HER3 dimerization interface antigen peptide, recombinant antigen peptide, encoding gene and application thereof
阅读说明：本技术 一种her3二聚化界面抗原肽、重组抗原肽、编码基因及其应用 (HER3 dimerization interface antigen peptide, recombinant antigen peptide, encoding gene and application thereof ) 是由 朱磊 孙金琳 张叶明 沈学彬 章圣朋 于 2020-06-01 设计创作，主要内容包括：本发明提供了一种HER3二聚化界面抗原肽、重组抗原肽、编码基因及其应用,与现有技术相比,本发明抗原肽是基于HER3结构功能选取的,在阻断HER3信号传导功能上更具有针对性。本发明的抗原肽位于非HER3配体结合区的二聚化界面区,而现有的抗体多靶向配体结合区或靶向在配体结合后构象改变的区域,靶向二聚化界面区可以避免和配体竞争结合部位；本发明的抗原肽为线性B细胞表位抗原肽,不会受HER3构象变化而造成脱靶效应。本发明在制备防治HER3过表达肿瘤和逆转靶向EGFR、HER2药物耐药中应用,主要用于制备治疗性疫苗,治疗成本远比抗体低,副作用比需大剂量使用的抗体低。(Compared with the prior art, the antigenic peptide is selected based on the structural function of HER3, and has more pertinence on the function of blocking HER3 signal conduction. The antigenic peptide of the invention is positioned in a dimerization interface region of a non-HER 3 ligand binding region, while the existing antibody multi-targets the ligand binding region or targets a region with changed conformation after ligand binding, and the targeting dimerization interface region can avoid competition with the ligand for a binding site; the antigenic peptide is a linear B cell epitope antigenic peptide, and cannot cause off-target effect due to HER3 conformational change. The invention is applied to the preparation of HER3 overexpression tumor prevention and treatment and reversal of drug resistance of targeting EGFR and HER2, is mainly used for preparing therapeutic vaccines, and has far lower treatment cost and lower side effect than antibodies which need to be used in large dose.)
1. An epidermal growth factor receptor 3 dimerization interface antigen peptide, which is characterized by comprising any one of the following amino acid sequences:
1) HER3183-227aa, and the amino acid sequence is shown as SEQ ID No. 1;
2) an antigenic peptide having dimerization activity against HER3 by substitution, deletion or addition of one or more amino acid residues in the amino acid sequence of SEQ ID No. 1;
3) HER3236-308aa, the amino acid sequence is shown in SEQ ID No. 2;
4) an antigenic peptide having dimerization activity against HER3 by substitution, deletion or addition of one or more amino acid residues in the amino acid sequence of SEQ ID No. 2;
5) HER3559-591aa, the amino acid sequence is shown as SEQ ID No. 3;
6) and SEQ ID No.3 by substitution, deletion or addition of one or more amino acid residues, thereby imparting dimerization activity against HER 3.
2. A recombinant antigenic peptide, which is constructed by fusing the dimeric interface antigenic peptide of the epidermal growth factor receptor 3 of claim 1 with MVF respectively by using a GPSL tetrameric peptide linker.
3. The recombinant antigenic peptide of claim 2, wherein said recombinant antigenic peptide is:
MVF-GPSL-HER3183-227aa, and the amino acid sequence is shown as SEQ ID No. 4.
4. The recombinant antigenic peptide of claim 2, wherein said recombinant antigenic peptide is:
MVF-GPSL-HER3236-308aa, and the amino acid sequence is shown as SEQ ID No. 5.
5. The recombinant antigenic peptide of claim 2, wherein said recombinant antigenic peptide is:
MVF-GPSL-HER3559-591aa, and the amino acid sequence is shown as SEQ ID No. 6.
6. The gene encoding the recombinant antigenic peptide of claim 3, wherein the method for synthesizing the encoding gene comprises:
the coding gene is obtained by amplification by taking a sequence SEQ ID No.7 as a template, taking a sequence SEQ ID No.10 and a sequence SEQ ID No.11 as primers, and taking PCR amplification conditions of 4 cycles of 95 ℃ for 5min, 95 ℃ for 30s, 53 ℃ for 30s and 72 ℃ for 30s, 26 cycles of 95 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 30s, and 72 ℃ for 7 min.
7. The gene encoding the recombinant antigenic peptide of claim 4, wherein the gene encoding is synthesized by: the coding gene is obtained by amplification by taking a sequence SEQ ID No.8 as a template, a sequence SEQ ID No.12 and a sequence SEQ ID No.13 as primers and taking PCR amplification conditions of 4 cycles of 95 ℃ for 5min, 30s at 95 ℃, 30s at 54 ℃ and 30s at 72 ℃, 26 cycles of 95 ℃ for 30s at 60 ℃ and 30s at 72 ℃ and 7min at 72 ℃.
8. The gene encoding the recombinant antigenic peptide of claim 5, wherein the method for synthesizing the encoding gene comprises: the coding gene is obtained by amplification by taking a sequence SEQ ID No.9 as a template, taking a sequence SEQ ID No.14 and a sequence SEQ ID No.15 as primers and taking PCR amplification conditions of 4 cycles of 95 ℃ for 5min, 95 ℃ for 30s, 52 ℃ for 30s and 72 ℃ for 30s, 26 cycles of 95 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 7 min.
9. The use of an epidermal growth factor receptor 3 dimerization interface antigen peptide according to claim 1, wherein the use is: the application in preparing the medicine for preventing and treating HER3 over-expressed tumor;
or after EGFR and HER2 targeting drug therapy, the application of the composition in treating tumors with drug resistance to EGFR and HER2 targeting drugs caused by high expression of HER3 occurs;
or as an active ingredient of a therapeutic polypeptide vaccine or as an antigen for preparing an antibody.
10. Use of the recombinant antigenic peptide of any one of claims 2 to 5, wherein said use is: the application in preparing the medicine for preventing and treating HER3 over-expressed tumor;
or after EGFR and HER2 targeting drug therapy, the application of the composition in treating tumors with drug resistance to EGFR and HER2 targeting drugs caused by high expression of HER3 occurs;
or as an active ingredient of a therapeutic polypeptide vaccine or as an antigen for preparing an antibody.
Technical Field
The invention relates to the field of preparation of polypeptides and drug carriers, in particular to HER3 dimerization interface antigen peptide, recombinant antigen peptide, encoding gene and application thereof.
Background
Human epidermal growth factor receptor 3(HER3, human epidermal growth factor receptor 3) and human epidermal growth factor receptor (EGFR/HER1, human epidermal growth factor receptor), human epidermal growth factor receptor 2(HER2, human epidermal growth factor receptor 2) belong to the epidermal growth factor receptor family members, and the receptor family plays an important role in the growth and development process of embryos and normal tissues. However, EGFR and HER2 expression up-regulation or activation mutation can be found in colorectal cancer, lung cancer and partial breast cancer respectively, is closely related to tumor proliferation, metastasis, anti-apoptosis and the like, and is a definite tumor treatment target molecule.
For two targets of EGFR and HER2, the currently marketed drugs include monoclonal antibodies Cetuximab and Panituzumab for targeting EGFR extracellular domain to treat colon cancer, and monoclonal antibodies Trastuzumab and Pertuzumab for targeting HER2 extracellular domain to treat breast cancer. Besides, the two drugs can obviously improve the survival time of sensitive patients and improve the treatment situation of the cancers. However, the higher rate of resistance that occurs after initial treatment of EGFR, HER2 positive tumor patients reduces the expected therapeutic effect of both classes of drugs. And the recurrent tumor has dual drug resistance of a monoclonal antibody and a tyrosine kinase inhibitor, so that the subsequent treatment faces the situation that no effective drug is available. Despite the complex and diverse causes, more and more studies show that the upregulation of HER3 expression and the abnormal activation of its signaling pathway are often seen in resistant non-small cell lung cancer, head and neck squamous cell carcinoma, breast cancer and metastatic colon cancer after treatment with EGFR, HER2 monoclonal antibody targeted and one-, two-, and three-generation tyrosine kinase inhibitors, suggesting that HER3 is associated with the development of drug resistance targeted to EGFR,
The HER family receptor with complete structure consists of an extracellular region, a transmembrane region and an intracellular region, wherein the intracellular region contains tyrosine kinase activity region and a plurality of phosphorylatable amino acid residues, the extracellular region can be divided into four subregions I-IV according to the functions of the extracellular region, the region I and the region III are positioned on the surface of a molecule, and a variable space structure can be formed between the region I and the region III for the recognition and combination of ligands; region II is a highly conserved domain of structure and is primarily involved in receptor dimerization, and region IV is primarily in a self-inhibitory resting state when ligand is not bound by embedding it within the extracellular domain through interaction with region II. When their ligands, such as Epidermal Growth Factor (EGF) and
Although the intracellular domain of HER3 has no intracellular tyrosine kinase domain, but has phosphorylatable amino acid residues, in drug-resistant tumor cells and tissues, HER3, which is up-regulated in expression, can be phosphorylated with cognate receptors having tyrosine kinase activity or other receptors, such as hepatocyte growth factor receptor MET, insulin-like growth factor-1 receptor IGF-1R, forming HER3/MET heterodimer or HER2/HER3/IGF-1R trimer, thereby activating signaling pathways such as PI3K/Akt that drive tumor cell proliferation, metastasis, anti-apoptosis and drug resistance. HER3 is therefore a key molecular target to overcome existing drug resistance targeting EGFR and
The currently reported drug development targeting HER3 mainly takes antibodies as main materials, and the antibody mainly has two types according to the binding regions, wherein one type is to compete with ligands to bind with a HER3 ligand binding region so as to block the signal path; another class of antibodies, such as LJM716, block its signaling pathway by binding to conformational epitopes formed by regions ii and iv when HER3 is not bound to the ligand, thereby preventing the formation of the conformation necessary for HER3 dimerization. Although both of these antibodies showed better inhibition of HER 3-highly expressed and EGFR/HER 2-targeted drug-resistant cell lines in vitro, they did not improve survival of the relevant patients in clinical trials compared to controls. Research shows that high-level autocrine and paracrine expression exists in HER3 ligands such as NRG in tumor patients with EGFR and HER2 drug resistance targeting and HER3 high-expression tumors, and the ligands are much smaller than antibody molecules, so that the antibody has the disadvantage of competing with the antibody for binding a receptor. Meanwhile, HER3 undergoes a change in spatial conformation upon ligand binding, which may result in off-target of the LJM 716-like antibody bound to a conformational epitope.
In addition, although the antibody drugs have good clinical efficacy, the antibody administration belongs to passive immunotherapy, long-term repeated administration is required, and the administration dosage is large, so the risk of toxic and side effects is increased, and the monoclonal antibody brings heavy economic burden to patients due to high production cost and high price. The therapeutic vaccine belongs to active immunization, has small administration dosage, only needs a few times of immunization, and is more compliant than antibody products. The malignant tumor therapeutic polypeptide vaccine takes tumor antigen peptide as an active component, can be produced at low cost by using genetic engineering or chemical synthesis means, and has better industrial development prospect.
Therefore, finding a suitable target on the HER3 molecule to develop a therapeutic vaccine and a high-efficiency antibody which are not influenced by the ligand and have the function of blocking the signal path of the HER3 molecule has important significance.
Studies have shown that tumor tissues express molecules that differ from normal tissues, called Tumor Associated Antigens (TAAs), such as highly expressed or mutated
The american scientist Kaumaya laboratory analysis yielded a series of linear B cell epitopes that had been marketed as targeting HER2 antibodies, including the B cell epitope of Pertuzumab antibody binding to the HER2 dimerization arm. They prepared a series of fusion peptides by linking these epitope peptides to a universal Th cell epitope peptide MVF (measles virus fusion protein amino acids 288-302) isolated and identified in this laboratory from measles fusion protein. In a first clinical trial, the fusion peptides can stimulate HER2 patients with high-expression breast cancer to generate high-titer functional antibodies similar to those of the marketed monoclonal antibodies, and generate better treatment effect.
Based on the same idea, the inventor of the plum gold and the like compares an EGFR dimerization arm region and a Pertuzumab antibody binding epitope (linear B cell epitope) positioned in a HER2 dimerization arm region, finds that the region is conserved among HER receptor family members, has the same main site amino acid, considers that the region also has the linear B cell epitope, and connects the region with MVF to construct an anti-EGFR dimerization fusion antigen peptide. The fusion antigenic peptide can stimulate the generation of antibodies targeting the dimerization interface of EGFR on animal models.
In addition, Guba scientists chemically couple EGF serving as EGFR ligand and an outer membrane protein P64k which is discovered by the Cuba scientists and is derived from Neisseria meningitidis and composed of 593 amino acid residues to prepare a CIMAvax-EGF (B cell epitope vaccine) which is an EGF over-expression therapeutic vaccine.
The above cases and scientific studies show that the molecules or parts of the molecules from the human body do not stimulate the immune response under normal conditions due to the restriction of MHC class II molecules, but when linked to a suitable carrier, such as P64k, MVF, the immunogenicity can be revealed and improved, and the body can be stimulated to generate a targeted immune response.
The immunogen of the existing tumor therapeutic B cell epitope vaccine is mostly selected from whole molecules or based on the combined epitope of the antibodies on the market. The former may cause the shielding of potential effective target epitope due to larger immunogenic molecule, and is difficult to stimulate the organism to generate effective immune response; the binding site of the antibody generated by the latter stimulating organism is basically the same as that of the antibody on the market, so that the disadvantage of poor competitive binding or off-target of the antibody on the market is difficult to avoid, and the antibody on the market has limitations.
Disclosure of Invention
The invention aims to provide a HER3 dimerization interface antigen peptide, which selects a plurality of peptide fragments positioned in a HER3 dimerization interface region, and uses an online B cell epitope prediction tool to predict and select a plurality of linear B cell epitope antigen peptides which have obvious antigen characteristics and are not reported at present.
The invention also aims to provide a recombinant antigen peptide constructed by fusing the antigen peptide and MVF.
Another object of the present invention is to provide a gene encoding a recombinant antigenic peptide.
The last aim of the invention is to provide an antigenic peptide or a recombinant antigenic peptide which is mainly applied to the treatment of HER3 over-expression tumors.
The specific technical scheme of the invention is as follows:
a HER3 dimeric interface antigen peptide comprising any one of the following amino acid sequences:
1) HER3183-227aa, and the amino acid sequence is shown as SEQ ID No. 1;
2) an antigenic peptide having dimerization activity against HER3 by substitution, deletion or addition of one or more amino acid residues in the amino acid sequence of SEQ ID No. 1;
3) HER3236-308aa, the amino acid sequence is shown in SEQ ID No. 2;
4) an antigenic peptide having dimerization activity against HER3 by substitution, deletion or addition of one or more amino acid residues in the amino acid sequence of SEQ ID No. 2;
5) HER3559-591aa, the amino acid sequence is shown as SEQ ID No. 3;
6) and SEQ ID No.3 by substitution, deletion or addition of one or more amino acid residues, thereby imparting dimerization activity against
A recombinant antigen peptide, which is constructed by fusing the HER3 dimerization interface antigen peptide with MVF by a GPSL tetramer peptide linker;
the method specifically comprises the following steps:
the recombinant antigen peptide constructed by fusing the antigen peptide HER3183-227aa and MVF is MVF-GPSL-HER3183-227aa, and the amino acid sequence of the recombinant antigen peptide is shown as SEQ ID No. 4;
the recombinant antigen peptide constructed by fusing the antigen peptide HER3236-308aa and MVF is MVF-GPSL-HER3236-308aa, and the amino acid sequence of the recombinant antigen peptide is shown as SEQ ID No. 5;
the recombinant antigen peptide constructed by fusing the antigen peptide HER3559-591aa and MVF is MVF-GPSL-HER3559-591aa, and the amino acid sequence of the recombinant antigen peptide is shown in SEQ ID No. 6.
The gene sequence of MVF-GPSL-HER3183-227aa is shown as SEQ ID No. 7.
The gene sequence of MVF-GPSL-HER3236-308aa is shown as SEQ ID No. 8.
The gene sequence of MVF-GPSL-HER3559-591aa is shown in SEQ ID No. 9.
The primers are as follows:
MVF-GPSL-HER3183-227aa gene primer:
F:GGAGCCATGGGTAAACTTCTTAGCCTTATCAAAGGTGTTATCG, as set forth in SEQ ID No. 10;
NcoI cleavage site
R:ATCCAAGCTTTTAGCACGCAAAGCAGTCGGTG, as shown in SEQ ID No. 11;
HindIII cleavage site
MVF-GPSL-HER3236-308aa gene primer:
F:GGAGCATATGAAACTTCTTAGCCTTATCAAAGGTGTTATCGTTCACC, as set forth in SEQ ID No. 12;
nde I cleavage site
R:ATCCCTCGAGGCATAAACCACCGCACGGCTC, as set forth in SEQ ID No. 13;
XholI cleavage site
MVF-GPSL-HER3559-591aa gene primers:
F:GGAGCCATGGGTAAACTTCTTAGCCTTATCAAAGGTGTTATCG, as set forth in SEQ ID No. 14;
NcoI cleavage site
R:ATCCAAGCTTTTAGCATTCGTTCTGAACGTCCGG, as shown in SEQ ID No. 15.
HindIII cleavage site
The synthesis method of the MVF-GPSL-HER3183-227aa coding gene with the endonuclease cleavage site comprises the following steps:
the coding gene is obtained by amplification by taking a sequence SEQ ID No.7 as a template, taking a sequence SEQ ID No.10 and a sequence SEQ ID No.11 as primers, and taking PCR amplification conditions of 4 cycles of 95 ℃ for 5min, 95 ℃ for 30s, 53 ℃ for 30s and 72 ℃ for 30s, 26 cycles of 95 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 30s, and 72 ℃ for 7 min.
The synthesis method of the MVF-GPSL-HER3236-308aa coding gene with the endonuclease cleavage site comprises the following steps:
the coding gene is obtained by amplification by taking a sequence SEQ ID No.8 as a template, a sequence SEQ ID No.12 and a sequence SEQ ID No.13 as primers and taking PCR amplification conditions of 4 cycles of 95 ℃ for 5min, 30s at 95 ℃, 30s at 54 ℃ and 30s at 72 ℃, 26 cycles of 95 ℃ for 30s at 60 ℃ and 30s at 72 ℃ and 7min at 72 ℃.
The synthesis method of the MVF-GPSL-HER3559-591aa coding gene with endonuclease cleavage sites comprises the following steps:
the coding gene is obtained by amplification by taking a sequence SEQ ID No.9 as a template, taking a sequence SEQ ID No.14 and a sequence SEQ ID No.15 as primers and taking PCR amplification conditions of 4 cycles of 95 ℃ for 5min, 95 ℃ for 30s, 52 ℃ for 30s and 72 ℃ for 30s, 26 cycles of 95 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 7 min.
The three polypeptides were submitted to chemical synthesis by commercial companies. The three recombinant peptide genes are chemically synthesized, the three recombinant peptide genes with endonuclease enzyme cutting sites are amplified by a PCR method, the three recombinant peptide genes are expressed by an escherichia coli prokaryotic expression system, and the three recombinant peptides are purified by an affinity chromatography method.
The HER3 dimerization interface antigen peptide or the recombinant antigen peptide is applied to the preparation of a medicine for preventing and treating HER3 over-expressed tumors, or the application of the HER3 dimerization interface antigen peptide or the recombinant antigen peptide to the treatment of the tumors with drug resistance to the targeted EGFR and HER2 due to the high expression of HER3 after the targeted EGFR and HER2 medicines are treated. The tumors comprise: breast cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, head and neck cancer, or prostate cancer.
Also includes the effective component of the therapeutic polypeptide vaccine or the antigen preparation antibody, wherein the antibody includes monoclonal antibody, polyclonal antibody or active fragment connected with the antigen peptide.
The invention utilizes the recombinant peptide to prepare the suspension with the final concentration of 2mg/L as an antigen to stimulate an animal model to generate antibodies with different titers, and the titer of the antibodies generated after the animal is immunized by the three kinds of recombinant peptide suspensions is higher than that generated after the corresponding solution type recombinant peptide is immunized.
The selection strategy of the HER3 dimerization antigenic peptide comprises the following steps:
the HER3 dimerization interface refers to the surface of molecules that contact each other when they form dimers with other receptors, and upon dimer formation, the HER3 intracellular tyrosine residues are phosphorylated by the intracellular tyrosine kinase of the receptor molecule with which they form dimers, thereby transmitting extracellular signals into the cell. The formation of dimers is therefore a key step in the signal transduction in which HER3 is involved. The invention selects all peptide fragments positioned in a dimerization interface region of HER3 based on a dimerization key step of HER3 participating in signal transduction, and uses a plurality of B cell epitope prediction tools for analysis and locking of B cell epitopes. The advantages of selecting B cell epitope antigen peptide by the strategy are three: firstly, the antibody can stimulate the organism to generate an antibody which is targeted to be combined with a HER3 dimerization interface, and further the formation of HER3 dimer is blocked spatially; secondly, the strategy selects short peptides, so that the shielding of potential effective target epitopes caused by large immunogenic molecules can be avoided, and the body is difficult to be stimulated to generate effective immune response; thirdly, the disadvantage that the known monoclonal antibody binding epitope is used as a vaccine to stimulate the organism to generate competitive binding disadvantage or off-target antibody which is the same as the monoclonal antibody is avoided.
The mechanism of action of the HER3 dimerization antigenic peptide of the invention is as follows:
the HER3 receptor consists of an extracellular region, a transmembrane region and an intracellular region, wherein the intracellular region contains an inactive tyrosine kinase activity region and a plurality of phosphorylatable amino acid residues, the extracellular region can be divided into four subregions I-IV according to the functions of the extracellular region, the region I and the region III are positioned on the surface of a molecule, and a variable space structure can be formed between the region I and the region III for the recognition and the combination of ligands; region II is a highly conserved domain of structure and is primarily involved in receptor dimerization, and region IV is primarily in a self-inhibitory resting state when ligand is not bound by embedding it within the extracellular domain through interaction with region II. When their ligands, such as
Compared with the prior art, the antigenic peptide is selected based on the functional structure of HER3, and has more pertinence in the function of blocking HER3 signal conduction. The antigenic peptide of the invention is positioned in a dimerization interface region of a non-HER 3 ligand binding region, while the existing antibody multi-targets the ligand binding region or targets a region with changed conformation after ligand binding, and the targeting dimerization interface region can avoid competition with the ligand for a binding site; the antigenic peptide is a linear B cell epitope antigenic peptide, and cannot cause off-target effect due to HER3 conformational change. The invention is applied to the preparation of HER3 overexpression tumor prevention and treatment and reversal of drug resistance of targeting EGFR and HER2, is mainly used for preparing therapeutic vaccines, and has far lower treatment cost and lower side effect than antibodies needing large dose use. The invention uses the recombinant antigen peptide suspension as an antigen and can stimulate an animal model to produce a method with higher titer antibodies than using the recombinant antigen peptide solution as the antigen. Compared with the method that the suspension and the solution of the same amount of recombinant peptide are respectively used as antigens to stimulate the same animal model to generate antibodies with different titers, the titer of the antibodies generated after the animal is immunized by the suspensions of the three types of recombinant peptide is higher than that generated after the corresponding solution type recombinant peptide is immunized, and the suspension type recombinant peptide particles prepared by the method can slowly release the recombinant peptide and can effectively and durably stimulate the experimental animal to generate related immune response.
Drawings
FIG. 1 shows a 188-236aa peptide fragment;
FIG. 2 shows the 234-312aa peptide fragment;
FIG. 3 is a 474-591aa peptide fragment;
FIG. 4 shows a selected HER3 dimerization interface B cell epitope peptide fragment;
FIG. 5 shows recombinant anti-HER 3 dimerization interface B cell epitope peptide;
FIG. 6 shows the double restriction enzyme identification of recombinant plasmids pET32a-MVF-GPSL-HER 3183-227aa, pET21b-MVF-GPSL-HER3236-308aa and pET32a-MVF-GPSL-HER 3559-591 aa;
m in A is DNA marker; 1, pET32a-MVF-GPSL-HER 3183-227aa plasmid enzyme digestion product;
m in B is DNA marker; 1, plasmid restriction enzyme products of pET21b-MVF-GPSL-HER3236-308 aa;
m in C is DNA marker; 1, plasmid restriction enzyme products of pET32a-MVF-GPSL-HER 3559-591 aa;
FIG. 7 is prokaryotic fusion expression of recombinant HER3 dimerization interface peptide;
in the figure, A: m is protein marker; 1-6, after induction, pET32a-MVF-GPSL-HER 3183-227aa positive clone thallus whole bacteria split protein;
b in the figure: m is protein marker; 1-8, after induction, pET21b-MVF-GPSL-HER3236-308aa positive clone thallus whole bacteria split protein;
c in the figure: m is protein marker; 1-6, after induction, pET32a-MVF-GPSL-HER 3559-591aa positive clone thallus whole bacteria split protein;
figure 8 purification of recombinant HER3 dimerization interface peptide;
in the figure, A is the purification of MVF-GPSL-HER3183-227aa, M-protein marker; 1-non-induced pre-mycoprotein; 2-induced mycoprotein; 3-cracking and precipitating; 4-lysis of the supernatant; 5-cleaved supernatant protein precipitated with 30% ammonium sulfate; 6-fusion protein; 7-thioredoxin (Trx) and other hetero proteins; 8-MVF-GPSL-HER 3183-227 aa;
in the figure, B represents the purification of MVF-GPSL-HER3236-308aa, M-protein molecular weight; 1-whole mycoprotein; 2-lysis of the supernatant; dissolving, cracking and precipitating by 3-8M/L urea; 4-flow through; 5-purified MVF-gps l-HER3236-308 aa;
in the figure, C is the purification of MVF-GPSL-HER3559-591aa, M-protein marker; 1-non-induced pre-mycoprotein; 2-induced mycoprotein; 3-cracking and precipitating; 4-lysis of the supernatant; 5-cleaved supernatant protein precipitated with 30% ammonium sulfate; 6-fusion protein; 7-Trx and other hetero proteins; 8-MVF-GPSL-HER 3559-591 aa;
figure 9 is an anti-recombinant HER3 dimeric interface peptide polyclonal antibody titer assay;
in the figure, A is the anti-MVF-GPSL-HER 3183-227aa antibody titer;
in the figure, B is the anti-MVF-GPSL-HER 3236-308aa antibody titer;
in the figure, C is the anti-MVF-GPSL-HER 3559-591aa antibody titer;
figure 10 is the antigen specificity of polyclonal antibodies against recombinant HER3 dimeric interfacial peptide;
in the figure, A is the antigen specificity analysis of the anti-MVF-GPSL-HER 3183-227aa polyclonal antibody, and in the figure, 1-0.8 mu g of MVF-GPSL-HER3183-227aa protein; 2-0.4 μ g MVF-GPSL-HER3183-227aa protein; 3-0.2 μ g MVF-GPSL-HER3183-227aa protein;
in the figure, B is the antigen specificity analysis of the anti-MVF-GPSL-HER 3236-308aa polyclonal antibody, and in the figure, 1-0.8 mu g of MVF-GPSL-HER3236-308aa protein; 2-0.4 μ g MVF-GPSL-HER3236-308aa protein; 3-0.2 μ g MVF-GPSL-HER3236-308aa protein;
in the figure, C is the antigen specificity analysis of the anti-MVF-GPSL-HER 3559-591aa polyclonal antibody, and in the figure, 1-0.8 mu g of MVF-GPSL-HER3559-591aa protein; 2-0.4 μ g MVF-GPSL-HER3559-591aa protein; 3-0.2 μ g of MVF-GPSL-HER3559-591aa protein;
figure 11 polyclonal antibody against recombinant HER3 dimeric interfacial peptide recognizes and precipitates non-denatured HER3 from MCF7 cell lysates;
in the figure, A: 1-MCF7 whole cell lysate; 2-PBS immunoprecipitation group; 3-negative serum immunoprecipitation group; 4-anti-MVF-GPSL-ErbB 3183-227aa serum immunoprecipitation group;
b in the figure: 1-MCF7 whole cell lysate; 2-PBS immunoprecipitation group; 3-negative serum immunoprecipitation group; 4-anti-MVF-GPSL-ErbB 3236-308aa serum immunoprecipitation group;
c in the figure: 1-MCF7 whole cell lysate; 2-PBS immunoprecipitation group; 3-negative serum immunoprecipitation group; 4-anti-MVF-GPSL-ErbB 3559-591aa serum immunoprecipitation group;
figure 12 is a polyclonal antibody against recombinant HER3 dimerization interface peptide that binds to HER3 on MCF7 cell membrane and targets its dimerization interface;
in the figure, A represents the combination of an anti-MVF-GPSL-HER 3183-227aa polyclonal antibody and MCF7 cells;
panel B shows anti-MVF-GPSL-ErbB 3236-308aa polyclonal antibody binding to MCF7 cells;
in the figure, C shows that the anti-MVF-GPSL-ErbB 3559-591aa polyclonal antibody is combined with MCF7 cells;
figure 13 is a polyclonal antibody against recombinant HER3 dimeric interfacial peptide inhibiting MCF7 cell proliferation;
in the figure, a shows that three polyclonal antibodies against recombinant HER3 dimerization interface peptide inhibit MCF7 cell proliferation;
in the figure, B shows the inhibition effect of three polyclonal antibodies against recombinant HER3 dimeric interface peptide on MCF7 cell proliferation stimulated by NRG1 beta.
Detailed Description
In the following examples, the amino acid sequence of HER3183-227aa antigen peptide is SEQ ID No.1, the amino acid sequence of HER3236-308aa antigen peptide is SEQ ID No.2, the amino acid sequence of HER3559-591aa antigen peptide is SEQ ID No.3, the amino acid sequence of MVF-GPSL-HER3183-227aa recombinant antigen peptide is SEQ ID No.4, the amino acid sequence of MVF-GPSL-HER3236-308aa recombinant antigen peptide is SEQ ID No.5, and the amino acid sequence of MVF-GPSL-HER3559-591aa recombinant antigen peptide is SEQ ID No. 6.