CN105200059B - The siRNA of targeted inhibition mouse UCP2 gene expressions and its structure of expression vector - Google Patents

Abstract

The present invention relates to an RNA interference recombinant lentiviral vector for targeting and inhibiting the expression of mouse neural stem cell UCP2 gene and its construction. The recombinant lentiviral vector pNL-EGFP/CMV/WPREdU3-sh mUCP2 targeting mouse neural stem cell UCP2 gene is constructed, Then, co-transfect 293T cells with the recombinant lentiviral vector combined with the second-generation lentiviral packaging plasmid pCD/NL-BH*DDD and the membrane protein expression plasmid pLTR-G to obtain the packaged recombinant lentiviral vector. Transfection of mouse primary neural stem cells with the recombinant lentiviral vector obtained in the present invention can efficiently and specifically inhibit the expression of UCP2 gene in mouse neural stem cells, laying a good experimental foundation for further functional research on mouse UCP2 gene.

Description

Construction of siRNA and its expression vector targeting to inhibit the expression of mouse UCP2 gene

technical field

The invention belongs to the technical field of bioengineering, and relates to an RNAi recombinant lentiviral expression vector for mouse neural stem cell UCP2 gene and the construction of the expression vector. The recombinant lentivirus described in the present invention can be applied to the research on the pathogenesis of neural tube defects.

Background technique

Neural Tube Defects (NTDs) refer to congenital birth defects with the central nervous system as the main site of disease. Shanxi Province, my country has the highest incidence rate, with a statistical result of 19.9‰. The high incidence of NTDs has become the main cause of lifelong disability and death of infants in many countries and regions around the world.

The closing process of the neural tube is tightly controlled by genes as well as regulated by environmental factors. According to the applicant's research results and literature search in recent years, the relationship between uncoupling protein 2 (uncoupling protein 2, UCP2) and neural tube defects has drawn preliminary relevant conclusions in population surveys, suggesting that the UCP2 gene is a candidate gene for NTDs . Based on the above conclusions, the applicant intends to use mouse neural stem cells (closer to early embryonic neural tube cells) to prove the connection between UCP2 and NTDs, and to explore the mechanism by which it causes neural tube defects. Neural stem cells are a kind of cells that are difficult to transfect by conventional methods, so a method with higher transfection efficiency is urgently needed to meet the needs of follow-up experiments. The establishment of the above technical platform will provide a UCP2 gene function research model and play an important role in the follow-up mechanism research. However, there is no research report on siRNA of UCP2 gene in mouse neural stem cells.

RNA interference (RNAi) is the process of using double-stranded RNA to efficiently and specifically block the expression of specific genes in vivo, and promote the degradation of target gene mRNA to cause gene silencing. In the initial stage of RNAi, the double-stranded RNA (dsRNA) introduced in various ways (electroporation, virus infection, etc.) is gradually cut into 21-23nt small interfering RNA fragments (siRNA) by Dicer enzyme. The siRNA duplex combines with the ribozyme complex to form the RNA-induced silencing complex (RISC). In the RISC, the siRNA is denatured, the sense strand is detached, and the antisense strand is still bound to the complex and guides RISC to bind to the homologous target RNA, inducing target mRNA degradation, thereby blocking gene expression. siRNA can also be used as a special primer to synthesize dsRNA molecules using target mRNA as a template under the action of RNA-dependent RNA polymerase, and the latter can enter the above cycle. The nascent dsRNA is repeatedly synthesized and degraded, and new siRNA is continuously formed, which reduces the target mRNA, leads to the silencing of the target gene, and presents the phenomenon of RNAi.

siRNA is the main effector of RNA interference. Up to now, the technical route of mammalian cell RNAi can be carried out in two ways: 1) Directly prepare siRNA fragments of 21-23 nt, and transfer siRNA into mammalian cells. 2) Transfect the DNA expression vector of short hairpin RNA (shRNA) into cells, express shRNA, and obtain siRNA after Dicer cleavage. The former is not suitable for long-term research projects, while the latter can last for a long time, which is conducive to the development of subsequent experiments.

The effect of RNAi on gene expression inhibition is largely limited by the transfection method and transfection efficiency. The key to solving the problem is to select the appropriate vector to introduce into the cells. At present, there are three ways to introduce siRNA, 1) chemically synthesized siRNAs transfection interference, 2) shRNA expression vector method, 3) viral vector method. The first two methods have low infection efficiency, especially for cells with poor interference effect of liposomes, such as neural stem cells.

Lentiviral vector is a gene therapy vector developed on the basis of HIV-1, which has the ability to infect both dividing cells and non-dividing cells, and is currently widely used in the research of expressing RNAi. Compared with plasmid vectors and other viral vectors, lentivirus-mediated RNA interference is characterized by high efficiency, stability, and strong specificity.

Contents of the invention

The purpose of the present invention is to provide a kind of siRNA targeting to inhibit the expression of UCP2 gene in mouse neural stem cells.

Another object of the present invention is to provide an RNA interference recombinant lentiviral vector that targets and inhibits the expression of UCP2 gene in mouse neural stem cells, so as to effectively inhibit the expression of UCP2 gene in primary mouse neural stem cells.

The siRNA targeted to inhibit the expression of UCP2 gene in mouse neural stem cells of the present invention has the nucleotide sequence shown in Seq ID No.1, which is denoted as LVT818.

According to the siRNA sequence designed above, the present invention synthesizes two DNA template single strands encoding shRNA, the DNA template single strands encoding shRNA are the sense strand with the nucleotide sequence shown in Seq ID No.2 and the positive strand with the nucleotide sequence shown in Seq The antisense strand of the nucleotide sequence shown in ID No.3 consists of 5' sticky end + 19nt target sequence + stem-loop structure + target sequence complementary sequence + transcription termination site + 3' sticky end structure.

LVT818-1 (justice chain):

5'-CcggCCTAATGGCTGCCTACCAAcTCAAGAGATTGGTAGGCAGCCATTAGGTTTTTTg-3'.

LVT818-2 (antisense strand):

5'-aattcaaaaaaCCTAATGGCTGCCTACCAATCTCTTGAgTTGGTAGGCAGCCATTAGG-3'.

The present invention also provides an RNA interference recombinant lentiviral vector containing said shRNA, which is a vector pNL-EGFP/CMV/ WPREdU3-sh mUCP2.

The specific construction method is to anneal the single-stranded DNA encoding shRNA to form a double-stranded DNA fragment, connect it to the multiple cloning site of the pMagic4.1 lentiviral vector (Shanghai sbo-bio company), and construct the recombinant lentiviral vector pNL- EGFP/CMV/WPREdU3-sh mUCP2, and then co-transfect 293T with the recombinant lentiviral vector combined with the second-generation lentiviral packaging plasmid pCD/NL-BH*DDD and membrane protein expression plasmid pLTR-G (both purchased from Addgene) cells to obtain the packaged recombinant lentiviral vector.

Wherein, in the construction method, a cohesive end connected to the site of the Age I and EcoR I enzyme cutting vector is introduced at the end of the DNA fragment, and the pMagic4.1 vector is digested with Age I and EcoRI to recover the large fragment After ligation with the double-stranded DNA fragment, the competent bacteria were transformed, and the recombinant positive clones were picked.

The RNA interference recombinant lentiviral vector constructed in the present invention can be applied to inhibit the expression of UCP2 gene in mouse neural stem cells, so as to effectively inhibit the expression of UCP2 gene in neural stem cells.

The present invention provides an siRNA sequence capable of specifically silencing mouse UCP2 gene, and it is confirmed by in vitro transfection experiments that the sequence can effectively inhibit the expression of UCP2 gene in primary mouse neural stem cells. The present invention further constructs a recombinant lentivirus vector capable of stably expressing the above siRNA sequence, and produces the recombinant lentivirus with a high infection rate in 293T cells. Through in vivo infection experiments, the recombinant lentivirus that efficiently and stably expresses the UCP2 gene shRNA sequence can significantly inhibit the UCP2 gene expression of primary neural stem cells. The above research results provide a cell model for further research on the role of UCP2 gene in the occurrence of neural tube defects.

The pMagic 4.1 vector used in the present invention contains a U6 promoter, which can continuously express small RNAs with interfering effects in host cells. At the same time, the plasmid can express the fluorescent protein EGFP driven by the CMV promoter, which facilitates the detection of the transfection efficiency when the virus is packaged and the infection efficiency when infecting the host cell. The second-generation lentiviral packaging plasmid pCD/NL-BH*DDD and the membrane protein expression plasmid pLTR-G used in the present invention provide enzymes and proteins required for virus packaging. Co-transfecting 293T cells with the above vectors can efficiently assemble self-inactivated lentiviral vectors. The use of helper plasmids to package virus eliminates the need for infectious adenovirus, and the use of only two plasmids increases transfection efficiency and vector yield.

Compared with chemically synthesized siRNA and shRNA based on transient expression vectors, shRNA vectors constructed using lentiviruses have the following advantages: on the one hand, they can be amplified instead of transient expression vectors, and can be inserted into the host cell genome and Stable expression without insertional inactivation; on the other hand, this viral vector can be used to infect non-dividing cell lines such as primary neural stem cells that are difficult to transfect with traditional transfection reagents, and can be integrated into the genome of infected cells after infection , for long-term stable expression, while other traditional forms of transfection experiments in the early stage could not achieve the expected goal. The invention integrates the target gene into the genome of the target cell for long-term expression and has a small immune response, and is an ideal interference carrier for introducing into neural stem cells and silencing UCP2 gene in neural stem cells.

The present invention utilizes the inactivated lentiviral vector to carry the DNA template of the UCP2 RNA interference fragment, which is transcribed into shRNA in vivo, which perfectly solves the problems of targeting, safety and expression persistence of RNA interference.

Description of drawings

Figure 1 is a schematic diagram of the structure of pMagic 4.1 lentiviral vector.

Figure 2 is the results of fluorescence microscope observation of virus-infected neural stem cells.

Fig. 3 is a graph showing the interference effect on mouse UCP2 gene mRNA expression after virus infection of neural stem cells for 72 hours.

Fig. 4 is the protein Western-blot detection result of mouse UCP2 gene after virus infection of neural stem cells for 72 hours.

Figure 5 is the gray value scan of the protein Western-blot detection results of the mouse UCP2 gene 72 hours after virus infection of the neural stem cells.

Detailed ways

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

Example 1: Design of an siRNA sequence that targets and inhibits the expression of the mouse UCP2 gene.

According to the mRNA sequence of UCP2 gene in mouse neural stem cells (NM_011671), a set of siRNA sequences and control siRNA sequences targeting mUCP2 gene expression were designed, and a siRNA sequence with the best interference effect was screened out, named LVT818.

The specific siRNA sequence is as follows, wherein LVT818 is an effective interference fragment group, and LVT4 is a negative control group.

LVT818: 5'-CCTAATGGCTGCCTACCAA-3'; GC52.6%.

LVT4: 5'-TTCTCCGAACGTGTCACGT-3'; GC52.6%.

Example 2: Design and synthesis of oligonucleotides.

The sense strand and antisense strand of shRNA were designed and synthesized with the siRNA sequence designed in the above-mentioned Example 1. The Loop structure in the shRNA was selected as "cTCAAGAGA" and "TCTCTTGAg", and Age I and EcoR I restriction site sticky ends were added to the 5' end respectively, and the sequence was synthesized by Invitrogen Company. The specific shRNA sequence is as follows.

LVT818-1 (justice chain):

5'- Ccgg CCTAATGGCTGCCTACCAAcTCAAGAGATTGGTAGGCAGCCATTAGGTTTTTTg -3'.

LVT818-2 (antisense strand):

5'- aattcaaaaaa CCTAATGGCTGCCTACCAATCTCTTGAgTTGGTAGGCAGCCATTAGG-3'.

LVT4-1 (justice strand):

5'-CcggTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTg-3'.

LVT4-2 (antisense strand):

5'-aattcaaaaaTTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAA-3'.

After the shRNA sequence synthesized above was dissolved to 20 μM with oligo announcement buffer, 30 μl of each complementary single strand was mixed, heated in a water bath at 95°C for 5 minutes, and naturally cooled to room temperature to form double-stranded oligo fragments. Take 1 μl of the annealed product for the subsequent ligation reaction, and store the rest at -20°C.

Example 3: Construction of an interfering lentiviral vector for targeted inhibition of mouse UCP2 gene expression.

The schematic diagram of the structure of the lentiviral vector pMagic 4.1 is shown in Figure 1. The vector contains a U6 promoter, which can continuously promote the expression of downstream genes in host cells and continuously express small RNAs with interfering effects. The vector also contains a CMV promoter, which can drive the expression of the fluorescent protein EGFP, which facilitates the detection of transfection efficiency and infection efficiency.

Digest the lentiviral vector pMagic 4.1 with Age I and EcoR I to linearize it. After gel purification and recovery, use T4 ligase to ligate the annealed product of Example 2 overnight at 16°C, transform competent bacteria, and pick recombinant positive clones , after transformation screening, sent to Invitrogen Company for sequencing identification.

(Positive clone sequencing results)

ATAGAAAATATTTGACTGTAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGCCTAATGGCTGCCTACCAACTCAAGGATTGGTAGGCAGTTGTAG .

(Control clone sequencing results)

TCGGGCCGGTTAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGTTCTTCGAACGTATGGATCG .

The sequencing results showed that the sequence was exactly the same as the designed one, and the obtained correct clone was the successful construction of an interference lentiviral vector targeting and inhibiting the expression of mouse UCP2 gene, named pNL-EGFP/CMV/WPREdU3-sh mUCP2.

Example 4: Packaging and titer determination of recombinant interference lentiviral vector.

Take the high-purity recombinant lentiviral vector pNL-EGFP/CMV/WPREdU3-sh mUCP2 prepared in Example 3, combined with the second-generation lentiviral packaging plasmid pCD/NL-BH*DDD and membrane protein expression plasmid pLTR-G (both purchased from Addgene) co-transfected 293T cells, and the specific operation steps were carried out according to the instructions of Lipofectamine 2000 from Invitrogen Company.

Prepare 2 293T cells in T10cm culture dishes in advance, the medium is DMEM+10%FBS, 1% Glutamax, 1% penicillin-streptomycin. Divide the cells into ten 10cm culture dishes with a cell density of about ¹⁰⁷ per bottle. The next day, the cells were examined under a microscope, and the degree of cell confluence was roughly 80-90%, and the distribution was uniform.

One hour before transfection, remove the cell plate, remove the original cell culture medium, add 9ml Opti-MEM medium, and return the cells to the incubator.

Take two sterile 15ml centrifuge tubes, add 100μg pNL-EGFP/CMV/WPREdU3-sh mUCP2 recombinant lentiviral vector, 65μg pCD/NL-BH*DDD lentiviral packaging plasmid and 35μg pLTR-G membrane protein expression in one Plasmid, make up to 5ml with Opti-MEM medium; add 500µl Trans-EZ solution and 4.5ml Opti-MEM medium to the other branch, and mix gently with an electric pipette.

Add the Trans-EZ dilution to the plasmid tube dropwise, shake gently while adding, and incubate at room temperature for 20 minutes to fully combine the DNA and Trans-EZ to form a transfection complex.

Take a 5ml pipette, and evenly drop the obtained DNA-Trans-EZ complex into the cell culture plate, 1ml per plate. Shake the culture plate back and forth, mix well and put it back into the 5% carbon dioxide incubator. After 6 hours, remove the cell supernatant and replace it with 10ml of DMEM complete medium. When the cells were observed one day after transfection, more than 95% of the cells were fluorescent. The cells were returned to the incubator to continue culturing for 2 days, and all supernatants were collected, centrifuged at 4°C and 4000 g for 10 minutes to remove cell debris, and the supernatant was filtered with a 0.45 μM filter to obtain packaged recombinant lentiviral vectors.

After concentrating and purifying the packaged recombinant lentiviral vectors, a concentrated solution of high-titer lentiviral vectors was obtained, which was subpackaged and stored at -80°C for a long time, and one of them was taken to measure the biological titer of the virus.

Example 5: Target cell invasion test and gene expression inhibition effect analysis.

1. Infect mouse neural stem cells with recombinant interfering lentiviral vectors.

1) Select mouse neural stem cells in good condition, blow gently with a pipette to disperse the cells, collect by centrifugation, resuspend in cell culture medium, count the cells, adjust the cell concentration to 3×10 ⁴ -5×10 ⁴ cells/ml, press 90 μl /well was added to a 24-well plate, and placed in an incubator for 24 hours.

2) Prepare the recombinant interfering lentiviral vector dilution virus solution with an MOI value of 40, absorb the culture medium in the 96-well plate, add 100 µl of the diluted virus solution to each well, and set up an invalid interference fragment viral vector dilution and an empty viral vector dilution , were used as negative control and blank control, respectively.

3) After 24 hours, remove the culture medium containing the virus, add new culture medium to continue the culture, and judge the transfection efficiency by observing the expression of GFP under an inverted fluorescence microscope after 72 hours. Figure 2 is the observation result of the fluorescence of virus-infected neural stem cells under a microscope. Since the pMAGic 4.1 vector contains the green fluorescent protein gene, it can be seen that more than 90% of the neural stem cells are infected by the virus vector, and the infection efficiency is high. Cell samples were collected, and the obtained cells were used for Real-time PCR (real-time quantitative PCR) and Western blot (Western blot) detection.

2. RT-QPCR detection of neural stem cells after infection.

72 hours after the virus infected the neural stem cells, the infected cells were collected, and the RNA of the cells was extracted by the conventional Trizol method, and the cDNA was obtained by reverse transcription. M-MLV reverse transcriptase and dNTP were purchased from PROMEGA, and Oligo dT was purchased from Shanghai Sangong. The specific steps were according to Promega's M-MLV operating instructions.

1) Add 1.0µl Oligo dT (0.5µg/µl) and 2.0µg total RNA to a small PCR tube, add DEPC-H ₂ O to 9µl, mix well, centrifuge, and incubate at 70°C for 10 minutes. Immediately thereafter, insert into a 0°C ice-water bath to anneal the Oligo dT and template.

2) According to the ratio in the table, calculate the required amount of reagents according to the number of reaction tubes. Mix the M-MLV enzyme and the like on ice to obtain the RT reaction solution.

3) Add 11µl RT reaction solution to each reaction tube, mix well and centrifuge.

4) The RT reaction was completed at 42°C for 1 hour, and then treated at 70°C for 10 minutes to inactivate the RT enzyme.

5) The obtained RT reaction product cDNA is used for PCR.

6) Real-time PCR detection.

The β-actin gene was used as an internal reference, and real-time fluorescent quantitative PCR was used for detection:

(1) The primer sequence is as follows:

(2) Configure the reaction system according to the following ratio:

(3) Set the program as two-step Real-time quantification, pre-denaturation at 95°C for 15S, followed by each step of denaturation at 95°C for 5S, annealing and extension at 60°C for 30S, a total of 45 cycles. Read the absorbance each time during the extension phase.

The results show that the designed specific interference fragment can effectively inhibit the expression of UCP2 gene, and the expression level of UCP2 in infected mouse neural stem cells is only 21% of that of the control, that is, the interference rate is 79%. The specific results are shown in Table 1 and Figure 3 , wherein pLVT4 is the cell group transfected with an empty virus control (negative control), and pLVT818 is the cell group transfected with pLVT818 target virus supernatant.

Fig. 3 is a diagram showing the effect of interference on mouse UCP2 gene mRNA expression after the recombinant interference lentiviral vector infected mouse neural stem cells for 72 hours. In the figure, the empty virus group is the control group transfected with empty virus (blank control), pLVT4 is the cell group transfected with pLVT4 virus control (negative control), and pLVT818 is the cell group transfected with pLVT818 target virus supernatant (positive group). It can be seen that the expression level of UCP2 gene mRNA in the infected mouse neural stem cells is only 21% of that of the blank control, that is, the interference rate is 79%.

3. Western-blot detection.

Antibody information in this experiment.

72 hours after virus transfection, the total protein of cells in each experimental group was extracted and detected by Western blot. The gray ratio of UCP2 and β-actin showed that the inhibition rate was 75%, indicating that the efficiency of the plasmid to inhibit UCP2 was high, as shown in Figure 4, 5.

Figure 4 shows the results of Western-blot detection of UCP2 gene protein after virus infection of neural stem cells for 72 hours. In the figure 1 is the empty virus control group, 2 and 3 are the negative control groups for pLVT4 virus interference, and 4 is the pLVT818 virus interference group, which can be seen in group 4 Protein expression bands were significantly reduced.

Figure 5 is a gray value scan of the Western-blot detection results of the mouse UCP2 gene protein after virus infection of the neural stem cells for 72 hours. 1 in the figure is the empty virus control group, 2 and 3 are the invalid interference groups, and 4 is the pLVT818 virus interference group. Gray-scale scanning showed that the expression interference in group 4 was 75%.

The above figures 3, 4, and 5 all show that the pLVT818 virus interference group can significantly reduce the expression of UCP2 in neural stem cells, and pLVT818 is an effective interference fragment.

SEQUENCE LISTING

<110> Shanxi Medical University

<120> Construction of siRNA and its expression vector targeting to inhibit mouse UCP2 gene expression

<160> 3

<170> Patent version 3.2

<210> 1

<211> 21

<212> RNA

<213> Artificial sequence

<223> siRNA targeting UCP2 gene expression in mouse neural stem cells

<400> 1

CCTAATGGCT GCCTACCAA 19

<210> 2

<211> 58

<212> RNA

<213> Artificial sequence

<223> The positive-sense strand of the shRNA sequence targeted to inhibit UCP2 gene expression in mouse neural stem cells

<400> 2

CCGGCCTAAT GGCTGCCTAC CAACTCAAGA GATTGGTAGG CAGCCATTAG GTTTTTTG 58

<210> 3

<211> 58

<212> RNA

<213> Artificial sequence

<223> Antisense strand of shRNA sequence targeted to inhibit UCP2 gene expression in mouse neural stem cells

<400> 3

AATTCAAAAA ACCTAATGGC TGCCTACCAA TCTCTTGAGT TGGTAGGCAG CCATTAGG 58

Claims (7)

1. a kind of siRNA of targeted inhibition mouse neural stem cells UCP2 gene expressions, the nucleotide sequence of the siRNA is such as Shown in Seq ID No.1.

2. a kind of DNA of coding shRNA, the shRNA is positive-sense strand or antisense strand containing siRNA described in claim 1 shRNA。

3. a kind of RNA interference recombinant lentivirus vectors of targeted inhibition mouse neural stem cells UCP2 gene expressions, are at itself After the DNA of shRNA described in the multiple cloning sites connection coding claim 2 of the second generation slow virus carrier pMagic 4.1 of inactivation The carrier pNL-EGFP/CMV/WPREdU3-sh mUCP2 of structure.

4. the construction method of RNA interference recombinant lentivirus vectors described in claim 3 is that the DNA of the coding shRNA is single-stranded Annealing forms double chain DNA fragment, is connected in the multiple cloning sites of pMagic4.1 carriers, structure recombined lentivirus vector pNL- EGFP/CMV/WPREdU3-sh mUCP2, then with recombined lentivirus vector joint second generation slow virus packaging plasmid pCD/ NL-BH*DDD and memebrane protein expression plasmid pLTR-G cotransfection 293T cells, obtain the recombined lentivirus vector of the packaging.

5. the construction method of RNA interference recombinant lentivirus vectors according to claim 4 is at the end of the DNA fragmentation End introduce withAge IWithEcoR IThe glutinous end that restriction enzyme site is connected.

6. the construction method of RNA interference recombinant lentivirus vectors according to claim 4, be withAge IWithEcoRIDigestion PMagic4.1 carriers, recycling large fragment are ligated and transformed into competence bacteria, picking recombinant clone with the double chain DNA fragment.

7. RNA interference recombinant lentivirus vectors described in claim 3 are on inhibiting mouse neural stem cells UCP2 gene expressions Using.