CN102924594B - Anti-tumor-protein-P63 monoclonal antibody and purpose thereof - Google Patents
Anti-tumor-protein-P63 monoclonal antibody and purpose thereof Download PDFInfo
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Abstract
The invention discloses a hybridoma cell line (with a collection number of CGMCC No.6131) and a monoclonal antibody 2B10 produced by the monoclonal antibody. The invention also related to an application of the monoclonal antibody 2B10 in preparing an immunoassay tool used for detecting a P63 protein, an immunohistochemical kit containing the monoclonal antibody 2B10, and the applications of the monoclonal antibody 2B10 in preparing kits used for identifying P63-positive cells or P63-positive-cell source tumors. The monoclonal antibody provided by the invention can be specifically combined with P63 protein, and has no cross-reaction with other proteins in the cell. Therefore, P63 protein immunoassay specificity and reliability are substantially improved.
Description
Technical field
The present invention relates to monoclonal antibody of anti-tumor protein P63 and uses thereof.
Background technology
P63 is one of p53 family member who in recent years finds, different from the single product of p53 genes encoding, due to the difference of promotor and alternative splicing, the isomer protein (TAp63 and △ Np63) that p63 genes encoding two class functions are different, in the property tumour of multiple tesselated epithelium source, occur to express to change, in close relations with tumor development.P63 plays a role by regulating downstream target gene and activating many A signal pathways as transcription factor.The major function of p53 is cell growth inhibiting and apoptosis-induced, and the generation of most of tumours is relevant with p53 transgenation, and p53 is the tumor suppressor gene that current function ratio is clearer and more definite.Although homology is up to 60% on protein sequence for p63 and p53, research finds that these two homologous proteins have different effects in vivo.P63 mainly occurs to play an important role in growth, differentiation and the form of various epithelium, for the growth of embryo's forming process mesectoderm, has great importance, and in tumour occurs, be more carcinogenesis rather than cancer suppressing action.
P63 is expressed in substrate/myoepithelical cell, mammary gland myoepithelical cell, prostate basic cell by immunohistochemistry. of basal cell, the cutaneous appendages of epidermis etc., clinically, can be used as the marker of rodent cancer, squama cancer, urinary tract transitional cell carcinoma, for the diagnosis and differential diagnosis of primary cutaneous tumour and adenocarcinoma metastatic, Benign Breast malignant change, prostate cancer.Existing bibliographical information, while using Immunohistochemical method to detect expression in prostate cancer, PIN and benign prostatic hyperplasia of p504S, p63, CK34 β E12, the 61 routine p504S positives in 72 routine prostate cancers, but p63 and CK34 β E12 are most of negative; The 5 routine p504S positives in 6 routine PIN, but p63 and CK34 β E12 are all positive; And hyperplasia of prostate shows as p504S feminine gender, p63 and the CK34 β E12 positive.Therefore, to the joint-detection of antigen p504S and p63, CK34 β E12, can greatly improve the accuracy rate of prostate cancer diagnosis.
Conventionally by the experiment of immunohistochemistry (IHC) pathology, detect clinically at present the expression situation of p63 in tumour cell.The core of IHC experiment is the monoclonal antibody of specific binding p63, and the quality of its performance is directly determining sensitivity and the specificity of whole detection.Therefore, developing a kind of high specific has great importance in conjunction with the anti-P63 monoclonal antibody of P63 albumen.
Summary of the invention
The technical problem to be solved in the present invention is for the monoclonal antibody of the anti-P63 albumen that a kind of binding specificity is higher is provided, and the application in the immunodetection instrument for the preparation of detection P63 albumen.
For solving the problems of the technologies described above, the invention provides a kind of hybridoma cell strain, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (referred to as CGMCC), preservation date is on May 16th, 2012, and deposit number is CGMCC No.6131.
The present invention also provides a kind of monoclonal antibody 2B10 of specific binding P63 albumen, by above-mentioned hybridoma cell strain, is produced.
The preparation method of monoclonal antibody of the present invention is as follows:
(1) structure of recombinant expression vector: carry out pcr amplification according to the ORF complete sequence of p63 gene (as shown in SEQ ID No.1) design primer, restriction endonuclease sites SgfI and MluI are introduced respectively in gene both sides, and introduce Myc-DDK sequence label (as shown in SEQ ID No.2) at its C-terminal, insert expression vector pCMV6-Entry, build p63 recombinant expression plasmid;
(2) expression and purification of P63 recombinant protein: with this plasmid transfection HEK293T cell, cracking centrifuging and taking supernatant, DDK affinity chromatography column purification, the P63 recombinant protein of acquisition purifying;
(3) screening of monoclonal antibody and preparation: adopt above-mentioned P63 recombinant protein immunity BALB/c mouse, get mouse spleen cell and SP2/0 cell merges, limiting dilution assay obtains mono-clonal, ELISA method screening positive hybridoma cell, obtain the hybridoma cell strain that can secrete anti-p63 specific antibody, called after 2B10, hypotype is accredited as IgG2a; Prepare mouse ascites, by affinity chromatography column purification p63 monoclonal antibody.By Western Blot, immunohistochemical experiment, immunofluorescence, verify respectively sensitivity and the specificity of this monoclonal antibody.
The specificity checking of said monoclonal antibody:
On OriGene high-density protein chip, comprise 10400 HEK293T cell proteins and cross expression lysate, every kind of protein lysate has the repetition of two copies on chip.Protein lysate by trace on nitrocellulose filter.The location of each clock protein lysate can accurately be located by Excel file.On protein chip, albumen is divided into 40 sub-matrixes, on each sub-matrix, has some references, by reference, can quantitative each chip point the content of upper albumen, monitor the repeatability of each immune response data and the direction of location positive signal.
Monoclonal antibody 2B10 of the present invention and said chip are hybridized and determine positive signal site, Western blot checking is carried out in obtained positive signal site, result shows: monoclonal antibody 2B10 specific binding P63 albumen of the present invention, and with other albumen no cross reactions.
The present invention also provides the application of monoclonal antibody 2B10 in the immunodetection instrument for the preparation of detection P63 albumen.
Particularly, described immunodetection instrument is test kit, chip or test paper.
In specific embodiment, the invention provides a kind of immunologic combined detection reagent kit, comprise said monoclonal antibody 2B10; Can detect the expression situation of P63 albumen in histocyte.
The present invention also provides the application of monoclonal antibody 2B10 in the test kit for the preparation of evaluation p63 positive cell or p63 positive cell source tumour.P63 is expressed in basal cell, myoepithelical cell, squamous cell, urinary tract transitional epithelial cell etc., and clinically, p63 can be used as the marker of above-mentioned cell; When above-mentioned cell generation canceration, p63 also can be used as the biomarker of above-mentioned cell carcinoma, for distinguishing, identify the primary source of tumour cell.
Described p63 positive cell is basal cell, myoepithelical cell, squamous cell, urinary tract transitional epithelial cell.
Described basal cell is epidermal basal cell, cutaneous appendages basal cell or prostate basic cell by immunohistochemistry.; Described myoepithelical cell is cutaneous appendages myoepithelical cell or mammary gland myoepithelical cell.
Described p63 positive cell source tumour is rodent cancer, myoepithelical cell cancer, squamous cell cancer, urinary tract transitional cell carcinoma.
Described rodent cancer is epidermal basal cell cancer, cutaneous appendages rodent cancer or prostate basic cell by immunohistochemistry. cancer; Described myoepithelical cell cancer is cutaneous appendages myoepithelical cell cancer or mammary gland myoepithelical cell cancer.
Preservation information
The scientific description that is used for the hybridoma cell strain of preservation is: anti-human TP63 monoclonal antibody hybridoma cell strain 2B10;
Depositary institution's full name: China Committee for Culture Collection of Microorganisms's common micro-organisms center;
Depositary institution is called for short: CGMCC;
Depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica;
Preservation date: on May 16th, 2012;
Deposit number: CGMCC No.6131.
Accompanying drawing explanation
Fig. 1 is the expression of Western blot checking p63 recombinant plasmid in HEK293T cell; The loading sample of left side swimming lane is the HEK293T cell pyrolysis liquid of transfection empty carrier, and the loading sample of the right swimming lane is the HEK293T cell pyrolysis liquid of transfection recombinant plasmid pCMV6-p63, and hybridizing primary antibodie used is DDK tag antibody;
Fig. 2 is the SDS-PAGE evaluation figure of p63 recombinant protein;
Fig. 3 is the results of hybridization of monoclonal antibody 2B10 in 9 kinds of different clones;
Fig. 4 is the expression (take 2B10 monoclonal antibody as primary antibodie) that immunohistochemical methods detects P63 albumen in prostate cancer tissue;
Fig. 5 is the combination situation that Western blot detects 2B10 antibody and TP63, C7orF31;
Left figure: the results of hybridization that 2B10 antibody is primary antibodie, left side swimming lane sample was the 293T cell pyrolysis liquid of expressing TP63 plasmid, right side swimming lane sample was the 293T cell pyrolysis liquid of expressing C7orF31 plasmid; Described TP63 plasmid and C7orF31 plasmid are all with DDK sequence label.
Right figure: the results of hybridization that DDK antibody is primary antibodie, left side swimming lane sample was the 293T cell pyrolysis liquid of expressing TP63 recombinant plasmid, right side swimming lane sample was the 293T cell pyrolysis liquid of expressing C7orF31 plasmid; Described TP63 plasmid and C7orF31 plasmid are all with DDK sequence label.
Embodiment
In order to make those skilled in the art understand better technical scheme of the present invention, below in conjunction with specific embodiment, the present invention is described in further detail.
The preparation of the anti-p63 monoclonal antibody of embodiment 1
One, the structure of p63 recombinant expression plasmid
Take the plasmid BC039815(obtaining from ATCC, contain p63ORF sequence shown in SEQ ID No.1) as template, design two primers and introduce respectively restriction enzyme site SgfI, MluI, and C-terminal Myc-DDK label (sequence is as shown in SEQ ID No.2), and be cloned into expression vector pCMV6-Entry, build p63 recombinant expression plasmid.
Two, the expression and purification of p63 recombinant protein
1, transfection HEK293T cell
HEK293T cell reaches with 1:3 in the Tissue Culture Dish of diameter 10cm and continues to cultivate; Get 7.5ml DMEM(serum-free and microbiotic) substratum to 50ml pipe, add 300 μ l PEI MegaTran1.0 to mix, then add the above-mentioned p63 recombinant plasmid dna of 75 μ g to mix and standing 30 minutes; Get respectively the above-mentioned mixed solution of 515 μ l to above-mentioned each Tissue Culture Dish, in 37 ℃, 5%CO
2in incubator, continue to cultivate.After transfection 24 hours, every culture dish cell adds 25 μ l2M Sodium propanecarboxylates to final concentration 5mM.
2, lysing cell
After transfection 48 hours, carry out lysis.Suck substratum, add 1ml PBS to carry out rinsing, suck PBS.Add 1ml lysis buffer, before use, add proteinase inhibitor PI and PMSF.Be placed in ice chest and vibrate on shaking table, collect the lysate in all culture dish, 4 ℃ centrifugal, collects supernatant.
3, DDK affinity chromatography column purification
Take aperture, be 0.45 μ M, pvdf membrane filter that diameter is 33mm filters the lysate supernatant after centrifugal and proceeds in 15ml pipe, adds the Sepharose Beads1ml mixing, and puts into 360 degree vortex mixers after sealing, in 4 ℃ in conjunction with 2 hours; Take out 15ml pipe, lysate is poured in BIO-RAD chromatography column, and catch and penetrate liquid, penetrate liquid sampling WB after dripping to the greatest extent to detect, see Fig. 1 (Fig. 1 shows: p63 recombinant plasmid is normal expression in HEK293T cell); With lysis buffer, rinse post material 1-2 time, with TBST, rinse Beads3 time again after dripping to the greatest extent, drip the most rear 0.1M Glycine(pH3.5 that uses) wash-out, 200 μ l for the first time, drip to the greatest extent and do not collect, second and third each 500 μ l, the 4th 250 μ l, are collected in a 1.5ml Tube, and add rapidly NaH
2pO
4(pH=11.0) be neutralized to about pH7.0, every pipe adds glycerine to final concentration be 10%, Tween-80 to final concentration be 0.1%.P63 recombinant protein after purifying is identified with SDS-PAGE, the results are shown in Figure 2.
Three, the screening of monoclonal antibody and preparation
1, animal immune: the P63 recombinant protein of above-mentioned purifying is with complete Freund's adjuvant emulsification, adopt subcutaneous or abdominal injection method immunity 6-8 BALB/c mouse in age in week, immunizing dose is 50 μ g/, immunity is for the second time carried out at interval after two weeks, with incomplete Freund's adjuvant emulsification, immunizing dose is 50 μ g/.After immune twice, get tail blood and measure serum titer with ELISA method gradient dilution; According to result, determine whether booster immunization, choose the mouse that antibody titer is the highest and carry out cytogamy.
2, cytogamy: myeloma cell adopts the sp2/0 in BALB/c mouse source, during fusion in logarithmic phase; Get the mouse spleen of above-mentioned immunity, make lymphocyte single cell suspension; Immune mouse splenic lymphocyte is mixed with 1:5-1:10 with myeloma cell, drip the 50%PEG(PH8.0 of 37 ℃) 1ml, add incomplete substratum and all the other stop buffers, centrifugal abandoning adds HAT substratum to suspend after supernatant to mix, MC constant volume is to 50ml, divide and install in 3.5cm culture dish, be put in wet box, be placed in 37 ℃, 5%CO
2in constant incubator, cultivate.
3, screening and clone: merge in 7-10 days and select cell clone, use the P63 recombinant protein of above-mentioned purifying to carry out ELISA test, labeled cell strain number.Positive porocyte is carried out to limiting dilution, within after each limiting dilution 5-6 days, measure ELISA value, limiting dilution is carried out in the higher mono-clonal hole of the positive value of picking OD280, until ELISA measures 96 orifice plates, entirely hardens really positive; The high mono-clonal of the positive value of picking is determined strain, and its corresponding fusion plate cell strain is 2B10.
4, the male BALB/c mouse abdominal injection 0.5ml pristane in age in the preparation and purification of ascites monoclonal antibody: 10-12 week, one week afterwards every mouse with 1ml syringe abdominal injection, through PBS, wash resuspended monoclonal cell suspension, cell consumption is 5 × 10
6/ only, every strain antibody is made a call to 2 mouse.After gathering, collects mouse ascites ascites, centrifuging and taking supernatant, and affinity chromatography carries out ascites purifying, according to antibody subtype, selects respective post material, and monoclonal antibody 2B10 hypotype is IgG2a, adopts protein G to carry out purifying.Monoclonal antibody concentration determination after purifying, packing, frozen at-20 ℃.
Take monoclonal antibody 2B10 as primary antibodie, protein lysate to HepG2, HeLa, HT29, A549, COS7, Jurkat, MDCK, PC12, nine kinds of clones of MCF7 is hybridized, is developed the color, its result as shown in Figure 3, as seen from Figure 3: P63 albumen has expression in various degree in clone HepG2, HeLa, HT29, A549, COS7, MDCK.
Embodiment 2 carries out immunohistochemical methods detection take monoclonal antibody 2B10 as primary antibodie to prostate cancer tissue
1, get prostate cancer tissue and carry out paraffin embedding, use Finesse histotome to cut into slices, tissue thickness is 6 μ m;
2, dewaxing and aquation: 3 times × 10min of analytical pure dimethylbenzene, 3 times × 10min of dehydrated alcohol, 95% ethanol 5min, 85% ethanol 5min, 75% ethanol 5min, deionized water soaks 3min × 3 time;
3, add antigen retrieval liquid (0.01M, pH6.0 sodium citrate buffer) pressure kettle hot high pressure to repair 2min, when pressure kettle temperature is down to approximately 90 ℃, open pressure kettle, take out sample, then naturally cool to room temperature.Deionized water soaks 3min × 3 time.
4, use 3% hydrogen peroxide deactivation to organize endogenous peroxydase, the standing 10min of room temperature.Deionized water soaks 5min × 3 time.
5, add confining liquid (PBS+5% skim-milk+5% normal goats serum), hatch 60min for 37 ℃.
6, remove confining liquid, do not rinse, add the monoclonal antibody 2B10 of the present invention of 1:150 dilution proportion; Be placed in wet box, hatch 60min for 37 ℃.PBST(is containing 0.1%Tween-20) wash 2 times, wash 5min at every turn.PB ST(is containing 0.02%Tween-20) wash 1 time, wash 5min at every turn.
7, drip Polink-test kit 2(Catlog No.D37-15) in 1,37 ℃ of reagent hatch 10-20 minute.Use PBS washing 3 times, each 5min.Drip 2,37 ℃ of reagent in Polink-2 test kit (Catlog No.D37-15) and hatch 10-20 minute, use PBS washing 3 times, each 5min.
8, application DAB solution (ZLI-9019 of Zhong Shan Golden Bridge) colour developing, colour developing 3 ~ 10min.Distilled water wash.
9, haematoxylin redyeing nucleus 2min, distilled water rinsing, 1% hydrochloric acid differentiation.Distilled water rinsing 3 times, the standing 1min of room temperature.
10, dehydration and transparent: 75% ethanol 5min, 100% ethanol 5min × 3 time, 85% ethanol 5min, 95% ethanol 5min, 100% ethanol 3 × 5min; Dimethylbenzene 3 × 5min, neutral gum mounting.
11, microscopy, is shown in Fig. 4.
As seen from Figure 4: visible a large amount of brown yellow granules in prostate cancer body of gland are P63 protein expression positive cell.
Embodiment 3, OriGene protein chip detect the specificity of monoclonal antibody 2B10
On OriGene high-density protein chip, comprise 10400 HEK293T cell proteins and cross expression lysate, every kind of protein lysate has the repetition of two copies on chip.Protein lysate by trace on nitrocellulose filter.The location of each clock protein lysate can accurately be located by Excel file.On protein chip, albumen is divided into 40 sub-matrixes, on each sub-matrix, has some references, by reference, can quantitative each chip point the content of upper albumen, monitor the repeatability of each immune response data and the direction of location positive signal.
Below for using OriGene albumen (OriGene Cat PA100001) chip 2B10 monoclonal antibody to be carried out to the concrete steps of specificity identification:
1, a protein chip is placed in 50ml centrifuge tube, uses 40ml deionized water to infiltrate chip, be placed on shaking table mixed at room temperature 30 minutes; Discard deionized water, use 10ml PBST balance chip, room temperature treatment 10 minutes.
2, to adding in 50ml centrifuge tube 40ml5% skimmed milk (diluting with PBST) to be placed on shaking table room temperature sealing 30 minutes.
3, use confining liquid (5% skimmed milk) dilution primary antibodie 2B10, Dilution ratio 1:200 is hybridized.
4, clean sealed membrane is pasted on experiment table, drip the above-mentioned dilution primary antibodie of 250-300 μ l on sealed membrane.
5, protein chip is extracted out from confining liquid, faced down one of protein chip NC film, from one side contact antibody of chip, slowly slide, rely on surface tension of liquid, antibody will slowly infiltrate chip NC film, until whole NC film infiltrates in primary antibodie solution.Whole operating process avoids producing bubble.Chip is moved on under 4 degree environment, standing, primary antibodie overnight incubation.On chip, add a cover culture dish lid, on it, stick a hygenic towelette, to prevent from hatching for a long time, cause antibody to evaporate.
6, chip was moved in 50ml centrifuge tube in second day, use PBST rinsing chip twice, remove unnecessary antibody.Use 40ml PBST(0.1%Tween-20) washing chip, be placed on shaking table and mix, wash three times, wash 5min at every turn.
7, use confining liquid (5% skimmed milk) dilution two anti-DyLight649-conjugated AffiniPure Fragment Goat-anti-Mouse IgG, Dilution ratio is 1:400.
8,, according to above-mentioned steps 4, step 5 is carried out two and anti-is hatched operation.Incubated at room 1 hour.Above chip, with aluminium-foil paper, hide, to prevent signal bleaching.
9,, according to above-mentioned steps 6, use PBST washing chip.
10, use deionized water rinsing chip, to remove remaining salinity and denaturing agent.
11, drying at room temperature chip, guarantees chip complete drying.
12, use chip scanner to read fluorescent signal.
13, according to B SA-Cy3 and BSA-Cy5, determine the site of chip direction and positive signal.
14, according to positive signal site, find out corresponding protein lysate ID, according to lysate database information, find corresponding protein name, NCBI typing number (accession number), protein I D, the information such as albumen size.
Chip results: occur two positive signal points on protein chip, corresponding albumen is respectively TP63 and C7orF31.
Embodiment 4, Western blot checking protein chip results of hybridization
Embodiment 3 acquired results are carried out to Western blot analysis verification.Cell pyrolysis liquid with the 293T cell of crossing expression TP63 recombinant plasmid, C7orF31 recombinant plasmid (C-terminal of two kinds of recombinant plasmids is all with DDK sequence label) carries out after loading, SDS-PAGE electrophoresis, transferring film, sealing, using respectively 2B10 monoclonal antibody of the present invention (Dilution ratio is 1:500) and anti-DDK tag antibody is that primary antibodie is hybridized, and results of hybridization is shown in Fig. 5.
Fig. 5 shows: uses the results of hybridization demonstration of 2B10 antibody of the present invention to two kinds of cell pyrolysis liquids, 2B10 antibody can with p63 protein binding, and be not combined with C7orF31 albumen; The DDK antibody results of hybridization of two kinds of cell pyrolysis liquids is all positive, shows that two kinds of plasmids all can normal expression in 293T cell.
Fig. 5 result shows, monoclonal antibody 2B10 of the present invention is specific binding P63 albumen only, with other albumen no cross reactions.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a monoclonal antibody 2B10 for specific binding P63 albumen, the hybridoma cell strain that is CGMCC No.6131 by deposit number produces.
2. a hybridoma cell strain, its deposit number is CGMCC No.6131.
3. the application of monoclonal antibody as claimed in claim 1 in the immunodetection instrument for the preparation of detection P63 albumen.
4. application according to claim 3, is characterized in that, described immunodetection instrument is test kit, chip or test paper.
5. an immunologic combined detection reagent kit, is characterized in that, comprises monoclonal antibody claimed in claim 1.
6. the application of monoclonal antibody as claimed in claim 1 in the test kit for the preparation of evaluation p63 positive cell or p63 positive cell source tumour.
7. application as claimed in claim 6, is characterized in that, described p63 positive cell is basal cell, myoepithelical cell, squamous cell, urinary tract transitional epithelial cell.
8. application as claimed in claim 7, is characterized in that, described basal cell is epidermal basal cell, cutaneous appendages basal cell or prostate basic cell by immunohistochemistry., and described myoepithelical cell is cutaneous appendages myoepithelical cell or mammary gland myoepithelical cell.
9. application as claimed in claim 6, is characterized in that, described p63 positive cell source tumour is rodent cancer, myoepithelical cell cancer, squamous cell cancer, urinary tract transitional cell carcinoma.
10. application as claimed in claim 9, it is characterized in that, described rodent cancer is epidermal basal cell cancer, cutaneous appendages rodent cancer or prostate basic cell by immunohistochemistry. cancer, and described myoepithelical cell cancer is cutaneous appendages myoepithelical cell cancer or mammary gland myoepithelical cell cancer.
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| CN108727496A (en) * | 2018-06-21 | 2018-11-02 | 南京基诺米医疗科技有限公司 | The anti-human p63 protein monoclonal antibodies of mouse prepare and its immunohistochemistry purposes |
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