CN-116515627-B - Bioreactor and application thereof

CN116515627BCN 116515627 BCN116515627 BCN 116515627BCN-116515627-B

Abstract

The invention provides a bioreactor, which is a self-mixing device and comprises an extrusion module, a flexible reaction cavity, wherein the flexible reaction cavity is internally provided with DNA hydrogel particles with magnetic cores, the extrusion module can extrude the flexible reaction cavity by a mechanical extrusion or pneumatic extrusion method to deform the flexible reaction cavity, the extrusion module repeatedly extrudes the flexible reaction cavity, and a reaction system in the flexible reaction cavity is uniformly mixed and can be used for RNA in-vitro transcription. By adopting the technical scheme of the invention, the reaction liquid in the reaction cavity is automatically and uniformly mixed in a self-mixing mode of mechanical extrusion or pneumatic extrusion, the contact of other uniform mixing machines to the reaction system is reduced, the pollution is avoided, and meanwhile, the products in the reaction cavity are discharged in an extrusion mode after the reaction is finished, so that the contact between the reaction cavity and hydrogel and the outside is effectively reduced.

Inventors

ZHANG YIHENG
CUI JINHUI
Request for anonymity
YANG XIUZHU

Assignees

苏州珀罗汀生物技术有限公司

Dates

Publication Date: 20260512
Application Date: 20230517

Claims (5)

1. The method for in vitro transcription of RNA is characterized in that the method is implemented based on a bioreactor for in vitro transcription of RNA, wherein the bioreactor is a self-mixing reactor and comprises an extrusion module, a flexible reaction cavity and a magnet capturing module, wherein magnetic core DNA hydrogel particles are arranged in the flexible reaction cavity; the extrusion module is used for deforming the flexible reaction cavity through a mechanical extrusion method, the mechanical extrusion method comprises the steps of converting circular motion into linear reciprocating motion of the extrusion module through a mechanical operation part and a limiting part to extrude the flexible reaction cavity, wherein the surface of the mechanical operation part is provided with a guide groove, the limiting part is movably arranged on the guide groove, the guide groove is provided with an irregularly-shaped closed-loop track taking the flexible reaction cavity as the center, the guide groove is provided with a plurality of limiting points, the distances between two adjacent limiting points and the outer wall of the flexible reaction cavity are unequal, when the mechanical operation part is operated, the limiting points are rotated to the position of the limiting part, the extrusion module can extrude the outer wall of the flexible reaction cavity, the limiting part is arranged between the flexible reaction cavity and the limiting part and is fixedly arranged with the limiting part, the limiting part is used for limiting the movement range of the extrusion module, and along the track along the linear motion of the hydrogel along the guide groove along with the rotation of the mechanical operation part, and the hydrogel can capture DNA particles in the flexible reaction cavity by using the hydrogel particles repeatedly moving along the linear motion of the hydrogel; The method comprises reacting the magnetic core DNA hydrogel particles as a template with T7 RNA polymerase as a catalytic enzyme in a system containing buffer solution and NTP to generate mRNA product in the bioreactor; And in the reaction process, the extrusion module is used for repeatedly extruding the flexible reaction cavity, so that the self-mixing of the reaction system in the flexible reaction cavity is realized.
2. The method according to claim 1, wherein the mechanical operation member is a gear or a turbine, and the extrusion module is linearly moved within a limited movement range of the limiting member by driving the circumferential movement of the mechanical operation member, so that the extrusion module approaches to or separates from the flexible reaction chamber, and the repeated extrusion of the outer wall of the flexible reaction chamber is achieved.
3. The method for in vitro transcription of RNA according to claim 1, wherein the limiting component comprises a limiting plate and a plurality of limiting rods, wherein the limiting plate is provided with limiting grooves corresponding to the limiting rods in number, and two ends of the limiting rods are respectively and movably arranged between the limiting grooves and the guide grooves; The limiting groove can limit the movement range of the limiting rod, and the movement range is equal to the difference value between the maximum radius and the minimum radius of the irregular track; The limiting component can reciprocate along the track of the guide groove, and the limiting groove can limit the movement range of the limiting component.
4. The method according to claim 3, wherein the movement of the pressing module on the orbit between the two limiting points allows the flexible reaction chamber to be continuously switched between the pressing and releasing modes instead of being constantly pressed or released when the limiting rod moves on the orbit between the two limiting points.
5. The method for in vitro transcription of RNA according to claim 1, wherein the preparation method of the magnetic core DNA hydrogel particles comprises the steps of preparing the magnetic core DNA hydrogel particles by adopting a high molecular compound of a functional group, magnetic particles and a DNA template, wherein the DNA template can be selectively modified with the functional group, and the functional group is any one or a combination of a plurality of amide, sulfhydryl, amino or azido groups.

Description

Bioreactor and application thereof Technical Field The invention relates to the technical field of RNA in-vitro transcription, in particular to a bioreactor and application thereof. Background MRNA is transcribed from DNA and functions to transfer genetic information from within the DNA to the protein. In recent years, mRNA vaccines are regarded as a potentially huge vaccine technology because of their biosafety, short production time and low cost compared with traditional vaccines. mRNA vaccine has great potential in preventing and treating infectious diseases, tumors and other hidden diseases. The first step of the traditional mRNA vaccine production technology is to obtain corresponding plasmid DNA by amplifying a large amount of plasmid DNA in escherichia coli bacteria, performing cleavage extraction, and then cutting a required DNA fragment in the plasmid by restriction endonuclease to obtain the required linear template DNA. Since large amounts of template DNA are required for large-scale production of mRNA vaccines in vitro, the time costs and raw material costs for culturing and linearizing the corresponding bacteria are relatively high. The Chinese patent CN112334579A discloses a bioreactor for RNA in vitro transcription, and discloses a RNA in vitro transcription reactor, wherein magnetic particles are used as cores, and linear DNA is fixed on the magnetic particles so as to achieve the effect of recycling. In the automated apparatus, the reaction chambers are of ellipsoidal internal geometry and have surrounding electromagnetic arrays around them. The reactor can achieve the effects of mixing the liquid in the reaction cavity and adsorbing the magnetic particles to discharge other liquid in the reaction cavity and removing the magnetic particles by changing the magnetism of the electromagnetic array. The technical scheme realizes the mixing and stirring of the reaction system solution through the electromagnetic array acting on the magnetic particles, namely, in the reactor, the DNA template fixed with the magnetic particles is used as the magnetic layer of stirring to realize the stirring function, but the shearing force generated by the eddy current or spiral operation of the magnetic particles in the reactor is higher. The Chinese patent CN 110272982A discloses a DNA composite hydrogel and a preparation method thereof, wherein the prepared gel particles with the magnetic core are applied to the production of cell-free protein, can be recycled, obviously, the DNA template particles with the magnetic core can be recycled, and the cost is obviously reduced. Based on the prior art, the application provides the in vitro transcription reactor with the flexible material as mRNA for further optimizing the process and reducing the cost of in vitro transcription of mRNA, and the self-mixing of the reaction system in the reactor is realized by using the flexible characteristic of the outer wall of the reactor through an external pressure extrusion method, so that the contact between the reaction system and a DNA template and the external environment are avoided, and the damage of internal shearing force to the reaction system is effectively reduced. Disclosure of Invention In view of the above, the present invention provides a bioreactor, which is a self-mixing flexible reactor, and adopts a self-mixing mode of mechanical extrusion or pneumatic extrusion to automatically mix the reaction solution in the reaction cavity, so as to effectively reduce the contact between the reaction cavity and the hydrogel and the outside, and reduce the damage of internal shearing force to the reaction system while effectively mixing. In order to achieve the aim, the invention provides a bioreactor which is a self-mixing device or a self-mixing flexible reactor and comprises an extrusion module, a driving module, a reaction module and a magnet capturing module, wherein the driving module can drive the extrusion module to reciprocate, the reaction module comprises a flexible reaction cavity, DNA hydrogel particles with magnetic cores are arranged in the flexible reaction cavity, the extrusion module can repeatedly extrude the flexible reaction cavity to uniformly mix a reaction system in the flexible reaction cavity so as to realize RNA in-vitro transcription, and the magnet capturing module is used for capturing the DNA hydrogel particles with the magnetic cores in the flexible reaction cavity and preventing the DNA hydrogel particles from falling out of the flexible reaction cavity, so that the DNA hydrogel particles can be reused. As a preferred embodiment, the flexible reaction cavity is made of PDMS. Preferably, the extrusion module deforms the flexible reaction cavity by a mechanical extrusion or pneumatic extrusion method, so that the reaction system is self-uniformly mixed. The driving component drives the mechanical operation component to perform circular motion and convert the circular motion into linear reciproca