Eukaryotic PCV promoter, vector and construction method and application thereof
By providing eukaryotic PCV promoters and their vectors, the problems of limited promoter types and low transcription efficiency are solved, enabling efficient expression of exogenous proteins in mammalian cells, simplifying the vector construction process, and improving expression efficiency and stability.
Patent Information
- Application Number
- CN202511729603.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-24
- Publication Date
- 2026-02-10
AI Technical Summary
There are few existing eukaryotic promoters and their transcription efficiency is low. Homologous recombination leads to instability in protein expression and gene therapy products, and there is a lack of diverse promoter options.
A eukaryotic PCV promoter and its vector are provided. The vector is constructed by homologous recombination and contains a PCV promoter, a linker fragment and a polyadenylate tailing signal. Enzyme digestion and ligation steps are omitted, and exogenous proteins are expressed directly and efficiently in mammalian cells.
The increased variety of promoters simplified the vector construction process, improved the expression efficiency of exogenous proteins, avoided homologous recombination problems, and achieved rapid, economical, and efficient expression of exogenous genes.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of bioengineering technology, and in particular to a eukaryotic PCV promoter, vector, its construction method, and its application. Background Technology
[0002] A eukaryotic promoter is a DNA sequence that is primarily responsible for initiating the transcription of specific downstream RNA transcripts. For example, if a downstream RNA transcript encodes a protein, the eukaryotic promoter can transcribe the open reading frame of the downstream protein, converting it into mRNA, and thus expressing it as a protein.
[0003] Eukaryotic promoters are essential components for gene transcription in eukaryotes, significantly influencing 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, especially since 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 variety of promoters to choose from and use. Summary of the Invention
[0004] To address the above technical problems, this invention provides a eukaryotic PCV promoter, vector, its construction method, and its application, which increases the types of promoters, and the vector pPCV containing this promoter can efficiently express exogenous proteins in mammalian cells.
[0005] This invention is achieved through the following technical solution:
[0006] In one aspect of the invention, a eukaryotic PCV (Papio ursinus cytomegalovirus) promoter is provided, having a nucleotide sequence as shown in SEQ ID NO.1, or a sequence having more than 80% homology with the nucleotide sequence shown in SEQ ID NO.1.
[0007] In another aspect of the invention, a carrier comprising the above-described eukaryotic PCV promoter is also provided.
[0008] Preferably, 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 the eukaryotic PCV promoter, a linker fragment, and a polyadenylated tailing signal. This vector is a circular vector, named pPCV.
[0009] More preferably, the polyadenylation signal is the bovine growth hormone gene polyadenylation sequence (BGHpolyA).
[0010] In another aspect of the present invention, a method for constructing a recombinant vector expressing a foreign gene is also provided, comprising:
[0011] Linearize the vector containing the eukaryotic PCV promoter described above, 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 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.
[0012] Preferably, the linearized vector is obtained by PCR amplification.
[0013] More preferably, the primers for PCR amplification are:
[0014] pPCV-F: 5'-CATGGACGAGCTGTACAAGTAActgtgccttctagttgcc-3' (SEQ ID NO. 8);
[0015] pPCV-R: 5'-CACCATGGTGGCttccaatggagctccaacgacgtccgc-3' (SEQ ID NO. 9).
[0016] 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.
[0017] 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.
[0018] In another aspect of the present invention, the application of the above-described eukaryotic PCV promoter in the preparation of protein expression vectors, genetically engineered vaccines, or gene therapy products is also provided.
[0019] In another aspect of the present invention, 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 is also provided.
[0020] The beneficial effects of this invention are as follows:
[0021] This invention provides a novel eukaryotic PCV promoter, which not only increases the variety of promoters but also enables the pPCV vector containing this promoter to efficiently express exogenous proteins in mammalian cells. This pPCV vector can also be used to construct recombinant vectors expressing exogenous genes via homologous recombination, avoiding traditional enzyme digestion and ligation steps. The entire construction process does not use any restriction enzymes or ligases, omitting 2-3 steps compared to traditional ligation processes, and achieving higher ligation efficiency. The pPCV vector of this invention provides a simple, rapid, economical, and efficient method for constructing recombinant vectors expressing exogenous genes, enabling the rapid construction of expression vectors for a large number of exogenous genes and showing great application potential. Attached Figure Description
[0022] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.
[0023] Figure 1 This is a pPCV carrier spectrum diagram of Embodiment 1 of the present invention.
[0024] Figure 2 This is a diagram showing the expression results of the pPCV-EGFP plasmid in avian and mammalian cells according to Example 2 of the present invention. In the diagram, A shows the fluorescence results of pPCV-EGFP plasmid transfected into LMH cells; B shows the fluorescence results of pPCV-EGFP plasmid transfected into 293T cells; C shows the negative control LMH cells; and D shows the negative control 293T cells. Detailed Implementation
[0025] In the following examples, experimental methods without specific conditions are generally performed under conventional conditions, such as those described in "A Concise Guide to Molecular Biology Experiments" (edited by FM Osber, RE Kingston, JG Seidman, et al., translated by Ma Xuejun and Shu Yuelong. Beijing: Science Press, 2004).
[0026] This invention provides a novel eukaryotic PCV promoter and constructs a protein expression vector based on this promoter. This vector can be cloned using homologous recombination, which is simple and efficient, and is expected to play an important role in gene therapy, genetically engineered vaccines and transgenic animal research.
[0027] The eukaryotic PCV promoter of this invention can effectively initiate the transcription and translation of downstream genes in eukaryotic cells, and has important application value and prospects.
[0028] Example 1: Construction of pPCV vector
[0029] This invention utilizes homologous recombination technology to construct a pPCV vector, comprising the following steps:
[0030] 1) Design upstream primer PCV Promotor-F and downstream primer PCV Promotor-R, upstream primer AMP / pBR322 / BGHPolyA-F and downstream primer AMP / pBR322 / BGHPolyA-R, with the corresponding nucleotide sequences as follows:
[0031] PCV Promotor-F:
[0032] 5'-CCGCCATGCATggccatggcactgggccaat-3' (SEQ ID NO. 2);
[0033] PCV Promotor-R:
[0034] 5'-acagGGTGGCttccaatggagctccaacgacgtccgca-3' (SEQ ID NO. 3);
[0035] AMP / pBR322 / BGHPolyA-F:
[0036] 5'-tccattggaaGCCACCctgtgccttctagttgc-3' (SEQ ID NO. 4);
[0037] AMP / pBR322 / BGHPolyA-R:
[0038] 5'-gccatggccATGCATGGCGGTAATACGGTTA-3' (SEQ ID NO. 5).
[0039] 2) Using Papio ursinus cytomegalovirus (PCV) DNA as a template, PCR amplification was performed using upstream primer PCV Promoter-F and downstream primer PCV Promoter-R to obtain promoter fragment A (the nucleotide sequence of fragment A is shown in SEQ ID NO.1); using pCDNA3.0 plasmid as a template, PCR amplification was performed using upstream primer AMP / pBR322 / BGHPolyA-F and downstream primer AMP / pBR322 / BGHPolyA-R to obtain fragment B.
[0040] 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.
[0041] Reaction conditions for promoter fragment A: 94℃ thermal denaturation for 5 min; 94℃ denaturation for 45 s, 53℃ annealing for 45 s, 72℃ extension for 1 min, for a total of 30 cycles; final extension at 72℃ for 10 min.
[0042] Reaction conditions for promoter fragment B: 94℃ thermal 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; final extension at 72℃ for 10 min.
[0043] PCR products were recovered using a DNA recovery kit (commercially available product, no specific model is specified in this application), and the method was described in the kit's instructions.
[0044] 3) Add fragments A and B to 50 μL of competent DH5α cells and mix well. The mass ratio of fragments A to B is 100 ng: 500 ng. After mixing, incubate the mixture on ice for 30 min. Heat shock in a 42℃ water bath for 90 seconds, then place on ice for 3 min. Add 900 μL of preheated LB medium and incubate at 37℃ with shaking at 180-200 rpm for 90 min. Collect the cells and resuspend them in LB liquid medium. Spread 200 μL of the resuspended solution onto LB plates (containing ampicillin) and incubate at 37℃ for 12-16 h. Pick single clones and extract the plasmid. This plasmid is a pPCV vector, and its plasmid map is shown below. Figure 1 As shown.
[0045] Example 2: The ability of pPCV vector to express EGFP protein
[0046] 1) Construction of pPCV-EGFP-Vector expressing green fluorescent protein
[0047] The upstream primer EGFP-F and the downstream primer EGFP-R were designed, with the sequences as follows:
[0048] EGFP-F: 5'-ccattggaaGCCACCATGGTGAGCAAGGGCGAGGAG-3' (SEQ ID NO. 6);
[0049] EGFP-R: 5'-ggcaactagaaggcacagTTACTTGTACAGCTCGTCCATG-3' (SEQ ID NO. 7).
[0050] Using pEGFP-N1 as a template, the EGFP gene fragment was amplified using upstream primer EGFP-F and downstream primer EGFP-R.
[0051] 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.
[0052] 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.
[0053] PCR products were recovered using a DNA recovery kit, following the instructions provided.
[0054] The pPCV vector was amplified using PCR, and the linearized pPCV vector was then recovered.
[0055] Preparation of upstream primers using pPCV linear vector:
[0056] pPCV-F: 5'-CATGGACGAGCTGTACAAGTAActgtgccttctagttgcc-3' (SEQ ID NO.8)
[0057] Preparation of downstream primers using pPCV linear vector:
[0058] pPCV-R: 5'-CACCATGGTGGCttccaatggagctccaacgacgtccgc-3' (SEQ ID NO. 9).
[0059] The DNA recovery kit recovers the target fragment. The recovered product is stored at -80°C for later use.
[0060] Add linearized pPCV vector and EGFP gene fragment to 50 μl of competent cells and mix well. The mass ratio of linearized pPCV vector to EGFP gene fragment is 100 ng: 500 ng. After mixing, incubate on ice for 30 min. Heat shock in a water bath at 42℃ for 90 s, then place on ice for 3 min. Add 900 μL of preheated LB medium and incubate at 37℃ with shaking at 180-200 rpm for 90 min. Collect the cells and resuspend them in LB liquid medium. Spread 200 μL of the resuspended solution onto LB plates (containing ampicillin) and incubate at 37℃ for 12-16 h. Pick single clones and extract the plasmid, which is named pPCV-EGFP.
[0061] 2) Detection of green fluorescent protein
[0062] 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 pPCV-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.
[0063] Fluorescence microscopy results as follows Figure 2 As shown, pPCV-EGFP showed strong fluorescence signals in 293T and LMH cells 24 hours after transfection, while the negative control group showed no fluorescence. This demonstrates that the pPCV-EGFP plasmid can express green fluorescent protein in mammalian and avian cells with good protein expression ability, and also indicates that the PCV promoter can efficiently express exogenous proteins in mammalian and avian cells. Figure 2 ).
[0064] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.
Claims
1. A eukaryotic PCV promoter having a nucleotide sequence as shown in SEQ ID NO.1, or a sequence having more than 80% homology with the nucleotide sequence shown in SEQ ID NO.
1.
2. A carrier comprising the eukaryotic PCV 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 the eukaryotic PCV 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 PCV 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: pPCV-F: 5'-CATGGACGAGCTGTACAAGTAActgtgccttctagttgcc-3' (SEQ ID NO. 8); pPCV-R: 5'-CACCATGGTGGCttccaatggagctccaacgacgtccgc-3' (SEQ ID NO. 9).
8. The construction method according to claim 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 PCV 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.