CN103013927A - Application of anti-cellular-prion-protein monoclonal antibody in preparing antitumor drugs - Google Patents
Application of anti-cellular-prion-protein monoclonal antibody in preparing antitumor drugs Download PDFInfo
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Abstract
The invention provides an application of an anti-cellular-prion-protein monoclonal antibody in preparing antitumor drugs. The anti-cellular-prion-protein monoclonal antibody is produced by a hybridoma cell line AbM-PRIO-3 with a collection number of CGMCC NO.4972. The antibody forms immunobinding with cellular prion protein, wherein an amino acid residue sequence of the cellular prion protein is represented by SEQ ID NO: 1. The monoclonal antibody is used for preparing antitumor drugs comprising anti-colorectal-cancer drugs, anti-colorectal-cancer-metastasis drugs, anti-rectal-cancer-cell-metastasis drugs, anti-rectal-cancer-cell-spreading drugs, anti-rectal-cancer-stem-cell-metastasis drugs, and anti-rectal-cancer-cell-growth drugs.
Description
Technical field
The present invention relates to a kind of native protein-Cellular Prion Protein (cellular prion protein) secreted with the human normal mucous membrane, the hybridoma of the monoclonal antibody that combines is by anti-cell type PrPC monoclonal antibody and the application in the preparation antitumor drug thereof of this hybridoma generation.
Background technology
Colorectal cancer is the large malignant tumour in third place in the world.Nearly two during the last ten years, and colorectal cancer incidence rate rises year by year, and especially Asian countries's colorectal cancer incidence rate ascendant trend is fairly obvious, and lethality rate is high, occupies the second of the cancer cause of the death.The main method of at present, treating in the world tumour is four kinds of operation, radiotherapy, chemotherapy and biotherapies etc.From medical angle, the so far treatment of tumour does not have a kind of means in full force and effect, the tumour patient that has especially shifted, the whole body diffusion of traditional uncontrollable tumour of methods for the treatment of.
The gene PRNP of Codocyte type PrPC is positioned karyomit(e) No. 20, and full length gene 20kb comprises 2 exons.The mRNA length of PRNP genetic transcription is 2.5kb (NM_000311), and the protein that 253 amino acid of coding form has 5 highly unstable series connection octapeptide iterons (5 tandem octapeptide iteron).Cellular Prion Protein has identical aminoacid sequence with the mad cow disease PrP Sc, for same gene PRNP coded.But aspect tertiary structure, Cellular Prion Protein has the tertiary structure of abundant α spiral, and pathogenic PrPC shows as β-pleated sheet structure.
With respect to the research of Protein virus, the function of Cellular Prion Protein not yet obtains clear and definite understanding.The symptoms such as irregular pulse, diel rhythm change can appear in mouse after the research prompting Cellular Prion Protein disappearance of prion protein gene-free mouse.Have been found that at present Cellular Prion Protein and kinds of tumors relevant (Mehrpour, the M. such as mammary cancer, prostate cancer, liver cancer and colorectal carcinoma; Codogno, P., 2009.Cancer Lett., 290:1-23), Cellular Prion Protein is expressed and is raised relevantly with Bcl-2 in cancer of the stomach and mammary cancer, and inhibited apoptosis function (Liang, J. are brought into play in participation; Pan, Y.L.; Ning, X.X.; Sun, L.J.; Lan, M.; Hong, L.; Du, J.P.; Liu, N.; Liu, C.J.; Qiao, T.D.; Fan, D.M., 2006.Tumour.Biol., 27:84-91) (Li, Q.Q.; Cao, X.X.; Xu, J.D.; Chen, Q.; Wang, W.J.; Tang, F.; Chen, Z.Q.; Liu, X.P.; Xu, Z.D., 2009.Cell Mol.Life Sci., 66:504-515).Cellular Prion Protein can be resisted Apoptosis of Breast Cancer (Diarra-Mehrpour, the M. that tumour necrosis factor RNF-α and TRAIL cause; Arrabal, S.; Jalil, A.; Pinson, X.; Gaudin, C.; Pietu, G.; Pitaval, A.; Ripoche, H.; Eloit, M.; Dormont, D.; Chouaib, S., 2004.Cancer Res., 64:719-727) (Meslin, F.; Hamai, A.; Gao, P.; Jalil, A.; Cahuzac, N.; Chouaib, S.; Mehrpour, M., 2007.Cancer Res., 67:10910-10919), also have in addition report to point out that Cellular Prion Protein rises to promote colon cancer cell existence (Li, Q.Q. by increasing glucose level; Sun, Y.P.; Ruan, C.P.; Xu, X.Y.; Ge, J.H.; He, J.; Xu, Z.D.; Wang, Q.; Gao, W.C., 2011.Cancer Sci., 102:400-406).The people such as Dodelet report that Cellular Prion Protein is necessary (Dodelet, V.C. for the self of CD34+ hemopoietic stem cell; Cashman, N.R., 1998.Blood, 91:1556-1561), the result of study prompting Cellular Prion Protein of Weinberg may form relevant (Liao, M.J. with the mammary gland cell spherule; Zhang, C.C.; Zhou, B.; Zimonjic, D.B.; Mani, S.A.; Kaba, M.; Gifford, A.; Reinhardt, F.; Popescu, N.C.; Guo, W.; Eaton, E.N.; Lodish, H.F.; Weinberg, R.A., 2007.Cancer Res., 67:8131-8138).Although above report prompting Cellular Prion Protein may be relevant with kinds of tumors, the concrete physiological function of Cellular Prion Protein in human tumor is not clear so far, has no report for the mechanism of action of tumorigenesis.
Increasing evidence shows tumor stem cell playing an important role for tumour.Tumor stem cell is the colony that has high self-renewal capacity in class existence and the tumor tissues, and is insensitive for radiation and chemotherapy, often becomes the root of tumor recurrence and transfer.The article that we have delivered shows that CD44 can promote generation (Du, the L. of tumour as the surface marker of tumor stem cell; Wang, H.; He, L.; Zhang, J.; Ni, B.; Wang, X.; Jin, H.; Cahuzac, N.; Mehrpour, M.; Lu, Y.; Chen, Q.2008.Clin.Cancer Res.14:6751-6760).The result of study showed cell type PrPC that we are nearest and the transfer of colorectal cancer cell have positive correlation.The two positive cells of result of study showed cell type PrPC and CD44 have higher tumour and form ability and transfer ability, and Cellular Prion Protein can significantly improve one-tenth knurl and the transfer ability of colorectal cancer stem cells.Therefore, be applied to the preparation of tumour medicine, for the treatment of tumour provides new strategy.
Summary of the invention
Therefore, the objective of the invention is for the deficiencies in the prior art, anti-cell type PrPC monoclonal antibody and the purposes of this mouse source anti-cell type PrPC monoclonal antibody in the preparation medicine for anti transfer of tumor of a kind of high specific and sensitivity is provided.The subclass of this antibody is IgG2a, energy specific combination Cellular Prion Protein recombinant antigen and natural antigen, the transfevent tumor stem cell of energy specific combination express cell type PrPC, the significantly transfer of inhibition tumor cell and tumor stem cell.
The present invention is used for realizing that the technical scheme of above-mentioned purpose is as follows:
On the one hand, the invention provides a kind of preserving number is the hybridoma cell line AbM-PRIO-3 (Classification And Nomenclature: mouse hybridoma cell of CGMCC No.4972, preservation date: on June 21st, 2011, preserving number is CGMCC No.4972, preservation ground: microbial strains preservation management committee of Chinese institute common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101).
On the other hand, the invention provides a kind of anti-cell type PrPC monoclonal antibody, is the generation of the hybridoma cell line of CGMCC No 4972 by preserving number.
Preferably, described anti-cell type PrPC monoclonal antibody can with the Cellular Prion Protein specific binding.
Preferably, the amino acid residue sequence of described Cellular Prion Protein is shown in SEQ ID NO:1.
Preferably, the nucleotide sequence of the encoding gene of described Cellular Prion Protein shown in SEQ ID NO:2.
Another aspect the invention provides a kind of method for preparing recombinant vectors, and described method comprises that gene described above inserts known carrier.
Another aspect, the present invention also provides a kind of recombinant vectors, and described recombinant vectors contains gene described above or is produced by method described above.
Preferably, described known carrier is pET-30a (+).
Again on the one hand, the invention provides a kind of organic method of restructuring for preparing, described method comprises that recombinant vectors described above imports host's organism.
Preferably, described host's organism is intestinal bacteria, and described intestinal bacteria are preferably the BL21 bacterial strain, and described BL21 bacterial strain is preferably BL21-Condon Plus (DE3).
Another aspect the invention provides a kind of anti-cell type PrPC monoclonal antibody for the preparation of the application in the anti-tumor drug.
Preferably, described antitumor drug comprises resistive connection rectum cancer medicine, resistive connection rectum cancer diversion medicaments, anti-rectum cancer cell diversion medicaments, anti-rectum cancer cell diffusion medicine, anti-rectum cancer stem cell diversion medicaments and anti-rectum cancer cell growth medicine.
Anti-cell type PrPC of the present invention antibody sensitivity and specificity are high, not only the Cellular Prion Protein antigen with restructuring has extremely strong specificity and susceptibility, and with Cellular Prion Protein in colorectal carcinoma tissue and the cell extremely strong specificity and susceptibility are arranged, thereby be applied to the clinical diagnosis of colorectal cancer tumor stem cell and the assessment of prognosis curative effect as molecular marker.
Description of drawings
Below, describe by reference to the accompanying drawings embodiment of the present invention in detail, wherein:
Fig. 1 is the agarose electrophoresis result schematic diagram behind the encoding gene pcr amplification of Cellular Prion Protein of the present invention, and 1 is the encoding gene of the Cellular Prion Protein of pcr amplification gained among the figure, and 2 is dna marker (Marker);
Fig. 2 is the collection of illustrative plates of pET-30a (+) carrier that is cloned in of the encoding gene of Cellular Prion Protein of the present invention and the collection of illustrative plates that this carrier cloning is expressed the zone;
Fig. 3 is the encoding gene clone's of Cellular Prion Protein of the present invention agarose electrophoresis result schematic diagram, and 1 is dna marker (Marker) among the figure, and 2-6 is respectively the gene clone of 1-5 Cellular Prion Protein;
Fig. 4 is the encoding gene clone's of 1-5 Cellular Prion Protein order-checking collection of illustrative plates;
Fig. 5 is for after changing host BL21-Codon Plus (DE3), the SDS-PAGE electrophoresis result schematic diagram of the Cellular Prion Protein of abduction delivering, 1 is protein labeling (Marker) among the figure, 2 are the bacterium of the encoding gene that contains Cellular Prion Protein of abduction delivering not, 3 supernatants for the bacterium of the encoding gene that contains Cellular Prion Protein of abduction delivering, 4 precipitations for the bacterium of the encoding gene that contains Cellular Prion Protein of abduction delivering;
Fig. 6 carries out SDS-PAGE electrophoresis result schematic diagram after the thick purification of the Cellular Prion Protein of great expression, 1 is protein labeling (Marker) among the figure, 2 precipitations for the bacterium of the encoding gene that contains Cellular Prion Protein of a large amount of abduction deliverings, 3 supernatants for the bacterium of the encoding gene that contains Cellular Prion Protein of a large amount of abduction deliverings;
Fig. 7 carries out SDS-PAGE electrophoresis result schematic diagram behind the Cellular Prion Protein purifying of great expression, and 1 is Cellular Prion Protein behind the purifying among the figure, and 2 is protein labeling (Marker);
Fig. 8 is that anti-cell type PrPC monoclonal antibody of the present invention is carried out the experimental result schematic diagram that immunohistochemical experiment detects the expression in the colorectal cancer pairing tissue, picture in picture 8-1 is the expression of Cellular Prion Protein in the transfevent colon cancer tissue, and Fig. 8-2 is the expression of Cellular Prion Protein in non-transfevent colon cancer tissue;
Fig. 9 is the purposes checking-immunoblot experiment result schematic diagram of the described anti-cell type PrPC monoclonal antibody of invention, among the figure among the A 1,2,5,6,7,8 is the expression of Cellular Prion Protein in the 6 routine transfevent rectum cancer and the expression in 3, the 4 routine non-transfevent rectum cancer, and B is the experimental result schematic diagram as the Actin muscle of positive control among the figure;
Figure 10 is that anti-cell type PrPC monoclonal antibody of the present invention is at the experimental result schematic diagram that carries out immunofluorescence location foreign cell type PrPC;
Figure 11 is that anti-cell type PrPC monoclonal antibody of the present invention suppresses the experimental result schematic diagram that the tumour liver cell shifts, Figure 11-1 is the test-results schematic diagram that LgG antibody inhibiting tumor stem cell shifts, and Figure 11-2 is that anti-cell type PrPC monoclonal antibody suppresses the test-results schematic diagram that tumor stem cell shifts.
Embodiment
Employed technology comprises gene amplification in following examples, gene clone, and cytogamy, and cell cultures, detection technique unless stated otherwise, are routine techniques known to those skilled in the art; Employed plant and instrument, reagent, cell etc. only specify in the specification sheets, are that those skilled in the art can obtain by public approach.
Following test mice is the Balb/c mouse, available from Beijing Vital River Experimental Animals Technology Co., Ltd..
Embodiment 1:The preparation of Cellular Prion Protein antigen
Cellular Prion Protein antigen is take normal certain tissue cDNA as template, and according to coding gene sequence (SEQ ID NO:2) the design special primer of Cellular Prion Protein, the primer two ends are connected into BamH I and Xho I restriction enzyme site.
Upstream primer: 5 '-CGGGATCCGGTGGTGGTATGGCGAACCTTGGCTGCTGGAT-3 ' (SEQ IDNO:3)
Downstream primer: 5 '-CCGCTCGAGTCCCACTATCAGGAAGATGAGGAAAG-3 (SEQ ID NO:4) '.
The encoding gene of pcr amplification Cellular Prion Protein (PCR parameter: 95 ℃ of 5min, 94 ℃ of 45S, 62 ℃ of 1min, 72 ℃ of 1min30s, 72 ℃ of 10min, reaction cycle number are 30) after carry out agarose electrophoresis, the result as shown in Figure 1, show amplification for the encoding gene of Cellular Prion Protein; Again the encoding gene of Cellular Prion Protein of amplification is connected with Xho I double digestion through BamH I and is connected with pET30a (+), chemical conversion competent cell BL21, picking 1-5 mono-clonal carries out agarose electrophoresis after extracting plasmid enzyme restriction, the result as shown in Figure 2, the fragment that evenly size is correct is inserted, it is checked order (Fig. 4 is the experimental result schematic diagram of order-checking), No. 4 clones are correct for authentication sequence again.Change expressive host BL21-Codon Plus (DE3), select No. 4 clones of the correct positive bacteria of order-checking, 0.4mmol/L IPTG abduction delivering spends the night.After inducing, collect thalline, centrifugal after ultrasonication, contrast as negative with the expression bacterium of not inducing, upper cleer and peaceful precipitation is not carried out the SDS-PAGE electrophoresis, and as shown in Figure 5, the product of Explicit Expression is non-solubility albumen as a result.
Carry out abduction delivering behind a large amount of culturing bacterium, collect the abduction delivering bacterium, after ultrasonication was centrifugal, upper cleer and peaceful precipitation was not carried out the SDS-PAGE electrophoresis, and as shown in Figure 6, the product of expression is non-solubility albumen, and size conforms to Cellular Prion Protein;
Again it is extracted Cellular Prion Protein, to carry out purifying through Ni-NAT affinity column (available from GEHealthcare), 50mM imidazoles wash-out Cellular Prion Protein, the SDS-PAGE electrophoresis, the result as shown in Figure 7, cut band and carry out MALDI-TOF/TOF evaluation (available from BRUKEROPTICS), the confirmation expression product is Cellular Prion Protein.
Embodiment 2: the preparation of anti-cell type PrPC hybridoma
1) immunity
The Cellular Prion Protein that will obtain through the Ni-NAT affinitive layer purification, subcutaneous abdomen immunity (eye socket is got 20 μ L serum and done negative control before the immunity) 3 4-6 age in week, female Balb/c mouse was numbered 1,2,3; Dosage is every mouse 60 μ g albumen+physiological saline to 200 μ L+CFA200 μ L.Per 14 days subcutaneous booster immunizations once, dosage is every mouse 30 μ g albumen+physiological saline to 200 μ L+IFA200 μ L.7 days eye sockets are got blood and are surveyed and tire behind the 3rd booster immunization, reach the demander and impact immunity, 50 μ g albumen+physiological saline to 100 μ L; Tail vein injection merged afterwards until 3 days.
2) merge
The mouse spleen of fresh cutting-out is put on the cell sieve pulverizes filtration, mix in 1: 5 ratio with sp2/0 cell (frozen available from magnificent larger protein center), 1500 rev/mins centrifugal 5 minutes.The centrifuge tube that centrifugal cell good and mixing is housed is put into 37 ℃ of warm water baths, and the limit is stirred the cell limit and is slowly added 1mL PEG1500.In water-bath, left standstill 1 minute.Slowly 1000 rev/mins of the IMDM (Sigma, H0262-10VL) of the serum-free of adding 10mL are centrifugal 5 minutes.Abandon supernatant, add careful cell is blown and beaten of serum of 10mL, and (dislocation of mouse cervical vertebra is put to death, and 75% alcohol immersion 3 minutes is taken out mouse and placed on the aseptic paper, and the outside of belly up to add thymocyte.Open the thoracic cavity and isolate thymus gland, put into culture dish, be caught broken with tweezers, add 5ml/ spleen of 400u/ml III Collagenase Type, nylon net filter is used in 37 degree digestion 20 minutes, obtains the thymus gland single cell suspension) and the 25mL semisolid medium, fully mixing is evenly poured in 20 Tissue Culture Dishs.Tissue Culture Dish is put in the wet box, put into 5%CO
2Cultivate in the incubator.
3) choose the clone
The last merging 7-10 days, observing clone cell, to roll into a ball big or small density moderate.Under anatomical lens, draw circle, real, large cloning cluster in the substratum that is prepared in advance 96 orifice plates of (containing feeder cell and 1%HT xanthoglobulin (hypoxantin) and Thymine deoxyriboside (thymidin) liquid).Put into 5%CO
2Cell culture incubator.
4) screening
A. one sieve: chose the clone rear about 3 days, and when the observation of cell amount accounts for floorage 2/3 greatly, got 100 μ L supernatant ELISA screening.Positive colony changes liquid, adds 200 μ L perfect mediums (containing 1%HT liquid).
B. two sieve: repeating step a carries out postsearch screening after 2 days.Positive strain changes the substratum that is prepared in advance 24 orifice plates of (containing feeder cell and 1%HT liquid) over to.
C. three sieve: after 5 days, with other recombinant protein wrapper sheet that contains label H is, get 100 μ L supernatant ELISA screening.Satisfactory clone changes 6 orifice plates or Tissue Culture Flask enlarged culturing over to.
5) cell cryopreservation
The logarithmic phase cell takes floorage about 80% can be frozen.Collect the impurity such as supernatant and removal dead cell, supernatant is stored in-20 ℃.Directly freeze-stored cell is put 4 ℃ of half an hour, then put-20 ℃ two hours, turn-80 ℃ of frozen spending the night, put liquid nitrogen container next day.
Embodiment 3: be that the hybridoma cell line preparation of CGMCC No.4972 is anti-thin by preserving number
Born of the same parents' type PrPC monoclonal antibody
The contriver is with hybridoma (called after: AbM-PRIO-3, Classification And Nomenclature: mouse hybridoma cell, preservation date: on June 21st, 2011, preserving number is CGMCC No.4972, preservation ground: microbial strains preservation management committee of Chinese institute common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101), for the preparation of mouse-anti Cellular Prion Protein monoclonal antibody, concrete operation step is as follows:
1) cell recovery
From-80 ℃ of refrigerators or liquid nitrogen container, take out rapidly cryopreservation tube; Put into rapidly 37 ℃ of water-baths and stir fast, make frozen storing liquid in 2 minutes, all be melted into liquid.In the 15mL centrifuge tube, add the 3mL blood serum medium, frozen storing liquid is sucked centrifuge tube, 1500 rev/mins, centrifugal 5 minutes.Abandon supernatant, hanged cell with perfect medium, be incubated in 6 orifice plates (3mL) or the bottle (5mL).
2) ascites preparation
The logarithmic phase cell washs with serum free medium and hangs; Count about 5 * 10
5, 1mL.The cell intraperitoneal injection of mice that suspends.Just collect ascites after 7-10 days.Collect about 3mL for the first time, repeated to get every 2,3 days, only finally can collect 8mL/.4000 rev/mins of each ascites of taking out, centrifugal 10 minutes; The centre is ascites.Careful sucking-off ascites is collected in the centrifuge tube 4 ℃ of preservations.
3) purifying of monoclonal antibody
With HiTrap rProtein A FF (GE Healthcare, 17-5079-01) affinity column monoclonal antibody purification.
With ascites under 4 ℃ of conditions, 10000 rev/mins centrifugal 10 minutes, remove lipid material.Centrifugal rear absorption supernatant, and dilute with 1: 3 (ascites: be coupled liquid) with being coupled damping fluid, with 0.45 μ m membrane filtration.Filtrate being loaded to is coupled the chromatography column that the damping fluid balance is crossed.With after being coupled damping fluid and washing pillar, with elution buffer wash-out antibody, be collected in the collection tube that neutralizer is housed in advance.
Embodiment 4: the evaluation of monoclonal antibody
1) mensuration of monoclonal antibody specificity
A. the mouse-anti PrPC monoclonal antibody that makes with the inventive method and commercialization mouse-anti PrPC monoclonal antibody (Cayman) are respectively with 1: 100,1: 200,1: 500,1: 1000, jointly hatched 10 minutes with colon cancer cell after the dilution in 1: 2000, then resist with phyenlephrinium (phenylephrine, PE) fluorescently-labeled two and hatched 10 minutes, in fluorescence microscopy Microscopic observation location and fluorescence intensity, detect specificity and the sensitivity of antibody.The mouse-anti PrPC monoclonal antibody that the inventive method makes is positioned on the cytolemma more specifically.
B. the mouse-anti PrPC monoclonal antibody that makes with the inventive method and commercialization mouse-anti PrPC monoclonal antibody are respectively with 1: 200,1: 500,1: 1000,1: 2000, jointly hatched 60 minutes with the colorectal carcinoma paraffin section after the dilution in 1: 5000, then resist and hatched 3 30 minutes with two, 3-diaminobenzidine (DAB) colour developing, mounting.Examine under a microscope location and the signal of positive cell, detect specificity and the sensitivity of antibody.The mouse-anti PrPC monoclonal antibody that the inventive method makes is positioned on the cytolemma more specifically, and sensitivity is 10 times of commercialization mouse-anti PrPC monoclonal antibody.
2) mensuration of antibody titer
The mouse-anti PrPC monoclonal antibody that makes with the inventive method and commercialization mouse-anti PrPC monoclonal antibody are respectively with 1: 200,1: 500,1: 1000,1: 2000,1: 5000, dilute rear and colon cancer cell lysate electrophoresis transferring film overnight incubation at 1: 10000, then resist with two of horseradish peroxidase-labeled and hatched 2 hours, hatch exposure imaging with the ECL luminous substrate, detect the sensitivity of antibody.The result shows that mouse-anti PrPC antibody titer that the inventive method makes is that (the mouse-anti PrPC antibody titer that the inventive method makes is 1000 for 20 times of commercialization mouse-anti PrPC monoclonal antibody, tiring of commercialization mouse-anti PrPC monoclonal antibody is 500, i.e. 10000/500=20).
3) the ELISA method is identified the monoclonal antibody subclass
A. experimental implementation
With coated sheep anti-mouse igg to the 0.5 μ g/mL of 100mM PBS (pH7.4) dilution, every hole adds 100 μ L, 4 ℃, spends the night.Be emptied liquid, wash 3 times with the PBS (PBST) that contains 0.05%Tween, every hole adds 200 μ L confining liquids, hatches 1 hour for 37 ℃.Be emptied liquid, clean 3 times with PBST.Every hole adds 0.1mL hybridoma supernatant, hatches 1 hour for 37 ℃.Being emptied liquid cleans 3 times with PBST.Sheep anti mouse (κ with confining liquid dilution in 1: 1000 HRP mark, λ) sheep anti mouse (the IgM of antibody (available from SouthernBiotech) or 1: 2000 dilution HRP mark, IgG1, IgG2a, IgG2b, IgG3, IgA) the every hole of antibody (available from Southern Biotech) 0.1mL, add respectively in the suitable hole, 37 ℃ with hatching 1 hour.Be emptied liquid, clean 3 times with PBS-T.Every hole adds 50 μ L substrate solutions, surveys the OD value under the 405nm wavelength in 10-20 minute.
B. experimental result
The experimental result demonstration, anti-cell type PrPC monoclonal antibody of the present invention is IgG2a type mouse resource monoclonal antibody.
Embodiment 5:ELISA method is measured the monoclonal antibody affinity costant
1) experimental procedure:
Coated immunity reconstitution cell type PrPC, coated concentration is 2 μ g/mL, 100 μ L/ holes, 4 ℃ of coated spending the night, 1 * PBST (phosphate buffered saline buffer adds polysorbas20) washes 3 times.Every hole adds 37 ℃ of sealings of 200 μ L confining liquids 2 hours, and 1 * PBST washes 3 times.Anti-cell type PrPC monoclonal antibody, since 1: 200 2 times of gradient dilution, the contrast that blanks of last 1 hole was hatched 1 hour for 37 ℃, and 1 * PBST washes 3 times.Sheep anti mouse two dilutions in anti-1: 20000 of horseradish peroxidase (HRP) mark, every hole 100 μ L were hatched 1 hour for 37 ℃, and 1 * PBST washes 3 times.Nitrite ion 100 μ L/ holes colour developing 10 minutes, 50 μ L/ hole stop buffer termination reactions.With microplate reader (making institute, model SM-3 available from Beijing celestial stone medical treatment product), measure wavelength 450/630 light absorption value.
2) data analysis: find out 〉=extension rate A corresponding to 1/2 " platform OD value ".
Affinity costant
Single IgG antibody molecular value is 150000, and antibody original concentration unit is 4.98mg/mL.
3) result: the affinity costant of monoclonal antibody of the present invention is 1.08 * 10
9
Embodiment 6: the purposes checking-immune group of anti-cell type PrPC monoclonal antibody of the present invention
Change (IHC)
1) material source:
The used Colorectal Carcinoma Specimen origin of the specific embodiment of the invention comprises colorectal cancer and the corresponding other healthy tissues sample of cancer altogether in Beijing Tumour Hospital tissue sample storehouse.Sample draw materials be the fresh knot Colorectal Carcinoma of the underwent operative excision cancer beside organism corresponding with it after the physiological saline washing, put into formalin and fix, the paraffin embedding preservation.Sample is all through proved by pathology, and patient's Informed Consent Form is arranged.
2) experimental procedure:
Roasting sheet, dimethylbenzene dewaxing, gradient ethanol aquation.0.3%H
2O
2Then methyl alcohol room temperature 10 minutes uses 0.01M PBS (phosphate buffered saline buffer) to wash 3 times * 5 minutes; 5% skim-milk, 37 ℃ were sealed 2 hours; Anti-cell type PrPC monoclonal antibody (dilution in 1: 2000), wet box is hatched 4 ℃ and is spent the night; Drip sheep anti mouse two anti-(middle China fir Golden Bridge, PV-6002), incubated at room 30 minutes; DAB-H
2O
2(control is 3-5 minute under the mirror, PBS rinsing, color development stopping for middle China fir Golden Bridge, ZLI-9031) colour developing; (middle China fir Golden Bridge ZLI-9040) redyes 45 seconds to Hematorylin, then 1% hydrochloride alcohol differentiation; The tap water flushing is anti-blue; 95% and 100% ethanol dehydration each 5 minutes, dimethylbenzene is transparent, the neutral gum mounting.
3) experimental result:
As shown in Figure 8, the immunohistochemical experiment result is carried out statistical study show, Cellular Prion Protein is not expressed (-) or weak positive expression (+) in 190 examples without shifting in the Colorectal Carcinoma; Positive result in 53 routine transfevent Colorectal Carcinomas (+++), weak positive expression (+) in the 18 routine transfevent colorectal cancers.The p value is less than 0.01.Therefore, the experimental result of organization chip is also supported the conclusion of Proteomic analysis fully, and namely Cellular Prion Protein is low in without Metastatic colon and rectum carcinoma expresses or do not express, but in the transfevent Colorectal Carcinoma high expression level.
Embodiment 7: the purposes checking-Diagnosis of Sghistosomiasis of anti-cell type PrPC monoclonal antibody of the present invention
Mark method (WB)
The inventor further uses immunoblotting WB and measures the protein expression of Cellular Prion Protein in Colorectal Carcinoma and 6 kinds of colorectal cancer cells systems.
1) experimental procedure:
Albumen 30 μ g samples in 2 kinds of Colorectal Carcinomas and the 6 kinds of colorectal cancer cells systems are splined on 12%SDS PAGE glue; After electrophoresis finishes, glue was soaked 10 minutes in 1 * transfer liquid; 350mA after pvdf membrane methyl alcohol is processed, transferring film 70 minutes; Film is placed 37 ℃ of the TBST confining liquids sealing 1 hour that contains 5% skim-milk; 37 ℃ of anti-cell type PrPC monoclonal antibodies (1: 1000 dilution) were hatched 1 hour or 4 ℃ of overnight incubation; Corresponding sheep anti mouse two anti-(middle China fir Golden Bridge, dilution in 1: 2500) incubated at room 1 hour; Hatch with Pierre's Si luminescence reagent (available from Pierce) and to expose with X-ray film afterwards.
2) experimental result:
As shown in Figure 9, in all eight pairs of colorectal cancers and cancer beside organism, Cellular Prion Protein is not expressed in healthy tissues substantially, high expression level in low differentiation tumor tissue.When the expression of Cellular Prion Protein is as positive control in Colorectal Carcinoma, become the high expression of cell lines Cellular Prion Protein of knurl ability in 6 kinds of colorectal cancer cells systems.
Embodiment 8: the purposes of anti-cell type PrPC monoclonal antibody of the present invention checking-immunity is glimmering
Light (IF)
For state after the secretion of analog cell type PrPC, the inventor has made up escherichia expression system, has expressed Cellular Prion Protein matter--the t-Cellular Prion Protein of removing signal peptide in e. coli bl21 (DE3).The inventor adopts immunofluorescence technique to detect the cellular localization of t-Cellular Prion Protein.
1) experimental procedure:
T-Cellular Prion Protein and colon cancer cell SW480 (available from ATCC) are cultivated altogether; 3.7% formaldehyde is fixed 15 minutes; 0.2%TritonX-100 punching 5 minutes; The PBS solution that contains 1%BSA sealed 1 hour; Add in special anti-cell type PrPC monoclonal antibody (dilution in 1: the 100) magazine and hatched 2 hours; Hatched 1 hour in the sheep anti mouse two of FITC mark anti-(middle China fir Golden Bridge, dilution in 1: the 200) magazine; 0.25 μ g/ μ L 4 ', 6-diamidino-2-phenylindone (DAPI) solution transfect cell 4 minutes; Cover glass taken out to cover dripping to be had on the slide glass of the anti-fluorescence decay mountant of 10 μ L, after the nail varnish mounting, observes under laser scanning co-focusing microscope.
2) experimental result:
As shown in figure 10, the t-Cellular Prion Protein is incorporated on the cytolemma, and the part Cellular Prion Protein is present in the golgi body.
Use to obtain conclusion, Cellular Prion Protein can combine with tumor stem cell, is a specifically expressing membrane protein of this cell.Cellular Prion Protein extracellular region in ripening process cuts, the N end that produces a 14KD comes off from cytolemma, this just means that Cellular Prion Protein is as the mark of tumor stem cell, can detect the existence of Cellular Prion Protein in serum, this detection for Cellular Prion Protein provides convenience.Specifically, because Cellular Prion Protein is a good tumor stem cell mark, it has advantages of that other tumor stem cell marks do not have, and the method for available ELISA detects the content of Cellular Prion Protein in the serum clinically.
Embodiment 9: the checking of the purposes of anti-cell type PrPC monoclonal antibody of the present invention-inhibition is swollen
The knurl stem cell shifts
The function of anti-cell type PrPC monoclonal antibody inhibition tumor stem cell transfer that the inventor has used the Transwell technology for detection.
1) experimental procedure:
Transwell is immersed in equilibrate overnight among the aseptic DMEM; Colorectal cancer stem cells is become single cell suspension with tryptic digestion from spherical clone, wash 2 times with PBS, counting.With 3 * 10
5Individual cell is resuspended among the 100 μ L DMEM, is inoculated into the upper strata of Transwell cell, adds the DMEM that 600 μ L contain 10%FBS simultaneously in Transwell lower floor.The anti-cell type PrPC monoclonal antibody of concentration 1 μ g/mL in all adding in the levels nutrient solution, 1 μ g/mL IgG is as negative control, cell cultures 24 hours.With 3.7% Paraformaldehyde 96 fixed cell, then use violet staining.Carefully wipe the cell that does not have transfer with cotton swab, PBS washes 3 times, and microscopically is taken a picture, the cell number that statistics shifts.
2) experimental result
As shown in figure 11, anti-cell type PrPC monoclonal antibody of the present invention significantly suppresses the tumor stem cell transfer.
3) anti-cell type PrPC monoclonal antibody and the mouse IgG of the present invention's preparation were hatched 2 hours altogether according to concentration and the colorectal carcinoma tumor stem cell of 1 μ g/mL respectively, operation method suppresses this cell under the mouse spleen coating.Put to death later on mouse in 30 days and detect cancer metastasis, find that anti-cell type PrPC monoclonal anti physical efficiency significantly suppresses tumor stem cell and shifts in Mice Body.
Embodiment 10: the checking of other purposes of anti-cell type PrPC monoclonal antibody of the present invention
1) the mouse-anti PrPC monoclonal antibody and the colorectal cancer cell that make with the inventive method were hatched 10 minutes jointly, then with PE fluorescently-labeled two anti-hatching 10 minutes, with the positive and negative CD44+ colorectal cancer cell of selected by flow cytometry apoptosis PrPC, orientation is expelled to nude mice by subcutaneous with the sorting cells of number, detects tumour and forms.The tumour formation ability that found that express cell type PrPC colorectal cancer cell is not express 4 times of PrPC cell.
2) with the cell operation transplantation of sorting under the spleen coating, sew up a wound, put to death mouse after 35 days and check whether liver metastasis of colorectal cancer cell, and get liver and fix, use paraffin section, the method affirmation metastasis of Hematorylin-Yihong (HE) dyeing.The transfer ability of the colon cancer cell of Explicit Expression Cellular Prion Protein is more than 20 times of colorectal cancer cell of not expressing PrPC as a result.
3) get 200 sorting cells and be inoculated in 0.35% the agarose, 20 days poststainings find that the clonality of the colorectal cancer cell of the Cellular Prion Protein positive is significantly higher than the PrPC negative cells.
Above experimental result shows the transfevent tumor stem cell in the single-minded identification colorectal cancer of anti-cell type PrPC antibody capable that the present invention prepares.
Therefore, Cellular Prion Protein tumor stem cell medicine has high application prospect to monoclonal antibody provided by the invention in the serum for the preparation of detecting clinically.
Claims (10)
1. hybridoma cell line AbM-PRIO-3 that preserving number is CGMCC No.4972.
2. an anti-cell type PrPC monoclonal antibody is the hybridoma cell line generation of CGMCC No.4972 by preserving number.
3. anti-cell type PrPC monoclonal antibody according to claim 2 is characterized in that, described anti-cell type PrPC monoclonal antibody and Cellular Prion Protein specific binding.
4. anti-cell type PrPC monoclonal antibody according to claim 3 is characterized in that, the amino acid residue sequence of described Cellular Prion Protein is shown in SEQ ID NO:1.
5. the encoding gene of Cellular Prion Protein according to claim 4 is characterized in that, the nucleotide sequence of the encoding gene of described Cellular Prion Protein shown in SEQ ID NO:2.
6. method for preparing recombinant vectors, described method comprise inserts known carrier with gene claimed in claim 5, and described known carrier is preferably pET-30a (+).
7. recombinant vectors, described recombinant vectors contain gene claimed in claim 5 or are produced by method claimed in claim 6.
8. one kind prepares the organic method of restructuring, described method comprises recombinant vectors importing host organism claimed in claim 7, described host's organism is preferably intestinal bacteria, described intestinal bacteria are preferably the BL21 bacterial strain, and described BL21 bacterial strain is preferably BL21-Condon Plus (DE3).
According to claim 2 to 4 each described anti-cell type PrPC monoclonal antibodies for the preparation of the application in the anti-tumor drug.
10. purposes according to claim 9, it is characterized in that, described antitumor drug comprises resistive connection rectum cancer medicine, resistive connection rectum cancer diversion medicaments, anti-rectum cancer cell diversion medicaments, anti-rectum cancer cell diffusion medicine, anti-rectum cancer stem cell diversion medicaments and anti-rectum cancer cell growth medicine.
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| GENBANK: "HM459606.1", 《GENBANK》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104031148A (en) * | 2014-05-15 | 2014-09-10 | 中国科学院动物研究所 | Monoclonal antibody aiming at human PrPc and application thereof |
| WO2015172708A1 (en) * | 2014-05-15 | 2015-11-19 | 中国科学院动物研究所 | Monoclonal antibody against human prpc and use thereof |
| CN104031148B (en) * | 2014-05-15 | 2018-03-13 | 中国科学院动物研究所 | Monoclonal antibody and its application for people PrPc |
| US10155808B2 (en) | 2014-05-15 | 2018-12-18 | Institute Of Zoology, Chinese Academy Of Sciences | Monoclonal antibody against human PrPc and use thereof |
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