A novel adeno-associated virus rapid purification device

Abstract

The utility model discloses a novel adenovirus rapid purification device, including lower casing, upper casing, centrifugal tube cover, filter adsorption subassembly and ultrafiltration membrane, the upper end of lower casing has an opening, the both ends of upper casing are respectively provided with an opening, and the opening of upper casing lower extreme is detachably arranged on the opening of lower casing, and the opening of upper casing upper end is detachably connected with centrifugal tube cover, filter adsorption subassembly is detachably arranged in upper casing, and ultrafiltration membrane is arranged in upper casing or lower casing, and is located the below of filter adsorption subassembly, the device is simple to operate, avoids the problem that the sample operation of multiple samples is easy to cause the confusion or addition error among samples to reduce the efficiency and accuracy of test, greatly solves the tediousness of traditional plasmid extraction kit on the time of operation, process.

Description

Technical Field

[0001] This utility model relates to the field of adeno-associated virus technology, and in particular to a novel rapid purification device for adeno-associated virus. Background Technology

[0002] Adeno-Associated Virus (AAV) belongs to the parvovirus family and is a non-enveloped, single-stranded linear DNA virus. The AAV genome is approximately 4700 bp long and includes two open reading frames (ORFs), located between two inverted terminal repeats (ITRs), each consisting of 145 nucleotides. Due to its wide host range, high safety, low immunogenicity, stable expression, and stable physical properties, AAV has been widely used in basic research and clinical trials, and the AAV vector has become one of the most commonly used gene therapy vectors worldwide.

[0003] When using AAV virions to infect animals and cultured cells for stable expression of exogenous genes, high-purity, high-titer AAV is required. AAV preparation and purification typically employ CsCl density gradient centrifugation and iodixanol gradient centrifugation. To simplify the plasmid extraction process, scientists have developed several commercially available rapid purification kits. Traditional AAV rapid extraction kits use PEG 8000 to precipitate AAV, followed by chloroform extraction, concentration, and salt displacement to obtain purified AAV samples. Traditional AAV rapid extraction kits require multiple transfers of lysed samples, a relatively cumbersome process that is prone to sample contamination or additive errors, reducing experimental efficiency and accuracy.

[0004] In summary, traditional commercial plasmid extraction kits have relatively cumbersome operation steps, and the operation of multiple samples can easily lead to sample confusion or errors in addition, reducing the efficiency and accuracy of the experiment. Utility Model Content

[0005] The purpose of this invention is to provide a novel rapid purification device for adeno-associated virus (AAV) to solve one or more technical problems existing in the prior art, or at least provide a beneficial option or create conditions.

[0006] To achieve the above-mentioned objectives, this utility model adopts the following technical solution:

[0007] This invention provides a novel rapid purification device for adeno-associated virus, comprising a lower sleeve, an upper sleeve, a centrifuge tube cap, a filtration and adsorption assembly, and an ultrafiltration membrane.

[0008] The upper end of the lower sleeve has an opening;

[0009] The upper sleeve has an opening at each end, the opening at the lower end of the upper sleeve is detachably disposed on the opening of the lower sleeve, and the opening at the upper end of the upper sleeve is detachably connected to the centrifuge tube cover.

[0010] The filter adsorption assembly is detachably disposed inside the upper sleeve;

[0011] The ultrafiltration membrane is disposed inside the upper sleeve or the lower sleeve, located below the filtration and adsorption assembly.

[0012] Furthermore, the filtration and adsorption assembly includes a centrifuge tube, a filter column, and an affinity chromatography medium;

[0013] The centrifuge tube is detachably installed inside the upper sleeve, and one end of the centrifuge tube has a feeding port and the other end has a discharging port.

[0014] The filter column and the affinity chromatography medium are both disposed inside the centrifuge tube, with the filter column positioned above the affinity chromatography medium.

[0015] Furthermore, a first protruding edge extends radially from the outer periphery of the feeding port of the centrifuge tube, and the first protruding edge overlaps the upper end opening surface of the upper sleeve.

[0016] Furthermore, the centrifuge tube cap is threaded onto the opening of the upper sleeve.

[0017] Furthermore, a second convex edge extends radially from the inner wall of the opening of the lower sleeve, and the ultrafiltration membrane overlaps the second convex edge of the lower sleeve.

[0018] Furthermore, the outer wall of the opening of the lower sleeve has a recessed portion extending radially, and the opening at the lower end of the upper sleeve is attached to the recessed portion of the lower sleeve.

[0019] Furthermore, the affinity chromatography medium is made of agarose microspheres.

[0020] Furthermore, the ultrafiltration membrane is made of polyethersulfone material with low protein and low DNA adsorption.

[0021] The beneficial effects of this utility model are:

[0022] This device is easy to operate, avoiding the problems of sample confusion or addition errors that can easily occur when operating multiple samples, thus reducing the efficiency and accuracy of the experiment. It greatly solves the cumbersome operation time and process of traditional plasmid extraction kits. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of a novel adeno-associated virus rapid purification device according to an embodiment of the present invention;

[0024] Figure 2 for Figure 1 A schematic diagram of the decomposition process;

[0025] Figure 3 for Figure 1 A cross-sectional schematic diagram;

[0026] Figure 4 for Figure 3 A schematic diagram of the decomposition process;

[0027] Figure 5 for Figure 4 A schematic diagram of the structure viewed from another angle.

[0028] In the picture:

[0029] 10. Lower sleeve; 11. Second convex edge; 12. Recessed portion;

[0030] 20. Install the sleeve;

[0031] 30. Centrifuge tube caps;

[0032] 40. Filtration and adsorption assembly; 41. Centrifuge tube; 411. First convex edge; 42. Filter column; 43. Affinity chromatography medium;

[0033] 50. Ultrafiltration membrane. Detailed Implementation

[0034] It should be noted that the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion, for example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product, or device.

[0035] Furthermore, the terms "installation," "setup," "equipped with," "connection," "linking," and "socketing" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral structure; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium, or an internal connection between two devices, components, or parts. Those skilled in the art can understand the specific meaning of these terms in this utility model based on the specific circumstances.

[0036] like Figures 1-5As shown, a novel adeno-associated virus rapid purification device includes a lower sleeve 10, an upper sleeve 20, a centrifuge tube cap 30, a filtration and adsorption assembly 40, and an ultrafiltration membrane 50. The lower sleeve 10 has an opening at its upper end; the upper sleeve 20 has openings at both ends, with the lower opening of the upper sleeve 20 detachably connected to the opening of the lower sleeve 10, and the upper opening of the upper sleeve 20 detachably connected to the centrifuge tube cap 30; the filtration and adsorption assembly 40 is detachably disposed within the upper sleeve 20; and the ultrafiltration membrane 50 is disposed within the lower sleeve 10, located below the filtration and adsorption assembly 40.

[0037] Specifically, the filtration and adsorption assembly 40 includes a centrifuge tube 41, a filter column 42, and an affinity chromatography medium 43. The centrifuge tube 41 is detachably disposed within the upper sleeve 20, and has a feed port at one end and a discharge port at the other end. The filter column 42 and the affinity chromatography medium 43 are both disposed within the centrifuge tube 41, with the filter column 42 positioned above the affinity chromatography medium 43. Here, the filter column is used to filter the lysed bacterial cells, facilitating subsequent centrifugation. Preferably, the affinity chromatography medium 43 is made of agarose microspheres, which can efficiently and specifically adsorb adeno-associated viruses and remove impurity proteins and other organic compounds from bacteria.

[0038] Based on the above structure, a first convex edge 411 extends radially from the outer periphery of the feeding port of the centrifuge tube 41, and the first convex edge 411 overlaps the upper end opening surface of the upper sleeve 20.

[0039] Based on the above structure, the centrifuge tube cap 30 is threaded onto the opening of the upper sleeve 20.

[0040] Specifically, a second protruding edge 11 extends radially from the inner wall of the opening of the lower sleeve 10, and the ultrafiltration membrane 50 overlaps the second protruding edge 11 of the lower sleeve 10.

[0041] Based on the above structure, a recessed portion 12 extends radially from the outer wall of the opening of the lower sleeve 10, and the opening at the lower end of the upper sleeve 20 is attached to the recessed portion 12 of the lower sleeve 10.

[0042] Preferably, the ultrafiltration membrane 50 is made of polyethersulfone material with low protein and low DNA adsorption, and can be used for the concentration and desalting of adeno-associated virus samples.

[0043] The working principle of this invention is as follows: First, the ultrafiltration membrane 50 in the lower sleeve 10 is removed, and the lysed AAV lysis buffer is added to the upper sleeve 20. Cellular impurities are collected on the upper surface of the filter column 42 by centrifugation, and the waste liquid flows into the lower sleeve 10. The AAV with cell and bacterial impurities removed specifically binds to the affinity chromatography medium 43. Affinity elution buffer is added to the upper sleeve 20, and the AAV is purified by centrifugation. Finally, the filter adsorption component 40 in the upper sleeve 20 is removed, and the ultrafiltration membrane 50 in the lower sleeve 10 is added. The purified AAV is added to the ultrafiltration membrane 50, and after centrifugation, concentration and solution replacement are performed on the ultrafiltration membrane 50 to completely achieve the purpose of rapid purification and replacement of AAV.

[0044] The preferred embodiments of this utility model have been described in detail above. Of course, this utility model may also take different forms than the above embodiments. Equivalent transformations or corresponding modifications made by those skilled in the art without departing from the spirit of this utility model should be within the protection scope of this utility model.

Claims

1. A novel rapid purification device for adeno-associated virus, characterized in that, Includes lower sleeve, upper sleeve, centrifuge tube cap, filtration and adsorption assembly, and ultrafiltration membrane: The upper end of the lower sleeve has an opening; The upper sleeve has an opening at each end, the opening at the lower end of the upper sleeve is detachably disposed on the opening of the lower sleeve, and the opening at the upper end of the upper sleeve is detachably connected to the centrifuge tube cover. The filter adsorption assembly is detachably disposed inside the upper sleeve; The ultrafiltration membrane is disposed inside the upper sleeve or the lower sleeve, located below the filtration and adsorption assembly.

2. The novel adeno-associated virus rapid purification device according to claim 1, characterized in that, The filtration and adsorption assembly includes centrifuge tubes, a filter column, and an affinity chromatography medium. The centrifuge tube is detachably installed inside the upper sleeve, and one end of the centrifuge tube has a feeding port and the other end has a discharging port. The filter column and the affinity chromatography medium are both disposed inside the centrifuge tube, with the filter column positioned above the affinity chromatography medium.

3. The novel adeno-associated virus rapid purification device according to claim 1, characterized in that, The centrifuge tube has a first protruding edge extending radially from the outer periphery of the feeding port, and the first protruding edge overlaps the upper end opening surface of the upper sleeve.

4. The novel adeno-associated virus rapid purification device according to claim 1, characterized in that, The centrifuge tube cap is threaded onto the opening of the upper sleeve.

5. The novel adeno-associated virus rapid purification device according to claim 1, characterized in that, The inner wall of the opening of the lower sleeve has a second convex edge extending radially, and the ultrafiltration membrane overlaps the second convex edge of the lower sleeve.

6. The novel adeno-associated virus rapid purification device according to claim 1, characterized in that, The lower sleeve has a recessed portion extending radially from the outer wall of its opening, and the opening at the lower end of the upper sleeve is attached to the recessed portion of the lower sleeve.

7. The novel adeno-associated virus rapid purification device according to claim 2, characterized in that, The affinity chromatography medium is made of agarose microspheres.

8. The novel adeno-associated virus rapid purification device according to claim 1, characterized in that, The ultrafiltration membrane is made of polyethersulfone material with low protein and low DNA adsorption.

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