A polynucleotide based on CMV promoter and its application

Abstract

The present invention belongs to the biological field, and specifically relates to a polynucleotide based on a CMV promoter and its application. The present invention constructs a plasmid containing a strong promoter for cell expression, introduces a random mutation CMV promoter through random mutation PCR technology, and screens to obtain a stronger CMV promoter sequence. The CMV promoter sequence obtained in the present invention can significantly increase the expression amount of an exogenous target protein, and provides a basis for increasing the expression amount of an exogenous protein in transient and stable transfection.

Description

Technical Field

[0001] The invention belongs to the biological field, and specifically relates to a polynucleotide based on a CMV promoter and an application thereof. Background Art

[0002] Recombinant protein expression system is a technology that uses genetic engineering technology to introduce target genes into host cells and express and produce specific proteins through the biosynthetic mechanism of host cells. Recombinant protein expression system is an important tool in the field of biopharmaceuticals, used for large-scale production of proteins with specific functions. By constructing and optimizing the expression system, the yield, purity and activity of proteins can be improved to meet the needs of biopharmaceuticals.

[0003] A strong promoter has an important influence on the efficiency of expressing foreign genes. A strong promoter can more effectively attract RNA polymerase, thereby increasing the frequency of transcription initiation and significantly improving the expression level of foreign genes.

[0004] The CMV promoter (CMV promoter) comes from human cytomegalovirus (CMV), has strong transcriptional activity, and is widely used in eukaryotic cells. It is a promoter sequence widely used in gene expression research and genetic engineering.

[0005] Promoters are key elements that affect the expression of foreign genes. Choosing a more appropriate promoter can significantly increase the expression level of foreign genes and directly increase the yield of target proteins, which is very important for industrial production and scientific research. High yield means higher production efficiency and lower costs.

[0006] In short, improving the expression of exogenous proteins is of great significance to scientific research, industrial production, medical applications and economic development. Summary of the invention

[0007] Error-prone PCR mainly uses low-fidelity Taq DNA polymerase to randomly introduce mutations during the PCR process. By adjusting the dosage of nucleotides, enzymes and magnesium ions, a mutant library with no sequence tendency is obtained. Random mutation PCR is a specific application of error-prone PCR. Random mutation PCR should use cDNA or double-stranded DNA fragments containing only the target sequence as templates. Generally, plasmid DNA and genomic DNA are not used. The random mutation PCR method requires the template length to be within about 1000 nucleotides. Among the key elements to be mutated in the present invention, the length of the CMV enhancer and promoter is 584bp. This fragment can be used by enzyme cutting and PCR to obtain linear DNA as a mutation template.

[0008] One of the advantages of random mutation PCR is that it can perform repeated random mutations on nucleic acid populations without the need to isolate clones and obtain sequence information. After obtaining the mutant library, individuals with special properties are screened out, and the individuals can also be directly used as templates for the second mutation PCR. Random mutagenesis is performed repeatedly and continuously, so that the small mutations obtained each time accumulate and produce important beneficial mutations.

[0009] In order to achieve the purpose of improving the expression level of exogenous genes and increasing the yield of exogenous proteins, the present invention provides the following technical solutions:

[0010] The first aspect of the present invention discloses a polynucleotide having promoter activity, wherein the nucleotide sequence of the polynucleotide is shown in any one of SEQ ID NO: 1, 2 or 3.

[0011] Wherein, the polynucleotide is obtained by random mutation PCR screening of CMV promoter.

[0012] The second aspect of the present invention discloses an expression cassette, which comprises the above-mentioned polynucleotide having promoter activity.

[0013] The third aspect of the present invention discloses a recombinant vector, which comprises the above-mentioned polynucleotide having promoter activity or expression cassette.

[0014] The fourth aspect of the present invention discloses a recombinant host cell, which contains the above-mentioned polynucleotide having promoter activity, expression cassette, or the above-mentioned recombinant vector.

[0015] The fifth aspect of the present invention discloses a method for enhancing the expression of a target gene, the method comprising operably linking the above-mentioned polynucleotide having promoter activity to the target gene.

[0016] The polynucleotide having promoter activity is constructed and screened as follows:

[0017] S1. Using error-prone PCR technology, a round of random mutation of the CMV promoter was performed followed by sequencing to construct a mutant library.

[0018] S2, transiently transfect the mutant library of S1 and measure the expression level, and select the mutant plasmid with high expression level;

[0019] S3, screening mutant plasmids with increased expression levels for a second round of random mutagenesis and sequencing to construct a mutant library;

[0020] S4, transiently transfect the mutant library of S3 and measure the expression level, and select the mutant plasmid with high expression level;

[0021] S5. Screen the mutant plasmids in S4 that have mutant CMV promoter and high expression, and determine the sequence of the polynucleotide after sequencing.

[0022] The sixth aspect of the present invention discloses the use of the above-mentioned polynucleotide having promoter activity, expression cassette, recombinant vector, or the above-mentioned recombinant host cell in enhancing the expression of target gene and producing target protein.

[0023] Compared with the prior art, the present invention has the following technical effects:

[0024] The present invention constructs a plasmid containing a strong promoter for cell expression, introduces random mutations based on the CMV promoter through error-prone PCR technology, and screens to obtain a stronger promoter sequence. The polynucleotide obtained in the present invention is used as a promoter to significantly increase the expression of an exogenous target protein, providing a basis for increasing the expression of transient and stable exogenous proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the expression vector GFP-V02 containing the CMV promoter (red) and green fluorescent protein (green).

[0026] Figure 2 Western Blot was used to detect the relative expression of Rspondin activated by the mutant promoter. The primary antibody was rabbit anti-his 1:25K at 4° overnight; the secondary antibody was goat anti-rabbit-HRP 1:20K at room temperature for 2h; Rspondin: 28.2kDa.

[0027] Figure 3 Schematic diagram of the relative expression level of Rspondin driven by mutant promoter detected by Fortiebio. DETAILED DESCRIPTION

[0028] 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.

[0029] Wherein, the "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide can be in the form of a single fragment or a component of a larger nucleotide sequence structure, which is derived from a nucleotide sequence separated at least once in quantity or concentration, and can be identified, manipulated, and restored by standard molecular biology methods (e.g., using a cloning vector) and its component nucleotide sequences. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), in which "U" replaces "T". In other words, "polynucleotide" refers to a nucleotide polymer removed from other nucleotides (a single fragment or an entire fragment), or can be a component or ingredient of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences, and the present invention mainly refers to DNA fragments with promoter activity.

[0030] The "mutant" refers to a polynucleotide that contains changes (i.e., substitutions, insertions and / or deletions) at one or more (e.g., several) positions relative to a "wild type" or "compared" polynucleotide or polypeptide, wherein substitution refers to replacing a nucleotide occupying a position with a different nucleotide. Deletion refers to the removal of a nucleotide occupying a position. Insertion refers to the addition of a nucleotide adjacent to and immediately following the nucleotide occupying the position. In a specific embodiment, the "mutation" is a "substitution", which is a mutation caused by the replacement of a base in one or more nucleotides by another different base, also known as a base substitution mutation (subsititution) or a point mutation (point mutation).

[0031] The "expression cassette" has a meaning generally understood by those skilled in the art, ie, an element containing a promoter, a target gene and capable of expressing the target gene.

[0032] The "vector" refers to a DNA construct containing a DNA sequence operably linked to an appropriate control sequence to express a target gene in a suitable host. The vector used in the present invention is not particularly limited and can be any vector known in the art, as long as it can be replicated in a host. That is, the vector includes but is not limited to plasmids and bacteriophages. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases integrate into the genome itself.

[0033] The "host cell" has a meaning generally understood by those of ordinary skill in the art, that is, a cell into which the nucleic acid having promoter activity of the present invention can be introduced, and after introduction, it is called a recombinant host cell. In other words, the present invention can utilize any host cell, as long as the cell contains the nucleic acid having promoter activity of the present invention and is operably linked to a gene to mediate the transcription of the gene. The host cell of the present invention can be a prokaryotic cell or a eukaryotic cell.

[0034] The "operably connected" means that the mutant of the glutamate dehydrogenase gene promoter of the present invention is functionally connected to the coding gene to start and mediate the transcription of the gene, indicating that the mutant of the glutamate dehydrogenase gene promoter of the present invention is operably connected to the coding gene to control the transcription activity of the operon gene. The operably connected method can be any method described by those skilled in the art.

[0035] Example 1

[0036] Through seamless cloning technology, the expression vector GFP-V02 vector ( Figure 1 The end sequence of the linearized vector was introduced into the 5' end of the forward / reverse PCR primer of the inserted fragment, so that the 5' and 3' ends of the PCR product respectively carried sequences consistent with the two ends of the linearized vector (15 - 20 bp). Finally, the PCR product and the linearized vector were mixed in a certain ratio, and the reaction was carried out at 37°C for 30 minutes under the catalysis of the recombinase to complete the directional cloning.

[0037] Through the above method, a mutant library was constructed and a series of mutant plasmids GFP-V02-m were obtained. After randomly picking clones for amplification, they were transfected into 293 cells, the end point fluorescence intensity was compared, clones with enhanced fluorescence intensity were selected for sequencing, and the DNA sequence of the sequencing results was analyzed.

[0038] Experimental Procedure

[0039] 1. Construction of mutant library

[0040] 1.1 Preparation of linearized vector:

[0041] First, the GFP-V02 linearized vector (including 2 fragments S and fragment 1) was obtained by double digestion with restriction endonucleases BglII and HindIII.

[0042] Design primers for PCR so that the 5' and 3' ends of the PCR product have sequences that are consistent with the two ends of the linearized vector (15 - 20 bp), as follows:

[0043] The vector sequence of the upstream partial fragment 2 of the CMV promoter was obtained by PCR with primers BglII-F+ I-R3; the vector sequence of the downstream partial fragment 3 of the CMV promoter was obtained by PCR with primers HindIII-F2+ HindIII-R2.

[0044] Table 1 BglII and HindIII double restriction enzyme system for linearization of GFP-V02 vector

[0045]

[0046] Table 2 Linearized vector primer information

[0047]

[0048] Table 3 Fragment 2 PCR system

[0049] Components Add volume <![CDATA[ddH2O]]> 12 μL 2×Phanta flash Master Mix (Dye Plus) 15 μL BglII-F (10 μM) 1 μL I-R3(10 μM) 1 μL GFP-V02 1 μL

[0050] Table 4 Fragment 3 PCR system

[0051] Components Add volume <![CDATA[ddH2O]]> 12 μL 2×Phanta flash Master Mix (Dye Plus) 15 μL HindIII-F2 (10 μM) 1 μL HindIII-R2 (10 μM) 1 μL GFP-V02 1 μL

[0052] 1.2 Obtaining insert fragments (CMV promoter fragment and CMV promoter random mutation fragment)

[0053] Primer design: Introduce homologous sequences at both ends of the linearized vector at the 5' end of the forward and reverse amplification primers of the inserted fragment, so that the 5' and 3' ends of the amplified inserted fragment respectively carry homologous sequences corresponding to the two ends of the linearized cloning vector (15 - 20 bp, excluding the restriction site). The results are shown in Table 5.

[0054] Table 5 Primer design for CMV promoter amplification region

[0055]

[0056] Insert fragment PCR amplification: Use high-fidelity polymerase to obtain the original CMV promoter target fragment through PCR method, and then amplify the original CMV promoter target fragment through error-prone PCR. The reaction system and procedure are shown in the table below.

[0057] High-fidelity PCR system and procedures:

[0058] Table 6 CMV promoter PCR system, obtaining CMV promoter fragment

[0059] Components Add volume <![CDATA[ddH2O]]> 12 μL 2×Phanta flash Master Mix (Dye Plus) 15 μL CMV-F (10 μM) 1 μL CMV-R (10 μM) 1 μL GFP-V02 1 μL

[0060] Table 7 CMV promoter PCR program, obtain CMV promoter fragment

[0061]

[0062] Table 8 Error-prone PCR system (obtaining random mutation fragments of CMV promoter)

[0063]

[0064] 1.3 Recombination reaction to obtain a complete plasmid containing random mutations of the CMV promoter

[0065] Prepare the following reaction system on ice and mix by gently pipetting. Centrifuge briefly to collect the reaction solution at the bottom of the tube. Incubate at 37℃ for 30 min; cool to 4℃ or immediately place on ice. See Table 9 for the reaction system.

[0066] Table 9 Recombination reaction system

[0067] Components Add volume (dosage) Linearized fragment 1 (5.3 kb) 3 μL (107 ng) Linearized fragment 2 (1.4 kb) 2 μL (28 ng) Linearized fragment 3 (1.0 kb) 1 μL (20 ng) Insert (Mutated CMV) (0.6 kb) 1 μL (13 ng) 5 × CE II Buffer 2 μL

[0068] Recombinant product transformation

[0069] Thaw the cloned DH5α Competent cell on ice, heat shock it in a 42℃ water bath for 45 seconds, and immediately cool it on ice for 2-3 minutes. Finally, use a sterile spreading stick to gently spread the above transformation system on the plate containing AMP resistance and place it in a ℃ incubator and invert it for culture for 12-16 hours.

[0070] Identification of recombinant products

[0071] Select several clones on the recombination reaction transformation plate for colony PCR identification. If the clone is correct, a band slightly larger than the insert size (3337 bp) should appear. Send directly to first generation sequencing. See Tables 10 and 11 below for colony PCR primers and sequencing primers.

[0072] Table 10 Colony PCR primers

[0073] Primer name sequence Mu-CMV-clone-F CAGAAGCCATCGTCCGCACATGCCT (SEQ ID NO: 10) Mu-CMV-clone-R CGGTGGTGCAGATGAACTTCAGGGT (SEQ ID NO: 11)

[0074] Table 11 Sequencing primers

[0075] Primer name sequence Seq-CMV TGACTTGGGCGATTCTGTGT (SEQ ID NO: 12)

[0076] 2. Transient transfection for expression determination

[0077] 2.1 Plasmid preparation

[0078] The constructed plasmid was inoculated into the E. coli DH5α strain, and after ice bathing for 5 minutes, it was heat-shocked in a 42℃ water bath for 60 seconds, and then ice bathed for 2 minutes again, inoculated into the sterilized corresponding resistance LB medium, and cultured at 220 rpm and 37℃ for 12-16 hours. The bacterial culture was centrifuged at 4500 rpm for 10 minutes, and the supernatant was discarded; after adding RES buffer to the precipitate to resuspend the bacteria, LYSbuffer was added to fully lyse the cells; NEU buffer was added to neutralize until colorless, and the liquid was applied to the DNA binding column (pre-balanced with EQUbuffer); EQU buffer was used to wash the column, WASH buffer was used to wash the column, and ELU buffer was used to elute the DNA; isopropanol was added, and the precipitate was transferred to NucleoBond® after being placed at room temperature for 5 minutes, and the precipitate was washed with 70% ethanol, dried at room temperature, and DNA was collected with sterile ddH2O; the plasmid concentration was detected using QuickDrop.

[0079] 2.2 Transient transfection and protein expression

[0080] Culture medium and cell preparation

[0081] 1 L OPM-293 CD05 Medium, stored in 4°C refrigerator, preheated in 37°C water bath before use

[0082] 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. After three passages, dilute the cell density to 0.4~0.7×106 cell / ml and culture in a shaker at 120 rpm, 37°C, 8% CO2, 80% humidity.

[0083] Cell transfection expression

[0084] Cell transfection Day 0

[0085] The cell transfection density should be between 3-5×10 6 cell / ml, take a sample to measure VCD and VIA. If the cell density is too high, dilute it with CD05 to an appropriate density. Filter 0.03 mg of plasmid through a 0.22 μm filter membrane and add it to 1.5 mL CD05 to prepare a plasmid dilution. Add 0.09 mg PEI to 1.5 mL CD05 to prepare a PEI dilution. Add the PEI dilution to the plasmid dilution and mix, and let it stand at room temperature for 20 min.

[0086] 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.

[0087] Protein expression Day1-Day6

[0088] 18-24h after transfection, add 5% of the initial volume of 293 ProFeed and culture in a shaker at 120rpm, 37°C, 8% CO2, and 80% humidity. On the 2nd to 5th day 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 6th day after transfection,

[0089] Harvest the cell supernatant by centrifugation.

[0090] 2.3 Transfection efficiency and expression level detection

[0091] 2.31 Transfection efficiency detection (flow cytometer BD Cytoflex)

[0092] Take cells for counting; calculate the cell volume required for 1E6 cells based on the counting results; take the corresponding volume of cells into a 1.5mL EP tube, add 1mL PBS to mix, centrifuge at 1000rpm for 5min, and discard the supernatant; add 500uL PBS to resuspend the cell pellet, store it at 4℃ in the dark, and perform flow cytometry detection.

[0093] The data were recorded according to the GFP positive rate in the histogram of the flow cytometer and recorded as the transfection efficiency.

[0094] 2.32 Expression level detection (ELISA reader Molecular Devices Spectramax M5)

[0095] Centrifuge at 1000rpm for 5min to collect the cell supernatant, add 180μL PBS to a black opaque 96-well plate, then take 20μL of cell supernatant and add it to the plate, mix well and wait for testing. Set the fluorescence intensity measurement mode of the microplate reader, the excitation wavelength is 405 nm, and the absorption wavelength is 488 nm. Select the detection area corresponding to the plate. Perform the test and save the results. According to the fluorescence intensity value of the sample * the dilution multiple, calculate the actual fluorescence intensity value of the final sample, which is the fluorescence expression.

[0096] Finally, the fluorescence expression level / transfection efficiency was used to calculate the expression level of a single cell and compared. In the first round, there were two groups of test results as shown in the following table, and clones with low expression levels were deleted.

[0097] Table 12

[0098] Shake tube number D2 transfection efficiency (%) D8 fluorescence expression (U) Fluorescence expression / transfection efficiency SF1 64.78 1.75E+04 2.70E+02 SF2 73.34 2.37E+04 3.23E+02 SF3 66.96 2.73E+04 4.08E+02 SF4 75.71 2.05E+04 2.71E+02 SF6 75.41 2.69E+04 3.57E+02 SF10 71.33 2.78E+04 3.89E+02 SF11 0.11 1.13E+02 1.02E+03 SF12 77.27 2.29E+04 2.96E+02 SF13 73.07 2.42E+04 3.31E+02 SF14 76.34 2.25E+04 2.94E+02 SF18 80.97 2.15E+04 2.65E+02 SF19 71.74 2.45E+04 3.41E+02 SF41 66.19 2.67E+04 4.04E+02 SF45 (control) 72.81 2.09E+04 2.87E+02

[0099] Table 13

[0100]

[0101] 3. Sequencing of mutant plasmids

[0102] According to the expression detection results, SF3, SF41, and N16 were selected for sequencing.

[0103] Table 14 Sequencing primers

[0104] Primer name sequence Seq-CMV TGACTTGGGCGATTCTGTGT (SEQ ID NO: 13)

[0105] The sequence is as follows:

[0106] REF (CMV enhancer + promoter sequence)

[0107] (SEQ ID NO: 14)

[0108] SF3 (first round mutation sequence)

[0109] GACATTGATTATTGACTAGTTATCAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT(SEQ ID NO:15)

[0110] N16

[0111] GACATTGATTAATGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTCCATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTAACATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGAT (SEQ ID NO:16)

[0112] SF41

[0113] (SEQ ID NO: 17)

[0114] 4. Perform a second round of random mutation on the mutant plasmid with increased expression in the first round, and the random mutation method is the same as the first round of mutation.

[0115] 5. The plasmid obtained by the second round of mutation was transiently transfected and the expression level was measured. The expression level of a single cell was calculated using the fluorescence expression level / transfection efficiency and compared. The method is the same as 2.2 and 2.3. The test results are shown in Table 15 below.

[0116] Table 15

[0117] Plasmid number 48H GFP+(%) Fluorescence value (10 times dilution) Fluorescence expression / transfection efficiency 3 98.15 25038.2 255.10 3-3 97.18 33301.67 342.68 3-14 94.8 30889.55 325.84 16-6 92.54 27733.64 299.69 16-8 90.54 28336.84 312.98 41 97.09 26791.7 275.95 41-7 94.97 30364.49 319.73 Comparison 97.71 23237.35 237.82

[0118] The mutant plasmid with increased expression was selected for sequencing. The sequencing primers were the same as 3, and the sequencing results after removing the repeated sequences were as follows:

[0119] 3-3

[0120] GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTACTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCATGTCTCCACCCCATTGACGTCAATGGGAGTTTGCTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT(SEQ ID NO:1)

[0121] 3-14

[0122] GACATTGATTATTGACTAGTTATCAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT(SEQ ID NO:2)

[0123] 41-7

[0124] (SEQ ID NO: 3)

[0125] Example 2 Verification of the effect of the mutant promoter.

[0126] In order to verify whether the mutant sequence has the effect of increasing the expression level, the synthetic company was commissioned to construct the mutant promoter in front of the exogenous protein Rspondin. The transient expression method was the same as 2.2

[0127] 1. Expression level detection

[0128] Western Blot

[0129] 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.

[0130] 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).

[0131] Transfer: Transfer the protein to the PVDF membrane at a constant current of 400 mA for 70 min.

[0132] Blocking: After transfer, immerse the PVDF membrane in rapid blocking solution and incubate at room temperature for 15-25 minutes.

[0133] 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.

[0134] 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 2 shown.

[0135] Conclusion: WB results showed that the promoter with mutation did increase the expression of protein Rspondin.

[0136] 2. Fortiebio

[0137] 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 minutes. The experiment used purified protein as the standard, and the gradient dilution was 20, 10, 5, 2.5, 1.25, 0.625, and 0.3125 μg / mL as the standard curve. Rspodin, Rspodin 3-3, Rspodin 3-14, and Rspodin 41-7 were diluted 20 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 experimental results are shown in Tables 16 and Figure 3 As shown, the final result was processed by taking the average value of parallel wells.

[0138] Table 16

[0139] Curve colour Name Dilute 20 folds Expression Green Rspodin 20.55mg / L Yellow Rspodin 3-3 32mg / L Purple Rspodin 3-14 31mg / L Pink Rspodin 41-7 40mg / L

[0140] Conclusion: Octet results showed that the expression level of 41-7 mutant promoter was 1.94 times that of the original promoter, and the expression levels of 3-3 and 3-14 mutant promoters were 1.56 and 1.50 times that of the original promoter, respectively.

[0141] WB results and Octet results showed that the promoter with mutation did increase the expression of protein Rspondin, and the results were consistent.

[0142] Note: Rspondin is a foreign protein with a his tag. The sequence is as follows

[0143] Rspondin protein DNA sequence

[0144] AGCAGAGGCATCAAGGGCAAGAGACAGAGAAGAATCAGCGCCGAGGGCAGCCAAGCCTGCGCCAAGGGCTGCGAGCTGTGCAGCGAGGTGAACGGCTGCCTGAAGTGCAGCCCTAAGCTGTTCATCCTGCTGGAGAGAAACGACATCAGACAAGTGGGCGTGTGCCTGCCTAGCTGCCCTCCTGGCTACTTCGACGCTAGAAACCCTGACATGAACAAGTGCATCAAGTGCAAGATCGAGCACTGCGAGGCCTGCTTCAGCCACAACTTCTGCACCAAGTGCAAGGAGGGCCTGTACCTGCACAAGGGCAGATGCTACCCTGCCTGTCCCGAGGGCAGCAGCGCCGCCAATGGCACCATGGAGTGCAGCAGCCCTGCTCAGTGCGAGATGAGCGAGTGGAGCCCTTGGGGCCCTTGCAGCAAGAAGCAGCAGCTGTGCGGCTTCAGAAGAGGCAGCGAGGAGAGAACAAGAAGAGTGCTGCACGCCCCTGTGGGCGACCACGCCGCCTGCAGCGACACCAAGGAGACAAGAAGATGCACCGTGAGAAGAGTGCCTTGCCCCGAGGGCCAAAAGAGAAGAAAGGGCGGCCAAGGCAGAAGAGAGAACGCCAACAGAAACCTGGCTAGAAAGGAGAGCAAGGAGGCCGGCGCCGGCAGCAGAAGGAGAAAAGGACAGCAACAGCAACAGCAACAAGGCACCGTGGGCCCTCTGACAAGCGCTGGCCCTGCCGA (SEQ ID NO: 18)

[0145] Amino acid sequence of Rspondin protein

[0146] SRGIKGKRQRRISAEGSQACAKGCELCSEVNGCLKCSPKLFILLERNDIRQVGVCLPSCPPGYFDARNPDMNKCIKCKIEHCEACFSHNFCTKCKEGLYLHKGRCYPACPEGSSAANGTMECS SPAQCEMSEWSPWGPCSKKQQLCGFRRGSEERTRRVLHAPVGDHAACSDTKETRRCTVRRVPCPEGQKRRKGGQGRRENANRNLARKESKEAGAGSRRRKGQQQQQQGTVGPLTSAGPA (SEQ ID NO: 19)

[0147] 3. In order to verify whether the mutant sequence also has the effect of increasing the expression amount on other vectors, the mutant promoter was constructed in front of the exogenous protein 042, and the vector used was a commercial pTT5 vector. The transient expression method was the same as 2.2. The expression supernatant on the sixth day was quantitatively detected, and the IgG in the cell fermentation broth was quantitatively determined using the IgG-Bio detection kit and the Cdex biochemical analyzer. 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).

[0148] The quantitative test results are as follows:

[0149] instrument Cedex Biochemical Analyzer Roche Reagent test kit IgG BIo cdex Roche

[0150] Expression improve PTT5-042 control 404 NA 3-ptt5-042 421 4.2 41-ptt5-042 425 5.2 3-3-ptt5-042 451 11.6 3-14-ptt5-042 445 10.1 41-7-ptt5-042 479 18.5

[0151] Note: 042 is a foreign protein with an Fc tag, the sequence is as follows

[0152] 042DNA sequence

[0153]

[0154] 042 Amino acid sequence

[0155] MGWSCIILFLVATATGAYAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGMIPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD* (SEQ ID NO: 21)

[0156] 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 polynucleotide having promoter activity, characterized in that: The nucleotide sequence of the polynucleotide is shown in any one of SEQ ID NO: 1, 2 or 3.

2. A polynucleotide having promoter activity according to claim 1, characterized in that: The polynucleotide is obtained by random mutation PCR screening of CMV promoter.

3. An expression cassette, characterized in that The expression cassette comprises the polynucleotide having promoter activity according to any one of claims 1 to 2.

4. A recombinant vector, characterized in that: The recombinant vector comprises the polynucleotide having promoter activity according to any one of claims 1 to 2, or the expression cassette according to claim 3.

5. A recombinant host cell, characterized in that The recombinant host cell contains the polynucleotide having promoter activity according to any one of claims 1 to 2, or the expression cassette according to claim 3, or the recombinant vector according to claim 4.

6. A method for enhancing the expression of a target gene, the method comprising operably linking the polynucleotide having promoter activity as claimed in any one of claims 1 to 2 to the target gene.

7. The polynucleotide having promoter activity according to any one of claims 1 or 2, characterized in that: The specific construction and screening methods are as follows: S1. Using error-prone PCR technology, a round of random mutation of the CMV promoter was performed followed by sequencing to construct a mutant library. S2, transiently transfect the mutant library of S1 and measure the expression level, and select the mutant plasmid with high expression level; S3, screening mutant plasmids with increased expression levels for a second round of random mutagenesis and sequencing to construct a mutant library; S4, transiently transfect the mutant library of S3 and measure the expression level, and select the mutant plasmid with high expression level; S5. Screen the mutant plasmids in S4 that have mutant CMV promoter and high expression, and determine the sequence of the polynucleotide after sequencing.

8. Use of the polynucleotide having promoter activity according to any one of claims 1 to 2, the expression cassette according to claim 3, the recombinant vector according to claim 4, or the recombinant host cell according to claim 5 in enhancing the expression of a target gene and producing a target protein.

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