CN109161545A - Inhibit the microRNA of chicken Sirt1 gene expression and its recombinates table plasmid and LMH cell line - Google Patents

Abstract

The invention discloses a microRNA that inhibits the expression of chicken Sirt1 gene, and discloses its recombinant overexpression plasmid and specific application, as well as the use of overexpressed miRNAs to construct an LMH cell line with stable low expression of Sirt1 gene. Compared with the prior art, the present invention interferes with the target gene with an artificially designed miRNA, and directly and rapidly constructs an overexpression vector of the miRNA through a simple PCR technology. The pLNCX-pmirG provided by the present invention is a miRNA overexpressed. It is a good vector and can carry out retrovirus packaging in turn. Combined with G418 pressurized screening and green fluorescent protein detection methods, a cell line that stably overexpresses miRNA can be quickly obtained, which can not only reveal the effect of Sirt1 gene on the nutritional status of liver for research It also provides a unique hepatocyte subclonal model for studying the function of human and animal livers in energy metabolism.

Description

Inhibit the microRNA of chicken Sirt1 gene expression and its recombinates table plasmid and LMH Cell line

Technical field

The present invention relates to cell engineerings, in particular to inhibit the microRNA of chicken Sirt1 gene expression and its recombinated table Plasmid and LMH cell line.

Background technique

SIRT1 is as dependence NAD⁺Deacetylase, cell metabolism state (energy state) factor NAD⁺Make SIRT1 at For an important molecule for contacting energetic supersession and genetic transcription, SIRT1 passes through NAD⁺It is connected to gene in metaboilic level and core The response that the network of transcription regulates and controls and body makes nutrition, hormone and environmental stimulus.Liver is Animal nutrition substance and energy The important place of metabolism is measured, the synthesis of Glycogen synthesis, gluconeogenesis, fatty acid and cholesterol and the generation of lipoprotein are responsible for and is divided It secretes, and nutritional status and hormone signal can be made a response, the intracorporal substance of machine and energy balance are adjusted, especially in glycolipid class Metabolism aspect plays an important role.When body takes in excessive energy matter, liver can be by these excessive energy objects Matter is converted into fat, and is transported to adipose tissue and is stored up.Excessive energy is taken in for a long time eventually results in fat, fatty liver With other fat relevant metabolic diseases.In poultry production, fat deposition excessively not only influences the health of animal, can also draw The problems such as playing efficiency of feed utilization and laying rate decline.The fat of deposition is in addition to a small amount of fat in feed, mainly from liver The dirty fat recombined.Therefore, study liver fat metabolism and its regulatory mechanism for control poultry fat deposition pole It is important.The present invention not only can be to grind using the LMH cell line for being overexpressed miRNAs building stable low-expression Sirt1 gene Study carefully and disclose Sirt1 gene influence liver to the acknowledgement mechanism of nutritional status, is also the research mankind, animal's liver in energetic supersession Function to provide a unique liver cell subclone be model.

Summary of the invention

Goal of the invention: being directed to technology vacancy of the existing technology, and this application provides inhibit chicken Sirt1 gene expression MicroRNA and its table plasmid and LMH cell line were recombinated, and provides corresponding construction method and application.

Technical solution: a kind of microRNA inhibiting chicken Sirt1 gene expression of the present invention is named as GSmiR30, sequence is as shown in SEQ ID NO:1.

MicroRNA of the present invention by clone chicken Sirt1 gene 3 ' UTR sequences, then choose base sequence in Lower site is as target spot: " CAGCATTAGAAGCTTTGGCAT ", design obtains；3 ' UTR sequence such as SEQ of the chicken Sirt1 gene Shown in ID NO:2.

A kind of microRNA inhibiting chicken Sirt1 gene expression of the present invention, is named as gSmiR70, sequence is such as Shown in SEQ ID NO:3；Then the gSmiR70 is chosen in base sequence by 3 ' UTR sequences of clone's chicken Sirt1 gene The site " TCTGGATTTAGAGCAAGGAAA " is obtained as shot design；3 ' UTR sequences such as SEQ the ID of the chicken Sirt1 gene Shown in NO:2.

A kind of microRNA inhibiting chicken Sirt1 gene expression of the present invention, is named as gSmiR91, sequence is such as Shown in SEQ ID NO:4；Then the gSmiR91 is chosen in base sequence by 3 ' UTR sequences of clone's chicken Sirt1 gene The site " CCATAAGGATGTGGTGTTTAT " is obtained as shot design；3 ' UTR sequences such as SEQ the ID of the chicken Sirt1 gene Shown in NO:2.

MicroRNA of the present invention crosses the construction method of table retroviral vector, comprising the following steps: utilizes pLNHX The basic sequence and pcDNA of plasmid^TMMicroRNA on 6.2-GW/EmGFP-miR plasmid expresses frame sequence construct pLNCX- PmiRG plasmid, design primer add insertion in the microRNA expression cassette of pLNCX-pmirG respectively using PCR amplification method GSmiR30/70/91 precursor DNA sequence obtains the plasmid for recombinating the microRNA of table: pLNCX-gSmiR30/70/91；Its In, the sequence of building gained pLNCX-pmiRG plasmid is as shown in SEQ ID NO:11.

Wherein, the pLNCX-gSmiR30 design primer gga-miR-SIRT1-30-F/R, sequence such as SEQ ID NO: Shown in 5-6；The pLNCX-gSmiR70 design primer gga-miR-SIRT1-70-F/R, sequence such as SEQ ID NO:7-8 institute Show；The pLNCX-gSmiR91 design primer gga-miR-SIRT1-91-F/R, sequence as shown in SEQ ID NO:9-10, See Table 1 for details:

1 PCR primer sequence of table

Building gained microRNA crosses table retroviral vector pLNCX-gSmiR30/70/91 also in protection of the invention In range.

The microRNA crosses table retroviral vector pLNCX-gSmiR30/70/91 and treats chicken Sirt1 base in preparation Because the application in abnormal expression related disease drug is also within the scope of the present invention.

A kind of LMH cell line of low expression Sirt1 gene of the present invention utilizes the above-mentioned microRNA structure of overexpression Build gained, comprising the following steps: after selecting the viral packaging of microRNA progress, infect LMH cell line；Then anti-using G418 Property pressurization screening and green fluorescent protein detection combine method screening obtain Sirt1 gene low expression LMH subcloned cells System.

Preferably, the concentration of the G418 pressurization screening is 1 μ g/mL, after screening 10 days, continues pressurization sieve after cell of transferring Choosing, repeat 3-5 times, finally using obtain egfp expression rate 98% or more cell as recombinant cell, and respectively from MRNA and protein expression level determine that the recombinant cell obtained is the LMH subclonal cell line of Sirt1 gene low expression.

The utility model has the advantages that compared with the existing technology, it is provided by the present invention that target gene is done with the miRNA of engineer It disturbs, and directly quickly constructs the over-express vector of the miRNA, pLNCX- provided by the present invention by simple round pcr PmirG is the good carrier that miRNA is overexpressed, and can successively carry out the packaging of retrovirus, in conjunction with G418 Pressurization screening and green fluorescent protein detection method can be quickly obtained cell line (the i.e. target gene quilt stablized and be overexpressed miRNA Stablize the cell line of interference).Its main advantage is: constructing stable low-expression Sirt1 gene using miRNAs is overexpressed LMH cell line, it is also research people that Sirt1 gene can be not only disclosed for research, which influences liver to the acknowledgement mechanism of nutritional status, It is model that the function of class, animal's liver in energetic supersession, which provides a unique liver cell subclone,.

Detailed description of the invention

Fig. 1 is the PCR and digestion result of miRNAs over-express vector, wherein the PCR of A:pLNCX-gSmiR30/70/91 As a result, B: the EcoR I digestion result of recombinant plasmid pLNCX-gSmiR30/70/91；M is DL5000 or DL10000；1,2,3 It is followed successively by pLNCX-gSmiR30/70/91；4,5,6 it is followed successively by pLNCX-gSmiR30/70/91 plasmid；

Fig. 2 is verifying of the miRNA to target spot function and effect, Control:pLNCX-pmirG；gSmiR30: pLNCX- gSmiR30；gSmiR70:pLNCX-gSmiR30；gSmiR91:pLNCX-gSmiR30；* indicates p < 0.01)；

Fig. 3 is miRNAs interference LMH cell Sirt1 gene as a result, Control:pLNCX-pmirG； gSmiR30: pLNCX-gSmiR30；2:gSmiR70:pLNCX-gSmiR30；GSmiR91:pLNCX-gSmiR30, * indicate p < 0.05；

Fig. 4 is the expression of results of GFP in LMH-gSmiR30 cell line, GFP in fluorescence microscope LMH-gSmiR30 Expression, A figure: light field (200 ×)；B figure: dark field (200 ×)；

Fig. 5 is the mRNA expression of Sirt1 gene in LMH-gSmiR30 cell line, LMH-pmirG: control；LMH- GSmiR30:SIRT1 gene low expression cell line, * * indicate p < 0.01；

Fig. 6 is the expression of SIRT1 albumen in LMH-gSmiR30 cell line；

Fig. 7 is the Western blot testing result that total protein of cell is extracted with Western/IP lysate, with β-Actin As internal reference, 1:LMH-pmirG (Control)；2:LMH-gSmiR30 is (for constructed Sirt1 gene low expression cell System).

Specific embodiment

The application is explained in detail combined with specific embodiments below.

Cell and plasmid origin: LMH cell is purchased from ATCC；PLNHX plasmid is purchased from Clontech； pcDNA^TM6.2-GW/ EmGFP-miR plasmid is purchased from Invitrogen.

Instrument and equipment:

Embodiment 1 inhibits the design of the microRNAs of chicken Sirt1 gene expression

3 ' the UTR sequences for cloning chicken Sirt1 gene obtained are inputted into Designed microRNA (the BLOCK- iT^TMRNAi Designer) on-line analysis tool (http://rnaidesigner.thermofisher.com/ Rnaiexpress/setOption.do? designOption=mirna &pid=-3453238029932242172), target position Point area is selected as " 3 ' UTR "；Option of species chicken, G/C content are selected as 35%-55%.3 different locis of picking, comment Divide 3 best miR RNAi, is respectively as follows: gSmiR30: " 5 ' AUGCCAAAGCUUCUAAUGCUG3 ' "；GSmiR70: " UUUCCUUGCUCUAAAUCCAGA " and gSmiR91: " AUAAACACCACAUCCUUAUGG ".

The building of embodiment 2miRNAs over-express vector

Utilize the basic sequence and pcDNA of pLNHX plasmid^TMMicroRNA expression on 6.2-GW/EmGFP-miR plasmid Frame sequence is by pcDNA^TMSequence between 17-1919 on 6.2-GW plasmid (includes CMV promoter, the site attB1, EmGFP The miR flanking sequence of gene order, 5 ' and 3 ', the site attB2 and TK polyadenosine acid signal) replace 2047- on pLNHX plasmid The segment (including HSP70 promoter and multiple cloning sites) in 2370 area, successfully constructs pLNCX-pmiRG plasmid, sequence is such as Shown in SEQ ID NO:11.

According to the microRNA design primer of design, using PCR amplification method respectively in the microRNA of pLNCX-pmirG The microRNA precursor DNA sequence that expression cassette addition insertion embodiment 1 designs, obtains the plasmid for recombinating the microRNA of table: pLNCX-gSmiR30、pLNCX-gSmiR70、 pLNCX-gSmiR91。

Method is as follows:

The plasmid for recombinating the microRNA of table is obtained using plasmid pLNCX-pmiG: pLNCX-pmiG plasmid divides as template Not Yong gga-miR-SIRT1-30-F/R, gga-miR-SIRT1-70-F/R, gga-miR-SIRT1-91-F/R tri- to primer expand Increase overall length plasmid sequence, be tapped and recovered DNA purpose band, then carries out 5 ' end phosphorylations, it is certainly a chain of finally by segment Change, secondary connection product is transferred in DH5 α Escherichia coli, prepares the plasmid for obtaining the microRNA for recombinating table: pLNCX- gSmiR30, pLNCX-gSmiR70,pLNCX-gSmiR91.Step:

(1) PCR reacts

Reaction system:

Reaction condition:

(2) 0.8% agarose gel electrophoresis detects, and is tapped and recovered target fragment；

(3) recycling segment 5 ' holds phosphatizing treatment:

System:

Reaction condition: 37 DEG C of 30min, 70 DEG C of 10min.

(4) product (3) is directly taken, self connection reaction is carried out:

(5) connection product is transformed into competent cell DH5 α, Amp resistance screening, the amplification of picking resistance positive bacterium colony After culture, plasmid is extracted, is identified with EcoR I digestion, positive plasmid send sequencing to identify.Sequencing is correctly constructed mistake Express the plasmid of microRNA: pLNCX-gSmiR30, pLNCX-gSmiR70, pLNCX-gSmiR91.

The PCR result and digestion result of building gained miRNAs over-express vector are as shown in Figure 1, wherein A:pLNCX- The PCR of gSmiR30/70/91 is as a result, B: (M is for the EcoR I digestion result of recombinant plasmid pLNCX-gSmiR30/70/91 DL5000 or DL10000；1,2,3 it is followed successively by pLNCX-gSmiR30/70/91；4,5,6 it is followed successively by pLNCX-gSmiR30/70/ 91 plasmids).By Fig. 1-A as it can be seen that using pLNCX plasmid as template, primer gga-miR-SIRT1-30-F/R, gga- are used respectively MiR-SIRT1-70-F/R, gga-miR-SIRT1-91-F/R carry out PCR amplification and obtain the piece greater than 5000bp (about 7000bp) Section, this is consistent with the theoretical value of segment of design amplification, and illustration purpose fragment amplification success, this 3 bar segment is cyclized through self After be transformed into DH5 α, the bacterium colony culture of the picking Amp resistance positive, and extract plasmid.By Fig. 1-B as it can be seen that institute's upgrading grain passes through 7000bp or so DNA fragmentation is respectively obtained after EcoR I digestion, it is consistent with the theoretical value of design, illustrate to connect and be converted to Then function send the clone of the digestion positive to sequencing respectively.

3 miRNAs effect test of embodiment

1, the 3 ' UTR sequence of chicken Sirt1 gene cloned according to this laboratory, with ggaSIRT1-UTR672-F (ATCTCGAGAGTGCTCACTGGTTACAGG)/ggaSIRT1-UTR672-R (ATGCGGCCGCAAGCTCAGTAACTGAAGC), expand that obtain include 3 from the genome of chicken by round pcr 3 ' the UTR sequence of Sirt1 gene of miRNAs (gSmiR30,70 and 91) target site point, and be cloned into pSicheck2 3 ' the area UTR of Synthetic Renilla luciferase gene (hRluc) gene obtains Dual-Luciferase report carrier pSicheck2-gSirt1-UTR672.Steps are as follows:

(1) phenol imitates the genome that method extracts chicken red blood cell；

(2) PCR amplification target fragment (gSirt1-UTR672):

Reaction system:

Reaction condition:

(3) target fragment is recycled after 1% agarose gel electrophoresis, digest recycling segment and report respectively with Xho I and Not I Accuse carrier pSicheck2 plasmid；

(4) it is tapped and recovered target fragment after agarose electrophoresis, then connects the two, is transformed into DH5 α, and PCR and digestion Identification extracts positive plasmid and send sequencing, obtains building correctly recombination report carrier pSicheck2-gSirt1-UTR672.

Construct the PCR and digestion qualification result such as Fig. 2, M:DL5000 of report carrier psiCHECH2-gSirt1-UTR672 Or DL10000；The amplification of 1:gSirt1-UTR672 segment；2: recombination reporter plasmid psiCHECH2-gSirt1-UTR672 Not I and Xho I double digestion result.By Fig. 2-A as it can be seen that with chicken genomic DNA template, with primer ggaSIRT1-UTR672- F/R, Successful amplification obtain the segment for being about 700bp, in the same size with estimated amplification.Fig. 2-B result explanation, target fragment success 3 ' UTR of Renilla luciferase gene (hRluc) gene are inserted into, positive plasmid is taken to be sequenced, it is correct to obtain building Report carrier psiCHECH2-gSirt1-UTR672.

2, the overexpression of 3 miRNAs of cotransfection the effect identification of miRNAs action target spot: is distinguished in CHO-K1 cell line Carrier pLNCX-gSmiR30/70/91 and report carrier pSicheck2- containing the 3 ' area UTR target site sequence of Sirt1 gene The activity of luciferase is detected after gSirt1-UTR672,48h to verify 3 miRNAs effects of effect.Steps are as follows:

(1), with F12K (containing 10% fetal calf serum, 1% is dual anti-) recovery culture CHO-K1, convergence degree is grown to 90% to it Passage cell, by 1.2 × 10⁵The cell concentration of/mL is laid in 24 well culture plates (hole 0.5mL/), and 37 DEG C of 5%CO2 cultivate 22h, more Not antibiotic F12K complete medium is changed, continues to cultivate 2h；

(2) cotransfection, prepare 32 μ L of transfection cocktail OptiMEM (viral expression plasmids pLNCX-gSmiR30 350ng and Report carrier plasmid pSicheck2-gSirt1-UTR672 50ng；X-tremeGENE 9DNA Transfection 1.2 μ L of Reagent), room temperature acts on 20min after mixing, mix, after 21 DEG C of standing 20min, mixed liquor be added dropwise to cell Continue to cultivate 48h in culture medium, after mixing；

(3) luciferase detection (Dual-Luciferase Reporter Assay System Kit), the method is as follows:

1. taking 24 well culture plates, culture medium is abandoned, the PBS that 37 DEG C of 1mL preheatings are added washed once；

2. detection process is carried out referring to Dual-Luciferase Reporter Assay System Kit specification；

3. calculating renilla luciferase activity and firefly luciferase activity ratio, that is, relative luciferase activity.

(4) in LMH cell, it is effective that verifying is transiently transfected with X-tremeGENE 9DNA Transfection Reagent 3 miRNAs over-express vectors pLNCX-gSmiR30/70/91,48h after fluorogenic quantitative detection Sirt1 gene mRNA water It is flat.

As seen from Figure 3,3 miRNAs can effectively inhibit renilla luciferase activity (inhibit efficiency reach 97% with On), illustrate that 3 designed miRNAs can may be expressed after the transcription by corresponding target site participation chicken Sirt1 gene Regulation.From fig. 4, it can be seen that being instantaneously overexpressed this 3 miRNAs, the mRNA level in-site of the Sirt1 gene in LMH cell is inhibited by 30% or so, further illustrate that these three 3 miRNAs can interfere the expression of Sirt1 gene from mRNA level in-site.

Embodiment 4 constructs the LMH subclonal cell line that recombinant cell is Sirt1 gene low expression

After selecting the viral packaging of gSmiR30 progress, LMH cell line is infected, constructs sub- gram of LMH of Sirt1 gene low expression Grand cell line.Method is as follows:

(1), with DMEM complete medium (containing 10% fetal calf serum, 1% is dual anti-) culture GP2-293 cell line, grown extremely to it Cell passage is carried out when 90% or more convergence degree, by 5 × 10⁵The cell concentration of/mL is inoculated in the culture dish (culture medium of 10cm For 10mL), 37 DEG C of 5%CO₂After cultivating 22h, the DMEM complete medium of antibiotic-free is changed into, continue to cultivate 2h；

(2) 500 μ L of transfection cocktail OptiMEM (pLNCX-gSmiR30 containing viral expression plasmids and packaging auxiliary matter are prepared Each 2.5 μ g of grain pVSV-G；15 μ L of X-tremeGENE 9DNA Transfection Reagent), room temperature acts on after mixing Then 20min is dropped evenly into culture dish, 37 DEG C of 5%CO₂Cultivate 72h；

(3) 4 DEG C of 500 × g are centrifuged 10min, collect supernatant, -80 DEG C are frozen after packing to get virion.

(4) LMH cell presses 3 × 10⁵/ hole is inoculated in 12 well culture plates, the Waymouth ' s complete medium in the hole 1mL/, 37 DEG C of 5%CO₂Not antibiotic Waymouth ' s complete medium is changed after culture 22h, continues to cultivate 2h；

(5) virus of collection pack after supernatant doubling dilution and polybrene mixing with the culture of the basis Waymouth ' s, to 0.5mL infection liquid (1/2 volume of culture medium) is added dropwise in cell culture medium, the final concentration of polybrene≤4 μ g/mL mixes；

(6) culture plate is placed in 32 DEG C, 1,200 × g is centrifuged 90min (improve efficiency of infection), then 37 DEG C of 5% CO₂ 18h is cultivated, fresh Waymouth ' s complete medium is replaced, removes polybrene；

(7) after continuing culture for 24 hours, G418 being added to final concentration 1 μ g/mL, daily 3d and changes liquid, the resistance for carrying out G418 continues (concentration of G418 pressurization screening is 1 μ g/mL, after screening 10 days, continues pressurization sieve after cell of transferring for sieve and subcloning culture Choosing repeats 3-5 times), screen recombinant cell；

(8) recombinant cell LMH-pLNCX-pmirG (LMH-pmirG) and LMH-pLNCX-gSmiR30 (LMH- are obtained GSmiR30) after (ratio of cell expressing green fluorescent protein is 98% or more), with the G418's containing 100ng/mL Waymouth ' s complete medium carries out secondary culture.

(9) recombinant cell is collected, total serum IgE and total protein is extracted, is carried out respectively with fluorescent quantitation and Western Blot The detection of Sirt1 gene mRNA and protein expression level.

It is pressurizeed using G418 resistance and screens table low with the method screening acquisition Sirt1 gene that green fluorescent protein detection combines The LMH subclonal cell line reached can be seen that from Fig. 5 result through the duplicate screening of multiple G418, the cell finally obtained it is glimmering The expression rate of photoprotein has reached 98% or more, illustrates that cell obtained has all successfully been transferred to fluorescent marker protein.From Fig. 6 Can be seen that in screened recombinant cell Sirt1 with the result of Fig. 7 is all had in mRNA and protein expression level The inhibition of effect illustrates that Sirt1 gene is stablized the LMH subclonal cell line of the recombinant cell LMH-gSmiR30 of low table and constructed successfully.

Sequence table

<110>Changshu Institute of Technology

<120>inhibit the microRNA of chicken Sirt1 gene expression and its recombinate table plasmid and LMH cell line

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tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 1320

ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 1380

ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 1440

agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 1500

cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 1560

caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 1620

tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 1680

ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 1740

ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 1800

gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 1860

gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 1920

tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 1980

cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 2040

gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 2100

ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat 2160

gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag 2220

tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac 2280

tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 2340

ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 2400

aggttttcac cgtcatcacc gaaacgcgcg aggcagccag cttacctccc ggtggtgggt 2460

cggtggtccc tgggcagggg tctcccgatc ccggacgagc ccccaaatga aagacccccg 2520

ctgacgggta gtcaatcact cagaggagac cctcccaagg aacagcgaga ccacaagtcg 2580

gatgcaactg caagagggtt tattggatac acgggtaccc gggcgactca gtcaatcgga 2640

ggactggcgc cccgagtgag gggttgtggg ctcttttatt gagctcgggg agcagaagcg 2700

cgcgaacaga agcgagaagc gaactgattg gttagttcaa ataaggcaca gggtcatttc 2760

aggtccttgg ggcaccctgg aaacatctga tggttctcta gaaactgctg agggctggac 2820

cgcatctggg gaccatctgt tcttggccct gagccggggc aggaactgct taccacagat 2880

atcctgtttg gcccatattc agctgttcca tctgttcttg gccctgagcc ggggcaggaa 2940

ctgcttacca cagatatcct gtttggccca tattcagctg ttccatctgt tcctgacctt 3000

gatctgaact tctctattct cagttatgta tttttccatg ccttgcaaaa tggcgttact 3060

taagctagct tgccaaacct acaggtgggg tctttcattc cccccttttt ctggagacta 3120

aataaaatct tttattttat ctatggctcg tactctatag gcttcagctg gtgatattgt 3180

tgagtcaaaa ctagagcctg gaccactgat atcctgtctt taacaaattg gactaatcga 3240

tgctatggca gggcctgccg ccccgacgtt ggctgcgagc cctgggcctt cacccgaact 3300

tggggggtgg ggtggggaaa aggaagaaac gcgggcgtat tggccccaat ggggtcacgg 3360

tggggtatcg acagagtgcc agccctggga ccgaaccccg cgtttatgaa caaacgaccc 3420

aacacccgtg cgttttattc tgtcttttta ttgccgtcat agcgcgggtt ccttccggta 3480

ttgtctcctt ccgtgtttca gttagcctcc cccatcagcg aaccgcgggc cctctagatc 3540

aaccactttg tacaagaaag ctgggtctag atatctcgag tgcggccaga tctgggccat 3600

ttgttccatg tgagtgctag taacaggcct tgtgtcctgc tttttgcggt cggcttgctg 3660

tcagtcagtg gccaaaacag caagcccaga ccgcaaaaag cagcatacag ccttcagcaa 3720

gcctccagga tccactggtc gactcactac ctccctttaa acgatcgacg gccacgaagt 3780

gcttagctta cttgtacagc tcgtccatgc cgagagtgat cccggcggcg gtcacgaact 3840

ccagcaggac catgtgatcg cgcttctcgt tggggtcttt gctcagggcg gactgggtgc 3900

tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt 3960

agtggtcggc gagctgcacg ctgccgtcct cgatgttgtg gcgggtcttg aagttcacct 4020

tgatgccgtt cttctgcttg tcggcggtga tatagacctt gtggctgttg tagttgtact 4080

ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc agctcgatgc 4140

ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt 4200

ccttgaagaa gatggtgcgc tcctggacgt agccttcggg catggcggac ttgaagaagt 4260

cgtgctgctt catgtggtcg gggtagcggg cgaagcactg cacgccgtag gtgaaggtgg 4320

tcacgagggt gggccagggc acgggcagct tgccggtggt gcagatgaac ttcagggtca 4380

gcttgccgta ggtggcatcg ccctcgccct cgccggacac gctgaacttg tggccgttta 4440

cgtcgccgtc cagctcgacc aggatgggca ccaccccggt gaacagctcc tcgcccttgc 4500

tcaccatggt tttaaagcct gcttttttgt acaaacttgt tgatagctta actagccagc 4560

ttgggactcc ctatagtgag tcgtattaat ttcgataagc cagtaagcag tgggttctct 4620

agttagttaa cgatctgatc tgacggttca ctaaaccagc tctgcttata tagacctccc 4680

accgtacacg cctaccgccc atttgcgtca atggggcgga gttgttacga cattttggaa 4740

agtcccgttg attttggtgc caaaacaaac tcccattgac gtcaatgggg tggagacttg 4800

gaaatccccg tgagtcaaac cgctatccac gcccattgat gtactgccaa aaccgcatca 4860

ccatggtaat agcgatgact aatacgtaga tgtactgcca agtaggaaag tcccataagg 4920

tcatgtactg ggcataatgc caggcgggcc atttaccgtc attgacgtca atagggggcg 4980

tacttggcat atgatacact tgatgtactg ccaagtgggc agtttaccgt aaatactcca 5040

cccattgacg tcaatggaaa gtccctattg gcgttactat gggaacatac gtcattattg 5100

acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg gccatttacc gtaagttatg 5160

taacgcggaa ctccatatat gggctatgaa ctaatgaccc cgtaattgat tactattaat 5220

aactagtcga ggggatcgag cccggggtgg gcgaagaact ccagcatgag atccccgcgc 5280

tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct ttcatagaag 5340

gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc ggtcatttcg 5400

aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg 5460

aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct 5520

cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc 5580

ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg 5640

catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg agcctggcga 5700

acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac 5760

cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc 5820

aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct 5880

cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc 5940

agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg 6000

ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg 6060

tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc 6120

agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag 6180

aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat 6240

cagatcggaa aacagcttta actaatccta ggctgagatg gatctattgg ctgcagcaga 6300

caagacgcgc ggcttcggtt ccaaaccgaa agcaaaaatt cagacggagg cgggaactgt 6360

tttaggttct cgtctcctac cagaaccaca tatcctgacg gggtcggatt ccacatcgac 6420

tcccttcctc aggtcgggcc acaaaaacgg cccccaaagt ccctgggacg tctcccaggg 6480

ttgcggccgg gtgttcagaa ctcgtcagtt ccaccacggg tccgccagat acagagctag 6540

ttagctaact agtacagacg caggcgcata acatcaaaca tagacactag acaatcggac 6600

agacacagat aagttgctgg ccagcttacc tcccggtggt gggtcggtgg tccctgggca 6660

ggggtctcca aatcccggac gagcccccaa atgaaagacc cccgtcgtgg gtagtcaatc 6720

actcagagga gaccctccca aggaacagcg agaccacgat tcggatgcaa acagcaagag 6780

gctttattgg gaatacgggt acccgggcga cgcagtctat cggaagactg gcgcgccgag 6840

tgaggggttg tgggctcttt tattgagctc ggagagcgga agcgcgcgaa cagaagcgag 6900

aagcgaactg attggttagt tcaaataagg tacagggtca ttttcaggtc cttggggcac 6960

cctggaaaca tctgatgatt cactagaaac tgctgagggc tggaccgcat ctggggacca 7020

tctgttcttg gccccgagcc ggggcaggaa ctgcttacca cagatatcct gtttggccca 7080

tcactcagct gtctcatctg ttcttggccc tgagccgggg caggaaccgc ttaccacaga 7140

tatcctgttt ggtattcagc tgtttctttg ttcctgacct tgatctgaac ttttctattc 7200

tcagttatgt atttttccat gccttgcaaa gtggcgttac ttaagctagc ttgccaccta 7260

cgggtggggt ctttcaaa 7278

Claims (10)

1. a kind of microRNA for inhibiting chicken Sirt1 gene expression, is named as gSmiR30, sequence such as SEQ ID NO:1 institute Show.

2. the microRNA according to claim 1 for inhibiting chicken Sirt1 gene expression, which is characterized in that the gSmiR30 By cloning 3 ' UTR sequences of chicken Sirt1 gene, site " CAGCATTAGAAGCTTTGGCAT " in base sequence is then chosen It is obtained as shot design.

3. a kind of microRNA for inhibiting chicken Sirt1 gene expression, is named as gSmiR70, sequence such as SEQ ID NO:3 institute Show；Then the gSmiR70 is chosen in base sequence by 3 ' UTR sequences of clone's chicken Sirt1 gene The site " TCTGGATTTAGAGCAAGGAAA " is obtained as shot design.

4. a kind of microRNA for inhibiting chicken Sirt1 gene expression, is named as gSmiR91, sequence such as SEQ ID NO:4 institute Show；Then the gSmiR91 is chosen in base sequence by 3 ' UTR sequences of clone's chicken Sirt1 gene The site " CCATAAGGATGTGGTGTTTAT " is obtained as shot design.

5. any microRNA crosses the construction method of table retroviral vector in a kind of claim 1-4, feature exists In, comprising the following steps: utilize the basic sequence and fpcDNA of pLNHX plasmid^TMOn 6.2-GW/EmGFP-miR plasmid MicroRNA expresses frame sequence construct pLNCX-pmiRG plasmid, design primer, using PCR amplification method respectively in pLNCX- The microRNA expression cassette addition insertion expression gSmiR30/70/91 precursor DNA sequence of pmirG, acquisition recombinated table The plasmid of microRNA: pLNCX-gSmiR30/70/91.

6. construction method according to claim 5, which is characterized in that the pLNCX-gSmiR30 design primer is gga- MiR-SIRT1-30-F/R, sequence is as shown in SEQ ID NO:5-6；The pLNCX-gSmiR70 design primer gga-miR- SIRT1-70-F/R, sequence is as shown in SEQ ID NO:7-8；The pLNCX-gSmiR91 design primer gga-miR-SIRT1- 91-F/R, sequence is as shown in SEQ ID NO:9-10.

7. the building gained of claim 5 or 6 microRNA crosses table retroviral vector.

8. microRNA described in claim 7, which crosses table retroviral vector, treats chicken Sirt1 abnormal gene expression phase in preparation Application in related disorders drug.

9. a kind of LMH cell line of low expression Sirt1 gene, which is characterized in that utilize any institute in overexpression claim 1-4 State microRNA building, comprising the following steps: after selecting the viral packaging of microRNA progress described in claim 1, infect LMH Cell line；Then Sirt1 gene is obtained using the method screening that G418 resistance pressurization screening and green fluorescent protein detection combine The LMH subclonal cell line of low expression.

10. the LMH cell line of low expression Sirt1 gene according to claim 9, which is characterized in that the G418 pressurization The concentration of screening is 1 μ g/mL, after screening 10 days, continues pressurization screening after cell of transferring, repeats 3-5 times, finally to obtain green Fluorescent protein expression rate is recombinant cell in 98% or more cell.