CN105734058A - SiRNA of silent human HAVCR2 (Hepatitis A virus cellular receptor 2) gene, recombinant vector, and applications of recombinant vector and SiRNA - Google Patents

Abstract

The invention relates to siRNA of a silent human HAVCR2 (Hepatitis A virus cellular receptor 2) gene and an application of the siRNA, wherein the siRNA has a sequence shown as SEQ ID NO.1. The invention further provides a recombinant vector comprising the siRNA of the silent human HAVCR2 gene, and an application of the recombinant vector; through transfection of the siRNA-containing recombinant vector to human HAVCR2 overexpression-type cells, a stable clone body is obtained; and the expression of HAVCR2 is blocked through the RNA interference technology. The siRNA of the silent human HAVCR2 gene and the recombinant vector can provide an effective technological tool for the research on the functions of the human HAVCR2 gene.

Description

The reticent siRNA of HAVCR2 gene of people, recombinant vector and purposes

Technical field

The invention belongs to molecular biology and biological pharmacy technical field, be specifically related to the reticent siRNA of HAVCR2 gene of people, recombinant vector and purposes.

Background technology

RNAi is a kind of PTGS mechanism, it passes through double-stranded RNA (doublestrandedRNA, dsRNA) mRNA (microRNAs) causing sequence homology degrades, and reduces the expression of target gene, and this phenomenon is conservative during evolution and extensive.Current RNAi technology has come into many fields of bioscience research, becomes a kind of main biological research tool, provides a kind of new research means for analyzing gene function and signal transduction pathway and gene therapy, obtained Nobel Prize in 2006.RNAi has following characteristics advantage: 1. the specificity of effect, only the mRNA with its homology is produced inhibitory action, and base mispairing can be substantially reduced suppression efficiency；2.RNAi has altitude validity, and " siRNA (smallinterferingRNA, the siRNA) " of relatively small amount just almost can suppress the expression of target gene completely.A kind of way carrying siRNA in active somatic cell is that as " bob folder ", siRNA sequence is cloned into plasmid vector.When sending in cyton, this hairpin is expressed, form bobby pin or the short hairpin RNA (asmallhairpinRNAorshorthairpinRNA of a double-strand, shRNA), and processed by RNAi passage, it is attached to RNA afterwards and induces silencing complex (RNA-inducedsilencingcomplex, RISC), this complex can in conjunction with and cut the mRNA mated with siRNA sequence, cause that mRNA cannot translate into respective egg white matter, the expression of corresponding protein in final silenced cell.ShRNA includes two short inverted repeat, and centre is stem ring (loop) sequence, and inverted repeat and stem ring sequence form a hairpin structure.

Hepatitis A virus receptor 2 (HAVCR2) i.e. Tim3 albumen, it is a kind of cell-membrane receptor albumen participating in cellular immunization regulation and control of TIM3 gene code, it it is the important negativity Molecular regulator of adaptive immunity reaction, playing a significant role in diseases associated with inflammation, it is expressed in the CD4+T cell of activation, CD8+T cell, NK cell and monocyte/macrophage etc..null(the JuY such as Ju,HouN,ZhangXN,etal.BlockadeofTim-3pathwayamelioratesinterferon-gammaproductionfromhepaticCD8+TcellsinamousemodelofhepatitisBvirusinfection[J].CellMolImmunol，2009，6 (1): 35-43.) research finds that in Peripheral Blood in Patients with Chronic Hepatitis B mononuclear cell (especially NK cell and CD8+T cell) and liver, on mononuclear cell, Tim-3 expression is significantly increased，This prompting Tim-3 albumen is likely to play an important role equally in the process that hepatitis B virus (HBV) infects.

Summary of the invention

Needs for gene studies, it is an object of the present invention to provide the siRNA of the HAVCR2 gene of a kind of reticent people, it can effectively suppress the expression of HAVCR2 gene in HAVCR2 process LAN type hepatocyte, can be that the research of people's HAVCR2 gene function is provided with effect technique instrument.

Second purpose of the present invention is to provide the purposes of the siRNA of the HAVCR2 gene of above-mentioned reticent people.

3rd purpose of the present invention is to provide the recombinant vector of the siRNA of the HAVCR2 gene of a kind of reticent people.

4th purpose of the present invention is to provide the purposes of the recombinant vector of the siRNA of the HAVCR2 gene of above-mentioned reticent people.

For reaching object above, the technical solution used in the present invention is: the siRNA of the HAVCR2 gene of reticent people, has the sequence such as SEQIDNO.1.

The purposes of the siRNA of the HAVCR2 gene of reticent people provided by the invention is its purposes in preparing HAVCR2 gene silencing cell model.

The recombinant vector of the siRNA of the HAVCR2 gene of reticent people provided by the invention, the siRNA of the HAVCR2 gene containing above-mentioned reticent people.

Further, described recombinant vector adopts two-step method to build and obtains, and concrete construction method is: the first step, and the siRNA expression template strand of iii vitro chemical synthesis HAVCR2 gene, in annealing buffer, annealing forms double-stranded DNA；Second step, is cloned into expression vector by the double-stranded DNA of formation, thus being built into recombinant vector.

Further, described 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 reticent people provided by the invention is its purposes in preparing HAVCR2 gene silencing cell model.

Provided by the present invention can the siRNA of HAVCR2 gene expression of effectively reticent people and recombinant vector containing this siRNA, can effectively suppress the expression of HAVCR2 gene in HAVCR2 process LAN type hepatocyte, can be that the research of people's HAVCR2 gene function is provided with effect technique instrument.

Accompanying drawing explanation

The X-ray development figure that Fig. 1 obtains when being the HAVCR2-GFP fusion protein of the HAVCR2 process LAN type 293T cell adopting WesteernBlot detection method detection people, GAPDH is the internal reference for measuring HAVCR2 relative expression quantity, " 1st " row HAVCR2 process LAN type 293T cell controls group place swimming lane corresponding to having transfected control plasmid pGC-NC-siRNA, " 2nd " row HAVCR2 process LAN type 293T cell experiment group place swimming lane corresponding to having transfected plasmid pGC-HAVCR2-siRNA.

Detailed description of the invention

Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in preferred embodiment, generally conventionally condition, for instance the Molecular Cloning: A Laboratory guide (third edition, J. the work such as Pehanorm Brooker, yellow training hall etc. is translated, Science Press, 2002) described in condition, or according to manufacturer it is proposed that condition.

Embodiment 1

The present embodiment is for illustrating the construction method of the siRNA of the HAVCR2 gene of reticent people provided by the invention.

According to the nucleotide sequence of HAVCR2 gene of report in Genbank, design 1 siRNA target sequence suppressing HAVCR2 gene expression with reference to siRNA design principle, for SEQIDNO.1, simultaneously design random controls sequence SEQIDNO.2, as follows:

SEQIDNO.1:UUGGGUUGUCGCUUUGCAA；

SEQIDNO.2:UUCUCCGAACGUGUCACGU.

Through Blast Genetic homology of carbapenem-resistant, not non-with any mankind HAVCR2 gene order homology of SEQIDNO.1 sequence, to guarantee the specificity of gene inhibition.

For above-mentioned target sequence, with reference to the layout strategy of siRNA, separately design a pair shRNA sequence (SEQIDNO.1 ') and a pair random controls sequence (SEQIDNO.2 '), as follows:

SEQIDNO.1 ':

Positive-sense strand:

5’-CcggTTGGGTTGTCGCTTTGCAACTCGAGTTGCAAAGCGACAACCCAATTTTTg-3’

Antisense strand:

5’-aattcaaaaaTTGGGTTGTCGCTTTGCAACTCGAGTTGCAAAGCGACAACCCAA-3’

SEQIDNO.2 ':

Positive-sense strand:

5’-ccggCATTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAATGTTTTTg-3’

Antisense strand:

5’-aattcaaaaaCATTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAATG-3’

Employing is chemically synthesized positive-sense strand and the antisense strand of sequence SEQIDNO.1 ' and SEQIDNO.2 ', and in its sequence, CTCGAG is loop ring sequence, and TTTTT is transcription terminator.

SEQIDNO.1 ' and SEQIDNO.2 ' is formed after transcribing in vivo respectively with the CTCGAG siRNA hairpin structure comprising SEQIDNO.1 and SEQIDNO.2 being loop stem ring, cuts the siRNA producing the HAVCR2 that sequence is the SEQIDNO.1 respectively and comparison siRNA that sequence is SEQIDNO.2 in vivo.

Embodiment 2

The present embodiment is for illustrating the construction method of the recombinant vector of the siRNA of the HAVCR2 gene of reticent people provided by the invention.

Carrier adopts slow virus carrier pGC-LV (purchased from Shanghai JiKai Gene Chemical Technology Co., Ltd).Its frame structure is:

hU6-MCS-Ubiquitin-EGFP-IRES-puromycin。

The construction method of recombinant vector is as follows:

By the positive-sense strand of the shRNA sequence SEQIDNO.1 ' of siRNA in embodiment 1 and antisense strand at annealing buffer (10mMTris-HCl, 1mMEDTA, 0.1mMNaCl) 90 DEG C of water-bath 15min, then room temperature is naturally cooled to, double-stranded DNA (dsDNA) sequence is synthesized, by double chain DNA sequence with EcoR I and Age I enzyme action, utilize EcoR I and Age I enzyme action slow virus carrier pGC-LV simultaneously, make it produce the sticky end identical with double chain DNA sequence, utilize T4DNA ligase linearizing for double digestion slow virus carrier and DNA fragmentation to be connected overnight in 16 DEG C.Connecting product transformed competence colibacillus cell DH5 α, picking transformant is resuspended in LB culture medium, takes 1 μ L as template, identifies positive colony by PCR.Positive colony student on commission work biological engineering (Shanghai) the limited company order-checking of PCR Screening and Identification being identified, sequencing result screens recombinant vector further.The recombinant vector built can direct transfection eukaryotic cell, it is also possible to for the packaging of slow virus.Build the recombinant vector containing shRNA sequence SEQIDNO.1 ' (plasmid) obtained and can be expressed as pGC-HAVCR2-siRNA.

The recombinant vector (plasmid) obtained containing random controls sequence SEQIDNO.2 ' by same method can be expressed as pGC-NC-siRNA, as comparison.

Embodiment 3

The present embodiment is used for the recombinant vector that the siRNA of the HAVCR2 gene of reticent people provided by the invention the is described inhibitory action to the HAVCR2 gene expression of the HAVCR2 process LAN type 293T cell of people.

1, cell is cultivated

The HAVCR2 process LAN type 293T cell of people in good condition is inoculated in 6 porocyte culture plates, (the U.S. in the DMEM culture medium containing 10% (v/v) calf serum, 100U/ml penicillin and 100mg/L streptomycin, Invitrogen company), in 37 DEG C, 5% (v/v) CO₂Cultivate under condition, treat that cell fusion degree reaches about 80%.Meanwhile, the 293T cell cultivated is divided into 2 groups, 1 group of experimental group and 1 group of matched group.

2, gene transfection

The plasmid containing shRNA sequence SEQIDNO.1 ' that embodiment 2 is obtained and the plasmid containing random controls sequence SEQIDNO.2 ', carry out transfection procedure according to InvitrogenLipofectamine2000 transfection reagent description respectively.Concrete grammar is as follows:

Culture fluid is replaced with the 2ml DMEM culture medium without serum, the plasmid and the 10 μ lLipofectamine2000 that take 4.0 μ g embodiments 2 are dissolved separately in the DMEM culture medium of 250 μ l serum-frees, mixing, room temperature stands 5min, then by plasmid-culture medium, Lipofectamine2000-culture medium mixed liquor mix homogeneously, room temperature places 20min, then added by mixed liquor in 293T cell (joins in experimental group 293T cell by the mixed liquor of the plasmid containing shRNA sequence SEQIDNO.1 ', the mixed liquor of the plasmid containing random controls sequence SEQIDNO.2 ' is joined in matched group 293T cell), in incubator 37 DEG C, 5% (v/v) CO₂After cultivating 12h under condition, change the complete medium containing fresh 10% (v/v) serum into, observe the expression of fluorescent marker gene after transfection 24h to judge transfection efficiency, collect cell after transfection 48h and carry out subsequent detection.

3, total protein extracting

Cell to the experimental group taken out from incubator and matched group, carries out following total protein extraction procedure respectively:

Discarding cell culture fluid, PBS washs 2 times；Discard PBS, add the 2 × LysisBuffer (1MTris-HCl (pH6.8,100mM), mercaptoethanol (2%), glycerol (20%), SDS (4%)) of appropriate pre-cooling；Scrape cell with cell, sample is transferred in 1.5mleppendorf pipe, cell lysis 10-15min on ice, Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000g (" g " is centrifugal unit of force), centrifugal 15min, take supernatant, measure protein concentration.

Each sample protein concentration is all adjusted to 2 μ g/ μ l, and-80 DEG C save backup.

4, immunoblotting

Each sample takes identical total protein concentration, add 2 × loadingbuffer sample-loading buffer (1MTris-HCl (pH6.8 of same volume, 100mM), mercaptoethanol (2%), glycerol (20%), SDS (4%), mercaptoethanol (2%)), mixing；Sample being carried out PAGE gel electrophoresis, after electrophoresis terminates, uses electrophoretic blotting device, at 4 DEG C, under 400mA constant current conditions, electricity turns 120min, by protein delivery to pvdf membrane；Electricity turned after with confining liquid (the TBST solution containing 5% skim milk) room temperature closing pvdf membrane 1h or 4 DEG C overnight；Primary antibodie (GFP antibody) is diluted with above-mentioned confining liquid, then with the pvdf membrane incubated at room 2h closed or 4 DEG C overnight, TBST washes film 3 times, each 10min, dilutes corresponding two anti-(mouseIgG-HRP) with above-mentioned confining liquid, incubated at room temperature pvdf membrane 2h, TBST washes film 3 times, each 10min, adopts Amersham company ECL+plusWesternblottingsystem test kit to develop the color, darkroom X-ray development, result is shown in Fig. 1.

5, interpretation of result

HAVCR2 expression is analyzed by gray value, with GAPDH for internal reference, and 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 having transfected pGC-NC-siRNA is: 0.90 ± 0.08, cell experiment group (" 2 " swimming lane) gray value having transfected pGC-HAVCR2-siRNA is: 0.03 ± 0.00, transfect the cell experiment group of pGC-HAVCR2-siRNA compared with having transfected pGC-NC-siRNA matched group, HAVCR2 expressing quantity is substantially lowered, difference has statistical significance (P < 0.05), this illustrates that the siRNA of reticent HAVCR2 provided by the invention efficiently can suppress the expression of HAVCR2 gene in HAVCR2 process LAN type 293T cell specifically.

Above-described embodiment is the illustration to the present invention, and the present invention can also implement with other ad hoc fashion or other particular form, without departing from idea of the invention or substitutive characteristics.Therefore, the embodiment of description from the viewpoint of any be regarded as illustrative but not determinate.The scope of the present invention should be illustrated by appended claims, and the change of any intention with claim and scope equivalence also should be within the scope of the present invention.

Claims (6)

1. the siRNA of the HAVCR2 gene of reticent people, it is characterised in that there is the sequence such as SEQIDNO.1.

2. the siRNA of the HAVCR2 gene of the reticent people described in claim 1 purposes in preparing HAVCR2 gene silencing cell model.

3. the recombinant vector of the siRNA of the HAVCR2 gene of reticent people, it is characterised in that described recombinant vector contains the siRNA of the HAVCR2 gene of the reticent people described in claim 1.

4. recombinant vector according to claim 3, it is characterized in that, described recombinant vector adopts two-step method to build and obtains, and concrete construction method is: the first step, the siRNA expression template strand of iii vitro chemical synthesis HAVCR2 gene, in annealing buffer, annealing forms double-stranded DNA；Second step, is cloned into expression vector by the double-stranded DNA of formation, thus being built into recombinant vector.

5. recombinant vector according to claim 4, it is characterised in that described 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 the arbitrary described reticent people of claim 3-5 purposes in preparing HAVCR2 gene silencing cell model.