CN107058330B - MZF1 protein bindable DNA fragment and application thereof in MZF1 activity detection - Google Patents
MZF1 protein bindable DNA fragment and application thereof in MZF1 activity detection Download PDFInfo
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Abstract
The invention relates to a DNA fragment capable of being combined by MZF1 protein, which comprises a plurality of MZF1 protein combination frames; also relates to the application of the DNA segment; also relates to a method for detecting the transcriptional regulation activity of MZF1 in cells. By using the DNA fragment and the method of the invention, the transcriptional regulation activity of MZF1 in cells can be directly and pertinently detected, rather than only the content of transcripts or proteins thereof, thereby enabling more accurate analysis of the role played by MZF1 as a transcription factor in the development of some pathologies.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a DNA fragment capable of being combined by MZF1 protein and application thereof in detecting the transcriptional regulation activity of MZF1 in cells.
Background
Medullary zinc finger protein 1(MZF1) is a protein having a zinc finger structure encoded by human chromosome 19 MZF1 gene, belongs to the family of zinc finger structure proteins of C2H2 class, and is abundantly present in human cells. The MZF1 protein has 734 amino acids in total length, about 82055Da, after combined with target gene, it combines with target gene promoter region DNA through its special zinc finger structure, and plays gene activation or inhibition effect in bone marrow hematopoiesis process.
After mutation of MZF1 gene, some regulation disorder and even disease may occur. However, currently, the detection of the activity of endogenous MZF1 of cells still depends on Western Blot or qRCR and other technologies, although the sensitivity is high, the detection is only the difference of the content of MZF1 protein, and the change of the activity after the MZF1 protein is combined with a target sequence cannot be directly reflected.
Therefore, it is necessary to design a new method for detecting the transcriptional regulation activity of MZF1 in cells.
Disclosure of Invention
The inventors found through research that there is a high degree of conservation in the DNA sequence bound by the MZF1 protein, with the core sequence of the bound DNA being 5'-AGTGGGGA-3' or 5'-CGGGNGAGGGGGAA-3' (N stands for A, T, C, G, as shown in SEQ ID NO: 1-4).
Based on the above research, the present invention provides a DNA fragment bindable by MZF1 protein, comprising a plurality of MZF1 protein binding boxes, wherein the sequence of at least one of the MZF1 protein binding boxes is 5'-AGTGGGGA-3', and the sequence of the remaining MZF1 protein binding boxes is 5'-CGGGNGAGGGGGAA-3' (N represents A, T, C, G, as shown in SEQ ID NO: 1-4).
Preferably, when comprising multiple MZF1 protein binding boxes, there is a spacer sequence between every two adjacent MZF1 protein binding boxes.
Preferably, the spacer sequence between every two adjacent MZF1 protein binding boxes is TA.
Preferably, the sequence of the DNA fragment is shown as SEQ ID NO. 5. In the process of research, we tested various combinations, and found that the DNA fragment of the sequence has higher binding efficiency to the MZF1 protein than other combination modes.
The invention also provides application of the DNA fragment in detecting the transcriptional regulation activity of MZF1 in cells.
The invention also provides a method for detecting the transcriptional regulation activity of MZF1 in a cell, which comprises the following steps:
s1: introducing into the cell a reporter gene system comprising the DNA fragment and a reporter gene expression cassette ligated downstream of the DNA fragment;
s2: calculating the intracellular MZF1 transcriptional regulatory activity by detecting the expression of the reporter gene. The expression intensity of the reporter gene can be used as an indicator indicating the transcriptional regulatory activity of MZF 1.
Preferably, the reporter gene system is a dual-luciferase reporter gene system, comprising a recombinant plasmid and a control plasmid, wherein the recombinant plasmid carries the DNA fragment and an expression frame of luciferase I connected to the downstream of the DNA fragment, and the control plasmid carries an expression frame of luciferase II, and the luciferase I is different from the luciferase II in fluorescence wavelength. MZF1 transcriptional regulation activity was expressed as the ratio of the intensity of fluorescence generated by luciferase I excitation to the intensity of fluorescence generated by luciferase II excitation.
Preferably, the recombinant plasmid is obtained by inserting the DNA fragment between Kpn I and Hind III sites of the plasmid pGL3.0-Basic.
Preferably, the control plasmid is phRL-TK.
Preferably, S1 specifically includes:
s11: culturing the cells to adhere to the wall and recovering the morphology;
s12: the recombinant plasmid and control plasmid were transfected into cells.
Preferably, S2 specifically includes:
s21: after transfection, the cells are continuously cultured for 24-36 hours, and the cells are washed;
s22: detecting the activity of luciferase I and luciferase II in the transfected cells respectively;
s23: luciferase II Activity normalized luciferase I Activity was used as an index for measuring the transcriptional regulation activity of MZF 1.
By using the DNA fragment and the method of the invention, the transcriptional regulation activity of MZF1 in cells can be directly and pertinently detected, rather than only the content of transcripts or proteins thereof, thereby enabling more accurate analysis of the role played by MZF1 as a transcription factor in the development of some pathologies.
Drawings
FIG. 1 is a photograph of agarose gel electrophoresis of a recombinant plasmid digested with Kpn I and Hind III;
FIG. 2 is a statistical graph showing the relative MZF1 activity (i.e., the ratio of firefly luciferase activity to Renilla luciferase activity) in human colon cancer cell line SW480, human cervical cancer cell line HeLa, and human breast cancer cell line MCF7 cells transfected with pGL3.0-Basic and pGL3.0-Basic-MZF1, respectively;
FIG. 3 is a statistical graph showing the relative MZF1 activities (i.e., the ratio of firefly luciferase activity to Renilla luciferase activity) in the human colon cancer cell line SW480, the human cervical cancer cell line HeLa, and the human breast cancer cell line MCF7 cells transfected with pcDNA3.1(+) empty vector and pcDNA3.1(+) -MZF1, respectively.
Detailed Description
The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
1. Construction of DNA fragment bindable by MZF1 protein
The inventors conducted research and analysis on MZF1, and found that the DNA sequences bound by MZF1 protein are 5'-AGTGGGGA-3', and 5'-CGGGNGAGGGGGAA-3' (N represents A, T, C, G, as shown in SEQ ID NO: 1-4), when synthesizing the DNA core sequence, the present embodiment was designed to synthesize the DNA core sequence by repeating the two DNA core sequences twice, and spacing the two DNA core sequences by TA bases to obtain a DNA fragment having the sequence shown in SEQ ID NO: 5.
To ensure the success of vector construction, cohesive ends of the corresponding restriction sites were added to the ends, in this case the 5 'cohesive end of Kpn I (CATGG) and the 3' cohesive end of Hind III (TTCGA), and the cohesive ends on both sides of the sequence were designed to facilitate ligation of the fragment to the vector.
The two oligonucleotide chains of the fragment, the sequences of which are shown in SEQ ID NO 6 and 7, were synthesized separately by de novo synthesis, and both were synthesized by Wuhan Strongylocentron Biotechnology, Inc. The two oligonucleotide strands are complementary and paired to form a double-stranded DNA fragment containing the cohesive end of the Kpn I cleavage site and the cohesive end of the Hind III cleavage site. The 50. mu.l annealing system was as follows: the antisense Buffer for DNA oligonucleotides (5X) 10. mu.l, oligonucleotide strands (50. mu.M) 10. mu.l each, and the balance ddH2O。
After fully mixing, setting a PCR instrument program to carry out annealing reaction to form a DNA double strand. The two synthesized DNA single strands were annealed with a DNA oligonucleotide annealing buffer (available from Biyuntian Co., Ltd., product No. D0251) to promote complementary pairing, thereby forming a double-stranded DNA having a cohesive end with a Kpn I cleavage site at the 5 'end and a cohesive end with a Hind III cleavage site at the 3' end. The specific procedures are as follows: annealing at 95 ℃ for 2 minutes (in order to allow the DNA oligo to be sufficiently denatured); the reaction was terminated after every 90 seconds by dropping 1 ℃ to 25 ℃. The DNA annealing product is identified by 1.5 percent common agarose gel electrophoresis (because the DNA fragment is small, a single and specific band can not be seen in the electrophoresis identification), and the DNA annealing product is placed on ice for standby after concentration measurement and can also be frozen at the temperature of 20 ℃ below zero for standby. If other reactions such as enzyme digestion and the like are intended to be carried out after the Annealing is finished, a purification kit is preferably used for purification, or the volume of an Annealing product in a reaction system is ensured not to exceed 5 percent so as to avoid the interference of an Annealing Buffer on a subsequent enzyme-linked reaction system.
2. Inserting DNA fragment capable of being combined by MZF1 protein into luciferase expression vector
The above-mentioned pGL3-Basic empty vector was double-digested with the endonucleases Kpn I and Hind III (available from TAKARA, Kpn I cat # 1618, Hind III cat # 1615), and 20. mu.l of the digestion system was as follows: 10 XQuickcut Greenbuffer 2. mu.l, Kpn I1. mu.l, Hind III 1. mu.l, pGL3-Basic empty vector 1. mu.g, the remainder being ddH2O。
After fully mixing, carrying out enzyme digestion reaction at 37 ℃ for 90 minutes, identifying the condition of plasmid after double digestion by using 1.5 percent common agarose gel electrophoresis, then cutting and recovering the gel (a DNA gel recovery kit is purchased from Tiangen Biochemical technology Co., Ltd., the product number is DP209), measuring the concentration of a sample after the recovery is finished, and placing the sample on ice for later use.
The annealed double-stranded DNA was ligated to pGL3-Basic luciferase expression vector. The 10. mu.l ligation system was as follows: DNA ligase (available from TAKARA, cat # 6022) 5. mu.l, total 5. mu.l of double-stranded DNA fragment and double-cleaved pGL3.0-Basic vector. To promote the enzyme ligation efficiency, the molar ratio of the DNA fragment to the vector was controlled to about 10: 1. After fully and uniformly mixing, the enzyme linked system is placed in a PCR instrument, the reaction is carried out for 1 hour at the temperature of 16 ℃, and the recombinant plasmid is obtained after the ligation reaction is finished and is placed on ice for standby.
The recombinant plasmid (pGL 3-Basic expression vector containing the DNA fragment to which the MZF1 protein binds) was transformed into E.coli. The transformation was carried out according to the classical heat shock method, as follows: taking out the escherichia coli competent cell DH5 alpha from a refrigerator at the temperature of-80 ℃, unfreezing the escherichia coli competent cell DH5 alpha on ice for 2-3 minutes, adding the connected recombinant plasmid, gently mixing the plasmids uniformly, incubating the plasmids on ice for 20 minutes, then thermally shocking the plasmids at the temperature of 42 ℃ for 60 seconds, quickly putting the plasmids on ice, standing the plasmids for 2 minutes, then adding 450 mu l of sterile LB liquid culture medium without antibiotics, putting the plasmids in a constant temperature shaking table for resuscitation for 1 hour under the resuscitation condition of 37 ℃ and 200 revolutions per minute. 100 mul of recovered bacterial liquid is evenly coated on LB solid selective medium with the diameter of 3.5 cm and containing ampicillin (the recombinant plasmid can tolerate the ampicillin). After 10 minutes of absorption, the cells were placed in an incubator at 37 ℃ overnight. On the next day, single colony is picked into 5-7ml LB liquid culture medium containing ampicillin, at 37 deg.C, 200 r/min, after 12 hours of bacteria reproduction, plasmid is extracted, and 1.5% common agarose gel electrophoresis is used for identification.
The above-extracted plasmid was identified by double digestion with the endonucleases Kpn I and Hind III (purchased from TAKARA, having a Kpn I designation of 1618 and a Hind III designation of 1615), and the identification of double digestion in the examples of the present invention is shown in FIG. 1 (lane 5 is marker, and lanes 1-4 are positive clones). The positive group was sent to Wuhan Strongylocentron Biotechnology GmbH for sequencing to confirm that the DNA fragment was integrated into pGL3-Basic vector. Thus, pGL3-Basic expression vector (hereinafter, all indicated as pGL3.0-Basic-MZF1) containing a DNA fragment to which the MZF1 protein binds was successfully constructed.
pGL3-Basic-MZF1 transfected cells
In the embodiment of the invention, the dual-luciferase experiment is carried out by adopting human colon cancer cell line SW480, human cervical cancer cell line HeLa and human breast cancer cell line MCF7 cells. Cells grown in logarithmic phase were each uniformly plated in 24-well plates, approximately 10 ten thousand cells per well, and cultured overnight in 10% fetal bovine serum, high-glucose DMEM medium. After the cells adhere and recover morphology, the reporter gene system-associated plasmid is transfected into the cells (preferably at a cell density of about 60-80% for transfection). The transfection reagent used was Neofect (purchased from Sokka Chuangzhi Biotech Co., Ltd., Cat. No. TF 20121201). The 50. mu.l transfection system was as follows: plasmid 0.5. mu.g, Neofect transfection reagent 0.5. mu.l, and the rest is serum-free medium.
The plasmids used in this experiment were: pGL3-Basic empty vector, pGL3.0-Basic-MZF1, phRL-TK (vector with Renilla luciferase gene), pcDNA3.1(+) -MZF1(MZF1 overexpression plasmid, hereinafter abbreviated as pcDNA3.1-MZF 1).
4. Calculation of the Activity of MZF1
MZF1 activity was calculated by normalization with renilla luciferase activity as an internal control. The specific experimental method is as follows: and (3) continuously culturing the transfected cells for 24-36 hours, removing the supernatant of the culture medium, cleaning the cells for 3 times by using 1xPBS (x-xps-based ligand) for 5 minutes each time, and carefully operating when cleaning the cells to avoid blowing the cells on the front side. The detection is carried out by using a dual-luciferase reporter gene detection kit (purchased from Ganning Biotech, New Inc., with the product number GN 201-01).
In order to verify whether the reporter gene system has activity or not, pGL3.0-Basic-MZF1 recombinant plasmids are transfected into cells, the activity of the MZF1 reporter gene system is detected, the determination result is shown in figure 2, and the fluorescence activity of a treatment group (transfected pGL3.0-Basic-MZF1) is obviously higher than that of a control group (transfected pGL3.0-Basic empty vector).
In order to verify whether the reporter gene system can specifically detect the activity of MZF1, pcDNA3.1(+) -MZF1 is transfected into cells, and the result is shown in FIG. 3, after the exogenous introduction of MZF1, the fluorescence activity of the positive treatment group (transfected with pcDNA3.1(+) -MZF1) is obviously higher than that of the control group (transfected with pcDNA3.1(+) empty vector).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> affiliated cooperation hospital of college of Tongji medical college of Huazhong university of science and technology
<120> MZF1 protein bindable DNA fragment and application thereof in MZF1 activity detection
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Claims (7)
1. A DNA fragment bindable by MZF1 protein, comprising a plurality of MZF1 protein binding boxes, wherein at least one MZF1 protein binding box has the sequence 5 '-AGTGGGGA-3'; the sequences of the other MZF1 protein binding boxes are 5'-CGGGNGAGGGGGAA-3', N represents A, C, T or G, a spacing sequence is arranged between every two adjacent MZF1 protein binding boxes, and the sequence of the DNA fragment is shown as SEQ ID NO. 5.
2. Use of the DNA segment of claim 1 to detect the transcriptional regulatory activity of MZF1 in a cell.
3. A method for detecting the transcriptional regulatory activity of MZF1 in a cell, comprising the steps of:
s1: introducing into said cell a reporter gene system comprising the DNA segment of claim 1 and a reporter gene expression cassette linked downstream of said DNA segment;
s2: calculating the intracellular MZF1 transcriptional regulatory activity by detecting the expression of the reporter gene;
the reporter gene system is a dual-luciferase reporter gene system and comprises a recombinant plasmid and a control plasmid, wherein the recombinant plasmid carries the DNA fragment and an expression frame of luciferase I connected to the downstream of the DNA fragment, the control plasmid carries an expression frame of luciferase II, and the luciferase I is different from the luciferase II in excitation and generates fluorescence.
4. The method according to claim 3, wherein the recombinant plasmid is obtained by inserting the DNA fragment between Kpn I and Hind III sites of the plasmid pGL 3-Basic.
5. The method of claim 4, wherein the control plasmid is phRL-TK.
6. The method according to any one of claims 3 to 5, wherein S1 specifically comprises:
s11: culturing the cells to adhere to the wall and recovering the morphology;
s12: the recombinant plasmid and control plasmid were transfected into cells.
7. The method according to claim 6, wherein S2 specifically comprises:
s21: after transfection, the cells are continuously cultured for 24-36 hours, and the cells are washed;
s22: detecting the activity of luciferase I and luciferase II in the transfected cells respectively;
s23: the value obtained by normalizing luciferase I activity with luciferase II activity was used as an index for measuring the transcriptional regulation activity of MZF 1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103382505A (en) * | 2013-08-07 | 2013-11-06 | 贵州大学 | Method for detecting promoter activity by utilizing double luciferase reporter genes |
| CN106399461A (en) * | 2016-09-14 | 2017-02-15 | 妙顺（上海）生物科技有限公司 | Method of detection of transcription factor expression activity by luciferase reporter gene system |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103382505A (en) * | 2013-08-07 | 2013-11-06 | 贵州大学 | Method for detecting promoter activity by utilizing double luciferase reporter genes |
| CN106399461A (en) * | 2016-09-14 | 2017-02-15 | 妙顺（上海）生物科技有限公司 | Method of detection of transcription factor expression activity by luciferase reporter gene system |
Non-Patent Citations (3)
| Title |
|---|
| 《Characterization of the DNA-binding properties of the myeloid zinc finger protein MZF1: two independent DNA-binding domains recognize two DNA consensus sequences with a common G-rich core》;MORRIS JF等;《MOLECULAR AND CELLULAR BIOLOGY》;19940331;第14卷(第3期);摘要、第1790页右栏倒数第2和4段 * |
| 《KIR基因表达调控机制及其对NK细胞功能的影响》;高晓宁;《中国博士学位论文全文数据库医药卫生科技辑》;20091015(第10期);第2.7节 * |
| MORRIS JF等.《Characterization of the DNA-binding properties of the myeloid zinc finger protein MZF1: two independent DNA-binding domains recognize two DNA consensus sequences with a common G-rich core》.《MOLECULAR AND CELLULAR BIOLOGY》.1994,第14卷(第3期),第1786-1795页. * |
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