Nucleic acid molecule capable of enhancing exogenous gene expression level and application of nucleic acid molecule

Abstract

The invention belongs to the technical field of biology, and particularly relates to a nucleic acid molecule capable of enhancing the expression level of an exogenous gene and application of the nucleic acid molecule. A nucleotide sequence is used, 34 amino acids expressed by the nucleotide sequence (hereinafter referred to as C peptides) are added to the N terminal of a target protein to be expressed, and the expression quantity of the target protein can be increased. Meanwhile, a nucleotide sequence is added behind the nucleotide to express a short peptide 2A peptide sequence derived from the virus, and the C peptide and the target protein are separated through self-cleavage of the 2A peptide, so that the complete target protein is obtained. The influence of the amino acid on the original function of the target protein is avoided. According to the method disclosed by the invention, the transient transfection expression quantity of the foreign protein in the 293 eukaryotic system can be improved. And a foundation is laid for protein early screening and function research.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a nucleic acid molecule capable of enhancing the expression level of an exogenous gene and an application thereof. Background Art

[0002] Recombinant protein is a protein gene that is introduced into host cells through vectors, and then expressed through the biological mechanisms of host cells. Various vector systems and host cells have been developed, including prokaryotes such as Escherichia coli and eukaryotes (such as yeast, insect cells and mammalian cells), to produce a wide spectrum of recombinant proteins close to their natural state, promoting the development of biochemical research, industrial catalysis, food processing, vaccines and treatment.

[0003] High-quality recombinant proteins are important starting materials for research and drug development. Protein expression and purification are often challenging due to the complexity of proteins themselves. Certain sequence or structural features of proteins may affect the yield and stability of recombinant protein products. Most proteins are fragile molecules that are susceptible to environmental stress during expression and purification. Therefore, recombinant protein expression often requires careful planning and process optimization to ensure high yields of recombinant proteins and minimize quality damage.

[0004] In transient expression, since the exogenous genes in the cells will be lost as the cells divide, the expression of the target protein can only be maintained for a few days to more than ten days, and there is a disadvantage of low expression. So how to increase the expression of transient transfection and obtain more recombinant proteins for drug efficacy and formulation research in the shortest time and at the lowest cost has become an urgent problem to be solved. Summary of the invention

[0005] The present invention aims to construct a nucleic acid molecule containing a function of enhancing the expression level of an exogenous gene, and utilizes the first 34 amino acids at the N-terminus of the capsid protein of Semliki forest virus (SFV) (hereinafter referred to as C-peptide), thereby increasing the recombinant protein in a transient expression system. The amino acid sequence of the translation enhancer is: MNYIPTQTFYGRRWRPRPAARPWPLQATPVAPVV (SEQ ID NO: 5). When studying proteins with low expression levels, adding this translation enhancer sequence in front can significantly promote protein translation.

[0006] In order to achieve the above-mentioned object, the first aspect of the present invention discloses a nucleic acid molecule for enhancing the expression level of an exogenous gene. The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 1, 2, 3 or 4.

[0007] The amino acid sequence of the protein expressed by the nucleic acid molecule is shown in SEQ ID NO:5.

[0008] The second aspect of the present invention discloses a recombinant expression vector, which comprises the above-mentioned nucleic acid molecule and a target protein gene.

[0009] Wherein, the recombinant expression vector also includes a DNA vector, an RNA vector, a plasmid, a transposon vector, a liposome, a CRISPR / Cas9 vector or a viral vector.

[0010] The target protein gene is also connected to a protein tag, and the protein tag is preferably a His tag.

[0011] A 2A peptide is also connected between the nucleic acid molecule and the target protein gene; the nucleotide sequence of the 2A peptide is shown in SEQ ID NO:6.

[0012] GGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAAGCCGGCGACGTGGAGGAGAAACCCGGCCCC (SEQ ID NO: 6).

[0013] The third aspect of the present invention discloses a recombinant host cell, wherein the above-mentioned recombinant expression vector is constructed into the genome of the host cell, so that the target protein gene is expressed in the host cell.

[0014] The fourth aspect of the present invention discloses the use of the above-mentioned nucleic acid molecules, recombinant expression vectors or host cells in a recombinant protein production system.

[0015] The system used to produce recombinant proteins is specifically the 293 expression system.

[0016] The recombinant expression vector prepared by the present invention is transfected into the 293 system, which can increase the expression amount of the exogenous target protein in the transient transfection system.

[0017] The present invention inserts a codon-optimized 2A peptide between the C peptide and the target protein, separates the C peptide and the target protein through self-cleavage of the 2A peptide, and obtains a complete target protein, thereby avoiding the influence of the exogenously inserted gene on the original function of the target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the protein expression amino acid sequence of the N-terminal C-peptide constructed in Example 1.

[0019] Figure 2 This is a schematic diagram of the nucleotide sequence of the protein nucleic acid molecule encoding the N-terminal C-peptide constructed in Example 1.

[0020] Figure 3Fortiebio was used to detect the expression levels of different samples of the synthetic expression vectors S1+042-PTT5, S2+042-PTT5, S3+042-PTT5, S4+042-PTT5 and 042-PTT5.

[0021] Figure 4 This is the staining result of SDS-PAGE in Example 2.

[0022] Figure 5 This is the band result diagram of Western blot (WB) in Example 3. DETAILED DESCRIPTION

[0023] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0024] Example 1

[0025] 1. Design a protein expression sequence with an N-terminal C-peptide, such as Figure 1-2 As shown, a 2A peptide is added between the C peptide and the protein sequence to self-cleave and remove the C peptide after protein expression, thereby ensuring the integrity of the protein N-terminus and synthesizing the related plasmid.

[0026] 2. Codon optimization The codons of the amino acid sequences of peptide C and peptide 2A were optimized, and the results were as follows: (1) Optimize sequence S1: TGAACTACATCCCCACACAGACCTTCTATGGCCGGAGATGGAGACCTAGACCTGCTGCTAGACCCTGGCCTCTGCAAGCCACCCCCGTGGCCCCCGTGGTGGGCAGCGGCCACAAACTTCTCCCTACTAAAGCAAGCAGGAGATGTTGAGGAGAACCCTGGGCCCC (SEQ ID NO: 1).

[0027] (2) Optimize sequence S2: ATGAATTACATCCCTACGCAAACGTTCTACGGCCGCCGGTGGCGCCCGCGCCCGGCGGCCCGTCCCTGGCCGTTGCAGGCCACTCCGGTGGCTCCCGTCGTCGGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAAGCCGGCGACGTGGAGGAGAAACCCGGCCCC (SEQ ID NO: 2).

[0028] (3) Optimize sequence S3: ATGAACTACATCCCCACACAGACCTTCTATGGCCGGAGATGGAGACCTAGACCTGCTGCTAGACCCTGGCCTCTGCAAGCCACCCCCGTGGCCCCCGTGGTGGGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAAGCCGGCGACGTGGAGGAGAAACCCGGCCCC (SEQ ID NO: 3).

[0029] (4) Optimize sequence S4: ATGAATTACATCCCTACGCAAACGTTCTACGGCCGCCGGTGGCGCCCGCGCCCGGCGGCCCGTCCCTGGCCGTTGCAGGCCACTCCGGTGGCTCCCGTCGTCGGCAGCGGGCCACAAACTTCTCCCTACTAAAGCAAGCAGGAGATGTTGAGGAGAACCCTGGGCCC (SEQ ID NO: 4).

[0030] 2A peptide sequence: GGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAAGCCGGCGACGTGGAGGAGAAACCCGGCCCC (SEQ ID NO: 6).

[0031] A synthesis company was commissioned to synthesize the above DNA sequences, and they were inserted into the expression plasmid ptt5 respectively to obtain expression vectors S1+042-PTT5, S2+042-PTT5, S3+042-PTT5, and S4+042-PTT5. At the same time, a control plasmid 042-PTT5 without C peptide and 2A peptide was constructed.

[0032] 042DNA sequence (stop codon is TGA) 042 Amino acid sequence: MGWSCIILFLVATATGAYAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGMIPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD* (SEQ ID NO: 8) 3. The present invention is used to transiently express foreign proteins in the 293 system, and the specific operations are as follows: The culture medium was OPM-293 CD05 Medium, stored in a 4°C refrigerator and preheated in a 37°C water bath before use.

[0033] Cell preparation: Resuscitate Expi293 cells into OPM-293 CD05, culture 30 mL in a 125 mL shake flask, 120 rpm, 37°C, 8% CO2, 80% humidity shaker, and inoculate after three or more passages. Dilute the resuscitated cells to a density of 0.4-0.7×10 6 cell / ml, 120 rpm, 37°C, 8% CO2, 80% humidity in a shaking incubator.

[0034] Cell transfection expression: Cell transfection Day 0 The cell transfection density should be between 3-5×10 6cell / ml, take a sample to measure VCD and VIA. If the cell density is too high, dilute it with OPM-293 CD05 to an appropriate density. When transfecting cells, add 0.03 mg of plasmid to 1.5 mL of OPM-293 CD05 after filtering through a 0.22 μm filter membrane to make a plasmid dilution. Add 0.09 mg of PEI to 1.5 mL of OPM-293 CD05 to make a PEI dilution. Add the PEI dilution to the plasmid dilution and mix, and let stand at room temperature for 20 min.

[0035] Pour 3 mL of the DNA-PEI mixture into 30 mL of Expi293 cells, mix well, and culture in a shaker at 120 rpm, 37°C, 8% CO2, and 80% humidity.

[0036] Protein expression Day1-Day7 18-24h after transfection, add 5% of the initial volume of OPM-293 ProFeed and culture in a shaker at 120rpm, 37°C, 8% CO2, and 80% humidity. 2-5 days after transfection, take samples regularly to record cell density and cell viability, measure the glucose concentration in the culture medium, and control the sugar content to 2-8g / L. On the 7th day after transfection, the cell supernatant was harvested by centrifugation, filtered, and packaged, stored at -80℃ for subsequent experiments.

[0037] Example 2 Expression level detection 1. Fortiebio The experiment used OctetRED96e (Sartorius) based on Bio-Layer Interferometry (BLI) technology for sample quantitative testing. The experiment used 0.05% PBST as the dilution buffer. Before the experiment started, the ProA sensor was placed in the buffer for pre-wetting for more than 10 minutes. The expression supernatants of S1+042-PTT5, S2+042-PTT, S3+042-PTT5, S4+042-PTT5, and 42-PTT5 were diluted 400 times and added to the sample plate. After the experiment started, the sensor was combined with the sample for 180 s, and the signal value at the binding end point was compared. The higher the signal value, the higher the expression level, and the expression levels of the five samples were roughly ranked.

[0038] Test results such as Figure 3 As shown, expression level: S3+042-PTT5>S4+042-PTT>S2+042-PTT5>S1+042-PTT5>042-PTT5 2. c-dex quantification Table 1 instrument Cedex Biochemical Analyzer Roche Reagent test kit IgG BIo cdex Roche The IgG-Bio test kit and Cdex biochemical analyzer were used to quantitatively determine the IgG in the cell fermentation broth. Based on the turbidimetric detection principle (the sample containing IgG Fc reacts with the specific antibody to form a precipitate that can be measured at 340nm) The quantitative test results are as follows: Table 2 concentration Control plasmid 042-PTT5 123mg / L S1+042-PTT5 216mg / L S2+042-PTT5 233mg / L S3+042-PTT5 428mg / L S4+042-PTT5 421mg / L in conclusion: Compared with the sequence without the peptide, the four codon sequences can increase the expression of foreign proteins. The results of the two detection methods are consistent.

[0039] The sequence S3 combination was the best, and the expression level of 042 increased by 248% compared with the sequence without the addition of this peptide.

[0040] 3. SDS-PAGE Protein sample preparation: Take 40 μL of cell supernatant and add 10 μL of 5×Sample Buffer, mix well, heat at 95℃ for 10 min, centrifuge and prepare for sample loading.

[0041] SDS-PAGE: After installing the precast gel plate into the electrophoresis device, add MOPS Running Buffer, take 10 μL of protein marker and appropriate amount of protein sample and load them in order. Perform SDS-PAGE at 130V for about 60-70min until the protein marker runs to the bottom of the separation gel, stain with Coomassie Brilliant Blue and take pictures. Figure 4 .

[0042] Example 3 In order to verify the combined effect of C+2A codon S3, we replaced the V02 vector and exogenous protein R, designed the expression plasmid C+2A+R-V02, and commissioned a synthetic company to synthesize the corresponding DNA sequence. At the same time, we constructed a control plasmid R-V02 without C peptide and 2A peptide.

[0043] R protein DNA sequence: AGCAGAGGCATCAAGGGCAAGAGACAGAGAAGAATCAGCGCCGAGGGCAGCCAAGCCTGCGCCAAGGGCTGCGAGCTGTGCAGCGAGGTGAACGGCTGCCTGAAGTGCAGCCCTAAGCTGTTCATCCTGCTGGAGAGAAACGACATCAGACAAGTGGGCGTGTGCCTGCCTAGCTGCCCTCCTGGCTACTTCGACGCTAGAAACCCTGACATGAACAAGTGCATCAAGTGCAAGATCGAGCACTGCGAGGCCTGCTTCAGCCACAACTTCTGCACCAAGTGCAAGGAGGGCCTGTACCTGCACAAGGGCAGATGCTACCCTGCCTGTCCCGAGGGCAGCAGCGCCGCCAATGGCACCATGGAGTGCAGCAGCCCTGCTCAGTGCGAGATGAGCGAGTGGAGCCCTTGGGGCCCTTGCAGCAAGAAGCAGCAGCTGTGCGGCTTCAGAAGAGGCAGCGAGGAGAGAACAAGAAGAGTGCTGCACGCCCCTGTGGGCGACCACGCCGCCTGCAGCGACACCAAGGAGACAAGAAGATGCACCGTGAGAAGAGTGCCTTGCCCCGAGGGCCAAAAGAGAAGAAAGGGCGGCCAAGGCAGAAGAGAGAACGCCAACAGAAACCTGGCTAGAAAGGAGAGCAAGGAGGCCGGCGCCGGCAGCAGAAGGAGAAAAGGACAGCAACAGCAACAGCAACAAGGCACCGTGGGCCCTCTGACAAGCGCTGGCCCTGCC (SEQ ID NO: 9) Amino acid sequence of the R protein: SRGIKGKRQRRISAEGSQACAKGCELCSEVNGCLKCSPKLFILLERNDIRQVGVCLPSCPPGYFDARNPDMNKCIKCKIEHCEACFSHNFCTKCKEGLYLHKGRCYPACPEGSSAANGTMECS SPAQCEMSEWSPWGPCSKKQQLCGFRRGSEERTRRVLHAPVGDHAACSDTKETRRCTVRRVPCPEGQKRRKGGQGRRENANRNLARKESKEAGAGSRRRKGQQQQQQGTVGPLTSAGPA (SEQ ID NO: 10).

[0044] The protein transient expression method is the same as in Example 1.

[0045] Expression detection: (1) Western Blot Protein sample preparation: Take 40 μL of cell supernatant and add 10 μL of 5×Sample Buffer, mix well, heat at 95℃ for 10 min, centrifuge and prepare for sample loading.

[0046] SDS-PAGE: After installing the precast gel plate into the electrophoresis device, add MOPS Running Buffer, take 10 μL protein marker and appropriate amount of protein sample and load them in order. Perform SDS-PAGE at 130V for about 60-70min until the protein marker runs to the bottom of the separation gel and prepare for transfer (the transfer solution needs to be pre-cooled in a 4℃ refrigerator in advance).

[0047] Transfer: Transfer the protein to the PVDF membrane at a constant current of 400 mA for 70 min.

[0048] Blocking: After transfer, immerse the PVDF membrane in rapid blocking solution and incubate at room temperature for 15-25 minutes.

[0049] Antibody incubation: Dilute the primary antibody (Anti-6X His tag® antibody) and rapid blocking solution at a concentration of 1:25000, cut the membrane according to the size of the target protein based on the protein marker position, put it in the diluted primary antibody, incubate at 37℃ for 1.5 hours or incubate in a refrigerator at 4℃ overnight. Rinse with TBST 3 times, 10 minutes each time. Dilute the secondary antibody (goat anti-rabbit IgG H&L-HRP) and rapid blocking solution at a concentration of 1:20000, put the PVDF membrane in the secondary antibody diluent, and incubate at 37℃ for 1.5 hours. Rinse with TBST 3 times, 10 minutes each time.

[0050] Development: Mix the Enhancer Buffer Solution and Peroxide Buffer of the ECL kit in a 1:1 ratio. Fully soak the PVDF membrane in the mixture and expose it in the fully automatic luminescence imaging system. The WB results are as follows: Figure 5 As shown in the figure, compared with the sequence without the addition of the polypeptide, the molecular weight of the exogenous protein R did not change, indicating that the 2A peptide sequence successfully self-cleaved and removed the added polypeptide sequence to avoid its influence on the exogenous protein. Compared with the sequence without the addition of the polypeptide, the expression level of the exogenous protein R was increased.

[0051] Table 3 Reagents Reagent name brand Anti-6X His tag® antibody Abcam Goat anti-rabbit IgG H&L (HRP) Abcam High-Sig ECL Western Blotting Substrate Tanon PVDF membrane Millipore (2) Fortiebio The experiment used OctetRED96e (Sartorius) based on Bio-Layer Interferometry (BLI) technology for sample quantitative testing. The experiment used 0.05% PBST as the dilution buffer. Before the experiment started, the HIS2 sensor was pre-wetted in the buffer for more than 10 min. The purified protein was used as the standard, and the gradient dilution was 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 μg / mL as the standard curve. R-V02-D7 and C+2A+R-V02-D7 were diluted 2 times and added to the sample plate, and duplicate wells were used as parallel controls. After the experiment started, the HIS2 sensor was combined with the sample for 180 s, and the data was analyzed using Data Analysis 9.0 analysis software. The final result was processed by taking the average value of the parallel wells.

[0052] The calculation results of the concentration test are shown in Table 4. It can be seen that the expression level of the exogenous protein R increased by 197% compared with the sequence without the addition of the polypeptide.

[0053] Table 4 Signal concentration R-V02 0.6nm 20.55mg / L C+2A+R-V02 1.78nm 60.97mg / L The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A nucleic acid molecule for enhancing the expression level of an exogenous gene, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 1, 2, 3 or 4.

2. The nucleic acid molecule according to claim 1, wherein The amino acid sequence of the protein expressed by the nucleic acid molecule is shown in SEQ ID NO:

5.

3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule according to claim 1 and a target protein gene.

4. The recombinant expression vector according to claim 3, characterized in that The recombinant expression vector also includes a DNA vector, an RNA vector, a plasmid, a transposon vector, a liposome, a CRISPR / Cas9 vector or a viral vector.

5. The recombinant expression vector according to claim 3, characterized in that The target protein gene is also connected to a protein tag, and the protein tag is selected as a His tag.

6. The recombinant expression vector according to claim 3, characterized in that A 2A peptide is also connected between the nucleic acid molecule and the target protein gene; the nucleotide sequence of the 2A peptide is shown in SEQ ID NO:

6.

7. A recombinant host cell, wherein the recombinant expression vector according to any one of claims 3 to 6 is constructed into the genome of the host cell, so that the target protein gene is expressed in the host cell.

8. Use of the nucleic acid molecule according to any one of claims 1 to 2, the recombinant expression vector according to any one of claims 3 to 6, or the host cell according to claim 7 in a recombinant protein production system.

9. The use according to claim 8, characterized in that The system used for producing recombinant protein is 293 expression system.

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