CN107488750B - Long-chain non-coding RNA lnc-ALVE1-AS1 detection of expression primer and kit - Google Patents
Long-chain non-coding RNA lnc-ALVE1-AS1 detection of expression primer and kit Download PDFInfo
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Abstract
The invention belongs to molecular biology fields, and in particular to a kind of long-chain non-coding RNA lnc-ALVE1-AS1 detection of expression primer and kit.The primer includes chain specific RT primer and fluorescence quantitative PCR detection primer；Wherein the sequence of chain specific RT primer is as shown in SEQ ID NO.8 and SEQ ID NO.9；The sequence of fluorescence quantitative PCR detection primer is as shown in SEQ ID NO.10 and SEQ ID NO.11.This research invention additionally provides lnc-ALVE1-AS1 detection of expression kit, provides method and basis for the analysis of lnc-ALVE1-AS1 expression rule, can be used for detecting the avian leukosis innate immunity state of chicken group in production practices.
Description
Technical field
The invention belongs to molecular biology fields, and in particular to a kind of long-chain non-coding RNA lnc-ALVE1-AS1 expression inspection
Survey primer and kit.
Background technique
Endogenous retrovirus (endogenous retroviruses) is genome ingredient, is considered derived from evolution
The retrovirus of middle infection biological body.As time immemorial retroviral infection in the intracorporal residual of host, pass by addition to it
Function not enough understand, be once regarded as junk DNA (Junk DNA).Some researches show that certain endogenous sex reversals as of late
Record virus not only plays a significant role the versatility of early embryonic development and embryonic stem cell, it is also possible to the cell innate immunity
It is related.
Current research discovery, endogenous retrovirus transcript is long-chain non-coding RNA (long non-coding
RNA, lncRNA) important sources, have activation interferon response reaction potential.In fact, lncRNA is in cell biology
Effect in regulation is increasingly taken seriously, and also has important regulative in antiviral natural is immune.Endogenous reverse transcription
LncRNA derived from virus will be gradually elucidated in the mechanism of action for fighting exogenous retrovirus proliferation, be expected to become and be resisted
The new generation vaccine or immunopotentiator of exogenous virus.
Fowl endogenous avian leukosis viruses ALVE is the endogenous retrovirus found earliest, may be with host immune response
It is related with genetic resistance.In particular, the endogenous retrovirus ALVE1 (length 7.5kb) on No. 1 chromosome of chicken
With avian leukosis virus very high homology, may have with the infection of avian leukosis virus and proliferation and centainly be associated with.
Summary of the invention
The present invention identifies a fowl endogenous retrovirus ALVE1 by high-flux sequence in chicken genome and derives
Antisense long-chain non-coding RNA, be named as lnc-ALVE1-AS1.Studies have shown that lnc-ALVE1-AS1 can be anti-with active cell
Viral interferon responsing reaction simultaneously inhibits to be proliferated with the exogenous J subgroup avian leucosis virus of its very high homology.Therefore, further
Understanding expression rule of the lnc-ALVE1-AS1 in different resistant strain chickens is particularly important.The object of the present invention is to provide
A kind of detection primer and kit that can detect lnc-ALVE1-AS1 expression.
The present invention provides a kind of primers for long-chain non-coding RNA lnc-ALVE1-AS1 detection of expression, including chain
Specific RT primer and fluorescence quantitative PCR detection primer；The sequence of the chain specific RT primer such as SEQ ID NO.8 and SEQ
Shown in ID NO.9；The sequence of the fluorescence quantitative PCR detection primer is as shown in SEQ ID NO.10 and SEQ ID NO.11.
The present invention further discloses above-mentioned primers to prepare the application in lnc-ALVE1-AS1 detection of expression kit.
A kind of long-chain non-coding RNA lnc-ALVE1-AS1 detection of expression kit, including chain specific RT primer, reverse
Record reagent, fluorescence quantitative PCR detection primer and quantitative PCR detecting reagent；The sequence of the chain specific RT primer such as SEQ ID
Shown in NO.8 and SEQ ID NO.9；The sequence of the fluorescence quantitative PCR detection primer such as SEQ ID NO.10 and SEQ ID
Shown in NO.11.
The detection of expression kit of detection lnc-ALVE1-AS1 expression rule provided by the invention, to detect lnc-
The expression rule of ALVE1-AS1 provides research method and basis.In addition, white in the fowl that production practices can be used for detecting chicken group
Blood disease innate immunity state.
Detailed description of the invention
Fig. 1 is lnc-ALVE1-AS1 fluorescence quantitative PCR detection result figure.
Fig. 2 is position view of the lnc-ALVE1-AS1 on No. 1 chromosome of chicken.
Fig. 3 is that lnc-ALVE1-AS1 activates antiviral interferon response response diagram.A: it is overexpressed lnc-ALVE1-AS1 pairs
The influence of cause of disease pattern recognition receptors gene expression；B: it is overexpressed the shadow that lnc-ALVE1-AS1 expresses interferon-stimulated gene
It rings；C: it is overexpressed influence of the lnc-ALVE1-AS1 to TLR3 protein expression.
Fig. 4 is that lnc-ALVE1-AS1 inhibits J subgroup avian leucosis virus vegetative map.A: it is overexpressed lnc-ALVE1-AS1 suppression
J subgroup avian leucosis virus p27 protein expression processed；B: it is overexpressed lnc-ALVE1-AS1 and inhibits J subgroup avian leucosis virus drop
Degree；C: the influence that indirect immunofluorescence confocal experiments analysis lnc-ALVE1-AS1 is proliferated J subgroup avian leucosis virus.
Specific embodiment
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case where spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
The measurement of 1 lnc-ALVE1-AS1 full length sequence of embodiment
(1) 5 ' and 3 ' end sequences of lnc-ALVE1-AS1 are identified
Mainly provided by Takara companyThe Kit of RACE 5 '/3 ' kit is completed.
Firstly, withReagent extracts total serum IgE, then removes genome with RNase-free DNase I.In
Respectively by the RNA of 1 μ g removal genome under the action of SMARTScribeReverse Transcriptase (kit offer)
Reverse transcription synthesizes 5 '-or 3 '-RACE products.
Then, according toThe operating instruction of the Kit kit of RACE 5 '/3 ' is distinguished using universal primer UPM
PCR amplification is carried out with 5 '-ends or 3 '-terminal gene specific primers (gene-specific primer, GSP), clone surveys
Sequence obtains 5 ' ends and the 3 ' end sequences of lnc-ALVE1-AS1.Wherein, 5 '-end used or 3 '-terminal gene specificity are drawn
The nucleotide sequence of object is as follows:
5 ' GSP:5 '-TGACGGGATGGGACACAACGCTAAACAGT-3 ' (SEQ ID NO.6)
3 ' GSP:5 '-GATTCGCCGCTCCGTTGCTGGTTTCTC-3 ' (SEQ ID NO.7).
(2) standard PCR amplification lnc-ALVE1-AS1 product.Wherein, the primer lnc-ALVE1-AS1-F and lnc-
The nucleotide sequence of ALVE1-AS1-R is as follows:
Lnc-ALVE1-AS1-F:5 '-ATCTTTATGTTCCATTGTCATCGC-3 ' (SEQ ID NO.2)
Lnc-ALVE1-AS1-R:5 '-GCCATTTTACCATCCACCAC-3 ' (SEQ ID NO.3)
Its reaction system includes: 100ng chicken embryo fibroblasts cDNA product as template, 1 μ L (10 μM) upstream primer
Lnc-ALVE1-AS1-F and 1 μ L (10 μM) lnc-ALVE1-AS1-R is respectively as amplimer, 1 μ L DNA Polymerase,
10 μ L 5x SF Buffer, 1 μ L (10 μM) dNTP Mix and 32 μ L ddH2O。
Its reaction condition are as follows: 95 DEG C of 3min；95 DEG C of 15s, 58 DEG C of 90s, 72 DEG C of 1min, 35x circulation；72℃7min； 4℃
It maintains.
(3) TA cloning and sequencing obtains the full length cDNA sequence of lnc-ALVE1-AS1, and the sequence is on No. 1 chromosome of chicken
Position view is shown in Fig. 2.Specific cDNA sequence such as SEQ ID NO.1.
The building of 2 lnc-ALVE1-AS1 over-express vector of embodiment
In the present embodiment 2, using from lnc-ALVE1-AS1 full length sequence obtained in embodiment 1, lnc- is constructed
ALVE1-AS1 is overexpressed plasmid.
Specifically comprise the following steps:
(1) it from the chicken embryo fibroblasts that AZA handles 96 hours, usesReagent extracts total serum IgE, then
Genome is removed with RNase-free DNase I.Using PrimeScript RT reagent Kit kit by its reverse transcription
At cDNA product.
(2) using 2 step of embodiment (1) cDNA product obtained as template, lnc-ALVE1- is expanded using high fidelity enzyme
AS1 full length sequence, wherein the nucleosides of the primer lnc-ALVE1-AS1-HindIII-F and lnc-ALVE1-AS1-XbaI-R
Acid sequence is as follows:
lnc-ALVE1-AS1-HindIII-F:
5′-aagcttATCTTTATGTTCCATTGTCATCGCTAAC-3′(SEQ ID NO.4)
lnc-ALVE1-AS1-XbaI-R:
5′-tctagaGCCATTTTACCATCCACCACATTG-3′(SEQ ID NO.5)
Its reaction system includes: 2 step of 100ng embodiment (1) cDNA product obtained as template, 1 μ L (10 μM)
Upstream primer lnc-ALVE1-AS1-HindIII-F and 1 μ L (10 μM) lnc-ALVE1-AS1-XbaI-R draws respectively as amplification
Object, 1 μ L DNA Polymerase, 10 μ L 5x SF Buffer, 1 μ L (10 μM) dNTP Mix and 32 μ L ddH2O。
Its reaction condition are as follows: 95 DEG C of 3min；95 DEG C of 15s, 58 DEG C of 90s, 72 DEG C of 1min, 35x circulation；72℃7min；4 DEG C of dimensions
It holds.
(3) agarose gel electrophoresis is carried out with pcr amplification product in 2 step of embodiment (2), then gel extraction product is simultaneously
It is connected to carrier T and carries out cloning and sequencing.Correct positive colony plasmid is sequenced and pcDNA3.1 (+) (Invitrogen) is overexpressed
Plasmid vector uses HindIII and XbaI enzyme cutting respectively.Finally, digestion recycling lnc-ALVE1-AS1 target fragment DNA and
PcDNA3.1 (+) carrier DNA is attached with T4DNA ligase, and connection product directly converts Escherichia coli.Recon warp
After PCR, digestion and cloning and sequencing identification correctly, it is named as pcDNA3.1-lnc-ALVE1-AS1.
3 lnc-ALVE1-AS1 of embodiment induces the analysis of antiviral natural immunocompetence
In the present embodiment 3, it is overexpressed plasmid using from pcDNA3.1-lnc-ALVE1-AS1 obtained in embodiment 2,
Lnc-ALVE1-AS1, which is had evaluated, by quantitative fluorescent PCR and Western-blot method induces antiviral natural immunocompetence.
Specifically comprise the following steps:
(1) chicken embryo fibroblasts are seeded to 6 porocyte culture plates, then transfect pcDNA3.1-lnc-ALVE1-AS1
It is overexpressed plasmid.Transfection of GFP is overexpressed plasmid as vehicle Control simultaneously.
(2) 36 hours after transfecting, cell is collected；
(3) total serum IgE is extracted, detects interferon signal path related gene using fluorescence quantifying PCR method.
(4) total protein is extracted, Western-blot analyzes TLR3 expression.By pcDNA3.1-lnc-ALVE1-AS1 and
GFP is overexpressed the cell pyrolysis liquid progress SDS-PAGE after plasmid transfection, then shifts according to Western-blot operating procedure
To nitrocellulose filter, the goat anti-rabbit igg that chicken TLR3 antibody and HRP label are separately added into after the closing of 5% skimmed milk is incubated
It educates, observes result.
It is analyzed by quantitative fluorescent PCR and Western-blot, it is observed that lnc-ALVE1-AS1 can be activated significantly
Antiviral natural gene involved in immunity expresses (Fig. 3).
4 lnc-ALVE1-AS1 J substock lymphoid leuoosis-resistant virus multiplication capability analysis of embodiment
In the present embodiment 4, it is overexpressed plasmid using from pcDNA3.1-lnc-ALVE1-AS1 obtained in embodiment 2,
It has evaluated lnc-ALVE1-AS1 J substock lymphoid leuoosis-resistant virus by ELISA, TCID50 and the burnt immunofluorescence method of copolymerization and increases
Grow ability.
Specifically comprise the following steps:
(1) chicken embryo fibroblasts are seeded to 6 holes or 24 porocyte culture plates, then infect J subgroup avian leucosis disease
Malicious (strain JS09GY3, GenBank accession number GU982308).
(2) 24 hours after virus infection, transfection lnc-ALVE1-AS1 is overexpressed plasmid；Transfection of GFP is overexpressed plasmid simultaneously
As vehicle Control.
(3) 96 hours after transfecting, supernatant is collected, it is white using IDEXX avian leukosis antigen detection kit detection J subgroup fowl
Blood disease virus p27 protein expression level.
(4) 96 hours after transfecting, cell and supernatant are collected, is dripped using TCID50 method measurement J subgroup avian leucosis virus
Degree.
(5) 96 hours after transfecting, the burnt immunofluorescence assay J subgroup avian leucosis virus Env protein expression of copolymerization is used.Letter
Want that steps are as follows: 4% paraformaldehyde room temperature fixes 20 minutes, and PBS is washed three times, and 5 minutes every time；0.5%Triton X-100
Room temperature penetrating 15 minutes, PBS was washed three times；2%BSA room temperature is closed 30 minutes, and PBS is washed three times；It is dense that addition is diluted to work
The specific monoclonal antibody JE9 of the ALVJ membrane glycoprotein of degree, 37 DEG C are incubated for 1 hour, and PBS is washed three times；It is added under the conditions of being protected from light dilute
The mountain sheep anti-mouse igg marked to working concentration Alexa Fluor 488 is released, 37 DEG C are incubated for 40 minutes, and PBS is washed three times；It is protected from light,
DAPI dyeing liquor dyes 10 minutes, mounting processing after PBS washing three times.Finally, being carried out using Leica SP8 Laser Scanning Confocal Microscope
Observation is taken pictures.
By ELISA, TCID50 and it is copolymerized burnt immunofluorescence experiment, it is observed that lnc-ALVE1-AS1 can be significant
Activation inhibits J subgroup avian leucosis virus proliferation (Fig. 4).
Embodiment 5lnc-ALVE1-AS1RT-qPCR detection
(1) it respectively from 5 ages in days and the different tissues of 30 age in days SPF chickens, usesReagent extracts total serum IgE,
Measure concentration.
(2) PrimeScript is usedTMThe reagent provided in RT reagent Kitwith gDNA Eraser kit
Remove genome.
Its reaction system includes: 1 μ g embodiment, 2 step (1) total serum IgE obtained as template, 2 μ L5 × gDNA
Eraser Buffer, 1 μ LgDNA Eraser, RNase Free dH2O polishing to 10 μ L.
Its reaction condition are as follows: 42 DEG C of 2min；4 DEG C of maintenances.
(3) PrimeScript is usedTMThe reagent provided in RT reagent Kitwith gDNA Eraser kit
The reaction of chain specific reverse transcriptase is carried out, by total serum IgE reverse transcription at cDNA product.Wherein, chain specific primer lnc- used
The nucleotide sequence of ALVE1-AS1-RT1 and lnc-ALVE1-AS1-RT2 is as follows:
Lnc-ALVE1-AS1-RT1 (SEQ ID NO.8): 5 '-GATGGACAGACCGTTGAG-3 '
Lnc-ALVE1-AS1-RT2 (SEQ ID NO.9): 5 '-CCTCATCCGTCTCGCTTA-3 '
Its reaction system includes: 2 step of embodiment (2), 10 μ L reaction product obtained as template, 5 μ Llnc-
ALVE1-AS1-RT1 and lnc-ALVE1-AS1-RT2 is as chain specificity RT mix primer, 1 μ L PrimeScript RT
Enzyme Mix I, 4 μ 5 × PrimeScript of L Buffer 2 and 4 μ LRNase Free dH2O.
Its reaction condition are as follows: 42 DEG C of 15min；85℃5s；4 DEG C of maintenances.
(4) it usesPremix Ex TaqTMThe reagent provided in kit carries out chain specificity fluorescent quantitative PCR
Reaction,.Wherein, the nucleotide sequence of specific primer lnc-ALVE1-AS1-F2 and lnc-ALVE1-AS1-R2 used is as follows:
Lnc-ALVE1-AS1-F2 (SEQ ID NO.10): 5 '-GACTACTGCCATAACTAAGG-3 '
Lnc-ALVE1-AS1-R2 (SEQ ID NO.11): 5 '-CAGAAGTCACAGCCAGAT-3 '
Its reaction system includes: 1 μ L embodiment, 2 step (3) cDNA product obtained as template, 0.4 μ L (10 μM)
Upstream primer lnc-ALVE1-AS1-F2 and 0.4 μ L (10 μM) lnc-ALVE1-AS1-R2 are respectively as amplimer, 10 μ LPremix Ex TaqTM(2 ×) and 8.2 μ L ddH2O。
Its reaction condition are as follows: 95 DEG C of 5min；95℃5s；60℃34s；40 circulations.
Chain specificity fluorescent quantitative PCR detection result as shown in Figure 1: long-chain non-coding RNA lnc-ALVE1-AS1 on 5th
High expression in age SPF chicken tissues organ and in 30 age in days SPF chicken tissues organs low expression.Therefore, this kit can be used for
Lnc-ALVE1-AS1 detection of expression.
SEQUENCE LISTING
<110>Yangzhou University
<120>long-chain non-coding RNA lnc-ALVE1-AS1 detection of expression primer and kit
<130>
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 2136
<212> DNA
<213>chicken
<400> 1
atctttatgt tccattgtca tcgctaacga gacggcaggc tgctcagaga cgggccacga 60
ccccgaagag aggcctcttc ctgggcgttg gccctggttg ccatcccgcc ttctgcactg 120
tttagcgttg tgtcccatcc cgtcacacag ctgacatcgc tcacggctgt ttcctgactt 180
tcgttttttc gggcactgcg cctgataatg tcccggggat ccacaagtgt agcagagccc 240
tcgggcacga ccacccgacc cagtttgtcc atccctctct ctattgacta ctgccataac 300
taagggctgg atagcagacg acatggccgc ggctatgcct tgatccgtaa gaggggcagt 360
cttctgcctg tctagcacat atttgattat ctctcctggg gtggtcagcg tggagggtgc 420
tgcccgtata agctgctgaa tatctggctg tgacttctgc ctaaagcagt caatgatcac 480
cggagcccgc gcggaaggtg ggagatctga cccttcaacc gcctttataa gacgattggc 540
aaaatccaca aaggactcag atggtccctg cgtaatttcc gcccacgggt ctgtgggttc 600
cgccaaccga gcgacctctc taaacgcctg gagagccgac gccgtaatag caaccagttc 660
ccccggtctt aataatgcag cctgaccctc tggattgccg gccattccat ccgccaaacc 720
ctttaaacga tccaagttag tccgttcccc ccgcccttga ccgttcgctg ggtgtcgggg 780
gtcgcgagtg gctgccgcta taaccgtctg tagttggact ccccaagcgt ccatccataa 840
ggcatatggg gcaggtccta aaataactct cattagattc gtgacgtcat gcggcagcag 900
cggggaggac atgagcgctt ccacttctgc catagtgatc ggggatcgta agcccttggt 960
cctgaccgta tcagccagtc ttgtgatcaa ttttggctcc agaggggtcc aggcgggtcc 1020
ctctgtctta atcactacag gcatggccac cacgggcgga cctgtactcg caagctcctc 1080
cctgatcctt gcccagtcag tcagggccgg cccaggggcc agacccgcgt gccctggctc 1140
cgcccttggc tgttccgccc cccaaggtgt gtcacccccc tggccctgct gctctcccac 1200
ccccgccagg gaaggataca aaccactccc cacataagga ggaggagggg ccgaggctgt 1260
ggcgcaatta cagccagtag ctgttccgca ctgatagcag gatgtgccaa cggttttagg 1320
tgtggccatt ttctccggcg ccatcttcgc atctcgctgc gcagttgttt ctcccacttc 1380
ctcccctttg tcgattcgcc gctccgttgc tggtttctcg atgcactccg gacctggggg 1440
agagaccctc cctcccccta atcccaacca aaactttgct tgctcagatg taacctgttc 1500
ctctcgagcc gccttcaatg cccccaaaac caatccccag gtttttaact ctcccgattt 1560
cccaagtacc attgcccgct gggagagcgc cgcggtaatg ggatcccagg accccgggga 1620
atataagtct gagggagaca taagcaaccc ttccttttgt aacagggaca acatggcccc 1680
tatttccttc ttagaaggag aggttttccc gcaataggtt ttacacgcgg acgaaatcac 1740
ctttatgacg gcttccatgc tagacccaca gggcgaccgg aatcgtgcct ggggtggact 1800
gctcagtcgt cgggcttcct tcccgtcttc caacgactct ctgagttctc ggtaggttat 1860
cttggccccc ggccgtggag ctccctccga cgtcactcag cttctgccct cctaagccgc 1920
agccccctct actagggtca tcgtcctcgc tccgttaagc gagacggatg agggcaggat 1980
cgccacgccg tctgtggccg accactattc cctaacgatc acgtcggggt caccaaatga 2040
agccttctgc ttcattcagg tgttcgcaat cgttagggac tcaacggtct gtccatctac 2100
ccaggtgcac accaatgtgg tggatggtaa aatggc 2136
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atctttatgt tccattgtca tcgc 24
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gccattttac catccaccac 20
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tctagagcca ttttaccatc caccacattg 30
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tgacgggatg ggacacaacg ctaaacagt 29
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<211> 27
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<213>artificial sequence
<400> 7
gattcgccgc tccgttgctg gtttctc 27
<210> 8
<211> 18
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<213>artificial sequence
<400> 8
gatggacaga ccgttgag 18
<210> 9
<211> 18
<212> DNA
<213>artificial sequence
<400> 9
cctcatccgt ctcgctta 18
<210> 10
<211> 20
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<400> 10
gactactgcc ataactaagg 20
<210> 11
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cagaagtcac agccagat 18
Claims (3)
1. for drawing for cDNA sequence long-chain non-coding RNA lnc-ALVE1-AS1 detection of expression as shown in SEQ ID NO.1
Object, which is characterized in that including chain specific RT primer and fluorescence quantitative PCR detection primer；The sequence of the chain specific RT primer
Column are as shown in SEQ ID NO.8 and SEQ ID NO.9；The sequence of the fluorescence quantitative PCR detection primer such as SEQ ID NO.10
With shown in SEQ ID NO.11.
2. a kind of cDNA sequence long-chain non-coding RNA lnc-ALVE1-AS1 detection of expression reagent as shown in SEQ ID NO.1
Box, including chain specific RT primer, reverse transcription reagents, fluorescence quantitative PCR detection primer and quantitative PCR detecting reagent；Its feature
It is, the sequence of the chain specific RT primer is as shown in SEQ ID NO.8 and SEQ ID NO.9；The quantitative fluorescent PCR inspection
The sequence of primer is surveyed as shown in SEQ ID NO.10 and SEQ ID NO.11.
3. primer described in claim 1 is preparing the application in lnc-ALVE1-AS1 detection of expression kit.
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