CN109627313B

CN109627313B - Mutant IRS-1 PTB structural domain protein, coding sequence and application thereof

Info

Publication number: CN109627313B
Application number: CN201710927849.7A
Authority: CN
Inventors: 曹轩; 刘安冬; 余悦琪; 邓婕; 罗丹妮
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2017-10-09
Filing date: 2017-10-09
Publication date: 2020-07-10
Anticipated expiration: 2037-10-09
Also published as: CN109627313A

Abstract

The invention carries out site-directed mutagenesis on amino acid residues of PTB structural domain wild type protein of IRS-1, mutates R at 99 th position to L, mutates S at 102 th position to A, constructs a MUTANT vector and carries out protein expression and purification, and expresses PTB fusion protein MUTANT GST-PTB-R99L/S102A of IRS-1 in escherichia coli, thereby greatly enhancing the specific binding affinity with IGF-1R phosphorylated by tyrosine residues, and constructs a recombinant plasmid to express PTB fusion protein MUTANT of IRS-1 and fusion protein (Arg)9-GST-PTB-MUTANT of cell penetrating peptide (Arg)9, which can penetrate and enter cancer cells, replace the combination of endogenous IRS-1 protein in the cancer cells and IGF-1R, block a plurality of signal paths related to the endogenous IRS-1 protein, inhibit the proliferation and invasion of the tumor cells, and have obvious anti-tumor effect.

Description

Mutant IRS-1 PTB structural domain protein, coding sequence and application thereof

Technical Field

The invention belongs to the technical field of biology, relates to a protein polypeptide with strong affinity for a phosphorylated tyrosine residue of an insulin-like growth factor receptor IGF-1R, relates to gene recombination, bioinformatics prediction, MUTANT construction and prokaryotic vector fusion protein expression and purification, and particularly relates to a PTB-MUTANT fusion gene and application of a prokaryotic expression vector thereof.

Background

General description of phosphorylated tyrosines and their anti-tumor applications: the antitumor drugs mainly comprise cytotoxic antitumor drugs, antitumor drugs taking cell signal transduction molecules as targets and the like, wherein the tyrosine kinase inhibitor can permeate cell membranes and target tumor cells, and the method becomes a hot spot of international new drug research. The phosphorylation modification of protein tyrosine is the basic mechanism for regulating cell signal transduction, and cancerated cells often show abnormally increased level of phosphorylated tyrosine (pY)¹. Conventional antitumor against pYThe drug is monoclonal antibody and kinase inhibitor for resisting pY. However, the monoclonal antibody has high preparation cost, high price, unstable quality of different batches, large molecular weight and poor cell penetration capability; the kinase inhibitor causes strong inhibition to the signal path related to the intracellular pY, has certain side effect, and generally has the condition of drug resistance caused by gene mutation, thereby losing the treatment effect²。

PTB overview: the PhosphoTyrosine Binding (PTB) domain has the property of specifically recognizing and Binding PhosphoTyrosine on ligands. A number of proteins in cells have such PTB domains, with the PTB domain of the linker protein Insulin Receptor Substrate 1(Insulin Receptor Substrate 1, IRS-1) having the property of specifically recognizing and binding to the Insulin-like Growth Factor Receptor (Insulin-like Growth Factor Receptor1, IGF-1R). Insulin-like growth factor-1 (IGF-1) binds to its receptor IGF-1R to cause autophosphorylation of tyrosine residues of the receptor, IRS-1 binds to phosphorylated tyrosine residues on IGF-1R through PTB domain as a linker protein, and can bind to a plurality of downstream proteins PI3K, SH-PTP2, Grb 2and the like through other domains or amino acid residues to mediate regulation of tumor cell proliferation, transformation, infiltration, metastasis and apoptosis³Reference is made to Smithgar, T.E., SH2and SH3 domains, potential targets for anti-cancer drug design, J Pharmacol Toxicol Methods 1995,34(3),125-32, 2 Tinti, M.J., Nardozza, A.P., Ferrari, E., Sacco, F., Corallino, S., Castagonoi, L., Cesareni, G.G., The 4G10, pY20and p-TYR-100 anti-dysfilcity, promoting by peptides microorganisms, New Biotechnol 571, 29(5), 577, Cai, W.W., 1487, M.K., coding peptides A.K., K.J., P.J., P.P., P.J.P.P.P., P.P.P.P.P.P., P.P., P., P.C.S., Castaginei.S. C. J., P., P., P., P. J. P. P.

Disclosure of Invention

The object of the present invention is to provide a mutated IRS-1 PTB domain protein and to provide a DNA molecule encoding the mutated IRS-1 PTB domain protein.

The PTB structural domain protein of IRS-1 is the phosphorylation tyrosine binding structural domain protein of insulin receptor substrate 1.

The PTB structural domain protein of the mutated IRS-1 is the phosphorylation tyrosine binding structural domain mutant protein of the insulin receptor substrate 1.

The invention also provides the application of the PTB structural domain protein of the mutant IRS-1 and the DNA molecule for encoding the PTB structural domain protein of the mutant IRS-1 in preparing antitumor drugs.

The technical scheme for realizing the invention is as follows:

compared with the PTB domain protein of a wild-type IRS-1, the PTB domain protein of the mutated IRS-1 provided by the invention has the advantages that the 99 th amino acid of the PTB domain protein of the mutated IRS-1 is mutated from R to L, and the 102 th amino acid is mutated from S to A.

The nucleotide sequence of the DNA molecule for coding the PTB structural domain protein of the mutated IRS-1 is shown as a sequence 2 in a sequence table.

The present invention also provides a fusion protein, which is composed of the above-mentioned PTB domain protein (PTB-MUTANT) of the MUTANT IRS-1 provided by the present invention and cell-penetrating peptide, wherein the cell-penetrating peptide can be composed of short peptide composed of 9 Arg (residues), and the preparation method comprises the following steps:

(1) the nucleotide sequence for constructing PTB-MUTANT MUTANT protein is amplified by using a PCR method, wherein a template is a wild-type PTB nucleotide sequence, primer pairs are F2 and R2 respectively, and the primer sequences are as follows:

F2:tcctggaggccatgctggccatggctgatgagttcTCGTG 40bp；

R2:CACGAgaactcatcagccatggccagcatggcctccagga 40bp；

the PCR reaction system is 2 XPCR Mix 10ul, F1 ul, R1 ul, H2O 6ul, template 2 ul. The PCR reaction program is 98 ℃ for 2min, 98 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 30s, 35cycle, 72 ℃ for 10 min;

the constructed PTB-MUTANT mutein containing the transmembrane peptide Arg9 has the nucleotide sequence amplified by a PCR method, wherein the template is the PTB-MUTANT nucleotide sequence, the primer pairs are respectively F3 and R3, and the primer sequences are as follows:

F3:GATCTGGTTCCGCGTcgtcgtcgccgccgtcgtcgccgccgtggcttcaaagaggtctg59bp；

R3:GTCAGTCAGTCACGAgaactcatcactcatggcc34bp；

the PCR reaction system is 2 XPCR Mix 10ul, F1 ul, R1 ul, H2O 6ul, template 2 ul. The PCR reaction program is 98 ℃ for 2min, 98 ℃ for 30s, 58 ℃ for 30s, 72 ℃ for 30s, 35cycle, 72 ℃ for 10 min;

(2) PTB-MUTANT and Arg9-PTB-MUTANT are respectively used for constructing a recombinant prokaryotic expression vector by utilizing the experimental principle of homologous recombination, the used vector is pGEX-4T2, and the enzyme cutting sites used for constructing the vector are Bam HI and Not I.

The invention aims to provide a recombinant protein MUTANT, namely a PTB fusion protein MUTANT (PTB-MUTANT or GST-PTB-MUTANT for short) and application of a related expression vector thereof. The fusion protein PTB-MUTANT can be competitively combined with the endogenous IRS-1 protein in cancer cells and IGF-1R, so that a plurality of signal paths related to the endogenous IRS-1 protein are blocked, and the proliferation and invasion of the tumor cells are inhibited, so that the anti-tumor effect is achieved, and the fusion protein PTB-MUTANT can be used for research and development of anti-tumor drugs.

The invention can be realized by the following technical scheme:

a recombinant PTB-MUTANT vector is constructed by cloning the PTB domain of IRS-1 from the cDNA of MCF7, double-point mutating, and connecting with empty vector pGEX-4T2, but not limited to this vector, and includes all empty vectors suitable for expression and purification, such as pGEX-4T3, pETM30, pETM11, etc., and other possible expression vectors (but not limited to) cosmids, plasmids, or modified viruses (e.g., replication defective retroviruses, adenoviruses, etc.), as long as the vector is compatible with the host cell used. Depending on the host cell chosen and the expression vector used, other additional sequences (e.g., origins of replication, additional DNA restriction endonuclease sites, enhancers, etc.) may be introduced into the expression vector. In addition, the invention encompasses nucleic acid vectors comprising a nucleic acid encoding at least one PTB-MUTANT monomer.

The gene nucleotide sequence and the corresponding amino acid sequence of the PTB-MUTANT are shown in figure 3.

The PTB-MUTANT is connected with an empty vector pGEX-4T2 through a molecular cloning means of homologous recombination to construct a prokaryotic expression vector pGEX-4T 2-PTB-MUTANT. Not limited to this vector, but including all empty vectors suitable for expression and purification, the constructed vector is Plasmid-PTB-MUTANT.

In the embodiment of the invention, 9 arginines (namely Arg) are inserted into pGEX-4T2-PTB-MUTANT to construct pGEX-4T2- (Arg)9-PTB-MUTANT, and the purified protein (Arg)9-GST-PTB-MUTANT can be directly added into a cell culture medium to be co-cultured with cells, and enters the cells by utilizing the membrane penetrating effect of (Arg)9 to further inhibit the proliferation of the cells. The use of cell-penetrating peptides is not limited to arginine-rich Arg9, but may also be TAT protein transduction domains, or liposomes, or nanoparticles, or any other carrier material that facilitates the delivery of PTB mutants to cells or tissues.

The invention uses PTB structure domain of linker protein Insulin Receptor Substrate 1(Insulin Receptor Substrate 1, IRS-1, hereinafter abbreviated as IRS-1) to have the property of being able to specifically recognize and bind to Insulin-like Growth Factor Receptor (Insulin-like Growth Factor Receptor1, IGF-1R, hereinafter abbreviated as IGF-1R), as shown in FIG. 1, the PTB structure domain of linker protein Insulin Receptor Substrate 1(IRS-1) is able to specifically recognize and bind to IGF-1R, Insulin-like Growth Factor-1 (IGF-1) binds to its Receptor IGF-1R, causing autophosphorylation of tyrosine residue of the Receptor, IRS-1 as linker protein binds to phosphorylated tyrosine residue on IGF-1R through PTB structure domain, and binds to phosphorylated tyrosine residue of multiple downstream proteins PI K, SH-2, beta 2 as well as the Receptor protein binds to tyrosine residue of IGF-1R, and binds to tyrosine residue of wild type protein Receptor kinase-1, so that the Receptor protein is easy to be purified and purified by introducing the Receptor protein of PTB-Receptor tyrosine-kinase-Receptor expression MUTANT protein, so long as compared with the wild type protein kinase-expression protein, and wild type tumor protein, the MUTANT of the cell is easy to be purified by introducing the affinity of the affinity protein of the affinity expression of the protein of the Receptor IGF-Receptor tyrosine-Receptor IGF-Receptor tyrosine-1, the MUTANT of IRS-protein of the cell expression of the MUTANT of the cell, the MUTANT of the cell, the MUTANT of IRS-Receptor IGF-type IRS-Receptor IGF-Receptor IRS-Receptor, the MUTANT of the MUTANT cell, the MUTANT of the MUTANT cell, the MUTANT of the MUTANT.

The research shows that the penetrating peptide composed of 9 Arg has the characteristic of directly penetrating through cell membranes to enter cells, so that the PTB fusion protein mutant of IRS-1 is conveniently introduced into living cells, the PTB fusion protein mutant of IRS-1 and the fusion protein (Arg)9-GST-PTB-MUATNT of cell penetrating peptide (Arg)9 are constructed by recombinant plasmids by a genetic engineering method, and the membrane penetrating capability and the anti-tumor effect of the mutant are researched. The cell-penetrating peptide used herein is arginine-rich (Arg)9, but is not limited to this, but may also be a TAT protein transduction domain, or a liposome, or a nanoparticle, or any other carrier material that facilitates delivery of the PTB mutant to cells or tissues. The experimental result shows that (Arg)9-GST-PTB-MUATNT can penetrate and enter cancer cells, replaces the combination of endogenous IRS-1 protein in the cancer cells and IGF-1R, blocks a plurality of signal paths related to the endogenous IRS-1 protein, inhibits the proliferation and invasion of the tumor cells and has obvious anti-tumor effect.

Compared with the prior art, the invention has the following excellent technical effects:

the fusion protein PTB-MUTANT of the invention can specifically recognize and bind to a phosphorylated tyrosine residue on IGF-1R. By using bioinformatics method, firstly, the crystal structure of IRS-1 is used as template to make molecular dynamics simulation, then the clustalw software is used to make homologous comparison analysis, and distance ligand is selected

The amino acid residues of PTB structural domain wild-type protein of IRS-1 are subjected to site-directed mutagenesis by a genetic engineering technology, R at the 99 th position is mutated into L, S at the 102 th position is mutated into A, a PTB-MUTANT MUTANT vector is constructed and protein expression and purification are carried out, and experiments prove that the PTB fusion protein MUTANT greatly enhances the affinity of the PTB fusion protein MUTANT for the specific binding with IGF-1R phosphorylated by tyrosine residues.

In order to introduce the PTB fusion protein MUTANT of IRS-1 into living cells, the characteristic that a penetrating peptide consisting of 9 Arg directly penetrates through a cell membrane to enter the cells is utilized, and a genetic engineering method is utilized to construct a fusion protein (Arg)9-GST-PTB-MUTANT containing the fusion protein MUTANT PTB-MUTANT and a cell penetrating peptide (Arg)9, wherein the fusion protein can penetrate and enter cancer cells, replace the combination of endogenous IRS-1 protein and IGF-1R in the cancer cells, block a plurality of signal paths related to the endogenous IRS-1 protein, inhibit the proliferation of the cancer cells and have a remarkable anti-tumor effect. Based on the characteristic of strong affinity of the PTB fusion protein MUTANT PTB-MUTANT and IGF-1R specific binding, the PTB fusion protein MUTANT has important indication significance in the aspects of application to living tumors and development of new drugs. And the mutant protein has low production cost, high yield, great application value and wide application prospect.

Drawings

FIG. 1: insulin-like growth factor receptor (IGF-1R) -mediated signaling pathways.

The PTB domain of insulin receptor substrate 1(IRS-1), an insulin-like growth factor receptor (IGF-1R) -mediated signaling pathway, a linker protein, is capable of specifically recognizing and binding IGF-1R. Extracellular insulin-like growth factor-1 (IGF-1) binds to receptor IGF-1R on its cell membrane, causing autophosphorylation of tyrosine residues of the receptor, intracellular IRS-1 as a linker protein binds to phosphorylated tyrosine residues on IGF-1R through PTB domain and binds to a plurality of downstream proteins PI3K, SH-PTP2, Grb2, etc. through other domains or amino acid residues, mediating the regulation of tumor cell proliferation, transformation, infiltration, metastasis and apoptosis.

FIG. 2: PTB fusion protein mutants block the pattern of action of insulin-like growth factor receptor (IGF-1R) -mediated signaling pathways.

The recombinant protein MUTANT, namely PTB-MUTANT, can be competitively combined with the endogenous IRS-1 protein in cancer cells with IGF-1R, so that a plurality of signal paths related to the endogenous IRS-1 protein are blocked, the signal paths mainly comprise the influence on the IGF-1R mediated signal paths, and the proliferation and invasion of tumor cells are inhibited, thereby achieving the anti-tumor effect.

FIG. 3: the nucleotides of the PTB domain of the protein IRS-1 and its corresponding amino acid sequence. Including wild-type PTB-WT and MUTANT protein PTB-MUTANT.

FIG. 4: and (3) an electrophoresis result of the successful construction of an expression vector of the PTB fusion protein MUTANT, namely pGEX-4T 2-IRS-1-PTB-MUTANT. Only the results of construction with pGEX-4T2 empty as expression vector are shown here, with the corresponding molecular weight changes if other suitable expression vectors are used.

FIG. 5: expression and purification results of wild-type PTB fusion protein, namely GST-PTB-WT fusion protein.

FIG. 6: and (3) successfully constructing a sequencing result of an expression vector of the PTB fusion protein MUTANT, namely pGEX-4T 2-IRS-1-PTB-MUTANT.

FIG. 7 PU LL DOWN experiments demonstrated that the PTB fusion protein mutants specifically bind with greater affinity than the wild type PTB fusion protein bound to insulin-like growth factor receptor (IGF-1R) phosphorylated at tyrosine residues.

FIG. 8 immunoblot experiments demonstrated that PTB fusion protein mutants can penetrate membranes into cancer cells.

FIG. 9 Co-immunoprecipitation experiments demonstrated that the PTB fusion protein MUTANT with 9 arginines (i.e., Arg9-GST-PTB-MUTANT) competed with the endogenous IRS-1 protein in cancer cells for binding to IGF-1R.

FIG. 10: MTT proliferation inhibition experiments prove that the PTB fusion protein MUTANT (namely Arg9-GST-PTB-MUTANT) with 9 arginines can penetrate and enter cancer cells to inhibit the proliferation of the cancer cells.

FIG. 11: histogram results of colony formation rates of MCF7 cell plates cultured with PTB fusion protein MUTANTs (i.e., Arg9-GST-PTB-MUTANT) having different concentrations of 9 arginines and a control group.

FIG. 12 is a graph showing the results of plate cloning of MCF7 cells cultured in different concentrations of the PTB fusion protein MUTANT with 9 arginines (i.e., Arg9-GST-PTB-MUTANT) and a control group.

Detailed Description

Example 1

Construction of pGEX-4T2-PTB fusion Gene expression vector

1.1 cloning of PTB and PTB-MUTANT Domain genes

Based on the PTB nucleotide sequence of IRS-1 in Genbank, as shown in FIG. 3, amplification primers were designed, and considering that the vector construction is a molecular cloning method using homologous recombination, appropriate homology arms, F1 and R1, respectively, were added when designing the primers, and specific primer sequences were as follows:

forward primer F1: GATCTGGTTCCGCGTttcaaagaggtctggcaag 34;

reverse primer R1: GTCAGTCAGTCACGAgaactcatcactcatggcc 34;

using cDNA of MCF7 cells as a template, respectively using F, R as amplification primers, carrying out PCR amplification on PTB structural domain genes, wherein a PCR system comprises 2x PCR Mix 10ul, F11 ul, R11 ul and H₂O6 ul, cDNA 2 ul. The PCR program was 98 ℃ for 2min, 98 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 30s, 35 cycles.

The primer sequences of PTB fusion protein MUTANT PTB-MUTANT of the double MUTANT are respectively F2 and R2 according to the amplified PTB nucleotide sequence, and the specific primer sequences are as follows:

F2:tcctggaggccatgctggccatggctgatgagttcTCGTG 40；

R2:CACGAgaactcatcagccatggccagcatggcctccagga 40；

1.2 prokaryotic expression vector construction of PTB and PTB-MUTANT Domain genes

The double enzyme digestion empty vector pGEX-4T2, the reaction system and the reaction conditions are as follows: cutsmart Buffer 3ul, BamHI HF0.5ul, NotI HF0.5ul, empty vector pGEX-4T 226 ul; the digestion experiment was carried out in a 37 ℃ water bath for 2h (endonuclease and related reagents from NEB).

Homologous recombination: pGEX-4T2 was linked to PTB and PTB-MUTANT reaction systems: 5xCE II Buffer 4ul, pGEX-4 T22.5ul, DNA 1ul, Exnase II 2ul, H₂O10.5 ul; reaction conditions are as follows: water bath at 37 deg.c for 30 min.

If other suitable expression vectors are used, the corresponding connection sites can be selected according to the specific situation of restriction enzyme sites and the basic principle of primer design, corresponding target fragment amplification primers can be designed, and the reaction system of homologous recombination can be adjusted. The principles used are consistent, but there may be slight differences in the amounts of materials used in the specific implementation.

After the ligation reaction is finished, the ligation product is transformed into DH5 α competent cells, after overnight culture, single clone is picked up and cultured by shaking bacteria, bacteria liquid PCR preliminary identification is carried out, and then sequencing verification is carried out, and the successfully constructed recombinant plasmids are named pGEX-4T2-PTB-WT and pGEX-4T2-PTB-MUTANT (recombinase and ligation reaction reagent are purchased from Nanjing Novonopraz company).

The successful construction of PCR amplified PTB nucleotide sequences, pGEX-4T2-PTB-WT and pGEX-4T2-PTB-MUTANT, results of electrophoresis are shown in FIG. 4. The successful sequencing results of pGEX-4T2-PTB-MUTANT construction are shown in FIG. 5.

If other suitable vectors are used, the successfully constructed vector names are named for the corresponding Plasmid-PTB-WT and Plasmid-PTB-TRM or according to their specific vector names.

1.3 construction of prokaryotic expression vector of (Arg)9-GST-PTB-MUTANT fusion Gene

Research already shows that the cell-penetrating peptide composed of 9 Arg has the characteristic of directly penetrating through a cell membrane to enter a cell, in order to introduce the PTB domain mutant protein of IRS-1 into a living cell, a gene engineering method is utilized to construct a recombinant plasmid to express the fusion protein (Arg) 9-GST-IRS-1-PTB-R99L/S102A of the PTB domain mutant of IRS-1 and the cell-penetrating peptide (Arg)9, a primer pair is F3 and R3, and the specific design is as follows, the enzyme digestion of an empty vector and homologous recombination are the same as before.

F3:GATCTGGTTCCGCGTcgtcgtcgccgccgtcgtcgccgccgtggcttcaaagaggtctg59；

R3:GTCAGTCAGTCACGAgaactcatcactcatggcc 34；

The penetrating peptide used herein is arginine-rich (Arg)9, but is not limited to this penetrating peptide, and may be a TAT protein transduction domain, and if a TAT protein transduction domain is used, a nucleotide sequence of TAT is added at the time of primer design. And the dosage of the recombination reaction and the ligation reaction is adjusted according to the corresponding principle. If liposomes, or nanoparticles, or any other carrier material facilitating the delivery of the PTB mutant to cells or tissues are used, they need to be in accordance with the principles of experimentation in the relevant field and are readily accomplished by one of ordinary skill in the art.

Expression and purification of pGEX-4T2-PTB fusion Gene

B L21 competent cells were transformed with pGEX-4T2-PTB, cultured overnight, monocloned, cultured with shaking, and absorbance was measured until OD600nm became 0.8, and then IPTG was added to a final concentration of 0.5M, and induced at 20 ℃ for 18h expression.GST-PTB protein was purified using GST Sepharose 4 FF.Coomassie blue staining of the protein before and after purification is shown in FIG. 6.

(Arg)9-GST-PTB-WT and (Arg)9-GST-PTB-MUTANT recombinant proteins can membrane-penetrate into MCF7 cells and specifically bind to IGF-1R phosphorylated at tyrosine residues, inhibiting cell proliferation.

3.1 PU LL DOWN experiments demonstrated that the mutant proteins bind with greater affinity than the wild-type PTB protein for the specific binding of IGF-1R phosphorylated at tyrosine residues.

MCF7 breast cancer cells at 2x10 per well⁶Cell density seedingThe cells were cultured in 6cm cell culture dishes (total 3 dishes) in DMEM (high-sugar) containing 10% fetal bovine serum in a carbon dioxide incubator (37 ℃). And extracting total cell protein when the cells grow to 75% the next day, adding GST-PTB-WT, GST-PTB-MUTANT and GST (control group) into whole cell lysate of 3 groups of cells according to a final concentration of 2 mu M, incubating for 3 hours at 4 ℃ with corresponding volume of GST agarose gel 4FF, discarding liquid, adding a proper amount of PBS and protein loading buffer into the GST agarose gel 4FF, boiling for denaturation, and performing Western blotting experiment detection. As shown in fig. 7.

anti-GST and anti-IGF 1R antibodies were used for immunoblot detection from CST.

3.2 immunoblotting experiments demonstrated that the mutant proteins could penetrate the membrane and enter the cancer cells.

MCF7 breast cancer cells at 2x10 per well⁶The cells were seeded at a cell density of 6cm in a cell culture dish (total of 3 dishes) in DMEM (high sugar) containing 10% fetal bovine serum and cultured in a carbon dioxide incubator (37 ℃). The next day when the cells grew to 85%, GST-PTB-MUTANT, (Arg)9-GST-PTB-MUTANT and (Arg)9-GST (control) were added to the 3 dishes of cells at a final concentration of 2. mu.M, respectively. After further incubation in the incubator for 2 hours, total cellular protein was extracted. The GST antibody was used for detection, and the band at about 36KD appeared in the experimental group, i.e. (Arg)9-GST-PTB-MUTANT, (Arg)9-GST (control group) showed a band at 26KD, while the GST-PTB-MUTANT group showed no band, thus demonstrating that (Arg)9-GST-PTB-MUTANT can penetrate into MCF7 cells (anti-GST antibody was purchased from CST). As shown in fig. 8.

anti-GST antibodies were purchased from CST when performing immunoblot assays.

3.3 Co-immunoprecipitation experiments demonstrated that the mutant proteins can replace the binding of endogenous IRS-1 protein to IGF-1R in cancer cells.

MCF7 breast cancer cells at 2x10 per well⁶The cells were seeded at a cell density of 6cm in a cell culture dish (3 dishes) in DMEM (high sugar) containing 10% fetal bovine serum and cultured in a carbon dioxide incubator (37 ℃). The next day when the cells grew to 75%, 3 dishes of cells were added with (Arg)9-GST-PTB-WT, (Arg)9-GST-PTB-MUTANT and (Arg)9-GST-PTB-MUTANT, respectively, at a final concentration of 2. mu.M(Arg)9-GST (control). After further incubation in the incubator for 2 hours, total cellular Protein was extracted, appropriate volumes of Protein A/G beads, antibody IGF-1R or IRS-1 were added, after 3 hours of incubation at 4 degrees, the liquid was discarded, and the beads were denatured by boiling with appropriate amounts of PBS and Protein loading buffer for detection by Western blotting experiments (anti-IGF 1R antibody from CST and anti-IRS-1 antibody from Santa Cruz). The results of the experiment are shown in FIG. 9.

anti-IGF 1R antibody was purchased from CST and anti-IRS-1 antibody was purchased from Santa Cruz for immunoblot detection.

3.3 MTT cell proliferation inhibition experiments and clone formation experiments detect that the fusion protein (Arg)9-GST-IRS-1-PTB-MUTANT can penetrate cell membranes to enter MCF7 cells and inhibit the proliferation of the cells, and the obvious anti-tumor effect is shown.

MTT cell proliferation inhibition assay: MCF7 cells were seeded into 96-well plates, ensuring that the number of cells per well was approximately 5000, with 3 sub-wells. After overnight incubation in a carbon dioxide cell incubator (37 ℃), 2 days after inoculation, (Arg)9-GST-PTB-MUTANT was added directly to MCF7 cell culture medium, using concentrations increasing from 0 μ M to 20 μ M in order, at a treatment time of 2h, 10ul MTT reagent was added per well, absorbance values were measured at a ═ 570nm, histograms were plotted according to the corresponding values, and cell proliferation of MCF7 was inhibited, and there was a significant difference from concentrations of 1 μ M to 20 μ M. The results of the experiment are shown in FIG. 10.

3.3.2. Plate clone formation experiment: MCF7 cells were seeded in 6-well plates to ensure approximately 100 cells per well, and 2 days after seeding, (Arg)9-GST-PTB-MUTANT was added directly to MCF7 cell culture medium using concentrations of 0. mu.M (notice) and 2. mu.M (Arg)9-GST as controls. After 14 days, visually apparent clones were counted and statistically analyzed after staining with crystal violet stain. The average number of cell-forming clones of MCF7 incubated with (Arg)9-GST-PTB-MUTANT was 15, the average number of clone-forming clones of No treatment group was 48, and the average number of clone-forming clones of (Arg)9-GST control group was 43. This experiment was repeated three times. The statistics of colony formation rate are shown in FIG. 11. The results of crystal violet staining are shown in FIG. 12.

Effect and evaluation: PTB fusion protein mutants of the inventionThe variant PTB-MUTANT is capable of specifically recognizing and binding to a phosphorylated tyrosine residue on IGF-1R. Performing molecular dynamics simulation by using crystal structure of IRS-1 as template, performing homologous comparison analysis by using clustalw software, and selecting distance ligand

The amino acid residues of a PTB structural domain wild-type protein of IRS-1 are subjected to site-directed mutagenesis by a genetic engineering technology, the 99 th R is mutated into L, the 102 th S is mutated into A, a PTB-MUTANT MUTANT vector is constructed and protein expression and purification are carried out, the affinity of the MUTANT protein for the specific combination of the MUTANT protein and IGF-1R phosphorylated by tyrosine residues is greatly enhanced, the combination of the IGF-1R and a plurality of downstream proteins PI3K, SH-PTP2, Grb 2and the like is blocked or influenced, and the physiological processes of proliferation, transformation, infiltration, transfer, apoptosis and the like of tumor cells are influenced.

The PTB fusion protein MUTANT of IRS-1 is introduced into cancer cells with over-activated IGF-1R, and the PTB fusion protein MUTANT is competitively combined with endogenous IRS-1 protein in the cancer cells to combine with the IGF-1R, so that a plurality of signal paths related to the endogenous IRS-1 protein are blocked, and the coprecipitation of PU LL DOWN and immune experiments proves that the MUTANT can be used for realizing the coprecipitation of the MUTANT by using the MUTANT of the PTB fusion protein of IRS-1.

In order to introduce the PTB structural domain MUTANT protein of IRS-1 into living cells, the characteristic that penetrating peptide consisting of 9 Arg directly penetrates cell membrane to enter cells is utilized, a gene engineering method is utilized to construct a recombinant plasmid-expressed PTB fusion protein MUTANT and fusion protein (Arg)9-GST-PTB-MUTANT of cell penetrating peptide (Arg)9, and the membrane penetrating capability and the anti-tumor effect of the MUTANT are researched. Experimental results show that (Arg)9-GST-PTB-MUTANT can penetrate and enter cancer cells, replaces the combination of endogenous IRS-1 protein in the cancer cells and IGF-1R, blocks a plurality of signal paths related to the endogenous IRS-1 protein, inhibits the proliferation and invasion of the tumor cells, and has a remarkable anti-tumor effect.

The (Arg)9-GST-PTB-WT and (Arg)9-GST-PTB-MUTANT recombinant proteins can penetrate into MCF7 cells and specifically bind to IGF-1R phosphorylated at tyrosine residues to inhibit cell proliferation PU LL DOWN experiments demonstrated that the MUTANT proteins have stronger affinity for binding specifically to IGF-1R phosphorylated at tyrosine residues than the wild-type PTB proteins (see FIG. 7). Immunotblotting experiments demonstrated that the MUTANT proteins can penetrate into cancer cells (see FIG. 8). Co-immunoprecipitation experiments demonstrated that the MUTANT proteins can replace the binding of endogenous IRS-1 proteins to IGF-1R in cancer cells (see FIG. 9).

MTT cell proliferation inhibition experiments, according to corresponding values, a histogram is drawn, and the cell proliferation of MCF7 is inhibited, and the PTB fusion protein mutant has significant difference from the use concentration of 1 mu M to 20 mu M. The results of the experiment are shown in FIG. 10.

Plate clone formation experiments were performed, stained with crystal violet stain, counted and statistically analyzed. The average number of cell-forming clones of MCF7 incubated with (Arg)9-GST-PTB-MUTANT was 15, the average number of clone-forming clones of No treatment group was 48, and the average number of clone-forming clones of (Arg)9-GST control group was 43. The experiment was repeated three times. The results of the colony formation experiments are shown in FIG. 11. The results of crystal violet staining are shown in FIG. 12. The cell proliferation of (a) is significantly inhibited.

The following are sequence listings of amino acid sequences and nucleotide sequences related to the present patent application, wherein the sequences 1 to 8 are in order: amino acid sequence of the PTB domain protein of mutated IRS-1; a nucleotide sequence encoding a PTB domain protein of mutated IRS-1; primer F1; primer R1; primer F2; primer R2; primer F3; primer R3.

SEQUENCE LISTING

<110> university of science and technology in Huazhong

<120> a PTB structural domain protein of mutated IRS-1, its coding sequence and application

<130>/

<160>8

<170>PatentIn version 3.3

<210>1

<211>105

<212>PRT

<213> Artificial sequence

<400>1

Phe Lys Glu Val Trp Gln Val Ile Leu Lys Pro Lys Gly Leu Gly Gln

1 5 10 15

Thr Lys Asn Leu Ile Gly Ile Tyr Arg Leu Cys Leu Thr Ser Lys Thr

20 25 30

Ile Ser Phe Val Lys Leu Asn Ser Glu Ala Ala Ala Val Val Leu Gln

35 40 45

Leu Met Asn Ile Arg Arg Cys Gly His Ser Glu Asn Phe Phe Phe Ile

50 55 60

Glu Val Gly Arg Ser Ala Val Thr Gly Pro Gly Glu Phe Trp Met Gln

65 70 75 80

Val Asp Asp Ser Val Val Ala Gln Asn Met His Glu Thr Ile Leu Glu

85 90 95

Ala Met Leu Ala Met Ala Asp Glu Phe

100 105

<210>2

<211>315

<212>DNA

<213> Artificial sequence

<400>2

ttcaaagagg tctggcaagt gatcctgaag cccaagggcc tgggtcagac aaagaacctg 60

attggtatct accgcctttg cctgaccagc aagaccatca gcttcgtgaa gctgaactcg 120

gaggcagcgg ccgtggtgct gcagctgatg aacatcaggc gctgtggcca ctcggaaaac 180

ttcttcttca tcgaggtggg ccgttctgcc gtgacggggc ccggggagtt ctggatgcag 240

gtggatgact ctgtggtggc ccagaacatg cacgagacca tcctggaggc catgctggcc 300

atggctgatg agttc 315

<210>3

<211>34

<212>DNA

<213> Artificial sequence

<400>3

gatctggttc cgcgtttcaa agaggtctgg caag 34

<210>4

<211>34

<212>DNA

<213> Artificial sequence

<400>4

gtcagtcagt cacgagaact catcactcat ggcc 34

<210>5

<211>40

<212>DNA

<213> Artificial sequence

<400>5

tcctggaggc catgctggcc atggctgatg agttctcgtg 40

<210>6

<211>40

<212>DNA

<213> Artificial sequence

<400>6

cacgagaact catcagccat ggccagcatg gcctccagga 40

<210>7

<211>59

<212>DNA

<213> Artificial sequence

<400>7

gatctggttc cgcgtcgtcg tcgccgccgt cgtcgccgcc gtggcttcaa agaggtctg 59

<210>8

<211>34

<212>DNA

<213> Artificial sequence

<400>8

gtcagtcagt cacgagaact catcactcat ggcc 34

Claims

1. A mutant PTB structural domain protein of IRS-1 is characterized in that compared with the PTB structural domain protein of wild-type IRS-1, the 99 th amino acid of the mutant PTB structural domain protein of IRS-1 is mutated from R to L, the 102 th amino acid is mutated from S to A, and the amino acid sequence of the mutant PTB structural domain protein of IRS-1 is shown as sequence 1 in a sequence table.

2. A DNA molecule encoding the PTB domain protein of mutated IRS-1 according to claim 1.

3. The DNA molecule according to claim 2, wherein the nucleotide sequence of the DNA molecule is represented by sequence 2 in the sequence listing.

4. A fusion protein consisting of the mutated IRS-1 PTB domain protein of claim 1 in combination with a cell-penetrating peptide.

5. The fusion protein of claim 4, wherein the cell-penetrating peptide is a short peptide consisting of 9 Arg's.

6. The fusion protein of claim 4 or 5, wherein the fusion protein comprises a GST tag.

7. A recombinant vector comprising the DNA molecule of claim 2 or 3.

8. The recombinant vector according to claim 7, wherein the vector used is pGEX-4T 2.

9. A host cell comprising the recombinant vector of claim 7 or 8.

10. A pharmaceutical composition characterized by being composition a or composition B as described below:

composition A comprising the protein of claim 1, 4, 5 or 6 and a pharmaceutically acceptable carrier or/and additive;

composition B comprising the DNA molecule of claim 2 or 3 and a pharmaceutically acceptable carrier or/and an additive.

11. Use of the protein of claim 1, 4, 5 or 6 for the preparation of a medicament for the treatment of tumors.

12. Use of the DNA molecule of claim 2 or 3 in the preparation of a medicament for use as an anti-tumor agent.

13. A preparation method of PTB-MUTANT protein containing cell-penetrating peptide Arg9 comprises the following steps:

F2:tcctggaggccatgctggccatggctgatgagttcTCGTG 40bp；

R2:CACGAgaactcatcagccatggccagcatggcctccagga 40bp；

the PCR reaction system is 2 XPCR Mix 10ul, F21 ul, R21 ul, H₂O6 ul, template 2ul, PCR reaction program 98 ℃ for 2min, 98 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 30s, 35cycle, 72 ℃ for 10 min;

F3:GATCTGGTTCCGCGTcgtcgtcgccgccgtcgtcgccgccgtggcttcaaagaggtctg 59bp；

R3:GTCAGTCAGTCACGAgaactcatcactcatggcc 34bp；

the PCR reaction system is 2 XPCR Mix 10ul, F31 ul, R31 ul, H₂O6 ul, template 2ul, PCR reaction program 98 ℃ for 2min, 98 ℃ for 30s, 58 ℃ for 30s, 72 ℃ for 30s, 35cycle, 72 ℃ for 10 min;

(2) PTB-MUTANT and Arg9-PTB-MUTANT are respectively used for constructing a recombinant prokaryotic expression vector by utilizing the experimental principle of homologous recombination, the used vector is pGEX-4T2, and the enzyme cutting sites used for constructing the vector are BamHI and NotI.