WO2023197365A1 - Recombinant protein carrying target protein and autonomously entering eukaryotic cell, recombinant expression vector, recombinant bacterium, and use - Google Patents

Abstract

The present invention relates to the technical field of fusion proteins. Provided are a recombinant protein carrying a target protein and autonomously entering a eukaryotic cell, a recombinant expression vector, a recombinant bacterium, and the use. The recombinant protein sequentially comprises, from the N-terminus to the C-terminus, a nuclear localization signal (NLS) that carries the recombinant protein into the nucleus, a target protein and short peptide AE1 having an ability of spanning the cell membrane, wherein AE1 nontoxically and efficiently guides the recombinant protein to span the cell membrane and enter a cell, and the target protein is carried into the nucleus by means of the NLS, thereby playing a role. Also provided is a recombinant photolyase capable of autonomously entering a human nucleus to repair DNA damage induced by UV radiation. The CPD photolyase used as a target protein has the effects of repairing a cyclobutane pyrimidine dimer (CPD) on the nuclear DNA, improving the activity of intracellular superoxide dismutase (SOD), and reducing the levels of intracellular reactive oxygen species (ROS) and a highly toxic molecule malondialdehyde (MDA).

Description

A recombinant protein, recombinant expression vector and recombinant bacteria that carry target proteins into eukaryotic cells autonomously and their applications

This application is required to be submitted to the China Patent Office on April 13, 2022. The application number is 202210384441.0 and the invention is titled "A recombinant protein, recombinant expression vector and recombinant bacteria that can carry a target protein autonomously into eukaryotic cells and their application" claim of priority, the entire contents of which are incorporated herein by reference.

Technical field

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.

Background technique

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. Studies have shown that skin cancer is the main pathological manifestation of overexposure to ultraviolet radiation. 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] .

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] . About 70 to 80% of the DNA damage induced by ultraviolet rays is CPDs, and the rest is 6-4PPs and Dewar isomers of 6-4PPs. Although 6-4PPs are significantly mutagenic in E. coli , mammalian cells repair 6-4PPs much faster than CPDs. Therefore, in mammalian cells, UV-induced CPDs cause significantly more mutations than 6-4PPs ^[5] . In addition to 6-4PPs and CPDs, 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] . In addition, 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). Lipids can be detected by detecting the level of MDA. The level of plasma oxidation indirectly reflects the degree of cell damage. In addition, 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] . There are two different types of photolyase in nature, namely CPD photolyase that repairs CPD and 6-4PP photolyase that repairs 6-4PPs. Although these two types of photolyases are similar in structure, they have strong substrate specificity ^[2] . However, human cells are not born with photolytic enzymes. For 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. Compared with the direct repair pathway of photolytic enzymes in other species, the efficiency of human cells in repairing CPD through NER is low, and the efficiency of NER-based CPD repair will further decrease with age, leading to the gradual accumulation of DNA damage and triggering a series of A variety of lesions, even cancer ^[7] .

Currently, people use a variety of physical methods to protect against UV radiation, including using protective equipment such as parasols and sun-protective clothing, applying sunscreen with appropriate sun protection factor (SPF) and protective spectrum, and adding UV absorbers to laundry detergent. Improve the UV protection factor of clothing, etc. In addition, studies have shown that adding liposomes containing DNA repair enzymes including the above-mentioned photolytic enzymes to sunscreen can transfer the repair enzymes into cells to repair UV-induced DNA damage ^{[2 ]} . Although cosmetics based on liposome technology have been widely used, they have problems such as high preparation technology requirements, poor stability of liposomes, and difficulty in compatibility with other raw materials in cosmetics. They are prone to precipitation, phospholipids are prone to oxidation and discoloration, and are prone to specificity. Undesirable phenomena such as odor.

references

[1]Jean Cadet, Thierry Douki, Jean-Luc Ravanat.Oxidatively Generated Damage to Cellular DNA by UVB and UVA Radiation[J].Photochemistry and Photobiology, 2015,91:140-155.

[2]Marie-Therese Leccia,Celeste Lebbe,Jean-Paul,et al.New Vision in Photoprotection and Photorepair[J].Dermatol Ther(Heidelb),2019,9:103-115.

[3] Cadet J, Sage E, Douki T. Ultraviolet radiation-mediated damage to cellular DNA. MutatRes.2005,571:3-17.

[4]George A Garinis, Judith Jans, et al.Photolyases: capturing the light to battle skin cancer[J]. Future Oncol, 2006, 2(2):191-199.

[5]Silvia Tornaletti,Gerd P.Pfeifer.UV damage and repair mechanisms in mammalian cells[J].BioEssays,1996,18(3):221-228.

[6]Navarrete-Dechent C,Molgo M.The use of a sunscreen containing DNA-photolyase in the treatment of patients with field cancerization and multiple actinic keratoses:a case-series[J].Dermatol Online.2017:15-23.

[7] de Laat WL, Jaspers NGJ, Hoeijmakers JHJ. Molecular mechanism of nucleotide excision repair. GenesDev.1999,13(7):768-85.

[8] Brem, R., M. Guven, P. Karran. Oxidatively-generated damage to DNA and proteins mediated by photosensitized UVA. Free Radical Biology and Medicine. 2017,107:101-109.

[9] Daniele Del Rio, Amanda J. Stewart, Nicoletta Pellegrini. A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutrition, Metabolism & Cardiovascular Diseases. 2005, 15: 316-328.

Contents of the invention

In view of this, 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. When the target protein is a recombinant photolyase, 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.

In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

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.

Preferably, 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. The structure of the recombinant photolyase, from the N-terminus to the C-terminus, sequentially includes: the nuclear localization derived from the SV40 virus signal, CPD photolyase and the short peptide AE1 from Drosophila melanogaster.

Preferably, the 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;

The amino acid sequence of the short peptide AE1 is shown in SEQ ID NO. 2.

Preferably, 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.

Preferably, 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.

Preferably, 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.

Beneficial effects: 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. Among the recombinant proteins provided by the present invention, 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.

Description of the drawings

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 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 μm;

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, 5: SVNLS::MrPHR1: after adding ULP1 enzyme to remove SUMO: :AE1 protein liquid;

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 6, 8: purified SUMO::MrPHR1::AE1 protein solution, 9: MrPHR1:: after adding ULP1 enzyme to remove SUMO. AE1 protein liquid;

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 UV radiation and then visible light irradiation;

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;

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;

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.

Detailed ways

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. Using the recombinant protein with the structure of the present invention, the target protein can be delivered into the cells and nuclei of animals. In the examples of the present invention, 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. In addition to the source of Metarhizium anisopliae in the examples, 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. .

In the present invention, the DNA damage preferably includes DNA damage of cyclobutane pyrimidine dimer (CPD). 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.

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 When increasing, 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. From the 5' end to the 3' end, 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. In the embodiment of the present invention, 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. In the embodiment, 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.

In the present invention, the concentration of kanamycin in the LB liquid culture medium is preferably 100 μg/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.

In the embodiment of the present invention, it is preferred to inoculate 100 μL of seed liquid into 1000 mL of kanamycin-containing LB liquid medium and culture it again. When the OD ₆₀₀ value reaches 0.6, 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.

After obtaining the crude protein extract, 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. On the nickel column; 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. In embodiments of the present invention, the recombinant photolyase can be used to successfully enter the nucleus and repair CPD damage caused by ultraviolet radiation.

The 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.

Example 1

Analysis of the ability of SVNLS and AE1 to bring foreign protein GFP into the nucleus

1) Construction and prokaryotic expression of SVNLS::GFP::AE1 protein expression vector.

When cloning the green fluorescent protein GFP coding sequence using PCR, 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. 6): CGGAATTCTTATCATTTTTTCCATTTCATGCGGCGGTTCTGAAACCAAATTTTAATCTGGCGGCCCTTGTACAGCTCGTCCA. Using the DNA fragment containing the GFP coding sequence as the template, the PCR amplification system (total volume 50 μL) is: ddH ₂ O 15 μL, 2× Phanta Max buffer (Vazyme) 25 μL, dNTP Mix (10 μM each, Vazyme) 1 μL, SVNLS-GFP -52μL (10μM), AE1-GFP-32μL (10μM), Phanta Max Super-Fidelity DNApolymerase (Vazyme) 1μL, DNA fragment template (GFP) 2μL. 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 μL 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.

2) Specific process of protein purification:

① After inducing the expression of SVNLS::GFP::AE1 according to the above method, centrifuge at 4°C and 4500rpm for 20 minutes to collect the cells. Use pH 7.0 lysis buffer to resuspend the bacterial cells and sonicate for 25 minutes (70kHz). After the crushing is completed, centrifuge at 4°C and 12,000 rpm for 60 min. Collect the broken fluid, supernatant and precipitate separately for SDS-PAGE analysis to detect whether the target protein is expressed in large amounts.

② Pass the supernatant, that is, the crude protein extract, through the nickel column. The target protein labeled with a SUMO tag of 6 histidines is adsorbed on the nickel column. Use a wash buffer with pH 7.0 to wash away the impurity proteins on the nickel column, and then use Elute the target protein with pH7.0 elute buffer. The eluted target protein was treated with 5mg ULP1 protease to remove the SUMO tag with 6 histidines. After overnight digestion, the protein was centrifuged using an Amino Ultra-15 (10kDa) ultrafiltration tube to concentrate the protein to 15mL. Pass 15 mL of the concentrated solution through the nickel column again to remove ULP1 and sumo tags. Use Amino Ultra-15 (10kDa) ultrafiltration tube to centrifuge and concentrate, and use pH 7.0 desalting buffer to remove imidazole. Finally, 50 mg of protein (volume of about 5 mL) was obtained, 10% glycerol was added, sterilized by filtration with a 0.2 μm filter, and stored at -80°C after aliquots.

3) Analysis of the ability of SVNLS::GFP::AE1 to enter HFF-1 cells

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 μL 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/μL) 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.

①Verification of SVNLS::GFP::AE1 entering cells

After the above-mentioned 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.

②Verification of SVNLS::GFP::AE1 entering the cell nucleus

After the above-mentioned HFF-1 cells were incubated with SVNLS::GFP::AE1 for 12 hours, the cell culture medium was poured out, and 1mL of 2μg/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.

Example 2

Expression and preparation of recombinant protein SVNLS::MrPHR1::AE1

In order to use SVNLS and AE1 short peptides to bring the CPD photolyase of Metarhizium anisopliae (MAA_05216, named MrPHR1) into mammalian cell nuclei and repair CPD damage on nuclear DNA, the present invention constructed the fusion protein SVNLS::MrPHR1:: AE1.

To this end, when cloning the MrPHR1 coding sequence (Genbank accession number: , and add a translation initiation codon ATG) in front of SVNLS. 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.

Using the same method, the 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.

Prokaryotic expression experiments were performed using the same method as above. The SDS-PAGE analysis results are shown in Figure 6. MrPHR1::AE1 and SVNLS::MrPHR1 proteins were successfully expressed and purified.

Example 3

Analysis of the ability of SVNLS::MrPHR1::AE1 to repair DNA CPD damage after entering the nucleus

1) Photorepair of UV-treated HFF-1 cells

(1) Cell culture and processing settings

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.

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.

(2) Extract genomic DNA from HFF-1 cells treated with different conditions:

Collect the above cells into Ep tubes, add 200 μL of DNA extraction solution (0.2M pH7.5Tris, 0.5M NaCl, 0.01M pH8.0EDTA, 1% SDS) to each tube, vortex thoroughly, and then add 200 μL of chloroform ( pH 7.0) Vortex thoroughly, centrifuge at 14000 rpm for 8 min at room temperature, transfer the supernatant to a new Ep tube, add 1.5 μL RNase to each tube, and incubate at 37°C for 1 h. Add 200 μL of chloroform (pH 7.0) and vortex thoroughly for extraction once, centrifuge at 14000 rpm for 8 min at room temperature, and transfer the supernatant to a new Ep tube. Add 500 μL of absolute ethanol, vortex thoroughly, place the Ep tube at -20°C for precipitation for 30 minutes, centrifuge at 14,000 rpm for 5 minutes at room temperature, and then remove the supernatant. Add 700 μL of 75% ethanol, vortex thoroughly, centrifuge at 14,000 rpm for 2 minutes at room temperature, remove the supernatant, open the Ep tube and place it in a 37°C oven for 5 minutes to completely evaporate the ethanol in the Ep tube. Add 30 μL of sterile water and vortex thoroughly to dissolve the precipitate. The resulting liquid is HFF-1 cell genomic DNA. Determine DNA concentration using absorbance method.

2) ELISA analysis of CPD content in DNA:

(1) Prepare a protamine-pretreated 96-well plate. Add 50 μL/well of sterile 0.003% (w/v) protamine sulfate solution to each well, dry at 37°C overnight, wash three times with 100 μL of ddH ₂ O, and store in the dark.

(2) CPD content determination. The measurement process was carried out according to the instructions of CPD antibody (Cosmo Bio Co. LTD. Japan). The DNA concentration is 50ng/μL. (Figure 7)

(3) Results. Compared with the negative control, the intracellular CPD content of HFF-1 increased approximately 150 times after UV irradiation. After irradiating the UV-damaged HFF-1 with visible light, it was found that only the SVNLS::MrPHR1::AE1 recombinant protein incubated and The CPD content in cells treated with visible light irradiation decreased by 35%, while the other three recombinant proteins (SVNLS::MrPHR1, MrPHR1::AE1 and SVNLS::GFP::AE1) had no significant effect on the CPD content in cells. This shows that SVNLS::MrPHR1::AE1 can successfully enter the nucleus and repair CPD damage caused by ultraviolet radiation.

Example 4

SVNLS::MrPHR1::AE1 reduces intracellular oxygen free radicals (ROS) levels after entering the cell nucleus 1) Photorepair of UV-irradiated HFF-1 cells

(1) Cell culture and processing settings. (Same as Example 3)

(2) Cell sample processing. The cells were resuspended in a certain amount of PBS to a concentration of 2×10 ⁷ cells/mL. Take 600 μL of 2×10 ⁷ cells/mL cell suspension into a 1.5 mL Ep tube, and sonicate for 3 minutes (70kHZ). After the crushing is completed, centrifuge at 4°C and 12,000 rpm for 10 min. Collect the supernatant and place it on ice for testing.

2) 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).

3) Results. Compared with negative control cells, UV radiation increased ROS levels in HFF-1 cells by 2.3 times. After irradiating the UV-damaged HFF-1 with visible light, it was found that only the cells incubated with the SVNLS::MrPHR1::AE1 recombinant protein had intracellular ROS levels that dropped to a level comparable to that of the negative control cells after visible light irradiation; while the other 2 cells The two recombinant proteins (SVNLS::MrPHR1, MrPHR1::AE1) had no effect on intracellular ROS levels. It shows that SVNLS::MrPHR1::AE1 scavenges ROS induced by UV radiation (Figure 8).

Example 5

SVNLS::MrPHR1::AE1 reduces the level of the intracellular toxic substance malondialdehyde (MDA) after entering the cell nucleus.

1) Photorepair of UV-treated HFF-1 cells

(1) Cell culture and processing settings. (Same as Example 3)

(2) Cell sample processing. Collect 5×10 ⁷ cells into a 1.5mL Ep tube, add 1mL of extraction solution (provided by the kit below), vortex and mix, and sonicate for 3 minutes (70kHZ). After the crushing is completed, centrifuge at 4°C and 12,000 rpm for 10 min. Collect the supernatant and place it on ice for testing.

2) MDA content determination: The determination process was carried out according to the instructions of the malondialdehyde (MDA) content detection kit (Solarbio, China).

3) Results. Compared with negative control cells, UV radiation increased the MDA content in HFF-1 cells by 5.4 times. After irradiating the UV-damaged HFF-1 with visible light, it was found that only the cells incubated with the SVNLS::MrPHR1::AE1 recombinant protein had a 51% decrease in intracellular MDA levels after visible light irradiation. The other two recombinant proteins (SVNLS::MrPHR1, MrPHR1::AE1) had no effect on intracellular MDA levels. This shows that SVNLS::MrPHR1::AE1 clears MDA induced by UV radiation (Figure 9).

Example 6

SVNLS::MrPHR1::AE1 increases intracellular superoxide dismutase SOD activity after entering the cell nucleus.

1) Photorepair of UV-treated HFF-1 cells

(1) Cell culture and processing settings. (Same as Example 3)

(2) Cell sample processing. (Same as Example 5)

2) SOD activity measurement: The measurement process was carried out according to the instructions of the superoxide dismutase (SOD) activity detection kit (Solarbio, China).

3) Results. The SOD enzyme activity in the negative control HFF-1 cells was at a low level, and UV radiation increased the SOD activity by 2.7 times. After irradiating the UV-damaged HFF-1 with visible light, it was found that only the SOD enzyme activity in cells incubated with the SVNLS::MrPHR1::AE1 recombinant protein further increased after visible light irradiation treatment, reaching 4 times that of the negative control. The other two recombinant proteins (SVNLS::MrPHR1, MrPHR1::AE1) have no effect on intracellular SOD enzyme activity (Figure 10), indicating that SVNLS::MrPHR1::AE1 can increase intracellular SOD enzyme activity, which may be the reason for this. One reason why recombinant proteins can reduce ROS levels in such cells.

Although the above embodiments describe the present invention in detail, they are only part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on this embodiment without any inventive step. These embodiments All belong to the protection scope of the present invention.

Claims (17)

A recombinant protein that carries a target protein autonomously into eukaryotic cells, characterized in that 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 order. The recombinant protein according to claim 1, wherein the eukaryotic cells include human cells or animal cells. The recombinant protein according to claim 1, wherein the target protein includes CPD photolyase. A recombinant photolyase that can autonomously enter the human cell nucleus to repair DNA damage induced by UV radiation. It is characterized in that the structure of the recombinant photolyase, from the N-terminus to the C-terminus, sequentially includes: the nuclear localization derived from the SV40 virus Signal NLS, CPD photolyase and short peptide AE1 derived from Drosophila melanogaster. The recombinant photolyase according to claim 4, wherein the amino acid sequence of the nuclear localization signal derived from the SV40 virus is shown in SEQ ID NO.1; The CPD photolytic enzyme includes CPD photolytic enzyme derived from Metarhizium anisopliae; The amino acid sequence of the short peptide AE1 derived from Drosophila melanogaster is shown in SEQ ID NO. 2. The recombinant photolyase according to claim 5, characterized in that the source of the CPD photolyase also includes CPD photolyase obtained from other strains or CPD photolyase after gene editing or modification. The recombinant photolyase according to claim 4, wherein the DNA damage includes DNA damage of cyclobutane pyrimidine dimers. A recombinant expression vector for expressing the recombinant protein according to any one of claims 1 to 3 or the recombinant photolyase according to any one of claims 4 to 7, characterized in that the recombinant expression vector includes the recombinant protein encoding the recombinant protein or the recombinant photolyase described in any one of claims 4 to 7. The recombinant photolyase gene is described. The recombinant expression vector according to claim 8, wherein the basic vector of the recombinant expression vector includes pET-28a-sumo vector. The construction method of the recombinant expression vector according to claim 8 or 9, characterized in that it includes the following steps: amplifying the nuclear localization signal NLS and the short peptide AE1 to the N-terminal and C-terminal of the target protein respectively by PCR method; When performing the PCR method, 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, and the coding sequence of the nuclear localization signal derived from the SV40 virus. and the first 17 bases of the CPD photolyase coding sequence. The construction method according to claim 10, characterized in that the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.3; the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.4. A recombinant bacterium expressing the recombinant protein according to any one of claims 1 to 3 or the recombinant photolytic enzyme according to any one of claims 4 to 7, characterized in that the genome of the recombinant bacterium includes a gene encoding the recombinant protein or The gene of the recombinant photolyase or the recombinant expression vector of claim 8 or 9. The recombinant bacterium according to claim 12, characterized in that the basic strain of the recombinant bacterium includes Escherichia coli. The method for preparing recombinant photolytic enzyme using the recombinant bacterium of claim 12 or 13, characterized in that it includes the following steps: inoculating a single colony of the recombinant bacterium into 5 mL of kanamycin-containing LB liquid culture medium for culturing , to obtain the seed liquid; inoculate the seed liquid into the LB liquid medium containing kanamycin and culture again, use IPTG to induce, and then culture at 18°C for 12 hours, collect the bacterial cells and break them, and centrifuge to collect the crude protein extract. The extract contains the recombinant photolytic enzyme. The method according to claim 14, wherein the concentration of kanamycin in the LB liquid culture medium is 100 μg/mL. Application of the recombinant photolyase according to any one of claims 4 to 7 in the preparation of a drug for treating DNA damage of cyclobutane pyrimidine dimers induced by UV radiation. A method for treating cells with DNA damage of cyclobutane pyrimidine dimers induced by UV radiation using the recombinant photolyase according to any one of claims 4 to 7, characterized in that it includes the following steps: combining the cells with the described Recombinant photolyase was co-incubated for 12 h.

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