CN106188292A - Anti-CD4 protein monoclonal antibody and application thereof - Google Patents
Anti-CD4 protein monoclonal antibody and application thereof Download PDFInfo
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- CN106188292A CN106188292A CN201510224780.2A CN201510224780A CN106188292A CN 106188292 A CN106188292 A CN 106188292A CN 201510224780 A CN201510224780 A CN 201510224780A CN 106188292 A CN106188292 A CN 106188292A
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Abstract
The present invention relates to biological technical field, disclose a kind of hybridoma cell strain (deposit number is CGMCC No.9576), and monoclonal antibody UMAB64 that thus hybridoma cell strain produces.The invention still further relates to monoclonal antibody UMAB64 and prepare the application in the immune detection instrument detecting CD4 albumen, immunohistochemical kit containing monoclonal antibody UMAB64, and the application that monoclonal antibody UMAB64 is in preparation is used for the test kit of marked tumor.Monoclonal antibody of the present invention can be combined with CD4 protein-specific, and with other albumen no cross reactions intracellular, significantly improve CD4 protein immunization detection specificity, accuracy and reliability.
Description
Technical field
The present invention relates to biological technical field, being specifically related to can the monoclonal antibody of specific bond CD4 albumen
UMAB64, produce the cell strain of described monoclonal antibody and apply the diagnostic method of this antibody and use
On the way.
Background technology
CD4 albumen is a kind of single transmembrane glycoprotein, the relative molecular weight of albumen about 55kD.Main expression
In auxiliary/induced T cell (CD4+) surface, such as: Th1/Th2, suppression or cytotoxic T are thin
Born of the same parents' subgroup.It is thin that CD4 is commonly applied to some t cell lymphoma, histocytic lymphoma, Langerhans
The detection of the diseases such as born of the same parents' histiocytosis and typing.
The most generally by CD4 in immunohistochemistry (IHC) Pathological experiment detection tumor cell
The expression situation of albumen.The monoclonal antibody that core is specific binding CD4 albumen of IHC experiment, its property
The quality of energy directly decides sensitivity and the specificity of whole detection.Therefore, a kind of combination spy is developed
The higher monoclonal antibody for CD4 albumen of the opposite sex has great importance.
Summary of the invention
In view of this, it is an object of the invention to provide the list of the higher CD4 albumen of a kind of binding specificity
Clonal antibody, and in preparation application in the immune detection instrument detecting CD4 albumen.
The invention provides a kind of hybridoma cell strain, be preserved in Chinese microorganism strain preservation conservator
Meeting common micro-organisms center (referred to as CGMCC), preservation date is on August 22nd, 2014, preservation
Numbered CGMCC No.9576.
Present invention also offers monoclonal antibody UMAB64 of a kind of specific binding CD4 albumen, by upper
State hybridoma cell strain to produce.
The preparation method of monoclonal antibody of the present invention is as follows:
(1) structure of recombinant expression carrier: according to CD4ORF nucleotide sequence (CD4ORF nucleoside
Acid sequence as shown in SEQ ID NO.1,1374bp；CD4 aminoacid sequence is as shown in SEQ ID NO.2)
Design primer carries out PCR amplification, and gene both sides introduce restriction endonuclease sites SgfI and MluI respectively,
Insert expression vector pCMV6-Entry, build CD4 recombinant expression plasmid；
(2) expression and purification of CD4 recombiant protein: CD4 recombinant expression plasmid is converted HEK293T
Cell, cracks centrifuging and taking supernatant, DDK affinity chromatograph column purification, it is thus achieved that the CD4 recombiant protein of purification；
(3) screening of monoclonal antibody and preparation: use the CD4 recombiant protein immunity of above-mentioned purification
BALB/c mouse, takes Mouse spleen cells and merges with SP2/0 cell, and limiting dilution assay obtains Dan Ke
Grand, ELISA method screening positive hybridoma cell, it is thus achieved that the hybridoma of anti-CD4 specific antibody can be secreted
Cell strain, named UMAB64, hypotype is accredited as IgG2b；Antibody is prepared by serum-free medium,
CD4 monoclonal antibody UMAB64 is obtained by affinity chromatograph column purification.Respectively by Western Blot,
Immunohistochemical experiment, immunofluorescence experiment verify sensitivity and the specificity of this monoclonal antibody.
Use OriGene high-density protein chip that the specificity of said monoclonal antibody is tested further
Card: comprise 10,000 HEK293T cell protein process LAN cracking on OriGene high-density protein chip
Thing, every kind of protein lysate has the repetition of two copies on chip.Protein lysate by trace at nitre
On acid cellulose film.The location of each clock protein lysate can be accurately positioned by Excel file.
On protein chip, albumen is divided into 40 sub-matrixes, and each sub-matrix has some references, by referring to, can
With the content of albumen on quantitative each chip point, monitor the repeatability of each immunoreation data, Yi Jiding
The direction of position positive signal.
Described monoclonal antibody UMAB64 and said chip are hybridized and determine positive signal position by the present invention
Point, result shows this stated clearly monoclonal antibody UMAB64 specific binding CD4 albumen, and and its
His albumen no cross reaction.
Present invention also offers monoclonal antibody UMAB64 in preparation for detecting the immunity inspection of CD4 albumen
Application in survey instrument.
Specifically, described immune detection instrument is test kit, chip or reagent paper.
In the particular embodiment, the invention provides a kind of immunologic combined detection reagent kit, including above-mentioned
Monoclonal antibody UMAB64, can detect the expression situation of CD4 in histiocyte.
Present invention also offers said monoclonal antibody preparation answering in the test kit of marked tumor
With.
Wherein said tumor specifically refers to the propagation of tumor cell and the closely-related tumor of expression of CD4,
Include but not limited to that t cell lymphoma, histocytic lymphoma, Langerhans cell tissue cell increase
Raw.
Compared with prior art, (deposit number is CGMCC to the invention provides a kind of hybridoma cell strain
No.9576), monoclonal antibody UMAB64 that and thus hybridoma cell strain produces.The present invention is also
Provide monoclonal antibody UMAB64 preparation answering in the immune detection instrument detecting CD4 albumen
With, the immunohistochemical kit containing monoclonal antibody UMAB64, and monoclonal antibody UMAB64
In preparation application in the test kit of marked tumor.Monoclonal antibody of the present invention can be with CD4 egg
The most specific binding, and with other albumen no cross reactions intracellular, significantly improve CD4 protein immunization
Specificity, accuracy and the reliability of detection, CD4 protein expression level in true reflection tumor cell,
Can be applicable to the diseases such as T cell leukemia, t cell lymphoma, Langerhans cell tissue hyperplasia
Auxiliary diagnosis and typing.
Preservation information
Classification And Nomenclature for the hybridoma cell strain UMAB64 of preservation is: CD4 mouse monoclonal antibody
Hybridoma cell strain；
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: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences's microorganism is ground
Study carefully institute；
Preservation date: on August 22nd, 2014；
Deposit number: CGMCC No.9576.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to reality
Execute the required accompanying drawing used in example or description of the prior art to be briefly described.
Fig. 1 shows embodiment 1 cloning site design such as figure, and wherein shading part is ORF district；
Fig. 2 shows embodiment 2CD4 albumen Western blot testing result figure, detects CD4 with anti-DDK
Albumen expression in HEK293T cell, wherein swimming lane L is the HEK293T cell of transfection empty carrier
Lysate be the testing result of antigen, swimming lane R be the HEK293T cell of transfection pCMV6-CD4 plasmid
The testing result of lysate antigen；
Fig. 3 shows embodiment 2CD4 protein SDS-PAGE result figure；
Fig. 4 shows that embodiment 3 identifies the Western blot of endogenous CD4 albumen with monoclonal antibody UMAB64
Testing result figure, wherein swimming lane HL-60 and swimming lane SW-620 is respectively (with monoclonal antibody
UMAB64 detects the expression in HL-60 Yu SW-620 cell of the CD4 albumen；
Fig. 5 shows that (one resists for CD4 monoclonal embodiment 4 people's Normal Lymph Nodes histogenic immunity group result figure
Antibody UMAB64)；
Fig. 6 shows that (one resists for CD4 monoclonal antibody embodiment 4 human lymphoma histogenic immunity group result figure
UMAB64)；
Fig. 7 shows that (one resists for CD4 monoclonal embodiment 4 people normal tonsil ImmunohistochemistryResults Results figure
Antibody UMAB64)；
Fig. 8 shows that (one resists for CD4 monoclonal antibody embodiment 5OriGene protein chip qualification result figure
UMAB64,1:100；Two resist for DyLight649-conjugated AffiniPure Fragment
Goat-anti-Mouse IgG, 1:400).
Detailed description of the invention
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Describe, it is clear that described embodiment is only a part of embodiment of the present invention rather than all wholely
Embodiment.Based on the embodiment in the present invention, those of ordinary skill in the art are not making creativeness
The every other embodiment obtained under work premise, broadly falls into the scope of protection of the invention.
Embodiment 1, the structure of CD4 recombinant expression plasmid
To obtain from American Type Culture collection warehousing (American type culture collection, ATCC)
Plasmid BC206453 (containing CD4ORF 1374bp) be template, design two primers and introduce respectively
Restriction enzyme site SgfI and MluI, is cloned into expression vector pCMV6-Entry, sets up CD4 recombinant expressed
Plasmid.Cloning site designs as shown in Figure 1.
Embodiment 2, the expression and purification of CD4 recombiant protein
1, transfection HEK293T cell: HEK293T cell reaches with 1:3 and continues in culture dish to cultivate；
Take 7.5mLDMEM (serum-free and antibiotic) in 50mL pipe, add 300 μ LPEI MegaTran
1.0 mixing；Add 75 μ g CD4 recombinant plasmid dnas in mixing liquid, mix and stand 30 minutes；
Take respectively 515 μ L in each culture dish in 37 DEG C of 5%CO2Incubator is cultivated.After transfecting 24 hours,
Every ware cell adds 25 μ L2M sodium butyrates to final concentration 5mM.
2, cell lysis: after transfecting 48 hours, carry out cell cracking.Suck culture medium, add 1mLPBS
Rinse, suck PBS.Add 1mL lysis buffer, before using, add protease inhibitor PI
And PMSF.Being placed in ice chest and vibrate on shaking table, collect and obtain lysate in all culture dishs, 4 DEG C are centrifuged,
Collect supernatant.Taking a small amount of supernatant uses WB to identify the expression of CD4, result Fig. 2.
3, DDK affinity chromatograph column purification: with 0.45 μM, after 33mm pvdf membrane filter filter centrifugation
Lysate supernatant and proceed to 15mL pipe, add the Beads 1mL that mixes, after sealing, put into 360 degree
In vortex mixer, combine 2 hours in 4 DEG C；Take out 15mL pipe, lysate is poured into BIO-RAD chromatography
In post, and catch and penetrate liquid, after dripping to the greatest extent, penetrate liquid sampling WB detection；Post material is rinsed with lysis buffer
1-2 time, rinse Beads 3 times with TBST again after dripping to the greatest extent, wash with 0.1M Glycine pH3.5 after dripping to the greatest extent
De-, 200 μ L for the first time, drip and do not collect to the greatest extent, second and third each 500 μ L, 250 μ L for the third time, collect
To a 1.5mL Tube, and it is rapidly added NaH2PO4(pH=11.0) it is neutralized to about pH7.0,
Often pipe adds glycerol extremely final concentration of 10%, Tween-80 to final concentration of 0.1%.CD4 after purification
Albumen SDS-PAGE identifies, sees Fig. 3.
From Fig. 2 result, WB in the HEK293T cell pyrolysis liquid of transfection pCMV6-CD4 plasmid
Having obvious specific band after detection at 50kD, molecular weight is consistent with CD4 expection molecular size range.
Show CD4 albumen specifically expressing in cell.
From Fig. 3 result, the albumen of purification has obvious specific band at PAGE glue 50kD,
Molecular weight is consistent with CD4 expection molecular size range.Show to obtain purity preferable CD4 albumen.
Embodiment 3, the preparation of CD4 monoclonal antibody and screening
According to standard method with the CD4 protein fragments of the purification of restructuring generation for B6/C57 mice (north
Capital laboratory animal Technology Co., Ltd. of dimension tonneau China) carry out immunity.Concrete grammar is as follows:
1, animal immune: purified T4 antigen, with complete Freund's adjuvant emulsifying, uses subcutaneous or abdomen
Chamber injecting method immunity 6-8 week old BALB/c mouse, immunizing dose be 50 μ g/ only, be spaced two weeks laggard
Row second time immunity, with incomplete Freund's adjuvant emulsifying, immunizing dose is 50 μ g/.Take after immune twice
Tail blood measures serum titer with ELISA method gradient dilution；Determine whether booster immunization according to result, choose
The mice that antibody titer is the highest carries out cell fusion.
2, cell merges: myeloma cell uses the sp2/0 that BALB/c originates, and is in logarithm raw during fusion
For a long time；Take immune mouse spleen, make lymphocyte single cell suspension；Mouse spleen lymphocyte and bone
Myeloma cells mixes with 1:5-1:10, drips 50%PEG (PH 8.0) 1mL of 37 DEG C, cannot add completely
Full culture medium and remaining stop buffer, be centrifuged after abandoning supernatant and add the suspension mixing of HAT culture medium, MC constant volume
To 50mL, it is dispensed in 3.5cm culture dish, is put in wet box, be placed in 37 DEG C, 5%CO2Constant temperature is trained
Support in case and cultivate.
3, screen and clone: in merging 7-10 days, selecting cell clone, using CD4 purification of recombinant proteins
Carry out ELISA test.Labeled cell strain number.Positive porocyte is carried out limiting dilution, each limited dilute
Releasing latter 5-6 days and measure ELISA value, the monoclonal hole that picking OD280 positive value is higher carries out limiting dilution,
Until ELISA measures 96 orifice plates and entirely hardens fruit for the positive.The picking positive is worth high monoclonal and determines strain.It is right
Should merge plate cell strain is UMAB64.
4, the preparation of ascites monoclonal antibody and purification: the male BALB/c mouse lumbar injection 0.5ml of 10-12 week old
Norphytane, after one week, every mice is thin through the monoclonal that PBS washing is resuspended with 1mL syringe lumbar injection
Born of the same parents' suspension, cell consumption is 5 × 106/ only, every strain antibody makes a call to 2 mices.Receive after mouse ascites gathers
Collection ascites, centrifuging and taking supernatant, affinity chromatography carries out ascites purification, selects corresponding post according to antibody subtype
Material, the monoclonal antibody that cell strain UMAB64 produces is IgG2b, uses protein G to be purified.After purification
Monoclonal antibody concentration mensuration, WB detection, subpackage, frozen at-20 DEG C.Wherein WB testing result is shown in Fig. 4.
Had special at molecular weight 50kD from Fig. 4 result, swimming lane HL-60 and swimming lane SW-620
Band, molecular weight is consistent with CD4 expection molecular size range, shows that monoclonal antibody UMAB64 can be special
The CD4 albumen that ground Western blot detection is endogenous.
Embodiment 4, monoclonal antibody UMAB64 are an anti-SABC detection
(1), experimental technique:
1, take the piece of tissue such as Normal Lymph Nodes, tonsil and lymphoma and carry out paraffin embedding, use Finesse
Histotome cuts into slices, and tissue thickness is 6 μm.
2, dewaxing and aquation: analytical pure dimethylbenzene 3 × 10min, dehydrated alcohol 3 × 10min, 95% ethanol
5min, 85% ethanol 5min, 75% ethanol 5min, deionized water soaks 3min × 3 time
3, antigen retrieval buffers (0.01M, pH6.0 sodium citrate buffer) pressure cooker high pressure hot repair is added
Multiple 2min, when pressure cooker temperature is down to about 90 DEG C, opens pressure cooker, takes out specimen, the coldest
But to room temperature.Deionized water soaks 3min × 3 time.
4, using 3% hydrogen peroxide deactivation tissue endogenous peroxydase, room temperature stands 10min.Go from
Sub-water soaking 5min × 3 time.
5, plus confining liquid (PBS+5% defatted milk powder+5% Normal Goat Serum), 60min is hatched for 37 DEG C.
6, remove confining liquid, do not rinse, add CD4 monoclonal antibody (UMAB64), thinner ratio: 1:150, make
It is diluted with confining liquid.It is placed in wet box, hatches 60min for 37 DEG C.PBST (0.1%Tween-20)
Wash 2 times, wash 5min every time.PBST (0.02%Tween-20) washs 1 time, washs 5min every time.
7, dropping Polink-test kit 2 (Catlog No.D37-15) reagent 1,37 DEG C is hatched 10-20 and is divided
Clock.PBS is used to wash 3 times, each 5min.Dropping Polink-2 test kit (Catlog No.D37-15)
Reagent 2, hatches 10-20 minute for 37 DEG C, uses PBS to wash 3 times, each 5min.
8, application DAB solution (Zhong Shan Golden Bridge ZLI-9019) colour developing, colour developing 3～10min.Distilled water
Washing.
9, haematoxylin redyeing nucleus 2min, distilled water rinses, 1% hydrochloric acid differentiation.Distilled water rinsing 3
Secondary, room temperature stands 1min.
10, dehydration and transparent: 75% ethanol 5min, 100% ethanol 5min x 3 times, 85% ethanol 5min,
95% ethanol 5min, 100% ethanol 3 × 5min；Dimethylbenzene 3 × 5min, neutral gum mounting.
11, microscopy, is shown in Fig. 5-7.
(2), experimental result:
From Fig. 5-7 result, in people normal lymphoid nodal tissue, lymphoma tissue and normal tonsil group
Visible specific membrane dyeing in knitting.Result and CD4 are in intracellular location and tissue expression specificity one
Cause, show that monoclonal antibody UMAB64 of the present invention can be used for Immunohistochemical detection CD4 albumen
Level.
Embodiment 5, the specific detection of monoclonal antibody UMAB64
10,000 HEK293T cell protein process LAN cracking are comprised on OriGene high-density protein chip
Thing, every kind of protein lysate has the repetition of two copies on chip.Protein lysate by trace at nitre
On acid cellulose film.The location of each clock protein lysate can be accurately positioned by Excel file.
On protein chip, albumen is divided into 40 sub-matrixes, and each sub-matrix has some references, by referring to, can
With the content of albumen on quantitative each chip point, monitor the repeatability of each immunoreation data, Yi Jiding
The direction of position positive signal.Below for using OriGene albumen (OriGene Cat PA100001) chip
Carry out the experimental technique of UMAB64 Identification of the antibodies experiment:
1, a protein chip is placed in 50mL centrifuge tube, uses 40mL deionized water infiltration core
Sheet, is placed on shaking table, mixed at room temperature 30 minutes.Discard deionized water, use 10mLPBST balance
Chip.Room temperature treatment 10 minutes.
2, in 50mL centrifuge tube, add 40mL5% skim milk (being diluted with PBST) to be placed in
On shaking table, room temperature is closed 30 minutes.
3, using confining liquid (5% skim milk) to dilute an anti-UMAB64, thinner ratio is classified as 1:200.
4, pasting in laboratory table by clean sealed membrane, dropping 250-300 μ L mono-resists on sealed membrane.
5, protein chip is extracted out from confining liquid, face down the one of protein chip NC film, from chip
While contact antibody, slowly slide, rely on surface tension of liquid, antibody will slowly infiltrate chip NC
Film, until whole NC film infiltrates in an anti-solution.Whole operating process is avoided producing bubble.By chip
Move on under 4 DEG C of environment, stand, an anti-overnight incubation.Chip is added a cover culture dish lid, it sticks one
Open wet paper towel, cause antibody to evaporate to prevent from hatching for a long time.
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 mixed
Close uniformly, wash three times, wash 5min every time.
7, confining liquid (5% skim milk) is used to dilute two anti-DyLight649-conjugated AffiniPure
Fragment Goat-anti-Mouse IgG, dilution ratio is 1:400.
8, according to above-mentioned steps 4, step 5 carries out two and anti-hatches operation.Incubated at room 1 hour.At core
Hide with aluminium-foil paper above sheet, to prevent signal bleaching.
9, according to above-mentioned steps 6, PBST is used to wash chip.
10, deionized water rinsing chip is used, to remove remaining salinity and denaturant.
11, drying at room temperature chip, it is ensured that chip is completely dried.
12, chip scanner is used to read fluorescence signal.
13, the site of chip direction and positive signal is determined according to BSA-Cy3 and BSA-Cy5.
14, corresponding protein lysate ID is found out according to positive signal site, according to lysate database information,
Finding corresponding protein name, NCBI typing number (accession number), protein I D, albumen is big
The information such as little.
As shown in Figure 8, monoclonal antibody UMAB64 can identify on OriGene protein chip result more specifically
CD4 albumen, shows that the specificity of monoclonal antibody UMAB64 of the present invention is preferable.
Claims (7)
1. a monoclonal antibody UMAB64 of specific binding CD4 albumen, deposit number be
The hybridoma cell strain of CGMCC No.9576 produces.
2. a hybridoma cell strain, its deposit number is CGMCC No.9576.
3. monoclonal antibody as claimed in claim 1 is used for detecting the immune detection of CD4 albumen in preparation
Application in instrument.
Application the most according to claim 3, described immune detection instrument is test kit, chip or examination
Paper.
5. an immunologic combined detection reagent kit, including the monoclonal antibody described in claim 1.
6. monoclonal antibody as claimed in claim 1 is preparation answering in the test kit of marked tumor
With.
Application the most according to claim 6, described tumor is t cell lymphoma, histiocyte pouring
Bar tumor, Langerhans cell tissue hyperplasia.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107236042A (en) * | 2017-07-05 | 2017-10-10 | 无锡傲锐东源生物科技有限公司 | Anti- EGFR L858R protein monoclonal antibodies and application thereof |
| CN108484770A (en) * | 2018-05-16 | 2018-09-04 | 武汉云克隆科技股份有限公司 | Recombinant rat anti-mouse CD4 monoclonal antibodies, preparation method and application |
| CN110577595A (en) * | 2019-08-09 | 2019-12-17 | 无锡傲锐东源生物科技有限公司 | anti-TTF 1 protein monoclonal antibody and application thereof |
| CN110903394A (en) * | 2019-12-27 | 2020-03-24 | 源道隆（苏州）医学科技有限公司 | Polypeptide capable of binding CD4 and application thereof |
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| CN1200737A (en) * | 1995-09-06 | 1998-12-02 | 艾德药品公司 | Recombinant anti-CD4 antibodies for human therapy |
| CN102649818A (en) * | 2011-02-25 | 2012-08-29 | 厦门大学 | CD4 protein-resistant monoclonal antibody and active fragment and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1200737A (en) * | 1995-09-06 | 1998-12-02 | 艾德药品公司 | Recombinant anti-CD4 antibodies for human therapy |
| CN102649818A (en) * | 2011-02-25 | 2012-08-29 | 厦门大学 | CD4 protein-resistant monoclonal antibody and active fragment and application thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107236042A (en) * | 2017-07-05 | 2017-10-10 | 无锡傲锐东源生物科技有限公司 | Anti- EGFR L858R protein monoclonal antibodies and application thereof |
| CN108484770A (en) * | 2018-05-16 | 2018-09-04 | 武汉云克隆科技股份有限公司 | Recombinant rat anti-mouse CD4 monoclonal antibodies, preparation method and application |
| CN108484770B (en) * | 2018-05-16 | 2020-11-13 | 武汉云克隆科技股份有限公司 | Recombinant rat anti-mouse CD4 monoclonal antibody, preparation method and application |
| CN110577595A (en) * | 2019-08-09 | 2019-12-17 | 无锡傲锐东源生物科技有限公司 | anti-TTF 1 protein monoclonal antibody and application thereof |
| CN110903394A (en) * | 2019-12-27 | 2020-03-24 | 源道隆（苏州）医学科技有限公司 | Polypeptide capable of binding CD4 and application thereof |
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