Eukaryotic APVP2 promoter, vector and construction method and application of eukaryotic APVP2 promoter
By using the eukaryotic APVP2 promoter and its vector, and constructing a protein expression vector through homologous recombination, the problems of limited promoter types and low transcription efficiency were solved, achieving efficient expression of exogenous proteins in mammalian and avian cells and simplifying the vector construction process.
Patent Information
- Application Number
- CN202610215710.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-14
- Publication Date
- 2026-05-19
AI Technical Summary
There are few existing promoter types and low transcription efficiency. Homologous recombination leads to instability in protein expression and gene therapy products, and there is a lack of diverse promoter options.
This invention provides a eukaryotic APVP2 promoter and its vector, constructs a protein expression vector through homologous recombination, omitting enzyme digestion and ligation steps, and constructs the pAPVP2 vector using PCR amplification and homologous recombination technology for use in genetic engineering vaccines and gene therapy.
It enables efficient expression of exogenous proteins in mammalian and avian cells, simplifies vector construction, and improves ligation efficiency and stability, making it suitable for genetically engineered vaccines and gene therapy.
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Figure CN122060734A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a eukaryotic APVP2 promoter, vector, its construction method and application. Background Technology
[0002] Promoters are essential components for gene transcription in eukaryotes, significantly impacting transcription efficiency. They are widely used in protein expression, gene therapy, genetically engineered vaccines, and breeding. However, the most commonly used promoters, including SV40, CMV, and Avian actin, offer limited choices, and some, like SV40, exhibit low transcription efficiency. With the rapid development of biotechnology, the need for promoters is increasing. In particular, repeatedly using the same promoter in the same animal or vector can lead to homologous recombination, resulting in instability in research products such as protein expression, gene therapy, genetically engineered vaccines, and breeding. Therefore, there is an urgent need for a wider variety of promoters to choose from and use. Summary of the Invention
[0003] The purpose of this invention is to overcome the shortcomings of the existing technology by providing a eukaryotic APVP2 promoter, vector, construction method, and application. This invention provides a novel eukaryotic protein expression promoter—the APVP2 promoter, which is the promoter for Avianpox virus (APV). A protein expression vector is constructed based on this promoter. This vector can be cloned using homologous recombination, exhibiting simplicity and efficiency. It is expected that this vector will play an important role in gene therapy, genetically engineered vaccines, and transgenic animal research.
[0004] The objective of this invention can be achieved through the following technical solutions:
[0005] The present invention provides a eukaryotic APVP2 promoter having a nucleotide sequence as shown in SEQ ID NO.1, or a sequence having more than 60% homology with the nucleotide sequence shown in SEQ ID NO.1.
[0006] The present invention also provides a carrier comprising the above-described eukaryotic APVP2 promoter.
[0007] Preferably, the vector is a protein expression vector comprising a prokaryotic origin of replication, a selection marker gene, and a foreign gene expression cassette. The foreign gene expression cassette, from upstream to downstream, sequentially includes the eukaryotic APVP2 promoter, a linker fragment, and a polyadenylated tailing signal. This vector is a circular vector, named pAPVP2.
[0008] More preferably, the polyadenylation signal is the bovine growth hormone gene polyadenylation sequence (BGHpolyA).
[0009] This invention also provides a method for constructing a recombinant vector expressing a foreign gene, comprising:
[0010] Linearize the vector containing the eukaryotic APVP2 promoter, add the linearized vector and exogenous gene fragment to competent cells, mix well, and incubate on ice for 25-40 minutes; heat shock at 42°C for 80-100 seconds, then place on ice for 2-5 minutes, add preheated LB medium, and incubate at 37°C with shaking at 180-200 rpm for 80-100 minutes; collect the bacterial cells, resuspend the cells in LB liquid medium, spread the resuspended solution on LB plates containing ampicillin, and incubate at 37°C for 12-16 hours; pick single clones to extract plasmids, which are the recombinant vectors for expressing the exogenous gene.
[0011] Preferably, the linearized vector is obtained by PCR amplification.
[0012] More preferably, the primers for PCR amplification are:
[0013] APVP2 Promotor-F:
[0014] 5'-TAACCGTATTACCGCCATGCATtacaaaaatcaaagtagatgcgcatgtat-3';
[0015] APVP2 Promotor-R:
[0016] 5'-GGTGGCgggccgcacgtcacaacaatgtgtagg-3'.
[0017] More preferably, the PCR amplification reaction system is as follows: 10×PCR Buffer 5µL, upstream and downstream primers 1µL each, DNA template 1µL, MgCl2 (25mM) 3µL, dNTPs (2.5mM each) 3µL, Pfu-Taq (5U / µL) 0.5µL, and finally add water to 50µL.
[0018] Reaction conditions: 94℃ heat denaturation for 5 min; 94℃ denaturation for 45 s, 53℃ annealing for 45 s, 72℃ extension for 6 min, for a total of 30 cycles; 72℃ final extension for 10 min.
[0019] This invention also provides the application of the above-mentioned eukaryotic APVP2 promoter in the preparation of protein expression vectors, genetically engineered vaccines, or gene therapy products.
[0020] This invention also provides the application of the above-mentioned vector in the preparation of shuttle vectors, exogenous gene expression vectors, genetically engineered vaccines, gene therapy products, or breeding products.
[0021] The beneficial effects of this invention are as follows:
[0022] Compared with existing technologies, the pAPVP2 vector of this invention can efficiently express exogenous proteins in mammalian cells. Furthermore, this vector can utilize homologous recombination to construct recombinant vectors expressing exogenous genes, avoiding traditional enzyme digestion and ligation steps. The entire construction process does not use any restriction enzymes or ligases, and it omits 2-3 steps compared to traditional ligation processes, while also achieving higher ligation efficiency. The process of constructing recombinant vectors expressing exogenous genes using the pAPVP2 vector of this invention is simple, rapid, economical, and efficient, allowing for the rapid construction of expression vectors for large quantities of exogenous genes. The pAPVP2 vector of this invention has excellent application prospects. Attached Figure Description
[0023] Figure 1 The pAPVP2 vector map.
[0024] Figure 2 The results are shown in the fluorescence microscopy observation of green fluorescent protein. A is the fluorescence result of 293T cells transfected with pAPVP2-EGFP plasmid; B is the fluorescence result of LMH cells transfected with pAPVP2-EGFP plasmid; C is the negative control 293T cells; D is the negative control LMH cells. Detailed Implementation
[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.
[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0027] Example 1. Construction of the pAPVP2 vector
[0028] The specific steps include:
[0029] The upstream primer APVP2 Promotor-F and the downstream primer APVP2 Promotor-R, and the upstream primer AMP / pBR322 / BGHPolyA-F and the downstream primer AMP / pBR322 / BGHPolyA-R, were designed with the following sequences:
[0030] APVP2 Promotor-F:
[0031] 5'-TAACCGTATTACCGCCATGCATtacaaaaatcaaagtagatgcgcatgtat-3' (SEQ ID NO. 2);
[0032] APVP2 Promotor-R:
[0033] 5'-GGTGGCgggccgcacgtcacaacaatgtgtagg-3' (SEQ ID NO. 3).
[0034] AMP / pBR322 / BGHPolyA-F:
[0035] 5'-acgtgcggcccGCCACCctgtgccttctagttgccagccatctgt-3' (SEQ ID NO. 4);
[0036] AMP / pBR322 / BGHPolyA-R:
[0037] 5'-gatttttgtaATGCATGGCGGTAATACGGTTA-3' (SEQ ID NO. 5).
[0038] Using APV viral DNA as a template, fragment A was amplified by PCR with upstream primer APVP2 Promotor-F and downstream primer APVP2Promotor-R. Using pCDNA3.0 plasmid as a template, fragment B was amplified by PCR with upstream primer AMP / pBR322 / BGHPolyA-F and downstream primer AMP / pBR322 / BGHPolyA-R.
[0039] PCR reaction system: 10×PCR Buffer 5µL, upstream and downstream primers 1µL each, DNA template 1µL, MgCl2 (25mM) 3µL, dNTPs (2.5mM each) 3µL, Pfu-Taq (5U / µL) 0.5µL, and finally add water to 50µL.
[0040] Reaction conditions: 94℃ heat denaturation for 5 min; 94℃ denaturation for 45 s, 53℃ annealing for 45 s, 72℃ extension for 6 min, for a total of 30 cycles; 72℃ final extension for 10 min.
[0041] PCR products were recovered using a DNA recovery kit, following the instructions provided.
[0042] Fragments A and B were added to 50 μL of competent cells and mixed thoroughly. The mass ratio of fragments A to B was 100 ng: 500 ng. After mixing, the mixture was incubated on ice for 30 min. The cells were then heat-shocked in a 42℃ water bath for 90 seconds, followed by incubation on ice for 3 min. 900 μL of preheated LB medium was added, and the mixture was incubated at 37℃ with shaking at 180–200 rpm for 90 min. The cells were collected and resuspended in LB liquid medium. 200 μL of the resuspended culture was plated on LB plates (containing ampicillin) and incubated at 37℃ for 12–16 h. Single clones were picked, and the plasmid was extracted. This plasmid was the pAPVP2 vector; the vector diagram is shown below. Figure 1 .
[0043] Example 2. The ability of the pAPVP2 vector to express EGFP protein
[0044] 1) Construction of pAPVP2-EGFP-Vector expressing green fluorescent protein
[0045] Design the upstream primer EGFP-F and the downstream primer EGFP-R. The primer sequences are as follows:
[0046] EGFP-F: 5'-gtgacgtgcggcccGCCACCATGGTGAGCAAGGGCGA-3' (SEQ ID NO. 6);
[0047] EGFP-R: 5'-ggcaactagaaggcacagTTACTTGTACAGCTCGTCCATG-3' (SEQ ID NO. 7).
[0048] Using pEGFP-N1 as a template, the EGFP gene fragment was amplified by PCR using upstream primer EGFP-F and downstream primer EGFP-R.
[0049] Reaction system: 10×PCR Buffer 5 µL, upstream and downstream primers 1 µL each, DNA template 1 µL, MgCl2 (25 mM) 3 µL, dNTPs (2.5 mM each) 3 µL, Pfu-Taq (5 U / µL) 0.5 µL, and finally add water to 50 µL.
[0050] Reaction conditions: 94℃ heat denaturation for 5 min; 94℃ denaturation for 45 s, 50℃ annealing for 45 s, 72℃ extension for 1 min, for a total of 30 cycles; final extension at 72℃ for 10 min.
[0051] PCR products were recovered using a DNA recovery kit, following the instructions provided.
[0052] The pAPVP2 vector was amplified using PCR to create a linearized vector, and the linearized pAPVP2 vector was then recovered.
[0053] Preparation of upstream primers for the pAPVP2 linear vector:
[0054] pAPVP2-F: 5'-TAACCGTATTACCGCCATGCATtacaaaaatcaaagtagatgcgcatgtat-3' (SEQ ID NO. 8);
[0055] Preparation of downstream primers using the pAPVP2 linear vector:
[0056] pAPVP2-R: 5'-GGTGGCgggccgcacgtcacaacaatgtgtagg-3' (SEQ ID NO. 9).
[0057] The DNA recovery kit recovers the target fragment. The recovered product is stored at -80°C for later use.
[0058] Linearized pAPVP2 vector and EGFP gene fragment were added to 50 μl of competent cells and mixed thoroughly. The mass ratio of linearized pAPVP2 vector to EGFP gene fragment was 100 ng: 500 ng. After mixing, the mixture was incubated on ice for 30 min. The cells were then heat-shocked in a 42℃ water bath for 90 s, followed by incubation on ice for 3 min. 900 μl of preheated LB medium was added, and the mixture was incubated at 37℃ with shaking at 180–200 rpm for 90 min. The cells were collected and resuspended in LB liquid medium. 200 μl of the resuspended culture was plated on LB agar plates (containing ampicillin) and incubated at 37℃ for 12–16 h. Single clones were picked, and the plasmid was extracted and named pAPVP2-EGFP.
[0059] 2) Detection of green fluorescent protein
[0060] Add 5 μL of Lipofectamine 2000 (Invitrogen) to 250 μL of OPTI-MEMI medium (Invitrogen) and incubate at room temperature for 5 minutes to obtain solution 1. Add 2 μg of pAPVP2-EGFP plasmid to 250 μL of OPTI-MEM medium to obtain solution 2. Mix solutions 1 and 2, incubate at room temperature for 20 minutes, and then add 500 μL of OPTI-MEM medium to obtain solution 3. Culture human renal epithelial cells (293T) and chicken hepatoma cells (LMH) in 6-well cell culture plates. When the cells reach 60-80% confluence, wash the cells twice with PBS, then add solution 3 and incubate at 37°C for 5 hours. Aspirate solution 3, add OPTI-MEM medium containing 10% fetal bovine serum, and continue culturing. After 24 hours of culture, observe the expression of fluorescent proteins under a fluorescence microscope.
[0061] Fluorescence microscopy results as follows Figure 2 As shown, pAPVP2-EGFP transfected cells exhibited strong fluorescence signals in 293T and LMH cells 24 hours after transfection, while the negative control group showed no fluorescence. This demonstrates that the pAPVP2-EGFP plasmid can express green fluorescent protein in mammalian and avian cells with good protein expression capabilities, and also indicates that the APVP2 promoter can efficiently express exogenous proteins in mammalian and avian cells. Figure 2 ).
[0062] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.
Claims
1. A eukaryotic APVP2 promoter having a nucleotide sequence as shown in SEQ ID NO.1, or a sequence having more than 60% homology with the nucleotide sequence shown in SEQ ID NO.
1.
2. A vector comprising the eukaryotic APVP2 promoter of claim 1.
3. The carrier according to claim 2, characterized in that, The vector is a protein expression vector containing a prokaryotic origin of replication, a selection marker gene, and a foreign gene expression cassette. The foreign gene expression cassette includes, from upstream to downstream, the eukaryotic APVP2 promoter, a linker fragment, and a polyadenylate tailing signal.
4. The carrier according to claim 3, characterized in that, The polyadenylation signal is the polyadenylation sequence of the bovine growth hormone gene.
5. A method for constructing a recombinant vector expressing a foreign gene, comprising: Linearize the vector containing the eukaryotic APVP2 promoter as described in claim 1, add the linearized vector and the exogenous gene fragment to competent cells, mix well, and incubate on ice for 25-40 minutes; heat shock in a 42°C water bath for 80-100 seconds, then place on ice for 2-5 minutes, add preheated LB medium, and incubate at 37°C with shaking at 180-200 rpm for 80-100 minutes; collect the bacterial cells, resuspend the bacterial cells in LB liquid medium, spread the resuspended solution on LB plates containing ampicillin, and incubate at 37°C for 12-16 hours; pick single clones to extract plasmids, which are the recombinant vectors for expressing the exogenous gene.
6. The construction method according to claim 5, characterized in that, Linearized vectors were obtained by PCR amplification.
7. The construction method according to claim 6, characterized in that, The primers for PCR amplification are: APVP2 Promotor-F: 5'-TAACCGTATTACCGCCATGCATtacaaaaatcaaagtagatgcgcatgtat-3'; APVP2 Promotor-R: 5'-GGTGGCgggccgcacgtcacaacaatgtgtagg-3'.
8. The construction method according to claim 6 or 7, characterized in that, The PCR amplification reaction system is as follows: 10×PCRBuffer 5µL, upstream and downstream primers 1µL each, DNA template 1µL, MgCl2 3µL, dNTPs 3µL, Pfu-Taq 0.5µL, and finally add water to 50µL. Reaction conditions: 94℃ heat denaturation for 5 min; 94℃ denaturation for 45 s, 53℃ annealing for 45 s, 72℃ extension for 6 min, for a total of 30 cycles; 72℃ final extension for 10 min.
9. The use of the eukaryotic APVP2 promoter according to claim 1 in the preparation of protein expression vectors, genetically engineered vaccines, or gene therapy products.
10. The use of the vector according to any one of claims 2 to 4 in the preparation of shuttle vectors, exogenous gene expression vectors, genetically engineered vaccines, gene therapy products, or breeding products.