CN106399378A - PLC (phospholipase C) epsilon gene targeted RNA-interference recombinant lentiviral vectors and construction method thereof - Google Patents

PLC (phospholipase C) epsilon gene targeted RNA-interference recombinant lentiviral vectors and construction method thereof Download PDF

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CN106399378A
CN106399378A CN201610979733.3A CN201610979733A CN106399378A CN 106399378 A CN106399378 A CN 106399378A CN 201610979733 A CN201610979733 A CN 201610979733A CN 106399378 A CN106399378 A CN 106399378A
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plc
homo
plv
gene
shrna
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房健民
郭佳
蒋明
尹衍新
于丽华
蒋韵
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SUZHOU RESEARCH INSTITUTE OF TONGJI UNIVERSITY
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Abstract

The invention relates to construction, screening and application of PLC (phospholipase C) gene targeted RNA-interference recombinant lentiviral vectors. Four lentiviral eukaryotic expression vectors capable of expressing SiRNA in 293 cells are constructed with an RNA interference technology for different target sequences of PLC genes, and the interference lentiviral vectors are obtained by culturing 293 T cells co-transfected by pLv-PLCshRNA, pMD2.G and pSPAX2 vectors. The PLC gene targeted RNA-interference recombinant lentiviral vectors can efficiently and specifically inhibit PLC gene expression and can be used for preparing drugs resisting tumor related genes.

Description

Targeting PLC ε gene RNA interference recombinant lentivirus vector and its construction method
Technical field
The present invention relates to the RNA interference recombinant lentiviral of targeting people source phospholipase C-epsilon (Phospholipase C, PLC ε) gene Viral vector and its structure, belong to biomedicine technical field.
Background technology
In tumor-related gene, Ras gene is most commonly seen, participates in cell proliferation signals conduction, encodes guanylic acid Associated proteins (G-protein).After Ras activation, G-protein only retains GTP binding activity and no proteolytic activity, therefore sustained activation, there is silk Heading signal is constantly incoming, cell hyperproliferation.In Ras signal path numerous downstream effect albumen, phospholipase C in recent years In (phospholipase C, PLC) family, a new hypotype PLC ε is found, and has typical structure domain except same with other PLC X, Y and C2, its c-terminus also has specific Ras binding structural domain and aminoterminal Guanine nucleotide exchange factor domain.PLCε Play facilitation during chemical carcinogen induction Mus cutaneous tumor, and relevant with tumor proliferation.Subsequent research table Bright, the facilitation of PLC ε may expand inflammatory reaction owing to it.PLC ε passes through to increase inflammatory reaction and vascularization promote Enter the generation of Mus intestinal canal tumour.PLC ε as the effector of Ras proto-oncogene, its tumor occur and progress effect to closing weight Will.
RNA perturbation technique energy selective degradation mRNA, silencing of target genes, in the expression of post-transcriptional level suppressor gene, from And can be utilized for the research of gene function and the research of drug targets.At present RNA in mammalian cell is induced by siRNA The operational approach of interference has two kinds, the respectively siRNA of the siRNA of chemosynthesis and cell inner expression.Chemical synthesiss are artificial Preparation siRNA, is usually respectively synthesized positive-sense strand and the antisense strand of 21 bases, in vitro suitably at a temperature of so that complementary strand is joined Right, form siRNA.Then, using the method for Conventional transfection, such as Lipofectamine transfection, calcium phosphate, electricity punching etc. will SiRNA imports cell, induction RNA interference.The method is simple to operate, quick, but the RNA interference effect causing is often temporarily It is difficult to persistently.Therefore, it is more likely to express siRNA at present in mammalian cell, effect is disturbed with the RNA remaining permanent Should.Cell inner expression method is by the vector-transfected cell of expression siRNA, expresses siRNA in the cell.Slow virus carrier is as normal One of viral vector, is characterized in that immunogenicity is low, can infect split coil method and nondividing phase cell, can self-contained fragment It is integrated into host cell gene group, and stably express siRNA in mammal various types of cells, long-term inhibition of gene expression.
Content of the invention
The present invention provides a kind of RNA interference recombinant viral vector of targeting PLC ε gene build, screen and application thereof.
, based on RNA perturbation technique, for the different target sequences of PLC ε gene, constructing four can be thin in 293T for the present invention The slow viruss carrier for expression of eukaryon pLv-PLC ε shRNA of siRNA is expressed, this carrier is the slow virus carrier of itself inactivation in born of the same parents, It illustrates collection of illustrative plates to see Fig. 1, can specific suppression PLC ε gene expression.
Technical scheme is as follows：
A kind of siRNA recombinant slow virus expression vector of targeting PLC ε gene, is for tumor PLC ε gene associations therapeutic Material, slow viruss are to be obtained by pLv-PLC ε shRNA, pMD2.G and pSPAX2 carrier cotransfection 293T cell culture；Wherein, institute The pLv-PLC ε shRNA recombinant vector stated be in the multiple clone site of plv-shRNA carrier connect double chain DNA fragment build and , restriction enzyme site is BamHI and EcoRI, and described double chain DNA fragment is one of following sequence：
(1) PLC ε-homo-U1 oligonucleotide sequence 1:
Positive-sense strand:
5’GATCCCCACTTTGTTAGTCAGGAGATCTCGAGATCTCCTGACTAACAAAGTGGTTTTTG3’
Antisense strand:
5’AATTCAAAAACCACTTTGTTAGTCAGGAGATCTCGAGATCTCCTGACTAACAAAGTGGG3’
(2) PLC ε-homo-U2 oligonucleotide sequence 2:
Positive-sense strand:
5’GATCCGCGGATTATTGGAACTCACTACTCGAGTAGTGAGTTCCAATAATCCGCTTTTTG3’
Antisense strand:
5’AATTCAAAAAGCGGATTATTGGAACTCACTACTCGAGTAGTGAGTTCCAATAATCCGCG3’
(3) PLC ε-homo-U3 oligonucleotide sequence 3:
Positive-sense strand:
5’GATCCCCATCATTTATCATGGACATACTCGAGTATGTCCATGATAAATGATGGTTTTTG3’
Antisense strand:
5’AATTCAAAAACCATCATTTATCATGGACATACTCGAGTATGTCCATGATAAATGATGGG3’
(4) PLC ε-homo-U4 oligonucleotide sequence 4:
Positive-sense strand:
5’GATCCGCTGCAATGTTTGAGGCAAATCTCGAGATTTGCCTCAAACATTGCAGCTTTTTG3’
Antisense strand:
5’AATTCAAAAAGCTGCAATGTTTGAGGCAAATCTCGAGATTTGCCTCAAACATTGCAGCG3’
According to the present invention, the construction method of the described siRNA recombinant slow virus expression vector of targeting PLC ε gene, including step As follows：
A. according to PLC ε mRNA sequence, design synthetic dsdna fragment, described double chain DNA fragment be described PLC ε- One of homo-U1, PLC ε-homo-U2, PLC ε-homo-U3 and PLC ε-homo-U4 nucleotide sequence；
B. and then described double chain DNA fragment is connected in the multiple clone site of plv-shRNA carrier and is built into pLv-PLC ε ShRNA recombinant vector；
C. again by described pLv-PLC ε shRNA recombinant vector and pMD2.G and pSPAX2 carrier cotransfection 293T cell culture, Obtain the recombined lentivirus vector system of targeting PLC ε gene RNA interference.
Recombined lentivirus vector in order to disturb to targeting PLC ε gene RNA reflects to the inhibition of PLC ε ε gene Fixed, carry out fluorescence quantitative PCR detection destination gene expression by after pLv-PLC ε ε shRNA and packaging plasmid cotransfection 293T cell Level, filters out effective interference plasmid.
Brief description
Fig. 1 is Lentiviral plv-shRNA result schematic diagram in embodiment.
Fig. 2 PLC ε RNA interfering slow virus carrier wink turns 293T cell 48h picture (fluorescence, visible ray).Wherein (a) is general Thang-kng mirror (100 ×)；B () is fluorescence light microscopic (100 ×).
PLC ε expression under Fig. 3 disturbance sequence.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than restriction the scope of the present invention.In addition, it is to be understood that after having read the content of present invention instruction, people in the art Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited Scope.
Main material：
293T cell is purchased from Shanghai Inst. of Cytobiology, Chinese Academy of Sciences；E. coli competent DH5 α is purchased from the full formula in Beijing Golden Bioisystech Co., Ltd；Slow virus carrier plv-shRNA is purchased from Clontech company, containing human U_6 promoter, encodes green Color fluorescent protein report gene；Various restriction endonuclease, T4DNA ligase and Taq archaeal dna polymerase are U.S. NEB Products；SYBR Green I q RT-PCR Mixs purchase and Shanghai Sheng Gong biotechnology service company；Hyclone And DMEM culture fluid is purchased from Gibico company of the U.S..
Embodiment 1
The slow virus carrier of targeting PLC ε gene builds
1. the design of nucleic acid oligomer and synthesis
Design targeting PLC ε gene（NM_016341.3）4 interference sequences（Table 1）, and synthesize corresponding single stranded DNA
Table 1 targeting PLC ε gene RNA interference sequence
Sequence names Sequence information Original position
U1 CCACTTTGTTAGTCAGGAGAT 965
U2 GCGGATTATTGGAACTCACTA 2999
U3 CCATCATTTATCATGGACATA 4343
U4 GCTGCAATGTTTGAGGCAAAT 5479
3 ' ends of shRNA oligonucleotide are TTTTT, and as the termination signal of RNA polymerase III type promoter U6, it 5 ' and 3 ' End is the restriction enzyme site of BamHI and EcoRI respectively, and the loop structure in plv-shRNA template has selected CTCGAG. Each single stranded DNA synthesis fragment is as follows：
(1) PLC ε-homo-U1 oligonucleotide sequence 1:
Positive-sense strand:
5’GATCCCCACTTTGTTAGTCAGGAGATCTCGAGATCTCCTGACTAACAAAGTGGTTTTTG3’
Antisense strand:
5’AATTCAAAAACCACTTTGTTAGTCAGGAGATCTCGAGATCTCCTGACTAACAAAGTGGG3’
(2) PLC ε-homo-U2 oligonucleotide sequence 2:
Positive-sense strand:
5’GATCCGCGGATTATTGGAACTCACTACTCGAGTAGTGAGTTCCAATAATCCGCTTTTTG3’
Antisense strand:
5’AATTCAAAAAGCGGATTATTGGAACTCACTACTCGAGTAGTGAGTTCCAATAATCCGCG3’
(3) PLC ε-homo-U3 oligonucleotide sequence 3:
Positive-sense strand:
5’GATCCCCATCATTTATCATGGACATACTCGAGTATGTCCATGATAAATGATGGTTTTTG3’
Antisense strand:
5’AATTCAAAAACCATCATTTATCATGGACATACTCGAGTATGTCCATGATAAATGATGGG3’
(4) PLC ε-homo-U4 oligonucleotide sequence 4:
Positive-sense strand:
5’GATCCGCTGCAATGTTTGAGGCAAATCTCGAGATTTGCCTCAAACATTGCAGCTTTTTG3’
Antisense strand:
5’AATTCAAAAAGCTGCAATGTTTGAGGCAAATCTCGAGATTTGCCTCAAACATTGCAGCG3’
2. nucleic acid oligomer annealing
The nucleic acid oligomer of synthesis is used respectively no enzyme water dissolution, concentration is 20 μM.Take corresponding positive-sense strand and antisense strand solution, according to Following proportioning prepares annealing reaction system
Component Volume（μL）
Annealing buffer 5
Positive-sense strand（20μM） 5
Antisense strand（20μM） 5
Water is to final volume 50
PCR instrument is made annealing treatment according to following program：95℃ 5min; 85℃ 5min; 75℃ 5min; 70 ℃5 min;4 DEG C of preservations.
3.pl-shRNA vector linearization
Take 2 μ g plv-shRNA carriers, carry out enzyme action process according to following system：
Component Volume（μL）
10 × buffer 5
BamHI 1
EcoRI 1
Water is to final volume 50
37 DEG C of enzyme action 1 hour, 1.5% sepharose electrophoresis, are reclaimed using DNA purification kit, nucleic acid-protein concentration mensuration instrument measures Reclaim quality.
The structure of 4.plv-PLC ε-shRNA carrier
Using T4DNA ligase, carry out carrier coupled reaction according to following system：
Component Volume（μL）
10 × T4 connects buffer 2
Carrier recovery 2
shDNA 2
T4DNA ligase 1
Water is to final volume 20
20 DEG C connect overnight, convert matter big pumping rod bacterium competence DH5 α, select positive colony and send to sequencing.The product that this step obtains Thing is plv-PLC ε-shRNA.
Embodiment 2
RNA interference turns 293T cell in slow viruss plasmid wink
4 kinds are disturbed slow viruss plasmid, the method using transient transfection transfects 293T cell.293T cell is using containing 10%FBS's DMEM in high glucose culture medium, is placed in 37 DEG C, culture in 5%CO2 incubator.Transfect first 1 day digestion 293T cell, count, cell is connect Plant to 35mm culture dish, each culture dish inoculation 6 × 105Individual cell；The secondary daily DMEM culture medium without FBS is prepared respectively Calcium turns reagent and RNA interference plasmid, adds 16 μ l calcium to turn reagent in every 84 μ l culture medium, adds 4 μ g matter in every 100 μ l culture medium Calcium is turned solution after mixing respectively and plasmid solution gently mixes by grain, and room temperature stands 10 minutes, is added dropwise in culture dish, 37 DEG C, culture 24h in 5%CO2 incubator, changes the fresh DMEM in high glucose culture medium containing 10% FBS and continues culture 48h, collect Cell, extracts cell RNA.
Embodiment 3
1. the RT-PCR detection of the extraction of total serum IgE, the synthesis of cDNA and RNA interference effect
Take 1 μ g RNA reverse transcription cDNA respectively, detect the expression of PLC ε using fluorescence quantitative PCR method, with beta-actin As internal reference, the primer sequence that quantitative fluorescent PCR is adopted is shown in Table 2, adopts 2-△△CTRelative quantitation method calculates different RNA and does Disturb the PLC ε expression of sequence sample.Filter out the minimum RNA interference sequence of expression.Testing result（See Fig. 3）Show The PLC ε gene expression dose of U4 sequence is minimum, illustrates that the RNA interference effect of U4 sequence is best.
2.PLC ε and the design of beta-actin primed probe
Primer Sequence
F-PLCε TCTTCGCACAATACCTACCTCAC
R-PLCε ATCAATGGCTTCAACCACTTCCT
F-actinb TATCGCCGCGCTCGTCGTC
R-actinb TCTTGCTCTGGGCCTCGTC
SEQUENCE LISTING
<110>Tongji University Suzhou academy
<120>Targeting PLC ε gene RNA interference recombinant lentivirus vector and its construction method
<130> 2016
<160> 8
<170> PatentIn version 3.5
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<212> DNA
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gatccccact ttgttagtca ggagatctcg agatctcctg actaacaaag tggtttttg 59
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<213>Synthetic
<400> 2
aattcaaaaa ccactttgtt agtcaggaga tctcgagatc tcctgactaa caaagtggg 59
<210> 3
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<212> DNA
<213>Synthetic
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gatccgcgga ttattggaac tcactactcg agtagtgagt tccaataatc cgctttttg 59
<210> 4
<211> 59
<212> DNA
<213>Synthetic
<400> 4
aattcaaaaa gcggattatt ggaactcact actcgagtag tgagttccaa taatccgcg 59
<210> 5
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<212> DNA
<213>Synthetic
<400> 5
gatccccatc atttatcatg gacatactcg agtatgtcca tgataaatga tggtttttg 59
<210> 6
<211> 59
<212> DNA
<213>Synthetic
<400> 6
aattcaaaaa ccatcattta tcatggacat actcgagtat gtccatgata aatgatggg 59
<210> 7
<211> 59
<212> DNA
<213>Synthetic
<400> 7
gatccgctgc aatgtttgag gcaaatctcg agatttgcct caaacattgc agctttttg 59
<210> 8
<211> 59
<212> DNA
<213>Synthetic
<400> 8
aattcaaaaa gctgcaatgt ttgaggcaaa tctcgagatt tgcctcaaac attgcagcg 59

Claims (3)

1. a kind of siRNA recombinant slow virus expression vector of targeting PLC ε gene, is for tumor PLC ε gene-correlation therapeutic Material, interference slow viruss are to be obtained by pLv-PLC ε shRNA, pMD2.G and pSPAX2 carrier cotransfection 293T cell culture；Its In,
Described pLv-PLC ε shRNA recombinant vector is connection double chain DNA fragment in the multiple clone site of pLv-shRNA carrier Build and obtain, restriction enzyme site is BamHI and EcoRI；Described double chain DNA fragment is one of following sequence：
(1) PLC ε-homo-U1 oligonucleotide sequence 1:
Positive-sense strand:
5’GATCCCCACTTTGTTAGTCAGGAGATCTCGAGATCTCCTGACTAACAAAGTGGTTTTTG3’
Antisense strand:
5’AATTCAAAAACCACTTTGTTAGTCAGGAGATCTCGAGATCTCCTGACTAACAAAGTGGG3’
(2) PLC ε-homo-U2 oligonucleotide sequence 2:
Positive-sense strand:
5’GATCCGCGGATTATTGGAACTCACTACTCGAGTAGTGAGTTCCAATAATCCGCTTTTTG3’
Antisense strand:
5’AATTCAAAAAGCGGATTATTGGAACTCACTACTCGAGTAGTGAGTTCCAATAATCCGCG3’
(3) PLC ε-homo-U3 oligonucleotide sequence 3:
Positive-sense strand:
5’GATCCCCATCATTTATCATGGACATACTCGAGTATGTCCATGATAAATGATGGTTTTTG3’
Antisense strand:
5’AATTCAAAAACCATCATTTATCATGGACATACTCGAGTATGTCCATGATAAATGATGGG3’
(4) PLC ε-homo-U4 oligonucleotide sequence 4:
Positive-sense strand:
5’GATCCGCTGCAATGTTTGAGGCAAATCTCGAGATTTGCCTCAAACATTGCAGCTTTTTG3’
Antisense strand:
5’AATTCAAAAAGCTGCAATGTTTGAGGCAAATCTCGAGATTTGCCTCAAACATTGCAGCG3’.
2. the construction method of the siRNA recombinant slow virus expression vector of targeting PLC ε gene according to claim 1,
As follows including step:
A. according to PLC ε mRNA sequence, design synthetic dsdna fragment, described double chain DNA fragment be described PLC ε- One of homo-U1, PLC ε-homo-U2, PLC ε-homo-U3 and PLC ε-homo-U4 nucleotide sequence;
B. and then described double chain DNA fragment is connected in the multiple clone site of plv-shRNA carrier and is built into pLv-PLC ε ShRNA recombinant vector;
C. again by described pLv-PLC ε shRNA recombinant vector and pMD2.G and pSPAX2 carrier cotransfection 293T cell culture, Obtain the recombined lentivirus vector system of targeting PLC ε gene RNA interference.
3. the siRNA recombinant slow virus expression vector of the targeting PLC ε gene described in claim 1 is related in preparation treatment tumor Application in property genomic medicine.
CN201610979733.3A 2016-11-08 2016-11-08 PLC (phospholipase C) epsilon gene targeted RNA-interference recombinant lentiviral vectors and construction method thereof Pending CN106399378A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103725682A (en) * 2013-12-30 2014-04-16 深圳先进技术研究院 Small interfering ribonucleic acid (RNA) for knocking down expression of estrogen related receptor (ERR), and recombinant vector and application of RNA
CN103834691A (en) * 2014-01-21 2014-06-04 宁波大学 Construction method of target RNA-interfered recombinant lentivirus vector for IL-33 gene
CN103882058A (en) * 2012-12-24 2014-06-25 苏州中同生物科技有限公司 Construction of lentivirus recombinant shuttle vector
CN104975044A (en) * 2015-05-22 2015-10-14 浙江大学 Construction of SRSF6-targeting lentivirus interference vector and applications of SRSF6-targeting lentivirus interference vector in colorectal cancer treatment

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103882058A (en) * 2012-12-24 2014-06-25 苏州中同生物科技有限公司 Construction of lentivirus recombinant shuttle vector
CN103725682A (en) * 2013-12-30 2014-04-16 深圳先进技术研究院 Small interfering ribonucleic acid (RNA) for knocking down expression of estrogen related receptor (ERR), and recombinant vector and application of RNA
CN103834691A (en) * 2014-01-21 2014-06-04 宁波大学 Construction method of target RNA-interfered recombinant lentivirus vector for IL-33 gene
CN104975044A (en) * 2015-05-22 2015-10-14 浙江大学 Construction of SRSF6-targeting lentivirus interference vector and applications of SRSF6-targeting lentivirus interference vector in colorectal cancer treatment

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHENG H等: "shRNA Targeting PLCε Inhibits Bladder Cancer Cell Growth In Vitro and In Vivo", 《UROLOGY》 *
KUN YANG等: "Effect of PLCε gene silencing on inhibiting the cancerous transformation of ulcerative colitis", 《EXPERIMENTAL AND THERAPEUTIC MEDICINE》 *
张彦懿等: "PLCε 基因shRNA重组腺病毒表达质粒的构建及鉴定", 《中国生物制品学杂志》 *
欧俐苹等: "阻断PLCE基因对膀胱癌细胞侵袭能力的影响", 《基础医学与临床》 *
郑传宜等: "人GLUT3基因RNA干扰病毒载体的构建与鉴定", 《中南大学学报》 *
郭永灿等: "PLCε shRNA质粒构建及功能鉴定", 《第三军医大学学报》 *

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