Equipment for producing shRNA (short hairpin ribonucleic acid) molecule by in-vitro transcription method

Abstract

The utility model discloses equipment for producing shRNA (short hairpin Ribonucleic Acid) molecules by utilizing an in-vitro transcription method, which comprises a heater, a reaction container, a first reagent tube, a second reagent tube, a second flow control pump, a third reagent tube, a third flow control pump, a temperature sensor and a control module, the control module is used for controlling the first flow control pump to extract a T7RNA polymerase solution in the first reagent tube into the reaction container, controlling the second flow pump to extract a DNA template solution in the second reagent tube into the reaction container, and controlling the third flow pump to extract a double-enzyme digestion system reagent solution in the third reagent tube into the reaction container; and the controller is also used for starting the heater, receiving the measurement signal output by the temperature sensor, and further controlling the working condition of the heater according to the measurement signal, so that shRNA molecules are generated in the reaction container. As the in-vitro transcription technology is adopted for producing the shRNA molecules by the equipment, the equipment can quickly produce a large amount of shRNA molecules, engineering bacteria do not need to be cultured, and the cost is reduced.

Description

Technical Field

[0001] The utility model relates to the technical field of biomedical engineering, in particular to a device for producing shRNA molecules by using an in vitro transcription method. Background Art

[0002] Short hairpin RNA (shRNA) molecules are the precursors and effective triggers of RNA interference (RNAi) in most organisms. RNA interference refers to the highly conserved phenomenon of highly efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA) or short hairpin RNA during evolution. In anti-pathogen research, exogenous dsRNA or shRNA can be actively absorbed by pathogens, processed into siRNA under the action of the Dicer complex, and the RNAi signaling pathway is activated, causing important gene silencing. If the target gene encodes a basic functional protein in the pathogen, its expression will be blocked, which will lead to the death of the pathogen. After research, it has been proved that RNA interference technology can be applied in the field of biological pesticides, and shRNA molecules can be used as active ingredients of RNA biological pesticides.

[0003] At present, shRNA molecules are usually produced through fermentation of engineered bacteria. For example, the constructed plasmid expression vector is transformed into the Escherichia coli strain HT115 (DE3), and isopropyl-β-D-thiogalactopyranoside (IPTG) is used to activate the expression of the corresponding gene to produce shRNA molecules. Although this method can produce shRNA molecules, the production yield is low and the engineered bacteria need to be artificially cultivated, resulting in high costs.

[0004] Therefore, the prior art still needs to be improved and developed. Utility Model Content

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the utility model is to provide a device for producing shRNA molecules using an in vitro transcription method, so as to solve the problem that the production of shRNA molecules by fermentation of engineered bacteria in the prior art has low yield and requires the cultivation of engineered bacteria, which further leads to high labor costs.

[0006] The utility model provides a device for producing shRNA molecules by using an in vitro transcription method, comprising:

[0007] Heater;

[0008] a reaction container, disposed on the heater; and

[0009] A first reagent tube for holding a T7 RNA polymerase solution;

[0010] a first flow control pump, wherein an inlet of the first flow control pump is communicated with the first reagent tube, and an outlet of the first flow control pump is communicated with the reaction container;

[0011] A second reagent tube for holding a DNA template solution;

[0012] a second flow control pump, wherein the inlet of the second flow control pump is communicated with the second reagent tube, and the outlet of the second flow control pump is communicated with the reaction container;

[0013] A third reagent tube for containing a double enzyme digestion system reagent solution;

[0014] a third flow control pump, wherein the inlet of the third flow control pump is communicated with the third reagent tube, and the outlet of the third flow control pump is communicated with the reaction container;

[0015] a temperature sensor connected to the reaction container and used to measure the temperature of the solution in the reaction container to output a measurement signal;

[0016] A control module is respectively connected to the first flow control pump, the second flow control pump, the third flow control pump, the temperature sensor and the heater. The control module is used to control the first flow control pump to extract T7 RNA polymerase solution into the reaction container, control the second flow pump to extract DNA template solution into the reaction container, control the third flow pump to extract double enzyme digestion system reagent solution into the reaction container, and control the opening and closing of the heater, and control the working condition of the heater according to the measurement signal to produce shRNA molecules in the reaction container.

[0017] According to a further configuration of the utility model, the first flow control pump, the second flow control pump and the third flow control pump are peristaltic pumps.

[0018] In a further configuration of the present invention, the control module comprises:

[0019] A pump driving unit connected to the peristaltic pump;

[0020] A heater driving unit connected to the heater;

[0021] The first control unit is respectively connected to the pump driving unit, the heater driving unit and the temperature sensor, the first control unit is used to output a first control signal to the pump driving unit, the pump driving unit is used to receive the first control signal and control the peristaltic pump corresponding to the first reagent tube to extract the T7 RNA polymerase solution into the reaction container according to the first control signal, control the peristaltic pump corresponding to the second reagent tube to extract the DNA template solution into the reaction container, and control the peristaltic pump corresponding to the third reagent tube to extract the double enzyme digestion system reagent solution into the reaction container; the first control unit is also used to output a second control signal, the heater driving unit is used to receive the second control signal and control the opening and closing of the heater according to the second control signal, and output a third control signal to the heater driving unit according to the measurement signal to control the working condition of the heater.

[0022] According to a further configuration of the utility model, the device further comprises:

[0023] A test tube rack, on which the first reagent tube, the second reagent tube and the third reagent tube are placed respectively.

[0024] In a further configuration of the present invention, the temperature sensor comprises:

[0025] A temperature probe for measuring the temperature of the solution in the reaction container, wherein the temperature probe is arranged in the reaction container;

[0026] A temperature transmitter is used to convert the solution temperature measured by the temperature probe into a measurement signal, and the temperature variable container is connected to the temperature probe and the first control unit respectively.

[0027] According to a further configuration of the utility model, the device further comprises:

[0028] A base, the peristaltic pump, the heater, the pump drive unit, the heater drive unit, the temperature transmitter and the test tube rack are respectively arranged on the base.

[0029] The utility model is further configured to include: a silicone hose;

[0030] The first reagent tube is connected to the inlet of the first flow control pump through the silicone hose;

[0031] The second reagent tube is connected to the inlet of the second flow control pump through the silicone hose;

[0032] The third reagent tube is connected to the inlet of the third flow control pump through the silicone hose.

[0033] In a further configuration of the utility model, the first flow control pump is connected to the reaction container via a silicone hose;

[0034] The second flow control pump is connected to the reaction container via the silicone hose;

[0035] The third flow control pump is connected to the reaction container via the silicone hose.

[0036] According to a further configuration of the utility model, the heater comprises a positive temperature coefficient constant temperature heater for heating the solution in the reaction container, and the reaction container is arranged on the positive temperature coefficient constant temperature heater and is connected to the control module.

[0037] Beneficial effects of the utility model:

[0038] The utility model provides a device for producing shRNA molecules by using an in vitro transcription method, comprising: a heater; a reaction container, which is arranged on the heater; and a first reagent tube for holding a T7RNA polymerase solution; a first flow control pump, the inlet of the first flow control pump is connected to the first reagent tube, and the outlet of the first flow control pump is connected to the reaction container; a second reagent tube for holding a DNA template solution; a second flow control pump, the inlet of the second flow control pump is connected to the second reagent tube, and the outlet of the second flow control pump is connected to the reaction container; a third reagent tube for holding a double enzyme digestion system reagent solution; a third flow control pump, the inlet of the third flow control pump is connected to the third reagent tube, and the outlet of the third flow control pump is connected to the reaction container. The port is connected to the reaction container; a temperature sensor is arranged in the reaction container, and the temperature sensor is used to measure the temperature of the solution in the reaction container to output a measurement signal; a control module is respectively connected to the first flow control pump, the second flow control pump, the third flow control pump, the temperature sensor and the heater, and the control module is used to control the first flow control pump to extract the T7RNA polymerase solution into the reaction container, control the second flow pump to extract the DNA template solution into the reaction container, and control the third flow pump to extract the double enzyme digestion system reagent solution into the reaction container; it is also used to control the opening and closing of the heater, and control the working condition of the heater according to the measurement signal to produce shRNA molecules in the reaction container. In the technical solution of the utility model, the device produces shRNA molecules using in vitro transcription technology. In vitro transcription is a technology that transcribes a large number of RNA molecules based on DNA templates outside the cell. Therefore, the device can quickly produce a large number of shRNA molecules without the need to cultivate engineering bacteria, which reduces costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the utility model or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the utility model. For ordinary personnel in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0040] Figure 1 It is a principle block diagram of the device for producing shRNA molecules by using an in vitro transcription method in the utility model.

[0041] Figure 2 This is a structural installation diagram of a device for producing shRNA molecules using an in vitro transcription method in one embodiment of the utility model.

[0042] Figure 3 It is a circuit structure diagram of the first control unit in one embodiment of the utility model.

[0043] Figure 4 This is a reset circuit diagram of the first chip in one embodiment of the utility model.

[0044] Figure 5 It is a circuit diagram of a program download interface in one embodiment of the utility model.

[0045] Figure 6 It is a program downloading circuit diagram in one embodiment of the utility model.

[0046] Figure 7 It is a circuit diagram of power conversion in one embodiment of the utility model.

[0047] Figure 8 The utility model is a power supply status indication circuit diagram in one embodiment of the utility model.

[0048] Fig. 9 It is a working status indication diagram of the first control unit in one embodiment of the utility model.

[0049] Fig.10 1 is a circuit diagram of a pump drive unit in one embodiment of the utility model.

[0050] Fig.11 It is a circuit diagram of a heater driving unit in one embodiment of the utility model.

[0051] Fig.12 This is a circuit diagram of a temperature sensor interface in one embodiment of the utility model.

[0052] The marks in the accompanying drawings are: 101, first reagent tube; 102, second reagent tube; 103, third reagent tube; 104, first flow control pump; 105, second flow control pump; 106, third flow control pump; 107, reaction container; 108, temperature sensor; 1081, temperature probe; 1082, temperature transmitter; 109, heater; 1091, positive temperature coefficient constant temperature heater; 110, control module; 1101, first control unit; 1102, heater drive unit; 1103, pump drive unit; 111, test tube rack; 112, base. DETAILED DESCRIPTION

[0053] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific implementation methods of the present invention are now described in detail with reference to the accompanying drawings. In the following description, it should be understood that the directions or positional relationships indicated by "front", "back", "up", "down", "left", "right", "longitudinal", "horizontal", "vertical", "horizontal", "top", "bottom", "inside", "outside", "head", "tail", etc. are based on the directions or positional relationships shown in the accompanying drawings, are constructed and operated in a specific direction, and are only for the convenience of describing the present technical solution, rather than indicating that the device or element referred to must have a specific direction, and therefore cannot be understood as a limitation to the present invention.

[0054] The utility model provides a device for producing shRNA molecules by using an in vitro transcription method, such as Figure 1 As shown, the device may include a heater 109, a reaction container 107, a first reagent tube 101, a first flow control pump 104, a second reagent tube 102, a second flow control pump 105, a third reagent tube 103, a third flow control pump 106, a temperature sensor 108 and a control module 110.

[0055] Specifically, the reaction container 107 is arranged on the heater 109, and the solution in the reaction container 107 is heated by the heater 109; the first reagent tube 101 is used to hold the T7 RNA polymerase solution; the inlet of the first flow control pump 104 is connected to the first reagent tube 101, and the outlet of the first flow control pump 104 is connected to the reaction container 107; the second reagent tube 102 is used to hold the DNA template solution; the inlet of the second flow control pump 105 is connected to the second reagent tube 102, and the outlet of the second flow control pump 105 is connected to the reaction container 107; the third reagent tube 103 is used to hold the double enzyme digestion system reagent solution; the inlet of the third flow control pump 106 is connected to the third reagent tube 103, and the outlet of the third flow control pump 106 is connected to the reaction container 107; the temperature sensor 108 It is connected to the reaction container 107 to measure the temperature of the solution in the reaction container 107 to output a measurement signal; the control module 110 is respectively connected to the first flow control pump 104, the second flow control pump 105, the third flow control pump 106, the temperature sensor 108 and the heater 109, and the control module 110 is used to control the first flow control pump 104 to extract the T7 RNA polymerase solution into the reaction container 107, control the second flow pump to extract the DNA template solution into the reaction container 107, and control the third flow pump to extract the double enzyme digestion system reagent solution into the reaction container 107. The control module 110 is also used to control the opening and closing of the heater 109, and control the working condition of the heater 109 according to the measurement signal output by the temperature sensor 108, so that shRNA molecules are produced in the reaction container 107.

[0056] Among them, the working conditions of the heater 109 include starting the heater 109 and shutting down the heater 109. When the heater 109 is started, the heater 109 heats the solution in the reaction container 107. When the heater 109 is shut down, the heater 109 no longer heats the solution in the reaction container 107. The reaction container 107 can be a container obtained by 3D printing.

[0057] In this embodiment, the device produces shRNA molecules using in vitro transcription technology. In vitro transcription is a technology that transcribes a large number of RNA molecules outside the cell based on a DNA template. In vitro transcription uses T7 RNA polymerase to synthesize RNA complementary to one strand of DNA from the downstream of the template DNA T7 promoter, which can easily and quickly obtain a large number of RNA molecules. Therefore, the device can quickly produce a large number of shRNA molecules without the need to cultivate engineered bacteria, thereby reducing costs.

[0058] In some embodiments, the first flow control pump 104 , the second flow control pump 105 , and the third flow control pump 106 are peristaltic pumps.

[0059] In this embodiment, the peristaltic pump may be, but is not limited to, a pump of model 104KA / BT, and the controller used by the peristaltic pump is a pulse-type stepping motor controller of model JBT-AP.

[0060] In some embodiments, Figure 2 , Figure 3 , Fig.10 and Fig.11 As shown, the control module 110 may include a pump driving unit 1103, a heater driving unit 1102 and a first control unit 1101; wherein the pump driving unit 1103 is connected to the peristaltic pump; the heater driving unit 1102 is connected to the heater 109; the first control unit 1101 is respectively connected to the pump driving unit 1103, the heater driving unit 1102 and the temperature sensor 108, the first control unit 1101 is used to output a first control signal to the pump driving unit 1103, the pump driving unit 1103 is used to receive the first control signal and control the peristaltic pump corresponding to the first reagent tube 101 to extract T7R according to the first control signal. NA polymerase solution into the reaction container 107, control the peristaltic pump corresponding to the second reagent tube 102 to extract the DNA template solution into the reaction container 107, and control the peristaltic pump corresponding to the third reagent tube 103 to extract the double enzyme digestion system reagent solution into the reaction container 107; the first control unit 1101 is also used to output a second control signal, the heater driving unit 1102 is used to receive the second control signal and control the opening and closing of the heater 109 according to the second control signal, and receive the measurement signal output by the temperature sensor 108, and then output the third control signal to the heater driving unit 1102 according to the measurement signal to control the working condition of the heater 109.

[0061] Here, it should be noted that the peristaltic pump in the peristaltic pump corresponding to the first reagent tube 101 refers to the first flow control pump 104, the peristaltic pump in the peristaltic pump corresponding to the second reagent tube 102 refers to the second flow control pump 105, and the peristaltic pump in the peristaltic pump corresponding to the third reagent tube 103 refers to the third flow control pump 106.

[0062] It should also be noted that the third control signal refers to a control signal for starting or shutting down the heater 109 during the shRNA production process. Specifically, after the first control unit 1101 outputs the second control signal to start the heater 109, when the heater 109 needs to be shut down, the signal output to the heater driving unit 1102 to shut down the heater 109 is the third control signal. After shutting down the heater 109, when the heater 109 needs to be restarted, the signal output to the heater driving unit 1102 to restart the heater 109 is the third control signal.

[0063] Specifically, Figure 3 As shown, it is a specific circuit structure diagram of the control module 110, wherein: Figure 3 1 is a specific circuit structure diagram of the first control unit 1101. The first control unit 1101 is a circuit composed of a first chip as a main component. The model of the first chip can be but is not limited to a chip of model ATMEGA328P-PU; Figure 4 It is the reset circuit of the first chip.

[0064] like Figure 5 As shown, this is a download interface used when downloading a program to the first chip. Figure 6 This is a specific circuit structure diagram used when downloading the program. In the circuit diagram of the downloading program, the chip used is the CH340C chip.

[0065] like Figure 7 As shown, it is a power conversion circuit. When a DC power supply is connected to the CON1 interface, one end of the sixth capacitor outputs +5V DC power, and the fourth pin of the third chip outputs +3.3V DC power.

[0066] like Figure 8 As shown, it is a status indication circuit when the power is connected. When +5V DC is connected, the light emitting diode LED4 in the figure is lit; Fig. 9 It is a status indication circuit when the first control unit 1101 is running. When the first control unit 1101 is running normally, the first chip outputs a high level to light up the light emitting diode LED1.

[0067] like Fig.10 As shown, it is a specific circuit structure diagram of the pump driving unit 1103. There are three pump driving units 1103, and each peristaltic pump is provided with a pump driving unit 1103, wherein the pump driving unit 1103 of the peristaltic pump corresponding to the first reagent tube 101 is connected to the D3 pin of the first chip, the pump driving unit 1103 of the peristaltic pump corresponding to the second reagent tube 102 is connected to the D5 pin of the first chip, and the pump driving unit 1103 of the peristaltic pump corresponding to the third reagent tube 103 is connected to the D6 pin of the first chip. During specific driving, when the D3 pin of the first chip outputs a high level, the peristaltic pump corresponding to the first reagent tube 101 starts working and transports the T7 RNA polymerase solution to the reaction container 107; when the D5 pin of the first chip outputs a high level, the peristaltic pump corresponding to the second reagent tube 102 starts working and transports the DNA template solution to the reaction container 107; when the D6 pin of the first chip outputs a high level, the peristaltic pump corresponding to the third reagent tube 103 starts working and transports the double enzyme digestion system reagent solution to the reaction container 107.

[0068] like Fig.11 , which is a specific circuit structure diagram of the heater driving unit 1102 . When driving the heater 109 , the D11 pin of the first chip outputs a high level to the heater driving unit 1102 , and the heater 109 starts to work for heating.

[0069] like Fig.12 As shown, it is a specific circuit structure diagram of the temperature sensor 108 interface. The measurement signal of the temperature sensor 108 is transmitted to the A1 pin of the first chip through the 2nd pin of the CN1 interface.

[0070] In order to solve the problem of placing reagent tubes (first reagent tube 101, second reagent tube 102 and third reagent tube 103) during the production of shRNA, the device is further provided with a test tube rack 111, wherein the first reagent tube 101, the second reagent tube 102 and the third reagent tube 103 are respectively placed on the test tube rack 111.

[0071] In this embodiment, the first reagent tube 101 , the second reagent tube 102 , and the third reagent tube 103 may be centrifuge tubes respectively.

[0072] In some embodiments, the temperature sensor 108 may include a temperature probe 1081 and a temperature transmitter 1082, wherein the temperature probe 1081 is used to measure the temperature of the solution in the reaction container 107, and the temperature probe 1081 is disposed in the reaction container 107;

[0073] The temperature transmitter 1082 is used to convert the solution temperature measured by the temperature probe 1081 into a measurement signal. The temperature transmitter 1082 is connected to the temperature probe 1081 and the first control unit 1101 respectively.

[0074] Specifically, the temperature probe 1081 may be, but is not limited to, a temperature probe 1081 of model PT100, and the temperature transmitter 1082 may be a transmitter of model HSTL-PT100G, wherein the measurement range of the temperature transmitter 1082 is 0-200°C, and the output signal type is 0-5V. When measuring, the temperature probe 1081 measures the temperature of the solution in the reaction container 107 and transmits the temperature measurement value to the temperature transmitter 1082, and the temperature transmitter 1082 converts the temperature measurement value into a standard electrical signal (equivalent to the above-mentioned measurement signal) and transmits it to the first control unit 1101, thereby realizing the monitoring of the temperature of the solution in the reaction container 107.

[0075] In this embodiment, the temperature probe 1081 is disposed in the reaction container 107 . Preferably, the temperature probe 1081 is in contact with the solution in the reaction container 107 , so that the measured value of the temperature of the solution in the reaction container 107 is more accurate.

[0076] In some embodiments, the device further includes a base 112 , wherein the peristaltic pump, the heater 109 , the pump drive unit 1103 , the heater drive unit 1102 , the temperature transmitter 1082 , and the test tube rack 111 are respectively disposed on the base 112 .

[0077] Specifically, the base 112 may be a square plate structure but is not limited to it. When setting, the test tube rack 111 may be fixedly set at one end of the base 112; the heater drive unit 1102 may be set at the other end of the base 112; the peristaltic pump and the heater 109 may be set between the test tube rack 111 and the heater drive unit 1102, the peristaltic pump may be close to the test tube rack 111, and three peristaltic pumps may be set side by side; the pump drive unit 1103 may be set adjacent to the peristaltic pump; the temperature transmitter 1082 may be set at one side of the heater 109 to facilitate the connection between the temperature transmitter 1082 and the temperature probe 1081.

[0078] In this embodiment, since the peristaltic pump, heater 109, pump drive unit 1103, heater drive unit 1102, temperature transmitter 1082 and test tube rack 111 are respectively arranged on the base 112, when the equipment is used, it is more convenient to carry. The peristaltic pump, heater 109, pump drive unit 1103, heater drive unit 1102, temperature transmitter 1082 and test tube rack 111 can be carried at the same time through the base 112.

[0079] In some embodiments, the device also includes a silicone hose; wherein the first reagent tube 101 is connected to the inlet of the first flow control pump 104 through a silicone hose, that is, the first reagent tube 101 is connected to the corresponding peristaltic pump through a silicone hose; the second reagent tube 102 is connected to the inlet of the second flow control pump 105 through a silicone hose, that is, the second reagent tube 102 is connected to the corresponding peristaltic pump through a silicone hose; the third reagent tube 103 is connected to the inlet of the third flow control pump 106 through a silicone hose, that is, the third reagent tube 103 is connected to the corresponding peristaltic pump through a silicone hose.

[0080] In a specific embodiment, the silicone hose has a specification of 1.6*4.8 (inner diameter 1.6 mm, outer diameter 4.8 mm).

[0081] In some embodiments, the first flow control pump 104 is connected to the reaction container 107 through a silicone hose, that is, the peristaltic pump corresponding to the first reagent tube 101 is connected to the reaction container 107 through a silicone hose; the second flow control pump 105 is connected to the reaction container 107 through a silicone hose, that is, the peristaltic pump corresponding to the second reagent tube 102 is connected to the reaction container 107 through a silicone hose; the third flow control pump 106 is connected to the reaction container 107 through a silicone hose, that is, the peristaltic pump corresponding to the third reagent tube 103 is connected to the reaction container 107 through a silicone hose.

[0082] In some embodiments, the heater 109 may include a positive temperature coefficient constant temperature heater 1091, which is used to heat the solution in the reaction container 107. The reaction container 107 is set on the positive temperature coefficient constant temperature heater 1091, and the positive temperature coefficient constant temperature heater 1091 is connected to the control module 110.

[0083] Specifically, the positive temperature coefficient constant temperature heater 1091 is disposed on the base 112, and the reaction container 107 is further disposed on the positive temperature coefficient constant temperature heater 1091. When the positive temperature coefficient constant temperature heater 1091 is connected to the control module 110, the positive temperature coefficient constant temperature heater 1091 is connected to the drain of the first MOS tube.

[0084] In this embodiment, the positive temperature coefficient constant temperature heater 1091 can be heated to 100° C., and the power supply voltage is DC +5V.

[0085] When the device for producing shRNA molecules using the in vitro transcription method is used in a specific application, first, a test tube rack 111, a peristaltic pump, a pump drive unit 1103, a positive temperature coefficient constant temperature heater 1091, a reaction container 107, a temperature probe 1081, a temperature transmitter 1082 and a heater drive unit 1102 are installed, and then three separate centrifuge tubes (a first reagent tube 101, a second reagent tube 102 and a third reagent tube 103) are placed on the test tube rack 111, and the basic reagents required for the in vitro transcription reaction are placed in the three separate centrifuge tubes, the inlet of each peristaltic pump is connected to the centrifuge tube through a silicone hose, and the outlet of each peristaltic pump is connected to the reaction container 107 through a silicone hose. During the reaction, the D3 pin of the first chip outputs a PWM wave with a specific duty cycle for 10s, driving the peristaltic pump corresponding to the first reagent tube 101 to deliver 1.2mL of T7RNA polymerase solution to the reactor. Further, the D5 pin of the first chip continuously outputs a PWM wave with a specific duty cycle for 15s, driving the peristaltic pump corresponding to the second reagent tube 102 to deliver 0.8mL of DNA template solution to the reactor to mix with the T7RNA polymerase solution. Then, the D11 pin of the first chip outputs a high level, and the positive temperature coefficient constant temperature heater 1091 starts to operate, heating the solution in the reactor to a certain temperature and then maintaining the temperature in the reactor within the range of 36-38°C for 120min. The temperature probe 1081 is used to collect the measured value of the solution temperature, and the temperature transmitter 1082 converts the measured value into an electrical signal for transmission. The signal is sent to the A1 pin of the first chip. When the temperature in the reaction container 107 is lower than 36°C, the D11 pin of the first chip outputs a high level, and the positive temperature coefficient constant temperature heater 1091 starts to run again. When the temperature of the solution in the reaction container 107 is higher than 38°C, the D11 pin of the first chip outputs a low level, so that the positive temperature coefficient constant temperature heater 1091 stops heating. After incubation for 120 minutes, the D6 pin of the first chip continuously outputs a PWM wave with a specific duty cycle for 9s, driving the peristaltic pump corresponding to the third reagent tube 103 to transport 2mL of the double enzyme digestion system reagent solution into the reaction container 107. Finally, the D11 pin of the first chip outputs a high level, and the positive temperature coefficient constant temperature heater 1091 starts to run again, and the temperature of the solution in the reaction container 107 is controlled within the range of 36-38°C for 30 minutes.

[0086] In summary, the utility model provides a device for producing shRNA molecules using an in vitro transcription method. The device produces shRNA molecules using in vitro transcription technology. In vitro transcription is a technology that transcribes a large number of RNA molecules based on a DNA template outside the cell. Therefore, the device can quickly produce a large number of shRNA molecules without the need to cultivate engineered bacteria, thereby reducing costs.

[0087] It can be understood that the above embodiments only express the preferred implementation methods of the utility model, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the patent scope of the utility model. It should be pointed out that, for ordinary technicians in this field, without departing from the concept of the utility model, the above-mentioned technical features can be freely combined, and several deformations and improvements can be made, which all belong to the protection scope of the utility model. Therefore, all equivalent changes and modifications made to the scope of the claims of the utility model should belong to the scope covered by the claims of the utility model.

Claims

1. A device for producing shRNA molecules using an in vitro transcription method, characterized in that: include: Heater; A reaction container, disposed on the heater; as well as A first reagent tube for holding a T7 RNA polymerase solution; a first flow control pump, wherein an inlet of the first flow control pump is communicated with the first reagent tube, and an outlet of the first flow control pump is communicated with the reaction container; A second reagent tube for holding a DNA template solution; a second flow control pump, wherein the inlet of the second flow control pump is communicated with the second reagent tube, and the outlet of the second flow control pump is communicated with the reaction container; A third reagent tube for containing a double enzyme digestion system reagent solution; a third flow control pump, wherein the inlet of the third flow control pump is communicated with the third reagent tube, and the outlet of the third flow control pump is communicated with the reaction container; a temperature sensor connected to the reaction container and used to measure the temperature of the solution in the reaction container to output a measurement signal; A control module is respectively connected to the first flow control pump, the second flow control pump, the third flow control pump, the temperature sensor and the heater. The control module is used to control the first flow control pump to extract T7 RNA polymerase solution into the reaction container, control the second flow control pump to extract DNA template solution into the reaction container, control the third flow control pump to extract double enzyme digestion system reagent solution into the reaction container, and is also used to control the opening and closing of the heater, and control the working condition of the heater according to the measurement signal to produce shRNA molecules in the reaction container.

2. The device for producing shRNA molecules using an in vitro transcription method according to claim 1, characterized in that: The first flow control pump, the second flow control pump, and the third flow control pump are peristaltic pumps.

3. The device for producing shRNA molecules using an in vitro transcription method according to claim 2, characterized in that: The control module comprises: A pump driving unit connected to the peristaltic pump; A heater driving unit connected to the heater; The first control unit is respectively connected to the pump driving unit, the heater driving unit and the temperature sensor, the first control unit is used to output a first control signal to the pump driving unit, the pump driving unit is used to receive the first control signal and control the peristaltic pump corresponding to the first reagent tube to extract the T7 RNA polymerase solution into the reaction container according to the first control signal, control the peristaltic pump corresponding to the second reagent tube to extract the DNA template solution into the reaction container, and control the peristaltic pump corresponding to the third reagent tube to extract the double enzyme digestion system reagent solution into the reaction container; the first control unit is also used to output a second control signal, the heater driving unit is used to receive the second control signal and control the opening and closing of the heater according to the second control signal, and output a third control signal to the heater driving unit according to the measurement signal to control the working condition of the heater.

4. The device for producing shRNA molecules using an in vitro transcription method according to claim 3, characterized in that: The device also includes: A test tube rack, on which the first reagent tube, the second reagent tube and the third reagent tube are placed respectively.

5. The device for producing shRNA molecules using an in vitro transcription method according to claim 4, characterized in that: The temperature sensor comprises: A temperature probe for measuring the temperature of the solution in the reaction container, wherein the temperature probe is arranged in the reaction container; A temperature transmitter is used to convert the solution temperature measured by the temperature probe into a measurement signal, and the temperature variable container is connected to the temperature probe and the first control unit respectively.

6. The device for producing shRNA molecules using an in vitro transcription method according to claim 5, characterized in that: The device also includes: A base, the peristaltic pump, the heater, the pump drive unit, the heater drive unit, the temperature transmitter and the test tube rack are respectively arranged on the base.

7. The device for producing shRNA molecules by in vitro transcription method according to any one of claims 1 to 6, characterized in that: Also includes: Silicone hose; The first reagent tube is connected to the inlet of the first flow control pump through the silicone hose; The second reagent tube is connected to the inlet of the second flow control pump through the silicone hose; The third reagent tube is connected to the inlet of the third flow control pump through the silicone hose.

8. The device for producing shRNA molecules using an in vitro transcription method according to claim 7, characterized in that: The first flow control pump is connected to the reaction container via the silicone hose; The second flow control pump is connected to the reaction container via the silicone hose; The third flow control pump is connected to the reaction container via the silicone hose.

9. The device for producing shRNA molecules using an in vitro transcription method according to claim 1, characterized in that: The heater comprises a positive temperature coefficient constant temperature heater for heating the solution in the reaction container. The reaction container is arranged on the positive temperature coefficient constant temperature heater and is connected to the control module.

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