Oligo-dT affinity chromatography filler and preparation method thereof

Abstract

The present invention provides an Oligo-dT affinity chromatography filler and a preparation method thereof. The chromatography material is prepared from vinyl sulfone-functionalized microspheres and Oligo-dT. The preparation method of the Oligo-dT affinity chromatography filler is as follows: 1) in a saline coupling buffer with a pH of 5-10, Oligo-dT that is not pre-modified at the 5' end and 3' end is reacted with vinyl sulfone-functionalized microspheres for 3 to 24 hours; 2) excess vinyl sulfone is blocked with a blocking reagent to obtain an Oligo-dT affinity chromatography filler. The Oligo-dT affinity chromatography filler prepared by the present invention has a high Oligo-dT density, a simple preparation method, high coupling efficiency, and does not require pre-modification of the 5' end and 3' end of Oligo-dT, thereby reducing production costs.

Description

Technical Field

[0001] The invention belongs to the technical field of chromatography filler preparation in the field of biochemical engineering, and particularly relates to an affinity chromatography filler with Oligo-dT as a ligand and a preparation method thereof. Technical Background

[0002] Oligo-dT (Oligo-dT)-based affinity chromatography is widely used in the purification of messenger RNA (mRNA). Messenger RNA (mRNA) is widely used in cDNA library construction, gene therapy, infectious disease treatment, and vaccine therapy. During mRNA production, chromatographic purification methods are required to remove impurities generated during the in vitro transcription (IVT) process. mRNA and the drugs it is constructed from typically contain a 3′-polyadenosine tail (polyA), which specifically binds to oligo-dT probes on solid supports, thereby achieving affinity purification.

[0003] Current methods for producing affinity chromatography media typically require functionalization of the 5' end of the oligo-dT prior to coupling to a solid support via a linker. This method of oligonucleotide functionalization is cumbersome and expensive, increasing the production cost of oligo-dT affinity chromatography media. Furthermore, few oligo-dT affinity chromatography media suitable for large-scale production are available on the market, making obtaining large quantities of mRNA a challenge. Summary of the Invention

[0004] The purpose of the present invention is to provide an affinity chromatography filler with Oligo-dT as a ligand and a preparation method thereof, which not only has a high target loading capacity but also has low cost and is easy to operate.

[0005] The Oligo-dT affinity chromatography filler of the present invention is prepared from vinyl sulfonated microspheres and Oligo-dT, wherein the 5' and 3' ends of the Oligo-dT do not require pre-modification. The preparation method comprises the following steps:

[0006] 1) reacting unmodified oligo-dT at the 5' and 3' ends with vinyl sulfone-functionalized microspheres in a saline coupling buffer at pH 5-10 at 15-40° C. for 3-24 hours to obtain oligo-dT-functionalized microspheres;

[0007] 2) Treating the Oligo-dT functionalized microspheres obtained in step 1) with a blocking reagent in a blocking buffer solution at 15-40° C. for 6-24 hours, blocking excess vinyl sulfone with the blocking reagent, and obtaining an Oligo-dT affinity chromatography filler.

[0008] Based on the technical solution described above, preferably, the preparation method of the vinyl sulfonated microspheres described in step 1) is: using an organic base as a catalyst, bis(vinylsulfonyl)methane, an organic base, and the microspheres after thorough dehydration are placed in an organic solvent, and the reaction is shaken at 10-60°C for 3-24 hours.

[0009] Based on the technical solution described above, preferably, the microspheres are hydrophilic microspheres having polyhydroxyl or polyamino structures on the surface.

[0010] In the technical solution described above, preferably, the hydrophilic microspheres are agarose microspheres, or polymethacrylate microspheres modified with hydroxyl or amino groups, polyethylene-divinylbenzene microspheres, silica microspheres or magnetic microspheres (such as ferromagnetic microspheres).

[0011] Based on the technical solution described above, preferably, the diameter of the microspheres is 1-100 μm.

[0012] In the technical solution described above, preferably, the organic base is selected from the group consisting of triphenylphosphine, tricyclohexylphosphine, triisopropylphosphine, tritolylphosphine, tri-p-tolylphosphine, and methylimidazole.

[0013] In the technical solution described above, the organic solvent is an aprotic solvent, preferably dichloromethane, N,N-dimethylformamide or acetonitrile.

[0014] For the technical solution described above, preferably, the molar ratio of the bis(vinylsulfonyl)methane to the organic base is 10-1000:1, preferably 10-200:1, and more preferably 10-80:1; the mass ratio of the bis(vinylsulfonyl)methane to the microspheres after dehydration is 1:1-1000, preferably 1-100:1, and more preferably 1-50:1.

[0015] In the technical solution described above, preferably, the concentration of the bis(vinylsulfonyl)methane in the organic solvent is 50-100 mM.

[0016] In the technical solution described above, the structure of the Oligo-dT without pre-modification at the 5' and 3' ends is as follows:

[0017] 5'-R-Oligo-dT-3',

[0018] Where R is the spacer arm and Oligo-dT is the affinity ligand;

[0019] The spacer arm is 5-20 deoxyribonucleotides, and the 5' end does not need to be modified; the Oligo-dT is a polydeoxythymidine with 10-40 bases, preferably 15-30 bases. For example, Oligo-dT is 5'-G5T 25 -3', that is, the structure from the 5' end to the 3' end is 5 deoxyguanosines and 25 deoxythymidines. 30 -3', A 30 It is oligodeoxyadenosine.

[0020] In the technical solution described above, preferably, the molar ratio of the unmodified Oligo-dT at the 5' and 3' ends in step 1) to the vinyl sulfone in the vinyl sulfone functionalized microspheres is 1:100-100000, preferably 1:1000-100000, more preferably 1:5000-50000.

[0021] In the technical solution described above, preferably, the concentration of the unmodified Oligo-dT at the 5' and 3' ends in step 1) in the saline buffer solution is 0.1-100 uM, preferably 1-50 uM.

[0022] For the technical solution described above, preferably, the salt in the salt-containing coupling buffer described in step 1) is one or more of sodium sulfate, sodium chloride, sodium oxalate, sodium acetate, ammonium sulfate, and sodium citrate, and the concentration of the salt in the salt-containing coupling buffer is 0.0i-2.5M, preferably 0.0i-1.5M.

[0023] In the technical solution described above, preferably, the pH in step 1) is 6-9.

[0024] In the technical solution described above, preferably, the coupling buffer in the salt-containing coupling buffer in step 1) is a phosphate buffer.

[0025] For the technical solution described above, preferably, the blocking reagent in step 2) is one or more of reduced glutathione, natural amino acids, 2-mercaptoethanol, ethanolamine, bovine serum albumin, and casein, and the natural amino acid is one or more of L-cysteine, L-glycine, L-arginine, and L-lysine.

[0026] In the technical solution described above, preferably, the blocking buffer in step 2) is phosphate buffer, citrate buffer, carbonate buffer or 4-hydroxyethylpiperazineethanesulfonic acid buffer (HEPES).

[0027] In the technical solution described above, preferably, the pH value of the blocking buffer in step 2) is 5-10.

[0028] In the technical solution described above, preferably, the molar ratio of the blocking reagent in step 2) to the vinyl sulfone in the Oligo-dT functionalized microspheres is 1-10:1.

[0029] In the technical solution described above, preferably, the concentration of the blocking reagent in step 2) in the blocking buffer is 10-1000 mM, preferably 10-200 mM.

[0030] Based on the technical solution described above, preferably, the method further comprises: 3) hybridizing the Oligo-dT affinity chromatography filler obtained in step 2) with poly A in a salt-containing hybridization buffer at 15-40° C. for 0.5-30 min.

[0031] Based on the technical solution described above, preferably, the salt concentration in the salt-containing hybridization buffer in step 3) is 0.1-1M, preferably 0.25-1M.

[0032] Based on the technical solution described above, preferably, the hybridization buffer in the salt-containing hybridization buffer in step 3) is a phosphate buffer or a carbonate buffer, and the pH value of the phosphate buffer or the carbonate buffer is 7-7.4.

[0033] In the technical solution described above, preferably, the molar ratio of the affinity chromatography filler of Oligo-dT to poly A in step 3) is 1-20:1, preferably 1-10:1, and more preferably 1-5:1.

[0034] In the technical solution described above, preferably, the concentration of the poly A in step 3) in the salt-containing hybridization buffer is 0.1-100 uM, preferably 1-50 uM.

[0035] The present invention also relates to the Oligo-dT affinity chromatography filler prepared by the protection method.

[0036] Beneficial effects:

[0037] The method for preparing the Oligo-dT affinity chromatography filler of the present invention has the following advantages over other methods:

[0038] (1) The target loading capacity is high. The reaction between hydrophilic microspheres and bis(vinylsulfonyl)methane can provide a large number of vinyl sulfone active sites for Oligo-dT coupling, thereby increasing the density of Oligo-dT on the microsphere surface.

[0039] The reaction steps are simpler, the reaction yield is high, and the synthesis cost is lower. The vinyl sulfone functionalized microspheres can directly (2) react with unmodified Oligo-dT at the 5' and 3' ends, eliminating the Oligo-dT functionalization modification step, thereby reducing the production cost of Oligo-dT affinity chromatography fillers.

[0040] In summary, the prepared Oligo-dT affinity chromatography filler has a high Oligo-dT density, a simple preparation method, high coupling efficiency, and does not require pre-modification of the 5' and 3' ends of Oligo-dT, thereby reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 : Reaction formula of agarose 6FF and BVS;

[0042] Figure 2 : Reaction formula of 6FF-BVS and blocking reagent;

[0043] Figure 3 : Reaction formula of 6FF-BVS and Oligo-dT;

[0044] Figure 4 : Preparation process of SiO2-NH2-BVS;

[0045] Figure 5 : IR spectra of agarose 6FF and BVS at different reaction times;

[0046] Figure 6 : Vinyl sulfone density after reaction of agarose 6FF with BVS for different times;

[0047] Figure 7 : Infrared spectrum of the reaction between aminosilica balls and BVS;

[0048] Figure 8 :Affinity chromatography media modified with different blocking reagents and hybridized with polyA;

[0049] Figure 9 : Affinity chromatography media modified under different pH conditions and hybridized with poly A;

[0050] Figure 10 : Oligo-dT affinity chromatography fillers with different structures;

[0051] Figure 11 : Oligo-dT affinity chromatography filler poly A hybridization prepared with 6FF-BVS of different densities;

[0052] Figure 12 :Oligo-dT affinity chromatography filler poly A hybridization of different T lengths;

[0053] Figure 13 :Effects of hybridization buffer with different salt concentrations on the loading capacity of Oligo-dT affinity chromatography medium. DETAILED DESCRIPTION

[0054] The Oligo-dT affinity chromatography filler of the present invention is prepared from vinyl sulfonated microspheres and Oligo-dT, wherein the 5' and 3' ends of the Oligo-dT do not need to be pre-modified. The preparation method comprises the following steps:

[0055] 1) In a saline coupling buffer at a pH of 5-10, reacting Oligo-dT that is not pre-modified at the 5' and 3' ends with vinyl sulfone-functionalized microspheres at 15-40° C. for 3-24 hours to obtain Oligo-dT-functionalized microspheres; wherein the vinyl sulfonated microspheres are prepared by placing bis(vinylsulfonyl)methane, an organic base, and microspheres with a diameter of 100 nm after thorough dehydration in an organic solvent, and reacting them at 10-60° C. for 3-24 hours;

[0056] 2) treating the Oligo-dT functionalized microspheres obtained in step 1) with a blocking reagent in a blocking buffer solution at 15-40° C. for 6-24 hours, blocking excess vinyl sulfone with the blocking reagent to obtain an Oligo-dT affinity chromatography filler; the blocking reagent is one or more of reduced glutathione, natural amino acids, 2-mercaptoethanol, ethanolamine, bovine serum albumin, and casein, and the natural amino acids are one or more of L-cysteine, L-glycine, L-arginine, and L-lysine.

[0057] The following specific embodiments are provided to further illustrate the present invention and should not be construed as limiting the present invention in any manner.

[0058] Example 1

[0059] 0.3 g of agarose microspheres (Bastarose 6FF: highly cross-linked 6% agarose from Boglong Biotechnology Co., Ltd., with an average particle size of 90 μm) were taken, filtered and washed with acetonitrile to remove moisture, and 2 ml of 50 mM acetonitrile solution of bis(vinylsulfonyl)methane (BVS) was added, followed by 2 ml of 2 mM acetonitrile solution of methylimidazole. The mixture was shaken at 25°C for 1, 3, 8, and 24 h, respectively. After the reaction, the mixture was washed with acetonitrile to obtain vinyl-functionalized agarose microspheres 6FF-BVS (modification time was 1, 3, 8, and 24 h, see Figure 1 Finally, the product was characterized by infrared spectroscopy (see Figure 5), the agarose microspheres 6FF-BVS obtained after reacting with BVS for 3-24h showed a peak at ~1130cm -1 and ~1300cm -1 New peaks appeared at 1130cm -1 Assigned to the symmetric stretching vibration of the sulfone group, ~1300 cm -1 The result was attributed to the asymmetric stretching vibration of the sulfone group, indicating that the vinyl sulfone functionalization was successfully achieved under catalytic conditions.

[0060] Weigh 0.1 g of vinyl sulfone functionalized agarose microspheres, add 30 ml of phosphate buffer (100 mM, pH = 7.4) with a cysteine concentration of 100 mM, and react at 25 ° C for 12 h to allow cysteine to fully react with the surface vinyl sulfone groups. The change in cysteine in the solution before and after the reaction was determined by Ellman's method to obtain the vinyl sulfone density on the surface of the agarose microspheres (see Figure 2 ), the surface VS density can reach up to 100mmol / g (see Figure 6 ), demonstrating that this modification method can increase the number of vinyl sulfone active sites for subsequent coupling with Oligo-dT.

[0061] Example 2

[0062] The preparation method of aminosilica spheres is as follows: 5 mL of silica microspheres (10 μm, Shanghai Aladdin Biochemical Technology Co., Ltd.) were ultrasonically dispersed in 100 mL of ethanol (95%), 1 M nitric acid was added dropwise to pH = 4.0, the reaction was carried out at 40 ° C for 30 minutes, and then the temperature was cooled to room temperature, 1 mL of APTES (3-aminopropyltriethoxysilane) was added, and the reaction was carried out at room temperature for 24 hours (see Figure 4 The resulting APTES-modified silica microspheres (SiO2-NH2) were collected by centrifugation and ultrasonically washed three times with ethanol.

[0063] Take 0.1g of the APTES-modified silica microspheres (SiO2-NH2) prepared above, add 2ml of 100mM N, N-dimethylformamide solution of bis(vinylsulfonyl)methane (BVS), then add 2ml of 5mM N, N-dimethylformamide solution of triphenylphosphine, and react at 25°C for 18h. After the reaction, wash with N, N-dimethylformamide to obtain vinyl sulfone functionalized amino silica spheres SiO2-NH2-BVS. Finally, the product was characterized by infrared spectroscopy (see Figure 7 ), vinyl sulfone functionalized aminosilica ball SiO2-NH2-BVS obtained after reaction with BVS has a peak of 1300 cm- 1 A new peak appeared at , which was attributed to the asymmetric stretching vibration of the sulfone group, indicating that the vinyl sulfone functionalization was successfully achieved under the catalyst conditions.

[0064] Example 3

[0065] 20 mg of vinyl functionalized agarose microspheres 6FF-BVS prepared in Example 1 with a modification time of 24 h were equilibrated in a coupling buffer (phosphate buffer, 100 mM, pH = 6) with a sodium chloride concentration of 0.15 M for 15 min. After being drained, 1 ml of Oligo-dT (5'-G5T 25 -3', Takara Biotechnology Co., Ltd.) with a concentration of 10uM coupling buffer (phosphate buffer, 100mM, pH=6), reacted at 25°C for 24h, and washed three times with water to obtain agarose microspheres with probes fixed on the surface (see Figure 3 ), and then the density of surface-immobilized Oligo-dT was calculated by measuring the change in solution absorbance before and after the reaction using an ultraviolet spectrophotometer.

[0066] Reduced glutathione (GSH, J&K), L-cysteine (Cys, J&K), 2-mercaptoethanol (BME, J&K), L-glycine (Gly, J&K), L-arginine (Arg, J&K), and L-lysine (Lys, J&K) were added to a buffer solution (phosphate buffer, 100 mM, pH = 7) to prepare a blocking reagent solution with a concentration of 80 mM. 50 ml of the blocking reagent solution was added to the 6FF-BVS -OdT, and reacted at 25°C for 8h to allow the blocking reagent to fully react with the remaining vinyl sulfone groups on the surface, and blocked Oligo-dT affinity chromatography packings were obtained in turn, which were respectively recorded as 6FF-BVS-OdI-GSH, 6FF-BVS-OdI-Cys, 6FF-BVS-OdI-BME, 6FF-BVS-OdI-Gly, 6FF-BVS-OdT-Arg, and 6FF-BVS-OdT-Lys.

[0067] The blocked Oligo-dT affinity chromatography fillers 6FF-BVS-OdT-GSH, 6FF-BVS-OdT-Cys, 6FF-BVS-OdT-BME, 6FF-BVS-OdT-Gly, 6FF-BVS-OdT-Arg, and 6FF-BVS-OdT-Lys were equilibrated in hybridization buffer (phosphate buffer, 100 mM, pH = 7, 0.25 M NaCl) for 15 min, centrifuged, and then 7 nmol poly A (5'-A) was added at 25°C. 30-3', Takara Biotechnology Co., Ltd.) hybridization buffer (phosphate buffer, 100 mM, pH = 7, 0.25 M NaCl) for 5 min, after which the cells were fully rinsed and washed three times with water. The absorbance in water was then measured using an ultraviolet spectrophotometer to calculate the amount of surface hybridized polyA and the corresponding density (see Figure 8 ).

[0068] Comparative Example 1

[0069] 6FF, 6FF-BVS, 6FF-BVS-GSH, 6FF-OdT-Cys, 6FF-BVS-BME, 6FF-BVS-Gly, 6FF-BVS-OdT-Arg, and 6FF-BVS-OdT-Lys were equilibrated in hybridization buffer (0.25 M NaCl) for 15 min, centrifuged, and then 7 nmol poly A (5'-A) was added at 25°C. 30 The 6FF-BVS were hybridized for 5 min in a hybridization buffer (phosphate buffer, 100 mM, pH = 7, 0.25 M NaCl) containing 5% paraformaldehyde (Pb) and 0.25% paraformaldehyde (Pb) at -3'. After hybridization, the surfaces were thoroughly rinsed and washed three times with water. The absorbance in water was measured using a UV spectrophotometer to calculate the amount of surface hybridized poly A. None of the 6FF-BVS showed poly A adsorption, indicating that the prepared Oligo-dT affinity chromatography medium can specifically bind to poly A.

[0070] Example 4

[0071] 20 mg of vinyl functionalized agarose microspheres 6FF-BVS prepared in Example 1 with a modification time of 24 h were equilibrated in a coupling buffer (phosphate buffer, 100 mM) with a sodium acetate concentration of 0.15 M at pH = 6, 7, 7.4, and 8 for 15 min, and then 1 ml of Oligo-dT (5'-G5T 25 The 6FF-BVS agarose microspheres were immobilized with the probe 6FF-BVS-OdT after the reaction. The absorbance change of the solution before and after the reaction was measured by ultraviolet spectrophotometry to calculate the density of surface-immobilized Oligo-dT, indicating that Oligo-dT can react with 6FF-BVS under different pH conditions.

[0072] Reduced glutathione (GSH) was added to HEPES buffer (100 mM, pH = 7) to prepare a blocking reagent solution with a concentration of 80 mM. 50 ml of the blocking reagent solution was added to 6FF-BVS-OdT and reacted at 25°C for 8 h to allow the reduced glutathione to fully react with the remaining vinyl sulfone groups on the surface to obtain a blocked Oligo-dT affinity chromatography medium 6FF-BVS-OdT-GSH.

[0073] The blocked Oligo-dT affinity chromatography medium 6FF-BVS-OdT-GSH was equilibrated in hybridization buffer (phosphate buffer, 100 mM, pH = 7, 0.25 M NaCl) for 15 min, centrifuged, and then 7 nmol poly A (5'-A 30 -3') hybridization buffer (phosphate buffer, 100mM, pH=7, 0.25M NaCl) was hybridized for 5 min. After the hybridization was completed, the surface was fully rinsed and washed three times with water. The absorbance in water was then measured using an ultraviolet spectrophotometer to calculate the amount of surface hybridized poly A. Oligo-dT affinity chromatography fillers prepared under different pH conditions can hybridize with poly A (see Figure 9 ).

[0074] Example 5

[0075] 20 mg of vinyl functionalized agarose microspheres 6FF-BVS prepared in Example 1 with a modification time of 24 h were equilibrated in a coupling buffer (phosphate buffer, 100 mM, pH = 6) with a sodium oxalate concentration of 0.15 M for 15 min. After being drained, 1 ml of oligo-dT containing different structures (5'-G5T 25 -3' (denoted as GT), 5'-A5T 25 -3' (denoted as AT), 5'-C5T 25 -3' (denoted as CT), 5'-T 30 -3' (denoted as TT, 10uM, Takara Biotechnology Co., Ltd.) in a coupling buffer (phosphate buffer, 100mM, pH=6) and reacted at 25°C for 24h. After the reaction, the microspheres were fully rinsed and washed with water three times to obtain agarose microspheres with probes fixed on the surface, 6FF-BVS-OdT. Subsequently, the absorbance change of the solution before and after the reaction was measured by ultraviolet spectrophotometer to calculate the density of Oligo-dT fixed on the surface. The reactivity of guanine is 6 times that of thymine, indicating that the reaction between guanine and 6FF-BVS has good reaction selectivity (see Figure 10 ).

[0076] Example 6

[0077] 20 mg of vinyl functionalized agarose microspheres 6FF-BVS prepared in Example 1 with different modification times (modification time: 3 h, 8 h, 24 h) were equilibrated in a coupling buffer (phosphate buffer, 100 mM, pH = 6) with a sodium citrate concentration of 0.15 M for 15 min. After being drained, 1 ml of Oligo-dT (5'-G 20 T 25 The 6FF-BVS-OdT agarose microspheres were immobilized with a 10 μM coupling buffer (phosphate buffer, 100 mM, pH 6) at 25°C for 24 hours. After completion of the reaction, the microspheres were rinsed thoroughly and then washed three times with water to obtain the probe-immobilized agarose microspheres. The absorbance change before and after the reaction was measured using a UV spectrophotometer to calculate the density of Oligo-dT immobilized on the microspheres. The results showed that when 6FF-BVS was modified for 24 hours, the coupling efficiency reached as high as 96%.

[0078] Bovine serum albumin (Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to a buffer solution (phosphate buffer, 100 mM, pH = 7) to prepare a blocking reagent solution with a concentration of 80 mM. 50 ml of the blocking reagent solution was added to 6FF-BVS-OdT and reacted at 25°C for 8 h to allow the bovine serum albumin to fully react with the remaining vinyl sulfone groups on the surface. After the reaction, the medium was washed three times with water to obtain a blocked Oligo-dT affinity chromatography medium.

[0079] The blocked Oligo-dT affinity chromatography medium was equilibrated in hybridization buffer (citrate buffer, 100 mM, pH = 7, 0.25 M NaCl) for 15 min, centrifuged, and then a mixture containing 7 nmol polyA (5'-A 30 -3') hybridization buffer (phosphate buffer, 100mM, pH=7, 0.25M NaCl) was hybridized for 5 min. After the hybridization was completed, the surface was fully rinsed and washed three times with water. The absorbance in water was then measured using an ultraviolet spectrophotometer to calculate the amount of surface hybridized poly A. Oligo-dT affinity chromatography fillers prepared under different pH conditions can hybridize with poly A (see Figure 11 ).

[0080] Example 7

[0081] 20 mg of vinyl functionalized agarose microspheres 6FF-BVS prepared in Example 1 with a modification time of 24 h were equilibrated in a coupling buffer (phosphate buffer, 100 mM, pH = 6) with an ammonium sulfate concentration of 0.15 M for 15 min. After being drained, 1 ml of oligo-dT containing different T lengths (5'-G5T10 -3' (denoted as GT10), 5'-G5T 25 -3' (denoted as GT25), 5'-G5T 40 -3' (denoted as GT40, 10uM) in coupling buffer (phosphate buffer, 100mM, pH=6) and reacted at 25°C for 24h. After the reaction was completed, the agarose microspheres 6FF-BVS-OdT with the probe immobilized on the surface were obtained after sufficient rinsing and washing three times with water. Subsequently, the absorbance change of the solution before and after the reaction was measured by ultraviolet spectrophotometer to calculate the density of surface-immobilized Oligo-dT.

[0082] L-cysteine was added to a buffer solution (phosphate buffer, 100 mM, pH = 7) to prepare a blocking reagent solution with a concentration of 80 mM. 50 ml of the blocking reagent solution was added to 6FF-BVS-OdT and reacted at 25°C for 8 h to fully react with the remaining vinyl sulfone groups on the surface. After the reaction, the solution was washed three times with water to obtain a blocked Oligo-dT affinity chromatography medium.

[0083] The blocked Oligo-dT affinity chromatography filler was equilibrated in hybridization buffer (phosphate buffer, 100 mM, pH = 7, 0.25 M NaCl) for 15 min, centrifuged, and then 7 nmol poly A (5'-A 30 -3') hybridization buffer (phosphate buffer, 100mM, pH=7, 0.25M NaCl) was hybridized for 5 min. After the hybridization was completed, the surface was fully rinsed and washed three times with water. The absorbance in water was then measured using an ultraviolet spectrophotometer to calculate the amount of surface hybridized poly A. Oligo-dT affinity chromatography fillers prepared under different pH conditions can hybridize with poly A (see Figure 12 ).

[0084] Example 8

[0085] 20 mg of vinyl functionalized agarose microspheres 6FF-BVS prepared in Example 1 with a modification time of 24 h were equilibrated in a coupling buffer (phosphate buffer, 100 mM, pH = 6) with a sodium chloride concentration of 0.15 M for 15 min. After being drained, 1 ml of Oligo-dT (5'-G5T 25 The reaction was continued at 25°C for 24 h. After the reaction was completed, the agarose microspheres 6FF-BVS-OdT with the probe immobilized on the surface were obtained by rinsing thoroughly and washing three times with water. The absorbance change of the solution before and after the reaction was measured by ultraviolet spectrophotometer to calculate the density of Oligo-dT immobilized on the surface.

[0086] Ethanolamine was added to a buffer solution (phosphate buffer, 100 mM, pH = 7) to prepare a blocking reagent solution with a concentration of 80 mM. 50 ml of the blocking reagent solution was added to 6FF-BVS-OdT and reacted at 25°C for 8 h to allow the ethanolamine to fully react with the remaining vinyl sulfone groups on the surface. After the reaction, the solution was washed three times with water to obtain a blocked Oligo-dT affinity chromatography medium.

[0087] The blocked Oligo-dT affinity chromatography medium was equilibrated in hybridization buffer (carbonate buffer, 100 mM, pH = 7) with NaCl concentrations of 0 M, 0.25 M, 0.5 M, 0.75 M, and 1 M for 15 min, centrifuged, and then 7 nmol poly A (5'-A 30 The hybridization was carried out for 5 min using hybridization buffer (carbonate buffer, 100 mM, pH = 7) with NaCl concentrations of 0 M, 0.25 M, 0.5 M, 0.75 M, and 1 M, respectively. After hybridization, the cells were fully rinsed and washed three times with water. The absorbance in the water was measured by UV spectrophotometer to calculate the amount of surface hybridized poly A. The prepared affinity chromatography filler can hybridize with poly A at different salt concentrations (see Figure 13 ).

Claims

1. A method for preparing an Oligo-dT affinity chromatography filler, characterized in that: The steps include: 1) In a pH 5-10 salt-containing coupling buffer, react unmodified Oligo-dT at the 5' and 3' ends with vinyl sulfone-functionalized microspheres at 15-40°C for 3-24 hours to obtain Oligo-dT-functionalized microspheres. 2) treating the oligo-dT functionalized microspheres obtained in step 1) with a blocking reagent in a blocking buffer solution at 15-40° C. for 6-24 hours to obtain an oligo-dT affinity chromatography medium; The preparation method of the vinyl sulfone functionalized microspheres is as follows: using an organic base as a catalyst, placing bis(vinylsulfonyl)methane, the organic base, and the dehydrated microspheres in an organic solvent, and shaking the reaction at 10-60°C for 3-24 hours; The microspheres are hydrophilic microspheres with hydroxyl or amino structures on the surface.

2. The preparation method according to claim 1, wherein: The catalyst is selected from the group consisting of triphenylphosphine, tricyclohexylphosphine, triisopropylphosphine, tritolylphosphine, tri-p-tolylphosphine, and methylimidazole; The organic solvent is an aprotic solvent.

3. The preparation method according to claim 2, wherein: The hydrophilic microspheres are agarose microspheres, or polymethacrylate microspheres with hydroxyl or amino groups modified on the surface, polyethylene-divinylbenzene microspheres, silicon spheres or magnetic microspheres, and the aprotic solvent is dichloromethane, N,N-dimethylformamide or acetonitrile.

4. The preparation method according to claim 1, wherein: The molar ratio of the bis(vinylsulfonyl)methane to the organic base is 10-1000:1, the mass ratio of the bis(vinylsulfonyl)methane to the microspheres after dehydration is 1:1-1000, and the concentration of the bis(vinylsulfonyl)methane in the organic solvent is 50-100 mM.

5. The preparation method according to claim 1, wherein: The structure of the Oligo-dT without pre-modification at the 5' and 3' ends is as follows: 5'-R-Oligo-dT-3' Where R is the spacer arm and Oligo-dT is the affinity ligand; The spacer arm is 5-20 deoxyribonucleotides, and the 5' end does not need to be pre-modified; the Oligo-dT is a polydeoxythymidine with 10-40 bases; The blocking reagent is one or more of reduced glutathione, natural amino acids, 2-mercaptoethanol, ethanolamine, bovine serum albumin, and casein, and the natural amino acids are one or more of L-cysteine, L-glycine, L-arginine, and L-lysine.

6. The preparation method according to claim 1, wherein: The molar ratio of the unmodified Oligo-dT at the 5' and 3' ends to the vinyl sulfone in the vinyl sulfone functionalized microspheres is 1:100-100000, and the concentration of the unmodified Oligo-dT at the 5' and 3' ends in the saline buffer solution is 0.1-100 uM; The molar ratio of the blocking reagent to the vinyl sulfone in the Oligo-dT functionalized microspheres is 1-10:1, and the concentration of the blocking reagent in the blocking buffer solution is 10-1000 mM.

7. The preparation method according to claim 1, wherein: The salt in the salt-containing coupling buffer is one or more of sodium sulfate, sodium chloride, sodium oxalate, sodium acetate, ammonium sulfate, and sodium citrate, the concentration of the salt in the salt-containing coupling buffer is 0.01-2.5M, and the coupling buffer in the salt-containing coupling buffer is a phosphate buffer; The blocking buffer solution is phosphate buffer, citric acid buffer, carbonate buffer or 4-hydroxyethylpiperazineethanesulfonic acid buffer, and the pH value of the blocking buffer solution is 5-10.

8. The preparation method according to claim 1, wherein: Also includes: 3) Hybridize the Oligo-dT affinity chromatography medium obtained in step 2) with poly A in a salt-containing hybridization buffer at 15-40°C for 0.5-30 min.

9. The preparation method according to claim 8, wherein: The molar ratio of the Oligo-dT affinity chromatography filler to polyA is 1-20:1, and the concentration of the poly A in the salt-containing hybridization buffer is 0.1-100 uM; The salt in the salt-containing hybridization buffer is one or more of sodium sulfate, sodium chloride, sodium oxalate, sodium acetate, ammonium sulfate, and sodium citrate. The salt concentration in the salt-containing hybridization buffer is 0.1-1M. The hybridization buffer in the salt-containing hybridization buffer is a phosphate buffer or a carbonate buffer. The pH value of the phosphate buffer or the carbonate buffer is 7-7.

4.

10. Oligo-dT affinity chromatography filler prepared by the method according to any one of claims 1 to 9.

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