Virus-mimicking microcapsules and microfluidic chip for preparing virus-mimicking microcapsules

Abstract

The application provides a virus-mimicking microcapsule and a microfluidic chip for preparing the virus-mimicking microcapsule, and the microcapsule structure is formed by chitosan, polylysine and oleic acid. The microcapsule structure contains integrase and a target DNA fragment inside, can realize gene modification of a host cell, simplifies the overall virus process and an operation process, and makes a gene improvement technology more practical.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, and in particular relates to a virus-like microcapsule and a microfluidic chip for preparing the virus-like microcapsule. Background Technology

[0002] Lentiviral vectors (defective HIV vectors) can effectively integrate exogenous genes or exogenous shRNA into the chromosomes of host cells, thereby achieving persistent expression of the target sequence. This is currently the most common gene-editing technology. After the lentiviral genome enters the cell, it is reverse transcribed into DNA in the cytoplasm, forming a pre-integration DNA complex. Upon entering the nucleus, the DNA integrates into the cell's genome. The integrated DNA is then transcribed into mRNA, returning to the cytoplasm to express the target protein; or it may produce small RNA.

[0003] As a representative of lentiviruses, HIV-1 has certain structural characteristics, which we will briefly introduce using HIV-1 as an example. HIV-1 is a double-stranded RNA virus composed of 9 genes. The gag, pol, and env genes encode the basic viral structure; the tat and rev genes are regulatory genes; and the vif, vpr, vpu, and nef genes are accessory genes. The gag gene encodes the core protein required by the virus; the pol gene encodes the enzyme required for viral replication; and the env gene encodes the viral envelope glycoprotein and determines the virus's targeting of the host. The rev gene encodes a protein that regulates the expression levels of gag, pol, and env; the tat gene encodes a protein involved in the regulation of RNA transcription; and both ends are long terminal repeats (LTRs), which contain cis-acting elements required for replication.

[0004] Currently used, relatively safe viral vectors delete all HIV accessory genes (vif, vpu, vpr, and nef) from the packaging plasmid to reduce the risk of recurrent viral infections (RCRs). The removal of these accessory genes does not affect viral titer or transfection ability, while increasing vector safety. Additionally, the tat gene is removed and replaced with a heterologous promoter sequence. Thus, only three of the nine genes in the original HIV genome (gag, pol, and rev) are retained in the lentiviral vector, making the lentiviral vector system even safer. However, the entire process involves cell culture, plasmid synthesis (combinations of three or four), and viral assembly, resulting in complexity and high cost. Summary of the Invention

[0005] In view of this, the present invention aims to propose a virus-like microcapsule and a microfluidic chip for preparing the virus-like microcapsule, so as to solve the problems of complex operation and high cost in the current production of lentivirus vectors.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] A virus-like microcapsule comprises a microcapsule structure formed by chitosan, polylysine, and oleic acid, wherein the microcapsule structure contains an integrase and a target DNA fragment.

[0008] Furthermore, the preparation of the microcapsule structure includes the following steps:

[0009] Step 1: Take low molecular weight chitosan aqueous solvent and treat it at high temperature to obtain chitosan with even lower molecular weight.

[0010] Step 2: Mix the chitosan obtained in Step 1 with the EDC / NHS solvent, then add polylysine and stir to accelerate the polymerization reaction;

[0011] Step 3: Add oleic acid and EDC / NHS solvent to the polymer obtained in Step 2, stir, and accelerate the polymerization reaction;

[0012] Step 4: Place the polymer obtained in Step 3 at room temperature for 4-6 hours to form 3 distinct liquid layers. Use a pipette to collect the bottom oil layer, which is the polymer with microcapsule structure obtained from the reaction.

[0013] Furthermore, the low molecular weight chitosan in step one has a molecular weight of 10,000-15,000 Dales, and the high-temperature treatment is performed at 100-150°C for 30-120 minutes.

[0014] Furthermore, in step two, the molar ratio of chitosan, EDC / NHS, and polylysine is 1:1-2:0.5-1.5, the stirring speed is 400-600 rpm, and the reaction time is 24-48 hours.

[0015] Furthermore, in step three, the molar ratio of chitosan and polylysine polymer, EDC / NHS, and oleic acid is 2.5:0.5-1.5:0.5-1.5, the stirring speed is 400-600 rpm, and the reaction time is 24-48 hours.

[0016] The second objective of this invention is to provide a microfluidic chip for preparing the aforementioned virus-like microcapsules, comprising a chip body, wherein the chip body has a sample inlet A and a sample inlet B at the front end and a sample outlet at the rear end, and microchannels A and B are provided inside the chip body, one end of microchannel A is connected to sample inlet B and the other end is connected to the sample outlet, one end of microchannel B is connected to sample inlet A and the other end is connected to microchannel A, and microfluidic flow rate control devices are provided at both sample inlet A and sample inlet B.

[0017] Lentiviral vector gene delivery technology is complex to prepare, requiring at least three gene vector plasmids, which are then delivered to 293T cells for virus preparation. After virus preparation, the virus contains the corresponding gene sequence RNA. The virus is then extracted and reinfected into target cells, where a series of tool enzymes, such as reverse transcriptase and integrase, are generated inside the cells to embed the RNA single strand into the target cell's genome. This process takes 5-7 days, is time-consuming, and costly. This invention creates a virus-like vector containing a double-stranded DNA fragment for embedding. By simply adding an integrase, it mimics the final step of viral integration, embedding DNA into the target genome. The process is simple, requiring only the mixing of DNA, enzymes, and biological materials. All steps can be completed within one day, resulting in high efficiency, speed, and a cost reduction of nearly 90%.

[0018] Furthermore, both microchannel A and microchannel B are connected to serpentine channels.

[0019] Furthermore, the microfluidic flow rate control device is an air compressor pump, and the chip body is made of PMMA material.

[0020] Furthermore, the method of using the microfluidic chip includes the following steps:

[0021] S1. After mixing the synthesized DNA double-stranded gene and integrase solution, place them into sample inlet B. Place chitosan + polylysine + oleic acid polymer into sample inlet A, add Span 80, and mix well.

[0022] S2. The liquid in the injection tank A enters the microchannel B through the air compressor pump, forming a stable oil phase liquid surface; the DNA double-stranded gene and integrase solution are introduced into the microchannel A through the injection tank B, forming a stable aqueous liquid surface, and are squeezed into the microchannel B to form an oil-in-water structure with the oil phase liquid surface, and form stable oil-in-water droplets.

[0023] S3. The formed water-in-oil droplets are rapidly squeezed into the sample outlet containing PBS buffer, forming an oil-in-water morphology and ultimately forming a W / O / W microcapsule structure.

[0024] A third objective of this invention is to propose the application of the aforementioned virus-like microcapsules and microfluidic chip in lentiviral vectors.

[0025] Compared with existing technologies, the virus-like microcapsules and the microfluidic chip for preparing virus-like microcapsules described in this invention have the following beneficial effects:

[0026] (1) This invention utilizes novel microfluidic chip microcapsule synthesis technology to form virus-like structures. Corresponding integrase and target DNA fragments are added inside, mimicking the final step in a viral system where integrase inserts exogenous DNA into the host sequence, thus achieving a novel synthetic virus-like microcapsule technology for gene delivery of exogenous DNA. This technology simplifies the overall viral process and operation, requiring only the mixing of DNA, enzymes, and biological materials. All steps can be completed within one day, resulting in high efficiency, rapid speed, and a cost reduction of nearly 90%, making gene modification technology more practical.

[0027] (2) This invention employs microfluidic technology, with microchannels embedded within the chip. A microfluidic flow rate control device compresses the liquid in the injection chamber into the built-in channels. Utilizing the immiscibility between the two liquid surfaces, an emulsified oil-in-water reaction is formed, resulting in water-insoluble, oily microcapsules containing the liquid. These microcapsules then enter the aqueous solvent, forming an oil-in-water state. This technology can better form oily microcapsules similar to viral frameworks, which can be loaded with small molecules such as proteases and DNA, facilitating assembly and enabling delivery to cell hosts. It replaces traditional viral vector methods, simplifying operation and increasing efficiency. Attached Figure Description

[0028] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0029] Figure 1 This is a diagram illustrating the gene integration process of a lentiviral vector.

[0030] Figure 2 This is a diagram of the lentivirus structure.

[0031] Figure 3 This is a diagram illustrating the gene integration process of microcarriers.

[0032] Figure 4 This is a schematic diagram of a lentiviral gene sequence;

[0033] Figure 5 This is a schematic diagram of the viral gene double-stranded DNA sequence (using EGFP as an example);

[0034] Figure 6 The pIN gene map of the constructed plasmid vector;

[0035] Figure 7 It is a polymer of chitosan, polylysine, and oleic acid;

[0036] Figure 8 A side view of a droplet microfluidic chip that can mimic antiviral microcapsules;

[0037] Figure 9 A schematic diagram of a droplet microfluidic chip capable of forming antiviral microcapsules;

[0038] Figure 10 A flowchart illustrating the operation of a droplet microfluidic chip capable of forming antiviral microcapsules;

[0039] Figure 11 A schematic diagram of the reaction that forms oil-in-water microcapsules in a microfluidic channel;

[0040] Figure 12 This is a schematic diagram of the microcapsule structure formed by the polymerization product of chitosan, polylysine, and oleic acid.

[0041] Figure 13 The image shows the observation and interpretation of the expression effect of GFP (green fluorescent protein) inside cells under a fluorescence microscope. In the image, A: 1 day after infection, B: passage 1, C: passage 2, D: passage 5, E: passage 10.

[0042] Explanation of reference numerals in the attached figures:

[0043] 1. Sample inlet A; 2. Sample inlet B; 3. Serpentine channel A; 4. Serpentine channel B; 5. Microchannel A; 6. Microchannel B; 7. Serpentine channel C; 8. Microchannel C; 9. Sample outlet. Detailed Implementation

[0044] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0045] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0046] Example 1

[0047] I. Preparation of the target gene

[0048] Normal lentiviral vector genes have long terminal repeats at both ends, generally referred to as LTRs (U3+R+U5): LTR 5' and LTR 3', containing non-protein-coding sequences and regulatory elements such as promoters and enhancers. This invention uses artificially synthesized double-stranded DNA fragments of the target gene, the specific gene sequence of which is shown in SEQ ID NO.1. Structurally, it adopts a gene construction scheme similar to that of the lentiviral vector (pRRLSIN.cPPT.PGK-GFP.WPRE). The 5' segment uses a lentiviral partial LTR 5' fragment (εU3) and an RSV promoter, while the 3' segment uses a lentiviral partial LTR 3' fragment (ΔU3) and SV40-polyA, forming a new transfected DNA double strand. Simultaneously, a eukaryotic hPKG promoter is used for the intracellular expression of the target protein. For example, EGFP fluorescent protein is used (see...). Figure 5 .

[0049] II. Synthesis of Integrase

[0050] The HIV integrase (IN) protein sequence is shown in SEQ ID NO.2.

[0051] The HIV integrase (IN) gene sequence is shown in SEQ ID NO.3.

[0052] expression carrier

[0053] The prokaryotic expression plasmid vector pcDNA3.1(+) (purchased from Shanghai Langjing Biotechnology Co., Ltd.) was used, and a 6HIS (CATCATCATCATCATCAC) tag was added to the protein end for purification.

[0054] plasmid construction

[0055] Plasmid modification is now highly commercialized, with preparation done by specialized companies; only the gene sequence is required. The plasmid construction of this invention was completed by Shanghai Langjing Biotechnology Co., Ltd., and the constructed plasmid gene sequence is shown in SEQ ID NO.4.

[0056] Methods for preparing integrase

[0057] 1. Culture 293T cells in HyClone RPMI-1640 liquid medium at 37°C and 5% CO2 under aseptic conditions in an ultra-clean laboratory.

[0058] 2. Mix the target plasmid vector pIN with polyethyleneimine (PEI) at a ratio of 0.1 ng / ml and add it to the culture supernatant of 293T cells (1 ml of the mixture is added to 10 ml of culture supernatant) for transfection.

[0059] 3. After culturing for 5 days, the culture supernatant was collected for purification.

[0060] Integrase purification methods

[0061] Equilibration buffer: 20 mM PB, 50 mM Tris-HCl, 500 mM NaCl, 20% ethanol, pH 7.4 Washing buffer: 20 mM MPB, 50 mM Tris-HCl, 500 mM NaCl, 1% ethanol, pH 7.4 Elution buffer: 20 mM PB, 50 mM Tris-HCl, 500 mM NaCl, 10% imidazole, pH 7.4

[0062] 1. Equilibrate the resin column containing HIS-tagged affinity (purchased from Zhongke Senhui Microsphere Technology (Suzhou) Co., Ltd., metal chelate affinity chromatography medium (Ni-NTA)) with equilibration buffer, and rinse twice.

[0063] 2. Add the buffer solution containing the integrase sample to the pre-equilibrated resin column.

[0064] 3. After loading the sample, continue rinsing with equilibration buffer until baseline is reached. At this point, the target protein integrase is adsorbed by the resin column.

[0065] 4. Wash with 10 column volumes of washing buffer, repeating three times, to remove impurity proteins.

[0066] 5. Elute with 3 column volumes of elution buffer to remove the target protein integrase adsorbed by the resin column and recover it.

[0067] 6. Place the eluent into a dialysis bag with a molecular weight of 1 kDa and dialyze at 4°C for 24 hours. The dialysate is 1XPBS.

[0068] 7. Remove the liquid containing integrase from the dialysis bag and store it at -80℃ for later use.

[0069] III. Microencapsulation Synthesis of Virus-like Structures

[0070] The microcapsule structure material is a composite of chitosan, polylysine, and oleic acid, such as... Figure 7 As shown.

[0071] Chitosan is a natural polysaccharide (Beta-[1f-4]-2-amino-2-deoxy-D-glucopyranose), a product of chitin production. Because it carries free amino groups, it is a cationic polymer and exhibits good biocompatibility.

[0072] Poly-L-Lysine (PLL) is a biodegradable cationic polypeptide with strong cationic properties. Its molecular weight ranges from 1000 to 5000 g / mol.

[0073] Oleic acid, with the chemical formula C18H34O2, is a monounsaturated Omega-9 fatty acid found in both plants and animals. Hydrogenation of oleic acid yields stearic acid. The trans isomer of oleic acid is called trans-oleic acid.

[0074] Synthesis method:

[0075] Step 1: Use 10000-15000 Dale low molecular weight chitosan (mass-volume ratio 3g / 100ml, 3g dissolved in 100ml) water solvent for high-temperature treatment, repeating twice at 120℃ for 40 minutes (first time: 120℃, 40 minutes, then once more at 120℃, 40 minutes). The purpose is to obtain chitosan with a lower molecular weight. After the reaction, the chitosan stock solution is dark yellow.

[0076] Step 2: Mix the chitosan and EDC / NHS obtained in Step 1, and then add polylysine. The molar ratio of chitosan, EDC / NHS and polylysine is 1:1.5:1. Use a magnetic stirrer to stir at a speed of 500 rpm to accelerate the polymerization reaction. The reaction time is 48 hours.

[0077] Step 3: Add oleic acid and EDC / NHS to the polymer obtained in Step 2. The molar ratio of chitosan and polylysine polymer, EDC / NHS and oleic acid is 2.5:1:1. Use a magnetic stirrer to stir at a speed of 500 rpm to accelerate the polymerization reaction. The reaction time is 36 hours.

[0078] Step 4: The chitosan + polylysine + oleic acid polymer obtained in Step 3 is an oily substance that is not easily soluble in water. Its density is higher than that of water molecules and oleic acid, so it remains at the bottom of the entire reaction solution. After stopping the polymerization reaction, it is left at room temperature for 5 hours, forming obvious liquid separation (3 layers). The bottom oil layer is collected with a pipette, which is the polymer obtained from the reaction.

[0079] Example 2

[0080] like Figure 8 and Figure 9As shown, another objective of this invention is to provide a microfluidic chip capable of forming a virus-like vector, comprising a chip body made of PMMA (polymethyl methacrylate) material. The chip body has a sample inlet A1 and a sample inlet B2 at its front end, with the bottoms of the inlet A1 and B2 embedded in the chip body. The chip body also has an outlet slot 9 at its rear end. The sample inlet A1 and B2 are cubical. The bottom of the sample inlet A1 is connected to a serpentine channel B4, which is connected to a microchannel B6 (60 μm aperture). The bottom of the sample inlet B2 is connected to a serpentine channel A3 (25 μm aperture), which is connected to a microchannel A5. The microchannel A5 is connected to a serpentine channel C7, which is connected to a microchannel C8. The microchannel C8 merges into the microchannel B6. The microchannel B6 communicates with the bottom of the outlet slot 9. The outlet slot 9 is cuboid. The main purpose of designing a serpentine channel is to alleviate and coordinate the liquid flow rate and pressure, thereby promoting the formation of a highly stable liquid with a uniform flow rate.

[0081] When using, mix the synthesized DNA double-stranded gene and integrase solution and place them into sample inlet B2. Place the chitosan + polylysine + oleic acid polymer into sample inlet A1, and add 30% of the polymer mass of Span 80. Mix well. Span 80 is an emulsifier that can separate oil and water to form a water-in-oil effect. Otherwise, stable microspheres cannot be formed. Liquid in sample inlet A1 flows into serpentine channel B4 at a flow rate of 100 μL / min via an air compressor pump, and then enters microchannel B6, forming a stable oil phase surface. DNA double-stranded gene and integrase solution are introduced into serpentine channel A3 via sample inlet B2 at a flow rate of 10 μL / min via an air compressor pump, and then sequentially pass through microchannel A5, serpentine channel C7, and microchannel C8, forming a stable aqueous phase surface. This aqueous phase is then squeezed into microchannel B6 to form a water-in-oil structure with the oil phase surface, forming stable droplets. The formed water-in-oil droplets are then rapidly squeezed into sample outlet 9 containing 50 ml of PBS buffer (5% Tween 20), forming an oil-in-water morphology, and ultimately forming a W / O / W microcapsule structure (see...). Figure 11 and Figure 12 ).

[0082] This chip utilizes microfluidic technology, featuring microchannels within its interior. An air compressor pump (purchased from Shanghai Pengzan Biotechnology Co., Ltd.) forces liquid from the sample inlet into these channels at a controlled flow rate. Leveraging the immiscibility between the two liquids, an emulsified water-in-oil reaction occurs, creating water-insoluble, oily microcapsules containing the liquid. These microcapsules then enter the aqueous solution, forming an oil-in-water emulsion. This technology allows for the formation of oily microcapsules resembling viral frameworks, capable of loading small molecules such as proteases and DNA. This facilitates assembly and enables delivery to cell hosts, replacing traditional viral vector methods with simplified and highly efficient operation.

[0083] In addition to microfluidic chips, liposome preparation can also be referenced, including ① injection method; ② thin film dispersion method; ③ ultrasonic dispersion method; ④ reverse phase evaporation method; ⑤ freeze drying method.

[0084] Application examples

[0085] Microcapsules containing double-stranded DNA with GFP (green fluorescent DNA) and integrase were prepared according to the procedure.

[0086] 293T cells were cultured in HyClone RPMI-1640 liquid medium with 10% fetal bovine serum added, under aseptic conditions of 37°C and 5% CO2 in an ultra-clean laboratory.

[0087] 293T cells were cultured at a density greater than 75%, and the culture medium in the culture dish was emptied.

[0088] Add the microcapsules to the serum-free culture medium at a ratio of 100 ng: 10 ml and mix well.

[0089] The culture medium containing microcapsules was added to a 293T cell culture dish and incubated at 37°C with 5% CO2 for 5 hours. After incubation, the culture medium was emptied and serum-containing culture medium was added again.

[0090] After two days of cultivation, the cells are passaged, with passage occurring every two days.

[0091] We tested the cells 1 day after infection, at passage 1, passage 2, passage 5, and passage 10 to determine whether the GFP gene was present in the cells and had been stably embedded in the gene sequence of the 293T cell line.

[0092] Detection methods include fluorescence microscopy observation and Q-PCR detection:

[0093] The expression effect of GFP (green fluorescent protein) inside cells was observed and analyzed using fluorescence microscopy. The detection results are shown in [Figure number missing]. Figure 13 .

[0094] Q-PCR was used to specifically detect the GFP gene in the extracted cells (the average number of cells extracted each time was basically the same) (repeated 3 times), mainly targeting the detection of its DNA content. The detection results are shown in Table 1.

[0095] Table 1. Results of DNA content detection

[0096] 1 0 <![CDATA[1.5X10 2 ]]> <![CDATA[2.1X10 3 ]]> <![CDATA[1.5X10 5 ]]> <![CDATA[1.5X10 6 ]]> <![CDATA[2.1X10 7 ]]> 2 0 <![CDATA[2.0X10 2 ]]> <![CDATA[4.7X10 3 ]]> <![CDATA[3.3X10 5 ]]> <![CDATA[5.0X10 6 ]]> <![CDATA[3.5X10 6 ]]> 3 0 <![CDATA[2.2X10 2 ]]> <![CDATA[1.3X10 3 ]]> <![CDATA[4.1X10 5 ]]> <![CDATA[8.2X10 5 ]]> <![CDATA[1.5X10 7 ]]>

[0097] The results showed that after passage, infected cells could significantly replicate the DNA fragment and effectively express the fluorescent protein GFP. However, if the DNA double strand is not inserted into the host 293T cell genome, it loses the opportunity for replication and amplification; therefore, after passage, the gene will be degraded and the fluorescence effect will gradually disappear. This experiment demonstrated that after several passages, a large number of cells still clearly expressed the target fluorescent GFP protein, and the amount of DNA gradually increased and tended to stabilize, indicating the presence of the GFP gene sequence in the host 293T cell genome, and also proving the reliability of this experiment. This demonstrates that this method can insert the target gene into the host cell gene sequence and maintain stability, with results similar to those of lentiviral vector infection, sufficiently proving the feasibility of this technology.

[0098] 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 within the protection scope of the present invention.

Claims

1. A virus-like microcapsule, characterized in that: The virus-like microcapsules are W / O / W microcapsule structures formed by an inner aqueous phase, an oil phase, and an outer aqueous phase; the inner aqueous phase contains integrase and target DNA fragments; the oil phase contains chitosan + polylysine + oleic acid polymer and Span 80; the outer aqueous phase is PBS buffer containing Tween 20. A DNA double-stranded gene and integrase solution were introduced into the microfluidic chip as the internal aqueous phase. Chitosan, polylysine, oleic acid polymer and Span 80 were mixed and introduced into the microfluidic chip as the oil phase, so that the internal aqueous phase and oil phase formed water-in-oil droplets. The water-in-oil droplets were then introduced into PBS buffer containing Tween 20 to form a W / O / W microcapsule structure.

2. The virus-like microcapsule according to claim 1, characterized in that: The preparation method of the chitosan + polylysine + oleic acid polymer includes the following steps: Step 1: Take low molecular weight chitosan aqueous solvent and treat it at high temperature; Step 2: Mix the chitosan obtained in Step 1 with the EDC / NHS solvent, then add polylysine and stir to accelerate the polymerization reaction; Step 3: Add oleic acid and EDC / NHS solvent to the polymer obtained in Step 2, stir, and accelerate the polymerization reaction; Step 4: Place the polymer obtained in Step 3 at room temperature for 4-6 hours to form 3 distinct liquid layers. Use a pipette to collect the bottom oil layer, which is the polymer with microcapsule structure obtained from the reaction.

3. The virus-like microcapsule according to claim 2, characterized in that: The low molecular weight chitosan in step one has a molecular weight of 10,000-15,000 Dales and is treated at 100-150°C for 30-120 minutes.

4. The virus-like microcapsule according to claim 2, characterized in that: In step two, the molar ratio of chitosan, EDC / NHS, and polylysine is 1:1-2:0.5-1.5, the stirring speed is 400-600 rpm, and the reaction time is 24-48 hours.

5. The virus-like microcapsule according to claim 2, characterized in that: In step three, the molar ratio of chitosan and polylysine polymer, EDC / NHS, and oleic acid is 2.5:0.5-1.5:0.5-1.5, the stirring speed is 400-600 rpm, and the reaction time is 24-48 hours.

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