CN107893085B - Dual-luciferase reporter gene plasmid for screening orphan receptor activity regulator and construction and application thereof - Google Patents
Dual-luciferase reporter gene plasmid for screening orphan receptor activity regulator and construction and application thereof Download PDFInfo
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Abstract
The invention discloses a dual-luciferase reporter gene plasmid for screening a regulator of orphan nuclear receptor activity, which is a firefly luciferase containing DR1 and an internal reference Renilla luciferase chimeric plasmid (p-TK- (DR1)3-Fluc-hRluc) is constructed by inserting the fragment shown in SEQ ID NO.1 into a commercial plasmid pmirGLO, and performing NheI/BglII double enzyme digestion to obtain a backbone plasmid with hRluc. The plasmid and the orphan nuclear receptor overexpression plasmid are co-transfected into cells, and the high-efficiency screening of the orphan nuclear receptor activity regulator can be realized by using the activity detection of the dual-luciferase gene.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to construction of a dual-luciferase reporter gene plasmid, wherein the dual-luciferase reporter gene plasmid can be used for screening a orphan nuclear receptor activity regulator.
Background
A orphan nuclear receptor is a nuclear receptor with no ligand or no ligand yet found. At present, more than 150 types of nociceptors have been discovered, and some of the nociceptors are involved in the metabolic regulation, embryonic development, cell differentiation, gene expression and the like of animals. In recent years, with the discovery of modulators of partial orphan nuclear receptor activity, particularly endogenous agonists, ligand-dependent nuclear receptors have been developed, such as retinoic acid X receptor, farnesoid X receptor, peroxisome proliferator-activated receptor γ, and the like. Therefore, the discovery of the regulator of the activity of the orphan nuclear receptor not only can deepen the understanding of the action of the orphan nuclear receptor, but also is beneficial to the development of new drugs for the targeted regulation of the orphan nuclear receptor.
Nocosomal receptor response elements (HREs) were first discovered when studying the effects of nuclear receptors such as vitamin D receptors, thyroid hormone receptors and vitamin a receptors. HREs are usually located in the promoter sequence of the target gene of nuclear receptors, to which the DBD of the nuclear orphan receptor can recognize and bind. HREs generally consist of two repetitive sequences, which results in better stability of DNA binding to the nuclear orphan receptor. Depending on the sequence of the combination, HREs can be divided into three forms, Direct Repeats (DRs), Everted Repeats (ERs), and Inverted Repeats (IRs), with DRs being the most common. Due to the difference in the number of bases between the two halves of the terminal sequences in the DRs, there are five DRs, namely DR1, DR2, DR3, DR4 and DR 5. The number of the spacing bases can influence the spatial configuration of the binding of the nuclear receptor and the DNA, so that the method has important significance on the binding force of the nuclear receptor and the DNA.
At present, the screening of the orphan nuclear receptor activity regulator is mainly realized by a three-plasmid system of a dual-luciferase reporter gene system. In experimental operation, the over-expression plasmid of the orphan nuclear receptor and the firefly luciferase plasmid pGL3- (DR1) expressed by the activity regulation of the orphan nuclear receptor need to be transiently transfected at the same time3And an internal reference plasmid pRL-TK. The method has poor stability, complex operation and high labor intensity, and can not be suitable for screening a large amount of medicines simultaneously. Therefore, the establishment of a stable and convenient screening method of the orphan nuclear receptor activity regulator has important practical significance.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a dual-luciferase reporter gene plasmid for screening a regulator of the activity of a nuclear receptor and the construction and the application thereof, and the invention constructs firefly luciferase containing a receptor DR1 and a chimeric plasmid (p-TK- (DR1) of internal reference Renilla luciferase)3-Fluc-hRluc), namely inserting the fragment shown as SEQ ID NO.1 into a commercial plasmid pmirGLO, and performing NheI/BglII double digestion to obtain a skeleton plasmid with the hRluc. The plasmid and the orphan receptor overexpression plasmid are co-transfected into cells, and the high-efficiency screening of the orphan receptor activity regulator is realized by using the activity detection of the dual-luciferase. The method has the advantages of simple operation, high sensitivity and strong specificity.
The construction method of the dual-luciferase reporter gene plasmid comprises the following steps:
1) carrying out amplification on an amplification mini-TK promoter primer with BglII and HindIII enzyme digestion site sequences by taking pGL3-basic plasmid as a template to obtain a promoter, and cloning the promoter into pGL3-basic plasmid after enzyme digestion to obtain pGL3-TK plasmid; the forward sequence of the primer is shown as SEQ ID NO.2, and the reverse sequence is shown as SEQ ID NO. 3;
2) will have NheI and BglII endonucleases (DR1)3The fragment was cloned into pGL3-TK plasmid to obtain pGL3-TK- (DR1)3A plasmid; said (DR1)3The fragment base sequence is a DNA double strand formed by SEQ ID NO.4 and SEQ ID NO. 5;
3) mixing pGL3-TK- (DR1)3The plasmid was digested with NheI/BamHI to give TK- (DR1)3A fragment;
4) the pmirGLO plasmid is subjected to double enzyme digestion by NheI/BglII to obtain a skeleton plasmid with hRluc;
5) the TK- (DR1) obtained in the step 3)3The fragment is connected with the skeleton plasmid obtained in the step 4) through T4 ligase to obtain the dual-luciferase reporter gene plasmid p-TK- (DR1)3-Fluc-hRluc。
The dual luciferase reporter plasmids described above may be used in the screening of modulators of orphan receptor activity. The method comprises the following specific steps:
(1) using dual luciferase reporter plasmid p-TK- (DR1)3-Fluc-hRluc cotransfects cells with a orphan nuclear receptor overexpression plasmid;
(2) and after 24-36 hours of transfection, adding the compound to be detected, cracking cells, detecting the ratio of the firefly luciferase activity to the renilla luciferase activity of cell lysate, and comparing with a control group without the compound to obtain the regulation and control effect of the compound to be detected on the activity of the orphan nucleus receptor.
The invention has the beneficial effects that:
modulators of the activity of the orphan receptor are of great significance for the study of the function of the orphan receptor and for the treatment of disorders associated with the orphan receptor. The firefly luciferase gene with the promoter region inserted into the orphan nuclear receptor reaction element and the renilla luciferase are integrated into one plasmid for the first time, the firefly luciferase gene is used as a main report gene to investigate the regulation effect of a small molecular compound on the orphan nuclear receptor activity, and the renilla luciferase is used as a reference report gene for normalization treatment, so that the influence of factors such as the experimental operation process, the transfection efficiency and the cell state difference on the result is overcome. The plasmid is applied to screening of the orphan nuclear receptor activity regulator, has the advantages of simple and convenient operation, high sensitivity, strong specificity and the like, and can realize semi-automatic and high-flux large-scale screening of candidate small molecules.
Drawings
FIG. 1 shows p-TK- (DR1)3-a plasmid map of Fluc-hRluc;
FIG. 2 shows p-TK- (DR1)3-the result of the enzymatic digestion of the Fluc-hRluc plasmid; wherein M is a molecular weight standard, 1 is a BamHI single enzyme digestion band, and the expected molecular weight is 7257 bp; 2 is a BamHI/NheI double enzyme digestion band, and the expected molecular weights are 2822bp +4435bp respectively; 3 is BglII/AseI, the expected molecular weight is 3482bp +3775 bp.
FIG. 3 shows the testis nucleus receptor 4(TR4), 9-cis-RA (TR4 agonist), metformin (TR4 inhibitor) and the test drug enzalutamide p-TK- (DR1)3Schematic representation of the regulatory role of the Fluc-hRluc reporter gene vector. Wherein COS-7 cells are transfected with TR4 and p-TK- (DR1)3After Fluc-hRluc, the ratio of firefly luciferase activity/renilla luciferase activity in the cell lysate is significantly increased; the ratio of firefly luciferase activity/renilla luciferase activity in the cell lysate of the 9-cis-RA added group was further increased; the firefly luciferase activity/renilla luciferase activity ratio in the cell lysate of the metformin-added group was significantly reduced; the ratio of firefly luciferase activity/Renilla luciferase activity in the cell lysate of the test drug, enzalutamide-added group, was significantly reduced.
Detailed Description
The following detailed description and claims of the present invention are made with reference to the accompanying drawings and specific examples, which are intended to illustrate the present invention and should not be construed as limiting the scope of the present invention.
Examples
Firstly, preparing strains, plasmids and reagents.
The strain is as follows: coli competent cell DH5a was purchased from TAKARA;
cell line: COS-7 cells were purchased from Shanghai cell Bank of Chinese academy;
plasmid: pGL3-Basic, pRL-TK, pmirGLO were purchased from Promega.
And (3) reagent sources: 2 XPCR Taq Mix, T4 DNA Ligase from Fermentas; endotoxin-free plasmid miniprep kit, high-purity plasmid miniprep kit and DNA extraction kit were purchased from Kangkang, a century company; restriction enzymes NheI, BglII, BamHI and HindIII were purchased from NEB, and the agarose gel recovery kit was purchased from Epigentics; luciferase assay kits were purchased from Promega; lipofectamine TM3000 from invitrogen; DMEM medium, fetal calf serum, trypsin, PBS buffer, OPTI-MEM medium, and streptomycin were purchased from Gkino biology, Inc., Hangzhou; other reagents are all domestic analytical purifiers.
Second, plasmid construction
(I) construction of pGL3-TK plasmid
1. Designing and synthesizing an amplification mini-TK promoter primer with BglII and HindIII enzyme cutting site sequences:
Forward(SEQ ID NO.2),5’-TCGAGATCTCTTCGCATATTAAGGTGAC-3’
Reverse(SEQ ID NO.3),5’-GCCAAGCTTGCATGCTGTTGACGCTGTT-3’。
sample addition parameters of 50 μ L PCR reaction system: mu.L (10pM) of each forward and reverse primer, 1. mu.L of 10mM dNTP, 5. mu.L of 10 XPCR buffer, 1. mu.L of LA Taq enzyme, 100ng of pRL-TK plasmid, and the amount of each forward and reverse primer was made up to 50. mu.L with ultrapure water. PCR operating parameters: pre-denaturation at 94 ℃ for 3 min; then denaturation at 94 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, extension at 72 ℃ for 20 seconds, and the cycle was repeated 34 times.
2. PCR products were purified and recovered using a gel recovery kit (ZYMO, cat # D4008) according to the protocol.
3. The purified product and pGL3-Basic empty vector plasmid were each cut 1. mu.g with the restriction enzymes BglII and HindIII. Enzyme digestion system (50 μ L): 10 Xdigestion buffer 5 u L, two restriction enzymes each 5 u L, DNA 1 u g, adding ultrapure water to make up to 50 u L. The enzyme digestion temperature is 37 ℃, and the enzyme digestion time is 2 hours.
4. And recovering the enzyme-digested fragments, and carrying out ligation reaction. Ligation reaction (20 μ L): 10 Xligation buffer 2. mu.L, vector backbone 50ng, insert 500ng, and ultrapure water to make up to 20. mu.L. Ligation was carried out overnight at 16 ℃.
5. And transferring the ligation product into escherichia coli, and selecting a monoclonal for sequencing to confirm that the vector is successfully constructed. Extracting plasmid with high purity plasmid small extraction kit (Kangji century, cat # CW0500S), measuring concentration, and storing under appropriate conditions for use. (II) pGL3-TK- (DR1)3Plasmid construction
1. Designed to synthesize a recombinant vector with NheI and BglII endonucleases (DR1)3Fragment (SEQ ID NO.4 SEQ ID NO.1 and SEQ ID NO.5 SEQ ID NO. 2):
5’-CTAGCCCGGGCTCGAGGAAGAGGTCAAAGGTCAAGGGTTCGAGGAAGAGGTCAAAGGTCAAGGGTTCGAGGAAGAGGTCAAAGGTCAAGGGTTCGA-3’，
5'-GATCTCGAACCCTTGACCTTTGACCTCTTCCTCGAACCCTTGACCTTTGACCTCTTCCTCGAACCCTTGACCTTTGACCTCTTCCTCGAGCCCGGG-3', annealing treatment: 200 μ M DNA single strand was placed in annealing buffer (10mM tris,50mM NaCl,1mM EDTA), left at 95 ℃ for 4 minutes, then placed on ice for 5-10 minutes, and stored at 4 ℃ until use.
2. The pGL3-TK plasmid 1 mu g is subjected to double enzyme digestion by using restriction enzymes NheI and BglII, and the enzyme digestion product is recovered for later use. The operation is the same as before.
3. Connecting the fragments obtained in 1 and 2, transforming the connecting product, and selecting monoclonal sequencing verification pGL3-TK- (DR1)3Whether plasmid construction was successful.
(III) p-TK- (DR1)3Construction of-Fluc-hRluc plasmid
1. The pmirGLO empty vector plasmid 1 μ g was digested by restriction enzymes BglII and NheI, and the hRluc-containing fragment (5120bp) was recovered by gel cutting for use.
2. pGL3-TK- (DR1) was digested simultaneously with restriction enzymes BamHI and NheI3Plasmid 1. mu.g, cut the gel and recover TK- (DR1)3Fragment of Fluc (2134bp), spare.
3. And (3) performing a ligation reaction on the fragments, transferring the ligation product into escherichia coli, selecting a monoclonal for sequencing, and determining that the vector is successfully constructed. Extracting plasmid, measuring concentration, and storing under proper conditions for later use.
Screening of modulators of orphan nuclear receptor activity
1. COS-7 cells (DMEM medium + 10% FBS (volume fraction)) were cultured in 96-well plates 1 day in advance, and plasmid transfection was performed until the cells grew to cover 70% of the area of action (about 24 hours).
2.96 well plates containing p-TK- (DR1) per well transfection system (100. mu.L)3-0.1 μ g of Fluc-hRluc reporter plasmid and 0.1 μ g of pcDNA3.1-vector/TR4 plasmid; the transfection reagent used was lipofectamine 3000(invitrogen, cat # L3000015), and the transfection method was according to the instructions of the reagent.
3. 24 hours after transfection, appropriate amounts of 9-cis-RA (TR4 agonist), metformin (TR4 inhibitor) or candidate small molecule compound were added to different experimental groups and incubated for 24 hours.
4. Cells were lysed and luciferase activity was detected. Cell lysis and Luciferase Activity detection Using the Dual-Luciferase Reporter Assay System (Promega, cat # E2920), the methods used follow the protocol.
5. The activity ratio of firefly luciferase/renilla luciferase is remarkably increased (about 15 times) compared with the vector group by the TR4 overexpression group through luciferase activity detection; the activity ratio of firefly luciferase/Renilla luciferase in the 9-cis-RA (20. mu.M) group was further increased by about 1.8 times based on the TR4 overexpression group; the activity ratio of firefly luciferase/Renilla luciferase in the metformin (500. mu.M) group was significantly inhibited by about 30% on the basis of the TR4 overexpression group; the test compound androgen receptor antagonist Enzalutamide (20. mu.M) group showed a lower ratio of firefly luciferase/Renilla luciferase activity than the TR 4-overexpressed group (see FIG. 3). Thus, the method of the present invention enables efficient and specific screening of modulators of the activity of the orphan receptor TR4 based on the detected activity ratio.
TABLE 1 plasmid transfection and regulator incubation protocol
". times.3" indicates that each group was made 3 replicates.
Sequence listing
<110> Zhejiang university
<120> dual-luciferase reporter gene plasmid capable of being used for screening orphan receptor activity regulator and construction and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2137
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cttatcgatt ttaccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc 60
cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct 120
tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca 180
ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgctcgaag 240
cggccggccg ccccgactct agaattatta cacggcgatc ttgccgccct tcttggcctt 300
aatgagaatc tcgcggatct tgcgggcgtc caacttgccg gtcagtcctt taggcacctc 360
gtccacgaac acaacaccac cgcgcagctt cttggcggtt gtaacctggc tggccacata 420
gtccacgatc tccttctcgg tcatggtttt accgtgttcc agcacgacga ctgcggcggg 480
cagctcgccg gcatcgtcgt cgggcaggcc ggcgaccccg gcgtcgaaga tgttggggtg 540
ttgcagcagg atgctctcca gttcggctgg ggctacctgg tagcccttgt atttgatcag 600
gctcttcagc cggtccacga tgaagaagtg ctcgtcctcg tcccagtagg cgatgtcgcc 660
gctgtgcagc cagccgtcct tgtcgatgag agcgtttgta gcctcggggt tgttaacgta 720
gccgctcatg atcatggggc cacggacgca cagctcgccg cgctggttca cacccagtgt 780
cttaccggtg tccaagtcca ccaccttagc ctcgaagaag ggcaccacct tgcctactgc 840
gccaggcttg tcgtcccctt cgggggtgat cagaatggcg ctggttgttt ctgtcaggcc 900
gtagccctgg cggatgcctg gtaggtggaa gcgtttggcc acggcctcac ctacctcctt 960
gctgagcggc gccccgccgc tggcgatctc gtgcaagttg cttaggtcgt acttgtcgat 1020
gagagtgctc ttagcgaaga agctaaatag tgtgggcacc agcagggcag attgaatctt 1080
atagtcttgc aagctgcgca agaatagctc ctcctcgaag cggtacatga gcacgacccg 1140
aaagccgcag atcaagtagc ccagcgtggt gaacatgccg aagccgtggt gaaatggcac 1200
cacgctgagg atagcggtgt cggggatgat ctggttgccg aagatggggt cgcgggcatg 1260
actgaatcgg acacaagcgg tgcggtgcgg tagggctacg cccttgggca atccggtact 1320
gccactactg ttcatgatca gggcgatggt tttgtcccgg tcgaagctct cgggcacgaa 1380
gtcgtactcg ttgaagccgg gtggcaaatg ggaagtcacg aaggtgtaca tgctttggaa 1440
gccctggtag tcggtcttgc tatccatgat gatgatcttt tgtatgatcg gtagcttctt 1500
ttgcacgttg aggatctttt gcagcccttt cttgctcacg aatacgacgg tgggctggct 1560
gatgcccatg ctgttcagca gctcgcgctc gttgtagatg tcgttagctg gggccacagc 1620
cacaccgatg aacagggcac ccaacacggg catgaagaac tgcaagctat tctcgctgca 1680
caccacgatc cgatggtttg tattcagccc atagcgcttc atagcttctg ccagccgaac 1740
gctcatctcg aagtactcgg cgtaggtaat gtccacctcg atatgtgcgt cggtaaaggc 1800
gatggtgccg ggcaccaggg cgtagcgctt catggctttg tgcagctgct cgccggcggt 1860
cccgtcttcg agtgggtaga atggcgctgg gcccttctta atgtttttgg catcttccat 1920
ggtggcttta ccaacagtac cggattgcca agcttgcatg ctgttgacgc tgttaagcgg 1980
gtcgctgcag ggtcgctcgg tgttcgaggc cacacgcgtc accttaatat gcgaagagat 2040
ctcgaaccct tgacctttga cctcttcctc gaacccttga cctttgacct cttcctcgaa 2100
cccttgacct ttgacctctt cctcgagccc gggctag 2137
<210> 2
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tcgagatctc ttcgcatatt aaggtgac 28
<210> 3
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gccaagcttg catgctgttg acgctgtt 28
<210> 4
<211> 96
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ctagcccggg ctcgaggaag aggtcaaagg tcaagggttc gaggaagagg tcaaaggtca 60
agggttcgag gaagaggtca aaggtcaagg gttcga 96
<210> 5
<211> 96
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gatctcgaac ccttgacctt tgacctcttc ctcgaaccct tgacctttga cctcttcctc 60
gaacccttga cctttgacct cttcctcgag cccggg 96
Claims (4)
1. A dual-luciferase reporter gene plasmid is characterized in that the plasmid is a firefly luciferase containing a receptor DR1 and a chimeric plasmid of an internal reference Renilla luciferase;
the plasmid is p-TK- (DR1)3-Fluc-hRluc，Is TK- (DR1)3The fragment is inserted into a backbone plasmid, the backbone plasmid is obtained by double digestion of pmirGLO plasmid with NheI/BglII to obtain a backbone plasmid with hRluc, TK- (DR1)3The base sequence of the fragment is shown as SEQ ID NO. 1.
2. A method of constructing the dual luciferase reporter plasmid of claim 1, comprising the steps of:
1) carrying out amplification on an amplification mini-TK promoter primer with BglII and HindIII enzyme digestion site sequences by taking pGL3-basic plasmid as a template to obtain a promoter, and cloning the promoter into pGL3-basic plasmid after enzyme digestion to obtain pGL3-TK plasmid; the forward sequence of the primer is shown as SEQ ID NO.2, and the reverse sequence is shown as SEQ ID NO. 3;
2) will have NheI and BglII endonucleases (DR1)3The fragment was cloned into pGL3-TK plasmid to obtain pGL3-TK- (DR1)3A plasmid; said (DR1)3The fragment is a DNA double strand formed by SEQ ID NO.4 and SEQ ID NO. 5;
3) mixing pGL3-TK- (DR1)3The plasmid was digested with NheI/BamHI to give TK- (DR1)3A fragment;
4) the pmirGLO plasmid is subjected to double enzyme digestion by NheI/BglII to obtain a skeleton plasmid with hRluc;
5) the TK- (DR1) obtained in the step 3)3The fragment is connected with the skeleton plasmid obtained in the step 4) through T4 ligase to obtain the dual-luciferase reporter gene plasmid p-TK- (DR1)3-Fluc-hRluc。
3. Use of the dual luciferase reporter plasmid of claim 1 for screening for modulators of orphan receptor activity.
4. Use according to claim 3, characterized in that it is specifically as follows:
(1) using dual luciferase reporter plasmid p-TK- (DR1)3-Fluc-hRluc cotransfects cells with a orphan nuclear receptor overexpression plasmid;
(2) and after 24-36 hours of transfection, adding the compound to be detected, cracking cells, detecting the ratio of the firefly luciferase activity to the renilla luciferase activity of cell lysate, and comparing with a control group without the compound to obtain the regulation and control effect of the compound to be detected on the activity of the orphan nucleus receptor.
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| CN103173481A (en) * | 2013-03-01 | 2013-06-26 | 上海中医药大学附属普陀医院 | Plasmid vector containing 3'UTR (untranslated regions) sequence of ABCB1 gene and reporter gene as well as construction method and use of plasmid vector |
| CN103266120A (en) * | 2013-04-18 | 2013-08-28 | 湖南师范大学 | Dual fluorescent reporter gene vector for identifying miRNA targets, preparation method and application thereof |
| CN103382505A (en) * | 2013-08-07 | 2013-11-06 | 贵州大学 | Method for detecting promoter activity by utilizing double luciferase reporter genes |
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| CN103266120A (en) * | 2013-04-18 | 2013-08-28 | 湖南师范大学 | Dual fluorescent reporter gene vector for identifying miRNA targets, preparation method and application thereof |
| CN103382505A (en) * | 2013-08-07 | 2013-11-06 | 贵州大学 | Method for detecting promoter activity by utilizing double luciferase reporter genes |
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