CN106854245A - Protein monoclonal antibody of AntiCD3 McAb 0 and application thereof - Google Patents

Abstract

The present invention relates to biological technical field, a kind of hybridoma cell strain (deposit number is CGMCC No.13287), and the monoclonal antibody UMAB256 that thus hybridoma cell strain is produced are disclosed.The invention further relates to applications of the monoclonal antibody UMAB256 in the immune detection instrument for being used for detecting CD30 albumen is prepared, immunohistochemical kit containing monoclonal antibody UMAB256, and applications of the monoclonal antibody UMAB256 in the kit for marked tumor is prepared.Monoclonal antibody of the present invention can be combined with CD30 protein-specifics, and with intracellular other albumen no cross reactions, significantly improve specificity, accuracy and the reliability of CD30 protein immunizations detection.

Description

Protein monoclonal antibody of AntiCD3 McAb 0 and application thereof

Technical field

The present invention relates to biological technical field, and in particular to can specific bond CD30 albumen monoclonal antibody UMAB256, Produce the cell line of the monoclonal antibody and apply the diagnostic method of the antibody and purposes.

Background technology

CD30 is tumor necrosis factor receptor super family (tumor necrosis factor receptor One of superfamily, TNFRSF) member, two-character given name TNFRSF8 is that a kind of molecular weight is the cell surface I type cross-films of 120kDa Glycoprotein.CD30 is distributed widely in activating T cell, B cell, the atypical lymphocyte of lymphocytic disease, undifferentiated big Cell etc., participates in cell activation and differentiation.By participating in the conduction of the activation signalses such as NF- κ B, the activation of cellular immunity is adjusted Cheng Zhong.CD30 can be combined with CD153 (CD30L), and limitation autoreactivity CD8+T cell propagation, protection body exempts from itself and exempts from Epidemic disease.

Found in the research of multinomial lymthoma pathogenic process, the expression of CD30 can be significantly risen higher than Hodgkin lymphoma (Hodgkin lymphoma, HL) and primary cutaneous type (anaplastic large cell lymphomas, ALCL), and low expression under non-pathological state activate T cell, B cell surface.Normal cell is not expressed leukocyte differentiation and is resisted 30 (CD30) of original.The targeted therapy for CD30 has turned into a potential remedy measures in recent years, research it is more be CD30 The exploitation of antibody and its related drugs.Become a molecular target for the Tumor Cell Lines immunization therapy for having much potentiality Point.The expression situation of albumen in immunohistochemistry (IHC) Pathological experiment detection tumour cell is clinically commonly used at present, but The core of IHC experiments is the monoclonal antibody of binding proteins specific, and the quality of its performance directly decides the spirit of whole detection Sensitivity and specificity.Therefore, a kind of binding specificity monoclonal antibody for CD30 albumen higher is developed to detect IHC CD30 expressions have great importance.

The content of the invention

In view of this, the monoclonal it is an object of the invention to provide a kind of binding specificity CD30 albumen higher resists Body, and its application in the immune detection instrument for being used for detecting CD30 albumen is prepared.

The invention provides a kind of hybridoma cell strain, China Committee for Culture Collection of Microorganisms is preserved in commonly micro- Bio-Centers (referred to as CGMCC), preservation date is on November 22nd, 2016, and deposit number is CGMCC No.13287.

Present invention also offers a kind of monoclonal antibody UMAB256 of specific binding CD30 albumen, by above-mentioned hybridoma Cell line is produced.

The preparation method of monoclonal antibody of the present invention is as follows：

(1) structure of recombinant expression carrier：According to CD30ORF nucleotide sequences (CD30ORF nucleotide sequences such as SEQ ID Shown in NO.1,1788bp；CD30 amino acid sequences are as shown in SEQ ID NO.2)

Design primer PCR expands CD30ORF 1222bp to 1785bp bit sequences, and gene both sides introduce limitation respectively Property restriction enzyme site SgfI and MluI, insert expression vector pET23a-N-His, build CD30 amino acid sequences the 408th to the The recombinant expression plasmid pET23a-N-His-rCD30 of 595；Upstream amplification primer sequence, SEQ ID NO.3: CACGCGATCGCCCACCGGAGGGCCTGCAGG downstream amplification primer sequence SEQ ID NO.4:ACCGACGCGT CTACTTTCCAGAGGCAGCTGT

(2) expression and purification of CD30 recombinant proteins：By CD30 recombinant expression plasmid Transformed E .coli cells, cracking centrifugation Supernatant is obtained, the purifying of affinity chromatography post obtains the CD30 recombinant proteins of purifying；

(3) screening of monoclonal antibody and preparation：Balb/c mouse are immunized using the CD30 recombinant proteins of above-mentioned purifying, are taken Mouse spleen cells are merged with SP2/0 cells, and limiting dilution assay obtains monoclonal, and ELISA method screening positive hybridoma is thin Born of the same parents, acquisition can secrete the hybridoma cell strain of the specific antibody of AntiCD3 McAb 0, be named as UMAB256, and hypotype is accredited as IgG1；Pass through Serum free medium prepares antibody, is purified by affinity column and obtains CD30 monoclonal antibodies UMAB256.Pass through respectively Western Blot, immunohistochemical experiment verify the sensitivity and specificity of the monoclonal antibody.

The specificity of said monoclonal antibody is verified using OriGene high-density proteins chip further： 10,000 HEK293T cell protein overexpression lysate, every kind of protein lysate are included on OriGene high-density protein chips There are two repetitions of copy on chip.Protein lysate is by trace on nitrocellulose filter.Each clock protein lysate Positioning can be accurately positioned by Excel file.Albumen is divided into 40 sub- matrixes on protein chip, on each sub- matrix There are some references, by referring to, the content of albumen on each chip point can be quantified, monitor the repetition of each immune response data Property, and the direction for positioning positive signal.

The monoclonal antibody UMAB256 and said chip are hybridized and determine positive signal site by the present invention, are as a result shown Show monoclonal antibody UMAB256 of the present invention specific binding CD30 albumen, and with other albumen no cross reactions.

Present invention also offers monoclonal antibody UMAB256 in the immune detection instrument for detecting CD30 albumen for preparing Application.

Specifically, the immune detection instrument is kit, chip or test paper.

In the particular embodiment, the invention provides a kind of immunologic combined detection reagent kit, including above-mentioned monoclonal is anti- Body UMAB256, can detect the expression situation of CD30 in histocyte.

Present invention also offers application of the said monoclonal antibody in the kit for marked tumor is prepared.Wherein institute State tumour and specifically refer to the propagation of tumour cell and the closely related tumour of expression of CD30, including but not limited between denaturation it is big thin Born of the same parents' lymthoma and Hodgkin lymphoma.

Compared with prior art, the invention provides a kind of hybridoma cell strain, (deposit number is CGMCC No.13287 the monoclonal antibody UMAB256 that), and thus hybridoma cell strain is produced.It is anti-present invention also offers monoclonal Applications of the body UMAB256 in the immune detection instrument for being used for detecting CD30 albumen is prepared, containing exempting from for monoclonal antibody UMAB256 Epidemic disease group kit, and applications of the monoclonal antibody UMAB256 in the kit for marked tumor is prepared.Institute of the present invention Stating monoclonal antibody can be combined with CD30 protein-specifics, and with intracellular other albumen no cross reactions, significantly improve Specificity, accuracy and reliability that CD30 protein immunizations are detected, CD30 protein expression levels in true reflection tumor tissues, can It is applied to primary cutaneous type and Hodgkin lymphoma.

Preservation information

Classification And Nomenclature for the hybridoma cell strain UMAB256 of preservation is：The hybridoma of CD30 mouse monoclonal antibodies is thin Born of the same parents' strain；

Depositary institution's full name：China Committee for Culture Collection of Microorganisms's common micro-organisms center；

Depositary institution is referred to as：CGMCC；

Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica；

Preservation date：On November 22nd, 2016；

Deposit number：CGMCC No.13287.

Brief description of the drawings

In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing The accompanying drawing to be used needed for having technology description is briefly described.

Fig. 1 shows the design of the cloning site of embodiment 1 as schemed, and wherein shading part is ORF areas；

Fig. 2 shows the recombinant C D30 albumen Western blot testing result figures of embodiment 2, with anti-HIS detection recombinant Cs D30 Expression of the albumen in E.coli cells, wherein the E.Coli cell pyrolysis liquids that left side swimming lane is transfection empty carrier are the inspection of antigen Survey the testing result that result, right lanes are the E.coli cell pyrolysis liquid antigens for transfecting pET23a-N-His-rCD30 plasmids；

Fig. 3 shows the recombinant C D30 protein SDS-PAGE result figures of embodiment 2, with affinity chromatography post purification of Recombinant CD30 eggs In vain, albumen after purification is by SDS-PAGE glue electricity arteries and veins, coomassie brilliant blue staining；

Fig. 4 shows that embodiment 3 recognizes complete CD30 (Full length CD30, CD30- with monoclonal antibody UMAB256 FL) the Western blot testing result figures of albumen.Swimming lane 1 is the cell pyrolysis liquid of transfection pCMV6-Entry；Swimming lane 2 is to turn Contaminate the cell pyrolysis liquid of pCMV6-CD30-FL；

Fig. 5 shows that (primary antibody is for the formalin fix of embodiment 4, the human lymphoma histogenic immunity group result figure of FFPE CD30 monoclonal antibody UMAB256)；

Fig. 6 shows normal person's tonsil ImmunohistochemistryResults Results figure (of the formalin fix of embodiment 4, FFPE It is CD30 monoclonal antibody UMAB256 to resist)；

Fig. 7 shows that (primary antibody is CD30 monoclonal antibodies UMAB256,1 to embodiment 5OriGene protein chip qualification results figure:100；Two Resist is DyLight649-conjugated AffiniPure Fragment Goat-anti-Mouse IgG, 1:400).

Specific embodiment

Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, Obviously, described embodiment is only a part of embodiment of the invention, rather than whole embodiments.Based in the present invention Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made, all Belong to the scope of protection of the invention.

The structure of embodiment 1, CD30 recombinant expression plasmids

With the plasmid RC219819 (1788bp of ORF containing CD30) obtained from bio tech ltd of Aureal Dongyuan County of the U.S. It is template, designs two primers and introduce restriction enzyme site SgfI and MluI respectively, be cloned into expression vector pET23a-N-His, builds Vertical CD30 recombinant expression plasmids.Cloning site design is as shown in Figure 1.

The expression and purification of embodiment 2, CD30 recombinant proteins

1st, Transformed E .coli cells：100ul competent cells are placed in after thawed on ice adds DNA gently to mix, ice 42 DEG C of heat shock 90s after bath 30min, are then continued ice bath 1-2min.The fresh nonreactive LB cultures of 500ul are added in super-clean bench Base, is spread evenly across on the flat board containing antibiotic in appropriate bacterium solution is taken after 37 DEG C of shaking tables incubation 45min, and culture dish is inverted in into 37 Overnight incubation in DEG C constant incubator.

2nd, cell lysis：Picking monoclonal in fresh culture, 37 DEG C, 200rpm cultivates to OD values and reach 0.4~0.6 When add IPTG (final concentration 1mM) Fiber differentiation 7h.Thalline is collected by centrifugation, then with the resuspended thalline of lysis buffer, ultrasound is broken Broken 20min collects supernatant after 12000rpm centrifugations 20min at 4 DEG C.Take a small amount of supernatant protein anti-His antibody and make WB mirror It is fixed, see Fig. 2.

3rd, affinity chromatography post purifying：With buffer solution balance nickel post, by supernatant with loading after 0.45 μm of membrane filtration simultaneously Outflow is collected, uncombined albumen is removed with buffer solution drip washing, finally with the elution containing various concentrations imidazoles, received respectively SDS-PAGE identifications are carried out after collection, satisfactory wash-out protein is merged and 10% glycerine is added, recombinant C D30 after purification Albumen SDS-PAGE identifications, are shown in Fig. 3.

From Fig. 2 results, in the E.coli cell pyrolysis liquids of transfection pET23a-N-His-rCD30 plasmids after WB detections There is obvious specific band at~25kD, be consistent substantially with the CD30 Argine Monohydrochlorides actual molecular weight of the 408 to 595th. Show recombinant C D30 albumen specifically expressings in cell.

From Fig. 3 results, the albumen of purifying has obvious specific band at PAGE glue~25kD, with CD30 albumen ammonia The actual molecular weight of base acid the 408 to 595th is consistent substantially.Show to have obtained the preferable CD30 recombinant proteins of purity.

Embodiment 3, the preparation of CD30 monoclonal antibodies and screening

The CD30 protein fragments of the purifying produced with restructuring according to standard method are used to (tie up tonneau in Beijing to Balb/c mouse Magnificent experimental animal Technology Co., Ltd.) it is immunized.Specific method is as follows：

1st, animal immune：Purified CD30 antigens are emulsified with complete Freund's adjuvant, using subcutaneous or intraperitoneal injection side Method is immunized 6-8 week old Balb/c mouse, and immunizing dose is 50 μ g/, and interval carries out second and is immunized after two weeks, with not exclusively not Family name's adjuvant emulsion, immunizing dose is 50 μ g/.The immune tail blood that takes afterwards twice determines serum titer with ELISA method gradient dilution；Root Determine whether booster immunization according to result, choosing antibody titer highest mouse carries out cell fusion.

2nd, cell fusion：Myeloma cell is in exponential phase using the sp2/0 in Balb/c sources during fusion；Take Immune mouse spleen, is made lymphocyte single cell suspension；Mouse spleen lymphocyte is with myeloma cell with 1:5-1:10 mixing, 37 DEG C 50%PEG (PH 8.0) 1mL is added dropwise, adds incomplete culture medium and remaining terminate liquid, centrifugation to add HAT after abandoning supernatant Culture medium suspends and mixes, and MC constant volumes are dispensed into 3.5cm culture dishes to 50mL, are put in wet box, are placed in 37 DEG C, 5%CO₂It is permanent Cultivated in warm incubator.

3rd, screen and clone：Fusion selects cell clone in 7-10 days, and ELISA surveys are carried out using CD30 purification of recombinant proteins Examination.Mark cell line number.Limiting dilution is carried out to positive hole cell, ELISA values, picking is determined within 5-6 days after each limiting dilution OD280 positive values monoclonal hole higher carries out limiting dilution, until it is the positive that ELISA determines the complete hardened fruit of 96 orifice plates.Picking Positive value monoclonal singling high.Its correspondence fusion plate cell line is UMAB256.

4th, the preparation and purification of ascites monoclonal antibody：The male Balb/c mouse peritoneal injections 0.5ml norphytanes of 10-12 week old, Every mouse washs resuspended monoclonal cell suspension with the intraperitoneal injection of 1mL syringes through PBS after one week, and cell consumption is 5 × 10⁶/ only, make a call to 2 mouse per strain antibody.After ascites is collected after mouse ascites accumulation, centrifuging and taking supernatant, affinity chromatography carries out abdomen Water is purified, and corresponding post material is selected according to antibody subtype, and the monoclonal antibody that cell line UMAB256 is produced is IgG1, is entered using protein G Row purifying.Monoclonal antibody concentration mensuration after purification, WB are detected, dispense, frozen at -20 DEG C.Wherein WB testing results are shown in Fig. 4.

From Fig. 4 results, swimming lane 2 has special band at molecular weight about 100kD, shows monoclonal antibody UMAB256 specifically can detect complete total length CD30 albumen by Western blot.

Embodiment 4, monoclonal antibody UMAB256 are detected for the SABC of primary antibody

(1), experimental technique：

The tonsil block of the human lymphoma tissue and normal person that the 1, take formalin fix carries out FFPE, uses Finesse histotomes are cut into slices, and tissue thickness is 6 μm.

2nd, dewaxing and aquation：Analyze pure 3 × 10min of dimethylbenzene, absolute ethyl alcohol 3 × 10min, 95% ethanol 5min, 85% Ethanol 5min, 75% ethanol 5min, deionized water immersion 3min × 3 time

3rd, add antigen retrieval buffers (0.01M, pH6.0 sodium citrate buffer) pressure cooker hot high pressure to repair 3min, treat height When pressure pot temperature is down to about 90 DEG C, pressure cooker is opened, take out sample, then naturally cool to room temperature.Deionized water soaks 3min × 3 times.

4th, using 3% hydrogen peroxide deactivation tissue endogenous peroxydase, it is stored at room temperature 10min.Deionized water is soaked 5min × 3 time.

5th, plus confining liquid (Normal Goat Serum of PBS+5% skimmed milk powers+5%), 37 DEG C of incubation 60min.

6th, confining liquid is removed, is not rinsed, add CD30 monoclonal antibodies (UMAB256), thinner ratio：1:150, carried out using confining liquid Dilution.It is placed in wet box, 37 DEG C of incubation 60min.PBST (0.1%Tween-20) is washed 2 times, and 5min is washed every time.PBST (0.02%Tween-20) is washed 1 time, and 5min is washed every time.

7th, 1,37 DEG C of 2 (Catlog No.D37-15) reagent of Polink- kits is added dropwise to be incubated 10-20 minutes.Use PBS Washing 3 times, each 5min.2,37 DEG C of Polink-2 kit (Catlog No.D37-15) reagent is added dropwise to be incubated 10-20 minutes, Washed 3 times using PBS, each 5min.

8th, developed the color using DAB solution (Zhong Shan Golden Bridge ZLI-9019), develop the color 3~10min.Distillation water washing.

9th, haematoxylin redyeing nucleus 2min, distilled water rinsing, the differentiation of 1% hydrochloric acid.Distilled water is rinsed 3 times, is stored at room temperature 1min。

10th, it is dehydrated and transparent：75% ethanol 5min, 100% ethanol 5min x 3 times, 85% ethanol 5min, 95% ethanol 5min, 100% 3 × 5min of ethanol；3 × 5min of dimethylbenzene, neutral gum mounting.

11st, microscopy, is shown in Fig. 5-6.

(2), experimental result：

From Fig. 5-6 results, the visible specific membrane dye in the tonsil of human lymphoma tissue and normal person Color.Result and CD30 positioning in the cell and tissue expression specificity are consistent, show that monoclonal antibody UMAB256 can be used to exempt from The level of CD30 albumen detects in epidemic disease histochemistry.

The specific detection of embodiment 5, monoclonal antibody UMAB256

10,000 HEK293T cell protein overexpression lysate is included on OriGene high-density protein chips, per hatching egg White lysate has two repetitions of copy on chip.Protein lysate is by trace on nitrocellulose filter.Each clock egg The positioning of white lysate can be accurately positioned by Excel file.Albumen is divided into 40 sub- matrixes on protein chip, each There are some references on sub- matrix, by referring to, the content of albumen on each chip point can be quantified, monitor each immune response number According to repeatability, and positioning positive signal direction.It is below to use OriGene albumen (OriGene Cat PA100001) Chip carries out the experimental technique of UMAB256 Identification of the antibodies experiments：

1st, a protein chip is placed in 50mL centrifuge tubes, chip is infiltrated using 40mL deionized waters, be placed on shaking table, Mixed at room temperature 30 minutes.Deionized water is discarded, chip is balanced using 10mLPBST.Room temperature treatment 10 minutes.

2nd, to adding 40mL5% skim milks (being diluted with PBST) to be placed on shaking table in 50mL centrifuge tubes, room temperature is sealed Close 30 minutes.

3rd, primary antibody UMAB256 is diluted using confining liquid (5% skim milk), thinner ratio is classified as 1:100.

4th, clean sealed membrane is pasted on experimental bench, dropwise addition 250-300 μ L primary antibodies are on sealed membrane.

5th, protein chip is extracted out from confining liquid, the one of protein chip NC films is faced down, contacted from one side of chip Antibody, slowly slides, and by surface tension of liquid, antibody will slowly infiltrate chip NC films, until whole NC films infiltration is in primary antibody In solution.Whole operation process avoids producing bubble.Chip is moved on under 4 DEG C of environment, is stood, primary antibody overnight incubation.In chip On add a cover culture dish lid, a hygenic towelette is sticked thereon, with prevent for a long time be incubated cause antibody to evaporate.

6th, in chip being moved into 50mL centrifuge tubes in second day, chip is rinsed twice using PBST, removes unnecessary antibody.Make Chip is washed with 40mL PBST (0.1%Tween-20), is placed on shaking table and is well mixed, washed three times, 5min is washed every time.

7th, secondary antibody DyLight649-conjugated AffiniPure are diluted using confining liquid (5% skim milk) Fragment Goat-anti-Mouse IgG, dilution ratio is 1:400.

8th, according to above-mentioned steps 4, step 5 carries out secondary antibody and is incubated operation.Incubation at room temperature 1 hour.Aluminium foil is used above chip Paper is covered, to prevent signal bleaching.

9th, according to above-mentioned steps 6, chip is washed using PBST.

10th, using deionized water rinsing chip, to remove the salinity and denaturant of remnants.

11st, drying at room temperature chip, it is ensured that chip is completely dried.

12nd, fluorescence signal is read using chip scanner.

13rd, the site of chip direction and positive signal is determined according to BSA-Cy3 and BSA-Cy5.

14th, correspondence protein lysate ID is found out according to positive signal site, according to lysate database information, it is right to find Answer the information such as protein name, NCBI typings number (accession number), protein I D, albumen size.

Result shows as shown in fig. 7, monoclonal antibody UMAB256 can specifically recognize CD30 albumen on OriGene protein chips The specificity of monoclonal antibody UMAB256 is preferably.

SEQUENCE LISTING

<110>Wuxi Origene Bio-tech Co., Ltd.

<120>Protein monoclonal antibody of AntiCD3 McAb 0 and application thereof

<210> 1

<211>1788

<212> DNA

<213>Artificial sequence

<400> 1

ORIGIN

//

1 ATGCGCGTCC TCCTCGCCGC GCTGGGACTG CTGTTCCTGG GGGCGCTACG AGCCTTCCCA

61 CAGGATCGAC CCTTCGAGGA CACCTGTCAT GGAAACCCCA GCCACTACTA TGACAAGGCT

121 GTCAGGAGGT GCTGTTACCG CTGCCCCATG GGGCTGTTCC CGACACAGCA GTGCCCACAG

181 AGGCCTACTG ACTGCAGGAA GCAGTGTGAG CCTGACTACT ACCTGGATGA GGCCGACCGC

241 TGTACAGCCT GCGTGACTTG TTCTCGAGAC GACCTCGTGG AGAAGACGCC GTGTGCATGG

301 AACTCCTCCC GTGTCTGCGA ATGTCGACCC GGCATGTTCT GTTCCACGTC TGCCGTCAAC

361 TCCTGTGCCC GCTGCTTCTT CCATTCTGTC TGTCCGGCAG GGATGATTGT CAAGTTCCCA

421 GGCACGGCGC AGAAGAACAC GGTCTGTGAG CCGGCTTCCC CAGGGGTCAG CCCTGCCTGT

481 GCCAGCCCAG AGAACTGCAA GGAACCCTCC AGTGGCACCA TCCCCCAGGC CAAGCCCACC

541 CCGGTGTCCC CAGCAACCTC CAGTGCCAGC ACCATGCCTG TAAGAGGGGG CACCCGCCTC

601 GCCCAGGAAG CTGCTTCTAA ACTGACGAGG GCTCCCGACT CTCCCTCCTC TGTGGGAAGG

661 CCTAGTTCAG ATCCAGGTCT GTCCCCAACA CAGCCATGCC CAGAGGGGTC TGGTGATTGC

721 AGAAAGCAGT GTGAGCCCGA CTACTACCTG GACGAGGCCG GCCGCTGCAC GGCCTGCGTG

781 AGCTGTTCTC GAGATGACCT TGTGGAGAAG ACGCCATGTG CATGGAACTC CTCCCGCACC

841 TGCGAATGTC GACCTGGCAT GATCTGTGCC ACATCAGCCA CCAACTCCTG TGCCCGCTGT

901 GTCCCCTACC CAATCTGTGC AGCAGAGACG GTCACCAAGC CCCAGGATAT GGCTGAGAAG

961 GACACCACCT TTGAGGCGCC ACCCCTGGGG ACCCAGCCGG ACTGCAACCC CACCCCAGAG

1021 AATGGCGAGG CGCCTGCCAG CACCAGCCCC ACTCAGAGCT TGCTGGTGGA CTCCCAGGCC

1081 AGTAAGACGC TGCCCATCCC AACCAGCGCT CCCGTCGCTC TCTCCTCCAC GGGGAAGCCC

1141 GTTCTGGATG CAGGGCCAGT GCTCTTCTGG GTGATCCTGG TGTTGGTTGT GGTGGTCGGC

1201 TCCAGCGCCT TCCTCCTGTG CCACCGGAGG GCCTGCAGGA AGCGAATTCG GCAGAAGCTC

1261 CACCTGTGCT ACCCGGTCCA GACCTCCCAG CCCAAGCTAG AGCTTGTGGA TTCCAGACCC

1321 AGGAGGAGCT CAACGCAGCT GAGGAGTGGT GCGTCGGTGA CAGAACCCGT CGCGGAAGAG

1381 CGAGGGTTAA TGAGCCAGCC ACTGATGGAG ACCTGCCACA GCGTGGGGGC AGCCTACCTG

1441 GAGAGCCTGC CGCTGCAGGA TGCCAGCCCG GCCGGGGGCC CCTCGTCCCC CAGGGACCTT

1501 CCTGAGCCCC GGGTGTCCAC GGAGCACACC AATAACAAGA TTGAGAAAAT CTACATCATG

1561 AAGGCTGACA CCGTGATCGT GGGGACCGTG AAGGCTGAGC TGCCGGAGGG CCGGGGCCTG

1621 GCGGGGCCAG CAGAGCCCGA GTTGGAGGAG GAGCTGGAGG CGGACCATAC CCCCCACTAC

1681 CCCGAGCAGG AGACAGAACC GCCTCTGGGC AGCTGCAGCG ATGTCATGCT CTCAGTGGAA

1741 GAGGAAGGGA AAGAAGACCC CTTGCCCACA GCTGCCTCTG GAAAGTAG

<210> 2

<211>595

<212> PRT

<213>Artificial sequence

<400> 2

ORIGIN

//

1 MRVLLAALGL LFLGALRAFP QDRPFEDTCH GNPSHYYDKA VRRCCYRCPM GLFPTQQCPQ

61 RPTDCRKQCE PDYYLDEADR CTACVTCSRD DLVEKTPCAW NSSRVCECRP GMFCSTSAVN

121 SCARCFFHSV CPAGMIVKFP GTAQKNTVCE PASPGVSPAC ASPENCKEPS SGTIPQAKPT

181 PVSPATSSAS TMPVRGGTRL AQEAASKLTR APDSPSSVGR PSSDPGLSPT QPCPEGSGDC

241 RKQCEPDYYL DEAGRCTACV SCSRDDLVEK TPCAWNSSRT CECRPGMICA TSATNSCARC

301 VPYPICAAET VTKPQDMAEK DTTFEAPPLG TQPDCNPTPE NGEAPASTSP TQSLLVDSQA

361 SKTLPIPTSA PVALSSTGKP VLDAGPVLFW VILVLVVVVG SSAFLLCHRR ACRKRIRQKL

421 HLCYPVQTSQ PKLELVDSRP RRSSTQLRSG ASVTEPVAEE RGLMSQPLME TCHSVGAAYL

481 ESLPLQDASP AGGPSSPRDL PEPRVSTEHT NNKIEKIYIM KADTVIVGTV KAELPEGRGL

541 AGPAEPELEE ELEADHTPHY PEQETEPPLG SCSDVMLSVE EEGKEDPLPT AASGK

Claims (7)

1. a kind of monoclonal antibody UMAB256 of specific binding CD30 albumen, is CGMCC No.13287's by deposit number Hybridoma cell strain is produced.

2. a kind of hybridoma cell strain, its deposit number is CGMCC No.13287.

3. monoclonal antibody as claimed in claim 1 prepare for detect CD30 albumen immune detection instrument in should With.

4. application according to claim 3, the immune detection instrument is kit, chip or test paper.

5. a kind of immunologic combined detection reagent kit, including the monoclonal antibody described in claim 1.

6. application of the monoclonal antibody as claimed in claim 1 in the kit for tagged tissue cell is prepared.

7. application according to claim 6, the histocyte includes but is not limited to primary cutaneous type and suddenly Strange gold lymthoma.