CN109652448A - The triple stable transfected cells strains of source of people OAT1-MRP2-UGT2B7 and its construction method - Google Patents
The triple stable transfected cells strains of source of people OAT1-MRP2-UGT2B7 and its construction method Download PDFInfo
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Abstract
The present invention relates to a kind of construction method of triple stable transfected cells strains of source of people OAT1-MRP2-UGT2B7 and functional verifications: the target gene of hOAT1 and pcDNA3.1/Hygro (+) expression vector are assembled, the target gene and pcDNA3.1/G418 (+) expression vector of hMRP2 assembles, the target gene and pcDNA3.1/Puromycin (+) expression vector of hUGT2B7 assembles, building pcDNA3.1/Hygro (+)-hOAT1 plasmid, pcDNA3.1/G418 (+)-hMRP2 plasmid and pcDNA3.1/Puromycin (+)-hUGT2B7 plasmid；Wherein, the nucleotide sequence of hOAT1, hMRP2 and hUGT2B7 target gene is respectively as shown in SEQ ID No.1-3；Three kinds of plasmids of building are successively transfected to MDCKII cell, Caco2 cell or HEK293 cell using liposome method, go out positive colony using special antibiotic-screening respectively, and it is transported through by using positive compound or metabolic function verifying, confirmation cell strain stablize expression OAT1, MRP2 and UGT2B7 gene and its albumen simultaneously.Triple stable transfected cells strains of the invention can be used for simulating the intake that OAT1 is mediated in liver, the outlet that MRP2 is mediated and the phase II metabolic function that UGT2B7 is mediated, and be the important in vitro study means for evaluating disposition of drug mechanism.
Description
Technical field
The present invention relates to cell strain building field more particularly to a kind of triple stable transfections of source of people OAT1-MRP2-UGT2B7
Cell strain construction method and functional verification.
Background technique
Drug metabolism transhipment is the key factor of internal disposition of drug, directly affects Drug safety and therapeutic effect.
With the development of biotechnology, establish external high expressing cell model have become research disposition of drug mechanism, interaction with
And the preferred model of safety.The cell model of currently reported research metabolism transhipment includes Caco2 cell (people's Colon and rectum
Cancerous cell line), high expression transporter MDCKII cell model (such as MDCKII-hOCTs, MDCKII-hOATPs, MDCKII-
HMDR1, MDCKII-hBCRP, MDCKII-MRP2, MDCKII-hMDR1-hBCRP etc.) and HEK293 cell model is (such as
HEK293-OATP1A2, HEK293-OATP1B1, HEK293-OCT etc.).Being established as of these cell models illustrates drug metabolism
Interaction between mechanism, research metabolic enzyme and transporter provides possibility.
In recent years, it is the weight obtained in medicament research and development process that disposition of drug mechanism is studied by establishing cell culture model
Gonna breakthrough and achievement.Application cell model can study transporting mechanism, metabolism, the drug medicine of candidate compound or clinical application
Object interaction, toxicity and safety evaluation etc..Cell model method is few with required medication amount, experimental method is simple,
The advantages such as the rapid, environmental condition controllable (temperature, pH etc.) of analysis, can be quickly obtained the transporting mechanism of drug permeable membrane, Ke Yida
To the purpose of external high flux screening.
In disposition of drug and interaction mechanism research, the cell strain of stable transfection is important investigative technique.Currently,
Lack a kind of OAT1-MRP2-UGT2B7 stable transfected cells strain.
Summary of the invention
In order to solve the above technical problems, the object of the present invention is to provide a kind of triple stabilizations of source of people OAT1-MRP2-UGT2B7
Transfection cell strain and its construction method provide technology platform for the transmembrane transport Mechanism Study work of Subsequent pharmacological.
The first purpose of the invention is to provide a kind of triple stable transfected cells strains of source of people OAT1-MRP2-UGT2B7
Construction method, comprising the following steps:
(1) target gene of hOAT1 and pcDNA3.1/Hygro (+) expression vector are assembled, the target gene of hMRP2 with
The assembling of pcDNA3.1/G418 (+) expression vector, the target gene and pcDNA3.1/Puromycin (+) expression vector of hUGT2B7
Assembling, building pcDNA3.1/Hygro (+)-hOAT1 plasmid, pcDNA3.1/G418 (+)-hMRP2 plasmid and pcDNA3.1/
Puromycin (+)-hUGT2B7 plasmid；Wherein, the nucleotide sequence of hOAT1, hMRP2 and hUGT2B7 target gene is respectively such as
Shown in SEQ ID No.1-3；
(2) it is successively transfected using three kinds of plasmids that liposome method constructs step (1) to MDCKII cell, Caco2 cell
Or HEK293 cell, cell transfecting for 24 hours after, filter out positive colony using specific antibiotic respectively, and by using the positive
Compound be transported through or metabolic function verifying, confirmation cell strain simultaneously stablize expression OAT1, MRP2 and UGT2B7 gene and its
Albumen constructs the triple stable transfected cells strains of source of people OAT1-MRP2-UGT2B7, wherein the antibiotic is G418
Antibiotic, hygromycin B antibiotic and Puromycin antibiotic.
Further, in step (1), using synthesis application of biological method, hOAT1, hMRP2 and hUGT2B7 purpose are synthesized
Gene.Compared with traditional gene clone technology, synthetic biology have the characteristics that it is quick, accurate, efficient, building gene table
It is more advantageous when up to system.
Further, in step (1), constructing used restriction endonuclease when plasmid is NheI and KpnI restriction enzyme
Or NheI and HindIII restriction enzyme.
It is time-consuming and laborious compared to physical transfection method, inefficient in step (2), artificial liposome in chemical transfection method
Method transfection efficiency with higher can transfect the cell line that other methods are not easy to transfect, DNA plasmid, interference tiny RNA
(microRNA, shRNA etc.) even albumen can be transfected by liposome method.In addition, lipofection is simultaneously
Suitable for transient expression and stablize expression, the former have the characteristics that it is quick, flexible, the latter have stablize, reproducibility by force etc. advantages.
Further, in step (2), when plasmid transfection, the concentration of three kinds of plasmids is 0.001 μ g/ μ L.
Further, in step (2), the concentration of G418 antibiotic is 800 μ g/mL, hygromycin B (hygromycin B)
The concentration of antibiotic is that the concentration of 400 μ g/mL, Puromycin (puromycin) antibiotic is 5 μ g/mL.Under concentrations above, no
Cell 2 days or so dead 60% containing corresponding resistant, and the cell containing related resistance in principle will not be dead.Excessive concentration can kill
Extremely contain resisting cell, and long-term cultured form malleable.
Further, in step (2), the cell of logarithmic growth phase carries out cell transfecting.
Further, it in step (2), when antibiotic-screening, when cell fusion degree is more than 25%, passes on immediately, every 3-4
The culture solution of its replacement antibiotic containing certain concentration is cultivated, and above step is repeated, until being passaged to 3-4 generation, picks out sun
Property monoclonal continue culture to growing up to cell strain.
Further, in step (2), plasmid transfection used medium is the DEME culture medium of serum-free.
Further, in step (2), plasmid transfection is carried out using PolyJet transfection reagent.
Further, it in step (2), is verified in terms of mRNA, protein expression and function three, it is steady to filter out
Surely tri- genes of hOAT1, hMRP2, hUGT2B7, and active positive colony cell strain are expressed.
Further, in step (2), transfection cell is preferably MDCKII cell.MDCKII is dog renal epithelial cell, with
The physiological characteristic of human normal cell line is close.It can be divided into polar columnar epithelium after culture and form brush border, between flanking cell
Close connection, forms complete cell monolayer, low to the permeability of substance.Lack big portion in biochemical characteristic inspection discovery MDCKII
Metabolic enzyme, intake transporter and the outlet transporter divided.Therefore, MDCKII cell is with cultivation cycle is short, background is lower, is easy to
The advantages that culture, the MDCKII cell strain of stable transfection are the effective means of drug metabolism transhipment research.
Further, the dosage of transfection reagent is the every 100 μ L culture medium of 40 μ L.
Second object of the present invention be claimed it is a kind of using source of people OAT1-MRP2- constructed by the above method
The triple stable transfected cells strains of UGT2B7, the cell strain can stable expression OAT1, MRP2 and UGT2B7 gene and its albumen simultaneously,
And there is corresponding transhipment and metabolic function.
Triple stable transfected cells strains that the present invention describes can be used for simulating the intake that OAT1 is mediated in liver, MRP2 is mediated
The phase II metabolic function that outlet and UGT2B7 are mediated is the important in vitro study means for evaluating disposition of drug mechanism.
According to the above aspect of the present invention, the present invention has at least the following advantages:
The present invention has successfully completed the triple stable transfections of hOAT1-hMRP2-hUGT2B7 by target gene synthetic technology
Cell strain building lays the foundation for the transmembrane transport and metabolic mechanism research work of Subsequent pharmacological.The present invention is quick, accurate, high
The building gene expression system of effect, transfection method is stable, reproducibility is strong.
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention and the accompanying drawings.
Detailed description of the invention
Fig. 1 is expression test result of the hMRP2 of the present invention in mRNA level in-site；
Fig. 2 is that the Steviol metabolin SV-G that UGT2B of the present invention is mediated is tied in the outlet rate test of monoclonal cell strain
Fruit.
Specific embodiment
With reference to the accompanying drawings and examples, specific embodiments of the present invention will be described in further detail.Implement below
Example is not intended to limit the scope of the invention for illustrating the present invention.
The building of 1 plasmid of embodiment
Using synthesis application of biological method, hOAT1, hMRP2, hUGT2B7 target gene are obtained by gene chemical synthesis, wherein
The nucleotide sequence of hOAT1, hMRP2 and hUGT2B7 target gene is respectively such as SEQ ID No.1, SEQ ID No.2, SEQ ID
Shown in No.3.By above-mentioned three kinds of genes respectively with pcDNA3.1/Hygro (+), pcDNA3.1/G418 (+), pcDNA3.1/
The connection of Puromycin (+) expression vector, after conversion amplification, extraction purification plasmid pcDNA3.1/Hygro (+)-hOAT1,
PcDNA3.1/G418 (+)-hMRP2, pcDNA3.1/Puromycin (+)-hUGT2B7 recombinant plasmid.Electricity is carried out to recombinant plasmid
Swimming identification, obtains corresponding purpose band, using Nhe I and Kpn I as restriction enzyme digestion sites (or Nhe I and Hind
III) double digestion identification, is carried out to recombinant plasmid, obtains corresponding carrier ribbon and target gene band, the results showed that three
Plasmid construction success, can be used for subsequent experimental.
The transfection of 2 plasmid of embodiment
(1) source of cell and culture:
Transfection cell is MDCKII cell, and from ECACC, (European cell strain/microorganism protects dog renal epithelial cell (MDCKII)
Hiding center) purchase.Cell is grown in 100mm Tissue Culture Dish, primary by 1:50 passage every three days, and culture medium is containing 10%
The DMEM (100U/mL penicillin, 100 μ g/mL streptomysins) of FBS is containing 5% carbon dioxide, 95% humidity, 37 DEG C of incubator
Middle culture.
(2) antibiotic concentration screens:
The MDCKII cell of logarithmic growth phase carries out antibiotic (G418, hygromycin B, Puromycin) concentration sieve
Choosing, antibiotic concentration are differed from 100 μ g/mL to 1000 μ g/mL, are cultivated 2-3 weeks, are selected all cells all to be killed minimum
The antibiotic concentration used when antibiotic concentration is as building stable cell strain.The result shows that the screening of MDCKII cell is dense
Degree G418 be that 800 μ g/mL, hygromycin B are 400 μ g/mL, and Puromycin is 5 μ g/mL.
(3) plasmid transfection:
A) the MDCKII cell of logarithmic growth phase spreads 6 orifice plates by 100,000/hole MDCKII, carries out transfection experiment afterwards for 24 hours.
B) according to PolyJet transfection reagent specification, by plasmid pcDNA3.1 empty carrier, pcDNA3.1/G418 (+)-
HMRP2 transfects MDCKII cell.Go out MDCKII-hMRP2 positive monoclonal using G418 antibiotic-screening, then to MDCKII-
It is transferred to pcDNA3.1/Hygro (+)-hOAT1 plasmid in hMRP2 positive monoclonal, filters out MDCKII--hOAT1-hMRP2 sun
Property monoclonal, then pcDNA3.1/puromycin (+)-hUGT2B7 plasmid is transferred in the positive monoclonal, and pass through metabolism function
It is able to verify that, confirmation cell strain stablizes expression OAT1, MRP2 and UGT2B7 gene and its albumen simultaneously.
Wherein, specific step is as follows for plasmid transfection:
The 2 above-mentioned plasmids of μ g are taken, are added in the DMEM containing 100 μ l serum-frees, 100 μ l are then added and turn containing 80 μ l liposomes
The serum-free DMEM of transfection reagent PolyJet is uniformly mixed, and is instilled in corresponding cell line 6 orifice plates, is transfected after standing 15min.
C) r for 24 hours after cell transfecting, replacement containing certain density selective antibiotic (G418 is 800 μ g/mL,
Hygromycin B is that 400 μ g/mL, Puromycin are screened for the DMEM cell training liquid of 5 μ g/mL).Cell fusion degree one
Denier is more than 25%, is passed on immediately, after the culture solution of every 3-4 days replacement antibiotic containing certain concentration loops back and forth like this 2-3 weeks, directly
To 3-4 generation is passaged to, selects monoclonal (about 50 or more) and continue to cultivate to 96 orifice plates, a culture solution was during which changed every three days
Until monoclonal cell strain grows up to, it is passaged to 24 orifice plates after 2 days and is expanded by cell, is tested for mRNA expression verifying, functional expression
Card and conservation.It chooses the wherein continuous surrounding of the higher cell strain of expression quantity and carries out functional verification experiment weekly, selected according to result
It is thin to be built into the triple stable transfections of source of people OAT1-MRP2-UGT2B7 for mrna expression amount highest, the most stable of cell strain of function
Born of the same parents' strain, as the cell model for stablizing overexpression.
The building of triple stable transfected cells strains and verifying: in step b) and c), first by pcDNA3.1/G418 (+)-
HMRP2 is transfected into MDCKII cell, does multiple groups parallel laboratory test, it is numbered for 1-119 respectively, is filtered out multiple MDCKII-
HMRP2 positive monoclonal.MRNA identification (quantitative fluorescent PCR) and protein expression are carried out to the positive monoclonal that parallel laboratory test obtains
(Western blot analysis).The cell clone total serum IgE selected is extracted using RNAiso Plus, and is divided with Q5000UV-Vis
The concentration and purity of photometer (Quawell) measurement RNA.According to PrimerScriptTM (Takara) kit method by RNA
Reverse transcription is reference gene at cDNA, selection GAPDH, uses iQTMSYBR Green Supermix (Bio-Rad) reagent and
CFX 96Real-Time fluorescence quantitative PCR instrument (Bio-Rad) quantifies hOAT1, hMRP2, hUGT2B7.PCR reacts item
Part are as follows: 95 DEG C of initial denaturations 5min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 2min are recycled 35 times, 72 DEG C of extensions
10min.After reaction, it takes 5 μ L PCR reaction products to carry out agarose gel electrophoresis analysis, confirms PCR reaction product.Detection
Expression of the hMRP2 in mRNA level in-site.As a result as shown in Figure 1, in figure, M refers to that the DNA marker of 100bp length, WT refer to
Be MDCKII wild-type cell, number represents MDCKII-hMRP2 positive monoclonal in figure.
The transport function of MDCKII-hMRP2 positive monoclonal uses MRP2 transporter substrate vincaleukoblastinum (Vinblastine)
It is tested, evaluates the function of every plant of monoclonal cell transporter.Positive colony cell after screening, by 1.0 × 105It is a thin
Born of the same parents/cm2Density be inoculated on 12 hole Transwell plates, culture 5 days after, measure MDCKII cell monolayer cross-film resistance
(TEER), in 180-200 Ω/cm2In the range of, cell monolayer is complete, that is, meets requirement of experiment.Using 10 μM of vincaleukoblastinum the bottom of as
Object, 37 DEG C of water-baths are incubated for 3hr and carry out transmembrane transport experiment, investigate outlet rate of the vincaleukoblastinum in each positive monoclonal cell strain,
The results are shown in Table 1.
Outer parallelism of 1 vincaleukoblastinum of table in each positive monoclonal cell strain
On the basis of the above-mentioned MDCKII-hMRP2 positive monoclonal picked out, number is re-started, then will
Into positive monoclonal cell, method according to step b) and c) carries out thin pcDNA3.1/Hygro (+)-hOAT1 plasmid transfection
Dysuria with lower abdominal colic dye, does multiple groups parallel laboratory test, filters out multiple MDCKII--hOAT1-hMRP2 positive monoclonals, replaces promise with OAT1 substrate
Fu Wei (Tenofovir) is tested, and the function of transporter is evaluated.Positive colony cell after screening, by 5.0 × 105It is a
Cell/cm2Density be inoculated on 24 orifice plates, culture 3 days after, using 10 μM of tenofovirs as substrate carry out intake experiment (37 DEG C,
10min), uptake ratio of the tenofovir in each monoclonal cell is investigated, the results are shown in Table 2.
Uptake ratio of 2 tenofovir of table in each positive monoclonal cell strain
On the basis of the above-mentioned MDCKII--hOAT1-hMRP2 positive monoclonal picked out, number is re-started, then will
PcDNA3.1/Puromycin (+)-hUGT2B7 plasmid transfection is into positive monoclonal cell, method according to step b) and c)
Cell transfecting is carried out, multiple groups parallel laboratory test is done, filters out multiple positive monoclonal MDCKII-hOAT1-hMRP2-hUGT2B7, is used
UGT2B7 substrate steviol (Steviol) is tested, and the steviol gluconic acid conjugate that evaluation UGT2B7 is mediated generates.Through
Positive colony cell after screening, by 5.0 × 105A cell/cm2Density be inoculated on 24 orifice plates, culture 3 days after, with 10 μM
Steviol is that substrate carries out cell metabolism experiment (37 DEG C, 10min), investigates metabolism speed of the Steviol in each monoclonal cell
Rate, the results are shown in Table 3.
Metabolic rate of 3 Steviol of table in each positive monoclonal cell strain
The positive monoclonal MDCKII--hOAT1-hMRP2-hUGT2B7 that will be filtered out, by 1.0 × 105A cell/cm2's
Density is inoculated on 12 hole Transwell plates, after culture 5 days, is measured the cross-film resistance (TEER) of MDCKII cell monolayer, is existed
180-200Ω/cm2In the range of, cell monolayer is complete, that is, meets requirement of experiment.Above-mentioned cell strain have MRP2, OAT1 and
It is reasonable in-vitro simulated can to transport progress to hepatic metabolism for UGT2B7 function.Using 10 μM of Steviol as substrate, 37 DEG C of water-baths are incubated
It educates, primarily looks at the rate for the metabolite SV-G that Steviol is generated under UGT2B7 mediation and in each monoclonal cell strain
Outlet rate.The metabolite SV-G of Steviol is the substrate of OAT1, while MRP2 participates in the transhipment of its outlet, therefore total outlet rate
(SV-G concentration in cell culture medium) is the synthesis result sent to some other department with the OAT1 intake mediated that MRP2 is mediated, as a result such as Fig. 2
It is shown.
The present invention has successfully completed the triple stable transfections of hOAT1-hMRP2-hUGT2B7 by target gene synthetic technology
Cell strain building lays the foundation for the transmembrane transport and metabolic mechanism research work of Subsequent pharmacological.The present invention is quick, accurate, high
The building gene expression system of effect, transfection method is stable, reproducibility is strong.Triple stable transfected cells strains that the present invention constructs are available
It is evaluation drug in the phase II metabolic function of OAT1 is mediated in simulation liver intake, the outlet that MRP2 is mediated and UGT2B7 mediation
The important in vitro study means of handling device.
The above is only a preferred embodiment of the present invention, it is not intended to restrict the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is several improvement and
Modification, these improvements and modifications also should be regarded as protection scope of the present invention.
Sequence table
<110>University Of Suzhou
<120>the triple stable transfected cells strains of source of people OAT1-MRP2-UGT2B7 and its construction method
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1692
<212> DNA
<213> (hOAT1)
<400> 1
atggccttta atgacctcct gcagcaggtg gggggtgtcg gccgcttcca gcagatccag 60
gtcaccctgg tggtcctccc cctgctcctg atggcttctc acaacaccct gcagaacttc 120
actgctgcca tccctaccca ccactgccgc ccgcctgccg atgccaacct cagcaagaac 180
ggggggctgg aggtctggct gccccgggac aggcaggggc agcctgagtc ctgcctccgc 240
ttcacctccc cgcagtgggg actgcccttt ctcaatggca cagaagccaa tggcacaggg 300
gccacagagc cctgcaccga tggctggatc tatgacaaca gcaccttccc atctaccatc 360
gtgactgagt gggaccttgt gtgctctcac agggccctac gccagctggc ccagtccttg 420
tacatggtgg gggtgctgct cggagccatg gtgttcggct accttgcaga caggctaggc 480
cgccggaagg tactcatctt gaactacctg cagacagctg tgtcagggac ctgcgcagcc 540
ttcgcaccca acttccccat ctactgcgcc ttccggctcc tctcgggcat ggctctggct 600
ggcatctccc tcaactgcat gacactgaat gtggagtgga tgcccattca cacacgggcc 660
tgcgtgggca ccttgattgg ctatgtctac agcctgggcc agttcctcct ggctggtgtg 720
gcctacgctg tgccccactg gcgccacctg cagctactgg tctctgcgcc tttttttgcc 780
ttcttcatct actcctggtt cttcattgag tcggcccgct ggcactcctc ctccgggagg 840
ctggacctca ccctgagggc cctgcagaga gtcgcccgga tcaatgggaa gcgggaagaa 900
ggagccaaat tgagtatgga ggtactccgg gccagtctgc agaaggagct gaccatgggc 960
aaaggccagg catcggccat ggagctgctg cgctgcccca ccctccgcca cctcttcctc 1020
tgcctctcca tgctgtggtt tgccactagc tttgcatact atgggctggt catggacctg 1080
cagggctttg gagtcagcat ctacctaatc caggtgatct ttggtgctgt ggacctgcct 1140
gccaagcttg tgggcttcct tgtcatcaac tccctgggtc gccggcctgc ccagatggct 1200
gcactgctgc tggcaggcat ctgcatcctg ctcaatgggg tgatacccca ggaccagtcc 1260
attgtccgaa cctctcttgc tgtgctgggg aagggttgtc tggctgcctc cttcaactgc 1320
atcttcctgt atactgggga actgtatccc acaatgatcc ggcagacagg catgggaatg 1380
ggcagcacca tggcccgagt gggcagcatc gtgagcccac tggtgagcat gactgccgag 1440
ctctacccct ccatgcctct cttcatctac ggtgctgttc ctgtggccgc cagcgctgtc 1500
actgtcctcc tgccagagac cctgggccag ccactgccag acacggtgca ggacctggag 1560
agcaggtggg cccccactca gaaagaagca gggatatatc ccaggaaagg gaaacagacg 1620
cgacagcaac aagagcacca gaagtatatg gtcccactgc aggcctcagc acaagagaag 1680
aatggactct ga 1692
<210> 2
<211> 4638
<212> DNA
<213> (hMRP2)
<400> 2
atgctggaga agttctgcaa ctctactttt tggaattcct cattcctgga cagtccggag 60
gcagacctgc cactttgttt tgagcaaact gttctggtgt ggattccctt gggcttccta 120
tggctcctgg ccccctggca gcttctccac gtgtataaat ccaggaccaa gagatcctct 180
accaccaaac tctatcttgc taagcaggta ttcgttggtt ttcttcttat tctagcagcc 240
atagagctgg cccttgtact cacagaagac tctggacaag ccacagtccc tgctgttcga 300
tataccaatc caagcctcta cctaggcaca tggctcctgg ttttgctgat ccaatacagc 360
agacaatggt gtgtacagaa aaactcctgg ttcctgtccc tattctggat tctctcgata 420
ctctgtggca ctttccaatt tcagactctg atccggacac tcttacaggg tgacaattct 480
aatctagcct actcctgcct gttcttcatc tcctacggat tccagatcct gatcctgatc 540
ttttcagcat tttcagaaaa taatgagtca tcaaataatc catcatccat agcttcattc 600
ctgagtagca ttacctacag ctggtatgac agcatcattc tgaaaggcta caagcgtcct 660
ctgacactcg aggatgtctg ggaagttgat gaagagatga aaaccaagac attagtgagc 720
aagtttgaaa cgcacatgaa gagagagctg cagaaagcca ggcgggcact ccagagacgg 780
caggagaaga gctcccagca gaactctgga gccaggctgc ctggcttgaa caagaatcag 840
agtcaaagcc aagatgccct tgtcctggaa gatgttgaaa agaaaaaaaa gaagtctggg 900
accaaaaaag atgttccaaa atcctggttg atgaaggctc tgttcaaaac tttctacatg 960
gtgctcctga aatcattcct actgaagcta gtgaatgaca tcttcacgtt tgtgagtcct 1020
cagctgctga aattgctgat ctcctttgca agtgaccgtg acacatattt gtggattgga 1080
tatctctgtg caatcctctt attcactgcg gctctcattc agtctttctg ccttcagtgt 1140
tatttccaac tgtgcttcaa gctgggtgta aaagtacgga cagctatcat ggcttctgta 1200
tataagaagg cattgaccct atccaacttg gccaggaagg agtacaccgt tggagaaaca 1260
gtgaacctga tgtctgtgga tgcccagaag ctcatggatg tgaccaactt catgcacatg 1320
ctgtggtcaa gtgttctaca gattgtctta tctatcttct tcctatggag agagttggga 1380
ccctcagtct tagcaggtgt tggggtgatg gtgcttgtaa tcccaattaa tgcgatactg 1440
tccaccaaga gtaagaccat tcaggtcaaa aatatgaaga ataaagacaa acgtttaaag 1500
atcatgaatg agattcttag tggaatcaag atcctgaaat attttgcctg ggaaccttca 1560
ttcagagacc aagtacaaaa cctccggaag aaagagctca agaacctgct ggcctttagt 1620
caactacagt gtgtagtaat attcgtcttc cagttaactc cagtcctggt atctgtggtc 1680
acattttctg tttatgtcct ggtggatagc aacaatattt tggatgcaca aaaggccttc 1740
acctccatta ccctcttcaa tatcctgcgc tttcccctga gcatgcttcc catgatgatc 1800
tcctccatgc tccaggccag tgtttccaca gagcggctag agaagtactt gggaggggat 1860
gacttggaca catctgccat tcgacatgac tgcaattttg acaaagccat gcagttttct 1920
gaggcctcct ttacctggga acatgattcg gaagccacag tccgagatgt gaacctggac 1980
attatggcag gccaacttgt ggctgtgata ggccctgtcg gctctgggaa atcctccttg 2040
atatcagcca tgctgggaga aatggaaaat gtccacgggc acatcaccat caagggcacc 2100
actgcctatg tcccacagca gtcctggatt cagaatggca ccataaagga caacatcctt 2160
tttggaacag agtttaatga aaagaggtac cagcaagtac tggaggcctg tgctctcctc 2220
ccagacttgg aaatgctgcc tggaggagat ttggctgaga ttggagagaa gggtataaat 2280
cttagtgggg gtcagaagca gcggatcagc ctggccagag ctacctacca aaatttagac 2340
atctatcttc tagatgaccc cctgtctgca gtggatgctc atgtaggaaa acatattttt 2400
aataaggtct tgggccccaa tggcctgttg aaaggcaaga ctcgactctt ggttacacat 2460
agcatgcact ttcttcctca agtggatgag attgtagttc tggggaatgg aacaattgta 2520
gagaaaggat cctacagtgc tctcctggcc aaaaaaggag agtttgctaa gaatctgaag 2580
acatttctaa gacatacagg ccctgaagag gaagccacag tccatgatgg cagtgaagaa 2640
gaagacgatg actatgggct gatatccagt gtggaagaga tccccgaaga tgcagcctcc 2700
ataaccatga gaagagagaa cagctttcgt cgaacactta gccgcagttc taggtccaat 2760
ggcaggcatc tgaagtccct gagaaactcc ttgaaaactc ggaatgtgaa tagcctgaag 2820
gaagacgaag aactagtgaa aggacaaaaa ctaattaaga aggaattcat agaaactgga 2880
aaggtgaagt tctccatcta cctggagtac ctacaagcaa taggattgtt ttcgatattc 2940
ttcatcatcc ttgcgtttgt gatgaattct gtggctttta ttggatccaa cctctggctc 3000
agtgcttgga ccagtgactc taaaatcttc aatagcaccg actatccagc atctcagagg 3060
gacatgagag ttggagtcta cggagctctg ggattagccc aaggtatatt tgtgttcata 3120
gcacatttct ggagtgcctt tggtttcgtc catgcatcaa atatcttgca caagcaactg 3180
ctgaacaata tccttcgagc acctatgaga ttttttgaca caacacccac aggccggatt 3240
gtgaacaggt ttgccggcga tatttccaca gtggatgaca ccctgcctca gtccttgcgc 3300
agctggatta catgcttcct ggggataatc agcacccttg tcatgatctg catggccact 3360
cctgtcttca ccatcatcgt cattcctctt ggcattattt atgtatctgt tcagatgttt 3420
tatgtgtcta cctcccgcca gctgaggcgt ctggactctg tcaccaggtc cccaatctac 3480
tctcacttca gcgagaccgt atcaggtttg ccagttatcc gtgcctttga gcaccagcag 3540
cgatttctga aacacaatga ggtgaggatt gacaccaacc agaaatgtgt cttttcctgg 3600
atcacctcca acaggtggct tgcaattcgc ctggagctgg ttgggaacct gactgtcttc 3660
ttttcagcct tgatgatggt tatttataga gataccctaa gtggggacac tgttggcttt 3720
gttctgtcca atgcactcaa tatcacacaa accctgaact ggctggtgag gatgacatca 3780
gaaatagaga ccaacattgt ggctgttgag cgaataactg agtacacaaa agtggaaaat 3840
gaggcaccct gggtgactga taagaggcct ccgccagatt ggcccagcaa aggcaagatc 3900
cagtttaaca actaccaagt gcggtaccga cctgagctgg atctggtcct cagagggatc 3960
acttgtgaca tcggtagcat ggagaagatt ggtgtggtgg gcaggacagg agctggaaag 4020
tcatccctca caaactgcct cttcagaatc ttagaggctg ccggtggtca gattatcatt 4080
gatggagtag atattgcttc cattgggctc cacgacctcc gagagaagct gaccatcatc 4140
ccccaggacc ccatcctgtt ctctggaagc ctgaggatga atctcgaccc tttcaacaac 4200
tactcagatg aggagatttg gaaggccttg gagctggctc acctcaagtc ttttgtggcc 4260
agcctgcaac ttgggttatc ccacgaagtg acagaggctg gtggcaacct gagcataggc 4320
cagaggcagc tgctgtgcct gggcagggct ctgcttcgga aatccaagat cctggtcctg 4380
gatgaggcca ctgctgcggt ggatctagag acagacaacc tcattcagac gaccatccaa 4440
aacgagttcg cccactgcac agtgatcacc atcgcccaca ggctgcacac catcatggac 4500
agtgacaagg taatggtcct agacaacggg aagattatag agtgcggcag ccctgaagaa 4560
ctgctacaaa tccctggacc cttttacttt atggctaagg aagctggcat tgagaatgtg 4620
aacagcacaa aattctag 4638
<210> 3
<211> 1574
<212> DNA
<213> (hUGT2B7)
<400> 3
atgtctgtga aatggacttc agtaattttg ctaatacaac tgagcttttg ctttagctct 60
gggaattgtg gaaaggtgct ggtgtgggca gcagaataca gccattggat gaatataaag 120
acaatcctgg atgagcttat tcagagaggt catgaggtga ctgtactggc atcttcagct 180
tccattcttt ttgatcccaa caactcatcc gctcttaaaa ttgaaattta tcccacatct 240
ttaactaaaa ctgagttgga gaatttcatc atgcaacaga ttaagagatg gtcagacctt 300
ccaaaagata cattttggtt atatttttca caagtacagg aaatcatgtc aatatttggt 360
gacataacta gaaagttctg taaagatgta gtttcaaata agaaatttat gaaaaaagta 420
caagagtcaa gatttgacgt catttttgca gatgctattt ttccctgtag tgagctgctg 480
gctgagctat ttaacatacc ctttgtgtac agtctcagct tctctcctgg ctacactttt 540
gaaaagcata gtggaggatt tattttccct ccttcctacg tacctgttgt tatgtcagaa 600
ttaactgatc aaatgacttt catggagagg gtaaaaaata tgatctatgt gctttacttt 660
gacttttggt tcgaaatatt tgacatgaag aagtgggatc agttttatag tgaagttcta 720
ggaagaccca ctacattatc tgagacaatg gggaaagctg acgtatggct tattcgaaac 780
tcctggaatt ttcagtttcc atatccactc ttaccaaatg ttgattttgt tggaggactc 840
cactgcaaac ctgccaaacc cctgcctaag gaaatggaag actttgtaca gagctctgga 900
gaaaatggtg ttgtggtgtt ttctctgggg tcaatggtca gtaacatgac agaagaaagg 960
gccaacgtaa ttgcatcagc cctggcccag atcccacaaa aggttctgtg gagatttgat 1020
gggaataaac cagatacctt aggtctcaat actcggctct acaagtggat accccagaat 1080
gaccttctag gtcatccaaa gaccagagct tttataactc atggtggagc caatggcatc 1140
tacgaggcaa tctaccatgg gatccctatg gtggggattc cattgtttgc cgatcaacct 1200
gataacattg ctcacatgaa ggccagggga gcagctgtta gagtggactt caacacaatg 1260
tcgagtacag acttgctgaa tgcattgaag agagtaatta atgatccttc atataaagag 1320
aatgttatga aattatcaag aattcaacat gatcaaccag tgaagcccct ggatcgagca 1380
gtcttctgga ttgaatttgt catgcgccac aaaggagcta aacaccttcg ggttgcagcc 1440
cacgacctca cctggttcca gtaccactct ttggatgtga ttgggttcct gctggtctgt 1500
gtggcaactg tgatatttat cgtcacaaaa tgttgtctgt tttgtttctg gaagtttgct 1560
agaaaagcaa agaa 1574
Claims (10)
1. a kind of construction method of the triple stable transfected cells strains of source of people OAT1-MRP2-UGT2B7, which is characterized in that including with
Lower step:
(1) target gene of hOAT1 and pcDNA3.1/Hygro (+) expression vector are assembled, the target gene of hMRP2 with
The assembling of pcDNA3.1/G418 (+) expression vector, the target gene and pcDNA3.1/Puromycin (+) expression vector of hUGT2B7
Assembling, building pcDNA3.1/Hygro (+)-hOAT1 plasmid, pcDNA3.1/G418 (+)-hMRP2 plasmid and pcDNA3.1/
Puromycin (+)-hUGT2B7 plasmid；Wherein, the nucleotide sequence of hOAT1, hMRP2 and hUGT2B7 target gene is respectively such as
Shown in SEQ ID No.1-3；
(2) the three kinds of plasmids constructed step (1) using liposome method successively transfect to MDCKII cell, Caco2 cell or
HEK293 cell, cell transfecting for 24 hours after, go out positive colony with antibiotic-screening, construct the source of people OAT1-MRP2-
The triple stable transfected cells strains of UGT2B7, wherein the antibiotic is G418, hygromycin B and puromycin.
2. construction method according to claim 1, it is characterised in that: in step (1), construct in used when plasmid
Enzyme cutting is III restriction enzyme of Nhe I and I restriction enzyme of Kpn or Nhe I and Hind.
3. construction method according to claim 1, it is characterised in that: in step (2), when plasmid transfection, three kinds of plasmids
Concentration be 0.001 μ g/ μ L.
4. construction method according to claim 1, it is characterised in that: in step (2), the concentration of the G418 is 800 μ
G/mL, the concentration of hygromycin B are 400 μ g/mL, and the concentration of puromycin is 5 μ g/mL.
5. construction method according to claim 1, it is characterised in that: in step (2), the cell of logarithmic growth phase into
Row cell transfecting.
6. construction method according to claim 1, it is characterised in that: in step (2), when antibiotic-screening, cell melts
It is right more than 25% when, pass on immediately, every 3-4 days replacement the antibiotic containing certain concentration culture solution cultivated, repeat more than
Step picks out positive monoclonal and continues culture to growing up to cell strain until be passaged to 3-4 generation.
7. construction method according to claim 1, it is characterised in that: in step (2), plasmid transfection used medium is
The DEME culture medium of serum-free.
8. construction method according to claim 1, it is characterised in that: in step (2), using PolyJet transfection reagent
Carry out plasmid transfection.
9. construction method according to claim 8, it is characterised in that: the dosage of transfection reagent is the every 100 μ L culture of 40 μ L
Base.
10. the triple stabilizations of source of people OAT1-MRP2-UGT2B7 constructed by a kind of method of any of claims 1-9
Transfection cell strain, it is characterised in that: cell strain stablizes expression OAT1, MRP2 and UGT2B7 gene and its albumen simultaneously.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002099088A1 (en) * | 2001-06-06 | 2002-12-12 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Double-transfected cell line useful for the identification of transport inhibitors |
-
2018
- 2018-12-29 CN CN201811642796.5A patent/CN109652448B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002099088A1 (en) * | 2001-06-06 | 2002-12-12 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Double-transfected cell line useful for the identification of transport inhibitors |
Non-Patent Citations (6)
| Title |
|---|
| AB009697.1: "Homo sapiens mRNA for hOAT1-1, complete cds", 《GENBANK》 * |
| C FAHRMAYR ET.AL: "Identification of drugs and drug metabolites as substrates of multidrug resistance protein 2 (MRP2) using triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells", 《BRITISH JOURNAL OF PHARMACOLOGY》 * |
| KAN CHEN ET.AL: "Abstract_ Construction and Characterization of Double-Transfected MDCKII cells Stably Expressing Human OAT1 and MRP2", 《ISSX ONLINE ABSTRACT 5TH ASIAN PACIFIC REGIONAL ISSX MEETING》 * |
| NM_001074.4: "Homo sapiens UDP glucuronosyltransferase family 2 member B7 (UGT2B7), transcript variant 1, mRNA", 《GENBANK》 * |
| U63970.1: "Human canalicular multispecific organic anion transporter (cMOAT) mRNA, complete cds", 《GENBANK》 * |
| 王美玉等: "甜菊苷代谢转运机制研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
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