CN102115492B - Vascular endothelial growth factor receptor partial polypeptide and application thereof - Google Patents
Vascular endothelial growth factor receptor partial polypeptide and application thereof Download PDFInfo
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- CN102115492B CN102115492B CN 201010033809 CN201010033809A CN102115492B CN 102115492 B CN102115492 B CN 102115492B CN 201010033809 CN201010033809 CN 201010033809 CN 201010033809 A CN201010033809 A CN 201010033809A CN 102115492 B CN102115492 B CN 102115492B
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Abstract
The invention discloses a vascular endothelial growth factor receptor partial polypeptide and application thereof. The amino acid sequence of the vascular endothelial growth factor receptor partial polypeptide is Sequence 1 in the sequence table. The polypeptide can be coupled with a carrier protein to form a compound protein. The carrier protein is a keyhole limpet hemocyanin or bovine serum albumin. The mixture of the compound protein and an immunological adjuvant can be used as an immunogen. The immunogen immunized animal acquires high-titer immune serum. The ELISA inspection indicates that the titer is higher than 1:60000; and the immunobiotting test proves that the immune serum has strong specificity when being combined with a chicken vascular endothelial growth factor receptor, and can be used for detecting the expression of the chicken vascular endothelial growth factor receptor on the cellular level.
Description
Technical field
The present invention relates to a kind of vascular endothelial growth factor receptor part of polypeptide and application thereof.
Background technology
Find that so far vascular endothelial growth factor (VEGF) receptor family comprises the acceptor with two kinds of signal transduction pathways: tyrosine kinase receptor and non-tyrosine kinase receptor, tyrosine kinase receptor comprises Flt-1 (fms-liketyrosine kinase), KDR Flk-1 (kinase insert domain containing fetal liverkinase) and VEGFR-3, Flt-1 claims again vegf receptor 1 (VEGFR-1), KDR Flk-1 claim again vegf receptor 2 (VEGFR-2), non-tyrosine kinase receptor comprises heparin-like molecule, neuropilin-1 acceptor (NRP-1) and Neuropilin-2 acceptor (NRP-2), wherein KDR is the high-affinity receptor of VEGF, the mitotic division of main participation vascular endothelial cell, propagation, migration, differentiation, the vital movements such as microtubule formation are the focuses of studying at present.KDR plays keying action in the tumor vessel forming process, high expression level is expressed and be low in the healthy tissues in endothelial cells in tumor neogenetic blood vessels and the high tumour cell of grade malignancy.KDR is expressed in vascular endothelial cell specifically, and its expression level and vascular endothelial cell renewal speed are proportionate.Healthy tissues vascular endothelial cell renewal speed is slow under the physiological conditions, and KDR expresses very low, and the vascular endothelial cell in the tumor tissues is in active vegetative state, fast 500 times than healthy tissues vascular endothelial cell of rate of propagation, and KDR expresses very high.The expression level of KDR can be used as the index of assessment oncobiology behavior and predicting tumors patient prognosis.The expression level of KDR can detect by the antibody with the KDR specific combination.
Summary of the invention
The purpose of this invention is to provide a kind of vascular endothelial growth factor receptor part of polypeptide.
Vascular endothelial growth factor receptor provided by the present invention (KDR) part of polypeptide, its aminoacid sequence are the sequence 1 in the sequence table.
Vascular endothelial growth factor receptor part of polypeptide of the present invention can be used to prepare immunogen, and this immunogen immune animal can obtain the polyclonal antibody with the KDR specific combination.
For producing in animal body immune response, the molecular weight of most of polypeptide is all too little, polypeptide and carrier proteins such as keyhole limpet hemocyanin or bovine serum albumin etc. are crosslinked after, can not only increase the size of antigen, also can strengthen immunity.
Therefore, the present invention also provides a kind of conjugated protein.
Conjugated protein provided by the present invention is the conjugated protein that described polypeptide and carrier protein couplet form.
Conjugated protein of the present invention, wherein, described carrier proteins is keyhole limpet hemocyanin or bovine serum albumin.
When immune animal, usually conjugated protein is mixed with immunological adjuvant in order to strengthen immunogenicity.
Immunogen provided by the present invention is the mixture of described conjugated protein and immunological adjuvant.
The polyclonal antibody of of the present invention and vascular endothelial growth factor specific combination is use the immunogen immune animal, takes the afterwards blood of animal of immunity, the serum of acquisition; Described immunogen is the mixture of conjugated protein and immunological adjuvant; Described conjugated protein is the conjugated protein that polypeptide and carrier protein couplet form, and the aminoacid sequence of described polypeptide is the sequence 1 in the sequence table.
The polyclonal antibody of of the present invention and vascular endothelial growth factor specific combination, wherein, described carrier proteins is keyhole limpet hemocyanin or bovine serum albumin.
Immunogen immune animal of the present invention has obtained the immune serum of more efficient valency, tire through ELISA check and to be higher than 1: 60000, and immunoblotting proves that the specificity that this immune serum is combined with the chicken vascular endothelial growth factor receptor is stronger, and immune serum can detect at cell levels the expression of chicken vascular endothelial growth factor receptor.
Description of drawings
Fig. 1 is the specificity that immunoblotting detects immune serum.
Fig. 2 is the picture that shows the result under the blank group laser confocal microscope;
A is for differing picture, and B is for differing and fluorescence stack picture, and C is the fluorescence picture.
Fig. 3 is the picture that shows the result under the control serum group laser confocal microscope;
A is for differing picture, and B is for differing and fluorescence stack picture, and C is the fluorescence picture.
Fig. 4 is the picture that shows the result under the immune serum group laser confocal microscope;
A is for differing picture, and B is for differing and fluorescence stack picture, and C is the fluorescence picture.
Embodiment
Embodiment 1, with the polyclonal antibody of KDR specific combination
(1) synthetic immunogen
Synthetic immune peptide, its aminoacid sequence are CAQEASPTLPRVHGLVHD.
Synthetic contrast polypeptide, its aminoacid sequence is AFGQVIEADAFGIDKTATC.
Polypeptide to synthetic detects.
The result shows, the immune peptide of synthetic is white lyophilized powder, and molecular weight is that to detect purity be 98% to 1931.13, HPLC (220nm, C18, linear gradient).
The contrast polypeptide is white lyophilized powder, and molecular weight is that 1957.19, HPLC (220nm, C18, linear gradient) detection purity is 95%.
(2) with the polyclonal antibody of chicken vascular endothelial growth factor receptor specific combination
Immune peptide and carrier proteins form polypeptide-carrier conjugated protein according to mass ratio coupling in 1: 1, and carrier proteins can be keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA), and the present embodiment is immune peptide-KLH conjugated protein.
Contrast polypeptide and KLH form contrast polypeptide-K LH conjugated protein according to mass ratio coupling in 1: 1.
Immune peptide-KLH conjugated protein mixes with Freund＇sadjuvantcomplete (Sigma), makes immunogen.
Contrast polypeptide-K LH conjugated protein mixes with Freund＇sadjuvantcomplete (Sigma), makes the contrast immunogen.
Immunogen and the immune SPF new zealand rabbit of contrast immunogen difference with above-mentioned preparation.
In 20 ages in week, the SPF new zealand rabbit of 2.5-3Kg is divided into two groups at random, immune peptide group and contrast polypeptide group, 2 every group.Immune peptide group injecting immune is former, contrast polypeptide group injection contrast immunogen.
The detailed process of immunity is as follows:
The 1st week, every day multiple spot back subcutaneous injection immunity SPF new zealand rabbit once, the total amount of every SPF new zealand rabbit injecting immune polypeptide-K LH conjugated protein or contrast polypeptide-K LH conjugated protein is 0.1mg;
In the 2nd week, the SPF new zealand rabbit is left intact;
Week in the 3rd week-the 7th, every day multiple spot back subcutaneous injection immunity SPF new zealand rabbit once, the total amount of every SPF new zealand rabbit injecting immune polypeptide-K LH conjugated protein or contrast polypeptide-K LH conjugated protein is 0.1mg;
The immunity of the 7th week finishes, and puts to death the SPF new zealand rabbit, takes 150ml blood from ear and heart, and short solidifying with setting accelerator, 3000 left the heart 20 minutes.
Immune peptide group adaptive immune serum 01 and 02.
Contrast polypeptide group obtains control serum 01 and 02.
ELISA detects tiring of immune serum and control serum, and concrete grammar is as follows:
1) above-mentioned chicken vascular endothelial growth factor receptor albumen is made into the solution of 100 μ g/ml with PBS, adds 96 hole enzyme plates by the every hole of 100 μ l/, 4 ℃ of absorption of spending the night;
2) protein solution of absorption that will spend the night next day in the coated enzyme plate is not outwelled, and washes 3 times with PBS.Add 3% (mass percent) BSA (not containing Tween20 with the PBS preparation) sealing, the every hole of 100 μ l/, 37 ℃, 1.5 hours;
3) thoroughly wash 2 with PBS) in precipitation 4 times after add immune serum or control serum (by volume be 1: 100,1: 500,1: 2500,1: 12500 or 1: 62500 dilution with PBS), the every hole of 100 μ l/, 37 ℃, 1.5 hours;
4) wash 3 with PBS) in precipitation 4 times, add two anti-(goat anti-rabbit igg is dilution in 1: 10000 with PBS by volume), the every hole of 100 μ l/, 37 ℃, 1hr;
5) wash 4 with PBS) in precipitation 4 times, add AP nitrite ion P-NPP (2mg/ml), the every hole of 80 μ l/, the lucifuge reaction adds equal-volume 0.2M NaOH termination reaction.
6) get 100 μ l 5) in solution 100 μ l after the termination reaction, measure OD value under the 405nm with microplate reader.
The experiment triplicate, the result represents with three times mean value.
Serum titer detected result such as table 1 and shown in Figure 1;
Table 1. serum titer detected result
| Extension rate | 100 | 500 | 2500 | 12500 | 62500 | Blank |
| Control serum 01 | 0.732 | 0.536 | 0.313 | 0.095 | 0.015 | 0.005 |
| Immune serum 01 | 1.529 | 1.642 | 1.557 | 1.244 | 0.694 | 0.005 |
| Control serum 02 | 0.780 | 0.595 | 0.294 | 0.079 | 0.027 | 0.006 |
| Immune serum 02 | 1.557 | 1.642 | 1.506 | 1.184 | 0.616 | 0.006 |
The immune serum bioactivity is the result show, immune serum energy and chicken vascular endothelial growth factor receptor specific combination, and it is tired and can be reached for 1: 60000; Control serum can not with chicken vascular endothelial growth factor receptor specific combination.
(3) specificity of immune serum
Extract the chicken endothelial progenitor cells total protein of vitro culture, immunoblotting detects the specificity (immune serum dilution in 1: 1000) of the chicken vascular endothelial growth factor receptor binding ability in immune serum and the chicken endothelial progenitor cells total protein.
Experimental result as shown in Figure 1, as seen obvious band, its size is about 200KD, very unconspicuous band is arranged in the position less than 200KD, chicken vascular endothelial growth factor receptor specific binding in immune serum and the chicken endothelial progenitor cells total protein is described, very weak with the bonding force of other albumen.
(4) expression of detection chicken vascular endothelial growth factor receptor on the cell levels
Use Immunohistochemical Method to detect the expression of the chicken vascular endothelial growth factor receptor of chicken endothelial progenitor cells.
Blank group, control serum group and immune serum group are set.
The blank group adopts PBS as primary antibodie, and goat anti-rabbit igg (the middle China fir Zf-0311 of Golden Bridge) is anti-as two;
The control serum group adopts control serum 01 as primary antibodie (dilution in 1: 500), and goat anti-rabbit igg (the middle China fir Zf-0311 of Golden Bridge) is anti-as two;
The immune serum group adopts immune serum 01 as primary antibodie (dilution in 1: 500), and goat anti-rabbit igg (the middle China fir Zf-0311 of Golden Bridge) is anti-as two;
The immunohistochemical methods concrete grammar is as follows:
1, sample preparation
Cell is inoculated in the coated cover glass of poly-lysine, treats that cell is adherent on cover glass, PBS cleans three times, and 4% Paraformaldehyde 96 is fixed.
2, dyeing
1) cover glass after fixing penetrating 15 minutes at 0.25%Tritonx-100;
2) wash 2min/ time * 3 times with PBS;
3) adding a sealing sealed on cover glass 30 minutes with lowlenthal serum;
4) outwell or blot liquid;
5) every cover glass adds the 100ul primary antibodie, hatches 30～60 minutes in the wet box;
6) wash 2min/ time * 3 times with PBS;
7) every cover glass add one two anti-, hatched 60 minutes;
8) wash 2min/ time * 3 times with PBS;
9) microscopy.
Observe ImmunohistochemistryResults Results under the laser confocal microscope, the result of blank group as shown in Figure 2, the result of control serum group as shown in Figure 3, all do not have fluorescence in microscopically blank group and control serum group, show PBS and control serum all can not with the chicken endothelial progenitor cells in chicken vascular endothelial growth factor receptor specific combination; The result of immune serum group has fluorescence at the cell of microscopically 95% as shown in Figure 4, show immune serum can with the chicken endothelial progenitor cells in chicken vascular endothelial growth factor receptor specific combination.
Comprehensive the above results shows, conjugated protein behind immune peptide and the carrier protein couplet is that the immunogen of main component can produce immune serum by stimulating animal, this immune serum can with the vascular endothelial growth factor receptor specific binding, and can detect at cell levels the expression of vascular endothelial growth factor receptor.
Above embodiment is described preferred implementation of the present invention; be not that scope of the present invention is limited; design under the prerequisite of spirit not breaking away from the present invention; various distortion and improvement that the common engineering technical personnel in this area make technical scheme of the present invention all should fall in the definite protection domain of claims of the present invention.
Sequence table
＜110〉Institute of Animal Sciences, Chinese Academy of Agricultural Sciences
＜120〉vascular endothelial growth factor receptor part of polypeptide and application thereof
<160>2
<210>1
<211>18
<212>PRT
＜213〉artificial sequence
<220>
<230>
<400>1
Cys Ala Gln Glu Ala Ser Pro Thr Leu Pro Arg Val His Gly Leu Val
1 5 10 15
His Asp
<210>2
<211>16
<212>PRT
＜213〉artificial sequence
<220>
<230>
<400>2
Ala Phe Gly Gln Val Ile Glu Ala Asp Ala Phe Gly Ile Asp Lys Thr
1 5 10 15
Ala Thr Cys
Claims (5)
1. a peptide species, its aminoacid sequence is the sequence 1 in the sequence table.
2. a conjugated protein is the conjugated protein that polypeptide and carrier protein couplet form, and the aminoacid sequence of described polypeptide is the sequence 1 in the sequence table; Described carrier proteins is keyhole limpet hemocyanin or bovine serum albumin.
3. an immunogen is the mixture of conjugated protein and immunological adjuvant; Described conjugated protein is the conjugated protein that polypeptide and carrier protein couplet form, and the aminoacid sequence of described polypeptide is the sequence 1 in the sequence table; Described carrier proteins is keyhole limpet hemocyanin or bovine serum albumin.
4. the application of immunogen claimed in claim 3 in the preparation polyclonal antibody.
5. with the polyclonal antibody of vascular endothelial growth factor specific combination, be to use the immunogen immune animal, take the blood of animal after the immunity, the serum of acquisition; Described immunogen is the mixture of conjugated protein and immunological adjuvant; Described conjugated protein is the conjugated protein that polypeptide and carrier protein couplet form, and the aminoacid sequence of described polypeptide is the sequence 1 in the sequence table; Described carrier proteins is keyhole limpet hemocyanin or bovine serum albumin.
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| CN 201010033809 CN102115492B (en) | 2010-01-05 | 2010-01-05 | Vascular endothelial growth factor receptor partial polypeptide and application thereof |
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| CN102115492B true CN102115492B (en) | 2013-03-06 |
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| CN107056950B (en) * | 2017-03-24 | 2020-10-20 | 攸归（上海）生物科技有限公司 | Fusion protein containing keyhole limpet hemocyanin fragment and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1694901A (en) * | 2002-09-12 | 2005-11-09 | 肿瘤疗法科学股份有限公司 | Kdr peptides and vaccines comprising the same |
| CN101070349A (en) * | 2007-05-22 | 2007-11-14 | 山西康宝生物制品股份有限公司 | Fusion protein with function of selective killing endothelial cells in tumor neogenetic blood vessels and use thereof |
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2010
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1694901A (en) * | 2002-09-12 | 2005-11-09 | 肿瘤疗法科学股份有限公司 | Kdr peptides and vaccines comprising the same |
| CN101070349A (en) * | 2007-05-22 | 2007-11-14 | 山西康宝生物制品股份有限公司 | Fusion protein with function of selective killing endothelial cells in tumor neogenetic blood vessels and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| Susana constantino rosa santos and sergio dias.Internal and external autocrine VEGF/KDR loops regulate survival of subsets of acute leukemia through distinct signaling pathways.《Blood》.2004,3883-3889. * |
| 李丽英等.抗KDR胞内区(KDR-CD)单克隆抗体的制备和鉴定.《细胞与分子免疫学杂志》.2008,第24卷(第4期),393-395. * |
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