CN1314414A - New activated T cell surface antigen of molecular weight 140 KDa - Google Patents
New activated T cell surface antigen of molecular weight 140 KDa Download PDFInfo
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- CN1314414A CN1314414A CN 01106793 CN01106793A CN1314414A CN 1314414 A CN1314414 A CN 1314414A CN 01106793 CN01106793 CN 01106793 CN 01106793 A CN01106793 A CN 01106793A CN 1314414 A CN1314414 A CN 1314414A
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Abstract
The present invention relates to biological technology. Immune cell membrane molecule has been used widely clinically in the diagnosis, prevention and treatment of some diseases. The present invention obtains A2 monoclone antibody through immunizing BALB/c mouse with activated T cell obtained through mixed lumphocyte culture to prepare splenocyte cultur liquid, fusion with myeloma cell NS-1, selective HAI culture and screening hybrid tumor supernatant. Analysis shows that the distinguished activated T cell surface antigen has unique distribution, expression rule and function and molecular weight 140 KDa.
Description
The present invention relates to biotechnology.
Body immune system is made up of central lymphoid organs, peripheral lymphoid organs, immunocyte and immune molecule.The immunne response process depends on intercellular interaction in the immunity system, comprises that iuntercellular directly contacts and pass through to discharge the interaction of cytokine or other medium.Between immunocyte or between cell and medium mutually the basic substance of identification be the immunocyte membrane molecule.The research of immunocyte membrane molecule is for understanding immunne response mechanism in depth, and diagnosis, prevention and the treatment of clinical some disease all had crucial meaning.By in August, 2000, the monoclonal antibody that U.S. FDA has been ratified 5 kinds of identification immunocyte membrane molecules is used for the treatment of clinical disease, and other has tens kinds of monoclonal antibodies carrying out clinical trial.
The activating T cell surface antigen that a kind of new molecular weight of the present invention is 140kDa, unique distribution is in mixed lymphocyte reacion activatory CD4
+, CD8
+, TCR γ δ
+T cell and activatory NK cell, and in other activated channel (stimulating as solid phase CD3 stimulation, PHA, PMA and PMA+A23187), this molecule is not expressed on the activating T cell surface.It can be worked in coordination with the CD3 molecule stimulates the T cell activation.The distribution of its uniqueness, expression rule and function show that it is a kind of recruit.
Technological line of the present invention:
Mixed lymphocytes is cultivated the activating T cell immunity BALB/c mouse obtain, and behind booster immunization 3 days, get spleen, preparation splenocyte suspension and myeloma cell NS-1 merge, and cultivate with the HAT selectivity.By indirect immunofluorescence mark and flow cytometry, screening can be cultivated the activating T cell bonded hybridoma supernatant that obtains with mixed lymphocytes, and the fused cell of secretion positive antibody has been obtained the A2 monoclonal antibody by cloning.Analyze the activating T cell surface antigen that the A2 monoclonal antibody is discerned by immunofluorescence dyeing, flow cytometry and the cellulotoxic experiment that heavily leads, show that it has unique distribution, expression rule and function.Identify that through biotin labeling, immunoprecipitation and chemoluminescence its molecular weight is 140kDa.
Wood invention molecular weight is that the activating T cell surface antigen of 140kDa is expressed in activated T cell subsets TCR γ δ
+Therefore the T cell, can be used as TCR γ δ
+One of T cell activation sign, significant in the research of transplantation immunity and clinical treatment graft-rejection.
The embodiment of the technology of the present invention route:
1. mixed lymphocytes is cultivated 10 milliliters of (MLC) aseptic extraction normal people peripheral bloods, and anticoagulant heparin adopts density gradient centrifugation to separate peripheral blood mononuclear cell (PBMC), by containing 1.5 * 10 in the 2ml perfect medium
6PBMC and 0.5 * 10
6The Daudi cell cultures that 3000Rad shone in 24 orifice plates, 37 ℃, 5%CO
2Incubator was cultivated 7 days, obtained the activated T cell.
2. the cellular immunization mixed lymphocytes is cultivated the activating T cell 1 * 10 that obtains
7Intraperitoneal injection immunity BALB/c mouse, 4 weeks are interior with method immunity 2 times, after 1 week, tail intravenous injection activating T cell 1 * 10
7Booster immunization after 3 days, is got spleen and is merged.
3. behind the MONOCLONAL ANTIBODIES SPECIFIC FOR booster immunization 3 days, get spleen, preparation splenocyte suspension and myeloma cell NS-1 merge, and cultivate with the HAT selectivity.By indirect immunofluorescence mark and flow cytometry, screening can be cultivated the activating T cell bonded hybridoma supernatant that obtains with mixed lymphocytes, and the fused cell of secretion positive antibody has been obtained the A2 monoclonal antibody by cloning.
4. research A2 monoclonal antibody is discerned the distribution of membrane molecule p140
4.1 human PBMC's activation: obtain the human PBMC with 1. methods, by containing 2 * 106 cell cultures in the 2ml perfect medium in 24 orifice plates, add PHA (4 μ g/ml), PMA (40ng/ml), PMA (40ng/ml)+A23187 (0.9 μ g/ml) respectively, other have a hole for be coated with CD3mAb (with 8 μ g/ml concentration wrap by).Wherein PMA, PMA+A23187 cultivate 48h for two groups, and PHA, CD3 mAb cultivate 72h for two groups, and the PBMC that other has PHA to stimulate keeps by adding rHuIL-2 (100u/ml) and is cultured to 12 days.Also have one group of mixed lymphocytes cultured cells to keep simultaneously and be cultured to 12 days by adding rHuIL-2 (100u/ml).The mixed lymphocytes culturing cell that is used for following immune double-colored analysis just adds rHuIL-2 (100u/ml) when stimulating first day.
4.2 immunofluorescence dyeing and flow cytometry: adjust various cell concns to 1 * 10 with the perfect medium that contains 10% calf serum
7/ ml respectively gets 50 μ l cell suspensions and adds in the pipe that is added with 50 μ l dilution in 1: 20 (diluting with DPBS) deactivation normal rabbit serum in advance, adds different mAb again, shake well, and 4 ℃ of effect 30min wash 2 times with washings.Abandon supernatant, add the sheep anti mouse FITC mark two anti-(dilution in 1: 20) of working concentration, shake well, 4 ℃ of effect 30min wash 2 times with washings, add an amount of stationary liquid, and FCM detects.In the double-colored experiment of direct immunofluorescence, adjust cell concn to 1 * 10
7/ ml, get 50 μ l cell suspensions and add and to be added with in advance in 50 μ l dilution in 1: the 20 deactivation normal rabbit serum, 4 ℃ hatch 10min after every again pipe add PE and FITC mark specific antibody, continue 4 ℃ again and hatch 30min, add an amount of stationary liquid after washing 3 times at last, FCM detects.
4.3 the result that obtains: the immunocyte membrane molecule p140 that the A2 monoclonal antibody is discerned does not express in static peripheral blood mononuclear cell (PBMC), the selective expression cultivates institute's activated T cell surface in mixed lymphocytes, other activated channel such as solid phase CD3 stimulation, PHA, PMA and PMA+A23187 stimulate, and can not induce its expression.The PBMC that PHA stimulates did not also express p140 in the time of 12 days, and mixed lymphocytes cultivation institute activated T cell promptly had than high expression level since the 3rd day.Find that through flow cytometry the p140 developed by molecule is in activated T and NK cell surface, especially at TCR γ δ
+The T cell expressing is higher.
5. study the function that the p140 molecule is participated in
The cytotoxicity test 5.1 heavily lead: collect logarithmic phase P815 cell, mark
51Cr (100 μ Ci/1 * 10
6Cell), hatch 2h, rock once for 37 ℃ every 15min.Finish after scouring 3 times of mark, adjusting target cell concentration is 1 * 10
6/ ml adds 96 hole U templates, 100 μ l/ holes.7 days activated lymphocyte action effect cell behind the collection MLC, adjusting cell concn is 1 * 10
6/ ml.Get 600 μ l cell suspensions and add corresponding antibody respectively and hatch 30min for every group at 37 ℃, centrifugal, resuspended with 600 μ l perfect mediums, carry out behind the doubling dilution adding respectively and contain in the 96 hole U templates of the P815 cell of mark 100 μ l/ holes, 37 ℃, 5%CO
2Incubator is cultivated 4h, collects supernatant, detects the cpm value with γ numeration instrument.
5.2 the result that obtains: the p140 molecule has the synergy with the CD3 molecule, is 10: 1 and 5: 1 o'clock imitating the target ratio, improves CD3 stimulated cells cytotoxic activity 12.1% and 9.2% respectively.
6. identify the molecular weight of p140 molecule
6.1 biotin labeling, immunoprecipitation and chemoluminescence: collect the JM cell 1 * 10 that is in logarithmic phase
7, to operate according to biotin labeling and the described step of immunoprecipitation test kit, the gained sample is transferred on the pvdf membrane behind the SDS-PAGE electrophoresis, with the colour developing that exposes of POD mark Avidin chemical luminescence reagent kit.
6.2 the result that obtains: the molecular weight of finally identifying the p140 molecule is 120-140kDa.
Claims (2)
1. activating T cell surface antigen that new molecular weight is 140kDa is characterized in that: mixed lymphocytes is cultivated the activating T cell immunity BALB/c mouse that obtains, behind booster immunization 3 days, get spleen, preparation splenocyte suspension and myeloma cell NS-1 merge, and cultivate with the HAT selectivity; By indirect immunofluorescence mark and flow cytometry, screening can be cultivated the activating T cell bonded hybridoma supernatant that obtains with mixed lymphocytes, the fused cell of secretion positive antibody is by cloning, obtained the A2 monoclonal antibody: analyze the activating T cell surface antigen that the A2 monoclonal antibody is discerned by immunofluorescence dyeing, flow cytometry and the cellulotoxic experiment that heavily leads, show that it has unique distribution, expression rule and function, identifies that through biotin labeling, immunoprecipitation and chemoluminescence its molecular weight is 140kDa.
2. the activating T cell surface antigen that a kind of new molecular weight according to claim 1 is 140kDa, it is characterized in that: this cell-surface antigens unique distribution is in mixed lymphocyte reacion activatory CD4
+, CD8
+, TCR γ δ
+T cell and activatory NK cell, and in other activated channel (stimulating as solid phase CD3 stimulation, PHA, PMA and PMA+A23187), this molecule is not expressed on the activating T cell surface, it can be worked in coordination with the CD3 molecule and stimulate the T cell activation, and its molecular weight is 140kDa.
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| CN 01106793 CN1314414A (en) | 2001-03-16 | 2001-03-16 | New activated T cell surface antigen of molecular weight 140 KDa |
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| CN 01106793 CN1314414A (en) | 2001-03-16 | 2001-03-16 | New activated T cell surface antigen of molecular weight 140 KDa |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101329230B (en) * | 2008-07-14 | 2010-10-20 | 中国人民解放军第三军医大学 | Improved method for dyeing immunofluorescence cell |
| CN101633907B (en) * | 2001-08-15 | 2012-09-05 | 宝生物工程株式会社 | Amplification culture method for antigen specific cytotoxic t lymphocytes |
-
2001
- 2001-03-16 CN CN 01106793 patent/CN1314414A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101633907B (en) * | 2001-08-15 | 2012-09-05 | 宝生物工程株式会社 | Amplification culture method for antigen specific cytotoxic t lymphocytes |
| CN101329230B (en) * | 2008-07-14 | 2010-10-20 | 中国人民解放军第三军医大学 | Improved method for dyeing immunofluorescence cell |
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