Anti- BTLA protein monoclonal antibodies and application thereofTechnical field
The present invention relates to biological technical field, and in particular to can specific bond BTLA albumen monoclonal antibody Umab61,Produce the cell line of the monoclonal antibody and apply the diagnostic method of the antibody and purposes.
Background technology
Very important effect is played in regulation of the activation of the accessory receptor on immunocyte surface to immunity of organism, whereinThe comparison of CD28/B7 ligand receptors family member research is more, such as：CTLA4, PD1 and B, T lymphocyte attenuators (B andT lymphocyte attenuator, BTLA) etc..They serve the effect of negative regulation immune response.
BTLA also known as CD272, is a member for the CD28 families being just found recently, belongs to Ig superfamily members.Main tableUp to B, T lymphocytic cell surface upon activation.The same with PD-1, also there is an immunity receptor Tyr suppression BTLA intracellular regionMotif, by the way that the activation of T cell can be suppressed after being combined with part HVEM.Thus in tumor-infiltrated lymphocyte, BTLA'sExpression quantity can reflect immunosuppressive state.It is thin that immunohistochemistry (IHC) Pathological experiment detection tumour is clinically commonly used at presentThe expression situation of albumen in born of the same parents, but the core of IHC experiments is the monoclonal antibody of binding proteins specific, the quality of its performanceDirectly decide the sensitivity and specificity entirely detected.Therefore, develop a kind of binding specificity it is higher be directed to BTLA eggsWhite monoclonal antibody has great importance to IHC detection BTLA expressions.
The content of the invention
In view of this, it is anti-it is an object of the invention to provide a kind of monoclonal of the higher BTLA albumen of binding specificityBody, and its preparing the application in being used to detect the immune detection instrument of BTLA albumen.
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 April 27th, 2016, and deposit number is CGMCC No.12284.
It is thin by above-mentioned hybridoma present invention also offers a kind of monoclonal antibody Umab61 of specific binding BTLA albumenBorn of the same parents' strain is produced.
The preparation method of monoclonal antibody of the present invention is as follows：
(1) structure of recombinant expression carrier：According to BTLA ORF nucleotide sequences (BTLA ORF nucleotide sequences such as SEQShown in ID NO.1,870bp；BTLA amino acid sequences are as shown in SEQ ID NO.2)
Primer PCR amplification BTLA ORF 1bp are designed to 867bp bit sequences, gene both sides introduce restriction enzyme respectivelyEnzyme site SgfI and MluI, inserts expression vector pCMV6-Entry, builds the restructuring table of BTLA amino acid sequences the 1st to the 289thUp to plasmid pCMV6-rBTLA；Upstream amplification primer sequence, SEQ ID NO.3:CACGCGATCGCATGAAGACATTGCCTGCCATGDownstream amplification primer sequence SEQ ID NO.4:ACCGACGCGTACTCCTCACACATATGGATGCA
(2) expression and purification of BTLA recombinant proteins：By BTLA recombinant expression plasmids convert HEK293T cells, crack fromThe heart obtains supernatant, and the purifying of DDK affinity columns obtains the BTLA recombinant proteins of purifying；
(3) screening and preparation of monoclonal antibody：Balb/c mouse are immunized using the BTLA recombinant proteins of above-mentioned purifying, takenMouse spleen cells are merged with SP2/0 cells, and limiting dilution assay obtains monoclonal, and ELISA method screening positive hybridoma is thinBorn of the same parents, acquisition can secrete the hybridoma cell strain of anti-BTLA specific antibodies, be named as Umab61, hypotype is accredited as IgG2a；Pass throughSerum free medium prepares antibody, and BTLA monoclonal antibodies Umab61 is obtained by affinity column purifying.Pass through respectivelyWestern Blot, immunohistochemical experiment verify the sensitivity and specificity of the monoclonal antibody.
Further the specificity of said monoclonal antibody is verified using OriGene high-density proteins chip：On OriGene high-density protein chips lysate, every kind of protein lysate are overexpressed comprising 10,000 HEK293T cell proteinThere is the repetition of two copies on chip.Protein lysate is by trace on nitrocellulose filter.Each clock protein lysatePositioning can be accurately positioned by Excel file.Albumen is divided on 40 sub- matrixes, each Asia matrix on protein chipThere are some references, by referring to can quantify the content of albumen on each chip point, monitor the repetition of each immune response dataProperty, and position the direction of positive signal.
The monoclonal antibody Umab61 and said chip are hybridized and determine positive signal site by the present invention, are as a result shownMonoclonal antibody Umab61 of the present invention specifically binds BTLA albumen, and with other albumen no cross reactions.
Prepared present invention also offers monoclonal antibody Umab61 for detecting in the immune detection instrument of BTLA albumenApplication.
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 Umab61, can detect the expression situation of BTLA in histocyte.
Present invention also offers application of the said monoclonal antibody in the kit for marked tumor is prepared.Wherein instituteState tumour and specifically refer to the propagation of tumour cell and the BTLA closely related tumour of expression, including but not limited to melanoma,Lung cancer, colon cancer, breast cancer.
Compared with prior art, the invention provides a kind of hybridoma cell strain, (deposit number is CGMCCNo.12284 the monoclonal antibody Umab61 that), and thus hybridoma cell strain is produced.Present invention also offers monoclonal antibodyUmab61 is preparing the application in being used to detect the immune detection instrument of BTLA albumen, the immune group of the Umab61 containing monoclonal antibodyChange kit, and applications of the monoclonal antibody Umab61 in the kit for marked tumor is prepared.List of the present inventionClonal antibody can be combined with BTLA protein-specifics, and with other intracellular albumen no cross reactions, significantly improve BTLA eggsBTLA protein expression levels in specificity, accuracy and the reliability of white immune detection, true reflection tumor infiltrating lymphocyte,It can be applied to the detection of the tumour immunity microenvironment such as melanoma, lung cancer, colon cancer, breast cancer.
Preservation information
Classification And Nomenclature for the hybridoma cell strain Umab61 of preservation is：The anti-human B and T lymphocyte attenuators of mouse(BTLA) monoclonal hybridoma 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 April 27th, 2016；
Deposit number：CGMCC No.12284.
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 existingThere is the accompanying drawing used required in technology description to be 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 that embodiment 2 recombinates BTLA albumen Western blot testing result figures, with anti-DDK detection restructuring BTLAExpression of the albumen in HEK293T cells, wherein the HEK293T cell pyrolysis liquids that left side swimming lane is transfection empty carrier are antigenTesting result, right lanes are the testing result of the HEK293T cell pyrolysis liquid antigens of transfection pCMV6-rBTLA plasmids；
Fig. 3 shows that embodiment 2 recombinates BTLA protein SDS-PAGE result figures, with anti-DDK affinity columns purification of Recombinant BTLAAlbumen, albumen after purification passes through the electric arteries and veins of SDS-PAGE glue, coomassie brilliant blue staining；
Fig. 4 shows that embodiment 3 recognizes complete BTLA (Full length BTLA, BTLA- with monoclonal antibody Umab61FL) the Western blot testing result figures of albumen.Swimming lane 1 is transfection pCMV6-Entry cell pyrolysis liquid；Swimming lane 2 is to turnContaminate pCMV6-BTLA-FL cell pyrolysis liquid；
Fig. 5 shows that the formalin of embodiment 4 is fixed, (primary antibody is BTLA to the human breast carcinoma ImmunohistochemistryResults Results figure of FFPEMonoclonal antibody Umab61)；
Fig. 6 shows that the formalin of embodiment 4 is fixed, (primary antibody is that BTLA is mono- to the human lung cancer ImmunohistochemistryResults Results figure of FFPEClonal antibody Umab61)；
Fig. 7 shows that (primary antibody is BTLA monoclonal antibodies Umab61,1 to embodiment 5OriGene protein chip qualification results figure:100；Secondary antibodyFor DyLight649-conjugated AffiniPure Fragment Goat-anti-Mouse IgG, 1:400).
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 inventionEmbodiment, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made, allBelong to the scope of protection of the invention.
The structure of embodiment 1, BTLA recombinant expression plasmids
It is with the plasmid RC219458 (867bp containing BTLAORF) obtained from bio tech ltd of Aureal Dongyuan County of the U.S.Template, designs two primers and introduces restriction enzyme site SgfI and MluI respectively, be cloned into expression vector pCMV6-Entry, set upBTLA recombinant expression plasmids.Cloning site design is as shown in Figure 1.
The expression and purification of embodiment 2, BTLA recombinant proteins
1st, HEK293T cells are transfected：HEK293T cells are with 1:3, which reach continuation in culture dish, cultivates；Take 7.5mLDMEM (nothingsSerum and antibiotic) into 50mL pipes, add 300 μ LPEI MegaTran1.0 and mix；Add 75 μ g BTLA recombination expression matterGrain DNA is mixed into mixing liquid and is stood 30 minutes；Take 515 μ L into each culture dish in 37 DEG C of 5%CO respectively2In incubatorCulture.After transfection 24 hours, 25 μ L2M sodium butyrates are added per ware cell to final concentration 5mM.
2nd, cell lysis：After transfection 48 hours, cell cracking is carried out.Culture medium is sucked, 1mLPBS is added and is rinsed, inhaledRemove PBS.1mL lysis buffers are added, preceding addition protease inhibitors PI and PMSF is used.It is placed in ice chest and is shaken on shaking tableSwing, collect and lysate is obtained in all culture dishes, supernatant is collected in 4 DEG C of centrifugations.A small amount of supernatant is taken to identify restructuring BTLA table using WBReach, result figure 2.
3rd, DDK affinity columns are purified：With 0.45 μM, the lysate supernatant after 33mm pvdf membrane filter filter centrifugations is simultaneously15mL pipes are transferred to, the Beads 1mL that mix is added, is put into after sealing in 360 degree of vortex mixers, in 4 DEG C of combinations 2 hours；Take out15mL is managed, and lysate is poured into BIO-RAD chromatographic columns, and is caught and penetrated liquid, drop to the greatest extent after penetrate liquid sampling WB detections；To crackWash buffer post material 1-2 times, Beads is rinsed 3 times after drop is most with TBST again, is washed after drop is most with 0.1M Glycine pH3.5De-, 200 μ L, are dripped and do not collect to the greatest extent for the first time, second and third each 500 μ L, and 250 μ L of third time are collected to a 1.5mL centrifuge tubeIn, and it is rapidly added NaH2PO4(pH=11.0) pH7.0 or so is neutralized to, often pipe adds glycerine to final concentration of 10%,Tween-80 to final concentration of 0.1%.Restructuring BTLA albumen after purification is identified with SDS-PAGE, sees Fig. 3.
In 30- after WB detections in Fig. 2 results, the HEK293T cell pyrolysis liquids of transfection pCMV6-rBTLA plasmidsThere is obvious specific band at 40kD, be consistent substantially with the theoretical molecular 33kD of polypeptide.Show to recombinate BTLA albumen in cellSpecifically expressing.
From Fig. 3 results, the albumen of purifying has obvious specific band at PAGE glue 30-40kD, the reason with polypeptideIt is consistent substantially by molecular weight 33kD.Show to have obtained the preferable BTLA recombinant proteins of purity.
The preparation and screening of embodiment 3, BTLA monoclonal antibodies
The BTLA protein fragments of the purifying produced according to standard method with restructuring are used to (tie up tonneau in Beijing to Balb/c mouseMagnificent experimental animal Technology Co., Ltd.) it is immunized.Specific method is as follows：
1st, animal immune：Purified BTLA antigens are emulsified with complete Freund's adjuvant, using subcutaneous or intraperitoneal injection side6-8 week old Balb/c mouse are immunized in method, and for 50 μ g/ only, interval carries out being immunized for second immunizing dose after two weeks, with not exclusively notFamily name's adjuvant emulsion, immunizing dose is 50 μ g/.It is immune to take tail blood to determine serum titer with ELISA method gradient dilution afterwards twice；RootDetermine whether booster immunization according to result, choose antibody titer highest mouse and carry out cell fusion.
2nd, cell fusion：Myeloma cell uses the sp2/0 that Balb/c originates, and exponential phase is in during fusion；TakeImmune 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, incomplete culture medium and remaining terminate liquid is added, centrifugation, which is abandoned, adds HAT after supernatantCulture medium, which suspends, to be mixed, and MC constant volumes are dispensed into 3.5cm culture dishes, are put in wet box to 50mL, are placed in 37 DEG C, 5%CO2It is permanentCultivated in warm incubator.
3rd, screen and clone：Fusion selects cell clone in 7-10 days, and ELISA surveys are carried out using BTLA purification of recombinant proteinsExamination.Mark cell line number.Positive hole cell is carried out to determine ELISA values, picking within 5-6 days after limiting dilution, each limiting dilutionThe higher monoclonal hole of OD280 positive values carries out limiting dilution, until it is the positive that ELISA, which determines the complete hardened fruit of 96 orifice plates,.PickingThe high monoclonal singling of positive value.Its correspondence fusion plate cell line is Umab61.
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 is injected intraperitoneally with 1mL syringes after one week wash the monoclonal cell suspension being resuspended through PBS, cell consumption for 5 ×106/ only, make a call to 2 mouse per strain antibody.After collecting ascites after mouse ascites accumulation, centrifuging and taking supernatant, affinity chromatography is carried outAscites is purified, and selects corresponding post material according to antibody subtype, the monoclonal antibody that cell line Umab61 is produced is IgG2a, using protein GPurified.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 50kD, shows monoclonal antibody Umab61Specifically complete total length BTLA albumen can be detected by Western blot.
Embodiment 4, monoclonal antibody Umab61 detect for the SABC of primary antibody
(1), experimental method：
1st, take human breast carcinoma tissue and human lung cancer tissue block that formalin is fixed to carry out FFPE, use FinesseHistotome is 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 and repair 3min, treat heightWhen pressure pot temperature is down to about 90 DEG C, pressure cooker is opened, sample is taken out, then naturally cools 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 soaked5min × 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, BTLA monoclonal antibodies (Umab61), thinner ratio is added：1:150, carried out using confining liquidDilution.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 PBSWashing 3 times, each 5min.2,37 DEG C of Polink-2 kits (Catlog No.D37-15) reagent is added dropwise and is incubated 10-20 pointsClock, is washed 3 times, each 5min using PBS.
8th, developed the color using DAB solution (Zhong Shan Golden Bridge ZLI-9019), develop the color 3~10min.Distill 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 temperature1min。
10th, it is dehydrated and transparent：75% ethanol 5min, 100% ethanol 5min x 3 times, 85% ethanol 5min, 95% ethanol5min, 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, visible specific membrane is dyed in human breast carcinoma tissue and human lung cancer tissue.As a result withBTLA positioning in the cell and tissue expression specificity are consistent, show that monoclonal antibody Umab61 can be used for immuning tissueLearn the level of detection BTLA albumen.
The specific detection of embodiment 5, monoclonal antibody Umab61
Lysate is overexpressed comprising 10,000 HEK293T cell protein on OriGene high-density protein chips, per hatching eggWhite lysate has the repetition of two copies on chip.Protein lysate is by trace on nitrocellulose filter.Each clock eggThe positioning of white lysate can be accurately positioned by Excel file.Albumen is divided into 40 sub- matrixes on protein chip, eachThere are some references on sub- matrix, by referring to can quantify the content of albumen on each chip point, monitor each immune response numberAccording to repeatability, and positioning positive signal direction.It is using OriGene albumen (OriGene Cat PA100001) belowChip carries out the experimental method of Umab61 Identification of the antibodies experiments：
1st, a protein chip is placed in 50mL centrifuge tubes, infiltrates chip 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, add 40mL5% skim milks (being diluted with PBST) into 50mL centrifuge tubes to be placed on shaking table, room temperature envelopeClose 30 minutes.
3rd, primary antibody Umab61 is diluted using confining liquid (5% skim milk), thinner ratio is classified as 1:100.
4th, clean sealed membrane is pasted on experimental bench, 250-300 μ L primary antibodies is added dropwise on sealed membrane.
5th, protein chip is extracted out from confining liquid, by the one of protein chip NC films down, contacted from one side of chipAntibody, is slowly slided, by surface tension of liquid, and antibody will slowly infiltrate chip NC films, until whole NC films infiltration is in primary antibodyIn solution.Whole operation process avoids producing bubble.Chip is moved on under 4 DEG C of environment, stood, primary antibody is incubated overnight.In chipUpper capping culture dish lid, sticks a hygenic towelette thereon, causes antibody to evaporate to prevent from being incubated for a long time.
6th, chip was moved in 50mL centrifuge tubes in second day, chip is rinsed twice using PBST, remove unnecessary antibody.MakeChip is washed with 40mL PBST (0.1%Tween-20), is placed on shaking table and is well mixed, wash 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 chipPaper 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 remnants salinity and denaturant.
11st, drying at room temperature chip, it is ensured that chip is completely dried.
12nd, fluorescence signal is read using chip scanner.
13rd, chip direction and the site of positive signal are 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, found pairAnswer protein name, NCBI typings number (accession number), protein I D, the information such as albumen size.
As a result as shown in fig. 7, monoclonal antibody Umab61 can specifically recognize BTLA albumen on OriGene protein chips, showMonoclonal antibody Umab61 specificity is preferably.
<110>Wuxi Origene Bio-tech Co., Ltd.
<120>Anti- BTLA protein monoclonal antibodies and application thereof
<210> 1
<211>870
<212> DNA
<213>Artificial sequence
<400> 1
1 ATGAAGACAT TGCCTGCCAT GCTTGGAACT GGGAAATTAT TTTGGGTCTT CTTCTTAATC
61 CCATATCTGG ACATCTGGAA CATCCATGGG AAAGAATCAT GTGATGTACA GCTTTATATA
121 AAGAGACAAT CTGAACACTC CATCTTAGCA GGAGATCCCT TTGAACTAGA ATGCCCTGTG
181 AAATACTGTG CTAACAGGCC TCATGTGACT TGGTGCAAGC TCAATGGAAC AACATGTGTA
241 AAACTTGAAG ATAGACAAAC AAGTTGGAAG GAAGAGAAGA ACATTTCATT TTTCATTCTA
301 CATTTTGAAC CAATGCTTCC TAATGACAAT GGGTCATACC GCTGTTCTGC AAATTTTCAG
361 TCTAATCTCA TTGAAAGCCA CTCAACAACT CTTTATGTGA CAGATGTAAA AGGTGCCTCA
421 GAACGACCCT CCAAGGACGA AGTGGCAAGC AGACCCTGGC TCCTGTATAG TTTACTTCCT
481 TTGGGGGGAT TGCCTCTACT CATCACTACC TGGTTCTGCC TGTTCTGCTG CCTGAGAAGG
541 CACCAAGGAA AGCAAAATGA ACTCTCTGAC ACAGCAGGAA GGGAAATTAA TCTGGTTGAT
601 GCTCACCTTA AGAGCGAGCA AACAGAAGCA AGCACCAGGC AAAATTCCCA AGTACTGCTA
661 TCAGAAGCTG GAATTTATGA TAATGACCCT GACCTTTGTT TCAGGATGCA GGAAGGGTCT
721 GAAGTTTGTT CTAATCCATG CCTGGAAGAA AACAAACCAG GCATTGTTTA TGCTTCCCTG
781 AACCATTCTG TCATTGGACT GAACTCAAGA CTGGCAAGAA ATGTAAAAGA AGCACCAACA
841 GAATATGCAT CCATATGTGT GAGGAGTTAA
//
<210> 2
<211>289
<212> PRT
<213>Artificial sequence
<400> 2
ORIGIN
1 MKTLPAMLGT GKLFWVFFLI PYLDIWNIHG KESCDVQLYI KRQSEHSILA GDPFELECPV
61 KYCANRPHVT WCKLNGTTCV KLEDRQTSWK EEKNISFFIL HFEPMLPNDN GSYRCSANFQ
121 SNLIESHSTT LYVTDVKGAS ERPSKDEVAS RPWLLYSLLP LGGLPLLITT WFCLFCCLRR
181 HQGKQNELSD TAGREINLVD AHLKSEQTEA STRQNSQVLL SEAGIYDNDP DLCFRMQEGS
241 EVCSNPCLEE NKPGIVYASL NHSVIGLNSR LARNVKEAPT EYASICVRS
//