CN102021190A - Method for screening effective shRNA of triacylglycerol hydrolase gene - Google Patents

Abstract

The invention discloses a method for screening effective shRNA of adipose triacylglycerol hydrolase gene. The method comprises the following steps of: A1, designing and synthesizing a single-chain DNA oligonucleotide segment for coded target shRNA; A2, constructing a pENTR/CMV-GFP/U6-shRNA carrier; A3, constructing a pDsRed1-C1-ATGL carrier; and A4, screening an effective sequence of the shRNA. Because an interference vector and a target gene are marked by red fluorescence and green fluorescence respectively, the process of screening interference sequences is time-saving and high in efficiency, the result is more intuitive and reliable, and a plurality of sequences can be screened at the same time.

Description

A kind of screening method of triglyceride hydrolysis enzyme gene effective shRNA

Technical field

The invention belongs to biological technical field, be specifically related to a kind of screening method of triglyceride hydrolysis enzyme gene effective shRNA.

Background technology

It is genetic intervention phenomenon by double-stranded RNA (dsRNA) mediation that RNA disturbs, its mRNA that can degrade special, effectively, thus cause the silence of gene.After reported first had successfully been induced the specific expressed silence of target gene on calendar year 2001 " Nature " magazine by siRNA in mammalian cell, the RNA perturbation technique just began as the widespread use on mammalian cell of the reticent instrument of a specific gene.Reports such as Rubinson in 2003 with viral system former generation cultured cells, stem cell and transgenic mice on all obtained RNA and disturbed successfully, expanded this The Application of Technology scope more greatly.At present, RNAi has developed into the genetics instrument that the regulation and control biological gene is expressed, is widely used in the post genome project researchs such as activity-dependent genetic screening, gene functional research, and is expected to become potential gene therapy instrument.

Triglyceride level (TG) is the main existence form of fat, and its hydrolytic process is the main energy derive of body.2004, come from three different breadboard Zimmermann, people such as Jenk ins and Villena, found a kind of lipase of similar performance simultaneously, difference called after: adiposetriglyceride lipas e (ATGL), desnutrin and phospholipase A2 ξ (iPLA2 ξ) studies show that through 26S Proteasome Structure and Function these three kinds of albumen are a kind of lipase in fact, and with its unify called after triglyceride hydrolysis enzyme (adipose triglyceride lipase, ATGL).Zimmermann etc. discover the ATGL of different plant species, all have a GXSXG (Padil-X-Serine-X-Padil) block in its aminoacid sequence, and its intermediary Serine site are relevant with the activity of ATGL.In addition, find also that at the N of ATGL end a α/βZhe Die zone (COG1752) and a Patatin structural domain (Pfam01734), these structures all are and the closely-related conservative property of lipase activity zone.To discovering of ATGL deficient mice, the body fat tissue content obviously improves, and shows the phenomenon that triglyceride level is piled up in a plurality of tissues; Simultaneously, heart causes the heart function disorder, and finally causes death owing to pile up excessive lipid.

Another key of using the RNA perturbation technique in zooblast is the screening of effective siRNA sequence.But so far, the sequence of screening effective RNAi makes up its adenovirus carrier then, still belongs to blank.

Therefore, there is defective in prior art, needs to improve perfect.

Summary of the invention

Technical problem to be solved by this invention is at the deficiencies in the prior art, and a kind of screening method of triglyceride hydrolysis enzyme gene effective shRNA is provided.

Method of the present invention may further comprise the steps: A1: the segmental design of single strand dna oligonucleotide of coding purpose shRNA, synthetic; The structure of A2:pENTR/CMV-GFP/U6-shRNA carrier; The structure of A3:pDsRed1-C1-ATGL carrier; The screening of A4:shRNA ordered sequence.

Described method, wherein preferred, described steps A 1 is specifically carried out following operation: clone the Xi Nongsa energy sheep ATGL sequence that obtains according to this laboratory, design three ATGL-siRNA and 1 negative control sequence, on the siRNA basis, with the oligonucleotide of 19bp with forward and reverse combination, middle hairpin loop sequence interval of adding GAGTACTG, make the inner hairpin structure that forms of oligonucleotide, every pair of oligonucleotide two ends have BamH I and Xho I restriction enzyme site respectively, wherein 3 ' of positive-sense strand end adds the TTTTTT termination signal, obtains the positive and negative adopted chain template of shRNA.

Described method, wherein preferred, described steps A 2 concrete following operation: the A21 that carry out: the single stranded oligonucleotide annealing reaction generates double-stranded; Synthetic single stranded oligonucleotide sequence is dissolved into the solution that concentration is 200 μ M with the sterilization deionized water, carry out annealing reaction then, annealing conditions is: 95 ℃ * 4 minutes, taking-up is put room temperature and is cooled off gradually, promptly obtaining concentration is the double chain oligonucleotide sequence of 50 μ M, is labeled as respectively: ds422, ds600, ds1148; Take a morsel respectively again and be diluted to the working concentration that concentration is 5nM, A22: ligation-structure shuttle vectors; With BamH I and Xho I double digestion pRNTR/CMV-GFP/U6 carrier and cut glue and reclaim, then described ds422, described ds600 and described ds 1148 are cut back to close the product ligation with pRNTR/CMV-GFP/U6 carrier enzyme respectively, reaction conditions is that 4 ℃ of connections are spent the night, and obtains connecting product; A23: connect product and transform; Get the described connection product of 10 μ L and add 100 μ L competence DH5 α bacteriums, ice bath 30 minutes, 42 ℃ of heat shocks 90 seconds, put ice bath 12 minutes, add 600 μ L LB nutrient solutions, 37 ℃ of gentle concussions were cultivated 45 minutes, and bacterium is coated on the agar plate that contains 50 μ g/mL kantlex, placed 37 ℃ of incubations; A24: screening shuttle vectors clone.

Described method, wherein preferred, described ligation system is as follows: pENTRTM/U6,2 μ L; Ds oligos (5nM), 1 μ L; 5 * annealing buffer, 4 μ L; T ₄Dna ligase, 1 μ L; The water that does not contain DNase/Rnase, 12 μ L; Cumulative volume 20.0 μ L.

Described method, wherein preferred, operation below described steps A 24 concrete the execution: (1) shuttle vectors clone's plasmid enzyme restriction is identified: the cohesive terminus complementation of the linear carrier pENTRTM/U6 that the cohesive terminus of ds oligos and test kit provide after ligation, forms circular plasmids; Picking mono-clonal bacterium colony on the kantlex agar plate, transferred species is in the LB nutrient solution that contains 50 μ g/mL kantlex of 4mL, 37 ℃ shaking culture 12-16 hour, get bacterium liquid 2mL and extract plasmid, size meets carries out Sca I and EcoR V double digestion is identified again, and product is observed with 1.2% agarose gel electrophoresis; (2) shuttle vectors clone's order-checking is identified: get the correct mono-clonal bacterium liquid of double digestion evaluation in (1), send Nanjing Jin Sirui Bioisystech Co., Ltd to carry out sequencing with the automatic fluorescence sequenator of ABI377 type DNA, primer is sequencing primer U6 or M13.

Described method, wherein preferred, described steps A 3 concrete following operation: the A31 that carry out: make up the pDsRed1-C1-ATGL cloning vector; The target gene fragment that reclaims is connected with the pGM-T carrier, 16 ℃ of incubated overnight, connect product transformed competence colibacillus E.coliDH5 α bacterial strain, coat in the LB culture dish that contains penbritin and X-gal/I PTG, in 37 ℃ of overnight incubation, the single bacterium colony of picking white shakes bacterium, carries out bacterium colony PCR simultaneously, gets 10 μ L reaction solutions after reaction finishes and carries out the evaluation of 1% agarose gel electrophoresis; Positive colony bacterium liquid is utilized alkaline lysis extracting plasmid, carry out double digestion with Xho I and Sal I restriction endonuclease then and identify that the endonuclease reaction system is as follows: 10 * H Buffer, 2.0 μ L; Xho I, 0.7 μ L; Sal I, 0.7 μ L; PGM-T-gATGL1A1,10.0 μ L; DdH ₂O, 6.6 μ L; Cumulative volume 20.0 μ L; 37 ℃ of water-baths spend the night, and get total overall reaction liquid electrophoresis on 1% sepharose, reclaim to have the target gene fragment that designs restriction enzyme site; A32: make up the pDsRed1-C1-ATGL expression vector.

Described method, wherein preferred, operation below described steps A 32 concrete the execution: (1) preparation pDsRed1-C1 endonuclease reaction system is also carried out 37 ℃ of water-baths and is spent the night; (2) after endonuclease reaction finishes, get total overall reaction liquid and carry out 1% agarose gel electrophoresis, reclaim the linearizing fragment after pDsRed1-C1 carrier enzyme is cut; (3) carrier segments is connected with target gene fragment, 16 ℃ of reactions are spent the night; (4) pDsRed1-C1-ATGL is converted into E.coli DH5 α competent cell, coating contains the plate of kantlex, 37 ℃ of overnight incubation, and picking list bacterium colony shakes bacterium, carries out bacterium colony PCR simultaneously and identifies, extracts plasmid then, carries out enzyme and cuts evaluation.

Described method, wherein preferred, described endonuclease reaction system is: 10 * H Buffer, 2.0 μ L; Xho I, 0.7 μ L; Sal I, 0.7 μ L; PDsRed1-C1-ATGL, 5.0 μ L; DdH ₂O, 11.6 μ L; Cumulative volume 20.0 μ L.

Described method, wherein preferred, described steps A 4 concrete following operation: the A41 that carry out: go the intracellular toxin plasmid to extract; A42: the recovery of cell; The A43 passage is cultivated: A44: cell transfecting.

Described method, wherein preferred, operation below described steps A 41 concrete execution the: (1) column equilibration: the balance liquid that in adsorption column, adds 500 μ L, adsorption column is put into collection tube, 12000rpm (13400 * g) centrifugal 1 minute, outwell the waste liquid in the collection tube, adsorption column is relay reclaim in the collector; (2) the bacterium liquid of getting the 5-15mL incubated overnight adds in the centrifuge tube, and 12000rpm (13400 * g) centrifugal 1 minute, absorb supernatant liquor; (3) in the centrifuge tube that leaves bacterial sediment, add the solution P1 that 500 μ L have added RNaseA, use the vortex vibrator bacterial cell precipitation that thoroughly suspends; (4) in centrifuge tube, add 500 μ L solution P2, leniently spin upside down and make the abundant cracking of thalline for 6-8 time; (5) add 700 μ L solution P3 in centrifuge tube, leniently spin upside down 6-8 time immediately, fully white flocks appears in mixing, and 12000rpm (13400 * g) centrifugal 10 minutes, form precipitation in the centrifuge tube bottom, the collection supernatant liquor; (6) the supernatant liquor gradation of collecting in (5) is added Filter column, Filter column is put into collection tube, and 12000rpm (13400 * g) centrifugal 2 minutes, the solution gradation that obtains in the collection tube of centrifugal back is added in the adsorption column; (7) 12000rpm (13400 * g) centrifugal 1 minute, outwell the waste liquid in the collection tube, adsorption column is put into collection tube; (8) in adsorption column, add 500 μ l protein liquid removals, and 12000rpm (13400 * g) centrifugal 1 minute, outwell the waste liquid in the collection tube, adsorption column is put into collection tube; (9) in rinsing liquid, add dehydrated alcohol according to 3 times of volumes, in adsorption column, add the described rinsing liquid of 700 μ L, and 12000rpm (13400 * g) centrifugal 1 minute, outwell the waste liquid in the collection tube, adsorption column is put into collection tube; (10) in adsorption column, add 500 μ L rinsing liquids, and 12000rpm (13400 * g) centrifugal 1 minute, outwell the waste liquid in the collection tube; (11) adsorption column is relay reclaim place in the collector 12000rpm (13400 * g) centrifugal 2 minutes, rinsing liquid remaining in the adsorption column is removed; (12) adsorption column is placed a clean centrifuge tube, to the unsettled dropping in the middle part of adsorption film 100-300 μ L elution buffer, room temperature was placed 2 minutes, and (13400 * g) collect plasmid solution in the centrifuge tube after centrifugal 1 minute 12000rpm.

Described method, wherein preferred, described steps A 42 is concrete carries out following operation: (1) takes out 1 frozen HEK293 cell from liquid nitrogen container, put into 38 ℃ of water-baths immediately, melts fully to cell; (2) frozen pipe content is changed in the 10mL centrifuge tube, add the DMEM that 4mL contains 10%FBS, the DMSO that adds when wash-out is frozen; (3) 1400rpm centrifugal 10 minutes, discards supernatant liquor; (4) add the DMEM that 2.0mL contains 10%FBS, blow evenly, be transferred in the plastic culture bottle, in described centrifuge tube, add the DMEM that 2.0mL contains 10%FBS once more, blow evenly, residual cells is changed in the described plastic culture bottle; (5) unscrew the culturing bottle bottle cap, put 37 ℃, 5%CO ₂Cultivate in the incubator.

Described method, wherein preferred, operation below described steps A 43 concrete the execution: (1) is inhaled substratum in the culturing bottle and is abandoned, and adds to inhale after the 1mL substratum cleans and abandons, to remove serum deprivation; (2) ATV of adding 1mL 0.25% places under the mirror and observes, and inhales after cell is full and abandons ATV; (3) add the DMEM that 2mL contains 10%FBS, the piping and druming attached cell makes the cell homodisperse, and the cell suspension branch is filled in 2-3 the new culturing bottle, puts 37 ℃, and the 5%CO2 incubator is cultivated.

Described method, wherein preferred, operation below described steps A 44 concrete execution the: will hang down serum Opti-MEM nutrient solution and Lipofectamine ^TM2000 under room temperature condition balance; In 2: 1 ratios with Lipofectamine ^TM2000 and plasmid add respectively among the low serum Opti-MEM of 250 μ L, flick tube wall and make mixing, room temperature leaves standstill 5min, and plasmid DNA/low serum Opti-MEM mixture is added Lipofectamine ^TMIn the 2000 low serum Opti-MEM mixtures, flick tube wall and make it mixing, incubated at room 20min; Transfection composite is added in the Tissue Culture Dish evenly, lentamente, gently revolve culture dish mixing liquid, cell is put into 37 ℃ of cell culture incubators hatch.

The present invention verified the interference effect of designed sequence earlier in HEK 293 cells before making up the recombinant adenovirus carrier, to save time and cost, interference carrier and target gene expression carrier are used green fluorescence and red fluorescence mark respectively, cotransfection HEK 293 cells, just can judge the transfection efficiency of interference carrier and the jamming effectiveness of target gene by the intensity of under fluorescent microscope, observing green fluorescence and red fluorescence, owing to adopt two kinds of different fluorescence of ruddiness and green glow mark interference carrier and target gene respectively, make interference sequence screening process time-saving and efficiency, the result is more intuitive and reliable, and can screen many sequences simultaneously.

Description of drawings

Fig. 1 is a pRNTR/CMV-GFP/U6-shRNA plasmid gel electrophoresis analysis

Fig. 2 cuts evaluation for the pRNTR/CMV-GFP/U6-shRNA enzyme

Fig. 3 identifies for the pGM-T-ATGL double digestion

Fig. 4 is that the PCR of pDsRed1-C1-ATGL positive transformant identifies;

Fig. 5 cuts evaluation for the pDsRed1-C1-ATGL enzyme;

Fig. 6 is the screening (200 *) of RNA interference sequence, and wherein A group, B group, C group, D group are respectively the interference effect detection of shRNA-NC, shRNA-422, shRNA-1148, shRNA-600 sequence; 1 (A1, B1, C1, D1), 2 (A2, B2, C2, D2) are respectively pENTR/CMV-GFP/U6-shRNA interference carrier and pDsRed1-C1-ATGL target gene expression carrier cotransfection HEK 293 cells green fluorescence and red fluorescence expression under the fluorescent microscope after 48 hours; 3 (A3, B3, C3, D3) are control group, i.e. independent transfection pDsRed1-C1-ATGL target gene expression carrier red fluorescence expression under the fluorescent microscope after 48 hours.

Embodiment

Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.

The first step: the segmental design of single strand dna oligonucleotide of coding purpose shRNA, synthetic;

Clone the Xi Nongsa energy sheep ATGL sequence that obtains according to this laboratory, utilize online siRNA select procedure (http://jura.wi.mit.edu/bioc/siRNAext/home.php) three ATGL-siRNA of design and 1 negative control sequence, i.e. s i RNA-422:TCCGGAGCTGCCTGCTGAA (SEQID NO:1); SiRNA-600:CCTCATCCCCCCTTCCTTC (SEQ ID NO:2); SiRNA-1148:CCACGGCCATGATGGTGCC (SEQ I D NO:3); Negative control siRNA-NC:ACTACCGTTGTTATAGGTG (SEQ ID NO:4).On the siRNA basis, with the oligonucleotide of 19bp with forward and reverse combination, the middle GAGTACTG that adds (contains Sca I restriction enzyme site, so that identify in the vector construction) the hairpin loop sequence at interval, make oligonucleotide inside can form hairpin structure, every pair of oligonucleotide two ends have BamH I and Xho I restriction enzyme site respectively, and wherein 3 ' of positive-sense strand end adds the TTTTTT termination signal, promptly obtain the positive and negative adopted chain template of shRNA, see Table 1:

The oligonucleotide sequence of the coding shRNA that table 1ATGL is special

Second step: the structure of pENTR/CMV-GFP/U6-shRNA carrier;

2.1 the single stranded oligonucleotide annealing reaction generates double-stranded;

Earlier synthetic single stranded oligonucleotide sequence is dissolved into the solution that concentration is 200 μ M with the sterilization deionized water, carry out annealing reaction then, the annealing reaction system is: annealing conditions is: 95 ℃ * 4min, taking-up is put room temperature and is cooled off gradually, promptly obtaining concentration is the double chain oligonucleotide sequence (dsoligos) of 50 μ M, is labeled as respectively: ds422, ds600, ds1148.Taking a morsel respectively is diluted to the working concentration that concentration is 5nM again, prepares for carrying out ligation, and all the other put-20 ℃ of preservations.

200 μ M positive-sense strands, 5 μ L

200 μ M antisense strands, 5 μ L

10 * annealing buffer, 2 μ L

The water 8 μ L that do not contain DNase/Rnase

Cumulative volume 20.0 μ L

2.2 ligation-structure shuttle vectors

With BamH I and Xho I double digestion pRNTR/CMV-GFP/U6 carrier and cut glue and reclaim, the production concentration of will annealing then is that ds422, the ds600 of 5nM, ds1148 cut back to close product with pRNTR/CMV-GFP/U6 carrier enzyme respectively and be connected, and the ligation system is as follows:

pENTRTM/U6 2μL

dsoligos(5nM) 1μL

5 * annealing buffer, 4 μ L

T ₄Dna ligase 1 μ L

The water 12 μ L that do not contain DNase/Rnase

Cumulative volume 20.0 μ L

Reaction conditions is that 4 ℃ of connections are spent the night, and transforms or put-20 ℃ of preservations afterwards.

2.3 connecting product transforms

Get 10 μ L and connect product and add 100 μ L competence DH5 α bacteriums, ice bath 30 minutes, 42 ℃ of heat shocks 90 seconds were put ice bath 1-2 minute fast, added 600 μ L LB nutrient solutions, and 37 ℃ of gentle concussions were cultivated 45 minutes.Bacterium is coated on the agar plate that contains 50 μ g/mL kantlex, placed 37 ℃ of incubations.

2.4 screening shuttle vectors clone

(1) shuttle vectors clone's plasmid enzyme restriction is identified

The cohesive terminus complementation of the linear carrier pENTRTM/U6 that the cohesive terminus of ds oligos and test kit provide after ligation, forms circular plasmids.Picking mono-clonal bacterium colony on the kantlex agar plate, transferred species is in the LB nutrient solution that contains 50 μ g/mL kantlex of 4mL, 37 ℃ shaking culture 12-16 hour, get bacterium liquid 2mL and extract plasmid, size meets carries out Sca I and EcoR V double digestion is identified again, and product is observed with 1.2% agarose gel electrophoresis.

(2) shuttle vectors clone's order-checking is identified

Get double digestion and identify correct mono-clonal bacterium liquid, send Nanjing Jin Sirui Bioisystech Co., Ltd to carry out sequencing with the automatic fluorescence sequenator of ABI 377 type DNA, primer is sequencing primer U6 (or M13).

The 3rd step: the structure of pDsRed1-C1-ATGL carrier

3.1 make up the pDsRed1-C1-ATGL cloning vector

The target gene fragment that reclaims is connected with the pGM-T carrier, 16 ℃ of incubated overnight, connect product transformed competence colibacillus E.coliDH5 α bacterial strain, coat in the LB culture dish that contains penbritin and X-gal/IPTG, in 37 ℃ of overnight incubation, the single bacterium colony of picking white shakes bacterium, carries out bacterium colony PCR simultaneously, gets 10 μ L reaction solutions after reaction finishes and carries out the evaluation of 1% agarose gel electrophoresis.Positive colony bacterium liquid is utilized alkaline lysis extracting plasmid, carries out double digestion with Xho I and Sal I restriction endonuclease then and identify that the endonuclease reaction system is as follows:

10×H?Buffer 2.0μL

Xho?I 0.7μL

Sal?I 0.7μL

pGEM-T-gATGL1A1?10.0μL

ddH2O 6.6μL

Total 20.0μL

37 ℃ of water-baths spend the night, and get total overall reaction liquid electrophoresis on 1% sepharose, reclaim to have the target gene fragment that designs restriction enzyme site.

3.2 make up the pDsRed1-C1-ATGL expression vector

Because the target gene fragment two ends have Xho I and Sal I endonuclease digestion site, pDsRed1-C1 is carried out same double digestion reaction, the recovery carrier segments also is connected with gene fragment.

(1) preparation pDsRed1-C1 endonuclease reaction system and carry out 37 ℃ of water-baths and spend the night;

10×H?Buffer 2.0μL

Xho?I 0.7μL

Sal?I 0.7μL

pDsRed1-C1 5.0μL

ddH2O 11.6μL

Cumulative volume 20.0 μ L

(2) after endonuclease reaction finishes, get total overall reaction liquid and carry out 1% agarose gel electrophoresis, reclaim the linearizing fragment after pDsRed1-C1 carrier enzyme is cut;

(3) carrier segments is connected with target gene fragment, 16 ℃ of reactions are spent the night;

(4) pDsRed1-C1-ATGL is converted into E.coli DH5 α competent cell, coating contains the plate of kantlex, 37 ℃ of overnight incubation, picking list bacterium colony shakes bacterium, carries out bacterium colony PCR simultaneously and identifies, extracts plasmid then, carry out enzyme and cut evaluation, the endonuclease reaction system as mentioned above.

The 4th step: the screening of shRNA ordered sequence;

4.1 picking pDsRed1-C1-ATGL positive monoclonal and 4 pRNTR/CMV-GFP/U6-shRNA interference carriers (comprising negative control) are inoculated in (10 μ g/mL kan) in the 4mL LB substratum respectively, 37 ℃, 200rpm shake bacterium and spend the night, shake to 10mL LB substratum in ratio commentaries on classics in 1: 50,37 ℃, 230rpm shake bacterium 16h, extract and remove the intracellular toxin plasmid.The concrete operations step is as follows:

Solution P1 adds RNa seA before use earlier, and mixing places 2-8 ℃ of preservation, should add earlier dehydrated alcohol in rinsing liquid according to the explanation of reagent bottle label before using for the first time.

(1) column equilibration step: the balance liquid that in adsorption column, adds 500 μ L, adsorption column is put into collection tube, and (13400 * g) centrifugal 1min outwell the waste liquid in the collection tube to 12000rpm, adsorption column is relay in the recovery collector, use the pillar of handling the same day as far as possible.

(2) get in the bacterium liquid adding centrifuge tube of 5-15mL incubated overnight, (13400 * g) centrifugal 1min absorb supernatant to 12000rpm as far as possible; Preferably, bacterium liquid can be collected bacterial sediment in the centrifuge tube by centrifugal several times more for a long time, and biomass is can fully be cracked into the insufficient extraction efficiency that can reduce plasmid of good, too much cellular lysate;

(3) add RNaseA among the solution P1, in the centrifuge tube that leaves bacterial sediment, add 500 μ L solution P1, use the vortex vibrator bacterial cell precipitation that thoroughly suspends;

Attention: the bacterial precipitation that must thoroughly suspend, if the not bacterium piece of thorough mixing is arranged, can influence cracking, cause extracted amount and purity on the low side;

(4) in centrifuge tube, add 500 μ L solution P2, leniently spin upside down and make the abundant cracking of thalline for 6-8 time.It is to be noted and attention will leniently mix in the above-mentioned steps, do not want concuss, in order to avoid contaminating genomic dna; This moment the bacterium liquid limpid thickness that should become, the used time should not surpass 5min, in order to avoid plasmid is damaged; Limpid if do not become, may be because thalline to be too much, cracking is not thorough, should reduce biomass;

(5) add 700 μ L solution P3 in centrifuge tube, leniently spin upside down 6-8 time immediately, fully mixing white flocks can occur at this moment, and (13400 * g) centrifugal 10min, form precipitation in the centrifuge tube bottom this moment to 12000rpm.Should be noted that: P3 should mix after adding immediately, avoids producing localized precipitation; If also have the minute white precipitation in the supernatant, but get supernatant behind the recentrifuge;

(6) the supernatant liquor gradation that previous step is collected adds Filter column, Filter column is put into collection tube, 12000rpm (13400 * g) centrifugal 2min, carefully the solution gradation that obtains in the collection tube of centrifugal back is added in the adsorption column, adsorption column is put into collection tube, in addition, if there is in the supernatant that remaining liquid description of step (5) draws impurity too much in the Filter column, can prolong the centrifugal time; If a spot of precipitation is arranged at collection tube bottom, centrifugal back, draw supernatant as best one can;

(7) (13400 * g) centrifugal 1min outwell the waste liquid in the collection tube to 12000rpm, and adsorption column is put into collection tube;

(8) in adsorption column, add 500 μ l protein liquid removals, 12, (13400 * g) centrifugal 1min outwell the waste liquid in the collection tube to 000rpm, and adsorption column is put into collection tube;

(9) add 700 μ L rinsing liquids in adsorption column, should add dehydrated alcohol according to 3 times of volumes before rinsing liquid uses, (13400 * g) centrifugal 1min outwell the waste liquid in the collection tube to 12000rpm, and adsorption column is put into collection tube; Preferably, behind the adding rinsing liquid, room temperature leaves standstill 2-5min, can remove impurity better;

(10) add 500 μ L rinsing liquids in adsorption column, (13400 * g) centrifugal 1min outwell the waste liquid in the collection tube to 12000rpm;

(11) adsorption column is relay reclaim and place 12000rpm in the collector (13400 * g) centrifugal 2min, purpose is that rinsing liquid remaining in the adsorption column is removed.Preferably, experiment that alcoholic acid is residual in the rinsing liquid can the follow-up enzyme reaction of influence (enzyme is cut, PCR etc.) is not subjected to the influence of residual ethanol for guaranteeing the downstream experiment, and adsorption column is uncapped, place room temperature to place several minutes, thoroughly to dry rinsing liquid remaining in the sorbing material;

(12) adsorption column is placed a clean centrifuge tube, to the unsettled dropping in the middle part of adsorption film 100-300 μ L elution buffer, room temperature is placed 2min, and 12000rpm (collects plasmid solution in the centrifuge tube behind 13400 * g) the centrifugal 1min; Preferably, in order to increase the organic efficiency of plasmid, the solution that obtains can be added again in the centrifugal adsorption column repeating step (12); The pH value of elutriant has a significant impact for elution efficiency; If water is cooked elutriant, should guarantee its pH value in the 7.0-8.5 scope, the pH value is lower than 7.0 can reduce elution efficiency; The elution buffer volume should not be less than 100 μ L, the too small organic efficiency that influences of volume, and the DNA product should be kept at-20 ℃, prevents degraded.

4.2 the recovery of cell;

(1) from liquid nitrogen container, takes out 1 frozen HEK293 cell, put into 38 ℃ of water-baths immediately, till melting fully to cell;

(2) frozen pipe content is changed in the 10mL centrifuge tube, add the DMEM that 4mL contains 10%FBS, the DMSO that adds when wash-out is frozen;

(3) 1200rpm, centrifugal 10min discards supernatant;

(4) add the DMEM that 2.5mL contains 10%FBS, blow evenly, being transferred to is in the plastic culture bottle, adds the DMEM that 2.5mL contains 10%FBS in centrifuge tube once more, blows evenly, and residual cells is changed in the same plastic culture bottle;

(5) unscrew the culturing bottle bottle cap, put 37 ℃, cultivate in the 5%CO2 incubator.

4.3 passage is cultivated

(1) substratum in the culturing bottle is inhaled abandoned, add the 1mL substratum and clean the back and inhale and abandon, to remove serum deprivation;

(2) ATV of adding 1mL 0.25% places under the mirror and observes, and inhales after cell is full and abandons ATV;

(3) add the DMEM that 2mL contains 10%FBS, the piping and druming attached cell makes the cell homodisperse, and the cell suspension branch is filled in 2-3 the new culturing bottle, puts 37 ℃, and the 5%CO2 incubator is cultivated.

4.4 cell transfecting

Transfection the day before yesterday, good 293 cells of inoculation upgrowth situation in six porocyte culture plates, every porocyte number about 5 * 10 ⁵Individual; Next day, treat 4 pRNTR/CMV-GFP/U6-shRNA interference carriers that will build when cell grows to 90% degrees of fusion respectively with pDsRed1-C1-ATGL plasmid co-transfection 293 cells that contain target gene, establish blank simultaneously, separately transfection only contains the contrast of target gene plasmid, whole screening repeats 3 times; Rotaring redyeing system is pRNTR/CMV-GFP/U6-shRNA plasmid 3 μ g, pDsRed1-C1-ATGL plasmid 1 μ g, and transfection process is with reference to the LipofectamineTM 2000 reagent working instructions of Invitrogen company; Behind the transfection 48h, under fluorescent microscope, observe the expression of fluorescence.

Transfection process is as follows: will hang down serum Opt i-MEM nutrient solution and Lipofectamine ^TM2000 under room temperature condition balance; The Lipofectamine of 10 μ L ^TM2000 and 5 μ g plasmids (in 2: 1 ratios, i.e. 2 μ L Lipofec tamine ^TM2000: 1 μ g plasmid DNA) add respectively among the low serum Opti-MEM of 250 μ L, flick tube wall and make mixing, room temperature leaves standstill 5min, and plasmid DNA/low serum Opti-MEM mixture is added Lipofectamine ^TMIn the 2000 low serum Opti-MEM mixtures, flick tube wall and make it mixing, incubated at room 20min; Transfection composite is added in the Tissue Culture Dish evenly, lentamente, gently revolve culture dish mixing liquid, cell is put into 37 ℃ of cell culture incubators hatch; 6-8h changes fresh medium after the transfection.

The 5th step: result verification;

5.1pRNTR/CMV-GFP/U6-shRNA the structure of carrier;

The shRNA-422 that obtains after the annealing, shRNA-600, shRNA-1148 and shRNA-NC double chain oligonucleotide are connected, transform, are coated with flat board with the pRNTR/CMV-GFP/U6 carrier, choose mono-clonal, behind the extracting plasmid, the results are shown in Figure 1; Through occurring 2 fragments behind Sca I and the EcoR V double digestion, size matches with expected results, sees Fig. 2; Sequencing result shows that sequence and the designed sequence inserted in pRNTR/CMV-GFP/U6-s hRNA-422, pRNTR/CMV-GFP/U6-s hRNA-600, pRNTR/CMV-GFP/U6-shRNA-1148, the pRNTR/CMV-GFP/U6-shRNA-NC carrier are in full accord, proves the vector construction success.

5.2pDsRed1-C1-ATGL the structure of carrier;

5.2.1pGEM-T-ATGL the bacterium liquid PCR of positive transformant screening is identified with double digestion;

Utilize the bacterium liquid of ATGL gene, utilize ATGLF1 and ATGLR1 primer to carry out bacterium liquid pcr amplification, through 1% agarose gel electrophoresis, the target gene fragment that 1461bp occurs behind positive colony extraction plasmid, utilizes SalI and XhoI restriction endonuclease to carry out the double digestion analysis, through 1% agarose gel electrophoresis, see Fig. 3, obtain the target gene fragment of 1461bp, reclaim the purpose fragment and it is connected with the pDsRed1-C1 carrier.

5.2.2pDsRed1-C1-ATGL the bacterium colony PCR of positive transformant screening is identified with double digestion;

Be connected to the ATGL gene on the pDsRed1-C1 carrier, utilize pDsRedATGLF1 and pDsRedATGLR1 primer to carry out colony PCR amplification after, the target gene fragment of 1461bp appears through 1% agarose gel electrophoresis, the results are shown in Figure 4; Carry out the double digestion analysis after extracting plasmid,, obtain target gene fragment, the results are shown in Figure 5, show successfully to make up the pDsRed1-C1-ATGL carrier through 1% agarose gel electrophoresis.

5.3shRNA the screening of sequence;

With express interference sequence the pRNTR/CMV-GFP/U6-shRNA carrier respectively with carrier (pDsRed1-C1-ATGL) the cotransfection 293 cell 48h that contain corresponding triglyceride hydrolysis enzyme gene fragment after, under fluorescent microscope, observe each the group in the egfp expression amount basic identical, but compare pRNTR/CMV-GFP/U6-shRNA-422 with negative control, pRNTR/CMV-GFP/U6-shRNA-1148 transfection group red fluorescent protein expression amount decreases, wherein the pRNTR/CMV-GFP/U6-shRNA-600 transfection group reduces the most obvious; The pRNTR/CMV-GFP/U6-shRNA-NC group is basic identical with the control group of not transfection interference carrier, sees Fig. 6; Red fluorescent protein and triglyceride hydrolysis enzyme Gene Partial functional domain form fusion rotein in the test, so the interference sequence of red fluorescent protein expression amount reduction explanation design has interference effect to triglyceride hydrolysis enzyme gene.

Should be understood that; this specification sheets is that the present invention is described in detail for example with regard to the concrete operations step; and for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.

Claims (10)

1. the screening method of a triglyceride hydrolysis enzyme gene effective shRNA is characterized in that, may further comprise the steps: A1: the segmental design of single strand dna oligonucleotide of coding purpose shRNA, synthetic; The structure of A2:pENTR/CMV-GFP/U6-shRNA carrier; The structure of A3:pDsRed1-C1-ATGL carrier; The screening of A4:shRNA ordered sequence.

2. method according to claim 1, it is characterized in that, described steps A 1 is specifically carried out following operation: according to the Xi Nongsa energy sheep ATGL sequence of GenBank announcement, design three ATGL-siRNA and 1 negative control sequence, on the siRNA basis, with the oligonucleotide of 19bp with forward and reverse combination, middle hairpin loop sequence interval of adding GAGTACTG, make the inner hairpin structure that forms of single strand dna oligonucleotide, every pair of single strand dna oligonucleotide two ends have BamH I and XhoI restriction enzyme site respectively, wherein 3 ' of positive-sense strand end adds the TTTTTT termination signal, is just obtaining shRNA, the antisense strand template.

3. method according to claim 2 is characterized in that, described steps A 2 concrete following operation: the A21 that carry out: the single strand dna oligonucleotide annealing reaction generates double-stranded; Synthetic single strand dna oligonucleotide sequence is dissolved into the solution that concentration is 200 μ M with the sterilization deionized water, carry out annealing reaction then, annealing conditions is: 95 ℃ * 4 minutes, taking-up is put room temperature and is cooled off gradually, promptly obtaining concentration is the double chain oligonucleotide sequence of 50 μ M, is labeled as respectively: ds422, ds600, ds1148; Take a morsel respectively again and be diluted to the working concentration that concentration is 5nM, A22: make up shuttle vectors; With BamH I and Xho I double digestion pRNTR/CMV-GFP/U6 carrier and cut glue and reclaim, then described ds422, described ds600 and described ds1148 are cut back to close the product ligation with pRNTR/CMV-GFP/U6 carrier enzyme respectively, reaction conditions is that 4 ℃ of connections are spent the night, and obtains connecting product; A23: connect product and transform; Get the described connection product of 10 μ L and add 100 μ L competence DH5 α bacteriums, ice bath 30 minutes, 42 ℃ of heat shocks 90 seconds, put ice bath 12 minutes, add 600 μ L LB nutrient solutions, 37 ℃ of gentle concussions were cultivated 45 minutes, and bacterium is coated on the agar plate that contains 50 μ g/mL kantlex, placed 37 ℃ of incubations; A24: screening shuttle vectors clone.

4. method according to claim 3 is characterized in that, described ligation system is as follows: pENTRTM/U6,2 μ L; Ds oligos (5nM), 1 μ L; 5 * annealing buffer, 4 μ L; T ₄Dna ligase, 1 μ L; The water that does not contain DNa se/Rnase, 12 μ L; Cumulative volume 20.0 μ L

5. method according to claim 3, it is characterized in that, operation below described steps A 24 concrete the execution: (1) shuttle vectors clone's plasmid enzyme restriction is identified: the cohesive terminus complementation of the linear carrier pENTRTM/U6 that the cohesive terminus of ds oligos and test kit provide, after ligation, form circular plasmids; Picking mono-clonal bacterium colony on the kantlex agar plate, transferred species is in the LB nutrient solution that contains 50 μ g/mL kantlex of 4mL, 37 ℃ shaking culture 12-16 hour, get bacterium liquid 2mL and extract plasmid, size meets carries out Sca I and EcoR V double digestion is identified again, and product is observed with 1.2% agarose gel electrophoresis; (2) shuttle vectors clone's order-checking is identified: get the correct mono-clonal bacterium liquid of double digestion evaluation in (1), send order-checking company to carry out sequencing with the automatic fluorescence sequenator of ABI377 type DNA, primer is sequencing primer U6 or M13.

6. method according to claim 1 is characterized in that, described steps A 3 concrete following operation: the A31 that carry out: make up the pDsRed1-C1-ATGL cloning vector; The target gene fragment that reclaims is connected with the pGM-T carrier, 16 ℃ of constant temperature spend the night, connect product transformed competence colibacillus E.coliDH5 α bacterial strain, coat in the LB culture dish that contains penbritin and X-gal/IPTG, in 37 ℃ of overnight incubation, the single bacterium colony of picking white shakes bacterium, carries out bacterium colony PCR simultaneously, gets 10 μ L reaction solutions after reaction finishes and carries out the evaluation of 1% agarose gel electrophoresis; Positive colony bacterium liquid is utilized alkaline lysis extracting plasmid, carry out double digestion with Xho I and Sal I restriction endonuclease then and identify that the endonuclease reaction system is as follows: 10 * H Buffer, 2.0 μ L; Xho I, 0.7 μ L; Sal I, 0.7 μ L; PGM-T-gATGL1A1,10.0 μ L; DdH2O, 6.6 μ L; Cumulative volume 20.0 μ L; 37 ℃ of water-baths spend the night, and get total overall reaction liquid electrophoresis on 1% sepharose, reclaim to have the target gene fragment that designs restriction enzyme site; A32: make up the pDsRed1-C1-ATGL expression vector.

7. method according to claim 6 is characterized in that, operation below described steps A 32 concrete the execution: (1) preparation pDsRed1-C1 endonuclease reaction system is also carried out 37 ℃ of water-baths and spent the night; (2) after endonuclease reaction finishes, get total overall reaction liquid and carry out 1% agarose gel electrophoresis, reclaim the linearizing fragment after pDsRed1-C1 carrier enzyme is cut; (3) carrier segments is connected with target gene fragment, 16 ℃ of reactions are spent the night; (4) pDsRed1-C1-ATGL is converted into E.coli DH5 α competent cell, coating contains the plate of kantlex, 37 ℃ of overnight incubation, and picking list bacterium colony shakes bacterium, carries out bacterium colony PCR simultaneously and identifies, extracts plasmid then, carries out enzyme and cuts evaluation.

8. method according to claim 7 is characterized in that, described endonuclease reaction system is: 10 * H Buffer, 2.0 μ L; Xho I, 0.7 μ L; Sal I, 0.7 μ L; PDsRed1-C1-ATGL, 5.0 μ L; DdH ₂O, 11.6 μ L; Cumulative volume 20.0 μ L.

9. method according to claim 1 is characterized in that, described steps A 4 concrete following operation: the A41 that carry out: go the intracellular toxin plasmid to extract; A42: the recovery of cell; The A43 passage is cultivated: A44: cell transfecting.

10. method according to claim 9, it is characterized in that, operation below described steps A 41 concrete execution the: (1) column equilibration: the balance liquid that in adsorption column, adds 500 μ L, adsorption column is put into collection tube, 12000rpm (13400 * g) centrifugal 1 minute, outwell the waste liquid in the collection tube, adsorption column is relay reclaim in the collector; (2) the bacterium liquid of getting the 5-15mL incubated overnight adds in the centrifuge tube, and 12000rpm (13400 * g) centrifugal 1 minute, absorb supernatant liquor; (3) in the centrifuge tube that leaves bacterial sediment, add the solution P1 that 500 μ L have added RNaseA, use the vortex vibrator bacterial cell precipitation that thoroughly suspends; (4) in centrifuge tube, add 500 μ L solution P2, leniently spin upside down and make the abundant cracking of thalline for 6-8 time; (5) add 700 μ L solution P3 in centrifuge tube, leniently spin upside down 6-8 time immediately, fully white flocks appears in mixing, and 12000rpm (13400 * g) centrifugal 10 minutes, form precipitation in the centrifuge tube bottom, the collection supernatant liquor; (6) the supernatant liquor gradation of collecting in (5) is added Filter column, Filter column is put into collection tube, and 12000rpm (13400 * g) centrifugal 2 minutes, the solution gradation that obtains in the collection tube of centrifugal back is added in the adsorption column; (7) 12000rpm (13400 * g) centrifugal 1 minute, outwell the waste liquid in the collection tube, adsorption column is put into collection tube; (8) in adsorption column, add 500 μ l protein liquid removals, and 12000rpm (13400 * g) centrifugal 1 minute, outwell the waste liquid in the collection tube, adsorption column is put into collection tube; (9) in rinsing liquid, add dehydrated alcohol according to 3 times of volumes, in adsorption column, add the described rinsing liquid of 700 μ L, and 12000rpm (13400 * g) centrifugal 1 minute, outwell the waste liquid in the collection tube, adsorption column is put into collection tube; (10) in adsorption column, add 500 μ L rinsing liquids, and 12000rpm (13400 * g) centrifugal 1 minute, outwell the waste liquid in the collection tube; (11) adsorption column is relay reclaim place in the collector 12000rpm (13400 * g) centrifugal 2 minutes, rinsing liquid remaining in the adsorption column is removed; (12) adsorption column is placed a clean centrifuge tube, to the unsettled dropping in the middle part of adsorption film 100-300 μ L elution buffer, room temperature was placed 2 minutes, and (13400 * g) collect plasmid solution in the centrifuge tube after centrifugal 1 minute 12000rpm.

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