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

Abstract

The invention discloses a preparation method and application of magnetic microspheres for purifying mRNA (messenger ribonucleic acid). The preparation method of the magnetic microspheres for purifying mRNA comprises the following steps: S1) washing the magnetic microspheres with active groups on the surfaces by using a coupling buffer solution; s2) Oligo dT is added to react with the magnetic microspheres, and then 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 magnetic microspheres for purifying mRNA. The surface of the magnetic microsphere is modified with amino, carboxyl, epoxy group, active ester (NHS) or benzenesulfonyl, so that the magnetic microsphere has reaction activity and can quickly react with Oligo dT, and Oligo dT is fixed on the surface of the magnetic microsphere, so that the magnetic microsphere has mRNA separation and purification capabilities, and because the magnetic microsphere has magnetism, in the presence of a magnetic field, the mRNA separation and purification capability of the magnetic microsphere is greatly improved. The magnetic microspheres can quickly move towards one side of a magnetic field, so that mRNA can be quickly separated and purified.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method and application of magnetic 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] A typical eukaryotic cell contains very little mRNA. Therefore, it is very challenging to isolate and purify mRNA. There are two traditional methods for isolating and purifying mRNA: 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 complicated and expensive, and the filter membranes used as consumables have various specificat...

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 equipment required 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 based on Oligo dT will specifically bind to the poly(A) tail at one end of the mRNA, thereby separating and purifying the mRNA. Although this method has been provided commercially, due to the cellulose itself, it cannot be used for rapid column chromatography. Time-consuming

Oligo dT coupled with magnetic microspheres usually uses expensive foreign brands, and the cut surface needs to be modified with streptavidin, which makes the use cost high and difficult to use on a large scale.

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