CN102492038A - 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,, also relate to this monoclonal antibody, and diagnosis and treatment Tim-3 high expression level cause the application in the disease in preparation Tim-3 detection reagent in particular to anti-people Tim-3 neutralizing monoclonal antibody L3D.
Background technology
Tim-3 structurally belongs to T cell immunoglobulin and Saliva Orthana (T cell Immunoglobulin domain and Mucin domain protein, Tim) family member.Since the wide participation of Tim family the process of immune regulation of body, its function receives people's attention just day by day.Tim-3 is specific expressed in activated T h1, Th17 effector cell surface, and is not expressed in the Th2 cell.(Galectin-9 Gal-9) is the native ligand of Tim-3 to existing known galactose-binding protein-9, and this molecule wide expression is in the periphery immunity system; Gal-9 combines with Tim-3 molecular specificity on Th1, the Th17 cell; Can cause the latter's accent and die, downward modulation immunoreation, inducing immune tolerance.At present, research confirm the Tim-3 molecule mainly the function wide participation through regulating different CD4+T cell subsets autoimmune disease, transplant rejection, immunne response process [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.].
Present research confirms that the unusual and numerous disease that Tim-3 expresses has confidential relation; As discover HIV; Patient and some tumour patients that HCV infects, Tim-3 expresses and raises on its T cell, dies because Tim-3 can mediate T effector cell's accent; Transmit the negativity conditioning signal, can cause the body's immunological function paralysis.Any blocking-up Tim-3/Gal-9 bonded factor 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; In diseases such as some autoimmune diseases such as systemic lupus erythematous (SLE), asthma; Because the rising that Gal-9 or Tim-3 express causes the function of Th1 cell to be suppressed, and then breaks intravital immunologic balance; Cause that pathologic Th2 cell activity strengthens, and causes the morbidity of disease.In this case, any blocking-up Tim-3/Gal-9 bonded factor all can help the recovery of immunologic balance in the body, alleviates the process of disease.All can be as inject anti-Tim-3 antibody or reorganization Tim-3 fusion rotein to the asthmatic model animal through blocking-up Tim-3/Gal-9 combination; Strengthen the Th1 cytoactive; Correction is by the cell-mediated SOA of Th2; Recover Th1/Th2 cell balance [Hu WK, 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 in the body; 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; In some autoimmune diseases such as inflammatory bowel and type i diabetes model; Discover blocking-up Tim-3 increased activity Th1 effector cell's function in the body; Further increased the weight of the autoimmunization damage, this result proves Tim-3 play a significant role in the keeping of immunologic balance in vivo [Sanchez-Fueyo A, Tian J once more; 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 that the Tim-3 path has important immunoloregulation function, and generation, the development of the unusual and multiple disease of its expression have substantial connection.Although research shows the neutralizing antibody of Tim-3 and also in the some diseases model, has brought into play good intervention effect; But the Tim-3 antibody that does not still have listing at present both at home and abroad; What therefore foundation and development had independent intellectual property right is the detection and the diagnosis and treatment articles for use on basis with antibody, has important practical significance for multiple diseases related intervention.
Summary of the invention
The invention discloses the monoclonal antibody L3D of a kind of anti-people Tim-3; Said monoclonal antibody comprises light chain and heavy chain; Shown in SEQ ID NO:1, SEQ ID NO:2 in the sequence table, its encoding sox is respectively shown in SEQ ID NO:3, SEQ ID NO:4 in the sequence table respectively for its amino acid variable region sequences.
The invention also discloses the purposes of anti-people Tim-3 neutralizing monoclonal antibody L3D, be included in preparation Tim-3 detection reagent, and the application in the disease that caused of diagnosis and treatment Tim-3 high expression level.
The disease that Tim-3 high expression level of the present invention caused comprises HIV and HCV virus infection, tumour, autoimmune disease such as systemic lupus erythematous (SLE), asthma etc.
The aminoacid sequence of the complementary determining region CDR1 of the light chain protein matter molecule variable region of monoclonal antibody of the present invention, CDR2, CDR3 is respectively shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 in the sequence table.The aminoacid sequence of the complementary determining region CDR4 of the heavy chain protein matter molecule variable region of said monoclonal antibody, CDR5, CDR6 is respectively shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 in the sequence table.
The invention also discloses the preparation method of said monoclonal antibody L3D, mainly comprise as follows:
1. the structure of anti-people Tim-3 monoclonal antibody hybridoma cell strain
At first, prokaryotic expression people Tim-3 albumen, immune Balb/c mouse carries out cytogamy with ordinary method.With indirect elisa method screening positive cell clone subclone repeatedly again, detecting up to all Hybridoma Cell Culture supernatants is 100% positive.Hybridoma L3D has been carried out chromosome karyotype analysis, and hybridoma L3D karyomit(e) average number is 106, uses the secreted Tegeline hypotype of two-phase agar diffusion experiment proof L3D hybridoma to be IgG2a.Fig. 1 shows that monoclonal antibody L3D can specificity combine people Tim-3 molecule.
2. the screening and the evaluation of the strain of anti-people Tim-3 monoclonal antibody hybridoma cell
Use the FACS method, the Tim-3 molecule on the people U937 cell is combined experiment with regard to people Tim-3 antibody.The result shows that L3D can combine the Tim-3 molecule on the U937, and shows and mouse Tim-3 bonded cross reactivity (Fig. 2).Use the neutralization of Lymphocyte Apoptosis experimental verification Tim-3 antibody active simultaneously.The result shows that L3D obviously suppresses the apoptosis (Fig. 3) of Gal-9 inductive people THP1 cell
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 is measured at ultraviolet spectrophotometer, calculates protein contnt.Set up the pyemia model with the C57BL/6 mouse, and autoimmunity inflammatory bowel animal model.After giving injected in mice L3D antibody or homotype control antibodies, observe the variation of mouse body weight or survival rate, analysis list clonal antibody L3D BA in vivo.The result shows that L3D antibody has significantly changed the survival rate (Fig. 4) of pyemia mouse, has influenced the inflammation degree (Fig. 5) of inflammatory bowel mouse, and The above results points out anti-people Tim-3 monoclonal antibody L3D to have good neutralization activity in vivo.
4. light, the angling of heavy chain gene of hybridoma L3D got
Extract neutralizing monoclonal antibody L3D cell RNA,, angle the weight chain gene of getting antibody with two pairs of Auele Specific Primers through RT-PCR.Conventional method connects into carrier, and the transformed competence colibacillus bacterium is cultivated the single bacterium colony of back picking, extracts and carries out the dna sequencing analysis after plasmid PCR identifies.
Pass through above-mentioned steps; Made up 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. light, heavy chain variable region gene sequence of monoclonal antibody L3D and aminoacid sequence confirms
With the online software of www.expasy.org light, the weight chain variable region nucleotide sequence of coding human Tim-3 neutralizing monoclonal antibody L3D is translated as its amino acid sequence coded, light, the weight chain variable region amino acid sequence of monoclonal antibody L3D is shown in SEQ ID NO:1 in the sequence table and SEQ ID NO:2.The aminoacid sequence of confirming complementary determining region CDR1, CDR2 and CDR3 in the light chain variable region sequence according to the Kabat DB is respectively shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 in the sequence table.The aminoacid sequence of complementary determining region CDR4, CDR5 and CDR6 in the weight chain variabl area sequence is respectively shown in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 in the sequence table.
The primer that the present invention uses an Analysis of Nested Design is successfully handed over from the people Tim-3 neutralizing monoclonal antibody L3D that cultivates and has been cloned the oncocyte that antibody is light, heavy chain variable region gene.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-people Tim-3 antibody.Monoclonal antibody of the present invention is light based on above-mentioned people Tim-3 neutralizing monoclonal antibody L3D of being cloned into, heavy chain variable region gene; Can make up and express multiple small molecules genetic engineering antibody, like single-chain antibody, single domain antibody, chimeric antibody, Fab antibody, antibody fusion protein etc.; Based on said gene encoded polypeptide or protein, can crosslinkedly multiple bioactive molecules, preparation is used for diagnosis or the medicine that the Tim-3 expression level detects, diagnoses and treat Tim-3 disease that high expression level causes.
Description of drawings
The screening of Fig. 1 ELISA method, identifier Tim-3 neutralizing monoclonal antibody L3D and antigenic specific binding activity.Contrast antigen is sulphur oxygen cyclase protein (Trx).
Fig. 2 flow cytometer (FACS) detect Tim-3 on monoclonal antibody L3D and the people U937 cell combine active, and with mouse RAW264.7 cell on the cross coupled activity of Tim-3.On A: monoclonal antibody L3D and the people U937 cell Tim-3 combine active; The cross coupled of Tim-3 is active on B: monoclonal antibody L3D and the mouse RAW264.7 cell.
The extracorporeal neutralizing activity analysis of Fig. 3 monoclonal antibody L3D.Gal-9 albumen can induce the accent of THP1 to die through the Tim-3 on the cell, this figure adopts PI and Annexcin-V to do apoptotic cell dyeing experiment respectively, observe Tim-3 antibody to Gal-9 induce transfer die in and barrier effect.
Fig. 4 monoclonal antibody L3D is to the influence of the pyemia course of disease.Set up the pyemia model the C57BL/6 mouse, give simultaneously and L3D or homotype control antibodies, observe the survival rate (A) of animal then, and use inflammatory mediator IL-6 (B) after the intervention of ELISA methods analyst antibody respectively, the expression of IL-1beta (C) changes.
Fig. 5 monoclonal antibody L3D is to the influence of the mouse inflammatory bowel course of disease.Set up the inflammatory bowel model the C57BL/6 mouse, give simultaneously and L3D or homotype control antibodies, observe changes of weight and the cytokine IL-17 of mouse, the variation of IFN-g then.A: the body weight of mouse is 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 heavy chain and light chain respectively), and C: molecular weight Marker.
Embodiment
Can more easily understand content of the present invention through consulting following embodiment, these embodiment are just for further specifying the present invention, and do not mean that qualification scope of the present invention.
The structure of embodiment one anti-people Tim-3 monoclonal antibody hybridoma cell strain
1. material
Fu Shi Freund's complete adjuvant and freund 's incomplete adjuvant, colouring reagents TMB:Sigma Company products; 20% foetal calf serum: Beijing Heng Shengma of unit biotechnology research institute product; Serum-free RPMI 1640:Gibco Company products; 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 ages in week for use, in the subcutaneous immunity of inguinal region, first pin adds the Fu Shi Freund's complete adjuvant with 100 μ g ricin, and second pin adds freund 's incomplete adjuvant, per 3 week immunity 1 time, immune 3 times altogether.The 3rd immunity back tail vein blood detects the antibody production with indirect ELISA, in fusion preceding 3 days, with 100 μ g ricin abdominal cavity booster immunizations once, merges in the 3rd day.
(2) cytogamy.Mice immunized plucked take off neck behind the eyeball and put to death, the aseptic mouse boosting cell of winning carries out cytogamy by ordinary method.Concrete grammar is: take off neck execution after 1. mice immunized being plucked the eyeball bloodletting, and 75% alcohol-pickled 3min, aseptic taking-up spleen grinds the individual cells suspension with 200 order steel meshes, and serum-free RPMI 1640 washes twice and numeration; 2. collect the SP2/0 cell of logarithmic phase, wash twice and numeration with serum-free RPMI1640; 3. press the SP2/0 cell: two kinds of cells of mixed of splenocyte=1: 5, wash 1 time with RPMI 1640, abandon most supernatant, gently cell is broken up; 4. slowly add 1ml50%PEG (M in the time at 1min
WBe 1500) solution, put 37 ℃ of water-bath 1min; 5. add serum-free RPMI16401ml, 5ml, 10ml, 10ml in the time at 1min, 2min, 5min; 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 that contains 20%FCS, the adjustment cell concn is 2 * 10
6/ ml drips behind the mixing and is being covered with nurse cell (1 * 10
4Cells/well) in 96 well culture plates (Gibco), 100 μ l/ holes place 37 ℃ 5%CO
2Cultivate in the incubator.
(3) screen anti-people Tim-3 monoclonal antibody hybridoma cell with indirect ELISA.Encapsulate elisa plate with 10 μ g/ml recombinant human Tim-3 (rhTim-3), spend the night and seal in 4 ℃.Add cell culture supernatant to be measured (37 ℃ of 1h, PBST wash plate 4 times) successively, and the 50 μ l HRP-GAM (37 ℃ of 45min, PBST wash plate 4 times) of dilution in 1: 500.After the tmb substrate colour developing, measure the OD value in the 450nm wavelength.
(4) hybridoma cell cloneization.Clone subclone repeatedly with indirect elisa method screening positive cells, detecting up to all Hybridoma Cell Culture supernatants is 100% positive again.Hybridoma cloning is used limiting dilution assay: 1. at the same day or preceding 1 day of cloning preparation nurse cell: take off neck and put to death kunming mice; 75% alcohol-pickled sterilization skin; The aseptic skin of abdomen of peeling off, syringe extract the 5ml RPMI-1640 and inject mouse peritoneal, repeatedly sucking-off abdominal cavity, flushing back washing lotion; With splashing into 96 orifice plates, the about 0.1ml in every hole after the RPMI-1640 dilution that adds 20% foetal calf serum.2. get a little and wait that the hybridoma of doing cloning moves in another sterile test tube, and accurate counting.3. limiting dilution assay carries out subclone.4. culture plate is placed 37 ℃ 5%CO
2Cultivate in the incubator, can be observed cell clone at microscopically after about 5 days.Change liquid in good time, detect, get the positive monoclonal cell strain and carry out enlarged culturing, freeze-stored cell strain in time.
(5) hybridoma Tegeline hypotype confirms.With sheep anti-mouse igg 1, IgG2a, IgG2b and IgG3; Culture supernatant after concentrating with regard to hybridoma is done the experiment of two-phase agar diffusion; The result shows that the Hybridoma Cell Culture supernatant can only combine band with the formation of sheep anti-mouse igg 2a antibody, proves that the secreted Tegeline hypotype of L3D hybridoma is IgG2a.
Through above-mentioned steps, screen people Tim-3 monoclonal antibody L3D.
Embodiment two has the screening and the evaluation of people Tim-3 neutralizing monoclonal antibody hybridoma cell strain
1. material
With embodiment one.
2. method and result
(1) with the screening of ELISA method, identifier Tim-3 neutralizing monoclonal antibody L3D and antigenic specific binding activity: encapsulate recombinant human Tim-3 albumen respectively; And sulphur oxygen cyclase protein (Trx) reference protein; Add different dilution anti-people Tim-3 antibody; And the anti-mouse IgG antibody of HRP mark, detect antigen-binding activity and the specificity of L3D with the method for indirect ELISA.The result shows that L3D can specificity combine people Tim-3 molecule, and titre is higher.
(2) stream Schwann Cells art (FACS) detect combining of Tim-3 on monoclonal antibody L3D and the people U937 cell and with mouse RAW264.7 cell on the cross coupled activity of Tim-3.Experiment is undertaken by conventional FACS method.The result shows that L3D can specificity combine people Tim-3 molecule, also has certain cross coupled active with mouse Tim-3 molecule.
(3) be monoclonal antibody L3D in and activation analysis.The Gal-9 that will recombinate is added to people THP1 cell cultures, induces the latter's accent to die, and in above-mentioned system, adds the L3D antibody of different concns respectively, cultivates after 24-48 hour, with the accent of the flow cytometer detection THP1 cell ratio of dying.The result shows that Gal-9 can induce people THP1 apoptosis (Annexcin-V
+PI
-Cell), and Gal-9 inductive apoptosis rate significantly descends after in above-mentioned system, adding L3D, and prompting monoclonal antibody L3D has neutralization preferably active external.
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 experiment in vitro; L3D can in GAL-9/Tim-3 inductive apoptotic effect, neutralizing monoclonal antibody L3D also can have certain cross reactivity with the Tim-3 of mouse.
Embodiment three people Tim-3 neutralizing monoclonal antibody hybridoma L3D are light, angling of heavy chain gene got
1. material
Primer: PHs1 sees SEQ ID NO:11 in the sequence table; PHs2 sees SEQ ID NO:12 in the sequence table; PHa1 sees SEQ ID NO:13 in the sequence table; PLs1 sees SEQ ID NO:14 in the sequence table, and PLs2 sees SEQ ID NO:15 in the sequence table; PLa1 sees SEQ ID NO:16 in the sequence table; Dna fragmentation purification kit: OMEGA biotechnology Company products; T4DNA ligase enzyme: New England Biolabs product; Carrier PGEM Teasy:Promega Company products; Competence bacterium JM109: available from Promega company.All the other are with embodiment two.
2. methods and results
The neutralizing monoclonal antibody L3D cell 5 * (10 of (1) taking the logarithm vegetative period
6～10
7) individual, centrifugal removal supernatant is evenly upspring cell.Add 1ml TRIzol (Invitrogen) and blow and beat repeatedly and make the abundant cracking of cell, vibrate after 5 minutes, add the 0.2ml chloroform; Vibrated 15 seconds, room temperature is placed 2～3min, 2 ℃～8 ℃ 12000r/min; Centrifugal 15 minutes; Get supernatant in another new pipe, add behind the 500 μ l Virahol mixings room temperature and placed 2 ℃～8 ℃ centrifugal 10min of 12000r/min 10 minutes.75% washing with alcohol deposition after the drying, does not have the deionized water dissolving deposition of RNA enzyme with 20 μ l.
(2) get the solution that contains the total RNA of 1 μ g, add AMV5 * damping fluid 4 μ l successively, Oligo (dT) (500ng/ μ l) 0.5 μ l; 2.5mmol/L dNTP 2 μ l; Rnasin (50U/ μ l) 0.5 μ l mends deionized water to 20 μ l, ThermoScript II 2～5U, and 42 ℃ were extended 1 hour.95 ℃ of sex change 5min put in the ice bath, and product is cDNA first chain.With two couples of Auele Specific Primer PHs1, PHa1 and PLs1, PLa1, in 20 μ l PCR reaction systems, add reverse transcription product 2 μ l respectively; Taq enzyme 10 * buffer 2 μ l, 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 are extended 10min after 72 ℃.
(3) with isolating the dna fragmentation that desire reclaims, under long wave ultraviolet light, downcut the blob of viscose that contains target DNA fragment, put into centrifuge tube, add the long-pending change glue of three times of colloids, blob of viscose is dissolved in 55 ℃ of water-baths fully.With the dna fragmentation purification kit reclaim dna fragmentation and with the dna fragmentation of purifying in the aqueous solution; With the PCR product that reclaims in T4DNA ligase enzyme damping fluid by after 2: 1 the ratio (mol ratio) and carrier PGEMTeasy mixing; The T4DNA ligase enzyme that adds 0.5U spends the night in 16 ℃ of connections, and the TV of ligation is 10 μ L.
(4) get connection liquid 10 μ l, be added among the 200 μ l competence bacterium JM109 and soft mixing, ice bath 30min; 42 ℃ of water-bath heat-shockeds 90 seconds change ice bath 2min rapidly over to, add 800 μ l LB substratum; Change 37 ℃ of constant temperature shaking tables over to, shake 45min, the centrifugal 1min of 4000r/min with the speed of 150r/min; Discard 800 μ l supernatants, get deposition and coat the solid LB flat board that contains Amp (final concentration is 100 μ g/ml), flat board is inverted in 37 ℃ of incubator 12～18h.
(5) the single clone of picking in above-mentioned flat board is inoculated in the LB substratum that contains acillin (100 μ g/ml).37 ℃ of constant temperature shaking table 170rpm, the concussion overnight cultures.Get 3ml bacterium liquid and add in the 1.5mlEppendorf pipe, the centrifugal 1min of 10000r/min abandons supernatant.To precipitate thalline and be resuspended in the 100 μ L solution I, and add freshly prepared solution II 200 μ L, light and slow ground is put upside down up and down for several times, to liquid become limpid till.Subsequently, add 150 μ L solution III again, softly put upside down up and down and make for several times the liquid mixing, occur a large amount of white flockss at this moment.4 ℃, the centrifugal 5min of 12000r/min gets supernatant and adds in another Eppendorf pipe, adds the saturated phenol of isopyknic Tris-HCl, and behind the concuss, the centrifugal 5min of 12000rpm moves to upper water in the one new pipe mutually.Add 500 μ L chloroforms again, extracting once again., carefully draw upper strata water, move in the new pipe, add the absolute ethyl alcohol mixing of 2 times of volumes, place 3h in-20 ℃ thereafter.4 ℃, the centrifugal 10min of 12000rpm abandons supernatant, washes deposition 2 times with 70% ethanol, and drying at room temperature 20min with the dissolving of 40 μ L aseptic double-distilled waters, carries out PCR and identifies and the dna sequencing analysis.
Made up through above-mentioned steps and to have contained that people Tim-3 neutralizing antibody L3D is light, the carrier of heavy chain gene; Through sequencing analysis, sequence alignment is that mouse immuning ball protein is light, heavy chain gene, and its corresponding amino acid sequence is respectively SEQ ID NO:1, SEQ ID NO:2.
The Protein A purifying of embodiment four monoclonal antibody L3D reaches the intervention experiment to pyemia, inflammatory bowel animal model
1. material
Protein A Sepharose CL 4B post albumen post: this yuan Zhenyang, Beijing Bioisystech Co., Ltd product; All the other are with embodiment two.
2. method and result
(1) will contain that people Tim-3 neutralizing antibody L3D is light, the carrier of heavy chain gene changes in the mammalian cell, express.Collect and express supernatant, add 1mL pH8.0, the 0.1moL/L phosphoric acid buffer is also with pH 9.0, and 1moL/L TRIS-HCL adjusts pH to 9.0.Used the adding of antibody expression supernatant in the good Protein A Sepharose CL 4B albumen post of 0.1moL/L phosphoric acid buffer pH 8.0 balances, washed pillar, in effluent, detected less than till the foreign protein with above-mentioned damping fluid.With the citrate buffer solution wash-out of pH 3.0, collect effluent, and neutralize with 1moL/L pH 8.5TRIS-HCL damping fluid immediately, with pH 7.2, the 0.01M PBS 72h that dialyses.OD is surveyed in sampling on ultraviolet spectrophotometer
260, OD
280, calculate protein contnt, after the 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 the C57BL/6 mouse.Concrete grammar: 1) pyemia model: at first dispose narcotic, speed is slept newly to mix with ketamine at 2: 1.5, dilutes one times with saline water again, injects 60 μ l to every C57 mouse peritoneal, can anaesthetize in about 1-2 minute.After treating the mouse holonarcosis, be fixed and lie on the back on operating table, dip in cotton balls and get 75% alcohol and sterilize at belly.Next refers to locate cut off the long otch of 2cm from top to bottom along hunter's line at xiphoid-process, carefully cuts off peritonaeum here, exposes belly; Carefully raise peritonaeum with tweezers; The bottom right fathom of otch is to caecum in the abdominal cavity, and generally its color is shallow slightly and expand, and it is expanded extracting gently of cecum; Apart from cecum 2/3rds places ligation, too tight (preventing that bowel necrosis from influencing experimental result) about 2/3 width is not wanted in ligation on caecum.On the outside of ligation puncture intestines wall 2 times, carefully extrude an amount of content with No. 8 syringe needles, caecum and content are put back in the abdominal cavity, keep the physiological location of caecum constant as far as possible.Successively close and close peritonaeum and skin, respectively with the ligation of band pin suture.The important indicator that modelling is successful is that mouse one all survival rates are 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.Observe the mouse changes of weight every day.
For the pyemia model; Mouse is divided into experimental group and control group, and 10 every group, (200ug/ only to give L3D antibody in preceding 12 hours respectively in modeling; Abdominal injection) or together measure the homotype control antibodies; The state of an illness of observing animal then comprises the variation of survival rate, body weight, and gets the spleen cell of experimental group and control animals at 5-7 days, with the methods analyst cytokine IL-1beta of quantitative PCR, IL-6, expression.The 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 the good body.
For the inflammatory bowel model, mouse is divided into experimental group and control group, 10 every group; (200ug/ only to give L3D antibody simultaneously respectively in modeling; Abdominal injection) or together measure the homotype control antibodies, observe the variation of the body weight of animal then, 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.The 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 the good body.
Claims (7)
1. an anti-people Tim-3 neutralizing monoclonal antibody or its fragment comprise light chain CDR1-3 and heavy chain CDR4-6, it is characterized in that the aminoacid sequence of said light chain CDR1-3 is:
CDR1: shown in SEQ ID NO:5 in the sequence table;
CDR2: shown in SEQ ID NO:6 in the sequence table;
CDR3: shown in SEQ ID NO:7 in the sequence table;
The aminoacid sequence of said heavy chain CDR4-6 is:
CDR4: shown in SEQ ID NO:8 in the sequence table;
CDR5: shown in SEQ ID NO:9 in the sequence table;
CDR6: shown in SEQ ID NO:10 in the sequence table.
2. monoclonal antibody as claimed in claim 1 or its fragment is characterized in that, the aminoacid sequence of said variable region of light chain is shown in SEQ ID NO:1 in the sequence table, and the aminoacid sequence of said variable region of heavy chain is shown in SEQ ID NO:2 in the sequence table.
3. like any described monoclonal antibody of claim 1-2 or its fragment; It is characterized in that; The encoding sequence of said variable region of light chain is shown in the nucleotide sequence of SEQ ID NO:3 in the sequence table, and the encoding sequence of said variable region of heavy chain is shown in the nucleotide sequence of SEQ ID NO:4 in the sequence table.
4. one kind contains any described monoclonal antibody of claim 1-3 or its segmental Tim-3 detection kit.
5. pharmaceutical composition, it comprises any described monoclonal antibody of claim 1-3 or its fragment.
6. any described monoclonal antibody of claim 1-3 or its fragment application in preparation Tim-3 diagnostic kit.
7. any described monoclonal antibody of claim 1-3 or its fragment are preparing by the application in the medicine of Tim-3 disease that high expression level causes.
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