CN106167819A - A kind of primer for detecting humanized's BAX gene expression dose combines and application - Google Patents
A kind of primer for detecting humanized's BAX gene expression dose combines and application Download PDFInfo
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- CN106167819A CN106167819A CN201610254111.4A CN201610254111A CN106167819A CN 106167819 A CN106167819 A CN 106167819A CN 201610254111 A CN201610254111 A CN 201610254111A CN 106167819 A CN106167819 A CN 106167819A
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Abstract
The invention belongs to gene technology field, the present invention relates to a kind of primer combination for detecting bak gene expression.The present invention, by designing specific primer and the specific primer of ribosomal protein RPL19 gene of humanized's bak gene, uses it for the detection of bak gene expression.The primer of present invention design, without non-specific amplification, the RPL19 gene not affected by er stress using expression, as house-keeping gene, can carry out accurate relative quantification to the expression of bak gene.The detecting system of the present invention is simply efficient, easy to use, and detection only needs 1.2h to complete.
Description
Technical field
The invention belongs to gene technology field, the present invention relates to a kind of for detecting drawing of humanized's BAX gene expression dose
Thing combines.
Background technology
BAX gene belongs to Bcl-2 gene family, is to regulate apoptotic important gene, and BAX gene is to be widely studied
Apoptosis-related genes, the BAX albumen of BAX gene code can antagonism Bcl-2 suppression apoptosis effect.BAX albumen is widely distributed
In human body many tissues and cell, especially at liver, renal tubules, islets of langerhans, gastric gland body, cardiac muscle, germinal center of lymph node and nerve
The tissue expressions such as unit are higher.Under the conditions of multiple physiological and pathological, the change such as anoxia, redox state all may interfere with including albumen
Piling up in matter net, produce er stress, the most too strong er stress can inducing cell apoptosis.Permitted cellulous apoptosis
During with the rising of BAX expression.BAX promotes apoptotic possible mechanism mainly: (1) mitochondria permeability turns
Change, the complex functionality of oxidative phosphorylation and ATP is destroyed；(2) cell Redox effect changes；(3) in mitochondrion
Apoptosis correlation molecule apoptosis enzyme activition factors A Paf-1 discharge, with cytochrome C interact, activate
Caspase mitochondrial apoptosis path.Homodimer can be formed after BAX overexpression, promote the release of cytochrome C, activate
Classical mitochondrial apoptosis path.Research discovery is induced in NO donor sodium nitroprusside (Sodium Nitroprusside, SNP)
During articular chondrocyte apoptosis, BAX protein expression significantly increases, and it can accelerate the process of articular chondrocyte apoptosis, and uses RNA to do
After the technology of disturbing makes BAX gene silencing, BAX mRNA and protein expression level are all decreased obviously, the work of SNP Induction of chondrocytes apoptosis
With being substantially suppressed.Equally, the research to cerebral ischemia/reperfusion injury of rats finds, BAX is table when cerebral ischemia reperfusion injury
Reach increase, form homodimer with Bcl-2 and promote apoptosis.
The change of BAX protein conformation or high expressed can the apoptosis of inducing mitochondrial mediation, and low expression or express lacks
Lose and occur with tumor or be in progress closely related.The research of carcinoma of endometrium is found, along with Surgical staging and histological grade
Increase, the intensification of muscle layer invasion and attack and the transfer of lymph node, BAX gene mRNA process LAN positive rate is gradually increased, and prompting is along with son
The increase of endometrial carcinoma grade malignancy and the reduction of differentiation degree, while undesired cell proliferation is more active, cell is spontaneous
Apoptosis also increases, and tumor also has higher invasive ability.Therefore the expression of BAX gene mRNA can be as judging height
One of index of danger factor and malignant behaviors, can assist the clinical comprehensive therapeutic plan working out personalization and follow up a case by regular visits to.
The research of BAX expression is entered from protein level except the method such as SABC and Western immunoblotting of employing
Outside row research, it is possible to from mRNA level in-site research, including sxemiquantitative reverse transcriptional PCR and fluorescence quantifying PCR method etc..With albumen for inspection
Survey mesh calibration method and generally operate comparatively laborious, time-consumingly.Sxemiquantitative reverse transcriptional PCR needs electrophoresis detection, and accuracy is not high enough.Glimmering
Fluorescent Quantitative PCR method is a kind of high sensitivity, simple and efficient detection method, simple to operate, can apply SYBR green dyestuff
Method detects, it is not necessary to use fluorescent probe, uses quantitative fluorescent PCR relative quantitation method by pipe metastable with expression
Family's genetic comparison reflects the expression of genes of interest, can well assist the research of BAX expression, and whole PCR reacts
Process only needs to complete for 1.2 hours.House-keeping gene need to select the gene maintaining cell bottom line function indispensable, in institute
There is in cell the genoid being intended to express, and expression is less by such environmental effects.Cell when stress some
The gene of normal expression can be suppressed, and there are some researches show that the more commonly used actin house-keeping gene is affected by er stress, table
The amount of reaching can reduce, and therefore is not suitable for being used as reference gene during er stress.Ribosomal protein gene product is to maintain cell
Necessary to vital movement, being expressed in all cells, it is expressed only by initiating sequence or promoter and RNA polymerase phase interaction
Impact, and do not regulated by other mechanism, can be used as reference gene during er stress.
Summary of the invention
The present invention is by designing specific primer and the specificity of ribosomal protein RPL19 gene of humanized's BAX gene
Primer, uses it for the detection of BAX gene expression dose.The operation principle of the present invention is application quantitative fluorescent PCR SYBR
The method of green fluorescent dye according to relative quantification principle, i.e. 2-△△CTMethod determines gene expression dose.
The present invention comprises two pairs of primers, and pair of primers is the specific primer for humanized's BAX gene；Another pair of primers
It it is the specific primer of the ribosomal protein RPL19 gene design for people.
MRNA sequence (serial number: NM_138761.3) design BAX primer sequence according to the human BAX that NCBI announces
It is shown in Table 1:
Table 1.BAX design of primers
| Primer | Sequence |
| F-bax | CGGGTTGTCGCCCTTTTCTA |
| R-bax | GTCCAATGTCCAGCCCATGA |
MRNA sequence (serial number: NM_000981.3) the design primer sequence of the human RPL19 announced according to NCBI is shown in Table 2:
Table 2.RPL19 design of primers
| Primer | Sequence |
| F-rpl | CGAGCGAGCTCTTTCCTTT |
| R-rpl | AGACCTTCTTCTTGCCACAGC |
Two pairs of primers of present invention design can distinguish the mrna expression level of specific detection BAX and RPL19 gene, without non-specific
Property amplification, selected house-keeping gene expression is not affected by er stress, the expression of BAX gene can be carried out standard
The amount of determination.The detecting system of the present invention uses SYBR green fluorescent dye method, it is not necessary to using probe, reagent is easy to use,
Only need to add coherent detection template, it is not necessary to loaded down with trivial details operating procedure, whole detection process only needs 1.2h to complete.
Accompanying drawing explanation
Fig. 1 is BAX and the RPL19 gene amplification product melt curve analysis in the embodiment of the present invention one, and wherein A is BAX gene
Amplified production melt curve analysis, B is RPL19 gene amplification product melt curve analysis.
Fig. 2 is the Manf albumen impact on PD cell model BAX gene expression dose in the embodiment of the present invention two.
Detailed description of the invention
Below in conjunction with the accompanying drawings and embodiment, the detailed description of the invention of the present invention is described in further detail.Hereinafter implement
Example is used for illustrating the present invention, but is not limited to the scope of the present invention.
The Acetaq SYBR supermix using promise only to praise in the embodiment of the present invention, uses 25 microlitre reaction systems.Glimmering
Fluorescent Quantitative PCR instrument uses ABI7500.
When utilizing specific primer sets and method detection humanized's BAX gene expression dose of the present invention, basic operation
For:
Step 1. cell RNA extracts
Using commercial kit to extract cell RNA, ultraviolet spectrophotometer measures RNA concentration, and controls to extract quality
OD260/280～2.0.The embodiment of the present invention uses sky root total RNA extraction reagent box to extract cell RNA, and concrete operations are illustratively
Book is carried out；
The reverse transcription of step 2.cDNA
Taking about 500ng RNA uses commercial kit to carry out cDNA reverse transcription.The present embodiment uses the commercialization of Takara
Test kit PrimeScriptTMRT Master Mix(Perfect Real Time) carry out reverse transcription, concrete operations are illustratively
Book is carried out；
Step 3. quantitative fluorescent PCR reacts
In experiment, every kind of template is separately added in two kinds of quantitative fluorescent PCR reaction systems and carries out the inspection of BAX and RPL19 gene respectively
Surveying, it is as follows that a kind of reaction system comprises component:
1×Acetaq SYBR supermix
Each 0.1～0.5 μM of forward primer (F-bax)
Each 0.1～0.5 μM of downstream primer (R-bax)
Template (cDNA of 1:5 dilution) 5 μ l
Cumulative volume 25 μ l
It is as follows that another kind of reaction system comprises component:
1×Acetaq SYBR supermix
Each 0.1～0.5 μM of forward primer (F-rpl)
Each 0.1～0.5 μM of downstream primer (R-rpl)
Template (cDNA of 1:5 dilution) 5 μ l
Cumulative volume 25 μ l
Quantitative fluorescent PCR reaction condition: 95 DEG C of 5min, then reruns 40 and circulates (concrete 95 DEG C of 10s, 60 DEG C of 34s),
Fluorescence signal is detected during annealing.Melt curve analysis analysis, the specificity of detection primer amplification can be carried out after end of run；
Step 4. Analysis of test results
Logarithmic (log) phase threshold value according to the automatic baseline of system and setting obtains the CT value of each reaction, calculates 2-△△CTValue, i.e. experimental group
The multiple that BAX mrna expression compares with matched group, △ △ CT=(CTbax(experimental group)-CTrpl19(experimental group))-
(CTbax(matched group)-CTrpl19(to group)).
The detection of embodiment one slow virus infected cell strain BAX gene expression dose
Slow virus infection SH-SY5Y cell by containing GRP78 gene interference sequence: the SH-SY5Y cell height containing 10%FBS
Sugar DMEM culture medium, is placed in 37 DEG C, cultivates in 5%CO2 incubator, and the SH-SY5Y cell of trophophase of taking the logarithm carries out digesting, counting
Number, is seeded to 96 porocyte culture plates, 5000 cells/well, uses next day the DMEM culture medium dilution RNAi without FBS the most sick
Poison concentrated solution, every hole adds the 10 μ l slow virus concentrated solution containing RNA interference sequence and the 90 μ l DMEM culture medium without FBS,
37 DEG C, in 5%CO2 incubator, cultivate 24h, change the fresh DMEM in high glucose culture medium containing 10% FBS and continue to cultivate 48h, collect
Cell extraction RNA, uses quantitative fluorescent PCR detection BAX expression after reverse transcription.Testing result is shown in Table 3:
Table 3. slow virus infected cell and the comparison of compared with control cells BAX expression
Table 3 result shows that slow virus infected cell BAX gene expression dose is 1.46 times of cellular control unit, and explanation is carried
Apoptosis can be promoted after the slow virus infected cell of GRP78 interference sequence.BAX and RPL19 gene amplification product melt curve analysis
It is respectively and the most single melts peak (see figure 1), primer amplification specificity be described preferably, without non-specific amplification band.
Embodiment two is by the protective effect of detection BAX gene expression dose research Manf protein on cells
Parkinson disease (Parkinson Disease, PD) are outside a kind of vertebral body to be Progressive symmetric erythrokeratodermia neurodegenerative disease, study table
Bright oxidativestress damage and the reaction of alpha-synapse nucleoprotein nerve immunity are to cause Parkinsonian main cause, and can produce stress
Reaction.Have proven to MANF albumen and dopaminergic neuron is had protective effect.By using 6-hydroxy dopamine (6-OHDA)
The parkinson disease cell model making SH-SY5Y cell injury can detect the Protective effects of Manf albumen.Experiment is divided into
Matched group (DMEM) group, 6-OHDA(50 μm ol/L) group and Manf+ 6-OHDA(50 μm ol/L) (4 g/ml) group.Give in advance
Add after giving Manf albumen 4h after 6-OHDA acts on 1h, 7h and 18h respectively and collect cell, extract RNA, after reverse transcription, use fluorescence
Quantitative PCR measures BAX gene expression dose.By comparing with matched group, calculate the relative expression levels of each group of BAX gene
(see figure 2), result shows that cell BAX gene expression dose does not raise at 1h and 18h, very in the case of Manf albumen exists
To slightly reducing, and 6-OHDA group has raised 7 hours and 18h BAX gene expression dose relatively matched group, and Manf egg is described
Protective effect to the SH-SY5Y cell injury that 6-OHDA induces in vain, may be realized by the expression of suppression BAX gene.
By the above embodiments it is recognised that the specific primer sets of the present invention and corresponding method, can be special
Property detection humanized's BAX gene expression, it is to avoid the interference of homologous genes, and not affected by er stress
RPL19, as internal reference, improves the detection accuracy of humanized's BAX gene expression dose, and detection process is simple, efficiently.
Being described in detail the specific embodiment of the present invention above, but it is intended only as example, the present invention does not limit
It is formed on particular embodiments described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and
Substitute the most all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and
Amendment, all should contain within the scope of the invention.
SEQUENCE LISTING
<110>Tongji University Suzhou academy
<120>a kind of primer for detecting humanized's BAX gene expression dose combines and application
<130> 2016
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>synthetic
<220>
<221> misc_feature
<223>pcr amplification primer thing
<400> 1
cgggttgtcg cccttttcta 20
<210> 2
<211> 20
<212> DNA
<213>synthetic
<220>
<221> misc_feature
<223>pcr amplification primer thing
<400> 2
gtccaatgtc cagcccatga 20
<210> 3
<211> 19
<212> DNA
<213>synthetic
<220>
<221> misc_feature
<223>pcr amplification primer thing
<400> 3
cgagcgagct ctttccttt 19
<210> 4
<211> 21
<212> DNA
<213>synthetic
<220>
<221> misc_feature
<223>pcr amplification primer thing
<400> 4
agaccttctt cttgccacag c 21
Claims (5)
1., for detecting a specific primer sets for humanized's BAX gene expression dose, this combination comprises following primer sequence
Row:
The primer combination of BAX gene test:
Forward primer F-bax:CGGGTTGTCGCCCTTTTCTA
Downstream primer R-bax:GTCCAATGTCCAGCCCATGA
The primer combination of RPL19 reference gene detection:
Forward primer F-rpl:CGAGCGAGCTCTTTCCTTT
Downstream primer R-rpl:AGACCTTCTTCTTGCCACAGC.
2. the method detecting humanized's BAX gene expression dose, it is characterised in that use as claimed in claim 1 special
Specific primer combines and carries out.
3. the test kit being used for detecting humanized's BAX gene expression dose, it is characterised in that comprise just like claim 1
Described specific primer sets.
Test kit the most according to claim 3, it is characterised in that also include archaeal dna polymerase, dNTP and buffer.
Test kit the most according to claim 4, it is characterised in that described polymerase is thermostable DNA polymerase.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102251050A (en) * | 2011-08-10 | 2011-11-23 | 青岛大学医学院附属医院 | Reagent for detecting cell apoptosis signal path and PCR method using same |
-
2016
- 2016-04-23 CN CN201610254111.4A patent/CN106167819A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102251050A (en) * | 2011-08-10 | 2011-11-23 | 青岛大学医学院附属医院 | Reagent for detecting cell apoptosis signal path and PCR method using same |
Non-Patent Citations (3)
| Title |
|---|
| HAFERKAMP B.等: "Homo sapiens Baxdelta2G9 (BAX) mRNA,complete cds, alternatively spliced", 《GENBANK DATABASE》 * |
| NOBUHIKO HIRAMATSU等: "Monitoring and Manipulating Mammalian Unfolded Protein Response,183-198", 《METHODS ENZYMOL》 * |
| STRAUSBERG R.L.等: "Homo sapiens ribosomal protein L19, mRNA (cDNA clone MGC:111176 IMAGE:30721753), complete cds", 《GENBANK DATABASE》 * |
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Application publication date: 20161130 |