Tank device of horizontal self-stirring in-vitro protein synthesis reactor

Abstract

The utility model discloses a tank device of a horizontal self-stirring in-vitro protein synthesis reactor, which comprises a horizontal tank body, and a self-stirring paddle component is distributed on the inner wall of the horizontal tank body. The self-stirring paddle assembly is suitable for stirring a protein synthesis reaction liquid in the horizontal tank body in the rotating process of the horizontal tank body. The reaction liquid can be fully mixed, the reaction efficiency is improved, the product activity is kept, and industrial amplification can be carried out.

Description

Technical Field

[0001] The utility model belongs to the technical field of biological reaction equipment, and in particular relates to a tank device of a horizontal self-stirring in vitro protein synthesis reactor. Background Art

[0002] At present, protein synthesis methods can be divided into two categories: one is the traditional cell culture method to synthesize proteins, and the other is the in vitro cell-free method to synthesize proteins. The traditional cell culture method refers to a molecular biology technology that expresses exogenous genes through model organisms such as bacteria, fungi, plant cells or animal cells. The in vitro cell-free method of synthesizing proteins mainly relies on the cell-free expression system, which uses exogenous target DNA as a template for protein synthesis. Through artificial control, the substrates required for protein synthesis and auxiliary factors of transcription and translation-related proteins are added to achieve the synthesis of target proteins. The expression of proteins in the in vitro translation system does not require plasmid construction, transformation, cell culture, cell collection and fragmentation steps. It is a relatively fast, time-saving and convenient way to express proteins.

[0003] However, due to the cumbersome preparation of the in vitro protein synthesis system and the complex addition of auxiliary factors, all in vitro protein synthesis products on the market are currently low in activity and extremely expensive (cost 100-1000 times that of traditional cell culture methods). Currently, only a few laboratories can prepare highly active proteins with a small amount of IVTT (In vitro transcription and translation) reaction solution. The reactor used in laboratory preparation is a 24-well plate, in which the IVTT reaction solution added is at the uL level.

[0004] Taking the uL-level reaction of a 24-well plate as a control, when the reactor is replaced with a container with a larger volume, such as a 16 cm diameter culture dish, a 20×26 cm plastic basin, a 30×40 cm plastic basin, a 1L beaker, an L-level microtube, an L-level vertical reaction tank, etc., and an mL-level or L-level IVTT reaction solution is added for amplified reaction, the activity of the final product is greatly reduced, and it has no industrial practical value. Conventional vertical reaction tanks use a vertical stirring paddle independent of the tank body. The reaction liquid is deposited at the bottom of the tank due to gravity. Even if there is a stirring paddle that keeps stirring, the thickness of the reaction liquid remains basically unchanged, that is, stirring cannot dilute the reaction liquid to make it more fully exposed to the air. It can be seen from this that it is not feasible to enlarge the size only from the perspective of the volume of the reactor. It is urgent to develop an industrial amplification equipment that can greatly enlarge the volume and maximize the activity of the final product. Utility Model Content

[0005] The utility model aims to solve the technical problem that the current in vitro protein synthesis can only be completed in small quantities in the laboratory and cannot be scaled up to industrial level.

[0006] To achieve the above-mentioned purpose, the utility model provides a tank device of a horizontal self-stirring in vitro protein synthesis reactor, comprising a horizontal tank body, on the inner wall of which a self-stirring blade assembly is arranged, and the self-stirring blade assembly is suitable for stirring the protein synthesis reaction liquid therein during the rotation of the horizontal tank body.

[0007] Furthermore, the height of the self-stirring blade assembly protruding from the inner wall of the tank body accounts for no more than 60% of the inner radius of the horizontal tank body.

[0008] Furthermore, the self-stirring blade assembly includes a plurality of blades, each of which extends along the axial direction of the horizontal tank body, and each of which is spaced apart along the inner circumference of the horizontal tank body.

[0009] Furthermore, the blades are arranged at equal intervals.

[0010] Furthermore, each of the blades is integrally formed, fixed to the inner wall of the horizontal tank body by welding, and the welds are polished.

[0011] Furthermore, the side of each of the blades away from the inner wall of the horizontal tank body is a concave arc structure.

[0012] Furthermore, there is an angle a between the length direction of each blade and the axial direction of the horizontal tank body.

[0013] Furthermore, the angle a is ≤20°.

[0014] Furthermore, an angle b is formed between the contour surface of each blade and a tangent plane of the horizontal tank body passing through the inner edge of the blade, and b<90°.

[0015] Furthermore, a plurality of through holes are arranged on the contour surface of each of the blades.

[0016] Furthermore, the total area of ​​the multiple through holes on each of the blades accounts for 30%-50% of the area of ​​the blade, and the spacing between adjacent through holes is equal.

[0017] Furthermore, the edge of each blade and the edge of the opening of each through hole have a rounded structure.

[0018] Furthermore, the volume of the horizontal tank body is 10L-10000L.

[0019] Furthermore, in a working state, the axial direction of the horizontal tank body is parallel to a horizontal plane.

[0020] Furthermore, a circle of rolling ribs is provided on the outer wall of the horizontal tank body.

[0021] Furthermore, a feed port is provided at one end of the horizontal tank body, and a rotating shaft is provided at the other end.

[0022] Compared with the prior art, the utility model has at least the following beneficial effects:

[0023] 1. A horizontal rotatable tank structure, with a customized self-stirring blade assembly on the inner wall of the tank cavity. Fresh air is continuously introduced into the tank cavity during operation, and the volume of the reaction liquid added into the tank cavity only accounts for a small part of the tank volume. In this way, during the reaction process, on the one hand, the reaction liquid can be diluted on the cavity wall by the rotation of the tank body, and the degree of dilution can be adjusted by controlling the volume of the added reaction liquid. The diluted reaction liquid can be more fully in contact with the air, thereby improving the reaction efficiency; on the other hand, the self-stirring blade assembly on the inner wall of the tank body during rotation can automatically stir the reaction liquid. Due to the angle design of the blade and the concave arc structure with wide ends and narrow middle, multi-directional turbulence and eddy currents will be formed, which can fully mix the reaction liquid and improve the reaction efficiency.

[0024] 2. Based on the tank device, an in vitro protein synthesis reactor can be formed. In vitro protein synthesis experiments were carried out on a variety of reactors with different tank volumes. The minimum tank volume was 10L and the maximum was 10,000L. The activity of the final product of each reactor was measured, which was equivalent to the activity prepared in the laboratory. It has industrial promotion value and is an efficient, convenient and low-cost in vitro protein synthesis reactor and synthesis method. It will undoubtedly become a steam locomotive to promote the future bio-industrial revolution, scientific and technological progress, medical research and development, and drug production, and will play a revolutionary role in the progress of the entire bio-industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a front view of an embodiment of the utility model;

[0026] Figure 2 A cross-sectional view of an embodiment of the utility model;

[0027] Figure 3 It is a left side view of an embodiment of the utility model;

[0028] Figure 4 A perspective view of the interior of a tank cavity according to an embodiment of the utility model;

[0029] Figure 5 This is a schematic structural diagram of a self-stirring blade assembly in one embodiment of the utility model;

[0030] Figure 6 This is a schematic diagram of the structure of a blade in one embodiment of the utility model;

[0031] Figure 7 This is a usage state diagram of an embodiment of the utility model;

[0032] Figure 8 This is a graph showing the activity test results of the target protein EGFP produced using a reactor composed of an embodiment of the utility model.

[0033] In the accompanying drawings, there are a frame 1; a horizontal tank body 2; a self-stirring paddle assembly 20; a paddle 21; a tank lock 22; a rolling rib 23; a rotating shaft 24; a feed port 25; an air inlet pipe 31; an exhaust pipe 33; a driving device 4; a bearing seat 5; a rotating wheel 6; and a pulley transmission mechanism 7. DETAILED DESCRIPTION

[0034] The following is an explanation of the implementation of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific implementations, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0035] like Figure 1-Figure 6 As shown, an embodiment of the tank device of the horizontal self-stirring in vitro protein synthesis reactor of the utility model includes a horizontal tank body 2, and a self-stirring blade assembly 20 is arranged on the inner wall of the horizontal tank body 2, and the self-stirring blade assembly 20 is suitable for stirring the protein synthesis reaction liquid therein during the rotation of the horizontal tank body 2.

[0036] In this embodiment, a horizontal tank body 2 is used to replace the commonly used vertical reaction tank, and the common independent vertical stirring paddle in the vertical reaction tank is replaced with a wall-connected horizontal stirring paddle fixed to the inner wall of the tank body. This horizontal tank body structure can dilute the reaction liquid on the cavity wall through the rotation of the tank body. The degree of dilution can be adjusted by controlling the volume of the added reaction liquid. The diluted reaction liquid has a larger surface area and can be more fully in contact with the air, thereby improving the reaction efficiency. In a conventional vertical reaction tank, the reaction liquid is deposited at the bottom of the tank due to gravity. Even if there is a stirring paddle that keeps stirring, the thickness of the reaction liquid remains basically unchanged, that is, stirring cannot dilute the reaction liquid to make it more fully in contact with the air. The structure of this embodiment can be said to have overturned the usual practice in this field.

[0037] In one embodiment of the utility model, the height of the self-stirring paddle assembly 20 protruding from the inner wall of the tank body accounts for no more than 60% of the inner radius of the horizontal tank body 2. Preferably, the ratio is less than 50%. The height of the self-stirring paddle assembly 20 should not be too high, and the specific height can be customized according to the volume of the reaction liquid in the tank. Generally, the more reaction liquid there is, the greater the height is, so that stirring can be more fully performed. Since the reaction liquid needs to be diluted, the volume of the reaction liquid cannot exceed a certain proportion of the tank volume. Accordingly, the height of the self-stirring paddle assembly 20 will be limited.

[0038] In one embodiment of the present invention, the self-stirring blade assembly 20 includes a plurality of blades 21 , each of the blades 21 extends along the axial direction of the horizontal tank body 2 , and each of the blades 21 is spaced apart along the inner circumference of the horizontal tank body 2 . Figure 4 and Figure 5 Six blades 21 are shown in the figure, which are arranged at intervals, so that the tank cavity can be divided into six areas. During operation, as the horizontal tank body 2 rotates, under the combined action of centrifugal force, gravity and blade stirring force, the reaction liquid will be diluted in each area. Since each area is surrounded by two blades 21 and the tank wall, the reaction liquid can be diluted to three surfaces in the area, so that the same volume of reaction liquid will be spread thinner, and the contact area with the air will be larger, thereby improving the reaction efficiency. The number of blades 21 can be increased or decreased according to the volume size of the horizontal tank body 2. Generally, the larger the volume, the more blades 21 there are, so that it can be divided into multiple areas with smaller volumes, which is conducive to the dilution of the reaction liquid.

[0039] In one embodiment of the present invention, Figure 4 As shown, the blades 21 are arranged at equal intervals. The six blades 21 at equal intervals can divide the tank cavity into six areas of equal volume, so that the dilution degree of the reaction liquid in each area is equivalent, that is, equivalent to the controlled contact area, the reaction efficiency is uniform, and the overall reaction effect is better.

[0040] In one embodiment of the present invention, Figure 6 As shown, each of the blades 21 is integrally formed, fixed to the inner wall of the horizontal tank body 2 by welding, and the weld is polished. The blade 21 is integrally formed in one piece, has a smooth surface, and is fixed to the tank wall by welding. The polishing treatment at the weld can improve the surface smoothness, reduce the obstruction to the flow of the reaction liquid, accelerate the mixing of the reaction liquid, and improve the reaction efficiency.

[0041] In one embodiment of the present invention, the side of each blade 21 away from the inner wall of the horizontal tank body 2 is a concave arc structure. Figure 4-Figure 6As shown, the side of each blade 21 away from the inner wall of the horizontal tank body 2 is a concave arc structure. It can be clearly seen from the figure that the blade 21 is wide at both ends and narrow in the middle, and the two ends are smoothly transitioned by a concave arc, which can facilitate the reaction liquid to flow between the divided areas and improve the overall mixing efficiency.

[0042] In one embodiment of the present invention, there is an angle a between the length direction of each blade and the axial direction of the horizontal tank body. Preferably, the angle a is ≤ 20°. Figure 4 As shown, the blade 21 is obviously not parallel to the central axis of the tank body, but has a certain angle a, that is, it is inclined to the axis, so that the reaction liquid in each divided area can flow from one end of the tank cavity to the other end. Since the end of the blade 21 is connected to the adjacent area, it can flow to the adjacent area to improve the mixing efficiency. The angle a should not be too large, that is, the blade 21 should not deviate too much from the axis, otherwise the reaction liquid will accumulate at one end of the tank cavity, which is not conducive to dilution.

[0043] In one embodiment of the utility model, the contour surface of each blade 21 and the tangent plane of the horizontal tank body 2 passing through the inner edge of the blade 21 have an angle b, b<90°. To explain, the blade 21 is not completely perpendicular to the tank wall, but deviates from the vertical plane by a certain angle b, which can generate a certain downward pressure or lifting force on the reaction liquid, which depends on the rotation direction of the tank body, and can improve the stirring effect. So as to obtain more complete mixing.

[0044] In one embodiment of the utility model, a plurality of through holes (not shown in the figure) are arranged on the contour surface of each blade 21. In this way, the reaction liquids in each divided area can interact with each other through these through holes, thereby improving the mixing effect.

[0045] In one embodiment of the utility model, the total area of ​​the plurality of through holes on each of the blades 21 accounts for 30%-50% of the area of ​​the blade 21, and the spacing between adjacent through holes is equal, so that the forward and reverse flows of equal speed and amount can be approximately formed, thereby improving the mixing effect.

[0046] In one embodiment of the present invention, the edges of each of the paddles 21 and the edges of the openings of each of the through holes have rounded corners, which can reduce the shear force of the paddles 21 on the active substance.

[0047] In one embodiment of the utility model, the volume of the horizontal tank body 2 is 10L-10000L. As the volume of the tank changes, the self-stirring blade assembly 20 in the tank will also change accordingly, such as increasing or decreasing the number of blades 21 therein to adapt to different working conditions. The specific size of the tank volume can be customized according to the production capacity, and the volume for industrial large-scale production is preferably 1000L.

[0048] In one embodiment of the present invention, Figure 7 As shown, in the working state, the axial direction of the horizontal tank body 2 is parallel to the horizontal plane. The completely horizontal tank structure can achieve maximum dilution of the reaction liquid therein, prevent the reaction liquid from depositing in a certain direction, and improve the reaction efficiency.

[0049] In one embodiment of the utility model, a circle of rolling ribs 23 is provided on the outer wall of the horizontal tank body 2. In the working state, one end of the horizontal tank body 2 is supported by a bearing seat 5 installed on the frame 1, and the other end is supported by a pair of rotating wheels 6 installed on the frame 1, and the rotating wheels 6 are in contact with the rolling ribs 23. In this way, when the horizontal tank body 2 rotates, the rolling ribs 23 roll along the pair of rotating wheels 6, and the rolling ribs protrude from the outer wall of the tank body by a certain thickness, so as to protect the horizontal tank body 2.

[0050] In one embodiment of the utility model, a feed port 25 is provided at one end of the horizontal tank body 2, and a rotating shaft 24 is provided at the other end. The feed port 25 is used for feeding the reaction liquid; the rotating shaft 24 is supported on the bearing seat 5 and connected to the driving device 4 through the pulley transmission mechanism 7 to drive the horizontal tank body 2 to rotate.

[0051] In one embodiment of the utility model, the horizontal tank body 2 is provided with a tank lock 22 for locking the tank cover so that the tank cover can tightly seal the feed port.

[0052] When a horizontal self-stirring in vitro protein synthesis reactor is formed by using the tank device of any embodiment of the tank device of the horizontal self-stirring in vitro protein synthesis reactor as described above to perform an in vitro protein synthesis reaction, the following steps are included: (1) introducing a protein synthesis reaction liquid that occupies 5%-30% of the volume of the horizontal tank body 2 into the horizontal tank body 2 and sealing it; (2) starting the driving device 4 to rotate the horizontal tank body 2 at a preset speed r for a preset time T. The smaller the protein synthesis reaction liquid that occupies the volume of the horizontal tank body 2, the thinner it will be spread during the rotation of the tank body, and the higher the reaction efficiency, but the yield will be reduced, and a certain balance needs to be made between yield and efficiency or activity. Preferably, in the step (2), while starting the driving device 4, the air source device is started, and air with a preset air pressure p is continuously input into the horizontal tank body 2 through the air inlet pipe 31, and the waste gas generated during the reaction is discharged through the exhaust pipe 33. More preferably, in step (2), r=20 rpm-200 rpm, T=1 h-10 h, p=10 kPa-300 kPa.

[0053] It should be noted that the direction of the horizontal tank body 2 can be adjusted by controlling the forward and reverse rotation of the driving device 4, and the mixing effect of the reaction liquid can be improved by intermittently or periodically adjusting the direction of the horizontal tank body 2. It is also possible to improve the mixing effect of the reaction liquid by combining the adjustment of the direction and the rotation speed according to actual needs, and finally achieve the effect of improving the reaction efficiency and the activity of the product.

[0054] Figure 8 The activity test results of the target protein EGFP prepared by the horizontal self-stirring in vitro protein synthesis reactor for in vitro protein synthesis for 1h, 3h and 6h respectively are shown. The volume of the tank device of the horizontal self-stirring in vitro protein synthesis reactor is 50L, the volume of the reaction liquid is 10L, r=50rpm, p=1 standard atmosphere, and the control group is the target protein prepared in the laboratory using a 24-well plate. Figure 8 It can be seen that under the three reaction times, the activity of the target protein EGFP obtained was equivalent to that of the control group, and the activity was well maintained.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A tank device of a horizontal self-stirring in vitro protein synthesis reactor, characterized in that: It includes a horizontal tank body, on the inner wall of which a self-stirring blade assembly is arranged, and the self-stirring blade assembly is suitable for stirring the protein synthesis reaction liquid in the horizontal tank body during the rotation of the horizontal tank body. The self-stirring blade assembly includes a plurality of blades, each of which is integrally formed and fixed to the inner wall of the horizontal tank body by welding.

2. The tank device of the horizontal self-stirring in vitro protein synthesis reactor according to claim 1, characterized in that: The height of the self-stirring blade assembly protruding from the inner wall of the tank body accounts for no more than 60% of the inner radius of the horizontal tank body.

3. The tank device of the horizontal self-stirring in vitro protein synthesis reactor according to claim 1, characterized in that: Each of the paddles extends along the axial direction of the horizontal tank body, and each of the paddles is arranged at intervals along the inner circumference of the horizontal tank body.

4. The tank device of the horizontal self-stirring in vitro protein synthesis reactor according to claim 3, characterized in that: The blades are arranged at equal intervals.

5. The tank device of the horizontal self-stirring in vitro protein synthesis reactor according to claim 3, characterized in that: Each of the blades is welded and fixed to the inner wall of the horizontal tank body and polished.

6. The tank device of the horizontal self-stirring in vitro protein synthesis reactor according to claim 3, characterized in that: The side of each blade away from the inner wall of the horizontal tank body is a concave arc structure.

7. The tank device of the horizontal self-stirring in vitro protein synthesis reactor according to claim 3, characterized in that: There is an angle a between the length direction of each blade and the axial direction of the horizontal tank body.

8. The tank device of the horizontal self-stirring in vitro protein synthesis reactor according to claim 7, characterized in that: The angle a is ≤20°.

9. The tank device of the horizontal self-stirring in vitro protein synthesis reactor according to claim 3, characterized in that: An included angle b is formed between the contour surface of each blade and a tangent plane of the horizontal tank body passing through the inner edge of the blade, and b<90°.

10. The tank device of the horizontal self-stirring in vitro protein synthesis reactor according to any one of claims 3 to 9, characterized in that: A plurality of through holes are arranged on the contour surface of each blade.

11. The tank device of the horizontal self-stirring in vitro protein synthesis reactor according to claim 10, characterized in that: The total area of ​​the multiple through holes on each of the blades accounts for 30%-50% of the area of ​​the blade, and the spacing between adjacent through holes is equal.

12. The tank device of the horizontal self-stirring in vitro protein synthesis reactor according to claim 10, characterized in that: The edge of each blade and the edge of the opening of each through hole have a rounded structure.

13. The tank device of the horizontal self-stirring in vitro protein synthesis reactor according to claim 1, characterized in that: The volume of the horizontal tank body is 10L-10000L.

14. The tank device of the horizontal self-stirring in vitro protein synthesis reactor according to claim 1, characterized in that: In the working state, the axial direction of the horizontal tank body is parallel to the horizontal plane.

15. The tank device of the horizontal self-stirring in vitro protein synthesis reactor according to claim 1, characterized in that: A circle of rolling ribs is arranged on the outer wall of the horizontal tank body.

16. The tank device of the horizontal self-stirring in vitro protein synthesis reactor according to claim 1, characterized in that: A feed port is arranged at one end of the horizontal tank body, and a rotating shaft is arranged at the other end.

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