CN107488750A - Long-chain non-coding RNA lnc ALVE1 AS1 detection of expression primers and kit - Google Patents
Long-chain non-coding RNA lnc ALVE1 AS1 detection of expression primers and kit Download PDFInfo
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Abstract
The invention belongs to biology field, and in particular to a kind of long-chain non-coding RNA lnc ALVE1 AS1 detection of expression primers 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 kits, provides method and basis for the analysis of lnc ALVE1 AS1 expression rules, can be used for the avian leukosis innate immunity state of detection chicken group in production practices.
Description
Technical field
The invention belongs to biology field, and in particular to a kind of long-chain non-coding RNA lnc-ALVE1-AS1 expression inspection
Survey primer and kit.
Background technology
Endogenous retroviruse (endogenous retroviruses) is genome composition, is considered as coming from evolution
The retroviruse of middle infection biological body.As residual of the time immemorial retroviral infection in host, pass by addition to it
Function not enough understand, be once regarded as junk DNA (Junk DNA).There are some researches show some endogenous sex reversals as of late
Record virus not only plays an important roll to the versatility of early embryonic development and embryonic stem cell, it is also possible to the cell innate immunity
It is relevant.
Current research finds that endogenous retroviruse 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 and control 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 with the mechanism of action for resisting exogenous retroviruse propagation, be expected to turn into resistance
The new generation vaccine or immunopotentiator of exogenous virus.
Fowl endogenous avian leukosis viruses ALVE is the endogenous retroviruse found earliest, may be with host immune response
It is relevant with genetic resistance.Particularly, the endogenous retroviruse 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 propagation and necessarily associate.
The content of the invention
The present invention identifies a fowl endogenous retroviruse ALVE1 in chicken genome by high-flux sequence and derived
Antisense long-chain non-coding RNA, be named as lnc-ALVE1-AS1.Research shows that lnc-ALVE1-AS1 can be resisted with active cell
Viral interferon responsing reaction simultaneously suppresses to breed with the exogenous J subgroup avian leucosis virus of its very high homology.Therefore, further
Understand expression rules of the lnc-ALVE1-AS1 in different resistant strain chickens to be particularly important.It is an object of the invention to provide
A kind of detection primer and kit that can detect lnc-ALVE1-AS1 expression.
The invention provides a kind of primer 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 application of the above-mentioned primer in lnc-ALVE1-AS1 detection of expression kits are prepared.
A kind of long-chain non-coding RNA lnc-ALVE1-AS1 detection of expression kits, 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 rules provided by the invention, to detect lnc-
ALVE1-AS1 expression rule provides research method and basis.In addition, it can be used for the fowl of detection chicken group white in production practices
Blood disease innate immunity state.
Brief description of the drawings
Fig. 1 is lnc-ALVE1-AS1 fluorescence quantitative PCR detection result figures.
Fig. 2 is position views 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 is expressed interferon-stimulated gene
Ring；C：It is overexpressed influences of the lnc-ALVE1-AS1 to TLR3 protein expressions.
Fig. 4 is that lnc-ALVE1-AS1 suppresses J subgroup avian leucosis virus vegetative maps.A：It is overexpressed lnc-ALVE1-AS1 suppressions
J subgroup avian leucosis virus p27 protein expressions processed；B：It is overexpressed lnc-ALVE1-AS1 and suppresses J subgroup avian leucosis virus drop
Degree； C：The influence that indirect immunofluorescence confocal experiments analysis lnc-ALVE1-AS1 breeds to J subgroup avian leucosis virus.
Embodiment
Following examples further illustrate present disclosure, but should not be construed as limiting the invention.Without departing substantially from
In the case of of the invention spirit and essence, the modifications or substitutions made to the inventive method, step or condition belong to the present invention
Scope.
Unless otherwise specified, the conventional meanses that technological means used in embodiment is well known to those skilled in the art.
The measure of the lnc-ALVE1-AS1 full length sequences of embodiment 1
(1) lnc-ALVE1-AS1 5 ' and 3 ' end sequences are identified
Mainly provided by Takara companiesRACE 5 '/3 ' Kit kits are completed.
First, useReagent extracts total serum IgE, then removes genome with RNase-free DNase I.
The RNA of genome is respectively provided 1 μ g in the presence of SMARTScribeReverse Transcriptase (kit offer)
Reverse transcription synthesizes 5 '-or 3 '-RACE products.
Then, according toThe operating instruction of the Kit kits of RACE 5 '/3 ', utilize universal primer UPM difference
Enter performing PCR amplification with 5 '-end or 3 '-terminal gene specific primer (gene-specific primer, GSP), clone surveys
Sequence obtains lnc-ALVE1-AS1 5 ' ends and 3 ' end sequences.Wherein, 5 '-end used or 3 '-terminal gene specificity are drawn
The nucleotide sequence of thing 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 products.Wherein, the primer lnc-ALVE1-AS1-F and lnc-
ALVE1-AS1-R nucleotide sequence 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 products are as template, 1 μ L (10 μM) sense 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 is：95℃3min；95 DEG C of 15s, 58 DEG C of 90s, 72 DEG C of 1min, 35x circulation；72℃7min； 4℃
Maintain.
(3) TA cloning and sequencings obtain lnc-ALVE1-AS1 full length cDNA sequence, 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 lnc-ALVE1-AS1 over-express vectors of embodiment 2 are built
In the present embodiment 2, using the lnc-ALVE1-AS1 full length sequences obtained in embodiment 1, lnc- is built
ALVE1-AS1 is overexpressed plasmid.
Specifically comprise the following steps：
(1) in the chicken embryo fibroblasts for handling 96 hours from AZA, useReagent extracts total serum IgE, then
Genome is removed with RNase-free DNase I.Using PrimeScript RT reagent Kit kits by its reverse transcription
Into cDNA products.
(2) the cDNA products obtained using the step of embodiment 2 (1) use high-fidelity enzymatic amplification lnc-ALVE1- as template
AS1 full length sequences, wherein, the primer lnc-ALVE1-AS1-HindIII-F and lnc-ALVE1-AS1-XbaI-R nucleosides
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：The cDNA products that the step of 100ng embodiments 2 (1) is obtained are as template, 1 μ L (10 μM)
Sense primer lnc-ALVE1-AS1-HindIII-F and 1 μ L (10 μM) lnc-ALVE1-AS1-XbaI-R draws respectively as amplification
Thing, 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 is：95℃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
Hold.
(3) row agarose gel electrophoresis are entered with pcr amplification product in the step of embodiment 2 (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 to be overexpressed with pcDNA3.1 (+) (Invitrogen)
Plasmid vector uses HindIII and XbaI enzyme cutting respectively.Finally, digestion recovery lnc-ALVE1-AS1 purpose fragments DNA and
PcDNA3.1 (+) carrier DNA is attached with T4DNA ligase, and connection product directly converts Escherichia coli.Recon passes through
After PCR, digestion and cloning and sequencing identification correctly, pcDNA3.1-lnc-ALVE1-AS1 is named as.
The lnc-ALVE1-AS1 of embodiment 3 induction antiviral natural immunocompetence analyses
In the present embodiment 3, plasmid is overexpressed using the pcDNA3.1-lnc-ALVE1-AS1 obtained in embodiment 2,
Lnc-ALVE1-AS1 have evaluated by quantitative fluorescent PCR and Western-blot methods and induce 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.GFP-transfected overexpression plasmid is as vehicle Control simultaneously.
(2) 36 hours after transfecting, cell is collected；
(3) total serum IgE is extracted, interferon signal path related gene is detected using fluorescence quantifying PCR method.
(4) total protein, Western-blot analysis TLR3 expressions are extracted.By pcDNA3.1-lnc-ALVE1-AS1 and
GFP is overexpressed the cell pyrolysis liquid progress SDS-PAGE after plasmid transfection, is then shifted according to Western-blot operating procedures
To nitrocellulose filter, the goat anti-rabbit igg that chicken TLR3 antibody and HRP marks are separately added into after the closing of 5% skimmed milk is incubated
Educate, observe result.
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).
The lnc-ALVE1-AS1 J substock lymphoid leuoosis-resistant virus multiplication capability analysis of embodiment 4
In the present embodiment 4, plasmid is overexpressed using the pcDNA3.1-lnc-ALVE1-AS1 obtained in embodiment 2,
Lnc-ALVE1-AS1 J substock lymphoid leuoosis-resistants virus is have evaluated by ELISA, TCID50 and the burnt immunofluorescence method of copolymerization to increase
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；GFP-transfected overexpression plasmid simultaneously
As vehicle Control.
(3) 96 hours after transfecting, supernatant is collected, it is white to detect J subgroup fowl using IDEXX avian leukosis antigen detection kit
The viral p27 protein expression levels of blood disease.
(4) 96 hours after transfecting, cell and supernatant are collected, determining J subgroup avian leucosis virus using TCID50 methods drips
Degree.
(5) 96 hours after transfecting, the burnt immunofluorescence assay J subgroup avian leucosis virus Env protein expressions of copolymerization are used.Letter
Want step as follows：4% paraformaldehyde room temperature fixes 20 minutes, and PBS is washed three times, 5 minutes every time；0.5%Triton X-100
Penetrating 15 minutes of room temperature, PBS are washed three times；2%BSA room temperatures are 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 glycoproteins of degree, 37 DEG C are incubated 1 hour, and PBS is washed three times；Added under the conditions of lucifuge dilute
The mountain sheep anti-mouse igg marked to working concentration Alexa Fluor 488 is released, 37 DEG C are incubated 40 minutes, and PBS is washed three times；Lucifuge,
DAPI dyeing liquors dye 10 minutes, mounting processing after PBS washings three times.Finally, carried out using Leica SP8 Laser Scanning Confocal Microscopes
Observation is taken pictures.
By ELISA, TCID50 and the burnt immunofluorescence experiment of copolymerization, it is observed that lnc-ALVE1-AS1 can be notable
Activation suppresses J subgroup avian leucosis virus propagation (Fig. 4).
Embodiment 5lnc-ALVE1-AS1RT-qPCR is detected
(1) respectively from 5 ages in days and the different tissues of 30 age in days SPF chickens, useReagent extracts total serum IgE,
Determine concentration.
(2) PrimeScript is usedTMThe reagent provided in RT reagent Kitwith gDNA Eraser kits
Remove genome.
Its reaction system includes：The total serum IgE that the step (1) of 1 μ g embodiments 2 is obtained is as template, 2 μ L5 × gDNA
Eraser Buffer, 1 μ LgDNA Eraser, RNase Free dH2O polishings to 10 μ L.
Its reaction condition is：42℃2min；4 DEG C of maintenances.
(3) PrimeScript is usedTMThe reagent provided in RT reagent Kitwith gDNA Eraser kits
Chain specific reverse transcriptase reaction is carried out, by total serum IgE reverse transcription into cDNA products.Wherein, chain specific primer lnc- used
ALVE1-AS1-RT1 and lnc-ALVE1-AS1-RT2 nucleotide sequence 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：The 10 μ L reaction products that the step of embodiment 2 (2) is obtained are as template, 5 μ Llnc-
ALVE1-AS1-RT1 and lnc-ALVE1-AS1-RT2 is as chain specificity RT mix primers, 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 is：42℃15min；85℃5s；4 DEG C of maintenances.
(4) usePremix Ex TaqTMThe reagent provided in kit carries out chain specificity fluorescent quantitative PCR
Reaction,.Wherein, specific primer lnc-ALVE1-AS1-F2 and lnc-ALVE1-AS1-R2 used nucleotide sequence 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：The cDNA products that the step (3) of 1 μ L embodiments 2 is obtained are as template, 0.4 μ L (10 μM)
Sense 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 is：95℃5min；95℃5s；60℃34s；40 circulations.
Chain specificity fluorescent quantitative PCR detection result is as shown in Figure 1：Long-chain non-coding RNA lnc-ALVE1-AS1 was 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
<210> 2
<211> 24
<212> DNA
<213>Artificial sequence
<400> 2
atctttatgt tccattgtca tcgc 24
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
gccattttac catccaccac 20
<210> 4
<211> 34
<212> DNA
<213>Artificial sequence
<400> 4
aagcttatct ttatgttcca ttgtcatcgc taac 34
<210> 5
<211> 30
<212> DNA
<213>Artificial sequence
<400> 5
tctagagcca ttttaccatc caccacattg 30
<210> 6
<211> 29
<212> DNA
<213>Artificial sequence
<400> 6
tgacgggatg ggacacaacg ctaaacagt 29
<210> 7
<211> 27
<212> DNA
<213>Artificial sequence
<400> 7
gattcgccgc tccgttgctg gtttctc 27
<210> 8
<211> 18
<212> DNA
<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
<212> DNA
<213>Artificial sequence
<400> 10
gactactgcc ataactaagg 20
<210> 11
<211> 18
<212> DNA
<213>Artificial sequence
<400> 11
cagaagtcac agccagat 18
Claims (3)
1. the primer for long-chain non-coding RNA lnc-ALVE1-AS1 detection of expression, it is characterised in that including chain specificity RT
Primer and fluorescence quantitative PCR detection primer；The sequence of the chain specific RT primer such as SEQ ID NO.8 and SEQ ID NO.9
It is shown；The sequence of the fluorescence quantitative PCR detection primer is as shown in SEQ ID NO.10 and SEQ ID NO.11.
2. a kind of long-chain non-coding RNA lnc-ALVE1-AS1 detection of expression kits, including chain specific RT primer, reverse transcription
Reagent, fluorescence quantitative PCR detection primer and quantitative PCR detecting reagent；Characterized in that,
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. application of the primer in lnc-ALVE1-AS1 detection of expression kits are prepared described in claim 1.
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| 朱文奇等: "禽内源性反转录病毒ALVE表观遗传学分析方法的建立及应用", 《中国动物传染病学报》 * |
| 胡序明等: "内源性反转录病毒衍生的长非编码RNA的功能", 《生命科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113930527A (en) * | 2021-12-09 | 2022-01-14 | 常州市动物疫病预防控制中心(常州市畜牧兽医站) | Non-coding RNA lnc-ALVE-env1 expression detection primer, kit and application |
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| CN107488750B (en) | 2019-11-05 |
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