CN101838312B - Mimic short peptide 12B of endothelial cell growth factor VEGF antigen epitope and application thereof - Google Patents
Mimic short peptide 12B of endothelial cell growth factor VEGF antigen epitope and application thereof Download PDFInfo
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- CN101838312B CN101838312B CN201010019511A CN201010019511A CN101838312B CN 101838312 B CN101838312 B CN 101838312B CN 201010019511 A CN201010019511 A CN 201010019511A CN 201010019511 A CN201010019511 A CN 201010019511A CN 101838312 B CN101838312 B CN 101838312B
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Abstract
The invention discloses a mimic short peptide 12B of endothelial cell growth factor VEGF antigen epitope and an application thereof. The amino acid sequence of the mimic short peptide 12B is KARQLELNERTCRCDK and the nucleotide sequence is AAAGCTCGTCAGCTGGAACTGAACGAACGTACCTGCCGTTGCGACAAA. In vitro experiments show that the mimic short peptide 12B of the invention has obvious inhibiting effect during the processes of endothelial cell proliferation and tube formation and the tumor cell proliferation and migration. Therefore, the mimic short peptide 12B of the invention can be used for preparing peptide vaccines, tumor angiogenesis inhibitor or tumor-oriented drugs, and has significant reference value on diagnosing and predicting tumor tumorigenesis by utilizing VEGF receptor as a target, discussing development of small molecular peptides on the tumor angiogenesis inhibitors, and developing and researching tumor-oriented drugs.
Description
Technical field
The invention belongs to biology field and medical field, particularly a kind of mimic short peptide 12B and application thereof of endothelial cell growth factor VEGF antigen epitope.
Background technology
VEGF be Ferrara in 1989 in Niu Chuiti folliculus stellate cell vitro culture liquid at first a kind of relative molecular weight of coming out of purifying at the heterodimeric protein of 34～42KD.Human VEGF gene structure is positioned on the chromosomal 6p21.3, alternately is made up of 8 exons and 7 introns.Cut mode according to its mRNA is different, has produced 5 kinds of different VEGF isomer, comprises VEGF206, VEGF189, VEGF165, VEGF145 and VEGF121.The isomer of these 5 kinds of VEGF all has the outgrowth activity of inducing endothelial cell, is a kind of effective vascularization and permeability inducible factor, its difference mainly be they with cell surface and extracellular matrix in avidity different of heparin.
Its corresponding vegf receptor of VEGF combines to play a role.The vegf receptor that clones at present mainly contains two kinds of Flt-1 and KDR/Flk-1; The two all has tyrosine kinase activity; Be a kind of gp of striding film; Optionally express and endothelial cell surface, bring into play biological action, promptly promote endothelial cell proliferation, the formation that promotes blood vessel and the increase of vascular permeability through starting the various signals path.
Display technique of bacteriophage (Phage Display Technology) is a kind of exogenous peptide or protein to be merged with specific phage capsid protein PIII or PVIII mutually; Be showed in phage surface and make up protein or polypeptide libraries, and therefrom filter out the genetically engineered new and high technology of target protein, polypeptide or antibody.Its advantage is and can phenotype and genotype be contacted directly together, guarantees conveniently to show target protein and this proteic gene order.Based on this, identify at peptide sequence in recent years, the research of the 26S Proteasome Structure and Function of polypeptide stand-in and that display technique of bacteriophage is being brought into play important irreplaceable effect aspect the research of multiple biological target tissue polypeptides such as human virus, cell, tumour.
Research shows, VEGF has high expression level with its acceptor in many tumor tissues, organizes in kinds of tumors such as mammary cancer, lung cancer, large bowel cancer, cancer of the stomach, melanoma, lymphoma, sarcoma, adenomas inside and outsidely all can detect a large amount of VEGF.In situ hybridization test shows that the mRNA of VEGF mainly is distributed in the tumour cell, and tumour cell also highly expresses Flk-1, and the prompting tumor by local exists autocrine and paracrine mechanism promotion angiogenic growth.The size of the expression degree of VEGF and the histological type of tumour, primary tumo(u)r, have or not multiple factors such as blood vessel or lymphatic vessel transfer relevant: the expression degree of VEGF is significantly higher than the squama cancer in the pulmonary adenocarcinoma; VEGF high expression level person, nodus lymphoideus transferring rate and metastasis obviously increase; Low differentiation tumor patient's vegf expression is higher than high differentiation person; The recurrence rate height of VEGF positive patient, poor prognosis, Infant Mortality height.And the age of tumour patient, sex, tumor location etc. are irrelevant with vegf expression.Given this; Investigators imagine; If can obtain the associated epitope sequence of a series of VEGF, process micromolecule polypeptide, for the research and development of antigen peptide vaccine from now on provide support through display technique of bacteriophage and computer modeling technique; In human body, produce special antibody, will play a great role the treatment of malignant tumour in the human body to VEGF.
At present in the polypeptide drug; At anti-tumor aspect; People such as Kathleen through artificial chemosynthesis 2 polypeptide; The result shows that this polypeptide is higher to the inhibiting rate of human cervical carcinoma cell, but this peptide section and humanized VEGF do not have tangible homology, and the conservative motif MXXP with anti-tumor activity that people such as Maruta obtain does not have homology with FGFs yet.If these peptide section synthetic expenses are high, and as vaccine, may in human body, induce to produce the specific antibody of this peptide section, thereby stop the performance of its antitumor effectiveness.
Summary of the invention
The shortcoming that primary and foremost purpose of the present invention is to overcome prior art provides a kind of mimic short peptide 12B of endothelial cell growth factor VEGF antigen epitope with not enough.
Another object of the present invention is to provide the application of the mimic short peptide 12B of said endothelial cell growth factor VEGF antigen epitope.
The object of the invention is realized through following technical proposals: a kind of mimic short peptide 12B of endothelial cell growth factor VEGF antigen epitope; Its aminoacid sequence is Lys Ala Arg Gln Leu Glu Leu Asn Glu ArgThr Cys Arg Cys Asp Lys, i.e. KARQLELNERTCRCDK;
The nucleotide sequence of the mimic short peptide 12B of said endothelial cell growth factor VEGF antigen epitope is as follows: AAAGCTCGTCAGCTGGAACTGAACGAACGTACCTGCCGTTGCGACAAA;
The mimic short peptide 12B of described endothelial cell growth factor VEGF antigen epitope is used to prepare polypeptide vaccine, growth inhibitor for tumor vessels or tumor targeting medicine;
Described tumour is preferably melanoma.
The present invention has following advantage and effect with respect to prior art: the mimic short peptide 12B of endothelial cell growth factor VEGF antigen epitope according to the invention designs through bioinformatics method according to people's VEGF 189 peptide section sequences, then obtains through display technique of bacteriophage.External relevant experiment proof mimic short peptide 12B has the obvious suppression effect in endothelial cell proliferation, one-tenth pipe, tumor cell proliferation and transition process, just it has significantly antitumor and anti-angiogenic rebirth effect.The mimic short peptide 12B of endothelial cell growth factor VEGF antigen epitope of the present invention inquires into micromolecule polypeptide the exploitation of neonate tumour blood vessel suppressor factor and the R and D of tumor targeting property medicine is had crucial reference value for the incidence and development that with the vegf receptor is target, diagnosis prediction tumour.
Description of drawings
Fig. 1 is that recombinant vectors 12B-pCANTAB5-E enzyme is cut the electrophorogram of evaluation.
Fig. 2 resists with the anti-people VEGF of rabbit phagotope peptide 12B is carried out the figure as a result that ELISA detects more.
Fig. 3 is the figure as a result that mimic short peptide 12B suppresses endotheliocyte HUVEC propagation.
Fig. 4 is the figure as a result that mimic short peptide 12B suppresses tumour cell B16 propagation.
Fig. 5 is that mimic short peptide 12B suppresses the microscopic examination figure that endotheliocyte HUVEC becomes pipe.
Fig. 6 is the figure as a result that mimic short peptide 12B suppresses tumour cell B16F10 migration.
Fig. 7 is the microscopic examination figure that mimic short peptide 12B suppresses tumour cell B16F10 migration.
Embodiment
Below in conjunction with embodiment and accompanying drawing the present invention is described in further detail, but embodiment of the present invention is not limited thereto.
Embodiment 1
(1) through bioinformatics method, the nucleotide sequence that simulation obtains the mimic short peptide 12B of endothelial cell growth factor VEGF antigen epitope is described below:
AAAGCTCGTCAGCTGGAACTGAACGAACGTACCTGCCGTTGCGACAAA；
(2) be building up to efficiently on the phage vector pCANTAB5-E for the mimic short peptide 12B that will have only 48 bases; Earlier according to base sequence on the pCANTAB5-E and restriction enzyme site; Design primer 1 carries out first round PCR with primer 2, both PCR products as second take turns PCR template.The product base sequence that obtains according to base sequence and the first round PCR of mimic short peptide 12B then, design primer A and primer B carry out second and take turns PCR.Amplification obtains base length and carries out next step experiment at 100～200bp, the product that includes mimic short peptide 12B.
Primer 1:AAGCTTTGGAGCCTTTTTTTTGGAGATTTTCAACGTGAAAAAATTATTATTCGC
Primer 2: GGCCGGCTGGGCCGCATAGAAAGGAACAACTAAAGGAATTGCGAATAATAATTTTT TCA
Primer A:TATGCGGCCCAGCCGGCCAAAGCTCGTCAGCTGGAACTGAACGAACGTACCTGC CG
Primer B:GCGGCCGCTTTGTCGCAACGGCAGGTACGTTCGTTC
First round PCR condition: 94 ℃ of 5min, 55 ℃ of 5min, 72 ℃ of 5min, 7 circulations.
| System | 50μl |
| Primer 1 | 20μl |
| Primer 2 | 20μl |
| Buffer | 5μl |
| dNTP | 4.75μl |
| The Taq enzyme | 0.25μl |
Second takes turns the PCR condition: 94 ℃ of preparatory sex change 5min, and 94 ℃ of sex change 40s, 56 ℃ of annealing 35s, 72 ℃ are extended 40s, 30 circulations.
| System | 50μl |
| First round PCR product | 2μl |
| Primer A | 1μl |
| Primer B | 1μl |
| Buffer | 5μl |
| dNTP | 4μl |
| The Taq enzyme | 0.25μl |
| Tri-distilled water | 36.75μl |
(3) take turns the PCR product with second and cut the glue recovery, be connected into T carrier (Takara company), change picking list bacterium colony in the DH5 α intestinal bacteria (Takara company) over to, the upgrading grain carries out enzyme and cuts evaluation, and the sample presentation order-checking.Order-checking is accomplished by Shanghai biotechnology Services Co., Ltd.
The structure of carrier, connection and conversion: carry out the double digestion reaction through HindIII and Not I polypeptide fragment is downcut from the T carrier; Be connected in the pCANTAB5-E carrier (Pharmacia company) with same restriction enzyme site, obtain recombinant vectors 12B-pCANTAB5-E.Enzyme tangent condition and system are following: 37 ℃ of water-bath enzymes are cut 16h.
Condition of contact is following with system: 16 ℃ of water-baths are connected 24h.
| The T4DNA ligase enzyme | 1μl |
| The pCANTAB5-E enzyme is cut product | 2μl |
| VEGF epitope peptide fragment | 15μl |
| Buffer | 2μl |
| Total system | 20μl |
The carrier 12 B-pCANTAB5-E that builds is transformed in the TG1 intestinal bacteria (Pharmacia company), and picking mono-clonal, upgrading grain carry out enzyme and cut evaluation.It is as shown in Figure 1 that enzyme is cut qualification result.
(4) VEGF epitope peptide phage display and evaluation: go to infect building the TG1 bacterium that is positioned at logarithmic phase with helper phage VCSM13 (Stratagene company), add kantlex to 70mg/L after leaving standstill 30min; The centrifugal acquisition of 8000rpm bacterium liquid supernatant under 4 ℃ of conditions adds sterilization saturated ammonium sulphate ice bath deposition 3h, and the 4 ℃ of centrifugal acquisition of following 8500rpm phage depositions are removed supernatant and do not flowed out state to there being supernatant, DMEM dissolving, 4 ℃ of preservations; Get 10 μ l dilution back and survey titre.The titre of mimic short peptide 12B is 3.7 * 10
12Pfu/mL.This phagotope peptide is carried out ELISA to be identified: in enzyme plate, encapsulate the anti-people VEGF of rabbit with 9ng/100 μ L and resist (available from Abcam company), 37 ℃, 2h more; Wash plate 3 times with 1 * PBS-T, each 3min; 37 ℃ of sealings of 200 μ L/ holes, 5% skim-milk 1h; Wash plate 3 times with 1 * PBS-T, each 3min; Add different dilution phagotope peptides and set up VCSM13 contrast, blank, 100 μ L/ holes, 37 ℃, 1h; Wash plate 3 times with 1 * PBS-T, each 3min; Add phage-resistance HRP ELIAS secondary antibody (1: 2500) 100 μ L/ holes, 37 ℃, 30min; Wash plate 5 times with 1 * PBS-T, each 3min; Add substrate colour developing liquid (TMB) 100 μ L/ holes, lucifuge 10min adds stop buffer 2M H
2SO
4The OD450nm value is surveyed in 50 μ L/ holes.As shown in Figure 2, can know that mimic short peptide 12B has been illustrated in phage surface.
(5) to endotheliocyte and the inhibiting detection of tumor cell proliferation
A, mtt assay are surveyed the inhibited proliferation of phagotope peptide to endotheliocyte: separation and the cultivation of (1) former generation Human umbilical vein endothelial cells HUVEC: get one section of neonatal umbilical cord (Guangzhou overseas Chinese hospital provides); With PBS vein is rinsed well, closed blood vessel one end with the hemostasis clamp; With 0.25% trysinization perfusion, room temperature digestion 15min; Use the nutrient solution flushing that contains 10% calf serum and stop tryptic digestion, washing fluid is collected in the aseptic centrifuge tube of 50mL the centrifugal 10min of 1500rpm; Add complete SFM (available from the Gibco company) nutrient solution that contains 20% foetal calf serum, be inoculated in the culturing bottle 37 ℃, 5%CO
2Cultivate 24h in the constant incubator, change liquid, removal red corpuscle and other are attached cell not; Treat that cell attachment grows to had digestive transfer culture behind the 90% fusion state.(2) the former generation Human umbilical vein endothelial cells HUVEC that takes the logarithm vegetative period; Abandon nutrient solution in the most hole; Add an amount of 0.025% pancreatin solution digestion, jog makes the Digestive system stepless action in cell, when cell circle in blocks contracts, is the individual cells state, removes Digestive system; Add an amount of substratum rapidly, blow and beat gently with suction pipe and make into single cell suspension; Get in concentration branch to 96 orifice plate of this single cell suspension counting back with 5000 cells in every hole, with the nutrient solution of the M199 that contains 10% calf serum in 37 ℃, contain 5%CO
2Constant incubator in cultivate 24h; Use the M199 nutrient solution that contains 0.2% calf serum instead and continue to cultivate 24h to the monolayer state; Add the contrast of different dilution phagotope peptides, phage, and set up blank, in 37 ℃, 5%CO2 constant incubator, cultivate 48h; Every hole adds 5g/L MTT solution 20 μ l, and 37 ℃, continue to hatch 3h, stop cultivating; The careful suction abandoned culture supernatant liquid in the hole, and every hole adds 150 μ l DMSO, and vibration 10min fully dissolves crystallisate; Select the 490nm wavelength, on enzyme-linked immunosorbent assay instrument, set up each hole receipts value, the record result.Experiment repetition 3 times.Experimental result shows that this phagotope peptide has the effectiveness of the former generation HUVEC propagation of obvious suppression, and is as shown in Figure 3, and under 1/30 epitope peptide extent of dilution, epitope peptide 12B surpasses 40% to the growth inhibition ratio of HUVEC.
B, mtt assay are surveyed the inhibited proliferation of phagotope peptide to tumour cell: the K-1735 B16 in the vegetative period of taking the logarithm (available from Wuhan University China typical culture collection center); Abandon nutrient solution in the most hole; Add an amount of 0.25% pancreatin solution digestion, jog makes the Digestive system stepless action in cell, when cell circle in blocks contracts, is the individual cells state, removes Digestive system; Add an amount of substratum rapidly, blow and beat gently with suction pipe and make into single cell suspension; Get in concentration branch to 96 orifice plate of this single cell suspension counting back with 5000 cells in every hole, with the nutrient solution of the DMEM that contains 10% calf serum in 37 ℃, contain 5%CO
2Constant incubator in cultivate 24h; Use the DMEM nutrient solution that contains 0.2% foetal calf serum instead and continue to cultivate 24h to the monolayer state; Add different dilution phagotope peptides, M13 phage, and set up blank, in 37 ℃, 5%CO
2Cultivate 48h in the constant incubator; Every hole adds 5g/L MTT solution 20 μ l, and 37 ℃, continue to hatch 3h, stop cultivating; The careful suction abandoned culture supernatant liquid in the hole, and every hole adds 150 μ l DMSO, and vibration 10min fully dissolves crystallisate; Select the 490nm wavelength, on enzyme-linked immunosorbent assay instrument, set up each hole receipts value, the record result.Experiment repetition 3 times.As shown in Figure 4, under 1/30 epitope peptide extent of dilution, epitope peptide 12B reaches 66.5% to the growth inhibition ratio of B16.
(6) endotheliocyte is become the inhibiting detection of pipe
The ECM gel that gets 450 μ l places 4 ℃ of activation to spend the night.With the DMEM mixing of ECM gel and 450 μ l, note not occurring bubble.Every hole is got 60 μ l and is laid in 96 orifice plates, and room temperature leaves standstill 5min, treat its pave be placed in 37 ℃ of CO2 incubators subsequent use.Get former generation human umbilical vein endothelial cell HUVEC, the trysinization with 0.025% becomes single cell suspension, blow even, with 1.5 * 10
4The amount of/50 μ l adds to be completed in 96 orifice plates of ECM gel.Get VEGF epitope peptide 12B and VCSM13 and be diluted to 1/100, add in 96 orifice plates, set up blank simultaneously, 96 orifice plates are placed 37 ℃ of CO with 1640 minimum mediums
2Observe behind the cultivation 5hr in the incubator and take pictures.(40 *) experimental result is as shown in Figure 5, shows that 12B can effectively suppress the one-tenth pipe effect of HUVEC.
(7) to the inhibiting detection of tumor cell migration:
The melanoma high-transfer cell strain B16F10 in A, the vegetative period of taking the logarithm (the biological ltd of the triumphant base in Nanjing) becomes single cell suspension with 0.25% trysinization, and counting is with 1640 minimum medium diluting cells number to 5 * 10
5/ ml.
B, put into 24 well culture plates of Transwell cell, adding 1640 perfect mediums, the 600 μ l that contain 10% calf serum in cell lower floor.
C, add B16F10 cell suspension 100 μ l on the cell upper strata, and the phagotope peptide of corresponding adding 12B, concentration is 10
10Pfu/ml, (concentration is 10 to set up VCSM13 simultaneously
10Pfu/ml) contrast and blank.
D, place 37 ℃ to contain 5%CO 24 orifice plates
2In the incubator, cultivate 18h.
E, cell is taken out,, put into fixedly 15min of 70% ethanol under the room temperature with 1640 minimum medium drip washing 2 times.
F, the cell that will fixedly finish are with the PBS drip washing of 0.015M pH7.4 3 times; Air-dry, to put back in 24 orifice plates, the cell upper strata adds 100 μ l Giemsa stains; Lower floor adds 800 μ l Giemsa stains; Dyeing is taken out behind the 15min, with the PBS drip washing of 0.015M pH7.4 3～4 times, with cotton swab gently with the cell on cell upper strata wipe, air-dry.
G, put into microscopically counting (40 *); Do contrast with Control group cell migration rate 100%; Cell count/Control that medication group cell migration rate=medication group is moved to cell lower floor organizes cell count * 100% calculating of moving to cell lower floor; As shown in Figure 6, can know that 12B epi-position Toplink suppresses the transporting action of high-transfer cell strain B16F10, suppress mobility and surpass 80%; It is as shown in Figure 7 to take pictures (100 *), can see intuitively that medication group and VCSM13 and Control group compares, and has obviously suppressed the transporting action of B16F10.
Above experimental result shows; The mimic short peptide 12B of endothelial cell growth factor VEGF antigen epitope of the present invention; To a certain extent can be at the proliferation function of vitro inhibition endotheliocyte and tumour cell, and can obviously suppress HUVEC become pipe with and the transporting action of B16F10.
The foregoing description is a preferred implementation of the present invention; But embodiment of the present invention is not restricted to the described embodiments; Other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; All should be the substitute mode of equivalence, be included within protection scope of the present invention.
SEQUENCE?LISTING
< 110>Ji'nan University
< 120>the mimic short peptide 12B and the application thereof of endothelial cell growth factor VEGF antigen epitope
<130>2
<160>6
<170>PatentIn?version?3.2
<210>1
<211>16
<212>PRT
<213>artificial?sequence
<220>
< 223>aminoacid sequence of the mimic short peptide 12B of endothelial cell growth factor VEGF antigen epitope
<400>1
Lys?Ala?Arg?Gln?Leu?Glu?Leu?Asn?Glu?Arg?Thr?Cys?Arg?Cys?Asp?Lys
1 5 10 15
<210>2
<211>48
<212>DNA
<213>artificial?sequence
<220>
< 223>nucleotide sequence of the mimic short peptide 12B of endothelial cell growth factor VEGF antigen epitope
<400>2
aaagctcgtc?agctggaact?gaacgaacgt?acctgccgtt?gcgacaaa 48
<210>3
<211>54
<212>DNA
<213>artificial?sequence
<220>
< 223>primer 1
<400>3
aagctttgga?gccttttttt?tggagatttt?caacgtgaaa?aaattattat?tcgc 54
<210>4
<211>59
<212>DNA
<213>artificial?sequence
<220>
< 223>primer 2
<400>4
ggccggctgg?gccgcataga?aaggaacaac?taaaggaatt?gcgaataata?attttttca 59
<210>5
<211>56
<212>DNA
<213>artificial?sequence
<220>
< 223>primer A
<400>5
tatgcggccc?agccggccaa?agctcgtcag?ctggaactga?acgaacgtac?ctgccg 56
<210>6
<211>36
<212>DNA
<213>artificial?sequence
<220>
< 223>primer B
<400>6
gcggccgctt?tgtcgcaacg?gcaggtacgt?tcgttc 36
Claims (4)
1. the mimic short peptide 12B of an endothelial cell growth factor VEGF antigen epitope, it is characterized in that: the aminoacid sequence of said mimic short peptide 12B is as follows:
Lys?Ala?Arg?Gln?Leu?Glu?Leu?Asn?Glu?Arg?Thr?Cys?Arg?Cys?Asp?Lys。
2. mimic short peptide 12B according to claim 1 is characterized in that: the nucleotide sequence of the said mimic short peptide 12B that encodes is as follows:
AAAGCTCGTCAGCTGGAACTGAACGAACGTACCTGCCGTTGCGACAA。
3. the application of the mimic short peptide 12B of claim 1 or 2 described endothelial cell growth factor VEGF antigen epitopes is characterized in that: said mimic short peptide 12B is used to prepare tumour polypeptide vaccine, growth inhibitor for tumor vessels or tumor targeting medicine.
4. application according to claim 3 is characterized in that: described tumour is a melanoma.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002046213A2 (en) * | 2000-11-07 | 2002-06-13 | Gpc Biotech Inc. | Methods and reagents for isolating angiogenic modulators |
| CN101429234A (en) * | 2008-12-11 | 2009-05-13 | 中国人民解放军第四军医大学 | Vaccine based on simulating human blood vessel endothelial cell growth factor VEGF epitope and preparation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002046213A2 (en) * | 2000-11-07 | 2002-06-13 | Gpc Biotech Inc. | Methods and reagents for isolating angiogenic modulators |
| CN101429234A (en) * | 2008-12-11 | 2009-05-13 | 中国人民解放军第四军医大学 | Vaccine based on simulating human blood vessel endothelial cell growth factor VEGF epitope and preparation method thereof |
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