COMPLEX FOR INTRACELLULAR DELIVERY OF MOLECULES

Abstract

A fusion protein and a complex containing same, capable of being used for the intracellular delivery of cargo molecules. The fusion protein and complex can implement the efficient release of cargo molecules from endocytic vesicles, thereby significantly improving the cytoplasmic delivery efficiency of the cargo molecules. One cargo molecules can be obtained in cytoplasms, they can exert any function related thereto. The fusion protein and complex provide effective means for affecting biological mechanisms and pathways of cells, and can be used in various fields such as research, treatment, and diagnosis.

Claims

1. A fusion protein, which comprises a cell-penetrating peptide, a pH-sensitive fusogenic peptide and a protease recognition sequence, wherein the protease is selected from furin and/or lysosomal cysteine protease.

2. The fusion protein according to claim 1, wherein the furin recognition sequence comprises R-X.sub.1-X.sub.2-R (SEQ ID NO: 1), wherein X.sub.1 is any amino acid, and X.sub.2 is K or R; preferably, the furin recognition sequence comprises R-R-X.sub.1-X.sub.2-R (SEQ ID NO: 2); preferably, the furin recognition sequence comprises a sequence shown in SEQ ID NO: 3; preferably, the furin recognition sequence comprises a sequence shown in SEQ ID NO: 4.

3. The fusion protein according to claim 1 or 2, wherein the lysosomal cysteine protease is selected from the group consisting of cathepsin B, cathepsin C, cathepsin X, cathepsin S, cathepsin L, cathepsin D or cathepsin H; preferably, the lysosomal cysteine protease is cathepsin L; preferably, the cathepsin L recognition sequence comprises a sequence shown in SEQ ID NO:6.

4. The fusion protein according to any one of claims 1 to 3, wherein the protease recognition sequence comprises a furin recognition sequence and a cathepsin L recognition sequence; preferably, the protease recognition sequence comprises SEQ ID NO: 3 and SEQ ID NO: 6; preferably, the protease recognition sequence comprises SEQ ID NO: 4 and SEQ ID NO: 6.

5. The fusion protein according to any one of claims 1 to 4, wherein the pH-sensitive fusogenic peptide is selected from influenza virus HA2 or its mutant (e.g., INF7, KALA or GALA), melittin, and any combination thereof; preferably, the pH-sensitive fusogenic peptide comprises INF7; preferably, the pH-sensitive fusogenic peptide comprises a sequence shown in SEQ ID NO: 8.

6. The fusion protein according to any one of claims 1 to 5, wherein the cell-penetrating peptide is selected from the group consisting of penetratin, Tat-derived peptide (e.g., Tat(48-60) or Tat(47-57)), Rev(34-50), VP22, transportan, Pep-1, Pep-7, and any combination thereof; preferably, the cell-penetrating peptide comprises a Tat-derived peptide, such as Tat(48-60); preferably, the cell penetrating peptide comprises a sequence shown in SEQ ID NO: 10.

7. The fusion protein according to any one of claims 1 to 6, wherein the fusion protein comprises the pH-sensitive fusogenic peptide, cell penetrating peptide, and protease recognition sequence from N-terminus to C-terminus; or, the fusion protein comprises the cell-penetrating peptide, pH-sensitive fusogenic peptide and protease recognition sequence from N-terminus to C-terminus; preferably, the protease recognition sequence comprises the furin recognition sequence and cathepsin L recognition sequence from N-terminus to C-terminus, or comprises the cathepsin L recognition sequence and furin-recognition sequence from N-terminus to C-terminus.

8. The fusion protein according to any one of claims 1 to 7, wherein the fusion protein comprises a sequence shown in any one of SEQ ID NOs: 12 to 14.

9. The fusion protein according to any one of claims 1 to 8, wherein the fusion protein further comprises a specific binding sequence, the specific binding sequence allows another molecule (e.g., polypeptide, protein, or nucleic acid) to specifically bind thereto; preferably, the specific binding sequence comprises a leucine zipper peptide which is able to form a heterodimer with its complementary peptide; preferably, the specific binding sequence comprises leucine zipper NZ or CZ; preferably, the specific binding sequence comprises a sequence shown in SEQ ID NO: 49 or 50; preferably, the specific binding sequence is located at the C-terminus of the protease recognition sequence.

10. A complex, which comprises the fusion protein according to any one of claims 1 to 9, and a cargo molecule; preferably, the cargo molecule is selected from the group consisting of nucleic acid, peptide or protein, carbohydrate, lipid, chemical compound and any mixture thereof; preferably, the nucleic acid is selected from the group consisting of DNA molecule, RNA molecule, siRNA, antisense oligonucleotide, ribozyme, aptamer and any combination thereof; preferably, the cargo molecule comprises a detectable label; preferably, the cargo molecule comprises an epitope tag, reporter gene sequence and/or nuclear localization signal (NLS) sequence.

11. The complex according to claim 10, wherein the fusion protein is fused with the cargo molecule, and the cargo molecule is a peptide or protein; preferably, the cargo molecule is fused to the C-terminus of the fusion protein; preferably, the complex comprises a single-chain polypeptide, and the single-chain polypeptide comprises the pH-sensitive fusogenic peptide, cell-penetrating peptide, protease recognition sequence and cargo molecule from N-terminus to C-terminus; preferably, the protease recognition sequence comprises the furin recognition sequence and cathepsin L recognition sequence from N-terminus to C-terminus, or comprises the cathepsin L recognition sequence and furin recognition sequence from N-terminus to C-terminus; preferably, the complex comprises a single-chain polypeptide, and the single-chain polypeptide comprises the cell-penetrating peptide, pH-sensitive fusogenic peptide, protease recognition sequence and cargo molecule from N-terminus to C-terminus; preferably, the protease recognition sequence comprises the furin recognition sequence and cathepsin L recognition sequence from N-terminus to C-terminus, or comprises the cathepsin L recognition sequence and furin recognition sequence from N-terminus to C-terminus.

12. The complex according to claim 10, wherein the fusion protein is chemically coupled to the cargo molecule; preferably, the chemical coupling is achieved through disulfide bond, phosphodiester bond, phosphorothioate bond, amide bond, amine bond, thioether bond, ether bond, ester bond or carbon-carbon bond; preferably, the cargo molecule is coupled to the N-terminus or C-terminus of the fusion protein.

13. The complex according to claim 10, wherein the fusion protein is non-covalently linked to the cargo molecule.

14. The complex according to claim 13, wherein the fusion protein is the fusion protein according to claim 9, and the cargo molecule comprises a domain capable of specifically binding to the specific binding sequence in the fusion protein; preferably, the domain capable of specifically binding to the specific binding sequence in the fusion protein is an amino acid sequence; preferably, the cargo molecule is a peptide or protein; preferably, the specific binding sequence in the fusion protein comprises a leucine zipper peptide, and the cargo molecule comprises the complementary peptide of the leucine zipper, so that the leucine zipper peptide and the complementary peptide are able to form a heterodimer; preferably, the specific binding sequence in the fusion protein comprises leucine zipper NZ (for example, a sequence shown in SEQ ID NO: 49), and the cargo molecule comprises leucine zipper CZ (for example, a sequence shown in SEQ ID NO: 50); preferably, the specific binding sequence in the fusion protein comprises leucine zipper CZ (for example, a sequence shown in SEQ ID NO: 50), and the cargo molecule comprises leucine zipper NZ (for example, a sequence shown in SEQ ID NO: 49).

15. The complex according to claim 13, wherein the fusion protein and the cargo molecule are conjugated by electrostatic interaction.

16. A composition, which comprises the fusion protein according to any one of claims 1 to 9, and a cargo molecule; preferably, the cargo molecule is selected from the group consisting of nucleic acid, peptide or protein, carbohydrate, lipid, chemical compound and any mixture thereof; preferably, the cargo molecule is selected from the group consisting of nucleic acid, peptide or protein; preferably, the nucleic acid is selected from the group consisting of DNA molecule, RNA molecule, siRNA, antisense oligonucleotide, ribozyme, aptamer and any combination thereof.

17. The composition according to claim 16, which comprises the fusion protein according to claim 9, and the cargo molecule comprises a domain capable of specifically binding to the specific binding sequence in the fusion protein; preferably, the domain capable of specifically binding to the specific binding sequence in the fusion protein is an amino acid sequence; preferably, the cargo molecule is a peptide or protein; preferably, the specific binding sequence in the fusion protein comprises a leucine zipper peptide, and the cargo molecule comprises the complementary peptide of the leucine zipper, so that the leucine zipper peptide and the complementary peptide are able to form a heterodimer; preferably, the specific binding sequence in the fusion protein comprises leucine zipper NZ (for example, a sequence shown in SEQ ID NO: 49), and the cargo molecule comprises leucine zipper CZ (for example, a sequence shown in SEQ ID NO: 50); preferably, the specific binding sequence in the fusion protein comprises leucine zipper CZ (for example, a sequence shown in SEQ ID NO: 50), and the cargo molecule comprises leucine zipper NZ (for example, a sequence shown in SEQ ID NO: 49).

18. An isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the fusion protein according to any one of claims 1 to 9, or the complex according to claim 11, or the composition according to claim 16 or 17.

19. A vector, which comprises the isolated nucleic acid molecule according to claim 18.

20. A host cell, which comprises the isolated nucleic acid molecule according to claim 18 or the vector according to claim 19.

21. A method for preparing the fusion protein according to any one of claims 1 to 9, or the complex according to claim 11, which comprises culturing the host cell according to claim 20 under a suitable condition, and recovering the fusion protein or complex from a culture of the cell.

22. A pharmaceutical composition, which comprises the fusion protein according to any one of claims 1 to 9, the complex according to any one of claims 10 to 15, the composition according to claim 16 or 17, the isolated nucleic acid molecule according to claim 18, the vector according to claim 19 or the host cell according to claim 20, and a pharmaceutically acceptable carrier and/or excipient; preferably, the pharmaceutical composition comprises the complex according to any one of claims 10 to 15, wherein the cargo molecule is a pharmaceutically active agent or a detectable label.

23. Use of the fusion protein according to any one of claims 1 to 9, or an isolated nucleic acid molecule, vector or host cell comprising a nucleotide sequence encoding the fusion protein, in the manufacture of a medicament.

24. Use of the complex according to any one of claims 10 to 15 or the composition according to claim 16 or 17, or an isolated nucleic acid molecule, vector or host cell comprising a nucleotide sequence encoding the complex or composition, in the manufacture of a medicament for treatment of a disease; wherein the cargo molecule contained in the complex or composition is capable of treating the disease; preferably, the disease is a disease related to programmed cell necrosis, and the cargo molecule comprises protein phosphatase 1B; preferably, the disease related to programmed cell necrosis comprises liver injury (e.g., drug-induced liver injury), inflammatory disease, ischemia-reperfusion injury and/or neurodegenerative disease.

25. A kit, which comprises the fusion protein according to any one of claims 1 to 9, the complex according to any one of claims 10 to 15, the composition according to claim 16 or 17, the isolated nucleic acid molecule according to claim 18, the vector according to claim 19, or the host cell according to claim 20; preferably, the kit further comprises an instruction for transfection and/or intracellular delivery.

26. Use of the fusion protein according to any one of claims 1 to 9, the complex according to any one of claims 10 to 15, the composition according to claim 16 or 17, the isolated nucleic acid molecule according to claim 18, the vector according to claim 19, or the host cell according to claim 20, as a delivery agent.

27. A method for delivering a cargo molecule into a cell, which comprises contacting the cell with the complex according to any one of claims 10 to 15, wherein the complex comprises the cargo molecule; preferably, the contacting the cell with the complex is carried out in vitro; preferably, the cargo molecule is selected from the group consisting of nucleic acid, peptide or protein, carbohydrate, lipid, chemical compound, and any mixture thereof preferably, the nucleic acid is selected from the group consisting of DNA molecule, RNA molecule, siRNA, and antisense oligonucleotide, ribozyme, aptamer and any combination thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0127] FIG. 1 shows a schematic diagram of the principle of the Split-GFP system for detecting the escape efficiency from endosome in Example 1.

[0128] FIG. 2 shows a schematic diagram of the structure of the delivery system-GFPβ1-10 protein complex in Example 1.

[0129] FIGS. 3 to 4 show the SDS-PAGE results of the delivery system-GFPβ1-10 complex in Example 1.

[0130] FIG. 5 shows the results of flow cytometry analysis of the delivery system for transducing GFPβ1-10 in Example 1.

[0131] FIG. 6 shows the results of fluorescence microscopy observation of the delivery system for transducing GFPβ1-10 in Example 1.

[0132] FIG. 7 shows the results of flow cytometry analysis of the delivery system containing mutation in enzymatic cleavage site for transducing GFPβ1-10 in Example 1.

[0133] FIG. 8 shows the Western blot detection results of the relative proportion of the cleaved and un-cleaved/complete GFPβ1-10 in the cell and the retention time thereof in the cell after transduction by the delivery system in Example 1.

[0134] FIG. 9 shows the Western blot detection results of the relative proportion of the cleaved and un-cleaved/complete GFPβ1-10 in the cell after transduction by the delivery system containing mutation in enzymatic cleavage site in Example 1.

[0135] FIG. 10 shows the results of flow cytometry analysis of the transduction by the delivery system containing mutation in enzymatic cleavage site but no pH-sensitive peptide in Example 1.

[0136] FIG. 11 shows the Western blot detection results of the relative proportion of the cleaved and un-cleaved/complete GFPβ1-10 in the cell after transduction by the delivery system containing mutation in enzymatic cleavage site but no pH-sensitive peptide in Example 1.

[0137] FIG. 12 shows a schematic diagram of the structure of the delivery system-ZFP9 complex in Example 2.

[0138] FIG. 13 shows the results of SDS-PAGE of the delivery system-ZFP9 complex in Example 2.

[0139] FIG. 14 shows the map of the eukaryotic expression plasmid containing ZFP9 binding site in Example 2.

[0140] FIGS. 15 to 16 show the results of flow cytometry analysis of the transduction of ZFP9 by the delivery system in Example 2.

[0141] FIG. 17 shows a schematic diagram of the structure of the delivery system-Ppm1b complex in Example 3.

[0142] FIG. 18 shows the results of SDS-PAGE of the delivery system-Ppm1b complex in Example 3.

[0143] FIG. 19 shows the results of flow cytometry analysis of the effect of the delivery system-Ppm1b complex on the ratio of TNF-α-induced cell necrosis in Example 3.

[0144] FIG. 20 shows a schematic diagram of the structure of the delivery system-Cas9 complex in Example 4.

[0145] FIG. 21 shows the results of SDS-PAGE of the delivery system-Cas9 complex in Example 4.

[0146] FIG. 22 shows a schematic diagram of the principle of detection of the editing efficiency of CRISPR/Cas9 by using HEK293T-RFP reporter cells in Example 4.

[0147] FIG. 23 shows the map of RFP reporter lentiviral plasmid in Example 4.

[0148] FIG. 24 shows the results of flow cytometry analysis of the gene editing efficiency of the delivery system-Cas9 complex in Example 4.

[0149] FIG. 25 shows the principle of the delivery system based on the adapter method in Example 5.

[0150] FIG. 26 shows the clone design of recombinant protein for the delivery system based on the adapter method in Example 5.

[0151] FIG. 27 shows the purification results of recombinant protein for the delivery system based on the adapter method in Example 5.

[0152] FIG. 28 shows the evaluation results of the delivery effect of the adapter-based delivery system by the Split-GFP endosome escape system in Example 5.

SEQUENCE INFORMATION

[0153] The information of some sequences involved in the present invention is provided in Table 1 below.

TABLE-US-00001 TABLE 1 Description of sequences SEQ ID NO Description 1 Furin recognition sequence-1 2 Furin recognition sequence-2 3 Furin recognition sequence-3 4 Furin recognition sequence-4 (Ne) 5 Nucleic acid sequence encoding Ne 6 CTSL recognition sequence N 7 Nucleic acid sequence encoding N 8 INF7 9 Nucleic acid sequence encoding INF7 10 Tat(48-60) 11 Nucleic acid sequence encoding Tat (48-60) 12 Fusion protein TIN 13 Fusion protein TINe 14 Fusion protein TINNe 15 NLS 16 CTSL recognition sequence Na 17 CTSL recognition sequence Nb 18 Furin recognition sequence Nc 19 Furin recognition sequence Nd 20 CTSD identification sequence Nf 21 Mutant N 22 Mutant Ne 23 GFPβ1-10-NLS 24 Nucleic acid sequence encoding GFPβ1-10-NLS 25 Nucleic acid sequence encoding Histone-H3 26 Nucleic acid sequence encoding GFPβ11 27 ZFP9-NLS 28 Nucleic acid sequence encoding ZFP9-NLS 29 Sequence encoding BFP 30 ZFP9 binding site sequence 31 Ppm1b 32 Nucleic acid sequence encoding Ppm1b 33 Cas9-NLS 34 Nucleic acid sequence encoding Cas9-NLS 35 Nucleic acid sequence encoding dsRed 36 Nucleic acid sequence encoding mCherry 37 DNA sequence of the recognition site for sgRNA 38 Influenza virus HA2 39 KALA 40 GALA 41 Melittin 42 Penetratin 43 HIV-TAT(47-57) 44 HIV-1 Rev(34-50) 45 VP22 46 Transportan 47 Pep-1 48 Pep-7 49 Leucine zipper NZ 50 Leucine zipper CZ 51 Nucleic acid sequence encoding leucine zipper NZ 52 Nucleic acid sequence encoding leucine zipper CZ 53 TrxA amino acid sequence 54 TrxA nucleic acid sequence

EXAMPLES

[0154] The present invention will now be described with reference to the following examples which are intended to illustrate the present invention rather than limit the present invention.

[0155] Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present invention were performed basically by referring to the methods as described in J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F M Ausubel et al., Compiled Molecular Biology Experiment Guide, 3rd edition, John Wiley & Sons, Inc., 1995; the restriction enzymes were used in accordance with the conditions recommended by the product manufacturer. Those skilled in the art know that the examples describe the present invention by way of example, and are not intended to limit the scope sought to be protected by the present invention.

[0156] The sources of the main reagents involved in the following examples are as follows: the materials required for cloning and construction were as follows: DNA polymerase (TaKaRa, R040A), DNA recovery kit (TianGen, DP214-03), plasmid mini-kit (TianGen, DP103-03), plasmid large-scale kit (QIAGEN, 12663), 5 tubes of Gibson assembly premix (NEB, E2611L), DNA marker (ThmeroFisher, SM0331), agarose (Biowest, BW-R0100),

[0157] the materials required for large-scale protein expression were as follows: peptone (BiSIGMA-ALDRICH, T7293-1KG), yeast powder (OXOID, LP0021B), sodium chloride (Xilong Chemical, 10011012AR), IPTG (Inalco, 1758-1400),

[0158] the media required for protein purification were as follows: SP SEPHAROSE FAST FLOW (GE Healthcare, 17-0729-01), NI SEPHAROSE (GE Healthcare, 17-5268-02),

[0159] the reagents required for protein purification and storage were as follows: glycerol/glycerol/C.sub.3H.sub.8O.sub.3 (SIGMA-ALDRICH, G5516), KCl (Xilong Chemical Industry, 1002007), Na.sub.2HPO.sub.4.12H.sub.2O (Xilong Chemical Industry, 1001067AR), KH.sub.2PO.sub.4 (Xilong Chemical Industry, 1002048AR500), imidazole (SIGMA-ALDRICH, V900153), Tris base (Seebio, 183995), glucose (Xilong Chemical Industry, 1064008AR500), BCA Protein Assay Kit (Thermo Scientific, 23227);

[0160] reagents required for cell culture: FBS (GIBCO, 10099-133), DMEM (GIBCO, 11965092), trypsin (AMRESCO, 0458);

[0161] reagents required for lentivirus packaging and infection: lentivirus packaging plasmid: pCMV-VSV-G (Addgene, 8454), pRSV-Rev (Addgene, 12253), pMDLg/pRRE (Addgene, 12251); X-tremeGENE transfection reagent (Roche, 06366244001), Puromycin (InvivoGen, ant-pr-5), Blasticidin (InvivoGen, ant-bl-5b), polybrene (Santa Cruz, sc-134220);

[0162] plasmids related to GFPβ1-10, ZFP9, Ppm1b, dsRed, mCherry, and Histone-H3 as used in the experiments were all synthesized by Biotech. The plasmid pCasKP-hph for amplifying the Cas9 sequence (Addgene, 117232);

[0163] other reagents: TNF-α (Novoprotein, CF09), PI (ThmeroFisher, P3566),

[0164] cell lines: HEK-293T (human renal epithelial cells), L929 (mouse fibroblasts), which were purchased from ATCC.

Example 1: Split-GFP System-Based Evaluation of Endosome Escape Efficiency of Delivery System

[0165] In the Split-GFP system, the eleven β-pleated sheets of GFP are split into a large fragment (β1-10) and a small fragment (β11), both of which lose the fluorescent activity; but if they meet, they can associate spontaneously and restore the fluorescence performance of GFP. Based on this, we constructed HEK293T cells stably expressing Histone-β11, and GFPβ1-10 with nuclear localization signal (NLS) as a cargo and an intracellular delivery system to be evaluated were expressed as a fusion protein, and subjected to transduction of the stable cell line. When GFPβ1-10 was transduced by the delivery system, it could bind to GFPβ11 and generate complete GFP only after successfully escaping from the endosome and entering the cytoplasm or nucleus, so that the endosome escape efficiency could be evaluated by the proportion and relative fluorescence intensity of ratio of GFP (FIG. 1).

[0166] 1.1 Construction of Delivery System-GFPβ1-10 Protein Complex Expression Vector

[0167] Construction of a recombinant protein expression vector of cargo molecule GFPβ1-10 (SEQ ID NO: 23) containing TAT (SEQ ID NO: 10), INF7 (SEQ ID NO: 8), protease cleavage site (Table 2) and nuclear localization signal (NLS) was performed, the schematic diagram of the structure of each recombinant protein was shown in FIG. 2, and the components from C-terminus to N-terminus and the amino acid sequences thereof were shown in Table 3 below. The construction method was as follows: first, the nucleic acid sequences of TAT, INF7, N, Na, Nb, Nc, Nd, Ne, Nf, mutant N, mutant Ne, and cargo molecule GFPβ1-10 in the delivery system were obtained by PCR amplification; these parts were linked through multiple rounds of PCR, and in the last round of PCR, the NdeI restriction site and the overlap on upstream of the corresponding NdeI restriction site in pET21b(+) were introduced at the 5′ end of the fragment through the forward primer, and the BamHI restriction site and the overlap on downstream of the corresponding BamHI restriction site in pET-21b(+) were introduced at the 3′ end of the fragment through the reverse primer. The pET-21b(+) plasmid was subjected to double digestion with NdeI and BamHI. The insert fragments with overlaps were ligated to the digested vector pET-21b(+) by GIBSON assembly.

TABLE-US-00002 TABLE 2 Protease recognition sequences Protease Recognition sequence SEQ ID NO: CTSL N 6 Mutant N 21 Na 16 Nb 17 Furin Nc 18 Nd 19 Ne 4 Mutant Ne 22 CTSD Nf 20

TABLE-US-00003 TABLE 3 Components contained in delivery system-cargo molecular complex components from N-terminal to C-terminal and their sequences pH- Protease sensitive recognition Complex name CPP peptide sequence Cargo T-GFP β1-10 Tat None None GFP β1-10-NLS TI-GFP β1-10 SEQ ID INF7 SEQ ID NO: 10 NO: 23 TIN-GFP β1-10 SEQ ID N NO: 8 TINa-GFP β1-10 Na TINb-GFP β1-10 Nb TINc-GFP β1-10 Nc TINd-GFP β1-10 Nd TINe-GFP β1-10 Ne TINf-GFP β1-10 Nf TINNe-GFP β1-10 N + Ne TNNE-GFP β1-10 None

[0168] 1.2 Expression and Purification of Delivery System-GFPβ1-10 Complex:

[0169] The expression plasmid described in 1.1 was transformed into the expression strain E. coli BL21 (DE3). A single colony was picked from the plate after transformation and inoculated in 5 ml of LB liquid medium containing ampicillin resistance and cultivated overnight, and then 1 ml of the overnight-cultured bacterial culture was transferred to 500 ml of LB liquid medium containing ampicillin resistance, followed by cultivation at 37° C. and 180 rpm until the bacterial culture had OD.sub.600 of about 0.6, and then the inducer IPTG was added to reach a final concentration of 0.2 mM, followed by induction at 25° C. for 8 h. After the induction, the bacterial cells were collected after centrifugation at 7000 g for 10 min at 4° C. Then the bacterial cells were re-suspended with 10 ml of equilibration buffer for protein purification (50 ml of glycerol, 8 g of NaCl, 0.201 g of KCl, 1.44 g of Na.sub.2HPO.sub.4, 0.24 g of KH.sub.2PO.sub.4, dissolved in 1 L of double-distilled water) and ultrasonically disrupted. Then the supernatant was collected by centrifugation and loaded on the protein purification column for polyhistidine-tagged protein of protein purification system. And then a desired protein was eluted with the elution buffer for protein purification system (50 ml of glycerol, 8 g of NaCl, 0.201 g of KCl, 1.44 g of Na.sub.2HPO.sub.4, 0.24 g of KH.sub.2PO.sub.4, 17 g of imidazole, dissolved in 1 L of double-distilled water). The protein concentration could be determined by a spectrophotometer or BCA Protein Assay Kit. Each purified fusion protein was aliquoted and stored at −20° C. The SDS-PAGE results of each protein were shown in FIGS. 3 to 4.

[0170] 1.3 Construction of HEK293T-GFPβ11 Cell Line

[0171] 1.3.1 Construction of Lentiviral Plasmid for GFPβ11 Cell Line:

[0172] The coding sequence of Histone-H3 (SEQ ID NO: 25) was obtained by PCR amplification, the coding sequence of GFPβ11 (SEQ ID NO: 26) was shorter and directly designed in the forward primer. The components were ligated through multiple rounds of PCR, and in the last round of PCR, the Hind III restriction site and the overlap on upstream of the corresponding Hind III restriction site on Lenti vector were introduced at the 5′ end of the fragment through the forward primer, and the BamHI restriction site and the overlap on downstream of the corresponding BamHI restriction site on Lenti vector were introduced at the 3′ end of the fragment through the reverse primer. The Lenti plasmid was subjected to double digestion with Hind III and BamHI. The insert fragments with overlaps were ligated to the digested Lenti vector by GIBSON assembly.

[0173] 1.3.2 Lentivirus Packaging, Infection and Resistance Screening of Cell Line:

[0174] HEK-293T cells were inoculated into a 6-well plate and cultured overnight, and it was ensured that the number of cells per well was about 2*10.sup.7/ml before plasmid transfection. Before transfection, the cells were transferred into serum-free DMEM medium. 1.5 μg of Lenti recombinant plasmid, 0.75 μg of pMDL plasmid, 0.45 μg of pVSV-G plasmid, 0.3 μg of pREV (mass ratio 5:3:2:1) were added to 300 μl of serum-free DMEM, followed by slowly blowing. 9 μl (1:3) of X-tremeGENE transfection reagent was added and slowly blown, and was set aside at room temperature for 15 minutes. The cell supernatant was added dropwise, and after 8 hours, the culturing was continued by changing the medium to DMEM containing 10% FBS. after 60 hours, the culture supernatant was collected for later infection.

[0175] HEK-293T cells were inoculated into a 12-well plate and cultured overnight, and it was ensured that the number of cells per well was about 2*10.sup.6/ml (50% density) before lentivirus infection. The original cell culture supernatant was discarded, followed by addition of 300 μl of lentivirus (moi=3) and 700 μl of 10% FBS DMEM, and polybrene was added at a concentration of 10 μg/ml. The cell plate was centrifuged at 2500 rpm for 30 min under aseptic conditions, and the culture was continued.

[0176] After 48 hours of lentivirus infection, the cells were passaged at a ratio of 1/3, and puromycin was added at a concentration of 2.5 μg/ml for resistance screening. The positive cells obtained from the screening were cloned to obtain HEK-293T-Hitone-GFPβ1 monoclonal cell line.

[0177] 1.4 Detection of Endosome Escape Efficiency of Delivery System-GFPβ1-10 Complex by Split-GFP System

[0178] The HEK-293T-Hitone-GFPβ1 cell line obtained in 1.3 was inoculated into a 12-well plate and cultured overnight, and it was ensured that the number of cells per well was about 5*10.sup.6/ml before protein treatment. After rinsing the cells with serum-free DMEM medium three times, 100 μl/5 μM of the delivery system-GFPβ1-10 complex obtained in 1.2 was added in serum-free medium, and incubation was performed for 3 h. Heparin solution was used for washing three times to remove the protein that had been adsorbed on the cell surface and had not yet been endocytosed into the cell, and the culturing was continued after changing the medium to 10% FBS DMEM medium. The observation with fluorescence microscope and the flow cytometry analysis of the expression of green fluorescent protein were performed at 12 h.

[0179] The results of flow cytometry analysis were shown in FIG. 5. The results showed that the average fluorescence intensity of the cells was increased when the pH-sensitive peptide INF7 was introduced on the basis of TAT, which proved that the pH-sensitive peptide had the effect of breaking endosome membrane. In particular, the cell fluorescence intensity was further increased when CTSL or Furin cleavage sites were introduced on the basis of TAT-INF7, in which the CTSL cleavage site N and the Furin cleavage site Ne showed the most significant effects. The fluorescence microscope observation was shown in FIG. 6, and the results showed that the combination of the above two cleavage sites (TINNe-GFP β1-10) could further significantly improve the endosome escape efficiency.

[0180] A mutation was introduced at a key site in the N or Ne cleavage site to obtain a delivery system-cargo molecule complex containing the mutation, wherein the mutant N had a sequence as shown in SEQ ID NO: 21, and the mutant Ne had a sequence as shown in SEQ ID NO: 22. The comparison of transfection efficiency between the mutant delivery system-cargo molecule complex (Mut) and the delivery system without mutation-cargo molecule complex (WT) was performed. The results were shown in FIG. 7. When the key amino acid in the cleavage site was mutated, whether it was a single cleavage site alone or a combination of two cleavage sites, the effect of enhancing the endosome escape efficiency was lost. The above results indicated that CTSL and Furin cleavage sites could indeed significantly improve the escape efficiency of cargo from endosome.

[0181] After washing three times with heparin solution to remove the protein that was adsorbed on the cell surface and had not yet been endocytosed into the cell, the cells were collected at different time points after the start of transduction. The cells were lysed to extract proteins and SDS-PAGE electrophoresis was performed, and then the monoclonal antibody (Abcam, ab32146) that recognized GFPβ1-10 was used to perform Western blot detection so as to analyze the cleavage and retention of the intracellular proteins. The results were shown in FIG. 8. At each time point before 6 hours, the total amounts of the intracellular proteins detected were comparable among the cells of the different groups, indicating that the pH-sensitive peptide and cleavage sites did not increase the endocytosis efficiency of cargo; the cleavage of the proteins (TIN-, TINe-, TINNe-) containing cleavage sites began within 30 minutes, and the amount of cleaved proteins no longer increased after 3 hours; wherein, about 40% of the proteins with single cleavage site was cleaved, while about 70% of the proteins with double cleavage sites was cleaved; since then, the proteins (T-, TI-) without cleavage site and the intact proteins whose cleavage site was not cleaved were introduced to lysosomes and began to degrade rapidly, and the corresponding bands almost disappeared at 12 h, while the proteins separated from TAT-INF7 after cleavage were still remained, and the TINNe group remained the most. The above results were consistent with the results of flow cytometry, that was, the higher the efficiency of enzyme cleavage, the more undegraded GFPβ1-10-NLS remained in the cell, the more intact GFP formed after GFPβ1-10-NLS entered into the nucleus and associated with GFPβ11, and the higher green fluorescence intensity.

[0182] Further, Western blot was performed to analyze the cleavage of the above-mentioned delivery system-cargo molecule complex containing mutations in the N or Ne cleavage site after transduction of the cells for 3 h. The results were shown in FIG. 9. The intracellular cleavage of the mutant delivery system-cargo molecular complexes (TINm-, TINNem- and TINNem-) could not be fulfilled. Therefore, combined with the flow cytometry results shown in FIG. 7, it could be seen that the reason of low delivery efficiency of the mutant delivery system-cargo molecule complexes was that the cleavage and the subsequent escape could not be could not be achieved. It also further confirmed that CTSL and Furin cleavage sites played a key role in the delivery system.

[0183] In addition, the delivery efficiency of the delivery system-complex (TNNe-GFPβ1-10-NLS) lacking the pH-sensitive peptide component was also detected by flow cytometry, and the results were shown in FIG. 10. We found that the average fluorescence intensity after 12 h of transduction was basically the same as that of T-GFPβ1-10-NLS, indicating that even if there was a cleavage site, the lack of pH-sensitive peptide could not achieve efficient delivery. And it was found by Western blot that the presence or absence of pH-sensitive peptide had no effect on the intracellular cleavage efficiency of the delivery system-complex (FIG. 11). Combining the above results, we confirmed that only the coexistence of pH-sensitive peptide and specific cleavage site in the delivery system could achieve the final high-efficiency delivery.

Example 2: Application of Delivery System in Transduction of Zinc Finger Protein ZFP

[0184] 2.1 Construction of Expression Vector for Delivery System-Zinc Finger Protein ZFP9 Complex

[0185] An expression vector of a recombinant protein containing TAT, INF7, protease cleavage site, and cargo molecule ZFP9 carrying nuclear localization signal (NLS) (SEQ ID NO: 27) was constructed, the schematic diagram of the structure of each recombinant protein was shown in FIG. 12, and the amino acid sequences of the components were shown in Table 4 below. The construction method was as follows: first, the nucleic acid sequences encoding the TAT, INF7, protease cleavage site, and cargo molecule ZFP9 in the delivery system were obtained by PCR amplification, and the parts were ligated by multiple rounds of PCR, and in the last round of PCR process, the NdeI restriction site and the overlap on upstream of the corresponding NdeI restriction site on pET-21b(+) were introduced at the 5′ end of the fragment through the forward primer, and the BamHI restriction site and the overlap on downstream of the corresponding BamHI restriction site on pET-21b(+) were introduced at the 3′ end of the fragment through the reverse primer. The pET-21b(+) plasmid was subjected to double digestion with NdeI and BamHI. The insert fragments with overlaps were ligated to the digested vector pET-21b(+) by GIBSON assembly.

TABLE-US-00004 TABLE 4 Components contained in delivery system-cargo molecular complexes components from N-terminal to C-terminal and their sequences pH- Protease sensitive cleavage Complex name CPP peptide sequence Cargo ZFP9 None None None ZFP9-NLS T- ZFP9 Tat None None SEQ ID NO: 27 TI- ZFP9 SEQ ID INF7 NO: 10 TINNe-ZFP9 SEQ ID N + Ne NO: 8

[0186] 2.2 Expression and Purification of Delivery System-ZFP9 Complex

[0187] The expression plasmid described in 2.1 was transformed into the expression strain E. coli BL21 (DE3). A single colony was picked from the plate after transformation and inoculated in 5 ml of LB liquid medium containing ampicillin resistance and cultivated overnight, and then 1 ml of the overnight-cultured bacterial culture was transferred to 500 ml of LB liquid medium containing ampicillin resistance, followed by cultivation at 37° C. and 180 rpm until the bacterial culture had an OD.sub.600 of about 0.6, and then inducer IPTG was added to a final concentration of 0.2 mM, followed by induction at 25° C. for 8 hours. After the induction, the bacterial cells were collected after centrifugation at 7000 g for 10 min at 4° C., and part of the bacterial cells were taken to detect the induction expression of protein. Then the bacterial cells were re-suspended with 10 ml of equilibration buffer for protein purification (50 ml of glycerol, 3.6342 g of Tris(hydroxymethyl)aminomethane, dissolved in 1 L of double-distilled water, adjusted pH to 8.0), and disrupted with ultrasonic. Then, the supernatant was collected by centrifugation and load on the Sulphopropyl (SP) cation exchange column of AKTA protein purification system; then desired proteins were obtained by gradient elution with the equilibration buffer and high-salt eluent (50 ml of glycerol, 116.88 g of NaCl, 3.6342 g of Tris(hydroxymethyl)aminomethane, dissolved in 1 L of double-distilled water, and adjusted pH to 8.0) in different proportions. The protein concentration could be determined according to spectrophotometer or BCA Protein Assay Kit. Each purified fusion protein was aliquoted and stored at −20° C. The SDS-PAGE results of each protein were shown in FIG. 13.

[0188] 2.3 Construction of Eukaryotic Expression Plasmid Containing ZFP9 Binding Site

[0189] An expression vector containing the coding sequence of blue fluorescent protein (BFP) and ZFP9 binding sites was constructed, the structure schematic diagram of which was shown in FIG. 14. The coding sequence of blue fluorescent protein (SEQ ID NO: 29) and the sequence of ZFP9 binding sites (6* binding sites) (SEQ ID NO: 30) were obtained by PCR amplification, and the two parts were ligated by two rounds of PCR; and in the second round of PCR, the Hind III restriction site and the overlap on upstream of the corresponding Hind III restriction site on pTT5 vector were introduced at the 5′ end of the fragment through the forward primer, and the BamHI restriction site and the overlap on downstream of the corresponding BamHI restriction site on pTT5 vector were introduced at the 3′ end of the fragment through the reverse primer. The pTT5 plasmid was double-digested with Hind III and BamHI. The insert fragments with overlaps were ligated to the digested pTT5 vector by GIBSON assembly to obtain pTT5-BFP-6BS plasmid.

[0190] 2.4 Detection of Delivery Efficiency of Transducing Zinc Finger Protein ZFP9 by Delivery System

[0191] HEK293T cells were inoculated in a 12-well plate and cultured overnight, and it was ensured that the number of cells in each well was about 5*10.sup.6/ml before protein treatment. 100 μL/5 of the delivery system-ZFP9 complex (ZFP9, T-ZFP9, TI-ZFP9, TINNe-ZFP9) obtained in 2.2 and 5 μg of the pTT5-BFP-6BS plasmid obtained in 2.3 were co-incubated at 37° C. for 30 minutes to fully form a complex, and X-tremeGENE transfection reagent (Roche) was used as a positive control (5 μg of plasmid and 15 μl of transfection reagent were mixed and used to transfect cells under serum-free conditions, and after 8 hours, the culturing was continued by changing the medium to 10% FBS DMEM), The cells were rinsed with serum-free DMEM medium three times, and then the complex was added and incubated for 3 h. Heparin solution was used to perform washing three times to remove the protein that was adsorbed on the cell surface and had not yet been endocytosed into the cell, then the culturing was continued after changing the medium to 10% FBS DMEM medium, and the flow cytometry analysis of blue fluorescent protein expression was performed at 12 h, 24 h, 36 h, and 48 h after the changing of medium.

[0192] The results of the flow cytometry analysis were shown in FIGS. 15 to 16. The plasmid bound to ZFP9 could complete the transcription process only when it entered the nucleus. Therefore, only when ZFP9 escaped from endosome, it could carry the bound plasmid to enter into the nucleus, and the transcription process could thus be started to express blue fluorescent protein. Compared with T-ZFP9, the introduction of pH-sensitive peptide INF7 (TI-ZFP9) could increase the blue fluorescence ratio by about 10% (p=0.035), but it was still at a low level, that was, most of the complex failed to escape and enter into the nucleus. While further introduction of a protease cleavage site into the system (TINNE-ZFP9), could significantly increase the proportion of blue fluorescent cells, reaching about 65% at 48 h (p=0.018), which was be equal to that of the Roche transfection reagent X-tremeGENE. It could be seen that during the endocytosis process of the delivery system carrying the ZFP9/pTT5-BFP-6BS complex into the cell, the introduction of CTSL and Furin-specific cleavage sites N and Ne significantly promoted the escape process of the carried ZFP9, more of the complex could enter into the nucleus to complete transcription, and then expressed BFP fluorescence.

Example 3: Application of Delivery System in Transduction of Protein Phosphatase Ppm1b

[0193] TNF-α binds to cell surface receptors to induce RIP3 phosphorylation and form necrosomes of multi-protein complexes, and the phosphorylated RIP3 in the necrosomes recruits and phosphorylates Mlk1 and then the cells enter the necrosis program. In this process, the intracellular protein phosphatase 1B (Ppm1b) can inhibit programmed cell necrosis (Necroptosis) by dephosphorylating RIP3. In view of the fact that programmed cell necrosis has been found to be closely related to the occurrence of inflammatory diseases, ischemia-reperfusion injury, neurodegenerative diseases and other diseases, Ppm1b protein has shown great potential in the treatment of the above-mentioned diseases related to programmed cell necrosis.

[0194] 3.1 Construction of Expression Vector for Delivery System-Ppm1b Protein Complex

[0195] An expression vector for a recombinant protein containing TAT, INF7, protease cleavage site, and cargo molecule Ppm1b (SEQ ID NO: 31) was constructed. The structure diagram of each recombinant protein was shown in FIG. 17, and the amino acid sequence of each component was shown in Table 6 below. The construction method was as follows: the nucleic acid sequences of TAT, INF7, protease cleavage site, and Ppm1b were obtained by PCR amplification, and the parts were ligated by multiple rounds of PCR, and in the last round of PCR, the NdeI restriction site and the overlap on upstream of the corresponding NdeI restriction site on pET-21b(+) were introduced at the 5′ end of the fragment by forward primer, and the BamHI restriction site and the overlap on downstream of the corresponding BamHI restriction site on pET-21b(+) were introduced at the 3′ end of the fragment through the reverse primer. The pET-21b(+) plasmid was subjected to double digestion with NdeI and BamHI. The insert fragments with overlaps were ligated to the digested vector pET-21b(+) by GIBSON assembly.

TABLE-US-00005 TABLE 5 Components contained in delivery system-cargo molecular complexes components from N-terminal to C-terminal and their sequences pH- Protease sensitive recognition Complex name CPP peptide sequence Cargo Ppm1b None None None Ppm1b T- Ppm1b Tat None None SEQ ID NO: 31 TI- Ppm1b SEQ ID INF7 NO: 10 TINNe- Ppm1b SEQ ID N + Ne NO: 8

[0196] 3.2 Expression and Purification of Delivery System-Ppm1b Complex

[0197] The expression plasmid described in 3.1 was transformed into the expression strain E. coli BL21 (DE3). a single colony was picked from the plate after transformation and inoculated in 5 ml of LB liquid medium containing ampicillin resistance and cultivated overnight, and then 1 ml of the overnight-cultured bacterial culture was transferred into 500 ml of LB liquid medium containing ampicillin resistance, followed by cultivation at 37° C. and 180 rpm until the bacterial culture had an OD.sub.600 of about 0.6, and then inducer IPTG was added to reach a final concentration of 0.2 mM, followed by induction at 25° C. for 8 h. After the induction, the bacterial cells were collected by centrifugation at 7000 g for 10 minutes at 4° C., and a part of the bacterial cells was taken to detect the induction expression of protein. Then the bacterial cells were re-suspended in 10 ml of equilibration buffer for protein purification (50 ml of C.sub.3H.sub.8O.sub.3, 3.6342 g of Tris(hydroxymethyl)aminomethane, dissolved in 1 L of double-distilled water, adjusted pH to 8.0), and disrupted by ultrasonic. Then the supernatant was collected by centrifugation and loaded on the Sulphopropyl (SP) cation exchange column of AKTA protein purification system; then the desired proteins were obtained by gradient elution with the equilibration buffer and high-salt eluent (50 ml of C.sub.3H.sub.8O.sub.3, 116.88 g of NaCl, 3.6342 g of Tris(hydroxymethyl)aminomethane, dissolved in 1 L of double-distilled water, adjusted to pH 8.0) in different proportions. The protein concentration could be determined by spectrophotometer or BCA Protein Assay Kit. Each purified fusion protein was aliquoted and stored at −20° C. The SDS-PAGE results of each protein were shown in FIG. 18.

[0198] 3.3 Effect of Delivery System-Ppm1b Complex on Rate of Necrosis Induced by TNF-α

[0199] L929 cells were inoculated in a 12-well cell culture plate and cultured overnight, and it was ensured that the number of cells per well was about 2*10.sup.6/ml before protein treatment. The cells were rinsed with serum-free DMEM medium three times, and then 100 μl/5 μM of the delivery system proteins (Ppm1b, T-Ppm1b, TI-Ppm1b, TINNe-Ppm1b) obtained in 3.2 were added in serum-free medium respectively, and incubated for 3 hours. Then 1 ml of 10% FBS DMEM containing 20 ng/ml TNF-α and 20 mM z-VAD was added, and incubated for 10 h. The cells were collected and subjected to PI staining, and the flow cytometry analysis was performed to observe the ratio of cell necrosis. Using lentivirus-transduced Ppm1b (Lenti-Ppm1b) and lentivirus (Lenti-vec) as controls, the lentivirus with Ppm1b expression sequence and the control lentivirus were packaged and collected on HEK-293T cells, and after infecting L929 cells for 24 h to make Ppm1b fully expressed in the cells, the infected L929 cells were re-plated for later TNF-α stimulation.

[0200] The results were shown in FIG. 19. Compared with T-Ppm1b, the introduction of pH-sensitive peptide INF7 (TI-Ppm1b) could merely slightly inhibit cell necrosis, but it was still at a low level. However, by further introduction of the protease cleavage site in the system (TINNe-Ppm1b), the TINNe group had an ability of inhibiting cell necrosis that was comparable to the level of the lentivirus group, and the cell necrosis rate can be greatly reduced to about 25%. Therefore, during the endocytosis process of the delivery system carrying the Ppm1b complex into the cell, the introduction of CTSL and Furin specific sites N and Ne significantly promoted the escape process of the carried Ppm1b, and more Ppm1b could enter into the cytoplasm and inhibit the phosphorylation process of RIP3, thereby reducing the rate of cell necrosis.

Example 4: Application of Delivery System in Gene-Editing Enzyme Cas9

[0201] In the CRISPR/Cas9 gene editing system, sgRNA binds to Cas9 protein, and sgRNA can specifically recognize a target site and Cas9 can bind and cut DNA double-stranded molecule, and the editing of a targeted gene is realized through non-homologous end recombination or homology-directed repair. In this system, Cas9 must enter the nucleus to complete its function. Based on this, we fused the delivery system with the Cas9 protein so as to achieve the gene editing on eukaryotic cells.

[0202] 4.1 Construction of Expression Vector for Delivery System-Cas9 Protein Complex

[0203] An expression vector for a recombinant protein containing TAT, INF7, protease cleavage site, and cargo molecule Cas9 carrying nuclear localization signal (NLS) (SEQ ID NO: 33) was constructed. The schematic diagram of the structure of each recombinant protein was shown in FIG. 20, and the amino acid sequence of each component was shown in Table 6 below. The construction method was as follows: the nucleic acid sequences encoding TAT, INF7, protease cleavage site N and Ne, Cas9 were obtained by PCR amplification, and the parts were ligated by multiple rounds of PCR; in the last round of PCR, the NdeI restriction site and the overlap on upstream of the corresponding NdeI restriction site on pET-21b(+) were introduced at the 5′ end of the fragment through the forward primer, and the BamHI restriction site and the overlap on downstream of the corresponding BamHI restriction site on pET-21b(+) were introduced at the 3′ end of the fragment through the reverse primer. The pET-21b(+) plasmid was subjected to double digestion with NdeI and BamHI. The insert fragments with overlaps were ligated to the digested vector pET-21b(+) by GIBSON assembly.

TABLE-US-00006 TABLE 6 Components contained in delivery system-cargo molecular complexes components from N-terminal to C-terminal and their sequences pH- Protease sensitive recognition Complex name CPP peptide sequence Cargo T- Cas9 Tat None None Cas9-NLS TI- Cas9 SEQ ID INF7 SEQ ID NO: 10 NO: 33 TINNe- Cas9 SEQ ID N + Ne NO: 8

[0204] 4.2 Expression and Purification of Delivery System-Cas9 Complex

[0205] Preliminary purification on nickel column: The expression plasmid described in 4.1 was transformed into the expression strain E. coli BL21 (DE3); a single colony was picked from the plate after transformation and inoculated into 5 ml of LB liquid medium containing ampicillin resistance and cultivated overnight; then 1 ml of the overnight-cultured bacterial culture was transferred into 500 ml of LB liquid medium containing ampicillin resistance, cultured at 37° C. and 180 rpm until the bacterial culture had an OD.sub.600 of about 0.6; then the inducer IPTG was added to reach a final concentration of 0.2 mM, followed by induction for 8 h at 25° C.; after the induction, the bacterial cells were collected by centrifugation at 7000 g and 4° C. for 10 minutes; then the bacterial cells were re-suspended with 10 ml of equilibration buffer for protein purification (5% glycerol, 30 mM TB8.0, 50 mM glycerol, 500 mM sodium chloride, 25 mM glucose) and disrupted with ultrasonic. Then the supernatant was collected by centrifugation and loaded on the protein purification column for polyhistidine-tagged protein of protein purification system; then the desired protein was eluted by protein purification system with the elution buffer for protein purification system (5% glycerol, 30 mM TB8.0, 50 mM glycerol, 500 mM sodium chloride, 25 mM glucose, 250 mM imidazole).

[0206] Final purification on cation exchange column: The desired protein collected from the preliminary purification on nickel column was dialysed into an equilibrium buffer (30 mM TB8.0, 50 mM glycerol, 250 mM sodium chloride, 25 mM glucose, adjusted pH to 7.2), loaded on the Sulphopropyl (SP) cation exchange column of AKTA protein purification system; then the desired protein was obtained by gradient elution with the equilibration buffer and high-salt eluent (30 mM TB8.0, 50 mM glycerol, 2 M sodium chloride, 25 mM Glucose, adjusted pH to 7.2) in different proportions. The protein concentration can be measured by spectrophotometer or BCA Protein Assay Kit. Each purified fusion protein was aliquoted and stored at −20° C. The SDS-PAGE results of each protein were shown in FIG. 21.

[0207] 4.3 Construction of HEK293T-RFP Reporter Cell Line

[0208] Cas9 can specifically recognize a target site by binding to sgRNA, and non-homologous end recombination will occur if a donor is not provided. Therefore, the sgRNA recognition site and two red fluorescent protein genes that are not in a reading frame (dsRed and mCherry are linked by G) are integrated into the genome of HEK-293T cells by means of lentivirus infection. In such case, if Cas9 causes the occurrence of homologous end recombination of DNA sequence on the sgRNA recognition site, it will cause the red fluorescent protein gene that is not in the reading frame to enter the reading frame and be expressed, which will change the cell from non-fluorescent to red fluorescent state, so that the efficiency of the delivery system for transduction of genetically engineered enzyme Cas9 can be evaluated by whether red fluorescence is produced and the number of red fluorescent cells (FIG. 22).

[0209] 4.3.1 Construction of Lentiviral Plasmid for RFP Reporter Cell Line

[0210] The coding sequence of dsRed (SEQ ID NO: 35) and the coding sequence of mCherry (SEQ ID NO: 36) were obtained by PCR amplification, the DNA sequence of sgRNA recognition site (SEQ ID NO: 37) was shorter and thus designed in the primer, and the components were ligated to by multiple rounds of PCR. In the last round of PCR, the Hind III restriction site and the overlap on upstream of the corresponding Hind III restriction site on Lenti vector were introduced at the 5′ end of the fragment through the forward primer, and the BamHI restriction site and the overlap on downstream of the corresponding BamHI restriction site on Lenti vector were introduced at the 3′ end of the fragment through the reverse primer. The Lenti plasmid was subjected to double digestion with Hind III and BamHI. The insert fragments with overlaps were ligated to the digested Lenti vector by GIBSON assembly. The map of successfully constructed plasmid is shown in FIG. 23.

[0211] 4.3.2 Lentivirus Packaging, Infection and Cell Line Resistance Screening

[0212] HEK293T cells were inoculated in a 6-well plate and cultured overnight, and it was ensured that the number of cells per well was about 2*10.sup.7/ml before plasmid transfection. Before transfection, the medium was replaced with serum-free DMEM medium. 1.5 μg of Lenti recombinant plasmid (RFP reporter), 0.75 μg of pMDL plasmid, 0.45 μg of pVSV-G plasmid, 0.3 μg of pREV plasmid (the mass ratio was 5:3:2:1) were added in 300 μl of serum-free DMEM, slowly blown well, and allowed to stand for 5 minutes. Then 9 μl of X-tremeGENE transfection reagent was added, slowly blown well, and allowed to stand at room temperature for 15 minutes. Then it was added to the cell supernatant. 8 hours later, the culturing was continued after changing the medium to DMEM containing 10% FBS. 60 hours later, the culture supernatant was collected and stored at 4° C.

[0213] HEK293T cells were inoculated into a 12-well plate and cultured overnight, and it was ensured that the number of cells per well was about 2*10.sup.6/ml (50% density) before lentivirus infection. The cell culture supernatant was discarded, and 300 μl of lentivirus (Moi=3), 700 μl of 10% FBS DMEM and polybrene at a concentration of 10 μg/ml were added, followed by centrifugation at 2500 rpm for 30 minutes under aseptic conditions, and then the culturing was continued.

[0214] After 48 hours of lentivirus infection, the cells were passaged at a ratio of 1/3, and puromycin at a concentration of 2.5 μg/ml was added to perform resistance screening; the positive cells obtained by the screening were cloned to obtain monoclonal cell strain of HEK293T-RFP reporter.

[0215] 4.3.3 Construction of GM3-gRNA Transcription Plasmid

[0216] The introduction of gRNA is performed by transfection of a transcription plasmid which will be transcribed into gRNA in the cell. The DNA sequence corresponding to the gRNA is generated by primer annealing and overlapping. In the process of primer design, the sticky end of the AflII restriction site was directly introduced; gRNA-cloning vector was treated with AflII single enzyme digestion; the vector and insert fragment were ligated by T4 DNA ligase using their respective sticky ends.

[0217] 4.4 Evaluation of Editing Efficiency of Delivery System-Cas9 Complex

[0218] Transfection of gRNA transcription plasmid: the HEK-293T-RFP reporter cell line was inoculated into a 12-well plate and cultured overnight, and it was ensured that the number of cells per well was about 2.5*10.sup.6/ml before transfection. Before transfection, the medium was replaced with serum-free DMEM. 1 μg of gRNA-GM3 transcription plasmid was added to 100 μl of serum-free DMEM, slowly blown well, allowed to stand for 5 min. 3 μl of X-tremeGENE transfection reagent was further added, blown well, and allowed to stand for 15 min. It was then added to the cell supernatant. After 8 hours, the medium was changed to 10% FBS DMEM.

[0219] Transduction of Cas9 by delivery system: 12 h after the transfection of gRNA transcription plasmid (4 h after changing to serum-containing DMEM), the cells were rinsed three times with serum-free DMEM, and 5 μM of the delivery system-Cas9 complex obtained in 4.2 was added under serum-free DMEM condition and incubated for 3 h. The medium was changed to 10% FBS DMEM and the culturing was continued, and the observation and flow cytometry analysis of red fluorescence protein expression were performed at 48 h.

[0220] The flow cytometry results were shown in FIG. 24. After transduction of T-Cas9, the proportion of red fluorescent cells was low (3.7%), that was, most of the Cas9 protein failed to escape and enter into the nucleus; the introduction of pH-sensitive peptide INF7 (TI-Cas9) could increase the proportion of red fluorescent cells to 9%; and the further introduction of a protease cleavage site into the system (TINNE-Cas9), significantly increased the number of cells expressing red fluorescent protein, which could reach about 14% in 48 h. Therefore, during the endocytosis process of the delivery system carrying Cas9 into the cell, the introduction of CTSL and Furin specific sites N and Ne significantly promoted the escape process of the carried Cas9, and more Cas9 entered into the nucleus under the action of the nuclear localization signal. After binding to the GM3 sgRNA formed by intracellular transcription, Cas9 specifically recognized the target sequence, and homologous end repair would occur near this site, so that the red fluorescent gene entered the reading frame and was expressed.

Example 5: Establishment of Delivery System Based on Non-Covalent Connection

[0221] In addition to fusion expression, the linking mode between the fusion protein of the present invention and cargo could also be non-covalent interaction through protein domains, which called adapter pairs, such as heterodimer leucine zipper with strong interaction (FIG. 25). Two antiparallel leucine zipper domains can spontaneously combine to form an oligomer due to the complementarity in their spatial structure and charge distribution. It is reported that, NZ and CZ are such a pair of leucine zippers that can be combined with each other. We fused the NZ domain to the intracellular delivery system of the present invention (TINNe-NZ), and fused CZ domain to GFPβ1-10 with nuclear localization signal (NLS) as Cargo (CZ-GFPβ1-10-NLS). The two fused proteins were mixed and incubated to combine with each other, and were subjected to transduction into the HEK293T cells stably expressing Histone-β11, and the endosome escape efficiency could be evaluated by the proportion of GFP and the relative fluorescence intensity. Therefore, the feasibility of using the adapter pair of NZ-CZ to link the fusion protein to the cargo was evaluated.

[0222] 5.1 Construction of Expression Vector for Delivery System-NZ Fusion Protein and CZ-GFPβ1-10 Fusion Protein

[0223] Based on the pET32a vector, the expression vector for recombinant protein containing TAT, INF7, protease cleavage site and NZ domain, and the expression vector for recombinant protein containing CZ domain, cargo molecule GFPβ1-10 with nuclear localization signal (NLS), were constructed, in which TrxA was introduced as a solubilizing label. The structural schematic diagram of each recombinant protein was shown in FIG. 26, and the amino acid sequence of the delivery system was shown in the following table. The construction method was as follows: first, the aforementioned vector was used as a template, the nucleic acid sequences encoding TAT, INF7, N, Ne, and NZ sequences in the delivery system, as well as the CZ sequence and the cargo molecule sGFPβ1-10, were obtained by PCR amplification. For TrxA-TINNe-NZ, in the last round of PCR process, the BamHI restriction site and the overlap on upstream of the corresponding BamHI restriction site on pET-32a(+) were introduced at the 5′ end of the fragment through the forward primer, the HIindIII restriction site and the overlap on downstream of the corresponding HIindIII restriction site on pET-32a(+) were introduced at the 3′ end of the fragment through the reverse primer. The pET-32a(+) plasmid was subjected to double digestion with BamHI and HIindIII. The insert fragments with overlaps were ligated to the digested vector pET-32a(+) by GIBSON assembly. For CZ-GFP (31-10, in the last round of PCR process, the NdeI restriction site and the overlap on upstream of the corresponding NdeI restriction site on pET21b(+) were introduced at the 5′ end of the fragment through the forward primer, and the BamHI restriction site and the overlap on downstream of the corresponding BamHI restriction site on pET-21b(+) were introduced at the 3′ end of the fragment through the reverse primer. The pET-21b(+) plasmid was subjected to double digestion with NdeI and BamHI. The insert fragments with overlaps were ligated to the digested vector pET-21b(+) by GIBSON assembly.

TABLE-US-00007 TABLE 7 Components contained in delivery system components from N-terminal to C-terminal and their sequences Solubi- pH- Protease Specific Delivery lizing sensitive recognition binding system tag CPP peptide sequence sequence TINNe-NZ TrxA Tat INF7 N + Ne NZ SEQ ID SEQ ID SEQ ID SEQ ID NO: 53 NO: 10 NO: 8 NO: 49

[0224] 5.2 Expression and Purification of Delivery System-NZ Fusion Protein and CZ-GFPβ1-10 Fusion Protein

[0225] The expression plasmid described in 5.1 was transformed into the expression strain E. coli BL21 (DE3); a single colony was picked from the plate after transformation and inoculated in 5 ml of LB liquid medium containing ampicillin resistance and cultivated overnight. 1 ml of the overnight-cultured bacterial culture was transferred to 500 ml of LB liquid medium containing ampicillin resistance, cultured at 37° C. and 180 rpm until the bacterial culture had an OD.sub.600 of about 0.6. Then the inducer IPTG was added to reach a final concentration of 0.2 mM for induction at 25° C. for 8 h. After the induction, the bacterial cells were collected by centrifugation at 7000 g for 10 min at 4° C.; then the bacterial cells were re-suspended in 10 ml of equilibration buffer for protein purification (50 ml of glycerol, 8 g of NaCl, 0.201 g of KCl, 1.44 g of Na.sub.2HPO.sub.4, 0.24 g of KH.sub.2PO.sub.4, dissolved in 1 L of double distilled water) and ultrasonically disrupted. The supernatant was collected by centrifugation and was loaded on the protein purification column for polyhistidine-tagged protein of protein purification system; then the desired protein was eluted by the protein purification system using the elution buffer for protein purification, Elution buffer 2 # (50 ml of glycerol, 8 g of NaCl, 0.201 g of KCl, 1.44 g of Na.sub.2HPO.sub.4, 0.24 g of KH.sub.2PO.sub.4, 17 g of imidazole, dissolved in 1 L of double-distilled water). The protein concentration could be determined by spectrophotometer or BCA Protein Assay Kit. Each purified fusion protein was aliquoted and stored at −20° C. The SDS-PAGE results of each protein were shown in FIG. 27.

[0226] 5.3 Detection of Delivery Efficiency of Adapter-Based Delivery System by Split-GFP System

[0227] The HEK-293T-Hitone-GFPβ11 cell line obtained above was inoculated into a 12-well plate and cultured overnight, and it was ensured that the number of cells per well was about 5*10.sup.6/ml before protein treatment; then the cells were rinsed with serum-free DMEM medium three times. The control groups and the experimental group and the amount of protein (obtained in 5.2) they used, were shown in the following table, in which before incubating with the cells, the two proteins in the Control Group 2 and the experimental group were mixed in a serum-free medium at room temperature for 10 min. After incubation in a serum-free medium condition for 3 hours, the cells were washed three times with heparin solution to remove the protein that was adsorbed on the cell surface and had not yet been endocytosed, then the culturing was continued after changing the medium to 10% FBS DMEM medium, and the fluorescence microscope observation and the flow cytometry analysis of the proportion of green fluorescence positive cells and MFI were performed at 12 hours.

[0228] The flow cytometry analysis results were shown in FIG. 28. The results showed that the MFI values of the two control groups (CZ-GFPβ1˜10/TrxA-TINNe+CZ-GFPβ1˜10) were both lower than 5, in which the Control Group 2 (TrxA-TINNe+CZ-GFPβ1˜10) had MFI slightly higher than that of the Control Group 1. We speculated that it was caused by the non-specific adsorption between TrxA-TINNe and CZ-GFPβ1˜10, which enabled the cargo CZ-GFPβ1˜10 to enter into the cell and escape from the endosome by TINNe. The MFI value of TrxA-TINNe-NZ+CZ-GFPβ1˜10 group was close to 20, which was significantly higher than that of the control groups, which indicated that under the action of NZ and CZ, TrxA-TINNe combined with GFPβ1˜10 in a certain degree, and through the action of TINNe, GFPβ1˜10 entered into the cell and escaped from the endosome. Although the fluorescence intensity of the adapter-based linking method was still lower than that of the TINNe-GFPβ1˜10 which was obtained by fusion expression, the experiment confirmed that the linking method based on NZ-CZ or similar adapter could be used to link the delivery system with the cargo, and could effectively deliver the cargo to enter into the cell and escape from endosome.

TABLE-US-00008 TABLE 8 Control and experimental groups protein to be added and its final concentration TrxA- TrxA- CZ-GFP TINNe-GFP Group TINNe TINNe-NZ β1-10 β1-10 Control Group 1 5 μM Control Group 2 5 μM 5 μM Control Group 3 5 μM Experimental 5 μM 5 μM Group

[0229] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details according to all the teachings that have been disclosed, and these changes are within the protection scope of the present invention. All of the present invention is given by the appended claims and any equivalents thereof