CN115404238A - Design and preparation method of BDNF mRNA with high translational stability - Google Patents

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

Design and preparation method of BDNF mRNA with high translational stability

technical field

The invention relates to the technical field of nucleic acid products, in particular to a method for designing and preparing BDNF mRNA with high translation stability.

Background technique

Mesenchymal stem cells (MSCs) are somatic stem cells derived from mesoderm, and exist in bone marrow, umbilical cord, and adipose tissue. MSCs can grow on plastic surfaces and have the potential to differentiate into osteoblasts, chondrocytes, and adipocytes. Therefore, MSCs are expected to be applied in the field of regenerative medicine such as reconstruction of bones, blood vessels, and cardiac muscle. In addition, MSCs are also considered to be a new treatment method for neurodegenerative diseases. While inhibiting neuroinflammation, they can also secrete a variety of neurotrophic factors in a limited manner for the treatment of nerve damage. Among them, brain-derived neurotrophic factor (BDNF) plays an important role in synaptic transmission. In situ injection or overexpression of BDNF can increase the production of newborn neurons, promote neurogenesis, and protect the motor nerves of patients with neurodegenerative diseases. Successful cases have been reported in terms of metabolization and delay of disease progression. However, two major problems that need to be solved urgently for neurotrophic factor therapy are: (1) too fast degradation rate; (2) inability to pass through the blood-brain barrier, and existing carriers have defects.

If the plasmid DNA containing gene-modified BDNF is directly transfected into the nucleus of human MSCs, there is a risk of contaminating the human genome, and liposome transfection of gene-modified BDNF mRNA can avoid this risk. However, wild-type human BDNF mRNA is easily degraded in vitro, and the half-life of BDNF protein expressed after transfection is short, so the open reading frame (ORF) of its mRNA must be optimized, and then capping/tailing reactions are performed to ensure its Stability in in vitro environment. The high cost and time-consuming problems of the previous mRNA synthesis process have restricted the development of related cellular drugs.

Contents of the invention

In order to solve the above problems, the present invention provides a method for designing and preparing BDNF mRNA with high translation stability. The method is convenient, quick, cost-effective, suitable for popularization and application, and effectively avoids the high cost and disadvantages of in vitro transcription operations in the prior art. Time-consuming and other problems restrict the progress of the research and development of related cell drugs. The cost and man-hours of mRNA synthesis in vitro can speed up the development of related cellular drugs.

In order to overcome the deficiencies in the prior art, the present invention provides a solution for the design and preparation of BDNF mRNA with high translation stability, as follows:

A design of highly translationally stable BDNF mRNA comprising:

On the basis of the wild-type human BDNF gene sequence, it is optimized as a stable BDNF sequence through mammalian cell codon preference design;

At the same time, the original 5'-/3'-untranslated region (UTR) in the wild-type human BDNF gene was discarded, and the 5'-UTR with a length shorter than 100nt and the 3'-UTR of globin were used to modify BDNF mRNA .

Further, the designed stable BDNF sequence was synthesized and ligated into an appropriate pcDNA3.1(+) plasmid, transformed into DH5α for massive amplification, and used for the preparation of BDNF mRNA.

A method for preparing BDNF mRNA with high translation stability, comprising the steps of:

Step 1: Take 1-10 μg of the plasmid for single enzyme digestion to linearize it, add 1-5 μl of endonuclease, 5-25 μl of endonuclease buffer and add ddH2O until the final system is 50-200 μl, at 37 Incubate at ℃ for 30min-2h;

Step 2: Add 1/100-1/50 volume of ethylenediaminetetraacetic acid (EDTA), 1/100-1/50 volume of ammonium acetate and 2-5 times the volume of absolute ethanol successively, mix well and place in low temperature Stand still, and then use low-temperature high-speed centrifugation to purify the linearized plasmid;

Step 3: in vitro capping transcription reaction;

Step 4: Remove nucleotides, short oligonucleotides, proteins and salts in the transcription and capping reaction solution;

Step 5: polyA tailing reaction;

Step 6: Remove proteins, buffer salts and nucleotides from the product obtained in step 5.

Further, the step 3 specifically includes: taking 0.5-5 μg linearized plasmid and adding an appropriate amount of nuclease-free water, and then sequentially adding 1-5 μl of 10× reaction buffer, 5-30 μl of 2×NTP/cap analog and 1- 5μl enzyme mixture, mix well and incubate at 37°C for 1-16h.

Further, the step 4 specifically includes: adding 0.1-1ml of concentrated binding solution, mixing, then adding 0.1-1ml of absolute ethanol for mixing, high-speed centrifugation through the column and discarding the filtrate; high-speed centrifugation with 0.1-1ml of washing solution for 2 washes Afterwards, 1-100 μl of eluent was used to dissolve the capped mRNA and eluted by high-speed centrifugation, and the filtrate was collected.

Further, the step 5 specifically includes: mixing RNase-free water, 1-10 μl of 10× polyadenylation polymerase reaction buffer, 1-30 μl of 10 mMATP, 20-100 μg of RNA, and 1-10 μl of polyadenylation Nucleic acid polymerase, mix well and incubate at 37°C for 10-30min, then add 5-15mM EDTA, place the reaction solution at low temperature for 10-30min to terminate the reaction and obtain the product.

Further, the step 6 specifically includes: adding 0.1-1ml of clear binding buffer, mixing, adding 0.1-1ml of absolute ethanol for mixing, high-speed centrifugation through the column and discarding the filtrate; high-speed centrifugation with 0.1-1ml of washing liquid to wash 2 After three times, add preheated 10-50 μl nuclease-free water to the center of the filter column to dissolve the capped/tailed mRNA and centrifuge it at high speed, and collect the filtrate to complete the preparation of BDNF mRNA with high translation stability.

The beneficial effects of the present invention are:

The present invention can complete the synthesis and purification of mRNA with translation stability in a short period of time by using a relatively low initial amount of plasmid, and does not require the input of additional reagents except for the use of several specific kits. Compared with expensive imported kits, the present invention can achieve the same production capacity when the two-step purification loss rate is lower than 5%, which greatly facilitates the flexible use of the mRNA product in downstream experiments and effectively solves the problem of It overcomes the high cost and time-consuming problems of the existing mRNA synthesis process in vitro, and speeds up the process of research and development of related cell drugs. It effectively avoids the problems of high cost and time-consuming in vitro transcription operation in the prior art, which restricts the progress of research and development of related cell drugs.

Description of drawings

Fig. 1 is the design schematic diagram of high translation stability BDNF mRNA of the present invention;

Fig. 2 is a flow chart of the preparation method of the present invention;

Fig. 3 is the efficiency figure of transfection MSC of embodiment 1 of the present invention;

Fig. 4 is the graph of BDNF concentration and activity rate change of the preparation transfected with MSC in 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 mice in different groups in Example 3 of the present invention.

Detailed ways

The present invention utilizes the method of gene modification to modify and optimize the BDNF mRNA sequence, and then realizes the expression of BDNF in MSCs through liposome transfection, which can greatly improve the BDNF secretion capacity of MSCs.

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Design of BDNF mRNA with high translational stability, including:

On the basis of the wild-type human BDNF gene sequence, it is optimized as a stable BDNF sequence through mammalian cell codon preference design;

At the same time, the original 5'-/3'-untranslated region (UTR) in the wild-type human BDNF gene was discarded, and the 5'-UTR with a length shorter than 100nt and the 3'-UTR of globin were used to modify BDNF mRNA .

The designed stable BDNF sequence was synthesized and ligated into an appropriate pcDNA3.1(+) plasmid, transformed into DH5α for massive amplification, and used for the preparation of BDNF mRNA.

A method for preparing BDNF mRNA with high translation stability, comprising the steps of:

Step 1: Take 1-10 μg of the plasmid for single enzyme digestion to linearize it, add 1-5 μl of endonuclease, 5-25 μl of endonuclease buffer and add ddH2O until the final system is 50-200 μl, at 37 Incubate at ℃ for 30min-2h;

Step 2: Add 1/100-1/50 volume of ethylenediaminetetraacetic acid (EDTA), 1/100-1/50 volume of ammonium acetate and 2-5 times the volume of absolute ethanol successively, mix well and place in low temperature Stand still, and then use low-temperature high-speed centrifugation to purify the linearized plasmid;

Step 3: in vitro capping transcription reaction;

step

4: Remove nucleotides, short oligonucleotides, proteins and salts in the transcription and capping reaction solution;

Step 5: polyA tailing reaction;

Step 6: Remove proteins, buffer salts and nucleotides from the product obtained in step 5.

The step 3 specifically includes: taking 0.5-5 μg of linear plasmid and adding an appropriate amount of nuclease-free water, the nuclease-free water is 1-5 μl of nuclease-free water, and then sequentially adding 1-5 μl of 10× reaction buffer, 5-30 μl 2×NTP/cap analog and 1-5 μl enzyme mixture, mix well and incubate at 37°C for 1-16h.

The step 4 specifically includes: adding 0.1-1ml of concentrated binding solution, mixing, adding 0.1-1ml of absolute ethanol and mixing, high-speed centrifugation through the column and discarding the filtrate; washing with 0.1-1ml of washing solution at high speed for 2 times, and then using 1 - 100 μl of eluate was used to dissolve the capped mRNA and eluted by high-speed centrifugation, and the filtrate was collected.

The step 5 specifically includes: mixing ribonuclease-free water, 1-10 μl of 10× polyadenylate polymerase reaction buffer, 1-30 μl of 10mMATP, 20-100 μg RNA, and 1-10 μl of polyadenylate polymerase , mix well and incubate at 37°C for 10-30min, then add 5-15mM EDTA, place the reaction solution at low temperature for 10-30min to terminate the reaction and obtain the product.

The step 6 specifically includes: adding 0.1-1ml of clearing binding buffer, mixing, adding 0.1-1ml of absolute ethanol to mix, high-speed centrifugation through the column and discarding the filtrate; washing with 0.1-1ml of washing liquid for high-speed centrifugation for 2 times, Add 10-50 μl of preheated nuclease-free water to the central filter membrane of the filter column to dissolve the capped/tailed mRNA and centrifuge it at high speed, and collect the filtrate to complete the preparation of BDNF mRNA with high translation stability.

A specific example is as follows:

Embodiment 1 (preparation of BDNF mRNA transfection MSC and MSC preparation)

(1) Subculture of MSC:

Human umbilical cord MSCs were seeded in culture flasks at a density of 1×10 ⁴ /cm ² . Place the cell culture flask in a 37°C, 5% CO ₂ saturated humidity incubator for culture, change the medium every three to four days, and implement subculture on the day before MSC transfection.

(2) Use the mRNA transfection kit to transfect MSC:

Cells were passaged at a density of 0.8-3.0×10 ⁴ cells/cm ² 18-24 hours prior to transfection to ensure that the cells reached an appropriate cell density (typically ≥80% confluency) to initiate transfection. Prepare the transfection system mixture by sampling in the following order: 1.9ml of Opti-MEM I Reduced-Serum Medium, 19.7μg of mRNA, 39.4μl of mRNA BoostReagent and 39.4μl of mRNA Boost Reagent, and incubate the mixture at room temperature for 2-4min Then add it to the T75 culture flask where the cells are cultured, and take it out after cultivating for an appropriate time for subsequent preparation of semi-finished products.

(3) Analysis of GFP-BDNF mRNA transfection MSC efficiency

In the embodiment of transfection efficiency detection, the carboxy terminus of the ORF of the design sequence of BDNF is inserted a section of green fluorescent protein (GFP) gene, then synthesizes mRNA with the preparation method of the present invention, and uses the method in embodiment 1 (2) MSCs were transfected, as shown in Figure 3, after transfection, the expression levels of GFP and BDNF were significantly higher than before transfection, and the transfection rate was greater than 30%.

Embodiment 2 (detection of MSC preparation cell viability and BDNF secretion ability)

In this example, the differences in the cell viability and BDNF secretion ability of the MSC preparation prepared according to the method of Example 1 compared with the non-transfected control group at 72 h after preparation were studied. The specific methods are as follows:

(1) Discard the medium after transfection, wash twice with PBS, add 5 ml of digestion solution to digest for 5 minutes, then add 5 ml of stop solution to stop digestion, pipette to make a cell suspension and collect it into a centrifuge tube, with 270g Centrifuge at a high speed for 5 minutes, discard the supernatant, add resuspension lotion and wash several times, and add an appropriate amount of excipient solution according to the number of cells, which is the prepared MSC-BDNF preparation.

(2) The MSC-BDNF preparation was sampled every 24 hours, and the cell viability of MSC in the preparation was detected by AO/PI staining. The obtained sample was rotated at 270 g for 5 minutes, and the cell pellet was discarded. The supernatant was used to detect the content of BDNF in the preparation by BDNF ELISA kit. Set 8 concentration gradients for the standard, take out the test sample plate, and add 50 μl Assay DiluentRD1-123 to each well. Standards and samples are added to the corresponding wells in an appropriate volume. After the cover plate is covered, set the shaker at 500±50rpm and incubate at room temperature for 2h; After the plate was covered with membrane, incubate on a shaker at room temperature for 1 hour; wash the plate four times after incubation, add 200 μl Substrate Solution, and incubate for 30 minutes at room temperature in the dark for color reaction. Then add 50 μl StopSolution to stop the color development, and use a microplate reader to measure the OD value of each well at a wavelength of 450 nm within 30 min.

In Figure 4a, the concentration of BDNF in the MSC-BDNF preparation of the experimental group transfected with BDNF mRNA was significantly higher than that of the MSC preparation of the control group without transfection treatment at each time period; The cell viability of group MSC preparations gradually approached with time.

Embodiment 3 (animal model experiment)

This embodiment has studied the effect of the transplantation of the MSC semi-finished preparation prepared by the method of Example 1 on the improvement of motor function and the prolongation of survival time of ALS (ALS) disease model mice, and the specific methods are as follows:

As experimental animals, male transgenic B6SJLTg(SOD1-G93A) 1Gur/J mice (experimental group) and their non-transgenic wild-type littermate mice (control group) were selected for 4-6 weeks, and they were raised at 60% humidity and 21-23°C , 12h light/12h dark cycle SPF environment. When the mice were 60 days old, they were grouped with 6 mice in each group. The mice in the control group and the mice in the experimental group were randomly divided into the model group (intrathecal injection of PBS), the MSC group (intrathecal injection of non-transfected MSC ) and MSC-BDNF group (intrathecal injection of MSCs transfected with BDNF mRNA). When performing MSC transplantation, MSCs were resuspended in PBS at a concentration of 10 ⁵ /μl, and 5 μl of the cell suspension was injected into the subarachnoid space in the midline between the vertebrae at the level of the cauda equina by lumbar puncture, and the intubation was maintained for 2 minutes , which is then slowly retracted. One month after the cell transplantation, the physiological indexes of the mice in each group were evaluated.

In Fig. 5a, the improvement of motor function of ALS model mice can be observed in ALS/MSC group and ALS/MSC-BDNF group. In this rotation test, ALS/MSC-BDNF group mice showed significantly more Compared with the ALS group and ALS/MSC group mice, they had a longer drop delay time; in addition, in Figure 5b, the ALS/MSC-BDNF group mice showed a longer drop delay than the ALS group and ALS group The mice in the ALS/MSC group had a longer lifespan (146.67 days), 136.67 days in the ALS group, and 142.33 days in the ALS/MSC group. The present invention has been described above in the manner of illustrating the embodiments. It should be understood by those skilled in the art that the present disclosure is not limited to the embodiments described above, and various changes can be made without departing from the scope of the present invention. change and replace.

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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