Composition system of protein modified effector and reverse transcriptor and application

Abstract

The invention provides a protein modification effector and reverse transcriptor composition system and application, and particularly provides a method for specifically modifying target protein, which comprises the following steps: in the presence of fusion protein and chimeric RNA (Ribonucleic Acid) or precursor ncRNA thereof, specifically modifying the target protein in a cell, the fusion protein and the chimeric RNA disclosed by the invention can be used for carrying out specific modification on a target protein.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a composition system of a protein modification effector and a reverse transcriptase and uses thereof. Background Art

[0002] Retromers are reverse transcription elements that are widely found in bacteria. Retromer operons include non-coding RNAs msd (multi-copy single-stranded RNA) and msd (multi-copy single-stranded DNA), as well as reverse transcriptase RT. Retromers can be expressed in hosts such as bacteria, yeast, and mammalian cells, and have been used in the fields of donor templates for gene editing, directed evolution, and molecular recording.

[0003] Targeted protein modification is a method of specifically modifying a target protein, often using small molecules or proteins, nucleic acids, etc. to bring the target protein and the protein modification effector closer in space, thereby achieving modification of the target protein. Currently, there is no research or report on the use of retrotransposons for targeted protein modification, and retrotransposons have not been able to modify proteins in human cells so far.

[0004] Therefore, there is an urgent need in the art to develop a composition system of a protein modification effector and a reverse transcriptase and its use; it can be used in the fields of gene therapy and crop breeding. Summary of the invention

[0005] The purpose of the present invention is to provide a composition system of a protein modification effector and a reverse transcriptase and its use.

[0006] In a first aspect of the present invention, a method for specifically modifying a target protein is provided, comprising the steps of:

[0007] (S1) in the presence of the fusion protein and the chimeric RNA or its precursor ncRNA, the target protein in the cell is specifically modified;

[0008] The fusion protein comprises: (a) a first fusion element, which is a protein modification effector or a recruitment element of a protein modification effector; and (b) a second fusion element, which is a reverse transcriptase, and the first fusion element and the second fusion element are fused together;

[0009] The chimeric RNA comprises a first functional module and a second functional module, wherein the first functional module binds to a reverse transcriptase; and the second functional module binds to a target protein;

[0010] When the chimeric RNA binds to the reverse transcriptase through the first functional module and binds to the target protein through the second functional module, the fusion protein specifically modifies the target protein.

[0011] In another preferred embodiment, the structure of the fusion protein is shown in Formula I below:

[0012] W0-W1-W2(I)

[0013] In the formula,

[0014] W0 is none, tag element, nuclear localization element, or a combination thereof;

[0015] W2 is none, a tag element, a nuclear localization element, or a combination thereof;

[0016] The structure of W1 is shown in the following formula Ia or Ib:

[0017] ALB(Ia)

[0018] BLA(Ib)

[0019] in,

[0020] A is a protein modification effector or a recruitment element of a protein modification effector, and the protein modification effector specifically modifies or demodifies the target protein adjacent to it depending on the effect of spatial distance;

[0021] B is reverse transcriptase;

[0022] L is none or a connecting peptide,

[0023] Each "-" is independently a peptide linker, a peptide bond, or a non-peptide bond.

[0024] In another preferred example, the first functional module is the MSR domain, and the second functional module is the MSD domain.

[0025] In another preferred example, the second functional module is the MSR domain, and the first functional module is the MSD domain.

[0026] In another preferred embodiment, the region binding to the target protein is the MSR domain and / or the MSD domain.

[0027] In another preferred embodiment, the MSR domain and / or MSD domain may be a wild-type or modified MSR domain and / or MSD domain.

[0028] In another preferred embodiment, the modified MSR domain and / or MSD domain is an MSR domain and / or MSD domain with a heterologous binding element.

[0029] In another preferred embodiment, the MSR domain and the MSD domain are not wild-type at the same time.

[0030] In another preferred embodiment, the chimeric RNA is an RNA-DNA hybrid molecule formed by processing the non-coding RNA (ncRNA).

[0031] In another preferred embodiment, the precursor ncRNA forms a chimeric RNA after processing.

[0032] In another preferred embodiment, the processing includes reverse transcription starting from guanosine (G), a reverse transcription start site in the MSR domain, to form the MSD domain.

[0033] In another preferred embodiment, the MSD domain in the chimeric RNA is connected to the reverse transcription start site guanosine (G) in the MSR domain (preferably, connected to the 2' end of the G).

[0034] In another preferred embodiment, the nucleic acid sequence of the chimeric RNA includes the structure shown in the following formula II from the 5' end to the 3' end:

[0035]

[0036] In the formula,

[0037] Z1 is the 5' end sequence to the left of the guanosine (G) at the start site of reverse transcription;

[0038] G is the reverse transcription start site guanosine (G);

[0039] Z2 is the 3' end sequence to the right of the guanosine (G) at the start of reverse transcription

[0040] Z1-G-Z2 constitutes the MSR sequence;

[0041] Z3 is the MSD sequence.

[0042] In another preferred embodiment, the MSR sequence is connected to the 5' end of the MSD sequence via a 2'-5' phosphodiester bond at the 2' position of the guanosine (G) residue at the reverse transcription start site.

[0043] In another preferred embodiment, the nucleic acid sequence of the chimeric RNA also includes a heterologous sequence.

[0044] In another preferred embodiment, the heterologous sequence specifically binds to the target protein.

[0045] In another preferred embodiment, the heterologous sequence replaces a partial segment of the MSD and / or MSR (such as an editable region sequence), thereby guiding the protein modification effector to specifically modify or demodify the target protein bound to the heterologous sequence.

[0046] In another preferred example, the stem-loop of the MSR or the stem-loop of the MSD may contain one or more mismatched nucleotides.

[0047] In another preferred example, the MSR sequence and the MSD sequence are provided in a trans arrangement or a cis arrangement.

[0048] In another preferred example, the nucleotide sequence encoding the reverse transcriptase (RT) is provided in a trans arrangement relative to the MSR sequence or the MSD sequence.

[0049] In another preferred embodiment, the reverse transcriptase (RT) is selected from the following group: Ec86RT, Ec67RT, Ec73RT, Ec107RT, Sa163RT or a combination thereof.

[0050] In another preferred embodiment, the protein modification effector is a protein or enzyme that can perform post-translational modification on a protein, and in particular refers to a protein or enzyme that modifies an adjacent target protein through a spatial distance effect.

[0051] In another preferred embodiment, the protein modification effector is selected from the following group: ubiquitinase, deubiquitinase, phosphorylase, dephosphorylase, glycosylase, deglycosylase, acetylase, methylase, biotinylase, or a combination thereof.

[0052] In another preferred embodiment, the recruitment element of the protein modification effector is a protein or polypeptide ligand that binds to the protein modification effector.

[0053] In another preferred embodiment, the protein or polypeptide ligand includes (but is not limited to): VHL E3 ubiquitin ligase ligand, P21 peptide, amino acids 167-374 of SPOP protein and other E3 ubiquitin ligases and truncated forms thereof.

[0054] In another preferred embodiment, the VHL E3 ubiquitin ligase ligand is selected from the following group: ALAPYIP, ALAAYAP, or a combination thereof.

[0055] In another preferred embodiment, the specifically modified protein modification effectors include (but are not limited to): VHL E3 ubiquitin ligase, CRBN E3 ubiquitin ligase, SPOP E3 ubiquitin ligase, E2 ubiquitin conjugating enzyme, histone acetyltransferase p300, adenylate-activated protein kinase (AMPK), protein kinase C (PKC), O-GlcNAc transferase, proteasome-targeting peptide (PTD), E3 ubiquitin protein ligase TRIM21, ABI1 (ABAinsensitive 1, ABA is abscisic acid) transcriptional regulatory factor.

[0056] In another preferred embodiment, the demodified protein modification effectors include (but are not limited to): glycosidase, phosphatase, deubiquitinating enzyme.

[0057] In another preferred embodiment, the length of the connecting peptide is 1-100 aa, preferably, 10-85 aa, and more preferably, 15-70 aa.

[0058] In another preferred embodiment, the amino acid sequence of the connecting peptide is selected from the following group:

[0059] (1) GS, SGS, SGGS, (SG) n or (GGGS) n , where n is 1-50;

[0060] (2) a polypeptide as shown in any one of SEQ ID NOs. 1-5;

[0061] (3) A polypeptide derived from a polypeptide having the function of the polypeptide as described in (2) formed by substituting, deleting or adding one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, and most preferably 1 amino acid residues in any one of the amino acid sequences as described in SEQ ID NOs. 1-5.

[0062] In another preferred embodiment, the protein modification effector includes a wild-type or mutant protein modification effector.

[0063] In another preferred embodiment, the amino acid sequence of the protein modification effector is selected from the following group:

[0064] (1) a polypeptide with an amino acid sequence as shown in any one of SEQ ID NOs. 6-10;

[0065] (2) A polypeptide derived from a polypeptide having the function of the polypeptide described in (1) (e.g., having double-stranded DNA cleavage activity) formed by replacing, deleting or adding one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, and most preferably 1 amino acid residues in the amino acid sequence shown in any one of SEQ ID NOs. 6-10.

[0066] In another preferred embodiment, the amino acid sequence of the reverse transcriptase is selected from the following group:

[0067] (1) A polypeptide with an amino acid sequence as shown in any one of SEQ ID NOs. 11-13;

[0068] (2) A polypeptide derived from a polypeptide having the function of the polypeptide described in (1) (e.g., having double-stranded DNA cleavage activity) formed by replacing, deleting or adding one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, and most preferably 1 amino acid residues in the amino acid sequence shown in any one of SEQ ID NOs. 111-13.

[0069] In another preferred embodiment, the amino acid sequence of the nuclear localization element is selected from the following group:

[0070] (1) the amino acid sequence shown in SEQ ID NO.14;

[0071] (2) A polypeptide derived from the polypeptide with the amino acid sequence shown in SEQ ID NO. 14 having the function of the polypeptide described in (1), formed by substituting, deleting or adding one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, and most preferably 1 amino acid residues in the amino acid sequence shown in SEQ ID NO. 14.

[0072] In another preferred embodiment, the amino acid sequence of the tag element is selected from the following group:

[0073] (1) the amino acid sequence shown in any one of SEQ ID NOs. 15-16;

[0074] (2) A polypeptide derived from a polypeptide having the function of the polypeptide described in (1) which is formed by substituting, deleting or adding one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, and most preferably 1 amino acid residues in the amino acid sequence shown in any one of SEQ ID NOs. 15-16.

[0075] In another preferred embodiment, the amino acid sequence of the fusion protein is selected from the following group:

[0076] (1) a polypeptide with an amino acid sequence as shown in any one of SEQ ID NOs. 17-21;

[0077] (2) A polypeptide derived from a polypeptide having the function of the polypeptide described in (1) (e.g., having double-stranded DNA cleavage activity) formed by substituting, deleting or adding one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, and most preferably 1 amino acid residues in the amino acid sequence shown in any one of SEQ ID NOs. 17-21.

[0078] In another preferred embodiment, the fusion protein has an amino acid sequence shown in any one of SEQ ID NOs. 17-21.

[0079] In another preferred embodiment, the editable region sequence of the MSD is selected from the following group:

[0080] (1) a sequence from position 95 to position 136 relative to the nucleotide sequence of SEQ ID NO: 22;

[0081] (2) a CTT sequence from position 79 to position 81 relative to the nucleotide sequence of SEQ ID NO: 23; or,

[0082] (3) The sequence from position 120 to position 215 relative to the nucleotide sequence shown in SEQ ID NO:24.

[0083] In another preferred embodiment, the editable region sequence of the MSR is selected from the following group:

[0084] (1) a TATCATTAAGGTC sequence from position 24 to position 36 relative to the nucleotide sequence of SEQ ID NO: 22;

[0085] (2) a GTTTC sequence from position 51 to position 55 relative to the nucleotide sequence shown in SEQ ID NO: 22;

[0086] (3) a GTGAATCGC sequence from position 27 to position 35 relative to the nucleotide sequence shown in SEQ ID NO: 23;

[0087] (4) a CCTCGAG sequence from position 43 to position 49 relative to the nucleotide sequence of SEQ ID NO: 24; or

[0088] (5) The CCTCGAG sequence from position 61 to position 67 relative to the nucleotide sequence shown in SEQ ID NO:25.

[0089] In another preferred embodiment, the retromer is selected from the group consisting of Ec86, Ec67, Ec73, Ec107, Sa163, or a combination thereof.

[0090] In another preferred embodiment, the nucleic acid sequence of the non-coding RNA of the reverse transcriptase is selected from the following group:

[0091] (i) a polynucleotide whose sequence is shown in any one of SEQ ID NOs. 22-24;

[0092] (ii) a polynucleotide having a nucleotide sequence homology ≥80%, preferably ≥95% (more preferably ≥98%, more preferably ≥99%) to any one of SEQ ID NOs. 22-24;

[0093] (iii) a polynucleotide having 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide shown in any one of SEQ ID NOs. 22 to 24;

[0094] (iv) a polynucleotide complementary to the polynucleotide described in any one of (i) to (iii).

[0095] In another preferred embodiment, the heterologous sequence refers to a DNA or RNA nucleotide sequence that specifically binds to the target protein.

[0096] In another preferred embodiment, the form of the heterologous sequence includes (but is not limited to): a double-stranded DNA form formed by a hairpin or stem-loop structure, an adaptor form, a G-quadruplex form, or a combination thereof.

[0097] In another preferred embodiment, the heterologous sequence is selected from the following group:

[0098] (i) a polynucleotide whose sequence is shown in any one of SEQ ID NOs. 25-26;

[0099] (ii) a polynucleotide having a nucleotide sequence homology ≥ 80%, preferably ≥ 95% (more preferably ≥ 98%, more preferably ≥ 99%) to any one of SEQ ID NOs. 25-26;

[0100] (iii) a polynucleotide having 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide shown in any one of SEQ ID NOs. 25-26;

[0101] (iv) a polynucleotide complementary to the polynucleotide described in any one of (i) to (iii).

[0102] In another preferred embodiment, the target protein is selected from the following group: P50, P65, cMYC, or a combination thereof.

[0103] In another preferred embodiment, the sequence of the chimeric RNA is selected from the following group:

[0104] (i) a polynucleotide whose sequence is shown in SEQ ID NO.27-28;

[0105] (ii) a polynucleotide having a nucleotide sequence homology ≥80%, preferably ≥95% (more preferably ≥98%, more preferably ≥99%) to the sequence shown in SEQ ID NO. 27-28;

[0106] (iii) a polynucleotide having 1 to 60 (preferably 1 to 30, more preferably 19 to 22) nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotides shown in SEQ ID NOs. 27-28;

[0107] (iv) a polynucleotide complementary to the polynucleotide described in any one of (i) to (iii).

[0108] In another preferred embodiment, the method utilizes a heterologous sequence to recruit a target protein, and recruits the protein modification effector through a reverse transcriptase RT-coupled protein modification effector or a reverse transcriptase RT-coupled polypeptide ligand, thereby specifically modifying the target protein.

[0109] In another preferred embodiment, the protein modification effector preferentially modifies the target protein.

[0110] In another preferred example, the heterologous sequence is located in the MSD region or the MSR region.

[0111] In another preferred embodiment, the method utilizes the heterologous sequence located in the MSD and the heterologous sequence located in the MSR to recruit the target protein and the protein modification effector respectively, shortening the spatial distance between the protein modification effector and the target protein, thereby specifically modifying the target protein.

[0112] In another preferred embodiment, the cells include human or non-human mammalian cells (such as primates, mice, rats or livestock) or plant cells.

[0113] In another preferred embodiment, the cells include cancer cells or normal cells.

[0114] In another preferred embodiment, the cells are selected from the following groups: mouse brain neuroma cells, human embryonic kidney cells, or human cervical cancer cells, human colon cancer cells, human osteosarcoma cells, kidney cells, liver cells, nerve cells, heart cells, epithelial cells, muscle cells, somatic cells, bone marrow cells, endothelial cells, or a combination thereof.

[0115] In another preferred embodiment, the cells are selected from the following group: 293 cells, A549 cells, SW626 cells, HT-3 cells, PA-1 cells, N2a cells, Hela cells, HCT116 cells, U2OS cells, or a combination thereof.

[0116] In another preferred embodiment, the cells are selected from the following group: N2a cells, HEK293FT cells, Hela cells, HCT116 cells, or U2OS cells.

[0117] In another preferred embodiment, the protein modification is carried out in an in vitro reaction system.

[0118] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0119] In another preferred embodiment, the cells are in vitro cells.

[0120] In the second aspect of the present invention, a fusion protein is provided, the structure of the fusion protein is shown in the following formula I:

[0121] W0-W1-W2(I)

[0122] In the formula,

[0123] W0 is none, tag element, nuclear localization element, or a combination thereof;

[0124] W2 is none, a tag element, a nuclear localization element, or a combination thereof;

[0125] The structure of W1 is shown in the following formula Ia or Ib:

[0126] ALB(Ia)

[0127] BLA(Ib)

[0128] in,

[0129] A is a protein modification effector or a recruitment element of a protein modification effector, and the protein modification effector specifically modifies or demodifies the target protein adjacent to it depending on the effect of spatial distance;

[0130] B is reverse transcriptase;

[0131] L is none or a connecting peptide,

[0132] Each "-" is independently a peptide linker, a peptide bond, or a non-peptide bond.

[0133] In another preferred embodiment, the non-peptide bond comprises PEG.

[0134] In the third aspect of the present invention, a chimeric RNA is provided, wherein the nucleic acid sequence of the chimeric RNA comprises the structure shown in the following formula II from the 5' end to the 3' end:

[0135]

[0136] In the formula,

[0137] Z1 is the 5' end sequence to the left of the guanosine (G) at the start site of reverse transcription;

[0138] G is the reverse transcription start site guanosine (G);

[0139] Z2 is the 3' end sequence to the right of the guanosine (G) at the start of reverse transcription

[0140] Z1-G-Z2 constitutes the MSR sequence;

[0141] Z3 is the MSD sequence.

[0142] In the fourth aspect of the present invention, a nucleic acid is provided, wherein the nucleic acid encodes the fusion protein described in the second aspect of the present invention and / or encodes the chimeric RNA described in the third aspect of the present invention.

[0143] In the fifth aspect of the present invention, an expression vector is provided, wherein the expression vector contains the nucleic acid described in the fourth aspect of the present invention.

[0144] In the sixth aspect of the present invention, an isolated host cell is provided, comprising the fusion protein of the second aspect of the present invention and / or encoding the chimeric RNA of the third aspect of the present invention, or the expression vector of the fifth aspect of the present invention.

[0145] In another preferred embodiment, the host cell is a prokaryotic cell, an archaebacterial or a eukaryotic host cell.

[0146] In another preferred embodiment, the eukaryotic host cell is a mammalian host cell.

[0147] In another preferred embodiment, the host cell is an artificial cell or a genetically modified cell.

[0148] In a seventh aspect of the present invention, a protein modification reagent is provided, the protein modification reagent comprising:

[0149] (a) the fusion protein according to the second aspect of the present invention, or a gene encoding the fusion protein or an expression vector thereof; and

[0150] (b) The chimeric RNA or its expression vector according to the third aspect of the present invention.

[0151] In another preferred embodiment, the expression vector comprises a viral vector.

[0152] In another preferred embodiment, the viral vector is selected from the following group: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.

[0153] In another preferred embodiment, the vector is selected from the following group: lentivirus, adenovirus, adeno-associated virus (AAV), or a combination thereof. Preferably, the vector is adeno-associated virus (AAV).

[0154] In the eighth aspect of the present invention, a kit is provided, which comprises the fusion protein described in the second aspect of the present invention and / or the chimeric RNA encoding the third aspect of the present invention, or the expression vector described in the fifth aspect of the present invention, or the host cell described in the sixth aspect of the present invention or the protein modification reagent described in the seventh aspect of the present invention.

[0155] In another preferred embodiment, the kit further comprises a label or contains instructions for using the method described in the first aspect of the present invention to specifically modify the target protein in the cell.

[0156] In a ninth aspect of the present invention, a composition is provided, comprising:

[0157] (a) the fusion protein according to the second aspect of the present invention, or a gene encoding the fusion protein or an expression vector thereof; and

[0158] (b) The chimeric RNA or its expression vector according to the third aspect of the present invention.

[0159] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0160] In another preferred embodiment, the expression vector comprises a viral vector.

[0161] In another preferred embodiment, the viral vector is selected from the following group: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.

[0162] In another preferred embodiment, the vector is selected from the following group: lentivirus, adenovirus, adeno-associated virus (AAV), or a combination thereof. Preferably, the vector is adeno-associated virus (AAV).

[0163] In another preferred embodiment, the gene encoding the fusion protein and the chimeric RNA are located in the same expression vector (such as an AAV vector) or different expression vectors.

[0164] In another preferred embodiment, the expression vector of the fusion protein and the expression vector of the chimeric RNA are the same expression vector (such as an AAV vector) or different expression vectors.

[0165] In another preferred embodiment, the dosage form of the composition is selected from the following group: a lyophilized preparation, a liquid preparation, or a combination thereof.

[0166] In another preferred embodiment, the composition is in the form of a liquid preparation.

[0167] In another preferred embodiment, the composition is in the form of an injection.

[0168] In another preferred embodiment, the composition is a cell preparation.

[0169] In another preferred embodiment, the expression vector of the fusion protein and the expression vector of the chimeric RNA are the same vector or different vectors.

[0170] In another preferred embodiment, the molar ratio of the component (a) to the component (b) is 100:1-0.01:1, preferably, 10:1-0.1:1, and more preferably, 2:1-0.5:1.

[0171] In another preferred embodiment, in the composition, the content of component (a) is 0.001%-99%, preferably 50%-99%, more preferably 90%-99%.

[0172] In another preferred embodiment, in the composition, the content of component (b) is 0.001%-99%, preferably, 0.1%-50%, more preferably, 0.1%-10%.

[0173] In another preferred embodiment, in the composition, the component (a) and the component (b) account for 0.01-99.99 wt %, preferably 0.1-90 wt %, and more preferably 1-80 wt % of the total weight of the composition.

[0174] In a tenth aspect of the present invention, a medicine kit is provided, comprising:

[0175] (a1) a first container, and the fusion protein according to the second aspect of the present invention, or a gene encoding it or an expression vector thereof, or a drug containing the fusion protein according to the second aspect of the present invention, or a gene encoding it or an expression vector thereof, located in the first container;

[0176] (b1) a second container, and the chimeric RNA or its expression vector according to the third aspect of the present invention, or a drug containing the chimeric RNA or its expression vector according to the third aspect of the present invention, located in the second container.

[0177] In another preferred embodiment, the medicine kit further comprises:

[0178] (c1) a third container, and other substances for improving protein modification efficiency and / or drugs containing other substances for improving protein modification efficiency located in the third container.

[0179] In another preferred embodiment, the first container, the second container and the third container are the same or different containers.

[0180] In another preferred embodiment, the drug in the first container is a single preparation containing the fusion protein described in the second aspect of the present invention, or its encoding gene or its expression vector.

[0181] In another preferred embodiment, the medicine in the second container is a single preparation containing the chimeric RNA or its expression vector described in the third aspect of the present invention.

[0182] In another preferred embodiment, the drug in the third container is a single preparation containing other substances that improve protein modification efficiency.

[0183] In another preferred embodiment, the dosage form of the drug is selected from the following group: a lyophilized preparation, a liquid preparation, or a combination thereof.

[0184] In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

[0185] In another preferred embodiment, the kit further contains instructions.

[0186] In the eleventh aspect of the present invention, there is provided a method as described in the first aspect of the present invention, or a fusion protein as described in the second aspect of the present invention, or a chimeric RNA as described in the third aspect of the present invention, or a nucleic acid as described in the fourth aspect of the present invention, or an expression vector as described in the fifth aspect of the present invention, or a host cell as described in the sixth aspect of the present invention, or a protein modification reagent as described in the seventh aspect of the present invention, or a composition as described in the ninth aspect of the present invention, or a use of the drug kit as described in the tenth aspect of the present invention for preparing a reagent or kit for specifically modifying a target protein.

[0187] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0188] Figure 1 Shown is a schematic diagram of targeted protein labeling using retrotransposons.

[0189] Figure 2 The diagram shows the coding DNA of the reverse transcriptase and the formation process of the reverse transcriptase. The coding gene of the reverse transcriptase RT is expressed to produce the reverse transcriptase RT protein, which catalyzes the non-coding RNA (ncRNA) to form msd, and RT forms a complex with msd and msr.

[0190] Figure 3It shows a schematic diagram of genetic engineering modification using reverse transcriptase for targeted protein labeling, wherein Figure A is a design diagram of RT fusion protein, the design of the fusion protein is to connect the reverse transcriptase and the protein modification effector through a linker, wherein the reverse transcriptase can be at the N-terminus of the fusion protein, and the protein modification effector is at the C-terminus of the fusion protein; or the reverse transcriptase can be at the C-terminus of the fusion protein, and the protein modification effector is at the N-terminus of the fusion protein; Figure B is a design diagram of non-coding RNA (ncRNA), wherein the editable region of msr ​​or msd in the ncRNA is replaced with a heterologous sequence that can bind to the target protein.

[0191] Figure 4 Shown are the msr and msd structures and DNA sequences of the retrotransposons Ec86. Figure A shows the msr and msd structures of the retrotransposons Ec86, with the red part being msr and the blue part being msd; the underlined GG is a mutation introduced in mammalian cells to prevent the polyT terminator, and the blue circled G is the reverse transcription start site; the red framed part is the region where msr can be engineered to introduce heterologous sequences, and the blue framed part is the region where msd can be engineered to introduce heterologous sequences; Figure B shows the ncRNA encoding DNA sequence of Ec86, where ncRNA is in red font and msd is in blue font. The red framed part is the region where msr can be engineered to introduce heterologous sequences, and the blue framed part is the region where msd can be engineered to introduce heterologous sequences. This figure takes Ec86 as an example to introduce the modification design of ncRNA.

[0192] Figure 5 It is shown that the MSD region of the engineered retrotransposons is used to form DNA hairpin / stem-loop structures or DNA aptamers for targeted protein labeling.

[0193] Figure 6 It is shown that the msr region of the engineered reverse transcriptase is used to form an RNA hairpin / stem-loop structure or an RNA aptamer for targeted protein labeling.

[0194] Figure 7 Shown is targeted protein labeling using engineered retrotransposons MSD and MSR to recruit target proteins and protein-modifying effectors.

[0195] Figure 8 The figure shows the use of engineered retrotransposons to target ubiquitination modification of p50 and p65 proteins for targeted protein degradation. The protein modification effector is an E3 ubiquitin ligase. This figure is based on the principles of the present invention, using E3 ubiquitin ligases to target ubiquitination modification of p50 and p65 proteins, thereby degrading p50 and p65 proteins, wherein PTD, VHL and SPOP all represent E3 ubiquitin ligases, CTRL represents a control, and α-GAPDH represents an internal reference.

[0196] Fig. 9 The figure shows that the cMYC protein is targeted for ubiquitination modification by using a fusion protein plasmid, thereby performing targeted protein degradation. The protein modification effector is an E3 ubiquitin ligase. This figure is based on the principle of the present invention, and uses an E3 ubiquitin ligase to target ubiquitinate modification on the cMYC protein in Hela cells, thereby degrading the cMYC protein.

[0197] Fig.10 Shown is the structure diagram of the chimeric RNA sequence composed of the retromer Ec86 and the E-BOX binding sequence, where Figure A is the structure diagram of the msr and msd of the retromer Ec86, the ncRNA is the red font part, and the blue font part is the msd; the underlined GG is a mutation introduced in mammalian cells to prevent the polyT terminator, and the G in the blue circle is the reverse transcription start site; the blue frame part is the region where msd can be engineered to introduce a heterologous sequence (E-BOX site sequence); Figure B is the ncRNA encoding DNA sequence of Ec86, where ncRNA is the red font part, and msd is the blue font part; the blue frame part is the region where msd can be engineered to introduce an E-BOX site sequence.

[0198] Fig.11 Shown is the structure diagram of the chimeric RNA sequence composed of the retrotransposon Ec86 and the NF-κB binding sequence, where Figure A is the structure diagram of the msr and msd of the retrotransposon Ec86, the ncRNA is the red font part, and the blue font part is the msd; the underlined GG is a mutation introduced in mammalian cells to prevent the polyT terminator, and the G in the blue circle is the reverse transcription start site; the blue frame part is the region where the msd can be engineered to introduce a heterologous sequence (NF-κB site sequence); Figure B is the ncRNA encoding DNA sequence of Ec86, where the ncRNA is the red font part, and the msd is the blue font part; the blue frame part is the region where the msd can be engineered to introduce a NF-κB site sequence. DETAILED DESCRIPTION

[0199] The inventors have conducted extensive and in-depth research and obtained unexpectedly a fusion protein and chimeric RNA through a large number of screenings. The fusion protein and chimeric RNA of the present invention can achieve arbitrary specific modification of the target protein. It is unexpectedly found that the present invention can degrade the target protein after ubiquitination modification of the target protein in the cell nucleus. On this basis, the inventors have completed the present invention.

[0200] Specifically, Figure 1As shown, the present invention provides a method for transforming the msd or msr part of a retron into a DNA or RNA that can bind to a protein, and coupling a protein modification effector or its recruiting ligand to the reverse transcriptase (RT) of the retron system, thereby achieving specific targeted protein modification.

[0201] the term

[0202] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0203] The terms "reverse transcriptase", "reverse transcriptase" and "RT" can be used interchangeably and refer to the reverse transcriptase described in the first aspect of the present invention.

[0204] The terms "msd" and "MSD" are used interchangeably to refer to multi-copy single-stranded DNA.

[0205] The terms "msr" and "MSR" are used interchangeably to refer to multiple copy single-stranded RNA.

[0206] The terms "editable region", "modification region" and "modifiable region" can be used interchangeably and refer to the region where the heterologous sequence is replaced.

[0207] The terms "connector", "connector peptide" and "connector peptide segment" can be used interchangeably and all refer to the connecting peptide described in the first aspect of the present invention. The length of the connecting peptide is 1-100 aa, preferably 10-85 aa, and more preferably 15-70 aa.

[0208] The terms "targeting protein", "target protein", "target protein", and "target protein" can be used interchangeably.

[0209] The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0210] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0211] Sequence identity (or homology) is determined by comparing two aligned sequences along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

[0212] As used herein, the term "complementary" refers to a polynucleotide that can form base pairs with each other. Base pairs are usually formed by hydrogen bonds between nucleotide units oriented in antiparallel between polynucleotide chains. Complementary polynucleotide chains can perform base pairing in a Watson-Crick manner (e.g., A and T, A and U, C and G) or in any other manner that allows the formation of a duplex. As known to those skilled in the art, when RNA is used instead of DNA, it is believed that the base complementary to adenosine is uracil (U) instead of thymine (T). However, unless otherwise stated, when uracil is represented in the context of the present invention, the ability to replace thymine is implied. "Complementarity" can exist between two RNA chains, two DNA chains, or between an RNA chain and a DNA chain. It is generally understood that two or more polynucleotides can be "complementary" and can form a duplex, although with incomplete or less than 100% complementarity. Two sequences are "fully complementary" or "100% complementary" if at least one contiguous portion of each polynucleotide sequence (including the region of complementarity) is perfectly base paired with the other polynucleotide without any mismatches or interruptions in such region. Two or more sequences are considered to be "fully complementary" or "100% complementary" even if one or both polynucleotides contain additional non-complementary sequences, as long as the contiguous region of complementarity in each polynucleotide is able to fully hybridize with the other. "Incomplete" complementarity refers to a situation where less than all of the contiguous nucleotides in such a complementary region are able to base pair with each other. Determining the percentage of complementarity between two polynucleotide sequences is a matter for those of ordinary skill in the art.

[0213] Protein modification effector

[0214] Protein modification effectors are proteins or enzymes that can perform post-translational modifications on proteins, especially proteins or enzymes that modify adjacent proteins through spatial distance effects.

[0215] In the present invention, the protein modification effector is selected from the following group: ubiquitinase, deubiquitinase, phosphorylase, dephosphorylase, glycosylase, deglycosylase, acetylase, methylase, biotinylase, or protein or polypeptide ligands bound thereto, or a combination thereof.

[0216] In another preferred embodiment, the protein or polypeptide ligand refers to a protein or polypeptide ligand that can bind to and recruit the protein modification effector.

[0217] In another preferred embodiment, the protein or polypeptide ligand includes (but is not limited to): VHL E3 ubiquitin ligase ligand, P21 peptide, amino acids 167-374 of SPOP protein (abbreviated as SPOP167-374) and other E3 ubiquitin ligases and truncated forms thereof.

[0218] In another preferred embodiment, the VHL E3 ubiquitin ligase ligand is selected from the following group: ALAPYIP, ALAAYAP, or a combination thereof.

[0219] In another preferred embodiment, the specifically modified protein modification effectors include (but are not limited to) VHL E3 ubiquitin ligase, CRBN E3 ubiquitin ligase, SPOP E3 ubiquitin ligase, E2 ubiquitin conjugating enzyme, histone acetyltransferase p300, adenylate-activated protein kinase (AMPK), protein kinase C (PKC), O-GlcNAc transferase, proteasome-targeting peptide (PTD), E3 ubiquitin protein ligase TRIM21, ABI1 (ABAinsensitive 1, ABA is abscisic acid) transcriptional regulatory factor.

[0220] In another preferred embodiment, the demodified protein modification effectors include (but are not limited to): glycosidase, phosphatase, deubiquitinating enzyme.

[0221] Retrons

[0222] Retron is a unique DNA sequence that encodes reverse transcriptase, similar to the reverse transcriptase produced by retroviruses. Retron consists of two parts: reverse transcriptase and ncRNA. Retron is widely present in many different bacterial genomes and is usually associated with prophage DNA. Retron consists of a unique DNA of about 2kb. The promoter sequence encoded by Retron DNA regulates the transcription of a simple operon consisting of three sites (msr, msd and ret).

[0223] like Figure 2 As shown, the retromer operon includes the non-coding RNAs msr (multi-copy single-stranded RNA) and msd (multi-copy single-stranded DNA), and the reverse transcriptase RT.

[0224] In another preferred embodiment, the retromer is selected from the group consisting of Ec86, Ec67, Ec73, Ec107, Sa163, or a combination thereof. Figure 4 Taking Ec86 as an example, the modification and design of ncRNA is introduced. Figure 4 A is the msr and msd structure diagram of the retromer Ec86, the red part is msr, and the blue part is msd; the underlined GG is a mutation introduced in mammalian cells to prevent the polyT terminator, and the G in the blue circle is the reverse transcription start site; the red framed part is the region where msr can be engineered to introduce heterologous sequences, and the blue framed part is the region where msd can be engineered to introduce heterologous sequences. Figure 4 B is the ncRNA coding DNA sequence of the retrotransposon Ec86, wherein ncRNA is the red font part and msd is the blue font part. The red frame part is the area where msr can be engineered to introduce heterologous sequences, and the blue frame part is the area where msd can be engineered to introduce heterologous sequences.

[0225] chimeric RNA

[0226] The present invention is the first to specifically modify or de-modify the target protein by combining the non-coding RNA (ncRNA) of the reverse transcriptor with the guide protein modification effector in a specific order. The non-coding RNA folds itself to form a specific structure, and is partially reverse transcribed by RT to generate about 20-200 bases of msd DNA, and the RNA sequence complementary to msd is removed by endogenous RNase H in the cell, finally forming msd single-stranded DNA. MSD includes a hairpin / stem-loop structure, and a part of the hairpin / stem-loop structure can be transformed into a heterologous sequence. Reverse transcriptase RT forms a protein-nucleic acid complex with msr and msd.

[0227] The chimeric RNA of the present invention comprises a msr pre-sequence, an msr gene encoding multiple copies of single-stranded RNA (msRNA); an msd gene encoding multiple copies of single-stranded DNA (msDNA); an msd post-sequence; a target heterologous sequence and a gene encoding a reverse transcriptase. The DNA synthesized by the reverse transcriptase result encoded by the retrotransposons can provide a DNA / RNA chimeric product consisting of a single-stranded DNA encoded by the msd gene and a single-stranded RNA encoded by the msr gene. The retrotransposons msr RNA contains a conserved guanosine residue (G) at the end of the stem-loop structure. The chain of the msr RNA is connected to the 5' end of the msd single-stranded DNA through a 2'-5' phosphodiester bond at the 2' position of the conserved guanosine residue.

[0228] In another preferred embodiment, the heterologous sequence is inserted into the editable region sequence of the msr gene or the msd gene, and the heterologous sequence guides the protein modification effector to specifically modify or demodify the target protein;

[0229] In another preferred embodiment, the retromer is selected from the group consisting of Ec86, Ec67, Ec73, Ec107, Sa163, or a combination thereof.

[0230] In another preferred embodiment, the editable region sequence of the MSD is selected from the following group:

[0231] (1) a sequence from position 95 to position 136 relative to the nucleotide sequence of SEQ ID NO: 22;

[0232] (2) a CTT sequence from position 79 to position 81 relative to the nucleotide sequence of SEQ ID NO: 23; or,

[0233] (3) The sequence from position 120 to position 215 relative to the nucleotide sequence shown in SEQ ID NO: 24. In another preferred embodiment, the editable region sequence of the MSR is selected from the following group:

[0234] (1) a TATCATTAAGGTC sequence from position 24 to position 36 relative to the nucleotide sequence of SEQ ID NO: 22;

[0235] (2) a GTTTC sequence from position 51 to position 55 relative to the nucleotide sequence shown in SEQ ID NO: 22;

[0236] (3) a GTGAATCGC sequence from position 27 to position 35 relative to the nucleotide sequence shown in SEQ ID NO: 23;

[0237] (4) a CCTCGAG sequence from position 43 to position 49 relative to the nucleotide sequence of SEQ ID NO: 24; or

[0238] (5) The CCTCGAG sequence from position 61 to position 67 relative to the nucleotide sequence shown in SEQ ID NO:24.

[0239] In another preferred embodiment, the nucleic acid sequence of the non-coding RNA of the reverse transcriptase is selected from the following group:

[0240] (i) a polynucleotide whose sequence is shown in any one of SEQ ID NOs. 22-24;

[0241] (ii) a polynucleotide having a nucleotide sequence homology ≥80%, preferably ≥95% (more preferably ≥98%, more preferably ≥99%) to any one of SEQ ID NOs. 22-24;

[0242] (iii) a polynucleotide having 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide shown in any one of SEQ ID NOs. 22 to 24;

[0243] (iv) a polynucleotide complementary to the polynucleotide described in any one of (i) to (iii).

[0244] Heterologous sequence

[0245] The msr and / or msd regions in the non-coding RNA are modified to insert a heterologous sequence, such as Figure 3 As shown in B, the heterologous sequence will eventually be included in msr and / or msd to recruit target proteins or endogenous effectors.

[0246] In another preferred embodiment, the MSD of the non-coding RNA is engineered to contain one or more heterologous sequences, the form of which includes but is not limited to double-stranded DNA formed by a hairpin / stem-loop structure, an aptamer, etc., which is used to recruit the target protein, and recruit the protein modification effector through a reverse transcriptase RT-coupled protein modification effector or a reverse transcriptase RT-coupled polypeptide ligand, thereby specifically modifying the target protein ( Figure 5 ).

[0247] In another preferred embodiment, the msr of the non-coding RNA is engineered to contain one or more heterologous sequences, the form of which includes but is not limited to double-stranded DNA formed by a hairpin / stem-loop structure, an aptamer, etc., which is used to recruit the target protein, and the protein modification effector coupled to the reverse transcriptase RT or the polypeptide ligand coupled to the reverse transcriptase RT is used to recruit the protein modification effector, thereby specifically modifying the target protein ( Figure 6 ).

[0248] In another preferred embodiment, the non-coding RNA msr and msd are respectively engineered to contain one or more heterologous sequences, wherein the heterologous sequences include but are not limited to double-stranded DNA formed by hairpin / stem-loop structures, aptamers, etc., and the heterologous sequences of msd and msr are used to recruit target proteins and protein modification effectors, respectively, to shorten the spatial distance between the protein modification effectors and the target proteins, thereby specifically modifying the target proteins ( Figure 7 ).

[0249] In another preferred embodiment, the heterologous sequence refers to a DNA or RNA nucleotide sequence that specifically binds to the target protein.

[0250] In another preferred embodiment, the heterologous sequence is in the form of (but not limited to): double-stranded DNA formed by a hairpin or stem-loop structure, an adaptor, a G-quadruplex, or a combination thereof.

[0251] In another preferred embodiment, the heterologous sequence is selected from the following group:

[0252] (i) a polynucleotide whose sequence is shown in any one of SEQ ID NOs. 225-26;

[0253] (ii) a polynucleotide having a nucleotide sequence homology ≥ 80%, preferably ≥ 95% (more preferably ≥ 98%, more preferably ≥ 99%) to any one of SEQ ID NOs. 25-26;

[0254] (iii) a polynucleotide having 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide shown in any one of SEQ ID NOs. 25-26;

[0255] (iv) a polynucleotide complementary to the polynucleotide described in any one of (i) to (iii).

[0256] In another preferred embodiment, the target protein is selected from the following group: P50, P65, cMYC, or a combination thereof.

[0257] In another preferred embodiment, the sequence of the chimeric RNA is selected from the following group:

[0258] (i) a polynucleotide whose sequence is shown in SEQ ID NO.27-28;

[0259] (ii) a polynucleotide having a nucleotide sequence homology ≥80%, preferably ≥95% (more preferably ≥98%, more preferably ≥99%) to the sequence shown in SEQ ID NO. 27-28;

[0260] (iii) a polynucleotide having 1 to 60 (preferably 1 to 30, more preferably 19 to 22) nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotides shown in SEQ ID NOs. 27-28;

[0261] (iv) a polynucleotide complementary to the polynucleotide described in any one of (i) to (iii).

[0262] Fusion Protein

[0263] As used herein, "the fusion protein of the present invention" or "polypeptide" refers to the fusion protein of the second aspect of the present invention. Figure 3 A, the structure is shown in the following formula I:

[0264] W0-W1-W2(I)

[0265] In the formula,

[0266] W0 is none, tag element, nuclear localization element, or a combination thereof;

[0267] W2 is none, a tag element, a nuclear localization element, or a combination thereof;

[0268] The structure of W1 is shown in the following formula Ia or Ib:

[0269] ALB(Ia)

[0270] BLA(Ib)

[0271] in

[0272] A is a protein modification effector or a recruitment element of a protein modification effector, and the protein modification effector specifically modifies or demodifies the target protein adjacent to it depending on the effect of spatial distance;

[0273] B is reverse transcriptase;

[0274] L is none or a connecting peptide,

[0275] Each "-" is independently a peptide linker, a peptide bond, or a non-peptide bond.

[0276] In another preferred embodiment, the reverse transcriptase (RT) is selected from the following group: Ec86RT, Ec67RT, Ec73RT, Ec107RT, Sa163RT or a combination thereof.

[0277] In the present invention, the length of the connecting peptide has an effect on the activity of the fusion protein, and the preferred length of the connecting peptide is 1-100aa, preferably 10-85aa, and more preferably 15-70aa. The amino acid sequence of the connecting peptide is selected from the following group: GS, SGS, SGGS, n*SG or n*(GGGS), wherein n is 1-50; or a polypeptide as shown in any one of SEQ ID NO.1-5. Or a polypeptide derived from a polypeptide having an amino acid sequence shown in SEQ ID NO.1-5, formed by replacing, deleting or adding one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, and most preferably 1 amino acid residues in the amino acid sequence shown in any one of SEQ ID NO.1-5.

[0278] As used herein, the term "fusion protein" also includes variant forms shown in any one of SEQ ID NO.17-21 having the above-mentioned activities. These variant forms include (but are not limited to): deletion, insertion and / or substitution of 1-3 (usually 1-2, preferably 1) amino acids, and addition or deletion of one or several (usually within 3, preferably within 2, and more preferably within 1) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed. For another example, adding or deleting one or several amino acids at the C-terminus and / or N-terminus generally does not change the structure and function of the protein. In addition, the term also includes the polypeptides of the present invention in monomeric and polymeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0279] The present invention also includes active fragments, derivatives and analogs of the above-mentioned fusion proteins. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially maintain the function or activity of the fusion protein of the present invention. The polypeptide fragments, derivatives or analogs of the present invention can be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of an antigenic peptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (fusion proteins formed by fusion with tag sequences such as a leader sequence, a secretory sequence or 6His). According to the teachings of this article, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.

[0280] A preferred class of active derivatives refers to polypeptides formed by replacing at most 3, preferably at most 2, and more preferably at most 1 amino acid with similar or similar properties compared to the amino acid sequence of Formula I. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0281] Table A

[0282]

[0283]

[0284] The present invention also provides analogs of the fusion protein of the present invention. The difference between these analogs and the polypeptides shown in any of SEQ ID NO.16-20 may be a difference in amino acid sequence, or a difference in modification form that does not affect the sequence, or both. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-natural or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0285] Modifications (usually without changing the primary structure) include: chemical derivatization of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the polypeptide or in further processing steps. This modification can be accomplished by exposing the polypeptide to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylase). Modifications also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to improve their resistance to proteolysis or optimize their solubility.

[0286] In a preferred embodiment, the amino acid sequence of the fusion protein of the present invention is shown in any one of SEQ ID NOs. 17-21.

[0287] Adeno-associated virus

[0288] Because adeno-associated virus (AAV) is smaller than other viral vectors, has no pathogenicity, and can transfect both dividing and non-dividing cells, gene therapy methods based on AAV vectors for genetic diseases have received widespread attention.

[0289] Adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus Dependinovirus of the family Parvoviridae. It is the simplest type of single-stranded DNA defective virus discovered so far, and requires a helper virus (usually adenovirus) to participate in replication. It encodes the cap and rep genes in the inverted repeat sequences (ITR) at both ends. ITRs play a decisive role in the replication and packaging of the virus. The cap gene encodes the viral capsid protein, and the rep gene is involved in the replication and integration of the virus. AAV can infect a variety of cells.

[0290] Recombinant adeno-associated virus vector (rAAV) is derived from non-pathogenic wild-type adeno-associated virus. Due to its good safety, wide range of host cells (dividing and non-dividing cells), low immunogenicity, and long-term expression of foreign genes in vivo, it is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant adeno-associated virus have been deeply understood, especially its application effects in various cells, tissues and in vivo experiments have been accumulated. In medical research, rAAV has been used in the study of gene therapy for a variety of diseases (including in vivo and in vitro experiments); at the same time, as a characteristic gene transfer vector, it is also widely used in gene function research, construction of disease models, preparation of gene knockout mice, etc.

[0291] In a preferred embodiment of the present invention, the vector is a recombinant AAV vector. AAV is a relatively small DNA virus that can be integrated into the genome of the cells they infect in a stable and site-specific manner. They can infect a large series of cells without any effect on cell growth, morphology or differentiation, and they do not seem to be involved in human pathology. The AAV genome has been cloned, sequenced and characterized. AAV contains an inverted terminal repeat (ITR) region of about 145 bases at each end, which serves as the origin of viral replication. The rest of the genome is divided into two important regions with encapsidation function: the left part of the genome containing the rep gene involved in viral replication and viral gene expression; and the right part of the genome containing the cap gene encoding the viral capsid protein.

[0292] AAV vectors can be prepared using standard methods in the art. Any serotype of adeno-associated virus is suitable. Methods for purifying vectors can be found in, for example, U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of hybrid vectors is described, for example, in PCT Application No. PCT / US2005 / 027091, the disclosures of which are incorporated herein by reference in their entirety. The use of vectors derived from AAV for transporting genes in vitro and in vivo has been described (see, for example, International Patent Application Publication Nos. WO91 / 18088 and WO93 / 09239; U.S. Patent Nos. 4,797,368, 6,596,535, and 5,139,941, and European Patent No. 0488528, all of which are incorporated herein by reference in their entirety). These patent publications describe various AAV-derived constructs in which the rep and / or cap genes are deleted and replaced by the gene of interest, and the use of these constructs to transport the gene of interest in vitro (into cultured cells) or in vivo (directly into an organism). Replication-deficient recombinant AAV can be prepared by co-transfecting the following plasmids into a cell line infected with a human helper virus (e.g., adenovirus): a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes). The resulting AAV recombinants are then purified by standard techniques.

[0293] In some embodiments, the recombinant vector is encapsidated into a viral particle (e.g., AAV viral particles including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16). Thus, the present disclosure includes recombinant viral particles (recombinant because they contain a recombinant polynucleotide) containing any of the vectors described herein. Methods for producing such particles are known in the art and are described in U.S. Pat. No. 6,596,535.

[0294] Expression vectors and host cells

[0295] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the coding sequence of the fusion protein of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

[0296] The polynucleotide sequence of the present invention can be used to express or produce a recombinant fusion protein by conventional recombinant DNA technology. Generally speaking, there are the following steps:

[0297] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding the fusion protein of the present invention, or a recombinant expression vector containing the polynucleotide;

[0298] (2) Host cells cultured in a suitable culture medium;

[0299] (3) Isolate and purify proteins from culture medium or cells.

[0300] In the present invention, the polynucleotide sequence encoding the fusion protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. As long as they can replicate and be stable in the host, any plasmid and vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0301] Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the fusion protein of the present invention and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: lac or trp promoters of Escherichia coli; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retroviral LTRs and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0302] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0303] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0304] The host cell can be a prokaryotic cell (such as Escherichia coli), or a lower eukaryotic cell, or a higher eukaryotic cell, such as a yeast cell, a plant cell, or a mammalian cell (including human and non-human mammals). Representative examples include: Escherichia coli, wheat germ cells, insect cells, SF9, Hela, HEK293, CHO, yeast cells, etc. In a preferred embodiment of the present invention, a yeast cell (such as Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cell includes: Kluyveromyces, more preferably Kluyveromyces marxianus, and / or Kluyveromyces lactis) is selected as the host cell.

[0305] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, which act on the promoter to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0306] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0307] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0308] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0309] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0310] The main advantages of the present invention include:

[0311] (1) The present invention first discovered that the fusion protein and chimeric RNA of the present invention can achieve arbitrarily specific modification or demodification of the target protein.

[0312] (2) The present invention provides for the first time an efficient protein modification tool, which contains one or more heterologous sequences on the msr and / or msd of non-coding RNA through engineering modification, and is used to recruit target proteins, and recruit protein modification effectors through reverse transcriptase RT-coupled protein modification effectors or reverse transcriptase RT-coupled polypeptide ligands, thereby specifically modifying the target protein.

[0313] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents involved in the present invention can be obtained from commercial channels unless otherwise specified.

[0314] Example 1: Detection of degradation levels of ubiquitinated P50 and P65 proteins

[0315] The reverse transcriptase is composed of reverse transcriptase RT and ncRNA. The reverse transcriptase can reverse transcribe the ncRNA as a template to produce single-stranded DNA (such as Figure 2). The present invention selects five reverse transcriptases (Ec86, Ec67, Ec73, Ec107, Sa163) that have been proven to have reverse transcription activity by in vitro experiments, and uses a connecting peptide to connect the reverse transcriptases (RT) of the reverse transcriptases Ec86, Ec67, Ec73, Ec107, and Sa163 to the amino or carboxyl termini of protein modification effectors (SPOP ubiquitinase (SEQ ID NO.6), VHL ubiquitinase (SEQ ID NO.7), PTD (SEQ ID NO.8), TRIM21 (PRSPR) (SEQ ID NO.9), ABI1 (SEQ ID NO.10)) to form a fusion protein. Among them, the amino acid sequences of reverse transcriptases Ec86RT, Ec67RT, and Sa163RT are shown in SEQID NO.11-13, respectively. V5 tags (SEQ ID NO.16) and FLAG tags (SEQ ID NO.15) can also be added to the fusion protein for subsequent Western blot detection. In this example, a V5 tag (SEQ ID NO.16) was added to the N-terminus of the fusion protein, and a FLAG tag (SEQ ID NO.15) and a nuclear localization signal NLS (SEQ ID NO.14) were added to the C-terminus of the fusion protein. The NLS sequence was used to localize to the cell nucleus. Taking the reverse transcriptase of the reverse transcriptase Ec86 connected to the carboxyl end of the protein modification effector as an example, the fusion proteins finally obtained were as follows: Ec86 RT and SPOP ubiquitinase fusion protein (SEQ ID NO.17), Ec86 RT and VHL ubiquitinase fusion protein (SEQ ID NO.18), Ec86 RT and PTD fusion protein (SEQ ID NO.19), Ec86 RT and TRIM21 (PRSPR) fusion protein (SEQ ID NO.20) and Ec86 RT and ABI1 fusion protein (SEQ ID NO.21); expression vectors of the above fusion proteins were constructed.

[0316] The present invention artificially synthesizes the non-coding RNA of the retrotransposons Ec86, Ec67 and Sa163. The non-coding RNA sequences of Ec86, Ec67 and Sa163 are shown in SEQ ID NO.22-24 respectively. Taking Ec86 as an example, the transformable region of msd (the bold region in SEQ ID NO.22) in the non-coding RNA of the retrotransposons Ec86 is replaced with a heterologous sequence (such as the NF-κB binding sequence (SEQ ID NO.26)), and finally forms a chimeric RNA. These chimeric RNAs are constructed into expression vectors called chimeric RNAs for subsequent experiments. Among them, the chimeric RNA sequence (SEQ ID NO.27) composed of Ec86 and the E-BOX binding sequence is shown in the schematic diagram. Fig.10As shown in Figures A and B in FIG; Ec86 and NF-κB binding sequence constitutes a chimeric RNA sequence (SEQ ID NO.28), the schematic diagram is as shown Fig.11 As shown in Figures A and B.

[0317] According to the commonly used mammalian cell culture method, human HEK293T cells were cultured in a 24-well cell culture dish, and the above 250 ng fusion protein expression vector and 250 ng chimeric RNA expression vector were transfected into the cells when the cell density reached an appropriate level. The transfection time was 48 hours. The transfected cells were cultured in a 37°C cell culture incubator with 5% CO2 for 48 hours, and then the samples were collected, and the expression levels of the target proteins P50 and P65 were detected by Western blot.

[0318] Since P50 and P65 proteins (target proteins) can bind to the double-stranded DNA of NF-κB, E3 ubiquitinase (PTD, VHL and SPOP) (protein modification effector) can further approach P50 and P65 proteins through the complex of Ec86RT (reverse transcriptase) and ncRNA connected to it, thereby ubiquitinating P50 and P65 and degrading them, thereby reducing the expression of P50 and P65 proteins ( Figure 8 As shown in Figure 2 , compared with the control, the fusion proteins of the present invention based on different ubiquitinating enzymes (PTD, VHL and SPOP) decreased the levels of P50 and P65 proteins by about 50%.

[0319] Example 2: Detection of degradation level of ubiquitinated cMyc protein

[0320] The operation of this example is the same as that of Example 1, except that in this example, the reverse transcriptase Ec86 is used, and the reverse transcriptase of the reverse transcriptase Ec86 is connected to the carboxyl end of the protein modification effector (VHL ubiquitinase (SEQ ID NO.7)) by a connecting peptide segment, and the fusion protein is finally obtained as follows:

[0321] The RT of Ec86 and VHL ubiquitinase fusion protein (SEQ ID NO.18) were used to construct the expression vector of the fusion protein.

[0322] The present invention artificially synthesizes the non-coding RNA of the retrotransposon Ec86. The non-coding RNA sequence of Ec86 is shown in SEQ ID NO.22. The modifiable region of msd (the bold region 8 in SEQ ID NO.22) in the non-coding RNA of the retrotransposon Ec86 is replaced with a heterologous sequence (E-BOX site sequence (SEQ ID NO.25)), and finally forms a chimeric RNA (SEQ ID NO.27), as shown in the schematic diagram. Fig.10As shown in Figures A and B in Figure 1. These chimeric RNAs were used to construct expression vectors called chimeric RNAs for subsequent experiments.

[0323] According to the commonly used mammalian cell culture method, human HEK293T cells were cultured in a 24-well cell culture dish, and the above-mentioned fusion protein and chimeric RNA expression vector (fusion plasmid) (800, 400, 200, 100 and 0 ng per well, respectively) were transfected when the cell density reached an appropriate level (6*10E4 / well). The transfected cells were cultured in a 37°C cell culture incubator with 5% CO2 for 48 hours, and then the samples were collected and the expression of the target protein cMYC protein was detected by Western blot.

[0324] Since cMYC protein can bind to the double-stranded DNA of E-BOX, the E3 ubiquitinase (VHL) can further approach the cMYC protein through the complex of Ec86RT and ncRNA connected to it, thereby ubiquitinating cMYC and degrading it, thereby reducing the expression of cMYC protein ( Fig. 9 As shown in Figure 2 , the fusion protein of the present invention degraded cMYC protein by more than 50% at a dose of 200ng-800ng per well.

[0325] The sequence information involved in the present invention is as follows:

[0326] SEQ ID NO.1: Connecting peptide sequence PAPAP

[0327] SEQ ID NO.2: Connecting peptide sequence SGSETPGTSESATPE

[0328] SEQ ID NO.3: Connecting peptide sequence AEAAAKEAAAKEAAAKEAAAKA

[0329] SEQ ID NO.4: Connecting peptide sequence GSGSSGGSSGSETPGTSESATPESSGGSSGGS

[0330] SEQ ID NO.5: Connecting peptide sequence SDSAGSAGSAGSGSSGS

[0331] SEQ ID NO.6: Amino acid sequence of SPOP ubiquitinase

[0332] VNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQS

[0333] SEQ ID NO.7: Amino acid sequence of VHL ubiquitinase

[0334] TLPVYTLKERCLQVVRSLVKPENYRRLDIVRSLYEDLEDHPNVQKDLERLTQE RIAHQRMGD

[0335] SEQ ID NO.8: Amino acid sequence of PTD ubiquitinase

[0336] ALAPYIPSGSALAPYIP

[0337] SEQ ID NO.9: Amino acid sequence of TRIM21 (PRSPR) ubiquitinase

[0338] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGEAAQEYQEKLQVALGELRRKQELAEKLEVIAIKR ADWKKTVETQKSRIHAEFVQQKNFLVEEEQRQLQELEKDEREQLRILGEKEAKLAQQSQALQELISELDRRCHSSALELLQEVIIVLERSESWNLKDLDITSPELRSVCHVPGLKKMLRTC

[0339] SEQ ID NO.10: Amino acid sequence of ABI1 ubiquitinase

[0340] TRVPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK

[0341] SEQ ID NO.11: Amino acid sequence of reverse transcriptase Ec86RT

[0342] MKSAEYLNTFRLRNLGLPVMNNLHDMSKATRISVETLRLLIYTADFRYRIYTVEKKGPEKRMRTIYQPSRELKALQGWVLRNILDKLSSSPFSIGFEKHQSILNNATPHIGANFILNIDLEDFFPSLTANKVFGVFHSLGYNRLISSVLTKICCYKNLLPQGAPSSPKLANLICSKLDYRIQGYAGSRGLIYTRYADDLTLSAQSMKKVVKARDFLFSIIPSEGLVINSKKTCISGPRSQRKVTGLVISQEKVGIGREKYKEIRAKIHHIFCGKSSEIEHVRGWLSFILSVDSKSHRRLITYISKLEKKYGKNPLNKAKT

[0343] SEQ ID NO.12: Amino acid sequence of reverse transcriptase Ec67RT

[0344] MTKTSKLDALRAATSREDLAKILDIKLVFLTNVLYRIGSDNQYTQFTIPKKGKGVRTISAPTDRLKDIQRRICDLLSDCRDEIFAIRKISNNYSFGFERGKSIILNAYKHRGKQIILNIDLKDFFESFNFGRVRGYFLSNQDFLLNPVVATTLAKAACYNGTLPQGSPCSPIISNLICNIMDMRLAKLAKKYGCTYSRYADDITISTNKNTFPLEMATVQPEGVVLGKVLVKEIENSGFEINDSKTRLTYKTSRQEVTGLTVNRIVNIDRCYYKKTRALAHALYRTGEYKVPDENGVLVSGGLDKLEGMFGFIDQVDKFNNIKKKLNKQPDRYVLTNATLHGFKLKLNAREKAYSKFIYYKFFHGNTCPTIITEGKTDRIYLKAALHSLETSYPELFREKTDSKKKEINLNIFKSNEKTKYFLDLSGGTADLKKFVERYKNNYASYYGSVPKQPVIMVLDNDTGPSDLLNFLRNKVKSCPDDVTEMRKMKYIHVFYNLYIVLTPLSPSGEQTSMEDLFPKDILDIKIDGKKFNKNNDGDSKTEYGKHIFSMRVVRDKKRKIDFKAFCCIFDAIKDIKEHYKLMLNS

[0345] SEQ ID NO.13: Amino acid sequence of reverse transcriptase Sa163RT

[0346] MTAKLESHVPAAPPVSAEAPAPTRPDAAKQEARRAHHEALRLRWKAIEEAGGTDAWVRQQLVAKGVAAEEVDFESSLSDKQKAAWKEKKKAEATERRAQKRLAWEAWKATHIHHLGVGVHW DEAGGPDKFDVAGREERAKANGLPEGLDSVEALAKALGISVSRLRWFSFHREVDTGTHYQTWEIPKRDGGKRTLTAPKRELKAVQRWVLANVVERLPVHGAAHGFVAGRSILTNALAHQG ADVVVKVDMKDFFPSVTWPRVKGLLRKGGLPENLATLLALLSTEAPREVVRFRGETLYVAKGPRALPQGAPTSPALTNALCLRLDKRLSALSKRLGFTYTRYADDLTFSWRRAKKSRQKE LPLADAPVALLLARVKGVLEAEGFTLHPDKTRVQRKGSRQRVTGLVVNEAPEGVPGARVPRDVVRRLRAAIHNREQGKPGPTGETLEQLKGLAAFLHMTDAEKGRAFLRRLEALEKRQTA

[0347] SEQ ID NO.14: Amino acid sequence of nuclear localization signal NLS

[0348] PKKKRKVDPKKKRKVDPKKKRKV

[0349] SEQ ID NO.15: Amino acid sequence of FLAG tag

[0350] DYKDHDGDYKDHDIDYKDDDDKEF

[0351] SEQ ID NO.16: Amino acid sequence of V5 tag

[0352] GKPIPNPLLGLDST

[0353] SEQ ID NO.17: Amino acid sequence of Ec86 RT and SPOP ubiquitinase fusion protein, wherein the italics are V5 tags, the bold is SPOP ubiquitinase, the underline is Ec86 RT, the double underline is the restriction site, and the wavy line is the FLAG tag and nuclear localization signal

[0354]

[0355]

[0356] SEQ ID NO.18: Amino acid sequence of Ec86 RT and VHL ubiquitinase fusion protein, wherein the italics are V5 tags, the bold is VHL ubiquitinase, the underline is Ec86 RT, the double underline is the restriction site, and the wavy line is the FLAG tag and nuclear localization signal

[0357]

[0358] SEQ ID NO.19: Amino acid sequence of RT and PTD fusion protein of Ec86, wherein the italic is V5 tag, the bold is PTD, the underline is RT of Ec86, the double underline is the restriction site, and the wavy line is the FLAG tag and nuclear localization signal

[0359]

[0360] SEQ ID NO.20: Amino acid sequence of Ec86 RT and TRIM21 (PRSPR) fusion protein, wherein the italics are V5 tags, the bold is TRIM21 (PRSPR), the underline is Ec86 RT, the double underline is the restriction site, and the wavy line is the FLAG tag and the nuclear localization signal

[0361]

[0362]

[0363] SEQ ID NO.21: Amino acid sequence of the fusion protein of Ec86 RT and ABI1, wherein the italics are V5 tags, the bold is ABI1, the underline is Ec86 RT, the double underline is the restriction site, and the wavy line is the FLAG tag and the nuclear localization signal

[0364]

[0365] SEQ ID NO.22: Non-coding RNA sequence of the reverse transcriptase Ec86, wherein the italic part is the msr transformable sequence; the bold part is the msd transformable sequence

[0366]

[0367] SEQ ID NO.23: Non-coding RNA sequence of the reverse transcriptase Ec67, wherein the italic part is the msr transformable sequence; the bold part is the msd transformable sequence

[0368]

[0369] SEQ ID NO.24: Non-coding RNA sequence of the reverse transcriptase Sa163, wherein the italic part is the msr transformable sequence; the bold part is the msd transformable sequence

[0370]

[0371] SEQ ID NO.25: E-BOX binding sequence

[0372] CCGGCTGACACGTGGTATTAATAGAAACTATTAATACCACGTGTCAGCC

[0373] SEQ ID NO.26: NF-KB binding sequence

[0374] TGGGGACTTTCCAGAAACTGGAAAGTCCCCA

[0375] SEQ ID NO.27: Chimeric RNA sequence composed of retrotransposons Ec86 and E-BOX, wherein the ncRNA sequence of the retrotransposons Ec86 is modified by msd to add the E-BOX binding sequence, and the bold part is the E-BOX binding sequence after msd modification

[0376]

[0377] SEQ ID NO.28: Chimeric RNA sequence composed of retrotransposons Ec86 and NF-κB binding sequences, wherein the ncRNA sequence of retrotransposons Ec86 is modified by adding msd to the NF-κB binding sequence, and the bold part is the NF-κB binding sequence after msd modification

[0378]

[0379] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for specifically modifying a target protein, characterized in that: Includes steps: (S1) in the presence of the fusion protein and the chimeric RNA or its precursor ncRNA, the target protein in the cell is specifically modified; The fusion protein comprises: (a) a first fusion element, which is a protein modification effector or a recruitment element of a protein modification effector; and (b) a second fusion element, which is a reverse transcriptase, and the first fusion element and the second fusion element are fused together; The chimeric RNA comprises a first functional module and a second functional module, wherein the first functional module binds to a reverse transcriptase; and the second functional module binds to a target protein; When the chimeric RNA binds to the reverse transcriptase through the first functional module and binds to the target protein through the second functional module, the fusion protein specifically modifies the target protein.

2. A fusion protein, characterized in that: The structure of the fusion protein is shown in Formula I below: W0-W1-W2(I) In the formula, W0 is none, tag element, nuclear localization element, or a combination thereof; W2 is none, a tag element, a nuclear localization element, or a combination thereof; The structure of W1 is shown in the following formula Ia or Ib: ALB(Ia) BLA(Ib) in A is a protein modification effector or a recruitment element of a protein modification effector, and the protein modification effector specifically modifies or demodifies the target protein adjacent to it depending on the effect of spatial distance; B is reverse transcriptase; L is none or a connecting peptide, Each "-" is independently a peptide linker, a peptide bond or a non-peptide bond.

3. A chimeric RNA, characterized in that The nucleic acid sequence of the chimeric RNA includes the structure shown in the following formula II from the 5' end to the 3' end: In the formula, Z1 is the 5' end sequence to the left of the guanosine (G) at the start site of reverse transcription; G is the reverse transcription start site guanosine (G); Z2 is the 3' end sequence to the right of the guanosine (G) at the start of reverse transcription Z1-G-Z2 constitutes the MSR sequence; Z3 is the MSD sequence.

4. A nucleic acid, characterized in that The nucleic acid encodes the fusion protein according to claim 2 and / or encodes the chimeric RNA according to claim 3.

5. An expression vector, characterized in that: The expression vector contains the nucleic acid according to claim 4.

6. An isolated host cell, characterized in that Comprising the fusion protein of claim 2 and / or encoding the chimeric RNA of claim 3, or the expression vector of claim 5.

7. A protein modification reagent, characterized in that The protein modification reagent comprises: (a) the fusion protein according to claim 2, or a gene encoding the fusion protein or an expression vector thereof; and (b) The chimeric RNA or its expression vector according to claim 3.

8. A composition, characterized in that The composition comprises: (a) the fusion protein according to claim 2, or a gene encoding the fusion protein or an expression vector thereof; and (b) The chimeric RNA or its expression vector according to claim 3.

9. A medicine box, characterized in that: The medicine kit comprises: (a1) a first container, and the fusion protein according to claim 2, or a gene encoding it, or an expression vector thereof, or a drug containing the fusion protein according to claim 2, or a gene encoding it, or an expression vector thereof, located in the first container; (b1) a second container, and the chimeric RNA or its expression vector according to claim 3, or a drug containing the chimeric RNA or its expression vector according to claim 3, located in the second container.

10. Use of the method according to claim 1, or the fusion protein according to claim 2, or the chimeric RNA according to claim 3, or the nucleic acid according to claim 4, or the expression vector according to claim 5, or the host cell according to claim 6, or the protein modification reagent according to claim 7, or the composition according to claim 8, or the kit according to claim 9 for preparing a reagent or kit for specific modification of a target protein.

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