CN115850441A - Recombinant protein for specifically targeted degradation of EGFR and mutant thereof, preparation method and application thereof - Google Patents
Recombinant protein for specifically 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 and degrading EGFR and a mutant thereof, a preparation method and application thereof, relating to the technical field of molecular biology. The invention provides a recombinant protein for specifically targeting and degrading EGFR and a mutant thereof, which is obtained by extracting 39 amino acid sequences of 304-342 combined by tumor suppressor Shf and EGFR and the mutant thereof and respectively adding His tag protein sequence and TAT sequence at the N end and the C end. Experiments prove that the recombinant protein P39 can enter cells to be combined with EGFR/EGFRvIII and promote ubiquitination modification and degradation of the EGFR/EGFRvIII, so that the biological phenotype of tumors carried by high 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 and degrading EGFR and a mutant thereof, a preparation method and application thereof.
Background
On 6 days 1 month 2022, IARCBIENNIAL REPORT2020-2021 (bi-annual Report 2020-2021) was published by International Agency of Research on Cancer (IARC) under the World health organization, and World Cancer Report2020 (Global Cancer Report 2020) was published by WHO in the last year, and the Report indicated that worldwide Cancer is a significant cause of non-natural death. The root cause of cellular carcinogenesis is gene mutation. With the rapid development of tumor molecular biology in recent years, the molecular mechanism for deeply understanding the tumor development and prognosis is helpful for the cognition and clinical decision of targeted therapy. Among them, abnormal activation or inhibition of protein kinases including Epidermal Growth Factor Receptor (EGFR) is very closely related to tumors, and protein kinase related inhibitors or agonists are the main choice for tumor targeted therapy.
EGFR, also known as ErbB1/HER1, is one of the epidermal growth factor receptor (HER) family members and is widely distributed in the cell membranes of various tissues of the human body. EGFR overexpression and/or mutation, which is present in most solid tumors (e.g., bladder cancer, non-small cell lung cancer, ovarian cancer, glioma, etc.), and makes cell growth out of control and malignant by means of signal transduction, is one of the important causes leading to tumor development and progression. EGFR is a transmembrane protein receptor with a relative molecular mass of 170kD and consists of three functional regions: the extracellular ligand connecting region, the single-chain transmembrane region and the intracellular tyrosine kinase region respectively comprise 621 amino acids, 23 amino acids and 542 amino acids, and the main functions of the three regions are ligand binding, anchoring on-membrane position and signal transduction relay respectively. The ligand of the polypeptide comprises Epidermal Growth Factor (EGF), transforming growth factor alpha (TGF-alpha), bidirectional regulator, neuregulin and the like, and once the extracellular region is connected with the ligand, the polypeptide can undergo homo-or hetero-dimerization, so that tyrosine residues in the intracellular region can be autophosphorylated, a series of downstream cascade signal transduction systems, such as RAS/RAF/MEK/ERK, PI3K/AKT/TOR, src kinase, STAT and other transcription factors, are activated to regulate the proliferation, differentiation and apoptosis of cells, and the polypeptide participates in the migration, invasion, metastasis, angiogenesis and the like of tumor cells. EGF and EGFR are combined to form a compound which is activated, then is subjected to ubiquitination modification and endocytosis to enter vesicles, enters cells through clathrin or non-clathrin mediated endocytosis pathway and is transported to early endosome and late endosome, and then is subjected to sorting and targeted lysosome degradation or returned to the surface of a plasma membrane for reuse. EGFR mutations generally occur in the extracellular region, and three extracellular EGFR deletion mutants are currently found: EGFRvI, EGFRvIII and EGFRvIII, with EGFRvIII being the most common. EGFRvIII lacks the ligand binding region and exhibits constitutive activation, resulting in sustained activation of EGFR and related pathways. The EGFR mutant is widely present in tumor cells and is not expressed in normal tissues, so that the EGFR mutant becomes a good molecular target. Currently, EGFR and its mutants are targeted with small molecule inhibitors, antibodies, vaccines, CAR-T and RNA-based therapeutic methods, and the like, and the anti-tumor purpose is achieved 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 mutation rate, so that the clinical treatment of tumors targeting EGFR and mutants thereof still has no great progress. High expression of EGFR and its mutants is mainly associated with gene amplification, but posttranslational dysregulation is also an important cause, such as persistent activation due to limited ubiquitination and reduced degradation after activation. Therefore, promoting ubiquitination modification and degradation of activated EGFR and mutants thereof is a potential targeting strategy.
Disclosure of Invention
In view of the above, the invention aims to provide a eukaryotic recombinant protein specifically targeting and degrading EGFR and a mutant thereof, which can enter cells to bind to EGFR/EGFRvIII and promote ubiquitination modification and degradation thereof, so as to inhibit the biological phenotype of a tumor carried by high EGFR or EGFRvIII.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a recombinant protein for specifically targeted degradation of EGFR and a mutant thereof, wherein the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1.
The invention provides a nucleotide sequence for coding the recombinant protein, and the nucleotide sequence is shown as SEQ ID NO. 2.
The invention provides a recombinant vector which comprises 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, converting to E.coli competent cells, culturing to obtain a GV219/His-P39-TAT plasmid, transfecting the plasmid to HEK293 cells, extracting protein from the cultured cells, and separating and purifying to obtain the recombinant protein.
Preferably, the method for constructing the GV219/HA-SHF vector comprises the following steps:
and (3) obtaining an HA-Shf gene sequence by using human mRNA as a template through amplification, and connecting the gene into a GV219 vector to obtain the GV219/HA-SHF vector.
Preferably, the modification comprises: introducing a His tag protein sequence at the N end of the P39 gene sequence obtained by amplification, and introducing a TAT sequence at the 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 mutant EGFRvIII protein thereof.
The invention also provides the application of the recombinant protein or the nucleotide sequence in preparing a medicament for inhibiting the activity of tumor cells.
The invention provides a recombinant protein P39 for specifically targeting and degrading EGFR and a mutant thereof, which is obtained by extracting 39 amino acid sequences of 304-342 combined by an anti-tumor factor Shf and the EGFR and the mutant thereof and respectively adding a His tag protein sequence and a TAT sequence to the N end and the C end. Experiments prove that the recombinant protein P39 can enter cells to be combined with EGFR/EGFRvIII and promote ubiquitination modification and degradation of the EGFR/EGFRvIII, so that the biological phenotype of tumors carried by high EGFR or EGFRvIII is inhibited, and the activity of tumor cells is inhibited.
Drawings
FIG. 1 shows that SHF binding to EGFR/EGFRvIII promotes ubiquitination degradation; wherein, A represents that SHF can bind to EGFR and promote ubiquitination degradation in cell experiments, and B represents that SHF can bind to EGFRvIII and promote ubiquitination degradation in cell experiments.
FIG. 2 shows the SHF-EGFR/EGFRvIII binding site analysis; wherein, A represents a schematic diagram of SHF-EGFR protein structure simulation, and B represents a schematic diagram of SHF key domain deletion mutant constructed according to the software analysis result.
FIG. 3 is SHF binding to EGFR/EGFRvIII through the 304-342aa domain; wherein, A represents that SHF binds to EGFRvIII through the 304-342aa domain, and B represents that SHF binds to EGFR through the 304-342aa domain.
FIG. 4 is a schematic diagram of the construction of GV219/His-P39-TAT plasmid.
FIG. 5 shows Westernblot to identify purified recombinant proteins.
FIG. 6 shows the cell localization of the recombinant protein P39 by immunofluorescence double staining.
FIG. 7 shows that P39 recombinant protein promotes ubiquitination degradation of EGFR/EGFRvIII; wherein, A represents that the P39 recombinant protein promotes the ubiquitination degradation of the EGFRvIII, and B represents that the P39 recombinant protein promotes the ubiquitination degradation of the EGFR.
FIG. 8 shows that P39 recombinant protein inhibits the formation of EGFRvIII overexpressing glioblastoma stem cell sphere; wherein the observation results are observed from the left to the right in turn on days 1, 3, 5, 7, 9 and 11.
FIG. 9 shows Westernblot to examine the effect of P39 recombinant protein on EGFR/EGFRvIII protein and downstream pathways.
FIG. 10 shows that P39 recombinant protein inhibits the growth of subcutaneous EGFRvIII overexpression glioblastoma in nude mice; where A represents the final tumor volume and B represents the tumor growth curve.
FIG. 11 shows that P39 significantly prolongs survival of intracranial EGFRvIII overexpressing glioblastoma nude mice (P < 0.01).
Detailed Description
The invention provides a recombinant protein for specifically targeting and degrading EGFR and a mutant thereof, wherein the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1.
In the invention, the recombinant protein is obtained by extracting 39 amino acid sequences of 304-342 combined by the tumor suppressor Shf, EGFR and mutants thereof through gene operation, respectively adding a His tag protein sequence and a TAT sequence at the N end and the C end, inserting a formed fusion gene into a multiple cloning site of a eukaryotic expression vector GV219, transfecting HEK293 cells, extracting total cell protein, and obtaining a final recombinant protein through an affinity purification small test and amplification, wherein the final recombinant protein is named as P39. In the invention, the amino acid sequence of the recombinant protein is as follows: mhhhhhPSSPLGEWTDPALPLENQVWYHGAISRTDAENLLRLCKEYGRKKRRQRRR (SEQ ID NO. 1); the total length of the recombinant protein is 57 amino acids, methionine (M) translated by initiation codon ATG is introduced into the N terminal, the underlined part is SHF protein 304-342 amino acid sequence, and the "hhhhh" is His label protein and is a screening and identifying label; "YGRKKRRQRRR" is a TAT sequence, a protein transduction signal peptide, and directs fusion proteins into cells.
The invention provides a nucleotide sequence for coding 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 a corresponding base sequence of the recombinant protein of the invention; the underlined sections are Xho I and Kpn I cleavage sites, respectively; the base "C" behind the Xho I restriction enzyme site and the base "GG" in front of the Kpn I restriction enzyme site are both endonuclease protection bases and are used for stabilizing the combination of the endonuclease and the DNA sequence to play a role; "GCCACC" is Kozak sequence, and can enhanceTranslation efficiency of eukaryotic genes.
The invention provides a recombinant vector which comprises 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, converting to E.coli competent cells, culturing to obtain a GV219/His-P39-TAT plasmid, transfecting the plasmid to HEK293 cells, extracting protein from the cultured cells, and separating and purifying to obtain the recombinant protein.
In the invention, the GV219/HA-SHF vector is preferably constructed by the following steps: using human mRNA as a template, adopting a primer F/R to amplify to obtain an HA-Shf gene, linearizing a GV219 vector by using XhoI and KpnI, then connecting the HA-Shf gene with the linearized GV219 vector, selecting a recombinant clone, and carrying out 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: TTAAACTTAAGCTTGGTACCTCTAAAGAGTCCGGATGGCCACAGGG (SEQ ID NO. 4).
In the present invention, the modification preferably includes: introducing a His tag protein sequence at the N end of the P39 gene sequence obtained by amplification, introducing a TAT sequence at the C end, and respectively introducing XhoI and KpnI endonuclease sites at the two ends.
The invention also provides application of the recombinant protein or the nucleotide sequence in promoting ubiquitination degradation of EGFR and mutant EGFRvIII protein thereof.
The invention also provides the application of the recombinant protein or the nucleotide sequence in preparing a medicament for inhibiting the activity of tumor cells.
In the invention, the GV219/HA-N-SHF vector is preferably constructed by the following steps: the commercial GV219 vector is linearized by using XhoI and KpnI, a primer F1/R1 (the primer sequence contains exchange pairing base and enzyme cutting site and contains a target gene 5' end part sequence for PCR fishing of a target gene) is used, an HA-N-SHF gene sequence (including a coding SHF protein 1-303aa gene sequence) obtained by amplification is connected with the linearized vector, and then recombinant clone is selected for enzyme cutting and sequencing identification to obtain the GV219/HA-N-SHF vector; the primer sequence during amplification is as follows: f1: ACGGGCCCTCCTAGACTCGAGCGCCACCATGTACCCTTATGATGTCCC (SEQ ID NO. 5), R1: TTAAACTTAAGCTTGGTACCCTCACTCCATGCTTAGGGGTTTGG (SEQ ID NO. 6).
In the present invention, the GV230/HA-C-SHF and GV230/HA-P39 vectors are preferably constructed by the following steps: the commercial GV230 vector is linearized by using XhoI and KpnI, primers (the primer sequence contains exchange pairing base and enzyme cutting site and contains a target gene 5' end part sequence used for PCR fishing target genes) are respectively used for amplifying the obtained HA-C-SHF gene sequence (including coded SHF protein 343-423aa gene sequence) and HA-P39 gene sequence (including coded SHF protein 304-342aa gene sequence), 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 vectors. In the invention, the sequence of a primer F2/R2 during HA-C-SHF amplification is as follows: f2: TACCGGACTCAGATCTCGAGCGCACCATGTACCCTTATGATGTCCCAG (SEQ ID NO. 7), R2: GATCCCGGGCCCGCGGTACCGTACGTAAGTGCCGGATGGCCACAGGGTAG (SEQ ID NO. 8); the sequence of the primer F3/R3 during HA-P39 amplification is as follows: f3: TACCGGACTCAGATCTCGAGCGCACCATGTACCCTTATGATGTCCC (SEQ ID NO. 9), R3: GATCCCGGGCCCGCGGTACCGTCTTTGCACAGCCGGAGCAGTTC (SEQ ID NO. 10).
In the invention, the GV141/Flag-EGFR and GV141/Flag-EGFRvIII vectors are preferably constructed by the following steps: the commercial GV141 vector is linearized by using XhoI and KpnI, the chemical synthesis of EGFR and EGFRvIII gene sequences is entrusted to the Shanghai Jikai gene chemical technology, xhoI and KpnI endonuclease sites are respectively introduced at the two ends of the sequences, the synthesized EGFR and EGFRvIII gene sequences are connected with the linearized GV141 vector, and then recombinant clones are selected for enzyme digestion and sequencing identification to obtain GV141/Flag-EGFR and GV141/Flag-EGFRvIII vectors.
In the inventionThe human glial blast cell U87EGFRvIII overexpression stable cell strain is constructed by the following steps: logarithmic phase growth of human glial cell U87 cells (purchased from the cell bank of the culture Collection of the Chinese academy of sciences, and expanded and stored in the laboratory) were collected at 2X 10 5 The density of each cell was plated in 6-well plates, after 24h EGFRvIII overexpression lentivirus (constructed by entrusted and Yuan Biotechnology (Shanghai) GmbH) was transfected, and after 72h transfection, selection was performed with 2. Mu.g/mL puromycin for 24h to obtain stable transfected cell lines.
In the invention, the GV219, GV141 and GV230 vectors are all purchased from Shanghai Jikai gene chemistry and technology GmbH, and the molecular cloning tool enzyme is purchased from Takara; the plasmid extraction kit and the glue recovery kit are purchased from Beijing kang, a century Biotechnology Co., ltd; ni-charged MagBeads was purchased from King-Ray Biotechnology Ltd; the D/F12, EGF, glutaMAX and B27 were all available from Thermo Fisher, USA, and bFGF was available from PeproTech, USA.
In the invention, the LB culture medium has the formula shown in molecular cloning. The methods mentioned in the present specification, plasmid extraction, PCR reaction, endonuclease digestion, DNA fragment recovery, ligation and transformation, are all conventional methods in the field of genetic engineering research, specifically referred to molecular cloning.
The present invention will be described in detail with reference to examples for better understanding the objects, technical solutions and advantages of the present invention, but they should not be construed as limiting the scope of the present invention.
In the following examples, unless otherwise specified, all methods are conventional.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Discovery of SHF-P39 recombinant protein
SHF binding to EGFR/EGFRvIII promotes ubiquitination and degradation thereof
GV141/Flag-EGFR, GV141/Flag-EGFRvIII and GV219/HA-SHF were grouped as shown in FIG. 1, HEK293 cells were transfected with the lipofectamine, proteins were collected after transfection for 24 hours, anti-DDDDK-tagmAb-Magnetic Beads were added, proteins on the Beads were collected overnight at 4 ℃ and expression of each protein was detected by Westernblot, and the results are shown in FIG. 1.
It can be seen that SHF can bind to EGFR and EGFRvIII and promote their ubiquitination degradation.
SHF-EGFR/EGFRvIII binding site analysis
For the SHF and EGFR protein structures, the 304-342aa sequence of SHF was extracted separately and domain deletion mutants were constructed (FIG. 2).
SHF binding to EGFR/EGFRvIII through 304-342aa Domain
GV141/Flag-EGFR, GV141/Flag-EGFRvIII, GV219/HA-N-P39, GV230/HA-C-P39 and GV230/HA-P39 were transfected into HEK293 cells by the lipofectamine as shown in FIG. 3, transfected for 24 hours, protein was collected, anti-DDK-tagmAb-Magnetic Beads were added, protein on the Beads was collected overnight at 4 ℃ and expression of each protein was detected by Western blot, as 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
Aiming at the structure and protein binding characteristics of the SHF protein (39 amino acids in total from 304 to 342aa are protein binding structural domains), a sequence expressing the structural domain is extracted, and a new recombinant gene with 39 amino acids in total from 304 to 342aa of the SHF is designed according to the existing experience and codon preference and named as P39.
A control group was also set: the control group recombinant protein adopts a 16-amino acid misordering control sequence (NLASPLPTEDPLEPG) at positions 304-320 of the SHF protein, and the rest connecting peptides are the same as those of the experimental group and are named as SC.
2.P39 recombinant Gene acquisition and expression vector construction
A GV219/HA-SHF vector constructed in a laboratory is used as a template, a high-fidelity PCR amplification kit of Takara company is utilized to amplify a P39 sequence containing 39 amino acids in total from 204 to 342 of SHF, his tag protein and TAT sequence are respectively introduced into the N end and the C end, and endonuclease (Xho I and Kpn I) sites are respectively introduced into the two ends (as shown in figure 4).
And (3) purifying a target fragment: the PCR product was digested with Xho I and Kpn I, and then the digested product was recovered with Kangji kit.
And (3) vector enzyme digestion recovery: the GV219 plasmid was digested with Xho I and Kpn I, and the plasmid was recovered from the kit of century using Kan after electrophoresis on 1% agarose gel.
Connecting: the PCR-digested product and the plasmid-digested product were ligated with T4DNA ligase from Takara at 4 ℃ overnight, transformed into E.coli Dh5. Alpha. Competent cells, plated on LB solid medium containing 50. Mu.g/ml kanamycin, and cultured at 37 ℃ overnight.
And (3) transformant identification: selecting a single colony, carrying out overnight culture on an LB liquid culture medium (containing 100 mu g/mL ampicillin), primarily screening positive clones by PCR, and extracting positive plasmids GV219/His-P39-TAT by a plasmid extraction kit; wherein, the forward primer of the PCR is as follows: CGCAAATGGGCGGTAGGCGTG (SEQ ID NO. 11), reverse primer: CGTCGCCGTCCAGCTCGACCAG (SEQ ID NO. 12).
And (3) plasmid sequencing identification: and (3) sending the positive plasmid to Shanghai workers for sequencing, and verifying the correctness of the gene clone by comparing sequencing results with software.
Example 3
Expression and purification of P39 fusion protein
Transfection and expression of HEK293 cells
The GV219/His-P39-TAT plasmid is transfected into HEK293 cells by a liposome transfection reagent (purchased from Life company), neomycin is added after 48 hours of transfection, and pressure screening is carried out to construct a stable transgenic cell strain expressing the P39 gene.
Separation and purification of P39 recombinant protein and Westernblot identification
After the HEK293 stable cell strain is expanded and cultured, the cells are collected and total cell protein is extracted, protein extract is filtered by a 0.45 mu m filter, recombinant protein with His label is purified by using Ni column, and related operation process is carried out according to the operation guide provided by the King of Murray company.
The specific process is as follows:
transferring the resin to the column, washing the nickel affinity chromatography column with double distilled water after complete precipitation,and prevent Ni in the next step 2+ Precipitating; by ddH 2 O washing to remove air in the matrix; binding buffer equilibrium of 10 x column volume; sampling; washing with a Bindingbuffer of 10 x column volume, and collecting effluent liquid; eluting with Elutionbuffer, and further performing ultrafiltration concentration to obtain the recombinant protein.
After SDS-PAGE electrophoresis and membrane transfer, the recombinant protein was identified by His monoclonal antibody, and the results are shown in FIG. 5.
As a result, the purified P39 recombinant protein was found to have a relative molecular mass of about 7kDa.
Example 4
The P39 eukaryotic recombinant protein can enter cells
Human glioblasts in logarithmic growth phase, U87 cells, were selected for digestion and counted at 1X 10 4 Inoculating the cells on cell slide, culturing for 24 hr, adding 500 μ M recombinant protein P39, and fixing with 4% PFA solution at room temperature for 15min for 6 hr; 0.3% TritonX100-PBS wash 3 times, 10min each time; sealing donkey serum at room temperature for 1 hr, incubating mouse anti-His at 4 deg.C in dark overnight, washing with PBS for 3 times, each for 10min; the donkey anti-mouse secondary antibody coupled with AlexaFlour594 was incubated for 1 hour at room temperature in the dark, and washed 3 times with PBS for 10min each time; use of DyLight which binds F-actin TM 488 Dye of Phalloidin for 20min; fluorocount (containing Hoechest10 ug/mL) was mounted and photographed by a fluorescence microscope, and the results are shown in FIG. 6.
The results showed that the recombinant protein P39 could smoothly enter the cells.
Example 5
P39 recombinant protein promotes ubiquitination degradation of EGFR and mutant protein thereof and activity of downstream pathway protein
GV141/Flag-EGFR, GV141/Flag-EGFRvIII and His-Ub were grouped as in FIG. 7, HEK293 cells were transfected by lipofectin, and the cells were treated with different concentrations of P39 recombinant protein, after 24 hours treatment 10. Mu. MMG132 was added for 4 hours to collect protein, anti-DDK-tagmAb-Magnetic Beads were added, after overnight at 4 ℃ protein on Magnetic Beads was collected and expression of each protein was detected by Westernblot, as shown in FIG. 7.
The result shows that the P39 recombinant protein promotes the ubiquitination degradation of the EGFR and the EGFRvIII mutant protein thereof.
Example 6
P39 recombinant protein inhibits the formation of glioblastoma stem cells cultured in vitro
Human glial blast cell U87EGFRvIII overexpressing stably transfected cells in logarithmic growth phase were selected, digested, counted, and cultured with neural stem cell culture medium (D/F12 + EGF + bFGF + B27+ GlutaMAX). Inoculating 1000 cells in each well by using a low-adsorption 96-well plate, simultaneously adding P39 recombinant protein and control protein SC, and arranging 3 multiple wells in each group; the capability of glioblastoma stem cell sphere formation was observed every other day during the culture and EVOS was used TM Photographs were taken by the FL Auto cell imaging system (40 ×) and recorded, with the results shown in fig. 8.
The results show that the P39 recombinant protein inhibits the formation of glioblastoma stem cells cultured in vitro.
When the stem cell balls are cultured for 11 days, the extracted protein is used for carrying out Westernblot to detect the expression levels of EGFR, EGFRvIII and downstream STAT3 proteins, and the result is shown in figure 9.
The results show that the P39 recombinant protein inhibits the expression of EGFR and EGFRvIII in glioblastoma stem cells cultured in vitro and inhibits the downstream STAT3 protein and the phosphorylation expression level thereof.
Example 7
P39 recombinant protein for inhibiting growth of glioblastoma in nude mice
1. According to 2X 10 of each balb/c nude mouse 6 U87EGFRvIII overexpression stable transfer cells are injected subcutaneously at the concentration of 100 mu l to establish the subcutaneous model of the nude mice. After 4 days, obvious tumor masses were formed subcutaneously, and were randomly grouped: (1) SC; (2) P39,6 per group, were administered with SC and P39 recombinant proteins (15. Mu.g/mouse, once on 2 days for 20 days), respectively, and the tumor size was measured every 2 days, and nude mice were sacrificed at the time of 22 days of feeding and the tumor size was weighed, and the results are shown in FIG. 10.
The results show that P39 significantly inhibits the growth of subcutaneous glioblastoma in nude mice.
2. According to 2X 10 of each balb/c nude mouse 6 100 mul concentration intracranial injection U87EGFRvIII overexpression stable cell to establish glioblastoma nude mouse intracranial model. Intracranial modelAfter 4 days of type establishment, random groupings were made: (1) SC; (2) P39, 10 per group, were given intraperitoneal injections of SC and P39 recombinant proteins (15 μ g/mouse, once in 2 days until death of nude mice), respectively, and the survival of nude mice was recorded, with the results shown in fig. 11.
The results show that P39 can significantly prolong the survival time of nude mice (P < 0.01).
The above description is only an embodiment of the present invention, and is not intended to limit the scope of the present invention, and all equivalent structures or equivalent processes performed by the present invention or directly or indirectly applied to other related technical fields are also included in the scope of the present invention.
Claims (10)
1. A recombinant protein for specifically targeting and degrading EGFR and a mutant thereof is characterized in that the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1.
2. A nucleotide sequence encoding the recombinant protein of claim 1, wherein the nucleotide sequence is set forth 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, comprising the steps of:
amplifying a P39 gene sequence by taking a GV219/HA-SHF vector as a template, modifying, converting to E.coli competent cells, culturing to obtain a GV219/His-P39-TAT plasmid, transfecting the plasmid to HEK293 cells, extracting protein from the cultured cells, and separating and purifying to obtain the recombinant protein.
6. The method according to claim 5, wherein the GV219/HA-SHF vector is constructed by the following steps:
and (3) obtaining an HA-Shf gene sequence by using human mRNA as a template for amplification, and connecting the gene into a GV219 vector to obtain the GV219/HA-SHF vector.
7. The method of claim 5, wherein the modifying comprises: introducing a His tag protein sequence at the N end of the P39 gene sequence obtained by amplification, and introducing a TAT sequence at the C end.
8. The method of 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 nucleotide sequence of claim 2 for promoting ubiquitination degradation of EGFR and its mutant EGFRvIII proteins.
10. Use of the recombinant protein according to claim 1 or the nucleotide sequence according to claim 2 for the preparation of a medicament for inhibiting the activity of tumor cells.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102219859A (en) * | 2011-05-20 | 2011-10-19 | 烟台荣昌生物工程有限公司 | Fusion protein for antagonizing angiogenesis inducible factor and application thereof |
| CN103755813A (en) * | 2014-01-07 | 2014-04-30 | 中国人民解放军第二军医大学 | Targeting anti-tumor fusion protein, and encoding gene and expression plasmid thereof |
| CN105367666A (en) * | 2015-12-14 | 2016-03-02 | 长春新悦然科技有限公司 | Recombinant targeting protein and preparation method and application thereof |
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| CN102219859A (en) * | 2011-05-20 | 2011-10-19 | 烟台荣昌生物工程有限公司 | Fusion protein for antagonizing angiogenesis inducible factor and application thereof |
| CN103755813A (en) * | 2014-01-07 | 2014-04-30 | 中国人民解放军第二军医大学 | Targeting anti-tumor fusion protein, and encoding gene and expression plasmid thereof |
| CN105367666A (en) * | 2015-12-14 | 2016-03-02 | 长春新悦然科技有限公司 | Recombinant targeting protein and preparation method and application thereof |
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| JINGJING WANG: "SHF Acts as a Novel Tumor Suppressor in Glioblastoma Multiforme by Disrupting STAT3 Dimerization", ADVANCED SCIENCE, vol. 9, no. 26, pages 2200169 * |
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