CN1162705C - Method of screening inhibiting new angiopoiesis medicine and cell clone used for said method - Google Patents
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Abstract
The present invention provides a method for filtering and restraining a neovascularization medicine, which comprises: biological molecules with CD146 combined activity are filtered by using CD46 protein molecules or cells used for expressing CD146 protein; whether the biological molecules with the CD146 combined activity can restrain or activate the cell growth or not is detected by the cells for naturally expressing the CD46 molecules. The medicine filtered by the present invention not only can be used for angiogenesis relative disease prevention, diagnosis and treatment but also can be used as a novel abortion and contraception medicine, and the present invention is used for population control.
Description
Technical field
The present invention relates to a kind of method that suppresses new angiopoiesis medicine and clone that is used for this method of screening.
Background technology
The CD146 molecule has another name called MUC18, A32 antigen, MCAM, Mel-CAM and S-Endo-1
[1-3]This molecule is accredited as the member of Ig gene superfamily recently.It is a kind of glycoprotein of striding film, and its extracellular region has the class Ig domain of a typical V-V-C-C-C, and beta sheet is made up of 43-70 amino acid.The effect of disulfide bond between the beta sheet makes domain very stable.The CD146 molecule only has one to stride the film district, and the kytoplasm part is shorter, and the recognition site of some potential protein kinases is arranged.CD146 is the protein of a high glycosylation, and about 35% molecular weight is made up of carbohydrates, comprises sialic acid and other carbohydrates.
At present, very limited to the understanding of CD146 function aspects.Confirmed that it is cell adhesion molecule and the inside and outside signal transduction molecule of cell born of the same parents
[4-6], to the set of sticking of part, the cytoskeleton reorganization, cell-cell interaction, cellular morphology safeguards that cell migration, hyperplasia control all play effect.In addition, CD146 implants at tumor development, blastocyst, the medium process of placentation plays an important role
[7-9]
Summary of the invention
An object of the present invention is to provide a kind of method that suppresses new angiopoiesis medicine of screening.
Another object of the present invention provides a kind of clone that is used for method of the present invention.
The invention provides a kind of method that suppresses new angiopoiesis medicine of screening, comprising:
(1) cell with CD146 protein molecular or expression CD146 albumen contacts washing then with a kind of biomolecule;
(2) sample after handling in the step (1) is contacted washing then with a kind of antibody of described biomolecule of detectable label mark;
(3) described detectable label is detected, obtain the biomolecule that can combine with described CD146 protein molecular;
(4) cell with natural expression CD146 molecule contacts with the biomolecule with the combination of CD146 protein molecular that obtains; If described biomolecule can suppress or activate the growth of the cell of this natural expression CD146 molecule, promptly be inhibited or promote the medicine that neovascularity generates.
In the method for the invention, described biomolecule can be part, antibody molecule or genetic engineering fragment, polypeptide or immunotoxin; The cell of described expression CD146 can be a 293-EBNA clone, and its deposit number is: CGMCC 0642; Described screening has CD146 and can be undertaken by competitive ELISA in conjunction with the biomolecule of activity.
The present invention also provides a kind of 293-EBNA clone that is used to screen the expression CD146 albumen that suppresses new angiopoiesis medicine.This clone be preserved in the biological center (CGMCC) of China Committee for Culture Collection of Microorganisms common micro-organisms October 8 calendar year 2001, the preservation centre address is China, Beijing, Zhong Guan-cun.Deposit number is CGMCC 0642.
The preparation method of clone of the present invention is as follows: at first the CD146 full-length gene is inserted into efficient expression vector pCEP-Pu, makes up a recombinant plasmid that contains the CD146 gene.With this plasmid transfection host cell is 293-EBNA.Selecting to screen the positive colony that contains recombinant plasmid on the nutrient culture media.Because the CD146 molecule can be expressed in the surface of cell membrane of host cell, therefore this cell can be used to screen various material that combines with CD146, and whether part, antibody, polypeptide or the proteotoxin that can screen by the cell detection of natural expression CD146 molecule have specific inhibition or activation effect.
This application of model can be illustrated in the following aspects: 1) by competitive combination screening CD146 targeted drug.Even the just more weak molecule of affinity that obtains is obtained the high medicine of affinity thereby also can improve molecule through further structural research.Its further evaluation and application comprise zoopery even clinical trial, all will be closely bound up with the efficient of initial screening model.2) detect the effect whether medicine that filters out has blocking-up or activation signal conduction.In the cell of natural expression CD146 molecule, add the drug molecule (antibody that filters out, polypeptide, immunotoxin etc.), if can suppress or the activation signal transduction, will cause the change of some phenotype of cell, as growth rate, cell polymorphism, cell colony formation, the growth conditions on soft agar, the antitumor activity of cell, the aspects such as expression of antigen, and its cellular control unit will not show this category feature.3) this model also provides a kind of method of selecting antibody from antiserum.The antiserum that synthetic peptide section immune animal is obtained screens through cell model, obtains the antibody of high-affinity, and then studies itself and CD146 the interact biological process of initiation and the related activity position of definite CD146.If it has potential pharmaceutically active, the toxicity that can be correlated with, pharmacology are measured.
Using value of the present invention is that (1) CD146 conjugate or CD146 engineering cell can be used as vaccine, be used for above-mentioned prevention and treatment of diseases, (2) the CD146 engineering cell can be used as a kind of drug screening molecular model, be used to screen various parts, antibody, polypeptide or proteotoxin, (3) native ligand of CD146 can be a kind of medicine and drug targeting, be used for prevention, diagnosis and the treatment of angiogenesis-associated diseases, and can be used as a kind of novel miscarriage contraceptive, be used for population control.
We develop the specific monoclonal antibody of CD146, the interaction of finding antibody and CD146 first can cause following result: (1) suppresses vascular endothelial cell proliferation and migration, (2) suppress angiogenesis, (3) cause tumor vascular atrophy and obstruction, suppress the growth and the transfer of kinds of tumors, its inhibiting rate is respectively leiomyosarcoma 50%, liver cancer 71.9%, cancer of pancreas 48.7%.(4) by suppressing the trophocyte, cause the pregnant mouse miscarriage, illustrate that CD146 is early stage key molecule of embryonic development and peculiar mark, most important for embryonic development and pregnancy maintenance.Our experimental result confirms that CD146 albumen is a kind of new vessel landmarks molecule, and it participates in angiogenesis, embryonic development and tumor growth and transfer.
Embodiment
Embodiment one
The foundation of CD146 drug screening cell model
1.CD146 the structure of expression vector
Extract total mRNA from the umbilical vein endothelium, by the synthetic first chain cDNA of reverse transcription.According to the gene order (P43121) of CD146 design contain restriction site 5 ' with 3 ' end primer.With cDNA is template, amplifies CD146 full-length gene fragment by the PCR reaction.Be inserted into the multiple clone site of efficient expression vector pCEP-Pu (Eddie Kohfeldt, 1996) by double digestion, make it to coincide with the open reading frame of carrier.By the DNA agarose gel electrophoresis PCR product and recombinant plasmid are identified.Enzyme cut recombinant plasmid as a result express foreign gene be cloned into the multiple clone site of carrier.Dna sequence analysis confirms that recombinant plasmid contains the CD146 gene.
2.CD146 the structure and the evaluation of engineering cell system
Preparation 293-EBNA competent cell, by the calcium phosphate precipitation method will build contain CD146 expression carrier pCEP-Pu import host cell be 293-EBNA (Invitrogen, CatNo.R620-07).Cells transfected is cultivated in containing the selection nutrient culture media of Puromycin and G418, and the picking cell colony continues selecting the frozen engineering cell in enlarged culture one week back on the nutrient culture media.
Identify the expression of CD146 molecule in 293-EBNA clone at gene and protein level.At first, determine with PCR method whether the CD146 gene is present in the 293-EBNA cell.Collect and cell lysis, add PCR reaction system and primer, the pcr amplification result shows that the gene band of a treaty 2000bp appears in the 293-EBNA cell of transfection CD146 gene, and the 293-EBNA cell of untransfected occurs without any the PCR product.Illustrate that the CD146 gene is present in the 293-EBNA cell.This clone be preserved in the biological center (CGMCC) of China Committee for Culture Collection of Microorganisms common micro-organisms October 8 calendar year 2001, the preservation centre address is China, Beijing, Zhong Guan-cun.Deposit number is CGMCC 0642.
Identify further whether the CD146 gene expresses in 293-EBNA clone.We add cell lysis buffer solution in cell culture fluid, scrape behind the cell centrifugal, identify that with SDS-PAGE and Westernblotting method the expression of CD146 molecule, result show that the CD146 molecule of expressing in the 293-EBNA cell can be discerned by the CD146 specific antibody.
Embodiment two
Adopt flow cytometer (FACS) to analyze the CD146 bond
Method is as follows: with 10
6Engineering cell be suspended in 100 μ l contain 10 μ g/mlCD146 specific antibodies nutrient culture media in, hatch 1h, it is inferior to give a baby a bath on the third day after its birth through nutrient culture media, add the FITC-sheep anti-mouse igg, hatched 45 minutes, give a baby a bath on the third day after its birth all over after, cell is suspended among the 200 μ l-500 μ l PBS again, on FACS, carry out immunofluorescence analysis and measure mean fluorecence density.Facs analysis shows, constructed cell can be at its surface expression CD146 molecule, and with antibody specific the combination taken place.The expressed CD146 molecule of cell that structure is described has been kept its native conformation preferably.
Embodiment three
CD146 molecular cell model discrimination specific antibody
1.ELISA the antibody of method screening and CD146 specific bond
The ELISA method can be used to screen the material that combines with the CD146-293-EBNA cell, as antibody.Method is as follows: with every hole 10
5Cell inoculation 96 porocyte culture plates, when treating cell 80% confluent cultures hole, use the methyl alcohol fixed cell, 2%BSA seals 2h, adds antibody sample to be detected (hybridoma supernatant, soluble antibody molecule fragment), corresponding cell culture medium or the negative contrast of damping fluid, hatch 2h for 37 ℃, PBS washes 3 times, adds the sheep anti mouse κ of AP mark, hatching 1h for 37 ℃, is the substrate colour developing with PNPP.The microplate reader reading.According to the result of chromogenic reaction, preliminary screening goes out the strong sample of reaction signal, carries out Western blot and identifies.
2. utilize the specific antibody and the binding peptide of antibody library and peptide storehouse screening CD146 molecule
With 10
5Cells/well is inoculated 96 porocyte culture plates, 37 ℃ of overnight incubation, and 4% paraformaldehyde is fixed, and 2%BSA seals 2h, and every hole adds 100 μ l 10
10Phage antibody library or peptide storehouse (being dissolved in 0.5%BSA) are hatched 2h for 37 ℃, and PBST and PBS respectively wash 10 times, again with 100 μ l 0.1MpH, 11.6 triethylamine wash-outs, collect eluent, dilute postoperative infection host bacterium in varing proportions, are coated with the LB flat board.Positive colony centrifuging and taking supernatant after enlarged culture carries out the next round screening.
The clone who obtains after the screening of number wheel suppresses one step of experiment by the ELISA competition and identifies.
Cell is inoculated in Tissue Culture Plate with 105 cells/well, cultivate 2h for 37 ℃, 4% paraformaldehyde is fixed, 2%BSA seals 2h, adds clone's supernatant to be checked and soluble cd 14 6 molecules, hatches 2h, through PBST and PBS washing, add the two anti-of HRP or AP mark, add the substrate colour developing, the microplate reader reading after hatching 1h.To present weak chromogenic reaction with the phage antibody hole of CD146 specific bond.
Confirm have the phage antibody of specific bond activity to transform the host bacterium again through above step, be prepared into plasmid DNA, will weigh, light chain clones in universal support, utilizes the universal primer order-checking.
Embodiment four
Detect the activation or the depression effect of CD146 cell model screening of medicaments
1.MTT method detects the activation or the depression effect of CD146 cell model screening of medicaments.
Endothelial cell with natural expression CD146 molecule is an object, adopts mtt assay to detect the activation or the depression effect of CD146 cell model screening of medicaments.Method is as follows: get the new vessels endothelial cell and cultivate, at exponential phase, trypsinization and the counting, with 3000 cell inoculations in every hole in 96 orifice plates.Add the medicine that variable concentrations filters out after 24 hours, continue to cultivate 24-72 hour.Abandon supernatant, every hole adds the MTT of 0.1mg/0.1ml, continues to cultivate to add 150 μ l DMSO vibration after 4 hours, surveys the OD value and calculates survival rate in 560nm.Blank is not for adding cell hole.Negative control is not for adding the medicine group.According to the influence of the survival rate analysis medicine pair cell of cell, judge that this medicine is the activator or the inhibitor of endothelial cell.
2.[
3H] method of mixing measures the effect of medicine to endothelial cell growth.
Method is as follows: get the endothelial cell of vigorous growth, inoculate 96 orifice plates with 3000 every holes of cell after the trypsinization, add the DMEM nutrient culture media that contains 10%FCS, hatch 2h, abandon nutrient solution, add serum free medium and spend the night, add the medicine of different amounts next day, hatches 48h.Add then [
3H] the mark thymine, incubation 4h, collecting cell is measured through liquid scintillation instrument, analyzes the effect of medicine cell growth.
3. detection of drugs influence that the trophocyte is sticked
Method is as follows: the trophocyte of separation and Culture natural expression CD146 of former generation molecule.Spread 96 orifice plates, ambient temperature overnight with extracellular matrix protein., with every hole about 1 * 10
5Individual cell inoculation, the medicine that variable concentrations is filtered out is hatched 2h in the trophocyte.With the cell that the PBS flush away does not stick, the cell that sticks is fixed 15 minutes with 4% paraformaldehyde, wash with PBS again, and then with 2%Giemsa dyeing 30 minutes, washing, methyl alcohol is fixed, and measures the O.D. value at 630nm wavelength place with microplate reader.Negative control is not for adding the medicine group.According to the comparison of O.D. value, judge that this medicine is activator or the inhibitor that the trophocyte is sticked.
List of references
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Claims (3)
1. one kind is screened the method that suppresses or promote new angiopoiesis medicine, comprising:
(1) cell with CD146 protein molecular or expression CD146 albumen contacts washing then with a kind of biomolecule;
(2) sample after handling in the step (1) is contacted washing then with a kind of antibody of described biomolecule of detectable label mark;
(3) described detectable label is detected, obtain the biomolecule that can combine with described CD146 protein molecular;
(4) cell with natural expression CD146 molecule contacts with the biomolecule with the combination of CD146 protein molecular that obtains; If described biomolecule can suppress or activate the growth of the cell of this natural expression CD146 molecule, promptly be inhibited or promote the medicine that neovascularity generates.
2. in accordance with the method for claim 1, wherein, described biomolecule is part, antibody molecule, polypeptide or immunotoxin.
3. in accordance with the method for claim 1, wherein, the cell of the expression CD146 described in the step (1) is a 293-EBNA clone, and its deposit number is: CGMCC 0642.
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| CN1916632B (en) * | 2005-08-18 | 2010-04-07 | 中国科学院生物物理研究所 | Detection method of molecules, and appliction |
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| CN1916632B (en) * | 2005-08-18 | 2010-04-07 | 中国科学院生物物理研究所 | Detection method of molecules, and appliction |
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