CN110499295A - A kind of CSF-1R reporter gene cell strain and its preparation method and application - Google Patents
A kind of CSF-1R reporter gene cell strain and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of CSF-1R reporter gene cell strain and its construction method and application, the reporter gene cell strain includes NF κ B reporter plasmid；It include luciferase reporter gene on the NF κ B reporter plasmid；The reporter gene cell strain further includes CSF-1R expression plasmid.Reporter gene cell strain of the invention, source of people CSF-1R is expressed on cell membrane, the luciferase of intracellular expression response NF κ B signal, CSF-1/IL-34 can dose-dependant the luciferase of specific activation reporter gene cell strain expression, the antibody drug for targeting CSF-1R simultaneously can block the combination of CSF-1/IL-34 and reporter gene cell strain surface C SF-1R, have important application in antibody drug Activity determination.
Description
Technical field
The invention belongs to field of biotechnology, it is related to a kind of reporter gene cell strain and its preparation method and application, especially
It is related to a kind of CSF-1R reporter gene cell strain and its preparation method and application.
Background technique
CSF-1 is initially considered as a kind of hemopoieticgrowth factor, by conjunction with unique cell surface receptor CSF-1R,
Play the biological function for promoting monocyte, macrophage and myeloid progenitor proliferation, differentiation and survival.CSF-1R is by former cancer
Gene c-Fms coding, is a kind of receptor tyrosine kinase, when CSF-1 or IL-34 are in conjunction with CSF-1R, CSF-1R dimerization is lured
Lead the activation of the transphosphorylation and downstream signal transmitting molecule MAPK and Akt of receptor.
CN201180017294 disclose measurement CSF-1R antibody drug biological activity method, the method includes to
It expresses and CSF-1 and CSF-1R antibody is added in the NKM-1 tumor cell line of CSF-1R, be put into incubator and be incubated for 3 days, according to measurement
Luminous signal, obtain the IC50 value of CSF-1R antibody, by comparing IC50 value evaluation monoclonal antibody activity.However the party
Method is long experimental period, and experimental result degree of variation is big, is not suitable for the Fast Evaluation of large batch of antibody activity.
Therefore it provides a kind of product and method for fast and accurately detecting CSF-1R antibody drug biological activity, is applicable in
In the biological activity of detection antibody drug on a large scale, have broad application prospects and huge market value.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of CSF-1R reporter gene cell strain and preparation method thereof and
Using the reporter gene cell strain detection time is short, and susceptibility is high, has to the activity of monitoring and evaluation monoclonal antibody important
Meaning.
To achieve this purpose, the present invention adopts the following technical scheme:
In a first aspect, the reporter gene cell strain includes NF κ B report the present invention provides a kind of reporter gene cell strain
Accuse gene plasmid；
It include luciferase reporter gene on the NF κ B reporter plasmid.
The reporter gene cell strain further includes CSF-1R expression plasmid.
In the present invention, transfection has the reporter gene cell strain of reporter plasmid and CSF-1R expression plasmid, in cell membrane
Upper expression source of people CSF-1R, intracellular expression respond NF κ B signal luciferase, CSF-1/IL-34 can dose-dependant it is special
Property activation reporter gene cell strain luciferase expression, while the antibody drug for targeting CSF-1R can block CSF-1/
The combination of IL-34 and reporter gene cell strain surface C SF-1R have important application in antibody drug Activity determination.
In the present invention, the antibody drug of the targeting CSF-1R includes S7-4, is had for precious ship biological medicine scientific and technological (Shanghai)
The self-control of limit company.
Preferably, the reporter gene cell strain is HEK293 cell.
Second aspect, the present invention provides a kind of preparation method of reporter gene cell strain as described in relation to the first aspect, institutes
The method of stating includes:
(1) NF κ B reporter plasmid transfection host cell, resistance screening are used；
(2) host cell obtained using CSF-1R expression plasmid transfection procedure (1), resistance screening obtain the report
Gene cell strain.
Preferably, step (1) resistance screening is carried out using hygromycin B.
Preferably, step (2) the CSF-1R expression plasmid preparation method the following steps are included:
CSF-1R genetic fragment is inserted into pcDNA3.3-TOPO carrier, is transformed into escherichia coli cloning bacterial strain, resistance sieve
Recombinant plasmid is obtained after choosing, sequencing, obtains CSF-1R expression plasmid.
Preferably, step (2) resistance screening is carried out using the mixed liquor of hygromycin B and G418.
As optimal technical scheme, the present invention provides a kind of preparations of reporter gene cell strain as described in relation to the first aspect
Method, which comprises
(1) NF κ B reporter plasmid transfected HEK 293 is used, resistance screening is carried out by hygromycin B；
(2) CSF-1R genetic fragment is inserted into pcDNA3.3-TOPO carrier, is transformed into escherichia coli cloning bacterial strain, resistance
Recombinant C SF-1R expression plasmid is obtained after screening, sequencing, and after the CSF-1R expression plasmid is mixed with transfection reagent, is transfected
The host cell that step (1) obtains carries out resistance screening by the mixed liquor of hygromycin B and G418, obtains the reporter gene
Cell strain.
The third aspect, the present invention provides a kind of detection reagent, the detection reagent includes report as described in relation to the first aspect
Accuse gene cell strain.
Preferably, the detection reagent further includes any one in pharmaceutically acceptable carrier, excipient or diluent
Kind or at least two combination.
Fourth aspect, the present invention provides a kind of detection methods of antibody drug biological activity, which comprises
Reporter gene cell strain as described in relation to the first aspect is added in CSF-1, IL-34 and CSF-1R antibody drug or such as the
In detection reagent described in three aspects, the biological activity of antibody drug is detected according to fluorescence signal.
5th aspect, the present invention provides a kind of reporter gene cell strain as described in relation to the first aspect, such as second aspect institute
The preparation method for the reporter gene cell strain stated, the detection reagent as described in the third aspect or the antibody medicine as described in fourth aspect
Application of the detection method of object biological activity in detection targeting CSF-1R antibody drug activity.
Compared with prior art, the invention has the following beneficial effects:
(1) reporter gene cell strain 1-4G of the invention expresses source of people CSF-1R on cell membrane, and intracellular expression responds NF κ
The luciferase of B signal；
(2) CSF-1 can dose-dependant the luciferase of specific activation reporter gene cell strain 1-4G expression；Together
When targeting CSF-1R antibody drug S7-4 can block the combination of CSF-1 and 1-4G surface C SF-1R；
(3) reporter gene cell strain 1-4G of the invention is suitable for evaluating the spy of CSF-1/IL-34 Yu its receptor CSF-1R
The opposite sex combines, the activity of monitoring and evaluation CSF-1/IL-34 and CSF-1R antibody, has significant specificity.
Detailed description of the invention
Fig. 1 is the result figure that TNF- ɑ stimulates NF κ B-HEK293 12-9E expressing luciferase；
The CSF-1R that Fig. 2 is 1-4G expresses streaming figure；
Fig. 3 is the result figure that CSF-1 stimulates 1-4G expressing luciferase；
Fig. 4 is the result figure that IL-34 stimulates 1-4G expressing luciferase；
Fig. 5 is that S7-4 blocks result figure of the CSF-1 in conjunction with 1-4G；
Fig. 6 is that S7-4 blocks result figure of the IL-34 in conjunction with 1-4G；
Fig. 7 is the specific outcome figure of 1-4G.
Specific embodiment
The technological means and its effect taken for the present invention is further explained, with reference to embodiments with attached drawing to this hair
It is bright to be further described.It is understood that the specific embodiments described herein are used only for explaining the present invention, rather than
Limitation of the invention.
In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art,
Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available from
The conventional products of acquisition.
Embodiment 1 constructs NF κ B reporter gene cell strain
The present embodiment by plasmid construction, cell transfecting, stable cell line screening and monoclonal and etc. building report
Gene cell strain, the specific method is as follows:
(1) reporter plasmid
(Promega is public using commercially available luciferase plasmids pGL4.32 (luc2/NF- κ B-RE/Hygro) for the present embodiment
Department) it is used as reporter plasmid, prepare reporter gene cell strain；
(2) cell transfecting
The last fortnight recovery HEK293 cell is mentioned, guarantees that cell is in logarithmic growth phase when using；It is collected in the day before transfection
Cell is inoculated in 6 orifice plates, 4E5 cells/well, culture medium 10%FBS+DMEM；
On the transfection same day, the culture medium of 6 orifice plates is changed in fresh 10%FBS+DMEM (hole 2mL/), prepares reporter gene matter
(ratio DNA:Lipo2000 is the mixed liquor of grain pGL4.32 (luc2/NF- κ B-RE/Hygro) and Lipofectamine 2000
4:10), it is added dropwise in 6 orifice plates, is placed in 37 DEG C of incubators and is incubated for 5h, cell culture medium is changed to fresh 10%FBS+
DMEM；
(3) stable cell line screens
Third day is transfected, cell culture medium is changed to 10%FBS+200 μ g/mL hygromycin B+DMEM, pressurization culture 16
After it, the cell strain for stablizing expression is obtained；
(4) monoclonal
The cell strain for stablizing expression is cloned using limiting dilution assay, i.e., is inoculated with cell with the density of 1.2 cells/wells
In 96 orifice plates；Second day after monoclonal, monoclonal cell strain is selected under microscope；Monoclonal cell strain is inoculated in 96 orifice plates
In, TNF- ɑ stimulation is added, obtains the window value of luciferase expression, the reporter gene monoclonal for filtering out high expression NF κ B is thin
Born of the same parents NF κ B-HEK293 12-9E；It takes monoclonal cell to expand culture, and freezes.
Fig. 1 stimulates the luciferase expression of NF κ B-HEK293 12-9E with showing TNF- ɑ dose-dependant.
The NF κ B reporter gene cell strain of the building expression of embodiment 2 CSF-1R
(1) building of CSF-1R plasmid
Design primer PCR amplification obtains CSF-1R full-length gene segment, and agarose gel electrophoresis uses after glue recycling
pcDNATM3.3-TOPOTMTA CloningTMCSF-1R genetic fragment is inserted into pcDNA3.3- by the connection of Kit kit, conversion
TOPO carrier is transformed into escherichia coli cloning bacterial strain, and picking monoclonal send sequence verification, obtains CSF-1R expression plasmid；
(2) cell transfecting
The last fortnight recovery monoclonal cell NF κ B-HEK293 12-9E cell is mentioned, guarantees that it is raw to be in logarithm for cell when using
For a long time；The day before transfection collects cell, is inoculated in 6 orifice plates, 1E6 cells/well, culture medium 10%FBS+DMEM；
Transfection the same day, the culture medium of 6 orifice plates is changed into Opti-MEM (hole 2mL/), prepare CSF-1R plasmid with
The mixed liquor of Lipofectamine 2000, is added dropwise in 6 orifice plates, is placed in 37 DEG C of incubators and is incubated for 5h, by cell culture
Base is changed to 10%FBS+DMEM；
(3) stable cell line screens
Transfection second day, is changed to+1200 μ g/mL G418+ of 10%FBS+50 μ g/mL hygromycin B for cell culture medium
DMEM, after pressurization culture 14 days, cell inoculation after CSF-1 stimulation 6h is added, obtains luciferase expression in white 96 orifice plates
Window value, filter out the maximum stable expression cell strain of window value；
(4) monoclonal
The maximum stable expression cell strain of the window value filtered out is used into two-dimensional finite dilution method, i.e. the hole A1 is 2000
Cell, later from the 1st twice of doubling dilution of column to the 12nd column, then from twice of doubling dilution of A row to H row；Second after monoclonal
It, selects monoclonal cell strain under microscope；Monoclonal cell strain is inoculated in white 96 orifice plates, 1E5 cells/well,
After CSF-1 stimulation 6h is added, the window value of luciferase expression is obtained, the maximum monoclonal cell 1-4G of window value is filtered out.
By the expression of flow cytomery 1-4G cell membrane CSF-1R, as shown in Fig. 2, the expression quantity of CSF-1R
It is 82.3%.It takes monoclonal cell 1-4G to expand culture, and freezes.
Embodiment 3CSF-1/IL-34 stimulates monoclonal cell 1-4G expressing luciferase to test
(1) experiment the previous day prepares culture medium: pre- heat analysis culture medium (2%FBS+DMEM) and culture completely before testing
Base (10%FBS+DMEM)；
(2) it collects cell: taking out Tissue Culture Dish, observe cell density under inverted microscope, it is ensured that cell is pollution-free；It abandons
Old culture medium is removed, with sterilizing PBS rinse cell, sodium citrate digests 2min, and is terminated and digested with complete medium；1000rpm,
After 5min centrifugation, cell is resuspended after the DMEM culture medium containing 2%FBS is added, carries out cell count；
(3) plating cells: cell is resuspended to 5E5/mL, and 100 holes μ L/ are laid in 96 orifice plates, is added in one collar aperture of surrounding
Enter PBS (100 hole μ L/), be temporarily placed in 37 DEG C of incubators, is incubated overnight；
(4) culture medium in white 96 orifice plates is removed, i.e., is adjusted to 95 μ L with the volley of rifle fire, cell conditioned medium is inhaled by experimental day
Out；
(5) CSF-1/IL-34 is added:
96 orifice plates are taken, CSF-1 are diluted to 400ng/mL, and with 4 times of doubling dilutions, that is, draw previous 75 μ L of hole mixing
Liquid is added into the 225 μ L analysis culture medium in the latter hole, and CSF-1 is added in white 96 orifice plates with 100 holes μ L/；
96 orifice plates are taken, IL-34 are diluted to 1000ng/mL, and with 3 times of doubling dilutions, that is, it is mixed to draw previous 110 μ L of hole
It closes liquid to be added into the 220 μ L analysis culture medium in the latter hole, IL-34 is added in white 96 orifice plates with 100 holes μ L/；
(6) 96 orifice plates of white are placed in incubator and are incubated for 6h, cell plates are balanced to room temperature, and 50 μ L are added in every hole
Microplate reader read plate after Bio-glo, 10min；
(7) result: data processing is carried out with Graphpad software, using 4- parametric equation.
From the figure 3, it may be seen that CSF-1 dose-dependently stimulates monoclonal cell 1-4G expressing luciferase；As shown in Figure 4,
IL-34 dose-dependently stimulates monoclonal cell 1-4G expressing luciferase.
Embodiment 4 evaluates the experiment of S7-4 biological activity
(1) experiment the previous day prepares culture medium: pre- heat analysis culture medium (2%FBS+DMEM) and culture completely before testing
Base (10%FBS+DMEM)；
(2) it collects cell: taking out Tissue Culture Dish, observe cell density under inverted microscope, it is ensured that cell is pollution-free；It abandons
Old culture medium is removed, with sterilizing PBS rinse cell, sodium citrate digests 2min, and is terminated and digested with complete medium；1000rpm,
After 5min centrifugation, cell is resuspended after the DMEM culture medium containing 1%FBS is added, carries out cell count；
(3) plating cells: cell is resuspended to 5E5/mL, and 100 holes μ L/ are laid in 96 orifice plates, and one collar aperture of surrounding is added
PBS (100 hole μ L/), is placed in 37 DEG C of incubators, is incubated overnight；
(4) culture medium in white 96 orifice plates is removed, i.e., is adjusted to 95 μ L with the volley of rifle fire, cell conditioned medium is inhaled by experimental day
Out；
(4) CSF-1/IL-34 and S7-4 is added: taking 96 orifice plates, S7-4 is diluted to 200 μ g/mL, and dilute with 3 times of multiple proportions
It releases, that is, draws previous 60 μ L mixed liquor of hole and be added into the 120 μ L analysis culture medium in the latter hole；Using analysis culture medium,
CSF-1 is diluted to 8ng/mL, IL-34 is added in 96 orifice plates after being diluted to 20ng/mL, 120 holes μ L/；By antibody and CSF-1/
IL-34 is uniformly mixed and is added in white 96 orifice plates, 100 holes μ L/；
(5) 96 orifice plates of white are placed in incubator and are incubated for 6h, cell plates are balanced to room temperature, and 50 μ L are added in every hole
Microplate reader read plate after Bio-glo, 10min；
(6) result: data processing is carried out with Graphpad software, using 4- parametric equation.
By Fig. 5,6 it is found that in the CSF-1/IL-34 of fixed concentration, S7-4 can with concentration dependant inhibit CSF-1/IL-
The combination of 34 and 1-4G illustrates that the reporter gene cell strain of building can be used to evaluate the life of CSF-1/IL-34&CSF-1R antibody
Object activity.
5 specific assay of embodiment
The present embodiment uses the specificity of the monoclonal antibody medicine examining report gene cell strain 1-4G of different target spots: S7-4 is (precious
Ship biological medicine science and technology (Shanghai) Co., Ltd., target spot CSF-1R), Tecentriq (target spot PD-L1), Erbitux (target
Point is EGFR) and Avastin (target spot VEGF).
(1) experimental day prepares culture medium: preheating complete medium (10%FBS+DMEM) and FACS before experiment
Buffer (1%BSA+PBS)；
(2) it collects cell: taking out Tissue Culture Dish, observe cell density under inverted microscope, it is ensured that cell is pollution-free；It abandons
Old culture medium is removed, with sterilizing PBS rinse cell, sodium citrate digests 2min, and is terminated and digested with complete medium；1000rpm,
After 5min centrifugation, FACS buffer is added, cell is resuspended, carry out cell count；
(3) plating cells: cell being laid in 96 hole U-shaped boards, 1E5/well, and 3000rpm, 4min centrifugation are spare；
(4) CSF-1 and S7-4 is added: taking 96 orifice plates, CSF-1 is diluted to 2 μ g/mL, and with 5 times of doubling dilutions, that is, inhale
Previous 36 μ L mixed liquor of hole is taken to be added into the 144 μ L FACS buffer in the latter hole；Using FACS buffer, by S7-
4, Erbitux, Avastin and Tecentriq are added in 96 orifice plates after being diluted to 20 μ g/mL, 144 holes μ L/；By antibody and
CSF-1 is uniformly mixed and is added in 96 hole U-shaped boards, 100 holes μ L/；
Board-washing: (5) 4 DEG C of incubation 1h use FACS buffer board-washing, wash twice；
(6) using goat-anti people's FC secondary antibody of FACS buffer dilution PE label, 1:150 times dilutes.96 are added after having diluted
In the U-shaped board of hole, 100 holes μ L/；
(7) 4 DEG C are incubated for 30 minutes, board-washing: using FACS buffer board-washing, wash twice；
(8) machine-readable plate on flow cytometer.
As a result as shown in fig. 7,1-4G to the antibody drug of EGFR, VEGF and PD-L1 target spot without dose-effect curve,
Illustrate that 1-4G is not suitable for the other drugs other than CSF-1/IL-34&CSF-1R target spot, it was demonstrated that 1-4G has significant
CSF-1/IL-34&CSF-1R specificity.
In conclusion reporter gene cell strain 1-4G of the invention expresses source of people CSF-1R, intracellular expression on cell membrane
Respond the luciferase of NF κ B signal；CSF-1/IL-34 can dose-dependant the luciferase of specific activation 1-4G table
It reaches；The antibody drug S7-4 for targeting CSF-1R simultaneously can block the combination of CSF-1/IL-34 and 1-4G；1-4G of the invention is suitable
For evaluating the specific binding of CSF-1/IL-34 Yu its receptor CSF-1R, at the same it is anti-suitable for CSF-1/IL-34&CSF-1R
Body blocks the combination of CSF-1/IL-34 itself and CSF-1R, the activity of monitoring and evaluation CSF-1/IL-34&CSF-1R antibody, specificity
Significantly, it has broad application prospects and huge market value.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office
Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
Claims (10)
1. a kind of reporter gene cell strain, which is characterized in that the reporter gene cell strain includes NF κ B reporter plasmid；
It include luciferase reporter gene on the NF κ B reporter plasmid.
2. reporter gene cell strain according to claim 1, which is characterized in that the reporter gene cell strain further includes
CSF-1R expression plasmid.
3. reporter gene cell strain according to claim 1 or 2, which is characterized in that the reporter gene cell strain is
HEK293 cell.
4. a kind of preparation method of reporter gene cell strain as described in any one of claims 1-3, which is characterized in that the side
Method includes:
(1) NF κ B reporter plasmid transfection host cell, resistance screening are used；
(2) host cell obtained using CSF-1R expression plasmid transfection procedure (1), resistance screening obtain the reporter gene
Cell strain.
5. the preparation method according to claim 4, which is characterized in that step (1) resistance screening using hygromycin B into
Row.
6. preparation method according to claim 5, which is characterized in that the preparation of step (2) the CSF-1R expression plasmid
Method the following steps are included:
CSF-1R genetic fragment is inserted into pcDNA3.3-TOPO carrier, is transformed into escherichia coli cloning bacterial strain, resistance screening, survey
Recombinant plasmid is obtained after sequence, obtains CSF-1R expression plasmid；
Preferably, step (2) resistance screening is carried out using the mixed liquor of hygromycin B and G418.
7. according to the described in any item preparation methods of claim 4-6, which is characterized in that the described method comprises the following steps:
(1) NF κ B reporter plasmid transfected HEK 293 is used, resistance screening is carried out by hygromycin B；
(2) CSF-1R genetic fragment is inserted into pcDNA3.3-TOPO carrier, is transformed into escherichia coli cloning bacterial strain, resistance screening,
Recombinant C SF-1R expression plasmid is obtained after sequencing, and after the CSF-1R expression plasmid is mixed with transfection reagent, transfection procedure
(1) host cell obtained carries out resistance screening by the mixed liquor of hygromycin B and G418, obtains the reporter gene cell
Strain.
8. a kind of detection reagent, which is characterized in that the detection reagent includes report base as described in any one of claims 1-3
Because of cell strain；
Preferably, the detection reagent further include in pharmaceutically acceptable carrier, excipient or diluent any one or
At least two combination.
9. a kind of detection method of antibody drug biological activity, which is characterized in that the described method includes:
Reporter gene cell strain as described in any one of claims 1-3 is added in CSF-1, IL-34 and CSF-1R antibody drug
Or in detection reagent as claimed in claim 8, the biological activity of antibody drug is detected according to fluorescence signal.
10. a kind of reporter gene cell strain as described in any one of claims 1-3, such as claim 4-7 are described in any item
The preparation method of reporter gene cell strain, detection reagent as claimed in claim 8 or antibody drug as claimed in claim 9
Application of the detection method of biological activity in detection targeting CSF-1R antibody drug activity.
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