CN112760291B - Cell line for detecting human interferon-kappa activity and construction and detection method thereof - Google Patents
Cell line for detecting human interferon-kappa activity and construction and detection method thereof Download PDFInfo
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Abstract
The invention discloses a cell line for detecting human interferon-kappa activity and a construction and detection method thereof. The cell line of the present invention is obtained by stably screening a monoclonal cell line by transfecting eukaryotic cells with an ISRE (interferon sensitive response element) luciferase reporter gene. The cell line constructed by the invention is easy to culture and amplify and has good passage stability, and the detection of the activity of the human interferon-kappa has the advantages of high sensitivity, short detection time, completion within 4-6 hours and capability of greatly improving the activity release efficiency of the human interferon-kappa in the production process.
Description
Technical Field
The invention belongs to the technical field of biological engineering, and particularly relates to a method for detecting the activity of human interferon-kappa by using a stably transfected cell line.
Background
Interferons (IFNs) are a class of cytokines with broad-spectrum antiviral, antitumor, immunomodulatory effects. It can exert antiviral activity by binding to cognate receptor complexes on target cells.
Interferon kappa (IFN-. Kappa.) is a new member of IFNs discovered in recent years, where IFN-. Kappa.consists of 207 amino acid residues, including a signal peptide of 27 amino acid residues at the N-terminus, with about 30% homology to other members of type I IFNs, and where the IFN-. Kappa.member is slightly larger than the other type I IFNs, with 12 amino acid residues inserted between the C and D helices. The IFN-kappa gene is located in the 9 th chromosome short arm, adjacent to other type I IFN genes, and the 3' end noncoding region of the IFN-kappa gene contains 1 intron. IFN-kappa shares a receptor protein with IFN-alpha and IFN-beta and belongs to the type I interferon. At present, researches show that the gene coding IFN-kappa is selectively expressed in epithelial keratin cells, and the recombinant hIFN-kappa is similar to interferons of other homotypes and can protect the cells from virus infection; and the expression has ethnic specificity.
After outbreak of new crown epidemic situation in 2019, interferon-alpha is listed as a broad-spectrum antiviral drug in a new crown treatment scheme under the condition of lack of specific drugs. However, studies have shown that various viruses have developed resistance mechanisms to interferon alpha, such as dengue fever, west nile virus, zika virus, and the like. Interferon- κ differs from interferon α in that the amino acid sequence is only 35% homologous, so many interferon α -resistant viruses remain sensitive to interferon κ. Therefore, the interferon-kappa (IFN-kappa) is expected to be an innovative medicine for treating the new coronary pneumonia. However, hIFN-. Kappa.is difficult to produce and has low yield, and the process of detecting the activity of the product is time-consuming. At present, the activity of interferon products is mostly detected by a type I interferon activity determination method recommended by Chinese pharmacopoeia, but the two methods described by the pharmacopoeia require the culture time of 18-24 hours. At present, the research on the method for quickly detecting the activity of hIFN-kappa in the prior art is rare.
Disclosure of Invention
The object of the present invention is to provide a cell line for the detection of interferon, in particular of hIFN- κ activity, by means of which a rapid and reliable detection of interferon activity is achieved.
Another objective of the present invention is to provide a method for constructing a cell line for interferon activity detection, which can be used for releasing interferon-kappa activity by constructing the stable monoclonal cell line, and can realize a sensitive and reliable activity detection result after a short culture time.
In order to achieve the purpose, the invention adopts the following technical scheme:
a cell line for the detection of human Interferon- κ (hIFN- κ) activity, said cell line being transfected with an ISRE (Interferon-sensitive response element) luciferase reporter element. In a specific embodiment, the cell line is a mammalian cell line, preferably a cell line of human origin, most preferably the human embryonic kidney cell line HEK293; in another specific embodiment, said ISRE luciferase reporter element is a cell line transfected with a plasmid, preferably said plasmid is a eukaryotic cell expression plasmid, most preferably PCBRG-ISRE; in another embodiment, the cell line is a monoclonal cell line obtained by stable selection; (ii) a In another specific embodiment, the cell line is human embryonic kidney HEK293-ISRE-Luc-JY with the preservation number of CCTCC NO: C2020256, and the cell line is deposited in China center for type culture Collection, address: china, wuhan university, the preservation date is 2020, 12, 8.
A method for constructing a cell line for detecting human interferon-kappa (hIFN-kappa) activity, comprising the following steps: 1) Cell recovery culture; 2) Transfecting a reporter gene plasmid; 3) Screening; 4) Mixed cloning test; 5) Limiting dilution and single cloning; 6) Testing the monoclonal function; 7) And (5) testing the monoclonal passage stability.
In a specific embodiment, the reporter plasmid contains an ISRE luciferase reporter element; preferably, the plasmid is a eukaryotic cell expression plasmid; more preferably, the plasmid is selected from any one of PCBRG-ISRE, pISRE-Luc and pGL 3-ISRE; further preferably, the plasmid is PCBRG-ISRE.
In a specific embodiment, the cell line is a mammalian cell line, preferably a cell line of human origin, more preferably the human embryonic kidney cell line HEK293.
In a specific embodiment, the transfection is a lipofection, preferably a Lipofectamin 3000 transfection.
In a specific embodiment, the screen is an antibiotic screen, preferably a hygromycin (hygromycin) resistance screen.
In a specific embodiment, the cell line is a monoclonal cell line obtained by stable selection.
In a specific example, the screened cells were tested for mixed clones, in which a gradient of diluted hIFN-. Kappa.was added to the mixed clone cell suspension, followed by luciferase assay for signal detection.
In a specific embodiment, the monoclonality is obtained by amplifying cells with good fluorescence signals detected by mixed clones, then carrying out passage and obtaining a monoclonal cell line by a limiting dilution method.
In a specific example, the obtained monoclonal cell lines were subjected to functionality and passaging stability tests; preferably, the obtained monoclonal cell line is diluted to 3x10 4 -5x10 5 Adding a gradient concentration of diluted hIFN-kappa into each living cell/ml, and then adding luciferase for signal detection.
In a specific example, the monoclonal antibodies with good signal detection were subcultured, and activity detection was performed by replacing part of the cells at P5, P10, P15 and P20.
In a specific embodiment, the cell recovery culture comprises: taking out HEK293 cells from liquid nitrogen tank, thawing in water bath at 36-38 deg.C, culturing in complete culture medium, and subculturing in cell culture box at 37 deg.C and 5% carbon dioxide for 2-3 generations.
In a specific embodiment, the transfection reporter plasmid comprises:
1) Taking the HEK293 cells after subculture, digesting with trypsin, stopping digestion with the complete culture medium, blowing to blow the cells, 800-Centrifuging at 1500rpm for 5-10 min, and adjusting cell concentration to 3 × 10 4 -5x10 5 Individual cells/ml;
2) Inoculating the cells into a 6-well plate, wherein each well is 100-120 mu l, and continuously culturing for 24-36 hours;
3) 2-4ug of PCBRG-ISRE plasmid was transfected into HEK293 cells according to Lipofectamin (Liposome) 3000 transfection reagent instructions, under the following conditions:
(a) Preparing a mixed solution of a tube A: 100-150 mul of serum reducing culture medium (preheated 37 ℃) +2-5 mug of PCBRG-ISRE +5-8ul Lipofectamin 3000 reagent, and mixing uniformly;
(b) Preparing mixed liquor of a tube B: 100-150 mul of reduced serum culture medium (preheated to 37 ℃) +8-12 mul of Lipofectamin 3000 transfection reagent, and mixing evenly;
(c) Uniformly mixing the mixed solution of the tube A and the mixed solution of the tube B to obtain a transfection solution, and standing for 3-10 minutes at room temperature;
(d) Adding the transfection solution into a HEK293 cell 6-pore plate, and uniformly mixing;
(e) The 6-well plate was placed in a cell incubator for further 24-36 hours.
In a specific embodiment, the reduced serum medium is Opti-MEM.
In a specific embodiment, the screening comprises: after 12 to 36 hours of cell transfection, DMEM medium containing 10% FBS and 200ug/ml hygromycin was added to the cells, and resistance was selected for 1 to 3 weeks.
In a specific embodiment, the mixed clone test comprises:
1) Digesting the HEK293-ISRE-Luc mixed clone cells subjected to amplification culture by using pancreatin, stopping digestion by using a screening culture medium, blowing and beating the cells, and centrifuging at 800-1500rpm for 3-10 minutes;
2) Discarding the supernatant, and blowing and resuspending the cells by using a screening culture medium to obtain a cell suspension;
3) Adjusting the cell density of the cell suspension to 3X10 4 -5x10 5 Each living cell is blown and beaten uniformly;
4) Adding 100-120 mul of the blown cell suspension obtained in the step 3) into each hole of a 96-hole plate, and putting the cell suspension into an incubator for continuous culture;
5) Diluting interferon with screening culture medium to obtain interferon diluent, adding 10-15 μ l interferon diluent into each well, setting negative control well, and culturing for 4-6 hr;
6) 80-100. Mu.l of Bright-Glo luciferase reagent was added to each well, reacted for 2-5 minutes, and luciferase signal was measured using a microplate reader.
In a specific embodiment, the limiting dilution monoclonality comprises:
1) The HEK293-ISRE-Luc mixed clone cells are subjected to subculture by using a screening culture medium, so that the cells are ensured to be in a good state and have the activity of more than 90% during monoclonation;
2) Taking cells with good growth state, digesting with pancreatin, stopping digestion with a screening culture medium, beating by blowing, and centrifuging at 800-1500rpm for 3-10 minutes;
3) Resuspending the cells in a selection medium to obtain a cell suspension, and adjusting the cell density of the cell suspension to 5X10 4 -5x10 5 Viable cells/ml;
4) The cells were monoclonalized by plating by limiting dilution to obtain 0.5X 10 2 -8×10 2 Viable cells/ml of cell suspension;
5) Diluting the cell suspension, blowing and beating uniformly, and adding 100-120 mu l of cell suspension into a 96-well plate;
6) Recording micropores of single cells in a 96-well plate, and replacing a fresh screening culture medium for the cells once every 3-5 days;
7) When the cell confluence degree in a 96-well reaches 40-60%, carrying out trypsinization, and carrying out amplification culture to obtain a 24-well plate;
8) Cells were continued to be cultured and expanded for functional and stability testing.
In a specific embodiment, the monoclonal functional and stability tests include:
1) Digesting HEK293-ISRE-Luc monoclonal cells with pancreatin, stopping digestion with a screening culture medium, blowing cells, and centrifuging at 800-1500rpm for 3-10 minutes;
2) Discarding the supernatant, and blowing and resuspending the cells by using a screening culture medium to obtain a cell suspension;
3) Adjusting the cell density of the cell suspension to 3x10 4 -5x10 5 Each living cell is blown and beaten uniformly;
4) Adding 100-110 mu l of the blown cell suspension obtained in the step 3) into each hole of a 96-hole plate, and putting the cell suspension into an incubator for continuous culture;
5) Diluting interferon with a screening culture medium in a gradient manner by 3-5 times to obtain an interferon diluent;
6) Adding 10-15 mul of interferon diluent into each hole, and continuously culturing for 4-6 hours;
7) Adding 80-100 μ l Bright-Glo luciferase reagent into each well, reacting for 2-5 min, and measuring luciferase signal with microplate reader;
8) The data is analyzed.
In particular, in the above method steps, the complete medium is a high-glucose DMEM medium containing 10% Fetal Bovine Serum (FBS).
In particular, in the above method steps, the selection medium is DMEM medium containing 10% Fetal Bovine Serum (FBS) and 200ug/ml hygromycin.
In particular, HEK293 cells in the logarithmic growth phase were trypsinized in step 1) of the transfection reporter plasmid procedure.
Specifically, in step 3) of the mixed clone test procedure, cell counting was performed with trypan blue staining, and cell density was adjusted to 3x10 with the screening medium according to the cell counting result 4 -5x10 5 Each live cell/ml, and the blow-beating is uniform.
In particular, in step 7) of the mixed clone test procedure, data were analyzed using graphpad. Prism.6 software.
In particular, in step 1) of the limiting dilution monoclonalization procedure, cells in a good state of logarithmic growth phase are taken for trypsinization.
Specifically, in step 3) of the limiting dilution monoclonalization procedure, cells were resuspended in 3-8ml of selection medium, cell counting was performed using trypan blue staining, and the cell density was adjusted to 5x10 by adding the selection medium according to the cell counting results 4 -1x10 5 Viable cells/ml.
In particular, in step 4) of the limiting dilution monoclonalization operation,screening culture medium is adopted according to the proportion of 1: (8-12) dilution of the cell line to obtain 1X 10 2 -8×10 2 Viable cells/ml of cell suspension.
In particular, in step 5) of the limiting dilution monoclonalization procedure, the selection medium is used to select the desired dna sequence at a rate of 1: (1-20) diluting the cell suspension.
Wherein, in the step 3) of the function and stability test, trypan blue staining is used for cell counting, and a screening culture medium is added according to the cell counting result to adjust the cell density to 3x10 4 -5x10 5 Viable cells/ml.
A method for detecting hIFN-kappa activity by using a cell line for detecting human interferon-kappa (hIFN-kappa) activity or a cell line prepared by using a method for constructing a cell line for detecting human interferon-kappa (hIFN-kappa) activity, comprising the steps of: 1) Culturing HEK293-ISRE-Luc monoclonal cells, digesting and centrifuging to obtain a cell suspension; 2) Mixing the cell suspension with a to-be-detected hIFN-kappa sample, and culturing; 3) Adding a detection reagent; 4) And detecting the fluorescent signal.
In a specific embodiment, in the above detection method, the HEK293-ISRE-Luc cell line is subcultured in a selection medium, trypsinized and centrifuged, and prepared to a concentration of 3 × 10 using the selection medium 4 -5x10 5 Individual cells/ml of cell suspension.
In a specific embodiment, in the above detection method, the obtained cell suspension is inoculated into a 96-well plate at 100-120 μ 1 per well, mixed with the hIFN-. Kappa.sample to be detected, and cultured at 37 ℃ under 5% carbon dioxide.
In one embodiment, the assay is performed by adding a luciferase assay reagent to each well, measuring the signal with a microplate reader, and recording the measurement.
The application of the cell line for detecting the activity of human interferon kappa (hIFN-kappa) or the cell line prepared by the construction method of the cell line for detecting the activity of human interferon kappa (hIFN-kappa) in preparing the medicine for quickly detecting the activity of human interferon kappa (hIFN-kappa).
The application of the cell line for detecting the activity of human interferon kappa (hIFN-kappa) or the cell line prepared by the construction method of the cell line for detecting the activity of human interferon kappa (hIFN-kappa) in preparing the medicine for the activity release detection of interferon kappa (hIFN-kappa).
The invention has the following beneficial effects:
(1) The invention provides a stable monoclonal cell strain capable of being used for releasing interferon-kappa activity, wherein the monoclonal cell strain is easy to culture and amplify, has good passage stability, and can sensitively and reliably monitor the interferon-kappa activity.
(2) The invention uses ISRE luciferase reporter gene system to detect the activity of interferon-kappa, and the method has large signal window, high sensitivity and short detection time, and can be completed within 4-6 hours. Compared with the prior art described in Chinese pharmacopoeia which requires cell culture for 18-24 hours for detection, the method described by the invention is more time-saving, and can greatly improve the activity release efficiency of the interferon-kappa in the production process.
Drawings
FIG. 1 shows the results of a HEK293-ISRE-Luc cell mixed clone assay;
FIG. 2 shows the results of the validation of the monoclonal activity of HEK293-ISRE-Luc cells;
FIG. 3 is a schematic flow chart of the method for detecting hIFN- κ activity of the invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments, and it is obvious that the described embodiments are for illustrative purposes only, and not for all purposes. Based on the embodiments of the present invention, those skilled in the art will better understand and appreciate the technical solutions claimed in the present invention and the technical effects achieved thereby.
In the following examples, reagents other than those specifically formulated were commercially available.
Example 1: monoclonal preparation of stably transfected cell lines
Resuscitating culture of HEK293 cells
Taking out HEK293 cells from a liquid nitrogen tank, thawing in water bath at 37 deg.C, culturing in complete culture medium (high-glucose DMEM medium (Gboco) containing 10% fetal calf serum), and culturing in cell culture box at 37 deg.C and 5% carbon dioxide; the cells are normally passaged for 3 generations to ensure that the cell state is good.
2. Transfection reporter gene plasmid
1) Taking HEK293 cells in logarithmic growth phase, digesting with pancreatin, stopping digestion with complete culture medium, blowing, adding into a centrifuge tube, centrifuging at 1000rpm for 5 min, and adjusting cell concentration to 3x10 with complete culture medium 5 Individual cells/ml;
2) Cells were seeded into 6-well plates, 100 μ l per well, and culture continued for 24 hours;
3) 3ug of PCBRG-ISRE plasmid was transfected into HEK293 cells as per the Lipofectamin 3000 transfection reagent instructions, under the following conditions:
(a) Preparing a mixed solution of a tube A: 125 mul of Opti-MEM (preheated to 37 ℃) +3 mug of PCBRG-ISRE +6ul of Lipofectamin 3000 reagent, and uniformly mixing by blowing and beating;
(b) Preparing a mixed solution of a tube B: 125 ul Opti-MEM (preheat 37 ℃) +9 ul Lipofectamin 3000 transfection reagent, blow beating and mixing well;
(c) Transferring all the reagents in the centrifugal tube B to the centrifugal tube A by using a pipettor, slightly blowing and beating the reagents, uniformly mixing the reagents to obtain transfection liquid, and standing the transfection liquid for 5 minutes at room temperature;
(d) Adding the transfection solution into a HEK293 cell 6-pore plate, and gently mixing uniformly;
(e) The 6-well plate was placed in a cell incubator for further 24 hours.
3. Antibiotic screening
24 hours after transfection, DMEM medium containing 10% Fetal Bovine Serum (FBS) and 200ug/ml hygromycin (hygromycin) was added and resistance selection was performed for 2 weeks.
4. Mixed cloning assay
1) After the HEK293-ISRE-Luc mixed clone cells after the expanded culture are digested by pancreatin, the digestion is stopped by using a screening culture medium, the cells are transferred into a 15ml centrifugal tube after being blown, and the cells are centrifuged for 5 minutes at 1000 rpm;
2) Discarding the supernatant, and blowing and beating the resuspended cells by using 1ml of cell screening culture medium;
3) Immediately, cell counting was performed using trypan blue staining, and cell density was adjusted to 3X10 with the selection medium according to the cell counting results 5 The living cells per ml are blown and beaten uniformly;
4) Adding 100 mul of the blown cell suspension obtained in the step 3) into each hole of a 96-hole plate, and putting the cell suspension into a carbon dioxide incubator for continuous culture;
5) Diluting interferon with screening culture medium in gradient, adding 12 μ l interferon diluent into each well, setting negative control well, and culturing for 5 hr;
6) Mu.l of Bright-Glo luciferase reagent was added to each well, reacted for 3 minutes, and the luciferase number was measured by placing it in a microplate reader.
7) The data were analyzed using graphpad. Prism.6 software, and the results are shown in figure 1.
5. Limiting dilution monoclonality
1) The HEK293-ISRE-Luc mixed clone cells are subjected to subculture by using a screening culture medium, so that the cells are ensured to be in a good state and have the activity of more than 90% during monoclonation;
2) Taking cells in a good growth logarithmic phase state, after trypsinization, stopping digestion by using a screening culture medium, blowing and beating the cells, adding the cells into a centrifugal tube, and centrifuging the cells for 5 minutes at 1000 rpm;
3) Resuspending the cells in 5ml of selection medium, immediately performing trypan blue staining for cell counting, and adjusting the cell density to 1X 10 by adding the selection medium according to the cell counting result 5 Viable cells/ml;
4) Cells were monoclonalized by plating by limiting dilution, using screening media at 1: the cell lines were diluted in 10 gradients to obtain 1X 10 2 Viable cells/ml of cell suspension;
5) Taking 0.65ml of 1X 10 2 Diluting the cell suspension of each living cell/ml to 12.35ml by using a screening culture medium, uniformly blowing, and adding into a 96-well plate, wherein each well is 100 mu l;
6) Recording the micropores of single cells in a 96-well plate, and replacing the fresh screening culture medium for the cells once every 4 days;
7) When the confluence degree of cells in the 96-well reaches about 50%, carrying out pancreatin digestion, and carrying out enlarged culture to a 24-well plate;
8) The cells continue to be expanded and enough cells are cultured for functional validation testing.
6. Monoclonal functional test and passage stability test
1) Digesting HEK293-ISRE-Luc monoclonal cells by pancreatin, stopping digestion by using a screening culture medium, blowing and beating, adding into a centrifuge tube, and centrifuging at 1000rpm for 5 minutes;
2) Discarding the supernatant, and blowing and beating the resuspended cells by using 1ml of cell screening culture medium;
3) Immediately, cell counting was performed using trypan blue staining, and cell density was adjusted to 3X10 with the selection medium according to the cell counting results 5 The living cells/ml are blown and beaten uniformly;
4) Adding 100 mul of the blown cell suspension obtained in the step 3) into each hole of a 96-hole plate, and putting the cell suspension into a cell culture box for continuous culture;
5) 4 times of gradient dilution is carried out on the interferon by adopting a screening culture medium;
6) Adding 12 mul of interferon with different concentrations into each hole, and continuously culturing for 5 hours;
7) Adding 88 mul Bright-Glo luciferase reagent into each hole, reacting for 3 minutes, and measuring luciferase signals by a microplate reader;
8) Data were analyzed using graphpad. Prism.6 software.
According to the determination result, the monoclonal cell line with the best detection result is preserved with the preservation number of CCTCC NO: C2020256, and the cell line is preserved in the China center for type culture Collection with the address: china, wuhan university, the preservation date is 2020, 12, 8.
Example 2: hIFN-kappa detection using HEK293-ISRE-Luc cell line
The HEK293-ISRE-Luc monoclonal cell line with the best signal prepared in example 1 was continuously subjected to stability test in the selection medium, and partial cells were substituted at P5, P10, P15 and P20, respectively, and interferon-. Kappa.activity was measured according to the same method as in the steps 1) -8) of "6. Monoclonal function test and passaging stability test" of example 1, and the results are shown in Table 1 and FIG. 2 below, and the process flow of hIFN-. Kappa.detection is shown in FIG. 3.
TABLE 1
As can be seen from the above examples, the HEK293-ISRE-Luc cell line of the present invention can effectively measure the human interferon-kappa activity when subcultured to 5 th generation. The EC50 values were lowest when the cell lines of the invention were subcultured to passage 15. In addition, the HEK293-ISRE-Luc cell line of the invention can stably measure the activity of interferon-kappa at 5 th, 10 th, 15 th and 20 th generations, and the excellent generation stability and reliable sensitivity are fully proved.
Although the present invention has been disclosed in terms of the preferred embodiment, it is not intended to limit the invention, and various changes and modifications can be made by one skilled in the art without departing from the spirit and scope of the invention, and those skilled in the art can make this invention within the scope of the invention as broadly described in conjunction with the general technology in the art.
Claims (2)
1. A cell line for detecting the activity of human interferon kappa (hIFN-kappa), which is transfected with ISRE (interferon sensitive response element) luciferase reporter gene vector and has a collection number of CCTCC NO: C2020256, wherein the cell line is collected in China center for type culture Collection, with the address: china, wuhan university, the preservation date is 2020, 12, 8 days.
2. Use of the cell line of claim 1 in the preparation of an agent for rapid detection of human interferon kappa (hIFN-kappa) activity.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4801685A (en) * | 1981-08-14 | 1989-01-31 | Hoffmann-La Roche Inc. | Microbial production of mature human leukocyte interferon K and L |
| US5766864A (en) * | 1994-11-01 | 1998-06-16 | Sumitomo Pharmaceuticals Company, Ltd. | Method of measuring interferon acitivity |
| CN108795982A (en) * | 2017-12-05 | 2018-11-13 | 安徽九川生物科技有限公司 | A kind of chicken interferon α biological activity detection methods |
| CN110499295A (en) * | 2019-09-11 | 2019-11-26 | 宝船生物医药科技(上海)有限公司 | A kind of CSF-1R reporter gene cell strain and its preparation method and application |
| JP2020127382A (en) * | 2019-02-08 | 2020-08-27 | 森永乳業株式会社 | Screening method and kit for interferon production-promoting substance |
-
2020
- 2020-12-25 CN CN202011560265.9A patent/CN112760291B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4801685A (en) * | 1981-08-14 | 1989-01-31 | Hoffmann-La Roche Inc. | Microbial production of mature human leukocyte interferon K and L |
| US5766864A (en) * | 1994-11-01 | 1998-06-16 | Sumitomo Pharmaceuticals Company, Ltd. | Method of measuring interferon acitivity |
| CN108795982A (en) * | 2017-12-05 | 2018-11-13 | 安徽九川生物科技有限公司 | A kind of chicken interferon α biological activity detection methods |
| JP2020127382A (en) * | 2019-02-08 | 2020-08-27 | 森永乳業株式会社 | Screening method and kit for interferon production-promoting substance |
| CN110499295A (en) * | 2019-09-11 | 2019-11-26 | 宝船生物医药科技(上海)有限公司 | A kind of CSF-1R reporter gene cell strain and its preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| Antiviral activity of transiently expressed IFN-kappa is cell-associated;Peter J Buontempo等;《J Interferon Cytokine Res》;20061231;第26卷(第1期);全文 * |
| CpG ODN增强人外周血单个核细胞对肝癌细胞系HepG-2杀伤活性的实验研究;韩刚等;《中国实验诊断学》;20080425(第04期);全文 * |
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