CN112574994B - Method for screening active region of pig miR-155 gene promoter and transcriptional regulatory element - Google Patents
Method for screening active region of pig miR-155 gene promoter and transcriptional regulatory element Download PDFInfo
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Abstract
The invention provides a method for screening a promoter active region and a transcription regulatory element of a pig miR-155 gene, which specifically comprises the following steps: (1) gram Long Zhu miR-155 gene promoter sequence; (2) Constructing a pig miR-155 gene 5' -end promoter series deletion fragment luciferase reporter gene vector; (3) The constructed pig miR-155 gene 5' -end promoter series deletion fragment luciferase reporter gene vectors are respectively transfected into cells, double luciferase activities are detected, if the luciferase activities between adjacent deletion fragments are found to be obviously changed, the pig miR-155 gene promoter active areas are indicated to be positioned between the adjacent deletion fragments, the pig miR-155 gene promoter active areas are identified through a double luciferase activity analysis method, regulatory elements for regulating and controlling the transcription activities of the pig miR-155 gene promoters are further identified, and the Foxp3 transcription factor binding site is determined to be a regulatory element for regulating and controlling the transcription activities of the pig miR-155 gene promoters.
Description
Technical Field
The invention relates to a pig miR-155 gene promoter and an active region screening method thereof, and also relates to a method for identifying a regulatory element for regulating and controlling the transcriptional activity of the pig miR-155 gene promoter.
Background
miR-155 is endogenous non-coding small-molecule RNA with regulation and control effects on activation of immune cells and expression of inflammatory factors, and plays an important role in promoting differentiation of immune cells of organisms, regulating inflammatory reaction and immune response of the organisms, resisting infection of the organisms and the like. Research shows that miR-155 is regulated and controlled by a transcription factor Foxp3, the transcription factor Foxp3 is necessary for regulatory T cells (Treg cells), and in human and mouse Treg cell whole genome research, foxp3 is found to be a direct target of miR-155, a binding site is located at a position about 500bp upstream of miR-155 precursor, and miR-155 can target and regulate Foxp3 expression so as to participate in regulating differentiation of the Treg cells. Further, research shows that miR-155 is regulated and controlled by nuclear transcription factors NF- κB, transcription factors AP-1, ets2 and ELK3, and research on the regulation and control of miR-155 is mostly concentrated on related diseases of human beings, and research on the regulation and control of miR-155 in pigs is freshly reported, so that the research on the aspects is developed and has important significance for explaining the functions of miR-155 genes in pigs.
Disclosure of Invention
(one) solving the technical problems
Aiming at the defects of the prior art, the invention provides a pig miR-155 gene promoter and an active region screening method thereof, and also provides a method for identifying a regulating element for regulating the transcriptional activity of the pig miR-155 gene promoter.
(II) technical scheme
In order to achieve the above purpose, the invention is realized by the following technical scheme:
in one aspect, the invention provides a pig miR-155 gene promoter, and the sequence of the pig miR-155 gene promoter is shown in a sequence table SEQ ID No. 1.
In another aspect, the invention is directed to determining the active region of a porcine miR-155 gene promoter and a regulatory element for regulating transcriptional activity of the porcine miR-155 gene promoter.
The method for determining the active region of the pig miR-155 gene promoter and the regulating element for regulating the transcriptional activity of the pig miR-155 gene promoter comprises the following steps:
(1) The sequence of a miR-155 gene promoter is shown in gram Long Zhu, wherein the sequence is marked as +1 by a pre-miR155 1 st base of a miR-155 precursor, 5000bp is extended to the 5' end, a specific primer is designed, amplification is carried out in two sections, target fragments are respectively from-4792 to-2377 and from-2270 to +119, and two sections of miR-155 promoter recombinant plasmids are constructed and are respectively named as pGL3-bicpro1 and pGL3-bicpro2; the sequences of the specific primers are shown in the following table:
(2) Constructing a pig miR-155 gene 5' -end promoter series deletion fragment luciferase report carrier: pGL3-bicpro1 (-4792-2377), pGL3-bicQ1 (-3428-2377), pGL3-bicQ2 (-3034-2377), pGL3-bicQ3 (-2822-2377), pGL3-bicpro2 (-2270- +119), pGL3-bicQ4 (-1832- +119), pGL3-bicQ5 (-1516- +119), pGL3-bicQ6 (-1203- +119), pGL3-bicQ7 (-409- +119);
(3) Respectively transfecting PK15 cells and 3D4/21 cells by using the vector constructed in the step (2), detecting the activity of the double luciferases of the pig miR-155 gene promoter and the deletion expression vector in the PK15 cells and the 3D4/21 cells by using a Promega bioluminescence detector, taking plasmid pRL-TK for expressing the sea cucumber luciferase as an internal reference, wherein the regulation activity of the promoter is the ratio of the activity of firefly luciferase to the activity of the internal reference sea cucumber luciferase, and verifying by test results: the active region of the pig miR-155 gene promoter is located in the region from-409 to +119, and an important element for regulating the activity of the pig miR-155 gene promoter exists in the active region;
(4) Analysis of the sequence from-409 to +119 shows that the sequence has one binding site region (-309 to-305) of Foxp3 and the other binding site region (-172 to-168) of Foxp 3; designing two binding site mutation specific primers of the Foxp3 transcription factor, respectively mutating the two binding sites of the Foxp3 transcription factor, and determining a regulating element for regulating the transcription activity of the pig miR-155 gene promoter according to the activity change condition of the mutated pig miR-155 gene promoter; experimental results show that the activity of the promoter is obviously reduced (P < 0.01) by site-directed mutagenesis of two binding sites of the Foxp3 transcription factor, and the Foxp3 transcription factor binding sites are regulatory elements necessary for maintaining the basic transcription activity of the pig miR-155 gene promoter.
Further, the sequence of the specific primer for constructing the pig miR-155 gene 5' -end promoter series deletion fragment luciferase report vector in the step (2) is shown in the following table:
further, the sequences of the primers specific for the Foxp3 transcription factor binding site mutation in step (4) are shown in the following table:
(III) beneficial effects
The invention provides a pig miR-155 gene promoter and a method for determining an active region and a regulating element for regulating transcriptional activity of the pig miR-155 gene promoter, wherein the active region of the pig miR-155 gene promoter is determined to be positioned in a-409- +119 region, and Foxp3 transcription factor binding sites are determined to be necessary regulating elements for regulating the transcriptional activity of the pig miR-155 gene promoter, so that research on the aspect of pig miR-155 transcriptional regulation is widened.
Drawings
FIG. 1 shows the enzyme digestion identification of a recombinant plasmid of a pig miR-155 promoter; wherein the left graph (i.e. graph A) is the double enzyme digestion identification of the recombinant plasmid with the first segment deletion of the pig miR-155 promoter; the right graph (namely graph B) is the double enzyme digestion identification of the recombinant plasmid with the second segment of deletion of the pig miR-155 promoter;
FIG. 2 luciferase activity assay of porcine miR-155 promoter 1 deleted fragment in PK15 cells;
FIG. 3 luciferase activity assay of porcine miR-155 promoter 2 deleted fragment in PK15 cells;
FIG. 4 luciferase activity assay of porcine miR-155 promoter 2 deleted fragment in 3D4/21 cells;
FIG. 5 Foxp3 transcription factor binding site mutation analysis.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Luciferase activity analysis of 1 pig miR-155 gene promoter
1.1 design of promoter primer
The precursor pre-miR155 of miR-155 is marked as +1, 5000bp is extended to the 5' end, a Primer Premier 5.0 is adopted to design a specific Primer, amplification is carried out in two sections, and target fragments are respectively from-4792 to-2377 and from-2270 to +119. The specific primers are shown in the following table:
extracting DNA of the large white pig ear margin tissue by using a DNA extraction kit (goods number: CW 2298) with the health century, and carrying out PCR amplification by using the extracted large white pig genome DNA as a template and using high-fidelity enzyme PrimeSTAR HS DNA Polymerase, wherein the PCR reaction solution comprises: 2. Mu.L of 5 XPrimeSTAR Buffer (Mg 2+ Plus), 0.8. Mu.L dNTP mix (2.5 mM each), 0.2. Mu.L Primer F1 (10. Mu.M), 0.2. Mu.L Primer R1 (10. Mu.M), 1. Mu.L DNA 1 (50 ng), 0.1. Mu. L PrimeSTAR HS DNA Polymerase (2.5U/. Mu.L), 3.7. Mu.L sterilized water, 10. Mu.L total volume, vortex mix, centrifuge and put into a PCR apparatus for reaction, the PCR procedure is: (two-step method) pre-denaturation at 94℃for 5min; cycle number 35cycles (denaturation 98 ℃ C. For 10s; extension 68 ℃ C. For 1 min/Kb); extending at 68deg.C for 10min; preserving at low temperature for 5min at 15 ℃.
1.2 detection, recovery and purification of amplified fragments of promoter by electrophoresis
Detecting PCR amplified products by 1% agarose gel electrophoresis, adding into a gel hole after fully mixing according to the proportion of 5 mu L of PCR products and 1 mu L of 6 Xloading buffer solution, adding 3mu L of DNA Marker (DL 2000) as a reference, setting an electrophoresis apparatus to 110V for 30min, and after electrophoresis, putting gel blocks into a gel imager, observing results under an ultraviolet lamp and photographing.
After comparing the target strip with the DNA Marker, which is accurate in position and negative and pollution-free, uniformly mixing the rest PCR product with a 6X sample buffer solution, and respectively adding different 1% concentration recovery gel holes with the DNA Marker, 110V and 30min; after electrophoresis, rapidly cutting off a gel block containing a target fragment under an ultraviolet lamp of a gel imager, and placing the gel block in a 2mL clean sterile EP tube; purification was performed with reference to the instructions of the Tiangen ordinary agarose gel DNA recovery kit (cat# DP 209). The purified DNA was measured for concentration and stored in a-20℃refrigerator.
1.3 ligation of amplified fragment of promoter to pMD19-T vector and transformation
1.3.1 reaction by adding A
Since most of the target fragment amplified by using the high-fidelity enzyme PrimeSTAR HS DNA Polymerase PCR is smooth, when the target fragment is ligated with the pMD19-T vector, it is necessary to add a base A to the 3' -end of the purified target fragment to make the target fragment become a cohesive end for ligation. According to the instructions of Tiangen plus A reaction (cat# RT 124), the following ingredients were added to a clean, sterile 0.2mL EP tube: mu.L of the purified blunt-ended DNA fragment, 4. Mu.L of the A-reaction solution, 1. Mu.L of Taq enzyme (2.5U/. Mu.L) were added. The components in the EP tube are gently mixed and centrifuged instantaneously and then are placed at 72 ℃ for 20-30min. The product after addition A can directly absorb part for ligation reaction.
1.3.2 ligation reactions
The following components were added to a sterile 0.2mL EP tube: mu.L of pMD19-T Vector, 4. Mu.L of post-A product, 5. Mu.L of Solution I. After being gently mixed and centrifuged instantaneously, the mixture is connected for 30 to 60 minutes at 16 ℃, and then is placed in a refrigerator at 4 ℃ for continuous overnight connection.
1.3.3 conversion
1. Taking out competent cells DH5 alpha stored in an ultralow temperature refrigerator at the temperature of minus 80 ℃ and placing the cells on ice for thawing, and then taking a clean sterile 1.5mL EP tube for marking and placing the marked cells on ice for precooling;
2. after the melted DH5 alpha is blown uniformly, 30 mu L of the mixture is removed into a precooled 1.5mL EP tube, 5 mu L of a connecting product is added into the mixture, and the mixture is stirred and lightly mixed by a gun head and then is kept on ice for 30min;
3. heat shock is carried out for 90s in a constant temperature water bath kettle at 42 ℃, and then the water is immediately placed on ice for 2-3 min; 400 mu L of LB liquid medium without antibiotics is added in an ultra-clean bench;
4. placing in 225rpm,37 ℃ constant temperature shaking table for 1h;
5. 4000rpm,3-4min, discarding 200 μl of supernatant in an ultra clean bench, blowing and mixing the precipitate with the rest LB liquid with a gun head, dripping 100 μl of the mixture on agar plate containing Amp antibiotics, spreading with a spreader, and standing in a constant temperature incubator at 37deg.C for 12-16 hr.
1.3.4 bacterial liquid PCR identification
Preparing a plurality of clean and sterile 1.5mL EP tubes in an ultra clean bench, and adding 1mL of LB liquid medium containing Amp antibiotics; clean and sterile white gun heads are clamped by tweezers burnt by an alcohol lamp, monoclonal bacterial colonies with neat edges are pricked on an Amp agar plate with bacterial colonies growing by the white gun heads, the white gun heads stained with bacterial liquid are placed into a prepared 1.5mL EP tube, marks are made, and the white gun heads are placed on a thermostatic shaking table at 225rpm and 37 ℃ for 6-8 hours. After turbidity of the bacterial solution in the 1.5mL EP tube, bacterial solution PCR was performed, and the following components were added to the sterile 0.2mL EP tube: 5 mu L Taq PCR Master Mix (2X), 1 mu L of bacterial liquid, 0.2 mu L of Primer F,0.2 mu L of Primer R,3.6 mu L of sterilized water; PCR procedure: 94 ℃ for 5min;35cycles (94 ℃ C. 30s;45-68 ℃ C. 30s;72 ℃ C. 1 Kb/min); 72 ℃ for 10min; 5min at 15 ℃.
Agarose gel electrophoresis detection: and (3) placing the prepared 1% agarose gel in an electrophoresis tank containing TAE buffer solution, respectively adding a bacterial liquid PCR product and a DNA Marker into a gel hole, setting the voltage of the electrophoresis apparatus to 110V for 30min, and comparing the positions of a strip of the DNA Marker and a target strip after electrophoresis is finished, wherein if the target strip is correct in position and a negative control is pollution-free, the corresponding bacterial liquid is positive bacteria.
And (3) sucking 400 mu L of bacterial liquid with positive PCR identification result into 1.5mL of clean and sterile EP, sending the bacterial liquid to Shanghai biological engineering Co., ltd for DNA sequencing, comparing the detected sequence with DNA sequence through on-line free analysis software NCBI, and carrying out identification analysis to ensure that the sequence is completely correct.
1.3.5 plasmid miniextraction
The bacterial liquid with completely correct sequencing was amplified overnight and then extracted according to the instructions of SanPrep column type plasmid DNA miniextraction kit (cat# B518191). The extracted plasmid is put in a refrigerator at the temperature of minus 20 ℃ for standby after concentration detection.
1.4 construction of recombinant plasmids of pGL3-Basic and promoter and deletion fragment
1.4.1 plasmid double cleavage and purification
Plasmids containing the miR-155 promoter fragment and empty pGL3-Basic plasmids were subjected to double digestion. Firstly, preparing a 1% concentration agarose gel, and carrying out double digestion by using two restriction enzymes of Kpn I and Xho I, wherein the reaction system is as follows: mu.L of 10 XQuickCut Buffer, 1. Mu.L of QuickCut Kpn I, 1. Mu.L of QuickCut Xho I, 1. Mu.g of plasmid DNA, and sterilized water was added to make up the system to 50. Mu.L. After gentle mixing, the mixture was centrifuged instantaneously, the PCR apparatus was set at 37℃for 10min, 10. Mu.L of 6 Xloading buffer was added, mixed uniformly, then the mixture was added to the wells, 5. Mu.L of DNA Marker (DL 5000) was added as a reference, the electrophoresis apparatus was set at 110V for 30min, and after electrophoresis, the target fragment was cut off rapidly under an ultraviolet lamp of the gel imager and purified.
1.4.2T4 ligation reaction
The linearized pGL3-Basic vector after double enzyme digestion and purification is quickly connected with a target fragment under the action of T4 DNA Ligase, and the reaction system is as follows: 1. Mu.L of 10×T4 DNA Ligase Buffer, 0.5. Mu. L T4.4 DNA Ligase, 1. Mu.L of pGL3-Basic double cleavage recovery, 4. Mu.L of target fragment double cleavage recovery, and adding sterilized water to 10. Mu.L; after gentle mixing, the mixture was centrifuged briefly and was kept at 16℃for 1-2h and then placed in a refrigerator at 4℃for further overnight connection.
1.4.3 transformation and identification of recombinant plasmids
The recombinant plasmid containing the target fragment connected by the T4 ligase is transformed into competent cells DH5 alpha (the step is the same as 1.3.3), and the identified positive bacteria (the step is the same as 1.3.4) are sent to Shanghai Biotechnology, inc. for sequencing, and the sequencing result is compared with a reference sequence.
1.4.4 recombinant plasmid endotoxinfree extraction
The plasmid was extracted from the bacterial solution with the completely correct sequencing result by referring to the Endo-Free Plasmid Mini Kit II (cat# D6950) instruction book of the endotoxin-free plasmid extraction kit. And the two sections of miR-155 promoter recombinant plasmids which are successfully constructed are named pGL3-bicpro1 and pGL3-bicpro2 respectively.
1.5 construction of report vector for deletion of luciferase from promoter series of pig miR-155 gene 5' end
1.5.1 primer design
And (3) taking the plasmid obtained in 1.4.4 as a template, and amplifying the pig miR-155 gene promoter series deletion fragments by adopting a nested PCR method. Specific primers are designed by adopting Primer Premier 5.0 software, and two restriction enzyme cutting sites of Kpn I and Xho I are respectively added at the 5' ends of the upstream Primer and the downstream Primer. Specific primers are shown in the following table:
1.5.2 nested PCR amplification of deletion fragments
And respectively using two segments of small promoter plasmids as templates, amplifying miR-155 promoter series deletion fragments by nested PCR, and using the same backward primer and different forward primers. Wherein the PCR reaction liquid comprises the following components: 2. Mu.L of 5 XPrimeSTAR Buffer (Mg 2+ Plus), 0.8. Mu.L dNTP mix (2.5 mM each), 0.2. Mu.L Primer F1 (10. Mu.M), 0.2. Mu.L Primer R1 (10. Mu.M), 1. Mu.L DNA 1 (50 ng), 0.1. Mu. L PrimeSTAR HS DNA Polymerase (2.5U/. Mu.L), 3.7. Mu.L sterilized water, 10. Mu.L total volume, vortex mix, centrifuge and put into a PCR apparatus for reaction, the PCR procedure is: (two-step method) pre-denaturation at 94℃for 5min; cycle number 35cycles (denaturation 98 ℃ C. For 10s; extension 68 ℃ C. For 1 min/Kb); extending at 68deg.C for 10min; preserving at low temperature for 5min at 15 ℃. The PCR amplification products were detected by 1% agarose gel electrophoresis.
1.5.3 series deletion luciferase reporter constructs
The serial deletion PCR products obtained by 1.5.2 amplification are respectively purified by a common agarose gel DNA recovery kit (product number: DP 209), and then are respectively subjected to double enzyme digestion by two restriction enzymes Kpn I and Xho I, and the reaction system is as follows: mu.L of 10 XQuickCut Buffer, 1. Mu.L of QuickCut Kpn I, 1. Mu.L of QuickCut Xho I, 1. Mu.g of DNA, and sterilized water was added to make up the system to 50. Mu.L. After gentle mixing, the mixture was centrifuged instantaneously, the PCR instrument was set at 37℃for 10min, 10. Mu.L of 6 Xloading buffer was added, mixed well, then added to the prepared agarose gel well, and 5. Mu.L of DNA Marker (DL 5000) was added as a reference, the electrophoresis instrument was set at 110V for 30min, and after electrophoresis, the target fragment was cut off rapidly under the UV lamp of the gel imager and purified. The linearized pGL3-Basic vector after double enzyme digestion and purification is quickly connected with a target fragment under the action of T4 DNA Ligase, and the reaction system is as follows: 1. Mu.L of 10×T4 DNA Ligase Buffer, 0.5. Mu. L T4.4 DNA Ligase, 1. Mu.L of pGL3-Basic double cleavage recovery, 4. Mu.L of target fragment double cleavage recovery, and adding sterilized water to 10. Mu.L; after gentle mixing, the mixture was centrifuged briefly and was kept at 16℃for 1-2h and then placed in a refrigerator at 4℃for further overnight connection. The recombinant plasmid containing the target fragment connected by the T4 ligase is transformed into competent cells DH5 alpha, and the identified positive bacteria are sent to Shanghai Biotechnology engineering Co., ltd for sequencing. The plasmid was extracted from the bacterial solution with the completely correct sequencing result by referring to the Endo-Free Plasmid Mini Kit II (cat# D6950) instruction book of the endotoxin-free plasmid extraction kit. Finally obtaining a report plasmid of the pig miR-155 gene with a promoter series deleted from the 5' end, which is named pGL3-bicQ1, pGL3-bicQ2 and pGL3-bicQ3 respectively; pGL3-bicQ4, pGL3-bicQ5, pGL3-bicQ6, pGL3-bicQ7.
1.6 cell culture and transfection of a porcine miR-155 Gene 5' -terminal promoter series luciferase reporter plasmid
PK15 cells were cultured in complete medium containing 89% DMEM, 10% Fetal Bovine Serum (FBS), 1% double antibody, 3D4/21 cells were cultured in complete medium containing 89% RPM 1640, 10% FBS, 1% MEM Non-Essential Amino Acids Solution, and cells with good growth state were cultured 1X 10 per well one day before transfection 5 Passaging to 24-well plate, placing at 37deg.C, 5% CO 2 Culturing in a cell culture box, and performing cell transfection when the cell density reaches about 80%. During transfection, pGL3-bicpro1, pGL3-bicpro2, pGL3-bicQ1, pGL3-bicQ2, pGL3-bicQ3, pGL3-bicQ4, pGL3-bicQ5, pGL3-bicQ6 and pGL3-bicQ7 were co-transfected with the internal reference plasmid pRL-TK, 3 to 4 replicates were made in each group, pGL3-Basic was used as a negative control. Transient transfection was performed according to the transfection procedure of JetPRIME transfection reagent, which was as follows:
(1) A plurality of clean, sterile 1.5mL EP tubes were first removed, each tube was added with 50. Mu. L jetPRIME Buffer, 0.5. Mu.g plasmid (pGL-basic+pRL-TK) and 1. Mu.L transfection reagent jetPRIME, gently swirled, mixed, and allowed to stand at room temperature for 10min.
(2) The old medium in the wells was discarded, washed 1-2 times with 500. Mu.L of PBS/DPBS, and then 450. Mu.L of low serum medium (containing 2% FBS) was added to each well.
(3) Transferring the mixed solution prepared in step (1) into 24-well plate, covering with cover, holding 24-well plate, shaking for 10 times to mix culture medium and transfection mixed solution, and placing into 37 deg.C and 5% CO 2 Cell incubator.
(4) After 6h of incubation of the transfected cells, the old medium was discarded, and after 1-2 washes with 500. Mu.L of PBS/DPBS, 500. Mu.L of fresh complete medium was added to each well. Then put into 37 ℃ and 5 percent CO 2 The cell culture incubator continues to culture for 48 hours.
1.7 Dual luciferase Activity assay
Firstly, preparing a new 1 Xcell lysate PLB by using sterile ultrapure water, and preparing a fluorescence detection Reagent LARII and Stop & Glo Reagent according to a proportion; discarding old culture medium in 24-well plate, washing with 500 μl PBS/DPBS for 1-2 times, discarding, adding 100 μl 1×cell lysate PLB, standing 24-well plate on shaking table at room temperature for 30-45min, and completely lysing cells; the lysed 24-well plate was placed on ice for 10min, then 10 μl of supernatant was pipetted from each sample and transferred to a clean sterile 1.5mL EP tube; opening the bioluminescence detector, firstly rapidly adding 50 mu L of LARII into 1.5mL of the EP tube, blowing and mixing uniformly, rapidly placing the mixture in the instrument, clicking and detecting, stopping the mixture after an OK interface appears, then taking out the EP tube, rapidly adding 50 mu L of Stop & Glo Reagent, blowing and mixing uniformly, placing the mixture in the instrument, clicking OK again, and respectively detecting the value M1 of firefly luciferase and the values M2 and M1/M2 of Renilla luciferase.
1.8 data processing
Each recombinant plasmid was subjected to 3 independent experiments, 3 replicates of each recombinant plasmid were performed, and the average was taken. The measured M1/M2 value of each well is the luciferase activity value of the well, and the values are expressed as mean value.+ -. Standard deviation. The significance test was tested with T test. * Represents P <0.05, and P <0.01.
1.9 results
Cloning of 1.9.1 pig miR-155 promoter region and restriction enzyme digestion identification of deletion recombinant plasmid
The DNA of the big white pig is used as a template, and host gene BIC promoter fragments of the pig miR-155 are amplified in two sections. The two sections of promoter amplification recovery products are respectively used as templates, the deletion fragments are amplified by nested PCR, and the promoter and the primers of the series of deletion fragments are respectively provided with KpnI and XhoI enzyme cutting sites. The length of a miR-155 first-segment promoter fragment is 2416bp, the miR-155 first-segment promoter fragment is named bicpro1, the length of a miR-155 second-segment promoter fragment is 2409bp, the miR-155 first-segment promoter fragment is named bicpro2, the miR-155 first-segment deletion fragment is 1052bp, 658bp and 446bp, and the miR-155 first-segment promoter fragment is named bicQ1, bicQ2 and bicQ3; the second deletion fragments were 1953bp, 1637bp, 1324bp and 528bp in length, designated as bicQ4, bicQ5, bicQ6 and bicQ7, respectively. The recombinant plasmids which were successfully constructed were identified by the double restriction enzyme method (as shown in FIG. 1) and named pGL3-bicpro1, Q2, Q3 and pGL3-bicpro2, Q4, Q5, Q6, Q7, respectively.
1.9.2 pig miR-155 promoter and deletion promoter activity analysis
And (3) transiently transfecting PK15 and 3D4/21 cells with recombinant plasmids of the constructed pig miR-155 promoter and deletion fragment respectively, taking pGL3-Basic as a negative control, taking plasmid pRL-TK as an internal reference, transfecting a transfection reagent JetPRIME for 48 hours, collecting cells, and detecting the double luciferase activities of the miR-155 promoter and the deletion expression vector in two tissue cells by using a bioluminescence detector. The regulation activity of the promoter is the ratio of the activity of firefly luciferase to the activity of internal reference Renilla luciferase. The results showed that (labeled +1 with 1 st base of pre-miR 155), the activity of the first segment and deletion fragment of the pig miR-155 promoter in PK15 cells was significantly lower than that of the negative control, and the activity was inactive (as shown in FIG. 2); the second promoter fragment showed more obvious relative activity in PK15 cells, wherein the Q7 fragment showed the strongest activity, and the activity was about 12 times higher than that of the negative control (as shown in FIG. 3); the second promoter fragment showed more pronounced relative activity in 3D4/21 cells with only the Q7 fragment, which was approximately 2-fold higher than the negative control (as shown in FIG. 4). Thus, it is assumed that the Q7 fragment located between-409 and +119 is a promoter having a regulatory function.
2 determining and controlling the transcriptional activity of the promoter of the pig miR-155 gene
(1) Pig miR-155 gene promoter site-directed mutagenesis test
In Foxp3 gene binding site region (-309 to-305, -172 to-168), primer Premier 5.0 is used to design mutation Primer, and the sequence is shown in the following table:
(2) Mutant sequence amplification
The experiment adopts an overlap extension PCR method to carry out site-directed mutation on the binding site of miR-155 and a target gene. Purifying and recovering the target fragment by adopting a Shanghai biological glue recovery kit, and carrying out PCR1 amplification by using a glue recovery product as a template, a forward primer F (a) and a mutagenesis primer b to obtain a product AB containing a mutation site and an upstream sequence; and then, carrying out PCR2 amplification by using a reverse primer R (d) and a mutagenesis primer c to obtain a product CD containing a mutation site and a downstream sequence. Product AB and product CD have 20bp fragments overlapping each other and carrying mutation sites.
The experiment is carried out by adopting high-fidelity enzyme Q5 (NEB), and a PCR system is adopted: mu. L Q5 Mix, 0.5. Mu.L Primer F, 0.5. Mu.L Primer R, 1. Mu.L DNA, 3. Mu.L sterilized water in a total volume of 10. Mu.L. The reaction conditions are as follows: pre-denaturation (98 ℃ C.: 30 s); denaturation (94 ℃ C.: 10 s), annealing (60 ℃ C.: 15 s), extension (72 ℃ C.: 15 s) for a total of 30 cycles; re-extending (72 ℃ C.: 2 min); preserving at 15 ℃ for 2min.
(3) Electrophoresis detection
The PCR products were detected by agarose gel at a concentration of 1.5%, and if the fragment position was correct, agarose gel recovery was performed according to the instructions of Shanghai Biogel recovery kit (B110092-0050), and the concentration and OD value thereof were detected.
(4) Post-mutation splicing
The product AB and the product CD are mixed according to a certain proportion according to the fragment size and the recovery concentration, and primer-free PCR, namely an overlapping extension stage, is carried out. The reaction conditions were 72℃for 10min. The target gene fragment containing the mutation site is obtained.
(5) Amplification of target Gene
And (3) carrying out amplification of the ad segment by using a primer-free PCR product as a template and adopting high-fidelity enzyme Q5 to obtain the target gene containing the mutation site. PCR system: mu. L Q5 Mix, 0.5. Mu.L Primer F, 0.5. Mu.L Primer R, 1. Mu.L DNA, 3. Mu.L sterilized water in a total volume of 10. Mu.L. The reaction conditions are as follows: pre-denaturation (98 ℃ C.: 30 s); denaturation (94 ℃ C.: 10 s), annealing (60 ℃ C.: 15 s), extension (72 ℃ C.: 15 s) for a total of 30 cycles; re-extending (72 ℃ C.: 2 min); preserving at 15 ℃ for 2min.
(6) The PCR product was detected by agarose gel with a concentration of 1.5%, and if the result was correct, the target fragment was purified and recovered by Shanghai biological gel recovery kit (B110092-0050).
(7) Adding A for reaction
The end-to-end addition A is carried out on the ad fragment of the target gene. The system comprises: the following ingredients were added to a clean, sterile 0.2mL EP tube: mu.L of the purified blunt-ended DNA fragment, 4. Mu.L of the A-reaction solution, 1. Mu.L of Taq enzyme (2.5U/. Mu.L) were added. The various components in the EP tube were gently mixed and centrifuged instantaneously and then placed at 72℃for 30min.
(8) Double digestion is carried out on the target gene product and pGL3-Basic vector by adopting restriction endonucleases KpnI and XhoI, and the target fragment is recovered by gel cutting and purification, and the enzyme digestion system is as follows: mu.L of 10 XQuickCut Buffer, 1. Mu.L of QuickCut Kpn I, 1. Mu.L of QuickCut Xho I, 1. Mu.g of DNA, and sterilized water was added to make up the system to 50. Mu.L. After gentle mixing, the mixture was centrifuged instantaneously, the PCR instrument was set at 37℃for 10min, 10. Mu.L of 6 Xloading buffer was added, mixed well, then added to the prepared agarose gel well, and 5. Mu.L of DNA Marker (DL 5000) was added as a reference, the electrophoresis instrument was set at 110V for 30min, and after electrophoresis, the target fragment was cut off rapidly under the UV lamp of the gel imager and purified. The linearized pGL3-Basic vector after double enzyme digestion and purification is quickly connected with a target fragment under the action of T4 DNA Ligase, and the reaction system is as follows: 1. Mu.L of 10×T4 DNA Ligase Buffer, 0.5. Mu. L T4.4 DNA Ligase, 1. Mu.L of pGL3-Basic double cleavage recovery, 4. Mu.L of target fragment double cleavage recovery, and adding sterilized water to 10. Mu.L; after gentle mixing, the mixture was centrifuged briefly and was kept at 16℃for 1-2h and then placed in a refrigerator at 4℃for further overnight connection. The recombinant plasmid containing the target fragment connected by the T4 ligase is transformed into competent cells DH5 alpha, and the identified positive bacteria are sent to Shanghai Biotechnology engineering Co., ltd for sequencing. The plasmid was extracted from the bacterial liquid with the completely correct sequencing result by referring to the Endo-Free Plasmid Mini Kit II (cat# D6950-01B) instruction book of the endotoxin-free plasmid extraction kit. Finally obtaining mutant plasmids: pGL3-Foxp3mut1, pGL3-Foxp3mut2.
(9) Cell culture and transient transfection
PK15 cell culture cells well grown in complete Medium containing 89% DMEM, 10% Fetal Bovine Serum (FBS), 1% double antibody were plated 1X 10 per well one day before transfection 5 Passaging to 24-well plate, placing at 37deg.C, 5% CO 2 Culturing in a cell culture box, and performing cell transfection when the cell density reaches about 80%. When in transfection, pGL3-Foxp3mut plasmid and internal reference plasmid pRL-TK are respectively co-transfected, 3-4 repeats are carried out on each group, and pGL3-Basic is used as a negative control. Transient transfection was performed following the transfection procedure of JetPRIME transfection reagent.
Experimental results:
through constructing a pig miR-155 gene promoter series deletion luciferase report carrier, transfecting PK15 and 3D4/21 cells and carrying out double luciferase activity analysis, the active region of the promoter is determined to be positioned in the-409- +119 region, and an important element for regulating and controlling the activity of the pig miR-155 gene promoter exists in the active region. The Gene Regulation is used for predicting potential transcription factor binding sites in a promoter region of a pig miR-155 Gene, and a plurality of transcription factor binding sites related to immunity, including Foxp3, ELK1, C-REL, HNF-1, NF-Y and the like, are found in the Gene regulatory sequence.
In order to further study the total effect of the transcription factor binding sites on the regulation of the activity of the promoter of the pig miR-155 gene, a corresponding primer pair pGL3-bicQ7 is designed according to the transcription factor binding sites to carry out site-directed mutagenesis, PK15 cells are transiently transfected, and the experimental result is shown in figure 5.
Experimental results show that the activity of the promoter is obviously reduced (P < 0.01) by site-directed mutagenesis of two binding sites of the Foxp3 transcription factor, and the Foxp3 transcription factor binding sites are regulatory elements necessary for maintaining the basic transcription activity of the pig miR-155 gene promoter.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Sequence listing
<110> academy of Henan animal husbandry economics
<120> method for screening promoter active region and transcriptional regulatory element of pig miR-155 gene
<130> 2020.12.11
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Claims (1)
1. A pig miR-155 gene promoter is characterized in that the miR-155 gene promoter sequence is shown in a sequence table SEQ ID No. 1.
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