CN103936853A - Kit used for detecting Tim-3, and application method thereof - Google Patents
Kit used for detecting Tim-3, and application method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biotechnology, and discloses a kit used for detecting Tim-3, and an application method thereof. The invention also discloses monoclonal antibodies used for detecting Tim-3, or fragments of the monoclonal antibodies; the monoclonal antibodies or the fragments comprise light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; amino acid sequences of light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are represented by SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, or SEQ ID No.6 respectively. The monoclonal antibodies are capable of realizing specific binding with human Tim-3; the kit which is prepared from the monoclonal antibodies and is used for detecting human Tim-3 possesses relatively high sensitivity, and can be used for detecting of human Tim-3 with low concentration.
Description
Technical field
The invention belongs to technical field of bioengineering, particularly the monoclonal antibody of anti-human Tim-3, its preparation method and detect in TIM-3 reagent and apply in preparation.
Background technology
Tim-3 structurally belongs to T cell immunoglobulin and Saliva Orthana (T cell Immunoglob μ Lin domain and Mucin domain protein, Tim) family member.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.Except the Tim-3 molecule of film combining form, whether the Tim-3 molecule of solubility, and how to affect process of immune regulation be the content it be not immediately clear.At present, studies confirm that Tim-3 molecule is mainly by regulating different CD4
+the function wide participation of T cell subsets autoimmune disease, transplant rejection, the immunne response process such as anti-infective.
At present, studies confirm that the abnormal and numerous disease that Tim-3 expresses has close relationship, as the expression rising of Tim-3 on patient that HIV infects, patient that HCV infects, patient, pyemic patient and some tumour patient T cells that HBV infects is found in research.For example, compared with normal mouse, in Sepsis mouse model abdominal cavity, on T cell, DC cell, Tim-3 expression level significantly increases.Tim-3 can mediate T effector cell's tune and die, and transmits negativity conditioning signal, can cause the immunologic function paralysis of body.Research shows, the factor of any blocking-up Tim-3/Gal-9 combination all can make Th1, Th17 cell avoid death, strengthens T effector cell's activity, recovers immunologic function.
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.Inject anti-Tim-3 antibody for example to asthmatic model animal or restructuring Tim-3 fusion rotein all can, by blocking-up Tim-3/Gal-9 combination, strengthen Th1 cytoactive, correct by the cell-mediated symptoms of asthma of Th2, recover the interior Th1/Th2 cell balance of body.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 increased the weight of autoimmunization damage, this result proving again Tim-3 plays a significant role in the maintaining of immunologic balance in vivo.
Above-mentioned research data shows, Tim-3 path has important immunoloregulation function, the generation of the abnormal and various diseases that Tim-3 expresses, develops and has substantial connection.Although research shows that the neutralizing antibody of Tim-3 brought into play good intervention effect in some disease model,, the Tim-3 antibody of listing not at present.Therefore, provide a kind of Tim-3 antibody of high specificity, study of pathogenesis, medical diagnosis on disease and treatment to some disease have great importance.
Summary of the invention
In view of this, the invention provides monoclonal antibody, its preparation method and the application of a kind of anti-human Tim-3, the monoclonal antibody of this anti-human Tim-3 or its fragment energy specificity are combined with people Tim-3, can be for the preparation of the test kit that detects people Tim-3, gained test kit has higher sensitivity, can detect the people Tim-3 of low concentration.
In order to realize goal of the invention of the present invention, the present invention adopts following technical scheme:
The monoclonal antibody or its fragment that the invention provides a kind of anti-human Tim-3, it comprises light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3;
Light chain CDR1 has aminoacid sequence shown in SEQ ID NO:1;
Light chain CDR2 has aminoacid sequence shown in SEQ ID NO:2;
Light chain CDR3 has aminoacid sequence shown in SEQ ID NO:3;
Heavy chain CDR1 has aminoacid sequence shown in SEQ ID NO:4;
Heavy chain CDR2 has aminoacid sequence shown in SEQ ID NO:5;
Heavy chain CDR3 has aminoacid sequence shown in SEQ ID NO:6.
Preferably, monoclonal antibody provided by the invention or its fragment, its variable region of light chain comprises light chain CDR1, light chain CDR2 and light chain CDR3, its variable region of heavy chain comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3;
Light chain CDR1 has aminoacid sequence shown in SEQ ID NO:1;
Light chain CDR2 has aminoacid sequence shown in SEQ ID NO:2;
Light chain CDR3 has aminoacid sequence shown in SEQ ID NO:3;
Heavy chain CDR1 has aminoacid sequence shown in SEQ ID NO:4;
Heavy chain CDR2 has aminoacid sequence shown in SEQ ID NO:5;
Heavy chain CDR3 has aminoacid sequence shown in SEQ ID NO:6.
Preferably, monoclonal antibody provided by the invention or its fragment, its variable region of light chain has aminoacid sequence shown in SEQ ID NO:7.
Preferably, monoclonal antibody provided by the invention or its fragment, its variable region of heavy chain has aminoacid sequence shown in SEQ ID NO:8.
In some embodiments of the invention, monoclonal antibody provided by the invention or its fragment are monoclonal antibody, and its variable region of light chain has aminoacid sequence shown in SEQ ID NO:7; Its variable region of heavy chain has aminoacid sequence shown in SEQ ID NO:8, called after L3A monoclonal antibody.
The present invention also provides the preparation method of a kind of monoclonal antibody or its fragment, comprises the following steps:
Step 1: obtain have nucleotide sequence shown in SEQ ID NO:9 DNA molecular, there is the DNA molecular of nucleotide sequence shown in SEQ ID NO:10;
Step 2: get that step 1 gained has the DNA molecular of nucleotide sequence shown in SEQ ID NO:9 and the first expression vector merges, build the first recombinant expression vector; Get that step 1 gained has the DNA molecular of nucleotide sequence shown in SEQ ID NO:10 and the second expression vector merges, build the second recombinant expression vector; Maybe the DNA molecular with nucleotide sequence shown in SEQ ID NO:9 is merged with DNA molecular and the 3rd expression vector with nucleotide sequence shown in SEQ ID NO:10, build the 3rd recombinant expression vector;
Step 3: get step 2 gained the first recombinant expression vector and step 2 gained the second recombinant expression vector, cotransfection proceeds to host cell; Or the 3rd recombinant expression vector transfection of step 2 gained is proceeded to host cell; Express, purifying, to obtain final product;
The variable region of light chain of this monoclonal antibody or its fragment has aminoacid sequence shown in SEQ ID NO:7; Variable region of heavy chain has aminoacid sequence shown in SEQ ID NO:8.
In some embodiments of the invention, in preparation method's step 2 provided by the invention, the first expression vector is the pcDNA3.1 plasmid that contains antibody light chain constant region gene.
In other embodiment of the present invention, in preparation method's step 2 provided by the invention, the second expression vector is the pcDNA3.1 plasmid that contains heavy chain of antibody constant region gene.
In other embodiment of the present invention, in preparation method's step 2 provided by the invention, the 3rd expression vector is the pcDNA3.1 plasmid that contains antibody constant region gene.
In other embodiment of the present invention, in preparation method's step 3 provided by the invention, host cell is mammalian cell.
In some embodiments of the invention, the preparation method of monoclonal antibody L3A provided by the invention comprises the following steps:
Step 1: obtain the DNA molecular of coding light chain, this DNA molecular comprises and has nucleotide sequence shown in SEQ ID NO:9; Obtain the DNA molecular of encoding heavy chain, this DNA molecular comprises and has nucleotide sequence shown in SEQ ID NO:10;
Step 2: get the DNA molecular of step 1 gained coding light chain, merge with the first expression vector, build the first recombinant expression vector; DNA molecular and the second expression vector of getting step 1 gained encoding heavy chain merge, and build the second recombinant expression vector;
Step 3: get gained step 2 gained the first recombinant expression vector, the second recombinant expression vector, cotransfection proceeds in mammalian cell, expresses, and purifying, to obtain final product.
The present invention also provides the method that obtains monoclonal antibody L3A variable region of light chain, weight chain variable region nucleotide sequence and aminoacid sequence, is specially:
1, the structure of anti-human Tim-3 monoclonal antibody hybridoma cell strain
First, obtain 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.Gained hybridoma has been carried out to chromosome karyotype analysis, and hybridoma karyomit(e) average number is 108, experimental results show that with two-phase agar diffusion the secreted immunoglobulin (Ig) hypotype of gained hybridoma is IgG2a.
2, the variable region of light chain of monoclonal antibody L3A, heavy chain variable region gene angle get with variable region of light chain, heavy chain variable region gene determine
The RNA that extracts gained hybridoma, through RT-PCR, angles respectively chain variable region gene and the heavy chain variable region gene of getting antibody with Auele Specific Primer.Adopt ordinary method that gained chain variable region gene is proceeded to carrier, transformed competence colibacillus bacterium, the single bacterium colony of picking after cultivating, after the qualification of extraction plasmid PCR is correct, carries out DNA sequencing; Derived heavy chain variable region gene is proceeded to carrier, transformed competence colibacillus cell, and the single bacterium colony of picking after cultivating, extracts after plasmid PCR qualification correctly, carries out DNA sequencing; Through sequential analysis, comparison, obtain chain variable region gene and the heavy chain variable region gene of monoclonal antibody L3A.The chain variable region gene sequence of gained monoclonal antibody L3A is nucleotide sequence shown in SEQ ID NO:9, and heavy chain variable region gene sequence is nucleotide sequence shown in SEQ ID NO:10.
3, determining of monoclonal antibody L3A light chain, weight chain variable region amino acid sequence
With the online software of www.expasy.org, the light chain of encoding human Tim-3 monoclonal antibody L3A, weight chain variable region nucleotide sequence are translated as to the aminoacid sequence of its coding, the light chain of monoclonal antibody L3A, weight chain variable region amino acid sequence are respectively shown in aminoacid sequence shown in SEQ ID NO:7, SEQ ID NO:8 shown in aminoacid sequence.The aminoacid sequence of determining complementary determining region light chain CDR1, light chain CDR2 in monoclonal antibody L3A light chain variable region sequence and light chain CDR3 according to Kabat database is respectively as shown in SEQ ID NO:1, SEQ ID NO:2 in sequence table and SEQ ID NO:3.Complementary determining region heavy chain CDR1, heavy chain CDR2 in weight chain variabl area sequence and the aminoacid sequence of heavy chain CDR3 are respectively as shown in SEQ ID NO:4, SEQ ID NO:5 in sequence table and SEQ ID NO:6.
The present invention also provides a kind of DNA molecular of encode monoclonal antibody provided by the invention or its fragment, and it has nucleotide sequence shown in SEQ ID NO:9, and the variable region of light chain of this monoclonal antibody or its fragment has aminoacid sequence shown in SEQ ID NO:7.
The present invention also provides a kind of DNA molecular of encode monoclonal antibody provided by the invention or its fragment, it has nucleotide sequence shown in SEQ ID NO:10, and the variable region of heavy chain of this monoclonal antibody or its fragment has aminoacid sequence shown in SEQ ID NO:8.
The present invention has successfully cloned from the people Tim-3 monoclonal antibody hybridoma cell of cultivating that antibody is light, heavy chain variable region gene.The mouse antibodies variable region that gained chain variable region gene and heavy chain variable region gene codified are correct.Based on above-mentioned people Tim-3 monoclonal antibody L3A chain variable region gene, the heavy chain variable region gene being cloned into, 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, the immunodetection instrument detecting for the preparation of people Tim-3 expression level, the medicine of preparation diagnosis and treatment Tim-3 disease that high expression level causes.
The present invention also provides a kind of monoclonal antibody or the application of its fragment in the immunodetection instrument for the preparation of detection people Tim-3, and this monoclonal antibody or its fragment comprise light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3;
Light chain CDR1 has aminoacid sequence shown in SEQ ID NO:1;
Light chain CDR2 has aminoacid sequence shown in SEQ ID NO:2;
Light chain CDR3 has aminoacid sequence shown in SEQ ID NO:3;
Heavy chain CDR1 has aminoacid sequence shown in SEQ ID NO:4;
Heavy chain CDR2 has aminoacid sequence shown in SEQ ID NO:5;
Heavy chain CDR3 has aminoacid sequence shown in SEQ ID NO:6.
Preferably, the immunodetection instrument in application provided by the invention is test kit, chip or test paper.
In some embodiments of the invention, the immunodetection instrument in application provided by the invention is test kit.
The present invention also provides a kind of test kit containing monoclonal antibody provided by the invention or its fragment, and this monoclonal antibody or its fragment comprise light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3;
Light chain CDR1 has aminoacid sequence shown in SEQ ID NO:1;
Light chain CDR2 has aminoacid sequence shown in SEQ ID NO:2;
Light chain CDR3 has aminoacid sequence shown in SEQ ID NO:3;
Heavy chain CDR1 has aminoacid sequence shown in SEQ ID NO:4;
Heavy chain CDR2 has aminoacid sequence shown in SEQ ID NO:5;
Heavy chain CDR3 has aminoacid sequence shown in SEQ ID NO:6.
In some embodiments of the invention, test kit provided by the invention comprises L3A monoclonal antibody, rabbit against human T im-3 polyclonal antibody and HRP mark goat anti-rabbit antibody.
Monoclonal antibody L3A high specificity provided by the invention, can specificity combine with people Tim-3, in the time that it is applied to the test kit of preparation detection people Tim-3, has significantly improved the sensitivity of test kit.Experimental result shows, test kit provided by the invention is the content of Tim-3 in detection of biological sample delicately, and its sensitivity reaches 0.1ng/mL～1000ng/mL.
In some embodiments of the invention, the preparation method of the rabbit against human T im-3 polyclonal antibody in test kit provided by the invention comprises:
Obtain people Tim-3 antigen;
After income earner Tim-3 immunize rabbit, gather antiserum(antisera), purifying, to obtain final product.
The present invention also provides the using method of mentioned reagent box, comprises the following steps:
Under step 1:4 DEG C of condition, coated L3A monoclonal antibody 16h～18h;
Step 2: add biological sample, hatch 1h for 37 DEG C;
Step 3: add rabbit against human T im-3 polyclonal antibody, hatch 1h for 37 DEG C;
Step 4: add HRP mark goat anti-rabbit antibody, after 37 DEG C hatch 0.5h, colour developing, stops, and detects;
Step 5: the concentration that judges people Tim-3 contained in biological sample according to detected result.
The invention provides monoclonal antibody or its fragment of a kind of anti-human Tim-3.The monoclonal antibody of this anti-human Tim-3 or its fragment comprise light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and wherein light chain CDR1 has aminoacid sequence shown in SEQ ID NO:1; Light chain CDR2 has aminoacid sequence shown in SEQ ID NO:2; Light chain CDR3 has aminoacid sequence shown in SEQ ID NO:3; Heavy chain CDR1 has aminoacid sequence shown in SEQ ID NO:4; Heavy chain CDR2 has aminoacid sequence shown in SEQ ID NO:5; Heavy chain CDR3 has aminoacid sequence shown in SEQ ID NO:6.Experimental result shows, monoclonal antibody provided by the invention or its fragment can specificity combine with people Tim-3, can be for the preparation of the test kit that detects people Tim-3, and gained test kit has higher sensitivity, can detect the people Tim-3 of low concentration.
Brief description of the drawings
Fig. 1 shows the result schematic diagram that adopts indirect ELISA method to detect the antiserum titre of the polyclonal antibody of the rabbit against human T im-3 that makes of the embodiment of the present invention 5, and wherein curve 1 shows monoclonal antibody and the people Tim-3 binding curve of the rabbit against human T im-3 that embodiment 5 makes; Curve 2 shows control group serum and people Tim-3 binding curve;
Fig. 2 shows the sensitivity detected result of the test kit that the embodiment of the present invention 6 makes, and wherein, curve 1 shows experimental group detected result, and curve 2 shows control group detected result;
Fig. 3 shows that the test kit that adopts embodiment 6 to make in the embodiment of the present invention 8 detects the result of people Tim-3 level in different biological specimens, wherein Fig. 3 A is for becoming human sample: sample 1: normal healthy controls, sample 2:TID (type 1 diabetes), sample 3:SLE (systemic lupus erythematous); Fig. 3 B is children: sample 4: normal healthy controls, sample 5: asthma.
Fig. 4 shows the result that adopts test kit that embodiment 6 makes to detect people Tim-3 level in pyemia patients serum in various degree in the embodiment of the present invention 9.
Embodiment
The invention discloses monoclonal antibody, its preparation method and the application of a kind of anti-human Tim-3.Those skilled in the art can be with reference to this paper content, implement this preparation method, obtain the monoclonal antibody of anti-human Tim-3, special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Preparation method of the present invention is described by preferred embodiment, and related personnel obviously can change this paper preparation method in content of the present invention, spirit and scope or suitably change and combination not departing from, and realizes and apply the technology of the present invention.
In monoclonal antibody, its preparation method and the application of anti-human Tim-3 provided by the invention, reagent used and raw material all can be buied by market.
In order to make those skilled in the art can understand better technical scheme of the present invention, below in conjunction with embodiment, further set forth the present invention:
The structure of the anti-human Tim-3 monoclonal antibody hybridoma cell of embodiment 1 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 RPMI1640: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; Recombinant human Tim-3 (rhTim-3) is prepared by Institute of Basic Medical Sciences, A Cademy of Military Medical Sciences.All the other reagent are commercial.
2. method and result
(1) BALB/c mouse immunity.Select 6 of the female BALB/c mouse in 4～6 week age, with 100 μ g ricins (Sigma company) 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 RPMI1640 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 RPMI1640, abandon most supernatant, gently cell is broken up; 4. in the 1min time, slowly add 1mL50%PEG (M
wbe 1500) solution, put 37 DEG C of water-bath 1min; 5. in the time of 1min, 2min, the 5min time, add respectively serum-free RPMI16401mL, 5mL, 10mL; 6. the centrifugal 7min of 800r/min, abandons supernatant, cell is hanged as far as possible gently; 7. add HAT (the Sigma)-RPMI1640 nutrient solution 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 DEG C
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 DEG C.Add successively cell culture supernatant to be measured (37 DEG C of 1h, PBST washes plate 4 times), and the 50 μ LHRP-GAM (37 DEG C 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 5mL1640 nutrient solution 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 DEG C
2in incubator, cultivate, after 5 days, examine under a 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 to gained hybridoma is done the experiment of two-phase agar diffusion, result shows that above-mentioned 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 the present embodiment gained hybridoma is IgG2a.By above-mentioned steps, screen people Tim-3 monoclonal antibody, called after L3A monoclonal antibody.
Embodiment 2 people Tim-3 monoclonal antibody L3A variable region of light chain, angling of heavy chain variable region gene are got
1. material
(1) variable region of light chain upstream and downstream primer
Light chain upstream primer MuLC5, MuLC6 and MuLC7, its sequence is shown in respectively sequence table SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13; Light chain downstream primer MuCK, its sequence is shown in sequence table SEQ ID NO:14, the working concentration of primer in the time of pcr amplification is 10pmol/ μ L.
(2) variable region of heavy chain upstream and downstream primer
Heavy chain upstream primer MuHC5, MuHC6, MuHC7 and MuHC8, its sequence is shown in sequence table SEQ ID NO:15, SEQ ID NO16, SEQ ID NO:17 and SEQ ID NO:18; Heavy chain downstream primer MuIgG2a is shown in SEQ ID NO:19 in sequence table, and the working concentration of primer in the time of pcr amplification is 10pmol/ μ L.
(3) 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 reagent sources are with embodiment 1.
2. methods and results
(1) cell strain 5 × (10 of the monoclonal antibody L3A that the embodiment 1 taking the logarithm vegetative period makes
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 DEG C～8 DEG C 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 DEG C～8 DEG C 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, Oligo (dT) (500ng/ μ L) 0.5 μ L, 2.5mmol/L dNTP2 μ L, RNasin (50U/ μ L) 0.5 μ L, mend deionized water to 20 μ L, ThermoScript II 2～5U, 42 DEG C are extended 1 hour.95 DEG C of sex change 5min, put in ice bath, and products therefrom is cDNA the first chain.Use respectively primer amplified chain variable region gene and heavy chain variable region gene.
Chain variable region gene amplification system: get above-mentioned products therefrom, be reverse transcription product 2 μ L, Taq enzyme 10 × buffer2 μ L, the each 1 μ L of light chain upstream primer MuLC5, MuLC6 and MuLC7, downstream primer MuCK3 μ L, 2.5mmol/L dNTP4 μ L, adds Taq enzyme 1～2U, mends deionized water to 50 μ L.Pcr amplification condition is: 95 DEG C of sex change 2 minutes, and loop parameter is: 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, totally 30 circulations, extend 10min after 72 DEG C, and gained amplified production is labeled as to amplified production 1.
Heavy chain variable region gene amplification system: get above-mentioned products therefrom, be reverse transcription product 2 μ L, Taq enzyme 10 × buffer2 μ L, the each 1 μ L of heavy chain upstream primer MuHC5, MuHC6, MuHC7 and MuHC8, downstream primer MuIgG2a3 μ L, 2.5mmol/L dNTP4 μ L, adds Taq enzyme 1～2U, mends deionized water to 50 μ L.Pcr amplification condition is: 95 DEG C of sex change 2 minutes, and loop parameter is: 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, totally 30 circulations, extend 10min after 72 DEG C, and gained amplified production is labeled as to amplified production 2.
(3) isolate respectively with agarose gel electrophoresis the DNA fragmentation that in amplified production 1 and amplified production 2, wish reclaims, it is target DNA fragment, under long wave ultraviolet light, cut respectively the blob of viscose containing target DNA fragment, put into centrifuge tube, mark one by one, add three times of change glues that colloid is long-pending, blob of viscose is dissolved in 55 DEG C of water-baths completely.Reclaim respectively in amplified production 1 in the object fragment in target DNA fragment and amplified production 2 and the DNA fragmentation of purifying is dissolved in the aqueous solution with DNA fragmentation purification kit, mark respectively, by the PCR product reclaiming in T4DNA ligase enzyme damping fluid by after the ratio (mol ratio) of 2: 1 and carrier PGEM Teasy mixing, add the T4DNA ligase enzyme of 0.5U to spend the night in 16 DEG C of connections, the cumulative volume of ligation is 10 μ L, and correspondence markings one by one.
(4) get above-mentioned gained connecting fluid 10 μ L, be added in 200 μ L competence bacterium JM109 and softly mix, ice bath 30min, 42 DEG C of water-bath heat-shockeds 90 seconds, proceed to rapidly ice bath 2min, add 800 μ LLB substratum, proceed to 37 DEG C of constant-temperature tables, with the speed shake 45min of 150r/min, the centrifugal 1min of 4000r/min, discards 800 μ L supernatants, gets precipitation and coats the solid LB flat board containing Amp (final concentration is 100 μ g/mL), flat-plate inverted is placed in to 37 DEG C of incubator 12～18h, and correspondence markings one by one.
(5) the single clone of picking in the above-mentioned flat board of gained, is inoculated in the LB substratum containing acillin (100 μ g/mL), and correspondence markings one by one.37 DEG C of constant-temperature table 170r/min, concussion overnight incubation.Get respectively 3mL bacterium liquid and add in 1.5mL Eppendorf pipe, the centrifugal 1min of 10000r/min, abandons supernatant.Adopt plasmid extraction kit, precipitation thalline be resuspended in 100 μ L solution I, add freshly prepared solution II 200 μ L, turn upside down for several times light and slowly, limpid to liquid change 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 DEG C, 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 12000r/min, 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 DEG C.4 DEG C, the centrifugal 10min of 12000r/min, 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 qualification and DNA sequencing analysis.
The carrier of the monoclonal antibody L3A chain variable region gene that contains people Tim-3, the carrier of heavy chain variable region gene are built by above-mentioned steps.Through sequence alignment, the nucleotide sequence of variable region of light chain of coding L3A monoclonal antibody and the nucleotide sequence of the variable region of heavy chain of coding L3A monoclonal antibody are obtained.The nucleotides sequence of the variable region of light chain of gained coding L3A monoclonal antibody is classified nucleotide sequence shown in SEQ ID NO:9 as; The nucleotides sequence of the variable region of heavy chain of gained coding L3A monoclonal antibody is classified nucleotide sequence shown in SEQ ID NO:10 as, and its corresponding aminoacid sequence is respectively aminoacid sequence shown in aminoacid sequence shown in SEQ ID NO:7, SEQ ID NO:8.
Expression and the purifying of embodiment 3 monoclonal antibody L3A
1. material
Protein A Sepharose CL4B post albumen post: Ben Yuan Zhenyang, Beijing Bioisystech Co., Ltd product; PcDNA3.1 plasmid: Invitrogen; The source of all the other reagent and material is with embodiment 1.
2. method and result
The monoclonal antibody L3A chain variable region gene that embodiment 2 is obtained, constant region of light chain gene packs pcDNA3.1 plasmid into, must be containing the carrier of monoclonal antibody L3A light chain gene; The monoclonal antibody L3A heavy chain variable region gene that embodiment 2 is obtained, weight chain constant area gene packs pcDNA3.1 plasmid into, obtains the carrier containing monoclonal antibody L3A heavy chain gene.The carrier that contains monoclonal antibody L3A light chain gene, the carrier cotransfection of heavy chain gene proceed in mammalian cell, express.Collect and express supernatant, add 1mL pH8.0,0.1mol/L phosphoric acid buffer and use 1mol/L TRIS-HCL to adjust pH to 9.0.Mouse ascites is added and used in the Protein A Sepharose CL4B albumen post that pH8.0,0.1mol/L phosphoric acid buffer balance 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 pH3.0, collect effluent liquid, and immediately with the neutralization of 1mol/L pH8.5TRIS-HCL damping fluid, with pH7.2, the PBS dialysis 72h of 0.01mol/L.OD260, OD280 are surveyed in sampling on ultraviolet spectrophotometer, calculate protein content, after freeze-drying in-20 DEG C of preservations.
The specific detection of embodiment 4 monoclonal antibody L3A
1. material
Sepsis patient serum is the treated acquisition of blood that is collected in PLA General Hospital septic; Normal human serum is the treated acquisition of blood that is collected in normal people; ELISA coating buffer, PBST, TMB, H2SO
4, TNF-a, sulphur oxygen cyclase protein be commercially available, the source of all the other reagent and material is with embodiment 1.
2. method and result
The specificity of the L3A monoclonal antibody that employing indirect ELISA method investigation embodiment 3 makes.Control group 1, control group 2, control group 3, experimental group, positive controls, negative control group are established in experiment.The wherein coated irrelevant antigen TNF-a1 μ g/mL of control group 1, coated irrelevant antigen sulphur oxygen cyclase protein (TRX) the 1 μ g/mL of control group 2; The coated BSA1 μ g/mL of control group 3; Experimental group is coated with septic serum; Positive controls is coated with people TIM-3 antigen (being recombinant human Tim-3) 1 μ g/mL; Negative control group is coated with normal human serum.Every group of sample dilutes with coating buffer, and each sample is coated with 2 holes in elisa plate, every hole 100 μ L, and 4 DEG C are spent the night.PBST washes 5 times, sealing, and 200 μ L confining liquids, 37 DEG C, seal 2 hours, PBST washes 5 times.Add L3A monoclonal antibody 50 μ L by the final concentration of 10 μ g/mL, 37 DEG C are reacted 1 hour, and PBST washes 5 times.Add 300 × dilution anti-mouse antibody (KPL company), 37 DEG C of reaction 45min.PBST washes 7 times, adds the TMB in 50 μ L/ holes, and room temperature lucifuge is hatched after 10min, adds the sulfuric acid termination reaction of the 1mol/L in 50 μ L/ holes, and microplate reader detects the OD of 450nm place value, calculates the mean value of every group of two hole count values.Monoclonal antibody L3A specific detection the results are shown in Table 1.
Table 1 monoclonal antibody L3A specific detection result
Known according to data in table 1, the OD450 value difference of four groups of negative control group, control group 1, control group 2 and control group 3 (three irrelevant albumen contrast) is different not remarkable, illustrate monoclonal antibody L3A that embodiment 3 makes not nothing to do with albumen combine; Compare with negative control group, positive controls and experimental group OD450 value difference are different and remarkable, present obvious positive findings.Compare with positive control, the OD450 value of experimental group and positive controls is suitable, in experimental group, contain people Tim-3 antigen, illustrate that the monoclonal antibody L3A that embodiment 3 makes can specificity combine with people Tim-3, and not with other protein binding, can be for the preparation of the immunodetection instrument that detects people Tim-3.
The preparation of embodiment 5 rabbit against human T im-3 polyclonal antibodies
1. material
Rabbit grows up: male, and body weight 2-3kg, Military Medical Science Institute's Experimental Animal Center provides; Freund's complete adjuvant and freund 's incomplete adjuvant: Sigma company product; All the other reagent and material source are with embodiment 1.
2. method and result
(1) rabbit immunity.People Tim-3 antigen (PBS dissolving) is mixed with adjuvant at 1: 1, and emulsification is complete.Use for the first time complete spoke formula adjuvant (CFA) immunity, second, third time full spoke formula adjuvant IFA immunity of toing many or too much for use, At intervals of two to three weeks.Select 2 of adult male rabbit, before immunity every rabbit through ear vein each 1mL that takes a blood sample, separation of serum, mark one by one, for check sample.Immunization method is subcutaneous multi-point injection, total dose 2mL (1mL people Tim-3 antigen+1mL adjuvant)/only.Latter the 7th day of last injection, ear vein blood sampling 1mL, separation of serum, mark one by one, tire with tube agglutination test titration, sero-fast the tiring that detected result is for both is respectively 1: 3000 and 1: 4000 (1: 2000 above available), all meets the requirements.
(3) antiserum(antisera) collection.Get antiserum titre and be the rabbit of 1: 4000, carotid artery bloodletting, and be collected in aseptic Erlenmeyer flask and solidify rear 4 DEG C of refrigerator overnight, separate upper serum.Then the centrifugal 10min of 2500r/min, collects upper serum.The serum of collecting after purification Identification, in a small amount packing ,-20 DEG C frozen.
(4) ELISA method detects antiserum titre.Experiment is divided into two groups, is experimental group and control group; Experimental group and control group are coated with people Tim-3 antigen 1 μ g/mL, every hole 100 μ L, and 4 DEG C are spent the night.PBST washes 5 times, sealing, and 200 μ L confining liquids, 37 DEG C, seal 2 hours, PBST washes 5 times.Then add respectively the antiserum(antisera) of different dilution (3) gained and control group serum to investigate the antiserum(antisera) 50 μ L of the rabbit against human T im-3 that the present embodiment obtains, 37 DEG C of reactions 1 hour, PBST washes 5 times.Add HRP mark goat anti-rabbit antibodies, 37 DEG C of reaction 60min.PBST washes 7 times, adds the TMB in 50 μ L/ holes, and room temperature lucifuge is hatched after 10min, adds the sulfuric acid termination reaction of the 1mol/L in 50 μ L/ holes, and microplate reader OD450nm detects.Deduct after the absorption value of blank hole, with the mapping of OD450nm value and antiserum(antisera) extension rate, obtain rabbit against human T im-3 antiserum(antisera) that the present embodiment makes and the combination situation of people Tim-3.With (sample light absorption value-blank absorbency)/(negative light absorption value-blank absorbency) > 2.1, think the positive, calculate the rabbit against human T im-3 antiserum titre of the present embodiment gained.The rabbit against human T im-3 antiserum(antisera) (being polyclonal antibody) that the present embodiment makes is shown in Fig. 1 with the people Tim-3 antigen situation of being combined, contrast control group, calculate according to above-mentioned formula, sero-fast the tiring that obtains the rabbit against human T im-3 of the present embodiment acquisition is 1: 3000, and the tiring of polyclonal antibody of the rabbit against human T im-3 that the present embodiment obtains is 1: 3000.
The preparation and application of embodiment 6 test kits
1. the preparation of test kit
Test kit provided by the invention comprises following component: L3A monoclonal antibody, rabbit against human T im-3 polyclonal antibody, horseradish peroxidase substrate buffer solution, protein standard substance recombinant human Tim-3 (100ug/mL, 0.1mL), negative control sample BSA.
Wherein L3A monoclonal antibody, the preparation method who is provided by the embodiment of the present invention 3 prepares; Rabbit against human T im-3 polyclonal antibody, the preparation method who is provided by the embodiment of the present invention 4 prepares.The collocation method of horseradish peroxidase substrate buffer solution is: take 10mg3,3 ', 5,5 '-tetramethyl benzidine (TMB) is dissolved in and in 5mL dehydrated alcohol, is prepared into TMB stock solution.Time to be used, get 0.5mL TMB stock solution and be added to 10mL phosphoric acid citric acid substrate buffer solution (0.2mol/L Na
2hPO
4, 0.1mol/L citric acid) in, then add 32 μ L0.75%H
2o
2mix, be configured to horseradish peroxidase substrate buffer solution.All the other reagent sources are with embodiment 1.
2. the using method of test kit
Adopt the method for test kit detection people Tim-3 provided by the invention as follows:
Step 1: Sheet clonal antibody L3A (10 μ g/mL) in high-affinity elisa plate, 4 DEG C spend the night after with the PBS damping fluid washing containing 0.2% tween 5 times;
Step 2: by suitable extent of dilution (as, 1: 2～1: 10) dilution normal people or patient's blood plasma, join in the elisa plate bar that is coated with L3A of step 1 gained, hatch 1 hour for 37 DEG C, with the PBS damping fluid washing containing 0.2% tween 5 times;
Step 3, add the polyclonal antibody (1: 5000) of rabbit against human T im-3, hatch 1 hour for 37 DEG C, with the PBS damping fluid washing containing 0.2% tween 5 times;
Step 4, add HRP mark goat anti-rabbit antibody (1: 1000), 37 DEG C hatch 0.5 hour after, with the PBS damping fluid washing containing 0.2% tween 5 times; Add TMB colour developing, after the sulfuric acid of 1mol/L stops, detect the value of OD450.
Step 5, judge the concentration of people Tim-3 contained in patient's blood plasma according to detected result.
Embodiment 7 test kit sensitivity detect
1. material
Test kit makes for the preparation method that embodiment 6 provides.Other reagent are commercial.
2. method
Experimental group and control group are established in experiment, and experimental group albumen to be detected is protein standard substance recombinant human Tim-3, and control group albumen to be detected is Trx albumen.Between different groups, except protein sample difference to be detected, other are in full accord, and concrete operations are as follows:
Step 1: Sheet clonal antibody L3A in high-affinity elisa plate, 4 DEG C spend the night after with the PBS damping fluid washing containing 0.2% tween 5 times;
Step 2: get protein standard substance recombinant human Tim-3 albumen, be configured to different concentration (concentration is respectively 0.0001ng/mL, 0.001ng/mL, 0.01ng/mL, 0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL and 10000ng/mL), join in the elisa plate bar that is coated with L3A of step 1 gained, hatch 1 hour for 37 DEG C, with the PBS damping fluid washing containing 0.2% tween 5 times;
Step 3: add the polyclonal antibody of rabbit against human T im-3, hatch 1 hour for 37 DEG C, with the PBS damping fluid washing containing 0.2% tween 5 times;
Step 4: add HRP mark goat anti-rabbit antibody, 37 DEG C hatch 0.5 hour after, with the PBS damping fluid washing containing 0.2% tween 5 times; Add TMB colour developing, after the sulfuric acid of 1mol/L stops, detect the value of OD450.
Step 5: be figure with OD450 value and protein concentration, judge the level of people Tim-3 contained in biological sample.
Detected result is shown in Fig. 2, it is analyzed known, in experimental group along with the increase of protein concentration, OD450 value increases gradually, and the OD450 value of all groups of control group is all lower, and do not change with protein concentration, illustrated test kit provided by the invention can specific detection biological sample in the content of people Tim-3, it detects and is limited to 0.1ng/mL to 1000ng/mL, compared with the minimum detectability 1ng/mL of the people Tim-3 reporting, detection limit and the sensitivity of people Tim-3 detection kit are significantly improved.
The level of people Tim-3 in embodiment 8 test kit detection of biological samples
1. material
Test kit is the test kit that embodiment 6 makes; Normal human serum comes from 10 normal peoples; Asthmatic patient serum comes from 10 asthmatic patients of hospital; Type i diabetes patients serum comes from 10 type i diabetes patients of hospital; The serum of Patients with SLE comes from 6 patients of PLA General Hospital systemic lupus erythematous; Other reagent are commercially available.
2. method and result
Experiment is divided into negative control, normal human serum group, asthmatic patient serologic group, type i diabetes patients serum group and Serum in Patients with SLE group.Between different groups, except biological sample difference to be detected, other are in full accord, and concrete operations are as follows:
Step 1: Sheet clonal antibody L3A in high-affinity elisa plate, respectively mark, 4 DEG C spend the night after with the PBS damping fluid washing containing 0.2% tween 5 times;
Step 2: get normal human serum, asthmatic patient serum, type i diabetes patients serum and Serum in Patients with SLE, join in the elisa plate bar that is coated with L3A of step 1 gained, hatch 1 hour for 37 DEG C, with the PBS damping fluid washing containing 0.2% tween 5 times;
Step 3: add the polyclonal antibody of rabbit against human T im-3, hatch 1 hour for 37 DEG C, with the PBS damping fluid washing containing 0.2% tween 5 times;
Step 4: add HRP mark goat anti-rabbit antibody, 37 DEG C hatch 0.5 hour after, with the PBS damping fluid washing containing 0.2% tween 5 times; Add TMB colour developing, after the sulfuric acid of 1mol/L stops, detect the value of OD450.
Step 5: add up the detected result of each group gained, analyze summary.
Experimental result shows that negative control absorption value is very low, illustrates that test kit reagent is normal, and data are reliable.The detected result of each group gained of the present embodiment is shown in Fig. 3, known according to Fig. 3, compare with adult normal human serum, in type i diabetes patients serum, the content of soluble T im-3 (sTim-3) significantly reduces, compared with soluble T im-3 in Children Normal human serum, in asthmatic children patients serum, the content of soluble T im-3 (sTim-3) significantly raises; In asthmatic patient serum, Serum in Patients with SLE there is variation in various degree in Tim-3, consistent with research report; These test kits that all illustrated that the present invention makes can be applied to scientific research and clinical detection.
The level of people Tim-3 in embodiment 9 test kit detection of biological samples
1. material
Test kit is the test kit that embodiment 6 makes; The patient's in different Sepsis stages serum: inflammation syndromes patient (its clinical disease is lighter than sepsis patient) serum comes from 10 patients of hospital; Sepsis patient serum comes from 10 patients of hospital; Patients with severe sepsis serum comes from 10 patients of hospital; Septic shock patient's serum comes from 10 patients of PLA General Hospital; Other reagent are commercially available.
2. method and result
Experiment is divided into inflammation syndromes group, Sepsis group, severe sepsis group and septic shock group.Each group is except biological sample difference to be measured, and other are in full accord, and concrete operations are as follows:
Step 1: Sheet clonal antibody L3A in high-affinity elisa plate, respectively mark, 4 DEG C spend the night after with the PBS damping fluid washing containing 0.2% tween 5 times;
Step 2: get inflammation syndromes serologic group, Sepsis serologic group, severe sepsis serologic group and septic shock serum, join in the elisa plate bar that is coated with L3A of step 1 gained, hatch 1 hour for 37 DEG C, with the PBS damping fluid washing containing 0.2% tween 5 times;
Step 3: add the polyclonal antibody of rabbit against human T im-3, hatch 1 hour for 37 DEG C, with the PBS damping fluid washing containing 0.2% tween 5 times;
Step 4: add HRP mark goat anti-rabbit antibody, 37 DEG C hatch 0.5 hour after, with the PBS damping fluid washing containing 0.2% tween 5 times; Add TMB colour developing, after the sulfuric acid of 1mol/L stops, detect the value of OD450.
Step 5: add up the detected result of each group gained, analyze summary.
Experimental result is shown in Fig. 4, can draw the content difference of soluble T im-3 (sTim-3) in different Sepsis stage patients serums from figure, and its content increases along with the increase of disease severity, consistent with research report.The test kit that we are described can be used for scientific research and disease clinical monitoring.
Below be only the preferred embodiment of the present invention, it should be pointed out that above-mentioned preferred implementation should not be considered as limitation of the present invention, protection scope of the present invention should be as the criterion with claim limited range.For those skilled in the art, without departing from the spirit and scope of the present invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. the monoclonal antibody of anti-human Tim-3 or its fragment, is characterized in that, it comprises light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3;
Described light chain CDR1 has aminoacid sequence shown in SEQ ID NO:1;
Described light chain CDR2 has aminoacid sequence shown in SEQ ID NO:2;
Described light chain CDR3 has aminoacid sequence shown in SEQ ID NO:3;
Described heavy chain CDR1 has aminoacid sequence shown in SEQ ID NO:4;
Described heavy chain CDR2 has aminoacid sequence shown in SEQ ID NO:5;
Described heavy chain CDR3 has aminoacid sequence shown in SEQ ID NO:6.
2. monoclonal antibody according to claim 1 or its fragment, is characterized in that, its variable region of light chain has aminoacid sequence shown in SEQ ID NO:7.
3. monoclonal antibody according to claim 1 or its fragment, is characterized in that, its variable region of heavy chain has aminoacid sequence shown in SEQ ID NO:8.
4. the monoclonal antibody as described in claims 1 to 3 any one or the preparation method of its fragment, is characterized in that, comprises the following steps:
Step 1: obtain have nucleotide sequence shown in SEQ ID NO:9 DNA molecular, there is the DNA molecular of nucleotide sequence shown in SEQ ID NO:10;
Step 2: DNA molecular and first expression vector described in getting with nucleotide sequence shown in SEQ ID NO:9 merge, and build the first recombinant expression vector; DNA molecular and second expression vector described in getting with nucleotide sequence shown in SEQ ID NO:10 merge, and build the second recombinant expression vector;
Step 3: get described the first recombinant expression vector and described the second recombinant expression vector, cotransfection proceeds to after host cell, expresses, and purifying, to obtain final product.
5. a DNA molecular for coding monoclonal antibody as claimed in claim 2 or its fragment, is characterized in that, it has nucleotide sequence shown in SEQ ID NO:9.
6. a DNA molecular for coding monoclonal antibody as claimed in claim 3 or its fragment, is characterized in that, it has nucleotide sequence shown in SEQ ID NO:10.
7. the monoclonal antibody as described in claims 1 to 3 any one or its fragment application in the immunodetection instrument for the preparation of detection people Tim-3.
8. application according to claim 7, is characterized in that, described immunodetection instrument is test kit, chip or test paper.
9. one kind containing the monoclonal antibody described in claim 1 to 3 any one or the test kit of its fragment.
10. a method that detects TIM-3, is characterized in that, comprises the following steps:
Step 1: the monoclonal antibody described in any one in claims 1 to 3 or its fragment are coated on enzyme linked immunological plate;
Step 2: add testing sample to hatch;
Step 3: add rabbit against human T im-3 polyclonal antibody to hatch;
Step 4: add HRP mark goat anti-rabbit antibody to hatch, colour developing, stops, and detects;
Step 5: the concentration that judges people Tim-3 contained in testing sample according to detected result.
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