CN104651390A - Method for building yeast two-hybrid cDNA library of bacterium - Google Patents
Method for building yeast two-hybrid cDNA library of bacterium Download PDFInfo
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Abstract
The invention discloses a method for building a yeast two-hybrid cDNA library of bacterium. The method comprises the following steps: extracting total RNA of the bacterium; removing 5s rRNA, tRNA and small-molecular RNA from the total RNA; enriching mRNA; synthesizing single-chain cDNA by performing reverse transcription PCR on the enriched mRNA by taking a random hexamer as a primer; amplifying by taking the single-chain cDNA as a template by means of LD-PCR to obtain double-chain cDNA; co-transforming a target protein yeast strain by the total-length double-chain cDNA and a library vector by means of homologous recombination to obtain the cDNA library of the bacterium. The invention provides the method for building the yeast two-hybrid cDNA library after synthesizing the cDNA by means of inverse transcription by taking the mRNA of the bacterium as the template. By taking the mRNA as the template, interference of rRNA, generated during the inverse transcription of the total RNA, can be avoided; use of restriction enzyme for locus deviation of the genomic DNA of the bacterium can be avoided as well; the accuracy of an open reading frame of the library is higher; quite good quality is realized.
Description
Technical field:
The invention belongs to biological field, be specifically related to a kind of method building the Yeast two-hybrid cDNA library of bacterium.
Background technology:
Two structural domains that the growth transcription factor GAL4 that yeast two-hybrid system make use of yeast contains, DNA binding domain (DNA binding domain, and transcription activating domain (activation domain BD), AD), known (bait gene) and target gene or the cDNA containing target gene are structured in containing on BD and AD plasmid vector respectively, when these two kinds of plasmid cotransformation competent yeast cells, if the protein-interacting that in the bait protein target protein that can be combined with AD that BD combines or library, some cDNA encodes, to each other in conjunction with time, BD and the AD being positioned at flank then can be caused spatially close, present the complete activity of GAL4 transcription factor, start the reporter gene in downstream as Ade, the expression of His and Lac etc., thus grow on specific defect substratum.Therefore, utilize yeast two-hybrid system can screen the interactional albumen with bait protein, can also be studied oneself and know interaction between albumen.
Wherein the structure of Yeast two-hybrid cDNA library generally adds homologous recombination sequence at cDNA two ends, then with target protein carrier if pGADT7-Rec cotransformation target protein yeast strain is as AH109, under the effect of yeast homologous recombinase, cDNA sequence is incorporated on carrier, completes the structure of cDNA library.Because most eukaryotic cell mRNA 3 ' end has Poly (A) tail, so by using with the homologous recombination sequence of Oligo (dT) and its pairing after extracting Eukaryotic total serum IgE, only mRNA can be inverted record, and the cDNA that reverse transcription simultaneously obtains adds homologous recombination sequence.
But the mRNA of the bacterium overwhelming majority is without Poly (A) tail, and mRNA only accounts for the 1-4% of total serum IgE, if use random hexamer as primer, in system, all RNA molecule all act as cDNA first chain template, in the cDNA of synthesis, 96% derives from rRNA, and composition is too complicated.The structure in existing directed toward bacteria library is generally by using restriction enzyme to carry out incomplete digestion to bacterial genomes DNA, then transformation of E. coli after being connected with carrier library, the skewed popularity of restriction endonuclease loci cannot be avoided, greatly reduce the abundance in library, therefore the cDNA library of bacterium structure and screening be limited always.
Summary of the invention:
The object of this invention is to provide a kind of method that energy high quality builds the Yeast two-hybrid cDNA library of the structure bacterium of the Yeast two-hybrid cDNA library of bacterium.
The method of Yeast two-hybrid cDNA library of the present invention, is characterized in that, comprises the following steps:
Extract bacterium total serum IgE, remove 5s rRNA, the tRNA in total serum IgE and microRNA again, again enrichment is carried out to mRNA, random hexamer is used to carry out the synthesizing single-stranded cDNA of reverse transcription PCR (RT-PCR) as the mRNA of primer pair enrichment, again by LD-PCR with the cDNA of strand for template amplification obtain double-strand cDNA, recycling homologous recombination, by total length double-strand cDNA and carrier library cotransformation target protein yeast strain, obtains the cDNA library of bacterium.
Preferably, 5s rRNA, tRNA in described removal total serum IgE and microRNA utilize test kit MEGAclear
tMkit removes.
It is preferably, described that to carry out enrichment to mRNA be utilize test kit MICROB ExpressTM Bacterial mRNAEnrichment Kit (Ambion) to carry out enrichment to mRNA.
Preferably, described yeast strain is yeast strain AH109.
Compared with existing method, the present invention proposes a kind of using bacterium mRNA as template, after reverse transcription synthesis cDNA, build the method for Yeast two-hybrid cDNA library.Owing to being be template with mRNA, not only avoid and produce interference with rRNA during total serum IgE reverse transcription, also can avoid using restriction enzyme to the site skewed popularity of bacterial genomes DNA, the exactness of the open reading frame in library is higher, and quality is very good.
Accompanying drawing illustrates:
Fig. 1 is the electrophoresis detection figure of bacterium total serum IgE;
Fig. 2 is the electrophoresis detection figure of the RNA sample removing 5s rRNA, tRNA and microRNA;
Fig. 3 is the electrophoresis detection figure of the mRNA of enrichment;
Fig. 4 is the electrophoresis detection figure of the double-strand cDNA that amplification obtains;
Fig. 5 filters with BD CHROMA SPIN TE-400 the electrophoresis detection figure that column purification reclaims double-strand cDNA;
Fig. 6 is the SD/-Leu flat board coating growth figure of yeast two-hybrid bacterial strain Y2HGOLD;
Fig. 7 is the electrophoresis detection figure of cDNA library quality examination.
Embodiment:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1: the extraction of bacterium total serum IgE
By the distortion Methionin bacillus (Lysinibacillus varians.) that activated, [by Methionin genus bacillus (Lysinibacillus sp.) GY32, (this bacterium is stored in China typical culture collection center (CCTCC) on September 7th, 2011 for it, address: Wuhan University of Wuhan, China city, deposit number is CCTCC NO:M 2011307, be disclosed in Chinese patent: ZL201110300903.8) rename] be transferred in LB liquid nutrient medium, 30 DEG C are cultured to logarithmic phase, 8000rpm 4 DEG C of centrifugal 2min, abandon supernatant.Add 1mL TRIZOL extracting solution, shake up, vortex oscillation 10min, add 200 μ L chloroforms, mixing 5min, room temperature places 5min, 4 DEG C, the centrifugal 10min of 13000rpm.Draw supernatant (about 600 μ L) in another clean centrifuge tube, add 400 μ L Virahols ,-20 DEG C of centrifugal 10min of precipitation 10min, 12000rpm.Outwell supernatant, add 1mL 75% ethanol, flick, the centrifugal 5min of 8000rpm, outwells supernatant, repeats 2 times.RNA room temperature is air-dry, and add 20-30 μ L DEPC process water dissolution, as shown in Figure 1 ,-80 DEG C of Ultralow Temperature Freezers save backup electrophoresis detection, obtain total serum IgE.
Embodiment 2: purifying also removes 5s rRNA, tRNA and microRNA, carries out enrichment to mRNA;
2.1 purifying also remove 5s rRNA, tRNA and microRNA
Use
the MEGAclear of company
tMkit process total serum IgE.Adding Elution Solution toward RNA sample is 100 μ L to volume, mixing; Add the Binding Solution of 350 μ L, mixing; Add the ethanol of 250 μ L 100%, mixing; Filter Cartridge is inserted in Collection and Elution Tube, above-mentioned mixed solution is transferred to FilterCartridge, the centrifugal 1min of 10000rpm, outwells waste liquid; Add 500 μ L Wash Solution toward Filter Cartridge, the centrifugal 30sec of 10000rpm, outwells waste liquid, repeats once; Filter Cartridge is inserted in new Collection/ElutionTube, 50 μ L Elution Solution are added toward Filter Cartridge center, 65-70 DEG C of process 10min, collected by centrifugation solution electrophoresis detection, as shown in Figure 2, total serum IgE sample has removed 5s rRNA, tRNA and microRNA, obtains the RNA sample removing 5s rRNA, tRNA and microRNA thus.
2.2 couples of mRNA carry out enrichment
Select
the MICROB ExpressTM Bacterial mRNA Enrichment Kit of company carries out enrichment to mRNA, this system to the quality of total serum IgE and purity requirement is: A260/A280>1.7, maximum is no more than 10 μ g, and maximum volume is no more than 15 μ L; Require that EDTA concentration is greater than 1mM, prevent RNA hydrolysis in heat-processed with chelating divalent positively charged ion.200 μ L Binding Buffer are added in 1.5mL Eppendorf pipe (test kit provides), then 2-10 μ gTotal RNA (<10 μ g is added, <15 μ L) (removal 5s rRNA, the tRNA obtained in step 2.1 and the RNA sample of microRNA), carefully mix.Add 4 μ L Capture Oligo Mix, carefully mix, low-speed centrifugal, at the bottom of liquid collecting to pipe.70 DEG C of heating 10min, make rRNA sex change destroy its secondary structure, so that rRNA and capture Oligonucleotide is fully hybridized.37 DEG C of annealing 15min, rRNA and capture oligonucleotide hybridization.Extend annealing time and fully have certain help to hybridization.Add required Oligo MagBeads at 1.5mL Eppendorf pipe, each sample needs 50 μ L.By 1.5mL Eppendorf pipe as on magnetic frame, make Oligo MagBeads be adsorbed onto magnetic one side completely, then, draw supernatant carefully and discard.Add isopyknic Nuclease-free Water, mix carefully, cleaning OligoMagBeads, then the same operation.Add isopyknic Binding Buffer, mix carefully, balance Oligo MagBeads, the same operation.Add isopyknic Binding Buffer, mix carefully, then put into 37 DEG C of water-baths.In RNA/CaptureOligo Mix, add the Oligo MagBeads that 50 μ L handle well, carefully mix, gently at the bottom of collected by centrifugation to pipe.Then 37 DEG C of incubation 15min.Use magnetic frame absorption Oligo MagBeads, then carefully supernatant (mRNA containing enrichment) is transferred to the Collection Tube being placed in precooling on ice.Add to Oligo MagBeads the Wash Solution that 100 μ L are preheating to 37 DEG C, mix carefully.Then use magnetic frame absorption Oligo MagBeads, finally carefully supernatant is transferred to containing in the Collection Tube of mRNA.The 3MSodium Acetate (sodium-acetate, 35 μ L) of 1/10 volume is added, the Glycogen (glycogen, 7 μ L) of the 5M of 1/50 volume in acquired 350 μ L elutriants.Add the ice-cold dehydrated alcohol (1175 μ L) of 3 times of volumes, fully mix ,-20 DEG C precipitate at least 1h.The centrifugal 30min of 13000rpm, carefully sucks supernatant.Add the ethanol 750 μ L washing precipitation of 70%, the centrifugal 10min of 13000rpm, then wash once.Ambient temperatare adds the Nuclease-free Water dissolution precipitation again of 50 μ L after putting 5min.Extraction portion is divided and is carried out electrophoresis detection as shown in Figure 3, mRNA by enrichment, remaining mRNA is put in-70 DEG C for subsequent use, obtain the mRNA of enrichment thus.
Embodiment 3 obtains double-strand cDNA
3.1 use random hexamer as the synthesizing single-stranded cDNA of primer
Sterile centrifugation tube adds 1-2 μ L RNA (0.025-1.0 μ g mRNA) (mRNA of the enrichment in embodiment 2), 1.0 μ LCDS III/6 primers, and adding sterilizing deionization ultrapure water to cumulative volume is 4.0 μ L; Mixing, 72 DEG C of water-baths 2 minutes; 2 minutes are cooled, gentle centrifugation in frozen water; Add following reagent: 2.0 μ L 5 × First-Strand Buffer, 1.0 μ L DTT (20mM), 1.0 μ L dNTPs (10mM), 1.0 μ L MMLV ThermoScript II; Mixing, 42 DEG C of water-baths 10 minutes; Add SMART III oligonucleotide of 1.0 μ L; 1 hour (adding Valelinum Liquidum) of 42 DEG C of water-baths; Amplification instrument is placed 10 minutes for 75 DEG C, stops building-up reactions, be cooled to room temperature.Add the RNase H of 2.0 μ L (2.0 unit), 37 DEG C of temperature are bathed 20 minutes.-20 DEG C of conditions transfer use of purchasing, and obtain strand cDNA.
3.2 obtain double-strand cDNA by LD-PCR amplification
PCR amplification instrument is preheated to 95 DEG C, following medicine is added: 2.0 μ L strand cDNA at centrifuge tube, 70 μ L deionization ultrapure waters, 10 μ L10 × BD Advantage 2PCR damping fluids, 2 μ L50 × dNTPs Mix, 2 μ L 5 ' PCR primer (primer sequence: 5'-TTCCACCCAAGCAGTGGTATCAACGCAGAGTGG-3'), 2 μ L 3 ' PCR primer (primer sequence: 5'-GTATCGATGCCCACCCTCTAGAGGCCGAGGCGGCCGACA-3'), 10 μ L10 × GC-Melt solution, 2 μ L50 × BD Advantage 2 polymerase mix, mixing, gentle centrifugation, drip sterilizing paraffin oil, amplification program: 95 DEG C of 30sec, 23 ~ 26 circulations, 95 DEG C of 10sec, 68 DEG C of 6min (note: each be circulated throughout after, annealing time increases by 5 seconds), 68 DEG C of 5min, get 7 μ L amplified productions, add Marker, at the agarose gel electrophoresis of 1.2%, detect amplification as Fig. 4,-20 DEG C of conditions transfer use of purchasing, and obtain double-strand cDNA.
3.3 filter column purification with BD CHROMA SPIN TE-400 reclaims double-strand cDNA
Take out filter post, teetertotter several times, every 95 μ L samples (the double-strand cDNA of step 3.2) filter post; Go out to filter post collapsible tube with forefinger and thumbscrew, filter column sleeve is entered collection tube, keeps two ends cap for subsequent use; Centrifugal 5 minutes of 700 × g; Take off filter post, abandon collection tube and filtrate; Filter post is inserted in the collection tube of another 2mL again, and careful filter post glue face that the cDNA sample of 95 μ L is added to central authorities, in order to avoid sample flows out from the inner side in glue face; Centrifugal 5 minutes of 700 × g; Take off filter post and collection tube, sublimed cDNA concentrates on the bottom of collection tube; From same sample, sublimed cDNA puts into unified centrifuge tube; Add following reagent: 1/10 volumes of acetic acid sodium (3M; PH 4.8), 2.5 times of volume 95% ethanol (-20 DEG C); Slightly shake up; Spend the night under-20 DEG C of conditions; Under room temperature, centrifugal 20 minutes of 14000rpm; Careful sucking-off supernatant liquor; Gentle centrifugation makes remaining liquid all focus on bottom centrifuge tube; Liquid more than careful sucking-off, the little agglomerate of cDNA dry 10 minutes in atmosphere; Dissolve the little agglomerate of cDNA with the deionization ultrapure water of 20 μ L, as shown in Figure 5 ,-20 DEG C of conditions transfer use of purchasing to electrophoresis detection, obtain the double-strand cDNA of purifying thus.
The structure of the Yeast two-hybrid cDNA library of embodiment 4GY32
The structure of 4.1 cDNA libraries
1) preparation of competent yeast cells
Inoculation yeast AH109 bacterial strain on YPDA substratum, incubation time≤4 week, colony diameter is 2-3mm; 15mL sterile centrifugation tube adds the YPDA liquid nutrient medium of 3mL, often pipe bacterium colony, 30 DEG C, 250rpm, 8h; Drawing 5 μ l seed liquor joins in the 250mL triangular flask containing 50ml YPDA; 30 DEG C, 250rpm, 16 ~ 20h survey OD600=0.15 ~ 0.3, nutrient solution are moved to 50ml centrifuge tube; Room temperature, 700rcf, 5min, abandon supernatant liquor; By the resuspended precipitation of 100mL YPDA, shake with 500ml triangular flask, 30 DEG C, 250rpm, 3 ~ 5h; Survey OD600=0.4 ~ 0.5, nutrient solution is dispensed into two 50mL centrifuge tubes, room temperature, 700rcf, 5min, abandon supernatant liquor; Often pipe adds 30mL deionization ultrapure water, resuspended precipitation, and 700rcf, 5min, remove supernatant; With 3mL 1.1*TE/LiAc suspension thalline, sucking-off and two 1.5mL centrifuge tubes, 12,000rpm, 15 ~ 30s; Abandoning supernatant, often pipe adds the resuspended precipitation of 600 μ L 1.1*TE/LiAc, (Caution: the competence of making must use at once) for subsequent use.
2) in the centrifuge tube of a 15mL, following medicine is added
20μL dscDNA
6μL pGADT7-Rec(0.54μg/μL)
20 μ L Herring Testes Carrier DNA (sex change)
3) in DNA, add the competence yeast cell of 600 μ L;
4) shake up gently;
5) the PEG/LiAc solution of 2.5mL is added;
6) shake up gently;
7) temperature bath 45min under 30 DEG C of conditions, mixing in every 15 minutes is once;
8) add 160 μ L DMSO, shake up, 42 DEG C of condition water-baths 20 minutes, shake up once in every 10 minutes;
9) centrifugal 5 minutes of 700 × g;
10) remove supernatant, each pipe adds the YPD Plus liquid nutrient medium (noting: the YPD substratum can not using standard) of 3mL;
11) 30 DEG C are shaken bacterium 90 minutes;
12) centrifugal 5 minutes of 700 × g;
13) remove supernatant, add the NaCl solution of 15mL 0.9%.
14) the bacterium liquid smearing 150 μ L on the culture dish of ready 150mm containing substratum (about needs 100, the culture dish of 100mmSD/-Leu containing substratum smears 1:10,1:100,1:1000 respectively, with the bacterium liquid that 1:10000 dilutes, to detect the efficiency of conversion)
15) culture dish be inverted, light culture under 30 DEG C of conditions until bacterium colony occur, (generally will cultivate 3-6 days);
16) transformation efficiency is calculated: general requirement>=1 × 10
6transformant/3 μ g pGADT7-Rec.
17) culture dish preserves 3-4 hour under 4 DEG C of conditions;
18) each culture dish adds the substratum (YPD of 25% glycerine) of 5mL cooling;
19) roll gently with the granulated glass sphere of sterilizing, yeast cell is driven feed liquor body;
20) mix in the triangular flask all liquid being imported a sterilizing;
21) hemocytometer detectable level is used.If concentration≤2 × 107 cell/mL, by the volume of centrifugal minimizing suspension liquid;
22) by the amount packing of 1mL/ pipe, save backup under-80 DEG C of conditions (shelf time can not more than 1 year);
23) smear by 1:100 respectively on the culture dish of 100mm SD/-Leu substratum, 1:1000, and the bacterium liquid 100 μ L that the rare ratio of 1:10000 has been released, cultivate until (generally will cultivate 2-3 days) appears in bacterium colony under 30 DEG C of conditions, number colony number, estimates the titer concentrations in library.
According to above-mentioned steps, use homologous recombination technique GY32 full-length cDNA (the double-strand cDNA of the purifying of step 3.3) transformed yeast double cross strains A H109, be coated with the SD/-Leu flat board (Fig. 6) that 50 diameters are 150mm, 50 2mL centrifuge tubes are dispensed into after whole mixed collection, often pipe 1mL, obtains the cDNA library of GY32.
4.2 cDNA library quality examinations
Extract 50 ~ 100 clones; Add at the sterile centrifugation tube of 0.5mL: 6.6 μ L PCR-grade deionized waters, 5 μ L10 × PCR buffer, 1 μ L AD 5'Primer (10 μMs), 1 μ L AD 3'Primer (10 μMs), 4 μ L dNTPs Mix (2.5mM), 2 μ L plasmids, 0.4 μ L rTaq (5U/ μ L, Takara), cumulative volume 20 μ L.Amplification condition: after 95 DEG C of first sex change of 3min, carry out 95 DEG C of 30s, 68 DEG C of 3min, totally 30 circulations; Last 68 DEG C of 5min that circulate.Get 10 μ L products, in the agarose gel electrophoresis detected result of 1.2%, the average gene of bacterium is 1Kb, building library is that excessive or too small fragment is all improper, and fragment is crossed conference and improved false positive in follow-up screening, and fragment is too small, is that the albumen that merges in library is too small, therefore suitable clip size is selected could to improve the diversity in library, as shown in Figure 7, the fragment major part of Ben Wenku is in 700-3000bp, size to fit.Random picking 13 clones carry out sequencing analysis and find that there is 10 clones and there is correct open reading frame, and the yeast two-hybrid library of the GY32 therefore built is successful.
Claims (4)
1. the method for a Yeast two-hybrid cDNA library, it is characterized in that, comprise the following steps: extract bacterium total serum IgE, remove 5s rRNA, the tRNA in total serum IgE and microRNA again, again enrichment is carried out to mRNA, random hexamer is used to carry out the synthesizing single-stranded cDNA of reverse transcription PCR as the mRNA of primer pair enrichment, again by LD-PCR with the cDNA of strand for template amplification obtain double-strand cDNA, recycling homologous recombination, by total length double-strand cDNA and carrier library cotransformation target protein yeast strain, obtains the cDNA library of bacterium.
2. method according to claim 1, is characterized in that, 5s rRNA, tRNA in described removal total serum IgE and microRNA utilize test kit MEGAclear
tMkit removes.
3. method according to claim 1, is characterized in that, described to carry out enrichment to mRNA be utilize test kit MICROB ExpressTM Bacterial mRNA Enrichment Kit (Ambion) to carry out enrichment to mRNA.
4. method according to claim 1, is characterized in that, described yeast strain is yeast strain AH109.
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| CN114990105A (en) * | 2022-06-09 | 2022-09-02 | 南京瑞源生物技术有限公司 | Yeast membrane library construction method based on plant sample |
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| CN111394800A (en) * | 2020-03-13 | 2020-07-10 | 南京农业大学 | Method for evaluating quality of ginseng-free species yeast two-hybrid library |
| CN111394800B (en) * | 2020-03-13 | 2022-04-08 | 南京农业大学 | Method for evaluating quality of ginseng-free species yeast two-hybrid library |
| CN114426968A (en) * | 2022-02-22 | 2022-05-03 | 江南大学 | Bacillus subtilis full-length cDNA library construction method and directional screening application thereof |
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Application publication date: 20150527 |