CN110484564A - A kind of pEGFP-N1-CXXC4 eukaryon expression plasmid, construction method and its application - Google Patents
A kind of pEGFP-N1-CXXC4 eukaryon expression plasmid, construction method and its application Download PDFInfo
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Abstract
The present invention relates to a kind of pEGFP-N1-CXXC4 eukaryon expression plasmid, construction method and its applications, comprising: pEGFP-N1-CXXC4 eukaryon expression plasmid, in pEGFP-N1 plasmid, containing CXXC4 nucleotide sequence, sequence is as shown in SEQ ID NO:1；It constructs the plasmid to need to use the CXXC4 amplimer containing ECORI and BamHI restriction enzyme site, primer sequence is respectively as shown in SEQ ID NO:2 and SEQ ID NO:3；The construction method of pEGFP-N1-CXXC4 eukaryon expression plasmid；PEGFP-N1-CXXC4 eukaryon expression plasmid is preparing the application in GBM targeted therapy biological agent.The beneficial effects of the present invention are: successfully constructing the recombinant gene expression plasmid pEGFP-N1-CXXC4 with CXXC4 gene.Using the CXXC4 amplimer for having ECORI and BamHI restriction enzyme site, the pEGFP-N1-CXXC4 recombinant expression plasmid with CXXC4 gene can efficiently, be easily constructed；It based on the targeted therapy biological agent of pEGFP-N1-CXXC4 eukaryon expression plasmid preparation, can be used for inhibiting GBM cell Proliferation, invasion, migration and one-tenth knurl ability, there is huge clinical value in preparing related targeted drug.
Description
Technical field
The invention belongs to technical field of biological genetic engineering, and in particular to a kind of pEGFP-N1-CXXC4 eukaryotic expression matter
Grain, construction method and its application.
Background technique
Glioblastoma multiforme (GBM) is that the primary malignancy that central nervous system (CNS) is most common, most fatal is swollen
Tumor.The prognosis for suffering from crowd is poor, and median survival interval is 12-16 months, and survival rate is only 5.5% or so within 5 years.
In recent years the study found that the abnormal change of GBM genome be cause its occurrence and development complexity and its draw
Rise various malignant activities root, further investigation research anomalous variation gene, and using corresponding molecular biology method into
Row is intervened, and is very necessary for conditions of patients control, the treatment of disease, the final prognosis of patient.
The mankind CXXC4 assignment of genes gene mapping encodes the albumen in the zinc finger of type containing a CXXC domain in 4q24, which makees in drosophila
It plays a role for typical case without the wing/integrated signal access antagonist.Coding albumen passes through big with postsynaptic density albumen/drosophila disk
Tumor suppressor/zonuls occludens interaction negativity is adjusted without the wing/integrated signal conduction, this is a kind of stable auxiliary activation of transcription
Scaffolding protein needed for object beta-catenin.In addition, the CXXC structural domain of the albumen has been found bis- core of CpG with non-methylation
Thuja acid combines, and navigates to promoter and the island CpG, and the catalyst structure domain phase with 2 type methylcystein 10-11 transposition dioxygenases
Interaction, the latter are a kind of iron of modifying DNA methylation state and the dioxygenase that alpha Ketoglutarate relies on.
Researcher's discovery, in the mankind, low expression exception and pernicious clear-cell carcinoma, intestinal cancer, the mammary gland of this gene of CXXC4
Cancer is related with the generation of prostate cancer；We also have found that CXXC4 high expresses the middle position of GBM patient in the preliminary stage of this research
Life cycle will be considerably longer than low expression patient.It is known that lasting proliferation signal, cell death are obstructed, infiltrate and shift
The characteristic of these tumour cells is an important factor for leading to tumour cell malignant activity, is to be present in malignant tumour generally
Phenomenon.Early-stage study shows that CXXC4 gene can be such that tumour cell occurs in terms of the abilities such as proliferation, invasion, migration and tumor formation
Change.CXXC4 gene expression can be such that cell proliferation rate reduces, increasing apoptosis is more, invasion migration reduces, one-tenth knurl ability weakens
Phenomena such as can be reversed.
Therefore, CXXC4 gene is overexpressed in tumour cell, the effect that tumour is precisely treated can be played, to be
Control growth of tumour cell provides new means.Currently, there is not yet building pEGFP-N1-CXXC4 gene eucaryon expression plasmid and
CXXC4 is overexpressed the report applied in GBM oncotherapy.
Summary of the invention
The purpose of the present invention is to overcome the above shortcomings and to provide a kind of buildings of pEGFP-N1-CXXC4 eukaryon expression plasmid
Method and its application.
PEGFP-N1-CXXC4 eukaryon expression plasmid contains CXXC4 nucleotide sequence, sequence in pEGFP-N1 plasmid
As shown in SEQ ID NO:1.
The plasmid is constructed to need to use the CXXC4 amplimer containing ECORI and BamHI restriction enzyme site, primer sequence point
Not as shown in SEQ ID NO:2 and SEQ ID NO:3.
The construction method of pEGFP-N1-CXXC4 eukaryon expression plasmid, comprising: following steps:
(1), the PCR amplification of CXXC4 gene C DS sequence, recycling,
(2), on the ECORI/BamHI restriction enzyme site and pEGFP-N1 plasmid on CXXC4 gene C DS sequence amplification product
ECORI/BamHI restriction enzyme site carry out double digestion in 20 μ l reaction systems；
(3), by after double digestion CXXC4 gene C DS sequence amplification product and pEGFP-N1 plasmid in 20 μ l reaction systems
In be attached.
Double enzyme digestion reaction system is 20 μ l systems；By the ECORI enzyme of 1 μ l, the BamHI enzyme of 1 μ l, 2 μ l CutSmart
The distilled water composition of buffer, the PCR product of 2 μ l or pEGFP-N1 plasmid, 14 μ l.
The reaction that CXXC4 gene C DS sequence amplification product after double digestion is connect with the pEGFP-N1 plasmid after double digestion
System is 20 μ l systems；By the CXXC4 gene C DS sequence amplification product of 2 μ l, T4 DNA Ligase of 2 μ l, 2 μ l
The distilled water composition of (100ng) pEGFP-N1 plasmid, 10 × buffer of 2 μ l, 12 μ l.
PEGFP-N1-CXXC4 eukaryon expression plasmid is preparing the application in GBM targeted therapy biological agent.
The beneficial effects of the present invention are: successfully constructing the recombinant gene expression plasmid pEGFP-N1- with CXXC4 gene
CXXC4.Using the CXXC4 amplimer for having ECORI and BamHI restriction enzyme site, can efficiently, easily construct and have
The pEGFP-N1-CXXC4 recombinant expression plasmid of CXXC4 gene.Target based on the preparation of pEGFP-N1-CXXC4 eukaryon expression plasmid
It to treatment biological agent, can be used for inhibiting GBM cell Proliferation, invasion, migration and one-tenth knurl ability, preparing related targeted drug
In have huge clinical value.
Detailed description of the invention
Fig. 1 pEGFP-N1 plasmid schematic diagram.
Fig. 2 WesternBlot verifying CXXC4 gene is transferred to the expression of albumen after cell.
Fig. 3 CXXC4 is overexpressed the influence to A172, U251 cell line proliferation ability.
Fig. 4 CXXC4 is overexpressed the influence to A172, U251 cell line transfer ability.
Fig. 5 CXXC4 is overexpressed the influence to A172, U251 cell line invasive ability.
Fig. 6 CXXC4 is overexpressed the influence to A172, U251 cell line one-tenth knurl ability.
Specific embodiment
The present invention is described further below with reference to embodiment.The explanation of following embodiments is merely used to help understand this
Invention.It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, also
Can be with several improvements and modifications are made to the present invention, these improvement and modification also fall into the protection scope of the claims in the present invention
It is interior.The reagent can be obtained from open market.
Embodiment 1
CXXC4 nucleotide sequence in pEGFP-N1-CXXC4 has sequence shown in SEQ ID NO:1.
Embodiment 2
The pEGFP-N1-CXXC4 eukaryon expression plasmid for constructing embodiment 1 is needed using containing ECORI and BamHI digestion
The CXXC4 amplimer in site, the sequence of primer primer F and primer R are respectively such as SEQ ID NO:2 and SEQ ID NO:
Shown in 3, the building of plasmid the following steps are included:
1) PCR amplification of CXXC4 gene:
1. PCR system (TOYOBO KOD-Plus-Neo kit):
2. PCR program:
2) gel electrophoresis and PCR product (OMEGA Gel Extraction Kit) is recycled:
1. loading and electrophoretic parameters:
Marker and sample, 120V electrophoresis 24min are added in loading wells；
2. cutting glue:
After electrophoretic band separates, purpose band is carefully cut using a clean, sharp knife blade, is removed as far as possible
Glue without target fragment keeps adhesive tape small as far as possible；
3. colloidal sol:
Adhesive tape is put into the microcentrifugal tube of clean 1.5ml and is weighed, to determine that the volume of adhesive tape (sets the close of glue
Degree is 1g/ml), isometric combination buffer (Binding Buffer, XP2) is added, mixture is incubated at 50 DEG C~55 DEG C
It educates 7 minutes or until gel dissolves completely, during which need to shake primary or vortex centrifugal pipe every 2~3min and accelerate dissolution；
4. adjusting pH value:
The color for observing mixed liquor, if the color of mixed liquor becomes orange or red (pH becomes larger), by the way that 5 μ l are added
The sodium acetate (pH 5.2) of 5M adjusts, and the color of mixed liquor should become light yellow；
5. collecting DNA:
By centrifugal column/DNA Mini Column in the 2ml collecting pipe of standard configuration, 700 μ l mixed liquors are added to centrifugation
In column, room temperature 10,000 × g is centrifuged 1min, discards centrifugate, by centrifugal column (if solution in used collecting pipe just now
Volume is greater than 700 μ l, then successively assembles centrifugal column, centrifugation with the amount of 700 μ l every time)；
6. in conjunction with:
It is added in 300 μ l combination buffers/Binding Buffer (XP2) to centrifugal column, room temperature 10,000 × g centrifugation
1min is to rinse centrifugal column；
7. cleaning:
700 μ l, which are added, has used the diluted SPW cleaning solution of dehydrated alcohol/SPW Wash Buffer to rinse centrifugal column, room temperature
10,000 × g is centrifuged 1min, and (SPW cleaning concentrate must be diluted before using with dehydrated alcohol to be added by every 25ml cleaning concentrate
The dilution proportion of 100ml dehydrated alcohol), this step is repeated once sufficiently to clean；
8. drying:
Centrifugate is discarded, empty centrifugal column is centrifuged 2min, drying centrifugal column (removal centrifugal column at full speed (>=13,000 × g)
Interior ethyl alcohol)；
9. elution:
Centrifugal column is placed in a clean 1.5ml microcentrifugal tube.By 15~30 μ l eluents/Elution
Buffer (heating effect is more preferable in 65-70 DEG C of water-bath in advance for eluent) is added directly into centrifugal column matrix, incubation at room temperature
1min, at full speed (>=13,000 × g) centrifugation 1min eluted dna (obtained solution can be rejoined in centrifugal column, again from
The heart increases DNA elution amount)；
10. measuring concentration:
After appropriate diluted sample, absorbance (DNA concentration=A260 × 50 × dilution times in 260mn and 280nm is measured
Number μ g/ml).
3) assembling of pEGFP-N1-CXXC4 eukaryon expression plasmid
1. the selection of restriction enzyme site:
The restriction enzyme site on restriction enzyme site and carrier, both selections shared ECORI and BamHI can be used by compareing target gene
Alternately, according to the restriction enzyme site of selection, corresponding restriction enzyme site protection base is searched；
2. PCR product and plasmid use double digestion (ECORI/BamHI/CutSmart buffer) simultaneously；
37 DEG C, after digestion 2h, 65 DEG C of metal bath 10min terminate reaction, run glue recycling.
3. connecting carrier:
Insertweight/volume:Vectorweight/volume=1:3~3:1,16 DEG C of connection 4-8h.
Embodiment 3
WesternBlot verifying CXXC4 gene is transferred to the expression of CXXC4 albumen after cell, comprising the following steps:
1) preparation of cell:
It is collected in 10cm culture dish respectively and has been transferred to blank/take CXXC4 Gene Lentiviral Vector in exponential phase of growth
U-251, A-172 cell, abandon culture medium, PBS wash 2 times after discard supernatant；
2) extraction of albumen
The RIPA lysate of 400 μ l is added to extract total protein in every ware, and after cell sufficiently cracks, lysate is moved to
In 1.5ml centrifuge tube, 12000rpm is centrifuged 5min at 4 DEG C, and supernatant is taken to be transferred to -20 DEG C of 0.5ml centrifuge tube preservations.
3) sample protein concentration is measured
Protein standard substance is made, according to protein standard substance through surveying absorbance at microplate reader measurement 562nm, draws sample standard
The concentration of sample is calculated in curve, the regression equation obtained according to standard curve.
4) WesternBlot verifies protein expression level
Albumen after gel electrophoresis is gone into pvdf membrane, is incubated for through two anti-binding of primary antibody, with horseradish peroxidase HRP-
ECL luminescence method is exposed in darkroom, analyzes, as a result such as Fig. 2.PEGFP-N1-CXXC4 group compared with pEGFP-N1-NC group,
CXXC4 protein expression is significantly raised, wherein *: p < 0.05 *: p < 0.05, *.
Embodiment 4
The targeted therapy biological agent of pEGFP-N1-CXXC4 eukaryon expression plasmid preparation based on embodiment 2, for pressing down
GBM cell Proliferation, invasion, migration and one-tenth knurl ability processed.
1. detection CXXC4 gene is transferred to GBM cell line proliferation capacity variation after cell:
1) A172, U251 cell for taking P2 to be overexpressed for A172, U251 cell and CXXC4, adjustment cell density is 4 ×
105/ ml, it is spare；
2) for A172 cell, 4 groups are set altogether, and 1 group is zeroing hole, remaining 3 groups are respectively as follows: common A172 groups of cells, transfection
The A172 groups of cells of empty plasmid, the A172 groups of cells for being transferred to CXXC4 gene；For U251 cell, 4 groups are set altogether, and 1 group is tune
Zero hole, remaining 3 groups are respectively as follows: common U251 groups of cells, the U251 groups of cells for having transfected empty plasmid, are transferred to CXXC4 gene
U251 cell is added in 96 well culture plates, 100 μ l suspensions of every hole addition, every group of 3 holes,；
3) respectively at 0h, for 24 hours, 48h, 72h and 96h when, first washed twice with without the DMED culture solution of FBS, then existed
100 μ l fresh cultures are added in each group culture plate, 10 μ l CCK-8 solution are then added, continue to be incubated in cell incubator
1h；
4) with spectrophotometric determination 450nm absorbance: making Detection wavelength with 450-490nm, 600-650nm makees reference wave
It is long；
5) cell growth curve is drawn according to the OD value of measurement, as a result such as Fig. 3.PEGFP-N1-CXXC4 group and pEGFP-
N1-NC group compares, and A172, U251 ability of cell proliferation of CXXC4 overexpression group are substantially reduced, wherein *: p < 0.05.
2. the variation that detection is transferred to the GBM cell migration ability of CXXC4 gene
Cell migration ability test is carried out using the Trans-well model that poly- carbon ester membrane micropore diameter is 8 μm.
1) A172, U251 cell and corresponding blank control cell that logarithmic growth phase is transferred to CXXC4 gene, no blood are collected
Clear culture medium prepares single cell suspension, adjusts cell concentration, is 1 × 106 cell/ml；
2) upper chamber is separately added into 100 μ l of plasma-free DMEM medium group of cells suspension, and lower room is the DMEM containing 30%FBS
600 μ l of culture medium co-cultures 48h；
3) insertion cell is carefully removed, the fixed 10min of 95% ethyl alcohol of pre-cooling is first sucked upper indoor liquid with cotton swab, then
Gently wipe the cell of Matrix matrigel and poly- carbon ester film upper surface.It is careful to take out upper chamber, and mark, with the first of pre-cooling
Alcohol fixes 30min.Routine hematoxylin dyes 1min.Graded ethanol is dehydrated (80%, 95%, 100%), and dimethylbenzene is transparent.Carefully
Poly- carbon ester film is cut from upper chamber substrate, sets resinene mounting on glass slide.(× 100) are random under high magnification microscope
It takes 6 visuals field to count, is averaged, experiment is repeated twice.As a result such as Fig. 4.PEGFP-N1-CXXC4 group and pEGFP-N1-NC group
Compare, A172, U251 the cell migration ability for being transferred to CXXC4 gene are substantially reduced, wherein *: p < 0.01 of *.
3. the variation that detection is transferred to the GBM cell invasion ability of CXXC4 gene:
1) EC Matrix matrigel, the Trans- for the insertion cell that poly- carbon ester membrane micropore diameter is 8 μm are covered with using preparatory
Well model carries out cell invasion ability test, the same Cell migration assay of specific steps.As a result such as Fig. 5.pEGFP-N1-CXXC4
Group is compared with pEGFP-N1-NC group, and A172, U251 the cell invasion ability for being transferred to CXXC4 genome are substantially reduced, wherein * *: p
< 0.01.
4. detection is transferred to CXXC4 gene pairs GBM tumour cell in the influence of nude mice by subcutaneous one-tenth knurl ability
1) animal packet:
Mouse is grouped according to random table, totally 4 groups: being transferred to the A172 cell transplantation tumor group of CXXC4 gene, be transferred to
The U251 cell transplantation tumor group of CXXC4 gene transfects the A172 cell transplantation tumor group of empty plasmid, transfects the U251 of empty plasmid
Cell transplantation tumor group；
2) tumor cell inoculation:
A172, U251 cell for being transferred to CXXC4 gene and its corresponding blank control cell of logarithmic growth phase are collected, is adjusted
Being made into concentration is 1 × 107Cells/ml is planted subcutaneous in nude mice right hind.
3) tumour tumor formation is observed:
The animations such as daily observation nude mice diet, activity, calculate transplantable tumor tumor formation rate, every 2d is by same observation after tumor formation
The major diameter (a) and minor axis (b) of person's vernier caliper measurement tumour, take the line of apsides average value of each group, count according to formula Vab2/2
Gross tumor volume is calculated, tumor volume change curve is drawn.After 5w, animal model euthanasia is given, and tumour is taken to claim weight in wet base.As a result such as
Fig. 6.PEGFP-N1-CXXC4 group is transferred to A172, U251 cell one-tenth knurl ability of CXXC4 gene compared with pEGFP-N1-NC group
It is substantially reduced, wherein *: p < 0.05.
Sequence table
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Claims (6)
1. a kind of pEGFP-N1-CXXC4 eukaryon expression plasmid, it is characterised in that: in pEGFP-N1 plasmid, contain CXXC4 core
Nucleotide sequence, sequence is as shown in SEQ ID NO:1.
2. pEGFP-N1-CXXC4 eukaryon expression plasmid according to claim 1, it is characterised in that: construct the plasmid needs
Using the CXXC4 amplimer containing ECORI and BamHI restriction enzyme site, primer sequence is respectively such as SEQ ID NO:2 and SEQ ID
Shown in NO:3.
3. a kind of construction method of pEGFP-N1-CXXC4 eukaryon expression plasmid as described in claim 1, it is characterised in that: packet
Include following steps:
1), the PCR amplification of CXXC4 gene C DS sequence, recycling；
2), on the ECORI/BamHI restriction enzyme site and pEGFP-N1 plasmid on CXXC4 gene C DS sequence amplification product
ECORI/BamHI restriction enzyme site carries out double digestion；
3), by after double digestion CXXC4 gene C DS sequence amplification product and pEGFP-N1 plasmid be attached.
4. construction method according to claim 3, it is characterised in that: the step 2 carries out double in 20 μ l reaction systems
Digestion；20 μ l reaction systems are produced by the PCR of the ECORI enzyme of 1 μ l, the BamHI enzyme of 1 μ l, CutSmart buffer of 2 μ l, 2 μ l
The distilled water of object or pEGFP-N1 plasmid and 14 μ l composition.
5. construction method according to claim 3, it is characterised in that: the step 3 is connected in 20 μ l reaction systems
It connects；20 μ l systems are by the CXXC4 gene C DS sequence amplification product of 2 μ l, (100ng) of T4DNA Ligase of 2 μ l, 2 μ l
The distilled water of pEGFP-N1 plasmid, 10 × buffer of 2 μ l and 12 μ l forms.
6. pEGFP-N1-CXXC4 eukaryon expression plasmid as described in claim 1 is preparing answering in targeted therapy biological agent
With.
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