DNA expression enhancer and its use

Abstract

This invention discloses a DNA expression enhancer, belonging to the fields of genetic engineering and DNA drug technology. The enhancer is a polypeptide with ribonuclease III activity and low affinity for ribonuclease inhibitors. It also provides a method for increasing the expression level of a target gene on a plasmid DNA eukaryotic expression vector using the enhancer, comprising the steps of: (a) constructing and preparing an enhancer expression plasmid for eukaryotic cells; and (b) co-transfecting host cells with the target gene expression plasmid and the enhancer expression plasmid. The enhancer increases the expression level and duration of the exogenous target gene in the organism and cells. The recombinant polypeptide enhancer provided by this invention can significantly increase the expression level of the exogenous target gene in mammalian cells, increasing the expression level by approximately 4 to 5 times. This technology, when applied to DNA vaccines, can improve antibody production levels, reduce drug dosage and cost, and greatly enhance the therapeutic effect of DNA drugs.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and DNA drug technology, and particularly to DNA expression enhancing factors and methods for using these factors to enhance the expression level of target genes on plasmid DNA eukaryotic expression vectors, as well as their application in the field of DNA drug formulation. Background Technology

[0002] Pathological angiogenesis or angiogenesis is a direct cause of many diseases, such as retinal neovascularization and capillary hemangiomas. Angiogenesis and fragmentation in tumor tissue play a crucial role in tumor development and metastasis, and angiogenesis in tumor tissue is directly related to pro-angiogenic factors such as VEGF and HGF. Plasmid-based gene therapy based on angiogenesis has been validated in numerous preclinical and clinical trials, and more than thirty gene therapy drugs have been approved for marketing. Furthermore, the gradual replacement of traditional inactivated vaccines with veterinary pDNA vaccines is also a trend in vaccine development. In 2018, my country's first DNA vaccine for the prevention of H5 subtype avian influenza in birds was approved. The U.S. Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA) have also approved several DNA-based vaccines for veterinary use, including a West Nile virus vaccine for horses and a melanoma vaccine for dogs.

[0003] Compared to viral vectors and mRNA drugs, DNA drugs have several unparalleled advantages, especially their weak immunogenicity, high safety, and ease of production and transportation. mRNA and adenovirus vector gene drugs require cryogenic storage at -20°C to -70°C, posing challenges to drug transportation and preservation. DNA drugs, on the other hand, can be stored at -2°C to 8°C, and even at room temperature (25°C), they exhibit good stability, which will greatly facilitate large-scale use. Furthermore, DNA drugs are simple to produce, requiring no mRNA transcription or modification, thus significantly reducing drug prices. However, the biggest problem currently facing DNA drugs is their typically low transfection efficiency, resulting in low expression levels and relatively short durations of expression of the target gene protein. In gene therapy, a single dose often requires milligrams of GMP-grade pDNA, posing challenges not only to plasmid production and use but also significantly limiting the development of pDNA drugs.

[0004] Furthermore, in higher organisms, genetic material is typically confined to the nucleus and mitochondria. DNA found in the cytoplasm either originates from damage to the nucleus or mitochondria, or from invading bacterial pathogens or DNA viruses. Studies have shown that the primary sensor triggering the DNA-induced innate immune response is cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP) synthase (cGAS). cGAMP activates the stimulator of interferon genes (STING), leading to an increase in type I interferon. However, cGAS has a limited ability to distinguish between microbial nucleic acids and endogenous nucleic acids. Therefore, innate immune responses triggered by exogenous DNA are unavoidable during the administration of DNA drug formulations. In addition, plasmid DNA contains small amounts of endotoxins, which can stimulate the synthesis of inflammatory factors IL-1, IL-6, IL-10, and TNF-α by activating TLR4. Although the National Medical Products Administration stipulates that the endotoxin content in DNA vaccine products must not exceed 0.01 EU / μg, endotoxin-induced innate immune responses in host cells are unavoidable, affecting the expression of target genes.

[0005] In other words, the current use of these DNA drugs and DNA vaccines generally suffers from problems such as low expression levels of exogenous proteins, large amounts of DNA used, significant individual differences in drug efficacy, and drug formulations triggering innate immune responses. Summary of the Invention

[0006] One of the objectives of this invention is to provide a DNA expression enhancing factor to solve the above-mentioned problems.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a DNA expression enhancing factor, wherein the enhancing factor is a polypeptide with ribonuclease III activity and low affinity for ribonuclease inhibitors.

[0008] As a preferred technical solution, the polypeptide is a leopard frog enzyme or a leopard frog enzyme derivative with one or more amino acids missing, substituted, inserted or added.

[0009] As a further preferred technical solution: the leopard frog enzyme derivative is formed by adding an amino acid Met to the N-terminus of the leopard frog enzyme. It has the amino acid sequence shown in SEQ ID NO: 1.

[0010] As a preferred technical solution, the polypeptide is a recombinant Amphinase or a recombinant Amphinase derivative with one or more amino acids missing, substituted, inserted, or added.

[0011] As a further preferred technical solution: the recombinant Amphinase is Amph 1, which has the amino acid sequence shown in SEQ ID NO.2.

[0012] A second objective of this invention is to provide a method for improving the expression level of a target gene on a plasmid DNA eukaryotic expression vector using the aforementioned enhancing factors. The technical solution adopted includes the following steps:

[0013] (a) Construct and prepare enhancement factor expression plasmids for eukaryotic cells.

[0014] (b) Co-transfect host cells with the target gene expression plasmid and the enhancer expression plasmid. The enhancer increases the expression level and duration of the exogenous target gene in the body and cells.

[0015] As a preferred technical solution: the encoding gene of the enhancing factor and the recombinant target protein gene are constructed in tandem within the same expression framework of the plasmid DNA, separated by a 2A self-cleaving peptide or an internal ribosome entry site. After transfection of host cells, the expressed enhancing factor increases the expression level and duration of the exogenous target gene.

[0016] The process of first inserting the DNA sequence of the protein of this invention and then inserting the DNA sequence of the exogenous protein when constructing the co-expression vector has the same effect on the expression of the exogenous protein in the host cells after transfection. The vector containing the DNA sequence of this invention and the vector containing the exogenous protein can be the same or different expression vectors. After transfection, they have the same effect on the expression of the exogenous protein in the host cells.

[0017] As a preferred technical solution, the co-transfection method is electrotransfection, liposome transfection, or calcium-mediated transfection.

[0018] As a preferred technical solution, the host cell is a eukaryotic cell.

[0019] Another object of the present invention is to provide an application of the above-mentioned enhancing factor in reducing the innate immune response mechanism triggered by drug formulation.

[0020] As a preferred technical solution, the pharmaceutical preparation is an injection.

[0021] Although the specific mechanism by which the rRanp recombinant polypeptide of the present invention promotes the expression of exogenous DNA is not yet clear, the ribonuclease III activity of rRanp and its immunity to RI inhibition are essential. Ranp has four disulfide bonds folded into a tertiary structure, of which the C-terminal disulfide bond formed by Cys87 and Cys104 is only found in amphibian RNases. His10, Lys31, and His97 are important components of the active site of the Ranp enzyme, as detailed in Example 6 below. Therefore, the nucleotide or amino acid sequence and length of the polypeptide encoded therein can vary to some extent, and those skilled in the art will understand that such variations are all within the scope of the present invention.

[0022] The frequency of synonymous codon usage varies among different species. When the frequency of synonymous codon usage in a foreign gene matches that of the expressing host, the expression level of the target gene will be significantly increased. Therefore, codon optimization based on this invention also falls within the scope of this invention.

[0023] The enhancing effect of this invention is not limited by the type of exogenously expressed protein, gene coding sequence, host cell type, or p53 genotype.

[0024] Regarding the issue of existing drug formulations triggering innate immune responses, the rRanp of this application may reduce the body's innate immunity triggered by exogenous DNA and endotoxins in DNA drugs through interaction with tRNA and miRNA precursors.

[0025] Compared with the prior art, the advantages of the present invention are as follows: the recombinant polypeptide enhancer provided by the present invention can significantly increase the expression level of exogenous target genes in mammalian cells, with the expression level increasing by about 4 to 5 times; the application of this technology to DNA vaccines can improve antibody production levels, reduce drug dosage and cost, and greatly enhance the therapeutic effect of DNA drugs. Attached Figure Description

[0026] Figure 1 Schematic diagram of the construction of expression plasmids for eGFP, Fluc, RNase I, and Amph 1 proteins. In the diagram, A. eGFP expression plasmid; B. Fluc expression plasmid; C. RNase I expression plasmid; D. Amph 1 expression plasmid;

[0027] Figure 2 Schematic diagram of the construction of CovS-P2A-rRanp and Fluc-P2A-rRanp expression plasmids. In the figure, A. CovS-P2A-rRanp expression plasmid; B. Fluc-P2A-rRanp expression plasmid;

[0028] Figure 3 Effects of rRanp recombinant protein expression on the growth of several cell types;

[0029] Figure 4 and Figure 5 The effect of rRanp recombinant protein on exogenous eGFP expression;

[0030] Figure 6 The effect of rRanp recombinant protein on Fluc gene expression in Balb / C mice;

[0031] Figure 7 The expression intensity of luciferase at the intramuscular injection site in mice of different drug administration groups from 6 h to 192 h after drug administration. Figure A shows the expression intensity of luciferase at the intramuscular injection site from 6 h to 48 h after drug administration; Figure B shows the expression intensity of luciferase at the intramuscular injection site from 72 h to 192 h after drug administration.

[0032] Figure 8 and Figure 9 The expression intensity of eGFP in cells 24-48 h after RNase I and Amph 1 transfection. Implementation

[0033] The invention will now be further described with reference to the accompanying drawings.

[0034] Definitions:

[0035] Before describing the invention, several definitions are provided to aid in understanding it. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] CovS: The SARS-CoV-2 Spike protein gene, a recombinant mRNA sequence of the Covid-19 S protein used in the Pfizer / BioNTech COVID-19 mRNA vaccine;

[0037] rRanp: Recombinant Ranpirnase;

[0038] pDNA: Plasmid DNA;

[0039] Fluc: Firefly luciferase;

[0040] eGFP: Enhanced Green Fluorescent Protein;

[0041] RNase III: Ribonuclease III;

[0042] ANG: Angiopoietin;

[0043] DNA vaccines typically involve recombining an antigen gene into a eukaryotic expression vector, which is then injected directly or via packaging into the body to express the corresponding antigen, inducing an immune response and thus providing immune protection.

[0044] rR3DE: This invention provides a class of recombinant polypeptides with ribonuclease III activity that can enhance the expression of exogenous DNA in host cells.

[0045] Example 1: Plasmid Construction and pDNA Preparation

[0046] 1.1 Plasmid Construction

[0047] The nucleotide sequences encoding firefly luciferase (Fluc) (M15077.1), human RNase I (FJ659112.1), and amphinase 1 (P85072.1) were downloaded from NCBI's GenBank. The nucleotide sequence encoding enhanced green fluorescent protein (eGFP) was obtained from the pEGFP-N1 vector (U55762.1). Expression plasmids for Flux, eGFP, RNase I, and Amph 1 were constructed, as follows: Figure 1 As shown. The target gene vectors used in this invention are all PUC 57 vectors with CMV promoters.

[0048] 2A peptides are short peptides derived from viruses, often referred to as "self-cleaving" peptides, capable of producing multiple proteins from a single transcript. Currently, there are four commonly used 2A peptides, derived from foot-and-mouth disease virus 2A (F2A), porcine swine fluke type 1 2A (P2A), *T. tylostella* virus 2A (T2A), and equine rhinitis virus 2A (E2A). P2A typically exhibits the highest cleavage efficiency; this example uses the short P2A peptide with the amino acid sequence: GSGATNFSLLKQAGDVEENPGP.

[0049] In this embodiment, "rRanp" is formed by adding the amino acid Met to the N-terminus of a mature Ranpirnase polypeptide sequence, resulting in the amino acid sequence shown in SEQ ID NO: 1. "CovS" is the recombinant mRNA sequence of the Covid-19 S protein used in the Pfizer / BioNTech COVID-19 mRNA vaccine, derived from the SARS-CoV-2 Spike protein gene.

[0050] The rRanp DNA sequence and the CovS DNA sequence are linked by a P2A cleavage peptide to form CovS-P2A-rRanp. Figure 2 As shown in Figure A, during transcription, the polypeptide is cleaved at position 21 of P2A, splitting the transcript into two polypeptide segments: CovS and rRanp.

[0051] The rRanp DNA sequence and the Fluc DNA sequence are linked by a P2A cleavage peptide to form Fluc-P2A-rRanp, as shown below. Figure 2 As shown in B, during transcription, the polypeptide is cleaved at position 21 of P2A, splitting the transcript into two polypeptide segments: Fluc and rRanp.

[0052] The CovS-P2A-rRanp and Flux-P2A-rRanp expression plasmids were sequenced and qualified before use.

[0053] 1.2 Obtaining pDNA

[0054] The plasmid DNA used in the embodiments of this invention was obtained by Qiagen EndoFree Plasmid Maxi Kit.

[0055] Example 2: Preparation of pDNA transfection formulation and pDNA vaccine formulation

[0056] There are various methods for introducing plasmid DNA into cells, including physical-mediated (electroporation, microinjection, and gene gun), chemical-mediated (calcium phosphate coprecipitation, vector transfection, etc.), and biological-mediated (protoplast transfection, virus-mediated transfection) pathways. Among these three techniques, vector transfection is the most commonly used. In this embodiment, liposomes are used as pDNA vectors. The liposomes in this embodiment are composed of non-ionized cationic lipids (specifically Dotap), ionized cationic lipids (specifically ALC-0315), cholesterol (Chol), and PEG2000-DMG in a molar ratio of Dotap:ALC-0315:Chol:PEG2000-DMG = 46.29:46.29:85.34:4. Liposomes were dissolved in anhydrous ethanol (to obtain an organic solution), and nucleic acids were dissolved in citric acid aqueous solution (10 mM, pH 4.0) (to obtain an aqueous solution). The aforementioned aqueous solution and organic solution were then mixed via a microfluidic chip at a 3:1 volume ratio, with a total flow rate greater than 3 ml / min. The liposome-pDNA formulation was dialyzed overnight with 1×PBS solution, and then transferred to glass vials for storage at 4°C or -20°C. Final pDNA concentration: 0.1–0.375 μg / µL.

[0057] Example 3: Effect of rRanp recombinant protein expression on cell growth.

[0058] The pcDNA3.1 vector backbone labeled with neomycin was used as a negative control to test the effect of rRanp recombinant protein expression on the expression of the tracer gene neomycin and cell growth.

[0059] The specific procedure was as follows: 2 × 10^4 cells / mL of H1299, H322, and A549 cells were seeded into 6-well plates. 24 hours after seeding, the medium was replaced with fresh 10% FBS-1640 medium. 1 mL of transfection reagent was added to each well. The transfection reagent was prepared as follows: pcDNA3.1 was mixed with eGFP, p53, and rRanp plasmid DNA at a weight ratio of 1:9, encapsulated in Dotap:Lecithin (molar ratio 20:9) liposomes, and then diluted with culture medium containing 10% FBS. Each mL of transfection reagent contained 1 μg of DNA for H1299, and 5 μg of DNA for H322 and A5495. Two days after transfection, the cells were replaced with culture medium containing different concentrations of G418: H1299 (400 μg / mL), H322 (100 μg / mL), and A549 (100 μg / mL). Once the cell density reaches 80% or higher, fix the cells with 10% formalin, stain them with Giemsa solution, and take photographs.

[0060] The results are as follows Figure 3 As shown: The control group p53 expression plasmid significantly killed H1299 cells lacking p53 expression and H322 cells with p53 gene mutation, and had a certain killing effect on wild-type p53 cells A549, which was in line with expectations. Compared with the EGFP expression plasmid and the p53 expression plasmid, there was no significant difference in the number of cells in 6-well plates expressing rRanp and those expressing eGFP protein. The rRanp expression plasmid had no effect on the growth of H1299, H322 and A549 cells and did not show significant cytotoxicity.

[0061] Example 4: Effect of rRanp recombinant protein on exogenous eGFP expression

[0062] The eGFP expression plasmid was used as the reporter gene plasmid, and it was co-transfected with Fluc, p53, and rRanp expression plasmids, respectively. The p53 and Fluc expression plasmids were used as controls to analyze the effect of ONC gene expression on eGFP gene expression in different cell lines.

[0063] The specific procedure was as follows: 2.5 × 10³ H1299 cells, 2.5 × 10³ H322 cells, and 3.0 × 10³ A549 cells were seeded into 96-well plates. The medium was replaced with fresh 10% FBS-1640 medium 24 hours after seeding for gene transfection. The medium was replaced again 24 hours after transfection, and then every 48 hours thereafter. Transfection reagents: eGFP expression vector was mixed 1:1 with Fluc, p53, and rRanp expression vectors, encapsulated in Dotap:Lecithin (molar ratio 20:9) liposomes, and then diluted with culture medium containing 10% FBS. Transfection dose: 1 μg pDNA / well. Photography and fluorescence counting: From 24 to 144 hours post-transfection, cell transfection was recorded by photographing every 24 hours using a green fluorescence microscope. Cell green fluorescence intensity was counted using Phtoshop.

[0064] The results are as follows Figure 4 and Figure 5 As shown: For H1299, H322, and A549 cells, from 24 to 144 hours post-transfection, the eGFP expression intensity in the eGFP-rRanp experimental group was significantly different from that in the eGFP-Fluc control group (p < 0.01). At each time point, the eGFP expression intensity in the eGFP-rRanp experimental group was significantly higher than that in the eGFP-Fluc control group, increasing by 1.71-4.17 times. From 24 to 144 hours post-transfection, in A549 cells, rRanp increased the expression of the co-transfected eGFP gene by an average of 3.09 times; in H1299 cells, rRanp increased the expression of the co-transfected eGFP gene by an average of 2.92 times; and in H1322 cells, rRanp increased the expression of the co-transfected eGFP gene by an average of 1.88 times (area below the zigzag line).

[0065] Example 5: Effect of rRanp recombinant protein on Fluc gene expression in Balb / C mice.

[0066] Seven-week-old female Balb / c mice were divided into six groups of three. Three groups were given a drug formulation consisting of liposome-coated rRanp-P2A-Fluc gene plasmids, while the other three groups were given a drug formulation consisting of liposome-coated Fluc gene plasmids. The drug particle size and encapsulation efficiency are shown in Table 1 below.

[0067] Table 1: Drug Particle Size and Encapsulation Efficiency

[0068] The two drug formulations were administered via intramuscular injection into the right lower extremity. Fluc gene expression was tested at three dosages: 30.4 μg / 50 µL, 15.2 μg / 50 µL, and 7.6 μg / 50 µL. In vivo IVIS imaging was performed at 6, 24, 48, 72, 96, 120, 144, and 168 hours after administration to analyze Fluc expression levels and duration.

[0069] The results are as follows Figure 6 As shown, the expression intensity of luciferin in the rRanp-P2A-Fluc and Fluc groups at three dosages (30.4 μg / 50 µL, 15.2 μg / 50 µL, and 7.6 μg / 50 µL) was positively correlated with the dosage. There were no significant differences in particle size and encapsulation efficiency between the rRanp-P2A-Fluc and Fluc groups. The gene expression duration in the Fluc group was 120 h, while a high level of Fluc gene expression was still detected in the rRanp-P2A-Fluc group 168 h after administration, indicating that rRanp prolonged Fluc gene expression for at least 2 days.

[0070] like Figure 7 As shown, at different time points, the expression intensity of luciferin at the intramuscular injection site in mice treated with rRanp-P2A-Fluc was significantly higher than that in mice treated with Fluc. At 48 h post-administration, rRanp in the 30.4 μg group increased Fluc protein expression by 8.22-fold, rRanp in the 15.2 μg group increased Fluc protein expression by 3.02-fold, and rRanp in the 7.6 μg group increased Fluc protein expression by 3.49-fold. At 6 h post-administration, the Fluc expression intensity in the 15.2 μg rRanp-P2A-Fluc group was similar to that in the 30.4 μg Fluc group. From 24 h to 48 h post-administration, the Fluc expression intensity in the 15.2 μg rRanp-P2A-Fluc group exceeded that in the 30.4 μg Fluc group.

[0071] Furthermore, as the administration time progressed, Fluc expression in the rRanp-P2A-Fluc group decreased slowly and gradually, with a smooth curve, while Fluc expression in the Fluc group decreased rapidly, with some groups showing fluctuations in the Fluc expression curve between 48h and 72h. During the experiment, the mice in all treatment groups showed good growth, and no adverse reactions were observed at the administration site.

[0072] Example 6 rRanp Activity Analysis

[0073] Ribonuclease III activity and low affinity for RI are essential conditions for rRanp to promote the expression of exogenous proteins, and also necessary for natural amphinase and Ranp to maintain tumor cell cytotoxicity. This invention compares and analyzes the amino acid sequences of proteins such as amphinase and Ranp that simultaneously possess low affinity for RI and ribonuclease III activity, RNase III with ribonuclease III activity, angiopoietin with high affinity for RI, and angiopoietin-ribonuclease 4 antibody (RNase 4), summarizing the common characteristics of these polypeptides with ribonuclease III activity and low affinity for RI. We collectively refer to these polypeptides with ribonuclease III activity and low affinity for RI as rR3DE (recombinant Ribonuclease III-associated DNA expression Enhancer, rR3DE).

[0074] .

[0075] Example 7: Effects of RI incompatibility and ribonuclease III activity on the enhanced expression of rR3DE

[0076] Amph 1 (with the amino acid sequence shown in SEQ ID NO: 2) is a variant of amphinase, belonging to the same amphibian ribonuclease family as Ranp, and both possess ribonuclease III activity. Amph 1 has conserved sites similar to Ranp His10, Lys31, Cys87, His97, and Cys104, exhibiting RI incompatibility. RNase I has sites similar to RanpHis10, Lys31, and His97, exhibiting RI affinity, but lacks ribonuclease III activity.

[0077] Using the eGFP expression plasmid as the reporter gene plasmid, eGFP expression plasmid was co-transfected with Fluc expression plasmid, p53 expression plasmid, RNase I expression plasmid, and Amph 1 expression plasmid, respectively. The promoting effects of Amph 1 and RNase I on eGFP gene expression were verified and compared. Specifically, 2.5 × 10^3 H1299 cells were seeded in 96-well plates. After 24 hours, the medium was replaced with fresh 10% FBS-1640 medium for gene transfection. The medium was replaced again 24 hours after transfection. Transfection reagents: The eGFP expression vector was mixed 1:1 with Fluc, p53, Amph 1, and RNase I expression vectors, respectively, and encapsulated in Dotap:Lecithin (molar ratio 20:9) liposomes, then diluted with culture medium containing 10% FBS. Transfection dosage: 1 μg pDNA / well. Cell transfection status was recorded by photographing under a green fluorescence microscope at 24 and 48 hours post-transfection, and the green fluorescence intensity of cells was counted using phtoshop.

[0078] The results are as follows Figure 8 and Figure 9 As shown, from 24 to 48 hours post-transfection, the eGFP expression intensity in the Amph 1 experimental group was 4.74-fold and 3.11-fold higher than that in the Fluc control group at the same time point. RNase I had no significant effect on cellular eGFP expression. Increasing RI incompatibility and ribonuclease III activity helps to enhance the rR3DE expression enhancement effect.

[0079] In summary, this invention provides a recombinant protein, rRanp, that can enhance the expression of exogenous DNA in organisms or cells. This polypeptide is characterized by ribonuclease III activity, and the enhancing effect of rR3DE is not limited by the target protein type, gene coding sequence, or host cell type. The nucleotide sequence of this recombinant protein can be added to any eukaryotic cell expression vector for independent or co-transfection with the target gene, thereby enhancing the expression level of the exogenous target gene in organisms and cells. This addresses the current challenges of low exogenous protein expression, large DNA dosage, and significant individual variability in drug efficacy during DNA drug administration. Furthermore, we have summarized the sequence, structure, and function of such polypeptides with ribonuclease III activity and RI incompatibility, and preliminarily verified the influence of RI incompatibility and ribonuclease III activity on the enhanced expression effect of rR3DE.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of DNA expression enhancers in improving the expression level of target genes on plasmid DNA eukaryotic expression vectors, characterized in that, The amino acid sequence of the DNA expression enhancer is shown in SEQ ID NO.1, or its amino acid sequence is shown in SEQ ID NO.2; the DNA expression enhancer is a polypeptide with ribonuclease III activity and low affinity for ribonuclease inhibitors.

2. The application according to claim 1, characterized in that, The DNA expression enhancer is co-transfected to generate a recombinant polypeptide, and the recombinant polypeptide generated by co-transfection is used as one of the components to make an injection.

3. A method for enhancing the expression level of a target gene on a plasmid DNA eukaryotic expression vector using the DNA expression enhancer described in claim 1, characterized in that, Includes the following steps: (a) Construct and prepare enhancement factor expression plasmids for eukaryotic cells. (b) Co-transfect host cells with the target gene expression plasmid and the enhancer expression plasmid. The enhancer increases the expression level and duration of the exogenous target gene in the body and cells.

4. The method according to claim 3, characterized in that: The enhancing factor encoding gene and the recombinant target protein gene are constructed in tandem within the same expression framework of the plasmid DNA, separated by a 2A self-cleaving peptide or an internal ribosome entry site. After transfection into host cells, the expressed enhancing factor increases the expression level and duration of the exogenous target gene.

5. The method according to claim 3, characterized in that: Codon optimization was performed on the gene encoding the enhancer.

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