CN103880960A - Monoclonal antibody for secreting anti-CD10 molecule and application thereof - Google Patents

Abstract

The invention relates to a preparation method of a monoclonal antibody capable of specifically recognizing human leukocyte differentiation antigens 10 (CD10) and aims to provide an application method for using the antibody in immunologic diagnosis of various tumors. A CD10 molecule is also known as a common acute lymphocytic leukemia antigen (CALLA), and is an associated factor participating in tumor-mesenchyme interaction; the protein has the enzymatic activity of neutral endopeptidase (NEP) or enkephalinase A; the antigen for preparing the antibody is a segment of optimized recombined protein which is expressed in escherichia coli and has immunogenicity activity; the finally obtained antibody belongs to IgG2a subtype, and sequences for coding a variable region of the antibody are obtained through a gene cloning way; an immunohistochemical detection on various of tumor tissues shows that the antibody can be used for well identifying occurrence of the tumors and can be applied to the immunologic diagnosis of the tumors.

Description

A kind of monoclonal antibody and application thereof of secreting anti-CD10 molecule

Technical field

The present invention relates to a kind of hybridoma cell line that can identify the mono-clonal molecule of people CD10 protein molecule and can secrete this antibody.Particularly, the invention provides a kind of monoclonal antibody of antitumor cell surface antigen CD10 molecule, DNA sequence dna to the aminoacid sequence of this heavy chain of antibody and variable region of light chain and coding variable region is determined, this antibody can be for passing through artificial or automatization mode, detect the expression status of CD10 in cell with immunohistochemical staining (IHC), Enzyme-linked Immunosorbent Assay (ELISA) or immunoblotting (Western Blot) mode, thereby for these tumours are carried out to diagnostic method, belong to field of biological detection.

Background technology

CD10 molecule is a kind of zinc dependency Zinc metalloproteinase (neutral metalloendopeptidase) that is present in cell surface, suppressed by EDTA, phosphamide (Phosphoramidon), be called again neutral endopeptidase (EC3. 4. 24. 11) or enkephalinase.This enzyme is made prothetic group with zine ion, can be hydrolyzed the peptide bond that the alpha-amino group of hydrophobicity nitrogen base acid forms, produce the polypeptide taking phenylalanine, α-amino-isovaleric acid or tyrosine as first residue, the effect substrate of this enzyme comprises enkephalin, bradykinin, angiotensinⅠ/II, neurotensin, pitocin, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 and chenotactic peptide (methionine(Met)-Ile-Phe tripeptides, Fmlp) etc.CD10 molecule is the antigen molecule of universality and acute lymphoblastic leukemia, therefore can be used as acute leukemia and the non-Hodgkin lymphoma conventional diagnosis index of classifying.Research and analyse, CD10 is not only expressed in marrow sexual cell, in non-marrow sexual cell, also there is expression, as mammary gland myoepithelical cell (breast myoepithelial cell), bile canaliculus (bile canaliculi), in inoblast (fibroblasts), particularly, at kidney, the brush border height of gastric epithelial cell is expressed.

Except common and acute leukemia, CD10 is also used to discriminating and the confirmation of other tumours in recent years, as uterus mesenchymoma (Gu Yang, Wang Shuwen, Liu Wen, the existing system evaluation for CD10 differential diagnosis endometrial stromal sarcoma by CD10 antibody at present for subsequent use, modern tumour medical science, 2011, 19(11): 2301-2304), rodent cancer (Yang Xichuan, Yan Heng, leaf celebrating a dance squad, clock white jade, Hao Fei, the meaning of CD10 immunohistochemical staining in rodent cancer and trichoepithelioma differential diagnosis, Chongqing Medical, 2009, 38(2): 142-143), colorectal cancer (Huang Wenbin, Zhou Xiaojun, Chen Jieyu, Meng Kui, Shi Qunli, Ma Henghui, Lu Zhenfeng, the expression of CD10 and clinical meaning thereof in Colorectal Carcinoma, clinical and experimental pathology magazine, 2005, 20 (4): 446-448), cancer of the stomach (Huang Wenbin, Zhou Xiaojun, Meng Kui, Chen Jieyu, Zhang Lihua, the expression of mesenchymal cell CD10 and clinical meaning thereof in stomach organization, diagnosis pathology magazine, 2005,12 (6): 447-449), the expression in primary lung cancer and thyroid carcinoma also has research.Recent findings, in soft tissue sarcoma the expression of CD10 also to some extent rise (Kemal Deniz, Ganime Coban, Turhan Okten.Anti-CD10 (56C6) expression in soft tissue sarcomas. Pathology – Research and Practice. 2012, (208): 281 – 285).CD10 molecule is that a part amount is the glycoprotein of 100kDa left and right, select different antigen to carry out immunity may to prepare the antibody of different binding characteristics, even if the antibody that same antigen obtains also can be identified glycosylation part or the protein portion of antigen, there is the multiple varient causing because of variable shearing or single nucleotide polymorphism in this molecule, finally cause different antibodies different with pattern to expressing the recognition capability of cell antigen simultaneously.In view of the importance of CD10 molecule in cancer pathology research, existing different antibody is for the preparation of at different immunological method CD10 molecules.Application number is that 201180018729.6 patent of invention " for method and the test kit of cancer diagnosis " has been described a kind of one or more antibody by CD13, CD59, CD10, CD26, CD142, CD143 and MHC I and is used alone or in combination, the method of carrying out tumor of prostate diagnosis with flow cytometry or ELISA mode, the CD10 monoclonal antibody that it uses is numbered MCA1556F, is prepared as immunogen by the white corpuscle purification membrane antigen of fresh preparation.Publication number is that the patent of invention " test kit and the application thereof of detection acute B Lymphocytic leukemia related immune phenotype " of CN103018463A also provides with the antibody of CD58, CD10, CD34, CD123, CD38, CD19 and CD45 and combined the method for the somatotype of B-lineage Acute Lymphocyte Leukemia, the CD10 antibody that it uses is numbered HI10a, be particularly suitable for flow cytometry, in diagnosing tumor, use the 56C6 antibody that maximum cell strains is CD10 at present.

Summary of the invention

First object of the present invention is to provide a kind of method of the CD10 of preparation recombinant protein, and this recombinant protein is by the extracellular region fragment of signal peptide, CD10 molecule and form for the histidine-tagged of purifying.

It is good that second object of the present invention is to provide a species specificity, the preparation method of the CD10 monoclonal antibody that avidity is high, this antibody capable specific combination CD10 recombinant antigen and natural antigen.

The 3rd object of the present invention is to provide a kind of using method that this antibody is detected for tumor tissue section's immunohistochemistry.

the technique means that technical solution problem adopts

The present invention is to the CD10 molecule of expressing on common and acute lymphoblastic leukemia medium size lymphocyte film, analyze according to the sequence of announcing, according to the structure on cytolemma, antigenicity, form amino acid whose hydrophilic and hydrophobic and secondary structure, select suitable soluble-expression to there is again good immunogenic region for recombinant expressed, for promoting the soluble-expression of recombinant protein, improve its expression behavior, and be convenient to purifying, the N end of recombinant protein and C end respectively fusion have the PelB signal peptide of intestinal bacteria polygalacturonase and histidine-tagged, through the synthetic rear clone of gene enter in expression plasmid carrier pET-pelB, carry out recombinant expressed, the soluble part of separation and purification expression product carries out after affinity purification as immunogen, immunity Balb/c mouse.Through cytogamy, recombinant C D10 screening and cloning, obtain the positive hybridoma cell system of efficient secrete monoclonal antibody.

Utilize this hybridoma cell line to carry out ascites preparation with mouse, Protein A/G post affinitive layer purification ascites, obtains mouse monoclonal antibody.The subclass of measuring this monoclonal antibody with elisa technique is IgG2 type monoclonal antibody, and affinity costant is 1.92 × 10 ⁹.The immunity marking (Western blotting) experiment shows the CD10 albumen of this antibody capable specific recognition restructuring and expresses the tumour cell of CD10 molecule.

Particularly, the present invention relates to the following aspects:

A kind of monoclonal antibody is 60656-7H5 secretion by mouse hybridoma cell.

Described monoclonal antibody, the antigen that its immune mouse is used is to have the nucleotide sequence coded recombinant C D10 antigen protein obtaining via Recombinant protein expression of SEQ ID No.1 in sequence table.

Described monoclonal antibody, its heavy chain and light chain variable region amino acid sequence are that the DNA sequence dna shown in SEQ ID NO.2 and SEQ ID NO.3 is coded.

Described monoclonal antibody, its heavy chain and light chain variable region amino acid sequence are respectively the aminoacid sequence shown in SEQ ID NO.4 and SEQ ID NO.5.

Described monoclonal antibody, it is mouse IgG 2a hypotype monoclonal antibody.

Described monoclonal antibody, it can identify the CD10 molecule of restructuring and tumor cell surface.

Described monoclonal antibody, it detects the CD10 expression of tumour and normal tissue cell for immunohistochemical method, immunoblotting and enzyme connection adsorption measurement.

Described monoclonal antibody, the application in immunohistochemical methods pathological diagnosis agent.

CD10 positive cell source tumour is precursor B lymphoblastic leukemia, follicular lymphoma, Burkitt lymphoma, angioimmunoblastic T cell lymphoma, endometrial stromal sarcoma, liver cancer, clear cell carcinoma of kidney

Described recombinant C D10 antigen protein, this recombinant protein is by impelling the signal peptide sequence of secreting, expressing, preferably have antigenic CD10 fragment and form for the albumen label of recombinant protein.

Described recombinant C D10 antigen protein, signal peptide sequence is intestinal bacteria polygalacturonase signal peptide sequence, CD10 fragment comprises the 411st fragment to the 750th amino acids, is made up of 5 to 7 Histidines for the label of purifying.

Described monoclonal antibody, is nucleotide sequence coded by shown in SEQ ID No:1, comprising the 411st fragment to the 750th amino acids sequence of CD10 molecule at expression in escherichia coli, and through nickel post affinity purification.

The heavy chain and the variable region of light chain that obtain the secreted antibody of hybridoma cell strain are coded by the DNA sequence dna shown in SEQ ID2 and SEQ ID3, further, the aminoacid sequence of heavy chain and variable region of light chain is as shown in SEQ ID4 and SEQ ID5, and this antibody is a kind of mouse IgG 2a hypotype monoclonal antibody.

Described monoclonal antibody, can be independently or with other antibody applied in any combination in immunoblotting or the enzyme-linked immune detection method of tumour cell prepared product, tumor tissue section.

advantage of the present invention and beneficial effect

(1) monoclonal antibody that hybridoma (60656-7H5) secretion that the present invention obtains produces, can identify recombinant protein c D10 molecule and express the lymphocyte of CD10, thering is the kinds of tumors tissue such as lymphoma, lobular carcinoma of breast, kidney clear cell tumor, breast ductal cancer, primary hepatocarcinoma and metastatic liver cancer that detects high expression level CD10 albumen.

(2) hybridoma (60656-7H5) that the present invention obtains is a kind of IgG2 antibody-like, with the extremely strong specificity of being combined with of CD10 albumen and susceptibility.

(3) monoclonal antibody that the hybridoma (60656-7H5) that the present invention obtains produces can be applicable to immunohistochemistry (IHC), the immune marking (Western blotting), indirect ELISA, antibody chip and detection and the examination, specificity and highly sensitive such as prepares.In the contrast experiment of the commercialization 56C5 cell strain generally to use at present, amount to more than 800 routine samples and comprised 342 routine lymphomas, 7 routine lobulars carcinoma of breast, 109 routine kidney clear cell tumors, 98 routine breast ductal cancers, 302 routine primary hepatocarcinoma and 23 routine hepatic metastases cancer samples, antibody prepared by cell strain of the present invention is respectively lymphoma 21.1% to the susceptibility of various diagnosing tumors, lobular carcinoma of breast 28.6%, kidney clear cell tumor 82.6%, breast ductal cancer 22.4%, primary hepatocarcinoma 37.4% and hepatic metastases cancer 26.1%, and the susceptibility of control antibodies is respectively 15.2%, 28.6%, 82.6%, 22.4%, 37.4% and 26.1%.The resolving ability of removing lobular carcinoma of breast, two kinds of antibody of breast ductal cancer and hepatic metastases cancer sample is similar, but staining power is higher than contrast.The susceptibility of other three kinds of tumours all exceeds 9%-38% than control antibodies 56C6, and therefore, CD10 monoclonal antibody of the present invention has wider range of application, can significantly improve the accuracy of diagnosis.

Brief description of the drawings

fig. 1: the CD10 recombinant antigen protein of purifying

1,2 is the result of expression product 300mM imidazoles wash-out, and Marker is molecular weight protein, and the size of expression product is about 41kDa, uses 12% SDS-PAGE gel.Purified recombinant C D10 albumen is solvable state, can partly protect native conformation, and its purity is in 90% left and right, and concentration is higher than 1.5mg/ml.

fig. 2: the evident characteristics of 60656-7H5 antibody and practical application effect

1 is molecular weight marker.60541 S-4 monoclonal antibodies of application of purified can detect specifically CD10 recombinant protein in western blot hybridization.

fig. 3: the result with CD10 monoclonal antibody to immunohistochemistry chip detection kinds of tumors tissue, existing attached is the picture of renal clear cell carcinoma.With 60656-7H5 monoclonal antibody, renal clear cell carcinoma is carried out to staining analysis, titre is 1:4000, and a left side is control antibodies 56C6, and right is CD10 antibody of the present invention, and the staining power of this antibody is higher than contrast, and specificity is consistent.

Embodiment

Below in conjunction with chart and the concrete mode of implementing, the present invention is further elaborated, so that those skilled in the art can more clearly learn technical scheme of the present invention, not limitation of the present invention.

the preparation of embodiment 1 recombinant C D10 protein fragments

One, gene clone

CD10 mono-clonal is according to the nucleotide sequence of NM_000902.3 in protein sequence that in Uniprot database, accession number is P08473.2 and corresponding Genbank, design specificity upstream primer CD10-F (5 '-CCTAAGCTTGGCAGTTGTGCAAACTATGTCAATGGGAATATGG-3 ') and downstream primer CD10-R (5 '-CTTGGGATCCCCAAACCCGGCACTTCTTTTCTGG-3 '), in the reverse transcription product from lymphocyte, amplification comprises and encodes the 411st to the 750th amino acid whose gene fragment.In the process of PCR, add respectively at gene 5 ' and 3 ' end hindIII and bamhI restriction enzyme site.PCR product through agarose gel electrophoresis separate after reclaim, respectively to reclaim antigen-4 fusion protein gene and carry out for the plasmid vector pET-pelB expressing hindIII and bamhI enzyme is cut, and electrophoresis reclaims again, connects with T4 DNA ligase.Connect product and transform competent escherichia coli cell BL21, the clone's inoculation on picking flat board, extracts plasmid DNA, carries out PCR qualification.PCR shows that the clone of the antigen-4 fusion protein gene positive carries out sequencing analysis, and sequence right-on clone use.

Two, protein expression and purifying

The bacterium that spends the night of single bacterium colony being cultivated in the ratio of 1:100 is forwarded to 100ml LB substratum, and adding final concentration is the kantlex of 50 μ g/ml, and 37 DEG C of shaking culture are to OD ₆₀₀be 0.6 ~ 0.8.Add the IPTG of 0.1 mol/L, 8h is cultivated in 25 DEG C of concussions, ultrasonication after receipts bacterium.This recombinant protein, with histidine-tagged, uses nickel post to carry out the affinity purification of protein.Carry out after wash-out with the imidazoles solution of different concns, each component and stream are worn to loading respectively and carry out SDS-PAGE separation detection, Fig. 1 merges the expression and the purification result that have pelB signal peptide and histidine-tagged recombinant C D10 albumen.The purity of recombinant C D10 albumen is more than 90%, and concentration is about 1-1.5 mg/mL, can meet the requirement of immune animal and antibody screening and qualification.

the foundation of embodiment 2 hybridoma cell lines

One, immunity

By Freund's complete adjuvant for polypeptide crosslinked in embodiment 1 (Sigma company) emulsification, immune 4-6 week female Balb/c mouse in age (purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.), every mouse of abdominal part hypodermic 6 points, dosage is 60 μ g/.Once, antigen uses the non-Freund's complete adjuvant of Fu Shi (Sigma company) emulsification to every 14 days booster immunizations, and dosage is 30 μ g/.Within after the 3rd booster immunization 7 days, detect in mice serum and resist immunogenic many anti-tiring with indirect ELISA (wavelength 450nm), the highest mouse of tiring is impacted immunity with tail vein injection, and antigen mixes with physiological saline, and dosage is 50 μ g/.

Two, cytogamy

Aseptic preparation immunity mouse boosting cell suspension up to standard, with murine myeloma cell sp2/0(ATCC) mix centrifugal 1500 rpm, 5min with 5:1 ratio.After abandoning supernatant, centrifuge tube is put into 37 DEG C of water-baths, slowly adds the PEG1500(Roche company of 1ml in 1 minute), and stir cell.In warm water, leave standstill after 1min, add the IMDM(Sigma company of 10ml serum-free), mix centrifugal 1000 rpm, 5min.Abandon after supernatant, add careful cell is blown and beaten of 10ml serum (PAA company), and add 5ml mixing 10xHAT(Sigma company) thymocyte, mix.The semisolid medium that adds again 25ml to contain 2.1% Nitrocellulose (Sigma company) fully mixes, and then pours into uniformly in 20 Tissue Culture Dishs.Tissue Culture Dish is put in wet box, put into 37 DEG C of 5%CO ₂in incubator, cultivate.

Three, choose clone

Merging latter 7 days clone cells, to roll into a ball big or small density moderate, under anatomical lens, draws round, real, large cloning cluster and squeeze in 96 well culture plates that are ready in advance substratum, puts into 37 DEG C of 5%CO ₂in incubator, cultivate.

Four, ELISA screening positive hybridoma cell

After 3 days, cell concentration accounts for greatly floorage 2/3, gets 100 μ l supernatant immunogens and synthetic polypeptide and carries out respectively ELISA screening.Positive colony changes liquid completely, adds 200 μ l containing feeder cell and 1%HT(Sigma company) perfect medium.Carry out two days later ELISA screening for the second time, positive colony proceeds to the 24 orifice plates cultivations that are ready in advance substratum (containing feeder cell and HT).After five days, get 100 μ l supernatants and carry out ELISA screening for the third time, positive colony successively proceeds to 6 orifice plates and Tissue Culture Flask enlarged culturing frozen.

embodiment 3 ascites induce legal system for monoclonal antibody

one, ascites preparation

Logarithmic phase cell washs with serum free medium and has hanged, counting 5 × 10 ⁵, 1ml.The cell abdominal injection suspending is used the mouse of paraffin oil sensitization in advance.After 7 days, start to collect ascites.The ascites of taking out is in 4 DEG C of centrifugal 4000 rpm, 10min.Ascites in the middle of careful sucking-off is collected in centrifuge tube, 4 DEG C or-20 DEG C of preservations.

two, the purifying of monoclonal antibody

With HiTrap rProtein A FF(GE company) affinity chromatography by specification antibody purification from ascites.SDS-PAGE glue qualification purity, Bradford method is measured concentration.The antibody of purifying is stored in-20 DEG C.

embodiment 4 monoclonal antibody CHARACTERISTICS IDENTIFICATION

one, subgroup identification

Use 100mM PBS(pH7.4) to 0.5 μ g/ml, every hole adds 100 μ l to the coated sheep anti-mouse igg (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge) of dilution, 4 DEG C, spends the night.Be emptied liquid, with the PBS(PBS-T containing 0.05% Tween) to wash 3 times, every hole adds 200 μ l confining liquids (containing the PBS of 2%BSA and 3% sucrose), hatches 1h for 37 DEG C.Be emptied liquid, with PBS-T cleaning 3 times.Every hole adds 0.1ml hybridoma supernatant, hatches 1h for 37 DEG C.Being emptied liquid PBS-T cleans 3 times.With sheep anti mouse (κ, the λ) antibody of confining liquid 1:1000 dilution HRP mark or sheep anti mouse (IgM, the IgG1 of 1:2000 dilution HRP mark, IgG2a, IgG2b, IgG3, IgA) antibody (Southern Biotech company) the every hole of 0.1ml adds respectively in suitable hole, hatches 1h for 37 DEG C.Be emptied liquid, with PBS-T cleaning 3 times.Every hole adds 50 μ l containing 0.15% ABTS(Southern Biotech company) and 0.03% H ₂o ₂citrate buffer solution (PH4.0) carry out color reaction, measure the OD value under 405nm wavelength in 10-20min.Result demonstration, monoclonal antibody of the present invention is IgG1 type mouse resource monoclonal antibody.

two, affinity costant is measured

CD10 albumen, coated concentration is 2 μ g/ml, 100 μ l/ holes, 4 DEG C of coated spending the night, PBS-T washes 3 times.Every hole adds 37 DEG C of sealing 2h of 200 μ l confining liquid, and PBS-T washes 3 times.The monoclonal antibody of purifying in embodiment 4, starts 2 times of gradient dilutions from 1:200, and the contrast that blanks of last 1 hole, hatches 1h for 37 DEG C, and PBS-T washes 3 times.The anti-1:20000 dilution of sheep anti mouse two of HRP mark, every hole 100 μ l, hatch 1h for 37 DEG C, and PBS-T washes 3 times.Every hole adds 100 μ l containing 0.1% TMB(Sigma company) and 0.03% H ₂o ₂citric acid-phosphoric acid buffer colour developing 10min, add 50 μ l 0.5M sulphuric acid soln termination reactions.Measure the light absorption value of wavelength 450nm by microplate reader.Draw the curve of the corresponding antibody dilution multiple of OD value, find out >=extension rate A corresponding to 1/2 " platform OD value ".Utilizing following formula to calculate affinity costant is 1.92 × 10 ⁹.

Affinity costant

three, monoclonal antibody atopic and effect

Select the CD10 albumen of restructuring, detect the identification specificity of monoclonal antibody of the present invention by the method for immunoblotting, immunoblot experiment process is as follows: every kind of about 5-10ng of albumen loading, carries out 12% polyacrylamide gel electrophoresis.Gel protein band is transferred to according to a conventional method to (Millipore company) on pvdf membrane in Bio-Rad electrotransfer system.Film is placed in containing 4 DEG C of the TBS-T confining liquids of 5% skim-milk and is spent the night.Add monoclonal antibody 60541 S-4(1:1000 dilutions) 4 DEG C of overnight incubation.Wash after film with TBS-T, add the sheep anti mouse two anti-(Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge) of 1:5000 dilution, incubated at room 1 hour.TBST washes film again, adds the super quick nitrite ion of ECL (Beijing Puli's lema gene Technology Co., Ltd.), carries out the collection of chemoluminescence view data with ChemiDoc MP multicolor fluorescence imaging system (Bio-Rad).

the variable region sequences of embodiment 4 antibody is measured

cultivate fresh hybridoma, get supernatant and carry out antigenic binding property checking, confirm that the cell strain for cloning can be secreted the antibody needing really, after having confirmed, centrifugal collection 10 ⁶above hybridoma.Trizol method is extracted the total RNA of hybridoma, get the total RNA of 9 μ L, add 2.5 μ L oligo (dT) 12 – 18 primer (10 mM), and 5 μ L dNTPs, mix, 5 minutes postposition of 70 DEG C of insulations 5 minutes on ice, or carry out sex change operation according to the reversed transcriptive enzyme using.Add subsequently 5 μ L RT buffer (5X), 2.5 μ L DTT (0.1 M) and 1 μ L reversed transcriptive enzyme, 42 DEG C are reacted 1 hour.Hatch 15 minutes with termination reaction for 70 DEG C, the cDNA of acquisition is kept at-20 DEG C.The the first chain cDNA obtaining is carried out to pcr amplification, in 50 μ L reaction systems, add each 25 pmol of primer, mouse monoclonal antibody primer sequence design and synthetic in " recombinant antibodies " (Science Press publishes for 2005) book that the sequence of primer doubly puts forth energy to edit according to Shen.All the other dNTPs and damping fluid, all according to being routinely added to, finally add cDNA template to add 1 μ L and 1U warm start Taq archaeal dna polymerase.Pcr amplification program is set, be generally 94 DEG C 40 seconds, 52 DEG C 40 seconds, 72 DEG C 40 seconds, carry out 20 to 25 respectively circulation, last 72 DEG C extend 3 minutes, product can be placed in 4 DEG C of electrophoresis for subsequent use or direct.Get 20 μ L PCR products and carry out electrophoretic analysis, on 1.5% sepharose, separate, the length of light chain (κ light chain) is between 320-340, and the length of heavy chain is between 340-370, while having the special product in this region, cut glue and reclaim, be cloned into T carrier or expression vector order-checking.

embodiment 5. organization chip dyeing and qualifications

one. chip preparation process

To the advanced row HE of each sample section statining, to determine tumor locus.Tumour target site is drawn a circle, preparation punching.Make when blank acceptor wax block, plastic processing frame is placed on mould, pour the paraffin (fusing point is at 56～58 DEG C) melting into mould, mould is put into-20 DEG C of refrigerator 6 min after being cooled to room temperature, wax stone is taken out from mould.On tissue sample machine, select the sample pin of 1mm diameter to punch on acceptor wax block, hole depth 3～4 mm, punch to gather at the mark position of wax stone with the perforating needle of another diameter 1mm and organize core, the shallow 0.1mm of the hole depth left and right of its Length Ratio acceptor wax block.By organizing in the direct insertion of core or the emptying aperture with the careful gripping insertion of tweezers acceptor wax block of collecting.So repeatedly until complete the preparation of whole sample spot.Finally with slide glass by a organized way core by flat, make organization chip wax stone flat-satin.The organization chip wax stone of making is put into wax stone again and make mould, put into 60 DEG C of baking box 15min, make to organize the wax of cured of core and acceptor to combine together, then from baking box, take out gently mould, allow and partly melt paraffin cooling approximately 30 min at ambient temperature of state, after putting into-20 DEG C of refrigerator freezing 6 min, organization chip wax stone is taken out from mould, cutting into slices or putting into 4 DEG C of refrigerators saves backup again.After repairing sheet, carry out serial section, thickness is decided to be 4 μ m, serial section is floated in cold water, allow it naturally launch, again the section separating is transferred in the warm water of 45 DEG C and opened up sheet 30 seconds, use the slide glass mount section of processing through 2 % APES acetone solutions, the organization chip of making is put into the roasting sheet of 60 DEG C of baking boxs 2 hours, taking-up room temperature is cooling, puts into-4 DEG C of Refrigerator stores.

two. IHC dyeing and analysis

Conventional dimethylbenzene dewaxing 3 times, each 6 minutes, aquation in 100%, 100%, 95%, 85% gradient ethanol, each 3 minutes, last tap water rinsed.Carry out antigen retrieval, then section is put into wet box, PBS rinses 3 × 3 minutes.Drip 3%H ₂o ₂hatch 10 minutes, PBS rinses 3 × 3 minutes.Get rid of PBS, drip confining liquid (1% BSA solution) incubated at room 10 minutes.Dry section, primary antibodie (diluting first the Dilution ratio of carrying out designerantibodies according to the antibody concentration) room temperature (25 DEG C) that drips suitable proportion dilution is hatched 1 hour, PBS rinses 3 × 3 minutes, drip two anti-incubated at room 20-30 minute, PBS rinses 3 × 3 minutes, get rid of PBS, with the DAB nitrite ion colour developing 3-10 minute of fresh configuration.Hematorylin is redyed 25 seconds, and PBS returns blue 30 seconds.According to 85%(3 minute)-95%(3 minute)-100%(3 minute)-100%(3 minute) alcohol gradient dewater successively, transparent 3 minutes of last dimethylbenzene, neutral gum mounting.

three. data statistics

Painted situation according to each antibody at sample point, adds up the tinctorial yield (painted sample number/total sample number) of each index, sees the following form.

SEQUENCE LISTING

<110> Fuzhou Maixin biotechnology Development Co., Ltd

Monoclonal antibody and the application thereof of the <120> anti-CD10 molecule of secretion

<130> 7

<160> 7

<170> PatentIn version 3.3

<210> 1

<211> 1032

<212> DNA

<213> nucleotide sequence

<400> 1

ggcagttgtg caaactatgt caatgggaat atggaaaatg ctgtggggag gctttatgtg 60

gaagcagcat ttgctggaga gagtaaacat gtggtcgagg atttgattgc acagatccga 120

gaagttttta ttcagacttt agatgacctc acttggatgg atgccgagac aaaaaagaga 180

gctgaagaaa aggccttagc aattaaagaa aggatcggct atcctgatga cattgtttca 240

aatgataaca aactgaataa tgagtacctc gagttgaact acaaagaaga tgaatacttc 300

gagaacataa ttcaaaattt gaaattcagc caaagtaaac aactgaagaa gctccgagaa 360

aaggtggaca aagatgagtg gataagtgga gcagctgtag tcaatgcatt ttactcttca 420

ggaagaaatc agatagtctt cccagccggc attctgcagc cccccttctt tagtgcccag 480

cagtccaact cattgaacta tgggggcatc ggcatggtca taggacacga aatcacccat 540

ggcttcgatg acaatggcag aaactttaac aaagatggag acctcgttga ctggtggact 600

caacagtctg caagtaactt taaggagcaa tcccagtgca tggtgtatca gtatggaaac 660

ttttcctggg acctggcagg tggacagcac cttaatggaa ttaatacact gggagaaaac 720

attgctgata atggaggtct tggtcaagca tacagagcct atcagaatta tattaaaaag 780

aatggcgaag aaaaattact tcctggactt gacctaaatc acaaacaact atttttcttg 840

aactttgcac aggtgtggtg tggaacctat aggccagagt atgcggttaa ctccattaaa 900

acagatgtgc acagtccagg caatttcagg attattggga ctttgcagaa ctctgcagag 960

ttttcagaag cctttcactg ccgcaagaat tcatacatga atccagaaaa gaagtgccgg 1020

gtttggggat cc 1032

<210> 2

<211> 380

<212> DNA

<213> DNA sequence dna

<400> 2

caggtgcagc tgcaggagtc tggggctgaa ctggtgaagc ctgggacttc agtgaagttg 60

tcctgcaagg cttctagaaa attcaccggt tactatttgt attgggtgaa gcagaggcct 120

ggacaaggcc ctgagtggat tggagagatt gtcaataaaa gcaatagagg tactaacctc 180

aatgagaagt tcaagagcaa ggccacactg actgtagaca aatcctccag tacagcatac 240

atgcaactca acagcctgac atctgaggac tctgcggtct attactgtac aagactgggc 300

ttctggggca gacaaggcaa aaccgtgcct ctctcctctg ccaaaacgac acccaagctt 360

gtctatccac tggcccctgg 380

<210> 3

<211> 383

<212> DNA

<213> DNA sequence dna

<400> 3

ggtgatatct tgctgaccca atctccactc tccctgactg tgacagcagg agagaaggtc 60

actatgagct gcaagtccag tagtgtcata aatagtacta atcaatcaaa ttacttgacc 120

tggtaccagc agaaaccagg gcagcctcct aaaatgttga tctactgggc aaggcgatcc 180

actagggaat ccggggtccc tgatcgcttc acaggcagtg gatctggaac agatttcact 240

ctcaccatca gcagtgtgca ggctgaagac ctggcagttt attactgtcc taatgataaa 300

ggaagtggcc ctctcacgtt cggtgctggg accaagctgg agctgaaacg ggctgatgct 360

gcaccaactg gatccatctt ccc 383

<210> 4

<211> 126

<212> PRT

<213> aminoacid sequence

<400> 4

Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Arg Lys Phe Thr Gly Tyr Tyr

20 25 30

Leu Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Pro Glu Trp Ile Gly

35 40 45

Glu Ile Val Asn Lys Ser Asn Arg Gly Thr Asn Leu Asn Glu Lys Phe

50 55 60

Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Leu Gly Phe Trp Gly Arg Gln Gly Lys Thr Val Pro Leu Ser

100 105 110

Ser Ala Lys Thr Thr Pro Lys Leu Val Tyr Pro Leu Ala Pro

115 120 125

<210> 5

<211> 127

<212> PRT

<213> aminoacid sequence

<400> 5

Gly Asp Ile Leu Leu Thr Gln Ser Pro Leu Ser Leu Thr Val Thr Ala

1 5 10 15

Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Ser Val Ile Asn Ser

20 25 30

Thr Asn Gln Ser Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Met Leu Ile Tyr Trp Ala Arg Arg Ser Thr Arg Glu Ser

50 55 60

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr

65 70 75 80

Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys

85 90 95

Pro Asn Asp Lys Gly Ser Gly Pro Leu Thr Phe Gly Ala Gly Thr Lys

100 105 110

Leu Glu Leu Lys Arg Ala Asp Ala Ala Pro Thr Gly Ser Ile Phe

115 120 125

<210> 6

<211> 43

<212> DNA

<213> artificial sequence

<400> 6

cctaagcttg gcagttgtgc aaactatgtc aatgggaata tgg 43

<210> 7

<211> 34

<212> DNA

<213> artificial sequence

<400> 7

cttgggatcc ccaaacccgg cacttctttt ctgg 34

Claims (10)

1. a monoclonal antibody, is characterized in that by mouse hybridoma cell be 60656-7H5 secretion.

2. monoclonal antibody claimed in claim 1, is characterized in that the antigen that its immune mouse is used is to have the nucleotide sequence coded recombinant C D10 antigen protein obtaining via Recombinant protein expression of SEQ ID No.1 in sequence table.

3. monoclonal antibody claimed in claim 1, is characterized in that its heavy chain and light chain variable region amino acid sequence are that the DNA sequence dna shown in SEQ ID NO.2 and SEQ ID NO.3 is coded.

4. monoclonal antibody claimed in claim 1, is characterized in that its heavy chain and light chain variable region amino acid sequence are respectively the aminoacid sequence shown in SEQ ID NO.4 and SEQ ID NO.5.

5. monoclonal antibody claimed in claim 1, is characterized in that it is mouse IgG 2a hypotype monoclonal antibody.

6. monoclonal antibody claimed in claim 1, is characterized in that its identification is recombinated and the CD10 molecule of tumor cell surface.

7. monoclonal antibody claimed in claim 1, is characterized in that it detects the CD10 expression of tumour and normal tissue cell for immunohistochemical method, immunoblotting and enzyme connection adsorption measurement.

8. monoclonal antibody claimed in claim 1, is characterized in that, the application in immunohistochemical methods pathological diagnosis agent.

9. according to recombinant antigen claimed in claim 2, it is characterized in that, this recombinant protein is by impelling the signal peptide sequence of secreting, expressing, preferably have antigenic CD10 fragment and form for the albumen label of recombinant protein.

10. according to recombinant antigen claimed in claim 2, it is characterized in that signal peptide sequence is intestinal bacteria polygalacturonase signal peptide sequence, CD10 fragment comprises the 411st fragment to the 750th amino acids, is made up of 5 to 7 Histidines for the label of purifying.

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