CN115404238A - Design and preparation method of BDNF mRNA with high translational stability - Google Patents
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Abstract
A method for designing and preparing BDNF mRNA with high translational stability comprises the following steps: on the basis of a wild human BDNF gene sequence, an optimized gene sequence is designed through the codon preference of a mammalian cell to be used as a stable BDNF sequence; meanwhile, the original 5'-/3' -untranslated region (UTR) in the wild-type human BDNF gene is abandoned, and the 5'-UTR with the length shorter than 100nt and the 3' -UTR of globin are adopted to modify BDNF mRNA. The BDNF mRNA under the design can be used for downstream application including transfection of mesenchymal stem cells after in vitro transcription synthesis and capping/tailing reaction. The method effectively avoids the problems of high cost, long time consumption and the like of in vitro transcription operation in the prior art and the defect of restricting the research and development progress of related cell medicaments.
Description
Technical Field
The invention relates to the technical field of nucleic acid products, in particular to a design and preparation method of BDNF mRNA with high translational stability.
Background
Mesenchymal Stem Cells (MSCs) are somatic stem cells derived from mesoblastic tissue, and are present in bone marrow, umbilical cord, adipose tissue, and other sites. MSCs can grow attached to plastic surfaces and have the potential to differentiate into osteoblasts, chondroblasts, and adipoblasts. Therefore, MSC is expected to be applied to the field of regenerative medicine such as reconstruction of bone, blood vessel, and cardiac muscle. In addition, MSC is also considered to be a current novel therapeutic means for neurodegenerative diseases, which can limitedly secrete various neurotrophic factors while inhibiting neuroinflammatory response, for treating nerve injury. Wherein, the brain-derived neurotrophic factor (BDNF) plays an important role in synaptic transmission, and the in-situ injection or overexpression of the BDNF can increase the yield of new neurons and promote the neurogenesis, and has successful case reports in the aspects of protecting motor neurons of neurodegenerative disease patients and suspending the disease progression. However, two major problems to be solved in neurotrophic factor therapy are: (1) excessive degradation rate; (2) The blood brain barrier cannot be passed, and the existing carrier has defects.
If the risk of polluting human genome exists when plasmid DNA containing the gene modification type BDNF is directly transfected to enter the human MSC cell nucleus, the risk can be avoided by transfecting the gene modification type BDNF mRNA by using a liposome method. However, wild-type human BDNF mRNA is easily degraded in an in vitro environment, the half-life of the BDNF protein expressed after transfection is short, and the Open Reading Frame (ORF) of its mRNA must be optimized and then capped/tailed to ensure its stability in an in vitro environment. The previous mRNA synthesis process has the problems of high cost, long time consumption and the like, and restricts the development process of related cell drugs.
Disclosure of Invention
In order to solve the problems, the invention provides a method for designing and preparing BDNF mRNA with high translation stability, which is convenient and rapid, has high cost performance, is suitable for popularization and application, and effectively avoids the defects that in the prior art, the in vitro transcription operation has the problems of high cost, long time consumption and the like and restricts the research and development progress of related cell drugs. The cost and man-hour of mRNA in vitro synthesis can accelerate the development of related cell drugs.
In order to overcome the defects in the prior art, the invention provides a solution for a design and preparation method of BDNF mRNA with high translational stability, which comprises the following steps:
a design of high translational stability BDNF mRNA comprising:
on the basis of a wild human BDNF gene sequence, the BDNF gene sequence is optimized into a stable BDNF sequence through the codon preference design of mammalian cells;
meanwhile, the original 5'-/3' -untranslated region (UTR) in the wild-type human BDNF gene is abandoned, and the BDNF mRNA is modified by adopting the 5'-UTR with the length shorter than 100nt and the 3' -UTR of globin.
Further, the designed stable BDNF sequence was synthesized and ligated into the appropriate pcDNA3.1 (+) plasmid, transformed into DH 5. Alpha. For mass amplification, and used for the preparation of BDNF mRNA.
A preparation method of BDNF mRNA with high translational stability comprises the following steps:
step 1: performing single enzyme digestion on 1-10 μ g of the plasmid to linearize the plasmid, adding 1-5 μ l of endonuclease, 5-25 μ l of endonuclease buffer solution and ddH2O to a final system of 50-200 μ l, and incubating at 37 ℃ for 30min-2h;
step 2: sequentially adding 1/100-1/50 volume of Ethylene Diamine Tetraacetic Acid (EDTA), 1/100-1/50 volume of ammonium acetate and 2-5 times volume of absolute ethyl alcohol, uniformly mixing, standing at low temperature, and purifying the linearized plasmid by adopting low-temperature high-speed centrifugation;
and step 3: in vitro capping transcription reaction;
and 4, step 4: removing nucleotides, short oligonucleotides, proteins and salts from the transcription and capping reaction;
and 5: polyadenylic acid tailing reaction;
step 6: removing the protein, buffer salt and nucleotide from the product obtained in step 5.
Further, the step 3 specifically includes: 0.5-5 μ g of the linear plasmid was added to an appropriate amount of nuclease-free water, followed by sequentially adding 1-5 μ l of 10 × reaction buffer, 5-30 μ l of 2 × NTP/cap analog and 1-5 μ l of the enzyme mixture, mixing well, and incubating at 37 ℃ for 1-16h.
Further, the step 4 specifically includes: adding 0.1-1ml of concentrated binding solution, mixing, adding 0.1-1ml of absolute ethanol, mixing, centrifuging at high speed, passing through a column, and removing the filtrate; centrifuging and cleaning with 0.1-1ml of cleaning solution at high speed for 2 times, dissolving the capped mRNA with 1-100 μ l of eluent, centrifuging and eluting at high speed, and collecting filtrate.
Further, the step 5 specifically includes: mixing non-ribonuclease water, 1-10 mul of 10 Xpolyadenylic acid polymerase reaction buffer solution, 1-30 mul of 10mMATP, 20-100 mu gRNA and 1-10 mul of polyadenylic acid polymerase, fully and uniformly mixing, placing at 37 ℃ for incubation for 10-30min, then adding 5-15mMEDTA, and placing the reaction solution at low temperature for 10-30min to stop the reaction to obtain the product.
Further, the step 6 specifically includes: adding 0.1-1ml of clearing and combining buffer solution, uniformly mixing, adding 0.1-1ml of absolute ethyl alcohol, uniformly mixing, centrifuging at high speed, passing through a column, and removing filtrate; and (3) carrying out high-speed centrifugal washing for 2 times by using 0.1-1ml of washing solution, adding 10-50 mu l of nuclease-free water which is preheated in advance to dissolve capped/tailed mRNA into the center of a filter membrane of the filter column, carrying out high-speed centrifugal elution, and collecting filtrate to finish the preparation of the BDNFmRNA with high translational stability.
The invention has the beneficial effects that:
the invention can complete the synthesis and purification of mRNA with translation stability in a short time by using plasmids with lower initial amount, and does not need the investment of additional reagents except for using a plurality of specific kits. Compared with an imported kit with high price, the method can achieve the same productivity under the condition that the two-step purification loss rate is lower than 5 percent, greatly facilitates the utilization of the mRNA product in downstream experiments, effectively solves the problems of high cost, long time consumption and the like of the existing mRNA in-vitro synthesis process, and accelerates the research and development process of related cell drugs. Effectively avoids the problems of high cost, long time consumption and the like of in vitro transcription operation in the prior art and overcomes the defect that the research and development progress of related cell drugs is restricted.
Drawings
FIG. 1 is a schematic diagram showing the design of BDNF mRNA with high translational stability according to the present invention;
FIG. 2 is a flow chart of the preparation method of the present invention;
FIG. 3 is a graph of the efficiency of transfecting MSCs according to example 1 of the present invention;
FIG. 4 is a graph of BDNF concentration and viability changes of the MSC-transfected preparations of example 1 of the present invention;
FIG. 5 is a graph showing the results of improvement of motor function and prolongation of survival time of different groups of mice in example 3 of the present invention.
Detailed Description
The invention utilizes a gene modification method to modify and optimize the BDNF mRNA sequence, and realizes the expression of BDNF in MSC by a liposome transfection mode, thereby greatly improving the BDNF secretion capacity of MSC.
The invention will be further described with reference to the following figures and examples.
Design of high translational stability BDNF mRNA, comprising:
on the basis of a wild human BDNF gene sequence, a stable BDNF sequence is designed and optimized through the codon preference of a mammal cell;
meanwhile, the original 5'-/3' -untranslated region (UTR) in the wild-type human BDNF gene is abandoned, and the BDNF mRNA is modified by adopting the 5'-UTR with the length shorter than 100nt and the 3' -UTR of globin.
The designed stable BDNF sequence is synthesized and connected into a proper pcDNA3.1 (+) plasmid, transformed into DH5 alpha for mass amplification, and used for preparing BDNF mRNA.
The preparation method of BDNF mRNA with high translational stability comprises the following steps:
step 1: performing single enzyme digestion on 1-10 μ g of the plasmid to linearize the plasmid, adding 1-5 μ l of endonuclease, 5-25 μ l of endonuclease buffer solution and ddH2O to a final system of 50-200 μ l, and incubating at 37 ℃ for 30min-2h;
and 2, step: sequentially adding 1/100-1/50 volume of Ethylene Diamine Tetraacetic Acid (EDTA), 1/100-1/50 volume of ammonium acetate and 2-5 times volume of absolute ethyl alcohol, uniformly mixing, standing at low temperature, and purifying the linearized plasmid by adopting low-temperature high-speed centrifugation;
and step 3: in vitro capping transcription reaction;
step (ii) of
4: removing nucleotides, short oligonucleotides, proteins and salts from the transcription and capping reaction;
and 5: polyadenylic acid tailing reaction;
and 6: removing the protein, buffer salt and nucleotide from the product obtained in step 5.
The step 3 specifically comprises: 0.5-5 mu g of linear plasmid is added into a proper amount of nuclease-free water, the nuclease-free water is 1-5 mu l of nuclease-free water, 1-5 mu l of 10 Xreaction buffer solution, 5-30 mu l of 2 XNTP/cap analogue and 1-5 mu l of enzyme mixture are sequentially added, and after uniform mixing, incubation is carried out for 1-16h at 37 ℃.
The step 4 specifically includes: adding 0.1-1ml of concentrated binding solution, mixing, adding 0.1-1ml of absolute ethyl alcohol, mixing, centrifuging at high speed, passing through a column, and removing the filtrate; washing with 0.1-1ml washing solution by high speed centrifugation for 2 times, dissolving the capped mRNA with 1-100 μ l eluent by high speed centrifugation, eluting, and collecting filtrate.
The step 5 specifically includes: mixing non-ribonuclease water, 1-10 mul of 10 Xpolyadenylic acid polymerase reaction buffer solution, 1-30 mul of 10mMATP, 20-100 mu gRNA and 1-10 mul of polyadenylic acid polymerase, fully and uniformly mixing, placing at 37 ℃ for incubation for 10-30min, then adding 5-15mM EDTA, and placing the reaction solution at low temperature for 10-30min to stop the reaction to obtain the product.
The step 6 specifically includes: adding 0.1-1ml of clearing and binding buffer solution, mixing uniformly, adding 0.1-1ml of absolute ethyl alcohol, mixing uniformly, centrifuging at high speed, passing through a column, and removing filtrate; and (3) carrying out high-speed centrifugal washing for 2 times by using 0.1-1ml of washing solution, adding 10-50 mu l of pre-preheated nuclease-free water to dissolve capped/tailed mRNA into the center of a central filter membrane of the filter column, carrying out high-speed centrifugal elution, and collecting filtrate to finish the preparation of the BDNFmRNA with high translational stability.
The specific embodiment is as follows:
example 1 (BDNF mRNA transfection of MSCs and preparation of MSC preparations)
(1) And (3) subculturing MSC:
human umbilical cord MSC 1 × 10 4 /cm 2 Is inoculated in a culture flask. Placing the cell culture flask at 37 deg.C and 5% CO 2 The culture was carried out in a saturated humidity incubator, the medium was changed every three to four days, and subculture was carried out the day before transfection of MSC.
(2) MSC transfection using mRNA transfection kit:
at 0.8-3.0X 10 at 18-24 hours before transfection 4 cells/cm 2 The cells are passaged to ensure that transfection begins when the cells reach the appropriate cell density (usually ≧ 80% confluence). Sequentially sampling and preparing a transfection system mixed solution according to the following sequence: 1.9ml of Opti-MEM I Reduced-Serum Medium, 19.7. Mu.g of mRNA, 39.4. Mu.l of mRNA Boost Reagent and 39.4. Mu.l of mRNA Boost Reagent, incubating the mixture at room temperature for 2-4min, adding the mixture to a T75 culture flask for culturing cells, and taking out the mixture after culturing for a proper time for preparing a subsequent semi-finished product.
(3) GFP-BDNF mRNA transfection MSC efficiency analysis
In the example of the transfection efficiency test, a section of Green Fluorescent Protein (GFP) gene was inserted into the carboxyl terminal of ORF of the designed sequence of BDNF, then mRNA was synthesized by the preparation method of the present invention, and MSC was transfected in example 1 (2), as shown in fig. 3, after transfection, the expression level of GFP and BDNF was significantly higher than that before transfection, and the transfection efficiency was more than 30%.
Example 2 (measurement of cell viability and BDNF secretion Capacity of MSC preparation)
This example investigated the differences in cell viability and BDNF secretion capacity of the MSC preparation prepared according to example 1 over 72 h after formulation compared to the untransfected control group as follows:
(1) And (3) discarding the culture medium of the transfected cells, washing the transfected cells twice by PBS, adding 5ml of digestion solution for digestion for 5min, adding 5ml of stop solution to stop digestion, blowing the cell suspension to prepare cell suspension, collecting the cell suspension in a centrifuge tube, centrifuging the cell suspension for 5min at the rotating speed of 270g, discarding the supernatant, adding a heavy suspension washing solution to wash the cell suspension for several times, and adding a proper amount of auxiliary material solution according to the number of the cells to obtain the prepared MSC-BDNF preparation.
(2) Sampling the MSC-BDNF preparation every 24h, detecting the cell viability of MSC in the preparation by AO/PI staining, taking the sample at the rotating speed of 270g for 5min, and discarding cell sediment. And (3) detecting the content of BDNF in the preparation by using the BDNF ELISA kit. The standard was set to 8 concentration gradients, the Assay panels were removed and 50. Mu.l Assay dilution RD1-123 was added to each well. Adding the standard substance and the sample into corresponding holes in proper volume, covering a membrane cover plate, arranging a shaking table at 500 +/-50 rpm, and incubating at room temperature for 2h; after the incubation is finished, adding washing liquid to wash the plate for four times according to requirements, then adding 200 mu l of Total BDNF Conjugate, covering a membrane cover plate, and incubating for 1h on a shaking bed at room temperature; after the incubation, the plate was washed four times, 200. Mu.l of Substrate Solution was added, and the plate was incubated at room temperature in the dark for 30min for color reaction. After the reaction, 50 μ l of Stop Solution was added to Stop the color development, and the OD value of each well was measured within 30min using a microplate reader at a wavelength of 450 nm.
In fig. 4a, the BDNF concentration in each time period of the experimental MSC-BDNF preparations transfected with BDNF mRNA was significantly higher than the control MSC preparation that was not transfected; in fig. 4b, the cell viability rates of the experimental MSC-BDNF preparation and the control MSC preparation gradually approached over time.
Example 3 (animal model experiment)
This example investigated the efficacy of transplantation of the MSC semi-finished preparation prepared according to the method of example 1 on improvement of motor function and prolongation of survival time of mice in the disease model of the progressive freezing disease (ALS), as follows:
experimental animals Male transgenic B6SJLTg (SOD 1-G93A) 1Gur/J mice (experimental group) and non-transgenic wild type littermate mice (control group) were selected for 4-6 weeks and were bred in SPF environment with 60% humidity, 21-23 deg.C, 12h light/12 h dark cycle. At 60 days of age, the mice were grouped into 6 groups, control group mice, experimental group mice were randomly divided into model group (intrathecally injected with PBS), MSC group (intrathecally injected with untransfected MSC), and MSC-BDNF group (intrathecally injected with MSC transfected with BDNF mRNA). When implementing MSC transplantation, the MSC is replaced by 10 5 Mu.l of the cell suspension was resuspended in PBS and 5. Mu.l of the cell suspension was injected into the subarachnoid space of the midline between the cauda equina vertebrae by means of lumbar punctureThe cannula will be maintained for 2 minutes and then slowly withdrawn. After one month of cell transplantation, various physiological indexes of each group of mice were evaluated.
In FIG. 5a, improvement in motor function was observed in ALS model mice in the ALS/MSC and ALS/MSC-BDNF groups, where the ALS/MSC-BDNF group mice exhibited significantly better rotarod performance in this rotatesting, and had longer drop delay than the ALS and ALS/MSC group mice; in addition, in FIG. 5b, the ALS/MSC-BDNF group mice showed a longer lifespan (146.67 days) than the ALS group and ALS/MSC group mice, 136.67 days for the ALS group and 142.33 days for the ALS/MSC group. The present invention has been described in an illustrative manner by the embodiments, and it should be understood by those skilled in the art that the present disclosure is not limited to the embodiments described above, but is capable of various changes, modifications and substitutions without departing from the scope of the present invention.
Claims (7)
1. A design of BDNF mRNA with high translational stability is characterized by comprising:
on the basis of a wild human BDNF gene sequence, the BDNF gene sequence is optimized into a stable BDNF sequence through the codon preference design of mammalian cells;
meanwhile, the original 5'-/3' -untranslated region (UTR) in the wild-type human BDNF gene is abandoned, and the 5'-UTR with the length shorter than 100nt and the 3' -UTR of globin are adopted to modify BDNF mRNA.
2. The design of BDNF mRNA with high translational stability of claim 1, wherein the designed stable BDNF sequence is synthesized and ligated into the proper pcDNA3.1 (+) plasmid, transformed into DH5 α for mass amplification, and used for preparing BDNF mRNA.
3. A preparation method of BDNF mRNA with high translational stability is characterized by comprising the following steps:
step 1: taking 1-10 mu g of plasmid, carrying out single enzyme digestion to linearize the plasmid, adding 1-5 mu l of endonuclease, 5-25 mu l of endonuclease buffer solution and ddH2O until the final system is 50-200 mu l, and incubating for 30min-2h at 37 ℃;
step 2: sequentially adding 1/100-1/50 volume of Ethylene Diamine Tetraacetic Acid (EDTA), 1/100-1/50 volume of ammonium acetate and 2-5 times volume of absolute ethyl alcohol, uniformly mixing, standing at low temperature, and purifying the linearized plasmid by adopting low-temperature high-speed centrifugation;
and step 3: in vitro capping transcription reaction;
and 4, step 4: removing nucleotides, short oligonucleotides, proteins and salts from the transcription and capping reaction;
and 5: polyadenylic acid tailing reaction;
step 6: removing the protein, buffer salt and nucleotide from the product obtained in step 5.
4. The method for preparing BDNF mRNA with high translational stability according to claim 3, wherein the step 3 specifically comprises the following steps: 0.5-5 μ g of the linear plasmid was added to an appropriate amount of nuclease-free water, followed by sequentially adding 1-5 μ l of 10 × reaction buffer, 5-30 μ l of 2 × NTP/cap analog and 1-5 μ l of the enzyme mixture, mixing well, and incubating at 37 ℃ for 1-16h.
5. The method for preparing BDNF mRNA with high translational stability according to claim 3, wherein the step 4 specifically comprises the following steps: adding 0.1-1ml of concentrated binding solution, mixing, adding 0.1-1ml of absolute ethanol, mixing, centrifuging at high speed, passing through a column, and removing the filtrate; washing with 0.1-1ml washing solution by high speed centrifugation for 2 times, dissolving the capped mRNA with 1-100 μ l eluent by high speed centrifugation, eluting, and collecting filtrate.
6. The method for preparing BDNF mRNA with high translational stability according to claim 3, wherein the step 5 specifically comprises the following steps: mixing non-ribonuclease water, 1-10 mul of 10 Xpolyadenylic acid polymerase reaction buffer solution, 1-30 mul of 10mMATP, 20-100 mu gRNA and 1-10 mul of polyadenylic acid polymerase, fully and uniformly mixing, placing at 37 ℃ for incubation for 10-30min, then adding 5-15mM EDTA, and placing the reaction solution at low temperature for 10-30min to stop the reaction to obtain the product.
7. The method for preparing BDNF mRNA with high translational stability according to claim 3, wherein the step 6 specifically comprises the following steps: adding 0.1-1ml of clearing and binding buffer solution, mixing uniformly, adding 0.1-1ml of absolute ethyl alcohol, mixing uniformly, centrifuging at high speed, passing through a column, and removing filtrate; and (3) carrying out high-speed centrifugal washing for 2 times by using 0.1-1ml of washing solution, adding 10-50 mu l of nuclease-free water which is preheated in advance to dissolve capped/tailed mRNA into the center of a filter membrane of the filter column, carrying out high-speed centrifugal elution, and collecting filtrate to finish the preparation of the BDNFmRNA with high translational stability.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104284979A (en) * | 2012-03-27 | 2015-01-14 | 库瑞瓦格有限责任公司 | Artificial nucleic acid molecules for improved protein or peptide expression |
| CN104762265A (en) * | 2015-04-22 | 2015-07-08 | 扬州大学 | Novel method for obtaining goat-induced multi-potent stem cells |
| CN108026537A (en) * | 2015-08-28 | 2018-05-11 | 库瑞瓦格股份公司 | Artificial nucleic acid molecule |
| CN109104870A (en) * | 2016-01-15 | 2018-12-28 | 丁恩雨 | A kind of general nucleic acid pharmaceutical carrier that can enhance mRNA stability He translating property |
| US20190002906A1 (en) * | 2017-05-31 | 2019-01-03 | Arcturus Therapeutics, Inc. | Synthesis and structure of high potency rna therapeutics |
| CN110218753A (en) * | 2019-05-27 | 2019-09-10 | 湖北爱济莱斯生物科技有限公司 | A method of improving external synthesis mRNA stability |
| CN112029773A (en) * | 2020-09-10 | 2020-12-04 | 武汉纽福斯生物科技有限公司 | Nucleic acids encoding BDNF and uses thereof |
-
2021
- 2021-12-22 CN CN202111580132.2A patent/CN115404238A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104284979A (en) * | 2012-03-27 | 2015-01-14 | 库瑞瓦格有限责任公司 | Artificial nucleic acid molecules for improved protein or peptide expression |
| CN104762265A (en) * | 2015-04-22 | 2015-07-08 | 扬州大学 | Novel method for obtaining goat-induced multi-potent stem cells |
| CN108026537A (en) * | 2015-08-28 | 2018-05-11 | 库瑞瓦格股份公司 | Artificial nucleic acid molecule |
| CN109104870A (en) * | 2016-01-15 | 2018-12-28 | 丁恩雨 | A kind of general nucleic acid pharmaceutical carrier that can enhance mRNA stability He translating property |
| US20190002906A1 (en) * | 2017-05-31 | 2019-01-03 | Arcturus Therapeutics, Inc. | Synthesis and structure of high potency rna therapeutics |
| CN110218753A (en) * | 2019-05-27 | 2019-09-10 | 湖北爱济莱斯生物科技有限公司 | A method of improving external synthesis mRNA stability |
| CN112029773A (en) * | 2020-09-10 | 2020-12-04 | 武汉纽福斯生物科技有限公司 | Nucleic acids encoding BDNF and uses thereof |
Non-Patent Citations (1)
| Title |
|---|
| ANDREA COLLIVA等: "Distinct role of 5\'UTR sequences in dendritic trafficking of BDNF mRNA: additional mechanisms for the BDNF splice variants spatial code", MOL BRAIN, vol. 14, no. 1 * |
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