CN101429234B - Vaccine based on simulating human blood vessel endothelial cell growth factor VEGF epitope and preparation method thereof - Google Patents

Abstract

The invention provides a vaccine based on an epitope simulating human vascular endothelial growth factor VEGF, as well as a preparation method thereof. A VEGF mimic epitope which is specifically affinitive with human-mouse chimeric monoclonal antibody Avastin is screened out by use of a phage random presentation technique, and the amino acid sequence of the mimic epitope is Asp-His-Thr-Leu-Tyr-Thr-Pro-Tyr-His-Thr-His-Pro; the mimic epitope has no homology with the protein sequence of VEGF. A vaccine which can induce a polypeptide epitope aiming at VEGF molecular autoantibody in vivo is constructed on the basis of the mimic epitope. The invention provides a strategy for developing and designing the tumor therapeutic vaccine, which is targeted at the VEGF. The VEGF is one of molecules which has the strongest effect of promoting vascular growth, and is an ideal target for resisting angiogenesis and treating tumors. Therefore, the vaccine replaces or replenishes monoclonal antibody passive immunotherapy with an active immunity mode, so as to lay foundations for overcoming the defects of monoclonal antibody therapy.

Description

Based on vaccine of anthropomorphic dummy's blood vessel endothelial cell growth factor VEGF epitope and preparation method thereof

Technical field

The invention belongs to the medical biotechnology field, be specifically related to a kind of based on anthropomorphic dummy's blood vessel endothelial cell growth factor VEGF mimic epitopes and can induce polypeptide epitope vaccine at VEGF molecule autoantibody and preparation method thereof in vivo with what this mimic epitopes made up.

Background technology

1, the correlative study of VEGF molecule and monoclonal antibody thereof

1.1VEGF molecule

The VEGF relative molecular weight is 34-42KD, is to be that the homodimer of 17-22KD connects by disulfide linkage and forms by molecular weight.The gene structure of human VEGF is positioned at chromosomal 6p21.3, and this full length gene is 28kb, and the gene of coding VEGF is about 1.4 ten thousand base pairs, alternately is made of 8 exons and 7 introns.Because the different cut modes of VEGF mRNA can make it that 5 kinds of VEGF isomer are arranged.According to amino acid whose big or small called after V EGF206 (comprising the 7th intron of all exons and part), VEGF189 (comprising 8 exons), VEGF165 (lacking exon 6), VEGF145 (lacking exon 7) and VEGF121 (lacking exon 6 and 7), comparatively common in the body with VEGF165.The isomer of 5 kinds of VEGF all has the outgrowth relative reactivity of inducing endothelial cell, and the difference on its function is that mainly they are different with the avidity of heparin in cell surface and the extracellular matrix.

The VEGF molecule is by bringing into play biological effect with vascular endothelial cell surface specific receptors bind.Its main biological function is to promote endothelial cell proliferation, promote angiogenic growth and increase vascular permeability.Now prove Ca in the cell that to increase sharply after VEGF and the receptors bind ²⁺Level by phosphoinositide specificity lecithinase C approach, makes IP in the cell ₃Level raises, and signal in the transfer cell is finally finished biological effect.

1.2VEGF the current situation of monoclonal antibody

In about 30 years of past, by

The monoclonal antibody technique that proposes with Milstein for a series of diseases in-vitro diagnosis opportunity is provided.Yet the antigenic characteristic of the specific recognition of monoclonal antibody was just just shown up prominently in nearly 10 years in the immunotherapy of human diseases.At present, monoclonal antibody (mAbs) is used to the diagnosis and the treatment of cancer in clinical, control autoimmune disease, graft-vs-host reaction and allograft rejection, and brain (ridge) marrow inflammation, myocardial damage and the reversible drug intoxication etc. of the initiation of treatment infectation of bacteria.

VEGF promotes one of molecule that the tumor vascular growth effect is the strongest, high this molecule of secretion of kinds of tumors; Therefore this molecule becomes the important target in the angiogenesis inhibitor treatment tumour method.A Wasiting (Avastin) is the anti-VEGF monoclonal antibody of a kind of humanization, can combine with people VEGF, the downstream signal path that mediates after blocking VEGF and the receptors bind, thereby the biologic activity that suppresses people VEGF, comprise and suppress endotheliocyte mitogenic activity, the active and short angiogenic activity of vascular permeability increase, reach antitumor purpose.

To be first be used for the monoclonal antibody formulation that antineoplastic vascular generates treatment by the FDA approval to Avastin, was used for the treatment of colorectal cancer and nonsmall-cell lung cancer by the FDA approval in 2004, and its antineoplastic vascular that appears as generates therapy and started new world.The Avastin advantage has: its action target spot directly is exposed in the blood, is convenient to medicine and directly acts on; Target spot genetic expression is stable, is difficult for developing immunity to drugs; Need not to consider tumor histology's characteristic; Can suppress metastases; The downstream scale effect is arranged; Untoward reaction is relatively little.Avastin with put, chemotherapy combined has synergy: (1) can make tumor area vasculogenesis normalizing, and high pressure alleviates between tissue, is beneficial to chemotherapeutic and carries to tumor area; (2) put, local tumor district anoxic can be urged vegf expression due to the chemotherapy, helps that the tumour cell opposing is put, the apoptosis induction mechanism of chemotherapy, and coupling Avastin will prevent the protective effect of this secondary, make the therapeutic response enhanced sensitivity.

The Avastin untoward reaction mainly is hemorrhage and gastric-intestinal perforation, also has weakness, pain, hypertension, diarrhoea, oligoleukocythemia, stomachache, headache in addition, feels sick, vomiting, anorexia, stomatitis, dvt form, the interior thrombus of abdomen, constipation, upper respiratory tract infection, exfoliative dermatitis, expiratory dyspnea, proteinuria etc.Rare serious adverse reaction has intestinal obstruction, bowel necrosis, mesenteric vein to close match, anastomotic ulcer, hyponatremia etc.Avastin dosage 375mg/m ², weekly 1 intravenous drip totally 4 times be 1 course of treatment, average 1 the course of treatment expense be about 100,000 Renminbi, spend high.

2, phage surface presents peptide storehouse technology at random

1985, Smith reported allogenic polypeptide in the result that the single stranded phage surface presents, and the phage surface that grows up thus presents technology and developed rapidly in recent years, becomes instrument effective and important in biological study and the Application Areas.This technology is that encoding exogenous polypeptide or proteic gene segment are inserted in the gene of phage capsid protein, thereby allogenic polypeptide or albumen are presented in phage surface.Owing to constitute the permutation and combination that the deoxynucleotide of exogenous genetic fragment can be at random, so can make up and present different allogenic polypeptides or proteic phage surface presents polypeptide or protein pool, and then be applied to the screening of enzyme substrates, the screening of antibody, the screening of part, the research of albumen interphase interaction, aspects such as the diagnosis of disease.

Filobactivirus is the phage that a class has screw mandrel shape form, belongs to Inoviridae (Inoviride) in classification, Inovirus (Inovirus).Being used for the phage that the surface presents is the colibacillary phage of a class specific infection, comprises M13, fd, f1, If1 and Ike etc., and they have similar structure.With the M13 phage is example, and its core is the single-stranded cyclic DNA of 6000bp, contains 10 genes, the 10 kinds of different albumen of encoding respectively.The gene VIII main capsid protein pVIII that encodes wherein, gene III, VI, VII, IX be coded auxiliary capsid protein pIII, pVI, pVII, pIX respectively.The M13 phage only infects the intestinal bacteria of tool F cilium with its less important capsid protein pIII.

Filobactivirus self has many characteristics that are suitable for making up peptide library.Can be received in as it and to insert allogenic polypeptide in the capsid protein, and will be presented in the virion surface behind the exogenous protein expression, be convenient to by corresponding acceptor or antibody recognition.Even the life cycle of exogenous array viral interference, also can be by dual-gene or phagemid system with expression of polypeptides in the surface, produce the chimeric phage that carries wild-type and recombinant capsid protein.Be easy to amplification, the phage after the reorganization can obtain amplification by ehec infection.For screen can specificity in conjunction with the polypeptide phage of a certain target molecule also ehec infection again, amplification back its nucleotide sequence of sequencing analysis.Phage is still very stable under various elution requirements (as low pH), can infect the very high titre of formation and (generally reach 10 ¹²), high like this titre is enough to represent clones all in the storehouse.

The appearance of phage display technology and develop into the extensive development of people and the screening new type antineoplastic medicine provides strong means.Along with the development of phage-displayed polypeptides storehouse technology, use the trend that little peptide analogue antigen antibody response has become development simultaneously.This be because: (1) contains the small peptide of the Key residues antigenic determinant on can simulated albumin matter; (2) in most cases, several Key residues and the formed non covalent bond of its binding molecule have constituted whole bonded major portions, i.e. interaction between the protein is to realize by the intersegmental interaction of partial oligopeptide.

The little peptide that screening combines with purpose screening thing from random peptide library, existing many pieces of documents report that successfully the success or not of screening depends on high quality peptide storehouse and highly purified screening thing.Bibliographical information is arranged, screening method is done modification slightly, promptly available mixture such as whole serum or other body fluid screen from the peptide storehouse and the little peptide of object bonded.Existing a plurality of research groups confirm that it is possible utilizing polyclonal serum to filter out the single-minded analogue antigen determinant of disease from random peptide library.This method filters out the phage that presents specificity analogue antigen determinant from a big peptide storehouse under the situation of not knowing any information of antigen in advance, this specific phage can only not react with normal serum with patients serum's reaction.And whether the sequence of analogue antigen determinant similar to initial molecule can not influence it and be applied to vaccine, thereby more meaningful aspect newfound disease.Cortese experiment group carries out a series of experiments, successfully screen can with antibodies among some disease patients serum, and not with the in combination little peptide of normal control.Usefulness such as Prezzi have infected the human serum of human hepatitis C (HCV) as the screening thing, and the little peptide of the phage of acquisition can react with the patients serum, and reactionless with control serum.With this type of peptide is that the antibody that the antigen immune rabbit obtains can be discerned HCV albumen.Now commercial human hepatitis B virus vaccine comprises that the genetically engineered recombinant human Hepatitis B virus vaccine of purifying all prepares by this method.

Phage small peptide storehouse technology has important significance for theories and using value.In the mutual identification of research between albumen, the prediction of protein folding and space conformation, the combining of interaction between peptide and organism and enzyme-to-substrate, antibody and antigenic interaction, aspects such as hormone and combining of acceptor demonstrate huge application potential.Having a extensive future such as the fields such as exploitation of the design of vaccine, the assignment of genes gene mapping, small-molecule drug.

3, therapeutic vaccine

3.1 progress

In recent years, in the process of the acute and chronic disease of treatment, demonstrated good effect at the proteic monoclonal antibody of mankind itself.But, the costliness of cost and the inconvenience of using have limited the widespread use of monoclonal antibody, therefore, turn to and seek proteic active immunity vaccine by accepting this antibody protein passively at the mankind itself, both the therapeutic modality with active immunity substituted passive immunization, became the developing direction of protein drug.At present, the research of therapeutic vaccine has become a focus, relates to a lot of diseases, as: chronic viral infection, allergy, tumour, Alzheimer thatch disease, diabetes, hypertension, obesity and rheumatic arthritis etc.

Therapeutic vaccine makes people's immunity system help reaction.Most vaccine can be divided into two big classes: a class is to induce body generation humoral immune reaction, produces antibody; Another kind of is to induce body generation cell immune response, produces cytotoxic T cell (CTLs).Back one class therapeutic vaccine is mainly used in the treatment of tumour and disease of viral infection.

Inducing antibody to treat infectious diseases is a kind of effective methods of treatment, and most preventative vaccines are all by inducing production of antibodies to protect body.Compare with preventative vaccine, the development of therapeutic vaccine will be many slowly, just see the hope of success up to therapeutic vaccine in recent years.Simultaneously, monoclonal antibody is indicating that in obtained immense success aspect the treatment disease therapeutic vaccine that can induce antibody to produce in vivo has vast potential for future development.In fact, it is possible that the endogenous specific antibody that has had animal experiment to show to induce certain level is treated disease, as: blocking-up TNF-α is with the treatment diseases associated with inflammation.It is very effective aspect treatment rheumatoid arthritis and Crohn ' s disease (the disconnected property ileitis of joint) that humanized anti-TNF-alpha monoclonal antibodies has been proved to be.The blocker listing of several TNF-α has been arranged at present, comprise two kinds of monoclonal antibody (infliximab, adalimumab) and a kind of receptor blocking agent (etanercept), they are helping thousands of patient to palliate the agonizing sufferings, and annual income reaches 2,000,000,000 dollars.So the TNF-α of blocking-up overexpression can reach the effect of treatment disease.Verifiedly in animal experiment can specificity induce the neutrality antibody of TNF-α, and the inductive antibody titers is enough to treat the scorching model of joint of animal by active immunity.

Other animal experiment shows and can treat following disease by producing high titre antibody in the inductor: the vaccine at Angiotensin can be treated hypertension; Can treat Eosinophilia's disease that pathogenic agent causes at the vaccine of IL-9; Vaccine at IL-5 can be treated asthma; Vaccine at N-methyl-D-aspartatereceptor-1 (NMDAR1) can be treated apoplexy.In addition, the immunity at some sexual hormoue such as human chorionic gonadotrophin (human chorionic gonadotro-pin HCG) can reduce the intravital hormonal readiness of women and reach contraceptive effect; Vaccine at gonadotropin-releasing hormone (GnRH) can be used for the treatment of advanced prostate cancer; Among the carcinoma of the pancreas patient, the antibody that utilizes therapeutic vaccine to induce at gastrin (gastrin) can prolong patient's life late.

Another kind of slightly different vaccine is lower to the requirement of security, and that is exactly the vaccine at habit-forming medicine.Vaccine at Cocaine and Nicotine in animal experiment has reduced the level of these medicines in brain, has eliminated their habituation symptom, and experimental animal is being accepted immune then medicine no longer the dependence.In testing in the clinical I phase, the Cocaine vaccine has been proved to be good tolerability, and can induce good antibody response, and now, people are longing for its validity result.

2.2 induce the theoretical investigation of autoantibody

The specific antibody that produces sufficiently high titre is with the treatment relative disease, and therapeutic vaccine must overcome three obstacles: T cell tolerance, Blymphocyte tolerance, induce antibody under the situation that does not have adjuvant and antigen prolonged action preparation.As everyone knows, the human immune system mainly starts to attack to external invader, and body itself is not attacked, and this may be because body has the ability that can discern " nonego " and " oneself ".Immune this specific character is commonly called tolerance or anergy, and tolerance occurs in B cell and T cell levels.In general, the T cell tolerance is stricter.Concerning many antigens, when the T cell tolerance took place, normal B cell strain but existed in vivo.In fact, have three kinds of mechanism to cause immunological tolerance: cell strain is rejected, and promptly specific lymphocyte is thoroughly rejected from lymphocyte populations; Immunity is incompetent, and promptly specific lymphocyte exists, but its function can not be activated; Immunity is ignored, and the lymphocyte that promptly has immunologic function exists, but owing to do not run into the autoantigen that exists with the antigen form, so can not be activated.Concerning the T cell, inducing tolerance and incompetent major organs is thymus gland (central tolerance), but induces tolerance also can carry out in periphery.Blymphocyte tolerance is mainly induced in marrow, but also can induce in periphery.Usually, antigenic immunological tolerance is easier to be illustrated for enriching of generally expressing.

In the immune response at exotic antigen, T cell and B cell are worked in coordination and could be produced antibody effectively: when being subjected to the exotic antigen immunity, specific B cell conjugated antigen produces initial activation signal.In addition, B cell endocytic antigen presents the mixture of antigen peptide and MHC II quasi-molecule on its surface.Usually, the B cell can not activate the TH cell.Activate the TH cell, dendritic cell is absolutely necessary, the dendritic cell antigen uptaking, and, activate the TH cell at the mixture of its cell surface antigen-presenting peptide and MHC II quasi-molecule.The antigen peptide that TH cell recognition B cell surface after the activation presents and the mixture of MHC II quasi-molecule cause other conversion of B cell proliferation, production of antibodies and antibody class.If lack the synergy of TH cell because of immunological tolerance, so just can not produce antibody.In design process at the vaccine of oneself protein, if with autoantigen and foreign protein or peptide carrier fusion, or be coupled at together, just might walk around the TH cell tolerance: the carrier proteins that the specific B cell of autoantigen just can absorb this autoantigen and link with it, and present the mixture of carrier peptides and MHC II quasi-molecule on its surface, because the TH cell does not have immunological tolerance to carrier proteins, so can be activated, thus collaborative from the specific antibody of the B of antigen-specific cell generation at autoantigen.

Compare with the T cell tolerance, it is much loose that Blymphocyte tolerance is wanted.In fact, in many cases, autospecific B cell occurs with normal frequency in vivo, will be activated if be subjected to the acting in conjunction of antigen and TH cell.So to many solubility oneself proteins, have its specific b cells strain in the body, especially when this albumen is not very expression more than needed especially like this.For this proteinoid or polypeptide, itself and carrier proteins are linked, walk around the T cell tolerance, just might produce effective vaccine.In fact, utilize this strategy, derived at the antibody response of multiple self hormone.

Summary of the invention

The object of the present invention is to provide a kind of vaccine based on anthropomorphic dummy's blood vessel endothelial cell growth factor VEGF epitope and preparation method thereof, utilize phage surface to present 12 peptides that peptide storehouse technology screening goes out to simulate the VEGF epi-position at random, make up the polypeptide epitope vaccine that can induce in vivo at VEGF molecule autoantibody on the basis of mimic epitopes, providing with VEGF is the tumor therapeutic vaccine exploitation of target spot and the strategy of design.

To achieve these goals, the present invention utilizes phage random to present technology screening to go out the special affine VEGF mimic epitopes with people mouse chimeric mAb A Wasiting (Avastin), and its aminoacid sequence is: Asp-His-Thr-Leu-Tyr-Thr-Pro-Tyr-His-Thr-His-Pro; The protein sequence of described mimic epitopes and VEGF does not have homology.

Structure can be induced the vaccine that produces autoantibody at the VEGF molecule, with above-mentioned mimic epitopes chemical coupling in protein carrier keyhole limpet hemocyanin (Keyhole limpet hemocyanin, KLH), its mode of connection is: KLH-Asp-His-Thr-Leu-Tyr-Thr-Pro-Tyr-His-Thr-His-Pro.

The preparation method of the mimic epitopes of described human vascular endothelial growth factor VEGF, carry out according to the following steps:

A) phage random presents the screening in 12 peptide storehouses

The mensuration of thalline titre at first:

Phage random presents dodecapeptide storehouse (Ph.D.-12 ^TMPhage display peptide library) behind the phage-infect host, adds the X-gal of 20mg/mL and the IPTG overnight incubation of 50mg/mL; Observe flat board, count blue plaque and multiply by this flat board pnagus medius dilution of sample multiple, obtain per 10 μ l Ph.D.-12 ^TMThe titre of phage, with blue plaque forming unit represent (plaque forming units, pfu);

Its secondary pollutant is eluriated:

(1) inoculates the phage host bacteria in the LB nutrient solution that contains tsiklomitsin, 37 ℃ of cultivations;

(2) with the NaHCO of 0.1mol/L ₃For solvent preparation pH is 8.6, the concentration of Avastin is the Avastin solution of 100 μ g/mL, Avastin solution bag is spent the night by the elisa plate hole; 96 orifice plates after will spending the night tip upside down on the clean paper handkerchief, eliminate residual liquid, are that the TBS solution of 1%BSA is hatched 2h for 4 ℃ with containing massfraction then; Adopting massfraction again is the TBST washing 6 times of 0.1% Tween 20, all 96 orifice plates will be tipped upside down on the clean paper handkerchief at every turn, eliminates residual liquid; The pH value that described TBS solution is 50mM is 7.5 Tris-HCl solution;

(3) add 200 μ l massfractions be 0.1% Tween 20 the TBST dilution contain 2 * 10 ¹¹The Ph.D.-12 of pfu ^TMPhage solution, incubated at room 1h; The liquid that inclines is removed unconjugated phage, is the TBST washing 10 times of 0.1% Tween 20 with massfraction again, all 96 orifice plates will be tipped upside down on the clean paper handkerchief at every turn, eliminates residual liquid; With the phage of 100 μ L elutriant elution of bound, and add the 15ml neutralizer; Get 1 μ L and survey titre, remaining liq adds 20mL and is in the early stage host's culture of logarithmic growth, cultivates 4.5h, the phage that obtains increasing in 37 ℃ of thermal agitations; Described elutriant is to contain the mixing solutions that the Glycine-HCl of 0.2mol/L and pH value are the BSA of 2.2 10g/L; Described neutralizer is that the pH value is the Tris-HCl solution of 9.1 1mol/L;

(4) will increase good phage and colibacillary mixed solution changes centrifuge tube over to, centrifugal 10000rpm * 10min under 4 ℃ of conditions; Supernatant liquor changes centrifuge tube over to, and 10000rpm * 10min is once centrifugal again under 4 ℃ of conditions; Get 80% supernatant liquor and change centrifuge tube over to, add 4 ℃ of standing over night of PEG8000/NaCl solution of 3ml; Next day, 4 ℃ of centrifugal 10000rpm * 15min, the centrifuged supernatant of inclining; With the resuspended precipitation of the TBS of 1mL,, precipitate residual cell in 4 ℃ of centrifugal 10000rpm * 5min; Supernatant adds the PEG-8000/NaCl ice bath 1h of 150ul, 4 ℃ of centrifugal 10000rpm * 15min, and the supernatant that inclines, containing massfraction with 200 μ L is 0.01% NaN ₃The resuspended precipitation of TBS, centrifugal 10000rpm * 1min; Supernatant liquor is the good Ph.D.-12 of amplification ^TMPhage phage elutriant, 1 μ L is used to survey titre, and remaining liquid is deposited in 4 ℃; Described PEG8000/NaCl solution contains the PEG-8000 of 200mg/mL and the NaCl of 2.5M;

(5) wrap again by elisa plate according to above-mentioned (2) step, carry out second and third and take turns screening; Second take turns, Avastin concentration is reduced to 50 μ g/m, 20 μ g/mL respectively in the third round screening, adds 2 * 10 respectively ¹¹Pfu takes turns the good Ph.D.-12 of amplification that filters out in the first round, two ^TMPhage, operation steps is undertaken by above-mentioned screening step (1)-(4); The Tween20 concentration of TBST all is increased to 0.5%; The eluted product of third round need not increase, and gets 1ul and measures titre;

(6) from screening back phage titre is measured for the third time, on the flat board of bacterial plaque number＜100 at random 80 of pickings separate good plaques and increase and purifying, the time that the plaque of picking is cultivated is no more than 18h; Described amplification and purifying: dip in 80 good blue plaques of separation getting with aseptic toothpick and put into host's culture that mid-log phase is cultivated respectively, after 37 ℃ of thermal agitations are cultivated 4.5h, the centrifugal 30s of 12000rpm, change supernatant liquor over to new centrifuge tube, the centrifugal again 30s of 12000rpm, get 80% supernatant, promptly get the phage suspension liquid that increases;

B) specific phage screening

In above-mentioned 80 clones that select, utilize phage E LISA further to select clone with the Avastin high-affinity;

(1) Avastin with 100mg/L wraps by 96 hole elisa plates, and every hole 200 μ L set up the negative contrast of interleukin 15 (IL-15) monoclonal antibody, 4 ℃ of overnight incubation to each Avastin hole simultaneously;

(2) coating buffer that inclines adds 4 ℃ of sealings of confining liquid 2h; The deblocking liquid that inclines, TBST cleans 6 times, all 96 orifice plates will be tipped upside down on the clean paper handkerchief at every turn, eliminates residual liquid; The phage of 80 purifying is pressed 1 * 10 ⁹/ hole adds Avastin, IL-15 monoclonal antibody bag respectively by incubated at room 2h in the hole; Described confining liquid contains the BSA of 5mg/L and the NaHCO of 0.1mol/L ₃Mixing solutions, the pH value of this confining liquid is 8.6;

(3) TBST cleans 6 times, and every hole adds 1: 5000 mouse anti M13 phage antibody, the 100 μ L of horseradish peroxidase (HRP) mark, incubated at room 1h; TBST cleans 6 times, and the light absorption value at A490nm place is measured in O-Phenylene Diamine (O-Phenylenediamine) colour developing back;

(4) according to the light absorption value at A490nm place, filtering out 42 has higher affinity with Avastin and carries out sequencing with the low clone of other contrast avidity; Screening method: phage combines back A490nm light absorption value greater than 0.5 with Avastin, simultaneously with the ratio of control group A 490nm light absorption value greater than 3;

C) phage presents the sequencing of peptide

(1) extraction of phage single-chain DNA:

After the amplification of phage clone, get the phage culture supernatant, extract test kit with single stranded DNA and carry out the extraction of phage single-chain DNA, quantitative its concentration of ultraviolet is greater than 100ng/ μ L;

(2) Sanger double deoxidating chain termination measuring dna sequence dna:

With phage single-chain DNA is template, and the sequencing primer that is provided in the phage random peptide library test kit is provided, and primer sequence is ccctc atagt tagcg taacg, carries out automatic sequencing on automatic dna sequencer;

(3) present the derivation of peptide ammino acid sequence at random:

According to the dna sequencing result, draw phage and present the aminoacid sequence of peptide at random, it is as follows to draw sequence: Asp-His-Thr-Leu-Tyr-Thr-Pro-Tyr-His-Thr-His-Pro.

The described preparation method that can induce at the vaccine of VEGF molecule generation autoantibody, adopt following steps:

(1) chemosynthesis mimic epitopes polypeptide as claimed in claim 1

Asp-His-Thr-Leu-Tyr-Thr-Pro-Tyr-His-Thr-His-Pro；

(2) adopt glutaraldehyde method will synthesize polypeptide epitope and KLH chemical coupling: it is to shake in 10 the borate buffer solution that KLH is melted into the pH value, add step (1) synthetic mimic epitopes polypeptide, slowly splash into massfraction and be 0.3% glutaraldehyde solution, shook 2 hours, the glycine that adds 0.25 milliliter of 1mol/L, termination reaction after 30 minutes; Separating, is 4 ℃ of dialysed overnight in 8.5 the borate buffer solution with conjugate in pH value, and exchange buffering liquid continues to dialyse and obtains the conjugate of purifying more than 4 hours; The mass ratio of KLH and mimic epitopes polypeptide is 2: 3, and the mol ratio of KLH and glutaraldehyde solution is 10: 3;

(3) getting trace (Dot Blot) ready and identify conjugate, can promptly be the polypeptide vaccine that people VEGF mimic epitopes makes up with Avastin bonded conjugate in getting trace ready.

Technique effect of the present invention: the commercial VEGF monoclonal antibody of utilization of the present invention Avastin filters out the peptide storehouse and Avastin bonded peptide for target molecule presents immediately from phage, this peptide of chemosynthesis, and with keyhole limpet hemocyanin (Keyhole limpet hemocyanin, KLH) carry out chemical coupling, successful structure vaccine.The associating freund's adjuvant immune health BALB/c mouse of growing up induces the antiserum(antisera) at people VEGF, and this antiserum(antisera) can bring into play the function consistent with Avastin, and the mimic epitopes polypeptide of preparation can be affine with the Avastin height; The mimic epitopes polypeptide vaccine that makes up uses the VEGF mimic epitopes that filters out to break through the autoimmunity tolerance in conjunction with carrier proteins KLH, produces the neutralizing antibody at VEGF; The adult BALB/c mouse of mimic epitopes polypeptide vaccine associating freund's adjuvant immune health can induce the antiserum(antisera) at this epi-position in the mouse body; This antiserum(antisera) can combine with VEGF; Can suppress vascular endothelial cell proliferation and migration.Thereby the method for using active immunity substitutes the passive immunotherapy mode of Avastin monoclonal antibody, has reduced the monoclonal antibody side effect, has alleviated the sufferer burden; Provide is the new exploitation of the tumor therapeutic vaccine of target spot and the strategy of design with VEGF.

Description of drawings

Fig. 1 be filter out 42 with Avastin higher affinity is arranged and with the ELISA result of contrast (human IL-15 monoclonal antibody) phage clone that avidity is low: the result that combines of the positive phage of black box and Avasdtin; White box is and the result that combines of human IL-15 monoclonal antibody that ordinate zou is the light absorption value at 490nm place, the positive phage numbering of X-coordinate;

Fig. 2 is that KLH-mimic epitopes 12 peptides (KLH-12P) are schemed with the Dot Blot evaluation of Avastin.

Fig. 3 be ELISA detect KLH-12P antiserum(antisera) and VEGF combine figure statistical graph as a result, black is the KLH-12P antiserum(antisera), white is the KLH antiserum(antisera), grey is that KLH-contrasts the 12P antiserum(antisera); 100 times of 1 serum dilutions, 2 are 50 times of serum dilutions, and 3 are 25 times of serum dilutions, and ordinate zou is a 490nm place light absorption value.

Fig. 4 is that mtt assay detects antiserum(antisera) inhibition vascular endothelial cell proliferation statistical graph as a result, black box is the KLH antiserum(antisera), white box is that KLH-contrast 12P antiserum(antisera), grey box are the KLH-12P antiserum(antisera), 1 for 100 times of serum dilutions, 2 are 25 times of serum dilutions for serum dilutes 50 times, 3, and ordinate zou is a 490nm place light absorption value.

Fig. 5 antiserum(antisera) suppresses migration of vascular endothelial cells statistical graph as a result, and 1 is 25 times of KLH antiserum(antisera)s of dilution, 2 KLH-contrast 12P antiserum(antisera)s for 25 times of dilutions, and 3 are 25 times of KLH-12P antiserum(antisera)s of dilution, ordinate zou is cell counting;

Fig. 6 is that antiserum(antisera) suppresses migration of vascular endothelial cells Electronic Speculum figure, and 6a figure is 25 times of KLH antiserum(antisera)s of dilution, and 6b figure is 25 times of KLH-contrasts of dilution 12P antiserum(antisera), and 6c figure is 25 times of KLH-12P antiserum(antisera)s of dilution.

Embodiment

The present invention is described in further detail below in conjunction with drawings and Examples.

At first using affine method for screening according to the VEGF therapeutic autovaccine of technical scheme preparation of the present invention utilizes Avastin to present 12 peptide storehouse (BioLabs for part at phage random New England) screening obtains 12 polypeptide mimic epitopes Asp-His-Thr-Leu-Tyr-Thr-Pro-Tyr-His-Thr-His-Pro (aspartic acid-Histidine-Threonine-leucine-tyrosine-Thr-Pro-tyrosine-Histidine-Threonine-Histidine-proline(Pro)) of high-affinity in, after this epi-position of chemosynthesis, use glutaraldehyde method itself and KLH are carried out chemical coupling, obtain the vaccine that to break the autoimmunity tolerance, produce autoantibody.

Realize the screening of peptide of the present invention storehouse, vaccine construction, and immune animal, the antibody calibration operation, finish according to the following steps:

3.1. phage random presents the screening in 12 peptide storehouses

Phage random presents dodecapeptide storehouse (Ph.D.-12 ^TMPhage display peptide library) available from U.S. New England Biolabs company, its titre is 1.5 * 10 ¹³Plaque forming unit (pfu)/mL; Diversity is 2.7 * 10 ⁹Transformants.The host bacterium is to have carrying of tetracyclin resistance can be formed the α complementation with the phage that has presented 12 peptides by the intestinal bacteria E.coli ER2738 of the F ' factor of phage-infect.The dna sequencing primer of target protein (96gIIIsequencing primer) sequence is: ccctcatagt tagcg taacg.

3.1.1 the mensuration of phage titre

(1) cultivation of E.coli ER2738 is with aseptic technique picking one transfering loop from the frozen thing of the colibacillary glycerine of ER2738, be inoculated in LB flat board (the Tryptones 10g/L of tetracyclin resistance, yeast extract 5g/L, agar 15g/L, NaCl 10g/L) on, cultivate 24h for 37 ℃, treat dull and stereotyped the bright bacterium colony of visible white and form after, basic culture plate is placed 4 ℃ of preservations.

(2) the single colony inoculation of picking E.coli ER2738 (Tryptones 10g/L in the LB nutrient solution that contains the 20mg/L tsiklomitsin from the flat board of above-mentioned steps (1), yeast extract 5g/L, NaCl 10g/L), 37 ℃ of shaking culture are to logarithmic growth mid-term (OD600 is about 0.5).

(3) with Ph.D.-12 ^TMPhage carries out 10 times of continuous gradient dilutions with the LB nutrient solution, for the dilution of the phage culture supernatant after the amplification 10 ⁸-10 ¹¹Doubly, the screening elutriant for not amplification dilutes 10 ¹-10 ⁴Doubly.

(4) Ph.D.-12 after the dilution ^TMThe phage sample is got 10 μ l, joins step (2) respectively and cultivates in the inoculum of 200 μ l, quick mixing, incubated at room 1-5min.

(5) bacterium that respectively infects phage moves into 45 ℃ of pre-temperature respectively, contains (Tryptones 10g/L, yeast extract 5g/L, NaCl 10g/L, MgCl in the culture tube of top-agar 3-5ml ₂6H ₂O1g/L, agarose 7g/L), add the X-gal of 40 μ l20mg/mL and the IPTG of 16 μ l50mg/mL, abundant mixing is poured the LB flat board of pre-temperature then rapidly into, tilts to make the top-agar uniform distribution.

(6) flat board is placed 5min, temperature is reduced, counter-rotating is dull and stereotyped then, 37 ℃ of overnight incubation.

Observe flat board, count blue plaque and multiply by this flat board pnagus medius dilution of sample multiple, obtain per 10 μ lPh.D.-12 ^TMThe titre of phage, with blue plaque forming unit represent (plaque forming units, pfu).

3.1.2. biological the elutriation

(1) inoculates ER2738 in advance in the LB nutrient solution that contains tsiklomitsin of 10mL (surveying titre uses) and 20mL (phage is used in amplification), 37 ℃ of cultivations.

(2) in 96 orifice plates, add 200 μ L, 100 μ g/mL Avastin solution (0.1mol/L NaHCO ₃, the pH8.6 dilution), place airtight wet box to shake gently, 4 ℃ of overnight incubation.

(3) 96 orifice plates in (2) are taken out, the liquid that inclines tips upside down on 96 orifice plates on the clean paper handkerchief, and light deduction is residual liquid to the greatest extent.

(4) massfraction of adding 200 μ L is 1% BSA-TBS confining liquid, hatches 2h for 4 ℃, and the pH value that described TBS solution is 50mM is 7.5 Tris-HCl solution;

(5) TBST (TBS+0.1%Tween 20) washing is 6 times, all 96 orifice plates will be tipped upside down on the clean paper handkerchief at every turn, and light deduction is residual liquid to the greatest extent.

(6) adding 200 μ lTBST (TBS+0.1%Tween 20) dilution contains 2 * 10 ¹¹The Ph.D.-12 of pfu ^TMPhage solution, incubated at room 1h.

(7) liquid that inclines is removed unconjugated phage, and TBST (TBS+0.1%Tween 20) washing 10 times all will tip upside down on 96 orifice plates on the clean paper handkerchief at every turn, and light deduction is residual liquid to the greatest extent.

(8), and add 15 milliliters of neutralizers (1mol/L Tris-HCl, pH 9.1) rapidly with the phage of 100 μ l elutriants (0.2mol/L Glycine-HCl, pH 2.2,10g/L BSA) elution of bound; Get 1 μ l and survey titre, the elutriant after the remaining neutralization adds 20mL and is in the early stage R2738 culture of logarithmic growth, cultivates 4.5h in 37 ℃ of thermal agitations, increases.

(9) amplification in (8) is good phage and colibacillary mixed solution are poured centrifuge tube into, in 4 ℃, and the centrifugal 10min of 10000rpm.Change centrifugal supernatant over to new centrifuge tube, once centrifugal again with method, get 80% centrifugal supernatant and go into new centrifuge tube.

(10) in centrifuge tube, add 3 milliliters of PEG8000/NaCl (200mg/mL PEG-8000,2.5MNaCl), in 4 ℃ of standing over night.

(11) take out PEG8000/NaCl next day and precipitate good liquid, centrifugal 10000rpm * 15min under 4 ℃ of conditions, the careful centrifugal supernatant that inclines; Once centrifugal fast with method again, exhaust residual supernatant with micropipet.

(12) with the resuspended centrifugation of 1mLTBS, resuspended liquid is changed in the little centrifuge tube, centrifugal 10000rpm * 5min under 4 ℃ of conditions is with residual cell precipitation.

(13) centrifugal supernatant is moved in the new little centrifuge tube, add the PEG8000/NaCl ice bath 1h of 150ul.Centrifugal 10000rpm * 15min under 4 ℃ of conditions, the careful centrifugal supernatant that inclines; Once centrifugal fast with method again, exhaust residual supernatant with micropipet.

(14) containing massfraction with 200 μ L is 0.01% NaN ₃The resuspended precipitation of TBS, centrifugal 10000rpm * 1min is with precipitate and separate.Supernatant liquor is moved into new centrifuge tube, be the good phage of amplification, 1 μ L is used to survey titre, and remaining liquid is deposited in 4 ℃.

(15) newly wrap with 200 μ L Avastin solution, be used for next round screening by 96 orifice plates, one hole, in order to improve the preciseness of screening peptide, second take turns with the third round screening in, the concentration of Avastin is reduced to 50 μ g/mL, 20 μ g/mL respectively.

(16) observe the result who surveys titre in the step 14, the quantity of counting locus coeruleus is to determine the titre of phage, and this phage is used for second screening of taking turns.In second screening of taking turns, the phagocytosis scale of construction of adding is 2 * 10 ¹¹Pfu, volume calculates according to the titre that step 14 is measured.

(17) carry out second and take turns screening, repeating step 2-15, and the concentration of Tween20 among the TBST is increased to 0.5%.

(18) with 200 μ L target protein solution, concentration is newly wrapped by 96 orifice plates, one hole, is used for the third round screening.Repeating step 2-8, the concentration of Tween is 0.5% among the TBST, and the eluted product of third round need not increase, and gets 1 μ L and carries out titer determination, and residue is deposited in 4 ℃.From titer determination, on the flat board of bacterial plaque number＜100 at random 80 of pickings separate good plaques and increase and purifying by following method, should notice that the time that the plaque of picking is cultivated can not surpass 18h this moment.Through the three-wheel screening, specific phage is further by enrichment, and the phage titre that obtains progressively raises.As shown in table 1.

Table 1: phage selection titer determination result

The screening number of times	Average titer
		The first round	1.5×10 9pfu
Second takes turns	4.3×10 9pfu
		Third round	1.1×10 10pfu

(19) amplification of positive bacteriophage is carried out dilution in 1: 100 with the overnight culture of ER2738 with the LB nutrient solution, and the 1mL/ pipe is sub-packed in the culture tube of 50mL.

(20) will dip in 80 good blue plaques of separation getting with aseptic toothpick and put into above-mentioned (19) culture tube respectively, 37 ℃ of thermal agitations are cultivated 4.5h and are increased.

(21) the good phage liquid that will increase is poured centrifuge tube into, centrifugal 30s of moment (12000rpm).Change centrifugal supernatant over to new centrifuge tube, once centrifugal again with method.Get 80% supernatant, be the phage liquid of amplification.Can deposit several weeks under 4 ℃, if will preserve for a long time, can add final concentration and be 50% aseptic glycerine and deposit in-20 ℃.

3.1.3. phage specificity screening (phage E LISA)

In 80 clones that this picks out, utilize phage E LISA further to select clone with the Avastin high-affinity.

(1) with 100mg/LAvastin (0.1mol/L NaHCO ₃, the pH8.6 dilution) and bag is by 96 hole elisa plates, and every hole 200 μ L are simultaneously to the negative contrast of each Avastin hole setter's interleukin 15 (IL-15) monoclonal antibody, 4 ℃ of overnight incubation.

(2) coating buffer that inclines adds confining liquid (5mg/L BSA, 0.1mol/L NaHCO ₃, pH 8.6), 4 ℃ of sealing 2h.

(3) the deblocking liquid that inclines, TBST washes 6 times, all 96 orifice plates will be tipped upside down on the clean paper handkerchief, gently deduction residual liquid to the greatest extent at every turn.

(4) with the phage (1 * 10 of 80 purifying ⁹/ hole) adds bag that step 1 sets up respectively by the hole.

(5) TBST washes 6 times, and every hole adds mouse anti M13 phage antibody (dilution in 1: 5000) the 100 μ L of horseradish peroxidase (HRP) mark, incubated at room 1h.

(6) TBST washes 6 times, and the light absorption value at A490nm place is measured in O-Phenylene Diamine (O-Phenylenediamine) colour developing back.

According to 80 clones' ELISA result, greater than 0.5, purpose OD value is benchmark with the ratio of contrast OD value greater than 3 with the OD value, and filtering out 42 has higher affinity with Avastin and contrast the low clone of avidity with other, and the result as shown in Figure 1.

3.1.4. have the phage of higher affinity to present the sequencing of peptide with Avastin to filtering out 42

(1) extraction of phage single-chain DNA

The amplification of phage clone is the same, gets the phage culture supernatant.The single stranded DNA extraction test kit of producing with Hua Shun company carries out the extraction of phage single-chain DNA, operate by process specifications: in containing the LB nutrient solution of single phage clone, add precipitated liquid, obtain the phage particle precipitation behind the high speed centrifugation, add lysate lytic phage coat protein, through resin purification, wash-out obtains phage single-chain DNA, and quantitative its concentration of ultraviolet is greater than 100ng/ μ L.

(2) Sanger double deoxidating chain termination measuring dna sequence dna

With the single stranded DNA is template, and the sequencing primer that is provided in the phage random peptide library test kit is provided, and primer sequence is 5 '-CCCTCATAGTTAGCGTAACG-3 ', carries out automatic sequencing on ABI 310 automatic dna sequencers.

(3) present the derivation and the amino acid identity analysis of peptide ammino acid sequence at random

According to the dna sequencing result, derive phage and present the aminoacid sequence of peptide at random, it is as follows to draw sequence:

Asp-His-Thr-Leu-Tyr-Thr-Pro-Tyr-His-Thr-His-Pro

3.2. the structure of vaccine

3.2.1. the chemosynthesis of epi-position

According to above-mentioned polypeptide epitope aminoacid sequence result, adopt the synthetic above polypeptide of solid-phase synthesis, synthetic work is finished by Xi'an China occasion biotechnology company, and purity is more than 98%.

3.2.2. the chemical coupling of polypeptide

Adopt glutaraldehyde method to carry out the chemical coupling of polypeptide and protein carrier KLH.

10mg keyhole limpet hemocyanin (Keyhole limpet hemocyanin, KLH) be melted in the borate buffer solution of 2ml pH10, gentle concussion, add the 15mg dodecapeptide, slowly splash into 1ml 0.3% glutaraldehyde solution, shake 2 hours (solution becomes yellow), add 30 minutes termination reactions of glycine effect of 1mol/L.With reactant 4 ℃ of dialysed overnight in the borate buffer solution of pH 8.5, exchange buffering liquid continues dialysis and gets final product more than 4 hours.

3.2.3. make up the evaluation of vaccine

Determine coupling effect with DOT Blot:

(1) with the link coupled vaccine, and KLH puts on nitrocellulose filter (NC film) so that same amount (about 3 μ g) is careful.

(2) seal the NC film more than 2 hours with 2%BSA-TBS (pH7.5).

(3) TBST (0.5%Tween20) washing NC film is 3 times, each 10 minutes.

(4) add the Avastin (4 μ g/ml) that TBST dilutes, the room temperature jog was hatched 1 hour.

(5) the unconjugated Avastin of flush away in kind.

(6) the anti-people two of alkaline phosphatase (AP) mark of adding TBST dilution is anti-, incubated at room 30 minutes.

(7) TBST flush away two is anti-, again with TBS flush away Tween 20, develops the color with alkaline phosphatase substrate developer (sigma) lucifuge.

The result as shown in Figure 2, KLH-mimic epitopes 12 peptides (KLH-12P) combine with the Avastin specificity, the combination of sigma colour developing performance antigen-antibody, circular patch as shown in Figure 2, and contrast 12 peptides (KLH-contrasts 12 peptides), and KLH all can not successfully combine with Avastin, and sigma colour developing does not show antigen-antibody in conjunction with (in Fig. 2 without any color reaction), promptly can not simulate the epi-position of VEGF, the success of vaccine coupling mimic epitopes is described.

3.3. animal immune

3.3.1 laboratory animal

BALB/c mouse, 4 ages in week are available from The Fourth Military Medical University's Experimental Animal Center.Be divided into 4 groups at random, 6 every group.

3.3.2 experiment material

Freund's complete adjuvant, Freund's incomplete adjuvant are all available from Beijing ancient cooking vessel state biological products company.Candidate vaccine KLH-12P, the KLH-contrast 12P and the reference protein KLH of above-mentioned preparation.

3.3.3 immune programme for children

(1) mouse is divided into 2 groups at random, 6 every group.

(2), and make it complete emulsification respectively with vaccine protein and carrier proteins contrast and freund's adjuvant mixing.

(3) the subcutaneous multi-point injection immune mouse in per two all backs once.Each every immunizing dose is 50 μ g albumen.

(4) being total to immunity four times, is Freund's complete adjuvant emulsification first, and the back is the emulsification of Freund twice, does not add adjuvant for the last time, carries out abdominal injection.

(5) the last immunity is back 10 days, and eyeball is got blood, and separation of serum.

3.4. antiserum(antisera) is identified

3.4.1.ELISA the mensuration antiserum(antisera) combines with VEGF's

3.4.1.1. material and reagent

96 hole elisa plates (Costar), NaHCO ₃PH8.6, KLH-12P, VEGF (sigma), the anti-people of HRP (horseradish peroxidase) mark, and anti-mouse two anti-(Wuhan Zhong Shan biological products company), ABTS (2,2 '-hydrazine-two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) two amine salt, sigma).

3.4.1.2. experimental technique

(1) with the vegf protein bag by elisa plate (0.1MNaHCO ₃PH8.6), the protein content of bag quilt is the 50ng/ hole, and 4 ℃ are spent the night.

(2) discard solution in the hole next day, uses 1%BSA-TBS, 4 ℃ of sealing 2h.

(3) thorough washing is (3min/ time, totally 6 times) after, add the antiserum(antisera) of KLH-12P immunity according to the gradient of serum content 1%, 2%, 4%, 2h is hatched in the room temperature vibration, and the antiserum(antisera) of the KLH-contrast 12P immunity that same gradient adds, the antiserum(antisera) of KLH immunity are as negative control.

(4) (3min/ time, totally 6 times) behind the thorough washing adds HRP mark goat-anti mouse two and resists, and 1h is hatched in the room temperature vibration.

(5) (3min/ time, totally 6 times) behind the thorough washing adds OPD colour developing liquid in each hole, behind the reaction 5min, and with the sulfuric acid termination reaction of 1M, ELISA readout instrument 490nm place reading.

The result as shown in Figure 3, the KLH-12P antiserum(antisera) combines best after diluting 25 times with VEGF, in 490nm place light absorption value maximum, and KLH-contrasts the 12P antiserum(antisera), the KLH antiserum(antisera) is constant substantially with combining of VEGF when diluting 25 times, 50 times, 100 times, at 490nm place light absorption value basically identical.

3.4.2. antiserum(antisera) suppresses the effect of vascular endothelial cell proliferation

3.4.2.1. material and reagent

(1) cell

Vascular endothelial cell (EVC) is all cultivated in RPMI1640 substratum (GIBCO Co).The penicillin, the Streptomycin sulphate that contain 10% calf serum (Hangzhou folium ilicis chinensis biological factory) and 100Uml-1 in the substratum.

(2) antiserum(antisera)

Antiserum(antisera) is the antiserum(antisera) of collecting in the animal immune experiment, and KLH immunity, KLH-contrast the negative contrast antiserum(antisera) of 12 peptides immunity.

(3) reagent:

MTT (3-(4,5-dimethylthiazole-2)-2,5-phenylbenzene tetrazole bromine salt), RPMI-1640, VEGF.

3.4.2.2. experimental technique

(1) inoculating cell: the vascular endothelial cell of trysinization monolayer culture with 0.25%, be mixed with the individual cells suspension with RPMI-1640, be inoculated in 96 well culture plates with every hole 104 cell count.

(2) cultivation was changed liquid after 12 hours, for containing the RPMI-1640 of 5ng/ml VEGF, be further divided into three groups and add Anti-KLH serum, Anti-KLH-contrast polypeptide serum, Anti-KLH-12P serum respectively, serum is that three gradients are 1%, 2%, 4%, continues to cultivate.

(3) continue to cultivate vascular endothelial cell 72 hours, every then hole adds MTT solution 20ul, cultivates 4 hours, stops cultivating, and careful the suction removed supernatant, and every hole adds 150ulDMSO (dimethyl sulfoxide (DMSO)), shakes 10 minutes.

(4) microplate reader 490nm place value of reading.

The result as shown in Figure 4, the effect of 1% and 2% KLH antiserum(antisera), KLH-contrast 12P antiserum(antisera), KLH-12P antiserum(antisera) inhibition vascular endothelial cell is all not obvious, 4% KLH antiserum(antisera), KLH-contrast 12P antiserum(antisera) still can not suppress vascular endothelial cell proliferation, and 4% KLH-12P antiserum(antisera) can suppress vascular endothelial cell proliferation.

3.4.3. antiserum(antisera) suppresses the effect of migration of vascular endothelial cells

3.4.3.1. material and reagent

(1) cell and cell cultures and antiserum(antisera) are as identical as described in the 3.4.2.1.

(2) reagent Mili-Cell, RPMI-1640,2% Paraformaldehyde 96, VEGF.

3.4.3.1. method and step

(1) RPMI-1640 preparation 1 * 106/ml vascular endothelial cell suspension is divided into three groups, contains 4%Anti-KLH-12P serum, 4%Anti-KLH serum, 4%KLH-contrast polypeptide antiserum(antisera) respectively.

(2) inoculate the above-mentioned cell suspension of 200ul respectively in the Mili-Cell epicoele, cavity of resorption all adds the RPMI-1640 that 600ul contains the VEGF of 5ng/ml, 37 ℃, cultivates 24 hours under 5% carbon dioxide conditions.

(3) the MILICELL epicoele is taken out, fix 10 minutes, wipe on the film not migrating cell gently away, used violet staining subsequently 5 minutes, drying at room temperature after the pure water rinsing with cotton swab with 2% Paraformaldehyde 96.

(4) take off filter membrane and place on the slide glass, microscopically is observed, count 5 at random and do not repeat 200 * cell count, calculating mean value.

Result such as Fig. 5, shown in Figure 6,4% KLH antiserum(antisera), KLH-contrast 12P antiserum(antisera) can not suppress migration of vascular endothelial cells, considerable change does not take place in (200 *) observation and cytometer number average under the mirror, and 4% KLH-12P antiserum(antisera) can suppress migration of vascular endothelial cells, and (200 *) observation and cytometer number average find that cell quantity obviously reduces under the mirror.

With KLH-Asp-His-Thr-Leu-Tyr-Thr-Pro-Tyr-His-Thr-His-Pro immunity BALB/c mouse, obtain antiserum(antisera), detect and to combine with the VEGF specificity through ELISA, titre can reach 1: 6000, and KLH-Asp-His-Thr-Leu-Tyr-Thr-Pro-Tyr-His-Thr-His-Pro (KLH-12P) antiserum(antisera) that dilutes 25 times can suppress vascular endothelial cell proliferation and migration.In sum, can be applied to the VEGF autovaccine of human body based on the vaccine (KLH-12P) of anthropomorphic dummy's blood vessel endothelial cell growth factor VEGF epitope, thereby substitute or additional monoclonal antibody passive immunotherapy in the active immunity mode, lay the first stone for overcoming monoclonal antibody treatment defective.

Claims (4)

1. the mimic epitopes of a human vascular endothelial growth factor VEGF, it is characterized in that: utilize phage random to present technology screening and go out the special affine VEGF mimic epitopes with people mouse chimeric mAb A Wasiting (Avastin), its aminoacid sequence is:

Asp-His-Thr-Leu-Tyr-Thr-Pro-Tyr-His-Thr-His-Pro。

2. the preparation method of a human vascular endothelial growth factor VEGF mimic epitopes is characterized in that, carries out according to the following steps:

A) phage random presents the screening in dodecapeptide storehouse

The mensuration of thalline titre at first:

Phage random presents dodecapeptide storehouse Ph.D.-12 ^TMThe phage-infect host after, add the X-gal of 20mg/mL and the IPTG overnight incubation of 50mg/mL; Observe flat board, count blue plaque and multiply by this flat board pnagus medius dilution of sample multiple, obtain per 10 μ l Ph.D.-12 ^TMThe titre of phage, with blue plaque forming unit represent (plaque forming units, pfu);

Its secondary pollutant is eluriated:

(1) inoculates the phage host bacteria in the LB nutrient solution that contains tsiklomitsin, 37 ℃ of cultivations;

(2) with the NaHCO of 0.1mol/L ₃For solvent preparation pH is 8.6, the concentration of Avastin is the Avastin solution of 100 μ g/mL, Avastin solution bag is spent the night by the elisa plate hole; 96 orifice plates after will spending the night tip upside down on the clean paper handkerchief, eliminate residual liquid, are that the TBS solution of 1%BSA is hatched 2h for 4 ℃ with containing massfraction then; Adopting massfraction again is the TBST washing 6 times of 0.1% Tween 20, all 96 orifice plates will be tipped upside down on the clean paper handkerchief at every turn, eliminates residual liquid; The pH value that described TBS solution is 50mM is 7.5 Tris-HCl solution;

(3) add 200 μ l massfractions be 0.1% Tween 20 the TBST dilution contain 2x10 ¹¹The Ph.D.-12 of pfu ^TMPhage solution, incubated at room 1h; The liquid that inclines is removed unconjugated phage, is the TBST washing 10 times of 0.1% Tween 20 with massfraction again, all 96 orifice plates will be tipped upside down on the clean paper handkerchief at every turn, eliminates residual liquid; With the phage of 100 μ L elutriant elution of bound, and add the 15ml neutralizer; Get 1 μ L and survey titre, remaining liq adds 20mL and is in the early stage host's culture of logarithmic growth, cultivates 4.5h, the phage that obtains increasing in 37 ℃ of thermal agitations; Described elutriant is to contain the mixing solutions that the Glycine-HCl of 0.2mol/L and pH value are the BSA of 2.2 10g/L; Described neutralizer is that the pH value is the Tris-HCl solution of 9.1 1mol/L;

(4) will increase good phage and colibacillary mixed solution changes centrifuge tube over to, centrifugal 10000rpm * 10min under 4 ℃ of conditions; Supernatant liquor changes centrifuge tube over to, and 10000rpm * 10min is once centrifugal again under 4 ℃ of conditions; Get 80% supernatant liquor and change centrifuge tube over to, add 4 ℃ of standing over night of PEG8000/NaCl solution of 3ml; Next day, 4 ℃ of centrifugal 10000rpm * 15min, the centrifuged supernatant of inclining; With the resuspended precipitation of the TBS of 1mL,, precipitate residual cell in 4 ℃ of centrifugal 10000rpm * 5min; Supernatant adds the PEG-8000/NaCl ice bath 1h of 150ul, 4 ℃ of centrifugal 10000rpm * 15min, and the supernatant that inclines, containing massfraction with 200 μ L is 0.01% NaN ₃The resuspended precipitation of TBS, centrifugal 10000rpm * 1min; Supernatant liquor is the good Ph.D.-12 of amplification ^TMPhage phage elutriant, 1 μ L is used to survey titre, and remaining liquid is deposited in 4 ℃; Described PEG8000/NaCl solution contains the PEG-8000 of 200mg/mL and the NaCl of 2.5M;

(5) wrap again by elisa plate according to above-mentioned (2) step, carry out second and third and take turns screening; Second take turns, Avastin concentration is reduced to 50 μ g/mL, 20 μ g/mL respectively in the third round screening, adds 2x10 respectively ¹¹Pfu takes turns the good Ph.D.-12 of amplification that filters out in the first round, two ^TMPhage, operation steps is undertaken by above-mentioned screening step (1)-(4); The Tween20 concentration of TBST all is increased to 0.5%; The eluted product of third round need not increase, and gets 1ul and measures titre;

(6) from screening back phage titre is measured for the third time, on the flat board of bacterial plaque number＜100 at random 80 of pickings separate good plaques and increase and purifying, the time that the plaque of picking is cultivated is no more than 18h; Described amplification and purifying: dip in 80 good blue plaques of separation getting with aseptic toothpick and put into host's culture that mid-log phase is cultivated respectively, after 37 ℃ of thermal agitations are cultivated 4.5h, the centrifugal 30s of 12000rpm, change supernatant liquor over to new centrifuge tube, the centrifugal again 30s of 12000rpm, get 80% supernatant, promptly get the phage suspension liquid that increases;

B) specific phage screening

In above-mentioned 80 clones that select, utilize phage E LISA further to select clone with the Avastin high-affinity;

(1) Avastin with 100mg/L wraps by 96 hole elisa plates, and every hole 200 μ L set up the negative contrast of interleukin 15 (IL-15) monoclonal antibody, 4 ℃ of overnight incubation to each Avastin hole simultaneously;

(2) coating buffer that inclines adds 4 ℃ of sealings of confining liquid 2h; The deblocking liquid that inclines, TBST cleans 6 times, all 96 orifice plates will be tipped upside down on the clean paper handkerchief at every turn, eliminates residual liquid; The phage of 80 purifying is pressed 1 * 10 ⁹/ hole adds Avastin, IL-15 monoclonal antibody bag respectively by incubated at room 2h in the hole; Described confining liquid contains the BSA of 5mg/L and the NaHCO of 0.1mol/L ₃Mixing solutions, the pH of this confining liquid is 8.6;

(3) TBST cleans 6 times, and every hole adds 1: 5000 mouse anti M13 phage antibody, the 100 μ L of horseradish peroxidase (HRP) mark, incubated at room 1h; TBST cleans 6 times, and the light absorption value at A490nm place is measured in O-Phenylene Diamine (O-Phenylenediamine) colour developing back;

(4) according to the light absorption value at A490nm place, filtering out 42 has higher affinity with Avastin and carries out sequencing with the low clone of other contrast avidity; Screening method: phage combines back A490nm light absorption value greater than 0.5 with Avastin, simultaneously with the ratio of control group A 490nm light absorption value greater than 3;

C) phage presents the sequencing of peptide

(1) extraction of phage single-chain DNA:

After the amplification of phage clone, get the phage culture supernatant, extract test kit with single stranded DNA and carry out the extraction of phage single-chain DNA, quantitative its concentration of ultraviolet is greater than 100ng/ μ L;

(2) Sanger double deoxidating chain termination measuring dna sequence dna:

With phage single-chain DNA is template, and the sequencing primer that is provided in the phage random peptide library test kit is provided, and primer sequence is ccctc atagt tagcg taacg, carries out automatic sequencing on automatic dna sequencer;

(3) present the derivation of peptide ammino acid sequence at random:

According to the dna sequencing result, draw phage and present the aminoacid sequence of peptide at random, it is as follows to draw sequence: Asp-His-Thr-Leu-Tyr-Thr-Pro-Tyr-His-Thr-His-Pro.

3. vaccine based on the anti-VEGF molecule of the mimic epitopes of the described human vascular endothelial growth factor VEGF of claim 1, it is characterized in that, described mimic epitopes chemical coupling is in protein carrier keyhole limpet hemocyanin (Keyhole limpet hemocyanin, KLH), its mode of connection is:

KLH-Asp-His-Thr-Leu-Tyr-Thr-Pro-Tyr-His-Thr-His-Pro。

4. the preparation method of the vaccine of the described anti-VEGF molecule of claim 3 is characterized in that, adopts following steps:

(1) chemosynthesis mimic epitopes polypeptide as claimed in claim 1

Asp-His-Thr-Leu-Tyr-Thr-Pro-Tyr-His-Thr-His-Pro；

(2) adopt glutaraldehyde method will synthesize polypeptide epitope and KLH chemical coupling: it is to shake in 10 the borate buffer solution that KLH is melted into the pH value, add step (1) synthetic mimic epitopes polypeptide, slowly splash into massfraction and be 0.3% glutaraldehyde solution, shook 2 hours, the glycine that adds 0.25 milliliter of 1mol/L, termination reaction after 30 minutes; Separating, is 4 ℃ of dialysed overnight in 8.5 the borate buffer solution with conjugate in pH value, and exchange buffering liquid continues to dialyse and obtains the conjugate of purifying more than 4 hours; The mass ratio of KLH and mimic epitopes polypeptide is 2: 3, and the mol ratio of KLH and glutaraldehyde solution is 10: 3;

(3) getting trace (Dot Blot) ready and identify conjugate, can promptly be the polypeptide vaccine that people VEGF mimic epitopes makes up with Avastin bonded conjugate in getting trace ready.

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