[0036] By adopting the above technical solution and using the design of elastic components, the connecting plate can drive the push plate to reset, thereby making it easier to insert the suction tube and the filter body into the insertion slot, improving the convenience of installation and disassembly of the suction tube and the filter body.

[0037] In summary, this application includes at least one of the following beneficial technical effects:

[0038] 1. The use of a conical pipette design can reduce the risk of injury to laboratory personnel, decrease the risk of viral infection, and improve the safety of supernatant extraction. Furthermore, compared to existing pipettes with spikes, the smoothly transitioned design of the pipette reduces slippage in the culture dish, improving the stability of supernatant extraction. It also allows the pipette tip to better adhere to the culture dish, enhancing the ease of supernatant extraction. Finally, it reduces the risk of abrasions to laboratory personnel, further improving the safety of supernatant extraction.

[0039] 2. Using a mounting bracket to secure the pipettes allows for easier placement and installation, improving the ease of use of the pipettes and reducing contact between laboratory personnel and the pipettes, thus enhancing the safety of supernatant extraction.

[0040] 3. After inserting the aspiration tube and filter body into the snap-fit ​​plate, the installation and removal of the aspiration tube and filter body can be more conveniently achieved by pushing the disassembly slide, reducing laboratory personnel's contact with the aspiration tube and filter body and improving the safety of virus supernatant extraction. Attached Figure Description

[0041] Figure 1 This is a schematic flowchart of a method for extracting viral supernatant for constructing plasmids according to Embodiment 1 of this application;

[0042] Figure 2 This is a schematic diagram illustrating the main structure of the mounting bracket in Embodiment 1 of this application;

[0043] Figure 3 This is a schematic diagram of Embodiment 1 of this application, mainly illustrating the suction tube and the filtering device;

[0044] Figure 4 This is a cross-sectional view of the suction tube, which is the main feature of Embodiment 1 of this application;

[0045] Figure 5 This is a cross-sectional view of Embodiment 1 of this application, which mainly illustrates the filtering device;

[0046] Figure 6 This is a schematic diagram illustrating the needle body and the disassembly slide of Embodiment 2 of this application;

[0047] Figure 7 This is a cross-sectional view of Embodiment 2 of this application, mainly showing the syringe body and the disassembly slide.

[0048] Reference numerals: 1. Suction tube; 2. Filter body; 3. Suction slot; 4. Filter slot; 5. Mounting bracket; 51. Fixing plate; 52. Support frame; 53. Base; 6. Removal plate; 7. Positioning pivot; 8. Removal hole; 9. Insertion hole; 10. Filter chamber; 11. Filter disc; 12. Filter screen; 13. Needle body; 14. Removal slide; 15. Sliding button; 16. Snap-fit ​​piece; 17. Insertion slot; 18. Connecting plate; 19. Elastic element; 20. Push plate. Detailed Implementation

[0049] The following is in conjunction with the appendix Figures 1-7 This application will be described in further detail.

[0050] This application discloses a method for extracting viral supernatant for constructing plasmids.

[0051] Example 1

[0052] Reference Figure 1 A method for extracting viral supernatant for constructing plasmids includes the following steps:

[0053] S1, culture virus supernatant. Use a culture dish to culture tool cells (usually 293T cells). Transfect the assembled virus elements into the tool cells and incubate them in a 37°C incubator for 24-48 hours to produce virus supernatant. This can be, but is not limited to, the culture of virus supernatant. This is the material required for this experiment. In the laboratory, it usually consists of a flat disc-shaped base and a lid made of polystyrene plastic.

[0054] S2, Extracting the supernatant: The supernatant in the culture dish is extracted using a syringe assembly. The syringe assembly includes a syringe body 13 and a suction tube 1 that is inserted into the syringe body 13. The suction tube 1 is inserted into the syringe body 13 for aspirating the supernatant. The suction tube 1 is annular in shape, and its outer diameter gradually decreases in the direction away from the syringe body 13. The end of the suction tube 1 away from the syringe body 13 has a chamfer to make the end of the suction tube 1 smoothly transition, thereby reducing the risk of injury to laboratory personnel. The suction tube 1 of this application is made of plastic material, which not only reduces the manufacturing cost of the suction tube 1, but also reduces the hardness of the suction tube 1, reducing the risk of injury to laboratory personnel. It also allows the suction tube 1 to aspirate more stably in the culture dish, improving the stability of extracting the supernatant using the suction tube 1.

[0055] S2.1, Assemble the syringe assembly. The suction tube 1 is placed on the mounting frame 5. The mounting frame 5 includes a bottom, a support frame 52, and a fixing plate 51. The support frame 52 is placed on the base 53 to raise the height, and the fixing plate 51 is installed on the top of the support frame 52. The fixing plate 51 has multiple insertion holes 9 for inserting the suction tube 1. After inserting the syringe body 13 and the suction tube 1, the suction tube 1 is removed from the insertion holes 9, thus facilitating the installation of the suction tube 1.

[0056] S2.2, Extract the supernatant. Insert the suction tube 1 into the culture dish and, under the suction of the syringe body 13, draw the supernatant into the syringe body 13.

[0057] S2.3, disassemble the syringe assembly, and finally remove the aspiration tube 1 to complete the aspiration of the supernatant.

[0058] S3, filter the supernatant. After removing the suction tube 1 from the plug ring, install the filter device onto the plug ring.

[0059] S4. Measure the supernatant, align the syringe body 13 equipped with the filter device with the graduated centrifuge tube, and inject the supernatant from the syringe into the graduated centrifuge tube to complete the collection and extraction of the supernatant.

[0060] The filtration device includes a filter body 2 and filter elements 11. The filter body 2 is inserted into a syringe body 13. The filter body 2 is annular, and its outer diameter increases and then decreases along its length. A filter chamber 10, cylindrical in shape, is provided within the filter body 2 for mounting the filter elements 11. This cylindrical design reduces supernatant residue in the syringe and improves the utilization rate of the extracted supernatant. The filter elements 11 consist of multiple layers of filter membranes, with the pore size gradually decreasing away from the syringe body 13. This allows the supernatant to be filtered sequentially, improving the filtration efficiency. Multiple filter screens 12 are provided within the filter chamber 10, with the inner diameter of each screen gradually decreasing away from the syringe body 13. The filter membranes are disposed on these filter screens 12.

[0061] Multiple disassembly plates 6 are rotatably connected to the fixed plate 51. The disassembly plates 6 are rectangular and made of plastic. Multiple positioning pivots 7 are provided on the fixed plate 51, fixed to the insertion hole 9 or next to the mounting hole. The disassembly plates 6 have positioning pivot holes that engage with the positioning pivots 7, and disassembly holes 8 that penetrate one side of the disassembly plate 6, allowing for easier insertion of the suction tube 1 or filter body 2. The suction tube 1 has a suction slot 3 on its side wall that engages with the disassembly hole 8, and the filter body 2 has a filter slot 4 on its side wall that engages with the disassembly hole 8. When it is necessary to disassemble the suction tube 1 or filter body 2, pressing the disassembly plate 6 and then pulling the syringe body 13 allows for more stable disassembly of the suction tube 1 or filter body 2.

[0062] The advantages of the viral supernatant extraction method for constructing plasmids according to the embodiments of this application are as follows: Through multifaceted design, the sharp edges of the aspirator 1 are reduced, and the softness of the aspirator 1 is decreased, thereby better reducing the risk of abrasions to laboratory personnel and improving the safety of supernatant extraction; from the perspective of ease of use, the mounting bracket 5 is used to place the aspirator 1 and the filter body 2, improving the convenience of installing the aspirator 1 and the filter body 2; finally, with the action of the disassembly plate 6, the aspirator 1 and the filter body 2 can be disassembled more easily, reducing contact between laboratory personnel and the aspirator 1 and improving the safety of supernatant extraction.

[0063] Example 2

[0064] Reference Figure 6 and Figure 7 The difference between this embodiment and embodiment 1 is that a disassembly slide 14 is slidably connected to the syringe body 13. The disassembly slide 14 is slidably arranged along the length direction of the syringe body 13. A snap-fit ​​piece 16 is fixed at one end of the disassembly slide 14 near the suction tube 1. The snap-fit ​​piece has an insertion slot 17. The suction slot 3 is engaged with the insertion slot 17, and the filter slot 4 is engaged with the insertion slot 17. The suction tube 1 and the filter body 2 are snapped into the insertion slot 17, so that the suction tube 1 and the filter body 2 can be installed and disassembled more conveniently, reducing the contact of staff with the virus and reducing the damage to staff.

[0065] A push plate 20 is slidably connected to the bottom of the insertion slot 17. The push plate 20 slides in a direction close to the suction tube 1 or the filter body 2, thereby driving the suction tube 1 and the filter body 2 to disengage from the insertion slot 17. A connecting plate 18 is slidably connected inside the disassembly slide 14. The connecting plate 18 is connected to the push plate 20 and slides in the same direction as the push plate 20. A sliding button 15 is slidably connected to the disassembly slide 14. The sliding button 15 is connected to the connecting plate 18 and slides in the same direction as the push plate. Several elastic elements 19, which are springs, are fixed inside the disassembly slide 14 to drive the connecting plate 18 away from the needle body 13.

[0066] The implementation principle of Example 2 is as follows: When in use, the suction tube 1 or the filter body 2 is snapped onto the snap-fit ​​piece 16, and then the disassembly slide 14 is pushed to easily install the suction tube 1 and the filter body 2. After use, the disassembly slide 14 is pushed and the sliding button 15 is pressed to make the suction tube 1 and the filter body 2 more easily detach from the snap-fit ​​piece 16, reducing the contact between laboratory personnel and the suction tube 1 and the filter body 2, and improving the safety of virus supernatant extraction.

[0067] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A method for extracting viral supernatant for constructing plasmids, characterized in that: Includes the following steps, S1, culture virus supernatant, culture tool cells in culture dish, transfect the assembled virus elements into tool cells, and incubate in a 37°C incubator for 24-48 hours; S2, extract the supernatant. Use a syringe assembly to extract the supernatant from the culture dish. The syringe assembly includes a syringe body (13) and a suction tube (1) that is inserted into the syringe body (13). The suction tube (1) is inserted into the syringe body (13) for suctioning the supernatant. The suction tube (1) is annular in shape, and its outer diameter gradually decreases in the direction away from the syringe body (13). The end of the suction tube (1) away from the syringe body (13) is smoothly transitioned. S3, filter the supernatant, remove the suction tube (1) from the plug ring, and then install the filter device on the plug ring; S4. Measure the supernatant, align the syringe body (13) with the filter device with the graduated centrifuge tube, and inject the supernatant in the syringe into the graduated centrifuge tube to complete the collection and extraction of the supernatant.

2. The method for extracting viral supernatant for constructing plasmids according to claim 1, characterized in that: The supernatant extraction process includes the following steps: S2.1, Assemble the syringe assembly, place the suction tube (1) on the mounting frame (5), the mounting frame (5) has a plurality of insertion holes (9) for inserting the suction tube (1); insert the syringe body (13) into the suction tube (1) and remove the suction tube (1) from the insertion hole (9); S2.2, extract the supernatant, insert the suction tube (1) into the culture dish, and under the suction of the syringe body (13), extract the supernatant into the syringe body (13); S2.3, disassemble the syringe assembly, and finally remove the aspiration tube (1) to complete the aspiration of the supernatant.

3. The method for extracting viral supernatant for constructing plasmids according to claim 2, characterized in that: The filtration device includes a filter body (2) and a filter plate (11). The filter body (2) is inserted into the syringe body (13). The filter plate (11) is disposed on the filter body (2) for filtering the supernatant. The mounting bracket (5) has a plurality of mounting holes for inserting and installing the filter body (2).

4. The method for extracting viral supernatant for constructing plasmids according to claim 3, characterized in that: The mounting bracket (5) is rotatably connected to multiple disassembly plates (6), and the mounting bracket (5) is provided with multiple positioning rotating posts (7). The positioning rotating posts (7) are located next to the insertion hole (9) or the mounting hole. The disassembly plate (6) is provided with a positioning rotating hole that is inserted and cooperates with the positioning rotating post (7). The disassembly plate (6) is provided with a disassembly hole (8). The disassembly hole (8) is opened through one side of the disassembly plate (6). The suction tube (1) is provided with a suction slot (3) that is inserted and cooperates with the disassembly hole (8) on its side wall. The filter body (2) is provided with a filter slot (4) that is inserted and cooperates with the disassembly hole (8) on its side wall.

5. The method for extracting viral supernatant for constructing plasmids according to claim 3, characterized in that: The filter body (2) is annular, and the outer diameter of the filter body (2) increases first and then decreases along its length. The filter body (2) has a filter cavity (10) for installing the filter sheet (11). The filter cavity (10) is cylindrical and is inserted into the needle body (13).

6. The method for extracting viral supernatant for constructing plasmids according to claim 5, characterized in that: The filter (11) is composed of multiple layers of filter membranes, and the pore size of the filter membranes gradually decreases in the direction away from the needle body (13).

7. The method for extracting viral supernatant for constructing plasmids according to claim 6, characterized in that: The filter chamber (10) is provided with a plurality of filter screens (12), the inner diameter of the filter screens (12) gradually decreases in the direction away from the needle body (13), and the filter membrane is disposed on the filter screens (12).

8. The method for extracting viral supernatant for constructing plasmids according to claim 3, characterized in that: The syringe body (13) is provided with a disassembly slide (14), which slides along the length of the syringe body (13). A snap-fit ​​piece (16) is provided at one end of the disassembly slide (14) near the suction tube (1). A insertion slot (17) is provided on the snap-fit ​​piece (16). The suction slot (3) is inserted into the insertion slot (17). The filter slot (4) is inserted into the insertion slot (17).

9. The method for extracting viral supernatant for constructing plasmids according to claim 8, characterized in that: A push plate (20) is slidably connected to the bottom of the insertion slot (17). The push plate (20) is used to push the suction tube (1) and the filter body (2) away from the snap-fit ​​piece (16). A connecting plate (18) is slidably connected inside the disassembly slide (14). The connecting plate (18) is connected to the push plate (20) and slides in the same direction as the push plate (20). A sliding button (15) is slidably provided on the disassembly slide (14). The sliding button (15) is connected to the connecting plate (18) and slides in the same direction as the push plate (20).

10. A method for extracting viral supernatant for constructing plasmids according to claim 9, characterized in that: The disassembly slide (14) is provided with several elastic elements (19) for driving the connecting plate (18) away from the needle body (13).

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