CN104894273A - Reporter gene detection method for biological activity of T cell activation regulator - Google Patents
Reporter gene detection method for biological activity of T cell activation regulator Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention relates to the technical field of biological pharmacy and discloses a reporter gene detection method for biological activity of a T cell activation regulator. According to the reporter gene detection method, a reporter gene vector driven by an IL-2 promoter transfects Jurkat cells and a stable monoclonal cell line Jurkat IL-2P Luc is obtained through pressurizing screening and monoclonal separation culture. A CD3 antibody and Raji cells expressing costimulatory molecules CD80/86 are added, meanwhile, a first pathway and a second pathway required by T cell activation are activated, so as to enable T cells to be activated and the regulator to be detected is added to detect expression of reporter gene luciferase driven by the IL-2 promoter. The biological activity of the T cell activation regulator is quantitatively detected through the dose-dependent relation between the dosage of the regulator and expression quantity of luciferase. According to the invention, the operation is simple, the sensitivity is high, repeatability is good, and the reporter gene detection method has important significance on quality control of the T cell activation regulator and clinical application.
Description
Technical field
The present invention relates to biological pharmacy technical field, specifically relate to a kind of reporter gene detection method of T cell activation conditioning agent biologic activity.
Background technology
T lymphocyte (Thymus-dependent lymphocyte, be called for short T cell) function suppression or activate play a part in tumour immunity, autoimmune disease and graft-rejection very important, T cell activation, not only need the first signal of φt cell receptor, and need second signal.First signal comes from CD3/TCR (the T cell receptor in T cell, φt cell receptor) major histocompatibility complex (major histocompatibility complex, MHC) on mixture and antigen presenting cell or other cell types and antigenic compound; Second signal and costimulatory signal derive from the combination of the CD28 in T cell and the CD80/86 on antigen presenting cell, this signal path is regulated, as the combination by CTLA-4 competitive inhibition CD28 and CD80/86, and then block second signal path, or by CD3 antibody competition in conjunction with CD3/TCR mixture, thus block the first signal path, the effect of adjustment (suppress or activate) T cell activity can be played.Based on this, emerge gone on the market in a large number or grind for T cell activation process in involved molecular events and the medicine that designs, be below referred to as T cell activation conditioning agent.Detecting T cell activation degree, is the important indicator assessed its drug effect when developing this type of medicine based on cell levels.
Existingly at cell levels, following a few class is divided into the method that T cell activation degree detects: first, detect the T cell multiplication effect (as patent CN 101427135 A) that T cell activation causes, the method needs to use primary T cells, material source relative difficulty and repeatability not do not ensure, duration of experiment is longer simultaneously, is generally 5-7 days; Second, associated molecule in direct-detection T cell activation process, the expression of such as mRNA or albumen is (as patent CN 101427135 A, US 6114129 A, US 5180662 A, CN 102811739 A and EP 2510948 A1, and reference: Stohl W et al., J Immunol.1990Aug 15; 145 (4): 1078-87.), the method experiment flow is complicated, and repeatability is short of relatively; 3rd, the problems such as detect T cell to the adhesivity (as patent CN 102811739 A and EP 2510948 A1) of the adhesion molecules such as ICAM1, this system needs separating primary cells equally and there is experiment flow complexity, the relative shortcoming of repeatability; 4th, detect the expression of the reporter gene of the promoters driven of associated molecule in T cell activation process (as patent US 5474897 A; And reference: Schwarz EM et al., Proc Natl Acad Sci U S A.1993Aug 15; 90 (16): 7734-8.; Qiu D et al., J Biol Chem.1999May 7; 274 (19): 13443-50.; Yang XY et al., J Biol Chem.2000Feb18; 275 (7): 4541-4.; Weaver JR et al., Mol Immunol.2007Apr; 44 (11): 2813-9.), the relatively above-mentioned three kinds of methods of these class methods, have quick, easy, repeatability feature preferably.
Based on the method detected T cell activation degree at cell levels of Reporter System, domestic there is no at present Patents report.In the 4th class detection means abroad had been reported, the CD28RE that have employed in the experimental system that US 5474897 A provides in IL-2 promotor drives the expression of reporter gene, CD28RE is positioned at-164 to-154, IL-2 transcription initiation site upstream (reference: Butscher WG et al., J Biol Chem.1998Jan 2; 273 (1): 552-60.), and IL-2 promotor comprises-300 to+40, containing the multiple response elements except CD28, the transcriptional expression of IL-2 is subject to the common regulation and control of these response elements, only can not well simulate with CD28E and the transcriptional expression situation of Inner source IL-2 after T cell activation under being reflected in external stimulus effect; Another kind of technique means in 4th class detection means is (as reference: Schwarz EM et al., Proc Natl Acad Sci U S A.1993Aug 15; 90 (16): 7734-8.; Qiu D et al., J Biol Chem.1999May 7; 274 (19): 13443-50.; Yang XY et al., J Biol Chem.2000Feb 18; 275 (7): 4541-4.; Weaver JR et al., Mol Immunol.2007Apr; 44 (11): 2813-9.), what use is the combination of stimulation mode of phorbol-12-myristate 13-acetate (PMA)+ionomycin class, PMA freely can enter cytolemma, directly acts on intracellular PKC (protein kinase C); Inonomycin is Calcium ionophore, makes stream in the Ca2+ of extracellular, the transcription factor of signal acting in conjunction in core that PKC and Ca2+ produces, and starts transcribing of T cell activation genes involved (as IL-2) and synthesizes with albumen; This stimulation mode effect is fast, but owing to directly acting on the middle and lower reaches of T cell activation signal path, cannot simulate the activation method of T cell under the pathological conditions such as tumour immunity, autoimmune disease and graft-rejection.
Summary of the invention
The object of the invention is to overcome weak point of the prior art, provide a kind of reporter gene detection method of T cell activation conditioning agent biologic activity, T cell activation conditioning agent can promote or suppressor T cell activates, if this type of conditioning agent possesses biologic activity, by detecting this type of conditioning agent the change of T cell activation degree to be detected, the present invention activates while needing the first signal and second signal according to T cell activation, and the critical event in this process is the mechanism of IL-2 up-regulated, utilizing when the first signal and second signal activate simultaneously can the T cell Jurkat of great expression IL-2, full genome synthesis IL-2 promotor-329 to+48, be connected to reporter gene luciferase (Luciferase) upstream, build the reporter gene luciferase expression vector of IL-2 promoters driven, by this expression vector electrotransfection Jurkat cell, detected by transfection efficiency, pressurization screening, obtain the stable cell line (hereinafter referred to as Jurkat IL-2P Luc cell strain) with the luciferase expression framework of IL-2 promoters driven, on this basis, the Raji cell of CD3 antibody and expression CD80/86 is used to stimulate as upstream, the effector function of this cell strain is detected, and this detection system is optimized, its specificity and repeatability are assessed.The present invention has quick, easy and simple to handle, the reproducible and highly sensitive feature of detection.
For achieving the above object, the technical solution adopted in the present invention is as follows:
The invention provides a kind of reporter gene detection method of T cell activation conditioning agent biologic activity; Comprise:
Step one: the structure of the Reporter gene vector of IL-2 promoters driven;
Step 2: with the structure of the stable cell line of the reporter gene of IL-2 promoters driven;
Step 3: the biologic activity of T cell activation conditioning agent is detected; Its detection method comprises:
At CD3 antibody with under the B cell system Raji acting in conjunction expressing CD80/86, aforementioned stable cell strain is activated, the luciferase report gene up-regulated of IL-2 promoters driven, synchronously add T cell activation conditioning agent, by examining report gene expression amount, qualitative assessment T cell activation conditioning agent biologic activity.
Preferably, in described step one, the construction process of the Reporter gene vector of described IL-2 promoters driven comprises: by IL-2 promotor, from-329 to+48, builds up to luciferase report gene upstream, expresses in order to drive IL-2.
More preferably, in described step 2, construction process with the described stable cell line of the reporter gene of IL-2 promoters driven comprises: described cell strain is T cell, after T cell is activated, IL-2 up-regulated, meanwhile, the T cell strain transfection luciferase report gene of IL-2 promoters driven, by pressurization screening and mono-clonal separation and Culture, the biologic activity for T cell activation conditioning agent detects;
Wherein, described Jurkat is people's Pancytopenia cell;
Described Raji is human B lymphocyte oncocyte;
Described PBMC, peripheral blood mononuclear cell is peripheral blood mononuclear cell;
Described CD is cluster of differentiation, determinant molecule;
Described PHA, phytohaemagglutinin are phytohaemagglutinin;
Described PMA, phorbol-12-myristate 13-acetate is phorbol exters;
Described Ionomycin is ionomycin;
Described IL-2, interleukin 2 is interleukin II;
Described CD28RE, CD28response element is CD28 response elemets;
Described CTLA4, cytotoxic T lymphocyte-associated antigen-4 is Cytotoxic T lymphocyte associated antigen-4;
Described Fc, crystalline fragment is crystallizable fragment.
The present invention compared with prior art has the following advantages:
Domesticly there is no the experimental system relevant report of by reporter gene detection method, T cell activation conditioning agent biologic activity being carried out to qualitative assessment, the present invention has filled up that this is blank, compared with the experimental system using primary T cells to carry out detecting, detection method provided by the present invention, without the need to end user's peripheral blood, solve current human peripheral source difficulty and this difficult problem poor of the experimental result consistence corresponding to peripheral blood in different people source, simultaneously without the need to being separated primary T cells, avoiding the flow processs such as loaded down with trivial details PBMC separation and T cell magnetic bead sorting, shortening experiment flow, simplify laboratory operating procedures, the upstream used stimulates as CD3 antibody and the Raji cell of expressing CD80/86, compared with the stimulus modality of traditional PHA or PMA+ionomycin, this stimulation mode of the present invention, closer to the truth in the pathologic condition lower bodies such as tumour immunity, autoimmune disease and graft-rejection during t cell activation, we have selected the critical event IL-2 in T cell activation process to be expressed as check position simultaneously, choose IL-2 promotor-329 to+48, in order to drive the expression of reporter gene luciferase, the CD28RE (being positioned at-164 to-154, IL-2 transcription initiation site upstream) adopted in the experimental system had been reported, IL-2 transcription factor action site contained by it is more comprehensive, the state that IL-2 activates at transcriptional level can be reflected more comprehensively really, qualitative assessment T cell activation degree, and then detection by quantitative is carried out to T cell activation conditioning agent biologic activity, this system has quick, easy and simple to handle, reproducible feature, to the quality control of T cell activation conditioning agent and clinical application significant.
Figure of description
Fig. 1 schematically illustrates the GFP expression schematic diagram of Jurkat cell by Flow cytometry of untransfected plasmid;
Fig. 2 schematically illustrates the GFP expression schematic diagram of GFP-transfected Jurkat cell by Flow cytometry;
Fig. 3 schematically illustrates and detects after CD3 antibody and Raji stimulate altogether, different clone's luciferase expression situation schematic diagram;
Fig. 4 schematically illustrates CTLA4-Fc fusion rotein and suppresses IL-2 to express dose-response curve schematic diagram;
Fig. 5 schematically illustrates elemental method checking-specificity schematic diagram;
Fig. 6 schematically illustrates elemental method checking-repeated schematic diagram.
Embodiment
Below in conjunction with drawings and Examples in detail the present invention is described in detail.
The structure of 1.pGL4.15-IL-2P plasmid
1.1 full genome synthesis IL-2 promotor (-329 to+48), wherein upstream is with KpnI restriction enzyme site GGTACC, downstream belt HindIII restriction enzyme site AAGCTT, and complete sequence is as follows:
GGTACCGAAAATTTTCTGAGTTACTTTTGTATCCCCACCCCCTTAAAGAAAGGA
GGAAAAACTGTTTCATACAGAAGGCGTTAATTGCATGAATTAGAGCTATCACCT
AAGTGTGGGCTAATGTAACAAAGAGGGATTTCACCTACATCCATTCAGTCAGTC
TTTGGGGGTTTAAAGAATTCCAAAGAGTCATCAGAAGAGGAAAAATGAAGGT
AATGTTTTTTCAGACAGGTAAAGTCTTTGAAAATATGTGTAATATGTAAAACATT
TTGACACCCCCATAATATTTTTCCAGAATTAACAGTATAAATTGCATCTCTTGTTC
AAGAGTTCCCTATCACTCTCTTTAATCACTACTCACAGTAACCTCAACTCCTGCCA
CAAAGCTT
1.2 full genome synthetic products and pGL4.15 plasmid are (purchased from Promega, article No. E6701) after KpnI and HindIII enzyme is cut, IL-2 promotor is connected into pGL4.15, build pGL4.15-IL-2P plasmid, Sinogenomax Co., Ltd. is sent to check order, entirely true.
The screening of 2 stable cell lines
2.1 transfection
(1) 2 days in advance, recovery Jurkat cell (purchased from ATCC, article No. TIB-152);
(2) (RPMI1640 is (purchased from GIBCO to get growth medium, article No. 61870-036500ML)+10% bovine serum is (purchased from ExCell, article No. FDN500)+1% dual anti-(purchased from Life, article No. 15070-063)) add in 2 holes of 6 orifice plates, 3ml/ hole, preheating in incubator; Separately get 220ul Cell line Nucleofector Solution V (purchased from Lonza, article No. VCA-1003)) preheating 10min in incubator;
(3) get 5ug pGL4.15-IL-2P plasmid respectively, 5ug pmaxGFP plasmid (transfection control) is for subsequent use in 2 aseptic 1.5ml centrifuge tubes;
(4) Nucleofector electroporation (Lonza) start, select procedure X-001;
(5) Jurkat cell counting, get 3,000,000 cells respectively in 2 aseptic 1.5ml centrifuge tubes, the centrifugal 5min of 90g, abandons most supernatant;
(6) often pipe adds the Cell line Nucleofector Solution V of 100ul preheating, and featheriness adds in the aseptic 1.5ml centrifuge tube containing plasmid, then featheriness adds in electric revolving cup;
(7) electric revolving cup is put into electroporation, press ' X ' key, complete after electricity turns, to add bottom electric revolving cup slightly sucking-off cell after pressure-vaccum with the growth medium that preheating got by the suction pipe that test kit provides, add in 6 orifice plate substratum, cultivate 24h.
2.2 detect transfection efficiency
Get 100,000, the cell of transfection pmaxGFP plasmid, Flow cytometry transfection efficiency.The fluorescence intensity being positioned at the cell of 1 passage after transfection obviously strengthens, and transfection efficiency is up to 88.5%, and transfection effectively (Fig. 1).
2.3 pressurization screening and Jurkat IL-2P Luc monoclonal cell strain separation and Culture
(1) 24h after transfection, the substratum that brings Selection In (growth medium+250ug/ml Totomycin), screen 3 weeks, every 3-4 days changes a Selective agar medium;
(2) treat that cellular control unit is entirely dead, screening completes.Counting, Selective agar medium is by cell dilution to 30 cell/15ml, 150ul/ hole paving 96 orifice plates at the bottom of U;
(3) often within 3-4 days, change a Selective agar medium, cultivate 2 weeks, basis of microscopic observation, select the cell hole of mono-clonal growth, and be transferred to 24 orifice plates, 1 Zhou Houzai reaches 6 orifice plate enlarged culturing.
3. effector function detects
3.1T the foundation of cell activation detection system
(1) count Jurkat IL-2P Luc cell, Selective agar medium is by cell dilution to 200 ten thousand cell/ml;
(2) count Raji cell, Selective agar medium is by cell dilution to 200 ten thousand cell/ml;
(3) the cell equal-volume mixing of having diluted above-mentioned two kinds, according to density paving 96 orifice plates at the bottom of V in 100ul/ hole;
(4) with Selective agar medium by CD3 antibody (purchased from BD Pharmingen, article No.: 555337) being diluted to final concentration is 0.4ug/ml, adds in 96 orifice plates at the bottom of above-mentioned V according to the volume in 100ul/ hole, pressure-vaccum makes itself and cell mix;
(5) cell cultivates 6h in CO2gas incubator;
(6) the centrifugal 5min of 3000rpm, sucks cell conditioned medium, and 50ul/ hole adds lysate Glo-lysis (purchased from Promega, article No.: E2661), lysis at room temperature 5min;
(6) draw 10ul cell lysate and proceed to white 384 orifice plates (purchased from PerkinElmer, model: ProxiPlate-384Plus), add luciferase substrate Bright-Glo Luciferase Assay System (purchased from Promega, article No. E2610), 10ul/ hole, reaction 5min, detects luciferase activity.
After CD3 antibody and Raji are total to irritation cell, cell be activated and express IL-2, compare IL-2 expression amount with non-stimulated control group obviously to increase, illustrate that CD3 antibody and Raji stimulate altogether can activate this transfectional cell and make it express IL-2, the synchronous expression (Fig. 2) stimulating reporter gene luciferase.
The foundation of 3.2T cell activation conditioning agent (for CTLA4-Fc fusion rotein) biologic activity detection system
Screen the IL-2P Luc positive cell strain obtained, according to 3.1 experimental results, choose No. 1 clone and carry out subsequent experimental.Detect the reactivity to CTLA4-Fc fusion rotein, cell density is 100,000/hole, and CTLA4-Fc initial concentration is 67ug/ml, 10 multiple proportions serial dilution 13 gradients, totally 14 concentration point.CTLA4-Fc acts on 6 hours and detects luciferase activity.
After adding the CTLA4-Fc fusion rotein of gradient dilution, along with CTLA4-Fc concentration increases, fluorescence intensity reduces gradually, namely IL-2 expression amount reduces gradually, the activation rate of visible cell reduces, and illustrates that this system can be used for the biologic activity (Fig. 3) of detection by quantitative T cell activation conditioning agent.
4 elemental method checkings
Select irrelevant IgG as negative control, the specificity of assessment the method; The detection of homogeneous Multi-example is carried out to the CTLA4-Fc fusion rotein of same batch, the repeatability of assessment the method.
Result shows, and adds irrelevant IgG, does not observe the suppression of T cell activation, and after adding CTLA4-Fc fusion rotein, T cell activation is significantly suppressed (Fig. 4).The display of repeatability result, CV% (variation coefficient) is 9.32%, has good repeatability (table 1, Fig. 5).
Table 1, elemental method checking-repeatability
| Sample name | IC 50(mol/L) |
| CTLA4-Fc 1 | 2.54E-09 |
| CTLA4-Fc 2 | 2.01E-09 |
| CTLA4-Fc 3 | 2.45E-09 |
| CTLA4-Fc 4 | 2.18E-09 |
| CTLA4-Fc 5 | 2.42E-09 |
| Mean value | 2.1816E-09 |
| Standard deviation | 2.62761E-10 |
| CV％ | 9.32％ |
For the ordinary skill in the art; specific embodiment is just to invention has been exemplary description; obvious specific implementation of the present invention is not subject to the restrictions described above; as long as have employed the improvement of the various unsubstantialities that method of the present invention is conceived and technical scheme is carried out; or design of the present invention and technical scheme directly applied to other occasion, all within protection scope of the present invention without to improve.
Claims (3)
1. the reporter gene detection method of a T cell activation conditioning agent biologic activity; Comprise:
Step one: the structure of the Reporter gene vector of IL-2 promoters driven;
Step 2: with the structure of the stable cell line of the reporter gene of IL-2 promoters driven;
Step 3: the biologic activity of T cell activation conditioning agent is detected, its detection method comprises:
At CD3 antibody with under the B cell system Raji acting in conjunction expressing CD80/86, aforementioned stable cell strain is activated, the luciferase report gene up-regulated of IL-2 promoters driven, synchronously add T cell activation conditioning agent, by examining report gene expression amount, qualitative assessment T cell activation conditioning agent biologic activity.
2. the reporter gene detection method of T cell activation conditioning agent biologic activity according to claim 1, in described step one, the construction process of the Reporter gene vector of described IL-2 promoters driven comprises: IL-2 promotor is built up to luciferase report gene upstream, expresses in order to drive IL-2.
3. the reporter gene detection method of T cell activation conditioning agent biologic activity according to claim 1, in described step 2, construction process with the described stable cell line of the reporter gene of IL-2 promoters driven comprises: described cell strain is T cell, after T cell is activated, IL-2 up-regulated, simultaneously, the T cell strain transfection luciferase report gene of IL-2 promoters driven, by pressurization screening and mono-clonal separation and Culture, the biologic activity for T cell activation conditioning agent detects.
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Cited By (6)
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| CN107916276A (en) * | 2017-12-04 | 2018-04-17 | 北京东方百泰生物科技有限公司 | A kind of novel T cell immunomodulator bioactivity detection method |
| CN108351344A (en) * | 2015-10-14 | 2018-07-31 | 耶鲁大学 | The T cell active array and its application method of Genome Scale |
| CN109975537A (en) * | 2019-04-09 | 2019-07-05 | 上海药明生物技术有限公司 | A kind of kit and method detecting TIM-3 antibody activity |
| CN112458149A (en) * | 2020-12-03 | 2021-03-09 | 北京东方百泰生物科技股份有限公司 | Method for detecting activation degree of antibody drug on T cells by taking CD3 as target |
| CN112921004A (en) * | 2021-02-05 | 2021-06-08 | 上海精翰生物科技有限公司 | Cell strain for detecting CD3 receptor agonist, construction method thereof and CD3 receptor agonist detection method |
| CN114292846A (en) * | 2021-12-30 | 2022-04-08 | 自然资源部第三海洋研究所 | Large yellow croaker interleukin 2 gene promoter sequence and application thereof |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108351344A (en) * | 2015-10-14 | 2018-07-31 | 耶鲁大学 | The T cell active array and its application method of Genome Scale |
| CN108351344B (en) * | 2015-10-14 | 2021-07-30 | 耶鲁大学 | Genome-scale T cell activity arrays and methods of use thereof |
| CN107916276A (en) * | 2017-12-04 | 2018-04-17 | 北京东方百泰生物科技有限公司 | A kind of novel T cell immunomodulator bioactivity detection method |
| CN107916276B (en) * | 2017-12-04 | 2019-11-08 | 北京东方百泰生物科技有限公司 | A kind of novel T cell immunomodulator bioactivity detection method |
| CN109975537A (en) * | 2019-04-09 | 2019-07-05 | 上海药明生物技术有限公司 | A kind of kit and method detecting TIM-3 antibody activity |
| CN109975537B (en) * | 2019-04-09 | 2022-05-24 | 上海药明生物技术有限公司 | Kit and method for detecting activity of TIM-3 antibody |
| CN112458149A (en) * | 2020-12-03 | 2021-03-09 | 北京东方百泰生物科技股份有限公司 | Method for detecting activation degree of antibody drug on T cells by taking CD3 as target |
| CN112921004A (en) * | 2021-02-05 | 2021-06-08 | 上海精翰生物科技有限公司 | Cell strain for detecting CD3 receptor agonist, construction method thereof and CD3 receptor agonist detection method |
| CN112921004B (en) * | 2021-02-05 | 2023-08-08 | 上海精翰生物科技有限公司 | Cell strain for detecting CD3 receptor agonist, construction method thereof and CD3 receptor agonist detection method |
| CN114292846A (en) * | 2021-12-30 | 2022-04-08 | 自然资源部第三海洋研究所 | Large yellow croaker interleukin 2 gene promoter sequence and application thereof |
| CN114292846B (en) * | 2021-12-30 | 2023-08-15 | 自然资源部第三海洋研究所 | Large yellow croaker interleukin 2 gene promoter sequence and application thereof |
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