CN102786595A - Anti-CD5 monoclonal antibody and purpose thereof - Google Patents
Anti-CD5 monoclonal antibody and purpose thereof Download PDFInfo
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Abstract
The invention discloses a hybridoma cell strain (the preservation number is CGMCC No. 6133), and a monoclonal antibody 2B8 derived from the hybridoma cell strain. The invention also relates to the application of the monoclonal antibody 2B8 in preparing an immunity testing tool for detecting the CD5 protein, actually, an immunohistochemical kit containing the monoclonal antibody 2B8, as well as the application of the monoclonal antibody 2B8 in preparing a kit for diagnosing tumors derived from B cells and rheumatoid arthritis. The monoclonal antibody provided by the invention can be combined with the CD5 protein specificity, has no cross reaction with other proteins in the cells, and greatly improves the specificity and reliability of the CD5 protein immunodetection.
Description
Technical field
But the present invention relates to the proteic monoclonal antibody of specific combination CD5, and the application in detecting CD5 albumen.
Background technology
CD5 is an I type gp, and is scavenging agent receptor family member.CD5 is expressed by thymocyte, mature T cells and part mature B cell (mainly being the B cell of secretion IgM), and lymphocyte activation is relevant with process of differentiation with regulating.CD5 antibody helps lymphoma is classified, and be the CD5 positive expression usually like the jacket cell lymphoma, and folliculus centrality lymphoma is often negative; The chronic lymphocytic leukemia that derives from the B cell expression that is positive, the lymphoma of other types such as follicular lymphoma, hairy cell leukemia, large celllymphoma etc. then are that CD5 is negative.Clinically, CD5 can be used as the biomarker of chronic lymphocytic leukemia, lymphoma mantle cell and diffuse large B cell lymphoma.
In addition; There is report to show; Rheumatoid arthritis is relevant with the lymphocytic level of CD5 positive B: the positive bone-marrow-derived lymphocyte level of rheumatoid arthritis patients CD5 is significantly higher than the positive bone-marrow-derived lymphocyte level of normal healthy controls group CD5, and the positive bone-marrow-derived lymphocyte level of height active rheumatoid arthritis patients CD5 is significantly higher than low active rheumatoid arthritis patients CD5+B lymphocyte level; Therefore, the detection of CD5 expression level can be used for the auxiliary diagnosis and the prognosis evaluation of rheumatoid arthritis.
Usually detect the expression situation of CD5 in the cell at present clinically through the experiment of immunohistochemistry (IHC) pathology.The core of IHC experiment is the monoclonal antibody that specificity combines CD5, and the quality of its performance is directly determining the sensitivity and the specificity of whole detection.Therefore, developing a kind of high specific combines the proteic anti-CD5 monoclonal antibody of CD5 to have great importance.
Summary of the invention
The technical problem that the present invention will solve is the proteic monoclonal antibody of anti-CD5 that provides a kind of binding specificity higher, and is used for detecting the application of the proteic immunodetection instrument of CD5 in preparation.
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 (abbreviating CGMCC as), preservation date is on May 16th, 2012, and deposit number is CGMCC No.6133.
The present invention also provides a specific specificity to combine the proteic monoclonal antibody 2B8 of CD5, is produced by above-mentioned hybridoma cell strain.
MONOCLONAL ANTIBODIES SPECIFIC FOR method according to the invention is following:
(1) structure of recombinant expression vector: carry out pcr amplification according to CD5 gene ORF complete sequence (shown in SEQ ID No.1) design primer; The gene both sides are introduced restriction endonuclease sites SgfI and MluI respectively; And at its C-terminal introducing Myc-DDK sequence label (shown in SEQ ID No.2); Insert expression vector pCMV6-Entry, make up the CD5 recombinant expression plasmid;
(2) CD5 Recombinant Protein Expression and purifying: with this plasmid transfection HEK293T cell, cracking centrifuging and taking supernatant uses DDK affinity chromatography column purification, obtains the CD5 recombinant protein of purifying;
(3) screening of monoclonal antibody and preparation: the CD5 recombinant protein immunity BALB/c mouse that adopts above-mentioned purifying; Get the mouse spleen cell and the SP2/0 cell merges; Limiting dilution assay obtains mono-clonal, ELISA method screening positive hybridoma cell, and acquisition can be secreted the hybridoma cell strain of anti-CD5 specific antibody; The anti-CD5 antibody of this hybridoma cell strain excretory called after 2B8, hypotype is accredited as IgG1; The preparation mouse ascites is through affinity chromatography column purification CD5 monoclonal antibody.Verify the sensitivity and the specificity of this monoclonal antibody respectively through Western Blot, immunohistochemical experiment, immunofluorescence.
The specificity checking of said monoclonal antibody:
Comprise 10400 HEK293T cellular proteinss on the OriGene high-density protein chip and cross the 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 through the Excel file.Albumen is divided into 40 inferior matrixes on the protein chip, on each inferior matrix some references is arranged, and through reference, quantitatively each chip point is gone up proteic content, monitors each immunoreation data repeatability, and the direction of location positive signal.
With the hybridization of monoclonal antibody 2B8 of the present invention and said chip and confirm the positive signal site, the result shows: monoclonal antibody 2B8 specificity of the present invention combines CD5 albumen, and with other albumen no cross reactions.
The present invention also provides monoclonal antibody 2B8 to be used for detecting the application of the proteic immunodetection instrument of CD5 in preparation.
Particularly, said immunodetection instrument is test kit, chip or test paper.
In concrete embodiment, the invention provides a kind of immunologic combined detection reagent kit, comprise said monoclonal antibody 2B 8; Can detect the expression situation of CD5 in the histocyte.
The present invention also provides said monoclonal antibody to be used for diagnosing the application of the test kit of B cell source tumour in preparation, and said B cell source tumour is chronic lymphocytic leukemia, lymphoma mantle cell or diffuse large B cell lymphoma.The CD5 molecule uses monoclonal antibody 2B8 of the present invention that the CD5 protein molecular is carried out immunodetection and can be used for diagnosing B cell source tumour as the molecular marked compound of B cell source tumour; At present, confirmed that clinically the CD5 molecule is the affinity tag of chronic lymphocytic leukemia, lymphoma mantle cell and diffuse large B cell lymphoma.
In view of the level of the positive B cell of CD5 and the dependency of rheumatoid arthritis, the present invention also provides said monoclonal antibody to be used for the application of the test kit of diagnostics classes rheumatic arthritis in preparation.
Preservation information
The scientific description that is used for the hybridoma cell strain of preservation is: anti-people CD5 monoclonal antibody hybridoma cell strain 2B8;
Depositary institution's full name: China Committee for Culture Collection of Microorganisms common micro-organisms center;
Depositary institution is called for short: CGMCC;
Depositary institution address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City;
Preservation date: on May 16th, 2012;
Deposit number: CGMCC No.6133.
Description of drawings
Fig. 1 is the expression of Western blot checking CD5 recombinant plasmid in the HEK293T cell; Last all article of left side swimming lane are the HEK293T cell pyrolysis liquid of transfection empty carrier, and last all article of the right swimming lane are the HEK293T cell pyrolysis liquid of transfection recombinant plasmid pCMV6-CD5, hybridize used one and anti-are the DDK tag antibody;
Fig. 2 is that the SDS-PAGE of CD5 recombinant protein identifies figure;
Fig. 3 expresses (is one to resist with the 2B8 monoclonal antibody) for the CD5 that immunohistochemical methods detects lymphatic cancer tissue;
Fig. 4 expresses (is one to resist with the 2B8 monoclonal antibody) for the CD5 that immunohistochemical methods detects tonsilla tissue;
Fig. 5 is the positive signal site of Western blot proofing chip;
Left figure: with 2B8 antibody (1:500 dilution) is an anti-results of hybridization, is followed successively by 293T cell pyrolysis liquid, albumen Marker, the wild-type 293T cell pyrolysis liquid of expressing the CD5 plasmid from left to right, crosses the 293T cell pyrolysis liquid of expressing CETN2, crosses the 293T cell pyrolysis liquid of expressing JUB;
Right figure: with the hybridoma culture supernatant of secretion 2B 8 antibody is an anti-results of hybridization, is followed successively by 293T cell pyrolysis liquid, albumen Marker, the wild-type 293T cell pyrolysis liquid of expressing the CD5 plasmid from left to right, crosses the 293T cell pyrolysis liquid of expressing CETN2, crosses the 293T cell pyrolysis liquid of expressing JUB.
Embodiment
In order to make those skilled in the art understand technical scheme of the present invention better, the present invention is done further detailed description below in conjunction with specific embodiment.
Embodiment 1 anti-CD5 MONOCLONAL ANTIBODIES SPECIFIC FOR
One, the structure of CD5 recombinant expression plasmid
Plasmid BC027901 (containing CD5 ORF sequence shown in the SEQ ID No.1) to obtain from ATCC is a template; Design two primers and introduce restriction enzyme site SgfI, MluI respectively; And C-terminal Myc-DDK label (sequence is shown in SEQ ID No.2); And be cloned into expression vector pCMV6-Entry, make up the CD5 recombinant expression plasmid.
Two, CD5 Recombinant Protein Expression and purifying
1, transfection HEK293T cell
The HEK293T cell reaches with 1:3 and continues in the Tissue Culture Dish that diameter is 10cm to cultivate; Get 7.5ml DMEM (serum-free and microbiotic) substratum to the 50ml pipe, add 300 μ lPEI MegaTran1.0 mixings, add the above-mentioned CD5 recombinant plasmid dna of 75 μ g then, mixing also left standstill 30 minutes; Getting the above-mentioned mixed solution of 515 μ l respectively adds in above-mentioned each Tissue Culture Dish, in 37 ℃, 5%CO
2Continue in the incubator to cultivate.After the transfection 24 hours, every petridish cell adds 25 μ l 2M Sodium propanecarboxylates to final concentration 5mM.
2, lysing cell
After the transfection 48 hours, carry out lysis.Substratum is removed in suction, adds 1ml PBS and carries out rinsing, inhales and removes PBS.Add the 1ml lysis buffer, add proteinase inhibitor PI and PMSF before using.Place ice chest on shaking table, to vibrate, collect the lysate in all petridish, 4 ℃ centrifugal, collects supernatant.
3, DDK affinity chromatography column purification
With aperture 0.45 μ m, the lysate supernatant after the filtration of the pvdf membrane filter of diameter 33mm is centrifugal also changes in the 15ml pipe, adds the Sepharose Beads 1ml that mixes, and puts into 360 degree vortex mixers after sealing, and combines 2 hours in 4 ℃; Take out the 15ml pipe, lysate is poured in the BIO-RAD chromatography column, and catch and penetrate liquid, penetrate liquid sampling WB after dripping to the greatest extent to detect, see Fig. 1; With lysis buffer flushing post material 1-2 time, drip to the greatest extent after again with TBST flushing Beads 3 times, drip to the greatest extent after with 0.1M Glycine (pH3.5) wash-out; 200 μ l for the first time; Drip to the greatest extent and do not collect, each 500 μ l of second and third time, the 4th time 250 μ l; Be collected among the 1.5ml Tube, and add NaH rapidly
2PO
4(pH=11.0) be neutralized to about pH7.0, it is 10% that every pipe adds glycerine to final concentration, Tween-80 to final concentration be 0.1%.CD5 recombinant protein behind the purifying identifies that with SDS-PAGE the result sees Fig. 2.
Three, the screening of monoclonal antibody and preparation
1, animal immune: the CD5 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/; Carry out the immunity second time at interval after two weeks, with incomplete Freund's adjuvant emulsification, immunizing dose is 50 μ g/.Get tail blood after immune twice and measure serum titer with ELISA method gradient dilution; Determine whether booster immunization according to the result, choose the highest mouse of antibody titer and carry out cytogamy.
2, cytogamy: the myeloma cell adopts the sp2/0 in BALB/c mouse source, is in logarithmic phase during fusion; Get above-mentioned mice immunized spleen, process the lymphocyte single cell suspension; The immune mouse SPL mixes with 1:5-1:10 with the myeloma cell; Drip 37 ℃ 50%PEG (PH 8.0) 1ml, add incomplete substratum and stop buffer thereof, centrifugal abandoning adds HAT substratum suspension mixing behind the supernatant; The MC constant volume is to 50ml; Divide to install in the 3.5cm petridish, be put in the wet box, place 37 ℃, 5%CO
2Cultivate in the constant incubator.
3, screening and clone: merges in 7-10 days and select cell clone, use the CD5 recombinant protein of above-mentioned purifying to carry out ELISA and test, labeled cell strain number.Positive porocyte is carried out limiting dilution, measured the ELISA value in 5-6 days behind each limiting dilution, limiting dilution is carried out in the higher mono-clonal hole of the positive value of picking OD280, measures 96 orifice plates until ELISA and hardens really positive entirely; The high mono-clonal of the positive value of picking is decided strain, and its corresponding fusion plate cell strain is 2B8.
4, the male BALB/c mouse abdominal injection 0.5ml pristane in age in the preparation of ascites monoclonal antibody and purifying: 10-12 week, every the mouse in a week back is washed resuspended monoclonal cell suspension with 1ml syringe abdominal injection through PBS, and the cell consumption is 5 * 10
6/ only, every strain antibody is made a call to 2 mouse.Treat that mouse ascites gathers the back and collects ascites, the centrifuging and taking supernatant, affinity chromatography carries out the ascites purifying, selects the respective post material according to antibody subtype, and monoclonal antibody 2B 8 hypotypes are IgG1, adopt proteinG to carry out purifying.Monoclonal antibody concentration determination behind the purifying, packing, frozen at-20 ℃.
Embodiment 2 is one anti-lymphatic cancer tissue to be carried out the immunohistochemical methods detection with monoclonal antibody 2B8
1, get the lymphatic cancer tissue and carry out paraffin embedding, use the Finesse histotome to cut into slices, tissue thickness is 6 μ m;
2, dewaxing and aquation: 3 times * 10min of analytical pure YLENE, 3 times * 10min of absolute ethyl alcohol, 95% ethanol 5min, 85% ethanol 5min, 75% ethanol 5min, deionized water soak 3min * 3 time;
3, add antigen retrieval liquid (0.01M, pH6.0 sodium citrate buffer) pressure kettle hot, high pressure and repair 2min, when treating that the pressure kettle temperature is reduced to about 90 ℃, open pressure kettle, take out sample, naturally cool to room temperature then.Deionized water soaks 3min * 3 time.
4, use 3% hydrogen peroxide deactivation to organize endogenous peroxydase, room temperature leaves standstill 10min.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, not flushing, the monoclonal antibody 2B8 of the present invention of adding 1:150 dilution proportion; Place wet box, hatch 60min for 37 ℃.5min is washed in PBST (containing 0.1%Tween-20) washing 2 times at every turn.5min is washed in PBST (containing 0.02%Tween-20) washing 1 time at every turn.
7, the reagent that drips among Polink-test kit 2 (the Catlog No.D37-15) was hatched 10-20 minute for 1,37 ℃.Use PBS washing 3 times, each 5min.The reagent that drips in the Polink-2 test kit (Catlog No.D37-15) was hatched 10-20 minute for 2,37 ℃, used PBS washing 3 times, each 5min.
8, use DAB solution (the middle China fir ZLI-9019 of Golden Bridge) colour developing, colour developing 3 ~ 10min.Distilled water wash.
9, haematoxylin redyeing nucleus 2min, zero(ppm) water rinsing, the differentiation of 1% hydrochloric acid.Zero(ppm) water rinsing 3 times, room temperature leaves standstill 1min.
10, dehydration and transparent: 75% ethanol 5min, 100% ethanol 5min * 3 time, 85% ethanol 5min, 95% ethanol 5min, 100% ethanol, 3 * 5min; YLENE 3 * 5min, the neutral gum mounting.
11, microscopy is seen Fig. 3.
Visible by Fig. 3: the visible a large amount of brown yellow granules of lymphatic cancer tissue are CD5 protein expression positive cell.
Embodiment 3 is one anti-tonsilla tissue to be carried out the immunohistochemical methods detection with monoclonal antibody 2B8
Use 8 pairs of tonsilla tissues of monoclonal antibody 2B of the present invention to carry out immunohistochemical methods and detect, detect step with embodiment 2, the result is as shown in Figure 4, and is visible by Fig. 4, and the visible a large amount of brown yellow granules of tonsilla tissue are CD5 protein expression positive cell.
Embodiment 4, OriGene protein chip detect the specificity of monoclonal antibody 2B8
Comprise 10400 HEK293T cellular proteinss on the OriGene high-density protein chip and cross the 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 through the Excel file.Albumen is divided into 40 inferior matrixes on the protein chip, on each inferior matrix some references is arranged, and through reference, quantitatively each chip point is gone up proteic content, monitors each immunoreation data repeatability, and the direction of location positive signal.
Below for using OriGene albumen (OriGene Cat PA100001) chip that the 2B8 monoclonal antibody is carried out the concrete steps that specificity is identified:
1, a protein chip is placed in the 50ml centrifuge tube, uses the 40ml deionized water to soak into chip, place on the shaking table mixed at room temperature 30 minutes; Discard deionized water, use 10mlPBST balance chip, room temperature treatment 10 minutes.
2, in the 50ml centrifuge tube, add 40ml 5% skimmed milk (diluting) and place on the shaking table, room temperature sealing 30 minutes with PBST.
3, use confining liquid (5% skimmed milk) dilution one anti-2B 8, Dilution ratio 1:200.
4, the film that seals with cleaning pastes on the experiment table, and the above-mentioned dilution one of dropping 250-300 μ l resists is sealing on the film.
5, protein chip is extracted out from confining liquid, one of protein chip NC film is faced down, one side contact antibody from chip slowly slides, and relies on surface tension of liquid, and antibody will slowly soak into chip NC film, soak in an anti-solution until whole NC film.The entire operation process avoids producing bubble.Chip is moved on under the 4 degree environment, leave standstill an anti-incubated overnight.On chip, add a cover the petridish lid, stick a hygenic towelette on it, cause antibody to evaporate to prevent to hatch for a long time.
6, chip moved in the 50ml centrifuge tube in second day, use PBST rinsing chip twice, remove unnecessary antibody.Use 40ml PBST (0.1%Tween-20) washing chip, place on the shaking table to 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, hide, to prevent the generation signals bleaching with aluminium-foil paper.
9,, use PBST washing chip according to above-mentioned steps 6.
10, use the deionized water rinsing chip, to remove remaining salinity and denaturing agent.
11, drying at room temperature chip is guaranteed the chip complete drying.
12, use chip scanner to read fluorescent signal.
13, confirm the site of chip direction and positive signal according to BSA-Cy3 and BSA-Cy5.
14, find out corresponding protein lysate ID according to the positive signal site,, find corresponding protein name, NCBI typing number (accession number), protein I D, information such as albumen size according to the lysate database information.
Chip results shows: occur 4 positive signal points on the protein chip, corresponding albumen is respectively CD5, CETN2, JUB, CACNG5.
Embodiment 5, Western blot checking protein chip results of hybridization
Use Western blot that embodiment 4 gained results are carried out analysis verification.Go up kind with the cell pyrolysis liquid of crossing the 293T cell express CD5 recombinant plasmid, CETN2 recombinant plasmid, JUB recombinant plasmid and wild-type 293T cell pyrolysis liquid respectively; After SDS-PAGE electrophoresis, commentaries on classics film, the sealing; Using the hybridoma supernatant of 2B 8 monoclonal antibodies of the present invention (Dilution ratio is 1:500) and secretion 2B8 antibody respectively is anti-hybridization, and its result is as shown in Figure 5.
Visible by Fig. 5: as no matter to be the 2B8 monoclonal antibody of the present invention or the Hybridoma Cell Culture supernatant of secretion 2B8 antibody; All only with the CD5 protein binding; Do not combine prompting and all have with CETN2, JUB proteantigen: monoclonal antibody 2B8 of the present invention only specificity combines CD5 albumen.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (8)
1. a specific specificity combines the proteic monoclonal antibody 2B8 of CD5, is the hybridoma cell strain generation of CGMCC No.6133 by deposit number.
2. hybridoma cell strain, its deposit number is CGMCC No.6133.
3. monoclonal antibody as claimed in claim 1 is used for detecting the application of the proteic immunodetection instrument of CD5 in preparation.
4. application according to claim 3 is characterized in that, said immunodetection instrument is test kit, chip or test paper.
5. an immunologic combined detection reagent kit is characterized in that, comprises the described monoclonal antibody of claim 1.
6. monoclonal antibody as claimed in claim 1 is used for diagnosing the application of B cell source tumor reagent box in preparation.
7. application according to claim 6 is characterized in that, said B cell source tumour is chronic lymphocytic leukemia, lymphoma mantle cell or diffuse large B cell lymphoma.
8. monoclonal antibody as claimed in claim 1 is used for the application of diagnostics classes rheumatic arthritis test kit in preparation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113105547A (en) * | 2021-05-18 | 2021-07-13 | 福州迈新生物技术开发有限公司 | anti-CD 5 protein monoclonal antibody and cell strain, preparation method and application thereof |
| WO2022152185A1 (en) * | 2021-01-12 | 2022-07-21 | 南京驯鹿医疗技术有限公司 | Cd5-targeting fully humanized antibody |
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| CN102137873A (en) * | 2008-08-29 | 2011-07-27 | 西福根有限公司 | Anti-CD5 antibodies |
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| WO2022152185A1 (en) * | 2021-01-12 | 2022-07-21 | 南京驯鹿医疗技术有限公司 | Cd5-targeting fully humanized antibody |
| CN113105547A (en) * | 2021-05-18 | 2021-07-13 | 福州迈新生物技术开发有限公司 | anti-CD 5 protein monoclonal antibody and cell strain, preparation method and application thereof |
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