CN115850441B - Recombinant protein for specific targeted degradation of EGFR and mutant thereof, preparation method and application thereof - Google Patents
Recombinant protein for specific targeted degradation of EGFR and mutant thereof, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a recombinant protein for specifically targeting degradation of EGFR and mutants thereof, a preparation method and application thereof, and relates to the technical field of molecular biology. The invention provides a recombinant protein for specifically targeting degradation of EGFR and mutants thereof, which is obtained by extracting 39 total amino acid sequences of 304-342 sites combined by tumor suppressor factor Shf and EGFR and mutants thereof, and respectively adding His tag protein sequences and TAT sequences at N terminal and C terminal. Experiments prove that the recombinant protein P39 can enter cells to bind EGFR/EGFRvIII and promote ubiquitination modification and degradation of the EGFR/EGFRvIII, so that biological phenotype of tumors carried by the EGFR or EGFRvIII is inhibited, and the activity of tumor cells is inhibited.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a recombinant protein for specifically targeting degradation of EGFR and mutants thereof, a preparation method and application thereof.
Background
On 1 and 6 days 2022, the international agency for research on cancer (IARC, international Agency for Research on Cancer) subordinate to the world health organization issued IARCBiennial Report2020-2021 (biennial report 2020-2021), WHO also issued World Cancer Report2020 (global cancer report 2020) in the last year, reporting that cancer is a significant cause of unnatural death worldwide. The root cause of the canceration of cells is a gene mutation. With the rapid development of tumor molecular biology in recent years, the molecular mechanism of in-depth understanding of tumorigenesis development and prognosis helps in the cognition and clinical decision making of targeted therapies. Among them, abnormal activation or inhibition of protein kinases including epidermal growth factor receptor (Epidermal growth factor receptor, EGFR) is extremely closely related to tumors, and inhibitors or agonists related to protein kinases are the mainstream choice for tumor-targeted therapies.
EGFR, also known as ErbB1/HER1, is one of the members of the epidermal growth factor receptor (HER) family, and is widely distributed on the cell membranes of various tissues in humans. EGFR overexpression and/or mutation is present in most solid tumors (e.g., bladder cancer, non-small cell lung cancer, ovarian cancer, glioma, etc.), and deregulation and malignancy of cell growth by means of signal transduction is one of the important causes of tumorigenesis and progression. EGFR is a transmembrane protein receptor of 170kD relative molecular mass, consisting of three functional regions: the extracellular ligand-binding domain, single-chain transmembrane domain, and intracellular tyrosine kinase domain, comprising 621, 23, and 542 amino acids, respectively, have the major functions of ligand binding, anchoring the position on the membrane, and signal transduction relays, respectively. The ligand includes epidermal growth factor (Epidermal growth factor, EGF), transforming growth factor alpha (TGF-alpha), bi-regulating factor, neuregulin, etc. once the extracellular region is connected with the ligand, homologous or hetero-dimerization can occur, so that tyrosine residues in the intracellular region are autophosphorylated, a downstream series of cascade signal transduction systems such as RAS/RAF/MEK/ERK, PI3K/AKT/TOR, src kinase, STAT, etc. transcription factors are activated to regulate proliferation, differentiation and apoptosis of cells, and the like, and the ligand participates in migration, invasion, metastasis, angiogenesis, etc. of tumor cells. EGF and EGFR are combined to form a complex, activated, subjected to ubiquitination modification and endocytosis, enter vesicles, enter cells through an endocytosis way mediated by clathrin or non-clathrin, are transferred to early endosomes and late endosomes, and are subjected to targeted lysosome degradation or returned to the surface of a plasma membrane for recycling after sorting. EGFR mutations typically occur in the extracellular domain, and three EGFR extracellular deletion mutants are currently found: EGFRvI, EGFRvII and EGFRvIII, most commonly EGFRvIII. Egfrvlll lacks ligand binding domains and exhibits constitutive activation, resulting in sustained activation of EGFR and related pathways. EGFR mutants are widely present in tumor cells but not in normal tissues, making them a good molecular target. At present, small molecule inhibitors, antibodies, vaccines, CAR-T, RNA-based treatment methods and the like of EGFR and mutants thereof are targeted, and the aim of resisting tumors is fulfilled mainly by targeted inhibition of EGFR kinase activity or induction of antibody and complement-mediated cytotoxicity. Although the treatment methods have the advantages of strong specificity, small toxic and side effects and the like, the defects of high EGFR mutation rate, acquired drug resistance, blood brain barrier and the like limit the further clinical application of the EGFR, so that the clinical treatment of tumors of targeted EGFR and mutants thereof still has no significant progress. High expression of EGFR and its mutants is mainly associated with gene amplification, but posttranslational dysregulation is also a significant cause, such as limited ubiquitination and reduced degradation after activation, which can lead to continued activation. Thus, ubiquitination modification and degradation of EGFR and its mutants that promote activation are potential targeting strategies.
Disclosure of Invention
In view of the above, the present invention aims to provide a eukaryotic recombinant protein specifically targeting and degrading EGFR and its mutant, which can enter cells to bind EGFR/EGFRvIII and promote its ubiquitination modification and degradation, thereby inhibiting the biological phenotype of high EGFR or EGFRvIII carrying tumor.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a recombinant protein for specifically targeting degradation of EGFR and mutants thereof, wherein the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1.
The invention provides a nucleotide sequence for encoding the recombinant protein, and the nucleotide sequence is shown as SEQ ID NO. 2.
The invention provides a recombinant vector comprising the nucleotide sequence.
The invention provides a recombinant strain, which comprises the recombinant vector.
The invention also provides a preparation method of the recombinant protein, which comprises the following steps:
amplifying a P39 gene sequence by taking a GV219/HA-SHF vector as a template, modifying, then converting into E.coli competent cells, culturing to obtain GV219/His-P39-TAT plasmids, transfecting the plasmids into HEK293 cells, extracting proteins from the cultured cells, and separating and purifying to obtain the recombinant proteins.
Preferably, the construction method of the GV219/HA-SHF vector comprises the following steps:
amplifying by taking human mRNA as a template to obtain an HA-Shf gene sequence, and connecting the gene into a GV219 vector to obtain the GV219/HA-SHF vector.
Preferably, the modification comprises: his tag protein sequence is introduced at N end of the amplified P39 gene sequence, and TAT sequence is introduced at C end.
More preferably, the modification further comprises introducing XhoI and KpnI endonuclease sites at both ends of the amplified P39 gene sequence.
The invention also provides application of the recombinant protein or the nucleotide sequence in promoting ubiquitination degradation of EGFR and EGFRvIII mutant thereof.
The invention also provides application of the recombinant protein or the nucleotide sequence in preparing medicaments for inhibiting the activity of tumor cells.
The invention provides a recombinant protein P39 for specifically targeting degradation of EGFR and mutants thereof, which is obtained by extracting 39 amino acid sequences in total from 304-342 positions combined by tumor-inhibiting factor Shf and EGFR and mutants thereof, and respectively adding His tag protein sequences and TAT sequences at N terminal and C terminal. Experiments prove that the recombinant protein P39 can enter cells to bind EGFR/EGFRvIII and promote ubiquitination modification and degradation of the EGFR/EGFRvIII, so that biological phenotype of tumors carried by the EGFR or EGFRvIII is inhibited, and the activity of tumor cells is inhibited.
Drawings
FIG. 1 is a graph showing that SHF binds EGFR/EGFRvIII to promote its ubiquitination degradation; wherein, A represents that SHF can bind EGFR and promote its ubiquitination degradation in cell experiments, and B represents that SHF can bind EGFRvIII and promote its ubiquitination degradation in cell experiments.
FIG. 2 is a SHF-EGFR/EGFRvIII binding site assay; wherein A represents a schematic diagram of SHF-EGFR protein structure simulation, and B represents a schematic diagram of constructing SHF key domain deletion mutants according to software analysis results.
FIG. 3 shows SHF binding to EGFR/EGFRvIII via the 304-342aa domain; wherein A represents SHF binding to EGFRvIII through the 304-342aa domain and B represents SHF binding to EGFR through the 304-342aa domain.
FIG. 4 is a schematic diagram of the construction of the GV219/His-P39-TAT plasmid.
FIG. 5 is a diagram of the Westernblot identification of purified recombinant proteins.
FIG. 6 shows the localization of recombinant protein P39 cells by immunofluorescence double-staining.
FIG. 7 is a graph showing that the P39 recombinant protein promotes ubiquitination degradation of EGFR/EGFRvIII; wherein A represents the P39 recombinant protein to promote the ubiquitination degradation of EGFRvIII, and B represents the P39 recombinant protein to promote the ubiquitination degradation of EGFR.
FIG. 8 shows that P39 recombinant protein inhibits the formation of glioblastoma stem cell pellets over-expressed by EGFRvIII; wherein, the observation results are sequentially from left to right on the 1 st day, the 3 rd day, the 5 th day, the 7 th day, the 9 th day and the 11 th day.
FIG. 9 shows the effect of Westernblot on EGFR/EGFRvIII proteins and downstream pathways detected by P39 recombinant proteins.
FIG. 10 shows that the P39 recombinant protein inhibits the growth of glioblastoma that overexpresses EGFRvIII in nude mice; wherein A represents the final volume of the tumor and B represents the growth curve of the tumor.
Fig. 11 shows that P39 significantly prolonged the survival of nude mice with intracranial egfrvlll over-expressed glioblastoma (P < 0.01).
Detailed Description
The invention provides a recombinant protein for specifically targeting degradation of EGFR and mutants thereof, wherein the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1.
In the invention, the recombinant protein extracts 39 total amino acid sequences of 304-342 sites combined with tumor-inhibiting factor Shf and EGFR and mutants thereof through gene operation, his tag protein sequence and TAT sequence are respectively added at N end and C end, fusion genes formed by the recombinant protein are inserted into the multicloning site of eukaryotic expression vector GV219,HEK293 cells were transfected, total cell protein was extracted and the final recombinant protein, designated P39, was obtained by affinity purification and amplification. In the invention, the amino acid sequence of the recombinant protein is as follows: mhhhhhhhhhhhPSSPLGEWTDPALPLENQVWYHGAISRTDAENLLRLCKEYGRKRRRQRRR (SEQ ID NO. 1); the recombinant protein has 57 amino acids in full length, an initiation codon ATG translated methionine (M) is introduced into the N end, an underlined part is an amino acid sequence of SHF protein 304-342, and 'hhhhhhhhh' is His tag protein which is a screening and identification tag; "YGRKRRQRRR" is TAT sequence, a protein transduction signal peptide, directing the fusion protein into cells.
The invention provides a nucleotide sequence for encoding the recombinant protein, and the nucleotide sequence is shown as SEQ ID NO. 2.
In the present invention, the nucleotide sequence is as follows:CTCGAGCGCCACC(ATGCATCATCATCATCATCATCCCAGCAGCCCCCTGGGGGAGTGGACAGATCCAGCACTGCCTCTGGAAAACCAGGTCTGGTATCACGGGGCCATCAGCCGAACCGACGCCGAGAACCTGCTCCGGCTGTGCAAAGAGTACGGCCGCAAGAAACGCCGCCAGCGCCGCCGCTAG)GGGGTACC(SEQ ID NO. 2); in the nucleotide sequence, the bracket part is the base sequence corresponding to the recombinant protein; the underlined parts are Xho I and Kpn I cleavage sites, respectively; the base "C" after the Xho I cleavage site and the base "GG" before the Kpn I cleavage site are both endonuclease protecting bases for stabilizing the combination of endonuclease to DNA sequence to play a role; "GCCACC" is a Kozak sequence, which can enhance the translation efficiency of eukaryotic genes.
The invention provides a recombinant vector comprising the nucleotide sequence.
The invention provides a recombinant strain, which comprises the recombinant vector.
The invention also provides a preparation method of the recombinant protein, which comprises the following steps:
amplifying a P39 gene sequence by taking a GV219/HA-SHF vector as a template, modifying, then converting into E.coli competent cells, culturing to obtain GV219/His-P39-TAT plasmids, transfecting the plasmids into HEK293 cells, extracting proteins from the cultured cells, and separating and purifying to obtain the recombinant proteins.
In the invention, the GV219/HA-SHF vector is preferably constructed by the following steps: and (3) taking human mRNA as a template, amplifying by adopting a primer F/R to obtain an HA-Shf gene, linearizing a GV219 vector by using XhoI and KpnI, connecting the HA-Shf gene with the linearized GV219 vector, and then selecting recombinant clones for enzyme digestion and sequencing identification to obtain the GV219/HA-SHF vector. In the invention, the amplification is preferably carried out by using a high-fidelity PCR amplification kit of Takara company; the sequence of the primer F/R during amplification is as follows: f: ACGGGCCCTCTAGACTCGAGCGCCACCATGTACCCTTATGATGTCCCAGACTATGCTATGCAGCAGGAGGGAGGACCC (SEQ ID NO. 3), R: TTAAACTTAAGCTTGGTACCCTAAAGAGTCCGGATGGCCACAGGG (SEQ ID NO. 4).
In the present invention, the modification preferably includes: his tag protein sequence is introduced at N end of the amplified P39 gene sequence, TAT sequence is introduced at C end, and XhoI and KpnI endonuclease sites are introduced at both ends respectively.
The invention also provides application of the recombinant protein or the nucleotide sequence in promoting ubiquitination degradation of EGFR and EGFRvIII mutant thereof.
The invention also provides application of the recombinant protein or the nucleotide sequence in preparing medicaments for inhibiting the activity of tumor cells.
In the invention, the GV219/HA-N-SHF vector is preferably constructed by the following steps: commercial GV219 vector is linearized by using XhoI and KpnI, primer F1/R1 (the primer sequence contains exchange pairing base, enzyme cutting site and 5' end part sequence of target gene for PCR fishing target gene) is used, amplified HA-N-SHF gene sequence (comprising gene sequence encoding SHF protein 1-303 aa) is connected with linearization vector, recombinant clone is selected for enzyme cutting and sequencing identification, and GV219/HA-N-SHF vector is obtained; the primer sequences during amplification are as follows: f1: ACGGGCCCTCTAGACTCGAGCGCCACCATGTACCCTTATGATGTCCC (SEQ ID NO. 5), R1: TTAAACTTAAGCTTGGTACCCTACTCCATGCTTAGGGGTTTGG (SEQ ID NO. 6).
In the invention, the GV230/HA-C-SHF and GV230/HA-P39 vectors are preferably constructed by the following steps: commercial GV230 vector is linearized by using XhoI and KpnI, primers (the primer sequence contains exchange pairing base and enzyme cutting site, and contains 5' -end part sequence of target gene for PCR fishing target gene) are used for amplifying obtained HA-C-SHF gene sequence (including SHF protein 343-423aa gene sequence) and HA-P39 gene sequence (including SHF protein 304-342aa gene sequence), and the two fragments are respectively connected with the linearized vector, and then recombinant clones are selected for enzyme cutting and sequencing identification to obtain GV230/HA-C-SHF and GV230/HA-P39 vector. In the invention, the primer F2/R2 sequence during HA-C-SHF amplification is as follows: f2: TACCGGACTCAGATCTCGAGCGCCACCATGTACCCTTATGATGTCCCAG (SEQ ID NO. 7), R2: GATCCCGGGCCCGCGGTACCGTAAGAGTCCGGATGGCCACAGGGTAG (SEQ ID NO. 8); the primer F3/R3 sequence during HA-P39 amplification is as follows: f3: TACCGGACTCAGATCTCGAGCGCCACCATGTACCCTTATGATGTCCC (SEQ ID NO. 9), R3: GATCCCGGGCCCGCGGTACCGTCTCTTTGCACAGCCGGAGCAGGTTC (SEQ ID NO. 10).
In the invention, the GV141/Flag-EGFR and GV141/Flag-EGFRvIII vectors are preferably constructed by the following steps: commercial GV141 vector is linearized by using XhoI and KpnI, EGFR and EGFRvIII gene sequences are chemically synthesized by adopting Shanghai Ji Kai gene chemistry technology, xhoI and KpnI endonuclease sites are respectively introduced at two ends of the sequences, the synthesized EGFR and EGFRvIII gene sequences are connected with the linearized GV141 vector, recombinant clones are selected for enzyme digestion and sequencing identification, and GV141/Flag-EGFR and GV141/Flag-EGFRvIII vectors are obtained.
In the invention, the human glioblast U87EGFRvIII over-expression stable transgenic cell strain is constructed by the following steps: human glioblast U87 cells (purchased from the cell bank of the national academy of sciences of China typical culture Collection, and amplified and stored by the present laboratory) grown in log phase were taken according to a 2X 10 protocol 5 Density of individual cells were seeded in 6-well plates, and EGFRvIII overexpressing lentivirus (commissioned and metabiotechnology (Shanghai) Co., ltd.) was transfected 24h later and screened with 2. Mu.g/mL puromycin 24h later after transfection to give stably transfected cell lines.
In the invention, the GV219, GV141 and GV230 vectors are all purchased from Shanghai Ji Kai gene chemical technology Co., ltd, and the molecular cloning tool enzyme is purchased from Takara company; plasmid extraction kit and gel recovery kit were purchased from Beijing kang as century biotechnology Co., ltd; ni-charged MagBeads available from Kirsry Biotechnology Co., ltd; the D/F12, EGF, glutaMAX and B27 were all purchased from Thermo Fisher, U.S.A., and bFGF was purchased from PeproTech, U.S.A.
In the invention, the formula of LB culture medium is shown in molecular cloning. In the method mentioned in the specification, plasmid extraction, PCR reaction, endonuclease digestion, DNA fragment recovery, ligation and transformation are all conventional operation methods in the field of genetic engineering research, and are specifically referred to as molecular cloning.
The present invention will be described in detail below with reference to examples for the purpose of making the objects, technical solutions and advantages of the present invention more apparent, but they should not be construed as limiting the scope of the present invention.
In the following examples, conventional methods are used unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
Discovery of SHF-P39 recombinant proteins
SHF binding EGFR/EGFRvIII promotes its ubiquitination degradation
HEK293 cells were transfected with GV141/Flag-EGFR, GV141/Flag-EGFRvIII and GV219/HA-SHF as in FIG. 1 by liposome transfection reagent, after 24 hours of transfection, anti-DDDDK-tagmAb-Magnetic Beads were added, after 4℃overnight, proteins on the Beads were collected and the expression of each protein was detected by Westernblot, and the results were shown in FIG. 1.
It can be seen that SHF binds EGFR and egfrvlll and promotes its ubiquitination degradation.
SHF-EGFR/EGFRvIII binding site assay
For SHF and EGFR protein structures, the 304-342aa sequence of SHF was extracted alone and domain deletion mutants were constructed (FIG. 2).
SHF binds EGFR/EGFRvIII through the 304-342aa domain
HEK293 cells were transfected with GV141/Flag-EGFR, GV141/Flag-EGFRvIII, GV219/HA-N-P39, GV230/HA-C-P39 and GV230/HA-P39 by liposome transfection reagent as in FIG. 3, for 24 hours, anti-DDDDK-tagmAb-Magnetic Beads were added after protein collection, proteins on the Beads were collected after overnight at 4℃and the expression of each protein was detected by Western blot, and the results are shown in FIG. 3.
It can be seen that SHF binds EGFR and EGFRvIII through its 304-342aa domain.
Example 2
Cloning of the P39 Gene
1. Design of recombinant human P39 gene
The sequence expressing the domain is extracted according to the SHF protein structure and protein binding property (39 amino acids are protein binding domain in total in 304-342 aa), and a new recombinant gene with 39 amino acids in total in 304-342aa of SHF is designed according to the prior experience and codon preference and is named as P39.
And simultaneously setting a control group: the recombinant protein of the control group adopts 16 amino acid disorder control sequences (NLASPLSPTEDWPLEPG) of the SHF protein 304-320, and the rest connecting peptide is the same as the experimental group and is named as SC.
Obtaining and expression vector construction of P39 recombinant Gene
The GV219/HA-SHF vector constructed in the laboratory is used as a template, a P39 sequence containing 39 amino acids of SHF204-342 is amplified by using a high-fidelity PCR amplification kit of Takara company, his tag protein and TAT sequences are respectively introduced at the N end and the C end, and endonuclease (Xho I and Kpn I) sites are respectively introduced at the two ends (as shown in figure 4).
Purification of the target fragment: the PCR product was digested with Xho I and Kpn I, and the digested product was recovered using the Kangji kit.
And (3) carrying out enzyme cutting and recovery of a carrier: the GV219 plasmid was digested with Xho I and Kpn I, subjected to 1% agarose gel electrophoresis, and recovered using the Kangji kit.
And (3) connection: the PCR cleavage product and plasmid cleavage product were ligated overnight at 4℃with T4DNA ligase from Takara, transformed into E.coli Dh5α competent cells, plated on LB solid medium containing 50. Mu.g/ml kanamycin, and cultured overnight at 37 ℃.
Identification of transformants: selecting single colony, culturing in LB liquid medium (containing 100 mug/mL ampicillin) overnight, and then primarily screening positive clone by PCR, and extracting positive plasmid GV219/His-P39-TAT by a plasmid extraction kit; wherein, the forward primer of PCR is: CGCAAATGGGCGGTAGGCGTG (SEQ ID NO. 11), reverse primer: CGTCGCCGTCCAGCTCGACCAG (SEQ ID NO. 12).
And (3) sequencing and identifying plasmids: the positive plasmid is sent to Shanghai worker for sequencing, and the sequencing result is compared by software, so that the correctness of gene cloning is verified.
Example 3
Expression and purification of P39 fusion proteins
Transfection and expression of HEK293 cells
HEK293 cells were transfected with GV219/His-P39-TAT plasmid by liposome transfection reagent (purchased from Life Co.) for 48 hours, and neomycin was added to perform pressure screening to construct stably transfected cell lines expressing P39 gene.
Separation and purification of P39 recombinant protein and Westernblot identification
After the HEK293 steady cell line is expanded and cultured, cells are collected and total proteins of the cells are extracted, the protein extract is filtered by a 0.45 mu m filter, a Ni column is used for purifying the recombinant protein with the His tag, and the related operation process is carried out according to the operation instruction provided by the Kirschner company.
The specific process is as follows:
transferring the resin to a column, washing the nickel affinity chromatography column with double distilled water after complete precipitation, and preventing the next Ni step 2+ Precipitating; with ddH 2 O washing to remove air in the matrix; bindingbuffer equilibrium for 10 column volumes; loading a sample; 10 times column volume of Bindingbuffer washing, collecting effluent; eluting by using an Elutionbuffer, and further performing ultrafiltration and concentration to obtain the recombinant protein.
The recombinant protein was subjected to SDS-PAGE electrophoresis and membrane transfer, and then identified by His monoclonal antibody, and the result is shown in FIG. 5.
As a result, it was found that the relative molecular mass of the purified P39 recombinant protein was about 7kDa.
Example 4
P39 eukaryotic recombinant protein can enter cells
Selecting human colloid in logarithmic growth phaseMaternal U87 cell digests were counted, 1X 10 4 Inoculating the individual cells to a cell climbing sheet, culturing for 24 hours, adding 500 mu M recombinant protein P39, and fixing for 6 hours at room temperature by 4% PFA solution for 15 minutes; washing with 0.3% TritonX100-PBS 3 times for 10min each; donkey serum was blocked for 1 hour at room temperature, and the mouse anti-His was incubated overnight at 4deg.C in the dark, washed 3 times with PBS for 10min each time; donkey anti-mouse secondary antibody coupled with AlexaFlour594 is incubated for 1 hour at room temperature in dark place, washed 3 times with PBS for 10min each time; dylight using F-actin-binding TM 488 Dye staining of Phalidin for 20min; fluomount (containing Hoechest10 ug/mL) was blocked and photographed by a fluorescence microscope, and the results are shown in FIG. 6.
The results showed that recombinant protein P39 was able to enter cells smoothly.
Example 5
P39 recombinant proteins promote ubiquitination degradation of EGFR and mutant proteins thereof and downstream pathway protein activity
GV141/Flag-EGFR, GV141/Flag-EGFRvIII and His-Ub were grouped as in FIG. 7, HEK293 cells were transfected with liposome transfection reagents, and cells were treated with different concentrations of P39 recombinant protein, treated for 24 hours, further treated with 10. Mu.MMG 132 for 4 hours, and then Anti-DDDDK-tagmAb-Magnetic Beads were added, and proteins on the Beads were collected after overnight at 4℃and the expression of each protein was detected by Westernblot, and the results were as in FIG. 7.
The results show that the P39 recombinant protein promotes ubiquitination degradation of EGFR and its mutant egfrvlll protein.
Example 6
P39 recombinant protein inhibits glioblastoma stem cell formation in vitro culture
Human glioblast U87EGFRvIII over-expressed stable transgenic cells in logarithmic growth phase were selected, counted after digestion, and cultured with neural stem cell culture medium (D/F12+EGF+bFGF+B27+GlutataMAX). Inoculating 1000 cells in each well by using a low adsorption 96-well plate, adding P39 recombinant protein and control protein SC, and arranging 3 compound wells in each group; the glioblastoma stem cell pellet formation was observed and EVOS was used in one day of culture TM The FL Auto cell imaging system (40X) was photographed and recorded, and the results are shown in FIG. 8.
The results show that the P39 recombinant protein inhibits glioblastoma stem cell formation in vitro culture.
The extract protein was used for Westernblot detection EGFR, EGFRvIII and downstream STAT3 protein expression levels when the stem cell pellet was cultured for 11 days, and the results are shown in fig. 9.
The results show that the P39 recombinant protein inhibits EGFR and egfrvlll expression and downstream STAT3 protein and phosphorylation expression levels thereof in glioblastoma stem cells cultured in vitro.
Example 7
P39 recombinant protein for inhibiting glioblastoma growth in nude mice
1. Each balb/c nude mouse was 2X 10 6 A subcutaneous injection of U87EGFRvIII over-expressed stably transformed cells was performed at a concentration of 100. Mu.l, and a nude mouse subcutaneous model was established. After 4 days of subcutaneous formation of distinct tumor masses, random groupings were made: (1) SC; (2) P39,6 per group, were given intraperitoneal injections of SC and P39 recombinant protein (15 μg/each, once every 2 days for 20 days), tumor size was measured every 2 days, nude mice were sacrificed and tumor size was measured at 22 days of feeding, and the results are shown in fig. 10.
The results show that P39 significantly inhibits the growth of glioblastoma in nude mice.
2. Each balb/c nude mouse was 2X 10 6 A glioblastoma nude mice intracranial model was established by intracranial injection of U87EGFRvIII over-expressing stably transformed cells at a concentration of 100. Mu.l. After 4 days of intracranial model establishment, random groupings: (1) SC; (2) P39, 10 animals per group, were given the SC and P39 recombinant proteins by intraperitoneal injection (15. Mu.g/animal, once every 2 days until nude mice die), and the survival of nude mice was recorded, and the results are shown in FIG. 11.
The results show that P39 can significantly prolong the survival of nude mice (P < 0.01).
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related arts are included in the scope of the present invention.
Claims (9)
1. A P39 recombinant protein for specifically targeting and degrading EGFR and a mutant EGFRvIII protein thereof is characterized in that the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1.
2. The polynucleotide for encoding the recombinant protein according to claim 1, wherein the polynucleotide sequence is shown in SEQ ID NO. 2.
3. A recombinant vector comprising the nucleotide sequence of claim 2.
4. A recombinant strain comprising the recombinant vector of claim 3.
5. The method for producing a recombinant protein according to claim 1, characterized in that said method comprises the steps of:
amplifying a P39 gene sequence by taking a GV219/HA-SHF vector as a template, modifying, then converting into E.coli competent cells, culturing to obtain GV219/His-P39-TAT plasmids, transfecting the plasmids into HEK293 cells, extracting proteins from the cultured cells, and separating and purifying to obtain the recombinant proteins.
6. The preparation method according to claim 5, wherein the construction method of the GV219/HA-SHF vector comprises the following steps:
amplifying by taking human mRNA as a template to obtain an HA-Shf gene sequence, and connecting the gene into a GV219 vector to obtain the GV219/HA-SHF vector.
7. The method of preparation of claim 5, wherein the modification comprises: his tag protein sequence is introduced at N end of the amplified P39 gene sequence, and TAT sequence is introduced at C end.
8. The method according to claim 7, wherein the modification further comprises introducing XhoI and KpnI endonuclease sites at both ends of the amplified P39 gene sequence.
9. Use of the recombinant protein of claim 1 or the polynucleotide of claim 2 for the preparation of a medicament for inhibiting glioblastoma cell activity.
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