Alkali cracking equipment and method for extracting plasmids by using same

Abstract

The invention provides alkali cracking equipment and a method for extracting plasmids by using the same. The alkali cracking equipment comprises a first pipeline, a second pipeline, a third pipeline, a cell lysis reactor and a neutralization standing tank, wherein the first pipeline is used for conveying a bacterial suspension, the second pipeline is used for conveying an alkali lysis solution, the third pipeline is used for conveying an acid solution, the cell lysis reactor is used as a reaction container of alkali lysis cells, and the neutralization standing tank is used for collecting a neutralized feed liquid and carrying out standing incubation; the first pipeline and the second pipeline are intersected and then connected with the inlet end of the cell dissolving reactor; the third pipeline is intersected with the outlet end of the cell dissolving reactor and then is connected with the inlet end of the neutralization standing tank. The alkali cracking equipment can realize fixed-proportion on-line mixing and continuous flow cracking of the bacterial suspension, the alkali cracking solution and the acid solution, and the method for extracting plasmids by using the alkali cracking equipment can obtain high-quality plasmids, and is simple and efficient.

Description

Alkaline lysis equipment and method for extracting plasmid using the same

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202310700226.1 filed on June 13, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The invention belongs to the fields of biotechnology and biomedicine, and particularly relates to an alkaline lysis device and a method for extracting plasmids using the device. Background Art

[0004] Plasmids are usually produced using Escherichia coli, and their purification production process uses alkaline lysis to extract plasmids. The alkaline lysis method generally includes three steps: the first step is resuspension of the bacteria, which is usually resuspended in a resuspension buffer by manual or magnetic stirring; the second step is alkaline lysis, which is usually performed by manual stirring to mix the resuspended bacteria with the alkaline lysis solution. After a certain period of time (usually 3 to 5 minutes), the cells are broken and release plasmids and other intracellular substances; the third step is neutralization, which is usually mixed by manual stirring after adding acid in the previous step to achieve the purpose of neutralization. At the same time, a white insoluble substance is produced through the replacement reaction, which carries denatured chromosomal DNA (deoxyribonucleic acid) and denatured proteins and cell fragments and precipitates after coagulation, while the plasmid DNA restores its natural configuration and dissolves in the clear liquid. However, the alkaline lysis method generally faces the following problems in large-scale production: 1. When adding alkali solution for lysis, it is usually completed in a stainless steel barrel or disposable liner bag. The manual stirring method will cause uneven mixing due to uneven force and uneven shear force, which will make the local pH (acidity and alkalinity) too high, resulting in irreversible deformation and destruction of the plasmid. 2. When the solution scale reaches hundreds of liters during the production process, the manual stirring method is time-consuming and the shear force is large, making it impossible to effectively scale up. 3. For the currently available lysis equipment, although mechanical lysis can be achieved, most of them are limited to laboratory scale and cannot be scaled up or produced under GMP (Good Manufacturing Practice) conditions.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to address the deficiencies of the prior art and provide an alkaline lysis device and a method for extracting plasmids using the device.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solution: an alkaline lysis equipment, comprising: a first pipeline, a second pipeline, a third pipeline, a lysis reactor and a neutralization and standing tank; the first pipeline is used to transport the bacterial suspension; the second pipeline is used to transport the alkaline lysis liquid; the third pipeline is used to transport the acid; the lysis reactor is used as a reaction container for alkaline lysis cells; the neutralization and standing tank is used to collect the neutralized liquid and perform static incubation; the first pipeline and the second pipeline are connected to the inlet end of the lysis reactor after intersecting; the third pipeline and the outlet end of the lysis reactor are connected to the inlet end of the neutralization and standing tank after intersecting.

[0008] In a specific embodiment, the alkaline lysis equipment includes: a first static mixer and a second static mixer; the first static mixer is arranged on the pipeline connected to the inlet end of the lysis reactor after the first pipeline and the second pipeline intersect, and is used to mix the material liquid in the corresponding pipeline; the second static mixer is arranged on the pipeline connected to the inlet end of the neutralization and static tank after the third pipeline and the outlet end of the lysis reactor intersect, and is used to mix the material liquid in the corresponding pipeline.

[0009] In a specific embodiment, the second pipeline includes: an alkali solution delivery pipeline, a surfactant delivery pipeline and an alkaline lysis liquid pipeline, the inlet end of the alkaline lysis liquid pipeline is connected to the alkali solution delivery pipeline and the surfactant delivery pipeline, and the outlet end of the alkaline lysis liquid pipeline intersects with the outlet end of the first pipeline; or an alkaline lysis liquid pipeline, the outlet end of the alkaline lysis liquid pipeline intersects with the outlet end of the first pipeline.

[0010] In a specific embodiment, the first pipeline is provided with a first valve, a first pump body and a first detection device, and the first detection device includes a first flow meter and a first pressure detector.

[0011] In a specific embodiment, the first pipeline connects the first purified water inlet and the second purified water inlet.

[0012] In a specific embodiment, a second valve, a second pump body and a second detection device are provided on the second pipeline, and the second detection device includes a second flow meter and a second pressure detector.

[0013] In a specific embodiment, the second pipeline is connected to the third purified water inlet.

[0014] In a specific embodiment, the third pipeline is provided with a third valve, a third pump body and a third detection device, and the third detection device includes a third flow meter and a third pressure detector.

[0015] In a specific embodiment, the third pipeline is connected to the fourth purified water inlet.

[0016] In a specific embodiment, the alkaline lysis equipment further includes a cleaning pipeline, which is connected to the lysis reactor, the neutralization and static tank, the first mixer, the second mixer, and each pipeline of the alkaline lysis equipment.

[0017] In a specific embodiment, the cleaning pipeline is provided with a fourth valve, a fourth pump body and a fourth detection device, and the fourth detection device includes a third conductivity detector.

[0018] In a specific embodiment, a first spraying portion is provided on the top of the neutralization and static tank, and the first spraying portion is connected to the cleaning pipeline and to the No. 5 purified water inlet.

[0019] In a specific embodiment, an explosion ring and / or a stirring device is provided at the bottom of the neutralization and static tank.

[0020] In a specific embodiment, a jacket is provided on the outside of the neutralization and static tank, liquid is provided in the jacket, the jacket is connected to an electric heating circulation device, the electric heating circulation device is used to heat the liquid, and a temperature transmitter is provided inside the neutralization and static tank.

[0021] In a specific embodiment, the electric heating circulation device includes: a tank body, in which an electric heating part is provided, and the tank body is connected to the liquid in the jacket through a circulation loop.

[0022] In a specific embodiment, the solution in the circulation loop and the liquid in the jacket are both purified water, the circulation loop is connected to the sixth purified water port, and a fifth pump body is provided on the circulation loop between the bottom of the tank body and the bottom of the jacket.

[0023] In a specific embodiment, a discharge port is provided at the bottom of the neutralization and static tank, and the discharge port is connected to the first clarification filter through a fourth pipeline and a deep filtration pipeline.

[0024] In a specific embodiment, the first clarifying filter includes: a primary clarifying filter connected to the discharge port and a secondary clarifying filter connected to the primary clarifying filter, and a sixth pump body is provided on the fourth pipeline.

[0025] In a specific embodiment, the alkaline lysis equipment includes a water storage tank, the inlet end of the water storage tank is connected to the water supply system, and the water storage tank is connected to the first pipeline through the second purified water inlet.

[0026] In a specific embodiment, the water storage tank is provided with a high liquid level detector and a low liquid level detector.

[0027] In a specific embodiment, a second spray portion is provided at the top of the water storage tank, and the bottom of the water storage tank is connected to the first waste pipe.

[0028] In a specific embodiment, the water supply system is connected to the No. 6 purified water inlet and the No. 7 purified water inlet through a fifth pipeline. The No. 7 purified water inlet is used to transport the purified water of the water supply system to the No. 1 purified water inlet, the No. 3 purified water inlet, the No. 4 purified water inlet, the No. 5 purified water inlet and the No. 8 purified water inlet. The No. 8 purified water inlet is connected to the second spray part.

[0029] In a specific embodiment, a seventh pump body and a fifth flow meter are provided on the pipeline connecting the No. 7 purified water inlet with the No. 1 purified water inlet, the No. 3 purified water inlet, the No. 4 purified water inlet, the No. 5 purified water inlet and the No. 8 purified water inlet.

[0030] In a specific embodiment, a first row of waste outlets is provided on the first pipeline, a second row of waste outlets is provided on the second pipeline, a third row of waste outlets is provided on the third pipeline, a fourth row of waste outlets is provided on the pipeline between the intersection of the third pipeline and the outlet end of the lysis reactor and the inlet end of the second static mixer, a fifth row of waste outlets is provided on the pipeline between the outlet end of the second static mixer and the neutralization and stasis tank, and a sixth row of waste outlets is provided at the bottom of the neutralization and stasis tank.

[0031] In a specific embodiment, the first waste outlet, the second waste outlet, the third waste outlet, the fourth waste outlet, the fifth waste outlet and the sixth waste outlet are all connected to the first waste outlet through the second waste outlet pipeline, and are discharged to the waste liquid collection end through the first waste outlet pipeline.

[0032] In a specific embodiment, a mixing valve is provided on the pipeline between the fifth waste outlet and the neutralization and stabilization tank.

[0033] In a specific embodiment, the interior of the neutralization and static tank is connected to instrument air via a sixth pipeline.

[0034] In a specific embodiment, the instrument air is connected to the first pipeline, the second pipeline, the third pipeline, the cleaning pipeline, the fourth pipeline, the circulation loop and the fifth pipeline.

[0035] In a specific embodiment, the first static mixer and the second static mixer each include a spiral mixer, a grid mixer and / or an X-type mixer.

[0036] In a specific embodiment, the lysis reactor comprises a disposable silicone tube, a pressure-resistant tube or a stainless steel tube.

[0037] A method for extracting plasmids using the alkaline lysis device comprises the following steps: obtaining a bacterial suspension and connecting the bacterial suspension to the first pipeline; obtaining an alkaline lysis solution and connecting the alkaline lysis solution to the second pipeline; obtaining an acid solution and connecting the acid solution to the third pipeline; setting the flow rate of the bacterial suspension in the first pipeline, the flow rate of the alkaline lysis solution in the second pipeline, and the flow rate of the acid solution in the third pipeline according to the flow ratio of the bacterial suspension, the alkaline lysis solution, and the acid solution; starting the lysis, and flowing the bacterial suspension in the first pipeline at the set flow rate. The alkaline lysis solution in the second pipeline is transported to the lysis reactor at a set flow rate for continuous flow lysis, and the acid solution in the third pipeline is transported to the neutralization and static tank together with the lysed feed liquid at a set flow rate for neutralization and static incubation for a certain time; the incubated feed liquid is clarified, filtered and concentrated, an impurity removal reagent is added, and the feed liquid is incubated at a specific temperature for a certain time and centrifuged, and the supernatant of the centrifugation is filtered to obtain a clarified concentrated feed liquid; the clarified concentrated feed liquid is loaded onto a molecular sieve chromatography column, and the first elution peak is collected to obtain the target plasmid.

[0038] In a specific embodiment, the step of obtaining the alkaline lysis solution includes: mixing the alkaline solution and the surfactant in a volume ratio of 1:1, thereby obtaining the alkaline lysis solution.

[0039] In a specific embodiment, the alkali solution contains 0.1 to 1 mol of sodium hydroxide, and the surfactant contains 1% to 5% of sodium lauryl sulfate.

[0040] In a specific embodiment, the flow ratio of the bacterial suspension, the alkaline lysis solution and the acid solution is (0.7-2):2:1.5.

[0041] In a specific embodiment, the flow rate of the bacterial suspension in the first pipeline is 21 to 45 liters / hour, the flow rate of the alkaline lysis solution in the second pipeline is 60 to 90 liters / hour, and the flow rate of the acid in the third pipeline is 45 to 67.5 liters / hour.

[0042] In a specific embodiment, the volume of the lysis reactor is 1.5 to 7.5 liters, and the lysis time of the lysis reactor is 1 to 5 minutes.

[0043] In a specific embodiment, the static incubation time is 0.5 to 12 hours.

[0044] In a specific embodiment, the incubated feed liquid is clarified, filtered and concentrated, an impurity removal agent is added, the feed liquid is incubated at a specific temperature for a certain time and centrifuged, and the centrifugal supernatant is filtered to obtain a clarified concentrated feed liquid. The steps include: conveying the incubated feed liquid to a first clarifying filter for clarification filtration, and collecting the clarified filtered feed liquid; concentrating the collected clarified filtered feed liquid, adding an impurity removal agent, the feed liquid is incubated at a specific temperature for a certain time and centrifuged, and the centrifugal supernatant is filtered through a second clarifying filter to obtain a clarified concentrated feed liquid.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] 1. The alkaline lysis equipment of the present invention can achieve online mixing and continuous flow lysis of bacterial suspension, alkaline lysis solution and acid solution in a certain proportion, and can obtain high-quality plasmids. It is simple, efficient, stable and reliable.

[0047] 2. The alkaline lysis equipment of the present invention is provided with a first static mixer and a second static mixer. The first static mixer can be used to mix the bacterial suspension and the alkaline lysis liquid in a certain proportion and then enter the lysis reactor for alkaline cell lysis reaction, thereby improving the efficiency, stability and reliability of the alkaline lysis reaction. The second static mixer can be used to mix the acid solution and the lysed liquid in a certain proportion and then undergo a neutralization reaction and transport it to a neutralization static tank for neutralization static incubation, thereby improving the efficiency, stability and reliability of the neutralization reaction.

[0048] 3. The alkaline lysis equipment of the present invention is provided with a cleaning pipeline, which can automatically clean the lysis reactor, neutralization and static tank, first mixer, second mixer and each pipeline of the alkaline lysis equipment with cleaning liquid as needed, which is fast, efficient, easy to use and low cost.

[0049] 4. The water supply system of the alkaline lysis equipment of the present invention is connected to the sixth purified water inlet, the seventh purified water inlet and the water storage tank through the fifth pipeline, which can easily provide water to multiple purified water inlets and water storage tanks.

[0050] 5. The alkaline cracking equipment of the present invention is provided with multiple waste outlets, which can facilitate the discharge of waste liquid, prevent environmental pollution, and have good safety.

[0051] 6. The alkaline cracking equipment of the present invention is connected to the instrument air, which can facilitate the discharge of liquid and the drying of the equipment by introducing compressed air.

[0052] 7. The alkaline cracking equipment of the present invention has a simple structure, is easy to use and has a wide range of applications.

[0053] 8. The method of extracting plasmids using alkaline lysis equipment of the present invention can obtain high-quality plasmids and is simple and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG1 shows a schematic structural diagram of an alkaline lysis device according to a specific embodiment of the present invention;

[0055] FIG2 shows a schematic structural diagram of a specific embodiment of the connection between the electric heating circulation device and the jacket of the alkaline lysis equipment of the present invention;

[0056] FIG3 shows a schematic structural diagram of a specific embodiment of the water supply system of the alkaline lysis equipment of the present invention connected to the sixth purified water inlet, the seventh purified water inlet and the water storage tank via a fifth pipeline;

[0057] FIG4 shows a schematic structural diagram of a specific embodiment of a cell lysis reactor of an alkaline lysis device of the present invention;

[0058] FIG5 shows a schematic structural diagram of a specific embodiment of the alkaline lysis equipment of the present invention in which the first static mixer and the second static mixer are both spiral mixers;

[0059] FIG6 shows a schematic structural diagram of a specific embodiment of the alkaline lysis equipment of the present invention in which the first static mixer and the second static mixer are both grid-type mixers;

[0060] FIG7 shows a schematic structural diagram of a specific embodiment of the alkaline lysis equipment of the present invention in which both the first static mixer and the second static mixer are X-type mixers;

[0061] FIG8 shows a schematic flow chart of a specific embodiment of the present invention for extracting plasmids using an alkaline lysis device.

[0062] Among them, 1-first pipeline; 11-first pump body; 12-first detection device; 2-second pipeline; 21-second pump body; 22-second detection device; 3-third pipeline; 31-third pump body; 32-third detection device; 4-lysis reactor; 5-neutralization and static tank; 51-first spray part; 52-explosion ring; 53-jacket; 54-temperature transmitter; 55-discharge port; 56-fourth pipeline; 57-deep filtration pipeline; 58-sixth pump body; 6-first static mixer; 7- -Second static mixer; 8-cleaning pipeline; 81-fourth pump body; 9-electric heating circulation device; 91-tank body; 92-electric heating part; 93-circulation loop; 94-fifth pump body; 95-liquid level gauge; 10-water storage tank; 101-high liquid level detector; 102-low liquid level detector; 103-second spray part; 13-first waste pipe; 14-fifth pipe; 15-seventh pump body; 16-fifth flowmeter; 17-second waste pipe; 18-mixing valve; 19-sixth pipe. DETAILED DESCRIPTION

[0063] The present invention will be further described below with reference to the embodiments shown in the accompanying drawings.

[0064] The directional terms used in the present invention, such as "inlet," "outlet," "inside," "outside," "top," and "bottom," are merely references to the accompanying drawings. Therefore, the directional terms used are for the purpose of illustrating and understanding the present invention, and are not intended to limit the present invention.

[0065] As shown in FIG1 , the alkaline lysis equipment of the present invention comprises: a first pipeline 1, a second pipeline 2, a third pipeline 3, a cell lysis reactor 4 and a neutralization and static tank 5.

[0066] The first pipeline 1 is used to transport the bacterial suspension.

[0067] The second pipeline 2 is used to transport the alkaline lysis solution.

[0068] The third pipeline 3 is used to transport acid solution.

[0069] The cell lysis reactor 4 is used as a reaction container for alkaline cell lysis.

[0070] The neutralization and static tank 5 is used to collect the neutralized liquid and perform static incubation.

[0071] The first pipeline 1 and the second pipeline 2 intersect and are connected to the inlet end of the lysis reactor 4. The third pipeline 3 and the outlet end of the lysis reactor 4 intersect and are connected to the inlet end of the neutralization and stabilization tank 5.

[0072] The bacterial suspension in the first pipeline 1 and the alkaline lysis solution in the second pipeline 2 are transported to the lysis reactor 4 at a set flow rate for continuous flow lysis. The acid solution in the third pipeline 3 is transported to the neutralization and static incubation tank 5 at a set flow rate together with the lysed liquid for neutralization and static incubation for a certain period of time. The device can achieve online mixing and continuous flow lysis of the bacterial suspension, alkaline lysis solution and acid solution in a fixed proportion, which is simple, efficient, stable and reliable.

[0073] In a specific embodiment, as shown in FIG1 , the alkaline lysis equipment includes: a first static mixer 6 and a second static mixer 7.

[0074] The first static mixer 6 is provided on the pipeline connected to the inlet end of the lysis reactor 4 after the first pipeline 1 and the second pipeline 2 intersect, and is used for mixing the feed liquids in the corresponding pipelines.

[0075] The second static mixer 7 is arranged on the pipeline connected to the inlet end of the neutralization and static tank 5 after the outlet end of the third pipeline 3 and the lysis reactor 4 intersect, and is used to mix the feed liquid in the corresponding pipeline.

[0076] Among them, the first static mixer 6 can be used to mix the bacterial suspension and the alkaline lysis solution in a certain proportion and then enter the cell lysis reactor 4 for alkaline cell lysis reaction, thereby improving the efficiency, stability and reliability of the alkaline lysis reaction. The second static mixer 7 can be used to mix the acid solution and the lysed liquid in a certain proportion and then perform a neutralization reaction and transport it to the neutralization static incubation tank 5 for neutralization static incubation, thereby improving the efficiency, stability and reliability of the neutralization reaction.

[0077] In a specific embodiment, the second pipeline 2 includes: an alkali solution delivery pipeline, a surfactant delivery pipeline, and an alkaline lysis liquid pipeline. The inlet end of the alkaline lysis liquid pipeline is connected to the alkali solution delivery pipeline and the surfactant delivery pipeline, and the outlet end of the alkaline lysis liquid pipeline intersects with the outlet end of the first pipeline 1. Among them, the alkali solution delivery pipeline is used to transport alkali solution, and the surfactant delivery pipeline is used to transport surfactant. The alkali solution delivery pipeline and the surfactant delivery pipeline are connected to the inlet end of the alkaline lysis liquid pipeline, which can facilitate the alkali solution and the surfactant to be mixed evenly according to the target volume ratio, and the alkaline lysis liquid that reaches the target ratio is output through the alkaline lysis liquid pipeline, which is convenient and efficient, and has good stability and reliability. In another specific embodiment, the second pipeline 2 can be a pipeline for transporting alkaline lysis liquid, and the outlet end of the alkaline lysis liquid pipeline intersects with the outlet end of the first pipeline 1. Among them, the alkaline lysis liquid pipeline is used to transport the alkaline lysis liquid formed by mixing the alkali solution and the surfactant in advance.

[0078] In a specific embodiment, a first valve and a first pump body 11 are provided on the first pipeline 1. The first valve can be used to open and / or close the first pipeline 1. The first pump body 11 can be used to control the flow rate of the bacterial suspension in the first pipeline 1.

[0079] In a specific embodiment, as shown in FIG1 , a first detection device 12 is provided on the first pipeline 1. The first detection device 12 includes a first flowmeter (FE) and a first pressure detector (pressure sensor PT). The first flowmeter can be used to conveniently detect the flow rate of the bacterial suspension in the first pipeline 1. The first pressure detector can be used to conveniently detect the pressure value of the bacterial suspension in the first pipeline 1, and has a simple structure and is easy to use.

[0080] In a specific embodiment, as shown in FIG1 , a second valve and a second pump body 21 are provided on the second pipeline 2. The second valve can be used to open and / or close the second pipeline 2. The second pump body 21 can be used to control the flow rate of the alkaline lysis solution in the second pipeline 2.

[0081] In a specific embodiment, as shown in FIG1 , a second detection device 22 is provided on the second pipeline 2. The second detection device 22 includes a second flow meter and a second pressure detector (pressure sensor PT). The second flow meter can be used to easily detect the flow rate of the alkaline lysis solution in the second pipeline 2. The second pressure detector can be used to easily detect the pressure value of the alkaline lysis solution in the second pipeline 2. In another specific embodiment, as shown in FIG1 , the second detection device 22 also includes a first conductivity detector (conductivity meter CT) and / or a first pH meter (pH). The first conductivity detector can be used to easily detect the conductivity of the alkaline lysis solution in the second pipeline 2. The first pH meter can be used to easily detect the pH value of the alkaline lysis solution in the second pipeline 2.

[0082] In a specific embodiment, as shown in FIG1 , a third valve and a third pump body 31 are provided on the third pipeline 3. The third valve can be used to open and / or close the third pipeline 3. The third pump body 31 can be used to control the flow rate of the acid solution in the third pipeline 3.

[0083] In one specific embodiment, as shown in FIG1 , a third detection device 32 is provided on the third pipeline 3. The third detection device 32 includes a third flowmeter and a third pressure detector. The third flowmeter can be used to easily detect the flow rate of the acid solution in the third pipeline 3. The third pressure detector can be used to easily detect the pressure value of the acid solution in the third pipeline 3. In another specific embodiment, as shown in FIG1 , the third detection device 32 also includes a second conductivity detector and / or a second pH meter. The second conductivity detector can be used to easily detect the conductivity of the acid solution in the third pipeline 3. The second pH meter can be used to easily detect the pH value of the acid solution in the third pipeline 3.

[0084] In a specific embodiment, as shown in Figure 1, the alkaline lysis equipment further includes a cleaning line 8 (CIP). The cleaning line 8 is connected to the cell lysis reactor 4, the neutralization and static tank 5, the first mixer 6, the second mixer 7 and each pipeline of the alkaline lysis equipment. Among them, the cleaning line 8 is connected to the interior of the neutralization and static tank 5 through the first pipeline 1, and can clean the first pipeline 1, the pipeline connecting the first pipeline 1 and the neutralization and static tank 5, and the interior of the neutralization and static tank 5. At the same time, the interior of the first mixer 6, the second mixer 7 and the cell lysis reactor 4 can also be cleaned. The cleaning line 8 can also use the neutralization and static tank 5 as the circulation center and clean the second pipeline 2, the third pipeline 3 and / or other pipelines by opening the corresponding valves of the second pipeline 2, the third pipeline 3 and / or other pipelines. The cleaning line 8 can be automatically cleaned as needed.

[0085] In a specific embodiment, as shown in Figure 1, the cleaning pipeline 8 includes an alkaline cleaning pipeline, which is fast and efficient, has significant effects, is easy to use, and has low cost. The cleaning pipeline 8 can also transport other cleaning liquids.

[0086] In a specific embodiment, as shown in FIG1 , a fourth valve and a fourth pump body 81 are provided on the cleaning pipeline 8. The fourth valve can be used to open and / or close the cleaning pipeline 8. The fourth pump body 81 can be used to control the flow rate of the cleaning liquid in the cleaning pipeline 8.

[0087] In a specific embodiment, as shown in FIG1 , a fourth detection device is provided on the cleaning pipeline 8 . The fourth detection device includes a third conductivity detector. The third conductivity detector can be used to easily detect the conductivity of the cleaning fluid in the cleaning pipeline 8 .

[0088] In a specific embodiment, as shown in Figures 1-3, a first spray unit (SB) 51 is provided at the top of the neutralization and static tank 5. The first spray unit 51 is connected to the cleaning pipeline 8 and facilitates alkaline cleaning of the interior of the neutralization and static tank 5 using alkaline solution in the cleaning pipeline 8. For example, the first spray unit 51 includes a spray ball, which has a good alkaline cleaning effect, a simple structure, and is easy to use.

[0089] In a specific embodiment, as shown in Figures 1 to 3, an explosion ring (ER) 52 and / or a stirring device is provided at the bottom of the neutralization and static tank 5 to facilitate the floating of the precipitate in the liquid in the neutralization and static tank 5.

[0090] In a specific embodiment, as shown in Figure 2, the outside of the neutralization and static tank 5 is provided with a jacket 53, and liquid is provided in the jacket 53. The jacket 53 is connected to the electric heating circulation device 9, which is used to heat the liquid, and the inside of the neutralization and static tank 5 is provided with a temperature transmitter (TT) 54. Among them, the electric heating circulation device 9 can circulate and heat the liquid in the jacket 53, and then the cleaning liquid (alkaline solution) in the neutralization and static tank 5 can be heated, thereby improving the cleaning effect during alkaline solution cleaning. The temperature transmitter 54 can be used to detect the temperature in the neutralization and static tank 5 in real time, and feed back to the circulating heating process of the electric heating circulation device 9. The temperature of the cleaning liquid in the cleaning pipeline 8 is 30 to 60°C (degrees Celsius), preferably 50°C.

[0091] In a specific embodiment, as shown in Figure 2, the electric heating circulation device 9 includes: a tank body 91, an electric heating part 92 is provided in the tank body 91, and the tank body 91 is connected to the liquid in the jacket 53 through a circulation loop 93, which can facilitate the circulation heating of the liquid in the jacket 53, and has a simple structure and is easy to use.

[0092] In a specific embodiment, as shown in FIG2 , the solution in the circulation loop 93 and the liquid in the jacket 53 are both purified water, which can be easily heated and circulated and has good economic efficiency.

[0093] In a specific embodiment, as shown in FIG2 , a fifth pump body 94 is provided on the circulation loop 93 between the bottom of the tank body 91 and the bottom of the jacket 53 , which can facilitate control of the flow rate of the solution in the circulation loop 93 and facilitate circulation of the solution in the circulation loop 93 .

[0094] In a specific embodiment, as shown in FIG2 , a liquid level gauge (LS) 95 is provided on the tank body 91 to facilitate measurement of the liquid level in the tank body 91 .

[0095] In a specific embodiment, as shown in FIG2 , a discharge port 55 is provided at the bottom of the neutralization and stabilization tank 5 . The discharge port 55 is connected to the first clarification filter via a fourth pipeline 56 , thereby facilitating the entry of the post-incubation liquid discharged from the neutralization and stabilization tank 5 into the first clarification filter for clarification filtration. For example, the fourth pipeline 56 is connected to the first clarification filter via a depth filtration pipeline 57 .

[0096] In a specific embodiment, the first clarifying filter includes: a primary clarifying filter connected to the discharge port 55 and a secondary clarifying filter connected to the primary clarifying filter, which can improve the clarifying and filtering effect.

[0097] In a specific embodiment, the filtration accuracy of the first-stage clarification filter is 50 μm (micrometers), and the filtration accuracy of the second-stage clarification filter is 0.6 μm.

[0098] In a specific embodiment, as shown in FIG2 , a sixth pump body 58 is provided on the fourth pipeline 56 , which can facilitate the delivery of the incubated liquid through the deep filtration pipeline 57 to the first clarification filter for clarification filtration and control the flow rate of the incubated liquid.

[0099] In a specific embodiment, as shown in Figure 3, the alkaline lysis equipment includes a water storage tank 10, the inlet of which is connected to the water supply system (industrial water PW). The water storage tank 10 is used to temporarily store water for the equipment and can provide a stable water source.

[0100] In a specific embodiment, as shown in FIG3 , the water tank 10 is provided with a high liquid level detector 101 and a low liquid level detector 102 , which can be used to control the water level in the water tank 10 and realize automatic replenishment of purified water in the water tank 10 .

[0101] In a specific embodiment, as shown in FIG3 , a second spray portion 103 is provided on the top of the water tank 10 to facilitate flushing and cleaning the interior of the water tank 10. For example, the second spray portion 103 includes a spray ball, which has a good flushing and cleaning effect, a simple structure, and is easy to use.

[0102] In a specific embodiment, as shown in FIG3 , the bottom of the water storage tank 10 is connected to the first waste pipe 13 , which facilitates discharging the wastewater in the water storage tank 10 to the waste liquid collection end through the first waste pipe 13 .

[0103] In a specific embodiment, as shown in Figures 1 and 3 , the first pipeline 1 connects to the first purified water inlet ① and the second purified water inlet ②. Specifically, the first purified water inlet ① is connected to the water supply system and is used to flush and clean the first pipeline 1. The second purified water inlet ② is connected to the water storage tank 10. The purified water flowing out of the second purified water inlet ② can be used to adjust the flow rate of the bacterial suspension in the first pipeline 1 to the target value and maintain the flow rate of the bacterial suspension in the first pipeline 1 in a balanced state. Furthermore, after the continuous flow lysis process of the device is completed, the purified water flowing out of the second purified water inlet ② can be used to push out the bacterial suspension in the first pipeline 1.

[0104] In a specific embodiment, as shown in Figures 1 and 3, the second pipeline 2 is connected to the third purified water port ③. The third purified water port ③ is connected to the water supply system for flushing and cleaning the second pipeline 2.

[0105] In a specific embodiment, as shown in Figures 1 and 3, the third pipeline 3 is connected to the fourth purified water port ④. The fourth purified water port ④ is connected to the water supply system for flushing and cleaning the third pipeline 3.

[0106] In a specific embodiment, as shown in Figures 1 and 3, the first spraying part 51 is connected to the fifth purified water port ⑤. The fifth purified water port ⑤ is connected to the water supply system for flushing and cleaning the neutralization and static tank 5.

[0107] In a specific embodiment, as shown in Figure 3, the water supply system is connected to purified water inlet No. 6 ⑥ and purified water inlet No. 7 ⑦ via a fifth pipeline 14. Purified water inlet No. 7 ⑦ is used to deliver purified water from the water supply system to purified water inlet No. 1 ①, purified water inlet No. 3 ③, purified water inlet No. 4 ④, purified water inlet No. 5 ⑤, and purified water inlet No. 8 ⑧. Purified water inlet No. 8 ⑧ is connected to the second spray section 103, facilitating flushing and cleaning of the water storage tank 10. Purified water inlet No. 6 ⑥ is connected to the circulation loop 93, facilitating the circulation of solution within the circulation loop 93.

[0108] In one specific embodiment, as shown in Figure 3 , a seventh pump 15 and a fifth flowmeter 16 are installed in the pipeline connecting the No. 7 purified water inlet ⑦ to the No. 1 purified water inlet ①, No. 3 purified water inlet ③, No. 4 purified water inlet ④, No. 5 purified water inlet ⑤, and No. 8 purified water inlet ⑧. The seventh pump 15 facilitates the delivery of purified water from the water supply system to the No. 1 purified water inlet ①, No. 3 purified water inlet ③, No. 4 purified water inlet ④, No. 5 purified water inlet ⑤, and No. 8 purified water inlet ⑧. The fifth flowmeter 16 facilitates the detection of the flow rate of purified water in the corresponding pipelines.

[0109] In a specific embodiment, as shown in FIG1 and FIG2, a first waste outlet is provided on the first pipeline 1. The second waste outlet is provided on the second pipeline 2. The waste liquid can be discharged as needed. The third waste outlet is provided on the third pipeline 3. A fourth waste outlet is provided on the pipeline between the junction of the third pipeline 3 and the outlet end of the lysis reactor 4 and the inlet end of the second static mixer 7. A fifth waste outlet is provided on the pipeline between the outlet end of the second static mixer 7 and the neutralization and static tank 5. The waste liquid can be discharged as needed. The bottom of the neutralization and settling tank 5 is provided with a sixth waste outlet. Able to discharge waste liquid as needed.

[0110] In a specific embodiment, as shown in FIG1 , the first row of waste outlets Second row of waste outlet Third row waste port Fourth row of waste outlet Fifth row waste outlet And the sixth row of waste outlet Both are connected to the first waste pipe 13 through the second waste pipe 17 and discharged to the waste liquid collection end through the first waste pipe 13, which can prevent environmental pollution and has good safety.

[0111] In a specific embodiment, the fifth row of waste outlet A mixing valve 18 is provided on the pipeline between the neutralization and static tank 5. When the flow rate of the bacterial suspension in the first pipeline 1, the flow rate of the alkaline lysis solution in the second pipeline 2, and the flow rate of the acid solution in the third pipeline 3 all reach the set value, the mixing valve 18 opens to start continuous flow lysis, which has good controllability.

[0112] In a specific embodiment, as shown in Figures 1 to 3, the interior of the neutralization and static tank 5 is connected to the instrument air (IA) through the sixth pipeline 19, which can be used to neutralize the neutralized liquid in the static tank 5. Compressed air is introduced through the sixth pipeline 19 to promote the floating of the sediment in the liquid, and the liquid can be completely discharged and the neutralization and static tank 5 can be blown dry by introducing compressed air.

[0113] In a specific embodiment, the instrument air is connected to the first pipeline 1, the second pipeline 2, the third pipeline 3, the cleaning pipeline 4, the pipeline between the first pipeline 1 and the second pipeline 2 and the neutralization and static tank 5, the fourth pipeline 56, the circulation loop 93 and the fifth pipeline 14, and can achieve the function of draining the liquid and drying the first pipeline 1, the second pipeline 2, the third pipeline 3, the cleaning pipeline 4, the pipeline between the first pipeline 1 and the second pipeline 2 and the neutralization and static tank 5, the fourth pipeline 56, the circulation loop 93 and the fifth pipeline 14 by introducing compressed air.

[0114] In a specific embodiment, as shown in FIG. 1 and FIG. 4 , the cell lysis reactor 4 includes a disposable silicone tube, a pressure-resistant tube or a stainless steel tube, which has a good cell lysis effect.

[0115] In a specific embodiment, as shown in Figure 1 and Figures 5 to 7, the first static mixer 6 and the second static mixer 7 both include a spiral mixer, a grid mixer and / or an X-type mixer, which can improve the uniform mixing effect of the liquid in the first static mixer 6 and the second static mixer 7 without damaging the plasmids.

[0116] In a specific embodiment, as shown in Figures 1 to 3, the first pump body 11, the second pump body 21, the third pump body 31, the fourth pump body 81, the fifth pump body 94, the sixth pump body 58 and the seventh pump body 15 are all diaphragm pumps, which have good pumping effect, are explosion-proof, have good safety, and are simple in structure and easy to use.

[0117] The alkaline lysis equipment of the present invention can achieve online mixing of a bacterial suspension, an alkaline lysis solution, and an acid solution in a predetermined proportion, as well as continuous flow lysis, with good stability and reliability. Furthermore, the cleaning line 8 can be automatically cleaned with good controllability. Furthermore, the line can be dried and stored using instrument air.

[0118] On the basis of the above embodiments, as shown in FIG1 and FIG8 , the present invention further proposes a method for extracting plasmids using the alkaline lysis device, comprising the following steps:

[0119] (1) Obtain a bacterial suspension and connect the bacterial suspension to the first pipeline 1. The bacterial suspension is obtained by resuspending the bacterial cells, and the bacterial suspension is transported by the first pipeline 1.

[0120] (2) Obtaining an alkaline lysis solution and connecting the alkaline lysis solution to the second pipeline 2. The alkaline lysis solution can be obtained by mixing an alkaline solution and a surfactant in a specific volume ratio. The alkaline lysis solution is then transported using the second pipeline 2.

[0121] (3) The acid solution is connected to the third pipeline 3. The acid solution serves as a neutralizing liquid and the third pipeline 3 is used for acid solution transportation.

[0122] (4) The flow rate of the bacterial suspension in the first pipeline 1, the flow rate of the alkaline lysis solution in the second pipeline 2, and the flow rate of the acid solution in the third pipeline 3 are set according to the flow ratio of the bacterial suspension, the alkaline lysis solution, and the acid solution. By controlling the flow rate of the bacterial suspension in the first pipeline 1, the flow rate of the alkaline lysis solution in the second pipeline 2, and the flow rate of the acid solution in the third pipeline 3, a fixed-proportion online mixing of the bacterial suspension, the alkaline lysis solution, and the acid solution can be achieved.

[0123] (5) Start lysis, transport the bacterial suspension in the first pipeline 1 and the alkaline lysis solution in the second pipeline 2 at a set flow rate to the lysis reactor 4 for continuous flow lysis, and transport the acid solution in the third pipeline 3 at a set flow rate together with the lysed liquid to the neutralization and static incubation tank 5 for neutralization and static incubation for a certain period of time. By controlling the flow rate of the bacterial suspension in the first pipeline 1 and the flow rate of the alkaline lysis solution in the second pipeline 2, the bacterial suspension and the alkaline lysis solution in the lysis reactor 4 can be fully mixed, thereby improving the online continuous flow lysis effect, and by controlling the flow rate of the acid solution in the third pipeline 3, the online neutralization reaction effect can be improved.

[0124] (6) The incubated liquid is transported to a first clarifying filter for clarification and filtration, and the clarified and filtered liquid is collected. The clarification and filtration of the incubated liquid facilitates the collection of the target plasmid.

[0125] (7) The collected clarified and filtered liquid is concentrated, an impurity removal agent is added, and the mixture is incubated at a specific temperature for a certain period of time and centrifuged. The supernatant of the centrifugation is filtered through a second clarifying filter to obtain a clarified concentrated liquid. The concentration of the clarified and filtered liquid further facilitates the collection of the target plasmid.

[0126] (8) The clarified concentrated liquid is loaded onto a molecular sieve chromatography column and the first elution peak is collected to obtain the target plasmid. The first elution peak is the target plasmid peak.

[0127] In a specific embodiment, the step of obtaining a bacterial suspension includes: weighing Escherichia coli cells, adding a bacterial resuspension buffer according to a certain weight-to-volume ratio, and stirring with a stirrer for a certain period of time to obtain a bacterial suspension. The plasmid content of the Escherichia coli cells should reach a certain amount.

[0128] In a specific embodiment, the weight-to-volume ratio of E. coli cells to cell resuspension buffer is 1:5 to 1:20, and the stirring time of the stirrer is 0.5 to 2 hours, which can facilitate obtaining a uniform bacterial suspension. Preferably, the stirrer is a magnetic stirrer for better stirring effect.

[0129] In a specific embodiment, the plasmid content of Escherichia coli is 1 to 10 mg / g, which can better meet the needs.

[0130] In a specific embodiment, the formula of the bacterial resuspension buffer is 50 mmol glucose, 25 mmol tris(hydroxymethyl)aminomethane and 10 mmol ethylenediaminetetraacetic acid at pH 8.0, which can better meet the needs.

[0131] In a specific embodiment, the step of obtaining the alkaline lysis solution includes: mixing the alkaline solution and the surfactant in a volume ratio of 1:1, thereby obtaining the alkaline lysis solution. The alkaline lysis solution is of high quality.

[0132] In a specific embodiment, the alkali solution formula is 0.1 to 1 mole of sodium hydroxide, and the surfactant formula is 1% to 5% of sodium lauryl sulfate, which can facilitate the production of high-quality alkaline lysis solution.

[0133] In a specific embodiment, the acid solution formula is 3 moles of potassium acetate and 2 moles of acetic acid, which can better meet the needs.

[0134] In a specific embodiment, the flow ratio of the bacterial suspension, alkaline lysis solution and acid solution is (0.7~2):2:1.5, which can better achieve a fixed proportion of online mixing of the bacterial suspension, alkaline lysis solution and acid solution, thereby improving the efficiency of the alkaline lysis reaction and the neutralization reaction.

[0135] In a specific embodiment, as shown in Figure 1, the flow rate of the bacterial suspension in the first pipeline 1 is 21 to 45 liters / hour, the flow rate of the alkaline lysis solution in the second pipeline 2 is 60 to 90 liters / hour, and the flow rate of the acid in the third pipeline 3 is 45 to 67.5 liters / hour, which can further better achieve a fixed proportion of online mixing of the bacterial suspension, alkaline lysis solution and acid, high alkaline lysis reaction efficiency, and high neutralization reaction efficiency.

[0136] In a specific embodiment, as shown in FIG1 and FIG4 , the volume of the lysis reactor 4 is 1.5 to 7.5 liters, and the lysis time of the lysis reactor 4 is 1 to 5 minutes, which can fully carry out the alkaline lysis reaction, thereby improving the alkaline lysis reaction effect.

[0137] In a specific embodiment, the static incubation time is 0.5 to 12 hours, which can facilitate the subsequent collection of the target plasmid.

[0138] In a specific embodiment, the collected clarified filtered liquid is concentrated, an impurity removal agent is added, and the liquid is incubated at a specific temperature for a certain time for centrifugation, and the supernatant of the centrifugation is filtered through a second clarifying filter to obtain a clarified concentrated liquid. The steps include: using a 5-100 kilodalton membrane package to concentrate the collected clarified filtered liquid, adding an equal volume of 4 molar ammonium sulfate and incubating at room temperature for 10-20 minutes for centrifugation, with a centrifugal force of 6000-10000 g acceleration and a centrifugation time of 10-20 minutes, and filtering the supernatant of the centrifugation through a second clarifying filter to obtain a clarified concentrated liquid, which can facilitate the subsequent collection of the target plasmid.

[0139] In a specific embodiment, for high-copy plasmids, the liquid after clarification and filtration is concentrated by more than or equal to 10 times and less than or equal to 50 times; for low-copy plasmids, the liquid after clarification and filtration is concentrated by more than or equal to 50 times and less than or equal to 100 times.

[0140] In a specific embodiment, the filtration accuracy of the second clarification filter is 0.2 to 0.65 microns, which can facilitate obtaining a clarified concentrated liquid.

[0141] Some specific examples are listed below

[0142] Example 1

[0143] S1. Weigh 808 g (grams) of Escherichia coli cells with a plasmid content of 4.21 mg / g (milligrams / gram), add cell resuspension buffer at a weight-to-volume ratio of 1:10 g / mL (grams / milliliter), the cell resuspension buffer formula is 50 mM (millimolar) Glucose (glucose), 25 mM Tris (trishydroxymethylaminomethane), and 10 mM EDTA (ethylenediaminetetraacetic acid), pH 8.0, use a magnetic stirrer to stir for 0.5 h (hour) to fully form a uniform bacterial suspension, and connect the obtained bacterial suspension to the first pipeline 1.

[0144] S2. Alkaline solution and surfactant are mixed in a volume ratio of 1:1 to obtain alkaline lysis solution, and the alkaline lysis solution is connected to the second pipeline 2. The alkaline solution formula is 400 mmol NaOH (sodium hydroxide) and the surfactant formula is 2% SDS (sodium dodecyl sulfate).

[0145] S3, connect the acid solution to the third pipeline 3. The acid solution formula is 3M (mole) CH3COOK (potassium acetate) and 2 moles CH3COOH (acetic acid).

[0146] S4. According to the flow ratio of bacterial suspension: alkaline lysis solution: acid solution of 1:2:1.5, the flow rate of the bacterial suspension in the first pipeline 1 is set to 30 L / h (liter / hour), the flow rate of the alkaline lysis solution in the second pipeline 2 is set to 60 L / hour, and the flow rate of the acid solution in the third pipeline 3 is set to 45 L / hour.

[0147] S5. Start lysis. The bacterial suspension in the first pipeline 1 and the alkaline lysis solution in the second pipeline 2 are transported to the lysis reactor 4 at a set flow rate for continuous flow lysis. The acid solution in the third pipeline 3 is transported to the neutralization and standing tank 5 at a set flow rate together with the lysed liquid. A neutralization reaction occurs after the acid solution and the lysed liquid come into contact. After that, the neutralization and standing incubation are carried out in the neutralization and standing tank 5 for 1 hour. The volume of the lysis reactor 4 is set to 1.5 liters, that is, the lysis time is 1 minute.

[0148] S6. The incubated liquid in the neutralization and static tank 5 is transferred to a first clarification filter through a discharge port 55 for clarification filtration, and the clarified and filtered liquid is collected. Discharge port 55 is connected to a 50-micron primary clarification filter and a 0.6-micron secondary clarification filter. The incubated liquid is filtered through the two-stage clarification filters and then collected into a disposable liner bag.

[0149] S7. Use a 100 kDa (kilodalton) membrane package to concentrate the clarified filtered liquid 22 times, add an equal volume of 4 M ammonium sulfate for incubation, incubate at room temperature for 20 min, and then centrifuge at 6000 g (gravity acceleration) for 20 min. Take the centrifugal supernatant and filter it through a 0.65 micron second clarification filter to obtain a clarified concentrated liquid.

[0150] S8. Load the clarified concentrated solution obtained in step S7 onto a molecular sieve chromatography column filled with Bestarose 6FF from Boglund. The loading flow rate is 60 cm / h (centimeter / hour), the loading volume is 0.23 CV (solution molar concentration multiplied by volume), the operating flow rate is 120 cm / h (centimeter / hour), and the first elution peak is collected.

[0151] Example 2

[0152] S1. Weigh 612 g of Escherichia coli cells with a plasmid content of 1.36 mg / g, add cell resuspension buffer at a weight-to-volume ratio of 1:10 g / ml, the cell resuspension buffer formula is 50 mM glucose, 25 mM tris(hydroxymethyl)aminomethane, and 10 mM ethylenediaminetetraacetic acid, pH 8.0, use a magnetic stirrer to stir for 0.5 h to fully form a uniform bacterial suspension, and connect the obtained bacterial suspension to the first pipeline 1.

[0153] S2. Alkaline solution and surfactant are mixed in a volume ratio of 1:1 to obtain alkaline lysis solution, and the alkaline lysis solution is connected to the second pipeline 2. The alkali solution formula is 400 mmol sodium hydroxide, and the surfactant formula is 2% sodium lauryl sulfate.

[0154] S3, connecting the acid solution to the third pipeline 3. The acid solution formula is 3 moles of potassium acetate and 2 moles of acetic acid.

[0155] S4. According to the flow ratio of bacterial suspension: alkaline lysis solution: acid solution of 1:2:1.5, the flow rate of the bacterial suspension in the first pipeline 1 is set to 30 L / h (liter / hour), the flow rate of the alkaline lysis solution in the second pipeline 2 is set to 60 L / hour, and the flow rate of the acid solution in the third pipeline 3 is set to 45 L / hour.

[0156] S5. Start lysis. The bacterial suspension in the first pipeline 1 and the alkaline lysis solution in the second pipeline 2 are transported to the lysis reactor 4 at a set flow rate for continuous flow lysis. The acid solution in the third pipeline 3 is transported to the neutralization and standing tank 5 at a set flow rate together with the lysed liquid. A neutralization reaction occurs after the acid solution and the lysed liquid come into contact. After that, the neutralization and standing incubation are carried out in the neutralization and standing tank 5 for 1 hour. The volume of the lysis reactor 4 is set to 1.5 liters, that is, the lysis time is 1 minute.

[0157] S6. The incubated liquid in the neutralization and static tank 5 is transferred to a first clarification filter through a discharge port 55 for clarification filtration, and the clarified and filtered liquid is collected. Discharge port 55 is connected to a 50-micron primary clarification filter and a 0.6-micron secondary clarification filter. The incubated liquid is filtered through the two-stage clarification filters and then collected into a disposable liner bag.

[0158] S7. Use a 100 kDa (kilodalton) membrane package to concentrate the clarified filtered liquid 66 times, add an equal volume of 4 M ammonium sulfate for incubation, incubate at room temperature for 20 min, and then centrifuge at 6000 g (gravity acceleration) for 20 min. Take the centrifugal supernatant and filter it through a 0.65 micron second clarification filter to obtain a clarified concentrated liquid.

[0159] S8. Load the clarified concentrated liquid obtained in step S7 onto a molecular sieve chromatography column filled with Bestarose 6FF from Boglund. The loading flow rate is 60 cm / h, the loading volume is 0.23 of the solution molar concentration multiplied by the volume, the running flow rate is 120 cm / h, and the first elution peak is collected.

[0160] The scope of protection of the present invention is not limited to the above-described embodiments. Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the scope and spirit of the present invention. If such modifications and variations fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. An alkaline lysis device, It is characterized in that include: A first pipeline (1), a second pipeline (2), a third pipeline (3), a lysis reactor (4) and a neutralization standing tank (5); wherein, The first pipeline (1) is used for conveying bacterial suspension; The second pipeline (2) is used for conveying alkaline lysis solution; The third pipeline (3) is used for conveying acid liquid; The cell lysis reactor (4) is used as a reaction container for alkaline cell lysis; The neutralization and static tank (5) is used to collect the neutralized liquid and perform static incubation; The first pipeline (1) and the second pipeline (2) are connected to the inlet end of the lysis reactor (4) after they intersect; the third pipeline (3) and the outlet end of the lysis reactor (4) are connected to the inlet end of the neutralization standing tank (5) after they intersect.

2. The alkaline lysis equipment according to claim 1, It is characterized in that The alkaline lysis equipment comprises: a first static mixer (6) and a second static mixer (7); The first static mixer (6) is arranged on the pipeline connected to the inlet end of the lysis reactor (4) after the first pipeline (1) and the second pipeline (2) meet, and is used to mix the feed liquids in the corresponding pipelines; The second static mixer (7) is arranged on the pipeline connected to the inlet end of the neutralization static tank (5) after the outlet end of the third pipeline (3) and the lysis reactor (4) intersect, and is used to mix the feed liquid in the corresponding pipeline.

3. The alkaline lysis equipment according to claim 1 or 2, It is characterized in that The second pipeline (2) comprises: an alkali solution delivery pipeline, a surfactant delivery pipeline and an alkali lysis solution pipeline, the inlet end of the alkali lysis solution pipeline is connected to the alkali solution delivery pipeline and the surfactant delivery pipeline, and the outlet end of the alkali lysis solution pipeline intersects with the outlet end of the first pipeline; or An alkaline lysis liquid pipeline, the outlet end of which intersects with the outlet end of the first pipeline.

4. The alkaline lysis equipment according to any one of claims 1 to 3, It is characterized in that The first pipeline (1) is provided with a first valve, a first pump body (11) and a first detection device (12), wherein the first detection device comprises a first flow meter and a first pressure detector.

5. The alkaline lysis equipment according to any one of claims 1 to 4, It is characterized in that The first pipeline (1) connects the first purified water inlet and the second purified water inlet.

6. The alkaline lysis equipment according to any one of claims 1 to 5, It is characterized in that The second pipeline (2) is provided with a second valve, a second pump body (21) and a second detection device (22), and the second detection device (22) comprises a second flow meter and a second pressure detector.

7. The alkaline lysis equipment according to any one of claims 1 to 6, It is characterized in that The second pipeline (2) is connected to the No. 3 purified water inlet.

8. The alkaline lysis equipment according to any one of claims 1 to 7, It is characterized in that The third pipeline (3) is provided with a third valve, a third pump body (31) and a third detection device (32), and the third detection device (32) comprises a third flow meter and a third pressure detector.

9. The alkaline lysis equipment according to any one of claims 1 to 8, It is characterized in that The third pipeline (3) is connected to the fourth purified water outlet.

10. The alkaline lysis equipment according to any one of claims 2 to 9, It is characterized in that The alkaline lysis equipment further comprises a cleaning pipeline (8), wherein the cleaning pipeline (8) is connected to the cell lysis reactor (4), the neutralization static tank (5), the first mixer (6), the second mixer (7) and the pipelines of the alkaline lysis equipment.

11. The alkaline lysis equipment according to claim 10, It is characterized in that The cleaning pipeline (8) is provided with a fourth valve, a fourth pump body (81) and a fourth detection device, and the fourth detection device includes a third conductivity detector.

12. The alkaline lysis equipment according to any one of claims 1 to 11, It is characterized in that A first spraying portion (51) is provided at the top of the neutralization and static tank (5), and the first spraying portion (51) is connected to the cleaning pipeline (8) and to the No. 5 purified water inlet. An explosion ring (52) and / or a stirring device is provided at the bottom of the neutralization and static tank (5).

13. The alkaline lysis equipment according to claim 12, It is characterized in that The neutralization and static tank (5) is provided with a jacket (53) on the outside, and liquid is provided in the jacket (53). The jacket (53) is connected to an electric heating circulation device (9), and the electric heating circulation device (9) is used to heat the liquid. The neutralization and static tank (5) is provided with a temperature transmitter (54) on the inside.

14. The alkaline lysis equipment according to claim 13, It is characterized in that The electric heating circulation device (9) comprises: a tank body (91), an electric heating part (92) is arranged in the tank body (91), and the tank body (91) is connected to the liquid in the jacket (53) through a circulation loop (93).

15. The alkaline lysis equipment according to claim 14, It is characterized in that The solution in the circulation loop (93) and the liquid in the jacket (53) are both purified water. The circulation loop (93) is connected to the sixth purified water port. A fifth pump body (94) is provided on the circulation loop (93) between the bottom of the tank body (91) and the bottom of the jacket (53).

16. The alkaline lysis equipment according to any one of claims 1 to 15, It is characterized in that The bottom of the neutralization and static tank (5) is provided with a discharge port (55), and the discharge port (55) is connected to the first clarifying filter through a fourth pipeline (56) and a deep filtration pipeline (57).

17. The alkaline lysis device according to claim 16, It is characterized in that The first clarifying filter comprises: a primary clarifying filter connected to the discharge port (55) and a secondary clarifying filter connected to the primary clarifying filter, and a sixth pump body (58) is arranged on the fourth pipeline (56).

18. The alkaline lysis equipment according to any one of claims 5 to 17, It is characterized in that The alkaline lysis equipment comprises a water storage tank (10), the inlet end of the water storage tank (10) is connected to a water supply system, and the water storage tank (10) is connected to the first pipeline (1) through the second purified water outlet.

19. The alkaline lysis device according to claim 18, It is characterized in that The water storage tank (10) is provided with a high liquid level detector (101) and a low liquid level detector (102).

20. The alkaline lysis device according to claim 18, It is characterized in that A second spraying portion (103) is provided at the top of the water storage tank (10), and the bottom of the water storage tank (10) is connected to a first waste discharge pipeline (13).

21. The alkaline lysis device according to claim 20, It is characterized in that The water supply system is connected to the No. 6 purified water inlet and the No. 7 purified water inlet through the fifth pipeline 14. The No. 7 purified water inlet is used to transport the purified water of the water supply system to the No. 1 purified water inlet, the No. 3 purified water inlet, the No. 4 purified water inlet, the No. 5 purified water inlet and the No. 8 purified water inlet. The No. 8 purified water inlet is connected to the second spray part (103).

22. The alkaline lysis device according to claim 21, It is characterized in that A seventh pump body (15) and a fifth flow meter (16) are provided on the pipeline connecting the No. 7 purified water inlet with the No. 1 purified water inlet, the No. 3 purified water inlet, the No. 4 purified water inlet, the No. 5 purified water inlet and the No. 8 purified water inlet.

23. The alkaline lysis device according to claim 20, It is characterized in that The first pipeline (1) is provided with a first row of waste outlets, the second pipeline (2) is provided with a second row of waste outlets, the third pipeline (3) is provided with a third row of waste outlets, a fourth row of waste outlets is provided on the pipeline between the intersection of the third pipeline (3) and the outlet end of the lysis reactor (4) and the inlet end of the second static mixer (7), a fifth row of waste outlets is provided on the pipeline between the outlet end of the second static mixer (7) and the neutralization and static tank (5), and a sixth row of waste outlets is provided at the bottom of the neutralization and static tank (5).

24. The alkaline lysis device according to claim 23, It is characterized in that The first waste outlet, the second waste outlet, the third waste outlet, the fourth waste outlet, the fifth waste outlet and the sixth waste outlet are all connected to the first waste outlet pipeline (13) via the second waste outlet pipeline (17), and are discharged to the waste liquid collection end via the first waste outlet pipeline (13).

25. The alkaline lysis device according to claim 23, It is characterized in that A mixing valve (18) is provided on the pipeline between the fifth waste outlet and the neutralization and static tank (5).

26. The alkaline lysis device according to claim 21, It is characterized in that The interior of the neutralization and static tank (5) is connected to instrument air via a sixth pipeline (19).

27. The alkaline lysis device according to claim 26, It is characterized in that The instrument air is connected to the first pipeline (1), the second pipeline (2), the third pipeline (3), the cleaning pipeline (8), the fourth pipeline (56), the circulation loop (93) and the fifth pipeline (14).

28. The alkaline lysis equipment according to claim 2, It is characterized in that The first static mixer (6) and the second static mixer (7) both include a spiral mixer, a mesh mixer and / or an X-type mixer.

29. The alkaline lysis equipment according to claim 1, It is characterized in that The cell lysis reactor (4) comprises a disposable silicone tube, a pressure-resistant tube or a stainless steel tube.

30. A method for extracting plasmids using the alkaline lysis device according to any one of claims 1 to 29, It is characterized in that The following steps are involved: Obtaining a bacterial suspension, and connecting the bacterial suspension to the first pipeline (1); Obtaining an alkaline lysis solution, and connecting the alkaline lysis solution to the second pipeline (2); Obtaining acid liquid, and connecting the acid liquid to the third pipeline (3); The flow rate of the bacterial suspension in the first pipeline (1), the flow rate of the alkaline lysis solution in the second pipeline (2), and the flow rate of the acid solution in the third pipeline (3) are set according to the flow ratio of the bacterial suspension, the alkaline lysis solution, and the acid solution; Start lysis, transport the bacterial suspension in the first pipeline and the alkaline lysis solution in the second pipeline to the lysis reactor at a set flow rate for continuous flow lysis, and transport the acid solution in the third pipeline to the neutralization and static incubation tank at a set flow rate together with the lysed liquid for neutralization and static incubation for a certain period of time; The incubated feed liquid is clarified, filtered and concentrated, an impurity removal agent is added, and the mixture is incubated at a specific temperature for a certain period of time and centrifuged, and the supernatant of the centrifugation is filtered to obtain a clarified concentrated feed liquid; The clarified concentrated feed solution is loaded onto a molecular sieve chromatography column, and the first elution peak is collected to obtain the target plasmid.

31. The method for extracting a plasmid according to claim 30, It is characterized in that The step of obtaining the alkaline lysis solution includes: mixing the alkaline solution and the surfactant in a volume ratio of 1:1 to obtain the alkaline lysis solution.

32. The method for extracting a plasmid according to claim 30, It is characterized in that The alkali solution contains 0.1 to 1 mol of sodium hydroxide, and the surfactant contains 1% to 5% of sodium dodecyl sulfate.

33. The method for extracting a plasmid according to claim 30, It is characterized in that The flow ratio of the bacterial suspension, the alkaline lysis solution and the acid solution is (0.7-2):2:1.

5.

34. The method for extracting a plasmid according to claim 33, It is characterized in that The flow rate of the bacterial suspension in the first pipeline (1) is 21 to 45 liters / hour, the flow rate of the alkaline lysis solution in the second pipeline (2) is 60 to 90 liters / hour, and the flow rate of the acid solution in the third pipeline (3) is 45 to 67.5 liters / hour.

35. The method for extracting a plasmid according to claim 30, It is characterized in that The volume of the cell lysis reactor (4) is 1.5 to 7.5 liters, the cell lysis time of the cell lysis reactor (4) is 1 to 5 minutes, and the static incubation time is 0.5 to 12 hours.

36. The method for extracting a plasmid according to claim 30, It is characterized in that The steps of clarifying, filtering and concentrating the incubated feed liquid, adding an impurity-removing agent, incubating at a specific temperature for a certain period of time, centrifuging, filtering the centrifuged supernatant, and obtaining a clarified concentrated feed liquid include: conveying the incubated feed liquid to a first clarifying filter for clarifying filtration, and collecting the clarified filtered feed liquid; concentrating the collected clarified filtered feed liquid, adding an impurity-removing agent, incubating at a specific temperature for a certain period of time, centrifuging, and filtering the centrifuged supernatant through a second clarifying filter to obtain a clarified concentrated feed liquid.

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