A nucleolar localization signal peptide and its application

Abstract

The present invention discloses a nucleolus localization signal peptide, and also provides a nucleolus localization marker of the nucleolus localization signal peptide and an application method of carrying a protein polypeptide into the nucleolus of a eukaryotic cell. Based on the fact that the present invention can be applied to the study of the physiological functions of eukaryotic cells and the pathogenic mechanism of pathogenic bacteria, and can also be used for the development of nucleolus targeted drug delivery technology, the present invention has great research and application value in the fields of biotechnology and medical technology.

Description

Technical Field

[0001] The invention relates to a nucleolus localization signal peptide and an application thereof, in particular to a nucleolus localization signal peptide sequence and a recombinant localization method, and the application of the nucleolus localization signal peptide to carry protein polypeptides into the nucleolus, belonging to the field of biotechnology. Background Art

[0002] The nucleolus is an important subcellular structure in eukaryotic cells and a non-membranous structure in the nucleus. In eukaryotic cells, the nucleolus is the site for the storage of ribosomal RNA (rRNA) genes, synthesis and processing of rRNA, and assembly of large and small ribosome subunits. It is involved in many important physiological processes such as the degradation of nuclear proteins, apoptosis, stress response, cell proliferation regulation, and tumorigenesis. The nucleolus is a highly dynamic structure, and the realization of its function is closely related to the localization and translocation of proteins in the nucleolus. The entry of proteins into the nucleolus mediated by nucleolar localization signals is an important part of the information exchange between nuclear and external substances in the intracellular signal transduction network. Most proteins entering the nucleolus require nucleocytoplasmic transport receptors to recognize and bind to the nucleolar localization signal sequence carried by nuclear proteins. Since the nucleolus does not have a membranous structure, there are certain difficulties in separation and research, which makes it difficult to study the function of the nucleolus and nucleolar proteins, such as specific nucleolar localization analysis. In the process of infecting eukaryotic host cells, pathogenic microorganisms in nature will use some effector proteins secreted by their secretion system to specifically target the nucleolus, and regulate the host by affecting the function of nucleolar proteins, rRNA synthesis and processing, and ribosome assembly and other physiological processes. The characteristics of these effector proteins entering the nucleolus are of great significance to the replication and virulence of pathogenic microorganisms. Research on the function of such proteins and the mechanism of entering the nucleolus is conducive to the discovery of new anti-pathogen targets. In addition, the rapid proliferation of cancer cells is also closely related to the structure and function of the nucleolus, so the positioning of the nucleolus and the development of nucleolar targeting drugs have important practical application value.

[0003] Up to now, more and more technologies are devoted to providing means for the movement and function tracking research of eukaryotic nucleolar proteins and effector proteins of pathogenic microorganisms that target the nucleolus to implement toxic functions, such as: development of specific nucleolar protein antibodies, localization, displacement and nucleolar function imaging technology of nucleolar proteins, FRAP and I-FRAP technology, etc. Among them, the research and development of nucleolar localization dyes mainly includes the development of antibodies for related proteins distributed in the nucleolus, fluorescent coupling compounds and nucleolar localization signal peptides, etc. The development of safe, efficient and low-cost nucleolar-specific localization indicator peptides provides a basis for the study of nucleolar localization of eukaryotic nucleolar proteins and pathogen effector proteins, so as to further explore the functional research. At the same time, the development of signal peptide fusion recombination technology that efficiently carries other proteins, small molecules or peptide drugs into the nucleolus also provides technical methods for the transportation of targeted drugs that inhibit the function of pathogen effector proteins, ribosomal rRNA and cell translation, and the delivery of nucleolar targeted drugs for cancer treatment. The development and application technology of nucleolar localization polypeptide sequences provide new ways for the study of eukaryotic cell functions, the defense of pathogenic microorganisms and the treatment of diseases. Summary of the invention

[0004] The purpose of the present invention is to provide a nucleolus localization signal peptide, and to recombinantly construct it into an expression vector for nucleolus localization labeling, and to use it to carry protein polypeptides into the nucleolus of eukaryotic cells. Specifically, the present invention is implemented by the following technical solutions:

[0005] The present invention discloses a nucleolar localization signal peptide (NoLS), which is a polypeptide with an amino acid sequence of SEQ ID NO.7, which is the 38-50 amino acid sequence of the Lpg0284 gene of the type IV secretion system effector protein of Legionella pneumophila strain Philadelphia 1. The polypeptide sequence can locate the nucleolar region of eukaryotic cells, and the coding gene sequence corresponding to the amino acid sequence SEQ ID NO.7 is SEQ ID NO.1.

[0006] The nucleolar localization signal peptide of the present invention can also be a polypeptide obtained by 2-8 tandem recombination of the sequence SEQ ID NO.7, and all of them have the function of nucleolar localization. More preferably, it can be a polypeptide obtained by 4 tandem recombination of the amino acid sequence SEQ ID NO.7, whose amino acid sequence is SEQ ID NO.8 and the coding gene sequence is SEQ ID NO.2.

[0007] The present invention also provides a nucleolar localization marker vector, which can be obtained by recombining the nucleolar localization signal peptide into the eukaryotic expression vector pCDNA3.1-EGFP. Among them, the present invention provides a method for constructing a nucleolar localization marker vector, which is to obtain SEQ ID NO.8 by recombining the amino acid sequence of SEQ ID NO.7 through four groups of series, and then construct it into the eukaryotic expression vector pCDNA3.1-EGFP to obtain the nucleolar localization marker vector pCDNA3.1-4xNoLS-EGFP. After four recombination, the nucleolar localization efficiency can be significantly enhanced, and it is the best series connection method that can meet the application requirements. The eukaryotic protein expression vector pCDNA3.1-4xNoLS-EGFP constructed by the construction method of the nucleolar localization marker vector of the present invention, after transient transfection and protein expression of eukaryotic cells, the 4xNoLS polypeptide can bring the green fluorescent protein EGFP in the vector into the nucleolus position of the eukaryotic cell, and emit green fluorescence in the nucleolus region after being excited by 480nm excitation light. The nucleolar localization marker vector can be used in the nucleolar marker of eukaryotic cells, and can be further used in the study of the physiological functions of eukaryotic cells and the pathogenic mechanism of pathogenic bacteria.

[0008] Obviously, the recombinant expression vector, expression cassette, transgenic cell line or recombinant bacteria containing the gene described in SEQ ID NO.1 or SEQ ID NO.2 naturally also fall within the protection scope of the present invention.

[0009] In addition, the present invention also provides the application of the nucleolar localization signal peptide in carrying protein polypeptides into the nucleolus of eukaryotic cells, wherein the protein polypeptide is a non-nucleolar targeted polypeptide, including but not limited to Flag-HA, Myc, and EGFP, which are commonly used label polypeptide sequences in biological research, and all three can be brought into the nucleolus region of the cell by SEQ ID NO.2 in the present invention. It should be noted that the application described in the present invention is only to confirm that the nucleolar localization signal peptide described in the present invention has the function of carrying protein polypeptides into the nucleolus of eukaryotic cells by using commonly used label polypeptides. Obviously, in practical applications, the target protein polypeptide is not limited to the commonly used label polypeptides described in the present invention. Among them, the Flag-HA and Myc sequences are sequence SEQ ID NO.3 and sequence SEQ ID NO.4, respectively. The present invention provides a method for constructing a recombinant plasmid capable of carrying a protein polypeptide into the nucleolus, wherein the sequence SEQ ID NO.2 is fused with the sequence SEQ ID NO.3 and the sequence SEQ ID NO.4 to form the sequence SEQ ID NO.5 and the sequence SEQ ID NO.6, respectively, and then the sequence SEQ ID NO.5 and the sequence SEQ ID NO.6 are recombined into the pCDNA3.1 vector, respectively, to obtain the recombinant plasmids pCDNA3.1-Flag-HA-4xNoLS and pCDNA3.1-Myc-4xNoLS capable of carrying a protein polypeptide into the nucleolus of a eukaryotic cell. The eukaryotic cell may be a human HeLa cell, a HEK293T cell line, or the like. Further: After the constructed expression vectors of pCDNA3.1-4xNoLS-EGFP, pCDNA3.1-Flag-HA-4xNoLS and pCDNA3.1-Myc-4xNoLS were transiently transfected into cells, the cells were cultured for 24 hours to express 4xNoLS-EGFP, Flag-HA-4xNoLS and Myc-4xNoLS proteins, and the immunofluorescence experimental method was used to observe the localization mark of the nucleolus by the nucleolus localization signal peptide, and the entry of the protein polypeptide Flag-HA and Myc carried by 4xNoLS into the nucleolus region of the cell nucleus. Therefore, it can be used for the development of nucleolus targeted drug delivery technology.

[0010] The present invention provides a nucleolus localization signal peptide, and also provides a nucleolus localization marker of the nucleolus localization signal peptide and an application method of carrying a protein polypeptide into the nucleolus of a eukaryotic cell. Based on the fact that the present invention can be applied to the study of the physiological functions of eukaryotic cells and the pathogenic mechanism of pathogenic bacteria, and can also be used for the development of nucleolus targeted drug delivery technology, the present invention has great research and application value in the fields of biotechnology and medical technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The nucleolar localization signal peptide NoLS is localized in the nucleolus of cells.

[0012] Figure A shows the localization of the nucleolar signal peptide NoLS encoded by SEQ ID NO.1 after being recombined into the pCDNA3.1-EGFP vector and transiently transfected into HEK293T cells for expression. Blue fluorescence indicates the cell nucleus labeled by the dye DAPI, green fluorescence indicates the expressed NoLS-EGFP, and red fluorescence indicates the eukaryotic nucleolar Nucleolin protein labeled by the Nucleolin protein-specific primary antibody recognized by the Cy3-coupled fluorescent secondary antibody. Merge represents the superposition of the first three images. Figure B shows the protein identification result of NoLS-EGFP after expression in HEK293T cells by Western Blot (WB).

[0013] Figure 2 The nucleolar localization of 4xNoLS in cells is obtained by 4-fold tandem recombination of the nucleolar localization signal peptide NoLS.

[0014] Figure A shows the tandem recombination construction method and primer design of 4xNoLS. Figure B shows the localization map after 4xNoLS of SEQ ID NO.2 is recombined into the pCDNA3.1-EGFP vector and transiently transfected into the HEK293T cell line after overexpression of 4xNoLS-EGFP protein. Blue fluorescence indicates the cell nucleus labeled by the dye DAPI, green fluorescence is the expressed 4xNoLS-EGFP, and red fluorescence indicates the Nucleolin protein in the nucleolus region of eukaryotic cells labeled by the Nucleolin protein-specific primary antibody recognized by the Cy3-coupled fluorescent secondary antibody. Merge represents the superposition of the first three images, and EGFP is the label control for positioning. Figure C shows the results of Western Blot identification of 4xNoLS-EGFP after expression in HEK293T cells. Figure D shows the localization of the nucleolar localization signal peptide 4xNoLS in HeLa cells.

[0015] Figure 3 To investigate the effect of signal peptide on cell proliferation, HEK293T cells were transiently transfected with pCDNA3.1-NoLS-EGFP and pCDNA3.1-4xNoLS-EGFP plasmids, and pCDNA3.1-EGFP was used as the control group. After 24 hours, the OD490 nm value was detected by MTT cell proliferation assay, and the relative cell proliferation rate was calculated based on the control group.

[0016] Figure 4The effect of signal peptide on cell death. pCDNA3.1-NoLS-EGFP and pCDNA3.1-4xNoLS-EGFP plasmids were transiently transfected in HEK293T cells, and pCDNA3.1-EGFP was used as the control group. After 24 hours, the cell apoptosis ratio was detected using a cell death detection kit with PI and Hoechst double staining. Figure A is a representative flow cytometric graph of the apoptosis detection results, and Figure B is a bar graph of the statistical results of three repeated experiments of the ratio of apoptotic cells to total cells (Apoptosis cells / %).

[0017] Figure 5 This is a localization diagram of the nucleolar localization signal peptide carrying the polypeptide Flag-HA and Myc protein into the nucleolus of the host cell. Figure A shows the tandem recombination construction method and primer design of 4xNoLS and Flag coupled to HA or Myc protein sequence, where the arrows indicate the primer sequence range, the boxes indicate the connected protein polypeptide and signal peptide sequence, and the straight line indicates the sequence connecting the protein polypeptide sequence and the signal peptide. Figure B shows the localization map of Flag-HA-4xNoLS and Myc-4xNoLS proteins overexpressed in HEK293T and HeLa cells after transient transfection of pCDNA3.1-Flag-HA-4xNoLS and pCDNA3.1-Myc-4xNoLS vectors 24 hours later. Blue fluorescence indicates cell nuclei labeled with the dye DAPI, green fluorescence is Flag-HA-4xNoLS and Myc-4xNoLS proteins labeled with FITC fluorescent antibody, red fluorescence indicates eukaryotic nucleolar Nucleolin protein labeled with Nucleolin protein-specific primary antibody recognized by Cy3-conjugated fluorescent secondary antibody, and Merge represents the superposition of the first three images. DETAILED DESCRIPTION

[0018] The following examples are further descriptions of the content of the present invention as an explanation of the technical content of the present invention, but the substantive content of the present invention is not limited to the following examples, and those of ordinary skill in the art can and should know that any simple changes or replacements based on the substantive spirit of the present invention should all belong to the protection scope claimed by the present invention. In addition, the following experimental methods, if not otherwise specified, are conventional methods. The test materials used in the following embodiments, if not otherwise specified, are purchased from conventional biochemical reagent stores. The quantitative tests in the following embodiments are the mean values ​​obtained from the results of three repeated experiments, and the significant differences between the groups are analyzed using t-tests. The following specific embodiments are described in further detail in conjunction with the accompanying drawings.

[0019] Experimental Materials

[0020] Cells: Human HEK293T and HeLa cell lines were purchased from ATCC (cell numbers: CRL-3216, CRM-CCL-2). Fast-cutting enzymes: HindⅢ and BamHI were purchased from Thermo. High-fidelity PCR enzymes were purchased from 2×Phanta Flash Master Mix (Dye Plus) from Novezan Technologies, product number: P520-02. DNA homologous recombination kits were purchased from ClonExpress Ultra One Step Cloning Kit V2 from Novezan Technologies, product number: C116-01. PI and Hoechst cell apoptosis and necrosis detection kits were purchased from Beyotime Biotechnology, product number: C1056. MTT cell proliferation and cytotoxicity detection kits were purchased from Beyotime Biotechnology, product number: C0009S. The signal peptide sequence, i.e., SEQ ID NO.1, was obtained from the gene sequence of the effector protein Lpg0284 of the Legionella pneumophila Lp02 strain retrieved from the NCBI website, and the upstream and downstream homology arm sequences containing the insertion site of the vector to be constructed were synthesized by General Biotech Co., Ltd. SEQ ID NO.3 and SEQ ID NO.4 and the relevant PCR primers in the present invention were directly synthesized by General Biotech Co., Ltd. The antibodies involved in the experiment were purchased from Proteintech Antibody Company. pCDNA3.1(+)-EGFP and pCDNA3.1(+) vector plasmids were purchased from Addgene.

[0021] Example 1

[0022] Localization of nucleolar localization signal peptides

[0023] (1) Construction of eukaryotic expression vector of nucleolar localization signal peptide

[0024] According to the gene sequence of Legionella effector protein Lpg0284 in NCBI, the gene sequence of amino acids 38-50 and the upstream and downstream homologous arm gene sequences of the vector pCDNA3.1-EGFP to be constructed were directly synthesized. The gene sequence is as follows: NoLS-F: 5'-GTTTAAACTTAAGCTTATGCAAGAAAGACGCTCCAAAAAAAGAGAACTTCACTGCAAA-3' NoLS-R: 5'-CCCTTGCTCACGGATCCACCTTTGCAGTGAAGTTCTCTTTTTTTGGAGCGTCTTTCTTG-3'

[0025] The high-fidelity PCR enzyme 2×Phanta Flash Master Mix (Dye Plus) was added to the primer pair for PCR reaction. The conditions are as follows:

[0026] Table 1: PCR reaction conditions

[0027]

[0028] The PCR product was recovered using a PCR product recovery kit. The pCDNA3.1-EGFP vector was double-digested with HindⅢ and BamHI, and the digested fragments were separated by agarose gel electrophoresis. The linearized vector fragment was recovered using a gel recovery kit. The signal peptide fragment PCR product and the linearized vector were connected according to the experimental method of the homologous recombination kit, and then transformed into DH5α competent medium. The next day, the positive clones in the ampicillin-resistant plate were picked for sequencing and identification. After the positive bacteria were expanded, the endotoxin-free plasmid extraction kit was used to extract the successfully constructed NoLS expression vector pCDNA3.1-NoLS-EGFP.

[0029] (2) Nucleolar localization of nucleolar localization signal peptide

[0030] The cultured HEK293T and Hela cells were digested and plated in a confocal dish. The next day, the expression vector pCDNA3.1-NoLS-EGFP was transiently transfected into the cells using Lipo8000 transfection reagent. After 24 hours, the culture medium was discarded, the cells were washed twice with PBS, and fixed with 4% paraformaldehyde at room temperature for 20 minutes. Next, 1 mL of 5% Triton-X100 was added to permeabilize the cells for 5 minutes, washed three times with PBS, and blocked with 1% bovine serum albumin at room temperature for 1 hour. The blocking solution was discarded, and a 1:200 Nucleolin protein-specific antibody (Proteintech Antibody Company) was added. The cells were incubated overnight at 4°C, washed three times with PBS, and a 1:200 Cy3 fluorescent antibody of the same species (Proteintech Antibody Company) was added. The nuclear dye DAPI was added and incubated at room temperature for 1 hour. After washing three times with PBS, an anti-fluorescence attenuation mounting medium was added. The cell samples were photographed with a confocal microscope for nucleolar localization images. The expression and localization results of the signal peptide in the cells are shown in Figure 2. Figure 1 and Figure 3 shown.

[0031] Example 2

[0032] Application method of nucleolar labeling using nucleolar localization signal peptide

[0033] Using the feature that the signal peptide of SEQ ID NO.1 can enter the nucleolus, SEQ ID NO.2 was constructed by four tandem recombination, and the 4xNoLS eukaryotic expression vector pCDNA3.1-4xNoLS-EGFP was obtained after further recombination. After four recombination, SEQ ID NO.1 can significantly enhance the efficiency of nucleolus localization. Combined with the green color of the green fluorescent protein EGFP after excitation light of a certain wavelength, this vector can be used for nucleolus localization identification. The 4xNoLS signal sequence was constructed by the fusion PCR method, and the construction schematic diagram is shown in FIG. Figure 2 As shown in Figure A, the primers for PCR are as follows:

[0034] 4xNoLS-F1: 5'-GTTTAAACTTAAGCTTATGCAAGAAAGACGCTCCAAAAAAGAGAACTTCACTGCA-3'

[0035] 4xNoLS-F2: 5'-CTTCACTGCAAACAAGAAAGACG-3'

[0036] 4xNoLS-R1: 5'-GCGTCTTTCTTGTTTGCAGTGAAG-3'

[0037] 4xNoLS-R2: 5'-CTTGCTCACGGATCCACCTTTGCAGTGAAGTTCTCTTTTTTTGGAGCGTCTTTCTTG-3'

[0038] First, using the NoLS sequence as a template, PCR reaction was performed using primers 4xNoLS-F1 and 4xNoLS-R1 (reaction conditions are the same as Table 1) to obtain the first signal peptide gene sequence, which contains the homology arms of the vector to be constructed and the second signal peptide. Using primers 4xNoLS-F2 and 4xNoLS-R1, PCR reaction was performed using the NoLS sequence as a template to obtain the second signal peptide, and the fragment was recovered and connected with the first signal peptide to obtain 2xNoLS. Using 2xNoLS as a template, primers 4xNoLS-F2 and 4xNoLS-R2 were added to perform PCR reaction, and the 2xNoLS signal peptide gene sequence containing the third and fourth signal peptides was directly obtained. The sequence contains the homologous gene sequence in the downstream sequence of the insertion site in the vector. Finally, the two groups of 2xNoLS obtained were connected to form the complete sequence of 4xNoLS, and then the obtained sequence product was constructed into the pCDNA3.1-EGFP vector linearized by double restriction digestion of HindⅢ and BamHI using a homologous recombination kit.

[0039] The constructed pCDNA3.1-4xNoLS-EGFP was transformed into a DH5α competent cell to obtain a plasmid, and the cell localization was characterized by the same method as the nucleolar localization experimental method of the nucleolar localization signal in Example 1 (2). The expression and localization results of the 4xNoLS-EGFP signal peptide protein in the cell are shown in Figure 2 After four times of tandem recombination, the above signal peptide has extremely strong nucleolus targeting and can be used as a nucleolus localization marker polypeptide in cell function research.

[0040] Example 3

[0041] Cytotoxicity of Nucleolar Localization Signal Peptides

[0042] In the present invention, the MTT cell proliferation assay and the cell death assay were used to test whether the nucleolar localization signal peptide NoLS and the polypeptide 4xNoLS in the localization marker vector had toxic effects on eukaryotic cells.

[0043] The MTT cell proliferation assay was performed as follows: HEK293T cells were trypsinized and 1×10 5 The cells were plated in 6-well cell culture plates and cultured overnight. The eukaryotic vector pCDNA3.1-NoLS-EGFP or

[0044] pCDNA3.1-4xNoLS-EGFP was digested with trypsin after 6 hours, and the cell suspension was plated in a 96-well culture plate with 5000 cells / well and 5 parallel wells per experimental group. The OD 490 value was determined using the MTT cytotoxicity detection kit after 24 hours. pCDNA3.1-EGFP was used as the label control group in the experiment, and the relative proliferation ratio was calculated based on the determination results. The experimental results are shown in Figure 3 shown.

[0045] The cell death test method is as follows: HEK293T cells were digested with trypsin and 1x10 5 The cells were plated in 6-well cell culture plates and cultured overnight. The eukaryotic vector pCDNA3.1-NoLS-EGFP or

[0046] pCDNA3.1-4xNoLS-EGFP, the experimental control group was HEK293T cells transiently transfected with pCDNA3.1-EGFP vector, and 24 hours later, the samples were processed according to the experimental instructions of the cell apoptosis and necrosis kit, and the death cell ratio was detected by flow cytometry. The experimental results are shown in Figure 4 shown.

[0047] Depend on Figure 3 and Figure 4It can be seen from the experimental results shown that the polypeptide expressed by the signal peptide sequence NoLS and its recombinant sequence 4xNoLS involved in the present invention is not toxic to eukaryotic cells in practical applications and can be widely used in research related to cellular functions.

[0048] Example 4

[0049] Nucleolar localization signal peptide carries protein polypeptide into the cell nucleolus

[0050] The above-mentioned successfully verified 4xNoLS gene sequence SEQ ID NO.2 with nucleolar localization marker was used to fuse it with the non-nucleolar targeted Flag-HA and Myc polypeptide gene sequences, namely SEQ ID NO.3 and SEQ ID NO.4, and constructed into the eukaryotic expression vector of pCDNA3.1 as an implementation case of the application method of the nucleolar localization signal peptide that can carry other protein polypeptides into the nucleolus of eukaryotic cells.

[0051] The gene sequences of 4xNoLS and protein polypeptide were connected into a complete fragment by homologous recombination. The construction diagram is shown in the figure. Figure 5 As shown in Figure A, the primer sequences used are as follows:

[0052] FH-F1: 5'-CCGAGCTCGGATCCATGGACTACAAGGACGACGATGAC-3'

[0053] FH-R1: 5'-CGTCTTTCTTGGAATTCAGGCGCGCCTCCGGCGTAGTCGGGC-3'

[0054] 4xNoLS-F3: 5'-AGGCGCGCCTGAATTCCAAGAAAGACGCTC-3'

[0055] 4xNoLS-R3: 5'-GCTGGATATCTGCAGAATTCTCATTTGCAGTGAAGTTCTC-3'

[0056] Myc-F1: 5'-CTTAAGCTTGGTACCATGGAGCAAAAGCTCATTTC-3'

[0057] Myc-R1: 5'-GAGCGTCTTTCTTGCAAGTCCTCTTC-3'

[0058] 4xNoLS-F4: 5'-GAAGAGGACTTGCAAGAAAGACGCTCC-3'

[0059] 4xNoLS-R4: 5'-GGTTTAAACGGGCCCTCATTTGCAGTGAAGTTCTC-3'

[0060] In the construction of all target protein vectors, the gene sequence in SEQ ID NO.2 is used as a template, and PCR reactions are performed using primers 4xNoLS-F3 and 4xNoLS-R3 and primers 4xNoLS-F4 and 4xNoLS-R4 (the reaction conditions are the same as those in Table 1, and the annealing temperature is 60°C) to obtain the 4xNoLS gene sequence containing homology arms. Then, the directly synthesized Flag-HA or Myc gene sequence is used as a template, and PCR reactions are performed using primers FH-F1 and FH-R1 or Myc-F1 and Myc-R1 (the reaction conditions are the same as those in Table 1, but the annealing temperature is changed to 61°C) to obtain the Flag-HA or Myc gene sequence containing homology arms. Next, a mixture of the above-mentioned Flag-HA or Myc sequence and the 4xNoLS gene sequence containing the homology arms was used as a template, and FH-F1 and 4xNoLS-R3 and Myc-F1 and 4xNoLS-R4 were used as primers for PCR reaction (the reaction conditions were the same as in Table 1, but the annealing temperature was 60°C) to obtain the Flag-HA-4xNoLS and Myc-4xNoLS gene sequence fragments containing the vector homology arms. The above-mentioned fragments were constructed into the pCDNA3.1 vector linearized by double restriction digestion with HindⅢ and BamHI using a homologous recombination kit, and finally the eukaryotic expression vectors pCDNA3.1-Flag-HA-4xNoLS and pCDNA3.1-Myc-4xNoLS were obtained.

[0061] The successfully constructed pCDNA3.1-Flag-HA-4xNoLS and pCDNA3.1-Myc-4xNoLS vectors were transformed into DH5α competent cells to obtain recombinant plasmids, and the two plasmids were transiently transfected into HEK293T and HeLa cells. The method was the same as the experimental description in Example 1 (2). Confocal microscopy was used to observe whether Flag-HA or Myc could be carried into the cell nucleolus by the SEQ ID NO.2 signal peptide of the present invention. Figure 5 The experimental results in show that the nucleolar localization signal peptide modified by four tandem recombination in the present invention has the function of carrying protein polypeptides into the nucleolus region of eukaryotic cells. Therefore, the above experiments show that the nucleolar localization signal peptide and its application method in the present invention have practical application value in carrying other polypeptides into the nucleolus.

[0062] It should be noted that the above-mentioned technical contents of the present invention are only for explanation and clarification to enable those skilled in the art to understand the technical essence of the present invention, so the technical contents are not used to limit the substantial protection scope of the present invention. The substantial protection scope of the present invention shall be subject to the claims. Those skilled in the art should know that any modification, equivalent substitution and improvement based on the substantial spirit of the present invention shall be within the substantial protection scope of the present invention.

Claims

1. A nucleolar localization signal peptide, which is a polypeptide with an amino acid sequence of SEQ ID NO.7, or a polypeptide obtained by tandem recombination of SEQ ID NO.7 2-8 times.

2. The coding gene of the nucleolar localization signal peptide according to claim 1, wherein the coding gene sequence corresponding to the amino acid sequence SEQ ID NO.7 is SEQ ID NO.

1.

3. A nucleolar localization signal peptide, which is a polypeptide obtained by tandemly recombining the amino acid sequence SEQ ID NO.7 of claim 1 four times, and its amino acid sequence is SEQ ID NO.

8.

4. The gene encoding the nucleolar localization signal peptide according to claim 3, whose sequence is SEQ ID NO.

2.

5. Use of the nucleolus localization signal peptide according to claim 1 or 3 in carrying protein polypeptides into the nucleolus of eukaryotic cells.

6. The use according to claim 5, wherein the gene encoding the protein polypeptide is at least one of the sequences SEQ ID NO.3 and SEQ ID NO.

4.

7. The use according to claim 6, wherein the method comprises the following steps: (1) Construction of recombinant plasmids carrying protein polypeptides into nucleoli: Sequence SEQ ID NO.2 was fused with sequence SEQ ID NO.3 and sequence SEQ ID NO.4 to form sequence SEQ ID NO.5 and sequence SEQ ID NO.6 respectively, and then sequence SEQ ID NO.5 and sequence SEQ ID NO.6 were recombined into pCDNA3.1 vector respectively, thereby obtaining recombinant plasmids pCDNA3.1-Flag-HA-4xNoLS and pCDNA3.1-Myc-4xNoLS carrying protein polypeptides into eukaryotic cell nucleoli; (2) Cell transfection and culture: The constructed recombinant plasmids pCDNA3.1-Flag-HA-4xNoLS and pCDNA3.1-Myc-4xNoLS were transiently transfected into eukaryotic cells and then cultured to express Flag-HA-4xNoLS and Myc-4xNoLS proteins in the cells. Immunofluorescence experiments were used to observe the localization of the nucleolus by the nucleolar localization signal peptide and the entry of the 4xNoLS-carrying protein polypeptides Flag-HA and Myc into the nucleolus region of the cell nucleus.

8. A nucleolar localization marker vector, which is obtained by recombinantly constructing the nucleolar localization signal peptide according to claim 1 or 3 into a eukaryotic expression vector pCDNA3.1 or pCDNA3.1-EGFP.

9. Use of the nucleolar localization marker vector according to claim 8 in nucleolar marking of eukaryotic cells.

10. A recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium containing the coding gene according to claim 2 or 4.

Images