Cytoplasm and nucleoplasm localization polypeptide for in vitro mammalian cell localization

Abstract

The invention relates to a cytoplasm and nucleoplasm localization polypeptide used for localization in mammalian cells in vitro. The amino acid sequence of the RfA1 protein is composed of conservative motifs and non-conservative connecting fragments between the motifs, the RfA1 protein is a natural block copolymer, the amino acid sequence of block arrangement of the RfA1 protein is redesigned, the motifs and the connecting fragments are recombined, and the modular design method of the protein tag is provided. According to the method, two protein tags for respectively and strictly locating foreign proteins in cytoplasm and nucleoplasm are obtained, the method and the two polypeptide tags can locate different functional proteins in cytoplasm or assist the different functional proteins to be transferred into nucleoplasm, the target capacity of the functional proteins can be effectively improved, and the method is expected to be applied to the fields of biotechnology, biological medicine and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of label proteins in biotechnology, and specifically relates to a cytoplasmic and nucleoplasmic localization polypeptide for localization in mammalian cells in vitro. Background Art

[0002] In the biological research of the post-genomic era, the functional analysis of proteins is an important aspect of in vitro experiments. The rapid, convenient and economical expression of recombinant proteins has become a prerequisite. Currently, commonly used protein expression systems include Escherichia coli, yeast, insect cells and mammalian cell systems.

[0003] In the process of heterologous expression, it is often necessary to detect the expression of the target protein, so a series of protein tags are needed for labeling and assistance. Protein expression tags play a vital role in promoting protein expression, assisting protein detection, and achieving protein purification. For example, after splicing the gene encoding the target protein with the gene encoding the fluorescent protein (eGFP, eCFP, eYFP), the subcellular localization and expression of the target protein in the cell can be directly observed in real time under a fluorescence microscope in the living cell state; for example, after connecting the target gene with macromolecular tags such as GST and TrxA, the solubility of the target protein can be promoted, the correct folding of the protein can be promoted, the stability can be improved, and the expression of the protein can be increased; for example, small molecule tags such as 6*His, Flag and c-Myc can provide a simple and effective purification route and detection and identification of the target protein.

[0004] Taking mammalian cells as an example, eukaryotic cells contain more than a dozen organelles such as nuclei, mitochondria, endoplasmic reticulum, Golgi bodies, as well as substructures such as cytoskeleton, cytoplasmic matrix, and cell membrane. The specific location of biological macromolecules in the above subcellular structures is called subcellular localization. Taking proteins as an example, proteins are first translated and synthesized in the cytoplasm, and then guided by various sorting signals to be localized in different subcellular structures such as cell membranes, lysosomes, and nuclei, participating in various life activities of cells. The function, metabolism, and interaction of proteins with other biological molecules are closely related to their subcellular localization. Mature proteins must be localized in specific subcellular structures to function normally. Once the wrong localization occurs, it may have a significant impact on cell function and even life individuals, such as developmental defects and tumors. Therefore, the study of protein subcellular localization is of great significance.

[0005] In the existing methods, NLS or NES is linked to the target protein by molecular cloning, and the target protein is localized in the nucleoplasm or cytoplasm through the nucleoplasm or cytoplasm localization characteristics of NLS or NES. However, the dose-effect relationship of this method cannot be accurately quantified, and it cannot guarantee that the target protein is specifically and specifically distributed in the nucleoplasm or cytoplasm. Summary of the invention

[0006] In view of the shortcomings that the prior art uses molecular cloning to connect NLS or NES to the target protein, and the nucleoplasm and cytoplasm localization characteristics of NLS or NES are used to achieve the nucleoplasm and cytoplasm localization of the target protein, but the dose-effect relationship cannot be accurately quantified, and the target protein cannot be specifically and specifically distributed in the nucleoplasm or cytoplasm. The present invention provides a cytoplasm and nucleoplasm localization polypeptide for localization in mammalian cells in vitro, which is a protein tag design and construction technology based on the squid reflective protein RfA1. This type of protein tag can strictly control the specific cytoplasm and nucleoplasm distribution of exogenous proteins in mammalian cells, and achieve a method for controllable cytoplasm and nucleoplasm localization of exogenous proteins in mammalian cells and its application.

[0007] Some nouns involved in the present invention are as follows:

[0008] The "reflectin RfA1" refers to a protein originally isolated and identified from the iris cells of the marine organism Doryteuthis opalescens, a full-length protein of the reflectin A1 (RfA1) derived from the membrane protein of the iris cells of the squid (Doryteuthis opalescens). The basic modules of the design are the conserved motifs (reflectin moitfs, hereinafter referred to as RM) in the amino acid sequence of RfA1 and the non-conserved linkers (hereinafter referred to as RL).

[0009] The "Rf-M" or "Rf-L" is a modular unit provided by the RfA1 template gene, which can be replaced, increased or decreased according to application requirements to adjust the cytoplasmic and nuclear localization of the target protein, or the cytoplasmic and nuclear distribution ratio.

[0010] The "cytoplasmic or nucleoplasmic localization" method refers to connecting the target protein to be studied to the C-terminus or N-terminus of the biomimetic polypeptide. For example, if specific cytoplasmic localization is required, the target protein is connected to a target polypeptide with a Rf-(M N +M1*5); if the target protein needs to be specifically localized in the cell nucleus, the target protein is connected to the nucleoplasmic localization polypeptide represented by Rf-(M1*3+L2*2).

[0011] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0012] A cytoplasmic and nucleoplasmic localization polypeptide for localization in mammalian cells in vitro, comprising two polypeptides as tags, respectively named Rf-(M N +M1*5) and Rf-(M1*3+L2*2). The former retains all the natural connection fragments of RfA1 and removes all the natural connection fragments except Rf-M N All motifs except for RfA1 were replaced by Rf-M1, which used three Rf-M1 modules and two Rf-L2 modules to construct a neat and regular block structure different from the template RfA1; Rf-(M N +M1*5) and Rf-(M1*3+L2*2) sequences were synthesized by chemical synthesis;

[0013] The Rf-M N The sequence structure and amino acid composition of , as shown in SEQ ID NO.1, are as follows:

[0014] MEPMSRMTMDFQGRYMDSQGRMVDP

[0015] The sequence structure and amino acid composition of the Rf-M1 are shown in SEQ ID NO.2, as follows:

[0016] PERYMDMSGYQMDMQGRWMDAQGR

[0017] The sequence structure and amino acid composition of the Rf-L2 are shown in SEQ ID NO.3, as follows:

[0018] FNNPFGQMWHGRQGHYPGYMSSHSMYGRNMYNPYHSHYASRHFDS

[0019] The Rf-(M N +M1*5) is shown in SEQ ID NO.4 as follows:

[0020] MNRYLNRQRLYNMYRNKYRGV MEPMSRMTMDFQGRYMDSQGRMVDP RYYDYYGRMHDHDRYYG

[0021] RSMFNQGHSMDSQRYGGWMDN PERYMDMSGYQMDMQGRWMDAQGR FNNPFGQMWHGRQGHYPG

[0022] YMSSHSMYGRNMYNPYHSHYASRHFDS PERYMDMSGYQMDMQGRWMDAQGR YVNPFNHHMYGR

[0023] NMCYPYGNHYYNRHMEHPERYMDMSGYQMDMQGRWMDAQGRHCNPFGQMWHNRHGYYPGHPHG

[0024] RNFQ PERYMDMSGYQMDMQGRWMDAQGR YVNPFSHNYGRHMNYPGGHYNYHHGRYMNH PERY

[0025] MDMSGYQMDMQGRWMDAQGR YIDNFDRNYYDYHMY

[0026] The sequence structure and amino acid composition of the Rf-(M1*3+L2*2) are shown in SEQ ID NO.5, as follows:

[0027] PERYMDMSGYQMDMQGRWMDAQGR FNNPFGQMWHGRQGHYPGYMSSHSMYGRNMYNPYHSHYA

[0028] SRHFDS PERYMDMSGYQMDMQGRWMDAQGR FNNPFGQMWHGRQGHYPGYMSSHSMYGRNMYNP YHSHYASRHFDS PERYMDMSGYQMDMQGRWMDAQGR ;

[0029] Connect the foreign protein to Rf-(M N +M1*5) and the N-terminus of Rf-(M1*3+L2*2) to obtain a recombinant plasmid. The foreign protein is at the N-terminus of Rf-(M N +M1*5) will be enriched in the cytoplasm, while under the action of Rf-(M1*3+L2*2) it will be localized in the nucleus.

[0030] Further, in step 4), the foreign protein is linked to Rf-(M N +M1*5) and the N-terminus of Rf-(M1*3+L2*2), preferably GFP protein is connected to Rf-(M N +M1*5) and the N-terminus of Rf-(M1*3+L2*2), that is, the GFP protein was seamlessly spliced ​​and constructed in the C segment of the GFP sequence of the pEGFP-C1 vector.

[0031] The above-mentioned method for preparing a cytoplasmic or nucleoplasmic localization polypeptide for localization in mammalian cells in vitro comprises the following steps:

[0032] 1) Using cytoplasmic and nucleoplasmic localization polypeptides derived from the RfA1 protein sequence as a design template for exogenous proteins with controllable cytoplasmic and nucleoplasmic localization in mammalian cells in vitro;

[0033] 2) The molecular weight, isoelectric point, and dissociation constant data of each motif and connected fragment of RfA1 were calculated using the ExPASy-ProtParam tool;

[0034] 3) Design two peptides as tags, named Rf-(M N +M1*5) and Rf-(M1*3+L2*2). The former retains all the natural connection fragments of RfA1 and removes all the natural connection fragments except Rf-M N All motifs except for RfA1 were replaced by Rf-M1, which used three Rf-M1 modules and two Rf-L2 modules to construct a neat and regular block structure different from the template RfA1; Rf-(M N +M1*5) and Rf-(M1*3+L2*2) sequences were synthesized by chemical synthesis;

[0035] 4) Connect the foreign protein to Rf-(M N +M1*5) and the N-terminus of Rf-(M1*3+L2*2) to obtain a recombinant plasmid. The foreign protein is at the N-terminus of Rf-(M N +M1*5) will be enriched in the cytoplasm, while under the action of Rf-(M1*3+L2*2) it will be localized in the nucleus;

[0036] Step 3) Rf-M N The sequence structure and amino acid composition are shown in SEQ ID NO.1;

[0037] The sequence structure and amino acid composition of Rf-M1 in step 3) are shown in SEQ ID NO.2;

[0038] The sequence structure and amino acid composition of Rf-L2 in step 3) are shown in SEQ ID NO.3;

[0039] Step 3) Rf-(M N +M1*5) has a sequence structure and amino acid composition as shown in SEQ ID NO.4;

[0040] The sequence structure and amino acid composition of Rf-(M1*3+L2*2) described in step 3) are shown in SEQ ID NO.5.

[0041] The present invention also relates to the use of the above-mentioned cytoplasmic or nucleoplasmic localization polypeptide for localization in mammalian cells in vitro, comprising the following steps:

[0042] By liposome transfection, the cytoplasmic and nucleoplasmic localization polypeptides obtained above for localization in mammalian cells in vitro are transferred to mammalian cells in vitro. While the exogenous protein is expressed, the C-terminus of the exogenous protein encoding gene is connected to the polypeptide tag Rf-(M N +M1*5) or Rf-(M1*3+L2*2), and then through cell transfection, cell nucleus staining, cell membrane staining, confocal microscopy was used to observe the intracellular localization of the exogenous protein after the tagged protein.

[0043] Furthermore, 12 hours after cell transfection, the cell nucleus was stained with DAPI and the cell membrane was stained with DiD.

[0044] The above-mentioned application of a cytoplasmic and nucleoplasmic localization polypeptide for localization in mammalian cells in vitro, after preliminary research, the inventors discovered for the first time that the full-length sequence of the RfA1 protein is specifically enriched in the cytotoxic cytoplasmic region in the form of aggregates after expression in mammalian cells, and its amino acid sequence can be modularly designed into a series of RfA1-M and RfA1-L. The combination of different types and quantities of RfA1-M and RfA1-L can strictly control the spatial distribution of the recombinant in the cytoplasm and nucleus. Based on this, the modular units RfA1-M, RfA1-L and their combination schemes can be used to change the localization of the target protein in the cell, which is of great value for studying the biological function of the target protein or improving its spatial directional distribution control characteristics, and has broad application prospects in the fields of basic biological research and molecular target delivery.

[0045] Compared with the prior art, the present invention has the following advantages:

[0046] 1. The cytoplasmic and nucleoplasmic localization polypeptide for localization in mammalian cells in vitro of the present invention is based on the sequence division of the RfA1 gene, and its fragments are used as selectable, replaceable and recombinant functional modules, and for the first time, a recombinant nucleic acid sequence that does not exist in nature is designed, and the nucleic acid sequence can respectively encode RfA1-(M N +M1*5) and RfA1-(M1*3+L2*2).

[0047] 2. The cytoplasmic and nucleoplasmic localization polypeptide for localization in mammalian cells in vitro described in the present invention, wherein the functional modules derived from the natural sequence of RfA1 are selectable and recombinant, and by designing and controlling the number of functional modules, its spatial selective distribution in the cell can be achieved, that is, efficient and controllable enrichment in the cytoplasm or nucleus, and in this process, other molecules connected in series can be pulled to perform spatial directional distribution in the cell. The nucleic acid sequence design scheme, nucleic acid sequence template and two nucleic acid tag demonstration examples designed by the present invention can realize the control of the spatial distribution (cytoplasm / nucleus) of the target protein molecule in the cell, and have potential application value in the fields of protein function realization, mechanism research and target efficiency of drug-like polypeptides / proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a diagram of the intracellular distribution of the GFP-RfA1 fusion protein in the embodiment of the present invention (a: cell nucleus (blue); b: GFP-RfA1 (green); c: cell membrane (red); d: fluorescence channel fusion);

[0049] Figure 2 is a schematic diagram of the arrangement of functional fragments in the RfA1 protein sequence in an embodiment of the present invention;

[0050] Figure 3 The polypeptide tag RfA1-(M N +M1*5) and RfA1-(M1*3+L2*2);

[0051] Figure 4 is GFP-RfA1-(M N +M1*5) fusion protein in the cell (a: cell nucleus (blue); b: GFP-RfA1-(M N +M1*5) (green); c: cell membrane (red); d: fluorescence channel fusion);

[0052] Figure 5 This is a diagram showing the distribution of GFP-RfA1-(M1*3+L2*2) fusion protein in cells in an embodiment of the present invention (a: cell nucleus (blue); b: GFP-RfA1-(M1*3+L2*2) (green); c: cell membrane (red); d: fluorescence channel fusion). DETAILED DESCRIPTION

[0053] The present invention is further described in detail below by way of examples, but these examples should not be considered as limiting the present invention.

[0054] Example:

[0055] A cytoplasmic and nucleoplasmic localization polypeptide for localization in mammalian cells in vitro:

[0056] The present invention uses GFP protein as a representative of the target protein to carry out the embodiment, and connects it to RfA1-(M N +M1*5) and the N-terminus of RfA1-(M1*3+L2*2), that is, splicing them to the C-terminus of the pEGFP-C1 vector;

[0057] First, the inventors have achieved the expression of natural RfA1 full-length protein in mammalian cells. The RfA1 protein is connected to the C-terminus of the GFP protein. The GFP protein is the target protein pulled by RfA1 and spatially localized.

[0058] like Figure 1 As shown in the figure, under the traction of RfA1, the GFP protein and its green fluorescence are almost completely localized in the cytoplasm of the cell, proving that RfA1 is exclusively and specifically present in the cytoplasm and is an extremely efficient cytoplasmic localization protein. Figure 2 As shown, RfA1 is a natural block copolymer, and its sequence is composed of amino acid sequences with different charged characteristics, among which the amino acid composition of RfA1-Ms is relatively conservative, while RfA1-Ls is positively charged under physiological conditions; these amino acid sequences are the basis of RfA1 structure and function, and are also the modular units for the design and construction of new artificial peptides;

[0059]

[0060] First, the present invention is based on the modular block structure of RfA1, RfA1-M 1，2，3，4 All of them were replaced by the RfA1-M1 sequence, i.e., the artificial polypeptide RfA1-(M N +M1*5); Furthermore, the modules RfA1-M1 and RfA1-L2 were used to design RfA1-(M1*3+L2*2) with higher repetitiveness, modularity and standardization in sequence structure; the sequences of the above two artificial polypeptides are fundamentally different from RfA1;

[0061] Peptide RfA1-(M N +M1*5) sequence structure and amino acid composition:

[0062] MNRYLNRQRLYNMYRNKYRG MEPMSRMTMDFQGRYMD SQGRMVDRYYDYYGRMHDHDRYYGRS

[0063] MFNQGHSMDSQRYGGWMDN PERYMDMSGYQMDMQGRWMDAQGR FNNPFGQMWHGRQGHYPGYM

[0064] SSHSMYGRNMYNPYHSHYASRHFD PERYMDMSGYQMDMQGRWMDAQGR YVNPFNHHMYGRNMC

[0065] YPYGNHYYNRHMEH PERYMDMSGYQMDMQGRWMDAQGR HCNPFGQMWHNRHGYYPGHPHGRNM

[0066] FQ PERYMDMSGYQMDMQGRWMDAQGR YVNPFSHNYGRHMNYPGGHYNYHHGRYMN PERYMDMS

[0067] GYQMDMQGRWMDAQGR YIDNFDRNYYDYHMY

[0068] The sequence structure and amino acid composition of the polypeptide RfA1-(M1*3+L2*2):

[0069] PERYMDMSGYQMDMQGR WMDAQGFNNPFGQMWHGRQGHYPGYMSSHSMYGRNMYNPYHSHYAS

[0070] RHFDS PERYMDMSGYQMDMQGR WMDAQGFNNPFGQMWHGRQGHYPGYMSSHSMYGRNMYNPYH

[0071] SHYASRHFDS PERYMDMSGYQMDMQGRWMDAQGR

[0072] Figure 3 It is a peptide tag Rf-(M N +M1*5) and Rf-(M1*3+L2*2) and their amino acid composition information, we can see:

[0073] In the prior art, derivatives or truncations based on RfA1 all use the natural sequence of RfA1 protein and the corresponding nucleotides as templates, and the starting point of the PCR reaction can be determined by the design of primers, that is, different derivatives can be obtained by PCR cloning technology; however, in the present invention, RfA1-(M N +M1*5) and RfA1-(M1*3+L2*2) are not simple truncations of RfA1, and therefore cannot be obtained by primer design and PCR reaction, and their coding sequences can only be obtained by redesigning the nucleotide sequence and chemical synthesis, which is the difference and innovation of the present invention compared with the prior art;

[0074] Ⅰ. Materials and Methods

[0075] 1) Design of GFP-RfA1 fusion protein expression vector: The RfA1 protein sequence was obtained from the NCBI database (ACZ57764.1). Since the working platform was HEK-293T cells, the corresponding nucleic acid coding sequence was optimized according to its amino acid sequence and mammalian codon preference and obtained by chemical synthesis;

[0076] After gene synthesis, it needs to be recombined into the pEGFP-C1 vector to achieve the fusion of RfA1 protein and GFP. Therefore, PCR primers are designed to amplify the RfA1 target fragment through PCR reaction, and restriction sites (EcoRI, BamHI) are added to both ends of its sequence. Then, restriction endonucleases are used to perform double enzyme digestion on RfA1 and pEGFP-C1 at the same time to produce complementary sticky ends, and T4 ligase is used to achieve splicing of the two.

[0077] 2) RfA1-(M N +M1*5) and RfA1-(M1*3+L2*2) encoding genes, and their fusion with the pEGFP-C1 vector: Using the modules and units in RfA1 as components, a new artificial polypeptide RfA1-(M N +M1*5) and RfA1-(M1*3+L2*2), and using their amino acid sequences as templates, directly design and synthesize coding nucleic acids that do not exist in nature according to the codon preference of the expression platform (mammalian cells or Escherichia coli cells), obtain the sequences by chemical synthesis, then design PCR primers, amplify the RfA1 target fragment by PCR reaction, and simultaneously add restriction sites (EcoRI, BamHI) at both ends of the sequence, and then use restriction endonucleases to cleave RfA1-(M N +M1*5) or RfA1-(M1*3+L2*2), pEGFP-C1 were double-digested to produce complementary sticky ends, and spliced ​​using T4 ligase;

[0078] 3) Amplification and concentration of recombinant plasmid vector: pEGFP-C1-RfA1, pEGFP-C1-RfA1-(M N+M1*5) and pEGFP-C1-RfA1-(M1*3+L2*2) plasmids were introduced into competent cells (DH5α) by conventional transformation, and screened on the surface of solid culture medium containing 100ng / μl Kanamycin; after culturing at 37°C overnight, single colonies were selected for PCR verification; single colonies of positive clones containing the target gene were selected and added to liquid culture medium containing Kanamycin for amplification, cultured at 37°C overnight, and then the plasmids were recovered using a plasmid extraction kit, and the plasmids were precipitated and concentrated using sodium acetate and ethanol to a concentration of more than 500ng / ul required for transfection;

[0079] 4) Artificial peptide RfA1-(M N +M1*5) and RfA1-(M1*3+L2*2) in the cell: In this example, the Lipo3000 transfection kit was used to transfect EGFP-C1-(Rf-(M N +M1*5)), pEGFP-C1-(Rf-(M1*3+L2*2)) concentrated plasmids were introduced into mammalian cells Hek-293T for expression, and the spatial position of the artificial polypeptide was studied using the GFP tag of the fusion protein;

[0080] First, the frozen cells were taken out of liquid nitrogen and placed in a 37°C water bath for 5 minutes, then DMEM complete medium was quickly added, and the supernatant was removed after low-speed centrifugation at 1050 rpm for 3 minutes, and DMEM medium was added again, and cultured overnight in the cell culture phase;

[0081] After 24 hours, the cell status was observed and the cells were cultured or trypsin-treated for passage according to the cell density;

[0082] After multiple passages, the cell status was observed. When the cell confluence reached 70-90%, lipofectamine 3000 (purchased from Thermo Fisher) was used for transfection. N +M1*5) and RfA1-(M1*3+L2*2) plasmids were introduced into mammalian cells (such as HEK-293T), and after transfection, they were cultured in a cell culture incubator and fluorescence observation was performed after 12-24 hours;

[0083] 4) Fluorescence localization analysis of artificial peptides: remove the cell culture medium, rinse with PBS three times, fix with 4% paraformaldehyde at room temperature for 20 minutes, remove the cell fixative and rinse with PBS three times, permeabilize with PBS solution containing 0.1% TritonX-100, then remove the permeabilizer and rinse with PBS three times, label the cell membrane with DiD (red fluorescence), and seal with sealing agent containing Dapi (cell nucleus is stained blue), and observe under fluorescence microscope after standing for 24 hours;

[0084] result:

[0085] Figure 4 GFP-RfA1-(M N +M1*5) fusion protein in HEK-293T cells (a: cell nucleus (blue); b: GFP-RfA1-(M N +M1*5) (green); c: cell membrane (red); d: fluorescence signal fusion effect), we can see:

[0086] The cell nucleus was stained with DAPI in the sealing medium and emitted blue fluorescence after laser stimulation; the artificial peptide RfA1-(M N +M1*5) was fused with GFP and expressed, showing green color, forming aggregates with a size of several hundred nanometers in the cytoplasm; the cell membrane was stained with Did, and emitted red fluorescence after laser stimulation; after the fluorescence signal was fused, the artificial polypeptide RfA1-(M N +M1*5) are specifically distributed in the cytoplasm and do not enter the nucleus;

[0087] Figure 5 This is the spatial distribution of GFP-RfA1-(M1*3+L2*2) fusion protein in HEK-293T cells (a: cell nucleus (blue); b: GFP-RfA1-(M1*3+L2*2) (green); c: cell membrane (red); d: fluorescence signal fusion effect). You can see:

[0088] The cell nucleus was stained with DAPI in the sealing medium, and it emitted blue fluorescence after laser stimulation. The artificial peptide RfA1-(M1*3+L2*2) was fused with GFP and expressed in green, forming aggregates of several hundred nanometers in size in the cell nucleus. The cell membrane was stained with Did, and it emitted red fluorescence after laser stimulation. After the fluorescence signals were fused, it was found that the artificial peptide RfA1-(M1*3+L2*2) N +M1*5) are specifically distributed in the cell nucleus but not in the cytoplasm.

[0089] Summarize:

[0090] According to the above results, in the method of the present invention, the embodiment RfA1-(M N+M1*5) can specifically enrich the target molecule (GFP in the present invention) in the cytoplasm, while the polypeptide tag represented by Example RfA1-(M1*3+L2*2) can specifically enrich the target molecule in the nucleus;

[0091] 1) Based on the block arrangement of RfA1 protein sequence, RfA1-Ms and RfA1-Ls in RfA1 were conceptualized as modular units, and recombinant design was performed using them as standardized components to obtain a cytoplasmic localization polypeptide RfA1-(M N +M1*5) and a nucleocytoplasmic localization polypeptide RfA1-(M1*3+L2*2);

[0092] 2) In the embodiment, green fluorescent protein GFP is a fusion protein with cytoplasmic localization polypeptide RfA1-(M N +M1*5) and the N-terminal cargo molecule of the nucleocytoplasmic localization polypeptide RfA1-(M1*3+L2*2), and its spatial distribution was successfully regulated; at the same time, the cargo molecule GFP also played a tracer role, marking the spatial distribution of the fusion protein;

[0093] 3) RfA1-(M N +M1*5) can efficiently accumulate the cargo molecule GFP in the cytoplasm, and RfA1-(M1*3+L2*2) can specifically enrich the cargo molecule GFP in the nucleus;

[0094] Based on the above embodiments, the natural block structure of RfA1 protein can be used as a template for functional fragment mining. With its RfA1-Ms and RfA1-Ls as functional units, localization peptides with different functions can be obtained through design and recombination. These artificial localization peptides can effectively enrich the target molecules in the cytoplasm or nucleus of the cell, or effectively regulate their enrichment ratio in the cytoplasm and nucleus. The above characteristics will be helpful for studying the functions of proteins in different subcellular regions, or for accurately controlling the intracellular spatial targeting ability of proteins / peptides and being applied to the field of drug delivery. It has broad application prospects in the fields of basic research in biotechnology and biomedical applications.

Claims

1. A cytoplasmic or nucleoplasmic localization polypeptide for localization in mammalian cells in vitro, characterized in that: It includes two polypeptides as tags, named RfA1-(M N +M1*5) and RfA1-(M1*3+L2*2). The former retains all the natural connection fragments of RfA1 and removes all the natural connection fragments except Rf-M N All motifs except for RfA1 were replaced by Rf-M1, which used three Rf-M1 modules and two Rf-L2 modules to construct a neat and regular block structure different from the template RfA1; RfA1-(M N +M1*5) and RfA1-(M1*3+L2*2) sequences were synthesized by chemical synthesis; The Rf-M N The sequence structure and amino acid composition of , as shown in SEQ ID NO.1, are as follows: MEPMSRMTMDFQGRYMDSQGRMVDP The sequence structure and amino acid composition of the Rf-M1 are shown in SEQ ID NO.2, as follows: PERYMDMSGYQMDMQGRWMDAQGR The sequence structure and amino acid composition of the Rf-L2 are shown in SEQ ID NO.3, as follows: FNNPFGQMWHGRQGHYPGYMSSHSMYGRNMYNPYHSHYASRHFDS The RfA1-(M N +M1*5) is shown in SEQ ID NO.4 as follows: MNRYLNRQRLYNMYRNKYRGV MEPMSRMTMDFQGRYMDSQGRMVDP RYYDYYGRMHDHDRYYGRSMFNQGHSMDSQRYGGWMDN PERYMDMSGYQMDMQGRWMDAQGR FNNPFGQMWHGRQGHYPGYMSSHSMYGRNMYNPYHSHYASRHFDS PERYMDMSGYQMDMQGRWMDAQGR YVNPFNHHMYGRNMCYPYGNHYYNRHMEHPERYMDMSGYQMDMQGRWMDAQGRHCNPFGQMWHNRHGYYPGHPHGRNMFQ PERYMDMSGYQMDMQGRWMDAQGR YVNPFSHNYGRHMNYPGGHYNYHHGRYMNH PERYMDMSGYQMDMQGRWMDAQGR YIDNFDRNYYDYHMY The sequence structure and amino acid composition of RfA1-(M1*3+L2*2) are shown in SEQ ID NO.5, as follows: PERYMDMSGYQMDMQGRWMDAQGR FNNPFGQMWHGRQGHYPGYMSSHSMYGRNMYNPYHSHYA SRHFDS PE RYMDMSGYQMDMQGRWMDAQGR FNNPFGQMWHGRQGHYPGYMSSHSMYGRNMYNP YHSHYASRHFDS PERYMDMS GYQMDMQGRWMDAQGR ; The foreign protein was linked to RfA1-(M N +M1*5) and the N-terminus of RfA1-(M1*3+L2*2) to obtain a recombinant plasmid. The foreign protein is at the N-terminus of RfA1-(M N +M1*5) will be enriched in the cytoplasm, while under the action of RfA1-(M1*3+L2*2) it will be localized in the nucleus.

2. A cytoplasmic or nucleoplasmic localization polypeptide for localization in mammalian cells in vitro according to claim 1, characterized in that: The foreign protein is connected to RfA1-(M N +M1*5) and the N-terminus of RfA1-(M1*3+L2*2), preferably GFP protein is connected to RfA1-(M N +M1*5) and the N-terminus of RfA1-(M1*3+L2*2), that is, the GFP protein was seamlessly spliced ​​and constructed in the C segment of the GFP sequence of the pEGFP-C1 vector.

3. A method for preparing a cytoplasmic or nucleoplasmic localization polypeptide for localization in mammalian cells in vitro according to claim 1 or 2, characterized in that: The steps include: 1) Using cytoplasmic and nucleoplasmic localization polypeptides derived from the RfA1 protein sequence as a design template for exogenous proteins with controllable cytoplasmic and nucleoplasmic localization in mammalian cells in vitro; 2) The molecular weight, isoelectric point, and dissociation constant data of each motif and connected fragment of RfA1 were calculated using the ExPASy-ProtParam tool; 3) Design two peptides as tags, named RfA1-(M N +M1*5) and RfA1-(M1*3+L2*2). The former retains all the natural connection fragments of RfA1 and removes all the natural connection fragments except Rf-M N All motifs except for RfA1 were replaced by Rf-M1, which used three Rf-M1 modules and two Rf-L2 modules to construct a neat and regular block structure different from the template RfA1; RfA1-(M N +M1*5) and RfA1-(M1*3+L2*2) sequences were synthesized by chemical synthesis; 4) Connect the foreign protein to RfA1-(M N +M1*5) and the N-terminus of RfA1-(M1*3+L2*2) to obtain a recombinant plasmid. The foreign protein is at the N-terminus of RfA1-(M N +M1*5) will be enriched in the cytoplasm, while under the action of RfA1-(M1*3+L2*2) it will be localized in the nucleus; Step 3) Rf-M N The sequence structure and amino acid composition are shown in SEQ ID NO.1; The sequence structure and amino acid composition of Rf-M1 in step 3) are shown in SEQ ID NO.2; The sequence structure and amino acid composition of Rf-L2 in step 3) are shown in SEQ ID NO.3; Step 3) RfA1-(M N +M1*5) has a sequence structure and amino acid composition as shown in SEQ ID NO.4; The sequence structure and amino acid composition of RfA1-(M1*3+L2*2) described in step 3) are shown in SEQ ID NO.

5.

4. The use of a cytoplasmic or nucleoplasmic localization polypeptide for localization in mammalian cells in vitro as claimed in claim 1, characterized in that: The steps include: By liposome transfection, the cytoplasmic and nucleoplasmic localization polypeptides obtained above for localization in mammalian cells in vitro are transferred to mammalian cells in vitro. While the exogenous protein is expressed, the C-terminus of the exogenous protein encoding gene is connected to the polypeptide tag RfA1-(M N +M1*5) or RfA1-(M1*3+L2*2), and then through cell transfection, cell nucleus staining, cell membrane staining, confocal microscopy was used to observe the intracellular localization of the exogenous protein after the tagged protein.

5. The use of a cytoplasmic or nucleoplasmic localization polypeptide for localization in mammalian cells in vitro according to claim 1, characterized in that: Twelve hours after cell transfection, the nuclei were stained with DAPI and the cell membranes were stained with DiD.

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