CN102492038B - Anti-human Tim-3 neutralized monoclonal antibody L3D and application thereof - Google Patents
Anti-human Tim-3 neutralized monoclonal antibody L3D and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-human Tim-3 neutralized monoclonal antibody L3D and an application thereof. Antibody light and heavy chain variable region genes are cloned from a prepared anti-human Tim-3 neutralized monoclonal antibody L3D hybridoma cell; and the obtained light chain and heavy chain variable region genes can be used for encoding a monoclonal antibody variable region. Based on the light and heavy chain variable region genes of the monoclonal antibody, a plurality of small molecular genetic engineering antibodies can be constructed and expressed. Polypeptides or proteins encoded on the basis of the genes can be cross-linked with a plurality of bioactive molecules, so that a Tim-3 expression level detection reagent is prepared for diagnosing and treating diseases caused by abnormal expression of Tim-3.
Description
Technical field
The present invention relates to a kind of monoclonal antibody, in particular to anti-human Tim-3 neutralizing monoclonal antibody L3D, also relate to this monoclonal antibody in preparation Tim-3 detection reagent, and diagnosis and treatment Tim-3 high expression level cause the application in disease.
Background technology
Tim-3 structurally belongs to T cell immunoglobulin and Saliva Orthana (T cell Immunoglobulin domain and Mucin domain protein, Tim) family member.Due to the wide participation of Tim family the process of immune regulation of body, its function is subject to people's attention just day by day.Tim-3 is specific expressed in Th1, the Th17 effector cell surface of activation, and is not expressed in Th2 cell.Existing known galactose-binding protein-9 (Galectin-9, Gal-9) be the native ligand of Tim-3, this molecule wide expression is in periphery immunity system, the Tim-3 molecular specificity of Gal-9 on Th1, Th17 cell is combined, can cause the latter's tune dies, lower immune response, inducing immune tolerance.At present, studies confirm that Tim-3 molecule mainly by regulate different CD4+T cell subsets function wide participation autoimmune disease, transplant rejection, immunne response process [the Anderson DE.TIM-3as a therapeutic target in human inflammatory diseases.Expert Opin Ther Targets.2007 such as anti-infective; 11 (8): 1005-9.Anderson AC, Anderson DE.TIM-3 in autoimmunity.Curr Opin Immunol.2006; 18 (6): 665-9.Degauque N, Mariat C, Kenny J, et al.Regulation of T-cell immunity by T-cell immunoglobulin and mucin domain proteins.Transplantation 2007; 84:S12-16.Zhu C, Anderson AC, Schubart A, et al.The Tim-3ligand galectin-9negatively regulates T helper type 1immunity.Nat Immunol 2005; 6:1245-1252.].
The current abnormal and numerous disease that Tim-3 expresses that studies confirm that has close relationship, find HIV as studied, patient and some tumour patients that HCV infects, on its T cell, Tim-3 expresses and raises, the tune that can mediate T effector cell due to Tim-3 is died, transmit negativity conditioning signal, can cause the immunologic function paralysis of body.The factor of any blocking-up Tim-3/Gal-9 combination all can make Th1, Th17 cell avoid death, strengthen T effector cell's activity, recover immunologic function [Kassu A, Marcus RA, D ' Souza MB, et al.Regulation of Virus-Specific CD4+T Cell Function by Multiple Costimulatory Receptors during Chronic HIV Infection.Immunol.2010; 185 (5): 3007-18.Huang X, et al.Lymphoma endothelium preferentially expresses Tim-3 and facilitates the progression of lymphoma by mediating immune evasion.Exp Med.2010; 207 (3): 505.N.Castelblanco, V. Kuchroo, D.R.Gretch, and H.R.Rosen.2009.Negative immune regulator Tim-3 is overexpressed on T cells in hepatitis C virus infection and its blockade rescues dysfunctional CD4+and CD8+T cells.Virol.83:9122-9130.].
On the one hand, some autoimmune diseases as diseases such as systemic lupus erythematous (SLE), asthma in, due to the rising of Gal-9 or Tim-3 expression, cause the function of Th1 cell to be suppressed, and then break the immunologic balance in body, cause the increased activity of pathologic Th2 cell, cause the morbidity of disease.In this case, the factor of any blocking-up Tim-3/Gal-9 combination all can be conducive to the recovery of immunologic balance in body, alleviates the process of disease.All can be by blocking-up Tim-3/Gal-9 combination as injected anti-Tim-3 antibody or restructuring Tim-3 fusion rotein to asthmatic model animal, strengthen Th1 cytoactive, correct by the cell-mediated symptoms of asthma of Th2, recover Th1/Th2 cell balance [Hu WK in body, Lu XX, Yang S et al.Expression of the Th1-specific cell-surface protein Tim-3 increases in a murine model of atopic asthma.Asthma.2009; 46 (9): 872.Pan HF, Zhang N, Li WX, Tao JH, Ye DQ.TIM-3 as a new therapeutic target in systemic lupus erythematosus.Mol Biol Rep.2010; 37 (1): 395-8.Wang Y, Meng J, Wang X, Expression of human TIM-1 and TIM-3 on lymphocytes from systemic lupus erythematosus patients.Scand Immunol.2008; 67 (1): 63-70.Kearley J, McMillan SJ, Lloyd CM.Th2-driven, allergen-induced airway inflammation is reduced after treatment with anti-Tim-3 antibody in vivo.Exp Med 2007; 204:1289; Fukushima A, Sumi T, Fukuda K, Kumagai N, et al.Antibodies to T-cell Ig and mucin domain-containing proteins (Tim)-1 and-3 suppress the induction and progression of murine allergic conjunctivitis.Biochem Biophys Res Commun.2007; 353 (1): 211.].On the other hand, some autoimmune diseases as inflammatory bowel and type i diabetes model in, research find blocking-up Tim-3 increased activity Th1 effector cell's function in body, further increase the weight of autoimmunization damage, this result proving again Tim-3 [Sanchez-Fueyo A that plays a significant role in the maintaining of immunologic balance in vivo, Tian J, Picarella D, et al.Tim-3inhibits T helper type 1-mediated auto-and alloimmune responses and promotes immunological tolerance.Nat Immunol 2003, 4:1093-1101.Li X, et al.Clinical Immunol.2010,134:169-177.].
Above-mentioned research data shows, Tim-3 path has important immunoloregulation function, the generation of the abnormal and various diseases of its expression, develops and has substantial connection.Although research shows the neutralizing antibody of Tim-3 and also brought into play good intervention effect in some diseases model, but the Tim-3 antibody still not going on the market both at home and abroad at present, therefore set up and develop have independent intellectual property right take antibody as basic detection and diagnosis and treatment articles for use, have important practical significance for multiple diseases related intervention.
Summary of the invention
The invention discloses the monoclonal antibody L3D of a kind of anti-human Tim-3, described monoclonal antibody comprises light chain and heavy chain, its amino acid variable region sequences is respectively as shown in SEQ ID NO:1, SEQ ID NO:2 in sequence table, and its encoding gene is respectively as shown in SEQ ID NO:3, SEQ ID NO:4 in sequence table.
The invention also discloses the purposes of anti-human Tim-3 neutralizing monoclonal antibody L3D, be included in preparation Tim-3 detection reagent, and application in the disease that causes of diagnosis and treatment Tim-3 high expression level.
The disease that Tim-3 high expression level of the present invention causes comprises that HIV and HCV virus infection, tumour, autoimmune disease are as systemic lupus erythematous (SLE), asthma etc.
Complementary determining region CDR1, the CDR2 of the light chain protein matter molecule variable region of monoclonal antibody of the present invention, the aminoacid sequence of CDR3, respectively as shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 in sequence table.Complementary determining region CDR4, the CDR5 of the heavy chain protein matter molecule variable region of described monoclonal antibody, the aminoacid sequence of CDR6 are respectively as shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 in sequence table.
The invention also discloses the preparation method of said monoclonal antibody L3D, mainly comprise as follows:
1. the structure of anti-human Tim-3 monoclonal antibody hybridoma cell strain
First, prokaryotic expression people Tim-3 albumen, immune Balb/c mouse, carries out cytogamy by ordinary method.With indirect elisa method screening positive cell clone subclone repeatedly again, be 100% positive until all Hybridoma Cell Culture supernatants detect.Hybridoma L3D has been carried out to chromosome karyotype analysis, and hybridoma L3D karyomit(e) average number is 106, experimental results show that with two-phase agar diffusion the secreted immunoglobulin (Ig) hypotype of L3D hybridoma is IgG2a.Fig. 1 shows that monoclonal antibody L3D can specific binding people Tim-3 molecule.
2. the screening and identification of anti-human Tim-3 monoclonal antibody hybridoma cell strain
Use FACS method, carry out combination experiment with regard to the Tim-3 molecule on people Tim-3 antibody on human U937 cell.Result shows that L3D can be in conjunction with the Tim-3 molecule on U937, and shows the cross reactivity (Fig. 2) of being combined with mouse Tim-3.Use the neutralization activity of Lymphocyte Apoptosis experimental verification Tim-3 antibody simultaneously.Result shows that L3D obviously suppresses the apoptosis (Fig. 3) of the people THP1 cell of Gal-9 induction
3. monoclonal antibody L3D is to intervention effect in the body of autoimmunization injury disease model
Affinity column purifying Balb/c mouse hybridoma cell ascites, measures at ultraviolet spectrophotometer, calculates protein content.Set up pyemia model with C57BL/6 mouse, and autoimmunity inflammatory bowel animal model.Give after injected in mice L3D antibody or homotype control antibodies, observe the variation of Mouse Weight or survival rate, analysis list clonal antibody L3D biologic activity in vivo.Result shows that L3D antibody has significantly changed the survival rate (Fig. 4) of pyemia mouse, affected the degree of inflammation (Fig. 5) of inflammatory bowel mouse, the above results points out anti-human Tim-3 monoclonal antibody L3D to have in vivo good neutralization activity.
4. light, the angling of heavy chain gene of hybridoma L3D got
Extract neutralizing monoclonal antibody L3D cell RNA, through RT-PCR, angle the weight chain gene of getting antibody with two pairs of Auele Specific Primers.Conventional method connects into carrier, transformed competence colibacillus bacterium, and the single bacterium colony of picking after cultivating, extracts after plasmid PCR is identified and carries out DNA sequencing analysis.
Pass through above-mentioned steps, build and contained that people Tim-3 neutralizing antibody L3D is light, the carrier of heavy chain gene, through sequential analysis, comparison, encoding sequence is that mouse immuning ball protein is light, heavy chain gene, and its aminoacid sequence is respectively SEQ ID NO:1 and SEQ ID NO:2.
5. determining of light, the heavy chain variable region gene sequence of monoclonal antibody L3D and aminoacid sequence
With the online software of www.expasy.org, light, the weight chain variable region nucleotide sequence of encoding human Tim-3 neutralizing monoclonal antibody L3D is translated as to the aminoacid sequence of its coding, light, the weight chain variable region amino acid sequence of monoclonal antibody L3D is as shown in SEQ ID NO:1 in sequence table and SEQ ID NO:2.The aminoacid sequence of determining complementary determining region CDR1, CDR2 in light chain variable region sequence and CDR3 according to Kabat database is respectively as shown in SEQ ID NO:5, SEQ ID NO:6 in sequence table and SEQ ID NO:7.Complementary determining region CDR4, CDR5 in weight chain variabl area sequence and the aminoacid sequence of CDR6 are respectively as shown in SEQ ID NO:8, SEQ ID NO:9 in sequence table and SEQ ID NO:10.
The primer that the present invention applies an Analysis of Nested Design is successfully handed over and oncocyte, has been cloned that antibody is light, heavy chain variable region gene from the people Tim-3 neutralizing monoclonal antibody L3D cultivating.The mouse antibodies variable region that gained light chain and heavy chain variable region gene codified are correct.Fig. 6 shows the molecular weight of anti-human Tim-3 antibody.Monoclonal antibody of the present invention is light based on the above-mentioned people Tim-3 neutralizing monoclonal antibody L3D being cloned into, heavy chain variable region gene, can build and express multiple small molecules genetic engineering antibody, as single-chain antibody, single domain antibody, chimeric antibody, Fab antibody, antibody fusion protein etc.; Based on the coded polypeptide of said gene or protein, can be cross-linked multiple bioactive molecules, detect, diagnose and treat diagnosis or the medicine of Tim-3 disease that high expression level causes for the preparation of Tim-3 expression level.
Accompanying drawing explanation
The specific binding activity of the screening of Fig. 1 ELISA method, identifier Tim-3 neutralizing monoclonal antibody L3D and antigen.Contrast antigen is sulphur oxygen cyclase protein (Trx).
Fig. 2 flow cytometer (FACS) detects the combination activity of Tim-3 on monoclonal antibody L3D and people U937 cell, and with mouse RAW264.7 cell on the cross coupled activity of Tim-3.The combination activity of Tim-3 on A: monoclonal antibody L3D and people U937 cell; The cross coupled activity of Tim-3 on B: monoclonal antibody L3D and mouse RAW264.7 cell.
The extracorporeal neutralizing activity analysis of Fig. 3 monoclonal antibody L3D.Gal-9 albumen can be by cell the tune of Tim-3 induction THP1 die, this figure adopts respectively PI and Annexcin-V to do apoptotic cell Coloration experiment, observe Tim-3 antibody to Gal-9 induction adjust die in and barrier effect.
The impact of Fig. 4 monoclonal antibody L3D on the pyemia course of disease.Set up pyemia model C57BL/6 mouse, give and L3D or homotype control antibodies simultaneously, then observe the survival rate (A) of animal, and use respectively inflammatory mediator IL-6 (B) after the intervention of ELISA methods analyst antibody, the expression of IL-1beta (C) changes.
The impact of Fig. 5 monoclonal antibody L3D on the mouse inflammatory bowel course of disease.Set up inflammatory bowel model C57BL/6 mouse, give and L3D or homotype control antibodies simultaneously, then observe the body weight change of mouse and the variation of cytokine IL-17, IFN-g.A: the body weight of mouse situation over time; The changing conditions of B: cytokine IL-17A; The changing conditions of C: cytokine IFN-g.
The protein electrophoresis collection of illustrative plates of Fig. 6 monoclonal antibody L3D.Each swimming lane is respectively A: non-reduced electrophoresis, B: reduction electrophoresis (showing respectively heavy chain and light chain), and C: molecular weight Marker.
Embodiment
Can more easily understand content of the present invention by consulting following embodiment, these embodiment are for further illustrating the present invention, and do not mean that the scope of the present invention that limits.
The structure of the anti-human Tim-3 monoclonal antibody hybridoma cell of embodiment mono-strain
1. material
Freund's complete adjuvant and freund 's incomplete adjuvant, colouring reagents TMB:Sigma company product; 20% foetal calf serum: Beijing Heng Shengma of unit biotechnology research institute product; Serum-free RPMI 1640:Gibco company product; SP2/0 cell: ATCC introduces, and Institute of Basic Medical Sciences, A Cademy of Military Medical Sciences, preserves; Balb/c and C57BL/6 mouse: Military Medical Science Institute's Experimental Animal Center provides; All the other reagent are commercial.
2. method and result
(1) Balb/c mouse immune.Select 6 of the female Balb/c mouse in 4～6 week age, with 100 μ g ricin in inguinal region subcutaneous inoculation, the first pin Freund Freund's complete adjuvant, the second pin Freund Freund's incomplete adjuvant, immunity in every 3 weeks 1 time, altogether immunity 3 times.Tail vein blood after the 3rd immunity, detects antibody production with indirect ELISA, in fusion first 3 days, with 100 μ g ricin abdominal cavity booster immunizations once, within the 3rd day, merges.
(2) cytogamy.After immune mouse is plucked to eyeball, de-neck is put to death, and the aseptic mouse boosting cell of winning, carries out cytogamy according to a conventional method.Concrete grammar is: after 1. immune mouse being plucked to eyeball bloodletting, de-neck is put to death, 75% alcohol-pickled 3min, and aseptic taking-up spleen, with 200 order steel meshes grinding individual cells suspensions, serum-free RPMI 1640 washes twice and numeration; 2. collect the SP2/0 cell of logarithmic phase, wash twice with serum-free RPMI1640 and also count; 3. in SP2/0 cell: splenocyte=1: 5 ratio is mixed two kinds of cells, wash 1 time with RPMI 1640, abandon most supernatant, gently cell is broken up; 4. in the 1min time, slowly add 1ml50%PEG (M
wbe 1500) solution, put 37 ℃ of water-bath 1min; 5. at 1min, 2min, in the 5min time, add serum-free RPMI16401ml, 5ml, 10ml, 10ml; 6. the centrifugal 7min of 800r/min, abandons supernatant, cell is hanged as far as possible gently; 7. add HAT (the Sigma)-RPMI RPMI-1640 containing 20%FCS, adjusting cell concn is 2 × 10
6/ ml, mixes rear dropping and is being covered with nurse cell (1 × 10
4cells/well) in 96 well culture plates (Gibco), 100 μ l/ holes, are placed in the 5%CO of 37 ℃
2in incubator, cultivate.
(3) screen anti-human Tim-3 monoclonal antibody hybridoma cell with indirect ELISA.With the coated elisa plate of 10 μ g/ml recombinant human Tim-3 (rhTim-3), spend the night and seal in 4 ℃.Add successively cell culture supernatant to be measured (37 ℃ of 1h, PBST washes plate 4 times), and the 50 μ l HRP-GAM (37 ℃ of 45min, PBST washes plate 4 times) of dilution in 1: 500.After tmb substrate colour developing, measure OD value in 450nm wavelength.
(4) hybridoma cell clone.Screening positive cell clone subclone repeatedly again with indirect elisa method, is 100% positive until all Hybridoma Cell Culture supernatants detect.The cloning limiting dilution assay of hybridoma: 1. prepared nurse cell on the same day or first 1 day of cloning: de-neck is put to death kunming mice, 75% alcohol-pickled sterilization skin, the aseptic skin of abdomen of peeling off, syringe extracts 5ml RPMI-1640 and injects mouse peritoneal, sucking-off abdominal cavity washing lotion after repeatedly rinsing, after diluting with the RPMI-1640 that adds 20% foetal calf serum, splash into 96 orifice plates, the about 0.1ml in every hole.2. the hybridoma of getting a little cloning to be done moves in another sterile test tube, and accurate counting.3. limiting dilution assay carries out subclone.4. culture plate is placed in to the 5%CO of 37 ℃
2in incubator, cultivate, after approximately 5 days, can be observed under the microscope cell clone.Change liquid in good time, detect, get positive monoclonal cell strain and carry out enlarged culturing, freeze-stored cell strain in time.
(5) determining of hybridoma immunoglobulin (Ig) hypotype.With sheep anti-mouse igg 1, IgG2a, IgG2b and IgG3, culture supernatant after concentrated with regard to hybridoma is done the experiment of two-phase agar diffusion, result shows that Hybridoma Cell Culture supernatant can only form in conjunction with band with sheep anti-mouse igg 2a antibody, proves that the secreted immunoglobulin (Ig) hypotype of L3D hybridoma is IgG2a.
By above-mentioned steps, screen people Tim-3 monoclonal antibody L3D.
Embodiment bis-has the screening and identification of people Tim-3 neutralizing monoclonal antibody hybridoma cell strain
1. material
With embodiment mono-.
2. method and result
(1) specific binding activity of use ELISA method screening, identifier Tim-3 neutralizing monoclonal antibody L3D and antigen: coated recombinant human Tim-3 albumen respectively, and sulphur oxygen cyclase protein (Trx) reference protein, add different dilution anti-human Tim-3 antibody, and the anti-mouse IgG antibody of HRP mark, use the method for indirect ELISA to detect antigen-binding activity and the specificity of L3D.Result shows, L3D energy specific binding people Tim-3 molecule, and titre is higher.
(2) stream Schwann Cells art (FACS) detect the combination of Tim-3 on monoclonal antibody L3D and people U937 cell and with mouse RAW264.7 cell on the cross coupled activity of Tim-3.The FACS method routinely of testing is carried out.Result demonstration, L3D energy specific binding people Tim-3 molecule, also has certain cross coupled activity with mouse Tim-3 molecule.
(3) be monoclonal antibody L3D in and activation analysis.Restructuring Gal-9 is added to people THP1 cell cultures, and induction the latter's tune is died, and adds respectively the L3D antibody of different concns in above-mentioned system, cultivates after 24-48 hour, by the tune of the flow cytometer detection THP1 cell ratio of dying.Result shows that Gal-9 can induce people THP1 apoptosis (Annexcin-V
+pI
-cell), and add L3D in above-mentioned system after, the apoptosis rate of Gal-9 induction significantly declines, prompting monoclonal antibody L3D has good neutralization active in vitro.
Pass through above-mentioned steps, the people Tim-3 semi-lactosi binding site neutralizing monoclonal antibody that has obtained called after L3D is handed over oncocyte, in vitro in experiment, L3D can in and the apoptotic effect of GAL-9/Tim-3 induction, neutralizing monoclonal antibody L3D also can have certain cross reactivity with the Tim-3 of mouse.
Embodiment tri-people Tim-3 neutralizing monoclonal antibody hybridoma L3D are light, angling of heavy chain gene got
1. material
Primer: PHs1, is shown in SEQ ID NO:11 in sequence table; PHs2, is shown in SEQ ID NO:12 in sequence table; PHa1, is shown in SEQ ID NO:13 in sequence table; PLs1 is shown in SEQ ID NO:14 in sequence table, and PLs2 is shown in SEQ ID NO:15 in sequence table; PLa1, is shown in SEQ ID NO:16 in sequence table; .DNA fragmentation purification kit: OMEGA biotechnology company product; T4DNA ligase enzyme: New England Biolabs product; Carrier PGEM Teasy:Promega company product; Competence bacterium JM109: purchased from Promega company.All the other are with embodiment bis-.
2. methods and results
(1) the neutralizing monoclonal antibody L3D cell 5 × (10 of taking the logarithm vegetative period
6～10
7) individual, centrifugal removal supernatant, evenly upsprings cell.Adding 1ml TRIzol (Invitrogen) repeatedly blows and beats and makes the abundant cracking of cell, vibrate after 5 minutes, add 0.2ml chloroform, vibrate 15 seconds, room temperature is placed 2～3min, 2 ℃～8 ℃ 12000r/min, centrifugal 15 minutes, get supernatant in another new pipe, add 500 μ l Virahols and mix rear room temperature placement 10 minutes, 2 ℃～8 ℃ centrifugal 10min of 12000r/min.75% washing with alcohol precipitation, after being dried, the deionized water dissolving precipitation with 20 μ l without RNA enzyme.
(2) get the solution containing the total RNA of 1 μ g, add successively AMV5 × damping fluid 4 μ l, (500ng/ μ is 0.5 μ l l) for Oligo (dT), 2.5mmol/L dNTP 2 μ l, (50U/ μ is 0.5 μ l l) for Rnasin, mend deionized water to 20 μ l, ThermoScript II 2～5U, 42 ℃ are extended 1 hour.95 ℃ of sex change 5min, put in ice bath, and product is cDNA the first chain.With two couples of Auele Specific Primer PHs1, PHa1 and PLs1, PLa1, in 20 μ l PCR reaction systems, add respectively reverse transcription product 2 μ l, Taq enzyme 10 × buffer 2 μ l, the each 1 μ l of upstream and downstream primer, 2.5mmol/L dNTP 1 μ l, add Taq enzyme 1～2U, mend deionized water to 20 μ l.95 ℃ of sex change 2 minutes, loop parameter is: 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, totally 30 circulations, extend 10min after 72 ℃.
(3) with the DNA fragmentation of isolating wish recovery, cut the blob of viscose containing target DNA fragment under long wave ultraviolet light, put into centrifuge tube, add three times of change glues that colloid is long-pending, blob of viscose is dissolved in 55 ℃ of water-baths completely.With DNA fragmentation purification kit reclaim DNA fragmentation and by the DNA fragmentation of purifying in the aqueous solution, by the PCR product reclaiming in T4DNA ligase enzyme damping fluid by after the ratio (mol ratio) of 2: 1 and carrier PGEMTeasy mixing, add the T4DNA ligase enzyme of 0.5U to spend the night in 16 ℃ of connections, the cumulative volume of ligation is 10 μ L.
(4) get connecting fluid 10 μ l, be added in 200 μ l competence bacterium JM109 and softly mix, ice bath 30min, 42 ℃ of water-bath heat-shockeds 90 seconds, proceed to rapidly ice bath 2min, add 800 μ l LB substratum, proceed to 37 ℃ of constant-temperature tables, with the speed shake 45min of 150r/min, the centrifugal 1min of 4000r/min, discard 800 μ l supernatants, get precipitation and coat the solid LB flat board containing Amp (final concentration is 100 μ g/ml), flat-plate inverted is placed in to 37 ℃ of incubator 12～18h.
(5) the single clone of picking in above-mentioned flat board, is inoculated in the LB substratum containing acillin (100 μ g/ml).37 ℃ of constant-temperature table 170rpm, concussion overnight incubation.Get 3ml bacterium liquid and add in 1.5mlEppendorf pipe, the centrifugal 1min of 10000r/min, abandons supernatant.Precipitation thalline is resuspended in 100 μ L solution I, adds freshly prepared solution II 200 μ L, turn upside down for several times light and slowly, to liquid become limpid till.Subsequently, then add 150 μ L solution III, gently turn upside down and make liquid blending for several times, now occur a large amount of white flockss.4 ℃, the centrifugal 5min of 12000r/min, gets supernatant and adds in another Eppendorf pipe, adds the saturated phenol of isopyknic Tris-HCl, and after concuss, the centrifugal 5min of 12000rpm, moves to upper water in one new pipe mutually.Add 500 μ L chloroforms, extracting once again again.Thereafter, carefully draw upper strata water, move in a new pipe, the dehydrated alcohol that adds 2 times of volumes mixes, and places 3h in-20 ℃.4 ℃, the centrifugal 10min of 12000rpm, abandons supernatant, washes precipitation 2 times with 70% ethanol, and drying at room temperature 20min, dissolves with 40 μ L aseptic double-distilled waters, carries out PCR evaluation and DNA sequencing analysis.
Build and contained that people Tim-3 neutralizing antibody L3D is light, the carrier of heavy chain gene by above-mentioned steps, be that mouse immuning ball protein is light, heavy chain gene through sequencing analysis, sequence alignment, its corresponding aminoacid sequence is respectively SEQ ID NO:1, SEQ ID NO:2.
The Protein A purifying of embodiment tetra-monoclonal antibody L3D and the intervention experiment to pyemia, inflammatory bowel animal model
1. material
Protein A Sepharose CL 4B post albumen post: Ben Yuan Zhenyang, Beijing Bioisystech Co., Ltd product; All the other are with embodiment bis-.
2. method and result
(1) by containing, people Tim-3 neutralizing antibody L3D is light, the carrier of heavy chain gene proceeds in mammalian cell, expresses.Collect and express supernatant, add 1mL pH8.0,0.1moL/L phosphoric acid buffer is also used pH 9.0, and 1moL/L TRIS-HCL adjusts pH to 9.0.Antibody expression supernatant is added and used in the Protein A Sepharose CL 4B albumen post that 0.1moL/L phosphoric acid buffer pH 8.0 balances are good, wash pillar with above-mentioned damping fluid, until can't detect foreign protein in effluent liquid.With the citrate buffer solution wash-out of pH 3.0, collect effluent liquid, and immediately with the neutralization of 1moL/L pH 8.5TRIS-HCL damping fluid, with pH 7.2, the 0.01M PBS 72h that dialyses.OD is surveyed in sampling on ultraviolet spectrophotometer
260, OD
280, calculate protein content, after freeze-drying in-20 ℃ of preservations (Fig. 6 antibody protein electrophorogram).
(2) reference method (Xu.R, et al.Eur.Immunol.2010.40:1079; Li X, et al.Clinical Immunol.2010,134:169). set up pyemia and inflammatory bowel model C57BL/6 mouse.Concrete grammar: 1) pyemia model: first configure narcotic, new speed dormancy and ketamine are mixed at 2: 1.5, then use one times of normal saline dilution, inject 60 μ l to every C57 mouse peritoneal, can anaesthetize for about 1-2 minute.After mouse holonarcosis, be fixed and lie on the back on operating table, dip 75% alcohol with cotton balls and sterilize at belly.At xiphoid-process, next refers to that place cuts off the long otch of 2cm from top to bottom along hunter's line, carefully cut off peritonaeum herein, expose belly, carefully raise peritonaeum with tweezers, at the bottom right of abdominal cavity otch fathom, to caecum, generally its color is slightly shallow and expand, and is expanded the extracting gently of cecum, distance cecum 2/3rds places ligation on caecum, too not tight (preventing that bowel necrosis from affecting experimental result) approximately 2/3 width of ligation.On the outside of ligation puncture intestines wall 2 times, carefully extrude appropriate content with No. 8 syringe needles, caecum and content are put back in abdominal cavity, keep the physiological location of caecum constant as far as possible.Successively close peritonaeum and skin, respectively with the ligation of band pin suture.The important indicator that model is successfully established is that one week survival rate of mouse is 20-30%.; 2) the C57 male mice in age in inflammatory bowel model: 6-8 week, is divided into 2 groups, 10 every group.Control group is normally drunk water, the 4%DSS solution of experimental group drink DSS preparation.Observing Mouse Weight every day changes.
For pyemia model, mouse is divided into experimental group and control group, every group 10, (200ug/ only within first 12 hours, to give respectively L3D antibody in modeling, abdominal injection) or with amount homotype control antibodies, the state of an illness of then observing animal comprised the variation of survival rate, body weight, and got the spleen cell of experimental group and control animals at 5-7 days, with methods analyst cytokine IL-1beta, the IL-6 of quantitative PCR, expression.Result shows that L3D has significantly increased the mortality ratio of pyemia animal pattern, has reduced the body weight of animal pattern, and the expression of cytokine, IL-1beta, IL-6 significantly raises, and prompting L3D has neutralization activity in good body.
For inflammatory bowel model, mouse is divided into experimental group and control group, every group 10, give respectively L3D antibody in modeling (200ug/ only simultaneously, abdominal injection) or with amount homotype control antibodies, then observe the variation of the body weight of animal, and got the spleen cell of experimental group and control animals at 5-7 days, with the methods analyst cytokine IL-17 of quantitative PCR, the expression of IFN-g.Result shows that L3D has significantly reduced the body weight of animal pattern, cytokine IL-17, and the expression of IFN-g significantly raises, and prompting L3D has neutralization activity in good body.
Claims (7)
1. an anti-human Tim-3 neutralizing monoclonal antibody, comprises light chain CDR1-3 and heavy chain CDR4-6, it is characterized in that, the aminoacid sequence of described light chain CDR1-3 is:
CDR1: as shown in SEQ ID NO:5 in sequence table;
CDR2: as shown in SEQ ID NO:6 in sequence table;
CDR3: as shown in SEQ ID NO:7 in sequence table;
The aminoacid sequence of described heavy chain CDR4-6 is:
CDR4: as shown in SEQ ID NO:8 in sequence table;
CDR5: as shown in SEQ ID NO:9 in sequence table;
CDR6: as shown in SEQ ID NO:10 in sequence table.
2. monoclonal antibody as claimed in claim 1, is characterized in that, the aminoacid sequence of described variable region of light chain is as shown in SEQ ID NO:1 in sequence table, and the aminoacid sequence of described variable region of heavy chain is as shown in SEQ ID NO:2 in sequence table.
3. the monoclonal antibody as described in any one in claim 1-2, it is characterized in that, the encoding sequence of described variable region of light chain is as shown in the nucleotide sequence of SEQ ID NO:3 in sequence table, and the encoding sequence of described variable region of heavy chain is as shown in the nucleotide sequence of SEQ ID NO:4 in sequence table.
4. a Tim-3 detection kit that contains the monoclonal antibody described in any one in claim 1-3.
5. a pharmaceutical composition, it comprises the monoclonal antibody described in any one in claim 1-3.
6. the application of the monoclonal antibody described in any one in preparation Tim-3 diagnostic kit in claim 1-3.
7. the application of the monoclonal antibody described in any one in preparation treatment pyemia or inflammatory bowel medicine in claim 1-3.
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