Zinc Finger Protein-Superoxide Dismutase Fusion Protein With Cell Membrane Penetrating Property

Abstract

The present disclosure belongs to the field of biotechnology, and particularly relates to a zinc finger protein-superoxide dismutase fusion protein with a cell membrane penetrating property, and a preparation and an application thereof. A domain of the fusion protein described in the present disclosure comprises a zinc finger protein and superoxide dismutase, and the fusion protein can enter cells to exert an antioxidant activity.

Claims

1. A fusion protein, wherein a domain of the fusion protein comprises a zinc finger protein and superoxide dismutase.

2. The fusion protein as defined in claim 1, wherein the zinc finger protein and superoxide dismutase are connected by a linker peptide, preferably, the domain of the fusion protein, from the N-terminus to the C-terminus, successively comprises the zinc finger protein, linker peptide, and superoxide dismutase.

3. The fusion protein as defined in claim 1, wherein the fusion protein further comprises a tag.

4. The fusion protein as defined in claim 1, wherein the fusion protein further comprises any one or two of the following features: (1) the amino acid sequence of the zinc finger protein is as shown in SEQ ID NO. 9; and (2) the amino acid sequence of the superoxide dismutase is as shown in SEQ ID NO. 11.

5. The fusion protein as defined in claim 1, wherein the amino acid sequence of the domain of the fusion protein is as shown in SEQ ID NO. 12.

6. An isolated polynucleotide encoding the fusion protein as defined in claim 1.

7. A recombinant expression vector comprising the isolated polynucleotide as defined in claim 6.

8. A host cell comprising the recombinant expression vector as defined in claim 7 or an exogenous, integrated in the genome of the host cell, isolated polynucleotide as defined in claim 6.

9. A method for preparing the fusion protein as defined in claim 1, comprising the following steps: (1) constructing a recombinant expression vector comprising the polynucleotide encoding the fusion protein, then transforming the recombinant expression vector into a host cell to induce expression, and isolating and obtaining the fusion protein from an expression product; or (2) culturing the host cell as defined in claim 8 under suitable conditions, such that the host cell expresses the fusion protein, and then isolating and purifying same to obtain the fusion protein.

10. Use of the fusion protein as defined in claim 1 in preparing antioxidant-related products, preferably, the antioxidant products are selected from cerebral ischemia therapeutic products, cancer therapeutic products, AIDS therapeutic products, amyotrophic lateral sclerosis therapeutic products, anti-inflammatory response products, Parkinson syndrome therapeutic products, and facial cosmetic products.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1: ZFP-SOD1 protein purification. (a) Schematic presentation of ZFP-hSOD1 construct. (b) SDS-PAGE of purified ZFP-hSOD1 protein. Arrow indicates the target protein band.

[0046] FIG. 2: Internalized SOD1 protein in Hela cells, as determined by SOD activity. n.s., not significant. *, P<0.05.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0047] The inventors of the present application identified the Cys.sub.2-His.sub.2 zinc-finger proteins (ZFPs) as a novel protein delivery system. ZFPs are inherently cell-permeable due to the constellation of six positively charged residues on the protein surface. We eliminated the DNA binding ability of ZFPs by mutating the residues responsible for DNA-binding in the a-helices. The engineered zinc-finger proteins (ZFPs) retained cell-permeability and can be used as a fusion tag to deliver cargo proteins. The cellular uptake efficacy is tunable by adjusting the number of tandem ZFP domains, which increases the plasticity for different applications. ZFP domains can mediate the efficient intracellular delivery of protein cargos such as green fluorescent protein (GFP) and Fok I nuclease. In addition to transformed cell lines, ZFPs can facilitate the delivery of cargo proteins into primary cells and stem cells, which is important for the therapeutic applications.

[0048] Superoxide dismutase (SOD) family include a group of well-studied antioxidant enzymes, i.e. SOD1, SOD2 and SOD3. SODs play a fundamental role in attenuating oxidative stress from cellular reactive oxygen species (ROS). The disorder of ROS can contribute to the occurrence and progression of a variety of diseases. Preclinical and clinical studies have shown great therapeutic potential of SODs. SODs have been employed for a wide range of medical indications such as ischemia reperfusion injury, transplant induced reperfusion injury, inflammation, Parkinson's disease, cancer and acquired immune deficiency syndrome (AIDS).

[0049] The present application provides the construction, expression, purification and cell activity assays of ZFP-SOD1 fusion protein for the first time, and proves that the ZFP-SOD1 can penetrate cell membranes and exert SOD1 antioxidant activity inside cells.

[0050] Before further describing the specific embodiments of the present disclosure, it is to be understood that the scope of protection of the present disclosure is not limited to the following specific particular embodiments; it is also to be understood that the terms used in the embodiments of the present disclosure are used for describing the specific particular embodiments, rather than limiting the scope of protection of the present disclosure. In the following embodiments, experimental methods without specifying specific conditions are generally carried out according to conventional conditions or conditions suggested by each manufacturer.

[0051] When numerical ranges are given in the embodiments, it is to be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected, unless specified otherwise in the present disclosure. Unless defined otherwise, all technical and scientific terms used in the present disclosure have the same meanings commonly understood by those of skill in the art. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge in the prior art and the description of the present disclosure, those of skill in the art can also use any prior art methods, devices, and materials which are similar or equal to the methods, devices, and materials described in the embodiments of the present disclosure to realize the present disclosure.

[0052] Unless specified otherwise, the experimental methods, detection methods, and preparation methods disclosed in the present disclosure all use conventional molecular biological, biochemical, chromatin structure and analysis, analytical chemical, cell culture, and recombinant DNA technology in the art, and other conventional technology in related fields. The technology has been completely described in existing documents. For details, reference can be made to: Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P.B. Becker, ed) Humana Press, Totowa, 1999, etc.

EXAMPLE 1

[0053] I. Materials

[0054] 1. Construction of Expression Plasmids

[0055] 1) pET28a plasmid encoding human SOD1 gene optimized for Escherichia coli expression (available from commercial gene synthesis service suppliers).

[0056] 2) ZFP containing plasmid.

[0057] 3) DNA polymerase.

[0058] 4) Deoxynucleotide mixture, including dATP, dCTP, dGTP and dTTP.

[0059] 5) PCR reaction buffers.

[0060] 6) Sterile water.

[0061] 7) DNA staining reagents.

[0062] 8) Homologous recombination enzymes.

[0063] 9) DH5α E. coli competent cells.

[0064] 10) Lysogeny broth (LB) medium.

[0065] 11) Agar, bacteriological grade.

[0066] 12) Plasmid DNA extraction kit.

[0067] 13) Gradient thermal cycler for PCR.

[0068] 14) Agarose gel electrophoresis reagents and equipment.

[0069] 15) UV trans-illuminator.

[0070] 16) Centrifuge.

[0071] 17) Water bath.

[0072] 2. Protein Expression and Purification

[0073] 1) Plasmids encoding recombinant ZFP-SOD1 proteins.

[0074] 2) BL21(DE3) competent E. coli cells.

[0075] 3) Agar, bacteriological grade.

[0076] 4) 50 mg/mL Kanamycin stock solution

[0077] 5) 1 M IPTG (isopropil-β-D-1-tiogalattopiranoside).

[0078] 6) Ni-NTA Agarose.

[0079] 7) 1M Tris-HCl pH 8.0.

[0080] 8) 4 M Imidazole stock solution.

[0081] 9) 100 mM ZnCl.sub.2 stock solution.

[0082] 10) 100 mM MgCl.sub.2 stock solution.

[0083] 11) Phenylmethylsulfonyl fuoride (PMSF) (100 mM in ethanol).

[0084] 12) Lysis buffer: 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 100 □M ZnCl2, 1 mM MgCl2, 1 mM PMSF and 5 mM imidazole.

[0085] 13) Wash buffer: 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 100 □M ZnCl2, 1 mM MgCl2 and 30 mM imidazole.

[0086] 14) Elution buffer: 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 100 □M ZnCl2, 1 mM MgCl2 and 300 mM imidazole.

[0087] 15) Storage buffer: 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 100 □M ZnCl2, 1 mM MgCl2 and 10% glycerol.

[0088] 16) Baffled cell culture flasks.

[0089] 17) Concentrator.

[0090] 18) 4-20% Tris-Glycine SDS-PAGE.

[0091] 19) SDS protein loading dye.

[0092] 20) BCA protein assay kit.

[0093] 21) Liquid nitrogen.

[0094] 3. Protein Transduction

[0095] 1) Purified ZFP-SOD1 proteins.

[0096] 2) Class II biosafety cabinet.

[0097] 3) Cell incubator.

[0098] 4) Bright field phase contrast microscope.

[0099] 5) Dulbecco's modified eagle's medium (DMEM).

[0100] 6) Fetal bovine serum (FBS).

[0101] 7) Penicillin and streptomycin solution.

[0102] 8) Phosphate-buffered saline (PBS).

[0103] 9) 9 mM ZnCl2.

[0104] 10) Triton X-100

[0105] 11) 0.05% trypsin-EDTA solution with phenol red.

[0106] 12) Hela cells.

[0107] 13) Tissue culture flasks.

[0108] 14) 24-well flat bottom tissue culture plates.

[0109] 15) Centrifuge.

[0110] 16) SOD assay kit.

[0111] II. Methods

[0112] 1. Construction of ZFP-SOD1 (zinc finger protein-superoxide dismutase fusion protein) Expression Plasmids

[0113] 1) Miniprep ZFP-encoding plasmid (pET28-ZiF1-EmGFP) and synthesize human SOD genes (superoxide dismutase genes) in pET28a vector (pET28a-hSOD1) using a commercial gene synthesis service.

[0114] 2) PCR amplify ZFP genes (zinc finger protein genes)

[0115] from the plasmid pET-1F-ZiF with the primers

TABLE-US-00009 ZFP-Fwd (5′-GCCTGGTGCCGCGCGGCAGCCCG AAAAAGAAACGCAAAGTGC-3′) and ZFP-SOD1-Rev (5′-GCTTTGGTGGCCATGGATCCACC GGTATGTGTTCTTTGATGG-3′).

[0116] 3) Prepare PCR mixture to amplify the genes encoding ZFP domain: use 5 ng of template DNA, 5 μL of 10× polymerase buffer, 1 Units (U) of Taq DNA polymerase, 0.2 mM dNTP mixture and 0.2 μM of each primer in a 50 μL solution. PCR conditions are cycled using the following settings: 95° C. for 5 min, 30 cycles of 95° C. for 30 sec, 58° C. for 30 sec and 72° C. for 1 min and final extension at 72° C. for 10 min. Purify the PCR product by gel extraction and determine DNA concentration using a spectrophotometer measuring Abs260×50 ng/μL.

[0117] 4) Digest the 1 μg of pET28a-hSOD1 plasmid with 10 U of each Ndel and BamHI in recommended buffer for 3 h at 37° C. Visualize DNA by agarose gel electrophoresis using a DNA staining dye, such as gel red.

[0118] 5) Purify the digested plasmid by gel extraction kit and determine DNA concentration by a spectrophotometer measuring Abs260×50 ng/μL.

[0119] 6) Perform homologous recombination reaction for constructing ZFP-SOD1 fusion protein as follows: 0.06 pmol ZFP PCR product, 0.03 pmol linearized pET28a-hSOD1 plasmid DNA, 2 μL recombination enzyme such as Exnase II, 4 μL 5× recombination buffer and deionized water up to 20 μL. Incubate the mixture at 37° C. for 30 min.

[0120] 7) Thaw 200 μL of chemically competent DH5α E.coli cells on ice, mix gently with 20 μL of recombination products and then incubate on ice for 30 min.

[0121] 8) Heat shock the mixture at 42° C. for 45-90 s and recover the cells in 900 μL LB medium for 1 h at 37° C. with shaking.

[0122] 9) Spread 100 μL of recovery culture on a LB agar plate supplemented with 50.Math.g/mL kanamycin and incubate overnight at 37° C.

[0123] 10) The following day, inoculate a single colony into 5 mL of LB culture containing 50 μg/mL kanamycin and culture overnight at 37° C.

[0124] 11) Miniprep pET28a-ZFP-SOD1 plasmid and confirm the construct (schematic presentation of ZFP-hSOD1 construct is shown in FIG. 1) by DNA sequencing using a primer binding to the T7 promoter (5′-TAATAC GAC T CAC TATAGGG-3′).

[0125] As can be seen from the results: the domain of the ZFP-SOD1 protein (zinc finger protein-superoxide dismutase fusion protein), from the N-terminus to the C-terminus, successively comprises the zinc finger protein, linker peptide, and superoxide dismutase, and full-length genes of the domain of the fusion protein all have correct sequences, and are all in line with expectations.

[0126] That is, in the domain of the fusion protein, the nucleotide sequence encoding the zinc finger protein is as shown in SEQ ID NO. 3, specifically is: gaaaaaccatacaaatgcccagaatgeggaaaatcttttagtgcctcagctgccctcgtcgcccatcaaagaacacatacc. The nucleotide sequence encoding the linker peptide is as shown in SEQ ID NO. 4, specifically is: ggtggatcc. The nucleotide sequence encoding the superoxide dismutase is as shown in SEQ ID NO. 5, specifically is:

TABLE-US-00010 atggccaccaaagcggtctgcgttttaaaagggga tggcccggtgcaaggcattattaatttcgaacaaa aagagagcaatggtccggttaaagtgtggggtagt atcaaaggcctgaccgagggtctgcatggctttca tgtgcatgaatttggcgataacaccgctggttgca cgtcagccggcccgcactttaatcctctgtcccgt aagcacggcggcccgaaggatgaggagcgtcacgt cggcgatctgggtaatgttactgccgataaggatg gggtggccgatgtttccattgaagattctgtcatc tcattgagtggggaccactgtatcattgggcgtac cttagtggtccatgaaaaggcagacgacctgggta agggcggaaatgaagaatccaccaaaacgggcaat gctggttcacgtttagcgtgtggtgtgattggtat cgcccaa.

[0127] The nucleotide sequence encoding the domain of the ZFP-SOD1 protein (zinc finger protein-superoxide dismutase fusion protein) is as shown in SEQ ID NO. 6, specifically is:

TABLE-US-00011 gaaaaaccatacaaatgcccagaatgcggaaaatc ttttagtgcctcagctgccctcgtcgcccatcaaa gaacacataccggtggatccatggccaccaaagcg gtctgcgttttaaaaggggatggcccggtgcaagg cattattaatttcgaacaaaaagagagcaatggtc cggttaaagtgtggggtagtatcaaaggcctgacc gagggtctgcatggctttcatgtgcatgaatttgg cgataacaccgctggttgcacgtcagccggcccgc actttaatcctctgtcccgtaagcacggcggcccg aaggatgaggagcgtcacgtcggcgatctgggtaa tgttactgccgataaggatggggtggccgatgttt ccattgaagattctgtcatctcattgagtggggac cactgtatcattgggcgtaccttagtggtccatga aaaggcagacgacctgggtaagggcggaaatgaag aatccaccaaaacgggcaatgctggttcacgttta gcgtgtggtgtgattggtatcgcccaa.

[0128] In order to purify the protein conveniently, an His tag is added to the N-terminus of the domain of the ZFP-SOD1 protein (zinc finger protein-superoxide dismutase fusion protein).

[0129] The nucleotide sequence encoding the His tag is as shown in SEQ ID NO. 7, specifically is: catcatcatcatcatcac.

[0130] The nucleotide sequence encoding the ZFP-SOD1 protein with the His tag is as shown in SEQ ID NO. 8, specifically is:

TABLE-US-00012 atgggcagcagccatcatcatcatcatcacagcag cggcctggtgccgcgcggcagcccgaaaaagaaac gcaaagtgctcgagcccggggaaaaaccatacaaa tgcccagaatgcggaaaatcttttagtgcctcagc tgccctcgtcgcccatcaaagaacacataccggtg gatccatggccaccaaagcggtctgcgttttaaaa ggggatggcccggtgcaaggcattattaatttcga acaaaaagagagcaatggtccggttaaagtgtggg gtagtatcaaaggcctgaccgagggtctgcatggc tttcatgtgcatgaatttggcgataacaccgctgg ttgcacgtcagccggcccgcactttaatcctctgt cccgtaagcacggcggcccgaaggatgaggagcgt cacgtcggcgatctgggtaatgttactgccgataa ggatggggtggccgatgtttccattgaagattctg tcatctcattgagtggggaccactgtatcattggg cgtaccttagtggtccatgaaaaggcagacgacct gggtaagggcggaaatgaagaatccaccaaaacgg gcaatgctggttcacgtttagcgtgtggtgtgatt ggtatcgcccaa.

[0131] 2. Expression and purification of ZFP-SOD1 protein (zinc finger protein-superoxide dismutase fusion protein)

[0132] 1) Thaw 50 μL of chemically competent BL21 E. coli cells on ice and mix gently with 100 ng of pET28a-ZFP-SOD1 plasmid. Perform heat shock transformation as described in steps 7)-9) of part II.1.

[0133] 2) The following day, inoculate a single colony into 10 mL of LB medium containing 50 μg/mL kanamycin and grow overnight at 37° C. with shaking.

[0134] 3) The following day, dilute the 10 mL of overnight culture into 1 L of LB medium supplemented with 50 μg/mL kanamycin. Grow the culture at 37° C. with shaking to an optical density at 600 nm (OD600) of 0.6-0.8 and induce protein expression with 0.5 mM IPTG. After 6 h of expression at 37° C., harvest cells by centrifugation at 5,000×g for 20 min at 4° C.

[0135] 4) Resuspend 1 g cell pellets in 5 mL of lysis buffer. Lyse the cells on ice with cell distributor or sonication.

[0136] 5) Centrifuge the cell lysate at 40,000×g for 30 min at 4° C. and transfer the supernatant into a fresh collection tube. For optimum results, perform all the following steps at 4° C.

[0137] 6) Flow the supernatant through a column pre-packed with 1 mL of equilibrated Ni-NTA agarose. Wash the resin with 20 mL of wash buffer.

[0138] 7) Elute the protein with 5 mL of elution buffer.

[0139] 8) Buffer exchange the eluted protein with storage buffer and concentrate the protein to at least 40 μM using a spin concentrator following manufacturer's instructions.

[0140] 9) Determine protein concentration by BCA or Bradford assay.

[0141] 10) Mix 2 μL of purified proteins with 2 μL 2×SDS-PAGE loading dye, boil at 95° C. for 10 min and then resolve on 4-20% Tris-Glycine SDS-PAGE to assess protein purity. The results are as shown in (b) of FIG. 1, and show that: the ZFP-SOD1 protein (zinc finger protein-superoxide dismutase fusion protein) can be successfully recombined and expressed in vitro, and a fusion protein with a relatively high purity can be obtained.

[0142] By means of N/C-terminal sequence analysis, the results show that the domain of the ZFP-SOD1 protein (zinc finger protein-superoxide dismutase fusion protein), from the N-terminus to the C-terminus, successively comprises the zinc finger protein, linker peptide, and superoxide dismutase, and the frames of the expressed fusion protein are all read correctly, and are consistent with the theoretical N/C-terminal amino acid sequences.

[0143] That is: in the domain of the fusion protein, the amino acid sequence of the zinc finger protein is as shown in SEQ ID NO. 9, specifically is EKPYKCPECGKSFSASAALVAHQRTHT. The amino acid sequence of the linker peptide is as shown in SEQ ID NO. 10, specifically is: GGS. The amino acid sequence of the superoxide dismutase is as shown in SEQ ID NO. 11, specifically is:

TABLE-US-00013 MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGS IKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR KHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVI SLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGN AGSRLACGVIGIAQ.
The amino acid sequence of the domain of the ZFP-SOD1 protein (zinc finger protein-superoxide dismutase fusion protein) is as shown in SEQ ID NO. 12, specifically is:

TABLE-US-00014 EKPYKCPECGKSFSASAALVAHQRTHTGGSMATKA VCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLT EGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGP KDEERHVGDLGNVTADKDGVADVSIEDSVISLSGD HCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRL ACGVIGIAQ.

[0144] In order to purify the protein conveniently, a His tag is added to the N-terminus of the domain of the ZFP-SOD1 protein (zinc finger protein-superoxide dismutase fusion protein).

[0145] The amino acid sequence of the His tag is as shown in SEQ ID NO. 13, specifically is: HEIREIHH. The amino acid sequence of the ZFP-SOD1 protein with the His tag is as shown in SEQ ID NO. 14, specifically is:

TABLE-US-00015 MGSSHHHHHHSSGLVPRGSPKKKRKVLEPGEKPY KCPECGKSFSASAALVAHQRTHTGGSMATKAVCVL KGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLH GFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEE RHVGDLGNVTADKDGVADVSIEDSVISLSGDHCII GRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGV IGIAQ.

[0146] 11. The protein is concentrated, rapidly frozen in liquid nitrogen, and stored at −80° C. .

[0147] 3. ZFP-SOD1 Protein (zinc finger protein-superoxide dismutase fusion protein) transduction

[0148] 1) Maintain HeLa cells in DMEM medium supplemented with 10% (v/v) FBS, 100 U/mL penicillin and 100 U/mL streptomycin at 37° C. in a fully humidified atmosphere with 5% CO.sub.2.

[0149] 2) Pre-coat a 24-well plate with 500 μL of 50 μg,/mL of poly-lysine for 30 to 60 min at 25° C. Seed Hela cells onto pre-coated plates at a density of 2×10.sup.5 cells per well.

[0150] 3) At 24 h after seeding, remove medium from each well and wash with 500 μL, of pre-warmed serum-free DMEM.

[0151] 4) Add to each well 250 μL, of SFM containing 2 μM of ZFP-SOD1 proteins and 100 μM ZnCl.sub.2. Incubate at 37° C. for 1 h.

[0152] 5) Remove media from cells and wash three times with 500 μL of calcium- and magnesium-free PBS supplemented with 0.5 mg/mL of heparin.

[0153] 6) Rinse cells with 0.05% trypsin-EDTA, remove trypsin solution and then incubate at 37° C. for 2 min.

[0154] 7) Lyse cells using 250 μL of PBS containing 0.1% (v/v) Triton X-100.

[0155] 8) Use SOD assay kit such as SOD Determination Kit (Sigma-Aldrich, St. Louis, Mo., USA) to determine internalized SOD proteins. The results are as shown in FIG. 2, and show that the ZFP-SOD1 protein (zinc finger protein-superoxide dismutase fusion protein) can enter cells to exert an antioxidant activity. The ZFP-SOD1 protein can penetrate the cell membrane and enter the cell, thereby improving the antioxidant activity of the cell itself (improved from 30 to 40, and improved by about ⅓).

COMPARATIVE EXAMPLE

[0156] The document of Kwon et al. (reference: Transduction of Cu, Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into mammalian cells) demonstrates that the cell presentation of SOD mediated by a cell membrane penetrating peptide Tat can only act on denatured SOD, but not act on SOD under physiological conditions. However, our results demonstrate that the zinc finger protein can present SOD under physiological conditions to a cell.

[0157] The above description merely relates to preferred embodiments of the present disclosure, but is not intended to limit the present disclosure in any formal and substantial ways. It should be noted that for those of skill in the art, without departing from the method of the present disclosure, several improvements and supplements can be further made, and these improvements and supplements also should fall within the scope of protection of the present disclosure. For those of skill in the art, without departing from the spirit and scope of the present disclosure, the equivalent changes of a little alterations, modifications and evolutions made according to the technical content revealed above are all equivalent embodiments of the present disclosure; meanwhile, any equivalent changes of alterations, modifications and evolutions on the above-mentioned embodiments according to the substantial technology of the present disclosure all still fall within the scope of the technical solution of the present disclosure.