CN102337247B - Recombinant HEK293 cell with high FGFR4 expression and application thereof - Google Patents
Recombinant HEK293 cell with high FGFR4 expression and application thereof Download PDFInfo
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- CN102337247B CN102337247B CN 201110269675 CN201110269675A CN102337247B CN 102337247 B CN102337247 B CN 102337247B CN 201110269675 CN201110269675 CN 201110269675 CN 201110269675 A CN201110269675 A CN 201110269675A CN 102337247 B CN102337247 B CN 102337247B
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Abstract
The invention discloses a recombinant HEK293 cell with high FGFR4 expression and application thereof. In the recombinant cell, an HEK293 cell serves as a host cell; and an exogenous expression vector for transfecting the host cell is an expression vector which comprises an FGFR4 full-length gene. The recombinant HEK293 cell with high FGFR4 expression is applied to screening of antitumor medicaments.
Description
Technical field
The invention belongs to the reconstitution cell technical field, relate to a kind of reorganization HEK293 cell and application thereof of FGFR4 high expression level.
Background technology
Receptor tyrosine kinase (receptor tyrosine kinase RTKs) is maximum class of enzymes connection acceptor, it be acceptor be again enzyme, can be with the part combination, and with the tyrosine residues phosphorylation of target protein.All RTKs are made up of three parts: extracellular domain, the single that contains ligand-binding site point striden the hydrophobic alpha helical region of film, contained the active cell intracellular domain of tyrosine protein kinase (RTK).Receptor tyrosine kinase not with signaling molecule in conjunction with the time exist with monomer, and do not have activity; In case signaling molecule is arranged is combined with the extracellular domain of acceptor, two monomeric acceptor molecules form dimer at film, the afterbody of the cell intracellular domain of two acceptors is in contact with one another, and activates the function of their protein kinase, and the result makes the tyrosine residues phosphorylation of afterbody.Phosphorylation causes the afterbody of recipient cell intracellular domain to be assembled into a signal mixture (signaling complex).Just the tyrosine position of phosphorylation becomes the binding site of signal protein (signaling protein) in the cell immediately, may have in 10～20 kinds of different cells to be activated after the combination of signal protein isoacceptor afterbody phosphorylation position.The signal mixture passes through several different signal transduction pathways, expansion information, a series of biochemical reaction in the activating cells; Perhaps different informixs is got up to cause comprehensive the replying (as cell proliferation) of cell.
The protein tyrosine kinase signal transduction is that the broad variety tumour cell is different from one of corresponding Normocellular key feature unusually.The known expression product of the oncogene about 50% of tumour generation, development that causes all has protein tyrosine kinase activity, and their unconventionality expression will cause the cell proliferation adjusting to get muddled, and then cause tumour to take place.In addition, the unconventionality expression of Tyrosylprotein kinase and acceptor is also closely related with the resistance of invasion and attack, transfer, tumor neovasculature generation and the chemotherapy of tumors of tumour.
Protein tyrosine kinase receptor is the protein that a class has tyrosine kinase activity, somatomedin (GF) as part and its in conjunction with after, stimulate GFR monomer dimerization (dimerization), phosphate on the catalysis ATP is transferred on the tyrosine residues, make Tyrosylprotein kinase district (TKD in its cytolemma born of the same parents, tyrosine kinase domain) specific site tyrosine generation phosphorylation, activate downstream signal transduction path, cause cell that corresponding biological function takes place and change, especially in mediation malignant tumour generating process, have vital role.
More than 20 aglucon of human fibroblast's growth factor receptors (fibroblast growth factor receptor FGFRs) family and they carries out multi-faceted regulation and control to cell under physiological situation, comprise that cell is grown, breaks up, divided a word with a hyphen at the end of a line, vasculogenesis etc.The transmission of FGF/FGFR signal is one of major reason of angiogenesis, fetal development, bone formation, wound healing, fibrosis and tumour formation.Four kinds of people FGFRs by the separate gene coding have been determined at present, i.e. FGFR1～FGFR4.
The generation of tumour, development and transfer depend on tumor vessel and form, only after new vessel forms, tumour could be grown, and tissue infiltration towards periphery, entering blood circulation shifts to other positions, fibroblast growth factor acceptor 4 (FGFR4) has than high expression level in tissues such as embryonic tissue, adult's tiny blood vessels wall and mammary cancer, carcinoma of the pancreas, kidney, and specific expressed in mature liver cells.
In recent years, numerous scholars carry out the research work of FGFR4 in tumor growth, generating process, result of study shows: FGFR4 expression amount in melanoma, prostate cancer, head and neck cancer, mammary cancer, liver cancer is higher, simultaneously, FGFR4 has mediated tumour cell chemotherapy resistance (chemoresistance), therefore, the effect of FGFR4 cell growth regulation and control has caused the concern of Chinese scholars.
Summary of the invention
The problem that the present invention solves is to provide a kind of reorganization HEK293 cell of FGFR4 high expression level, and utilizes this cell to carry out the screening of antitumor drug, can be used as to adopt micromolecular inhibitor blocking-up FGFR4 to express to treat the cell model of drug resistance of tumor.
The present invention is achieved through the following technical solutions:
A kind of reorganization HEK293 cell of FGFR4 high expression level, this reconstitution cell are to be host cell with the HEK293 cell, exogenous expression's carrier of transfection host cell be the expression vector that comprises the FGFR4 full-length gene.
The nucleotide sequence of described FGFR4 full-length gene is shown in SEQ.ID.NO.1.
The expression vector of the described FGFR4 of comprising full-length gene is pcDNA3.1 (+)-FGFR4, and this expression vector is by the BamHI restriction enzyme site FGFR4 full-length gene to be cloned into carrier for expression of eukaryon pcDNA3.1 (+).
The HEK293-FGFR1 reconstitution cell of described FGFR4 high expression level is applied to screening anti-tumor medicine.
Described application is that the HEK293-FGFR1 reconstitution cell with the FGFR4 high expression level is built into the cytolemma chromatographic column, is indication with the retention time, and screening is the antitumor drug of target spot with FGFR4.
Compared with prior art, the present invention has following beneficial technical effects:
1, the present invention has made up carrier for expression of eukaryon pcDNA3.13.1 (+)/FGFR4 of fibroblast growth factor acceptor 4 (FGFR4), confirms that through cloning and sequencing sequence is with the people FGFR4 sequence unanimity in the ncbi database; Through liposome transfection HEK293 cell, the G418 resistance screening obtains can stable growth, the cell strain of survival, detects through Western blot, filters out reorganization HEK293/FGFR4 cell strain stable, high expression level FGFR4;
The reorganization HEK293/FGFR4 cell strain that comprises the FGFR4 full length gene that the present invention makes up can stably express FGFR4, has complete molecular structure.
2, the HEK293 cell as host cell is the immortalized cells of primary human embryonic kidney cell's transfection V-type adenovirus (Ad5) DNA, the various expression of receptor amounts that this cell also has itself are relatively low, and the relative expression quantity of the FGFR4 of the HEK293/FGFR4 cell expressing after the reorganization obviously raises than HEK293 is unloaded, occupy the predominant expression amount with respect to other cell surface molecules, be in sepcific ligands in conjunction with superiority, so just having improved greatly is specificity and the susceptibility of drug screening target with FGFR4.
3, the FGFR4 of the HEK293/FGFR4 cell expressing after the reorganization utilizes immunofluorescence to detect its expression on cytolemma, has FGFR4 to express on the showed cell film, and embodies the molecular activity of FGFR4.
4, because the recognition capability of the expressed FGFR4 of reconstitution cell, and the FGFR4 expression amount is enough, make the binding ability of itself and part become the foundation of its screening: successfully to set up the drug screening pattern that the HEK293/FGFR4 cytolemma chromatographic column of stable high expression level FGFR4 is carried out, the compound that can combine with FGFR4 or its retention time of activeconstituents obviously prolong, this explanation is indication with the retention time, this cytolemma chromatogram can be applied to the screening of antitumor drug, especially to the screening of the more antitumor active ingredient of Chinese herbs of activeconstituents.
Description of drawings
Fig. 1-a～Fig. 1-b is amplification FGFR4 gene and linearized vector electrophoresis detection collection of illustrative plates;
Behind Fig. 2 pcDNA3.1 (+)/FGFR4 recombinant vectors transfection Escherichia coli DH5, extract plasmid bacterium liquid PCR qualification result;
Fig. 3 is the positive cell cultivation figure through the G418 resistance screening;
Fig. 4 is that Western blot analyzes positive cell FGFR4 expression amount;
Fig. 5 is that immunofluorescence analysis FGFR4 locatees at the FGFR4/HEK293 cell expressing;
Fig. 6 is the HEK293/FGFR4 cell growth curve;
Fig. 7 is that Gefitinib is in the color atlas contrast of HEK293/FGFR4 and HEK293 cytolemma chromatogram.
Embodiment
The present invention is on the basis of carrier for expression of eukaryon pcDNA3.1 (+)/FGFR4 that makes up the FGFR4 full-length gene, transfection HEK293 cell, obtain reorganization HEK293/FGFR4 cell strain stable, high expression level FGFR4, be to do to such an extent that describe in detail to this aspect below, the explanation of the invention is not limited.
The present invention is to be carrier is carrier with pcDNA3.1 (+), specifically selects HindIII and XhoI restriction enzyme site as the multiple clone site that is connected foreign gene.
1) FGFR4 gene clone
Selection comprises FGFR4 full length gene cDNA sequence pOTB7/FGFR4 plasmid clones as template, adopts the corresponding Auele Specific Primer of Primer Premier5.0 software design, and adds the HindIII/XhoI restriction enzyme site respectively at two ends:
FGFR4-HindIII：5’-AGT
AAGCTTATGCGGCTGCTGCTGGCCCT-3’
FGFR4-XhoI：5’-GAT
CTCGAGTCATGTCTGCACCCCAGACC-3’
Adopt the Takara LA Taq of company to increase, the PCR reaction system is: archaeal dna polymerase 0.2 μ L, 10 * LAbuffer, 2.5 μ L, dNTP 4 μ L, upstream primer (10 μ mol/L) 2 μ L, downstream primer (10 μ mol/L) 2 μ L, template DNA (pOTB7/FGFR4 plasmid) 1 μ L, BSA (5 μ mol/L), ddH2O 11.3 μ L, totally 25 μ L.95 ℃ of sex change 3min first, again through 35 Circulations (94 ℃ of sex change 1min, 60 ℃ of renaturation 1min, 72 ℃ are extended 3min), last 72 ℃ are extended 10min, obtain a large amount of purpose fragments, adopt commercialization PCR purification kit with the amplified production purifying to product, the FGFR4cDNA amplification detects shown in Fig. 1-a, and M is marker, and the result shows that amplification arrived the gene of expection.
2) recombinant expression vector makes up
Utilize HindIII/XhoI double digestion carrier for expression of eukaryon pcDNA3.1 (+) and FGFR4 isogeneity product, the enzyme system of cutting is: the FGFR4 fragment of HindIII 1 μ L, XhoI 1.5 μ L, 10 * M buffer, 2 μ L, amplification and ring-type pcDNA3.1 (+) 10 μ L, ddH
2O 5.5 μ L37 ℃ of reaction 3h cut product to pcDNA3.1 (+) carrier enzyme and adopt commercialization PCR product purification test kit with the amplified production purifying, and pcDNA3.1 (+) carrier enzyme is cut the product electrophoresis result shown in Fig. 1-b, and M is marker.
Under 4 ℃ of conditions of FGFR4 amplified fragments after linearized vector cut with enzyme, the connection of spending the night under the T4 DNA ligase effect, reaction system is, FGFR4 fragment 6 μ L after linear pcDNA3.1 (+) 2 after enzyme is cut, enzyme are cut, T4DNA ligase 1 μ L, T4buffer 1 μ L, the pcDNA3.1 (+) that obtains recombinating/FGFR4 expression vector.
Prepare the bacillus coli DH 5 alpha competent cell according to the molecular cloning experiment guide, pcDNA3.1 (+)/FGFR4 plasmid of will recombinating adds in the 200 μ L competent cells, ice bath 30min, 42 ℃ of heat shock 90s, add no amicillin resistance LB liquid nutrient medium, 37 ℃ of jolting 1h, the centrifugal 3min of 3000rpm, with bacterial sediment piping and druming evenly, add and contain in amicillin resistance (100mg/mL) the LB solid medium, 37 ℃ of incubated overnight, the picking mono-clonal is enlarged culturing in containing amicillin resistance (100mg/mL) LB liquid nutrient medium, adopt commercial kit to obtain the high purity plasmid, bacterium liquid PCR preliminary evaluation positive colony (result as shown in Figure 2), and send ABI PRISM 3730 automatic dna sequencers to check order, checking sequence base, DNAstar Seqman carries out sequence assembly, the sequencing result that obtains is shown in SEQ.ID.NO.1, comparison in sequencing result and the gene pool (NCBI), sequence identity is 100%, contains correct sequence and is pcDNA3.1 (+)/FGFR4 recombinant expression vector.
3) high expression level FGFR4 reorganization HEK293 cell construction
1. use the lipofectamine LipofectAMINE of Invitrogen company 2000 to carry out transfection: the conventional HEK293 of cultivation cell, in 6 orifice plates, inoculate 3 * 10
5Cells/well adds the 2mL perfect medium, puts CO
237 ℃ of overnight incubation in the incubator treat to carry out when cell grows to 50-80% transfection.In aseptic centrifuge tube, be formulated as follows solution:
Solution A: 1-2 μ g is treated that the ultrapure DNA of transfection is diluted in the 100 μ L serum free mediums.
Solution B: 2-25 μ L LipofectAMINE is diluted in the 100 μ L serum free mediums.
Mixed solution A and B, mixing gently, room temperature is placed 15～45min.With 2mL serum free medium washed cell gently, add 0.8mL serum free medium/hole, liposome complex is added drop-wise in the hole, jiggle mixing, put CO
2Hatch 8h for 37 ℃ in the incubator.Replace transfection liquid with perfect medium, continue to cultivate.The screening concentration of HEK293 cell is 600 μ g/mL.Transfection after 72 hours the ratio in 1: 10 transfectional cell is gone down to posterity in 6 orifice plates, be changed to the selection substratum that contains G418.Visible individual cells in 6 orifice plates continues to cultivate about 14 days, and visible individual cells division growth forms single resistance colony, as shown in Figure 3.
2. limiting dilution assay: do 10 times of continuous dilutions (10 after the resistance granulocyte colony digests
-2～10
-10), each dilution cell being added drop-wise in 96 orifice plates cultivating, left and right sides cell covered with whole hole in 15 days, and trysinization is passaged to big bottle and cultivates, and frozen, culturing cell is twice repeatedly.
4) high expression level FGFR4 reorganization HEK293 identifies
1. under Western Blot:4 ℃ of condition, with HEK293 cell and the HuH7 cell strain of transfection FGFR4, transfection pcDNA3.1 (+), by 1.5 * 10
6Individual cell adds the 200 μ L cell pyrolysis liquids ratio lysing cell of (adding proteinase inhibitor PMSF), add 150 μ L loading buffer, boiling water bath 10min, extract total protein of cell and carry out the SDS-PAGE electrophoresis, finish complete wet electric transfer printing with pvdf membrane, pvdf membrane after shift with the TBST room temperature sealing that contains 5% skim-milk the back, wash specificity primary antibodie and the 4 ℃ of night incubation of target protein that add through the TBST dilution of 5%BSA through TBST, after the TBST washing 3 times, horseradish peroxidase two anti-that adds the TBST dilution of 5%BSA again, with ECL chemiluminescence detection protein expression, detect HEK293/FGFR4 cell FGFR4 protein expression.After testing, as shown in Figure 4, F6, F9, F12, F24, F28 for the transfection of screening the cell clone of pcDNA3.1 (+)/FGFR4, P13 is the reorganization HEK293 cell of transfection zero load, the expression amount that the FGFR4 expression amount of clone F28, clone F24, clone F6 is cloned P13 is improved, and the highest with the FGFR4 expression amount of clone F28.
2. immunofluorescence analysis FGFR4 expression and localization: the unloaded cell of pcDNA3.1 (+) in the vegetative period of taking the logarithm and pcDNA3.1 (+)/FGFR4 cell, 0.25% tryptic digestion is by 1 * 10
5Be inoculated in about individual/hole in 6 orifice plates that are placed with the pretreated sterility cover slide of poly-lysine, cultivate the 24h cell attachment after, discard substratum, wash the cover glass three times of climbing cell gently with PBS.4% with PBS (pH=7.4) dissolving Paraformaldehyde 96 1mL fixed cell 15min, after discard.The soft washed cell of PBS 2 times adds 1mL and contains 1% bovine serum albumin (bovine serum albumin, PBST incubated cell 30min BSA).(bovine serum albumin, PBST BSA) is added on the cover glass incubated at room 45min with 1: the 50 anti-people FGFR4 of dilution rabbit polyclonal antibody with containing 1% bovine serum albumin.PBS washs three times gently.Each 5min.Anti-with 1: 100 dilution FITC mark goat anti-rabbit igg two with the PBST that contains 1%BSA, be added drop-wise on the creep plate, the room temperature lucifuge is hatched 1h.In the dark place, PBS washing three times, each 5min.Drip 90% glycerine mounting at slide glass, note not producing bubble.With fluorescence microscope and take pictures, the result as shown in Figure 5, wherein A is pcDNA3.1 (+)/FGFR4 cell clone 28 immunofluorescence results, B is pcDNA3.1 (+) cellular immunofluorescence result, both contrast obvious discovery, and the former has the green fluorescence of FGFR4 immunofluorescence, and the latter does not have; This shows that FGFR4 has obtained effectively expressing in the reconstitution cell.
4. get well-grown and approach the cell that converges, use 0.25% trysinization, make cell suspension with new substratum, counting.According to cell counting as a result diluting cells suspension to concentration be 1 * 10
4Individual/mL, every 25mL bottle inoculation 2mL cell suspension is inoculated 30 bottles of cells altogether.Begin counting cells behind the 24h, later on every the 24h counting once, continuous counter 10d.Get 3 bottles of cells at every turn, count calculating mean value respectively.Change liquid for the cell of no count every 3d.According to the cell counting result, be X-coordinate with time, cell count is ordinate zou, draw growth curve, the result compares with the control cells that does not have transfection FGFR4 as shown in Figure 6, transfection cell speed of growth after cultivating 6d of FGFR4 obviously accelerate, show the distinctive effect of stimulation of FGFR4, further can prove also can being activated accordingly based on the FGFR4 signal path of reconstitution cell, thereby be embodied in the quickening of the speed of growth; The reconstitution cell that just can obtain some amount after cultivating through the short time is described on the other hand.
5) the cytolemma chromatogram detects the retention time of HEK293/FGFR4 and the Gefitinib of HEK293
Because the HEK293 cell as host cell is the immortalized cells of primary human embryonic kidney cell's transfection V-type adenovirus (Ad5) DNA, the various expression of receptor amounts that this cell also has itself are relatively low, and the relative expression quantity of the FGFR4 of the HEK293/FGFR4 cell expressing after the reorganization obviously raises than HEK293 is unloaded, occupy the predominant expression amount with respect to other cell surface molecules, be in sepcific ligands in conjunction with superiority, so just having improved greatly is specificity and the susceptibility of drug screening target with FGFR4.And the cell of reorganization shows FGFR4 activity accordingly.
So, the HEK293/FGFR4 cell of high expression level FGFR4 can be used as and adopts micromolecular inhibitor blocking-up FGFR4 to express to treat the cell model of drug resistance of tumor, further, can be applied to the screening of antitumor drug:
The HEK293-FGFR1 reconstitution cell of FGFR4 high expression level is built into the cytolemma chromatographic column, is indication with the retention time, and screening is the antitumor drug of target spot with FGFR4.
Carry out sample detection (with sample on the methyl alcohol that comprises Gefitinib (1.16mg/ml)) after the HEK293/FGFR4 cytolemma chromatographic column balance of high expression level FGFR4, be moving phase with 5mmol/L (pH 7.4) ammonium acetate buffer, found that Gefitinib has good reservation at this cytolemma, simultaneously with the HEK293 cell of untransfected as negative control.
Gefitinib is shown in the contrast as shown in Figure 7 of the reservation color atlas of HEK293/FGFR4 and HEK293 cytolemma stationary phase, X-coordinate be the time (minute).Be moving phase with 5mmol/L (pH 7.4) ammonium acetate buffer, Gefitinib both retention time be respectively 12min and 6min.This be since on the HEK293/FGFR4 cytolemma chromatographic column FGFR4 and the Gefitinib of high expression level combination has taken place, postponed the eluted time of Gefitinib, shown the ability to the specific combination of the antitumor drug that can combine with FGFR4, and as indication, can be used as the mark of screening with retention time.
Therefore, the HEK293/FGFR4 cell strain is on cytolemma stratographic analysis screening active compound, have susceptibility height, advantage that bonding force is strong, can be applied to the screening at the antitumor drug of FGFR4 target spot, especially be applied in the screening of the more antitumor active ingredient of Chinese herbs of activeconstituents.
Claims (3)
1. the reorganization HEK293 cell of a FGFR4 high expression level, it is characterized in that, the reorganization HEK293 cell of this FGFR4 high expression level is to be host cell with the HEK293 cell, exogenous expression's carrier of transfection host cell be the expression vector that comprises the FGFR4 full-length gene;
The nucleotide sequence of described FGFR4 full-length gene is shown in SEQ.ID.NO.1;
The expression vector of the described FGFR4 of comprising full-length gene is pcDNA3.1 (+)-FGFR4, and this expression vector is by XhoI and HindIII restriction enzyme site the FGFR4 full-length gene to be cloned into carrier for expression of eukaryon pcDNA3.1 (+).
2. to be applied to FGFR4 be the screening of the antitumor drug of target spot to the reorganization HEK293 cell of the described FGFR4 high expression level of claim 1.
3. application as claimed in claim 2 is characterized in that, the reorganization HEK293 cell construction of FGFR4 high expression level is become the cytolemma chromatographic column, is moving phase with the ammonium acetate buffer, is indication with the retention time, and screening is the antitumor drug of target spot with FGFR4.
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| CN103116006B (en) * | 2013-01-25 | 2015-09-30 | 重庆医科大学附属第一医院 | A kind of Novel screen choosing method of cancer therapy drug |
| CN104250241B (en) * | 2013-11-15 | 2016-10-26 | 重庆三峡医药高等专科学校 | The preparation method of a kind of CB1 antagonist EGCG and the medicine of preparation |
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