CN109811007A - A kind of over-express vector FYJNa significantly increasing cell ABCC1 gene expression - Google Patents
A kind of over-express vector FYJNa significantly increasing cell ABCC1 gene expression Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A kind of over-express vector FYJNa significantly increasing cell ABCC1 gene expression, belongs to gene engineering technology field, it is characterised in that one kind can significantly increase the nucleotide sequence of the over-express vector FYJNa of cell ABCC1 gene expression as shown in Seq ID No:1.It is cDNA using reverse transcription after carrying out Total RNAs extraction to A549/DDP cell, the area CDS and corresponding mRNA sequence, the design primer of CREB1 gene is found, in addition I restriction enzyme site of Hind III and EcoR, expands the area CREB1 gene C DS；Then neutral mutation is carried out, digestion carrier connects target fragment and carrier, recombinant expression carrier, point mutation is carried out after the completion of subclone, detects the expression of FYJNa and ABCC1 gene mRNA and albumen respectively, is overexpressed the expression that FYJNa can raise the mRNA and protein level of ABCC1 as the result is shown.
Description
Technical field
The present invention relates to the over-express vector FYJNa that one kind can significantly increase cell ABCC1 gene expression, belong to gene work
Journey technical field.
Background technique
ABCC1 gene was one of C subtribe member in ATP combination box (ABC) drug transporter superfamily, in head in 1992
Secondary identify from drug resistance lung cancer cell line H69AR comes (Cole et al., 1992)；The assignment of genes gene mapping is in chromosome
16p13.11, its length are about 200,000 base-pair, which contains 34 exons, encode the egg of 1531 amino acid
White matter, molecular weight are 190kDa (é va Bakos et al., 2007).The protein has 3 hydrophobic transmembrane domains
(TMD), also referred to as transmembrane domain (MSD), referred to as TMD0 (end N-), TMD1 (centre) and TMD2 (end C-) (omka
M et al.,2015).ABCC1 is using ATP combination/hydrolysis energy as main active transporter (Sharom et
Al., 2015), ABCC1 has extensive substrate specificity, and overexpressing cell can transport a large amount of substrates, such as antifolic
Object, antifolate, anthracycline, vinca alkaloids etc. (omka M et al.,2015)；Inorganic anion, it is such as sub-
Arsenate and arsenate, ABCC1 are also endogenous compound in extensive range, including glutathione (GSH), glucuronic acid and are had
Machine anion, such as the active transport C4 or estradiol -17- β-d glucosiduronic acid (E of inflammatory mediator leukotriene C sulfate conjugate
217βG).Therefore, the organic anion that ABCC1 is played, anionic drugs, the conjugate of xenobiotic pump out effect, thus ABCC1
It has been determined one of polyspecific organic anion transporter (MOATs) or glutathione conjugate (GS-X) pump (é va
Bakos et al., 2007,Omka M et al., 2015, Lu et al., 2015, ConseilG et al.,
2005))；So ABCC1 gene product is one of the key gene of body discharge medicine protection normal cell.CAMP response element
Binding protein (cAMP response element binding protein, CREB), is nuclear factor.The friendship of the gene
Lead to several transcript variants for encoding not isotype for montage；It is conservative on CREB1 and promoter as activating transcription factor
CAMP response element (CRE) combines, and mediates the responsive transcription to a variety of stimulations, including hormone, membrane depolarization, growth and nerve
Trophic factors, to serve as the medium (Xie et al., 2015) between unlike signal access.Recent studies have found that CREB egg
White level, in high expression (Tan et al., 2012), participates in proliferation, the survival of tumour cell in part of cancerous tissue and cell
And the adjusting of the various aspects such as transfer.Therefore CREB1 gene how is transformed, building one can significantly increase cell ABCC1 gene
The over-express vector of expression becomes a great problem urgently to be solved, so a kind of can significantly increase cell ABCC1 gene expression
Over-express vector FYJNa is cDNA using reverse transcription after carrying out Total RNAs extraction to A549/DDP cell, is found on the website NCBI
The area CDS of CREB1 gene and corresponding mRNA sequence；According to design of primers principle 5.0 software Design primers of Primer, divide
I restriction enzyme site of Hind III and EcoR is not added in upstream and downstream；The area PCR amplification CREB1 gene C DS is carried out by template of people cDNA；
Then 905,908,911 and 914 sites in the area CREB1 gene C DS are mutated；Digestion pcDNA3.1 (+) carrier；Connect mesh
Segment and pcDNA3.1 (+)；PcDNA3.1-FYJNa is named as after recombinant expression carrier conversion, detection and identification；It is prominent
It is as follows to become information: 905 T sports C；908 T sports C；911 C sports A；914 T sports C and can be obtained；So
Afterwards, the expression of FYJNa and ABCC1 gene mRNA and albumen, hair are detected by qRT-PCR and Western blot method respectively
The over-express vector FYJNa that bright one kind can significantly increase cell ABCC1 gene expression is necessary.
Summary of the invention
In order to overcome the difficulty for how constructing the over-express vector FYJNa that one can significantly increase cell ABCC1 gene expression
Topic, the present invention provides the over-express vector FYJNa that one kind can significantly increase cell ABCC1 gene expression, this kind of one kind can be shown
Write enhancing cell ABCC1 gene expression over-express vector FYJNa utilize to A549/DDP cell progress Total RNAs extraction after invert
Record be cDNA, found on the website NCBI CREB1 gene the area CDS and corresponding mRNA sequence；It is used according to design of primers principle
5.0 software Design primers of Primer add I restriction enzyme site of Hind III and EcoR in upstream and downstream respectively；Using people cDNA as template into
The area row PCR amplification CREB1 gene C DS；Then 905,908,911 and 914 sites in the area CREB1 gene C DS are mutated；Enzyme
Cut pcDNA3.1 (+) carrier；Connect target fragment and pcDNA3.1 (+)；By it after recombinant expression carrier conversion, detection and identification
It is named as pcDNA3.1-FYJNa；Abrupt information is as follows: 905 T sports C；908 T sports C；911 C sports A；
914 T sports C and can be obtained；Then, FYJNa and ABCC1 base is detected by qRT-PCR and Western blot method respectively
Because of the expression of mRNA and albumen；It is overexpressed the expression that FYJNa can raise the mRNA and protein level of ABCC1 as the result is shown.
The technical solution adopted by the present invention to solve the technical problems is:
One kind of the present invention can significantly increase the over-express vector FYJNa of cell ABCC1 gene expression, and concrete scheme is as follows:
1.A549/DDP cell recovery and culture
1.1 cell recoveries: (1) taking out freeze-stored cell in liquid nitrogen, quickly rocks in 37 DEG C of water-baths；(2) to its whole
Melt, be slowly injected into the centrifuge tube of 1640 culture medium containing 5mL, 1000g is centrifuged 5min and abandons supernatant；(3) 1640 culture medium of 5mL
Suspend precipitating, moves to cell bottle, 37 DEG C, the interior culture cell of 5%CO2 incubator.
1.2 A549/DDP cell secondary cultures: (1) former culture in glassware base is abandoned, is gently washed cell 2 times with 5mL PBS；
(2) 1mL pancreatin is added to change to cellular morphology under the microscope；(3) 5mL culture medium is added and terminates digestion, repeatedly slightly
Cell is blown and beaten, all attached cells are blown down；(4) it adds 10mL culture medium after mixing, takes out 5mL culture medium respectively and be added
In two new bottles, 37 DEG C, the interior culture cell of 5%CO2 incubator.
1.3 A549/DDP cell normal morphology figures: after lung cancer A549/DDP cell recovery and secondary culture, growth
For state as shown in Fig. 2, visible cell adherent growth, index of refraction is good, and growth conditions are good.
2. passing through regulation of the point mutation detection FYJNa to MRP1 gene expression
The building of 2.1 CREB1 over-express vectors: (1) on NCBI website find CREB1 gene (Gene ID:
692871) the area CDS and corresponding mRNA (NM_134442.4) sequence；(2) according to design of primers principle Primer 5.0
Software Design primers add I restriction enzyme site of Hind III and EcoR, primer sequence: CREB 1-F:5 '-in upstream and downstream respectively
CCCAAGCTTATGACCATGGAATCTGGAGC-3'；CREB1-R:5 '-CCGGAATTCTTAATCTGATTTGTGGCAG-3 '；
(3) area PCR amplification CREB1 gene C DS is carried out by template of people cDNA；(4) digestion pcDNA3.1 (+) carrier；(5) purpose is connected
Segment and pcDNA3.1 (+)；(6) pcDNA3.1-CREB1 is named as after recombinant expression carrier conversion, detection and identification.
The building of 2.2 FYJNa activation tagging vectors: (1) area CDS of CREB1 gene and right on NCBI website is found
MRNA sequence (the number: NM_134442.4 answered；Homo sapiens cAMP responsive element binding
protein 1(CREB1),VERSION NM_134442.4)；(2) according to design of primers principle 5.0 software design of Primer
Primer adds I restriction enzyme site of Hind III and EcoR in upstream and downstream respectively；(3) PCR amplification CREB1 is carried out by template of people cDNA
The area gene C DS；(4) then 905,908,911 and 914 sites in the area CREB1 gene C DS are mutated；(5) digestion
PcDNA3.1 (+) carrier；(6) connection target fragment and pcDNA3.1 (+)；(7) after recombinant expression carrier conversion, detection and identification
It is named as pcDNA3.1-FYJNa；Abrupt information is as follows: 905 T sports C；908 T sports C；911 C mutation
For A；914 T sports C.
2.3 PEI infection protocols transfect lung cancer A549/DDP cell: (1) growing to convergence degree to cell is 60% or so Shi Kai
Begin to transfect.(2) in 1.5EP pipe, 300 μ L Opti-DMEM is added and are mixed with 12 μ g recombinant plasmids, in yet another 1.5EP pipe
Every pipe is added 300 μ L Opti-DMEM and mixes with 24 μ L PEI, stationary incubation 8min.(3) above-mentioned two pipe is mixed well, is stood
It is incubated for 15min.(4) culture medium in cell bottle is drawn, is washed once with PBS, is added and is trained without dual anti-and serum 1640 culture mediums
It supports.(5) above-mentioned mixed 600 μ L of mixed liquor is added in the blood-free medium in cell bottle, light shake mixes up and down.It is placed in
37 DEG C, after cultivating 4h in 5%CO2 incubator, former culture medium is sopped up, complete medium is changed into and continues to cultivate.
2.4 qRT-PCR detect FYJNa and ABCC1mRNA expression
2.4.1 lung cancer A549/DDP cell total rna extracts: (1) abandoning former culture in glassware base, gently washed with 5mL PBS thin
Born of the same parents 2 times；(2) 1mL pancreatin is added to change to cellular morphology under the microscope；(3) 5mL culture medium is added and terminates digestion, instead
Multiple slight piping and druming cell, blows down all attached cells；(4) it moves in centrifuge tube, 1000g is centrifuged 5min, abandons supernatant；(5)1mL
PBS suspension cell moves in 1.5Ep pipe, and 5000g is centrifuged 2min, abandons supernatant；(6) 1mL Trizol is added in each Ep pipe, hangs
Floating cell is stored at room temperature 10min；(7) 0.2mL chloroform is added, acutely shakes 30s, is placed at room temperature for 3min, 12000r4 DEG C of centrifugation
10min；(8) upper layer is transferred in clean 1.5Ep pipe, 1/2 times of volume of dehydrated alcohol is added, mixes well；It (9) will be above-mentioned
Mixed liquor is transferred in adsorption column, stands 2min, and 4 DEG C of centrifugation 3min of 12000r outwell liquid in collecting pipe；(10) 500 are added
4 DEG C of centrifugation 30s of μ L RPE Solution, static 2min, 10000r, outwell liquid in collecting pipe, are repeated once；(11) suction
Attached column is put into collecting pipe, 4 DEG C of centrifugation 2min of 10000r；(12) adsorption column is put into clean 1.5mL centrifuge tube, is being inhaled
4 DEG C of centrifugation 2min of 30 μ L DEPC-treated ddH2O, static 5min, 12000r are added in membrane center.2.4.2 reverse transcription is anti-
It answers: according to reverse transcription reagent box specification, using two-step method by acquired Total RNA reverse transcription for cDNA.(1) by reversion
Record reaction system I: following ratio Total RNA or Poly (A) RNA, 0.2-2 μ L；Oligo dT or Random
Primer (50 μM), 1 μ L；Dntp Mixture (10mM each), 1 μ L；14 μ L of Rnase Free H2O, Up to is prepared anti-
Responsive transcription liquid；(2) following reaction is carried out in PCR instrument: 65 DEG C, 5min is subsequently placed in chilling on ice；(3) in above-mentioned PCR pipe
Middle II reaction solution of addition reverse transcription reaction system (20 μ L system): reaction solution after above-mentioned denaturation, annealing, 14 μ L；5Xfirst-
Strand Buffer, 4 μ L；M-MuLV Reverse Transcriptase (200U/ μ L), 1 μ L；Rnase Inhibitor, 1
μL.(4) by the following conditions progress reverse transcription reaction in PCR instrument: 42-50 DEG C, 45-60min；70 DEG C, 10min；- 20 DEG C of guarantors
It deposits.
2.4.4 qRT-PCR reaction system: (1) by gained cDNA Power 2 × SYBR Real-Time PCR
Premixture kit carries out Real Time PCR, and reaction system is 20 μ L, qRT-PCR reaction systems: cDNA, 1 μ L；
Sense primer, 0.4 μ L；Anti-sense primer, 0.4 μ L；2 × Mix, 10 μ L；Rox, 0.4 μ L；DdH2O, 7.8 μ L.
(2) reaction condition are as follows: 95 DEG C of 2min, 40 × (95 DEG C of 15s, 60 DEG C of 40s), 95 DEG C of 15s, 60 DEG C of 1min；It carries out after circulation terminates
Solubility curve detection.(3) data are with 2-ΔΔCtMethod is calculated.(4) each gene qRT-PCR upstream and downstream primer sequence: F:5 '-
CCAGGGAGGAGCAATACAGC-3'；R:5 '-TGTCCATCAGTGGTCTGTGC-3 '；ABCC1F:5 '-
GGGGTCCTCATTATCTTCTGG-3'；ABCC1R:5 '-TGGTCTCAGGGTAGGGGTT AG-3 '；β-actin F:5 '-
AGCGAGCATCCCCCAAAGTT-3'；β-actin R:5 '-GGGCACGAAGGCTC ATCATT-3 '.
2.5 Western blot detect FYJNa and ABCC1 protein expression level
2.5.1 lung cancer A549/DDP cell holoprotein extracts: (1) abandoning former culture in glassware base, gently washed with 5mL PBS thin
Born of the same parents 2 times；(2) 1mL pancreatin is added to change to cellular morphology under the microscope；(3) 5mL culture medium is added and terminates digestion, instead
Multiple slight piping and druming cell, blows down all attached cells；(4) it moves in centrifuge tube, 1000g is centrifuged 5min, abandons supernatant；(5)1mL
PBS suspension cell moves in 1.5Ep pipe, and 5000g is centrifuged 2min, abandons supernatant；(6) add 300 μ L protein lysates, be placed in 4 DEG C and shake
30min on bed；(7) 12000g is centrifuged 20min, takes supernatant, -20 DEG C of preservations.
2.5.2 protein denaturation: protein sample and sample-loading buffer are mixed in 4:1 ratio, boiling water bath heat 5min~
10min, to its near room temperature, -20 DEG C save or continue to test.
2.5.3 SDS- polyacrylamide gel electrophoresis: (1) offset plate is combined；(2) by separation glue formula: water, 8.2mL；
30% acrylamide, 6.6mL；10%SDS, 0.2mL；Tris-Cl (PH8.8), 5mL；10%APS, 200 μ l；TEMED, 20 μ l,
Prepare separation gel: (3) inject separation gel in layer glass plate, and after appropriate location is added, 1mL isopropanol is added in sliding pipette tips,
After being gelled, isopropanol is poured out, water is exhausted；(4) by concentration glue formula: water, 5.7mL；30% acrylamide, 1.7mL；10%
SDS, 0.1mL；Tris-Cl (PH6.8), 2.5mL；10%APS, 100 μ l；TEMED, 10 μ l prepare concentration glue, by concentration glue note
Enter in layer glass plate, until liquid level overflows, is inserted into comb.(5) after being gelled, comb is extracted, is assembled with electrophoresis tank, electrophoresis is added
Liquid；(6) 30 μ l samples are added in point sample, every hole, and Maker applied sample amount is 10 μ l；(7) constant current 100mA clicks start button, bromine phenol
The bottom end that indigo plant reaches glue nearby stops electrophoresis.
2.5.4 transferring film: (1) offset plate is taken out, glue is placed in transferring film liquid and is impregnated；(2) clip black flour is swept away, by sponge, filter
Paper, glue, pvdf membrane, filter paper, sponge sequence put well, grip clamp；(3) it is put into transferring film slot, it is black to be put well to black, it is put into ice
Pot pours into transferring film liquid；(4) transferring film slot is assembled, constant pressure 100V carries out transferring film.
2.5.5 close: (1) Ponceaux contaminates film 5min；(2) clear water gently rinses Ponceaux；(3) it is indicated according to maker big
It is small to cut film；(4) 5% skimmed milk power (confining liquid) is added, room temperature shaking table is incubated for 1h.
2.5.6Western hybridize: I. primary antibody is incubated for: (1) according to specification, diluting antibody；(2) primary antibody is incubated for, and is placed in 4
DEG C shaking table shakes overnight；(3) primary antibody is recycled, PBST washes film 10min, three times.II. secondary antibody is incubated for (being protected from light operation): (1) according to explanation
Book dilutes antibody；(2) secondary antibody is incubated for, and is placed in 4 DEG C of shaking table 1h；(3) secondary antibody is recycled, PBST washes film 10min, three times.III. albumen
Upper machine testing: Fig. 5 is seen using the bis- infrared laser imaging system detection protein band variations of Odyssey, analysis result figure is shown in Fig. 6.
Beneficial effects of the present invention are a kind of over-express vector FYJNa benefit for significantly increasing cell ABCC1 gene expression
It is cDNA with reverse transcription after carrying out Total RNAs extraction to A549/DDP cell, the area CDS of CREB1 gene is found on the website NCBI
And corresponding mRNA sequence；According to design of primers principle 5.0 software Design primers of Primer, added respectively in upstream and downstream
I restriction enzyme site of Hind III and EcoR；The area PCR amplification CREB1 gene C DS is carried out by template of people cDNA；Then to CREB1 gene
905,908,911 and 914 sites in the area CDS are mutated；Digestion pcDNA3.1 (+) carrier；Connect target fragment with
pcDNA3.1(+)；PcDNA3.1-FYJNa is named as after recombinant expression carrier conversion, detection and identification；Abrupt information is such as
Under: 905 T sports C；908 T sports C；911 C sports A；914 T sports C and can be obtained；Then, respectively
The expression of FYJNa and ABCC1 gene mRNA and albumen is detected by qRT-PCR and Western blot method；As the result is shown
The expression of mRNA and protein level of ABCC1 can be raised by being overexpressed FYJNa.
Detailed description of the invention
The following further describes the present invention with reference to the drawings.
Fig. 1 is that the FYJNa overexpression for the over-express vector that one kind of the present invention can significantly increase cell ABCC1 gene expression is prominent
The base sequence figure of variable load body.
Fig. 2 is the normal light of the FYJNa for the over-express vector that one kind of the present invention can significantly increase cell ABCC1 gene expression
Learn microscopically observation A549/DDP cell (10 × 10) cell state figure.
Fig. 3 is the FYJNa's for the over-express vector that one kind of the present invention can significantly increase cell ABCC1 gene expression
PcDNA3.1-CREB1 is overexpressed plasmid figure.
Fig. 4 is the FYJNa of the FYJNa for the over-express vector that one kind of the present invention can significantly increase cell ABCC1 gene expression
With 1 gene mRNA expression variation diagram of ABCC.A is FYJNa mRNA expression in figure；B is ABCC1mRNA expression (* * *
p<0.001)。
Fig. 5 is the FYJNa's for the over-express vector that one kind of the present invention can significantly increase cell ABCC1 gene expression
Western blot testing result
Fig. 6 is the FYJNa of the FYJNa for the over-express vector that one kind of the present invention can significantly increase cell ABCC1 gene expression
With ABCC1 expression of gene protein variation diagram.A is FYJNa protein expression level in figure；B be ABCC1 protein expression level (* * * p <
0.001)。
Specific embodiment
Embodiment one: as shown, one kind of the present invention can significantly increase the over-express vector of cell ABCC1 gene expression
FYJNa experimental method and result are as follows:
2.A549/DDP cell recovery and culture
1.1 cell recovery
(1) freeze-stored cell is taken out in liquid nitrogen, is quickly rocked in 37 DEG C of water-baths；
(2) all melt to it, be slowly injected into the centrifuge tube of 1640 culture medium containing 5mL, 1000g is centrifuged in 5min abandoning
Clearly；
(3) 1640 culture medium of 5mL, which suspends, precipitates, and moves to cell bottle, 37 DEG C, the interior culture cell of 5%CO2 incubator.
1.2A549/DDP cell secondary culture
(1) former culture in glassware base is abandoned, is gently washed cell 2 times with 5mL PBS；
(2) 1mL pancreatin is added to change to cellular morphology under the microscope；
(3) 5mL culture medium is added and terminates digestion, slightly blow and beat cell repeatedly, blow down all attached cells；
(4) it adds 10mL culture medium after mixing, takes out 5mL culture medium respectively and be added in two new bottles, 37 DEG C,
Cell is cultivated in 5%CO2 incubator.
1.3 A549/DDP cell normal morphology figures
After lung cancer A549/DDP cell recovery and secondary culture, growth conditions are as shown in Fig. 2, the adherent life of visible cell
Long, index of refraction is good, and growth conditions are good.
2. passing through regulation of the point mutation detection FYJNa to ABCC1 gene expression
The building of 2.1 CREB1 over-express vectors
(1) found on the website NCBI CREB1 gene (Gene ID:692871) the area CDS and corresponding mRNA (NM_
134442.4) sequence；
(2) according to design of primers principle 5.0 software Design primers of Primer, Hind III is added in upstream and downstream respectively
With I restriction enzyme site of EcoR, primer sequence is shown in Table 1；
(3) area PCR amplification CREB1 gene C DS is carried out by template of people cDNA；
(4) digestion pcDNA3.1 (+) carrier；
(5) connection target fragment and pcDNA3.1 (+)；
(6) pcDNA3.1-CREB1 is named as after recombinant expression carrier conversion, detection and identification.
1 CDS amplimer of table
The building of 2.2FYJNa over-express vector
(1) area CDS and the corresponding mRNA sequence (number: NM_ of CREB1 gene are found on the website NCBI
134442.4)；
(2) according to design of primers principle 5.0 software Design primers of Primer, Hind III is added in upstream and downstream respectively
With I restriction enzyme site of EcoR, primer sequence is shown in Table 1；
(3) area PCR amplification CREB1 gene C DS is carried out by template of people cDNA；
(4) neutral mutation then is carried out to the binding site in the shRNA interference site of CREB1 gene；
(5) digestion pcDNA3.1 (+) carrier；
(6) connection target fragment and pcDNA3.1 (+)；
(7) pcDNA3.1-FYJNa is named as after recombinant expression carrier conversion, detection and identification；Abrupt information is such as
Under: 905 T sports C；908 T sports C；911 C sports A；914 T sports C.Homo sapiens cAMP
responsive element binding protein 1(CREB1),VERSION NM_134442.4
2.3 PEI infection protocols transfect lung cancer A549/DDP cell
(1) it is grown to when convergence degree is 60% or so when cell and starts to transfect
(2) in 1.5EP pipe, 300 μ L Opti-DMEM is added and are mixed with 12 μ g recombinant plasmids, yet another 1.5EP pipe
In every pipe 300 μ L Opti-DMEM be added mixed with 24 μ L PEI, stationary incubation 8min.
(3) above-mentioned two pipe is mixed well, stationary incubation 15min
(4) culture medium in cell bottle is drawn, is washed once with PBS, the 1640 culture medium cultures without dual anti-and serum are added.
(5) above-mentioned mixed 600 μ L of mixed liquor is added in the blood-free medium in cell bottle, light shake is mixed up and down
It is even.37 DEG C are placed in, after cultivating 4h in 5%CO2 incubator, former culture medium is sopped up, complete medium is changed into and continues to cultivate.
2.4 qRT-PCR detect FYJNa and ABCC1mRNA expression
2.4.1 lung cancer A549/DDP cell total rna extracts
(1) former culture in glassware base is abandoned, is gently washed cell 2 times with 5mL PBS；
(2) 1mL pancreatin is added to change to cellular morphology under the microscope；
(3) 5mL culture medium is added and terminates digestion, slightly blow and beat cell repeatedly, blow down all attached cells；
(4) it moves in centrifuge tube, 1000g is centrifuged 5min, abandons supernatant；
(5) 1mL PBS suspension cell moves in 1.5Ep pipe, and 5000g is centrifuged 2min, abandons supernatant；
(6) 1mL Trizol is added in each Ep pipe, suspension cell is stored at room temperature 10min；
(7) 0.2mL chloroform is added, acutely shakes 30s, is placed at room temperature for 3min, 4 DEG C of centrifugation 10min of 12000r；
(8) upper layer is transferred in clean 1.5Ep pipe, 1/2 times of volume of dehydrated alcohol is added, mixes well；
(9) above-mentioned mixed liquor is transferred in adsorption column, stands 2min, 4 DEG C of centrifugation 3min of 12000r outwell collecting pipe
Middle liquid；
(10) 4 DEG C of centrifugation 30s of 500 μ L RPE Solution, static 2min, 10000r are added, outwell liquid in collecting pipe
Body is repeated once；
(11) adsorption column is put into collecting pipe, 4 DEG C of centrifugation 2min of 10000r；
(12) adsorption column is put into clean 1.5mL centrifuge tube, 30 μ L DEPC-treated is added in adsorbed film center
4 DEG C of centrifugation 2min of ddH2O, static 5min, 12000r.
2.4.2 reverse transcription reaction is anti-by acquired Total RNA using two-step method according to reverse transcription reagent box specification
It is transcribed into cDNA.
(1) inverse transcription reaction liquid is prepared by table 2；
2 reverse transcription reaction system I of table
(2) following reaction is carried out in PCR instrument: 65 DEG C, 5min is subsequently placed in chilling on ice；
(3) 3 inverse transcription reaction liquid of table (20 μ L system) is added in above-mentioned PCR pipe；
3 reverse transcription reaction system II of table
(4) by the following conditions progress reverse transcription reaction in PCR instrument: 42-50 DEG C, 45-60min；70 DEG C, 10min；-20
DEG C save.
2.4.4 qRT-PCR reaction system
(1) gained cDNA is subjected to Real with Power 2 × SYBR Real-Time PCR Premixture kit
Time PCR, reaction system are 20 μ L, and system is shown in Table 4；
4 qRT-PCR reaction system of table
(2) reaction condition are as follows: 95 DEG C of 2min, 40 × (95 DEG C of 15s, 60 DEG C of 40s), 95 DEG C of 15s, 60 DEG C of 1min；Circulation knot
Solubility curve detection is carried out after beam；
5 qRT-PCR upstream and downstream primer of table
(3) data are with 2-ΔΔCtMethod is calculated；
(4) each gene qRT-PCR upstream and downstream primer sequence is shown in Table 5:
2.5 Western blot detect FYJNa and ABCC1 protein expression level
2.5.1 lung cancer A549/DDP cell holoprotein extracts
(1) former culture in glassware base is abandoned, is gently washed cell 2 times with 5mL PBS；
(2) 1mL pancreatin is added to change to cellular morphology under the microscope；
(3) 5mL culture medium is added and terminates digestion, slightly blow and beat cell repeatedly, blow down all attached cells；
(4) it moves in centrifuge tube, 1000g is centrifuged 5min, abandons supernatant；
(5) 1mL PBS suspension cell moves in 1.5Ep pipe, and 5000g is centrifuged 2min, abandons supernatant；
(6) add 300 μ L protein lysates, be placed in 30min on 4 DEG C of shaking tables；
(7) 12000g is centrifuged 20min, takes supernatant, -20 DEG C of preservations.
2.5.2 protein denaturation protein sample and sample-loading buffer are mixed in 4:1 ratio, and boiling water bath heating 5min~
10min, to its near room temperature, -20 DEG C save or continue to test.
2.5.3 SDS- polyacrylamide gel electrophoresis
(1) offset plate is combined；
(2) 6 groups of assignment system separation gels of table are pressed:
Table 6 separates glue formula
(3) separation gel is injected in layer glass plate, after appropriate location is added, 1mL isopropanol is added in sliding pipette tips, to glue
After solidifying, isopropanol is poured out, water is exhausted；
(4) glue is concentrated by 7 groups of assignment systems of table, by concentration glue injection layer glass plate, until liquid level overflows, is inserted into comb.
Glue formula is concentrated in table 7
(5) after being gelled, comb is extracted, is assembled with electrophoresis tank, electrophoresis liquid is added；
(6) 30 μ l samples are added in point sample, every hole, and Maker applied sample amount is 10 μ l；
(7) constant current 100mA, clicks start button, and the bottom end that bromophenol blue reaches glue nearby stops electrophoresis.
2.5.4 transferring film
(1) offset plate is taken out, glue is placed in transferring film liquid and is impregnated；
(2) clip black flour is swept away, and is put well by the sequence of sponge, filter paper, glue, pvdf membrane, filter paper, sponge, grip clamp；
(3) it is put into transferring film slot, it is black to be put well to black, it is put into curling stone, pours into transferring film liquid；
(4) transferring film slot is assembled, constant pressure 100V carries out transferring film.
2.5.5 closing
(1) Ponceaux contaminates film 5min；
(2) clear water gently rinses Ponceaux；
(3) indicate that size cuts film according to maker
(4) 5% skimmed milk power (confining liquid) is added, room temperature shaking table is incubated for 1h.
2.5.6 Western hybridizes
I. primary antibody is incubated for
(1) according to specification, antibody is diluted；
(2) primary antibody is incubated for, and is placed in 4 DEG C of shaking tables and is shaken overnight；
(3) primary antibody is recycled, PBST washes film 10min, three times.
II. secondary antibody is incubated for (being protected from light operation)
(1) according to specification, antibody is diluted；
(2) secondary antibody is incubated for, and is placed in 4 DEG C of shaking table 1h；
(3) secondary antibody is recycled, PBST washes film 10min, three times.
III. machine testing on albumen
Fig. 5 is seen using the bis- infrared laser imaging system detection protein band variations of Odyssey, and analysis result figure is shown in Fig. 6.
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.The technology of the industry
Personnel are it will be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this hairs
Bright principle, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these variations
It all fall within the protetion scope of the claimed invention with improvement, its is equivalent by appended claims for the claimed scope of the invention
Object defines.
Nucleotide and/or amino acid sequence table
<110>Qiqihar University
<120>a kind of over-express vector FYJNa that can significantly increase cell ABCC1 gene expression
<160>9
<210>1
<211>2000
<212>DNA
<213>artificial sequence
<400>1
1 tgtttccgtg cgcggccgct gcgcactcgg cactgggcgg cgctggctgg ctccctggct
61 gcggctcctc agtcggcggc ggctgctgct gcctgtggcc cgggcggctg ggagaagcgg
121 agtgttggtg agtgacgcgg cggaggtgta gtttgacgcg gtgtgttacg tgggggagag
181 aataaaactc cagcgagatc cgggccgtga acgaaagcag tgacggagga gcttgtacca
241 ccggtaacta aatgaccatg gaatctggag ccgagaacca gcagagtgga gatgcagctg
301 taacagaagc tgaaaaccaa caaatgacag ttcaagccca gccacagatt gccacattag
361 cccaggtatc tatgccagca gctcatgcaa catcatctgc tcccaccgta actctagtac
421 agctgcccaa tgggcagaca gttcaagtcc atggagtcat tcaggcggcc cagccatcag
481 ttattcagtc tccacaagtc caaacagttc agtcttcctg taaggactta aaaagacttt
541 tctccggaac acagatttca actattgcag aaagtgaaga ttcacaggag tcagtggata
601 gtgtaactga ttcccaaaag cgaagggaaa ttctttcaag gaggccttcc tacaggaaaa
661 ttttgaatga cttatcttct gatgcaccag gagtgccaag gattgaagaa gagaagtctg
721 aagaggagac ttcagcacct gccatcacca ctgtaacggt gccaactcca atttaccaaa
781 ctagcagtgg acagtatatt gccattaccc agggaggagc aatacagctg gctaacaatg
841 gtaccgatgg ggtacagggc ctgcaaacat taaccatgac caatgcagca gccactcagc
901 cgggcaccac aatcctacag tatgcacaga ccactgatgg acagcagatc ttagtgccca
961 gcaaccaagt tgttgttcaa gctgcctctg gagacgtaca aacataccag attcgcacag
1021 cacccactag cactattgcc cctggagttg ttatggcatc ctccccagca cttcctacac
1081 agcctgctga agaagcagca cgaaagagag aggtccgtct aatgaagaac agggaagcag
1141 ctcgagagtg tcgtagaaag aagaaagaat atgtgaaatg tttagaaaac agagtggcag
1201 tgcttgaaaa tcaaaacaag acattgattg aggagctaaa agcacttaag gacctttact
1261 gccacaaatc agattaattt gggatttaaa ttttcacctg ttaaggtgga aaatggactg
1321 gcttggccac aacctgaaag acaaaataaa cattttattt tctaaacatt tctttttttc
1381 tatgcgcaaa actgcctgaa agcaactaca gaatttcatt catttgtgct tttgcattaa
1441 actgtgaatg ttccaacacc tgcctccact tctcccctca agaaattttc aacgccagga
1501 atcatgaaga gacttctgct tttcaacccc caccctcctc aagaagtaat aatttgttta
1561 cttgtaaatt gatgggagaa atgaggaaaa gaaaatcttt ttaaaaatga tttcaaggtt
1621 tgtgctgagc tccttgattg ccttagggac agaattaccc cagcctcttg agctgaagta
1681 atgtgtgggc cgcatgcata aagtaagtaa ggtgcaatga agaagtgttg attgccaaat
1741 tgacatgttg tcacattctc attgtgaatt atgtaaagtt gttaagagac ataccctcta
1801 aaaaagaact ttagcatggt attgaaggaa ttagaaatga atttggagtg ctttttatgt
1861 atgttgtctt cttcaatact gaaaatttgt ccttggttct taaaagcatt ctgtactaat
1921 acagctcttc catagggcag ttgttgcttc ttaattcagt tctgtatgtg ttcaacattt
1981 ttgaatacat taaaagaagt
<210>2
<211>29
<212>DNA
<213>artificial sequence
<400>2
CCCAAGCTTA TGACCATGGA ATCTGGAGC 29
<210>3
<211>28
<212>DNA
<213>artificial sequence
<400>3
CCGGAATTCT TAATCTGATT TGTGGCAG 28
<210>4
<211>20
<212>DNA
<213>artificial sequence
<400>4
CCAGGGAGGA GCAATACAGC 20
<210>5
<211>20
<212>DNA
<213>artificial sequence
<400>5
TGTCCATCAG TGGTCTGTGC 20
<210>6
<211>21
<212>DNA
<213>artificial sequence
<400>6
GGGGTCCTCA TTATCTTCTG G 21
<210>7
<211>21
<212>DNA
<213>artificial sequence
<400>7
TGGTCTCAGG GTAGGGGTTA G 21
<210>8
<211>20
<212>DNA
<213>artificial sequence
<400>8
AGCGAGCATC CCCCAAAGTT 20
<210>9
<211>21
<212>DNA
<213>artificial sequence
<400>9
GGGCACGAAG GCTCATCATT 20
Claims (1)
1. the over-express vector FYJNa that one kind can significantly increase cell ABCC1 gene expression, it is characterised in that one kind can significantly increase
The nucleotide sequence of the over-express vector FYJNa of strong cell ABCC1 gene expression is as shown in Seq ID No:1.
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