CN109134654B

CN109134654B - Single-chain antibody targeting EGFR dimerization interface and application thereof

Info

Publication number: CN109134654B
Application number: CN201810872308.3A
Authority: CN
Inventors: 赵林; 李黄金; 蔡金艳; 郭土敬
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2018-08-02
Filing date: 2018-08-02
Publication date: 2021-08-03
Anticipated expiration: 2038-08-02
Also published as: CN109134654A

Abstract

The invention discloses a single-chain antibody targeting an EGFR dimerization interface and application thereof. The fusion protein comprises amino acid sequences which are connected from 5 'to 3' in sequence and are shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain variable region protein, the connecting peptide and the amino acid sequence are shown as SEQ ID NO: 3 in sequence table 3. The gene of the single-chain antibody targeting the EGFR dimerization interface and the pichia pastoris engineering bacteria are constructed and optimized, the single-chain antibody is obtained through fermentation, separation and purification, namely the single-chain antibody targeting the EGFR dimerization interface is successfully and efficiently prepared through a gene engineering technology, the antibody has small relative molecular mass and good penetrability, the tumor inhibition effect is greatly improved in the treatment of solid tumors, the proliferation and growth of EGFR abnormal expression tumor cells can be effectively inhibited, a foundation is laid for the development of a subsequent EGFR novel inhibitor, and the application value is huge.

Description

Single-chain antibody targeting EGFR dimerization interface and application thereof

Technical Field

The invention relates to the technical field of treatment of EGFR (epidermal growth factor receptor) abnormally expressed tumors, in particular to a single-chain antibody targeting an EGFR dimerization interface and application thereof.

Background

Epidermal growth factor receptor (EGFR for short) is a member of the tyrosine kinase type i receptor family, the other members of which include HER2, HER3 and HE R4. The EGFR receptor is divided into three parts, an extracellular ligand binding region, a transmembrane immobilization region, and an intracellular tyrosine kinase region. After the specific ligand is combined with an EGFR receptor, the receptor is converted from an inactive monomer state into an activated dimerization state, the EGFR forms a dimer with itself or other members of the family, further, an intracellular tyrosine kinase is activated, a tyrosine residue of an intracellular segment of the EGFR receptor is autophosphorylated or cross-phosphorylated, a signal transduction protein or a signal protein containing an SH2 structural domain is recruited and tightly combined with the residue to form a multi-protein complex for signal transduction, and the downstream signal pathways including PI3K-Akt, MAPK-Erk and the like are activated to regulate the growth and proliferation of cells. Normal EGFR expression is closely related to basic physiological activities of cells, but when EGFR is abnormally expressed, it often promotes growth, migration, infiltration, and anti-apoptosis of tumor cells. EGFR has become an important target for tumor therapy. To date, two broad classes of monoclonal antibodies (MAbs) and small molecule tyrosinase inhibitors (TKIs) have been developed against EGFR. Monoclonal antibodies currently marketed in China include Cetuximab (Cetuximab), Panitumumab (Panitumumab), Nimotuzumab (Nimotuzumab), and the like; the small molecule inhibitors include Gefitinib (Gefitinib), Erlotinib (Erlotinib) and lapatinib (Lapat inib). The clinical effective rate of the two drugs is only 10-20%, and the analysis reasons include that the ligand has stronger competitive combination and the targeted area has high mutability. Therefore, there is a need to develop new targeted drugs to improve clinical efficacy.

Currently, there are two main constitutive forms of HER family receptor dimerization: homo-dimerization and hetero-dimerization. As shown in fig. 1: when no ligand binds to the receptor, the EGFR receptor exists as a monomer, and is biologically inactive; when a specific ligand binds to a receptor, its conformation changes, exposing the dimerization arm to form a dimer with another molecule of an EGFR or other receptor of the HER family, thereby initiating tyrosine phosphorylation of the intracellular region of the receptor, activating a second messenger of signal transduction, initiating a downstream series of biochemical reactions. Thus, "dimerization" is a common and essential activation step for EGFR family receptors, and the selection of a highly conserved region at the dimerization interface as a targeting site for an antibody may effectively prevent homo-or heterodimerization of the receptor by steric hindrance of the antibody, thereby inhibiting activation of EGFR signaling.

In the previous research, the inventor obtains a monoclonal antibody 5G9 targeting an EGFR dimerization interface, and in vitro and in vivo experiments prove that the monoclonal antibody can be specifically combined with the EGFR on the surface of tumor cells to inhibit the growth of tumors, and has good application prospect as a novel EGFR inhibitor.

The traditional monoclonal antibody is composed of 2 completely identical heavy chains and 2 completely identical light chains, has large relative molecular mass and poor penetrability, and has greatly influenced tumor inhibition effect in the treatment of solid tumors.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a new generation of anti-EGFR antibody, namely a single-chain antibody targeting an EGFR dimerization interface. The gene engineering technology is utilized to construct the single-chain antibody targeting the EGFR dimerization interface, realize the expression of the single-chain antibody in yeast, compare and analyze the functions of the single-chain antibody, study the binding activity and the anti-tumor activity of the single-chain antibody, and provide a new treatment method for the treatment of tumors with EGFR abnormal expression.

It is a first object of the present invention to provide a heavy chain variable region protein of a single chain antibody targeting the EGFR dimerization interface.

It is a second object of the present invention to provide a heavy chain variable region gene of a single chain antibody targeting EGFR dimerization interface.

It is a third object of the present invention to provide a light chain variable region protein of a single chain antibody targeting EGFR dimerization interface.

It is a fourth object of the present invention to provide a light chain variable region gene of a single chain antibody targeting EGFR dimerization interface.

It is a fifth object of the present invention to provide a fusion protein of a single chain antibody targeting the EGFR dimerization interface.

The sixth object of the present invention is to provide a fusion gene of a single-chain antibody targeting EGFR dimerization interface.

The seventh object of the present invention is to provide the use of the above-mentioned heavy chain variable region protein, the above-mentioned heavy chain variable region gene, the above-mentioned light chain variable region protein, the above-mentioned light chain variable region gene, the above-mentioned fusion protein and/or the above-mentioned fusion gene in the preparation of a medicament for treating tumors in which EGFR is abnormally expressed.

It is a seventh object of the present invention to provide a medicament.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the gene composition of the single-chain antibody targeting the EGFR dimerization interface is composed of a heavy chain variable region and a light chain variable region of a monoclonal antibody through a connecting peptide (Linker), a His label is added at the downstream end of the gene for gene construction and protein purification, and restriction endonuclease enzyme cutting sites are added at the upstream end and the downstream end. In order to realize the efficient preparation of the single-chain antibody, a constitutive expression vector pGAPZ alpha A plasmid is selected to assist the target gene to be introduced into a yeast genome, and therefore, both ends of the gene are also connected with enzyme cutting sites.

The invention therefore claims the following:

a heavy chain variable region protein of a single-chain antibody targeting an EGFR dimerization interface, wherein the amino acid sequence of the protein is shown as SEQ ID NO: 1 is shown.

A heavy chain variable region gene of a single-chain antibody targeting EGFR dimerization interface, which can encode the above heavy chain variable region protein; preferably, the nucleotide sequence is as shown in SEQ ID NO: 2, respectively.

A light chain variable region protein of a single-chain antibody targeting an EGFR dimerization interface, wherein the amino acid sequence of the protein is shown as SEQ ID NO: 3, respectively.

A light chain variable region gene of a single-chain antibody targeting an EGFR dimerization interface, which can encode the heavy chain variable region protein; preferably, the nucleotide sequence is as shown in SEQ ID NO: 4, respectively.

A fusion protein targeting EGFR dimerization interface single chain antibody, which comprises the amino acid sequence of the above heavy chain variable region protein, the above connecting peptide and the above light chain variable region protein which are connected in sequence from 5 'to 3'.

Preferably, the amino acid sequence of the connecting peptide is as shown in SEQ ID NO: 5 is shown in the specification; that is, the amino acid sequence of the fusion protein SEQ ID NO: shown at 7.

Preferably, the fusion protein is further connected with a signal peptide at the upstream of the 5' end.

Preferably, the amino acid sequence of His tag is connected with the downstream of 3' end of the fusion gene.

More preferably, the amino acid sequence of the signal peptide is as set forth in SEQ ID NO: 9, so that the fusion protein can be expressed in a pichia pastoris expression system.

More preferably, the amino acid sequence of the His-tag is 6 His, i.e. the amino acid sequence is: HHHHHHHH.

Most preferably, the amino acid sequence of the fusion protein SEQ ID NO: shown at 11.

The invention also provides a fusion gene of a single-chain antibody targeting an EGFR dimerization interface, which encodes the fusion protein, namely the fusion gene comprises the nucleotide sequence of the heavy chain variable region protein, the connecting peptide and the light chain variable region protein which are sequentially connected from 5 'to 3', and belongs to the protection scope of the invention.

Preferably, the nucleotide sequence of the linker peptide is as set forth in SEQ ID NO: 6 is shown in the specification; that is, the nucleotide sequence of the fusion gene is SEQ ID NO: shown in fig. 8.

Preferably, the fusion gene is further linked to a signal peptide upstream of the 5' end.

Preferably, the nucleotide sequence of His tag is connected with the downstream of 3' end of the fusion gene.

More preferably, the nucleotide sequence of the signal peptide is as set forth in SEQ ID NO: 10 is shown in the figure;

more preferably, the nucleotide sequence of the His-tag is 6 His, i.e. the nucleotide sequence is:

CACCACCACCACCACCAC。

preferably, the 3' -most downstream of the fusion gene further comprises a stop codon.

Most preferably, the nucleotide sequence of the fusion gene is SEQ ID NO: shown at 12.

A recombinant vector is a plasmid containing the fusion gene and a recombinant engineering bacterium is a host bacterium containing the recombinant vector, which belong to the protection scope of the invention.

Preferably, the plasmid is a pGAPZ α a plasmid.

Preferably, the host bacterium is escherichia coli or/and yeast.

More preferably, the host bacterium is e.coli DH5 a escherichia coli or/and X33 pichia pastoris.

The use of the above-mentioned heavy chain variable region protein, the above-mentioned heavy chain variable region gene, the above-mentioned light chain variable region protein, the above-mentioned light chain variable region gene, the above-mentioned fusion protein and/or the above-mentioned fusion gene in the preparation of a medicament for treating tumors in which EGFR is abnormally expressed.

The recombinant vector and/or the recombinant engineering bacteria are/is within the protection scope of the invention.

Preferably, the EGFR aberrantly expressing tumor is an EGFR aberrantly expressing lung cancer.

The medicament contains the fusion protein and also belongs to the protection scope of the invention.

The agent is capable of treating tumors with aberrant expression of EGFR.

Compared with the prior art, the invention has the following beneficial effects:

the invention creates a novel small molecule antibody, namely a single-chain antibody EGFR-scFv, by artificially modifying the gene of a novel monoclonal antibody targeting an EGFR dimerization interface. The invention has the following beneficial effects: (1) the molecular weight is about 28.5kDa, which is only 1/6 of the original intact antibody, the permeability is good, the antibody is easier to enter the interior of solid tumor than the original intact antibody, and the injection dosage is smaller; (2) the immunogenicity is low, and the anti-foreign protein reaction is not easy to generate when the vaccine is used for a human body; (3) the production efficiency is high, and the method is simple, easy and efficient from the fermentation of the engineering bacteria to the later purification; (4) constitutive expression, methanol induction is not needed, the nutrition requirement is simple, and the production environment is friendly; (5) the targeting property of the dimerization interface of the EGFR and the activity of inhibiting the growth of EGFR abnormal expression tumor cells are consistent with those of the original complete antibody, and the EGFR abnormal expression tumor targeting antibody has great potential for clinical treatment of EGFR abnormal expression tumors.

The gene of the single-chain antibody targeting the EGFR dimerization interface and the pichia pastoris engineering bacteria are constructed and optimized, the single-chain antibody is obtained through fermentation, separation and purification, namely the single-chain antibody targeting the EGFR dimerization interface is successfully and efficiently prepared through a gene engineering technology, the antibody has small relative molecular weight and good penetrability, the tumor inhibition effect is greatly improved in the treatment of solid tumors, the proliferation and growth of EGFR abnormal expression tumor cells can be effectively inhibited, a foundation is laid for the development of a subsequent EGFR novel inhibitor, and the application value is very high.

Drawings

FIG. 1 is a graph of the difference between activation of dimerization and inhibition by antibodies after opening of the EGFR dimerization interface.

FIG. 2 is a diagram of a first PCR amplification electrophoresis; the 1-10 holes are complex holes, the brighter band located at a higher point of 750bp is the target band, and the rest are the impurity bands.

FIG. 3 is a photograph of an adjusted PCR amplification electrophoresis; the number 1-6 holes are multiple holes, the target band is located near 750bp, the rest are miscellaneous bands, and the reduction of the miscellaneous bands is seen.

FIG. 4 is an electrophoretogram of pGAPZ α A plasmid extraction; wherein 1 and 2 are repeated experimental groups.

Fig. 5 is a plate image of e.coli DH5 α transformed and plated, from which larger colony strains were selected for inoculation and simultaneously PCR-identified.

FIG. 6 is a PCR identification electropherogram of recombinants; 1-16 are lanes of PCR amplification of different colonies.

FIG. 7 is an electrophoretogram of recombinant plasmid extracted by the miniprep kit; wherein lanes 1-8 are different repeat groups extracted from the same Erlenmeyer flask.

FIG. 8 is a recombinant plasmid restriction enzyme electrophoresis detection diagram; lane 1 is the recombinant plasmid not digested; lane 2 shows the digested recombinant plasmid.

FIG. 9 is a photograph of plates coated with electrotransformed X-33 yeast.

FIG. 10 is an SDS-PAGE electrophoresis of recombinant yeast expression identification; wherein the "empty" is X-33 empty host, the No. 1-11 is selected X-33-EGFR-scFv recombinant yeast, and the bright band around 29.0kDa is target protein EGFR-scFv.

FIG. 11 is a photograph of an electrophoretic test of the supernatant and the undissolved precipitate after concentration; 1 is the electrophoresis result of the sample which is centrifuged at 10000rpm and then concentrated by 10 times; 2 is the result of electrophoresis after resuspension of the undissolved precipitate with 1ml of water.

FIG. 12 shows the results of electrophoretic detection of EGFR-scFv protein purification; 1 is a front sample of a nickel column; 2 is a penetration sample of the nickel column; 3 is an eluted sample of the nickel column; 4 is the sample after Sephadex-G25 molecular sieve chromatography displacement buffer.

FIG. 13 is a scatter plot of a BCA protein concentration assay standard curve.

FIG. 14 is a bar graph of the inhibition rate of EGFR-scFv on H292 human lung cancer cells.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Main apparatus and equipment:

PCR instrument, horizontal electrophoresis tank, vertical electrophoresis tank, electrophoresis instrument, super clean bench, bench centrifuge, high speed freezing centrifuge, electronic balance, constant temperature shaking table, constant temperature incubator, uniform speed stirrer, high performance liquid chromatography system, G25 molecular sieve, nickel column, refrigerator, -80 deg.C super low temperature refrigerator, liquid nitrogen tank, digital display constant temperature water bath, cell incubator, enzyme labeling instrument, inverted microscope.

Main reagents and materials:

2 XT 5Super PCR Mix, endonuclease (Xho I, Xba I, BlnI), T4 ligase, E.coli DH5 alpha containing pGAPZ alpha A plasmid, E.coli DH5 alpha empty host, X-33 Pichia pastoris empty host, H292 human lung cancer cell, small extract kit, gel recovery kit, PBS powder, bleomycin, BCA kit, 1640 culture medium, Fetal Bovine Serum (FBS), 0.25% Trypsin-EDTA Solution (0.02% EDTA), CCK-8.

The reagent formula comprises:

(1) LB Medium

Peptone (Tritone) 1g

Yeast extract (Yeast) 0.5g

NaCl 1g

Add 100ml ddH₂Dissolving O, sterilizing under high pressure, and storing at room temperature.

(2) YPD medium

Peptone (Tritone) 2g

Yeast extract (Yeast) 1g

90ml of ddH were added₂Dissolving in O, autoclaving, adding 10ml 20% glucose solution, mixing, and storing at room temperature for one week.

(3) Ampicillin

Ampicillin dry powder 1g

By ddH₂Dissolving O, diluting to 10ml, filtering with 0.22 μm filter membrane for sterilization, and storing at-20 deg.C.

(4)1M Tris-HCl (pH 7.5 and pH8.3)

Tris 12.12g

Addition of ddH₂Dissolving O80 ml, adjusting pH to 7.5 and 8.3 with concentrated hydrochloric acid, and adding ddH₂And (4) metering the volume of O to 100ml, and storing at normal temperature.

(5) Phenol-chloroform-isoamyl alcohol

Phenol 250. mu.l

Chloroform 240. mu.l

Isoamyl alcohol 10. mu.l

Mixing at a ratio of 25:24: 1.

(6)3M NaAc(pH 5.2)

40.82g of sodium acetate trihydrate

80ml of ddH were added₂Dissolving O, adjusting pH to 5.2 with glacial acetic acid, diluting to 100ml, and storing at room temperature.

(7) Yeast competent treatment (10mM lithium acetate, 10mM DTT, 0.6M sorbitol, 10mM Tri-HCl, pH7.5)

By ddH₂O to 20ml, filtering with 0.22 μm filter membrane for sterilization, packaging into 1.5ml EP tube, and storing at-20 deg.C.

(8) Sorbitol suspension (0.6M sorbitol, 10mM Tri-HCl, pH7.5)

1M sorbitol 600. mu.l

1M Tri-HCl(pH7.5) 20μl

By ddH₂O is added to the solution to be 1ml, and the solution is sterilized and filtered by a 0.22 mu m filter membrane and stored at 4 ℃.

(9) Saturated ammonium sulfate

Ammonium sulfate 76.7g

Dissolving in 100ml, and storing at room temperature.

(10)Stripping Buffer(20mM Tris-HCl，50mM EDTA·2Na，0.5mM NaCl，pH 8.3)

1M Tris-HCl(pH8.3) 2ml

EDTA·2Na 1.862g

2.922g of sodium chloride

By ddH₂Dissolving O, diluting to 100ml, filtering with 0.45 μm filter membrane, and storing at room temperature.

(11)Charge Buffer(50mM NiSO₄)

1.314g of nickel sulfate hexahydrate

(12) Nickel column equilibration buffer (20mM Tris-HCl, 0.5M NaCl, 20mM imidazole, pH8.3)

1M Tris-HCl(pH8.3) 2ml

2.922g of sodium chloride (NaCl)

Imidazole 0.120g

Addition of ddH₂Dissolving O, diluting to 100ml, filtering with 0.45 μm filter membrane, and storing at room temperature.

(13) Nickel column elution buffer I (20mM Tris-HCl, 0.5M NaCl, 250mM imidazole, pH8.3)

1M Tris-HCl(pH8.3) 2ml

2.922g of sodium chloride (NaCl)

Imidazole 1.702g

(14) Nickel column elution buffer II (20mM Tris-HCl, 0.5M NaCl, 500mM imidazole, pH8.3)

1M Tris-HCl(pH8.3) 2ml

2.922g of sodium chloride (NaCl)

Imidazole 3.404g

Example 1 Gene and codon optimization for construction of Single chain antibody

First, obtaining of EGFR dimerization interface targeting monoclonal antibody light chain gene and heavy chain gene

1. Experimental procedures

Previous studies by the inventors have resulted in the monoclonal antibody anti-mer 5G9 targeting the EGFR dimerization interface and preserved its hybridoma cells.

Hybridoma cells targeting the EGFR dimerization interface were cultured, collected, and RNA was extracted by the Trizol method. After reverse transcription, the heavy chain variable region gene and the light chain variable region gene of the target EGFR dimerization interface monoclonal antibody 5G9 are respectively amplified by using universal degenerate primers, and are constructed on a cloning vector pMD18-T vector through AT cloning. Sending to a sequencing company for sequencing analysis. The sequence was further optimized for pichia codon bias.

2. Results of the experiment

The heavy chain variable region gene of the EGFR dimerization interface targeted monoclonal antibody Antidimer 5G9 is optimized through pichia pastoris codon preference optimization to obtain the optimized heavy chain variable region gene of the EGFR dimerization interface targeted single-chain antibody, and the nucleotide sequence of the heavy chain variable region gene is shown as SEQ ID NO: 2, the amino acid sequence of the encoded protein is shown as SEQ ID NO: 1 is shown.

The light chain variable region gene of the EGFR dimerization interface targeted monoclonal antibody anti-dimer 5G9 is optimized through pichia pastoris codon preference optimization to obtain the optimized light chain variable region gene of the EGFR dimerization interface targeted single-chain antibody, and the nucleotide sequence of the light chain variable region gene is shown as SEQ ID NO: 4, the amino acid sequence of the encoded protein is shown as SEQ ID NO: 3, respectively.

II, obtaining of EGFR dimerization interface targeted monoclonal antibody single-chain antibody gene

1. Experimental procedures

The heavy chain variable region gene and the light chain variable region gene obtained in the previous step are connected via a linker peptide (G)₄S)₃Ligation, wherein the peptide (G) is ligated₄S)₃The nucleotide sequence of (a) is shown as SEQ ID NO: 6, the amino acid sequence of the encoded protein is shown as SEQ ID NO: 5, obtaining the fusion gene of the coding single-chain antibody EGFR-scFv, wherein the nucleotide sequence of the fusion gene is shown as SEQ ID NO: 8, the amino acid sequence of the encoded protein is shown as SEQ ID NO: shown at 7.

Further, in order to realize secretory expression and subsequent affinity purification in pichia pastoris, a signal peptide (the nucleotide sequence is shown as SEQ ID NO: 10, and the amino acid sequence of the encoded protein is shown as SEQ ID NO: 9) is connected to the upstream of the 5 'end of the obtained fusion gene, a His tag and a stop codon are connected to the downstream of the 3' end (the nucleotide sequence of the His tag is 6 His, namely the nucleotide sequence is CACCACCACCACCACCAC, the amino acid sequence of the encoded protein is HHHHHHHH, and the nucleotide sequence of the stop codon is TGA), so that the final fusion gene which can realize secretory expression in a yeast expression system and can realize affinity purification is obtained, and the nucleotide sequence of the fusion gene is shown as SEQ ID NO: 12, the amino acid sequence of the encoded protein is shown as SEQ ID NO: shown at 11.

And (3) sending the designed sequence to a company for synthesis, connecting the sequence to a cloning vector, and transforming escherichia coli to obtain a positive strain.

2. Results of the experiment

And obtaining the Escherichia coli containing the fusion gene of the single-chain antibody EGFR-scFv which is correctly sequenced for subsequent experiments.

Example 2 construction of recombinant expression vector of fusion Gene of Single-chain antibody EGFR-scFv

First, experiment operation

1. PCR amplification of Single chain antibodies

Escherichia coli containing a fusion gene of a single-chain antibody EGFR-scFv (EGFR-scFv gene) was inoculated into 2ml of LB medium (containing 0.1% AMP), and cultured at 37 ℃ and 180rpm for 14 to 16 hours to amplify the bacteria. The amplified Escherichia coli containing the single-chain antibody gene is used for extracting plasmid DNA in the Escherichia coli, and the plasmid DNA is used as a template chain for PCR amplification to carry out PCR amplification.

The upstream primers for PCR amplification were: 5' -GACACTCGAGAAGAGAGAGGCTGAGGC-3', the downstream primer is: 5' -ACTCTCTAGATCAGTGGTGGTGGTGG-3'. Increase 5' upstream of EGFR-scFv GeneXhoI3' downstream of the cleavage siteXbaIThe enzyme cutting site is cut, and protective bases are added at two ends.

Preparing a PCR amplification system:

PCR conditions were as follows:

94℃，5min；

∞。

and after electrophoresis, detecting the PCR product by electrophoresis, and purifying by using an enzyme digestion recovery kit.

2. Construction of recombinant plasmid and transformation of E.coli DH5 alpha

And amplifying the yeast containing pGAPZ alpha A plasmids, extracting the pGAPZ alpha A plasmids by using a small extraction kit, and carrying out electrophoresis detection. According to the design, the target gene and pGAPZ alpha A are subjected to double enzyme digestion by using Xho I and Xba I, and then are connected by using T4DNA ligase to obtain the recombinant plasmid

Preparing a double enzyme digestion system:

carrying out water bath for 2h at 37 ℃ after enzyme digestion, carrying out gel electrophoresis on the product after the enzyme digestion is finished, and then recycling the product by using a gel recycling kit.

Preparing a connecting system:

after the preparation, the mixture is subjected to instantaneous centrifugation and placed in a water bath kettle at 16 ℃ for constant temperature connection overnight. The next day, 5. mu.l of the ligation product was added to DH 5. alpha. competent cells, ice-washed for 30min, quickly placed in a 42 ℃ water bath for heat shock for 1.5 min, quickly placed in ice, ice-washed for 2min, added with 1ml of LB medium, and left to stand at 37 ℃ for 1 h. The EP tube was removed, centrifuged at 10000rpm for 2min, 800. mu.l of the supernatant was discarded, and the remaining 200. mu.l was resuspended and added to LB plate containing 25. mu.g/ml bleomycin and incubated overnight at 37 ℃. After the bacteria grow out, PCR identification is carried out, the bacteria colony is picked by toothpick, washed in LB culture medium containing 50 mug/ml bleomycin and washed into a PCR tube of a prepared system at the same time. And (3) putting the inoculated test tube into a shaker at 37 ℃ and 180rpm for culturing for 14-16 h while carrying out PCR.

Preparing a PCR identification system:

PCR conditions were as follows:

94℃,5min；

∞。

the retained recombinant e.coli DH5 α was selected by PCR identification.

Second, experimental results

1. Single chain antibody PCR amplification

Carrying out PCR amplification on the target gene by using the designed primer to obtain a large number of target bands (as shown in figure 2), wherein the target bands contain different sizes of hybrid bands and are not few; the amount of the template in a PCR system is reduced, the annealing temperature of 1-2 ℃ is slightly increased (53 ℃ for the first time), and the number of the impurity bands is reduced (shown in figure 3); the question is that the specificity of the primer is not strong enough, and there are many non-specific binding sites in the gene of the Escherichia coli, so that there are many bands with different sizes. The solution is as follows: (1) the amount of the template is reduced, the number of non-specific binding sites is correspondingly reduced, and the primers are combined with the two ends of the target gene as far as possible; (2) slightly raising the annealing temperature reduces the combination of the primer and the template and greatly reduces the non-specific combination, so that the PCR amplification can obtain purer target genes.

2. Coli DH 5. alpha. transformation

pGAPZ alpha A plasmid is extracted from DH5 alpha containing pGAPZ alpha A purchased, and pGAPZ alpha A plasmid is extracted from 4ml of bacterial liquid by using a miniprep kit and is detected by electrophoresis (as shown in figure 4). Since the plasmid size was 3147bp, it was not in the DNAmarker range of DL2000, above 2000 pb. After transformation, the plates were plated with bleomycin and viewed again (see FIG. 5). Colonies were picked for PCR identification (see FIG. 6), and most of the colonies picked contained the desired gene, but only 2, 4, 5, 9, 10, 14, and 16 were propagated in LB medium containing bleomycin, so strains with relatively good copy numbers, i.e., Nos. 4 and 10, were selected for sequencing, and no gene mutation was observed. Both strains were propagated in large numbers and given a stock protection.

The reason why the band of interest identified by PCR but not grown in liquid medium is suspected to be false positive. The recombinant plasmid does not enter the cell completely, and only remains in the periplasm of the Escherichia coli, and is lost in a liquid medium.

Example 3 extraction of recombinant plasmid and Yeast transformation

First, experiment operation

1. Extraction of recombinant plasmid

100ml of the recombinants were shaken and pGAPZ. alpha.A-EGFR-scFv plasmid (about 20. mu.g) was extracted with a miniprep kit and the recombinant plasmid was linearized with BlnI.

Enzyme digestion system:

carrying out enzyme digestion for 2h in water bath at 37 ℃, sampling and carrying out gel electrophoresis detection. The obtained enzyme digestion product is desalted by phenol-chloroform. The method comprises the following steps:

(1) by ddH₂And O, complementing 500 mu l of the enzyme-cut plasmid, adding 500 mu l of phenol-chloroform-isoamylol, and turning upside down and mixing uniformly. The mixture was centrifuged at 10000rpm at 4 ℃ for 5min, and the supernatant was carefully aspirated and transferred to a 2ml EP tube.

(2) The ratio of sample to NaAc (pH5.2) is 10: 1, adding 3M NaAc into the sample, adding 2 times of anhydrous ethanol precooled at-20 ℃, uniformly blowing, and precipitating at-20 ℃ for 1 h. After the precipitation, the mixture was centrifuged at 4 ℃ and 10000rpm for 15min, and the supernatant was discarded.

(3) Adding 700 μ l of normal temperature 75% ethanol, resuspending, centrifuging at 4 deg.C and 10000rpm for 15min, and discarding the supernatant. Repeat 2 times.

(4) After the ethanol in the precipitate was dried in a clean bench, it was dissolved in 10. mu. lddH2O, and the sample was diluted for electrophoresis.

2. Electrotransformation of yeast

Preparation of X33 competent cells:

(1) the Pichia pastoris empty host X33 was activated at 30 ℃ and 180rpm and inoculated into 50ml YPD medium and shaken to mid-log growth (about 8h with activated bacteria).

(2) The bacterial liquid is evenly distributed in two sterile round-bottom centrifuge tubes, centrifuged for 2min at 4 ℃ and 10000rpm, and the supernatant is discarded.

(3) 20ml of precooled ddH2O was added to the cells, and the cells were resuspended, centrifuged at 10000rpm for 2min at 4 ℃ and the supernatant was discarded.

(4) Adding 20ml of pre-cooled treatment fluid into the thallus, re-suspending the cells, standing at 30 ℃ for 30min, centrifuging at 4 ℃ and 10000rpm for 2min, and discarding the supernatant.

(5) 10ml of precooled sorbitol is added into the thalli, cells are resuspended, the mixture is centrifuged at 10000rpm for 2min at 4 ℃, and the supernatant is discarded. Repeat 2 times.

(6) 1ml of precooled sorbitol is added into the thalli to resuspend the thalli, the thalli is transferred into precooled 1.5ml of EP, the thalli is centrifuged for 2min at the temperature of 4 ℃ and the rotating speed of 10000rpm, and the supernatant is discarded. The pellet was resuspended with 100. mu.l of pre-cooled sorbitol.

And transferring the prepared competent cells into the purified recombinant plasmid, uniformly mixing, transferring into an electrode cup, and carrying out ice bath for 5 min. And arranging an electrotransfer instrument, wiping the electrode cup dry and putting the electrode cup into the electrotransfer instrument, and starting to electrically shock and transform the cells. After the shock was completed, 1ml of sorbitol solution was quickly added and the lid was quickly closed. And transferring the electrode cup to a super clean bench, uniformly blowing the thalli, transferring the thalli to a 1.5ml EP tube, and standing for 1-2 h at 30 ℃. 200. mu.l of the bacterial suspension was applied to YPD solid culture plates containing 500. mu.g/ml bleomycin, and the plates were cultured in a 30 ℃ incubator for 3 days.

After the bacterial colonies are separated out, the bacterial colonies are picked and respectively preserved.

3. Expression characterization of recombinant Yeast

The recombinant yeast and the empty host were activated, inoculated into 20ml YPD medium containing 0.1% ampicillin, and fermented at 30 ℃ and 180rpm for 72 hours. After the fermentation was completed, the mixture was centrifuged at 4 ℃ and 10000rpm, the supernatant was taken, saturated ammonium sulfate was added in the same volume and shaken well, and the mixture was precipitated overnight in a refrigerator at 4 ℃. The next day, centrifugation was carried out at 10000rpm for 10min at 4 ℃ and the supernatant was discarded, and 1ml dH2O was added to dissolve the precipitate (20-fold concentration). Sampling, performing SDS-PAGE electrophoresis detection, comparing with an empty host, selecting a strain which can express and has relatively high expression quantity, performing mass propagation and preserving the strain.

Second, experimental results

Yeast electroporation requires a large amount of recombinant plasmid, so the plasmid is extracted by successive repetitions using a miniprep kit until 20. mu.g is reached, and electrophoretically detected (see FIG. 7). Entering the enzyme cutting stage, adding BlnI, performing electrophoresis on the enzyme cutting product (as shown in figure 8), and compared with the plasmid which is not cut, moving the position of the strip upwards because the chain DNA is blocked in the micropores of the gel relative to the circular DNA and the moving speed is slower in the same electric field, so that the strip is moved upwards. After enzyme digestion, phenol-chloroform purification is carried out to remove salts in the environment, and in the electric conversion process, the salts can reduce electrophoresis between electrodes, so that current is directly conducted through liquid, and the breakdown efficiency is reduced.

After the yeast was electrically transformed, the cells were spread on YPD plates containing bleomycin and ampicillin, and after three days, colonies were observed to grow (see FIG. 9), and since the number of colonies was small, the largest possible colonies were picked up and the number of colonies was large, and inoculated into a YPD liquid medium containing bleomycin and ampicillin, and cultured, and expression fermentation was performed the next day. The bacterial suspension was concentrated and subjected to SDS-PAGE (FIG. 10), and it was confirmed that the

strains

3, 5 and 11 had a high expression level of the target protein, and therefore, these three strains were selected, stored, and then subjected to expression fermentation.

EXAMPLE 4 expression fermentation and purification of Single chain antibody

The pGAPZ alpha A plasmid is a high copy Pichia pastoris expression vector and is a common vector for expressing foreign proteins by yeast. This is also a shuttle plasmid, which can be retained in different vectors, so that the target gene can be transformed into the E.coli DH5 alpha cloning vector after being connected to the multiple cloning sites thereof, and a large number of recombinant plasmids are cloned and transformed into yeast cells by electrotransformation. The enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is stably expressed at high levels in many biological cells, including Pichia pastoris. The GAP promoter (pGAP) expresses slightly more recombinant protein than the AOX1 promoter. pGAPZ alpha A (3.1KB) vector stably expresses recombinant proteins in Pichia using the GAP promoter. The recombinant protein is expressed as a fusion protein with C-terminal myc epitope and C-terminal His tag

First, experiment operation

1. Yeast fermentation

2ml of YPD medium containing 0.1% ampicillin was placed in a test tube, and a recombinant yeast glycerol seed was inoculated into the test tube at an inoculum size of 1%. The tube was placed in a constant temperature shaker and incubated at 30 ℃ for 16h at 180 rpm. After the two stages of activation, the cells were inoculated in a 1L shake flask containing 300ml of YPD medium containing 0.1% ampicillin at an inoculum size of 1%, placed in a constant temperature shaker and cultured at 30 ℃ for 72 hours at 180 rpm.

2. Concentrating the fermentation broth

Pouring the fermentation liquor into a centrifuge cup, and centrifuging for 10min at 4000rpm and 4 ℃ by using a vertical refrigerated centrifuge. And (3) taking the supernatant, measuring the volume of the supernatant, calculating the mass of the ammonium sulfate solid according to the semi-saturated ammonium sulfate concentration of 383.5g/L, and grinding the ammonium sulfate solid into powder. Placing the supernatant in a large beaker, continuously adding a small amount of ammonium sulfate solid powder under the stirring of a uniform speed stirrer to precipitate the protein, and finally placing the beaker in a refrigerator at 4 ℃ to precipitate overnight. The next day, the liquid was poured into a centrifuge cup, centrifuged at 4000rpm at 4 ℃ for 30min using a vertical refrigerated centrifuge, and the supernatant was discarded. The precipitate was dissolved in the equilibration buffer of a nickel column. Centrifuging at 10000rpm and 4 deg.C for 10min in a high-speed refrigerated centrifuge, and collecting supernatant.

3. Column chromatography purification of the body

Activating the nickel column, loading the nickel column with the balance buffer solution to flush the base line, and zeroing the equipment. And (3) flushing the baseline with a protein concentrate by pass and then with an equilibrium buffer solution, starting to load the sample, and flushing the baseline with the equilibrium buffer solution again after the sample is loaded. And (4) eluting by using the nickel column elution buffer solution I, collecting the peak sample, continuously replacing the nickel column equilibrium buffer solution II, and collecting the peak sample. After the nickel column was purified, the eluted I sample was passed through a G25 molecular sieve to replace the protein environment with 10mM PBS buffer.

4. BCA protein concentration assay

The BCA working solution a and the working solution B were mixed at a ratio of 50:1 to prepare a working solution. Standard line preparation: 1mg/ml BSA solution was placed in wells of a microplate reader at 10. mu.l, 9. mu.l, 8. mu.l, 7. mu.l, 6. mu.l, 4. mu.l, 2. mu.l and 0. mu.l, respectively, and each well was filled with 10. mu.l. The sample to be tested was added in 5. mu.l, 2. mu.l and 1. mu.l at 2-fold, 5-fold and 10-fold dilutions, respectively, and filled with 10. mu.l. After preparation, 200. mu.l of working solution was added to each well. Incubating in an incubator at 37 ℃ for 30min, detecting the light absorption value in the wavelength of 590nm, drawing a standard curve, displaying a curve equation, and calculating the concentration of the protein solution to be detected.

Second, experimental results

The expression of the target protein is expressed by a yeast expression system, and the fermentation product is finally left in the fermentation liquid. Experiment to avoid contamination, shake flask fermentations were then selected and 0.1% ampicillin was added to the medium. After fermentation, the fermentation broth is concentrated by using half-saturated ammonium sulfate precipitation, and after centrifugation, the precipitate is dissolved by using a nickel column equilibrium buffer solution, but the centrifuged supernatant is still turbid, and the precipitate is not dissolved when protein is redissolved after centrifugation. Sampling for electrophoresis (see FIG. 11), centrifuging the supernatant (10-fold concentrated relative to the fermentation broth) to obtain a small amount of the desired protein and larger foreign proteins, because the centrifugal force is insufficient or the time is insufficient, resulting in incomplete precipitation of some macromolecular proteins; however, undissolved precipitates (only 1ml of water is used for resuspension, and the concentration is 1000 times relative to the fermentation broth), which are also found to have bands of target proteins, and some impure proteins, are suspected to be caused by that when ammonium sulfate powder is added during the concentration of semi-saturated ammonium sulfate, the amount added at one time is too large, so that the local ammonium sulfate concentration is too high, and the protein coagulation is caused.

The reconstituted supernatant was purified by nickel column chromatography, and the liquid environment of the protein was replaced by PBS using G25 molecular sieves, and samples were electrophoresed (see FIG. 12). All samples pass through a nickel column at one time, only the obtained penetrating sample is remained with the foreign protein, and the target protein is specifically combined with the nickel ions of the column through the His tag, so that a good purification effect is achieved; it can be seen from the nickel column elution that the target protein was highly purified and concentrated, which brings favorable conditions for the desalting of added G25; the miscellaneous band around 66.2kDa is suspected of contamination by Lording Buffer, resulting in each lane containing a band with a gray scale approximation.

After purification, protein concentration was measured with BCA kit, incubated for 30min, and a value of a standard curve was obtained by a microplate reader at a wavelength of 570nm, and a scatter plot (see FIG. 13) was drawn and brought into each dilution reading to obtain a concentration of 2.10 mg/ml. Adding glycerol to obtain protein solution containing 10% glycerol, and storing in-80 deg.C refrigerator.

Example 5 measurement of cytostatic Rate of Single-chain antibody

First, experiment operation

1. Cell resuscitation

Adding a large amount of tap water into a large beaker, and adjusting the temperature of the water to 42 +/-1 ℃ by using boiling water. A strain of H292 cells (human lung cancer cells) is taken out of a liquid nitrogen tank and is clamped by tweezers to be rapidly put into warm water to be continuously stirred until the cells are completely melted. The cell line was transferred to a clean bench. A small culture flask was taken, 4.5ml of 1640 medium and 500. mu.l of FBS were added, and finally the cells in the cryopreserved tube were transferred to the culture flask, the flask lid was put on fire, the flask lid was closed and shaken up crosswise. Taking out of the clean bench, and introducing 5.0% CO at 37 deg.C₂The bottle cap is slightly unscrewed, and the incubator is closed for 24 hours.

2. Cell exchange liquid

The cell state (number of cells, adhesion condition, amount of suspended cells, cell morphology, culture environment cleanliness, etc.) of the cells cultured for 24 hours is observed in a microscope, and whether the cells are continuously cultured is judged. And (3) putting the cells with good cell state into a super clean bench, discarding the culture medium, quickly returning to the side of an alcohol lamp, and over-firing the bottle mouth. Washing the bottle with 2-3 ml PBS (or culture medium), discarding, burning the bottle mouth, and repeating for more than two times. Adding 3.6ml 1640 culture medium and 400 μ l FBS (7 ml complete medium in the middle bottle and 10ml complete medium in the large bottle), shaking, adding 5.0% CO at 37 deg.C₂The incubator is used for 24 h.

3. Cell passage and plating

And (3) transferring the culture bottle to a super clean bench after the cells grow over the culture bottle, removing the culture medium, enabling the bottle mouth to be over-fired, and cleaning the culture bottle with PBS for 2-3 times. 1ml of pancreatin was added, shaken to cover all the cells, and placed in an incubator to be left to digest for 1 minute. After digestion, complete medium was added to stop digestion and the cells in the flask were pipetted down. The cell suspension was transferred to a centrifuge tube, centrifuged at 1200rpm for 5min and the supernatant discarded. Add 4ml of medium with a pipette and blow and mix repeatedly, approximately 50 times. The cell concentration was calculated by pipetting 10. mu.l of the cell suspension into a hemocytometer using a 10. mu.l pipette and counting under the microscope (four large cells on the plate), and the formula was calculated:

a small amount of the cell suspension was diluted to 35000 cells/ml with 1640 medium, and FBS was added to make the content 10%. The cells were pipetted into a sterile 96-well plate at 100. mu.l/well, and the cells were re-pipetted once for each approximately 9 wells, PBS was added to the edge, and the plate was covered with 5.0% CO at 37 ℃₂The incubator is used for 24 h. The remaining cell suspension was added to a new flask, and 10% FBS was added, and the mixture was placed in an incubator for culture.

4. Cell starvation culture and drug treatment

After plating for 24h, the medium in the 96-well plate was slowly aspirated out and discarded, and the wells were washed 1-2 times with PBS. 1640 medium containing 0.5% FBS was prepared, and 200. mu.l/well of the medium was added to the wells containing the cells, and the wells were covered with a lid and placed at 37 ℃ in 5.0% CO₂The incubator is used for 24 h. The culture medium is discarded the next day, and the wells are washed 1-2 times with PBS. EGFR-scFv was prepared in 1640 medium at 10, 20, 40, 80, 160 and 200. mu.g/ml, respectively, and a dilution was prepared in the same amount with PBS as a negative control. Adding diluent at 200 μ l/well, repeating 3 wells per gradient, selecting 3 empty wells, adding 200 μ l 1640 culture medium as zero-setting well, covering with a cover, placing at 37 deg.C and 5.0% CO₂Incubate for 1 h. Diluting 10 μ/ml EGF and 10% FBS in 1640 medium, adding 10 μ l per well, placing at 37 deg.C and 5.0% CO₂The incubator is used for 72 h.

5. Cell activity detection and single-chain antibody inhibition rate calculation

After three days of incubation, 110. mu.l of medium was discarded from each well, 10. mu.l of CCK-8 was added to each well, the lid was closed, and 5.0% CO was placed at 37 ℃₂The incubator is used for 2 h. After dyeing, the obtained product is put into an enzyme-labeling instrument to measure the absorbance at the wavelength of 450 nm. Data processing:

second, experimental results

WB detection is carried out on H292 human lung cancer cell surface receptors in a laboratory, H292 is an EGFR high expression cell, and the cell is selected to carry out an inhibition experiment. CCK-8 was used to analyze the proliferation inhibition of H292 by different doses of EGFR-scFv. The results show that different concentrations of EGFR-scFv have proliferation inhibitory effects on H292 cells and appear dose-dependent (as in FIG. 14), but with increasing dose, they also peak, approaching saturation, with 37% inhibition measured at 200. mu.g/ml and still increasing slowly with higher concentration.

Sequence listing

<110> university of Guangdong department of pharmacy

<120> single-chain antibody targeting EGFR dimerization interface and application thereof

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Gly Val Ile Asn Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe

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Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

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Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys

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Ala Arg Tyr Gly Asn Tyr Glu Asp Tyr Trp Gly Gln Gly Thr Thr Leu

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Thr Val Ser Ser

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gaggttcaat tgcaacaatc tggtgctgag ttggttagac caggtacttc tgttaaggtt 60

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tctcgtgttg aggctgagga cgttggtgtt tactactgtg ctcaaaactt ggagttgcca 300

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Gly Val Ile Asn Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe

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Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

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Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys

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Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro

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Gly Gln Ser Pro Gln Pro Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser

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Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr

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Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

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gaggttcaat tgcaacaatc tggtgctgag ttggttagac caggtacttc tgttaaggtt 60

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ccaggtcaag gtttggagtg gatcggtgtt atcaacccag gttctggtaa cactaagtac 180

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atgcaattgt cttctttgac ttctgacgac tctgctgttt acttctgtgc tagatacggt 300

aactacgagg actactgggg tcaaggtact actctcactg tgagctctgg tggtggtggt 360

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

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Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly

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Gly Ala Gly Thr Lys Leu Glu Leu Lys His His His His His His

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gagtgggtta agcaaagacc aggtcaaggt ttggagtgga tcggtgttat caacccaggt 180

tctggtaaca ctaagtacaa cgagaagttc aagggtaagg ctactttgac tgctgacaag 240

tcttcttcta ctgcttacat gcaattgtct tctttgactt ctgacgactc tgctgtttac 300

ttctgtgcta gatacggtaa ctacgaggac tactggggtc aaggtactac tctcactgtg 360

agctctggtg gtggtggttc tggtggtggt ggttctggtg gtggtggttc tgacatcgtt 420

atgactcaag ctgctttctc taacccagtt actttgggta cttctgcttc tatctcttgt 480

agatcttcta agtctttgtt gcactctaac ggtatcactt acttgtactg gtacttgcaa 540

aagccaggtc aatctccaca accattgatc taccaaatgt ctaacttggc ttctggtgtt 600

ccagacagat tctcttcttc tggttctggt actgacttca ctttgagaat ctctcgtgtt 660

gaggctgagg acgttggtgt ttactactgt gctcaaaact tggagttgcc attgactttc 720

ggtgctggta ctaagttgga gttgaagcac caccaccacc accactga 768

Claims

1. The fusion protein of the single-chain antibody targeting the EGFR dimerization interface is characterized by comprising the amino acid sequences of a heavy chain variable region protein, a connecting peptide and a light chain variable region protein which are sequentially connected from 5 'to 3', wherein the amino acid sequence of the heavy chain variable region protein is shown as SEQ ID NO: 1, the amino acid sequence of the light chain variable region protein is shown as SEQ ID NO: 3, respectively.

2. The fusion protein of claim 1, wherein the amino acid sequence of the linker peptide is as set forth in SEQ ID NO: 5 is shown in the specification; the amino acid sequence of the fusion protein is shown as SEQ ID NO: shown at 7.

3. A fusion gene of a single-chain antibody targeting an EGFR dimerization interface, which encodes the fusion protein of claim 1, wherein the fusion gene comprises a nucleotide sequence of a heavy chain variable region protein, a linker peptide and a light chain variable region protein which are connected in sequence from 5 'to 3', and the amino acid sequence of the heavy chain variable region protein is as shown in SEQ ID NO: 1, the amino acid sequence of the light chain variable region protein is shown as SEQ ID NO: 3, respectively.

4. The fusion gene of claim 3, wherein the nucleotide sequence of the linker peptide is as set forth in SEQ ID NO: 6 is shown in the specification; the nucleotide sequence of the fusion gene is shown as SEQ ID NO: shown in fig. 8.

5. Use of the fusion protein of claim 1 and/or the fusion gene of claim 3 for the preparation of a medicament for the treatment of tumors with aberrant expression of EGFR.

6. An agent comprising the fusion protein of claim 1.