CN102153644A

CN102153644A - Antigenic peptide for dimerization of epidermal growth factor receptor

Info

Publication number: CN102153644A
Application number: CN 201010618851
Authority: CN
Inventors: 李黄金; 朱磊; 吴梅芝
Original assignee: Guangdong Pharmaceutical University
Priority date: 2010-12-31
Filing date: 2010-12-31
Publication date: 2011-08-17
Anticipated expiration: 2030-12-31
Also published as: CN102153644B

Abstract

The invention discloses an antigenic peptide for the dimerization of an epidermal growth factor receptor, and relates to the field of the preparation of therapeutic polypeptide vaccines and antibody medicaments. An amino acid sequence of the antigenic peptide is SEQ ID NO: 1 or SEQ ID NO: 2 or a polypeptide which is subjected to the substitution and/or deletion and/or addition of one or more of amino acid residues and has the antigenic activity of the dimerization of the epidermal growth factor receptor. The invention also discloses a fusion antigenic peptide for the dimerization of the epidermal growth factor receptor, and the fusion antigenic peptide is a fusion peptide formed by the antigenic peptide and measles virus of fusion protein (MVF) serving as a general T cell epitope peptide. The antigenic peptide or the fusion antigenic peptide is mainly used for preparing the therapeutic vaccines. The preparation cost of the vaccines is far lower than that of various humanized or human antibodies, and large-dose and long-term repeat administration of the humanized or human antibodies is avoided, so the using cost is low, and the toxic and side effects are small. Compared with a chemical synthesis method, the recombination deoxyribonucleic acid (DNA) technology for preparing the antigenic peptide has the advantages that: the cost is low, and industrial amplification and quality control are easy.

Description

A kind of EGF-R ELISA dimerization antigen peptide

Technical field

The present invention relates to therapeutical peptide vaccine and antibody drug preparation field, be specifically related to a kind of EGF-R ELISA dimerization antigen peptide and preparation method thereof and antibody.

Background technology

Target medicine research and development are improving the malignant tumor medicine curative effect, are reducing and be significant aspect the toxic side effects.EGF-R ELISA (Epidermal growth factor receptor, EGFr) family (comprises EGFr, HER2/ErbB-2, HER3/ErbB-3 and HER4/ErbB-4 etc.) in the growing of embryo and healthy tissues, play an important role, but they are in a series of epithelial cancers, as nonsmall-cell lung cancer, mammary cancer, head and neck cancer, glioma, kidney, overexpression in the tumour cell such as cervical cancer and carcinoma of endometrium, and cause the fast breeding of malignant cell, shift, neovascularization, resistance and anti-apoptosis have become one of the most popular tumour medicine target molecule at present.

The EGFr receptoroid is a transmembrane glycoprotein, by extracellular region, stride zone such as three of film district and intracellular regions etc. and form, complete acceptor molecule extracellular region contains ligand binding domain, intracellular region contains the tyrosine kinase activity district, the two is the main target site of antitumor drug, and mainly is the monoclonal antibody (chimeric antibody or humanized antibody) of anti-extracellular domain and the micromolecular inhibitor that suppresses tyrosine kinase activity at the product form of this two target spot.At present existing anti-EGFr antibody (C225/Erbitux, chimeric antibody), Anti-HER 2 (Trastuzumab/Herceptin, humanized antibody) and small molecules tyrosine kinase inhibitor official listings such as (ZD1839, OSI-774).Its target of monoclonal antibody series products is stronger, and toxic side effect is littler; In addition, kill the tumour cell the cell-mediated cytotoxicity that antibody product still can utilize ADCC(antibody to rely on by the disabling signal path except direct as tyrosine kinase inhibitor) effect poisoning tumour cell, net effect is better.Therefore, the monoclonal antibody series products is more favored in new drug development and clinical application than tyrosine kinase inhibitor.

There is polymorphism in the up-regulated of EGFr family receptors between all kinds of epithelial cancer cells.The acceptor specy difference of up-regulated in different epithelial cancer types even the different subtype of the same race has about 30% to cross expression EGFr or ErbB-2 approximately, and other has about 5% to cross expression ErbB-3, has limited the antitumor spectra of this antibody-like.In addition, need to adopt complicated molecular biology and immunology means to identify the acceptor type of expressing before the medication, therefore brought great trouble to medication clinically.In the signal transduction process of EGFr family receptors mediation, the outer ligand binding domain of extracellular signaling molecule (part) and born of the same parents combines and causes conformational change and cause " Dimerized " between acceptor molecule, the Dimerized activation that further causes Tyrosylprotein kinase district in the born of the same parents, thereby excite the conduction of downstream signal, produce series of biologic effects such as cell proliferation.Dimerized can between acceptor molecule of the same race or the heterologous receptor of same family is intermolecular carrying out, form homodimer or heterodimer respectively.HER2 lacks ligand binding domain, but the Dimerized respective interface of its participation is in exposed state all the time, therefore need not part and induces and can form heterodimer with other member of this family.Tyrosylprotein kinase district in the HER3 disappearance born of the same parents can form heterodimer with other member and come conducted signal.Therefore, " Dimerized " is early stage committed step and the total incident of EGFr family signal conduction.Pertuzumab and 806 is antibody products of new generation of developing in recent years, has been in the III clinical trial phase stage at present.The two is respectively at the Dimerized interface region of HER2 and HER3, HER2 is crossed expression for the former and the non-HER3 positive tumor cell of expressing of crossing all has remarkable toxicity, and crossing the expressing tumor cell to EGFr and HER3, the latter all has remarkable killing activity, obviously, this type of antibody at the dimerization interface has more anti-knurl broad spectrum.Because EGFr is the most perfect, the member the most widely that distributes of structure in its receptor family, and place, Dimerized interface sequence is highly conserved sequence, therefore has reason to believe that the antibody at EGFr " Dimerized " interface may have more antitumor broad effect spectrum.

Although the monoclonal antibody series products has shown good curative effect clinically, this series products mainly is chimeric antibody or humanized antibody, needs the large scale and high density cell cultures, complex process, production cost height.In addition, Antybody therapy belongs to passive immunization therapy,, transformation period weak point big because of the antibody molecule amount, so need long-term, heavy dose of repeat administration (weekly, each hundreds of milligram), therefore bring bigger security risk (can cause acute myocardial injury) and heavy economical load (about 50,000 dollars of annual cost) as having found Herceptin at present to the patient.Active immunity treatment can be brought out body interior humoral immune reaction or t cell immune response at tumor associated antigen (TAA), and can form the immunological memory sexual cell, therefore need not the repeat administration as the passive immunization.In theory, TAA is not an antigen truly, because they belong to autoantigen, human body has immunotolerance to it.But in the tumour patient body, can detect specific antibody and the t cell immune response of TAA, and the EGF vaccine the fact such as official listing show, might break the immunological tolerance of patient by external source TAA administration, thereby excite body fluid and cell immune response TAA.Therefore, exploitation becomes possibility at the therapeutical peptide vaccine at EGFr receptoroid " Dimerized " interface.

Traditional polypeptide vaccine is in big carrier proteins, with the enhancement antigen peptide based immunogens with the chemical coupling of small molecules antigen peptide.Carrier proteins commonly used has bSA (BSA), oralbumin (OVA) and key hole keyhole limpet hemocyanin (KLH) etc., but such carrier proteins complex structure, and belonging to natural extract, molecular size and quality homogeneity are poor, are difficult to use in clinical.Branch oligomerization Methionin is the class novel carriers molecule that developed recently comes out, and is little and to be branch dendritic because of molecular weight, but the equal antigen peptide in the coupling of the ε-NH2 of nearly all Methionin forms so-called multiple antigenic peptide (MAP).MAP has stronger immunogenicity, but the too intensive polymerization of epitope may cause the change of epi-position conformation and influence this type of antigenic specificity, and may there be stronger cytotoxicity in poly-lysine.P64K albumen be by the Cuba scientist find a kind of from meningococcal outer membrane protein, this albumen has strong immunogenicity, and molecular size is moderate, and is simple in structure, bacillus coli gene engineering system mass production be can pass through, low-coat scale production and Quality Control therefore are easy to.At present, by Cuba exploitation with recombinant human EGF(hEGF) with the polypeptide vaccine approved listing of reorganization P64K chemical coupling, and just carrying out the II clinical trial phase in the North America.This vaccine is used for nonsmall-cell lung cancer (EGFr crosses expression) patient Shi Ke and induces the anti-hEGF antibody of high titre, and significantly increases the The median survival time rate, and only shows slight fash reaction aspect untoward reaction.This vaccine is the polypeptide vaccine of first official listing in the world.

Difference by the epitope type that adopts can be divided into polypeptide vaccine T cell vaccine and B cell vaccine two big classes roughly.The past polypeptide vaccine of development is mainly the T cell vaccine, and its reason is that T cellular immunization is considered to topmost immunization route always.Although existing numerous CTL and Th cell epitope peptide are used for clinical trial (195,197,221) as vaccine, do not get permission listing up to still having the t cell epitope polypeptide vaccine at present, its reason is that mainly there are specificity HLA restriction in CTL or Th cell epitope.B cell epitope vaccine does not have the such HLA restriction of t cell epitope, and the successful listing of antibody class product and EGF vaccine humoral immunization series products such as (B cell epitope vaccines) shows further that then the B cell vaccine has more DEVELOPMENT PROSPECT than T cell vaccine.At present, analyze the B cell epitope that two kinds of anti-" Dimerized " antibody such as Pertuzumab and 806 grades separate, identify HER2 and HER3 respectively by experiment, such epi-position is " linearizing " epi-position, is positioned at " Dimerized " interface zone conservative, that disulfide linkage enriches.

Forming fusogenic peptide with B cell epitope and " general-purpose Th epi-position " is a kind of new B cell polypeptide vaccine construction form that U.S. scientist Kaumaya proposes in recent years.Th epi-position wherein is from the micromolecule polypeptide of Measles virus fusion rotein (MVF) or Toxoid,tetanus (TT), is made up of the amino-acid residue about 18, can effectively break the restriction of MHC II quasi-molecule, therefore is called as " general-purpose Th epi-position ".The laboratory of Kaumaya utilizes a few strain Anti-HER 2 experiment Analysis to obtain a series of B cells " linearity " epi-position of HER2, comprising the B cell epitope from " Dimerized " interface.They have made up a series of B cell epitopes-MVF fusogenic peptide with chemical synthesis process.In cross the I clinical trial phase that the expressing tumor patient done with HER2, these fusogenic peptide vaccines have all shown good immunogenicity, and 3 subcutaneous vaccination immunity can be induced high titre Anti-HER 2 reaction in vivo, and produce notable therapeutic effect.

Summary of the invention

The object of the present invention is to provide a kind of EGF-R ELISA dimerization antigen peptide.

Another object of the present invention provides the fusogenic peptide of above-mentioned antigen peptide and Universal T-cell epitopes peptide MVF composition.

The present invention is achieved through the following technical solutions above-mentioned purpose:

A kind of EGF-R ELISA dimerization antigen peptide has the Dimerized antigenic activity of EGF-R ELISA, and following (a) and (b), (c) or aminoacid sequence (d) are arranged:

（a）SEQ?ID?NO:?1；

(b) with the aminoacid sequence of SEQ ID NO:1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have EGF-R ELISA dimerization antigenic activity;

（c）SEQ?ID?NO:?2；

(d) with the aminoacid sequence of SEQ ID NO:2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have EGF-R ELISA dimerization antigenic activity.

A kind of EGF-R ELISA dimerization fused antigen peptide is the fusogenic peptide that above-mentioned any antigen peptide and Universal T-cell epitopes peptide MVF form.

Above-mentioned fused antigen peptide, the fusogenic peptide of forming according to SEQ ID NO:1 sequence and t cell epitope peptide MVF has the aminoacid sequence of following (e):

(e) SEQ ID NO:3; According to the sub-degeneracy of amino acid code, its sequence can also have following (f) described version:

(f) aminoacid sequence of SEQ ID NO:3 had identical function through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and with the protein of (e).

The fusogenic peptide of forming according to SEQ ID NO:2 sequence and t cell epitope peptide MVF has the aminoacid sequence of following (g), also can do the version described in (h) simultaneously:

（g）SEQ?ID?NO:?4；

(h) aminoacid sequence of SEQ ID NO:4 had identical function through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and with the protein of (g).

The method for synthesizing gene of above-mentioned fused antigen peptide SEQ ID NO:3 is: with sequence SEQ ID NO:5 and SEQ ID NO:6 primer and template each other, carry out first round PCR; Being template with first round PCR product then, is primer with SEQ ID NO:7 and SEQ ID NO:8, carries out second and takes turns PCR; Taking turns the PCR product with second again is template, is that primer carries out third round PCR with SEQ ID NO:9 and SEQ ID NO:10, obtains product.

The method for synthesizing gene of above-mentioned fused antigen peptide SEQ ID NO:4 is: with sequence SEQ ID NO:11 and SEQ ID NO:12 primer and template each other, carry out first round PCR; Being template with first round PCR product then, is primer with SEQ ID NO:13 and SEQ ID NO:14, carries out second and takes turns PCR; Taking turns the PCR product with second again is template, is that primer carries out third round PCR with SEQ ID NO:15 and SEQ ID NO:16; With third round PCR product is template, is that primer carries out four-wheel PCR with SEQ ID NO:17 and SEQ ID NO:18; With four-wheel PCR product is template, is that primer carries out the 5th and takes turns PCR with SEQ ID NO:9 and SEQ ID NO:19, obtains product S EQ ID NO:4.

A kind of fused antigen peptide, be the epi-position HER2(266-296 that anti-HER2 dimerization antibody pertuzumab isolation identification arrives) fusogenic peptide formed with Universal T-cell epitopes peptide MVF, aminoacid sequence is SEQ ID NO:20, its method for synthesizing gene is: with sequence SEQ ID NO:21 and SEQ ID NO:22 primer and template each other, carry out first round PCR; Being template with first round PCR product then, is primer with SEQ ID NO:7 and SEQ ID NO:23, carries out second and takes turns PCR; Taking turns the PCR product with second again is template, is that primer carries out third round PCR with SEQ ID NO:9 and SEQ ID NO:24, obtains product S EQ ID NO:20.

Above-mentioned antigen peptide or fused antigen peptide are crossed application in the epithelial cancer medicine of expression at preparation treatment EGFr.

Above-mentioned application can be as the active ingredient of therapeutical peptide vaccine or as antigen prepd antibody.Antibody comprises monoclonal antibody and polyclonal antibody.

Above-mentioned epithelial cancer comprises nonsmall-cell lung cancer, head and neck cancer, mammary cancer, carcinoma of endometrium, prostate cancer, kidney, cancer of the stomach etc.

The EGF-R ELISA dimerization antigen peptide mechanism of action of the present invention: EGF-R ELISA (EGFr) is a class transmembrane receptor, mainly by extracellular domain, stride the film district and born of the same parents' internal area is formed, extracellular domain contains ligand binding domain, and born of the same parents' internal area contains tyrosine kinase activity.Ligand molecular with can cause that homology between acceptor molecule (between two EGFr) or allos are (with other member of family after the ligand binding domain of extracellular domain combines, as HER2, HER3 and HER4) dimerization, cause the activation of born of the same parents' internal area Tyrosylprotein kinase, thereby cause the transmission of downstream signal, cause cell proliferation, anti-apoptosis and induce relevant genetic expressions such as neovascularization.The EGFr family member crosses and expresses and epitheliomatous generation, development and prognosis height correlation.Antigen peptide of the present invention can stimulate the antibody that produces anti-EGFr dimerization in the body, and blocking-up EGFr crosses the signal path expressing tumor cell, relevant with anti-apoptosis with propagation, thereby reaches the result of treatment of control tumor cell proliferation, transfer and neovascularization.

Compared with prior art, the present invention has following beneficial effect:

(1) antigen peptide of the present invention be at EGFr cross express epitheliomatous, and existing antigen peptide is respectively at the B cell epitope of HER2(antibody Pertuzumab) and the B cell epitope of HER3(antibody 806).Application and scope all are different from existing antigen peptide.

(2) antibody that produces of antigenic stimulation of the present invention is primarily aimed at homologous dimerizationization (between the EGFr) between the EGFr acceptor molecule and allos dimerization (EGFr and with other member of family, as HER2, HER3 and HER4), none is Dimerized at EGFr to be used for clinical antibody at present.The antibody that is produced mainly combines with the dimerization interface, and the exposure at dimerization interface belongs to the middle transition conformation, relative less with the chance of antibody contact, the cell that has only EGFr to cross expression just has more dimerization interface to expose, so anti-Dimerized antibody or vaccine are less relatively to the normal cell toxicity that acceptor does not have up-regulated, and because of the anti-Dimerized signal path that can block other member's mediation of EGFr receptor family simultaneously, so anticancer effect is better.

(3) antigen peptide of the present invention is mainly used in preparation therapeutic type vaccine.Because of the vaccine manufacturing cost far below all kinds of humanizations or total man source antibody, and do not need the such heavy dose of the latter, repeat administration over a long time, so use cost is low, toxic side effects is little.

(4) the present invention prepares antigen peptide with recombinant DNA technology, and is lower than the chemical synthesis cost of routine, be easier to industrialization amplification and Quality Control.

Description of drawings

Fig. 1: the B cell epitope compare of analysis at the Dimerized interface of anti-EGFr family;

Fig. 2: the B cell epitope peptide that anti-EGFr is Dimerized;

Fig. 3: the antigen peptide of the MVF-GPSL-B cell epitope peptide form that anti-EGFr is Dimerized;

Fig. 4: the antigen peptide gene order of the MVF-GPSL-B cell epitope peptide form that anti-EGFr is Dimerized;

Fig. 5: the antigen peptide gene electrophorogram of the MVF-GPSL-B cell epitope peptide form that anti-EGFr is Dimerized, wherein 1 is DNA Maker, 2 and 3 is MVF-GPSL-Her2 (266-296), 4 and 5 is MVF-GPSL-EGFr (262-292), 6 is MFV-GPSL-EGFr (311-326), and 7 is MVF-GPSL-EGFr (262-326);

Fig. 6: the Dimerized B cell epitope of anti-EGFr antigen peptide gene expression product soluble analysis, M is a molecular weight standard, and 1 is the broken supernatant of MVF-GPSL-EGFr (262-292) engineering bacteria, and 2 is the fragmentation precipitation of MVF-GPSL-EGFr (262-292) engineering bacteria; 3 is the broken supernatant of MVF-GPSL-EGFr (311-326) engineering bacteria, 4 is the fragmentation precipitation of MVF-GPSL-EGFr (311-326) engineering bacteria, 5 is the broken supernatant of MVF-GPSL-EGFr (262-326) engineering bacteria, and 6 is the fragmentation precipitation of MVF-GPSL-EGFr (262-326) engineering bacteria;

Fig. 7: the Dimerized B cell epitope of anti-EGFr antigen peptide immunogenicity analytical results figure, wherein HER is MVF-GPSL-Her2 (266-296), EGFr-1 is MVF-GPSL-EGFr (262-292), EGFr-2 is MFV-GPSL-EGFr (311-326), and EGFr-3 is MVF-GPSL-EGFr (262-326);

Fig. 8: anti-antigen peptide antibody is to squamous cell carcinoma A431 inhibition analysis (mtt assay) figure as a result, Anti-HER is anti-MVF-GPSL-Her2 (266-296) antibody, anti-EGFr-1 is antibody MVF-GPSL-EGFr (262-292) antibody, anti-EGFr-2 is anti-MFV-GPSL-EGFr (311-326) antibody, and anti-EGFr-3 is anti-MVF-GPSL-EGFr (262-326) antibody.

Embodiment

Among the following embodiment, EGFr(262-292) be SEQ ID NO:1, EGFr(262-326) be SEQ ID NO:2, MVF-GPSL-EGFr(262-292) be SEQ ID NO:3, MVF-GPSL-EGFr(262-326) being SEQ ID NO:4, MVF-GPSL-Her2(266-296) is SEQ ID NO:20.

Embodiment 1 EGFr family anti-Dimerized interface B cell epitope compare of analysis

According to the Dimerized antibody Pertuzumab of anti-HER2 and Dimerized antibody 806 isolation identification of anti-HER3 to known epi-position be HER2(266-296) and HER3(287-303), aminoacid sequence (GenBank ID:AAX41033 with this two epi-position and hEGFr, BAI46646) carry out the homologous sequence compare of analysis, result such as Fig. 1.The result shows, EGFr(262-292) and HER2(266-296) homologous sequence each other, homology is 53%, the two all contains 2 cysteine residues, the position of this two residue and near sequence thereof or residue character high conservative.EGFr(311-326) sequence then with HER3(287-302) homology.In addition, EGFr(262-326) sequence is a highly conserved sequence, and corresponding sequence 100% homology with mouse, rabbit, chicken contains 3 pairs of disulfide linkage, is positioned at the abundant district of disulfide linkage of acceptor extracellular region.

According to above-mentioned analytical results, EGFr(262-326) should be the receptor dimerization interface region, choose EGFr(262-292), EGFr(311-326) and, see Fig. 2 EGFr(262-326) as the Dimerized candidate B cell epitope peptide of anti-EGFr.

The antigen peptide of embodiment 2 preparation MVF-GPSL-B cell epitope peptide forms

Can adopt chemosynthesis or recombinant DNA technology to prepare the antigen peptide of MVF-GPSL-B cell epitope peptide form, above-mentioned B cell epitope peptide MVF-GPSL-EGFr(262-292), MVF-GPSL-EGFr(262-326 MVF-GPSL-EGFr(311-326)), MVF wherein is " a general-purpose Th epitope sequences ", and GPSL is a resilient connector.Simultaneously with MVF-GPSL-HER2(266-296) be contrast (Fig. 3).Chemical synthesis can adopt conventional peptide synthesis technology to carry out.DNA recombinant technology method is artificial antigen peptide-coding sequence (goal gene), and it is cloned in intestinal bacteria, yeast or other expression systems, induces goal gene to efficiently express in a suitable manner, again through conventional method separation and purification promptly.

The structure of the Dimerized B cell epitope of the anti-EGFr antigen peptide of recombinating:

Ordinary method makes up recombinant antigen peptide gene, and the Dimerized B cell epitope of anti-EGFr antigen peptide gene as shown in Figure 4.

(1) MFV-GPSL-EGFr (311-326) gene is synthetic

By following 3 stepping performing PCRs, the reaction conditions of PCR1 and PCR2 is 94 ℃ of 30 s, 68 ℃ of 30 s, and totally 25 circulations, the reaction conditions of PCR3 is 94 ℃ of 5 min, 94 ℃ of 30 s, 55 ℃ of 30 s, 72 ℃ of 30 s, totally 25 circulations, 72 ℃ of 7 min.

PCR1：SEQ?ID?NO:25+SEQ?ID?NO:26

PCR2：SEQ?ID?NO:7+?SEQ?ID?NO:26+PCR1

PCR3: SEQ?ID?NO:9+?SEQ?ID?NO:27+PCR2

(2) MFV-GPSL-EGFr (262-326) gene is synthetic

By following 5 stepping performing PCRs, the reaction conditions of PCR1-PCR4 is 94 ℃ of 30 s, 68 ℃ of 30 s, and totally 25 circulations, the reaction conditions of PCR5 is 94 ℃ of 5 min, 94 ℃ of 30 s, 55 ℃ of 30 s, 72 ℃ of 30 s, totally 25 circulations, 72 ℃ of 7 min.

PCR1:?SEQ?ID?NO:11+?SEQ?ID?NO:12

PCR2:?SEQ?ID?NO:13+?SEQ?ID?NO:14+PCR1

PCR3:?SEQ?ID?NO:15+?SEQ?ID?NO:16+PCR2

PCR4:?SEQ?ID?NO:17+?SEQ?ID?NO:18+PCR3

PCR5:?SEQ?ID?NO:9+?SEQ?ID?NO:19+PCR4

The Dimerized B cell antigen peptide of anti-EGFr gene assembling result as shown in Figure 5.The result shows that amplified production is consistent with theoretical size.

(3) gene clone

Above-mentioned amplified production is cloned according to a conventional method in the same loci of expression vector pET32a after with the NcoI-HindIII double digestion, so as with the Trx amalgamation and expression.Get fusion expression vector pET32a-MVF-EGFr(311-326 behind the clone) and pET32a-MVF-EGFr(262-326).The structure of all expression vectors all is the host bacterium with the bacillus coli DH 5 alpha, and institute's expression vector that obtains entrusts the big gene company limited of Shenzhen China to carry out the sequential analysis of destination gene expression box.Expression cassette is through sequencing analysis, and the result shows consistent with theoretical sequence.Expression vector plasmid after the sequential analysis conclusive evidence transforms expressive host bacterium e. coli bl21 (DE3), must express engineering bacteria.

(4) genetic expression is induced

Engineering bacteria is inoculated in the LB substratum that contains 50 μ g/mL penbritins, and 37 ℃ of shaking culture are spent the night, in 1%(V/V) the ratio fresh LB substratum of transferring, continue to cultivate 2.5h, add IPTG and carry out induced expression to final concentration 1mmol/L, coinduction 4h.Centrifugal 5 min of 6000 r/min collect thalline.The engineering bacteria that obtains after inducing with IPTG the big or small Trx-His6-antigen peptide fusion rotein consistent with theoretical value.

(5) expression product separation and purification

Through thalline that induced expression obtains by 10%(W/V) be suspended in the 20 mmol/L sodium phosphate buffers (pH7.5) that contain 500 mmol/L NaCl and 20mmol/L imidazoles, in ice bath with 300 watts of output rating intermittent types ultrasonication, 10 min, centrifugal 10 min of 12000 r/min.Get supernatant and cross above-mentioned broken bacterium damping fluid equilibrated HisTrap FF post, same buffer is washed flat back with 20 mmol/L sodium phosphate buffer (pH7.5) wash-outs that contain 500 mmol/L NaCl and 200mmol/L imidazoles, collects the target peak sample.The institute sample that obtains to 20mmol/L Tris-HCl(pH7.5) the damping fluid dialysed overnight, during change liquid 3 times.The desalination sample that obtains add recombinant enterokinase-His6 in the ratio of 80 μ g albumen/unit enzymes, 37 ℃ of insulation 8hr.Enzymolysis product is crossed HisTrap FF post, collects to penetrate liquid and be MVF-EGFr(311-326) and MVF-EGFr(262-326) fusogenic peptide, cross after the Sepharose G25 desalination-25 ℃ frozen standby.

(6) protein s DS-PAGE and gel scanning analysis

Carry out the SDS-PAGE of various samples with reference to ordinary method.Protein sample in yeast culture thing or the purge process boils 5 min with 2 times of sample-loading buffers, and reduction electrophoresis sample-loading buffer does not contain beta-mercaptoethanol.Gel that electrophoresis obtains is taken pictures and expression level or lipidated protein analysis by producer's explanation with " day energy board " gel density scan instrument.

The SDS-PAGE analytical results shows, except that the MVF-GPSL-EGFr(262-326 that contains 6 halfcystines) expression product almost all be the inclusion body, all the other are all based on solubility expression product (Fig. 6).

Expression product downcuts the Trx-His6 label with enteropeptidase behind the Ni column purification, after the Ni post and collect and penetrate liquid and be the antigen peptide of not being with any label.

The analysis of the Dimerized B cell antigen peptide based immunogens of the embodiment 3 anti-EGFr of reorganization

Purified antigen peptide immune mouse and rabbit by the following method behind determination of protein concentration.

1. antigen emulsification

Use the 2-5mL syringe, get 1.25mL and contain antigenic PBS, another syringe is got (equal-volume) Fu Shi Freund's complete adjuvant or Freund, connect with the stainless steel jointing, push-and-pull mixed about 10-30 minute repeatedly, kept flat and left standstill 30 minutes, no longer separate mutually as water and milk, i.e. the injectable animal.

2. immune programme for children

Get female BALB/c mouse and new zealand white rabbit breeding time, use for the first time Fu Shi Freund's complete adjuvant, every 100 μ g of mouse, subcutaneous multi-point injection, total dose 0.2mL/ only, every 1000 μ g of White Rabbit, subcutaneous multi-point injection, total dose 0.5mL/ are only, reinstate for the second time freund 's incomplete adjuvant, dosage at interval 3 weeks, is injected back 10 days with ELISA method survey serum antibody titer for the third time with for the first time identical.Do not reach 1 * 10 as titre ⁴More than, then continue immunity.

3. antibody titers is measured

Get 56 ℃ of insulations of antiserum(antisera) 30min, with the deactivation complementary interaction.With the PBS solution bag that contains 2 μ g/mL antigen peptide of 50 μ l by 96 orifice plates, 4 ℃ spend the night or 37 ℃ leave standstill 2hr.Abandon most coating buffer, every hole is with the PBS sealing that contains 1% BSA of 100 μ L, 4 ℃ spend the night or 37 ℃ leave standstill 2hr.Abandon most confining liquid, the antiserum(antisera) of getting the mouse-anti human IgG according to 1:1000,1:2000 ... doubling dilution adds in the 50 μ L respective aperture, adds the normal mouse serum of 1:1000 or PBS as negative control, and the mouse-anti human IgG of 1:100 is as positive control.Hatch 1hr for 37 ℃, wash 5 times, pat dry on the thieving paper, add " two is anti-" (sheep anti-mouse igg-HRP), hatch 1hr for 37 ℃ with the PBT washing lotion.Wash 5 times with the PBT washing lotion, thieving paper pats dry, and adds substrate colour developing about 10 minutes, and the H2SO4 termination reaction of 1N is surveyed OD410 with microplate reader, deduct that OD410 reaches behind the background 〉=and 0.2 o'clock maximum dilution multiple is titre.If 3 repetitions.

Every kind of antigen peptide has 5 mouse, and the data of each cylinder are from the titer determination value of 1 mouse resisting anteserum.The antiserum(antisera) that obtains get the titre of antibody that each antigen peptide produces through elisa assay, see Fig. 7.The result shows, 5 mouse of all antigen peptide inoculations have all produced and have been higher than 40000 titer antibody, wherein MVF-GPSL-EGFr(262-326) antibody titers a little more than other antigen peptide, the highest titre reaches 160000.

Embodiment 4 anti-recombinant anti-epidermal growth factor receptors Dimerized B cell epitope antigen peptide antibody purification and antibody activity analysis

Get 56 ℃ of insulations of White Rabbit antiserum(antisera) 30min among the embodiment 3, with the deactivation complementary interaction.Carry out purifying in order to HiTrap Protein G post according to shop instruction, wherein column balance buffering liquid is 20mmol/L sodium phosphate buffer (pH7.4), elution buffer be 100mmol/L glycine-HCl(pH2.7), purpose peak sample is collected in and presets neutralization buffer 1mol/L Tris-HCl(pH9.0).Survey the protein concentration of antibody ,-20 ℃ of preservations are standby.

The antibody purification that obtains detect to suppress the activity that EGFr crosses the squamous cell carcinoma A431 growth of expression with mtt assay.Get the squamous cell carcinoma strain A431 that EGFr crosses expression, by 1 * 10 ⁴Cells/well is inoculated 96 orifice plates, and 37 ℃ of overnight incubation add the anti-peptide antibody of rabbit of 1.9 described purifying of 0.1 μ g/mL, and IgG makes negative control with mouse, cultivates 72 hr for 37 ℃.Survey OD570, calculate inhibiting rate (%) by following formula: (the anti-peptide antibody of OD normal rabbit IgG-OD) * normal rabbit igg of 100/ OD.If 3 repetitions.Detected result as shown in Figure 8, the result shows that prepared anti-peptide antibody can effectively suppress the growth that EGFr crosses expressing tumor cell A431.

The result shows, but produces the specific antibody of high titre by EGFr dimerization B cell epitope fused antigen peptide inducing mouse disclosed by the invention and rabbit, and the growth that this antibody-like can be crossed the human tumor cells A431 of expression at external effective inhibition EGFr.Because the B cell epitope that is adopted has 100% homology between people, mouse and rabbit, therefore can infer that above-mentioned antigen peptide also can induce specific antibody high titre, that anti-EGFr is Dimerized to produce in human body, as long as and have high titre antibody to produce to reach and be similar to anti-Dimerized antibody Pertuzumab and 806 such therapeutic effect of malignant tumour.

SEQUENCE?LISTING

＜110〉Guangdong Pharmaceutical University

＜120〉a kind of EGF-R ELISA dimerization antigenic peptide sequence table

<130>

<160> 27

<170> PatentIn?version?3.3

<210> 1

<211> 31

<212> PRT

＜213〉artificial sequence

<400> 1

Asp?Thr?Cys?Pro?Pro?Leu?Met?Leu?Tyr?Asn?Pro?Thr?Thr?Tyr?Gln?Met

1 5 10 15

Asp?Val?Asn?Pro?Glu?Gly?Lys?Tyr?Ser?Phe?Gly?Ala?Thr?Cys?Val

20 25 30

<210> 2

<211> 65

<212> PRT

＜213〉artificial sequence

<400> 2

Asp?Thr?Cys?Pro?Pro?Leu?Met?Leu?Tyr?Asn?Pro?Thr?Thr?Tyr?Gln?Met

1 5 10 15

Asp?Val?Asn?Pro?Glu?Gly?Lys?Tyr?Ser?Phe?Gly?Ala?Thr?Cys?Val?Lys

20 25 30

Lys?Cys?Pro?Arg?Asn?Tyr?Val?Val?Thr?Asp?His?Gly?Ser?Cys?Val?Arg

35 40 45

Ala?Cys?Gly?Ala?Asp?Ser?Tyr?Glu?Met?Glu?Glu?Asp?Gly?Val?Arg?Lys

50 55 60

Cys

65

<210> 3

<211> 53

<212> PRT

＜213〉artificial sequence

<400> 3

Lys?Leu?Leu?Ser?Leu?Ile?Lys?Gly?Val?Ile?Val?His?Arg?Leu?Glu?Gly

1 5 10 15

Val?Glu?Gly?Pro?Ser?Leu?Asp?Thr?Cys?Pro?Pro?Leu?Met?Leu?Tyr?Asn

20 25 30

Pro?Thr?Thr?Tyr?Gln?Met?Asp?Val?Asn?Pro?Glu?Gly?Lys?Tyr?Ser?Phe

35 40 45

Gly?Ala?Thr?Cys?Val

50

<210> 4

<211> 89

<212> PRT

＜213〉artificial sequence

<400> 4

Pro?Thr?Leu?Ser?Glu?Ile?Lys?Gly?Val?Ile?Val?His?Arg?Leu?Glu?Gly

1 5 10 15

Val?Glu?Gly?Pro?Ser?Leu?Asp?Thr?Cys?Pro?Pro?Leu?Met?Leu?Tyr?Asn

20 25 30

Pro?Thr?Thr?Tyr?Gln?Met?Asp?Val?Asn?Pro?Glu?Gly?Lys?Tyr?Ser?Phe

35 40 45

Gly?Ala?Thr?Cys?Val?Lys?Lys?Cys?Pro?Arg?Asn?Tyr?Val?Val?Thr?Asp

50 55 60

His?Gly?Ser?Cys?Val?Arg?Ala?Cys?Gly?Ala?Asp?Ser?Tyr?Glu?Met?Glu

65 70 75 80

Glu?Asp?Gly?Val?Arg?Lys?Cys?Lys?Lys

85

<210> 5

<211> 59

<212> DNA

＜213〉artificial sequence

<400> 5

ttcaccgtct?tgaaggtgtt?gaaggtccgt?ctcttgatac?ttgtccgccg?cttatgctt 59

<210> 6

<211> 59

<212> DNA

＜213〉artificial sequence

<400> 6

ttccgggtta?acatccatct?ggtaagtagt?cgggttgtaa?agcataagcg?gcggacaag 59

<210> 7

<211> 51

<212> DNA

＜213〉artificial sequence

<400> 7

aaacttctta?gccttatcaa?aggtgttatc?gttcaccgtc?ttgaaggtgt?t 51

<210> 8

<211> 50

<212> DNA

＜213〉artificial sequence

<400> 8

aacacaagta?gcaccgaagg?agtatttacc?ttccgggtta?acatccatct 50

<210> 9

<211> 46

<212> DNA

＜213〉artificial sequence

<400> 9

ggagccatgg?gtaaacttct?tagccttatc?aaaggtgtta?tcgttc 46

<210> 10

<211> 46

<212> DNA

＜213〉artificial sequence

<400> 10

atccaagctt?ttaaacacaa?gtagcaccga?aggagtattt?accttc 46

<210> 11

<211> 59

<212> DNA

＜213〉artificial sequence

<400> 11

ggaaggtaaa?tactccttcg?gtgctacttg?tgttaagaaa?tgtccgcgta?actacgttg 59

<210> 12

<211> 59

<212> DNA

＜213〉artificial sequence

<400> 12

caacgtagtt?acgcggacat?ttcttaacac?aagtagcacc?gaaggagtat?ttaccttcc 59

<210> 13

<211> 49

<212> DNA

＜213〉artificial sequence

<400> 13

ccgtctcttg?atacttgtcc?gccgcttatg?ctttacaacc?cgactactt 49

<210> 14

<211> 49

<212> DNA

＜213〉artificial sequence

<400> 14

agcacgaaca?caagaaccgt?gatcagtaac?aacgtagtta?cgcggacat 49

<210> 15

<211> 49

<212> DNA

＜213〉artificial sequence

<400> 15

tcgttcaccg?tcttgaaggt?gttgaaggtc?cgtctcttga?tacttgtcc 49

<210> 16

<211> 49

<212> DNA

＜213〉artificial sequence

<400> 16

tcttccattt?cgtaggaatc?agcaccacaa?gcacgaacac?aagaaccgt 49

<210> 17

<211> 48

<212> DNA

＜213〉artificial sequence

<400> 17

ccgactcttt?ctgaaatcaa?aggtgttatc?gttcaccgtc?ttgaaggt 48

<210> 18

<211> 45

<212> DNA

＜213〉artificial sequence

<400> 18

ctttttacat?ttacgaacac?cgtcttcttc?catttcgtag?gaatc 45

<210> 19

<211> 44

<212> DNA

＜213〉artificial sequence

<400> 19

ggagaagctt?ttacttttta?catttacgaa?caccgtcttc?ttcc 44

<210> 20

<211> 182

<212> DNA

＜213〉artificial sequence

<400> 20

ggagcatatg?aaacttctta?gccttatcaa?aggtgttatc?gttcaccgtc?ttgaaggtgt 60

tgaaggtccg?tctcttcttc?actgtccggc?tcttgttacc?tacaacaccg?ataccttcga 120

atctatgccg?aacccggaag?gtcgctacac?cttcggtgct?tcctgtgttt?aaaagcttgg 180

at 182

<210> 21

<211> 59

<212> DNA

＜213〉artificial sequence

<400> 21

ttcaccgtct?tgaaggtgtt?gaaggtccgt?ctcttcttca?ctgtccggct?cttgttacc 59

<210> 22

<211> 59

<212> DNA

＜213〉artificial sequence

<400> 22

ttccgggttc?ggcatagatt?cgaaggtatc?ggtgttgtag?gtaacaagag?ccggacagt 59

<210> 23

<211> 50

<212> DNA

＜213〉artificial sequence

<400> 23

aacacaggaa?gcaccgaagg?tgtagcgacc?ttccgggttc?ggcatagatt 50

<210> 24

<211> 46

<212> DNA

＜213〉artificial sequence

<400> 24

atccaagctt?ttaaacacag?gaagcaccga?aggtgtagcg?accttc 46

<210> 25

<211> 51

<212> DNA

＜213〉artificial sequence

<400> 25

ttcaccgtct?tgaaggtgtt?gaaggtccgt?ctctttgtgg?tgctgattcc?t 51

<210> 26

<211> 52

<212> DNA

＜213〉artificial sequence

<400> 26

acatttacga?acaccgtctt?cttccatttc?gtaggaatca?gcaccacaaa?ga 52

<210> 27

<211> 47

<212> DNA

＜213〉artificial sequence

<400> 27

atccaagctt?ttaacattta?cgaacaccgt?cttcttccat?ttcgtag 47

Claims

1. EGF-R ELISA dimerization antigen peptide has following (a) and (b), (c) or aminoacid sequence (d):

（a）SEQ?ID?NO:?1；

（c）SEQ?ID?NO:?2；

2. an EGF-R ELISA dimerization fused antigen peptide is the fusogenic peptide that described antigen peptide of claim 1 and Universal T-cell epitopes peptide MVF form.

3. fused antigen peptide according to claim 2 has following (e) or aminoacid sequence (f):

（e）SEQ?ID?NO:?3；

4. fused antigen peptide according to claim 2 has following (g) or aminoacid sequence (h):

（g）SEQ?ID?NO:?4；

5. according to the described fused antigen peptide of claim 3, it is characterized in that the method for synthesizing gene of SEQ ID NO:3 is:, carry out first round PCR with sequence SEQ ID NO:5 and SEQ ID NO:6 primer and template each other; Being template with first round PCR product then, is primer with SEQ ID NO:7 and SEQ ID NO:8, carries out second and takes turns PCR; Taking turns the PCR product with second again is template, is that primer carries out third round PCR with SEQ ID NO:9 and SEQ ID NO:10, obtains product.

6. according to the described fused antigen peptide of claim 4, it is characterized in that the method for synthesizing gene of SEQ ID NO:4 is:, carry out first round PCR with sequence SEQ ID NO:11 and SEQ ID NO:12 primer and template each other; Being template with first round PCR product then, is primer with SEQ ID NO:13 and SEQ ID NO:14, carries out second and takes turns PCR; Taking turns the PCR product with second again is template, is that primer carries out third round PCR with SEQ ID NO:15 and SEQ ID NO:16; With third round PCR product is template, is that primer carries out four-wheel PCR with SEQ ID NO:17 and SEQ ID NO:18; With four-wheel PCR product is template, is that primer carries out the 5th and takes turns PCR with SEQ ID NO:9 and SEQ ID NO:19, obtains product S EQ ID NO:4.

7. EGF-R ELISA dimerization fused antigen peptide, it is characterized in that the epi-position HER2(266-296 that anti-HER2 dimerization antibody pertuzumab isolation identification arrives) fusogenic peptide formed with Universal T-cell epitopes peptide MVF, nucleotides sequence is classified SEQ ID NO:20 as, its method for synthesizing gene is: with sequence SEQ ID NO:21 and SEQ ID NO:22 primer and template each other, carry out first round PCR; Being template with first round PCR product then, is primer with SEQ ID NO:7 and SEQ ID NO:23, carries out second and takes turns PCR; Taking turns the PCR product with second again is template, is that primer carries out third round PCR with SEQ ID NO:9 and SEQ ID NO:24, obtains product S EQ ID NO:20.

8. the described antigen peptide of claim 1 is crossed application in the epithelial cancer medicine of expression at preparation treatment EGFr.

9. application according to claim 8 is characterized in that as the active ingredient of therapeutical peptide vaccine or as antigen prepd antibody.

10. application according to claim 8 is characterized in that described epithelial cancer is nonsmall-cell lung cancer, head and neck cancer, mammary cancer, carcinoma of endometrium, prostate cancer, kidney or cancer of the stomach.