Method for removing endotoxin in biosynthetic recombinant small RNA (Ribonucleic Acid) and application

Abstract

The invention discloses a method for removing endotoxin in biosynthetic recombinant small RNA (Ribonucleic Acid), which comprises the following steps of: separating and purifying total RNA expressed by escherichia coli in FPLC (Fluorescence Programmable Logic Controller) by using an anion exchange column, desalting and concentrating by using an ultrafiltration membrane, and removing the endotoxin in a recombinant small RNA solution by using an ultrafiltration method. Meanwhile, the effects of different endotoxin removal methods are also compared, and finally, the effects of different methods are comprehensively evaluated from the three aspects of sample flux, the recovery rate of the recombinant small RNA and the endotoxin removal rate. Other impurities are not introduced in the process of removing the endotoxin by the ultrafiltration method, the flux is high, the durability is good, the stability is excellent, and the content of the endotoxin is 1.2-5.8 EU / ml, which is lower than the endotoxin limit requirement of biosynthetic protein preparations similar to the production process in Pharmacopoeia. The invention provides an economical, efficient and rapid method for removing the endotoxin of the recombinant small RNA, and powerful support is provided for wide application of the recombinant small RNA.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for removing endotoxin in biosynthesized recombinant small RNA and its application. Background Art

[0002] RNAi technology can effectively and specifically inhibit the expression of specific genes and is widely used in the field of gene therapy for exploring gene functions and infectious diseases and malignant tumors. As of 2022, there are 17 RNA drugs approved for marketing worldwide, and hundreds of RNA drugs are in clinical trials. However, the study of RNA functions and the development of RNA-based therapies depend on the efficient acquisition of large amounts of safe, homogeneous RNA raw materials and efficient and durable RNA quantitative analysis methods.

[0003] The patent with application number CN201810595579.9 discloses a method for producing recombinant small RNA. The method uses tRNA as a scaffold, chimeric miRNA precursor, and expresses the target small RNA in Escherichia coli. The method is economical, practical, convenient, effective, easy to scale and has a short production cycle. It also provides a set of useful tools for in vitro / in vivo RNA structure, interaction and function research, which helps to promote the functional research of non-coding RNA and the development of new RNA drugs.

[0004] However, this method uses recombinant RNA expressed by E. coli, which is a biological preparation and has the problem of endotoxin contamination. Endotoxins can cause a variety of pathological and physiological effects in the body, such as high fever, diarrhea, septic shock, and even death. At present, there are no biosynthetic RNA preparations on the market in China. How to remove endotoxins in recombinant small RNA in an economical, efficient and fast way is also a problem of great concern to manufacturers. In order to improve the safety of recombinant small RNA products, it is necessary to establish an efficient and durable method to remove endotoxins in recombinant small RNA raw materials. Summary of the invention

[0005] In view of the endotoxin problem of biosynthetic RNA preparations including recombinant small RNA, the purpose of the present invention is to provide a method and application for removing endotoxins in recombinant small RNA by comparing and screening different endotoxin methods. The endotoxin content in the recombinant small RNA solution prepared by this method meets the endotoxin limit requirements of the "Chinese Pharmacopoeia" for biosynthetic protein preparations with similar production processes. At the same time, a set of efficient, convenient and durable ultrafiltration methods is established for removing endotoxins in recombinant small RNA preparations.

[0006] The technical solution provided by the present invention is as follows:

[0007] The present invention provides a method for removing endotoxin in biosynthesized recombinant small RNA, comprising the following steps:

[0008] S1, extracting total RNA from Escherichia coli, separating and purifying the total RNA using a strong anion exchange column in FPLC to obtain recombinant small RNA;

[0009] S2, using a first ultrafiltration membrane with a molecular weight of 20 kD to desalt and concentrate the recombinant small RNA to obtain a recombinant small RNA concentrate;

[0010] S3. Ultrafiltration is performed on the recombinant small RNA concentrate using a second ultrafiltration membrane with a molecular weight of 120 kD to obtain the recombinant small RNA with endotoxin removed.

[0011] Further, in step S1, the recombinant small RNA includes a tRNA scaffold, a miRNA precursor sequence and a target small RNA sequence;

[0012] The target small RNA sequence is miRNA, siRNA, shRNA or RNA aptamer.

[0013] Furthermore, the tRNA scaffold includes human serine tRNA, human glutamate tRNA, human cysteine ​​tRNA, human leucine tRNA, human lysine tRNA, human glutamine tRNA, human tyrosine tRNA and bacterial methionine tRNA.

[0014] Furthermore, the recombinant small RNA includes a human serine tRNA scaffold, a miR-34a precursor sequence and a chimeric miR-34a mature sequence, that is, the recombinant small RNA is a tRNA ser -34a;

[0015] In step S1, before extracting total RNA from Escherichia coli, the tRNA ser -34a is expressed as follows:

[0016] tRNA ser The -34a expression plasmid was used to transform HST08 competent bacteria, and LB medium was added for culture. The bacterial sludge was collected and resuspended in magnesium acetate-Tris·HCl solution. Saturated phenol was added, the aqueous phase was collected, NaCl solution was added to precipitate macromolecular impurities, the supernatant was collected, anhydrous ethanol was added to the supernatant, and total RNA was obtained after centrifugation.

[0017] Further, in step S1, the total RNA is separated and purified using a strong anion exchange column in FPLC, specifically:

[0018] The total RNA was separated and purified using an ion exchange column, wherein the mobile phase A, mobile phase B and flow rate were as follows:

[0019] Mobile phase A: 10 mM NaH2PO4 solution, pH 7.0;

[0020] Mobile phase B: 10 mM NaH2PO4 solution, 1 M NaCl solution, pH 7.0;

[0021] The flow rate was 2.0 mL / min, and the column was flushed alternately with DEPC water, mobile phase A, and mobile phase B;

[0022] The following procedure was used to isolate total RNA:

[0023] 0% mobile phase B is used for separation for 0 to 5 minutes, 55% mobile phase B is used for separation for 5 to 12 minutes, 55 to 75% mobile phase B is used for separation for 12 to 45 minutes, 75 to 85% mobile phase B is used for separation for 45 to 70 minutes, 100% mobile phase B is used for separation for 70 to 80 minutes, and 0% mobile phase B is used for separation for 80 to 85 minutes to obtain tRNA ser -34a.

[0024] Further, in step S2, the first ultrafiltration membrane is a regenerated cellulose membrane;

[0025] In step S2, the desalting and concentrating conditions of the recombinant small RNA are as follows: adding 2 volumes of ethanol to the recombinant small RNA solution, placing it under low temperature conditions for 10-60 minutes, and then centrifuging it at 4°C and 8000-10000 rpm for 10 minutes to obtain the recombinant small RNA solid; then using a 20kD first ultrafiltration membrane, centrifugation is performed at 4°C and 8000-10000 rpm to obtain the recombinant small RNA concentrate.

[0026] Further, in step S3, the second ultrafiltration membrane is a regenerated cellulose membrane;

[0027] The method of ultrafiltration the recombinant small RNA concentrate using a second ultrafiltration membrane with a molecular weight of 120 kD comprises the following steps:

[0028] S301, Preparation of tRNA ser -34a solution, tRNA ser -34a solution was diluted to 200-800 μg / mL with DEPC water;

[0029] S302, take tRNA ser -34a solution into 120kD ultra Centrifugal Filters, centrifuge for 10 min at 8000-10000 rpm and 4°C, and collect the first filtrate;

[0030] S303, DEPC water is added to the 120kD ultra Centrifugal Filters, and the mixture is centrifuged at 8000-10000 rpm and 4°C for 10 min to collect the second filtrate, and the first filtrate and the second filtrate are mixed to obtain tRNA with endotoxin removed. ser -34a.

[0031] Furthermore, in step S303, tRNA with endotoxin removed is obtained. ser -34a and later, also includes:

[0032] S304, detection of tRNA before and after ultrafiltration ser -34a solution concentration and endotoxin level (EU / mL) were calculated for different batches of tRNA according to the following formula ser -34a solution bacterial endotoxin removal rate Q and effective component recovery rate R:

[0033]

[0034] In the formula, C 超 is the content of bacterial endotoxin in the solution after ultrafiltration (EU / mL); C 原 is the bacterial endotoxin content in the stock solution before ultrafiltration (EU / mL);

[0035]

[0036] In the formula, C 超 tRNA in the aqueous phase after ultrafiltration ser -34a content (μg / mL); C 原 tRNA in the stock solution before ultrafiltration ser -34a content (μg / mL), V 超 V is the volume of the aqueous solution collected after ultrafiltration (mL); 原 Add tRNA before ultrafiltration ser - Volume of 34a solution (mL).

[0037] Furthermore, the endotoxin removal rate in the recombinant small RNA is above 72%;

[0038] The recovery rate of the recombinant small RNA is above 93%.

[0039] At the same time, the present invention also provides an application of the above-mentioned method for removing endotoxins in biosynthesized recombinant small RNA to removing endotoxins in RNA derived from bacterial fermentation.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The method for removing endotoxins in recombinant small RNA provided by the present invention, after the constructed recombinant small RNA expression plasmid is transformed into Escherichia coli for cultivation, total RNA is extracted, and a strong anion exchange column is used for separation and purification in FPLC, and finally an ultrafiltration membrane is used for desalting, concentration and ultrafiltration to remove endotoxins in the recombinant small RNA solution again. At the same time, the effects of different endotoxin removal methods are also compared, and finally the effects between different methods are comprehensively evaluated from three aspects: sample flux, recovery rate of recombinant small RNA, and endotoxin removal rate. The ultrafiltration method does not introduce other impurities in the process of removing endotoxins, and has high flux, good durability, and excellent stability. The content of endotoxins is 1.2-5.8EU / ml, which is lower than the endotoxin limit requirements of the Pharmacopoeia for biosynthetic protein preparations with similar production processes. The present invention provides an economical, efficient and rapid method for removing endotoxins from recombinant small RNA, which provides strong support for the wide application of recombinant small RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a graph showing the expression results of urea polyacrylamide electrophoresis to identify the expression of recombinant small RNA using endotoxin-free strain Clear coli BL21 (DE3) and Escherichia coli HST08 in the examples of the present invention;

[0043] Figure 2 In another embodiment of the present invention, tRNA is removed by affinity chromatography after endotoxin is removed. ser -34a chromatogram (A) and recovery result graph (B);

[0044] Figure 3 In another embodiment of the present invention, a commercial endotoxin removal kit is used to remove tRNA ser -34a sample recovery results after endotoxin;

[0045] Figure 4 In another embodiment of the present invention, tRNA is removed by ultrafiltration ser -34a sample recovery results after endotoxin;

[0046] Figure 5 The ultrafiltration method, affinity chromatography, and commercial endotoxin removal kit used in the present invention are used to remove tRNA. ser -34a Endotoxin effect comparison chart, where A is the treatment flux comparison chart, B is the recovery rate comparison chart, and C is the endotoxin removal result comparison chart;

[0047] Figure 6 Three batches of tRNA were treated by ultrafiltration in the present invention. ser -34a sample recovery (A) and removal of different batches of tRNA ser-34a endotoxin effect comparison chart (B);

[0048] Figure 7 tRNA after ultrafiltration to remove endotoxins for different storage times in the embodiment of the present invention ser -34a solution endotoxin content detection chart. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the embodiments described below are part of the embodiments of the present application, rather than all of the embodiments. The components of the embodiments of the present application described and shown in the drawings here can be arranged and designed in various different configurations.

[0050] See also Figure 1 The present invention provides a method for removing endotoxin in biosynthesized recombinant small RNA, comprising the following steps:

[0051] S1. Total RNA was extracted from Escherichia coli, and the total RNA was separated and purified using a strong anion exchange column in FPLC to obtain recombinant small RNA;

[0052] S2, using a first ultrafiltration membrane with a molecular weight of 20 kD to desalt and concentrate the recombinant small RNA to obtain a recombinant small RNA concentrate;

[0053] S3. Ultrafiltration is performed on the recombinant small RNA concentrate using a second ultrafiltration membrane with a molecular weight of 120 kD, and high-speed centrifugation is performed to obtain the filtrate to obtain the recombinant small RNA with endotoxin removed.

[0054] Optionally, in step S1, the recombinant small RNA includes a tRNA scaffold, a miRNA precursor sequence and a target small RNA sequence.

[0055] The target small RNA sequence is miRNA, siRNA, shRNA or RNA aptamer.

[0056] Optionally, the tRNA scaffold includes human serine tRNA, human glutamate tRNA, human cysteine ​​tRNA, human leucine tRNA, human lysine tRNA, human glutamine tRNA, human tyrosine tRNA and bacterial methionine tRNA.

[0057] Optionally, the recombinant small RNA includes a human serine tRNA scaffold, a miR-34a precursor sequence, and a chimeric miR-34a mature sequence, that is, the recombinant small RNA is a tRNA ser -34a.

[0058] In step S1, before extracting total RNA from E. coli, tRNA ser -34a is expressed, specifically: tRNA ser The -34a expression plasmid was used to transform HST08 competent bacteria, the bacterial sludge was collected and resuspended in magnesium acetate-Tris·HCl solution, saturated phenol was added, the aqueous phase was collected by centrifugation, NaCl solution was added to precipitate macromolecular impurities, the supernatant was collected, anhydrous ethanol was added to the supernatant, and total RNA was obtained after centrifugation.

[0059] Optionally, in step S1, a strong anion exchange column is used to separate and purify the total RNA in FPLC, specifically: a strong anion exchange column is used to separate and purify the total RNA.

[0060] The mobile phase A, mobile phase B and flow rates are as follows:

[0061] Mobile phase A: 10 mM NaH2PO4 solution, pH 7.0;

[0062] Mobile phase B: 10 mM NaH2PO4 solution, 1 M NaCl solution, pH 7.0;

[0063] The flow rate was 2.0 mL / min, and the column was flushed alternately with DEPC, mobile phase A, and mobile phase B;

[0064] The following procedure was used to separate total RNA: 0-5 min (0% B), 5-12 min (55% B), 12-45 min (55-75% B), 45-70 min (75-85% B), 70-80 min (100% B), 80-85 min (0% B) to obtain tRNA ser -34a.

[0065] Optionally, in step S2, the first ultrafiltration membrane is a regenerated cellulose membrane. In step S2, the desalting and concentrating conditions of the recombinant small RNA are as follows: adding 2 volumes of ethanol to the recombinant small RNA solution, placing it under low temperature conditions for 10-60 minutes, and then centrifuging it at 4°C and 8000-10000 rpm for 10 minutes to obtain the recombinant small RNA solid. After dissolving the solid with DEPC water, centrifuging it with a first ultrafiltration membrane with a molecular weight of 20kD to obtain the recombinant small RNA concentrate.

[0066] Optionally, in step S3, the second ultrafiltration membrane is a regenerated cellulose membrane, and the dilution of the recombinant small RNA concentrate is ultrafiltered using the second ultrafiltration membrane with a molecular weight of 120 kD, and high-speed centrifugation is performed, specifically comprising the following steps:

[0067] S301, Preparation of tRNAser -34a solution, tRNA ser -34a solution was diluted to 800-200 μg / mL with DEPC water.

[0068] S302, take tRNA ser -34a solution was added to 120kD ultra Centrifugal Filters, and centrifuged at 8000-10000 rpm and 4°C for 10 min to collect the first filtrate.

[0069] S303, DEPC water is added to the 120kD ultra Centrifugal Filters, and centrifuged at 8000-10000 rpm and 4°C for 10 min to collect the second filtrate, and the first filtrate and the second filtrate are mixed to obtain tRNA with endotoxin removed. ser -34a.

[0070] Optionally, in step S303, tRNA with endotoxin removed is obtained ser -34a and later, also includes:

[0071] S304, detection of tRNA before and after ultrafiltration ser -34a solution concentration and endotoxin level (EU / mL) were calculated for different batches of tRNA according to the following formula ser -34a solution bacterial endotoxin removal rate Q and effective component recovery rate R:

[0072]

[0073] (1) In the formula, C 超 is the content of bacterial endotoxin in the solution after ultrafiltration (EU / mL); C 原 is the bacterial endotoxin content in the stock solution before ultrafiltration (EU / mL);

[0074]

[0075] (2) In the formula, C 超 tRNA in the aqueous phase after ultrafiltration ser -34a content (μg / mL); C 原 tRNA in the stock solution before ultrafiltration ser -34a content (μg / mL), V 超 V is the volume of the aqueous solution collected after ultrafiltration (mL); 原 Add tRNA before ultrafiltration ser - Volume of 34a solution (mL).

[0076] The following is a recombinant small RNA with human serine tRNA scaffold, miR-34a precursor sequence and chimeric miR-34a mature sequence - has-tRNA ser -miR-34a(tRNA ser -34a) as an example for detailed description.

[0077] Example 1: tRNA ser -34a expression

[0078] (1) 200 ng tRNA ser After the -34a expression plasmid was transformed into HST08 competent bacteria, 5mL LB medium was added and cultured overnight at 37°C and 200rpm. After the bacterial solution was centrifuged at 8000rpm for 2min, the precipitate was collected. 200μL 10mM magnesium acetate-Tris·HCl solution was added to the precipitate to resuspend it, and then 200μL saturated phenol was added and shaken at room temperature for 30min. After centrifugation at 10000rpm for 10min, the aqueous phase was collected and 0.1 times the volume of the aqueous phase of 5M NaCl was added to precipitate macromolecular impurities. 2 times the volume of anhydrous ethanol was added to the supernatant, and after centrifugation at 10000rpm for 10min, the supernatant was discarded. After the RNA was dried, DEPC water was added to dissolve the RNA, the concentration was determined, and it was stored in a refrigerator at -80℃.

[0079] Example 2: FPLC purification of tRNA ser -34a

[0080] (1) Purification of tRNA using a strong anion exchange column ser -34a.

[0081] Mobile phase A: 10 mM NaH2PO4 solution, pH 7.0.

[0082] Mobile phase B: 10 mM NaH2PO4 solution, 1 M NaCl solution, pH 7.0.

[0083] The flow rate is 2.0 mL / min. The column is alternately flushed with DEPC water, mobile phase A, and mobile phase B for about 0.5 h, 3 column volumes each time.

[0084] Run the following program to separate the total RNA: 0-5 min (0% B), 5-12 min (55% B), 12-45 min (55-75% B), 45-70 min (75-85% B), 70-80 min (100% B), 80-85 min (0% B). Detect RNA at an absorbance of 260 nm and collect the peak corresponding to the recombinant RNA. Use denaturing polyacrylamide gel electrophoresis to identify the purity.

[0085] (2) RNA sample processing method

[0086] The steps of total RNA extraction were the same as above. After the extracted total RNA solution was filtered through a 0.22 μm microporous filter membrane, 5 mg was injected each time.

[0087] (3) FPLC component collection and concentration and desalting

[0088] Denaturing polyacrylamide gel electrophoresis was used to identify the purity of the collected components. After mixing, the components were precipitated with 2 volumes of anhydrous ethanol, placed in a -80°C refrigerator for about 30 minutes, and centrifuged at 9000 rpm at 4°C for 10 minutes to collect RNA. The resulting RNA precipitate was dissolved in DEPC water, centrifuged at 9000 rpm at 4°C for 10 minutes using Ultra Centrifugal Filters, and the filtrate was removed. This step was repeated until all solutions were centrifuged, and the concentration was measured and stored at -80°C.

[0089] Example 3: Expression of tRNA using the endotoxin-free strain Clear coli BL21 (DE3) ser -34a

[0090] In order to directly obtain endotoxin-free recombinant small RNA, we first tried to use endotoxin-free strains to ferment and produce recombinant small RNA. The steps are as follows:

[0091] (1) 200 ng tRNA ser After the -34a expression plasmid was transformed into DE3 competent bacteria, 5 mL of LB medium was added and cultured overnight at 37°C and 200 rpm. After the bacterial solution was centrifuged at 9000 rpm for 2 minutes, the precipitate was collected. 180 μL of 10 mM magnesium acetate-Tris·HCl solution was added to the precipitate for resuspending, and then 200 μL of saturated phenol was added and shaken at room temperature for 30 minutes. After centrifugation at 9000 rpm for 10 minutes, the aqueous phase was collected and 0.1 times the volume of the aqueous phase of 5M NaCl was added to precipitate macromolecular impurities. 2 times the volume of anhydrous ethanol was added to the supernatant, and after centrifugation at 9000 rpm for 10 minutes, the supernatant was discarded. After the RNA was dried, DEPC water was added to dissolve the RNA, the concentration was determined, and it was stored in a refrigerator at -80°C.

[0092] (2) Identification by denaturing polyacrylamide gel electrophoresis

[0093] Mix 2 μg RNA sample with 2× RNA loading buffer and add to the sample well of denaturing gel. After electrophoresis at 120-150V for 40-60min, place in a solution containing 0.5 μg / mL ethidium bromide and shake gently for 20-30min, observe with gel imaging system, and take photos for preservation.

[0094] Figure 1 This example uses an endotoxin-free strain to express tRNAser -34a, and tRNA was expressed using HST08 strain ser -34a was used as a control, and the extracted RNA was identified by urea polyacrylamide gel electrophoresis. The results showed that Clearcoli BL21 also had an additional band between 150-300 nt (box), but compared with HST08, tRNA in ClearcoliBL21 ser The expression level of -34a was much lower than that of HST08 (box); the results also showed that after transformation of the recombinant MSA (Sephadexaptamer tagged methionyl-tRNA) expression plasmid, Clear coli BL21 added a band between 80-150nt, but the expression level of the aptamer (MSA, box) in the Clear coli BL21 group was also much lower than that of HST08 (box). The above results show that the endotoxin strain Clear coli BL21 cannot be directly used for large-scale production of endotoxin-free recombinant small RNA.

[0095] Example 4: Removal of tRNA by affinity chromatography ser -Endotoxin in 34a

[0096] (1) Rinse and balance the liquid phase instrument system and Endotoxin rem Tanrose 4FF column at a flow rate of 0.25 mL / min. The mobile phase is as follows: 2 column volumes of DEPC water, 2 column volumes of regeneration solution, 2 column volumes of buffer, 2 column volumes of regeneration solution, and 5 column volumes of buffer.

[0097] (2)tRNA ser -34a sample solution preparation: Take 3 batches of tRNA ser -34a concentrated solution, each batch was diluted with DEPC water to three different concentrations of 300μg / mL, 800μg / mL, and 1600μg / mL. The samples were centrifuged at 15000g for 10 minutes before loading and filtered with a 0.22μm filter membrane to reduce impurities, improve purification efficiency and prevent clogging of the column. 100μL of each concentration was separated into enzyme-free sterile centrifuge tubes before loading.

[0098] (3) After the Endotoxin rem Tanrose 4FF column is well balanced, inject 1 mL of sample, adjust the flow rate to 0.25 mL / min, detect at a wavelength of 260 nm, and collect tRNA ser The peak corresponding to -34a, each batch of samples with different concentrations were injected three times.

[0099] (4) Simultaneous detection of tRNA before and after injection ser-34a solution concentration, volume and endotoxin level (EU / mL), and calculate tRNA of different batches and injection amounts according to formula (3) and (4). ser -34a solution bacterial endotoxin removal rate Q and effective component recovery rate R:

[0100]

[0101] (3) In the formula, C 吸附 is the content of bacterial endotoxin in the solution after affinity chromatography (EU / mL); C 原 is the bacterial endotoxin content in the stock solution before affinity chromatography treatment (EU / mL).

[0102]

[0103] (4) In the formula, C 吸附 tRNA in solution after affinity chromatography ser -34a content (μg / mL); C 原 tRNA in the stock solution before affinity chromatography ser -34a content (μg / mL), V 吸附 is the volume of the solution after affinity chromatography treatment (mL); V 原 Add tRNA before affinity chromatography ser - Volume of 34a solution (mL).

[0104] (5) If the endotoxin content in the sample is still higher than the required value, repeat 1-3.

[0105] (6) Flushing out tRNA remaining in the column ser -34a and endotoxin, and the chromatographic column was stored in 20% ethanol, and the elution procedure was as follows: 2 column volumes of regeneration solution, 2 column volumes of buffer, 2 column volumes of regeneration solution, 2 column volumes of DEPC water, and 2 column volumes of 20% ethanol.

[0106] Figure 2 This example uses affinity chromatography to remove tRNA ser Post-endotoxin tRNA in -34a ser -34a recovery results. tRNA ser After -34a enters the chromatographic column, the peak shape is better, there is no tailing and the time is shorter ( Figure 2 A); tRNA ser The recovery rate measurement results of -34a showed that when the sample amount was large, the recovery rate decreased, and when the sample amount was small, the recovery rate increased ( Figure 2 B), The experiment was performed with three replicates.

[0107] Example 5: Removal of tRNA using a commercial endotoxin removal kit ser -34a endotoxin

[0108] The tRNA was purified by commercial endotoxin removal kit (Clean All DNA / RNA Clean-Up and Concentration Micro Kit, Xiamen Limulus Reagent Biological Company). ser -34a for endotoxin removal.

[0109] (1) Take 3 batches of tRNA ser -34a solution, diluted with DEPC water to a concentration of about 200 μg / mL.

[0110] (2) Take 200 μL tRNA ser -34a in a 1.5mL nonpyrogenic & DNase / RNase-free centrifuge tube, add 500μL Binding Buffer H, vortex mix. Then add 400μL anhydrous ethanol, vortex mix for 10 seconds.

[0111] (3) Assemble the collection column on the collection tube, add 600 μL of the RNA mixture in step 2, centrifuge for 1 min, and discard the filtrate.

[0112] (4) Repeat step 3 until all RNA mixture is collected.

[0113] (5) Add 500 μL Wash Solution K to the column, centrifuge for 1 min, and discard the filtrate.

[0114] (6) Add 500 μL Wash Solution K again, centrifuge for 2 min, and discard the filtrate.

[0115] (7) Replace the lower collection tube with the 1.7 mL centrifuge tube provided with the kit.

[0116] (8) Add 40 μL Elution Buffer, let stand at room temperature for 2 min, centrifuge at 200 g for 2 min, then centrifuge at 14,000 g for 1 min. If there is still solution in the upper column, centrifuge again at 14,000 g for 1 min.

[0117] (9) Collect the filtrate, measure the volume and concentration, and simultaneously detect tRNA before and after treatment ser -34a solution endotoxin level (EU / mL), calculated according to the following formula for different batches of tRNA ser -34a solution bacterial endotoxin removal rate Q and effective component recovery rate R:

[0118]

[0119] (5) In the formula, C kit is the content of bacterial endotoxin in the solution after being treated with the endotoxin removal kit (EU / mL), C 原 is the bacterial endotoxin content in the original solution before treatment (EU / mL).

[0120]

[0121] (6) In the formula, C kit To collect tRNA in the solution after treatment with the endotoxin removal kit ser -34a content (μg / mL); C 原 tRNA in the solution before treatment ser -34a content (μg / mL), V kit is the volume of solution collected after endotoxin removal (mL), V 原 The loading volume for endotoxin removal is 0.20 mL.

[0122] Figure 3 This example uses a commercial endotoxin removal kit to remove tRNA ser The results of the recovery of recombinant small RNA after endotoxin removal in -34a. The results showed that the recovery rates of the three batches of samples were between 88% and 91.5%, but the column volume of the kit was small and the upper limit of the sample loading was 200μg, so it was not suitable for large-scale endotoxin removal.

[0123] Example 6: Removal of tRNA by ultrafiltration ser -Endotoxin in 34a

[0124] The recombinant small RNA was treated for endotoxin removal using a 120kD ultrafiltration column. The specific operation is as follows:

[0125] (1) Take 3 batches of tRNA ser The -34a solution was diluted to about 200 μg / mL with DEPC water.

[0126] (2) Take tRNA ser -34a solution 3 mL was added to 120 kD ultra Centrifugal Filters, centrifuged at 10000 rpm, 4 °C for 10 min, and the filtrate was collected;

[0127] (3) Add 3 mL of DEPC water to the 120 kD ultra Centrifugal Filters and centrifuge at 10,000 rpm at 4°C for 10 min. Collect the filtrate and combine it with the filtrate from the second step to determine the concentration and volume.

[0128] (4) Simultaneously detect the RNA solution concentration and endotoxin level (EU / mL) before and after ultrafiltration, and calculate the tRNA of different batches according to formula (1) and (2). ser -34a solution, the removal rate Q of bacterial endotoxins and the recovery rate R of effective ingredients.

[0129] Figure 4 This example uses ultrafiltration to remove tRNA ser -34a tRNA after endotoxin in solution ser The results showed that the recovery rates of the three batches of recombinant small RNAs all reached more than 96%, indicating that the ultrafiltration endotoxin removal method has a high recovery rate.

[0130] Example 7 tRNA ser Detection of endotoxin in -34a

[0131] In Example 3, tRNA ser -34a is expressed at a low level in endotoxin-free strains and is not suitable for large-scale production of recombinant small RNA. To further compare the differences between different endotoxin removal methods, the three different methods of Example 4, Example 5 and Example 6 were evaluated for tRNA removal from three aspects: sample processing throughput, endotoxin removal rate and recovery rate of effective components. ser -34a, and then find a large-scale endotoxin removal method suitable for recombinant small RNA.

[0132] like Figure 5 The results showed that in terms of the recombinant small RNA processing flux, the ultrafiltration method had a processing flux of 0.8 mg, the affinity chromatography method had a processing flux of 0.3 mg, and the commercial kit had a processing flux of 0.04 mg, so the ultrafiltration method had the largest processing flux ( Figure 5 A); In terms of the recovery rate of recombinant small RNA, after ultrafiltration, affinity chromatography, and commercial kit treatment, tRNA ser -34a recovery rates were high, 97.6%, 94.3%, 90.0% ( Figure 5 B); Endotoxin removal rate is the most important indicator for evaluating the applicability of a method. The results showed that the endotoxin removal rates of ultrafiltration, affinity chromatography, and commercial kit samples were 75.7%, 33.8%, and 13.5%, respectively. Therefore, the ultrafiltration method had the highest endotoxin removal rate ( Figure 5 C, **: P<0.01). Based on the results of the three evaluation indicators, ultrafiltration method has the best effect in removing endotoxin.

[0133] To further verify the ultrafiltration method to remove tRNA ser -34a Robustness and stability of the endotoxin method using ultrafiltration to treat three batches of tRNA ser -34a, detection of tRNAser -34a recovery rate and endotoxin removal rate. The results showed that the ultrafiltration method was used to treat three batches of tRNA ser The recovery rate of -34a samples was above 93% (n=3) ( Figure 6 A); Three batches of tRNA ser The endotoxin removal rates of the samples of -34a were 86.1%, 78.3%, and 72.6%, respectively, and the removal rates were all above 72% (n=3) ( Figure 6 B). Therefore, the method of removing endotoxin by ultrafiltration is efficient, durable and reproducible. In addition, multiple sets of tRNA treated by ultrafiltration ser The endotoxin content of -34a was tested to be between 1.2 and 5.8 EU / mL, which is lower than the endotoxin limit required by the pharmacopoeia for biosynthetic recombinant protein preparations with similar production processes.

[0134] At the same time, in order to verify the ultrafiltration method to remove endotoxins after tRNA ser -34a solution during normal storage (-80℃) will introduce endotoxins, three batches of tRNA were treated by ultrafiltration ser -34a, tRNA detection after 0, 4, and 8 months ser -34a endotoxin content. Results Figure 7 As shown in the figure, compared with the test immediately after ultrafiltration treatment, the endotoxin content detected after 4 months was almost unchanged, and the endotoxin content detected after 8 months did not increase significantly, indicating that ultrafiltration can remove tRNA from different batches. ser -34a endotoxin, under normal storage conditions, there was no increase in sample endotoxin within 8 months. In summary, ultrafiltration can remove tRNA ser -34a No impurities are introduced during the endotoxin removal process, and this method has the characteristics of high throughput, high recovery rate, high endotoxin removal rate and good reproducibility, which is suitable for large-scale removal of recombinant small RNA endotoxins.

[0135] After screening the above-mentioned methods for removing endotoxin from recombinant small RNA, we finally determined that ultrafiltration is the most suitable method for removing endotoxin from recombinant small RNA through a comprehensive evaluation of sample processing flux, endotoxin removal rate, and recovery rate of effective ingredients. The ultrafiltration method allows the endotoxin content in recombinant small RNA to be controlled at 1.2-5.8EU / mL, which is lower than the endotoxin limit requirements of the pharmacopoeia for biosynthetic recombinant protein preparations with similar production processes. The ultrafiltration method not only has the advantages of large sample processing flux, high recovery rate, high endotoxin removal rate, and good reproducibility, but also has simple steps, saves time, and does not introduce impurities. It is a method that can be specifically used to remove endotoxin from recombinant small RNA.

[0136] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for removing endotoxins in biosynthesized recombinant small RNA, characterized in that: The following steps are involved: S1, extracting total RNA from Escherichia coli, separating and purifying the total RNA using a strong anion exchange column in FPLC to obtain recombinant small RNA; S2, using a first ultrafiltration membrane with a molecular weight of 20 kD to desalt and concentrate the recombinant small RNA to obtain a recombinant small RNA concentrate; S3. Ultrafiltration is performed on the recombinant small RNA concentrate using a second ultrafiltration membrane with a molecular weight of 120 kD to obtain the recombinant small RNA with endotoxin removed.

2. The method for removing endotoxins in biosynthesized recombinant small RNA according to claim 1, characterized in that: In step S1, the recombinant small RNA includes a tRNA scaffold, a miRNA precursor sequence and a target small RNA sequence; The target small RNA sequence is miRNA, siRNA, shRNA or RNA aptamer.

3. The method for removing endotoxins in biosynthesized recombinant small RNA according to claim 2, characterized in that: The tRNA scaffolds include human serine tRNA, human glutamate tRNA, human cysteine ​​tRNA, human leucine tRNA, human lysine tRNA, human glutamine tRNA, human tyrosine tRNA and bacterial methionine tRNA.

4. The method for removing endotoxins in biosynthesized recombinant small RNA according to claim 3, characterized in that: The recombinant small RNA includes a human serine tRNA scaffold, a miR-34a precursor sequence and a chimeric miR-34a mature sequence, that is, the recombinant small RNA is a tRNA ser -34a; In step S1, before extracting total RNA from Escherichia coli, the tRNA ser -34a is expressed as follows: tRNA ser The -34a expression plasmid was used to transform HST08 competent bacteria, and LB medium was added for culture. The bacterial sludge was collected and resuspended in magnesium acetate-Tris·HCl solution. Saturated phenol was added, the aqueous phase was collected, NaCl solution was added to precipitate macromolecular impurities, the supernatant was collected, anhydrous ethanol was added to the supernatant, and total RNA was obtained after centrifugation.

5. The method for removing endotoxins in biosynthesized recombinant small RNA according to claim 4, characterized in that: In step S1, the total RNA is separated and purified using a strong anion exchange column in FPLC, specifically: A strong anion exchange column was used to separate and purify the total RNA, wherein the mobile phase A, mobile phase B and flow rate were as follows: Mobile phase A: 10 mM NaH2PO4 solution, pH 7.0; Mobile phase B: 10 mM NaH2PO4 solution, 1 M NaCl solution, pH 7.0; The flow rate was 2.0 mL / min, and the column was flushed alternately with DEPC water, mobile phase A, and mobile phase B; The following procedure was used to isolate total RNA: 0% mobile phase B is used for separation for 0 to 5 minutes, 55% mobile phase B is used for separation for 5 to 12 minutes, 55 to 75% mobile phase B is used for separation for 12 to 45 minutes, 75 to 85% mobile phase B is used for separation for 45 to 70 minutes, 100% mobile phase B is used for separation for 70 to 80 minutes, and 0% mobile phase B is used for separation for 80 to 85 minutes to obtain tRNA ser -34a.

6. The method for removing endotoxin from biosynthesized recombinant small RNA according to any one of claims 1 to 5, characterized in that: In step S2, the first ultrafiltration membrane is a regenerated cellulose membrane; In step S2, the desalting and concentrating conditions of the recombinant small RNA are as follows: adding 2 volumes of ethanol to the recombinant small RNA solution, placing it under low temperature conditions for 10-60 minutes, and then centrifuging it at 4°C and 8000-10000 rpm for 10 minutes to obtain the recombinant small RNA solid; then using a 20kD first ultrafiltration membrane, centrifugation is performed at 4°C and 8000-10000 rpm to obtain the recombinant small RNA concentrate.

7. The method for removing endotoxins in biosynthesized recombinant small RNA according to claim 5, characterized in that: In step S3, the second ultrafiltration membrane is a regenerated cellulose membrane; The method of ultrafiltration the recombinant small RNA concentrate using a second ultrafiltration membrane with a molecular weight of 120 kD comprises the following steps: S301, Preparation of tRNA ser -34a solution, tRNA ser -34a solution was diluted to 200-800 μg / mL with DEPC water; S302, take tRNA ser -34a solution into 120kD ultra Centrifugal Filters, centrifuge at 8000-10000 rpm, 4°C for 10 min, and collect the first filtrate; S303, DEPC water is added to the 120kD ultra Centrifugal Filters, and the mixture is centrifuged at 8000-10000 rpm and 4°C for 10 min to collect the second filtrate, and the first filtrate and the second filtrate are mixed to obtain tRNA with endotoxin removed. ser -34a.

8. The method for removing endotoxins in biosynthesized recombinant small RNA according to claim 7, characterized in that: In step S303, tRNA with endotoxin removed is obtained. ser -34a and later, also includes: S304, detection of tRNA before and after ultrafiltration ser -34a solution concentration and endotoxin level (EU / mL) were calculated for different batches of tRNA according to the following formula ser -34a solution bacterial endotoxin removal rate Q and effective component recovery rate R: In the formula, C 超 is the content of bacterial endotoxin in the solution after ultrafiltration (EU / mL); C 原 is the bacterial endotoxin content in the stock solution before ultrafiltration (EU / mL); In the formula, C 超 tRNA in the aqueous phase after ultrafiltration ser -34a content (μg / mL); C 原 tRNA in the stock solution before ultrafiltration ser -34a content (μg / mL), V 超 V is the volume of the aqueous solution collected after ultrafiltration (mL); 原 Add tRNA before ultrafiltration ser - Volume of 34a solution (mL).

9. The method for removing endotoxins in biosynthesized recombinant small RNA according to claim 8, characterized in that: The endotoxin removal rate in the recombinant small RNA is above 72%; The recovery rate of the recombinant small RNA is above 93%.

10. Use of the method for removing endotoxins in biosynthesized recombinant small RNA as claimed in any one of claims 1 to 9 in removing endotoxins in RNA derived from bacterial fermentation.

Images