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Preparation method and application of microspheres for purifying mRNA (messenger ribonucleic acid)

A microsphere and purification column technology, applied in the biological field, can solve the problems of inability, expensive, complicated equipment and so on

Pending Publication Date: 2022-04-12
SUZHOU KNOWLEDGE & BENEFIT TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the TFF method is to filter impurities according to the different molecular weight cut-offs of the filter membranes. The required equipment is complex and expensive, and the filter membranes used as consumables have various specifications, and the replacement is cumbersome; the oligo(dt) modified cellulose chromatography column method is According to Oligo(dt) will specifically bind to the poly(A) tail at one end of the mRNA, so as to separate and purify the mRNA
Although this method has been provided commercially, due to the cellulose itself, fast column chromatography cannot be performed, and the process is time-consuming and laborious.

Method used

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  • Preparation method and application of microspheres for purifying mRNA (messenger ribonucleic acid)
  • Preparation method and application of microspheres for purifying mRNA (messenger ribonucleic acid)
  • Preparation method and application of microspheres for purifying mRNA (messenger ribonucleic acid)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] A kind of preparation method of the microsphere that is used to purify mRNA, comprises the steps, gets the rigid polystyrene microsphere (knowledge benefit) that surface has epoxy active group brand), wash 5 times with coupling buffer, add Oligo dT according to the ratio of 5nmol / mL polystyrene microspheres, mix well at 25°C, and react for 5 hours. After the reaction, use coupling buffer Wash the microspheres to wash away unreacted Oligo dT; add blocking reagent, mix well at 25°C, and react for 5 hours. After the reaction, wash the microspheres with washing buffer, and then place the prepared Oligo dT The microspheres were added to the storage buffer for later use.

[0027] The application of the microspheres prepared above for the purification of mRNA is as follows: take an empty column tube of appropriate size, use gravity sedimentation or use a column packing machine, etc., and fill the microspheres with Oligo dT coupled on the surface into the empty column tube to ...

Embodiment 2

[0030] A kind of preparation method of the microsphere that is used to purify mRNA, comprises the steps, gets the rigid polystyrene microsphere (knowledge benefit) that surface has benzenesulfonyl active group brand), wash 3 times with coupling buffer, add Oligo dT according to the ratio of 1nmol / mL polystyrene microspheres, mix well at 25°C, and react for 3 hours. After the reaction, use coupling buffer Wash the microspheres to wash away the unreacted Oligo dT; add the blocking reagent, mix well at 25°C, and react for 3 hours. The microspheres were added to the storage buffer for later use.

[0031] The application of the microspheres prepared above for the purification of mRNA is as follows: take an empty column tube of appropriate size, use gravity sedimentation or use a column packing machine, etc., and fill the microspheres with Oligo dT coupled on the surface into the empty column tube to obtain mRNA purification column.

[0032] The specific purification method is: d...

Embodiment 3

[0034] A kind of preparation method of the microsphere that is used to purify mRNA, comprises the steps, gets the rigid polystyrene microsphere (knowledge benefit) that surface has active ester (NHS) active group brand), wash 3 times with coupling buffer, add Oligo dT according to the ratio of 1nmol / mL polystyrene microspheres, mix well at 37°C, and react for 3 hours. After the reaction, use coupling buffer Wash the microspheres to wash away the unreacted Oligo dT; add the blocking reagent, mix well at 37°C, and react for 2 hours. The microspheres were added to the storage buffer for later use.

[0035] The application of the microspheres prepared above for the purification of mRNA is as follows: take an empty column tube of appropriate size, use gravity sedimentation or use a column packing machine, etc., and fill the microspheres with Oligo dT coupled on the surface into the empty column tube to obtain mRNA purification column.

[0036] The specific purification method is...

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Abstract

The invention discloses a preparation method and application of microspheres for purifying mRNA (messenger Ribonucleic Acid), and the preparation method of the microspheres for purifying mRNA comprises the following steps: S1) washing polystyrene microspheres with active groups on the surface by using a coupling buffer solution; s2) Oligo dT is added to react with the polystyrene microspheres, and after the reaction is finished, a coupling buffer solution is used for cleaning; and S3) adding a blocking reagent for reaction, and after the reaction is finished, cleaning with a coupling buffer solution to prepare the microspheres for purifying mRNA. The surface of the rigid polystyrene microsphere is modified with amino, carboxyl, epoxy group, active ester (NHS) or benzenesulfonyl, so that the rigid polystyrene microsphere has reaction activity and can quickly react with Oligo dT, and Oligo dT is fixed on the surface of the microsphere, so that the microsphere has mRNA separation and purification capabilities; and the microspheres have rigid structures, so that the microspheres can bear a certain pressure, and the Oligo dT microspheres are loaded into a chromatographic column, so that mRNA can be rapidly separated and purified on equipment such as liquid chromatography and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method and application of microspheres for purifying mRNA. Background technique [0002] In eukaryotes, mature mRNA molecules are transferred from the nucleus to the cytoplasm and used as a direct template for protein synthesis. Therefore, mRNA is of great significance in the fields of molecular biology and vaccine preparation. [0003] The total amount of various types of RNA in a typical eukaryotic cell is about 15pg, and mRNA only accounts for about 3% of it. Therefore, it is extremely challenging to isolate and purify mRNA. Traditional methods for isolating and purifying mRNA include the following two methods: (1) tangential flow filtration (TFF); (2) oligo(dt) modified cellulose chromatography column method. Among them, the TFF method is to filter impurities according to the different molecular weight cut-offs of the filter membranes. The required equipment is co...

Claims

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Application Information

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IPC IPC(8): B01J20/26B01J20/28B01J20/30B01D15/08C12N15/10
Inventor 阳承利李雪
Owner SUZHOU KNOWLEDGE & BENEFIT TECH CO LTD
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