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WO2023197365A1 - Protéine recombinante portant une protéine cible et entrant de manière autonome dans une cellule eucaryote, vecteur d'expression recombinant, bactérie recombinante et utilisation - Google Patents

Protéine recombinante portant une protéine cible et entrant de manière autonome dans une cellule eucaryote, vecteur d'expression recombinant, bactérie recombinante et utilisation Download PDF

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WO2023197365A1
WO2023197365A1 PCT/CN2022/088658 CN2022088658W WO2023197365A1 WO 2023197365 A1 WO2023197365 A1 WO 2023197365A1 CN 2022088658 W CN2022088658 W CN 2022088658W WO 2023197365 A1 WO2023197365 A1 WO 2023197365A1
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recombinant
protein
photolyase
cells
cpd
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方卫国
包玉婷
张明祥
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浙江大学
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/51—Lyases (4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/99—Other Carbon-Carbon Lyases (1.4.99)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of fusion proteins, and specifically relates to a recombinant protein that carries a target protein and enters eukaryotic cells autonomously, a recombinant expression vector, a recombinant bacterium, and their applications.
  • Ultraviolet radiation can cause many harms to humans.
  • Ultraviolet rays in sunlight are divided into three categories according to wavelength.
  • One is short-wave ultraviolet rays (UVC: 200 ⁇ 280nm), which are almost all absorbed by the atmospheric ozone layer.
  • the second is mid-wave ultraviolet rays (UVB: 290-320nm), which account for only about 5% of ultraviolet rays in sunlight.
  • Epidermal damage, including acute sunburn and pigmentation, is mainly caused by UVB.
  • the third is long-wave ultraviolet rays (UVA: 320 ⁇ 400nm), which accounts for up to 95% of ultraviolet rays in sunlight.
  • Long-wave ultraviolet rays have strong penetrating ability and can reach deep layers of the skin, easily causing skin blackening and photoaging.
  • UVA and UVB in surface ultraviolet radiation are related to most skin cancers, including squamous cell carcinomas (SCCs) and cutaneous malignant melanomas (CMMs). etc., among which non-melanoma skin cancers (NMSCs) account for more than 90% of all skin cancers [1,2] .
  • SCCs squamous cell carcinomas
  • CMMs cutaneous malignant melanomas
  • NMSCs non-melanoma skin cancers
  • the nuclear DNA photoproducts (photoproducts) produced by ultraviolet radiation are the main cause of inducing gene mutations and skin cancer.
  • UVB radiation is the most energetic and highly mutagenic component of sunlight. It is directly absorbed by DNA and induces the formation of dimer photoproducts between two adjacent pyrimidine bases [3] .
  • Ultraviolet radiation mainly induces the production of two types of DNA dimer photoproducts: cyclobutane pyrimidine dimer (CPD) and (6-4) pyrimidinone photoproduct (6-4photoproduct, 6-4PPs). Both types of light damage can distort DNA helices and interfere with cellular transcription and replication processes, thereby causing DNA mutations, inhibition of RNA synthesis, cell cycle arrest, and apoptosis [4] .
  • UV-induced CPDs cause significantly more mutations than 6-4PPs [5] .
  • UV rays may also cause DNA damage indirectly by producing singlet oxygen or free radicals, for example, by activating small molecules such as riboflavin, tryptophan, and porphyrins that can activate cellular oxygen to produce reactive oxygen species (ROS). .
  • ROS can promote DNA single-strand breaks by inducing oxidative base damage in irradiated cells or directly attacking the sugar-phosphate backbone of DNA molecules [8] .
  • oxygen free radicals can act on the unsaturated fatty acids of lipids to generate peroxidized lipids, which gradually decompose into a series of complex compounds, including malondialdehyde (MDA).
  • MDA malondialdehyde
  • Lipids can be detected by detecting the level of MDA.
  • the level of plasma oxidation indirectly reflects the degree of cell damage.
  • MDA is also a highly toxic molecule that can damage various physiological mechanisms of the human body by reacting with molecules such as DNA and proteins. For example, some of the adducts formed by the interaction between MDA and nucleic acid bases are mutagenic, Cross-linking of MDA and collagen may significantly promote the hardening of cardiovascular tissue [9] .
  • Photolyase also known as photolyase, is a flavoprotein that can repair DNA photoproducts induced by ultraviolet radiation by absorbing blue light [6] .
  • CPD photolyase that repairs CPD
  • 6-4PP photolyase that repairs 6-4PPs.
  • these two types of photolyases are similar in structure, they have strong substrate specificity [2] .
  • human cells are not born with photolytic enzymes.
  • DNA photoproducts caused by ultraviolet radiation humans can only repair them through other complex mechanisms involving multiple proteins, such as the nucleotide excision repair (NER) pathway.
  • NER nucleotide excision repair
  • the purpose of the present invention is to provide a recombinant protein, a recombinant expression vector and a recombinant bacterium that can carry a target protein autonomously into eukaryotic cells and their applications.
  • the recombinant protein can autonomously enter the nucleus of eukaryotes and directly achieve functional targets. Proteins perform functions in cells.
  • the recombinant photolyase that enters the cell nucleus can be used to repair the cyclobutane pyrimidine dimers produced in genomic DNA induced by ultraviolet radiation, repair DNA damage, and at the same time increase intracellular superoxide dismutase (SOD) ) activity, as well as reducing the levels of intracellular oxygen free radicals (ROS) and the highly toxic molecule malondialdehyde (MDA), which is expected to reduce the chance of cancer.
  • SOD superoxide dismutase
  • ROS oxygen free radicals
  • MDA highly toxic molecule malondialdehyde
  • the invention provides a recombinant protein that carries a target protein and enters eukaryotic cells autonomously.
  • the structure of the recombinant protein, from the N-terminus to the C-terminus, includes the nuclear localization signal NLS, the target protein and the short peptide AE1 in sequence.
  • the target protein includes CPD photolyase.
  • the invention also provides a recombinant photolyase that can autonomously enter the nucleus of human cells to repair DNA damage induced by UV radiation.
  • amino acid sequence of the nuclear localization signal is shown in SEQ ID NO.1;
  • the CPD photolytic enzyme includes CPD photolytic enzyme derived from Metarhizium anisopliae;
  • amino acid sequence of the short peptide AE1 is shown in SEQ ID NO. 2.
  • the DNA damage includes DNA damage of cyclobutane pyrimidine dimers.
  • the present invention also provides a recombinant expression vector for expressing the above-mentioned recombinant protein or the above-mentioned recombinant photolyase, and the recombinant expression vector includes a gene encoding the above-mentioned recombinant protein or the above-mentioned recombinant photolyase.
  • the basic vector of the recombinant expression vector includes pET-28a-sumo vector.
  • the present invention also provides a recombinant bacterium expressing the above recombinant protein or the above recombinant photolyase, and the genome of the recombinant bacterium includes a gene encoding the recombinant protein or the above recombinant photolyase or the above recombinant expression vector.
  • the basic strain of the recombinant bacteria includes Escherichia coli.
  • the present invention also provides the application of the above-mentioned recombinant photolyase in preparing a drug for treating DNA damage of cyclobutane pyrimidine dimers induced by UV radiation.
  • the present invention provides a recombinant protein that carries a target protein and enters eukaryotic cells autonomously. From the N-terminus to the C-terminus, it includes the nuclear localization signal NLS, the target protein and the short peptide AE1 with the ability to cross the cell membrane.
  • the localization signal NLS can bring the recombinant protein into the nucleus.
  • AE1 leads the target protein across the cell membrane into the cell in a non-toxic and efficient manner, and the target protein is further brought into the cell nucleus by NLS to act.
  • the present invention also provides a recombinant photolyase that can autonomously enter human cell nuclei to repair DNA damage induced by UV radiation. It uses CPD photolyase as a target protein and has the function of repairing CPD damage on cell nuclear DNA.
  • Figure 1 is the vector map of pET-28a-sumo-SVNLS-GFP-AE1, pET-28a-sumo-SVNLS-MrPHR1-AE1, pET-28a-sumo-MrPHR1-AE1, and pET-28a-sumo-SVNLS-MrPHR1;
  • Figure 2 shows SDS-PAGE analysis of SVNLS::GFP::AE1 protein expression and purification
  • M Marker
  • 1 pET-28a-sumo empty strain total protein crude extract
  • 2 SUMO::SVNLS::GFP: : AE1 total protein crude extract
  • 3 supernatant of crude extract
  • 4 purified SUMO::SVNLS::GFP::AE1 protein solution
  • 5 SVNLS::GFP: after adding ULP1 enzyme to remove SUMO. :AE1 protein liquid
  • M Marker
  • 1 pET-28a-sumo empty strain total protein crude extract
  • 2 SUMO::SVNLS::GFP: : AE1 total protein crude extract
  • 3 supernatant of crude extract 2
  • 4 purified SUMO::SVNLS::GFP::AE1 protein solution
  • 5 SVNLS::GFP: after adding ULP1 enzyme to remove SUMO. :AE1 protein liquid
  • Figure 3 shows the use of flow cytometry to analyze the entry of SVNLS-GFP-AE1 protein and GFP protein into HFF-1 cells;
  • Figure 4 shows the observation of the entry of SVNLS::GFP::AE1 protein into the nucleus of HFF-1 cells using a fluorescence microscope, the scale bar is 10 ⁇ m;
  • Figure 5 shows SDS-PAGE analysis of SVNLS::MrPHR1::AE1 protein expression and purification
  • M Marker
  • 1 pET-28a-sumo empty strain total protein crude extract
  • 2 SUMO::SVNLS::MrPHR1: : AE1 total protein crude extract
  • 3 supernatant of crude extract 2
  • 4 purified SUMO::SVNLS::MrPHR1::AE1 protein solution
  • ULP1 enzyme ULP1 enzyme
  • Figure 6 shows SDS-PAGE analysis of MrPHR1::AE1 and SVNLS::MrPHR1 protein expression and purification
  • M Marker
  • 1 pET-28a-sumo empty strain total protein crude extract
  • 2 SUMO::SVNLS:: MrPHR1 total protein crude extract
  • 3 supernatant of crude extract 2
  • 4 purified SUMO::SVNLS::MrPHR1 protein solution
  • 5 SVNLS::MrPHR1 protein solution after adding ULP1 enzyme to remove SUMO
  • 6 SUMO::MrPHR1::AE1 total protein crude extract
  • 7 supernatant of crude extract
  • 8 purified SUMO::MrPHR1::AE1 protein solution
  • 9 MrPHR1:: after adding ULP1 enzyme to remove SUMO.
  • AE1 protein liquid M: Marker
  • 1 pET-28a-sumo empty strain total protein crude extract
  • 2 SUMO::SVNLS::
  • Figure 7 is an ELISA test for the ability of SVNLS::MrPHR1::AE1 protein to repair CPD damage to nuclear DNA; 1-8 represent genomic DNA extracted from HFF-1 cells treated with different conditions, 1: negative control without any treatment; 2: UV irradiation; 3: UV irradiation followed by visible light irradiation; 4: Cells incubated with SVNLS::GFP::AE1 protein were treated with UV irradiation followed by visible light irradiation; 5: Cells incubated with SVNLS::MrPHR1 protein were UV radiation followed by visible light irradiation treatment; 6: Cells incubated with MrPHR1::AE1 protein were treated with UV radiation followed by visible light irradiation; 7: Cells incubated with SVNLS::MrPHR1::AE1 protein were treated with UV radiation; 8: With Cells co-incubated with SVNLS::MrPHR1::AE1 proteins were treated with
  • Figure 8 shows that SVNLS::MrPHR1::AE1 protein reduces the level of oxygen free radicals (ROS) induced by UV radiation in cells, where 1-6 represents HFF-1 cells treated with different conditions, 1: without any treatment Negative control; 2: UV radiation; 3: UV radiation followed by visible light irradiation treatment; 4: Cells incubated with SVNLS::MrPHR1 protein were treated with UV radiation followed by visible light irradiation; 5: Cells co-incubated with MrPHR1::AE1 protein were treated with UV radiation followed by visible light irradiation treatment; 6: Cells incubated with SVNLS::MrPHR1::AE1 protein were UV irradiated followed by visible light irradiation treatment;
  • ROS oxygen free radicals
  • Figure 9 shows that SVNLS::MrPHR1::AE1 protein reduces the level of malondialdehyde (MDA) induced by UV radiation in cells, where 1-6 represent HFF-1 cells treated with different conditions, the same as Figure 8;
  • MDA malondialdehyde
  • Figure 10 shows that the SVNLS::MrPHR1::AE1 protein further increases the activity of superoxide dismutase (SOD) in cells exposed to UV radiation, where 1-6 represent HFF-1 cells treated with different conditions, the same as Figure 8.
  • SOD superoxide dismutase
  • the invention provides a recombinant protein that carries a target protein and enters eukaryotic cells autonomously.
  • the structure of the recombinant protein, from the N-terminus to the C-terminus, includes the nuclear localization signal NLS, the target protein and the short peptide AE1 in sequence.
  • the present invention has no special limitations on the method of constructing a fusion protein using the NLS, target protein and AE1, and it can be constructed using conventional fusion protein construction methods in the field.
  • the eukaryotic cells of the present invention preferably include animal cells.
  • the target protein can be delivered into the cells and nuclei of animals.
  • CPD photolyase is used as an example to illustrate the recombinant protein, but it cannot be regarded as the entire protection scope of the present invention.
  • the present invention also provides a recombinant photolyase that can autonomously enter human cell nuclei to repair DNA damage induced by UV radiation (the embodiment is also called fusion protein SVNL::GFP::AE1).
  • the structure of the recombinant photolyase is from N From end to C end, it includes in sequence: nuclear localization signal NLS derived from SV40 virus - CPD photolyase - short peptide AE1 derived from Drosophila melanogaster.
  • the amino acid sequence of the nuclear localization signal (SVNLS) derived from the SV40 virus of the present invention is preferably as shown in SEQ ID NO.1: PKKKRKV; the CPD photolyase is preferably derived from Metarhizium anisopliae, and its Genbank accession number: XP_007821405;
  • the amino acid sequence of the short peptide AE1 derived from Drosophila melanogaster is preferably shown in SEQ ID NO. 2: GRQIKIWFQNRRMKWKK, which has the ability to cross cell membranes.
  • the source of the CPD photolytic enzyme of the present invention can also be derived from other sources, such as CPD photolytic enzymes obtained from other strains or CPD photolytic enzymes after gene editing or modification. .
  • the DNA damage preferably includes DNA damage of cyclobutane pyrimidine dimer (CPD).
  • CPD cyclobutane pyrimidine dimer
  • the CPD photolyase is sent into human cells using AE1, and is led into the nucleus by SVNLS, thereby promoting Exogenous CPD photolyase targets DNA damage caused by CPD; increases intracellular superoxide dismutase (SOD) activity, and reduces intracellular oxygen free radicals (ROS) and the highly toxic molecule malondialdehyde (MDA) )level.
  • SOD superoxide dismutase
  • ROS oxygen free radicals
  • MDA highly toxic molecule malondialdehyde
  • the present invention also provides a recombinant expression vector for expressing the above-mentioned recombinant protein or the above-mentioned recombinant photolyase, and the recombinant expression vector includes a gene encoding the above-mentioned recombinant protein or the above-mentioned recombinant photolyase.
  • the basic vector of the recombinant expression vector of the present invention preferably includes the pET-28a-sumo vector.
  • the present invention has no special limitation on the construction method of the recombinant expression vector, which preferably includes: amplifying SVNLS and AE1 to the N-terminal and C-terminal of the CPD photolyase respectively by PCR method; and performing the PCR amplification
  • the upstream primer sequentially includes two protective bases GG from the 5' end to the 3' end, the recognition site of the restriction endonuclease BamHI, the coding sequence of SVNLS and the 17 starting points of the CPD photolyase coding sequence. base.
  • the downstream primer includes two protective bases GG, the recognition site of the restriction endonuclease XbaI, the coding sequence of the short peptide AE1, and the last 17 coding sequences of Metarhizium anisopliae photolyase. base (excluding stop codon); the resulting product was cloned into the E. coli expression vector pET-28a-sumo.
  • the primers designed for the PCR amplification preferably include: upstream primer SVNLS-MrPHR1-5 (SEQ ID NO.3): CGGGATCCATGCCAAAAAAGAAGAGAAAGGTCAGTCCTGACCACACCAACG; downstream primer AE1-MrPHR1-3 (SEQ ID NO.4 ): CGGAATTCTTATCATTTTTTTCCATTTCATGCGGCGGTTCTGAAACCAAATTTTAATCTGGCGGCCCATGCCATTGGCGATTCC.
  • the present invention also provides a recombinant bacterium expressing the above recombinant protein or the above recombinant photolyase, and the genome of the recombinant bacterium includes a gene encoding the recombinant protein or the above recombinant photolyase or the above recombinant expression vector.
  • the basic strain of the recombinant bacteria of the present invention preferably includes Escherichia coli.
  • Escherichia coli BL21 strain is used as an example for explanation, but it cannot be regarded as the entire protection scope of the present invention.
  • the present invention has no particular limitation on the construction method of the recombinant bacteria. It is preferred to use the above recombinant expression vector to transform the Escherichia coli BL21 strain to obtain the Escherichia coli recombinant strain that expresses and produces the above recombinant photolyase.
  • the present invention also provides a method for preparing the recombinant photolytic enzyme using the above recombinant bacteria, which preferably includes: inoculating a single colony of the above recombinant bacteria into 5 mL of LB liquid culture medium containing kanamycin and culturing it to obtain a seed liquid; The seed liquid was inoculated into LB liquid medium containing kanamycin and cultured again, induced by IPTG, and then cultured at 18°C for 12 hours. The bacterial cells were collected and broken, and the crude protein extract was collected by centrifugation. The crude protein extract contained all the The recombinant photolyase enzyme.
  • the concentration of kanamycin in the LB liquid culture medium is preferably 100 ⁇ g/mL; the culture using the LB culture medium containing kanamycin is preferably carried out on a shaking table.
  • the temperature is preferably 37°C, 220 rpm.
  • IPTG is added to the culture medium for induction.
  • the final concentration of IPTG added is preferably 0.8mmol/L.
  • the present invention does not specifically limit the method of collecting and disrupting the bacterial cells after culturing at 18°C for 12 hours. Conventional methods in the field are used to collect the bacterial cells, disrupt the cells, and obtain a crude protein extract through centrifugation.
  • the present invention preferably further includes purification, and more preferably includes passing the crude protein extract through a nickel column to adsorb the recombinant photolyase labeled with a SUMO tag of 6 histidines.
  • the protein on the nickel column is eluted and treated with ULP1 protease to remove the SUMO tag with 6 histidines, and is further passed through the nickel column and desalted to obtain the purified recombinant photolyase.
  • the present invention also provides the application of the above-mentioned recombinant photolyase in preparing a drug for treating DNA damage of cyclobutane pyrimidine dimers induced by UV radiation.
  • the recombinant photolyase can be used to successfully enter the nucleus and repair CPD damage caused by ultraviolet radiation.
  • recombinant protein, recombinant expression vector and recombinant bacteria provided by the present invention that carry the target protein into eukaryotic cells autonomously and their applications will be described in detail below with reference to the examples. However, they should not be understood as limiting the scope of the present invention.
  • the coding sequences of SVNLS and AE1 were connected to the N-terminus and C-terminus of the GFP protein respectively (the stop codon in the GFP coding sequence was removed and added in front of SVNLS A translation initiation codon (ATG).
  • the upstream primer SVNLS-GFP-5 (SEQ ID NO. 5) used for PCR amplification: CGGGATCCATGCCAAAAAAGAAGAGAAAGGTCGTGAGCAAGGGCGAGGA; the downstream primer AE1-GFP-3 (SEQ ID NO.
  • the PCR amplification system (total volume 50 ⁇ L) is: ddH 2 O 15 ⁇ L, 2 ⁇ Phanta Max buffer (Vazyme) 25 ⁇ L, dNTP Mix (10 ⁇ M each, Vazyme) 1 ⁇ L, SVNLS-GFP -52 ⁇ L (10 ⁇ M), AE1-GFP-32 ⁇ L (10 ⁇ M), Phanta Max Super-Fidelity DNApolymerase (Vazyme) 1 ⁇ L, DNA fragment template (GFP) 2 ⁇ L.
  • the reaction program is: 95°C 3min ⁇ (95°C 15sec ⁇ 56 ⁇ 72°C 15sec ⁇ 72°C 1min/kb) cycle 30 times ⁇ 72°C 5min.
  • the PCR product was connected to the BamHI and EcoR I sites of the vector pET-28a-sumo. After sequencing to verify that there were no mutations, the prokaryotic expression vector pET-28a-sumo-SVNLS-GFP-AE1 of the protein SVNLS::GFP::AE1 was obtained, and into Escherichia coli BL21 strain.
  • Escherichia coli BL21 strain expresses SVNLS::GFP::AE1 protein induced by IPTG. Inoculate a colony of the recombinant Escherichia coli strain into 5 mL of LB liquid medium containing kanamycin. After culturing overnight at 37°C, take 100 ⁇ L of the bacterial liquid into In 1000 mL of LB liquid medium containing kanamycin, culture again at 37°C until the OD 600 value of the bacterial solution reaches about 0.6, and then add IPTG (final concentration: 0.8 mmol/L) to the culture medium. Continue culturing at 18°C for 12 hours to express the recombinant protein. SDS-PAGE analysis showed that the SVNLS::GFP::AE1 protein was successfully expressed and existed in the supernatant of the extract as shown in Figure 2.
  • Human skin fibroblast HFF-1 was cultured in a 100 mm diameter culture dish for 4 days. After trypsin treatment, the cells were collected and resuspended in 1 mL of cell culture medium. Then 100 ⁇ L of the above cell suspension was inoculated into a 30 mm diameter culture dish containing 2 mL of culture medium. After culturing at 37°C for 3-4 days, filter-sterilized SVNLS::GFP::AE1 protein (final concentration: 100ng/ ⁇ L) was added to the cell culture medium, and another group was set up to add GFP protein as a control. After incubation in a 37°C incubator for 12 hours, the culture medium was poured out, and gently washed three times with 1 ⁇ PBS to remove residual proteins on the cell surface before use in subsequent experiments.
  • HFF-1 cells were incubated with the SVNLS::GFP::AE1 protein for 12 hours, they were digested with trypsin, and the collected cells were detected with a flow cytometer to analyze the entry of the above-mentioned protein into the cells and the intracellular GFP fluorescence intensity.
  • the results are shown in Figure 3.
  • the fluorescence intensity of GFP in HFF-1 cells co-incubated with SVNLS::GFP::AE1 protein was higher than that in HFF-1 cells co-incubated with GFP protein, indicating that SVNLS::GFP::AE1 protein and Compared with the GFP protein without SVNLS and AE1, it has a stronger ability to enter cells.
  • HFF-1 cells were incubated with SVNLS::GFP::AE1 for 12 hours, the cell culture medium was poured out, and 1mL of 2 ⁇ g/mL DAPI staining solution (stained cell nuclei) was added to the culture dish to cover the cells. Leave it at room temperature for 5 minutes in the dark and aspirate. Remove the DAPI staining solution and wash 3 times with 1 ⁇ PBS for 5 minutes each time. After sealing, use a fluorescence microscope to observe. The fluorescence observation results are shown in Figure 4. The green fluorescence shows the SVNLS::GFP::AE1 protein transported into the cells, and the blue fluorescence shows the nucleus of HFF-1 cells. Figure The green fluorescence and blue fluorescence overlap, indicating that SVNLS::GFP::AE1 successfully entered the nucleus of HFF-1.
  • the present invention constructed the fusion protein SVNLS::MrPHR1:: AE1.
  • MrPHR1 coding sequence Genbank accession number: , and add a translation initiation codon ATG
  • the primers used were SEQ ID NO.3 and SEQ ID NO.4, and the template was the cDNA of Metarhizium anisopliae mycelium.
  • the amplification system and amplification procedure are the same as in Example 1, except that the upper and lower primers in the amplification system are changed to SEQ ID NO.3 and SEQ ID NO.4.
  • the PCR product was connected to the BamHI and EcoR I sites of the vector pET-28a-sumo. After sequencing to verify that there were no mutations, the prokaryotic expression vector pET-28a-sumo-SVNLS-MrPHR1-AE1 of SVNLS::MrPHR1::AE1 was obtained and transformed into Escherichia coli strain BL21.
  • the IPTG-induced expression and purification of SVNLS::MrPHR1::AE1 protein in E. coli BL21 strain are the same as in Example 1.
  • the SDS-PAGE analysis results are shown in Figure 5.
  • the SVNLS::MrPHR1::AE1 protein was successfully expressed and purified.
  • control proteins MrPHR1::AE1 and SVNLS::MrPHR1 were constructed.
  • the construction method is the same as Example 2 except for the different amplification primers;
  • the sequence of the upstream primer MrPHR1-5 (SEQ ID NO.7) used to construct the protein MrPHR1::AE1 is CGGGATCCATGGCTCGAAAATCATCG; the sequence of the downstream primer AE1-MrPHR1-3 (SEQ ID NO.4) is the same as above.
  • the sequence of the upstream primer SVNLS-MrPHR1-5 used to construct the protein SVNLS::MrPHR1 is the same as above (SEQ ID NO.3); the sequence of the downstream primer MrPHR1-3 (SEQ ID NO.8) is CGGAATTCCTACATGCCATTGGCGA.
  • HFF-1 cells were passaged into Corning culture dishes with a diameter of 30 mm, cultured in a carbon dioxide incubator at 37°C for 3 days, and then processed as follows: negative control without any treatment (untreated), cells co-incubated with recombinant protein (Cell+ protein), UV irradiated cells (UV/cell), visible light irradiated UV-treated cells (Light+UV/cell), cells incubated with recombinant proteins treated with UV irradiation (UV+protein/cell), cells co-incubated with recombinant proteins The incubated cells were UV irradiated and then treated with visible light (Light+UV+protein/cell), with 3 replicates for each setting.
  • recombinant proteins For cells co-incubated with recombinant proteins, they are treated with visible light after UV irradiation (Light+UV+protein/cell). In this setting, there are 4 types of recombinant proteins (SVNLS::MrPHR1::AE1, SVNLS::MrPHR1, MrPHR1:: AE1 or SVNLS::GFP::AE1), in other settings the recombinant protein was only SVNLS::MrPHR1::AE1.
  • the recombinant protein was added to the cell culture medium after the cells were cultured for 60 hours (the final protein concentration was 100ng/mL), and the culture was continued for 12 hours at 37°C before subsequent treatment.
  • the dosage of UV treatment is 1J/m 2 .
  • the visible light treatment process is as follows: place a glass piece on the cell culture dish (to filter UV and reduce evaporation of the culture medium), and irradiate it with visible light for 3 hours in a carbon dioxide incubator at 37°C (the light source is a 10W fluorescent tube). The treated cells were taken out and washed three times with PBS, and then collected into 1.5mL Ep tubes.
  • SVNLS::MrPHR1::AE1 reduces intracellular oxygen free radicals (ROS) levels after entering the cell nucleus 1) Photorepair of UV-irradiated HFF-1 cells
  • ROS content determination The determination process was carried out according to the instructions of the Oxiselect in vitro ROS/RNS assay kit (Cell Biolabs, USA).
  • SVNLS::MrPHR1::AE1 reduces the level of the intracellular toxic substance malondialdehyde (MDA) after entering the cell nucleus.
  • MDA malondialdehyde
  • MDA content determination The determination process was carried out according to the instructions of the malondialdehyde (MDA) content detection kit (Solarbio, China).
  • SVNLS::MrPHR1::AE1 increases intracellular superoxide dismutase SOD activity after entering the cell nucleus.
  • SOD activity measurement The measurement process was carried out according to the instructions of the superoxide dismutase (SOD) activity detection kit (Solarbio, China).

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Abstract

La présente invention relève du domaine technique des protéines de fusion. L'invention concerne une protéine recombinante portant une protéine cible et entrant de manière autonome dans une cellule eucaryote, un vecteur d'expression recombinant, une bactérie recombinante et une utilisation. La protéine recombinante comprend séquentiellement, de l'extrémité N-terminale à l'extrémité C-terminale, un signal de localisation nucléaire (NLS) qui transporte la protéine recombinante dans le noyau, une protéine cible et un peptide court AE1 ayant la capacité de couvrir la membrane cellulaire, AE1 guidant de manière non toxique et efficace la protéine recombinante pour couvrir la membrane cellulaire et entrer dans une cellule, et la protéine cible étant transportée dans le noyau au moyen du NLS, jouant ainsi un rôle. L'invention concerne également une photolyase recombinante capable d'entrer de manière autonome dans un noyau humain pour réparer un dommage à l'ADN induit par un rayonnement UV. La photolyase de CPD utilisée en tant que protéine cible a des effets de réparation d'un dimère de cyclobutane pyrimidine (CPD) sur l'ADN nucléaire, d'amélioration de l'activité de la superoxyde dismutase intracellulaire (SOD), et de réduction des taux d'espèces d'oxygène réactif intracellulaire (ROS) et d'une molécule hautement toxique de malondialdéhyde (MDA).
PCT/CN2022/088658 2022-04-13 2022-04-24 Protéine recombinante portant une protéine cible et entrant de manière autonome dans une cellule eucaryote, vecteur d'expression recombinant, bactérie recombinante et utilisation WO2023197365A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119751643A (zh) * 2025-03-07 2025-04-04 中国农业大学 一种拉合尔钝缘蜱的唾液腺载脂蛋白、基因、载体、重组菌及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116120453B (zh) * 2023-01-03 2023-09-26 华南理工大学 一种基于蛋白多聚体的间充质干细胞工程化改造方法及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030229202A1 (en) * 2000-08-25 2003-12-11 Yong Guo Membrane penetrating peptides and uses thereof
US20050014263A1 (en) * 1998-05-29 2005-01-20 Moyer Richard W. Materials and methods for delivery and expression of heterologous DNA in vertebrate cells
US20090054828A1 (en) * 2007-08-22 2009-02-26 Cardiac Pacemakers, Inc. Systems for transient conduction control
US20090130034A1 (en) * 2005-09-27 2009-05-21 Emory University Re-engineered uv damage endonuclease, compositions and methods
US20200283745A1 (en) * 2017-11-14 2020-09-10 Oregon Health & Science University Nuclear-targeted dna repair enzymes and methods of use
CN111936624A (zh) * 2017-11-30 2020-11-13 阿米库斯治疗学公司 Cdkl5表达变体和cdkl5融合蛋白
CN113454103A (zh) * 2018-12-14 2021-09-28 阿斯特里昂治疗学有限公司 紫外线诱导dna损伤的修复

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3526235A4 (fr) * 2016-10-12 2021-03-10 Feldan Bio Inc. Agents navette peptidiques synthétiques conçus de manière rationnelle pour administrer des cargos polypeptidiques d'un espace extracellulaire au cytosol et/ou au noyau d'une cellule eucaryote cible, leurs utilisations, méthodes et kits associés
WO2020240248A1 (fr) * 2019-05-25 2020-12-03 Helalat Seyed Hossein Endonucléases de réparation des dommages d'ultraviolets
CN113406331A (zh) * 2020-03-16 2021-09-17 上海交通大学 基于绿色荧光蛋白的真菌免疫调节蛋白细胞定位方法
CN112143752B (zh) * 2020-10-10 2021-12-14 华中农业大学 真菌细胞核荧光标记的表达载体、试剂、制备方法及应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050014263A1 (en) * 1998-05-29 2005-01-20 Moyer Richard W. Materials and methods for delivery and expression of heterologous DNA in vertebrate cells
US20030229202A1 (en) * 2000-08-25 2003-12-11 Yong Guo Membrane penetrating peptides and uses thereof
US20090130034A1 (en) * 2005-09-27 2009-05-21 Emory University Re-engineered uv damage endonuclease, compositions and methods
US20090054828A1 (en) * 2007-08-22 2009-02-26 Cardiac Pacemakers, Inc. Systems for transient conduction control
US20200283745A1 (en) * 2017-11-14 2020-09-10 Oregon Health & Science University Nuclear-targeted dna repair enzymes and methods of use
CN111936624A (zh) * 2017-11-30 2020-11-13 阿米库斯治疗学公司 Cdkl5表达变体和cdkl5融合蛋白
CN113454103A (zh) * 2018-12-14 2021-09-28 阿斯特里昂治疗学有限公司 紫外线诱导dna损伤的修复

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119751643A (zh) * 2025-03-07 2025-04-04 中国农业大学 一种拉合尔钝缘蜱的唾液腺载脂蛋白、基因、载体、重组菌及其应用

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