CN1995065B - Fusion protein capable of inhibiting PTTG expression, its coding gene and application - Google Patents

Abstract

The invention discloses a fusion protein capable of inhibiting PTTG expression, its coding gene and application. The purpose is to provide a fusion protein capable of inhibiting PTTG expression, its coding gene and its application in the preparation of drugs for treating PTTG-positive tumors. The fusion protein is a fusion protein in which amino acid residues 1-256 of the amino terminal of β-TrCP are connected at the amino (N) terminal of PBF or a polypeptide consisting of amino acid residues 1-30 of the carboxyl terminal of PBF through a connecting peptide. The fusion protein β-TrCP-PBF and its coding gene of the present invention will play an important role in the functional research of PTTG and the preparation of medicines for treating PTTG positive tumors and diseases related to PTTG overexpression, and have broad application prospects.

Description

Fusion protein capable of inhibiting PTTG expression, its coding gene and application

technical field

The present invention relates to a fusion protein and its encoding gene and application, in particular to a fusion protein and its encoding gene capable of inhibiting the expression of PTTG, as well as the expression method of the fusion protein and the preparation and treatment of PTTG positive tumors and PTTG overexpression-related drugs. Application in the medicine of disease.

Background technique

Pituitary tumor transforming gene (PTTG gene) is a proto-oncogene expressed in a variety of tumors. Research results in recent years have shown that PTTG gene is a powerful tumor transformation gene, which can participate in the formation and development of tumors through the following mechanisms: inducing cell transformation; promoting the expression of other proto-oncogenes and tumor-promoting factors; interfering with sister staining Monomer separation; promote tumor angiogenesis involving basic fibroblast growth factor (bFGF), etc. Among them, bFGF has the closest relationship with PTTG, and some scholars even believe that some biological effects of PTTG are realized through bFGF. bFGF can promote tumor neovascularization and play an important role in tumor growth and invasion, while tumor invasion and metastasis are often the reasons for treatment failure. Therefore, it is of great significance to use PTTG as a target for research and treatment of tumors.

Selective protein degradation is a characteristic of cell growth and maintenance of self-stabilization, and proteasome is a protein degradation system responsible for clearing intracellular instructions for degradation of proteins. Among them, the ubiquitin-proteasome pathway (Ubiquitin-Proteasome Pathway) is the most important efficient protein degradation pathway in vivo. This pathway has two steps: 1) multiple ubiquitins are covalently attached to the target protein; 2) the polyubiquitinated target protein is degraded by the 26S proteasome complex. The process of ubiquitin binding to the target protein requires at least three enzymes: ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin ligase (E3). The Skp1-Cullin1-F-box (SCF) complex is an important E3 enzyme, which consists of four parts: Skp1, Cul1, Rbx1 and F-box protein, the first three are invariable components, and the F-box protein is a variable change components. F-box protein is a huge protein family, at least 70 kinds have been found so far, and its common feature is that one end has an F-box motif to bind to Skp1, and the other end has a complex binding region, which can bind to different substrate target proteins Binding, thereby mediating the degradation of the target protein after ubiquitination by SCF complex type E3.

To study and treat specific oncogenes by inhibiting the expression of relevant oncogenes at the mRNA level is currently the most commonly used method. Faster and more reliable. The high level of PTTG protein stays in each cell cycle for a long time, which is one of the reasons for the abnormal transformation of cells.

β-TrCP protein (gene sequence GenBank number is Y14153, protein GenBank number is CAA74572.1) is a member of the F-box protein family, consisting of 569 amino acid residues, its protein structure schematic diagram is shown in Figure 1; from the amino terminal Amino acids 142 to 194 are F-box motifs, which can bind to skp1; amino acids 195 to 256 are connecting peptides, which provide a favorable spatial structure for effective ubiquitination; amino acids 257 to 569 are 7 WD The -40 repeat sequence constitutes a complex spatial structure, which is conducive to binding to different substrate proteins. β-TrCP protein widely exists in a variety of human cells and is involved in the regulation of multiple signaling pathways and cell cycle in vivo.

PBF (GenBank number of gene sequence: BC019295, GenBank number of PBF protein: AAH19295.1) is the abbreviation of PTTG binding factor (PTTG-binding-factor). It is ubiquitously expressed in tissues and can specifically interact with PTTG both in vivo and in vitro. PBF contains two NLS (nuclear localization signal, nuclear localization signal) sequences between amino acids 149-166, and NLS mediates protein entry into the nucleus; PTTG does not contain NLS sequences, and PTTG requires the participation of NLS sequences of PBF to enter the nucleus and function . Another experiment proved that the C-terminus of PBF protein contains 30 amino acids of nuclear localization signal, which are necessary for the combination of PBF and PTTG, and are sufficient for the combination of PTTG.

Ubiquitin ligase E3 is responsible for specific recognition and binding of substrates during ubiquitination. In recent years, several research groups have applied recombinant technology to construct recombinant chimeric ubiquitin ligases, which successfully degrade certain proteins that are natural substrates of non-ubiquitinated systems under normal conditions. For example, by modifying the chimeric F-box protein composed of the substrate-binding subunit F-box of the ubiquitin ligase complex Skp1-Cullin1-F-box (SCF), it can target the degradation of pRB family protein members, β- catenin and cyclin A-Cdk2. These findings suggest that recombinant ubiquitin ligases can be used for targeted degradation of some disease-associated molecules.

Contents of the invention

The purpose of the present invention is to provide a fusion protein capable of inhibiting the expression of PTTG.

The fusion protein provided by the present invention has the effect of inhibiting the expression of PTTG, named β-TrCP-PBF, which is obtained by linking the peptide at the 1-30 amino acid residues (including the nuclear localization sequence) at the carboxyl (C) terminal of PBF The amino (N) terminal of the composed polypeptide is connected to the fusion protein of β-TrCP amino acid residues 1-256 from the amino terminal (including the F-box motif).

The selection of the connecting peptide is varied, preferably a flexible peptide, and its amino acid residue sequence can be represented by SEQ ID NO: 1 (GSGSGSGSGS) or 1-3 SEQ ID NO: 2 (GGGGS) in the sequence listing. the repeated sequence shown. Flexible peptides are easily bendable, allowing the expressed protein to fold correctly without affecting the respective active binding domains of the fusion protein.

Specifically, the fusion protein that inhibits the expression of PTTG is one of the following amino acid residue sequences:

1) SEQ ID NO: 3 in the sequence listing;

2) SEQ ID NO: 4 in the sequence listing;

3) SEQ ID NO: 5 in the sequence listing;

4) SEQ ID NO: 6 in the sequence listing;

5) SEQ ID NO: 7 in the sequence listing.

SEQ ID NO: 3 in the sequence listing is composed of 446 amino acid residues, the 1-256 amino acid residues from the amino terminal are β-TrCP amino acid residues 1-256 from the amino terminal, and the 257-266 amino acid residues from the amino terminal It is a connecting peptide sequence, the 267th-446th position from the amino terminal is PBF; the SEQ ID NO: 4 in the sequence table is composed of 451 amino acid residues, and the 1st-256th position from the amino terminal is β-TrCP from the 1st amino terminal -256 amino acid residue sequence, the 257-271 from the amino terminal is the connecting peptide sequence, and the 272-451 from the amino terminal is PBF; SEQ ID NO: 5 in the sequence table is composed of 446 amino acid residues, from The 1-256th position of the amino terminal is the amino acid residue sequence of β-TrCP from the 1-256th position of the amino terminal, the 257-266 position of the amino terminal is the connecting peptide sequence, and the 267-446 position of the amino terminal is PBF; the sequence list The SEQ ID NO: 6 is composed of 441 amino acid residues, the 1-256th position from the amino terminal is the amino acid residue sequence from the 1-256th position of β-TrCP from the amino terminal, and the 257-261 position from the amino terminal is the connection Peptide sequence, 262-441 from the amino terminal is PBF; SEQ ID NO: 7 in the sequence table consists of 296 amino acid residues, β-TrCP from the 1-256 of the amino terminal The sequence of amino acid residues at position 257-266 from the amino terminal is the connecting peptide sequence, and the position 267-296 from the amino terminal is the amino acid residue at position 1-30 at the carboxyl terminal of PBF.

The gene (β-TrCP-PBF) encoding the fusion protein with the effect of inhibiting the expression of PTTG is one of the following nucleotide sequences:

1) The DNA sequence of SEQ ID NO: 8 in the sequence listing;

2) DNA sequence of SEQ ID NO: 9 in the sequence listing;

3) The DNA sequence of SEQ ID NO: 10 in the sequence listing;

4) the DNA sequence of SEQ ID NO: 11 in the sequence listing;

5) DNA sequence of SEQ ID NO: 12 in the sequence listing;

6) The DNA sequence of SEQ ID NO: 3-7 in the coding sequence listing.

SEQ ID NO: 8 in the sequence listing consists of 1341 bases, and its coding sequence is the 1-1338th base from the 5' end, encoding a protein with the amino acid residue sequence of SEQ ID NO: 3 in the sequence listing, The 1st-768th base from the 5' end is the coding sequence of the 1-256th amino acid residue sequence from the amino terminal of β-TrCP, and the 769-798th base from the 5' end is the coding sequence of the connecting peptide, from The 799th-1338th base at the 5' end is the coding sequence of PBF; the SEQ ID NO: 9 in the sequence table is composed of 1356 bases, and its coding sequence is from the 1st-1353rd base at the 5' end, and the encoding has The protein of the amino acid residue sequence of SEQ ID NO: 4 in the sequence listing, the 1-768th base from the 5' end is the coding sequence of the 1-256th amino acid residue sequence from the 5' end of β-TrCP The 769-813 bases at the end are the coding sequence of the connecting peptide, and the 814-1356 bases from the 5' end are the coding sequence of PBF; SEQ ID NO: 10 in the sequence table consists of 1341 bases, and its encoding The sequence is bases 1-1338 from the 5' end, encoding a protein with the amino acid residue sequence of SEQ ID NO: 5 in the sequence listing, and bases 1-768 from the 5' end are β-TrCP from the amino terminal The coding sequence of the 1-256th amino acid residue sequence, the 769-798th base from the 5' end is the coding sequence of the connecting peptide, and the 799-1338th base from the 5' end is the coding sequence of PBF; Sequence Listing The SEQ ID NO: 11 in the sequence consists of 1326 bases, and its coding sequence is the 1-1323 bases from the 5' end, which encodes a protein with the amino acid residue sequence of SEQ ID NO: 6 in the sequence listing. Bases 1-768 at the 'end are the coding sequence of amino acid residues at positions 1-256 from the amino terminal of β-TrCP, and bases 769-783 at the 5' end are the coding sequence of the connecting peptide. The 784-1323 bases at the end are the coding sequence of PBF; the SEQ ID NO: 12 in the sequence listing is composed of 891 bases, and its coding sequence is the 1-888 bases from the 5' end, and the encoding has the sequence listing In the protein of the amino acid residue sequence of SEQ ID NO: 7, the 1st-768th base from the 5' end is the coding sequence of the 1-256th amino acid residue sequence from the 5' end of β-TrCP. Bases 769-798 are the coding sequence of the connecting peptide, and bases 799-888 from the 5' end are the coding sequence of amino acid residues 1-30 at the carboxyl end of PBF.

The expression vector, transgenic cell line and host bacteria containing the fusion protein gene of the present invention all belong to the protection scope of the present invention.

For the convenience of purification, the 5' end of the fusion protein gene that inhibits PTTG expression in the above-mentioned recombinant expression vector is also connected with a His-tag or FLAG-tag coding sequence.

The starting vector for constructing the recombinant expression vector containing the fusion protein gene of the present invention is any prokaryotic or eukaryotic expression vector; the prokaryotic expression vector can be pET-32a, pET-28a, pET-28b, pET-28c, pET-21a (+) or pET-30a etc.; The eukaryotic expression vector can be pCMV5, pSilence1.0-U6 (Ambion, Austin, TX, USA), pcDNA, pEGFP-N1, pSV40, pCI-neo (purchased from Promega), pTEF1, pPICZa, pAM82 or pAAh5, etc.

The above-mentioned recombinant expression vectors can be constructed according to conventional methods.

The pair of primers for amplifying any fragment of the above-mentioned fusion protein coding gene is also within the protection scope of the present invention.

The present invention also provides a method for expressing the above-mentioned fusion protein capable of inhibiting the expression of PTTG.

The method for expressing the above-mentioned fusion protein capable of inhibiting PTTG expression provided by the present invention is to introduce the above-mentioned recombinant expression vector containing the fusion protein gene capable of inhibiting PTTG expression into host cells, and express the fusion protein capable of inhibiting PTTG expression.

The host can be Escherichia coli, yeast, mammalian cells, insect cells or Bacillus subtilis, etc., preferably Escherichia coli.

The Escherichia coli can be E.coli DH5α, E.coli BL ₂₁ (DE ₃ ) or E.coli Top10, etc.

The yeast is preferably Pichia pastoris. Wherein, the Pichia pastoris is preferably Pichia pastoris GS115, KM71 (purchased from Invitrogen, USA) or SMD1168 (purchased from Invitrogen, USA).

The above-mentioned recombinant bacteria can be constructed according to conventional methods.

The medium and culture conditions for cultivating the host cells containing the fusion protein gene capable of inhibiting the expression of PTTG can be the medium and culture conditions for cultivating the starting host. Wherein, when cultivating the recombinant Escherichia coli host, it is necessary to add an inducer, such as IPTG, etc., the concentration of the added IPTG is 0.5-1.0mmol/L, preferably 1mmol/L, and the induction temperature is 16-37°C, preferably 28°C , the induction time is 2-4 hours, preferably 4 hours.

The invention also provides a medicine for treating PTTG positive tumors and diseases related to PTTG overexpression.

The therapeutic drug provided by the present invention has an active ingredient of the fusion protein or its coding gene which can inhibit the expression of PTTG.

The fusion protein gene capable of inhibiting the expression of PTTG may exist in a eukaryotic expression vector.

When necessary, one or more pharmaceutically acceptable carriers can also be added to the above drugs. The gene drug carrier includes (but not limited to) a carrier carrying a gene, such as liposome, recombinant virus vector, etc.; a carrier carrying a protein drug, such as (but not limited to) other protein or polypeptide components; the carrier also includes Common diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, etc. in the pharmaceutical field.

The dosage of the above-mentioned protein drugs and gene drugs can adopt the conventional doses of protein drugs and gene drugs in the field of pharmacy, and can be adjusted according to the actual situation.

The invention provides a fusion protein β-TrCP-PBF capable of inhibiting the expression of PTTG and its coding gene. The fusion protein retains the part of the N-terminal of the β-TrCP protein that includes the F-box motif, and replaces the substrate protein binding region at the C-terminus with PBF that can specifically bind PTTG or its C-terminal 1-30 amino acid residues (including nuclear localization sequence), and connecting the two with a flexible linker peptide (such as "GSGSGSGSGS", etc.) can improve the flexibility of the fusion protein in space docking. The PBF region in β-TrCP-PBF is responsible for the targeted binding of PTTG, and the F-box motif is responsible for binding with Skp1 and then forming SCF-type E3, thereby specifically mediating the ubiquitination and degradation of PTTG protein. Experiments have proved that β-TrCP-PBF of the present invention can not only significantly down-regulate the expression level of exogenous PTTG protein, but also the expression level of endogenous PTTG protein in PTTG positive cells (such as Hela cells) transfected with β-TrCP-PBF gene eukaryotic expression vector The level was significantly lower than that before transfection. In addition, the proliferation ability of cells (with a certain ability to promote apoptosis) and the ability to form cell clones were also inhibited. The fusion protein β-TrCP-PBF and its coding gene of the present invention will play an important role in the functional research of PTTG and the preparation of medicines for treating PTTG positive tumors and diseases related to PTTG overexpression, and have broad application prospects.

The present invention will be described in further detail below in conjunction with specific embodiments.

Description of drawings

Fig. 1 is the structural representation of β-TrCP and fusion protein β-TrCP-PBF of the present invention

Figure 2 is the physical map of vector pCMV5

Figure 3 is the result of double enzyme digestion identification of pCMV5-β-TrCP-PBF vector

Figure 4 is the experimental results of co-transfecting COS-7 cells with pCMV5-β-TrCP-PBF and pEGFP-N ₃ -PTTG to detect the effect of the fusion protein β-TrCP-PBF of the present invention on the expression level of exogenous PTTG protein

Fig. 5 is the immunoblotting detection result of fusion protein β-TrCP-PBF of the present invention to the expression level of endogenous PTTG protein of PTTG-positive cervical cancer cell line Hela cell

Fig. 6 is the statistical result of the influence of fusion protein β-TrCP-PBF of the present invention on the proliferation of PTTG-positive cervical cancer cell line Hela cells

Fig. 7 is the microscopic observation result of fusion protein β-TrCP-PBF of the present invention on the proliferation ability of PTTG-positive cervical cancer cell line Hela cell clone

Detailed ways

The methods used in the following examples are conventional methods unless otherwise specified, and the specific steps can be found in: "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3 ^rd edition, 2001, NY, Cold Spring Harbor). The primers and DNA sequences used were synthesized by Shanghai Handsome Biotechnology Co., Ltd.

Embodiment 1, the acquisition of fusion protein gene β-TrCP-PBF and the construction of its eukaryotic expression vector

1. Amplification of β-TrCP linked to the Flag-tag sequence at the N-terminus from the coding sequence of amino acid residues 1-256 at the N-terminus

Extract the total RNA of the MCF-7 cell line (purchased from Shanghai Cell Institute), synthesize its cDNA by reverse transcription and use it as a template, in the primer TRC7 (upstream primer): 5'

-GTCTCTAGAGCCACC ATGGACTACAAGGACGACGATGACAAA ATGGACCCGGCCGAGG-3' (the underlined base is the Flag-tag coding sequence) and TRC6 (downstream primer): 5'

- Under the guidance of GTCGGATCCACTATGTCTTCCACATTCTCCAATTAGATTC-3', PCR amplifies the coding sequence of amino acid residues 1-256 from the amino-terminus of β-TrCP connected to the Flag-tag sequence at the amino-terminus, and introduces restriction enzyme Xba at both ends of the amplified fragment I and BamH I restriction sites. After the reaction, the PCR amplification product was detected by 1% agarose gel electrophoresis. As a result, the target fragment with a length of 819bp was obtained by PCR amplification. The sequencing results showed that the correct 5' end connection Flag-tag coding sequence was obtained. The β-TrCP gene is a gene fragment composed of bases 1-768 at the 5' end, that is, the coding sequence of amino acid residues 1-256 at the amino-terminus of β-TrCP connected to the Flag-tag sequence at the N-terminus. The fragment of interest is recovered and purified.

2. Cloning of the PBF coding sequence linked to the "GSGSGSGSGS" connecting peptide sequence at the amino terminal

Using the MCF-7 cell line cDNA as a template, primer PBF3 (upstream primer): 5'

-CTG GGATCCGGCTCAGGCTCAGGATCAGGCTCA ATGGCGCCCGGAGTGG-3' (underlined bases are "GSGSGSGSGS" linker peptide coding sequence) and PBF2 (downstream primer): 5'

-CTTCCCGGGTTAGTTGTTTTCAAATCTAGCATACG-3'-guided PCR amplification of the PBF coding sequence whose amino terminal is connected to the "GSGSGSGSGS" connecting peptide (SEQ ID NO: 1 in the sequence listing) sequence, and simultaneously introduces restriction endonuclease BamH I at both ends of the amplified fragment and Sma I restriction site. After the reaction, the PCR amplification product was detected by 1% agarose gel electrophoresis, and the target fragment with a length of 585bp was obtained by PCR amplification. The sequencing results showed that the 5' end connection "GSGSGSGSGS" connecting peptide with the correct sequence was obtained The PBF gene fragment of the coding sequence, that is, the PBF coding sequence connected to the "GSGSGSGSGS" connecting peptide sequence at the amino terminal. The fragment of interest is recovered and purified.

In addition, the following DNA fragments were also obtained by ordinary PCR amplification (simultaneously introducing restriction endonucleases BamH I and Sma I restriction sites at both ends of the amplified fragment): 1) three "GGGGS" were connected to the amino terminal 2) the PBF coding sequence of the connecting peptide connecting two "GGGGS" repeating sequences at the amino terminal; 3) the PBF coding sequence connecting the "GGGGS" connecting peptide at the amino terminal; 4) the amino terminal The coding sequence of the polypeptide consisting of amino acid residues 1-30 at the carboxy terminus of PBF of the "GSGSGSGSGS" connecting peptide.

3. Construction of recombinant chimeric molecule β-TrCP-PBF eukaryotic expression vector

As shown in Figure 2, the vector pCMV5 (GenBank number: AF239249) contains CMV: CMV promoter, 842-913 bases; MCS: multiple cloning site, 902-986 bases; SV40: SV40 start site, 1817-1894 bases; T7: T7 promoter, 2023-2005 bases; pBR322_origin: pBR322 start site, 3043-2424 bases; Ampicillin: ampicillin resistance gene, 4058-3198 bases; f1_origin: f1 start site, 4207-4513 bases. After the carrier pCMV5 was double-digested with restriction endonucleases Xba I and Sma I, the β-TrCP gene that was connected to the Flag-tag coding sequence at the 5' end of Example 1 step 1 was amplified from 5 with T4 DNA ligase. The gene fragment consisting of bases 1-768 at the ' end (with Xba I and BamH I restriction sites added at both ends) and the PBF of the 5' end connected to the "GSGSGSGSGS" linker peptide coding sequence amplified in Step 2 of Example 1 The gene fragment (with BamH I and Sma I restriction sites added at both ends) was ligated with linearized pCMV5, the ligated product was transformed into Escherichia coli JM109 competent cells, positive single clones were screened, plasmids were extracted, and Xba I and Sma I were respectively used to I, Xba I and BamH I, BamH I and Sma I carry out double enzyme digestion identification to the recombinant plasmid, and the enzyme digestion identification result is shown in Figure 3 (swimming lane M is DNA Marker, and swimming lane 1 is the Xba I and Sma I of recombinant plasmid The result of double enzyme digestion identification, lane 2 is the double enzyme digestion identification result of Xba I and BamH I of the recombinant plasmid, and swimming lane 3 is the double enzyme digestion identification result of BamH I and Sma I of the recombinant plasmid), the recombinant plasmid was passed through Xba I and Sma I I double-enzyme digestion obtained a DNA fragment of about 1386bp, the recombinant plasmid obtained a DNA fragment of about 813bp through XbaI and BamHI double-enzyme digestion, and the recombinant plasmid obtained a DNA fragment of about 579bp through BamHI and SmaI double-enzyme digestion, and Expected results match. Sequencing and identification of the above recombinant plasmid, except for the Flag-tag coding sequence and restriction site, the foreign DNA fragment inserted in the vector has the nucleotide sequence of SEQ ID NO: 8 in the sequence table, consisting of 1341 bases , its coding sequence is base 1-1338 from the 5' end, encoding a protein with the amino acid residue sequence of SEQ ID NO: 3 in the sequence listing, base 1-768 from the 5' end is β-TrCP The coding sequence of the 1-256th amino acid residue sequence from the amino terminal, the 769-798th base from the 5' end is the coding sequence of the connecting peptide, and the 799-1338th base from the 5' end is the coding sequence of PBF . The constructed eukaryotic expression vector of β-TrCP-PBF was named pCMV5-β-TrCP-PBF.

In addition, the β-TrCP gene amplified in Step 1 of Example 1 is connected to the β-TrCP gene of the Flag-tag coding sequence by the above-mentioned method from the 1st to 768th base of the 5' end (adding Xba I at both ends) and BamH I restriction site) and the PBF gene fragment of the 5' end amplified in step 2 of Example 1 to connect three "GGGGS" repeat sequences to connect the peptide coding sequence (with BamHI and SmaI restriction sites added at both ends) , the PBF gene fragment of the two "GGGGS" repeat sequences connecting the peptide coding sequence at the 5' end (with BamH I and Sma I restriction sites added at both ends), and the "GGGGS" repeat sequence connecting the peptide coding sequence at the 5' end The coding sequence and linear Ligated in the pCMV5 of bc to obtain the following recombinant eukaryotic expression vectors: 1) the eukaryotic expression vector containing the fusion protein gene of three "GGGGS" repeated sequences as the connecting peptide, named pCMV5-β-TrCP-PBF3, the fusion The protein gene has the nucleotide sequence of SEQ ID NO: 9 in the sequence listing, consisting of 1356 bases, and its coding sequence is the 1st-1353 bases from the 5' end, and the coding sequence has SEQ ID NO: 4 in the sequence listing The amino acid residue sequence of the protein, the 1-768th base from the 5' end is β-TrCP, the coding sequence of the 1-256th amino acid residue sequence from the amino terminal, the 769-813th base from the 5' end It is the coding sequence of the connecting peptide, and the 814th-1356th base from the 5' end is the coding sequence of PBF; 2) The eukaryotic expression vector containing the fusion protein gene of two "GGGGS" repeated sequences of the connecting peptide is named pCMV5 -β-TrCP-PBF2, the fusion protein gene has the nucleotide sequence of SEQ ID NO: 10 in the sequence table, consists of 1341 bases, and its coding sequence is the 1st-1338th base from the 5' end, encoding The protein having the amino acid residue sequence of SEQ ID NO: 5 in the sequence listing, the coding sequence of the 1-256 amino acid residue sequence from the amino terminal of β-TrCP from the 1st-768th base at the 5' end, from 5' The 769-798th base at the 'end is the coding sequence of the connecting peptide, and the 799-1338th base at the 5' end is the coding sequence of PBF; 3) The eukaryotic expression of the fusion protein gene containing the connecting peptide as "GGGGS" The vector is named as pCMV5-β-TrCP-PBF1, and the fusion protein gene has the nucleotide sequence of SEQ ID NO: 11 in the sequence table, consisting of 1326 bases, and its coding sequence is from 1-1323 at the 5' end base, encoding a protein with the amino acid residue sequence of SEQ ID NO: 6 in the sequence table, from 1-768 at the 5' end The bases are the coding sequence of the 1-256th amino acid residue sequence from the amino terminal of β-TrCP, the 769-783 bases from the 5' end are the coding sequence of the connecting peptide, and the 784-1323 bases from the 5' end The base is the coding sequence of PBF; 4) the amino terminal of the polypeptide containing the linking peptide "GSGSGSGSGS" consisting of the 1-30 amino acid residues at the carboxyl terminal of PBF is connected to β-TrCP from the 1-256 amino acids at the amino terminal The eukaryotic expression vector of the fusion protein gene of residue, named as pCMV5-β-TrCP-PBF30, this fusion protein gene has the nucleotide sequence of SEQ ID NO: 12 in the sequence list, is made up of 891 bases, and its encoding The sequence is bases 1-888 from the 5' end, encoding a protein with the amino acid residue sequence of SEQ ID NO: 7 in the sequence listing, bases 1-768 from the 5' end are β-TrCP from the amino terminal The coding sequence of the 1-256th amino acid residue sequence, the 769-798th base from the 5' end is the coding sequence of the connecting peptide, and the 799-888th base from the 5' end is the 1st-30th base of the PBF carboxy terminal coding sequence for amino acid residues. The fusion protein gene of the present invention is collectively referred to as β-TrCP-PBF, and its encoded protein is collectively referred to as β-TrCP-PBF. The structural schematic diagram of the protein is shown in FIG. 1 .

Example 2, detection of the inhibitory effect of the fusion protein β-TrCP-PBF of the present invention on PTTG gene expression

1. Detection of the influence of the fusion protein β-TrCP-PBF of the present invention on the expression of the exogenous PTTG-EGFP fusion protein

The eukaryotic expression vector pCMV5-β-TrCP-PBF constructed in Example 1 carrying the fusion protein gene of the present invention and the plasmid pEGFP-N 3 -PTTG (pEGFP-N ₃ -PTTG (pEGFP-N ₃ - PTTG is a eukaryotic expression vector of the PTTG-EGFP fusion protein gene. After transfection into eukaryotic cells, PTTG protein with green fluorescent protein label can be expressed. This fusion protein can emit green fluorescence under blue excitation light. Plasmid pEGFP-N ₃ -PTTG construction method: extract the total RNA of the MCF-7 cell line, synthesize its cDNA by reverse transcription and use it as a template, in the primer: MP5 (upstream primer):

5'-CGTCAATTCGCCACCATGGCTACTCTGATCTATGTTG-3' and MP6 (downstream primers):

Under the guidance of 5'-CAGGGATCCAATATCTATGTCACAGCAAACAG-3', the PTTG gene fragment (Genbank No.: AF095287) was amplified by PCR, and EcoR I and BamH I were added at both ends of the amplified fragment as points, and then the vector pEGFP-N ₃ ( CLONTECH Biotechnology Company) performed double digestion with restriction endonucleases EcoR I and BamH I, then used T4 DNA ligase to connect the PTTG gene fragment amplified by PCR above with linearized pEGFP-N3, and transform the ligated product into Escherichia coli JM109 competent cells were screened for positive monoclonals, plasmids were extracted, identified by enzyme digestion and sequencing, and finally the target plasmid pEGFP-N ₃ -PTTG carrying the PTTG-EGFP fusion protein gene was obtained. ) transiently co-transfect COS-7 cells, and detect the influence of the fusion protein of the present invention on the expression of PTTG. The specific method is: inoculate COS-7 cells in the logarithmic growth phase in a 24-well culture plate with 1×10 ⁵ cells per well Inside, the culture medium is DMEM medium (GIBCO company) containing 10% fetal bovine serum (HYCLONE company), and cultured in a 5% CO ₂ , 37°C incubator. When the cells grew to 90-95% the next day, use liposome Lipofectamine ^TM 2000 reagent (Gibco Company) and refer to the instruction manual to co-compound the plasmid pCMV5-β-TrCP-PBF and pEGFP-N ₃ -PTTG at a ratio of 1:1. COS-7 cells were transfected, and control cells were co-transfected with pCMV5 empty vector and pEGFP-N ₃ -PTTG (1:1) in the same way, and observed and photographed with a fluorescence microscope (OLYMPUS IX51) 48 hours after transfection, with Image-pro plus v5.02 Sofeware was used to analyze the expression of PTTG in transfected cells. The results are shown in Figure 4 (A: pCMV5 empty vector and pEGFP-N ₃ -PTTG co-transfected cells, B: pCMV5-β-TrCP-PBF and pEGFP-N ₃ -PTTG co-transfected cells), pCMV5-β- The fluorescence brightness of cells co-transfected with TrCP-PBF and pEGFP-N ₃ -PTTG is significantly weaker than that of cells co-transfected with empty plasmid pCMV5 and pEGFP-N ₃ -PTTG, indicating that the fusion protein β-TrCP-PBF of the present invention can Significantly down-regulated the expression of exogenous PTTG protein.

Using the same method as above, pCMV5-β-TrCP-PBF3, pCMV5-β-TrCP-PBF2, pCMV5-β-TrCP-PBF1, pCMV5-β-TrCP-PBF30 were co-transfected with pEGFP-N ₃ -PTTG respectively to COS- 7 cells, to detect the fusion protein β-TrCP-PBF whose linking peptide is 1-3 "GGGGS" repeat sequences of the present invention and the linking peptide of "GSGSGSGSGS" consisting of amino acid residues 1-30 at the carboxyl end of PBF The effect of the fusion protein whose amino terminal is connected with β-TrCP from amino acid residues 1-256 of the amino terminal on the expression of exogenous PTTG protein, and the result is the fluorescence brightness of the cells transfected with the eukaryotic expression vector carrying the fusion protein gene of the present invention It is obviously weaker than the fluorescence brightness of cells co-transfected with empty plasmid pCMV5 and pEGFP-N ₃ -PTTG, indicating that the fusion protein β-TrCP-PBF of the present invention can significantly down-regulate the expression of exogenous PTTG protein.

2. Detection of the influence of the fusion protein β-TrCP-PBF of the present invention on the proliferation of PTTG positive cancer cells and the expression level of endogenous PTTG in cells

Taking the human cervical cancer cell line Hela cells as an example, the eukaryotic expression vector pCMV5-β-TrCP-PBF constructed in Example 1 carrying the fusion protein gene of the present invention was transfected into PTTG positive cancer cells to detect the fusion protein β of the present invention -The effect of TrCP-PBF on the proliferation of PTTG-positive cancer cells and the expression level of endogenous PTTG in the cells. The specific method is: inoculate the Hela cells in the logarithmic growth phase in a 24-well culture plate with a cell volume of 2×10 ⁵ per well, and culture The liquid is DMEM medium containing 10% fetal bovine serum, cultured in 5% CO ₂ , 37°C incubator. The next day, when the cells grew to 90-95%, the plasmid pCMV5-β-TrCP-PBF was transfected into Hela cells with liposome Lipofectamine ^TM 2000 reagent, and the control cells were transfected with pCMV5 empty vector in the same way. The cells were harvested 48 hours after transfection, and the effect of the fusion protein β-TrCP-PBF on the PTTG protein level was first detected by Western blot. The primary antibody was anti-PTTG antibody (purchased from SANTA CRUZ), and the secondary antibody was goat anti-rabbit (purchased from SANTA CRUZ), the test results are as shown in Figure 5, compared with the control cells transfected with pCMV5, the PTTG protein level was significantly down-regulated in the Hela cells transfected with pCMV5-β-TrCP-PBF, indicating that the fusion protein β of the present invention -TrCP-PBF can down-regulate the expression level of endogenous PTTG protein. At the same time, the cell numbers of the two groups of transfected cells were counted on the 1st, 2nd, 3rd, and 4th days after transfection. The statistical results of the cell numbers are shown in Figure 6. The proliferation of the Hela cells of -TrCP-PBF is obviously inhibited, shows that the fusion protein β-TrCP-PBF of the present invention can effectively inhibit the proliferation of cells (PTTG positive tumor cells) related to the overexpression of PTTG, and has the effect of promoting apoptosis . In addition, Hela cells transfected with pCMV5 and pCMV5-β-TrCP-PBF were inoculated in a 24-well culture plate at a volume of 1×10 ³ cells per well, and the formation of cell clones was observed under a microscope on the 7th day. As shown in Figure 7, compared with the control cell of transfection pCMV5, the Hela cell cell colony formation of transfection pCMV5-β-TrCP-PBF is significantly reduced, shows that fusion protein β-TrCP-PBF of the present invention can effectively inhibit and PTTG Clonal proliferation ability of overexpression-related cells (PTTG-positive tumor cells).

Also pCMV5-β-TrCP-PBF3, pCMV5-β-TrCP-PBF2, pCMV5-β-TrCP-PBF1, pCMV5-β-TrCP-PBF30 were transfected into Hela cells with the same method as above, to detect the connecting peptide of the present invention as The fusion protein β-TrCP-PBF of 1-3 repeating sequences of "GGGGS" and the connecting peptide of "GSGSGSGSGS" are connected to the amino-terminal of the polypeptide consisting of the 1-30 amino acid residues at the carboxy-terminal of PBF to connect β-TrCP from the amino group The influence of the fusion protein of amino acid residues 1-256 at the terminal end on the expression level of endogenous PTTG protein, the proliferation ability of PTTG positive cells and the ability to form cell clones, the results were transfected with the eukaryotic expression vector carrying the fusion protein gene of the present invention. The expression level of PTTG in Hela cells is significantly lower than the expression level of PTTG in cells transfected with empty plasmid pCMV5, and the cell proliferation number and cell clone formation are also significantly reduced compared with the ratio of cells transfected with empty plasmid pCMV5, indicating that the fusion of the present invention The protein β-TrCP-PBF can significantly down-regulate the expression of endogenous PTTG protein, the proliferation ability of PTTG-positive cells and the ability to form cell clones, and can be used to prepare drugs for treating PTTG-positive tumors and diseases related to PTTG overexpression.

Embodiment 3, prokaryotic expression of fusion protein β-TrCP-PBF of the present invention

After the prokaryotic expression vector pET-32a (Novagen Company) was double-digested with restriction endonucleases Xba I and Sma I, the 5' end amplified in step 1 of Example 1 was ligated with the Flag-tag code using T4 DNA ligase The sequence of the β-TrCP gene is a gene fragment consisting of bases 1-768 at the 5' end (with Xba I and BamH I restriction sites added at both ends) and the 5' end connection amplified in Step 2 of Example 1" The PBF gene fragment (with BamH I and Sma I restriction sites added at both ends) of the GSGSGSGSGS" connecting peptide coding sequence is connected with the linearized vector pET-32a to obtain the prokaryotic expression vector of the fusion protein β-TrCP-PBF of the present invention, Named as pET32a-βTrCP-PBF [A prokaryotic expression vector (named pET32a-βTrCP-PBF3) of the β-TrCP-PBF gene containing the connecting peptide as three "GGGGS" repeat sequences was obtained by the same method, and the connecting peptide was The prokaryotic expression vector of the β-TrCP-PBF gene with two "GGGGS" repeat sequences (named pET32a-βTrCP-PBF2), and the prokaryotic expression vector of the β-TrCP-PBF gene containing the connecting peptide as "GGGGS" (named pET32a -βTrCP-PBF1), containing the linking peptide of "GSGSGSGSGS" at the amino terminal of the polypeptide consisting of the 1-30 amino acid residues at the carboxyl end of PBF, connecting the β-TrCP from the 1-256 amino acid residues at the amino terminal - the prokaryotic expression vector of the TrCP-PBF gene (named pET32a-βTrCP-PBF30)], and then the above prokaryotic expression vectors were respectively transformed into E. Inoculate the positive recombinant bacteria into 1000 mL LB liquid medium containing 100 mg/mL ampicillin for mass culture at 37 °C until the logarithmic growth phase (OD ₆₀₀ value is about 0.8), add the chemical inducer IPTG to the final concentration to 1 mmol/L, and culture was continued for 4 hours at 37°C. After the cultivation, collect the bacteria by centrifugation at 4°C and 6000rpm for 15 minutes, resuspend the bacteria pellet with 100mL TE buffer (20mmol/L, pH8.0), add lysozyme (1mg/mL) and stir magnetically at room temperature for 10min; ice bath Medium-ultrasonic (30s/on, 20s/off, 15 cycles) disrupted the bacteria; centrifuged at 4°C, 12000rpm for 20min to collect the inclusion body precipitate and supernatant respectively. Wash the inclusion body pellet once with 1mol/L NaCl (prepared in TE), centrifuge at 4°C, 12000rpm for 20min to remove impurities in the inclusion body, discard the supernatant and wash twice with TE buffer, 4°C, 12000rpm Centrifuge for 20 minutes to collect the precipitate, use Flag-tag to purify the expressed fusion protein β-TrCP-PBF, and obtain the purified β-TrCP-PBF with the connecting peptide of the present invention as "GSGSGSGSGS", and the connecting peptide as three repeats of "GGGGS" Sequence of β-TrCP-PBF, β-TrCP-PBF with two "GGGGS" repeating peptides, β-TrCP-PBF with "GGGGS" as the connecting peptide, and "GSGSGSGSGS" as the connecting peptide at the carboxyl end of PBF The amino terminal of the polypeptide consisting of amino acid residues 1-30 is connected with β-TrCP from β-TrCP-PBF of amino acid residues 1-256 at the amino terminal.

sequence listing

<160>12

<210>1

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>1

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

1 5 10

<210>2

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>2

Gly Gly Gly Gly Ser

1 5

<210>3

<211>446

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>3

Met Asp Pro Ala Glu Ala Val Leu Gln Glu Lys Ala Leu Lys Phe Met

1 5 10 15

Asn Ser Ser Glu Arg Glu Asp Cys Asn Asn Gly Glu Pro Pro Arg Lys

20 25 30

Ile Ile Pro Glu Lys Asn Ser Leu Arg Gln Thr Tyr Asn Ser Cys Ala

35 40 45

Arg Leu Cys Leu Asn Gln Glu Thr Val Cys Leu Ala Ser Thr Ala Met

50 55 60

Lys Thr Glu Asn Cys Val Ala Lys Thr Lys Leu Ala Asn Gly Thr Ser

65 70 75 80

Ser Met Ile Val Pro Lys Gln Arg Lys Leu Ser Ala Ser Tyr Glu Lys

85 90 95

Glu Lys Glu Leu Cys Val Lys Tyr Phe Glu Gln Trp Ser Glu Ser Asp

100 105 110

Gln Val Glu Phe Val Glu His Leu Ile Ser Gln Met Cys His Tyr Gln

115 120 125

His Gly His Ile Asn Ser Tyr Leu Lys Pro Met Leu Gln Arg Asp Phe

130 135 140

Ile Thr Ala Leu Pro Ala Arg Gly Leu Asp His Ile Ala Glu Asn Ile

145 150 155 160

Leu Ser Tyr Leu Asp Ala Lys Ser Leu Cys Ala Ala Glu Leu Val Cys

165 170 175

Lys Glu Trp Tyr Arg Val Thr Ser Asp Gly Met Leu Trp Lys Lys Leu

180 185 190

Ile Glu Arg Met Val Arg Thr Asp Ser Leu Trp Arg Gly Leu Ala Glu

195 200 205

Arg Arg Gly Trp Gly Gln Tyr Leu Phe Lys Asn Lys Pro Pro Asp Gly

210 215 220

Asn Ala Pro Pro Asn Ser Phe Tyr Arg Ala Leu Tyr Pro Lys Ile Ile

225 230 235 240

Gln Asp Ile Glu Thr Ile Glu Ser Asn Trp Arg Cys Gly Arg His Ser

245 250 255

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Ala Pro Gly Val Ala

260 265 270

Arg Gly Pro Thr Pro Tyr Trp Arg Leu Arg Leu Gly Gly Ala Ala Leu

275 280 285

Leu Leu Leu Leu Ile Pro Val Ala Ala Ala Gln Glu Pro Pro Gly Ala

290 295 300

Ala Cys Ser Gln Asn Thr Asn Lys Thr Cys Glu Glu Cys Leu Lys Asn

305 310 315 320

Val Ser Cys Leu Trp Cys Asn Thr Asn Lys Ala Cys Leu Asp Tyr Pro

325 330 335

Val Thr Ser Val Leu Pro Pro Pro Ala Ser Leu Cys Lys Leu Ser Ser Ala

340 345 350

Arg Trp Gly Val Cys Trp Val Asn Phe Glu Ala Leu Ile Ile Thr Met

355 360 365

Ser Val Val Gly Gly Thr Leu Leu Leu Gly Ile Ala Ile Cys Cys Cys

370 375 380

Cys Cys Cys Arg Arg Lys Arg Ser Arg Lys Pro Asp Arg Ser Glu Glu

385 390 395 400

Lys Ala Met Arg Glu Arg Glu Glu Arg Arg Ile Arg Gln Glu Glu Arg

405 410 415

Arg Ala Glu Met Lys Thr Arg His Asp Glu Ile Arg Lys Lys Tyr Gly

420 425 430

Leu Phe Lys Glu Glu Asn Pro Tyr Ala Arg Phe Glu Asn Asn

435 440 445

<210>4

<211>451

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>4

Met Asp Pro Ala Glu Ala Val Leu Gln Glu Lys Ala Leu Lys Phe Met

1 5 10 15

Asn Ser Ser Glu Arg Glu Asp Cys Asn Asn Gly Glu Pro Pro Arg Lys

20 25 30

Ile Ile Pro Glu Lys Asn Ser Leu Arg Gln Thr Tyr Asn Ser Cys Ala

35 40 45

Arg Leu Cys Leu Asn Gln Glu Thr Val Cys Leu Ala Ser Thr Ala Met

50 55 60

Lys Thr Glu Asn Cys Val Ala Lys Thr Lys Leu Ala Asn Gly Thr Ser

65 70 75 80

Ser Met Ile Val Pro Lys Gln Arg Lys Leu Ser Ala Ser Tyr Glu Lys

85 90 95

Glu Lys Glu Leu Cys Val Lys Tyr Phe Glu Gln Trp Ser Glu Ser Asp

100 105 110

Gln Val Glu Phe Val Glu His Leu Ile Ser Gln Met Cys His Tyr Gln

115 120 125

His Gly His Ile Asn Ser Tyr Leu Lys Pro Met Leu Gln Arg Asp Phe

130 135 140

Ile Thr Ala Leu Pro Ala Arg Gly Leu Asp His Ile Ala Glu Asn Ile

145 150 155 160

Leu Ser Tyr Leu Asp Ala Lys Ser Leu Cys Ala Ala Glu Leu Val Cys

165 170 175

Lys Glu Trp Tyr Arg Val Thr Ser Asp Gly Met Leu Trp Lys Lys Leu

180 185 190

Ile Glu Arg Met Val Arg Thr Asp Ser Leu Trp Arg Gly Leu Ala Glu

195 200 205

Arg Arg Gly Trp Gly Gln Tyr Leu Phe Lys Asn Lys Pro Pro Asp Gly

210 215 220

Asn Ala Pro Pro Asn Ser Phe Tyr Arg Ala Leu Tyr Pro Lys Ile Ile

225 230 235 240

Gln Asp Ile Glu Thr Ile Glu Ser Asn Trp Arg Cys Gly Arg His Ser

245 250 255

Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met

260 265 270

Ala Pro Gly Val Ala Arg Gly Pro Thr Pro Tyr Trp Arg Leu Arg Leu

275 280 285

Gly Gly Ala Ala Leu Leu Leu Leu Leu Ile Pro Val Ala Ala Ala Gln

290 295 300

Glu Pro Pro Gly Ala Ala Cys Ser Gln Asn Thr Asn Lys Thr Cys Glu

305 310 315 320

Glu Cys Leu Lys Asn Val Ser Cys Leu Trp Cys Asn Thr Asn Lys Ala

325 330 335

Cys Leu Asp Tyr Pro Val Thr Ser Val Leu Pro Pro Ala Ser Leu Cys

340 345 350

Lys Leu Ser Ser Ala Arg Trp Gly Val Cys Trp Val Asn Phe Glu Ala

355 360 365

Leu Ile Ile Thr Met Ser Val Val Gly Gly Thr Leu Leu Leu Gly Ile

370 375 380

Ala Ile Cys Cys Cys Cys Cys Cys Arg Arg Lys Arg Ser Arg Lys Pro

385 390 395 400

Asp Arg Ser Glu Glu Lys Ala Met Arg Glu Arg Glu Glu Arg Arg Ile

405 410 415

Arg Gln Glu Glu Arg Arg Ala Glu Met Lys Thr Arg His Asp Glu Ile

420 425 430

Arg Lys Lys Tyr Gly Leu Phe Lys Glu Glu Asn Pro Tyr Ala Arg Phe

435 440 445

Glu Asn Asn

450

<210>5

<211>446

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>5

Met Asp Pro Ala Glu Ala Val Leu Gln Glu Lys Ala Leu Lys Phe Met

1 5 10 15

Asn Ser Ser Glu Arg Glu Asp Cys Asn Asn Gly Glu Pro Pro Arg Lys

20 25 30

Ile Ile Pro Glu Lys Asn Ser Leu Arg Gln Thr Tyr Asn Ser Cys Ala

35 40 45

Arg Leu Cys Leu Asn Gln Glu Thr Val Cys Leu Ala Ser Thr Ala Met

50 55 60

Lys Thr Glu Asn Cys Val Ala Lys Thr Lys Leu Ala Asn Gly Thr Ser

65 70 75 80

Ser Met Ile Val Pro Lys Gln Arg Lys Leu Ser Ala Ser Tyr Glu Lys

85 90 95

Glu Lys Glu Leu Cys Val Lys Tyr Phe Glu Gln Trp Ser Glu Ser Asp

100 105 110

Gln Val Glu Phe Val Glu His Leu Ile Ser Gln Met Cys His Tyr Gln

115 120 125

His Gly His Ile Asn Ser Tyr Leu Lys Pro Met Leu Gln Arg Asp Phe

130 135 140

Ile Thr Ala Leu Pro Ala Arg Gly Leu Asp His Ile Ala Glu Asn Ile

145 150 155 160

Leu Ser Tyr Leu Asp Ala Lys Ser Leu Cys Ala Ala Glu Leu Val Cys

165 170 175

Lys Glu Trp Tyr Arg Val Thr Ser Asp Gly Met Leu Trp Lys Lys Leu

180 185 190

Ile Glu Arg Met Val Arg Thr Asp Ser Leu Trp Arg Gly Leu Ala Glu

195 200 205

Arg Arg Gly Trp Gly Gln Tyr Leu Phe Lys Asn Lys Pro Pro Asp Gly

210 215 220

Asn Ala Pro Pro Asn Ser Phe Tyr Arg Ala Leu Tyr Pro Lys Ile Ile

225 230 235 240

Gln Asp Ile Glu Thr Ile Glu Ser Asn Trp Arg Cys Gly Arg His Ser

245 250 255

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Ala Pro Gly Val Ala

260 265 270

Arg Gly Pro Thr Pro Tyr Trp Arg Leu Arg Leu Gly Gly Ala Ala Leu

275 280 285

Leu Leu Leu Leu Ile Pro Val Ala Ala Ala Gln Glu Pro Pro Gly Ala

290 295 300

Ala Cys Ser Gln Asn Thr Asn Lys Thr Cys Glu Glu Cys Leu Lys Asn

305 310 315 320

Val Ser Cys Leu Trp Cys Asn Thr Asn Lys Ala Cys Leu Asp Tyr Pro

325 330 335

Val Thr Ser Val Leu Pro Pro Pro Ala Ser Leu Cys Lys Leu Ser Ser Ala

340 345 350

Arg Trp Gly Val Cys Trp Val Asn Phe Glu Ala Leu Ile Ile Thr Met

355 360 365

Ser Val Val Gly Gly Thr Leu Leu Leu Gly Ile Ala Ile Cys Cys Cys

370 375 380

Cys Cys Cys Arg Arg Lys Arg Ser Arg Lys Pro Asp Arg Ser Glu Glu

385 390 395 400

Lys Ala Met Arg Glu Arg Glu Glu Arg Arg Ile Arg Gln Glu Glu Arg

405 410 415

Arg Ala Glu Met Lys Thr Arg His Asp Glu Ile Arg Lys Lys Tyr Gly

420 425 430

Leu Phe Lys Glu Glu Asn Pro Tyr Ala Arg Phe Glu Asn Asn

435 440 445

<210>6

<211>441

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>6

Met Asp Pro Ala Glu Ala Val Leu Gln Glu Lys Ala Leu Lys Phe Met

1 5 10 15

Asn Ser Ser Glu Arg Glu Asp Cys Asn Asn Gly Glu Pro Pro Arg Lys

20 25 30

Ile Ile Pro Glu Lys Asn Ser Leu Arg Gln Thr Tyr Asn Ser Cys Ala

35 40 45

Arg Leu Cys Leu Asn Gln Glu Thr Val Cys Leu Ala Ser Thr Ala Met

50 55 60

Lys Thr Glu Asn Cys Val Ala Lys Thr Lys Leu Ala Asn Gly Thr Ser

65 70 75 80

Ser Met Ile Val Pro Lys Gln Arg Lys Leu Ser Ala Ser Tyr Glu Lys

85 90 95

Glu Lys Glu Leu Cys Val Lys Tyr Phe Glu Gln Trp Ser Glu Ser Asp

100 105 110

Gln Val Glu Phe Val Glu His Leu Ile Ser Gln Met Cys His Tyr Gln

115 120 125

His Gly His Ile Asn Ser Tyr Leu Lys Pro Met Leu Gln Arg Asp Phe

130 135 140

Ile Thr Ala Leu Pro Ala Arg Gly Leu Asp His Ile Ala Glu Asn Ile

145 150 155 160

Leu Ser Tyr Leu Asp Ala Lys Ser Leu Cys Ala Ala Glu Leu Val Cys

165 170 175

Lys Glu Trp Tyr Arg Val Thr Ser Asp Gly Met Leu Trp Lys Lys Leu

180 185 190

Ile Glu Arg Met Val Arg Thr Asp Ser Leu Trp Arg Gly Leu Ala Glu

195 200 205

Arg Arg Gly Trp Gly Gln Tyr Leu Phe Lys Asn Lys Pro Pro Asp Gly

210 215 220

Asn Ala Pro Pro Asn Ser Phe Tyr Arg Ala Leu Tyr Pro Lys Ile Ile

225 230 235 240

Gln Asp Ile Glu Thr Ile Glu Ser Asn Trp Arg Cys Gly Arg His Ser

245 250 255

Gly Gly Gly Gly Ser Met Ala Pro Gly Val Ala Arg Gly Pro Thr Pro

260 265 270

Tyr Trp Arg Leu Arg Leu Gly Gly Ala Ala Leu Leu Leu Leu Leu Ile

275 280 285

Pro Val Ala Ala Ala Gln Glu Pro Pro Gly Ala Ala Cys Ser Gln Asn

290 295 300

Thr Asn Lys Thr Cys Glu Glu Cys Leu Lys Asn Val Ser Cys Leu Trp

305 310 315 320

Cys Asn Thr Asn Lys Ala Cys Leu Asp Tyr Pro Val Thr Ser Val Leu

325 330 335

Pro Pro Ala Ser Leu Cys Lys Leu Ser Ser Ala Arg Trp Gly Val Cys

340 345 350

Trp Val Asn Phe Glu Ala Leu Ile Ile Thr Met Ser Val Val Gly Gly

355 360 365

Thr Leu Leu Leu Gly Ile Ala Ile Cys Cys Cys Cys Cys Cys Cys Arg Arg

370 375 380

Lys Arg Ser Arg Lys Pro Asp Arg Ser Glu Glu Lys Ala Met Arg Glu

385 390 395 400

Arg Glu Glu Arg Arg Ile Arg Gln Glu Glu Arg Arg Ala Glu Met Lys

405 410 415

Thr Arg His Asp Glu Ile Arg Lys Lys Tyr Gly Leu Phe Lys Glu Glu

420 425 430

Asn Pro Tyr Ala Arg Phe Glu Asn Asn

435 440

<210>7

<211>296

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>7

Met Asp Pro Ala Glu Ala Val Leu Gln Glu Lys Ala Leu Lys Phe Met

1 5 10 15

Asn Ser Ser Glu Arg Glu Asp Cys Asn Asn Gly Glu Pro Pro Arg Lys

20 25 30

Ile Ile Pro Glu Lys Asn Ser Leu Arg Gln Thr Tyr Asn Ser Cys Ala

35 40 45

Arg Leu Cys Leu Asn Gln Glu Thr Val Cys Leu Ala Ser Thr Ala Met

50 55 60

Lys Thr Glu Asn Cys Val Ala Lys Thr Lys Leu Ala Asn Gly Thr Ser

65 70 75 80

Ser Met Ile Val Pro Lys Gln Arg Lys Leu Ser Ala Ser Tyr Glu Lys

85 90 95

Glu Lys Glu Leu Cys Val Lys Tyr Phe Glu Gln Trp Ser Glu Ser Asp

100 105 110

Gln Val Glu Phe Val Glu His Leu Ile Ser Gln Met Cys His Tyr Gln

115 120 125

His Gly His Ile Asn Ser Tyr Leu Lys Pro Met Leu Gln Arg Asp Phe

130 135 140

Ile Thr Ala Leu Pro Ala Arg Gly Leu Asp His Ile Ala Glu Asn Ile

145 150 155 160

Leu Ser Tyr Leu Asp Ala Lys Ser Leu Cys Ala Ala Glu Leu Val Cys

165 170 175

Lys Glu Trp Tyr Arg Val Thr Ser Asp Gly Met Leu Trp Lys Lys Leu

180 185 190

Ile Glu Arg Met Val Arg Thr Asp Ser Leu Trp Arg Gly Leu Ala Glu

195 200 205

Arg Arg Gly Trp Gly Gln Tyr Leu Phe Lys Asn Lys Pro Pro Asp Gly

210 215 220

Asn Ala Pro Pro Asn Ser Phe Tyr Arg Ala Leu Tyr Pro Lys Ile Ile

225 230 235 240

Gln Asp Ile Glu Thr Ile Glu Ser Asn Trp Arg Cys Gly Arg His Ser

245 250 255

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Arg Ala Glu Met Lys Thr

260 265 270

Arg His Asp Glu Ile Arg Lys Lys Tyr Gly Leu Phe Lys Glu Glu Asn

275 280 285

Pro Tyr Ala Arg Phe Glu Asn Asn

290 295

<210>8

<211>1341

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>8

atggacccgg ccgaggcggt gctgcaagag aaggcactca agtttatgaa ttcctcagag 60

agagaagact gtaataatgg cgaaccccct aggaagataa taccagagaa gaattcactt 120

agacagacat acaacagctg tgccagactc tgcttaaacc aagaaacagt atgtttagca 180

agcactgcta tgaagactga gaattgtgtg gccaaaacaa aacttgccaa tggcacttcc 240

agtatgattg tgcccaagca acggaaactc tcagcaagct atgaaaagga aaaggaactg 300

tgtgtcaaat actttgagca gtggtcagag tcagatcaag tggaatttgt ggaacatctt 360

atatcccaaa tgtgtcatta ccaacatggg cacataaact cgtatcttaa acctatgttg 420

cagagagatt tcataactgc tctgccagct cggggattgg atcatatcgc tgagaacatt 480

ctgtcatacc tggatgccaa atcactatgt gctgctgaac ttgtgtgcaa ggaatggtac 540

cgagtgacct ctgatggcat gctgtggaag aagcttatcg agagaatggt caggacagat 600

tctctgtgga gaggcctggc agaacgaaga ggatggggac agtatttatt caaaaacaaa 660

cctcctgacg ggaatgctcc tcccaactct ttttatagag cactttatcc taaaattata 720

caagacattg agacaataga atctaattgg agatgtggaa gacatagtgg atccggctca 780

ggctcaggat caggctcaat ggcgcccgga gtggcccgcg ggccgacgcc gtactggagg 840

ttgcgcctcg gtggcgccgc gctgctcctg ctgctcatcc cggtggccgc cgcgcaggag 900

cctcccggag ctgcttgttc tcagaacaca aacaaaacct gtgaagagtg cctgaagaac 960

gtctcctgtc tttggtgcaa cactaacaag gcttgtctgg actacccagt tacaagcgtc 1020

ttgccaccgg cttccctttg taaattgagc tctgcacgct ggggagtttg ttgggtgaac 1080

tttgaggcgc tgatcatcac catgtcggta gtcgggggaa ccctcctcct gggcattgcc 1140

atctgctgct gctgctgctg caggaggaag aggagccgga agccggacag gagtgaggag 1200

aaggccatgc gtgagcggga ggagaggcgg atacggcagg aggaacggag agcagagatg 1260

aagacaagac atgatgaaat cagaaaaaaa tatggcctgt ttaaagaaga aaacccgtat 1320

gctagatttg aaaacaacta a 1341

<210>9

<211>1356

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>9

atggacccgg ccgaggcggt gctgcaagag aaggcactca agtttatgaa ttcctcagag 60

agagaagact gtaataatgg cgaaccccct aggaagataa taccagagaa gaattcactt 120

agacagacat acaacagctg tgccagactc tgcttaaacc aagaaacagt atgtttagca 180

agcactgcta tgaagactga gaattgtgtg gccaaaacaa aacttgccaa tggcacttcc 240

agtatgattg tgcccaagca acggaaactc tcagcaagct atgaaaagga aaaggaactg 300

tgtgtcaaat actttgagca gtggtcagag tcagatcaag tggaatttgt ggaacatctt 360

atatcccaaa tgtgtcatta ccaacatggg cacataaact cgtatcttaa acctatgttg 420

cagagagatt tcataactgc tctgccagct cggggattgg atcatatcgc tgagaacatt 480

ctgtcatacc tggatgccaa atcactatgt gctgctgaac ttgtgtgcaa ggaatggtac 540

cgagtgacct ctgatggcat gctgtggaag aagcttatcg agagaatggt caggacagat 600

tctctgtgga gaggcctggc agaacgaaga ggatggggac agtatttatt caaaaacaaa 660

cctcctgacg ggaatgctcc tcccaactct ttttatagag cactttatcc taaaattata 720

caagacattg agacaataga atctaattgg agatgtggaa gacatagtgg cggaggtgga 780

tctggtggcg gaggaagcgg aggtggcgga tctatggcgc ccggagtggc ccgcgggccg 840

acgccgtact ggaggttgcg cctcggtggc gccgcgctgc tcctgctgct catcccggtg 900

gccgccgcgc aggagcctcc cggagctgct tgttctcaga acacaaacaa aacctgtgaa 960

gagtgcctga agaacgtctc ctgtctttgg tgcaacacta acaaggcttg tctggactac 1020

ccagttacaa gcgtcttgcc accggcttcc ctttgtaaat tgagctctgc acgctgggga 1080

gtttgttggg tgaactttga ggcgctgatc atcaccatgt cggtagtcgg gggaaccctc 1140

ctcctgggca ttgccatctg ctgctgctgc tgctgcagga ggaagaggag ccggaagccg 1200

gacaggagtg aggagaaggc catgcgtgag cgggaggaga ggcggatacg gcaggaggaa 1260

cggagagcag agatgaagac aagacatgat gaaatcagaa aaaaatatgg cctgtttaaa 1320

gaagaaaacc cgtatgctag atttgaaaac aactaa 1356

<210>10

<211>1341

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>10

atggacccgg ccgaggcggt gctgcaagag aaggcactca agtttatgaa ttcctcagag 60

agagaagact gtaataatgg cgaaccccct aggaagataa taccagagaa gaattcactt 120

agacagacat acaacagctg tgccagactc tgcttaaacc aagaaacagt atgtttagca 180

agcactgcta tgaagactga gaattgtgtg gccaaaacaa aacttgccaa tggcacttcc 240

agtatgattg tgcccaagca acggaaactc tcagcaagct atgaaaagga aaaggaactg 300

tgtgtcaaat actttgagca gtggtcagag tcagatcaag tggaatttgt ggaacatctt 360

atatcccaaa tgtgtcatta ccaacatggg cacataaact cgtatcttaa acctatgttg 420

cagagagatt tcataactgc tctgccagct cggggattgg atcatatcgc tgagaacatt 480

ctgtcatacc tggatgccaa atcactatgt gctgctgaac ttgtgtgcaa ggaatggtac 540

cgagtgacct ctgatggcat gctgtggaag aagcttatcg agagaatggt caggacagat 600

tctctgtgga gaggcctggc agaacgaaga ggatggggac agtatttatt caaaaacaaa 660

cctcctgacg ggaatgctcc tcccaactct ttttatagag cactttatcc taaaattata 720

caagacattg agacaataga atctaattgg agatgtggaa gacatagtgg cggaggtgga 780

tctggtggcg gaggaagcat ggcgcccgga gtggcccgcg ggccgacgcc gtactggagg 840

ttgcgcctcg gtggcgccgc gctgctcctg ctgctcatcc cggtggccgc cgcgcaggag 900

cctcccggag ctgcttgttc tcagaacaca aacaaaacct gtgaagagtg cctgaagaac 960

gtctcctgtc tttggtgcaa cactaacaag gcttgtctgg actacccagt tacaagcgtc 1020

ttgccaccgg cttccctttg taaattgagc tctgcacgct ggggagtttg ttgggtgaac 1080

tttgaggcgc tgatcatcac catgtcggta gtcgggggaa ccctcctcct gggcattgcc 1140

atctgctgct gctgctgctg caggaggaag aggagccgga agccggacag gagtgaggag 1200

aaggccatgc gtgagcggga ggagaggcgg atacggcagg aggaacggag agcagagatg 1260

aagacaagac atgatgaaat cagaaaaaaa tatggcctgt ttaaagaaga aaacccgtat 1320

gctagatttg aaaacaacta a 1341

<210>11

<211>1326

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>11

atggacccgg ccgaggcggt gctgcaagag aaggcactca agtttatgaa ttcctcagag 60

agagaagact gtaataatgg cgaaccccct aggaagataa taccagagaa gaattcactt 120

agacagacat acaacagctg tgccagactc tgcttaaacc aagaaacagt atgtttagca 180

agcactgcta tgaagactga gaattgtgtg gccaaaacaa aacttgccaa tggcacttcc 240

agtatgattg tgcccaagca acggaaactc tcagcaagct atgaaaagga aaaggaactg 300

tgtgtcaaat actttgagca gtggtcagag tcagatcaag tggaatttgt ggaacatctt 360

atatcccaaa tgtgtcatta ccaacatggg cacataaact cgtatcttaa acctatgttg 420

cagagagatt tcataactgc tctgccagct cggggattgg atcatatcgc tgagaacatt 480

ctgtcatacc tggatgccaa atcactatgt gctgctgaac ttgtgtgcaa ggaatggtac 540

cgagtgacct ctgatggcat gctgtggaag aagcttatcg agagaatggt caggacagat 600

tctctgtgga gaggcctggc agaacgaaga ggatggggac agtatttatt caaaaacaaa 660

cctcctgacg ggaatgctcc tcccaactct ttttatagag cactttatcc taaaattata 720

caagacattg agacaataga atctaattgg agatgtggaa gacatagtgg cggaggtgga 780

tctatggcgc ccggagtggc ccgcgggccg acgccgtact gaggttgcg cctcggtggc 840

gccgcgctgc tcctgctgct catcccggtg gccgccgcgc aggagcctcc cggagctgct 900

tgttctcaga acacaaacaa aacctgtgaa gagtgcctga agaacgtctc ctgtctttgg 960

tgcaacacta acaaggcttg tctggactac ccagttacaa gcgtcttgcc accggcttcc 1020

ctttgtaaat tgagctctgc acgctgggga gtttgttggg tgaactttga ggcgctgatc 1080

atcaccatgt cggtagtcgg gggaaccctc ctcctgggca ttgccatctg ctgctgctgc 1140

tgctgcagga ggaagaggag ccggaagccg gacaggagtg aggagaaggc catgcgtgag 1200

cgggaggaga ggcggatacg gcaggaggaa cggagagcag agatgaagac aagacatgat 1260

gaaatcagaa aaaaatatgg cctgtttaaa gaagaaaacc cgtatgctag atttgaaaac 1320

aactaa 1326

<210>12

<211>891

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>12

atggacccgg ccgaggcggt gctgcaagag aaggcactca agtttatgaa ttcctcagag 60

agagaagact gtaataatgg cgaaccccct aggaagataa taccagagaa gaattcactt 120

agacagacat acaacagctg tgccagactc tgcttaaacc aagaaacagt atgtttagca 180

agcactgcta tgaagactga gaattgtgtg gccaaaacaa aacttgccaa tggcacttcc 240

agtatgattg tgcccaagca acggaaactc tcagcaagct atgaaaagga aaaggaactg 300

tgtgtcaaat actttgagca gtggtcagag tcagatcaag tggaatttgt ggaacatctt 360

atatcccaaa tgtgtcatta ccaacatggg cacataaact cgtatcttaa acctatgttg 420

cagagagatt tcataactgc tctgccagct cggggattgg atcatatcgc tgagaacatt 480

ctgtcatacc tggatgccaa atcactatgt gctgctgaac ttgtgtgcaa ggaatggtac 540

cgagtgacct ctgatggcat gctgtggaag aagcttatcg agagaatggt caggacagat 600

tctctgtgga gaggcctggc agaacgaaga ggatggggac agtatttatt caaaaacaaa 660

cctcctgacg ggaatgctcc tcccaactct ttttatagag cactttatcc taaaattata 720

caagacattg agacaataga atctaattgg agatgtggaa gacatagtgg atccggctca 780

ggctcaggat caggctcaag agcagagatg aagacaagac atgatgaaat cagaaaaaaa 840

tatggcctgt ttaaagaaga aaacccgtat gctagatttg aaaacaacta a 891

Claims (7)

1. A fusion protein with the effect of inhibiting the expression of PTTG, which is one of the following amino acid residue sequences: 1) SEQ ID NO: 3 in the sequence listing; 2) SEQ ID NO: 4 in the sequence listing; 3) SEQ ID NO: 5 in the sequence listing; 4) SEQ ID NO: 6 in the sequence listing; 5) SEQ ID NO: 7 in the sequence listing. 2. the gene of the fusion protein described in coding claim 1 that has the effect of inhibiting PTTG expression. 3. The gene according to claim 2, characterized in that: the gene is one of the following nucleotide sequences: 1) The DNA sequence of SEQ ID NO: 8 in the sequence listing; 2) DNA sequence of SEQ ID NO: 9 in the sequence listing; 3) The DNA sequence of SEQ ID NO: 10 in the sequence listing; 4) the DNA sequence of SEQ ID NO: 11 in the sequence listing; 5) The DNA sequence of SEQ ID NO: 12 in the sequence listing. 4. An expression vector containing the gene of claim 2 or 3. 5. A transgenic cell line containing the gene of claim 2 or 3. 6. The host bacterium containing the gene of claim 2 or 3. 7. a method for expressing the fusion protein with the effect of suppressing PTTG expression described in claim 1, is that the expression vector described in claim 4 is introduced into host cell, expresses and obtains the fusion protein with the effect of suppressing PTTG expression; Said host Escherichia coli, yeast, mammalian cells, insect cells or Bacillus subtilis.

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