CN105734058B - SiRNA, recombinant vector and the purposes of the HAVCR2 gene of silencing people - Google Patents
SiRNA, recombinant vector and the purposes of the HAVCR2 gene of silencing people Download PDFInfo
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- CN105734058B CN105734058B CN201410748574.7A CN201410748574A CN105734058B CN 105734058 B CN105734058 B CN 105734058B CN 201410748574 A CN201410748574 A CN 201410748574A CN 105734058 B CN105734058 B CN 105734058B
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Abstract
The present invention relates to s iRNA of HAVCR2 gene of silencing people and application thereof, the s iRNA has the sequence such as SEQ ID NO.1.The present invention also provides s iRNA recombinant vectors of the HAVCR2 gene containing above-mentioned silencing people and application thereof, by the recombinant vector containing s iRNA being transfected into the HAVCR2 overexpression type cell of people, stable clone is obtained, the expression of HAVCR2 is blocked using RNA perturbation technique.S iRNA, the recombinant vector of the HAVCR2 gene of silencing people provided by the invention can provide effective technical tool for the research of the HAVCR2 gene function of people.
Description
Technical field
The invention belongs to molecular biology and biopharmaceutical technology, and in particular to the s of the HAVCR2 gene of silencing people
IRNA, recombinant vector and purposes.
Background technique
RNAi is a kind of posttranscriptional gene silencing mechanism, it passes through double-stranded RNA (double stranded RNA, dsRNA)
The mRNA (microRNAs) of sequence homology is caused to degrade, to reduce the expression of target gene, this phenomenon is protected during evolution
It keeps and extensively.RNAi technology has come into many fields of bioscience research at present, becomes a kind of main biology
Research tool is learned, provides a kind of new research means for analysis gene function and signal transduction pathway and gene therapy, in
The acquisition Nobel Prize in 2006.RNAi has following characteristics advantage: the specificity of 1. effects, only generates to the mRNA homologous with it
Inhibiting effect, base mispairing will be greatly reduced inhibition efficiency；2.RNAi has the altitude validity, " siRNA of relatively small amount
(smal l interfering RNA, s iRNA) " can almost inhibit the expression of target gene.It is defeated in active somatic cell
Sending a kind of method of s iRNA is, is cloned into plasmid vector for s iRNA sequence as " short hair clip ".When being sent into cell body,
The hairpin is expressed, and forms the bobby pin or short hairpin RNA (a small hairpin RNA or of a double-strand
Short hairpin RNA, shRNA), and handled by the channel RNAi, it is integrated to RNA induction silencing complex (RNA- later
Induced silencing complex, RISC), this compound can be combined and be cut and s iRNA sequences match
MRNA causes mRNA that can not translate into corresponding protein, the expression of corresponding albumen in final silenced cell.ShRNA includes two
Short inverted repeats, centre are stem ring (loop) sequence, and inverted repeats and stem ring sequence form a hairpin structure.
Hepatitis A virus receptor 2 (HAVCR2) i.e. Tim3 albumen is a kind of participation cellular immunity of TIM3 gene coding
The cell-membrane receptor albumen of regulation is that the important negativity of adaptive immunity reaction adjusts molecule, weight is played in diseases associated with inflammation
It acts on, is expressed in CD4+T cell, CD8+T cell, NK cell and monocyte/macrophage of activation etc..(the Ju such as Ju
Y,Hou N,Zhang XN,et al.Blockade of Tim-3pathway ameliorates interferon-gamma
production from hepatic CD8+T cells in a mouse model of hepatitis B virus
Infection [J] .Cell Mol Immunol, 2009,6 (1): 35-43.) research discovery Peripheral Blood in Patients with Chronic Hepatitis B monokaryon
Tim-3 expression is significantly increased on monocyte in cell (especially NK cell and CD8+T cell) and liver, this prompt Tim-3
Albumen is likely to equally play an important role during hepatitis B (HBV) infects.
Summary of the invention
For the needs of gene studies, it is an object of the present invention to provide a kind of s of the HAVCR2 gene of silencing people
IRNA can effectively inhibit the expression of HAVCR2 gene in HAVCR2 overexpression type liver cell, can be people HAVCR2 gene function
Research effective technical tool is provided.
A second object of the present invention is to provide the purposes of the s iRNA of the HAVCR2 gene of above-mentioned silencing people.
Third object of the present invention is to provide the recombinant vectors of the s iRNA of the HAVCR2 gene of silencing people a kind of.
Fourth object of the present invention is to provide the use of the recombinant vector of the s iRNA of the HAVCR2 gene of above-mentioned silencing people
On the way.
To achieve the above objectives, the technical solution adopted by the present invention is that: the siRNA of the HAVCR2 gene of silencing people has
Such as the sequence of SEQ ID NO.1.
The purposes of the siRNA of the HAVCR2 gene of silencing people provided by the invention is that it is thin in preparation HAVCR2 gene silencing
Purposes in born of the same parents' model.
The recombinant vector of the siRNA of the HAVCR2 gene of silencing people provided by the invention contains above-mentioned silencing people's
The siRNA of HAVCR2 gene.
Further, the recombinant vector is obtained using two-step method building, and specific construction method is: the first step, iii vitro chemical
The siRNA expression template for synthesizing HAVCR2 gene is single-stranded, and annealing forms double-stranded DNA in annealing buffer；Second step will be formed
Double-stranded DNA be cloned into expression vector, to be built into recombinant vector.
Further, the expression vector is siRNA carrier for expression of eukaryon Psilencer2.1-U6, pGCsi-U6/Neo/
GFP, pGenesil-1.1 or slow virus carrier pGC-LV.
The purposes of the recombinant vector of the siRNA of the HAVCR2 gene of silencing people provided by the invention is it in preparation HAVCR2
Purposes in gene silencing cell model.
It is provided by the present invention can effective reticence people HAVCR2 gene expression siRNA and recombination containing the siRNA
Carrier can effectively inhibit the expression of HAVCR2 gene in HAVCR2 overexpression type liver cell, can be people HAVCR2 gene function
Research provides effective technical tool.
Detailed description of the invention
Fig. 1 is the HAVCR2- that the HAVCR2 overexpression type 293T cell of people is detected using Westeern Blot detection method
The X-ray development figure obtained when GFP fusion protein, GAPDH are the internal reference for measuring HAVCR2 relative expression quantity, " 1 " column
Corresponding to swimming lane where having transfected the HAVCR2 overexpression type 293T cell controls group of control plasmid pGC-NC-siRNA, " 2 "
Column correspond to the HAVCR2 overexpression type 293T cell experiment group place swimming lane for having transfected plasmid pGC-HAVCR2-siRNA.
Specific embodiment
Hereinafter reference will be made to the drawings, and a preferred embodiment of the present invention will be described in detail.It is not specified in preferred embodiment
The experimental method of actual conditions, usually according to normal condition, such as Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brooker
Deng writing, Huang Peitang etc. translated, Science Press, 2002) described in condition, or according to the normal condition proposed by manufacturer.
Embodiment 1
The present embodiment is used to illustrate the construction method of the siRNA of the HAVCR2 gene of silencing people provided by the invention.
According to the nucleotide sequence for the HAVCR2 gene reported in Genbank, 1 is designed with reference to siRNA design principle
Inhibit the siRNA target sequence of HAVCR2 gene expression, is SEQ ID NO.1, while designing random controls sequence SEQ ID
NO.2, as follows:
SEQ ID NO.1:UUGGGUUGUCGCUUUGCAA；
SEQ ID NO.2:UUCUCCGAACGUGUCACGU.
Through Blast Genetic homology of carbapenem-resistant, SEQ ID NO.1 sequence is not same with the non-HAVCR2 gene order of any mankind
Source, to ensure the specificity of gene inhibition.
A pair of of shRNA sequence (SEQ ID is separately designed with reference to the layout strategy of s iRNA for above-mentioned target sequence
NO.1 ') and a pair of of random controls sequence (SEQ ID NO.2 '), it is as follows:
SEQ ID NO.1 ':
Positive-sense strand:
5’-Ccgg TTGGGTTGTCGCTTTGCAA CTCGAGTTGCAAAGCGACAACCCAA TTTTTg-3’
Antisense strand:
5’-aattcaaaaa TTGGGTTGTCGCTTTGCAA CTCGAGTTGCAAAGCGACAACCCAA-3’
SEQ ID NO.2 ':
Positive-sense strand:
5’-ccgg CA TTCTCCGAACGTGTCACGT CTCGAGACGTGACACGTTCGGAGAA TG TTTTTg-3’
Antisense strand:
5’-aattcaaaaa CA TTCTCCGAACGTGTCACGT CTCGAGACGTGACACGTTCGGAGAA TG-3’
Using the positive-sense strand and antisense strand for being chemically synthesized sequence SEQ ID NO.1 ' and SEQ ID NO.2 ', sequence
Middle CTCGAG is loop ring sequence, and TTTTT is transcription terminator.
SEQ ID NO.1 ' and SEQ ID NO.2 ' forms the packet with CTCGAG for loop stem ring after transcribing in vivo respectively
The s iRNA hairpin structure of ID containing SEQ NO.1 and SEQ ID NO.2, cutting generate sequence respectively in vivo is SEQ ID
The s iRNA and sequence of the HAVCR2 of NO.1 is the control siRNA of SEQ ID NO.2.
Embodiment 2
The present embodiment is used to illustrate the building of the recombinant vector of the siRNA of the HAVCR2 gene of silencing people provided by the invention
Method.
Carrier is using slow virus carrier pGC-LV (being purchased from Shanghai JiKai Gene Chemical Technology Co., Ltd).Its frame structure
Are as follows:
hU6-MCS-Ubiquitin-EGFP-IRES-puromycin。
The construction method of recombinant vector is as follows:
By the positive-sense strand of the shRNA sequence SEQ ID NO.1 ' of s iRNA and antisense strand in embodiment 1 in annealing buffer
(10mM Tris-HCl, 1mM EDTA, 0.1mM NaCl) 90 DEG C of water-bath 15min, then cooled to room temperature, reaction synthesize
Double-stranded DNA (dsDNA) sequence, by double chain DNA sequence I digestion of EcoR I and Age, and meanwhile it is sick slowly using EcoR I and I digestion of Age
Poisonous carrier pGC-LV makes it generate cohesive end identical with double chain DNA sequence, using T4DNA ligase that double digestion is linear
The slow virus carrier and DNA fragmentation of change are stayed overnight in 16 DEG C of connections.Connection product transformed competence colibacillus cell DH5 α, picking transformant weight
It is suspended from LB culture medium, takes 1 μ L as template, positive colony is identified by PCR.The positive colony of PCR screening and identification is entrusted
Sangon Biotech's sequencing identification, sequencing result further screen recombinant vector.The recombination of building
Carrier can direct transfection eukaryocyte, can be used for the packaging of slow virus.Construct the SEQ of sequence containing the shRNA ID obtained
The recombinant vector (plasmid) of NO.1 ' can be expressed as pGC-HAVCR2-siRNA.
It obtains to be expressed as containing the recombinant vector (plasmid) of random controls sequence SEQ ID NO.2 ' in the same way
PGC-NC-siRNA, as control.
Embodiment 3
The present embodiment is used to illustrate the recombinant vector of the siRNA of the HAVCR2 gene of silencing people provided by the invention to people's
The inhibiting effect of the HAVCR2 gene expression of HAVCR2 overexpression type 293T cell.
1, cell culture
By the HAVCR2 overexpression type 293T cell inoculation of people in good condition in 6 porocyte culture plates, containing 10%
(v/v) (U.S., Invitrogen are public in the DMEM culture medium of calf serum, 100U/ml penicillin and 100mg/L streptomysin
Department), in 37 DEG C, 5% (v/v) CO2Under the conditions of cultivate, reach about 80% to cell fusion degree.Meanwhile it is the 293T cultivated is thin
Born of the same parents are divided into 2 groups, 1 group of experimental group and 1 group of control group.
2, gene transfects
To the plasmid and the SEQ of sequence containing random controls ID of the SEQ ID of sequence containing the shRNA NO.1 ' that embodiment 2 obtains
The plasmid of NO.2 ' carries out transfection procedure according to Invi trogen Lipofectamine2000 transfection reagent specification respectively.
The specific method is as follows:
Culture solution is replaced with the DMEM culture medium that 2ml is free of serum, takes the plasmid and 10 μ l of 4.0 μ g embodiments 2
Lipofectamine2000 is dissolved separately in the DMEM culture medium of 250 μ l serum-frees, is mixed, is stored at room temperature 5min, then will
Plasmid-culture medium, Lipofectamine2000- culture medium mixed liquor are uniformly mixed, and 20min are placed at room temperature for, then by mixed liquor
Be added in 293T cell (mixed liquor for containing the plasmid of shRNA sequence SEQ ID NO.1 ' is added in experimental group 293T cell,
The mixed liquor for containing the plasmid of random controls sequence SEQ ID NO.2 ' is added in control group 293T cell), in the incubator
37 DEG C, 5% (v/v) CO2Under the conditions of cultivate 12h after, change the complete medium containing fresh 10% (v/v) serum into, transfection is for 24 hours
It observes the expression of fluorescent marker gene afterwards to judge transfection efficiency, collects cell progress subsequent detection after transfecting 48h.
3, total protein extracts
To the cell of the experimental group and control group taken out from incubator, following total protein extraction procedure is carried out respectively:
Cell culture fluid is discarded, PBS is washed 2 times；PBS is discarded, 2 × LysisBuffer (the 1M Tri being pre-chilled in right amount is added
S-HCl (pH 6.8,100mM), mercaptoethanol (2%), glycerol (20%), SDS (4%))；Cell is scraped with cell scraper, by sample
Product are transferred in 1.5ml eppendorf pipe, on ice lytic cell 10-15min, Ultrasonic Cell Disruptor smudge cells, and 4 DEG C,
12000g (unit that " g " is centrifugal force) is centrifuged 15min, takes supernatant, measures protein concentration.
Each sample protein concentration is adjusted to 2 μ g/ μ l, and -80 DEG C save backup.
4, immunoblotting
Each sample takes identical total protein concentration, and 2 × loading buffer sample-loading buffer (1M of same volume is added
Tris-HCl (pH 6.8,100mM), mercaptoethanol (2%), glycerol (20%), SDS (4%), mercaptoethanol (2%)), it mixes；
PAGE gel electrophoresis is carried out to sample, after electrophoresis, using electrophoretic blotting device, under 4 DEG C, 400mA constant current conditions
Electricity turns 120min, will be on protein delivery to pvdf membrane；Electricity uses confining liquid (the TBST solution containing 5% skim milk) room after the completion of turning
Temperature closes pvdf membrane 1h or 4 DEG C are stayed overnight；Dilute primary antibody (GFP antibody) with above-mentioned confining liquid, then with the pvdf membrane room closed
Temperature is incubated for 2h or 4 DEG C overnight, and TBST is washed film 3 times, each 10min, dilutes corresponding secondary antibody (mouse IgG- with above-mentioned confining liquid
HRP), it is incubated for pvdf membrane 2h at room temperature, TBST is washed film 3 times, each 10min, using Amersham company ECL+plus
Western blotting system kit develops the color, the development of darkroom X-ray, the result is shown in Figure 1.
5, interpretation of result
HAVCR2 expression quantity is analyzed by gray value, using GAPDH as internal reference, relative quantification HAVCR2 expression.
HAVCR2 relative expression quantity is defined as: HAVCR2 gray value/GAPDH gray value.
In Fig. 1, cell controls group (" 1 " swimming lane) gray value of pGC-NC-siRNA has been transfected are as follows: 0.90 ± 0.08, transfection
The cell experiment group of pGC-HAVCR2-siRNA (" 2 " swimming lane) gray value are as follows: 0.03 ± 0.00, transfect pGC-HAVCR2-
The cell experiment group of siRNA is compared with having transfected pGC-NC-siRNA control group, and HAVCR2 expressing quantity is obviously lowered, difference
With statistical significance (P < 0.05), this illustrates that the siRNA of silencing HAVCR2 provided by the invention efficiently can specifically press down
The expression of HAVCR2 gene in HAVCR2 overexpression type 293T cell processed.
Above-described embodiment is only to of the invention for example, the present invention can also be with other ad hoc fashions or others
Particular form is implemented, without departing from the gist of the invention or substantive characteristics.Therefore, the embodiment of description is in any way
It is regarded as illustrative and non-limiting.The scope of the present invention should illustrate by appended claims, any and claim
Intention and the equivalent variation of range should also be included in the scope of the present invention.
Claims (6)
1. the siRNA of the HAVCR2 gene of silencing people, which is characterized in that sequence is as shown in SEQ ID NO.1.
2. the siRNA of the HAVCR2 gene of silencing people described in claim 1 is in preparation HAVCR2 gene silencing cell model
Purposes.
3. the recombinant vector of the siRNA of the HAVCR2 gene of silencing people, which is characterized in that the recombinant vector contains claim
The siRNA of the HAVCR2 gene of silencing people described in 1.
4. recombinant vector according to claim 3, which is characterized in that the recombinant vector is obtained using two-step method building,
Specific construction method is: the first step, the siRNA expression template that iii vitro chemical synthesizes HAVCR2 gene is single-stranded, in annealing buffer
Middle annealing forms double-stranded DNA；The double-stranded DNA of formation is cloned into expression vector, to be built into recombinant vector by second step.
5. recombinant vector according to claim 4, which is characterized in that the expression vector is siRNA carrier for expression of eukaryon
Psilencer2.1-U6, pGCsi-U6/Neo/GFP, pGenesil-1.1 or slow virus carrier pGC-LV.
6. the recombinant vector of the siRNA of the HAVCR2 gene of silencing people as claimed in claim 3 to 5 is in preparation HAVCR2 base
Because of the purposes in silenced cell model.
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| CN104185681A (en) * | 2012-02-01 | 2014-12-03 | 卡姆普根有限公司 | C10RF32 antibodies, and uses thereof for treatment of cancer |
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| CN104185681A (en) * | 2012-02-01 | 2014-12-03 | 卡姆普根有限公司 | C10RF32 antibodies, and uses thereof for treatment of cancer |
Non-Patent Citations (3)
| Title |
|---|
| 《Tim-3在HBV慢性感染中的表达及作用研究》;鞠瑛;《中国优秀博士学位论文全文数据库医药 卫生科技辑》;20100515(第5期);第E061-2页 * |
| 《Tim-3在人肝癌细胞中的表达及其对细胞增殖与迁移能力的影响》;魏东等;《重庆医学》;20140630;第43卷(第16期);第1975-1982页 * |
| 《Tim-3在肿瘤相关巨噬细胞极化及肝细胞肝癌进展中的作用及机制研究》;阎文江等;《中国优秀硕士学位论文全文数据库医药 卫生科技辑》;20141115(第11期);第E072-508页 * |
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